Triterpene compositions and methods for their applying

FIELD: organic chemistry, natural compounds, medicine, oncology.

SUBSTANCE: invention represents new saponin mixtures used for inhibition of initiation and activation of mammalian epithelial cell in pre-malignant or malignant state, for stimulation of apoptosis of mammalian malignant cell, prophylaxis of anomalous proliferation of mammalian epithelial cell, for treatment of inflammatory and regulation of angiogenesis in mammal. These mixtures are isolated form plants of species Acacia victoriae. Also, invention relates to methods for their applying. These compounds can comprise triterpene component, such as acacic or oleanolic acid to which oligosaccharides and monoterpenoid components are joined. Mixtures and compounds elicit properties associated with regulation of apoptosis and cytotoxicity of cells and strong anti-tumor effect with respect to different tumor cells.

EFFECT: valuable medicinal properties of compositions.

43 cl, 53 tbl, 50 dwg, 44 ex

 

Background of the invention

The technical field to which the invention relates.

The present invention essentially relates to the field of medicine. More specifically the present invention relates to methods of obtaining new plant compounds that have therapeutic application to mammals.

The state of the problem

Plants are a valuable source for the identification of new biologically active molecules. One of the various classes of molecules that have been identified in plants, is the class of saponins. Saponins are high-molecular compounds, which are glycosides in which the sugar component is attached to the triterpene or steroid aglycone. In particular, triterpene saponins are subject to higher interest due to their biological properties.

Were investigated pharmacological and biological properties of triterpene saponins produced by different species of plants, including fungicidal, antiviral, antimutagenic, or spermicidal contraceptives, cardiovascular and anti-inflammatory activity (Hostettmann et al., 1995). As you know, in the process of binding to the lipids in the blood saponins form complexes with cholesterol, thereby changing the dynamics of its metabolism (Oakenfull et al., 1983). Triterpene glycosides, on the anticipated by so food, been shown to reduce cholesterol in the blood and tissues of experimental animals (Cheeke, 1971). It was found that in many countries saponins are part of the medicines folk medicine and some newly developed herbal remedies.

It is known that triterpen, glycyrrhetic acid and some of its derivatives possess anti-ulcer, anti-inflammatory, antiallergic, protivogerpetical and antiviral activity. For example, some derivatives of glycyrrhetinic acid can be a means of prevention or treatment of gastric ulcer (Doll et al., 1962). Among such compounds are well known in this field are carbenoxolone (U.S. patent No. 3070623)derived complex ester of glycyrrhetinic acid, substituted at the 3’-position (U.S. patent No. 3070624), amino acid salts of glycyrrhetinic acid (publication of the Japan patent-44-32798), amide derivatives of glycyrrhetinic acid (Belgium patent No. 753773) and amide derivatives of 11-desoxycortisol acid (patent of great Britain No. 1346871). It was shown that glycyrrhetinic acid inhibits the enzymes involved in the biosynthesis of leukotrienes, including 5-lipoxygenase activity, and it is believed to be responsible for anti-inflammatory activity (Inoue et al., 1986).

It was reported that betulin acid, Pyh triterpen, is a selective inhibitor of the growth of human melanoma in xenotransplantation models Nude mice, and, as shown, is the cause of cytotoxicity due to induction of apoptosis (Pisha et al., 1995). It was demonstrated that triterpene saponin, derived from the Chinese plant family Cucurbitaceae, and antitumor activity (Kong et al., 1993). It is known that monoglycoside triterpenes show potent and selective cytotoxicity against human leukemia cell MOLT-4 (Kasiwada et al., 1992), and some triterpene glycosides of plants of family chastikovyh inhibit tumor growth and increase the lifespan of mice implanted ascitic Ehrlich carcinoma (Nagamoto et al., 1988). The preparation of saponin originating from dolichos Dolichos falcatus, belonging to the legume family, as reported effective against cells of sarcoma-37 in vitro and in vivo (Huang et al., 1982). It was shown that the soybean saponin, also representing the legume family, is effective in several types of tumors (Tomas-Barbaren et al., 1988). Oleanolic acid and gipsokartonovy glycosides, exhibiting hemolytic and molluscicidal activity, were isolated from crushed fruits legumes Swartzia madagascariensis (Leguminosae) (Borel & Hostettmann, 1987).

Genistein, a natural isoflavonoid granted the military from soy products, is an inhibitor of tyrosinekinase, and it is found that inhibits proliferation of estrogen receptor-positive and estrogen-negative cell lines (breast carcinoma (Akiyama et al., 1987). Inositoltrifosfata (phytic acid), widespread in the plant world and is a natural food component of cereals and pulses products, as we have seen, leads to terminal differentiation of the cell line carcinoma of the colon. Phytic acid also exhibits antitumor activity against experimental carcinogenesis of the colon and mammary gland in vivo (Yang et al., 1995). It was also shown that some triterpene aglycones have cytotoxic or cytostatic activity: for example, it was shown that the stem bark of the plant Crossopteryx febrifuga (Rubiaceae) has cytotoxic activity against a cell line carcinoma of the colon of a person With a 115 in the range of ng/ml (Tomas-Barbaren et al., 1988).

Although in the previous messages were identified triterpene compounds, widely used, still in the art, there is an urgent need to identify new biologically active triterpene compounds. Many of these compounds are toxic in normal mammalian cells. In addition, the biological activity of R is it identified triterpenes varies widely and many of them, apparently, characterized by limited or varying degrees of effectiveness in the treatment of any given condition of a human or mammal. A large variety of different triterpenes that were previously identified, and a wide range of variability and unpredictability in the manifestation of the biological activities observed even in closely related triterpene compounds, creates difficulties faced in obtaining triterpenes that represent potential therapeutic agents. The difficult task of identifying new triterpenes with useful biological activity should open new perspectives in the treatment of various human diseases for which therapeutic options are currently limited.

Summary of invention

The present invention relates to a new use beans and roots of acacia Victoria Acacia victoriae (Benth.) (Leguminosae) for the separation of biologically useful compounds. Seeds of Acacia victoriae are used as food by local residents of Australia for many generations (Lister et al., 1996). However, beans and roots previously discarded as waste. Therefore, the applicants of the present invention have demonstrated a new antitumor and other biologically useful compounds in those frequent the x specified plants, not previously used. For example, new biologically active saponins described in the present invention, often specific cytotoxic against tumour cells.

In one embodiment, the present invention represents a new saponine compounds and their mixtures, which can be isolated from the plant Acacia victoriae, and methods of their use. In this regard, one embodiment of the present invention is sapojnikova composition comprising triterpene or other aromatic terpenoid composition. Described here saponins may also include glycosidic group.

In the preferred embodiments, which includes saponin triterpenoid component, such triterpene component is usually acacia acid or oleanolic acid (carovillano), or other structurally similar triterpene component. Triterpene or triterpenoid connection can basically enable monoterpenoid(s) component(s), and to a person skilled in the art should be obvious that the described saponine compounds may be further substituted by other chemical functional groups. Thus, described herein saponine connection may include triterpene component that is attached, at the very the least, one, preferably two, three or more monoterpenoid components. When there is more than one monoterpenoid component, each of them can be attached (i) directly to the triterpene component; (ii) to sugar or other linking group which is attached to the triterpene component; or (iii) to monoterpenoid component, which, in turn, is attached to the triterpene component directly or through sugar or other linking group. Linking groups include sugar, acyl, amide, alkoxy, catiline, alkyl, alkylene and other similar chemical groups that are known to specialists in this field of technology. Described here triterpene glycosides usually have a molecular weight in the range of 1800-2600 Amu or from at least 1800, 1900, 2000, 2100 to about 2200, 2300, 2400 or 2600 u

An important aspect of the present invention is the separation of mixtures containing one or more selected saponins or triterpene glycosides, which can be characterized by the following properties: (a) the selectability of the tissues of Acacia victoriae: (b) the presence of at least one triterpene glycoside having a molecular weight of from about 1800 to 2600 Amu; (C) the ability to induce cytotoxicity against Jurkat cells; and (d) the ability of inducirovani apoptosis of Jurkat cells.

In specific embodiments of the present invention, the triterpene composition can be characterized by the following properties: the ability to induce cytotoxicity against Jurkat cells with IC50from about 0.12 µg/ml to about 0.40 in µg/ml. In other embodiments of the present invention induce apoptosis by introducing into the Jurkat cell concentration of approximately 100 to 400 ng/ml In other embodiments of the present invention induce apoptosis by introducing into cells Jurkat concentration from about 200 to about 250, 300, 350 or 400 ng/ml, or from about 300 to about 350 or 400 ng/ml.

In the following embodiments of the present invention, the level of apoptosis measured by the reorganization of the plasma membrane of Jurkat cells binding annexin. This can be assessed using flow cytometry, and the level of induced apoptosis may be 16-18%.

In other embodiments, the present invention relates to a mixture containing one or more selected triterpene glycosides characterized by the following properties: (a) the selectability of the tissues of Acacia victoriae; (b) the presence of at least one triterpene glycoside with a molecular weight of from about 1800 to about 2600 Amu; and (C) the ability to induce the release of cytochrome C from mitochondria Jurkat cells.

In the following embodiments of the present from which retene seems to be a mixture, containing one or more selected triterpene glycosides characterized by the following properties: (a) the selectability of the tissues of Acacia victoriae; (b) the presence of at least one triterpene glycoside with a molecular weight of from about 1800 to about 2600 Amu; and (C) the ability to activate caspase-3 in Jurkat cell, where caspana activity is in the range of about from 0.3 to 1.6 fluorescence units/min/mg.

In other embodiments of the present invention, a mixture containing one or more selected triterpene glycosides, can be characterized by the following properties: (a) the selectability of the tissues of Acacia victoriae; (b) the presence of at least one triterpene glycoside with a molecular weight of from about 1800 to about 2600 Amu; and (C) the ability to cause the cleavage of PARP protein in Jurkat cell.

In other embodiments of the present invention, a mixture containing one or more selected triterpene glycosides, can be characterized by the following properties: (a) the selectability of the tissues of Acacia victoriae; (b) the presence of at least one triterpene glycoside with a molecular weight of from about 1800 to about 2600 Amu; and (C) the ability to suppress the activity of PI3 kinase in Jurkat cell.

In other embodiments of the present invention, a mixture containing one or more private is the R triterpene glycosides, can be characterized by the following properties: (a) the selectability of the tissues of Acacia victoriae; and (b) the ability to inhibit the initiation and activation of epithelial cells of the mammal to be precancerous or malignant status.

In other embodiments of the present invention, a mixture containing one or more selected triterpene glycosides, can be characterized by the following properties: (a) the selectability of the tissues of Acacia victoriae; and (b) the ability to induce apoptosis of malignant cells in mammals.

In his important aspect of the present invention relates to natural pharmaceutical compositions containing triterpene glycoside in a pharmaceutically acceptable medium such as a buffer, a solvent, a diluent, an inert carrier oil, cream or suitable for food material. In one embodiment of the present invention the nutraceutical composition may contain dried and ground roots, beans Acacia victoriae or combinations thereof in a pharmacologically acceptable medium. Disclosed here nutraceutical compositions can take the form of tablets, capsules or ointments.

In another aspect, the present invention relates to a method of obtaining a composition containing a mixture of one or more selected triterpene glycosides, including: (a) the capture plant tissues of Acacia victoriae; (b) EC is the traction of the fabric with a solvent to obtain an extract; and (C) obtaining one or more triterpene glycosides from this extract. Usually used in this way fabrics are beans, roots, seedlings, or a mixture thereof. The solvent used for extraction may be any organic solvent that is suitable for extraction, often by dissolving need tapaninaho connection. Applicable for extraction solvents are methanol, ethanol, isopropyl alcohol, dichloromethane, chloroform, ethyl acetate, water, glycerin and mixtures thereof.

This method may further include stage.

For example, the method may additionally include a selection of songs from bagasse by filtration after extraction. In another embodiment, the method further includes a step of degreasing plant tissue using an organic solvent before extraction. The organic solvent can be any suitable for degreasing solvent, such as hexane, dichloromethane, chloroform, ethyl acetate, or mixtures thereof. In another embodiment, the allocation method further includes evaporation of the solvent after the extraction.

This method may also include receiving a mixture of triterpene compositions by chromatographic separation, at least one triterpene glycoside from the unity. Examples of methods of chromatography is liquid chromatography, GHSD or HPLC. Although the solvents that can be used for chromatographic separation, must be determined by the specialist in the art, examples of such solvents are methanol, acetonitrile, water and mixtures.

In another aspect, the present invention relates to a method of obtaining a composition containing a mixture of one or more selected triterpene glycosides, including: (a) obtaining a tissue culture comprising cells of plants Acacia victoriae; and (b) extraction of triterpene compositions of these cells with a solvent, thereby extragere at least the first triterpene compound of this tissue. In one aspect, this tissue culture includes culture of hairy roots. In another aspect of the present invention, the tissue culture are produced by infected cells Acacia victoriae cells Agrobacterium rhizogenes strain R-1000. In a related aspect of the present invention, a tissue culture includes culture medium containing sucrose in an amount of from about 3% to about 4% by weight. In another aspect of the present invention used for extraction of the composition with a solvent is methanol, ethanol, isopropyl alcohol, dichloromethane, ethyl acetate, in the a or mixtures thereof.

In another aspect of the present invention, this method also includes additional stages, such as the filtration plant bagasse from a mixture triterpenes, the allocation of mixed triterpene composition by liquid chromatography and / or evaporation of the solvent after extraction.

In one aspect describes a method of continuous playback tissues of Acacia victoriae, which can be extracted active compounds according to the present invention. In one embodiment of the present invention, describes a tissue culture hairlike roots, including cells Acacia victoriae, which were infected with Agrobacterium rhizogenes R-1000 in the appropriate culture medium. A close variant of this medium for culturing tissue contains from about 3% to about 4% sucrose.

In another aspect of the present invention, described by way of continuous collection of plant tissues of Acacia victoriae, including: (a) the cultivation of Acacia victoriae hydroponics; and (b) collection of plant tissues from approximately one to approximately 4 times a year, and this collection fabric does not cause the death of the plant. A close variant of the present invention, the system of cultivation is Aeroponics. In another similar embodiment of the present invention used for culturing tissue Acacia victoriae is the root tissue.

An important aspect of N. the present invention is a method of suppressing the initiation and activation of epithelial cells of the mammal to be precancerous or malignant status, includes introduction into the cell of a mammal a therapeutically effective amount of the nutraceutical compositions described above. In one embodiment, such epithelial cell is a skin cell, the cell colon cell cancer, cell, ovarian cell, a pancreatic cancer, a prostate cancer cell, a kidney cell, a cell of the lung, cell bladder or breast cancer cell. A close variant of the present invention, the specified mammal is man. In another similar embodiment of the present invention by way of introduction nutraceutical composition is oral administration. In an alternative embodiment of the present invention, a method of introducing a nutraceutical composition is a local introduction.

The present invention also relates to a method of inducing apoptosis of malignant cells in a mammal, comprising the processing of this cell a therapeutically effective amount of the nutraceutical compositions described above. In one embodiment, the specified cell is a skin cell, the cell colon cell cancer, cell, ovarian cell, a pancreatic cancer, a prostate cancer cell, a kidney cell, a cell of the lung, cell bladder or breast cancer cell. In a similar embodiment, the mammal is man. In another similar variations is the way of introducing a nutraceutical composition is oral administration. In an alternative embodiment of the present invention, a method of introducing a nutraceutical composition is a local introduction.

The present invention also relates to a method of preventing abnormal proliferation of epithelial mammalian cells in vitro or in the body of a mammal, including treatment of mammalian cells or the introduction of this mammal a therapeutically effective amount of the nutraceutical compositions described above. In one aspect of the present invention epithelial cells are cells of the intestinal crypts. In another aspect of the present invention, the epithelial cells are cells of the colon. A close variant of the present invention mammal is man. In another similar embodiment of the present invention, a method of introducing a nutraceutical composition in vivo is oral administration.

The present invention also provides a method of treatment of a mammal from inflammation involving the introduction of this mammal a therapeutically effective amount of the nutraceutical compositions described above. A close variant of the present invention, such mammal is man.

The present invention also is purified triterpene compound, including triterpene component connection is nnow with monoterpenoid component of the following formula:

or pharmaceutical drug, where (a) R1and R2selected from the group consisting of hydrogen, C1-5-alkyl or oligosaccharide;

(b) R3selected from the group consisting of hydrogen, hydroxyl, C1-5-alkyl, C1-5-alkylene, C1-5-alkylsulphonyl, sugar and monoterpenoid group; and (C) this formula further includes R4where R4selected from the group consisting of hydrogen, hydroxyl, C1-5-alkyl, C1-5-alkylene, C1-5-alkylsulphonyl, sugar, C1-5-Olkiluoto of ester and monoterpenoid group, and where R4can be connected to the triterpene component or monoterpenoid component. The present invention also relates to the compound in which R3is sugar. In related embodiments of the present invention, the sugar is selected from the group consisting of glucose, fucose, ramnose, arabinose, xylose, Hinojosa, maltose, glucuronic acid, ribose, N-acetylglucosamine and galactose. In other similar embodiments of the present invention, the compound further includes a monoterpenoid component connected with sugar. The present invention is a compound in which R3has the formula:

where R5selected from the group consisting of bodoro is a, hydroxyl, C1-5-alkyl, C1-5-alkylene, C1-5-alkylsulphonyl, sugar, C1-5-Olkiluoto of ester and monoterpenoid group.

In one embodiment of the present invention, R5represents hydrogen or hydroxyl. In another embodiment of the present invention, each of R1and R2includes oligosaccharide. In another embodiment of the present invention, each of R1and R2includes a monosaccharide, a disaccharide, trisaccharide or tetrasaccharide. In related embodiments of the present invention, each of R1and R2includes sugar, which separately and independently from each other selected from the group consisting of glucose, fucose, ramnose, arabinose, xylose, maltose, Hinojosa, glucuronic acid, ribose, N-acetylglucosamine and galactose. In other aspects of the present invention, at least one of the methylated sugars.

In one embodiment of the present invention, R4connected with triterpene component via one of the carbon of the methylene connected to the triterpene component. In another embodiment of the present invention, triterpene component is oleanolic acid instead acacia acid.

In another embodiment, the present invention relates to compositions containing triterpene glycoside following formula:

or pharmaceutical drug, where (a) R1represents oligosaccharide comprising N-acetylglucosamine, fucose and xylose;

and (b) R2represents oligosaccharide comprising glucose, arabinose and rhamnose. In its close variant, the present invention relates to a compound of the formula:

or pharmaceutical product.

In another aspect, the present invention relates to cleaning compositions containing triterpene glycoside having the molecular formula:

or a pharmaceutical product where (a) R1represents oligosaccharide comprising N-acetylglucosamine, fucose and xylose; and (b) R2represents oligosaccharide comprising glucose, arabinose and rhamnose. In their close aspect, the present invention relates to the purification and characterization of compounds of the formula:

or a pharmaceutical product.

In another aspect, the present invention relates to cleaning compositions containing triterpene glycoside having the molecular formula:

or a pharmaceutical product where (a) R1represents oligosaccharide comprising N-acetylglucosamine, glucose, fucose and xylose; and (b) R2the present is the focus of the oligosaccharide, comprising glucose, arabinose and rhamnose. In their close aspect, the present invention relates to the purification and characterization of compounds having the molecular formula:

In another aspect, the present invention relates to compounds, including triterpene component, oligosaccharide and three monoterpenoid unit. In one embodiment, triterpene component is acacia acid or oleanolic acid.

An important aspect of the present invention to provide pharmaceutical preparations containing purified and characterized compounds. In one embodiment, the pharmaceutical drug is a pharmacologically acceptable medium comprising a buffer, a solvent, a diluent, an inert carrier oil, cream or suitable for food material. In some aspects of the present invention, consider the pharmaceutical composition further contains an agent for targeted delivery. In related aspects of the present invention, the agent directed delivery is capable of direct delivery of the pharmaceutical composition to the epithelial cell. A close variant of the present invention, this agent directed delivery is the antibody to bind with the epithelial Clelia

In some variants of the present invention, the pharmaceutical composition contains at least one compound that can destroy the epithelial cell.

For compounds of the present invention was demonstrated chemoprotective effect in mice affected by the carcinogen DMBA. Therefore, the present invention relates to a method of suppressing the initiation and activation of the epithelial cells of the mammal to be precancerous or cancerous state in a mammal, comprising the introduction of this mammal a therapeutically effective amount of the pharmaceutical compositions described above. In one embodiment of the present invention, the epithelial cell is a skin cell, a cell, colon cell cancer, cell ovarian cell pancreatic cancer, cell lung cell bladder, a prostate cancer cell, a kidney cell or a cell of the mammary gland. A close variant of the present invention, the mammal is man. In another similar embodiment of the present invention, the method of administration of the pharmaceutical composition is an oral introduction. And in another similar embodiment of the present invention a method of administration of the pharmaceutical composition is a topical introduction. In another embodiment of the present invention, by way of introduction, the pharmacist is ical composition is intratumoral injection. In another embodiment of the present invention, the method of administration of the pharmaceutical composition is intravenous. In another embodiment of the invention, the method of administration of the pharmaceutical composition is an inhalation aerosol.

The present invention also relates to the introduction of the pharmaceutical compositions of the present invention in combination with other treatment modalities. In one embodiment, such other treatment methods are x-ray, ultraviolet, γor microwave irradiation of epithelial cells.

The present invention also relates to a method of stimulating apoptosis of malignant mammalian cells, implying that the mammal a therapeutically effective amount of the pharmaceutical compositions described in this application. In one embodiment of the present invention, such a cell is a skin cell, the cell colon cell cancer, cell, ovarian cell, pancreas cell, lung cell, a bladder, a prostate cancer cell, a kidney cell or a cell of the mammary gland.

In one of its important aspects, the present invention relates to a method of preventing abnormal proliferation of epithelial cells in a mammal, implying that the mammal therapeu the automatic effective amount of the pharmaceutical compositions described above. In one embodiment, such epithelial cell is a cell of the intestinal crypt. In another embodiment of the present invention, such epithelial cell is a cell of the colon. A close variant of the present invention, such mammal is man. In another similar embodiment of the present invention, the method of administration of the pharmaceutical composition is an oral introduction. In another embodiment of the present invention, by way of introduction is the local introduction. In another embodiment of the present invention, by way of introduction is intratumoral injection. In another embodiment of the present invention, the method of administration of the pharmaceutical composition is intravenous. In the following embodiment of the present invention, the method of administration of the pharmaceutical composition is an inhalation aerosol. The present invention also relates to the use of pharmaceutical compositions of the present invention in combination with other treatment modalities. In one embodiment, such other treatment methods are x-ray, ultraviolet, γor microwave irradiation of epithelial cells.

The present invention also relates to a method of treatment of a mammal from inflammation involving the introduction of this mammal terapeutiche the key effective amounts described herein, the pharmaceutical composition, contains triterpene compounds. A close variant of the present invention, such mammal is man. In another embodiment of the present invention, the method of administration of the pharmaceutical composition is an oral introduction. In another embodiment of the present invention, by way of introduction is the local introduction. In another embodiment of the present invention, the method of administration of the pharmaceutical composition is an inhalation aerosol.

Another important aspect of the present invention is a method of regulating angiogenesis in a mammal, involving the introduction of this mammal therapeutically effective amounts described herein, the pharmaceutical compositions. This method can be applied, if this mammal is man.

Although some of the described methods are applied in vitro, however, it is assumed that in vivo triterpene glycoside compounds will exhibit similar effects.

In addition to introducing the methods of prevention and treatment of cancer using the compounds of the present invention, the applicants are several other ways of using compounds of the present invention. In particular, the compounds of the present invention can be used as solvents, antioxidants, n is otography and antiviral agents, ichthyocides or molluscicides, contraceptives, anthelminthic, regulators of angiogenesis, means for protection against UV-radiation, means salt, diuretics, anti-inflammatory agents, regulators of cholesterol metabolism, cardiovascular effectors, antiulcer funds, painkillers, sedatives, immunomodulators, antipyretics, anti-fragility of capillaries, anti-aging manifestations, the means for increasing the content of collagen in the skin, means to enhance male sexual function and means to improve cognitive abilities and memory.

Brief description of drawings

The following drawings are part of the description of the present invention and are included to further illustrate some of its aspects. The present invention may be better understood with reference to one or more drawings in combination with the detailed description of specific options.

Figure 1. The impact of UA-BRF-004-DELEP-F001 on line human tumor cells. Figure 1 illustrates the growth inhibition of cell lines ovarian (SK-OV-3, HEY, OVCAR-3), breast (MDA-468), melanoma (A-M, Hs294t) and epidermoid cells (A431), treated with crude extract of bean plants.

Figure 2. The impact of UA-BRF-004-DELEP-F023 (fraction 23) on the transformed and not rasformirovaniya cell lines. Figure 2 illustrates the cytotoxicity exhibited by fraction 23, in the cells of the ovary (SK-OV-3, OCC1, HEY, OVCAR-3)cells, T-cell leukemia (Jurkat), prostate (LNCaP), primary tumor cells of human ovarian (FTC), human fibroblasts (FS) and endothelial cells (HUVEC). The level of cytotoxicity in normal cells was only 15-17%, while against tumor cells it was 50-95%.

Figure 3. The effect of fraction 35 (“UA-BRF-004-DELEP-F035 or F035) on line human tumor cells. Figure 3 shows the cytotoxicity manifested processed by the fraction of 35 cells of the ovary (HEY, OVCAR-3, C-1, SK-OV-3), pancreatic (PANC-1) and kidney (769-P, 786-O, A) person. The value of the IC50for these cell lines ranged from 1-6 mg/ml

Figure 4. The impact of factions on line 35 leukemia cells. Figure 4 shows that the fraction of 35 exhibits strong cytotoxicity against Jurkat cells (T cell leukemia) with IC50130 ng/ml and IC50for REH cells, KG-1 and NALM-6 (b-cell leukemia) in the range of 1-3 mg/ml

Figure 5. The effect of fraction 35 on the proliferation of endothelial cells. Figure 5 shows that the fraction of 35 is a strong inhibitor of proliferation of endothelial cells upon stimulation with bFGF or without it.

6. The effect of fraction 35 on the migration of endothelial cells of capillaries. Figure 6 shows the lack of influence is and the migration of endothelial cells of the capillaries, that confirms the absence of toxicity.

7. Shows the results of thin-layer chromatography of extracts of seedlings and calluses. Track 1 - stem calluse formed in the environment BA-IAA; track 2 - root calluse formed in the environment BA-IAA; lane 3 - callus cells of the hypocotyl; lane 4 - seedlings treated with methylammonium (100 μm) in semi-solid medium; lane 5 control seedlings grown in semi-solid medium; lane 6 - standard F023; track 7 - escape, grown in an environment VA; lane 8 - seedlings treated with 50 μm metilammonia; track 9 - seedlings treated with 100 μm metilammonia; track 10 - sprout treated with 200 μm metilammonia; track 11 - the control seedlings; and track 12 - standard F023.

Fig. See the photo SENCAR mouse (left) and hybrid mouse SENCARxC57B1 (right), Both mice repeatedly injected dose of 100 nmol DMBA within 8 weeks. For the 15th week in both mice developed multiple papillomas, however, in hybrid mice SENCAR×C57B1 papillomas were smaller and their number was less. Line C57B1 resistant to carcinogenesis and she did not develop tumors.

Figa-f Shows the slices of the epidermis of mice treated with acetone, DMBA or DMBA + UA-BRF-004-DELEP-F035. Figa: treatment with acetone for 4 weeks. Figw: treatment with acetone for 8 weeks. Pigs: treatment of DMBA on the 4th week. Fig.9D: education is denied DMBA at the 8th week. Five: treatment of DMBA + UA-BRF-004-DELEP-F035 on the 4th week. Fig.9F: treatment of DMBA + UA-BRF-004-DELEP-F035 on the 8th week.

Figa-Century Demonstrated the antioxidant effect of UA-BRF-004-DELEP-F035 against DNA after 4 weeks. Figa: shows antioxidant effect after treatment with low concentrations of UA-BRF-004-DELEP-F035 (0.1 mg in 0.2 ml). Figw: shows antioxidant effect after processing a high concentration of UA-BRF-004-DELEP-F035 (0.3 mg 0.2 ml).

Figa-Century Shows the thickness of the epidermis through 4 weeks after DMBA treatment and UA-BRF-004-DELEP-F035. Figa: shows the effect of treatment with low concentrations of UA-BRF-004-DELEP-F035 (0.1 mg in 0.2 ml) to the thickness of the epidermis. Figw: shows the effect of processing high concentration of UA-BRF-004-DELEP-F035 (0.3 mg 0.2 ml) to the thickness of the epidermis.

Fig. Shows the degree (percentage) changes in the thickness of the epidermis through 4 weeks after DMBA treatment at low (0.1 mg in 0.2 ml) or high (0.3 mg 0.2 ml) concentrations of UA-BRF-004-DELEP-F035.

Fig. Shows the degree (percentage) reduction of papillomas 8 weeks after DMBA treatment at low (0.1 mg in 0.2 ml) or high (0.3 mg 0.2 ml) concentrations of UA-BRF-004-DELEP-F035.

Fig. Shows autoradiogram PCR reaction, illustrating the amplification of mouse gene H-ras mutant 61-th codon.

Fig. Shows the initial strategy used for purification and separation of biologically active triterpene compounds from Acacia victorae.

Fig. Shown superior General scheme of the purification, isolation and characterization of active components Acacia victoriae.

Figa-Century Figa: shows the HPLC spectrum of the acetylated sugars isolated from hydrolyzed to the active ingredients found in the faction 94 (“UA-BRF-004Pod-DELEP-F094 or F094”). Figw: shows the HPLC spectrum of the acetylated sugars isolated from hydrolyzed active components found in F094.

Figa-F. Figa: shows the HPLC spectra of UA-BRF-004-DELEP-F035 and F035-B2. Figw: shows the HPLC spectra of UA-BRF-004Pod-DELEP-F094. Figs: shows the HPLC spectra F140. Fig.18D: shows the HPLC spectra F142. File: shows the HPLC spectra F144. Fig.18F: shows the HPLC spectra F145.

Figa-Century Analysis of the cell cycle cells OVCAR-3 before and after treatment (48 hours) fraction 35. The figure shows approximately 8%increase in the number of cells at the stage of G1and about 10%fewer cells in S-phase of the cell cycle after treatment with fraction 35, which indicates the lock on G1-stage. Figa: analysis of cell cycle in untreated cells OVCAR-3. Figw: analysis of cell cycle in cancer cells OVCAR-3-treated fraction 35.

Fig. Data EMSA show significant inhibition of TNF activation of NF-KB as a result of processing cells of UA-BRF-004-DELEP-F035 and UA-BRF-004Pod-DELEP-F094. The processing parameters were as follows: track 1 - no treatment; lane 2, TNF (100 RMB); is orozca 3 - UA-BRF-004-DELEP-F035 (1 μg/ml); lane 4 - TNF + F035 (1 μg/ml); lane 5 - F035 (2 μg/ml); lane 6, TNF + F035 (2 μg/ml); lane 7 - F094 (1 μg/ml); lane 8 - TNF + F094 (1 μg/ml); lane 9 - F094 (2 μg/ml); lane 10 - TNF + F094 (2 μg/ml).

Fig. Libidinously test, demonstrating inhibition of PI3 kinase action of UA-BRF-004-DELEP-F035 and wortmannin.

Fig. Analysis of gel-ordinator-page by the method of Western blotting with chemiluminescence method using antibodies specific against phosphorylated akt and total AKT. After treatment cells 1 and 2 µg/ml of UA-BRF-004-DELEP-F035 have experienced a marked suppression ACT-phosphorylation (the ACT), which was similar to the level observed after 2-hour treatment of cells 1 μm wortmannin.

Fig. Presents the results of PCR amplification of the gene GOV of four independently transformed root clones (lanes from left to right: 1 - scale KBP; 2 - positive control (plasmid DNA of strain R1000); 3 - negative control (DNA nontransgenic roots); 4-7 - four independently transformed root clones. It should be noted amplification of a fragment in the 645 BP on the material of the positive control and transformed roots.

Fig. Structure ellipsoid and ellipsoid-E (Beutler, 1997).

Fig. HPLC-separation of components F094.

Fig. HPLC-separation of components F035.

Fig. The first fractionation F94 prepreparation HPLC.

Fig. The second fractionation F094 using prepreparation HPLC.

Fig. Preparative fractionation F094.

Fig. Analysis preparative fraction D.

Fig. Analysis preparative fraction G/H.

Fig. Connection G1 after the second treatment on PFP-column.

Fig. Connection G1 after final purification on a column of C-18.

Fig. Connection D1 after purification on column (Waters C-18.

Fig. Connection D1 after final purification on a column of C-18-Aq.

Fig. Shows the connections after splitting connection D1.

Fig. Shows the connections after splitting G1.

Fig. Shows the connections after cleavage of compound B1.

Fig. The structure of the triterpene glycoside D1.

Fig. The structure of the triterpene glycoside G1.

Fig. The structure of the triterpene glycoside B1.

Fig. The effect of a mixture of triterpene glycosides (F035) on line cancerous and normal cells: F035 were evaluated for cytotoxicity in accordance with the procedures described in the examples. Activity F035 evaluated on the panel lines cancerous and normal cells, as shown in this figure. For cancer cells, the size of the IC50varied in the range of 0.2 to 5.8 µg/ml was Not established significant cytotoxicity (IC50from 15 µg/ml to more than 25 μg/ml) in normal and immortalized cell lines.

Figure 4. The cytotoxicity profiles of purified triterpene glycosides D1 and G1 in relation to lines of cancerous human cells. Purified extracts were evaluated for their activity against the following lines cancerous human cells: Jurkat (T cell leukemia), option-2 HEY (ovary), 769-P (kidneys), MDA-MB-231, MDA-MB-453 (breast). The results are presented as mean values ± standard deviation.

Figa-that is, the Effect of purified compounds D1 and G1 and a mixture of triterpene glycosides (F035) on apoptosis. The level of apoptosis was determined using test binding of annexin-V in which the cells were stained with annexin V-FITZ and the DNA content was measured with the use of iodide of propecia (PI) and analyzed by flow cytometry. Cells were incubated for 16 hours in the presence of 0.5-1.0 µg/ml of extract. After 16 hours of treatment, there was a 3 cell population. Cells that died or were at an advanced stage of apoptosis (positive FITZ-annexin-V and PI)undergoing apoptosis cells (positive for FITZ-annexin-V and negative for PI) and viable cells were not subjected to apoptosis (negative and FITZ-annexin-V and PI: bottom left graph).

Figa-Century Suppression of P13-kinase activity and ACT-phosphorylation. The ability to fosforilirovanii the phosphatidyl-Inositol (PI) was determined for immunoprecipitation Belk is R from cell lysates. Autoradiographically tested the results of kinase test in vitro after separation by thin-layer chromatography of immunoprecipitates R on the material of Jurkat cells. Figw: Suppression of phosphorylation ACT residues Ser-473 and Thr-308 purified and crude triterpene glycosides. The Jurkat cells were incubated with untreated (F035) and purified extracts of D1 and G1 for 16 hours at 37°C. Cell lysates were dispersed by the method of electrophoresis in 9%of the LTO-page and analyzed by the method of Western blotting-ECL using as probes of antibodies to Ser-473, Thr-408 and to the General ACT.

Figa-D. Suppression of TNF-induced factor NF-KB and induction of iNOS triterpene glycosides. The Jurkat cells were subjected to treatment by different concentrations of F035 (1-4 µg/ml; figa) and 2 μg/ml of purified extracts (D1 and G1; figv) for 16 hours, and NF-KB activated 100 PKM TNF for 15 min at 37°C. the DNA-protein complex was separated in a 7.5%native polyacrylamide gel and isotope labeled bands were visualizable and tested quantitatively using a Phospholmager. NOS induced in cells U-937 (figs) and Jurkat (fig.46D), as described in the Methods section. Cellular protein was separated by electrophoresis in LTO-page and tested by the method of Western blotting-ECL using antibodies against iNOS.

Fig. The effect of F035 and D1 on PARP cleavage in Jurkat cells.

p> Fig. The effect of z-vad fmk on F035-induced PARP cleavage in Jurkat cells.

Fig. The effect of F035, F094, D1 and G1 on caspase activity in Jurkat cells.

Fig. The effect of F035 on the release of cytochrome from mitochondria of Jurkat cells.

Detailed description of the invention

The present invention allows to overcome the limitations present in the art by providing new biologically active triterpene glycosides. In particular, applicants have identified and purified triterpene compounds plants Acacia victoriae. Identified compounds have strong anti-tumor activity concentrations at which there is low or no cytotoxicity against normal human cells.

Triterpene compounds of the present invention were identified as a result of targeted screening of extracts from 60 plants selected from legumes grown in arid and semi-arid regions. When selecting one of the extracts, denoted as UA-BRF-004-DELEP-F001 and isolated from tissues of acacia Acacia victoriae (Benth.) (Leguminosae), found a strong antitumor activity against various lines of human tumor cells. Then, the extract was further purified to obtain a series of fractions. In two rounds of the process was identifitsirovannykh, the contained data antitumor compounds in pure form. This extract was identified as containing saponins type triterpene glycosides. After this has developed a procedure for effective isolation of active components.

Further testing with a more purified extract has additionally enabled to detect the biological activity of this extract. The purified extract showed higher antitumor activity compared to the crude extract at concentrations that were found low toxicity or no detected toxicity against normal human cells. In addition, it was shown that this extract has chemoprotective effect in mice exposed to carcinogens.

The plant which was highlighted in this extract, Acacia victoriae, was selected based on such factors as the natural environment and weak knowledge of this species. Acacia victoriae is from Australia, but as the garden culture of this species was common around the world and is widely known as prickly acacia or graceful acacia. The trees grow at a rate of 60 to 120 cm per year, are characterized by late listopadu in the dry season and can withstand temperatures down to -15°C. Adult plants grow up to 30-50 meters in height and characterized the bluish-green tweakability leaves. In the South-Western United States, this tree usually blooms from April to may, and the beans ripen in June. Acacia victoriae finds various applications in agriculture, including use as a windbreak plantings, planting shelterbelts, as a food product, to stabilize the soil in the critical regions, and as an ornamental plant, with a low level of water consumption. The local population of Australia for many generations consume seeds of various species of acacia (Lister et al., 1996). Among acacia Acacia victoriae is the most common and widespread species, found throughout Australia: so it is the most “used” in food. Seeds of shares, usually called “wattleseed”, are in great demand as a natural component of confectionery and bread, as well as an aromatic ingredient for desserts, especially ice cream. They are also used for making high-quality coffee substitute, and among species of acacia, Acacia victoriae (Benth.) generally considered the owner of the most pleasant aroma (Lister et al., 1996). However, there's no information about how to use beans and roots of this plant.

The present invention relates to a new use beans and roots of Acacia victoriae for the purpose of isolating biologically active compounds. Applicants of this image the plants showed the presence of new anticancer and other biologically active compounds in those parts of the crop species, not previously used.

II. Purification and identification of triterpenes according to the present invention

An important aspect of the use of plant extracts as pharmaceutical compositions associated with the characterization and determination of their individual active components. The same applies to drugs triterpene saponins, which often requires sophisticated ways to highlight, recognize patterns, and analysis of their components and glycosides. For biological testing of purified compounds, it is necessary to allocate them in sufficient quantity and at sufficient level of purity.

As triterpenes and other related saponins have a relatively high molecular weight and high polarity may be necessary for their selection. A problem associated with the separation of purified saponins, is the presence of complex mixtures of closely related compounds, slightly different, or the nature of the aglycone or sugar component (nature, number, position and chirality attached monosaccharides). Difficulties also arise from the presence of labile substituents such as esters. For example, the main soybean saponin in its pure form, which is γ-mironovym derivative (BOA), is extracted exclusively water dissolve the om ethanol at room temperature. Extraction with heating (up to 80° (C) leads to the splitting of the ester component and the formation of soybean saponin-1 (Bb) (Kudou et al., 1992). In plants, saponins are accompanied by highly polar compounds such as sugars and coloring matter, including phenolic compounds, etc. and therefore they are difficult to crystallize and may be hygroscopic, making it even harder to obtain crystals.

Characterization of purified saponins also necessary due to the absence of crystalline material. The melting point does not have a precise meaning and often melting associated with decomposition. Therefore, the determination of the purity of the sample can not be based only on the determination of the melting point, the constant optical rotation or other physical parameters. A more effective test for the purity of the saponin can be developed on the basis of TLC or HPLC, and if possible, on the basis of simultaneous chromatography with an authentic sample. The color of the spots on the TLC plates after spraying of suitable reagents is an additional indicator of the alleged individual components. For example, one of the triterpene glycosides of the present invention (D1) has a retention time in HPLC 15.2 minutes. This value differs from the value for another close connection, ellipsoid-E, isolated from Arhidendron ellipticum (J.Beutler et al., 1997), which has a retention time in HPLC 12.5 minutes. Further characterization triterpenes of the present invention showed that this difference in retention time due to, at least, the difference in chirality and dual relationships in D1 compared with the data published for ellipsoid-E.

(1) Chemical cleaning

Dry-cleaning methods well known to specialists in this field of technology. These methods involve the fractionation of the crude plant extract on triterpene glycoside compounds described in this application. After baseline separation of compounds from plant material according to the present invention of interest triterpene glycosides can be further purified using methods described here, for example, by chromatography, with the aim of achievement of partial or complete purification (or purification to homogeneity). Analytical methods specifically applicable to the production of purified triterpene glycoside compounds described in the text below.

Some aspects of the present invention concern the purification, and in a particular embodiment, the substantial purification of triterpene glycosides from plant material. In a preferred embodiment of the present invention triterpene glycoside purified from the plant, classifying the of egasa to the legume family, or, more preferably, from plants of the genus acacia, and most preferably from a plant species Acacia victoriae, namely species Acacia victoriae (Benth.). The term “isolated triterpene glycoside”as used herein, means a composition that is separated from other components, where this composition is purified to any degree relative to its naturally occurring state.

Generally speaking, the term “isolated” refers to an organic molecule or group of similar molecules, which have been subjected to fractionation to remove various other components, and this song basically retains its biological activity. Used the term “purified substantially” refers to a composition which triterpene glycosides constitute the main component of the composition, so that it is about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more molecules of this composition.

In principle, there is no need to triterpene compositions of the present invention have always been isolated and is represented in its most purified state. Indeed, it is assumed that less refined products will find their application in some embodiments. For example, applicants consider the use of dried roots and beans Acacia victoria and their extracts as nutraceutical compositions. By definition, nutraceutical compositions contain a mixture of various biologically active compounds that have a beneficial synergistic effect on the body. Nutraceutical compositions of the present invention can be in the form of tablets or capsules and can be administered orally, or, alternatively, they may contain extracts of the present invention in the composition of ointments that can be applied topically. Partial purification can be achieved using fewer stages of purification in combination with (or only) different forms of the same General pattern of cleaning. For example, it is clear that chromatography on cation-exchange columns, carried out on the apparatus for HPLC, in General, enables the purification of a higher level compared with the same method, using the chromatography system of low pressure. Ways, providing a low degree of relative purification may have some benefits in the total amount of product or preservation of biological activity of triterpene compounds.

(ii) Extraction and pre-treatment

The extraction procedure should be soft as possible, because some saponins undergo changes, including catalytic hydrolysis during the aq is th extraction, the esterification of acidic saponins with alcohol extraction, hydrolysis of labile ester groups and perebazirovano. Therefore, the accuracy required in the conduct of the individual stages of the extraction procedure, for example, when conducting thin-layer chromatography.

Although there are numerous changes, known General procedures for obtaining the crude mixtures of saponins are usually based on extraction using methanol, ethanol, water or a water-alcohol solution; conduct before extraction or in respect of the extract phase degreasing, usually using petroleum ether; the dissolution or suspendirovanie obtained extract in water; shaking or washing the specified solution or suspension saturated aqueous n-butanol; and deposition (optional) saponins diethyl ether or acetone. Can also be enabled stage of dialysis in order to remove small water-soluble molecules, such as sugars (see, for example, Zhou et al., 1981; Massiot et al., 1988).

The most efficient extraction of dried plant material can be achieved using methanol or aqueous methanol. Methanol is also used in the case of fresh plant material. Although the water is usually less effective solvent for the extraction of saponins (except con the specific feasibility of obtaining water-soluble glycosides), she has a number of advantages due to more easy liabilityennosti and more pure extract. Depending on the amount of water used for extraction can be obtained either monogenoidea or bidesmosidic saponins (Domon and Hostettmann, 1984; Kawamura et al., 1988). Fresh plant material contains active enzymes (esterase), which in the case of homogenizing solvent is able to convert bidesmosidic in monodispersity. Even in the dry material can be esterase activated in the presence of water. If MOMORDICA - I (monodispersity saponin with oleanolic acid), it was found that the conversion in momartin-II (corresponding bridesmaid) occurs in water and in 30%and 60%solutions of methanol, but not in 80%of his solution and 100%methanol. In contrast, homogenates of fresh roots in methanol retain catalytic activity. However, enzymes can be inactivated first by soaking fresh roots in 4%solution of hydrochloric acid, after which, as shown, bridesmaid becomes the main component. Therefore, it is obvious that the right choice of extraction procedure is extremely important.

Methods commonly used for protein purification, such as dialysis, ion exchange chromatography and gel chromatography, applicable for the partial separation of saponin is in aqueous solution from niezapominajek components, but, usually, they are ineffective in the separation of the individual saponins because of the trend of saponins to form mixed micelles. Therefore, normally, the effective separation requires the use of organic solvents or systems “water-solvent”, which would solubilizers amphiphilic saponins in the form of monomers, which ensured that there would be no interference with the separation caused by the formation of mixed micelles.

Common problem associated with furostanolic saponins, is the formation of derivatives 22-och3in the process of extraction with methanol. However, the original 22-hydroxypentanal can be obtained either by extraction with another solvent (e.g. pyridine), or by handling methoxylamine artifacts boiling water acetone (Konishi & Shoji, 1979).

(iii) Thin-layer chromatography (TLC)

Qualitative analysis of triterpene saponins with TLC important from all points of view, the study of saponins. Plates for TLC (usually silica gel) suitable for purified saponins, and for the crude extracts, inexpensive, can be used quickly and efficiently and does not require special equipment. For spraying on these plates there are a number of available reagents for visualization (PL. 2). There are the following methods of cooking most of razprostranenieto:

Vanillin-sulfuric acid (reagent Gaudin). 1%solution of vanillin in ethanol are mixed in a ratio of 1:1 with a 3% solution of perchloro acid in water and sprayed on the TLC plate. Then add a 10%solution of sulfuric acid in ethanol and heated to 110°C.

- Reagent Lieberman-Burchard. Concentrated sulfuric acid (1 ml) is mixed with acetic anhydride (20 ml) and chloroform (50 ml). As a result of heating up to 85-90°achieved the desired color of the TLC plate.

- Chloride of antimony (III). On the TLC plate sprayed 10%solution of antimony chloride in chloroform and heated to 100°C.

- Anisic aldehyde-sulfuric acid. Anisic aldehyde (0.5 ml) is mixed with glacial acetic acid (10 ml), methanol (85 ml) and concentrated sulfuric acid (5 ml). This solution is sprayed on the TLC plate, which is then heated to 100°C.

Spraying with vanillin-sulphuric acid in the presence of ethanol and perchloro acid, for example, gives a blue or purple coloration of triterpene saponins. The use of anisic aldehyde-sulfuric acid by heating the TLC plate leads to the development of a blue or blue-violet color. Spraying the TLC plates with a solution of cerium sulfate in sulfuric acid gives a violet-red, blue, or green fluorescent areas with UV light of 365 nm (Kitagawa et al., 1984b). In some cases, the ROSTO spraying water on the TLC plate sufficient to detect the present of saponins. Additional description of the spray reagents can be found, for example, Stahl (1969).

The most commonly used in TLC solvent is a mixture of chloroform-methanol-water (65:35:10), although can be used and other solvents. The mixture of the solvents n-butanol-ethanol-ammonia (7:2:5) is applicable, in particular, for glycosides containing residues of uronic acids, i.e. it is applicable for highly polar compounds. Other commonly used solvents include a mixture of n-butanol-acetic acid-water (4:1:5, upper layer) or chloroform-methanol-acetic acid-water(60:32:12:8).

The system used for TLC of glycoalkaloids, usually include a mixture of ethyl acetate-pyridine-water (30:10:30; the upper phase). The visualization is carried out with the use of steroid reagents (anisic aldehyde-sulfuric acid) or alkaloid reagents (reagent Dragendorff, sulfate, cerium (IV). Other solvents and visualizeus reagents for TLC described by Jadhav et al., 1981 and Baerheim Svendsen & Verpoorte, 1983.

When conducting TLC is possible to carry out quantitative analysis of different types. For example, the density of points obtained by spraying a suitable reagent can be directly determined using a densitometer. Alternatively, a quantitative assessment is possible by holding the TLC separation, the scraping of interest strip plates (placed, for example, in a pair of the ode), elution of saponin and measure the UV absorption after addition of the appropriate reagent (e.g., concentrated sulfuric acid).

Plates for TLC with reversed phases are commercially available and are accurate analytical method for the study of saponins, which complements TLC on silica gel. For the manifestation of reversed-phase plates using almost exclusively a mixture of methanol-water and acetonitrile-water (for example, plates Merck RP-8 or RP-18 for high-performance TLC). Alternatively, it may be used plate glass-based DIOL HPTLC. They can be used solvents for normal TLC on silica gel and methanol-water and acetonitrile-water solvents used in OF TLC.

Examples of reagents for detection by TLC and spectral and colorimetric analysis of saponins below in table 2.

1. Thin-layer chromatography centrifugation (TSHC)

Method TSHC is planar method, close to preparative thin-layer chromatography (TLC), but there is no necessity of scraping strips with TLC-plates (Hostettmann et al., 1980). TSHC based on centrifugal force to accelerate the movement of the mobile phase through the annular TLC-plate. Plate coated with a suitable sorbent (with thickness of 1, 2 or 4 m is), rotate with a speed of about 800 rpm with the electric motor, the sample contribute to the center plate and the eluent is served by the pump through the sorbent. The elution solvent leads to the formation of concentric bands on the plate. They are removed from the edges and collect for TLC analysis. 2-mm layer of sorbent is also possible to divide the mixture in an amount of 50-500 mg.

The combination TSHC with a mixture of chloroform-methanol-water (100:30:3) and column chromatography was described in the allocation of ginsenosides (ginseng glycosides) (Hostettmann et al., 1980). Saponins have also been obtained using a mixture of chloroform-methanol-water at silikagelevye plates. Two glycoside protoparmelia-And from the roots of Eleutherococcus sentlcosus (aralia family, Araliaceae) were purified by the method TCHZ (chloroform-methanol-water, 65:35:7) after column chromatography on silica gel, and gel filtration on Sephadex LH-20 (Segiet Is represented by & Kaloga, 1991). To highlight cycloartane glycosides Passiflora Passiflora quadrangularis (Passifloraceae), the solvent system ethyl acetate-ethanol-water (8:2:1 or 16:3:2) was used at a flow rate of 1 ml/min (Orsini et al., 1987) or 1.5 ml/min (Orsini & Verotta, 1985).

Centrifugal liquid chromatograph Hitachi, model CLC-5, has been described as applicable for the separation of saponins. Chromatography on this device, make use of silikagelevye plates, as well as eluent take a mixture of cloroform-methanol-water (7:3:1 (lower phase) → 65:35:10 (lower phase)). Using this method, in General, 1 g of procedendo zapanikoval fraction was subjected to chromatography on a ring plate (Kitagawa et al., 1988; Taniyama et al., 1988).

(iv) Chromatography on an open column

He previously been described by a number of classical systems of solvents for chromatography saponins on a column of silica gel that can be found, for example, Woitke et al., 1970 and Adler & Hiller, 1985. Chromatography on an open column is often used as the initial fractionation of the crude mixture of saponins, although in some cases, it can help to get clean products. In General, however, the separation is not high, and the complex mixture are separated only partially. Other problems associated with loss of material due to irreversible adsorption and long time required for such divisions.

Chromatography on silica gel with a mixture of chloroform-methanol-water as eluent is one of the most common methods. When using two-phase system, the eluent is a saturated solution of chloroform in water. Therefore, the gradient of a mixture of chloroform-methanol-water (e.g., 65:35:5→65:40:10) can be used for source separation on silica gel methanol extract of the plant tissue. Further chromatography on columns at low pressure can be is used to obtain, for example, monogenoidea molluscicidal saponin, while bidesmosidic saponin can be isolated by chromatography on a column of silica gel with the solvent system in the form of a mixture of acetone-n-propanol-water (35:35:5) (Borel et al., 1987).

A complex mixture of triterpene glycosides were isolated from the bulbs of iris plant Crocosmia crocosmiiflora (Iridaceae). Three of them containing 2,9,16-trihydroxypyrimidine acid glycosides Polygalaceae acid, was isolated by using strategies that include chromatography of the crude mixture of saponins on an open column with silica gel-60 (60-230 μm) using a mixture of n-butanol-ethanol-water (5:1:4, upper layer) and a mixture of chloroform-methanol-water (60:29:6) as eluents. For final treatment was applied HPLC (Asada et al., 1989).

The wide use of chromatography on silica gel also possible to divide the dammarane glycosides, echinostomatidae A-D pumpkin plants Actinostemma lobatum (Cucurbitaceae). After using polystyrene gel columns MCI (Mitsubishi Chemical Industries) the appropriate fraction was subjected to chromatography with different solvents: chloroform-methanol-water(7:3:0,5; 32:8:1), chloroform-methanol (9:1; 1,1), chloroform-ethanol (17:3), ethyl acetate-methanol (4:1) and chloroform-methanol-ethyl acetate-water (3:3:4:1,5; bottom layer). In this way there was obtained pure echinostomes With, whereas selection is ctenostomata and required additional phase chromatography at low pressure, and for echinostomatidae D was necessary final separation on a column of C-18 with elution with 70%methanol (Iwamoto et al., 1987).

Some ester saponins were purified chromatographically on silica gel impregnated with 2% boric acid (Srivastava & Kulshreshtha, 1986, 1988).

Currently, in addition to the standard silica gel in the allocation of saponins using chromatography on an open column using coarse fraction sorbents inverted phases. Due to the heterogeneity of the granular composition and not too large in size speakers fit can be a gravity column. Chromatography with reversed phase, in principle, be used after the initial stage of separation on silica gel and allows you to change the selectivity in respect of the shared substances. Another possibility is the inclusion of reversed-phase separation after step KTH (DCCC, Higuchi et al., 1988).

1. Chromatography on an open column with polymeric sorbents

For many years in chromatographic practice was used dextranase media, for example, in the nozzle columns of Sephadex. The greatest distribution was received by Sephadex LH-20, although it is interesting and a series of polymers “G”.

In a recent work on the allocation of saponins was used by a new generation of polymers, in particular, in Japan. For example, the polymer Diaion HP-20 (Mitsubishi ChemicalIndustries, Tokyo) is a highly porous polymer, which is widely used at the initial stages of treatment.

Typically, polymeric carriers washed with water after sample loading order of elution of monosaccharides, small charged molecules, such as amino acids, and other substances with high solubility in water. Elution with a gradient mixture of water-methanol (or methanol) then allows to obtain fractions of saponins. Other chromatographic methods are used for allot of pure saponins.

It was also reported about the elution gels HP-20 mixtures of acetone-water. For example, when selecting bidesmosidic glycosides Quilichao acid from tubers pumpkin plants Thladiantha dubia (Cucurbitaceae) methanol extracts were passed through a column of Diaion CHP-20P and washed with water. The crude saponins were suirable 40%acetone. Further separation was carried out using chromatography on silica gel (elution with a mixture of ethyl acetate-methanol-water (6:2:1)and by HPLC (Nagao et al., 1990).

To highlight fibrinolytic saponins from the seeds of sponge-loofah Luffa cyilndrica (Cucurbitaceae), an aqueous extract was subjected to chromatography on a column of Amberlite XAD-2 with elution with methanol, followed by elution of the second column of XAD-2 40-70%methanol. Active particles were obtained in a purified state after chromatography on columns is e silica gel with elution with a mixture of chloroform-methanol-water (65:35:10, the bottom layer → 65:40:10) (Yoshikawa et al., 1991).

(v) Liquid chromatography with an average pressure (GHSD)

When required relatively large amounts of purified saponins, can be effective IHSD. Unlike commercially available equipment for liquid chromatography high pressure (ghvd), gram quantities of sample can be loaded on the column and the separation is carried out at a pressure up to 40 bar. Granular composition of such media is usually 25-40 μm, and the separation takes place quickly, which requires significantly less time than by chromatography on an open column. Direct transfer of share analytical HPLC on GHSD can be achieved using a reversed-phase media, which facilitates the choice of solvent (Hostettmann et al., 1986).

For example, molluscicide saponins of cossonii spiciform Cussonia spicata (Araliaceae) were obtained in sufficient quantities for biological testing method GHSD sorbent-8 with elution with a mixture of methanol-water (2:1) (Gunzinger et al., 1986). Indeed, this approach to the allocation of saponins from butanole extract of the stem bark requires 2 stages (one on silica gel, another on OF the material).

The selection of saponins can also be achieved by a combination GHSD, for example, on a column of LiChroprep RP-8 (25-40 μm, 46×2.6 cm) with mixtures IU anal - water, rotary localarray countercurrent chromatography (RLP) (Dorsaz & Hostettmann, 1986). In another embodiment, in IHSD use columns with axial compression Jobin-Yvon (Elias et al., 1991).

Examples of combinations of carriers and solvents that were used to highlight triterpenes from plant extracts, are shown below in table. 1.

Table 1

The application of the method GHSD for separation of triterpene saponins
PlantMediaSolventsBibliography
Cussonia spicatasilica gell3-Meon-N2O (6:4:1)Gunzinger et al., 1986
 C-8Meon-N2About (2:1)Gunzinger et al., 1986
Calendula arvensisS-8MeOH-H2O(65:35; 73:27)Chemli et al., 1987
.officinalissilica gelCHCl3-MeOH-H2O (61:32:5)Vidal-Ollivier et al., 1989
 S-18MeOH-H2O(60:40; 80:20)Vidal-Ollivier et al., 1989
Polygala chamaebuxussilica gelCH2CL2-Meon-N2O (80:20:2)Hamburger & Hostettmann, 1986
 S-8MeOH-H O (55:45)Hamburger & Hostettmann, 1986
Swartzia madagascariensisS-8MeOH-H2O (65:35)Borel & Hostettmann, 1987
Talinum tenuissimumS-8MeOH-H2O (60:40)Gafner et al., 1985
Sesbania sesbanS-8MeOH-H2O(55:45; 60:40)Dorsaz et al., 1988
Tetrapleura tetrapteraS-8MeOH-H2O (70:30)Maillard et al., 1989
Albizzia lucidaS-8Meon-N2O (6:4→9:1)Orsini et al., 1991
 S-18Meon-N2O (7:3)Orsini et al., 1991
Passiflora quadrangularisS-18MeOH-H2O (17:3)Orsini & Verotta, 1985
Hedera helixS-18the gradient Meon-N2AboutElias et al., 1991
Primula verisS-18MeOH-H2O (5:5→7:3)Calis et al., 1992
-‘-silica gell3-Meon-N2(61:32:7)Calis et al., 1992
Steroid saponins
Balanites aegyptiacasilica gelCHCl3-MeOH-H2O (80:20:1→25:25:2 and 70:30:3)Hosny et al., 1992

(vi) high-performance liquid chromatography (VAG is)

HPLC is a powerful technique from the point of view of obtaining multiharmonic quantities of mixtures of closely related compounds and in this sense it is often used as a final treatment stage. While in the method GHSD are relatively large particles (25-100 μm), granular fraction for prepreparation HPLC constitute 5-30 μm, which, therefore, provides a higher degree of separation.

Prepreparation HPLC was used to separate triglycosides oleanolic acid from the products of their partial hydrolysis. This was necessary in order to determine whether attached galactose component of the position of the glucose residue of the C-3 or C-4. The selection of isomeric saponins were carried out on a column of LiChrosorb RP-8 (7 μm; 250×16 mm) with elution with a mixture of acetonitrile-water (38:62) at a flow rate of 10 ml/min. and Detection was carried out at 206 nm and from 50 mg of the mixture (Decosterd et al., 1987).

Large-scale separation of saikosaponins a, C and d from the roots of Bupleurum Bupleurum falcatum (family Umbelliferae, Umbrellifeae) was achieved on columns with axial compression size h cm (EXT. the diameter). Preliminary purification of the methanol extract was performed by distributing the solvent and chromatography on resin HP-20. For preparative HPLC column was Packed with silica gel C-18 (particle size 0 μm; 5 kg) and was suirable at a flow rate of 210 ml/min Paladino gradient of aqueous acetonitrile. Download sample weighing 10 g was sufficient to allocate 400 mg saikosaponin, 1200 mg saikosaponin and 1600 mg saikosaponin d (Sakuma & Motomura, 1987).

Ginsenosides have been isolated from ginseng Rapach trifolius (Araliaceae) using a two-step procedure, including chromatography in the system Waters Prep 500 (column radial compression) with three successive cartridges with silica gel (300×57 mm). The eluent was the upper phase of a mixture of n-butanol-ethyl acetate-water (4:1:5), and the size of the injection charge was 4, For final purification was used prepreparation HPLC with a hydrocarbon column (Waters, 300×7.8 mm) with elution with a mixture of acetonitrile : water (86:14 80:20) at a flow rate of 2 ml/min (Lee & der Marderosian, 1988).

The only serious problem with the detection of components in the eluent when HPLC is the lack of a suitable chromophore for UV detection of the majority of saponins, although this can usually be overcome by using methods such as the determination of the refractive index, molecular weight determinations and derivatization.

However, given the small changes in gradient, in principle, can be applied UV detection at wavelengths 203-210 nm with suitable clean solvent. Successful times the population were also carried out using a gradient mixture of acetonitrile-water with UV detection. Acetonitrile is preferable to methanol at low values of the wavelength due to the lower absorption of ultraviolet radiation. If in a series of test saponins differences in polarity is not too large (for example, due to small differences in the sugar chain)may isocratic elution.

Method of separating mixtures of saponins include separation on activating column (C8) using elution gradient of aqueous acetonitrile. The amount of acetonitrile increasing from 30% to 40% within 20 minutes, resulting in a relatively weak shift of the base amount of UV absorption. More polar bidesmosidic saponins usually suiryudan faster than monogenoidea saponins, and a glucuronidase held less time than other glycosides. Nonpolar octillery media can be used for selection of the lipophilic component of saponins. Using this method glycosides hederagenin were suirvey previously, such as the less polar glycosides of oleanolic acid (Domon et al., 1984).

Application derivatizing triterpenes

Detection at short wavelengths associated with the problem of the instability of the background values due to interference caused by the presence of trace quantities of highly UV-active material may be improved by ASH analysis derivatizing triterpenes. One such possible method is the functionalization of free carboxyl groups found in the saponin was found during the quantitative determination monodesmethyl saponins. Processing glycosides of oleanolic acid 4-bromophenethylamine in the presence of potassium bicarbonate and crown ether leads to the formation of brompheniramine derivatives. These 4-brompheniramine derivatives are absorbed at 254 nm, and the detection can be performed at this wavelength due to the lack of solvent interference (Slacanin et al., 1988). Such derivatization is shown below:

An alternative method of analysis associated with obtaining a fluorescent coumarin derivatives via the formation of ester to carboxylic acid residue. Thus, the soybean saponins were analyzed and quantified for different grades and different organs of soybean using anthracene as internal standard (Kitagawa et al., 1984a; Tani et al., 1985).

2. Cleaning sample

In order to remove foreign material, which is often characterized by a high level of absorption of ultraviolet light, it may be necessary to carry out the pre-cleaning. This can be achieved, for example, by using a cartridge Sep-Pak® C18 (Guedon et al., 1989) or Extrelut® (Sollorz, 1985).

In the analysis of ionic compounds, such as compounds containing a free carboxyl group in the aglycone or residues of glucuronic acid, it is necessary to use any method of suppression of ionization in order to achieve prevention of the expansion of the peak. This can be achieved by adding to the stream of acid with low UV absorption, such as phosphoric acid or triperoxonane acid. Another possible method is the use of ionophores HPLC, in which the sliding phase add the counterion. Efficiency evaluation of ionic compounds increases due to the formation of ionic complexes with monoamine reagents. The derivatization of carboxylic groups (as listed above) is an alternative method for additives in the mobile phase, which results in a significant improvement in resolution of the peak.

The advantage of quantitative HPLC before photometric methods is that in this case can be determined the number of individual saponins in the mixture or extract. Many of the examples in HPLC get better results than when colorimetry, gas chromatography or TLC-fluorimetry.

In cases where the peak resolution of mixtures of saponins on columns HPLC with reversed phase is insufficient, can be used a number of methods, including the use of columns with hydroxicut is that chemically modified columns of porous glass column with silica gel and HPLC using borate complexes.

3. Hydroxyapatite

Hydroxyapatite (CA10[RHO4]6[HE]2) is more hydrophilic compared with silica gel and can be used together with a simple binary systems, water solvents, which facilitates UV detection. It is stable in neutral and alkaline environments. Recently received solid particles of hydroxyapatite, resistant to high pressure (up to 150 kg/cm2), which expanded the scope of application of HPLC. Saponins, distinguished only by the end pentabromo link and not multiple methods RP-HPLC, can be separated in this way (Kasai et al., 1987b). The separation of ginsenosides ginseng Rapach ginseng (Araliaceae) was achieved isocratically (acetonitrile-water, 80:20) or that gave the best results in a linear gradient mixture of acetonitrile-water (70:30→90:10) (Kasai et al., 1987b). As was established using silica gel, glycosides suiryudan as increasing polarity, i.e. in the opposite observed in RP-HPLC direction.

4. Borate ion-exchange HPLC

This method finds its application in the analysis of mono - and oligosaccharides. The best results in this case get on anion-exchange column, for example column Asahipa ES-502N™ (100×7.6 mm) of the company Asahi Kasei Kogyo Co., with 0.4 M H3IN3in 20% (V/V) acetonitrile (pH 8) at 75°C. the chromatography Conditions depend on the formation of borate complexes with CIS-dialami in sacharides component. After separation, the borate can be removed in the form of volatile mutivariate by repeated co-distillation of the resulting eluate with methanol.

5. Chemically modified porous glass

Microporous glass (MPs) is characterized by high chemical resistance and is stable in the pH range 2-12. Octadecene porous glass (IPS-OD) was obtained as a nozzle for HPLC with reversed phase and used for the rapid and efficient separation of saponins. For example, as ginsenosides and saikosaponin can be distinguished from both extracts combination medicines containing ginseng and Bupleurum, using for separating a mixture of acetonitrile-water (25,5:74,5) (Kanazawa et al., 1990a). Comparison of the MPs-OD and OD-silikagelevye columns used in HPLC for ginseng extract and a mixture of ginsenosides showed that the nature of retention was similar, however, the coefficient of performance speakers MPs-OD was less. The resolution of some pairs of ginsenosides was higher on speakers MPs-OD (Kanazawa et al., 1993).

6. Silica gel

Using mobile phases containing the DN, often intrinsic to the separation of saponins, therefore, HPLC silica gel is usually not suitable for such eluents. However, modification of the packing column makes possible the separation of water-soluble glycosides without deterioration of the column. This procedure involves first washing the column with methanol, then with a mixture of chloroform-methanol-ethanol-water (62:16:16:6) and, finally, the solvent system, which will be used for the separation (Kaizuka & Takahashi, 1983). Using, for example, 5-μm column with silica gel and aqueous eluent hexane-ethanol-water (8:2:0.5 to) can be carried out effective analysis of ginseng saponins and saikosaponins of Bupleurum Bupleurum falcatum.

(vii) Other chromatographic methods

Isolation of pure saponins requires one or, more usually, several stages of chromatographic separation in order to remove other polar components of the alcohol or aqueous plant extracts.

Has been described a number of separation methods that can be used for separation of triterpene saponins, including flash chromatography, KTH, liquid chromatography, low pressure (GHND), liquid chromatography medium pressure (IHSD), HPLC and standard chromatography on an open column (see, for example, Hostettmann et al., 1986, 1991; Marston & Hostettmann, 1991b). Information about the conditions of separation, solvent systems and Tphotos known to experts in the art in connection with the described invention. Best results are usually obtained with the use of strategies based on a combination of methods, such as methods specifically described below.

As some saponins are acidic, can be obtained and their salts, and after chromatographic purification may be necessary processing ion exchange resin with the purpose of allocation of free saponin. Examples of suitable resins are Dowex 50Wx8 (H+-form) (Kitagawa et al., 1988; Yoshikawa et al., 1991), Amberlite IRC84 (Okabe et al., 1989; Nagao et al., 1990) and Amberlite MB-3 (Mizutani et al., 1984). However, if to prevent decomposition of the necessary neutrality or careful control of the pH stages of filtration, ion-exchange resins should be excluded.

In some cases, the crude fractions of saponins were methylated (on the assumption that the presence of free carboxyl groups) in order to achieve satisfactory separation of closely related saponins (Okabe et al., 1989; Nagao et al., 1989, 1990).

1. Flash chromatography

Flash chromatography is a method of preparative liquid chromatography under pressure, which helps to save much time compared to standard chromatography on an open column. Using conventional glass column, however, the eluent is directed through the sorbent with compressed air or nitrogen, reaching the maximum pressure is about 2 bar at the top of the column. The particle size of the sorbent to some extent mitigated by the fact that the solvent is supplied under pressure, respectively, the resolution is improved.

Flash chromatography can be used as a quick alternative method in relation to methods of chromatography in open column, used for pre-fractionation. Using this method, the separation of the sample size from 10 mg to 10 g can be done in just 10 minutes. For example, this method was selected hederagenin, biogenesis and medicative glucosides from the roots of Dolichos kilimandscharicus (Leguminosae), with molluscicides and fungicidal activity. Methanol extract (3.3 grams) was fractionated on silica gel (grain size distribution 63-200 μm) column size 60×4 cm with a solvent system “chloroform-methanol-water (50:10:1) at a flow rate of 15 ml/min was sufficient for the removal of polluting materials and obtain two fractions that are rich in saponins. Purified triterpene glycosides was obtained by combining the methods KTH and GHND on C8-media (Marston et al., 1988a).

Although most applications use a silica gel sorbents, there is a distinct trend towards the use of materials for reversed-phase chromatography. Flash chromatography with reversed phase provides a separation of CA is onenow from others, the more polar components such as oligosaccharides.

2. Liquid chromatography low pressure (GHND)

Method GHND used for separation of purified saponins due to the high speed separation and ease of operation. In GHND used column sorbents which are characterized by a particle size of 40-60 microns. At pressure up to 10 bar possible high flow speed and speakers usually make glass. Commercially available pre-Packed columns of different size (for example, a series of "Lobar" Merck) are ideal for preparative chromatography saponins when the sample volume in the 50-500 mg. of High and uniform packing density columns ensures high separation efficiency. In addition, the analytical conditions HPLC can be relatively easily used for the separation method GHND considering the fact that chemistry sorbents similar (Marston & Hostettmann, 1991b).

Most of the methods carried out on a reversed-phase sorbents with elution with a mixture of methanol - water. In this case, usually made only pre-treated samples. A good example GHND is the allocation mollusciciding and hemolytic glycosides of oleanolic acid and hypsogenia of Swartzia madagascariensis (Leguminosae). Dried crushed beans were extracted with water and the extract obtained was fractionally between n-butanol and the ode. After separation of the organic phase by chromatography on an open column saponins were separated on a column of Lobar LiChroprep C-8 (40-63 μm; 27×2.5 cm) by elution with a mixture of methanol-water (75:25) (Borel & Hostettmann, 1987).

Association columns for GHND in a row allows you to increase the amount of downloads and (or) power division. This approach was used during the selection dammarane glycosides from Actinostemma lobata (Cucurbitaceae), where three Lobar column, size 27×2.5 cm were combined with each other. Also eluent contained a small amount of water (a mixture of ethyl acetate-n-propanol-water, 20:3:0,3) (Iwamoto et al., 1987).

3. Countercurrent chromatography

Separation methods in the system “liquid-liquid”, as has been confirmed, is ideal for its application in respect of saponins. Highly polar saponins especially suitable for countercurrent chromatographic separation because in this case there is no loss of material due to irreversible adsorption on the packing material of the column. This can be a significant argument in favor of this approach for the direct fractionation of the crude extracts.

4. Droplet counter-current chromatography (KTH)

Method KTH based on continuous transmission of droplets of the mobile phase through the immiscible liquid stationary phase contained in a large number of VERTIC is selected glass tubes. Sorbate (permeate) is subjected to a continuous distribution between two phases. Depending from the top or bottom was made the mobile phase in these tubes, chromatography is defined as, respectively, “descending” or “ascending”. Using the method CPTH you can separate closely related saponins and even to isolated pure products (Hostettmann et al., 1984). Indeed, some variants of separation, not adivasis using idcategory chromatography can be carried out with the use of this method. In the method KTH managed to separate the isomers of saponins, which differ only by the position of substitution of the acetate groups of the sugar residues (Ishii et al., 1984).

The number of solvent system was used for separation of saponins method KTH (see, for example, Hostettmann et al., 1986), and among them, the greatest application has found a mixture of chloroform-methanol-water (7:13:8). A mixture of chloroform-methanol-water can be used either for ascending chromatography high-polar saponins, or for descending chromatography saponins with one or two sugars and several free hydroxyl groups.

Was described by the large-scale procedure KTH intended for pre-treatment, 18 columns (300×10 mm inner diameter) using as the stationary phase of water-saturated n-BU is anola, and as mobile phase saturated n-butanol-water (Komori et al., 1983). Sometimes to obtain pure saponins spend two (or more) stage KTH.

5. Distribution chromatography centrifugation (storage complex)

A recently developed method of the storage complex are promising due to its speed and versatility (Marston et al., 1990). The method of the storage complex based on the retention of stationary phase field of centrifugal forces during the rotation of 800-2000 or more rpm, and not gravitational field. The principle of this method is a continuous process of non-equilibrium distribution of the sorbate between the two immiscible phases contained in the rotating coils or cartridges.

In the apparatus, the design of which includes a rotating coils, can be used as orbital, and neorbitalnyh movement around the Central axis. In one such apparatus, high-speed countercurrent chromatograph, has a Teflon tube of inner diameter of 1.6 or 2.6 mm, is wound in a spiral around the bobbin. The core unit consists of one, two or three of these reels. In the case of cassette design of the device cartridges are located at the periphery of the rotor so that their longitudinal axis parallel to the direction of centrifugal force. The number and volume of cartridges can vary depending on the specific use of this equipment is the same. Compared to CPD and GPD, in which the separation can last for 2 days or longer, in the method of the storage complex the same results can be obtained within a few hours. Capacity units with coils and cartridges is within gram quantities. The device with multilayer orbital series of coils used, for example, for pre-treatment cycloartane glycosides Abrus Abrus frutlculosus (Leguminosae) (Fullas et al., 1990). Molluscicide triterpene glycosides were isolated from ivy Hedera helix (Araliaceae) on another machine, the chromatograph Sanki LLN (6 cartridges with a total volume of 125 ml). A methanol extract of the fruits were distributed between n-butanol and water. Botanology fraction was injected directly into the machine in quantities of 100 mg using as mobile phase lower layer solvent mixture chloroform-methanol-water(7:13:8).

Two main saponin, asiaticoside and madecassoside, were isolated from Centella asiatica Centella asiatica (Umbrellifeae) using the separator-extractor multilayer coils Ito (RPS.), equipped with a column size 66 m 2.6 mm (inner diameter) (capacity 350 ml), rotating at 800 rpm, the Sample size of 400 mg can be separated using the solvent system chloroform-methanol-2-butanol-water (7:6:3:4; mobile phase is the bottom layer). For detection was used to T The X (Diallo et al., 1991). The same apparatus was used during the selection of triterpenoid sesame Sesamum alatum (Pedaliaceae). The bottom layer of a mixture of chloroform-methanol-isopropanol-water (5:6:1:4) was selected as mobile phase, and the load was 1.25 g (Potterat et al., 1992).

6. Combination methods

Rarely for a single phase chromatography to separate from extract pure saponin. As a rule, to obtain the desired product requires the use of several preparative methods. Using a combination of classical methods such as chromatography on an open column) and modern high-resolution techniques (such as HPLC), as proved, is sufficient to separate most of the saponins.

An example is the combination GHSD on silica gel with OF material, GHND and TLC by centrifugation for separation of saponins (Hamburger & Hostettmann, 1986). Similarly, the allocation of five triterpene saponins from Swartzia madagascariensis (Leguminosae) requires the application of chromatography in open column, GHND and IHSD (Borel & Hostettmann, 1987).

The storage complex was used in combination with flash chromatography and OPLC to highlight triterpene glycosides from Abrus fruticulosus (Leguminosae). The device with coils, arranged in several layers (solvent chloroform-methanol-water 7:13:8, the mobile phase is the bottom layer)was used for the initial cleaning, the while flash chromatography and OPLC are effective for obtaining pure substances (Fullas et al., 1990).

Sometimes for the purification of saponins may be sufficient to direct a combination of flash chromatography on unmodified silica gel flash chromatography or chromatography on an open column with reversed phases (Schopke et al., 1991).

Another strategy is based on the transmission of the extracts (after pre-separation) through highly porous polymers with subsequent fractionation of the crude sapojnikova mixtures. This approach was used for isolation of glycosides 3β-hydroxylean-12-ene-28,29-dieval acid from Nothopanax delavayi (Araliaceae). A methanol extract from the leaves and stems were distributed between hexane and water. The aqueous layer was chromatographically on a column of Diaion HP-20 and suirable water, 15% methanol, 50% methanol, 80% methanol, absolute methanol and chloroform. Glycosides was obtained by sequential column chromatography of the eluate in 80%methanol using silica gel and a mixture of ethyl acetate-ethanol-water (7:2:1) (Kasai et al., 1987). To highlight triterpene and matricectomy saponins Acanthopanax senticosus (Araliaceae), this procedure was started with fractionation of the methanol extract of the leaves on the polymer Diaion HP-20. Elyuirovaniya methanol fraction was chromatographically on silica gel (chloroform-methanol-water, 30:10:1) and all the resulting fractions were subjected to chromatography on columns LiChroprep RP-8. Okonchatelno the purification was performed by HPLC on a TSK-GEL ODS-120T (300× 21 mm; methanol-water,70:30; 6 ml/min; radioimmunoassay detection) or chromatography on a column of hydroxyapatite (acetonitrile-water, 85:15) (Shao et al., 1988).

In the method of separation of glycosides of oleanolic acid used a combination of Sephadex LH-20 (methanol), KTH (chloroform-methanol-water, 7:13:8) and HPLC (C-18; methanol-water, 65:35) (De Tommasi et al., 1991).

(viii) reaction of the coloring

The reaction triterpenes with any number of agents can be used to obtain colored compounds with the purpose of the quantitative or qualitative determination of these triterpenes. For example, aromatic aldehydes, such as anisic aldehyde or vanilla in a strong mineral acid such as sulfuric, phosphoric and perchloro acids, interacting with the aglycones, give colored products with a maximum absorption at wavelengths of 510 and 620 nm. In these reactions are considered, dehydration takes place, resulting in the formation of unsaturated methylene groups that give colored condensation products with aldehydes. When using vanillin and sulfuric acid triterpene saponins with a hydroxyl group in position C-23 give a peak absorption between 460-485 nm (Hiai et al., 1976).

Unsaturated and gidroksilirovanii triterpenes and steroids give red, blue or green staining with acetic anhydride and sulfuric acid (Abisch & Reichstein, 1960). Because the RUB is panowie saponins tend to give a pink or purple color, and steroid saponins - blue-green color, these classes of saponins can be identified by this method.

For detection triterpenes can be used a large number of other reagents, including: sulfate, cerium (IV) or salts of iron (III) and inorganic acids such as sulfuric acid, which gives a violet-red colour of the solution; 30%solution of chloride of antimony (III) in a mixture of acetic anhydride - acetic acid, which gives the reaction staining with hydroxytriazine and hydroxysteroids; chloride of antimony (III) in a mixture of nitrobenzene-methanol, which can be used to identify 5,6-dihydrobromide steroid glycosides (diosgenine and solasodine glycosides) and 5αor 5β-H-derivatives (for example, Tomatina); and carbazole, which in the presence of borate and concentrated sulfuric acid allows to detect the presence of uronic acids (Bitter & Muir, 1962).

Examples of reagents for detection and spectrophotometric and colorimetric detection of saponins below in table 2.

Table 2

Reagents for visualization of triterpene saponins
ReagentBibliography
Vanillin-sulfuric acidGodin, 1954
Vanilla-phospho what Naya acid Oakenfull, 1981
Reagent Lieberman-Burchard (acetic anhydride-sulfuric acid)Abisch & Reichstein, 1960; Wagner et al., 1984
1% cerium Sulfate in 10% sulfuric acidKitagawa et al., 1984b
10% Sulfuric acid in ethanolPrice et al., 1987
50% Sulfuric acidPrice et al., 1987
p-Anisic aldehyde-sulfuric acidWagner et al., 1984
Reagent Komarovsky (p-hydroxy-Benzylalcohol-sulfuric acid)Wagner et al., 1985
Chloride of antimony (III)Wagner et al., 1984
BloodWagner et al., 1984
Water 

(ix) Isolation of triterpene glycosides from Acacia victonae

The beans extracts were prepared by extraction with a mixture of chloroform-methanol or dichloromethane-chloroform in the University of Arizona in Tucson (Tucson, AZ). Applicants have identified a mixture of triterpene glycosides from acacia Victoria Acacia victoriae (Benth.) (Leguminosae). The first collection of UA-BRF-004-DELEP-F001 was obtained as follows: (1) grinding in a mortar of Wiley to particle size of 3 mm; (2) available in two-liter containers for percolation; (3) extraction of the crushed biomass with a mixture of dichloromethane-methanol (1:1) for 4 hours, followed by settling in for the night, then United faction which was dried in vacuum to obtain UA-BRF-004-DELEP-F001 (52 g). Next F001 (51,5 g) were extracted with ethyl acetate to obtain active insoluble material (34,7 g), denoted F004. Flash chromatography on silica gel (1.7 kg; Merck; particle size 23-220 μm) was used for fractionation F004 (34,2 g), then 51 fraction 670 ml was suirable a mixture of dichloromethane-methanol (stepwise gradient from 95% to 0% dichloromethane and 5% to 100% methanol). The column is washed with 9 l of methanol, then 6 l of a mixture of methanol - water (80:20) and, finally, 6 l of the same solvent with addition of 1% formic acid. The TLC fractions 23-34 and 39-40 were combined in and 17.2 g of the drug F023. Liquid chromatography medium pressure (GHSD; system Buchi-632) was applied twice, each time with 8 g F023, column $ 14.9×46 cm, Packed sorbent LiChropep C18 (particle size of 15-25 μm) using a gradient mixture of acetonitrile - water(0, 10, 20, 30, 50% acetonitrile in water) followed by rinsing with absolute methanol. 16 g of 0-20% acetonitrile received seven grams faction F027, which turned out to be inactive. The rest of the material were combined and subjected to repeated GHSD in the same system using 30-40% acetonitrile to minimize the interleaved and obtaining fractions F028-F036. Although most of these fractions showed antitumor activity, for further analysis and evaluation of selected F035 (fraction 35) (maximum output 2,19 g).

III. Structural characteristics of the as triterpenes

For quantitative and qualitative analysis triterpenes and their activity can be used a number of methods, including: analysis ichthyocides activity, gravimetric analysis, spectrophotometry, TLC, GC, HPLC, HMQC, HMBC, NOESY, COSY, NMR, rentgenocraniology etc. Analysis based on the classical properties of triterpene saponins (surface activity, toxicity to fish) mostly replaced by photometric methods such as densitometry, colorimetry derivatives, and, more recently, GC, HPLC and, in particular, NMR. Spectrophotometric methods are characterized by very high sensitivity, but usually unsuitable for analysis triterpenes in the composition of the crude plant extracts because these reactions are not specific and painted products can form compounds accompanying triterpenes, such as phytosterols and flavonoids. Another problem commonly encountered in the analysis of saponins, associated with incomplete extraction of the plant material. However, there is a wide range of suitable methods for the quantitative analysis triterpenes.

When determining the structure of saponins is necessary to solve a number of basic tasks: to determine the structure of natural aglycone; to establish the structure and sequence of monosaccharides in the carbohydrate component; to determine the type of tie is th monosaccharide links between them; to determine the anomeric configuration of each glycoside-linked monosaccharide level; to determine the position of the carbohydrate component in the aglycone.

For a final conclusion about the structure you need to use different methods. Structural analysis usually has a stepped manner in which the saponin is gradually divided into smaller fragments that become separate objects spectrometric analysis. By impartial data on individual pieces, you can build the basic structure of saponin.

Typically, the amount of purified saponins little, so when determining their structure is preferable to use high-sensitivity, high-resolution and, if possible, undamaged methods. Modification methods NMR spectroscopy and mass spectroscopy (MS) provide the necessary capabilities to analyze complex of saponins. By combinations of these and other methods may be implemented effective structural analysis. For example, the method FAB-MS receive information about the molecular weight, and in many cases, the sequence of sugars, while the methods of one - and two-dimensional NMR enable you to map the sugar of communication and help to define the structure of the aglycone. Such structural analysis and chemical analysis are discussed in detail in absolutepage & Kasai, 1984.

(i) Nuclear magnetic resonance (NMR)

Of all the modern methods of structure analysis of oligosaccharides and glycosides NMR spectroscopy allows you to get the most complete information, regardless of the presence or absence of prior information about the structure (Agrawal, 1992). This is the only approach that, in principle, can provide a full definition of the structure without the use of any additional method.

1.13C-Nuclear magnetic resonance

13C-NMR spectroscopy, which is widely used at present for determining the structure of the saponins, is a fast and nondestructive method, although it requires a sufficiently large number of sample (milligramme quantities). Analysis of the obtained spectra allows to make conclusions about the positions in which the glycosidic chain attached to the aglycone; sequence, the nature and number of monosaccharides; configuration and conformation militating relations; the presence of acylglycerides in chains; on the nature of the aglycone and the structure attached esters of acids.

To determine the chemical shifts, it is useful to compare the obtained data with the data published for standards and related compounds. Reference values of chemical shifts in the spectra of13C-NMR for triterpene saponin can use the SQL data received for glycoside myogenin (Domon & Hostettmann, 1984). In addition, data were collected (Nakanishi et al., 1983)13C-NMR signals set for Oleanna (Patra et al., 1981; Agrawal & Jain, 1992), ursan, Lupan (Wenkert et al., 1978; Sholichin et al., 1980), hopane (Wenkert et al., 1978; Wilkins et al., 1987) and lanostane (Parrilli et al., 1979). Similar data for dammarane glycosides in a systematic review (Tanaka & Kasai, 1984); and are described13C-NMR spectroscopy nicogenin (Tori et al., A) and saikosaponins (Tori et al., 1976b). Were investigated sapogenin ginseng and related the dammarane glycosides (Asakawa et al., 1977). Also described13C-NMR spectroscopy acacia acid (Kinjo et al., 1992).

When13C-NMR in the case of derivatization of hydroxyl groups, i.e. glycosylation, methylation or acetylation), αand βcarbon atoms in the sugar and the aglycone exhibit characteristic changes. For example, α-CH-signals are shifted in the direction of weak fields, while signals βmoved apart in a strong field, and the shift is a result γ-high field shift. Thus, glycosylation of the aglycone gives the shift towards weak fields γand high field shift (shifts glikozidirovaniya) adjacent carbon atoms (Tori et al., 1976b; Kasai et al., 1977). In cleaning glikozidirovanie group 3βHE gives the shift of C-3 in the weak field primernaya 8,0-to 11.5 ppm, atoms C-2 and C-4 to +0,9 or from -0,9 -1,9 to ppm, atom-23 in a strong field of 0.5 to 5.1 ppm and atom-24 between-0.2 to +1,6 ppm Glikozidirovanie group 28-COOH gives the shift of the carbon carboxyl group in strong fields (2.5 to 5.0 ppm)and the signal of the C-17 in the direction of weak fields (1.0 to 2.5 ppm) (Agrawal & Jain, 1992). Therefore, comparison of data13C-NMR of saponin aglycone and allows you to define the place of attachment of sugar (Seo et al., 1978; Tanaka, 1985).

Similarly, data13C-NMR allows to determine (in a simpler saponins) position militating relations on the basis of the comparison shift chemical shift data for model compounds (Konishi et al., 1978). Data13C-NMR for methyl-β--fucopyranoside were brought from Seo et al., 1978, while13C-NMR signals for more complex sugars are Gorin & Mazurek (1975) and Dorman & Roberts (1970). Apiose gives typical13C-NMR data, and these data were registered (Sakuma & Shoji, 1982; Adinolfi et al., 1987; Reznicek et al., 1990).

2.1H-Nuclear magnetic resonance

Although the analysis of spectra13C-NMR and estimation of signals in them is a particularly effective method of determining the structure of the saponins, however, the full interpretation of the spectra of the1H-NMR is rare. Proton1H-NMR spectra of complex and tedious to analyze. The vast majority of the proton resonances of the carbohydrate composition of the Commissioner is in a very narrow spectral range is 3.0-4.2 ppm, that creates a serious problem of their overlapping. This is due to the total mass of nanomeric marinovich and methylene protons of the sugars, which are characterized by very similar values of chemical shift in different monosaccharide residues.

However, methyl peaks triterpenes easily distinguishable, so most of the proton resonances in structures oleanane, Ursina and family structures were determined after 1960 with the use of different methods (Kojima & Ogura, 1989). For example, the full interpretation of the spectra of1H and13C-NMR for sapogenols In soybean (33) and the configuration of its hydroxymethylene substituent at C-4 were established by a combination of methods13C-DEPT,13C-ART, two-dimensional correlation spectroscopy (COSY:1H/13C-COSY;1H/1H-COSY) and1H/1H-ROESY (two-dimensional nuclear strengthening the Noe in the rotating coordinate system) (Baxter et al., 1990). The results of decoding Quaternary carbon resonances in this sapogenin confirmed using1H-heteronuclear spectroscopy with multiple (NMS) and single bonds (HMQC) (Massiot et al., 1991b). Also achieved a complete interpretation of spectra1H-NMR of diosgenin and solasodine (Puri et al., 1993).

Some useful data can be obtained from spectra1H-NMR for anomeric configurations and relationships sakh the nuclear biological chemical (NBC chain. For example, the constant spin-spin interaction of the proton With-1 α-linked glucose units is about 3 Hz, while β-related links have constant interaction 6-7 Hz. Other details about the interaction constants of the anomeric protons in sugars can be found in various sources (Lemiex et al., 1958; Capon & Thacker, 1964; Kizu & Tomimori, 1982).

If you have any difficulties in determining the configuration of the hydroxyl groups at C-2, C-3 and C-23, C-24 leanedover and arsenevich triterpenes, valuable information can be obtained in the analysis of peaks1H-NMR signals of protons oxygen-bearing carbon atoms (Kojima & Ogura, 1989).

(ii) Methods of one-dimensional and two-dimensional NMR

In practice, some spectra1H and13C-NMR can be identified and decoded on the basis of the parameters changes, however, for accurate interpretation of the results of NMR studies requires unambiguous decoding of the NMR spectrum, which will establish the relationship of individual peaks with the corresponding carbon atoms and / or hydrogen in the structure. In many cases, such information cannot be obtained on the material of the one-dimensional spectra1H and13C-NMR, but can be set by using the two-dimensional study. Such studies facilitate spectral analysis by distributing information on the two frequency dia is asanam and define interactions between nuclei. Although the mechanisms of formation of different sequences of pulses can be very confusing, interpretation of two-dimensional NMR spectra are usually very simple. To establish the chemical structures was a significant number of studies using two-dimensional NMR spectra. Examples of such methods, as well as other NMR methods specifically addressed by the applicants in connection with the use for chemical analysis of triterpene saponins of the present invention, described below and in table 3.

1. NMFS, HMQC

Using spectra multiquantum coherence13C HMQC and NMFS informative not only in connection with the identification of the aglycone, but also for verification of the sequence of sugars. Application of methods of HMQC and NMFS similar heteronuclear correlation spectroscopy13C1H (HETCOR), but instead surveillance13Since there is more abundant1H. for Example, in the case of ellisonian, vydeleny from Daisy Bellis perennis (Asteraceae), it was possible to identify all chemical shifts for spectra1H-NMR by reviewing data13C-NMR-spectrum in combination with two-dimensional1H-detectivesyme HMQC spectra and NMFS. For almost all possible correlations in the molecule were observed cross-peaks corresponding to the interaction of the TLD is or three links. Similarly, large-scale correlation1H13C HMQC spectra and NMS can be used to determine the sequence and conditions attaching sugar residues (et al., 1991).

2. Two-dimensional NOESY

This method was applied, for example, to determine the sugar sequence in glycosides cycloserine-a (artisically a and b) (Jansakul et al., 1987) and the sequence of monosaccharides in saxifragales And isolated from Polanica'androsace saxifragifolia (Primulaceae) (Waltho et al., 1986). Position rumeilah and glucosimine links on arabinose component of kalopanagiotis With confirmed using NOESY-based analysis of the sugar sequence permitieron saponin. Observed specific peaks for N-1 remoteley component and N-2 arabinosyl component, and N-1 glucosamine component and N-3 arabinosyl component (Shao et al., 1989b). The structure of the sugar components furostanolic saponins isolated from Balanites aegyptiaca (Balanitaceae), was determined using two-dimensional NOESY on the NMR spectrometer at 400 MHz (Kamel et al., 1991).

Consistent application of the methods of two-dimensional NMR allowed us to interpret the spectra of13C and1H for oligosaccharide segment sarsapogenin of glycoside 3-O-[{α-L-rhamnopyranose (1→4)}{#x003B2; -D-glyukopiranozil (1→2)}-β-D-glyukopiranozil]-(255)-5β-spirostan-3β-Ola. The combination of methods DEPT, HETCOR, long range HETCOR, various homoatomic methods, NOESY and INEPT was used for structural analysis in order to resolve problems associated with excessive saturation of the proton spectrum (Pant et al., 1988d).

Identification and determination of the sequence of sugars in pentasaccharides the saponin 3-O-[β-D-xylopyranosyl (1→3)-α-L-arabinopyranosyl] (1→4)-β-D-glyukopiranozil (1β3)-α-L-rhamnopyranose (1→2)-α-L-arabinopyranosyl] hederagenin isolated from plants Blighia welwitschii (Sapindaceae), became possible when using only methods NMR using a spectrometer with a frequency of 500 MHz. First saponin has azetilirovanie, and subsequent analysis of the spectra DQF-COSY, NOESY and ROESY allowed to determine the structure. The most valuable information to establish the sequence of sugars was obtained using NOE (Penders et al., 1989).

Saponin, including 10 sugar residues isolated from giant goldenrod Solidago gigantea (Asteraceae), was identified by NMR, based on multi-stage RCT analysis. This analysis included methods COSY, heteronuclear COSY, H-N-s-coherent transfer COSY-type and two-dimensional NOESY. Thus, were excluded analysis, coupled with the significant Khimich the ski destruction of the molecule, and to establish patterns only took 30 mg of product (Reznicek et al., 1989a, 1989b). Similar methods were used in the structural analysis of four other glycosides, gigantospenos 1-4 (bidesmosidic myogenin, including 9-10 sugar units)produced by the same plant species (Reznicek et al., A).

The combination of NMR methods two-dimensional COSY, HMBC and ROESY was effective to establish sequence and site links in hexasaccharide, part of limonoid, which is a saponin oleanolic acid isolated from Mimosa small-flowering Mimosa tenuiflora (Leguminosae) (Jiang et al., 1991).

Method two-dimensional NMR perceiving and rederivation horizantally-And allowed us to map the protons and to determine the sequence of sugars. As shown, esterified residue xylose is β-shape and has a1C4-conformation. In relation perceiving derived methods used HMQC, NMFS and ROESY (or, more precisely, the method CAMELSPIN (transverse relaxation in mesomolecular emulated localized spins), and in relation to native saponin used methods NONANE and TOCSY. In particular, for determining the sequence of sugars used the method ROESY (Massiot et al., 1991a).

The sequence of sugars and migliozzi communication triterpene glycosides of sea organisms were fitted the us according to NT 1in the analysis method NOESY (Miyamoto et al., 1990), however, this approach has been limited due to the complexity of the obtained spectra1H-NMR in the range of 3-5 ppm, which usually prevents the determination of NOE when a large number of protons. However, the combination of methods COSY, NOESY, direct NMR and XHCORR-NMR allowed us to decipher the signals and to determine the structure pentasaccharide triterpene saponins isolated from the sea cucumbers Holothuria forskalii (Rodriguez et al., 1991).

In the structural analysis of santiaguito, which astroshamanism isolated from Antarctic sea stars Neosmilaster georyianus, applied the methods of COSY, TOCSY, HMQC and ROESY NMR and method data ROESY used to determine the exact sequence of carbohydrates, the place of their connection and stereochemical structure (Vazquez et al., 1992).

3. COSY

There are two fundamental types of two-dimensional NMR spectroscopy: J-resolution spectroscopy, in which one frequency axis characterizes the spin pair (J), and the other carries information about the chemical shift; and correlation spectroscopy, in which both the frequency axis characterize chemical shifts (value δ) (Agrawal, 1992). One of the main advantages of the two-dimensional analysis is that it allows you to overcome the problem of overflow of the spectra. This is particularly important in relation to the range of 2.5 to 4.0 ppm in the analysis of high field H-COSY, as it allows to simplify the mapping of the protons of the sugars. Under favorable conditions can be identified all the protons present in the composition of the carbohydrate residue.

On the basis of COSY spectra it is possible to draw some General conclusions. For example, the position of Vice monosaccharide units can be identified by the presence or absence of the corresponding hydroxyl protons; the ring size of the monosaccharide can be determined directly; and the nature of matching peaks reflects multipletness overlapping peaks, which gives the opportunity to evaluate the constant of spin-spin interaction.

In some cases the definition of the structure of saponin, along with the sequence of sugars, can only be performed using two-dimensional and one-dimensional spectroscopy1H-NMR (Massiot et al., 1986, 1988b). First saponin will peracetylated and when sufficient field strength (>300 MHz) spectrum of the proton resonance of sugars is split into two areas: one in the range of 4.75-of 5.40 ppm, corresponding to Seas, and another in the range from 3.0 to 4.3 ppm, corresponding to the CH2SLA, CHOR and CH2'OR. Anomeric protons are between the two fields in the presence of ether bonds, and in excess of 5.5 ppm in the case of ester bonds.

When parazitirovanii also receive a derivative that is soluble is in chloroform, benzene or acetone. Equivalent perdetermined solvents mobility of the molecules is such that the signals detected more clearly and constants of the interaction can be determined with high accuracy. Analysis of acetylated saponin alfalfa roots methods COSY and COSY long range was sufficient to identify its structure, Aga2Glc-2Aga-3hederagenin28-GLC (Massiot et al., 1986).

Patterns additionally peracetylated saponins isolated from the leaves of alfalfa seed Medicago sativa (Leguminosae) and Tridesmostemon claessenssi (Sapotaceae), were determined using the same methods mentioned above. Confirmation of position and sequence of the sugars obtained using HMQC (1J-mates) and NMFS (2J and3J-mates) and homoatomic relayed by the Hartmann-Hahn (NONANE) methods COSY and ROSEY (Massiot et al., 1990, 1991b). Ester of a sugar chain saponins .claessenssi include balance β-D-xylose in an unusual configurationlC4(all Vice-axial). At 600 MHz spectrum1H-NMR can be effectively resolved with the determination of the chemical shifts of all protons without paramilitary (Schopke et al., 1991).

4. COSY long range

This method was used for mapping of the sugar protons in steroid saponin isolated from onion Allium vinele (Liliaceae) (Chen & Snyder, 1987, 1989). Method1H13C COSY long range is also used for analyzing the structure of the aglycone in cyclooctanone the saponin (Wang et al., 1989b) and to determine the position of miscarrage4The J-coupling between the anomeric protons in the internal balance of glucose and N-2 internal balance of arabinose in the above saponin Medicago sativa (Massiot et al., 1986).

5. Phase-sensitive COSY with two-quantum filtered (DQF-COSY, DQ-COSY)

This method was applied for analysis of the sugar protons in steroid saponins bows of the genus Allium (Chen & Snyder, 1987, 1989) and determination of the proton chemical shifts of glycosides 16α-hydroxyprednisolone acid from the roots of Crossopteryx febrlfuga (Rubiaceae) (Gariboldi et al., 1990). The same method was used for the complete analysis of the protons of the saccharide in the acetylated derivative hederagenin of the fruits sapinda Sapindus rarak (Sapindaceae) (Hamburger et al., 1991). For analysis militating links used NOE spectroscopy (Hamburger et al., 1992).

6. NONANE

System proton mate aglycones glycosides hypsogenia and Quilichao acid was completely decrypted using the method NONANE. It is similar to COSY (and close to the method pornocastellano spectroscopy - TOCSY), except that the observed match correlation peaks are in the same phase, thereby predotvrasheniye zeroing overlapping peaks. To determine the carbohydrate chains constants vicinal pair obtained in the method NONANE, allow us to estimate the relative stereochemistry of the asymmetric center, thereby providing identification of individual monosaccharides. Heteronuclear1H13C-correlation analysis can be used to analyze the resonance13With the sugar components and sugar relationships defined by the spectra NMS (Frechet et al., 1991).

7. FLOCK, COLOC and NOE

Heteronuclear correlation spectroscopy long range, including bilinear spin-decoupling pulses (FLOCK)was used to analyze remote mates1H13C AutoSize And isolated from sesame Sesamum alatum (Pedaliaceae). Thus, it was found the interaction of the proton at C-18 and carbon-13, C-17 and C-28. In combination with heteronuclear13C1H correlation analysis of long range (XHCORR) most information was obtained about the structure of the new skorenovac aglycone (Potterat et al., 1992).

An example of the application of two-dimensional correlation spectroscopy13C1H (COLOC) for analysis of long-range mate (2JCHand3JCHis the elucidation of the structure of saponins isolated from Crossopteryx febrifuga (Rubiaceae) (Gariboldi et al., 1990).

NOE finds wide application in the analysis of the structure of saponins, for example, when Cartaromana sharidny protons and sequencing sugars superside-I (Okabe et al., 1989) and Kamaldinov-I and II (Nishino et al., 1986). NOE between H-2 arabinose and anomeric proton ramnose helped confirm the presence of disaccharide glycosides of communication Rha-2Ara in sisitine (Yoshikawa et al., 1991b). This method is widely used due to the frequent detection of coherence between proton aglycone and anomeric proton at the binding site. Negative NOE was observed between the proton at C-3 and the anomeric proton of the 3-O-glycoside residue of cycloartenol and other saponins (Wang et al., 1989b).

(iii) Spectroscopy and other methods of structural analysis

Structural analysis of saponins and their corresponding aglycones is based not only on chemical methods, but the spectroscopic and close methods, for example, infrared, ultraviolet, NMR, mass spectroscopy (MS), x-ray analyses, the analysis of optical rotary dispersion (ORD) and circular dichroism (CD). Modern advances in these methods, especially noticeable in NMR spectroscopy and mass spectroscopy, facilitated the analysis of saponins and their respective fragments after cleavage reactions, and thus the information obtained can be generalized and can be identified patterns. Moreover, NMR spectroscopy is nedestro the active method, while NMR and MS allow you to analyze intact saponin.

The necessary integrated approach to the elucidation of the structure of saponins with the inclusion of various spectroscopic methods, each of which contributes to the data.

1. Mass spectroscopy (MS)

The choice of method of ionization for MS depends on the polarity, availability, and molecular weight of the analyzed compounds. The principal is that the so-called “soft” ionization methods, such as FAB and desorption chemical ionization (DCI), used to obtain data about the sequence of sugars in the molecular mass of natural glycosides (Wolfender et al., 1992). This allows you to explore glycosides without derivatization. In some cases, fragmentation occurs aglycones, and for this purpose more applicable mass spectra of electron impact (EI-MS).

2. MS, fast atom bombardment (FAB-MS)

In FAB-analysis of the accelerated beam of atoms (or ions) are released from the ion gun toward the target, which was pre-loaded in a viscous liquid (so-called “matrix”, usually glycerol or 1-triglycerin)containing the analyzed sample (Barber et al., 1981, 1982). When the beam of atoms is faced with the matrix, its kinetic energy is transferred to surface molecules, a significant number of which is sprayed from the liquid in a high vacuum, stanova the ü ion sources. Ionization of many of these molecules occurs in the process of spraying, resulting in the formation of positive and negative ions. Any of them can be registered by selecting the appropriate settings on your device, though, as it has been shown that negative ions are generally more applicable when working with saponins.

3. Mass spectrometry of secondary ions (SIMS)

This is another method of desorption caused by the accelerated particles: Kev ions falling on the surface of a thin film of biological molecules, induce the same desorption ionization, as in the method PD-MC (Benninghoven & Sichtermann, 1978). Confirmed the applicability of this method in structural analysis of three new bidesmosidic, acetylcodeine A1, A2and a3soy, isolated from seeds of soybean Glycine max (Leguminosae). Ion peaks of the main fragments gave information about the nature of acetylation monosaccharide units, and sequencing of these links (Kitagawa et al., 1988).

4. Laser desorption (LD)

When LD was shown that excitation with short laser pulses (<10 NS) gives a picture of the desorbed molecular ions, similar to the pattern produced when PD and SIMS. Mass spectrometry laser desorption, Fourier transform (LD/FTMS), a method that is also applicable for the analysis of complex glycosides, p is adequate to obtain spectra, which differ from the spectra FAB-MC, but complementary to them.

5. MS method field desorption (FDMS)

This method is applicable for the determination of molecular masses of saponins along with the determination of the number, nature and sequence of the sugar residues (Komori et al., 1985).

However, the experimental complexity of the FDMS and the fact that the spectra FAB-MC require a longer time to measure, determined the decrease in recent years the popularity of the method FDMS. Mass spectra of FD are characterized by an additional disadvantage associated with their complication in the presence of nationsbank fragments, which significantly complicates the interpretation of results. At the same time, the method FDMS was with great success used in the structural analysis of saponins (Hostettmann, 1980).

(iv) Liquid chromatography with mass spectrometry (LC-MS)

Currently there are several types of effective interfaces (connecting devices) for the direct or indirect supply effluent with HPLC-columns for mass spectrometry. For example, qualitative analysis of the fractions of the crude saponins were carried out by combining polymicro-HPLC interface for fast atom bombardment (FRIT-FAB) (Hattori et al., 1988). For this analysis use the NH2-column (such as column μS-Finepak SIL NH2, Jasco; 25cm × 1.5 mm inner diameter), which is better C18 column with octadec what silicagel when the dividing ratio output stream 1:20 (i.e. 100 μl/min → 5 μl/min). Elution in a linear gradient of acetonitrile and water containing 1% glycerol, should be the norm to provide better clarity of the peaks compared to by isocratic elution. Mass spectra of negative FAB was registered for saponins with a molecular weight of up to 1235 u With the application of this method revealed pseudomolecular ions [M-1]-and fragment ions associated with splitting of sugar components (Hattori et al., 1988).

Has also been described system FRIT-FAB LC-MS, intended for separating a mixture of isomers of saponins, rasamoelina and araneina (molecular masses of both amount to 650 Amu), selected from the hips Rs rugosa (Rosaceae). And rosemullion (glycoside of ursan), and eruptin (glycoside oleanane) include only the glucose residue at the C-28: researched in negative and positive types FAB with the inert gas xenon. HPLC was performed on columns with octadecylsilane (250×1.5 mm) with a mixture of acetonitrile-water (7:3, containing 0.5% glycerol) as solvent at a flow rate of 1 ml/min Were identified pseudomolecular ions [M+1]-and [M+1]+along with powerful peaks arising from the original aglycones in the mass spectra with negative FAB (Young et al., 1988).

It is also possible to detect the saponins by dynamic mass spectroscopy secondary and the new (SIMS), by a method similar to dynamic FAB, in which the eluent run directly in the ion source. Thus, the combination of HPLC with UV (206 nm) and S IMS-analysis was used to analyze mixtures of one monogenoidea and two bidesmosidic triterpene glycosides (Marston et al., 1991).

The disadvantage of methods FRIT-FAB and CF-FAB is the need to ensure a low flow rate (approximately 1-5 ál/min). After HPLC separation, it is necessary to split the exit stream. However, the interface in the form of thermocapillary (TSP) (Blackley & Vestal, 1983) is quite simple and can operate with flow rates of 1-2 ml/min This makes this method more attractive for solving problems related to the analysis of plant components. The essence of the method of the TSP is a soft ionization of molecules with similar chemical ionization in MS. This enables the analysis of non-volatile and thermolabile mono-, di -, and even triglycosides. Receive information about the molecular weight of saponin, the nature and sequence of chains of sugars. Method TSP LC-MS was used in the study mollusciciding saponins, extracted with methanol from the fruit of Tetrapleura tetraptera (Leguminosae) (Maillard & Hostettmann, 1993). In poleconomy eluent was added 0.5 M ammonium acetate (0.2 ml/min) to obtain the volatile buffer for ionization by ion evaporation: the total flow of ions in the TSP LC-MS (mass is the range of 450-1000 Amu) well that was what is observed when analyses HPLC-UV at 206 nm. Traces of ions at m/z 660, 676, 822 880 and gave signals representing pseudomolecular ions [M+H]+the main saponins. Mass spectra TSP obtained for each of saponins in the extract gave a main peak for pseudomolecular ion [M+H]+. Fragmentation of carbohydrate residues was observed in the analysis molluscicidal saponin Arianna, whose loss of N-acetylglucosamine component led to the formation of the peak [A+N]+corresponding to the aglycone (Maillrad & Hostettmann, 1993).

Method LC-MS used for the analysis of saponins, promising as a method GC-MS has little practical application, and in HPLC, used separately, the identity of the peaks can only be confirmed by retention time. Method LC-MS not only applicable for the analysis of triterpene glycosides in plant extracts, but also can be valuable (as a method of MS-MS) for the structural analysis of individual saponins in these extracts.

(v) Infrared spectroscopy (IRS)

In addition to the regular use of ICS, it has one or two features that are important for the structural analysis of saponins. X applicable for the study of steroid sapogenins due to the fact that the presence of several bands of high intensity between the frequencies 1350 and 875 cm-1is dia is diagnostic for spirochaetales side chain (Jones et al., 1953). Four lanes, 980 (A-band), 920 (In-band), 900 (band) and 860 cm-1(D-band), were identified as characteristic bands for rings E and F. In the case of 25R-sapogenins In-band is characterized by a stronger absorption than C-band, while in 25R-series, this ratio is reversed. The sapogenins with deputies of oxygen in the rings E and F, or position 27, the parameters of these bands substantially changed (Takeda, 1972).

The presence of ionized carboxyl groups in the saponins can be detected by the bands in the IR spectrum at 1610 and 1390 cm-1(Numata etc al., 1987). This information is applicable in the process of selection is important when figuring out whether ionized carboxyl groups in the molecule.

(vi) X-ray crystallography

X-ray crystallography was used to study the molecular geometry trisaccharides triterpene asiaticoside isolated from Centella asiatica (Umbrelliferae). Crystallization was performed from dioxane (Mahato et. al., 1987). Analysis of x-ray diffraction has also been successful from the point of view of the confirmation of the structure of 3-β-D-glucoside millevoi acid (Pegel & Rogers, 1985).

X-ray crystallography is particularly effective in the study of the structure of the aglycones. Valuable information for establishing the structure of the aglycone of altosid And isolated from Sesamum alatum (Pedaliaceae), sex is or by the method of x-ray diffraction of crystalline triacetate artifact, formed after acid hydrolysis (Potterat et al., 1992). Rentgenotomograficheskie analysis of the source aglycone 3-O-glycoside medicamentos acid, isolated from tubers Dolichos killmandscharicus (Leguminosae)showed that this molecule had a CIS-condensed rings D and E. the Ring was characterized by slightly broken conformation “sofa”, while the rings a, b, D and E had conformation “chair” (Stoeckti-Evans, 1989).

(vii) the cleavage Reaction

Triterpene saponins are glycosides in which hydroxypyrimidine group of sugars in their cyclic pyranose or furanose forms form acetals with triterpene or steroid residue. The ester linkage between hydroxypyrazoles and triterpene or steroid known as a glycosidic bond. Monosaccharide components of oligosaccharides are also linked by ether bonds (migliaia communication).

Upon completion of the hydrolysis of the glycoside, glycosidic bond is cleaved, releasing the constituent monosaccharides and neugebaude component (aglycone or Genin). Neugeborne part after hydrolysis of saponins referred to as “sapogenols” or “sapogenin”. All known saponins are O-glycosides with ether or ester bonds.

Numerous chemical reactions and methods were used for the separation of saponins into smaller components necessary to conduct a more precise analysis (see, for example, Kitagawa, 1981). Such methods will find particular application in structural analysis of triterpene saponins.

1. Acid hydrolysis

Acid hydrolysis can be carried out by heating under reflux saponin in the acid for a certain period of time, for example, for 4 hours at 2-4 M hydrochloric acid. Remaining after hydrolysis of the aqueous solution is extracted with diethyl ether, chloroform or ethyl acetate to obtain the aglycone. Extraction of sugars from the aqueous layer carried pyridine after neutralization solution (alkaline or basic ion exchange resin) (Tschesche & Forstmann, 1957; Sanberg & Michel, 1962) and evaporation to dryness. Using this method, the saponins are completely broken down into components that provides information allowing identification of the aglycone, and to determine the amount and nature of monosaccharides present. If acid hydrolysis subjected to prosapogenin (obtained after cleavage of the ester bonds in alkaline hydrolysis), it is possible to determine the nature of the sugar chains that are attached to the aglycone by ether bonds. Water of reaction may be replaced by alcohol or dioxane.

In addition to hydrochloric acid, to carry out hydrolysis of the saponins is also possible to use sulfuric acid. When using sulfuric acid less likely is atrocity or rebuild this molecule, however, the cleavage of ether bonds is not so effective. The standard method of obtaining hypsography acid of saponins carnations of the genus Dianthus, for example, involves the hydrolysis of 1 M sulfuric acid in dioxane (Oshima et al., 1984). A comparative analysis of the hydrolytic conditions for hydrochloric acid and sulfuric acid in water and water - ethanol mixture showed that the best allocation of sugars is achieved by heating saponin for 2 hours in a solution of 5% sulfuric acid - water sealed in a vacuum ampoule (Kikuchi et al., 1987). Somewhat milder hydrolysis can be carried out using triperoxonane acid, for example, by heating under reflux for 3 hours in 1 M triperoxonane acid.

Alternative hydrolysis of saponins in the solution is their hydrolysis directly on the plate for TLC by processing pairs of hydrochloric acid. After evaporation of the acid give standard elution TLC-solvent for the purpose of identification available monosaccharides (Kartnig & Wegschaider, 1971; 't, 1987). In this method, limit sugar, xylose and galactose were identified after partial hydrolysis of ageusia-Century TLC plates showed a mixture of chloroform-methanol-water (8:5:1) and detection used a mixture of aniline-diphenylamine-N3RHO4-methanol(1:1:5:48) (Uniyal et al., 1990).

2. Basic hydrolysis

RA is salanie On-acyclicity sugar chains is carried out in the conditions of the basic hydrolysis, usually by heating under reflux in 0.5 M potassium hydroxide. Alternatively, you can use the solutions of 1-20% potassium hydroxide in ethanol or methanol, although there is a risk of methylation, especially carboxyl group of triterpene acids. Ion-exchange resin such as Dowex-I provide the soft terms of the basic hydrolysis (Bukharov & Karlin, 1970). Another method involves the use of lithium iodide in collidine (Kochetkov et al., 1964).

By careful control of reaction conditions is possible selective cleavage of various ester components. For example, the hydrolysis of the saponin of kisutu To11when heated under reflux in 0.5 M potassium hydroxide in half an hour resulted in the removal of the sugar at C-28 of bidesmosidic. However, mixing this saponin for 20 hours in 0.1 M potassium hydroxide at room temperature to selectively remove the acetate group at C-28 ester glycoside chain (Kizu et al., 1985b).

3. Partial hydrolysis

In some cases, when saponins are characterized by significantly branched or long sugar chains, it is necessary the procedure of partial hydrolysis, with the aim of obtaining fragments, more suitable for structural analysis. This can be achieved using acid or, if necessary enzymes. Oligos arid and/or other parts of saponin isolated and then characterize them.

For example, saponin isolated from Phytolacca dodecandra (Phytolaccaceae), hydrolyzed in 0.1 M hydrochloric acid for 45 minutes to obtain a mixture of three products. These compounds were separated by the method OF GHND and sequences of sugars was determined by the method MS13C-NMR and GC-MS of articolate. The compilation of all the information received has allowed to establish the chemical formula of this compound as a derivative of oleanolic acid (Dorsaz & Hostettmann, 1986).

Hydrolysis in dioxane provides a softer conditions, and therefore may be used for partial hydrolysis. In this example, the saponin was heated under reflux for 6 hours in dioxane with 0.1 M hydrochloric acid (1:3) (Ikram et al., 1981). Another method of partial hydrolysis of the saponins is processing solution triterpene glycoside in alcohol alkali metal (sodium or potassium), and then adding a trace amount of water (Ogihara & Nose, 1986).

4. Hydrothermalis

Hydrothermalis triterpene glycosides leads to the formation of the corresponding aglycones and thereby help to facilitate structure determination. This method involves the heating of the glycoside with water or with a mixture of water and dioxane at 100-140°for 10-140 hours depending on the specific sample. For example, Hydrothermalis triterpene 3,28-O-bellicose leads to the formation of the corresponding 3-O-glycoside is (Kim et al., 1992).

5. Enzymatic hydrolysis

Very efficient and mild method for the removal of sugar residues from saponins without creating artifacts is enzymatic hydrolysis. Although the corresponding hydrolases for all sugars are not commercially available, the removal of residues β-glucose using β-glucosidase is currently promising. An additional advantage of cleavage by specific enzymes is due to the fact that the anomeric configuration of the sugar component is confirmed automatically. Some enzymes, which are particularly applicable for use in the hydrolysis of triterpene glycosides, are hydrolases β-galactosidase, hemicellulase, untreated hesperidins, pectinase and naringinase.

Systematic analysis of the crude drugs hesperidins, naringinase, pectinase, cellulase, amylase and emulsin showed that hesperidins, naringinase and pectinase are the most effective in the hydrolysis of ginsenosides (Kohda & Tanaka, 1975).

(viii) Analysis of aglycones after hydrolysis

Upon completion of hydrolysis, the aglycones can be separated from the hydrolysate or by simple filtration, or by possibility in a mixture of water - organic solvent and analyzed by known triterpenes. The most known method is what I TLC using a solvent, as a mixture of diisopropyl ether - acetone (75:30). Nabilelshami reagents are those reagents that are commonly used for analysis of saponins (see table 2).

For gas-liquid chromatography is required derivatization triterpenes. For example, the methyl esters of oleanolic and ursolic acids were separated by GC on a glass column Packed with 30% OV-17 or SE-30 (Fokina, 1979). Triterpenes can be determined by method GC after derivatization using N,O-bis(trimethylsilyl)ndimethylacetamide and chlorotrimethylsilane, as, for example, in the case of sapogenins a-E soy or metagenomes acid alfalfa (Jurzysta & Jurzysta, 1978).

Method GC-MS also has value from the point of view of the characteristics of sapogenins. Usually get trimethylsilyl derivatives, and then analyze them in the spectrometer. An example is the application of this method to study triterpenes oleanolova and uranovogo series. Nine similarbank triterpenes shared method on GC column with OV-101 and analyzed the parameters of their mass spectra: those that included a 12-envoy double bond, were typical retroreective Diels-alder reaction (Burnouf-Radosevich et al., 1985). This method is also used to determine triterpenes isolated from licorice (Bombardelli et al., 1979).

HPLC-analysis does not require derivatization and is characterized by high reproducibility rez is lettow and sensitivity analysis triterpenes. Can be used HPLC with normal phase (analysis sapogenins quinoa Burnouf-Radosevich & Delfel, 1984) and with reversed phase (Lin et al., 1981), however, the disadvantage OF HPLC is related to the fact that the compounds tend to precipitate in the mobile aqueous phase.

(ix) Analysis of sugars after hydrolysis

Analysis of monosaccharides can be carried out by TLC, for example, on silikagelevye plates with strong solvents such as a mixture of ethyl acetate-methanol-water-acetic acid (65:25:15:20) and n-butanol-ethyl acetate-isopropanol-acetic acid-water(35:100:60:35:30) (Shiraiwa et al., 1991). Detection is usually done with a phthalate p-anisidine, naftoresursi, timalsina acid (Kartnig & Wegschaider, 1971) or chloride of triphenyltetrazolium (Wallenfels, 1950; Kamel et al., 1991). Alternatively, quantitative analysis of monosaccharides can be carried out using the methods of GC or HPLC.

A number of HPLC methods have been described in connection with the analysis of sugars, including: analysis of NH2-related columns with elution with a mixture of acetonitrile-water (75:25) (Glombitza & Kurth, 1987); analysis on columns C-18 (acetonitrile-water, 4:1) determination of refractive index required for the purposes of quantitative analysis, by comparing the integrated HPLC-peaks with standards (Adinolfi et al., 1987); analysis on ion-exclusion column (Aminex HPX-87H (BioRad) with elution with a solution of 0.005 M sulfuric acid (0.4 ml/min) (Adinolfi et al., 1990); and analysis of p-bromobenzoate is in the sugar series (formed by methanolysis of the saponin mixture of 5%hydrochloric acid-methanol and subsequent p-bromobenzylamine methylated sugars) by HPLC and identified by comparison with authentic derived (Kawai et al., 1988; Sakamoto et al., 1992).

In the GC method used perselisihan sugar (Wulff, 1965), or spend GC-MS additonality derivatives. An alternative procedure is to analysis appropriately derivatizing monosaccharides combined method GC-Fourier-converted X (FTIR) (Chen & Snyder, 1989).

The most frequently detected sugars are D-glucose, D-galactose, L-arabinose, D-xylose, D-fucose, L-rhamnose, D-Hinojosa, D-glucuronic acid and D-ribose.

IV. Derivatives of the compounds of the present invention

As described in detail in this application, it is believed that several advantages can be achieved through manipulation of triterpene glycosides with giving them new properties, longer half-life in vivo, or other preferred properties. Such methods include, but are not limited to, manipulation or modification of mixtures triterpene glycosides or their individual triterpene molecules, modification or destruction of sugars and conjugation triterpene compounds with inert carriers such as various proteins or non-protein components, including immunoglobulins or Fc fragments. It should be noted that the long duration of half-life does not match the duration of the “slow release pharmaceutical compositions. The prep is ATA slow release usually create so to ensure the delivery of a constant level of the drug over a longer period of time. The increase in the time of half-life drugs, such as triterpene glycoside of the present invention, gives a higher plasma levels after its introduction, and these levels are maintained for a long time, but the nature of the reduction of these levels depends on the pharmacokinetic parameters of this connection.

(i) Conjugates triterpenes and related molecules

As described above, triterpene compounds of the present invention, identified herein may be connected with specific molecules to improve the efficiency of triterpene glycosides in the treatment of patients suffering from any disease that can be treated using compounds of the present invention.

Illustrative variants of such molecules include monitoring agents and agents who need to increase the half-life of triterpene compounds in vivo. Triterpene compounds can be combined with secondary molecules in any functional way, so that he has provided to each segment of the execution of the inherent functions without substantial disturbance of biological activity, for example, protivo the holeva activity claimed here connections.

Triterpene compositions of the present invention can be directly connected to the second connection, or they can be linked via a linker group. The term “linker (linker group)” means one or more bifunctional molecules that can be used for covalent joining triterpene compounds or mixtures triterpenes to the agent and which will not impair the biological activity of these triterpene compounds. The linker can be attached to any part of triterpene, provided that the area of merger does not violate the biological activity, e.g., antitumor activity of the compounds of the present invention.

Typical option for connection via a linker triterpene compounds of the present invention the second agent is receiving active complex ester triterpene with subsequent reaction of the specified complex ester with nucleophilic functional group of the agent, the adherence to which is. Active esters can be obtained, for example, by reaction of the carboxyl group triterpene with alcohol in the presence of a dehydrating agent, such as dicyclohexylcarbodiimide (DCC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide the iodide (EDCI). The use of EDC to obtain conjugates are described in U.S. patent No. 4526714, in the patent application PCT WO 91/01750 and Arnon et al., 1989: these sources in their full scope is shown here for information in the form of bibliographic references. Agent to join triterpene, for example, tumor-specific antibody, then mixed with the activated complex ester in aqueous solution with the receipt of this conjugate.

When the linker between triterpenes and the agent is desirable, active ester triterpene glycoside can be obtained as described above and subjected to a reaction with a linker group, for example, 2-aminoethanol, alkylenediamines, amino acid such as glycine, or a protected carboxyl group of the amino acid, such as tert-butyl ester of glycine. If the composition of the linker has a protected carboxyl group, the protective group is removed and get active ester linker (as described above). The active ester is then subjected to reaction with a second molecule of obtaining conjugate. Alternatively, the second agent can be derivatization succinic anhydride with obtaining conjugate agent-succinate, which can be condensed in the presence of EDC or EDCI with triterpen-linker derivative, in which the linker has a free amino - or Hydra is xinnuo group (see, for example, patent application PCT WO 91/01750, full details of which are given here as a reference).

You can also get the conjugate triterpene glycoside comprising a linker with a free amino group, and cross-sew it with a free amino group heterobifunctional crosslinker, such as sulfosuccinimidyl-4-(N-multimediamagazin)-1-carboxylate, which will react with the free sulfhydryl groups of protein antigens.

Triterpene glycoside also can be attached to the linker group by reaction of the aldehyde group with aminolink with the formation of the intermediate imine conjugate, followed by reduction with sodium borohydride or cyanoborohydride sodium. Examples of such linkers include aminoalcohols such as 2-aminoethanol, and diamino-compounds such as Ethylenediamine, 1,2-Propylenediamine, 1,5-pentanediamine, 1,6-hexanediamine and the like. Triterpene glycoside can then be connected to the linker with obtaining first succinylamino derived using succinic anhydride and subsequent condensation conjugate triterpene glycoside-linker with DCC, EDC or EDCI.

In addition, the triterpene glycoside or aglycone can be oxidized periodate, and received from him dialdehyde can condense with the above aminoalcohols or diamine is connections. Free hydroxyl or amino group on the linker may be condensed with succinate derived antigen in the presence of DCC, EDC or EDCI. In the art it is known a large number of types of linkers that can be used to obtain triterpene conjugates. The list of typical linkers for use in the present invention are shown below in table 4.

Table 4

Heterobifunctional cross-linkers
LinkerWith what respondsThe advantages and applicabilityThe length of the spacer elements of the shoulder after cross-linkage
SMTPPrimary amines, sulfhydryl groupsGreater stability11,2
SPDPPrimary amines, sulfhydryl groupsMilirovanie Split cross stitch6,8
LC-SPDPPrimary amines, sulfhydryl groupsElongated spacer elements shoulderthe 15.6
Sulfo-SPDPPrimary amines, sulfhydryl groupsElongated spacer elements shoulder solubilitythe 15.6
SMCCPrimary amines, sulfhydryl groupsStable maleimide reactive group Conjugation enzyme - antibody conjugation of the hapten - protein-media11,6
Sulfo-SPrimary amines, sulfhydryl groupsStable maleimide reactive group of the water-solubility of Conjugation enzyme - antibody11,6
MBSPrimary amines, sulfhydryl groupsConjugation enzyme - antibody conjugation of the hapten - protein-media9,9
Sulfo-MBSPrimary amines, sulfhydryl groupsThe solubility9,9
Fairs are forthcoming-SiabPrimary amines, sulfhydryl groupsConjugation enzyme - antibody10,6
Sulfo-fairs are forthcoming-SiabPrimary amines, sulfhydryl groupsThe solubility10,6
SMPBPrimary amines, sulfhydryl groupsElongated spacer elements shoulder Conjugation f is rent - antibody14,5
Sulfo-SMPBPrimary amines, sulfhydryl groupsElongated spacer elements shoulder solubility14,5
EDC/Sulfo-NHSPrimary amines, carboxyl groupConjugation of the hapten-carrier0
ABHCarbohydrates (indiscriminately)Reacts with sugar groups11,9

(ii) streamlining the creation of medicines

The goal of rational methods of drugs is to obtain structural analogs of biologically active compounds. By creating such analogs, it is possible to get drugs, which are more active or stable than the natural molecules, characterized by different sensitivity to changes or the ability to disrupt the function of various other molecules. In one of the approaches it is possible to determine the spatial structure of the triterpene compounds of the present invention or their fragments. For this you can use the data of x-ray crystallography, computer modeling, or a combination of both these methods. Another method is randomized samedaycashloans groups in the molecule triterpene and the resulting impact on the functional group.

Also, the antibodies specific triterpene compounds selected by the method of functional analysis and subsequent determination of its crystal structure. In principle, this method allows to obtain the core of a pharmaceutical product on the basis of which you can create new medicine. You can avoid crystallographic analysis of protein by creating antiidiotypic antibodies specific functional pharmacologically active antibody. Using the situation of “double mirror reflection”, the binding sites antiidiotypic antibodies, as you might expect, are analogues of the sites of the original antigen. Antiidiotypic antibody can then be used to identify and isolate peptides from banks of peptides obtained by chemical or biological means. Selected peptides can then serve as the core of pharmacological drug. Antiidiotypic antibodies can be obtained by the methods described herein to generate antibodies using antibodies as antigen.

Thus, it is possible to create drugs with improved biological activity, for example, antitumor activity, compared to the original triterpene compound. Using procedures hee the practical selection and descriptions given here, a significant amount of triterpene compounds of the present invention can be obtained in order to conduct crystallographic analysis. In addition, the establishment of the chemical properties of these compounds will enable computational prediction of structure-function relationships.

V. Treatment of cancer using the triterpene compounds of the present invention

In the process of development of cancer, mammalian cells undergo a series of genetically determined changes that result in abnormal cell proliferation. This can occur, usually in several stages: (1) initiation: an external agent or stimulus induces a genetic change in one or more cells; and (2) activation: involvement of other genetic and metabolic mechanisms, one of which may be inflammation. At the stage of “activation” in the cell begin metabolic transformation and transition in the growth stage, in which blocks apoptosis.

Cancer cells are characterized by loss of apoptotic control in addition to the loss of regulatory control over the stages of the cell cycle. Cancer cells (malignant cells) lack of regulatory mechanisms of normal growth due to a number of metabolic disorders that occur at the stages of initiation and is ctively at an early stage of malignancy. These abnormalities are a consequence of genetic changes in the cell. Data genetic changes may include: (i) activating mutations and/or increased expression of proto-oncogene; and/or (ii) inactivating mutations and/or the attenuation of the expression of one or more tumor-suppressor genes. Most of the products encoded by oncogenes and tumor-suppressor genes are components of signal transduction pathways that control the start or end of the cell cycle, activation, differentiation, damage semantic DNA and initiation of DNA repair mechanisms, and/or regulation of programmed cell death. Almost all tumors have mutations in multiple oncogenes and tumor suppressor genes. We can conclude that in cells operate multiple parallel mechanisms of control of cell growth, differentiation, DNA repair and apoptosis.

Triterpene compounds of the present invention can be introduced to the needy in this patient in the prevention of cancer or treatment after the diagnosis of such disease. To suppress the initiation and activation of cancer to kill cancer/malignant cells, to suppress cell growth, induction of apoptosis, to inhibit metastasis, decrease tumor size and other ways reversively reduction manifestations of tumor cells of malignant phenotype, using the methods and compositions of the present invention can provide contact cells“targets” as described here triterpene compounds. This can be achieved by contact of the tumor or tumor cells with a single compound or pharmaceutical composition that contains triterpene compounds of the present invention, or by contact of the tumor or tumor cells with more than one compound or pharmaceutical compositions simultaneously, where one song contains triterpene of the present invention, and the other includes the second agent.

Preferred from the viewpoint of exposure according to the present invention, cancer cells are cells of epithelial carcinomas, such as tumor cells, colon, uterus, ovaries, pancreas, lung, bladder, breast, kidney and prostate. Other cancer target cells are cells of brain tumors, liver, stomach, esophagus, head and neck, testicular, cervical, lymph system, larynx, esophagus, parotid glands, bile ducts, colon, uterus, endometrium, kidney, bladder and thyroid gland; including squamous cell carcinoma (epidermis), adenocarcinoma, small cell carcinoma, glioma, neuroblastoma, etc. However, the list provided is illustrative only, but not restrictive, since potentially any tumor cell can be treated using the triterpene compounds of the present invention. Methods of testing the relative effectiveness of the compounds of the present invention from the point of view of impact on the above tumor cells and other tumor cells specifically presented in this description and will be clear to experts in the art in light of the present application.

Compounds of the present invention is preferably introduced in the form of a nutraceutical composition or a pharmaceutical composition containing a pharmaceutically or pharmacologically acceptable solvent or carrier. The nature of the medium depends on the chemical properties of the applied(s) connection(s), including the parameters of solubility, and/or method of administration. For example, if it is desirable oral administration, it can be selected solid media, whereas for intravenous administration may be used a liquid salt solution.

The expression “pharmaceutically or pharmacologically acceptable” refers to molecular components and compositions that do not cause an adverse, allergic or other adverse reactions caused by the introduction of an animal or person. Used in this text is “pharmaceutically acceptable carriers” are any and all solvents, dispersing medium, coatings, antibacterial and antifungal agents, isotonic and slow absorption agents and the like, the Use of such media and agents for pharmaceutically active substances is well known in the art. They can be used in therapeutic compositions, with the exception of the cases of incompatibility of any of these standard environments or agents with a given active ingredient. Such compositions may also include additional active ingredients.

A. Nutraceutical composition

Nutraceutical compositions are the drugs of natural ingredients, which are multicomponent systems containing preferably Energetichesky active natural products and supplements that promotes healing. Nutraceutical compositions can be prepared from medicinal plants. Information about the many plants and herbs used for the preparation of nutraceutical compositions can be obtained from scientific publications, including the "German Commission E Monographs", "Botanical Safety Handbook" and "HerbalGram", a quarterly publication of the American Botanical Council, which describes the results of numerous clinical trials nutraceutical compositions.

Information about the descriptions and components, I is m use dosages (depending on form), action, contraindications, side effects, interactions with standard drugs, routes of administration, duration of application, official status, the rating of the Botanical security Agency ANR USA and the comments are available for a variety of plants, including, without limitation, blueberries, roster, pithecolobium, cat's claw, Cayenne pepper, cranberries, Buttercup field, Chinese dagel (dong quai) Angelica sinensis, sweet grass, primrose oil, guayule, garlic, ginger, gingko, ginseng, Chinese ginseng, Siberian, Hydrastis (Golden seal”), Cola, grape seeds, green tea, hawthorn, torrau, licorice, milk Thistle sea, Palma, Serenoa, St. John's wort and Valerian.

The action of such nutraceutical compositions can be fast and/or short-term, or may contribute to the improvement of health for a long time. The present invention is focused on herbal remedies derived from acacia Victoria Acacia victoria. The invention relates to nutraceutical compositions containing the dried and ground roots and beans Acacia victoriae or extracts of these tissues in a pharmacologically acceptable medium, designed as a natural means, inter alia, for the prevention and treatment of cancer. Nutraceutical composition can be used is to prevent the initiation and activation of carcinogenesis, and for the induction of apoptosis of malignant cancer cells. Declare here nutraceutical compositions can also be used as anti-inflammatory, antifungicide, antiviral, antimutagenic, or spermicidal contraceptives, cardiovascular and controlling cholesterol metabolism funds. Nutraceutical composition may be contained in such an environment, as a buffer, a solvent, a diluent, an inert carrier oil, cream or suitable for food stuff.

The nutraceutical composition may be administered orally and can be prepared in the form of tablets or capsules. Oral administration may be preferred for the treatment of colon cancer and other tumors of the internal organs.

Alternatively, the nutraceutical composition may be prepared in the form of ointments containing extracts of roots or beans Acacia victoriae in oil or cream is intended for topical application to the skin. This form of nutraceutical compositions can be used to prevent the initiation of cancer of the skin. Application data nutraceutical compositions provides a method of suppressing the initiation and activation of epithelial cells of a mammal in pre-malignant or malignant state by introducing into the cell of a mammal a therapeutically E. the effective amount of the nutraceutical composition. This is especially applicable in cases of cancer of epithelial cells, such as skin cancer.

b. The pharmaceutical composition

The present invention also relates to selected compositions Acacia victoriae, which partially or completely purified and characterized for their structure. Purification and analysis of these triterpene glycoside compounds described in the examples. D1, G1 and B1 are three songs that were purified and its structure was almost completely characterized (Fig, 40 and 41). In biological tests on the activity of these compounds against lines of cancer cells has been demonstrated suppression of cell growth and induction of apoptosis of malignant cells (Fig, 44A-E). Moreover, the partially purified composition of these saponins isolated from Acacia victoriae also show chemoprotective effect in mice that were exposed to the carcinogen DMBA (Fig, 9, 11, 12 and 13). Thus, these compositions have anti-cancer activity and there are a number of mechanisms that induce apoptosis of cancer cells. Pharmaceutical compositions based on these compounds are represented as powerful chemotherapeutic agents that can be used by themselves or in combination with other forms of cancer treatment such as chemotherapy, radiotherapy, surgical the e intervention gene therapy and immunotherapy. This combination therapy are described in detail hereinafter. Specialist in the art will be able to determine effective doses and modes of combination therapy.

C. Methods of introduction

(i) Injecting

In one of its variants, the present invention relates to preparations for parenteral administration triterpene compositions, for example, prepared for injection intravenous, intramuscular, subcutaneous or other similar means, including direct delivery to the tumor or in the affected area. On the basis of the present description, a person skilled in the art can prepare aqueous compositions that contain triterpene compound. Typically, such compositions can be obtained in the form of liquid solutions or suspensions for injection; can also be obtained in solid forms suitable for solutions or suspensions by the addition of a liquid prior to injection; and these preparations can also be emulsified.

Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surface-active substance, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols and their mixtures, as well as in oil. Standartnyh conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions; preparations containing sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the preparation of injectable solutions or dispersions for immediate admission. In all cases, the dosage form must be sterile and should be enough liquid to easily flow through the syringe. It must be stable under the conditions of industrial manufacture and storage and must be protected from contaminating action of microorganisms, such as bacteria and fungi.

Triterpene compounds can be included in the composition in a neutral form or in the form of salts. Pharmaceutically acceptable salts are additive acid salt (formed with free amino groups of the protein), formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric, almond acid and the like. Salts formed free carboxyl groups can also be formed with inorganic bases, such as, for example, hydroxides of sodium, potassium, ammonium, calcium or ferric glands is, and organic bases, such as Isopropylamine, trimethylamine, histidine, procaine and the like.

The media can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g. glycerol, propylene glycol and liquid polyethylene glycol and the like), suitable mixtures and vegetable oil. Sufficient fluidity can be achieved, for example, by use of a coating such as lecithin, by the maintenance of the required particle size in the case of suspension, and by the use of surfactants. To prevent the action of microorganisms can be used various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like.

Often preferred is the inclusion isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of injectable compositions can be achieved by use of these compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by adding active compounds in the required amount in the appropriate solvent with various other ingredients enumerated above, if they are needed, with subsequent sterile is filtered. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle, which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders intended for the preparation of sterile injectable solutions, the preferred methods of obtaining them are drying in the vacuum drying and freezing, giving the active ingredient in powder form plus any additional desired components from a previously sterile-filtered their solution.

(ii) Other methods of introduction

Other methods of introduction will also find application in connection with the present invention. For example, triterpene compounds of the present invention can be prepared in the form of suppositories, and in some cases in the form of aerosol and intranasal compositions. For suppositories, the filler composition should include traditional binders and carriers such as polyalkylene glycols or triglycerides. Such suppositories can be prepared from mixtures containing the active ingredient in an amount of from about 0.5% to about 10% (by weight), preferably from about 1% to about 2%.

Compositions for oral administration can be prepared in the form of solutions, suspensions, tablets, ilul, capsules, sustained-release preparations or powders. Such compositions can be introduced, for example, by ingestion or inhalation. If the pharmaceutical composition is intended for inhalation, it preferably should be of the aerosol. Examples of procedures for the preparation of aqueous aerosols for use in accordance with the present invention can be found in U.S. patent No. 5049388, the full content of which is introduced into the present description by reference. The preparation of dry aerosol preparations are described, for example, in U.S. patent No. 5607915, the full content of which is introduced into the present description by reference.

Also applicable to the introduction of the compounds of the present invention directly in the form of transdermal preparations containing amplifiers permeability, such as DMSO. These compositions can also contain any other suitable carriers, excipients or diluents. To treat some symptoms of the disease may be introduced other drugs for local use. For example, can be prepared intranasal medications that contain fillers, not irritating the mucosa of the nasal cavity and, basically, do not violate cilliary functions. In connection with the present invention can be used diluents, such as water, water-salt Rast is the PR or other known substances. Preparations for introduction into the nose can also contain preservatives, such as, without limitation, chlorbutanol and benzylaniline. May contain a surfactant, enhances the absorption of these compounds nasal mucous membrane.

(iii) Preparation and introduction

After preparation, the solutions must be introduced by that is compatible with the finished dosage form and in such amount that is therapeutically effective. The selected method of preparation can be carried out using a variety of fillers, including, for example, mannitol pharmaceutical purity, lactose, starch, magnesium stearate, sodium-containing sharedzilla, magnesium carbonate, etc.

Typically, compounds of the present invention should contain from less than 1% to approximately 95% active ingredient, preferably from about 10% to about 50%. Preferably, the patient is administered from about 10 mg/kg of body weight per day to about 25 mg/kg of body weight per day. Frequency of administration must be determined by the doctor based on observations of the patient's response. Other effective dose can be easily determined by the person skilled in the art using standard tests by constructing a curve of dependence “dose-response”.

Regardless of the route of administration, suitable pharmaceutical compositions in accordance the present invention should in principle include the number of triterpene compositions mixed with a suitable pharmaceutical diluent or excipient such as sterile aqueous solution, to obtain the final concentrations in the range depending on the purpose of application. Methods for the preparation of drugs in General are well known in the art and examples can be found in the guide: Remington''s Pharmaceutical Sciences, 16th ed.. Mack Publ. Co., 1980, the full content of which is introduced into the present description by reference. It should be noted that contamination with endotoxins should be at the minimum safe level, for example, less than 0.5 ng/mg protein. Moreover, intended for the introduction of human drugs must meet the requirements of sterility, progenote, General safety and purity in accordance with standards adopted by the Federal Department of biological standards of the United States.

A therapeutically effective dose can be easily determined using the animal model in accordance with the studies described here. For example, often experimental animals with solid tumors used for optimization of suitable therapeutic doses before using them in clinical practice. It is known that such models are quite reliable from the point of view of determining effective anticancer strategies.

In some embodiments, may is be desirable to provide continuous delivery of therapeutic compositions to a patient. When intravenous and intraarterial introduction this is done using droppers. For local use must be made of re-introduction. For different purposes can be used drugs extended release, which emit a small but constant amount of therapeutic agent over an extended period of time. For internal use preferred may be continuous perfusion of the area of interest. This can be done catheterization, in some cases after surgery, followed by continuous introduction of a therapeutic agent. The duration of perfusion should be determined by the attending physician for a specific patient and a specific case, and this time should vary from about 1-2 hours to about 2-6 hours, to about 6-10 hours to about 10-24 hours, to about 1-2 days to 1-2 weeks or more. In General, the dose of therapeutic composition during continuous perfusion should be equivalent dose, administered in single or multiple injections calculated for that period of time for which such injections are performed. It is believed, however, that when perfusion can be used in higher doses.

1. The treatment Protocol

The applicants of the present invention consider two basic ways, and the use of the triterpene compounds of the present invention separately and in combination therapy. The first one aims to metastatic cancer in patients who had not previously held chemo-, radio - or biotherapy, or in patients receiving such pre-treatment. Such patients a systematic introduction by intravenous, subcutaneous, oral administration or injection directly into the tumor. Input pharmaceutical doses are preferably 10-25 mg triterpene compounds of the present invention per 1 kg of body weight of the patient per day, including 13, 16, 19 and 22 mg/kg / day. Alternative for treatment of the patient can be used in pharmaceutical compositions containing from about 1 mg/kg / day triterpene compositions of the present invention to about 100 mg/kg / day, including 3, 6, 9, 12, 15, 18, 21, 28, 30, 40, 50, 60, 70, 80 and 90 mg/kg / day triterpene compositions of the present invention.

Treatment usually includes daily introduction, at least within eight weeks or weekly injection, at least within eight weeks. In accordance with the prescription of the medical doctor, the treatment can be continued according to the same scheme as long as the observed tumor growth or until there is no further response to treatment.

Another use of the compounds of the present invention is associated with the treatment of patients with ill the deposits were treated surgically, using chemotherapy and / or radiotherapy. Adjuvant therapy should be carried out in the same mode, which is described above, for at least one year to prevent relapse.

2. Prevention of cancer using the compounds of the present invention

Another use of the compounds and mixtures according to the present invention is associated with cancer prevention in high-risk groups. In these patients (for example, those who have a genetic predisposition to tumors, such as breast cancer, colon cancer, skin cancer and other) introduction must be done through the mouth (tumors of the gastrointestinal tract), by local application on the skin (skin tumors) or systemically for at least one year, and probably longer for the prevention of cancer. Such method can be applied to patients and precisely defined precancerous lesions, such as polyps of the rectum and other pre-malignant lesions of the skin, breast, lung or other organs.

3. Clinical Protocol

The clinical Protocol was designed by the applicants in such a way as to ensure the treatment of cancer using triterpene compounds of the present invention. In accordance with t is Kim Protocol selected patients, for which histological methods confirmed the diagnosis of cancer, for example ovarian cancer, pancreatic cancer, kidney cancer, prostate cancer, lung cancer or bladder cancer. Patients may have previously used (although not necessarily) chemo-, radio -, and gene therapy. Optimally, so that such patients remained normal function of the bone marrow (defined by the absolute number of peripheral granulocytes >2000 1 mm3and platelet count of 100,000 to 1 mm3), adequate liver function (bilirubin ≤1.5 mg/DL) and renal (creatinine < 1.5 mg/DL).

This Protocol provides for the introduction by intratumoral injection of the pharmaceutical composition, containing about 10-25 mg triterpene compounds of the present invention per 1 kg of body weight of the patient. For tumor size ≥4 cm injected volume should be 4-10 ml, preferably 10 ml), while for smaller tumors <4 cm should be used volume of 1-3 ml (preferably 3 ml). Multiple injections, built on the basis of the volume of a single dose of 0.1-0.5 ml, should be conducted at a distance of 1 cm or more.

Treatment includes approximately 6 doses, administered within two weeks. For the purpose of physician this course can be extended in 6 doses every two weeks or less frequently in the month, 2 month, quarter etc).

If patients have shown surgery, the tumor they should be treated the same way as described above, during at least two consecutive two-week courses of treatment. One week after the completion of the second year (or more, for example, the third, fourth, fifth, sixth, seventh, eighth, etc.) the patient should be carried out surgery. Before suturing incision in 10 ml of pharmaceutical composition containing triterpene compounds of the present invention, should be applied to the surgical site (operational bed), contact with which should last at least 60 minutes. After that, the wound closed, leaving the catheter or drainage tube. On the 3rd day after the operation, an additional 10 ml of the pharmaceutical composition is injected through this tube and left in contact with the operating bed at least 2 hours. Then it is removed by suction and remove the catheter in a clinically appropriate time.

4. Treatment of artificial and natural body cavities

One of the main sources of recurrent tumors are microscopic residual lesions, which, after its removal remain in the place of the primary tumor, as well as local and peripheral lesions. In addition, analogion the e situation we have then, when the natural cavity of the body are contaminated microscopic tumor cells. Effective treatment of such microscopic lesions should have a significant advantage over therapeutic regimens.

Thus, in some embodiments, the cancer can be removed surgically, resulting in a “cavity”. And during surgery, and after him (periodically or continuously) therapeutic composition of the present invention is injected into the body cavity. This is essentially a “local” application to the surface of the cavity. The amount of the composition should be sufficient to ensure that the entire surface of the cavity was precontractual with the input composition.

In one embodiment, the introduction should be carried out by simple injection of therapeutic composition into the cavity formed by the remote tumor. In another embodiment, may be desirable to use mechanical subject: sponge, swab, etc. Any of these approaches can be used after removal of the tumor, and also during the first surgery. In another embodiment, the catheter is introduced into the cavity before closing the surgical incision. Then the cavity can be continuously perfusion within the desired period of time.

The other way l the ing local application therapeutic compositions directed to the natural body cavity, such as the oral cavity, pharynx, esophagus, larynx, trachea, thoracic cavity, abdominal cavity or cavities of hollow organs, including the bladder, the colon and other internal organs. In this case, in the cavity of the primary tumor may be present or absent. This treatment is aimed at the microscopic lesions in this cavity, but sometimes it can also affect the mass of the primary tumor, if it has not been before this is removed, or precancerous lesions that may be present in this cavity. In this case, several methods can be used for “local” use in relation to these internal organs or abdominal surfaces. For example, the cavity of the pharynx just may be treated by washing and rinsing. However, for local processing of the larynx and trachea may require endoscopy and local delivery of therapeutic composition. In relation to internal organs, such as the mucous membranes of the bladder or bowel, you may have internal catheter for infusion through the catheter or direct visualization with the help of a cystoscope or other endoscopic instrument. Such cavity, as the chest cavity or abdominal cavity, can be processed using internal catheters or surgical methods that provide access to these areas.

(iv) T is rapitinka sets.

The present invention also relates to therapeutic kits comprising the described triterpene composition. These kits generally contain a pharmaceutically acceptable drug, at least one triterpene compounds of the present invention, placed in a suitable container. The data sets also include other pharmaceutically acceptable agents, such as drugs, containing components for delivery triterpene compounds in specific areas of the body of a patient in need of treatment or any one or more drugs, for example, chemotherapeutic agents, which can act with triterpene compounds consistently.

These kits can contain a single package that contains triterpene compounds of any additional components or without them, or they may contain separate containers for each desired agent. If the components of the kit are presented in the form of one or more liquid solutions, such liquid solution is an aqueous solution, and particularly preferred is a sterile aqueous solution. However, the components in this kit may be present in the form of dry(a) powder(s). If the reagents or components are present in the form of a dry powder, the powder can be R Sweden by adding a suitable solvent. It is envisaged that the solvent may also be in a separate container. The container in this set, in principle, can be at least one vial, test tube, flask, vial, syringe or other container form, which can be placed triterpene glycoside and any other desired agent, preferably in the form of an aliquot. In the case of the inclusion of additional components, this kit may also contain a second vial or other container in which these components are placed so that you can enter separately prepared dose. These kits can also include a second/third container for a sterile, pharmaceutically acceptable buffer or other diluent.

Data sets can also include a device through which the triterpene composition is administered to an animal or person, for example, one or more needles or syringes, or eye dropper, pipette, or other such device with which a particular drug can be introduced to the animal or applied to the desired area of the body. The kits of the present invention also typically include the installation of a device for vials or TPI other components, taking into account the restrictions imposed by the conditions of commercial sale, such as, for example, injection or inhalation plastic capacity is, in which place and retain desirable ampoules and other devices.

VI. Chemotherapeutic combinations and treatment

In some embodiments of the present invention may be desirable introduction triterpene compositions of the present invention with one or more other agents with antitumor activity, including chemotherapy and radiotherapy funds, as well as therapeutic proteins or genes. This can enhance the overall antitumor activity, achieved by treatment using only the compounds of the present invention, or it can be used to prevent or suppress the resistance of tumors to multiple drugs.

The use of the present invention in combination with the introduction of a second chemotherapeutic agent provides a simple introduction animal triterpene composition with a second chemotherapeutic agent such that it was effectively to achieve their combined anti-tumor effects in this animal. Therefore, these agents should be presented in an effective amount and for a period of time effective to achieve their joint presence in tumor tissue and their joint impact on the environment of this tumor. D what I achieve this the triterpene composition and the chemotherapeutic agents can be introduced to the animal simultaneously as part of the overall composition, and in the form of two separate compositions using different routes of administration.

Alternatively, the introduction of triterpene compositions can anticipate the application of chemotherapeutic agent, radiotherapy or protein or gene therapy or follow him at intervals from minutes to weeks. In embodiments in which the second agent and triterpene composition is administered to the animal separately, in General, need to ensure that the time interval between each of the injections was not too large in order to have additional agent and triterpene composition has not lost the ability to exercise the preemptive effect together against this tumor. In these cases, it is envisaged that the contact of this tumor with each of both agents proceeded for about 5 minutes to about one week for each of them or, more preferably, for about 12-72 hours for each, when the delay time is about 24-48 hours. In some cases, it may be desirable significant increase in treatment time, where the intervals between the respective introductions are a few days(2, 3, 4, 5, 6 or 7) or even a few weeks(1, 2, 3, 4, 5, 6, 7 and 8). It is also assumed that desirable may be repeated introduction or triterpene glycoside or the second agent. To achieve regression op is Halsey, both agents are delivered in a total amount effective to suppress their growth, regardless of the time of introduction.

A number of agents suitable for use in the described methods of combination therapy. Chemotherapeutic agents are given as examples, are, for example, etoposide (VP-16), adriamycin, 5-fluorouracil (5-FU), camptothecin, actinomycin-D, mitomycin-C and cisplatin (CDDP).

As should be clear to experts in the field, appropriate doses of chemotherapeutic agents, in General, should be in the ranges that are already used in clinical treatments, in which chemotherapeutic agents administered separately or in combination with other chemotherapeutics. As an example, such agents can lead to cisplatin; can also be used for other DNA alkylating agents. Cisplatin is widely used for the treatment of cancer, and the effective dose used in clinical practice, is 20 mg/m2for 5 days every three weeks for three courses. Cisplatin is not absorbed when administered orally, and therefore, it is administered by intravenous, subcutaneous, intratumoral or intraperitoneal injection.

Additional applicable agents are compounds that inhibit DNA replication, mitosis and segregation XP is the ILAC. Such chemotherapeutic compound is adriamycin, also known as doxorubicin, etoposide, verapamil, podophyllotoxin etc. Being widely used in clinical practice for the treatment of tumors, these compounds are administered by bolus intravenous injection at doses ranging from 25-75 mg/m221-day intervals for adriamycin up to 35-50 mg/m2for etoposide, with doubling of the dose by oral administration.

Can also be used by agents that disrupt the synthesis and the polynucleotide according predecessors, In particular, applicable are the agents that are extensively tested and are easily accessible. With this in mind, such agents as 5-fluorouracil (5-FU), are preferably used tumor tissue, making this agent particularly suitable for targeted delivery to tumor cells. Although 5-FU is toxic, it can be used in a wide range of media, including local injection, and, most of all carry out intravenous doses in the range of 3-15 mg/kg / day.

Examples of chemotherapeutic agents that are used in connection with combined therapy are listed in table 5.

Each of these in it, agents are given by way of example and is not limiting. In this regard, you can send specially the East to the directory "Remington''s Pharmaceutical Sciences", 15th ed., Chapter 33, in particular, str-652. It may be necessary for some varying dosages depending on the status of a particular patient, whose treatment. In any case responsible for the application the person must install the appropriate dose for each individual patient. Moreover, sterility, progenote, overall levels of safety and purity of the preparations for the introduction of a person must meet the standard requirements of the Federal Department of biological standards of the United States.

Table 5

Chemotherapeutic agents used against cancer
ClassType agentNonproprietary names (other names)Disease
Alkylating agentsChloroethyl-amines (“nitrogen mustards”)Mechlorethamine (HN2)Hodgkin's disease, non-Hodgkin lymphoma
Cyclophosphamide and phosphamideAcute and chronic lymphomatosa, Hodgkin's disease, non-Hodgkin lymphoma, multiple myeloma, neuroblastoma, breast cancer, ovarian, lung, Williams cancer, sarcoma of the cervix, testis, soft tissue
Melphalan (L-zarqali is in) Multiple myeloma, breast cancer, ovarian

Alkylating agents ChlorambucilChronic lymphocytic leukemia, primary macroglobulinemia, Hodgkin's disease, non-Hodgkin lymphoma
Ethylenimine and methylmelamineHexamethylmelamineOvarian cancer
thio-TERABladder cancer, breast cancer, ovarian
The alkyl sulphonatesThe busulfanChronic granulocytic leukemia
Derivative nitrosoanatabineCarmustin (BCNU)Hodgkin's disease, non-Hodgkin lymphoma, primary brain tumors, multiple myeloma, malignant myeloma
Lomustin (CCNU)Hodgkin's disease, non-Hodgkin lymphoma, primary brain tumors, small cell lung cancer
Semustine (methyl-CCNU)Primary brain tumors, stomach cancer, colon cancer
Streptozocin (streptozotocin)Malignant pancreatic insulinoma, malignant carcinoid
TriazineDacarbazine (DTIC: dimethyltriptamine acarbose) Malignant melanoma, Hodgkin's disease, soft tissue sarcoma
AntimetabolitesAnalogues of folic acidMethotrexate (amethopterin)Acute lymphocytic leukemia, horiokartsinoma, mushroom avium, breast cancer, cancer of the head and neck, lung cancer, osteosarcoma
Pyrimidine analogsFluorouracil (5-fluorouracil, 5-FU) Floxuridine (fortecortin, FudR)Breast cancer, head and neck, colon, pancreas, ovary, bladder, precancerous skin lesion (local)
Cytarabine (citizenoriented)Acute granulocytic and acute lymphocytic leukemia
Purine analogs and related inhibitorsMercaptopurine (6-mercaptopurine, 6-MP)Acute lymphocytic, acute granulocytic and chronic granulocytic leukemia

  Tioguanin (6-tioguanin, TG)Acute lymphocytic, acute granulocytic and chronic granulocytic leukemia
Pentostatin (2-deoxycoformycin)Volokolamsky leukemia, mushroom fungal infection, chronic is s lymphocytic leukemia
Natural productsThe Vinca alkaloidsVinblastine (VLB)Hodgkin's disease, non-Hodgkin lymphoma, breast cancer, testicular
VincristineAcute lymphocytic leukemia, neuroblastoma, nephroblastoma, Hodgkin's disease, rhabdomyosarcoma, non-Hodgkin lymphoma, small cell lung cancer
EpipodophyllotoxinEtoposide CentipaidTesticular cancer, breast cancer, small cell lung cancer and other tumors of the lung, Hodgkin's disease, non-Hodgkin's lymphomas, acute granulocytic leukemia, Kaposi's sarcoma
AntibioticsDactinomycin (actinomycin-D)Horiokartsinoma, neuroblastoma, rhabdomyosarcoma, testicular cancer, Kaposi's sarcoma
Daunorubicin (daunomycin, rubinomics)Acute granulocytic and acute lymphocytic leukemia
DoxorubicinSarcoma of soft tissue, osteo - and other sarcomas, Hodgkin's disease, non-Hodgkin lymphoma, acute leukemia, breast cancer, urinary tract, thyroid, lung, stomach, neuroblastoma
BleomycinTesticular cancer, head and neck, skin, esophagus, lung and urogenital tract; Hodgkin's disease, non-Hodgkin lymphoma
Plicamycin (mithramycin)Testicular cancer, malignant hypercalcemia
Mitomycin (MITOMYCIN-C)Cancer of the stomach, cervix, colon, pancreas, bladder, head and neck

 EnzymesL-asparaginaseAcute lymphocytic leukemia
Bioactive modifiersα-interferonVolokolamsky leukemia, Kaposi's sarcoma, melanoma, carcinoid, renal cancer cells, ovarian, bladder, non-Hodgkin lymphoma, mushroom mycosis fungoides, multiple myeloma, chronic granulocytic leukemia
Agents of different natureCoordination of the platinum complexesCisplatin (CIS-DDP), carboplatinTesticular cancer, ovarian, bladder, head and neck, lung, thyroid, cervix, endometrium, neuroblastoma, osteosarcoma
AnthracenedioneMitoxantroneAcute granulocytic leukemia, breast cancer
Substituted ureaHydroxyureaChronic granulocytic leukemia, true polycythemia, primary thrombocytosis, PLN is qualitative melanoma
Derived methylhydrazineProcarbazine (N-methylhydrazine, MIH)Hodgkin's disease
Supressant of adrenocorticoidsMitotane (o,p’-DDD)Cancer of the adrenal cortex
AminoglutethimideBreast cancer
Hormones and antagonistsAdrenocorticosteroidPrednisone (some other equivalent means)Acute and chronic lymphomatosa, non-Hodgkin lymphoma, Hodgkin's disease, breast cancer
ProgestinsHydroxyprogesterone Medroxyprogesterone MagistralataEndometrial cancer, breast cancer
EstrogensDiethylstilbestrol Ethinylestradiol (available other drugs)Breast cancer, prostate
The antagonist of the estrogenTamoxifenBreast cancer
AndrogensTestosterone propionate Fluoxymesterone (available other drugs)Breast cancer
Antagonists of the androgenFlutamideProstate cancer

Similar releasing factor gonadotropinLeuprolideProstate cancer

Other factors that cause DNA damage, are widely used γ-irradiation, x-irradiation and (or) targeted delivery of radioactive isotopes in tumor cells. Other factors that cause DNA damage, are microwave and ultraviolet radiation. It is most likely that all these factors cause a wide range of DNA damage, DNA precursors, the disruption of the processes of replication and DNA repair, Assembly and stability of chromosomes. Dose x-ray irradiation range from 50 to 200. X-rays per day for long periods of time (3-4 weeks), and a single dose is 2000-6000 X-rays. The dose ranges for radioactive isotopes vary widely, depending on the time of the half-life of the isotope, the strength and type of the emitted radiation and the absorptivity of their cancer cells.

VII. Targeted therapy of cancer

Described here triterpene compounds may be associated with one or more molecules that direct connection directly into the cancer cells. Such targeting has the advantage that it can be used to increase the overall concentration of the drug is spent funds directly to the place of treatment, for example, in the tumor, thereby minimizing system “flow” of the drug. As in the case discussed above chemotherapeutic agents directed triterpene compound may be used in combination with a second agent such as a chemotherapeutic agent. And triterpen, and the second agent can be directed to the same or different targets in tumor tissue. In this case, can be achieved in total than the sum or even an apparent synergistic effect.

Examples directing agents used in combination with the triterpene compounds of the present invention, will be those guides agents that are able to deliver triterpene compounds in the region of the tumor, i.e. able to be localized in the place of its location. Also desirable are agents targeting the vascular system of the tumor. In particular, note that targeting triterpene glycoside compounds allows the use of more efficient concentration in the tumor area with no side effects or minimal side effects that can occur when a wider or systemic distribution of triterpene compounds. In particular, the target of the sending agent can be: components of tumor cells; components of tumor vascular the system; components that communicate or usually associated with a tumor; components that communicate or usually associated with the vascular system of the tumor; the components of the extracellular matrix of the tumor or stremi or related to them, and even cell types that are found in the vascular system of the tumor.

(i) Tumor target cells and antibodies

Malignant cells that comprise the tumor, can be marked using bispecific antibodies, which includes the binding site with a relatively specific marker or antigen such tumor cells. For example, specific inhibition or destruction of tumor cells can be achieved by binding of the conjugate to the antibody-triterpene composition with a tumor cell target.

There are a lot of so-called “tumor antigens”, any of which may be selected as a target in communication with the guide tagging according to the present invention. Here the following are the significant number of antigens associated with solid tumors. The preparation and use of antibodies against such antigens are well known to experts in the art and, in particular, described herein. Examples of antibodies are antibodies to tumors of the female reproductive system (see, for example, the catalogue of the collection of ATSS): OS 125; OS 133; OMI; Mo v1; Mo v2; 3C2; S; I 3; DU-PAN-2; F 36/22; 4F7/7A10; OV-TL3; W; DF3; 2C8/2F7; MF 116; Mov18; CEA 11-H5; CA 19-9 (1116NS 19-9); n-E2; T/36; NDOG2; H317; 4D5, 3H4, 7C2, 6E9, 2C4, 7F3, 2H11, 3E8, 5B8, 7D3, SB8; HMFG2; 3.14.A3; tumors of the breast: DF3; NCRC-11; 3C6F9; MVE; CLNH5; MAC 40/43; EMA; HMFG1; HFMG2; 3.15.C3; M3, M8, M24; M18; 67-D-11; D547Sp, D75P3, H222; anti-EGF; LR-3; TA1; H59; 10-3D-2; HmAB1,2; 1,2 MBR·3; 24·17·1; 24·17·2 (3E1·2); F36/22.M7/105; C11, G3, H7; B6·2; B1·1; Cam 17·1; SM3; SM4; C-Mul (566); 4D5 3H4, 7C2, 6E9, S, 7F3, N, E, W, 7D3, V; OS 125; MO v2; DU-PAN-2; 4F7/7A10DF3; B72·3; cccccCEA 11; H17·E2; 3·14·A3; FO23C5; tumors of the colon and rectum: B72·3; (17·1A) 1083·17·1A; S·1A; ZCE·025; AB2; NT·29·15; 250·30.6; H; A7; GA73·3; T/36; A; 28.19.8; X MSO-791; DU-PAN-2; ID3; CEA 11-H5; 2C8/2F7; CA-19-9 (1116NS 19·9); PR5C5; PR4D2; PR4D1; melanomas: 4·1; 8·2 M17; 96·5; 118·1; 133·2, (113·2); L1, L10, R10(R19); I12; K5; 6·1; R24; 5·1; 225.28S; 465.12S; 92·27; F11; 376.96S; 465.12S; 15·75; 15·95; Mel-14; Mel-12; Me3-TB7; 225.28SD; 763.24TS; 705F6; 436910; M; tumors of the gastrointestinal tract: ID3; DU-PAN-2; OV-TL3; B72-3; CEA 11-H5; 3-14-A3; COLI; CA-19-9 (1116NS 19-9) and SA; OS; tumors of the lung: 4D5 N, S, E, S, 7F3, N, SE, W, 7D3, SB8; MO v2; B72-3; DU-PAN-2; CEA 11-H5; MUC 8-22; MUC 2-63; MUC 2-39; MUC 7-39; and heterogeneous tumor types: b 240; PAb 246; b 1801; ERIC-1; M148; FMH25; 6-1; CA1; 3F8; 4F7/7A10; 2C8/2F7; CEA 11-H5.

Another method for identifying and labeling the tumor is associated with n is mediocre characteristics of the tumor itself, not with the biochemical properties of an antigen expressed by a tumor cell. A known number of lines of tumor cells that can be used for directing agents. For example, intact cells or cell homogenates obtained from known cell lines, can be used to produce anti-tumor antibodies, aiming the corresponding types of tumors. Similarly, such lines of tumor cells can be used in carrying out various tests in vitro. In this case, the technician can access the directory of ADS to select the characteristic lines of human tumor cells, to which there is open access (from directory ATSS). Examples of such lines are: J82; RT4; ScaBER; T24; TCCSUP; 5637; SK-N-MC; SK-N-SH; SW 1088; SW 1783; U-87 MG, U-118 MG; U-138 MG, U-373 MG; Y79; BT-20 and BT-474; MCF7; MDA-MB-134-VI; MDA-MD-157; MDA-MB-175-VII; MDA-MB-361; SK-BR-3; C-33 A; HT-3; ME-180; MS751; SiHa; JEG-3; Caco-2; HT-29 and SK-CO-1; HuTu 80; A-253; FaDu; A-498; A-704; Caki-1, And Caki-2; SK-NEP-1; SW 839; SK-HEP-1; A-427; Calu-1; Calu-3; Calu-6; SK-LU-1; SK-MES-1; SW 900; EB1; EB2; P3HR-1; HT-144; Malme-3M; RPMI-7951; SK-MEL-1; SK-MEL-2; SK-MEL-3; SK-MEL-5 and SK-MEL-24; SK-MEL-28; SK-MEL-31; Caov-3; Caov-4; SK-OV-3; SW 626; Capan-1, And Capan-2; DU 145; A-204; Saos-2; SK-ES-1; SK-LMS-1; SW 684; SW 872; SW 982; SW 1353; U-2 OS; Malme-3; KATO III; Cate-1B; Tera-1; Tera-2; SW 579; AN3 CA; HEC-1-A; HEC-1-B; SK-UT-1; SK-UT-1B; SW 954; SW 962; NCI-H69; NCI-H128; BT-483; BT-549; DU4475; HBL-100; Hs 578Bst; Hs 578T; MDA-MB-330; MDA-MB-415; MDA-MB-435S; MDA-MB-436; MDA-MB-453; MDA-MB-468, T-47D; Hs 766T; Hs 746T; Hs 695T; Hs 683; Hs 294T; Hs 602; JAR; Hs 445; Hs 700T; H4; Hs 696; Hs 913T; Hs 729; FHs 738Lu; FHs 173We; FHs 738B1; NIH:OVCAR-3; Hs 67; RD-ES; ChaGo K-1; WRI-Rb-1; NCI-H446; NCI-H209; NCI-H146; NCI-H441; NCI-H82; H9; NCI-H460; NCI-H596; NCI-H676B; NCI-H345; NCI-H820; NCI-N; NCI-H661; NCI-H510A; D283 Med; Daoy; D341 Med; AML-193 and MV4-11.

You can find the directory ADS for any subsequent year to identify other suitable cell lines. Also, if it is desirable for a specific type of cell types, each specialist known methods for producing such cells and/or their immediate source. Therefore, in the analysis of scientific literature, you can easily choose the source of the tumor cells of any type, it is desirable to identify as a target.

As explained above, antibodies represent a direct means of recognition of tumor target antigen. It is known that a very large number of antibodies directed against solid tumors. Some of the applicable antitumor antibodies listed above. However, as should be known to experts in the art, some of these antibodies have the desired biochemical properties or insufficiently specific in relation to the tumor, so that they can be used as a therapeutic agent. An example is the antibody MUC8-22 that recognizes a cytoplasmic antigen. The use of such antibodies may represent only experimental interest, for example, in model systems or in screening tests.

In General, the antibody is for use in these aspects of the present invention must be able to recognize antigens, presented on the cell surface and which preferentially, or even specifically expressed by tumor cells.

Such antibodies should preferably be characterized by a high affinity, For example, ad<200 nm, and preferably Kd<100 nm, and should not show significant reactivity in relation to vital normal tissues, such as one or more tissues selected from the tissues of the heart, kidneys, brain, liver, bone marrow, colon, breast, prostate, thyroid, gall bladder, lung, adrenal gland, skeletal muscle, nerve fibers, pancreas, skin, or other vital organs or tissues in the human body. Among the vital tissues of the most important tissues from the point of view of the purposes of the present invention and on the basis of the low in their reactivity are the heart tissue, kidney, Central and peripheral nervous system and liver. Used in this text, the term “no significant reactivity” refers to the antibody or antibody fragment that, in case of their application in relation to a specific tissue suitable for immunohistochemical analysis conditions, will not be called staining at all or will be caused barely noticeable staining on CTD is determined as being positive cells, dispersed in the mass of the vast majority of negative cells.

Specifically promising antibodies provided for use in the present invention are those antibodies that are selective against solid tumors. For example, antibodies that bind with the proto-oncogene TAG72 and HER-2, which selectively detected on the surface of many cancer of the breast, lung and colon (Thor et al., 1986; Colcher et al., 1987; Shepard et al., 1991); MOv18 and OV-TL3 and antibodies that bind to the core mucinosa milk protein and fat balls milk man (Miotti etc al., 1985; Burchell et al., 1983); and the antibody 9.2.27, which binds to the antigen of melanoma with high Mr(Reisfeld et al., 1982). Also applicable are antibody against folate-binding protein, which are known to be homogeneous expressed in almost all carcinomas of the ovary; antibodies against oncogene erb family that sverkhekspressiya in squamous tumors and in most gliomas; and other antibodies known as objects of preclinical and clinical testing.

Antibodies B3, KSI/4, CC49, 260F9, HMSO-791, D612 and SM3 are considered suitable for use in clinical practice after the standard preclinical testing, usually conducted in the clinic. Antibody B3 (U.S. patent No. 5242813; Brinkmann et al., 1991) has custody No. HB10573 in ATSS; EN is Italo KSI/4 can be obtained as described in U.S. patent No. 4975369; and antibody D612 (U.S. patent No. 5183756) has custody No. NW in ATSS.

Another way to define associated with tumor targets associated with the direct characteristics of the tumor itself and not with the definition of the biochemical properties of an antigen expressed by a tumor cell. Therefore, applicants believe that any antibody that preferentially binds to a tumor cell can be used as a guide component of the conjugate with triterpene compound. Preferred binding to a tumor cell-based antibody that has a high degree of affinity with respect to that of the tumor cells and does not show significant reactivity in relation to vital normal cells or tissues that have been defined above.

Also, the present invention presents a number of methods for producing antibodies for use in delivery purposes triterpene glycosides to tumor cells as described in the text. To obtain specific for tumor cells, the antibodies can give the animal a composition comprising an antigen of a tumor cell, and select (which will be described in more details below) resulting from the antibody with the appropriate specificity. This immunizing composition may contain completely settled or partially purified product of any of the above antigens; it can be such a composition as the membrane preparation, enriched by any of the above antigens; any of the cells listed above; or contain a mixture or cell population, including any of the cell types listed above.

Obviously, regardless of the source of antibodies, in the practical application of the present invention in medicine may be given preference prior confidence in that which is the clinical object tumor expresses the antigen, which is ultimately selected. This can be achieved by direct test, including testing on the antigenicity of a sample of tumor tissue taken, for example, by surgical biopsy, or perhaps testing of circulating antigen. This can easily be done by immunological screening, for example, using ELISA method (enzyme-linked immunosorbent assay), in which antibodies from the hybridoma Bank test on the affinity of binding to the tumor. Then antibodies with the appropriate selectivity and affinity in relation to the tumor are selected to obtain bispecific antibodies of the present invention.

Thanks to a well-known phenomenon of cross-reactivity, it is assumed that the used antibodies, can be obtained in accordance with the scheme immun the organization, where the source of the antigens come from an animal, such as a mouse or a Primate, in addition to the immunizations, for which use the original antigens on human cells. When using human-derived antigens, they can be obtained from the line human tumor cells, or they can be obtained by taking the assay-specific examined the patient. Indeed, the known methods for producing antibodies, which are tumor specific patient “cut to order” (Stevenson et al., 1990), and they are presented in connection with the present invention.

1. Methods for producing antibodies

As noted above, antibodies can be used in particular embodiments of the present invention. For example, can be obtained antibodies specific against a particular site in a patient's body or a specific type of tissue. Such antibodies can then be attached to the triterpene compound of the present invention, thus a specific way to refer (“align”) data triterpene compounds on the fabric, in respect of which this specific antibody. An example of such an antibody is the antibody that binds to a tumor cell. In a preferred embodiment of the present invention the antibody is a monoclonal antibody. The ways in which the teachings of monoclonal and polyclonal antibodies are well known in the art and, in particular, described further here (see, e.g., Howell & Lane, 1988).

Briefly, a polyclonal antibody obtained by immunization of an animal with the immunogen, comprising the desired antigen, and the capture of the antisera from that immunized animal. To obtain antisera can be used in a wide range of animal species. Typically an animal used for obtaining antisera that is non-human animal including rabbits, mice, rats, hamsters, pigs or horses. Due to the relatively large amount of blood, the preferred choice for obtaining polyclonal antibodies are rabbits.

Both polyclonal and monoclonal antibodies that are specific against the antigen isoforms, can be obtained using standard methods of immunization, which is generally well known in the art. A composition containing antigenic epitopes of specific cell types, or, on the other hand, the compounds of the present invention, can be used to immunize one or more animals, such as rabbit or mouse, which are then used to produce antibodies specific for these antigens. Polyclonal antisera can be obtained after a certain period of time required for the formation of antibodies, simply by taking the rovi these animals and obtaining serum samples on the material of whole blood.

We can assume that the monoclonal antibodies of the present invention will find practical use in immunochemical procedures that can be used to test for the presence of triterpene compounds of the present invention in other types of plants, in addition to Acacia victoriae, or other procedures using antibodies specific for particular antigens. As discussed above, an example of the use of antibodies in connection with the present invention is to obtain antibodies specific against tumor antigens, the binding of the antibody with the triterpene compounds of the present invention and the treatment of patients using conjugate the antibody-triterpen thanks triterpene compounds in a specific way are delivered to tumor cells or other cells involved in the condition, the treatment of which can be performed using the triterpene compounds of the present invention. In General, in various embodiments of the present invention can be used polyclonal and monoclonal antibodies to various antigens. For example, you can use them for cleaning triterpene compounds on affinity columns. Methods of obtaining and characterization of these antibodies is well known in this field and described, for example, Harow & Lane, 1988; full contents of this guide are included in the present description by reference.

As is well known in this field, the immunogenicity of a particular connection may vary. Therefore, it is often necessary to stimulate the immune system of the recipient, which can be achieved by binding the peptide or polypeptide immunogen to a carrier. Examples and preferred carriers are hemocyanin lymph snails (KLH) and bovine serum albumin (BSA). As carriers can also be used other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin. Methods of conjugation of the polypeptide to a protein carrier are well known in this area and include the use of glutaraldehyde, of ester m-maleimidomethyl-N-hydroxysuccinimide, carbodiimide and bis-butterbeans of benzidine.

Also, as is well known in this field, the immunogenicity of specific immunogenic compositions may be enhanced by the use of non-specific stimulators of the immune response, called adjuvants. Examples and preferred adjuvants include complete beta-blockers (non-specific stimulator of the immune response containing killed tubercle bacilli Mycobacterium tuberculosis), n is the complete beta-blockers and aluminum hydroxide.

The number of immunogenic compositions used to obtain polyclonal antibodies varies depending on the nature of the immunogen, as well as species used for immunization of the animal. Can be used by various routes of administration of the immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal). Production of polyclonal antibodies can be monitored by sampling blood of the immunized animal after various time intervals after immunization. Can also be held the second booster injection. Boosting and titration of antibodies continue up until you reach the desired title. Upon reaching the desired level of immunogenicity, the immunized animal can be killed, and the serum isolated and stored, and/or the animal can be used to generate monoclonal antibodies (mAbs).

Monoclonal antibodies can be easily obtained by using well-known techniques such as described in U.S. patent No. 4196265, the full content of which is incorporated into this description by reference. Typically, this technique involves immunizing a suitable animal selected immunogenic composition, for example, a purified or partially purified tumor antigen, polypeptide or peptide, or tumor cells is Oh. Immunizing composition is administered so as to provide effective stimulation of cells that produce antibodies. Preferred animals are rodents, such as mice and rats, however, also possible to use cells of rabbits, sheep or frogs. Using rats has some advantages (Coding, 1986), but the mouse is preferable, and most preferable are mouse strain BALB/c, because it is the most widely used standard line, giving a high percentage of stable cell hybrids.

Following immunization, somatic cells with the potential for generation of antibodies, in particular, b-lymphocytes (b-cells), are selected for use in accordance with the Protocol for production of monoclonal antibodies. These cells can be taken by biopsy of the spleen, tonsils or lymph nodes, or from samples of peripheral blood. Preferred splenocytes and blood cells: the first is a rich source producing antibodies cells undergoing fission lymphoblasts, and the latter is preferred because of easy availability of blood samples. Must often be preimmunization group of animals to highlight the spleen of the animal, characterized by a higher titer of antibodies, after which the spleen lymphocytes obtained by homogenizing the spleen with a syringe. Usually when lisence immunized mouse contains approximately 5× 107-2×108the lymphocytes.

Then b-lymphocytes immunized animal, producing antibodies, hybridized cells immortalizing myeloma line, usually from the same animal species, specimens of which have been immunized. The preferred lines myeloma cells suitable for hybridization with obtaining hybridomas are cells that do not produce antibodies, characterized by high hybridization ability and deficient on the enzyme that makes them incapable of growing in certain selective media which support the growth of only the desired hybrid cells (hybridomas).

Can be used any of a number of myeloma cells, which are well known in the art. For example, for immunization of mice, you can use cells P3-H/AG, P3-H-Ad, NS1/1.Ag-4 1, Sp210-Ag14, FO, NSO/U, MPC-11, MPC-11-X45-GTG 1.7 and S194/5XXO Bul; for rats, you can use cell R210.RCY3, Y3-Ag 1.2.3, IR983F and V; and in all cases, to merge cells, you can use the cells U-266, GM-1500-GRG2, LICR-LON-HMy2 and UC729-6 (see, for example, Goding, 1986; Campbell, 1984; catalogue of ATSS).

Methods of obtaining hybrids between producing antibodies by cells in the spleen or lymph nodes and myeloma cells usually involves mixing somatic cells with myeloma cells in a ratio of 2:1, although this ratio can vary from about 20: to about 1:1, accordingly, in the presence of an agent or agents (chemical or electrical)that promote the fusion of cell membranes. Were described hybridization methods using Sendai virus (Kohler &Milstein, 1975, 1976) and with the use of polyethylene glycol (PEG), for example, 37%PEG (o/o) (Gefter et al., 1977). Suitable methods are also electrically induced fusion (Coding, 1986).

In the procedures of merger, viable hybrids usually formed at low frequency, about 1×10-6up to 1×10-8. However, this does not create problems because viable, i.e. fused hybrids are differentiated from the parent Nikitich cells (in particular, from Nikitich myeloma cells, which normally must continue indefinitely to share) under cultivation in selective medium. A selective medium is a medium containing an agent that blocks the de novo synthesis of nucleotides in the culture medium. Preferred examples of such agents are aminopterin, methotrexate and azaserine. Aminopterin and methotrexate block de novo synthesis as purine and pyrimidine bases, while azaserine blocks only purine synthesis. When using aminopterin or methotrexate in the environment include gipoksantin and thymidine as a source of nucleotides (NAT-environment). In using the implement azaserine in the culture medium was added gipoksantin.

Preferred selective environment is a NAT environment. Only those cells that are able to act on the path to “salvation” of nucleotides that are able to survive in the NAT environment. The myeloma cells are defective in key enzymes of this mechanism, for example, hypoxanthineguanine (HPTR), so they cannot survive. B cells can act on this mechanism, but they have a limited lifespan in culture and usually die within 2 weeks. Therefore, in selective media can only survive a cell hybrids myeloma and b-cells.

As a result of such cultivation are hybrid population from which to select specific hybridoma. Typically, the hybrid selection is carried out by culturing the cells in a dilution of monoclonal microtonally tablets with subsequent testing supernatants individual clones (approximately 2-3 weeks) for the desired activity. This test should be sensitive, simple and rapid, such as radioimmunoassay test, enzyme immunological test, test cytotoxicity test on plaque formation, the dot-blot immunoassay, etc.

Selected hybridoma must then be subjected to serial dilution and cloned in a separate line of antibody-producing cells, and then these clones unlimited FOTS is izvodyat obtaining monoclonal antibodies (mAbs). Cell lines can be used to obtain mAb in two main ways. A sample of the hybridoma cells can be injected (usually in the abdominal cavity) histocompatible animal of that kind, which was used as a source of somatic and myeloma cells for the original hybridization. The injected animal develop tumors secreting specific monoclonal antibody produced fused hybrid cells. Then the physiological fluid of the animal, such as serum and ascitic fluid may be selected for receipt of these mAbs in high concentrations. Individual cell lines can be cultured in vitro so that the mAbs are naturally released into the culture medium from which they can easily extract at high concentrations. mAbs obtained by any of these methods can be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography.

(iii) Additional target tumor cells and bind ligands

In addition to antibodies for the delivery of the triterpene compounds of the present invention to the site of localization of the tumor by binding to the antigen of tumor cells, can be used and other ligands. If the op is for antigens which sverkhekspressiya receptors (e.g. estrogen receptor, EGF-receptor) or mutant receptors that guide agents can serve as the appropriate ligands.

Similarly, the ligands of the receptors of endothelial cells, there may be components that are specifically or preferentially bind tumor cells. For example, if a tumor antigen is sverhagressivnym receptor, the tumor cell may be covered with a specific ligand in vivo. Hence, then, such a ligand may be labeled either-specific antibody, or by using the receptor. Specific examples of this type aiming (guides) agents are antibodies against ligands of TIE-1 or TIE-2, antibodies against platelet factor-4 protein and leukocyte adhesion.

(iv) Toxins

In some embodiments, it is envisaged that the second therapeutic agents used in combination with triterpene compounds described in this application should be pharmacological agents connected with antibodies or growth factors, in particular, cytotoxic or with other protivoglistotnoe active agents, capable to destroy, inhibit the growth or division of endothelial cells. In General terms, the present invention relates to the use of ubago pharmacological agent, together with and in addition to the described triterpene compounds, which can be anywhereman with directing agent, preferably with an antibody, and delivered in active form to tumor target cells. Examples antileech agents are chemotherapeutic agents, radioactive isotopes, and cytotoxins. In the case of chemotherapeutic agents, applicants believe that especially preferred are such agents as steroid hormones; an antimetabolite such as citizenoriented, fluorouracil, methotrexate or aminopterin; anthracycline; mitomycin-C; Vinca alkaloids;

demecolcine; etoposide; mithramycin; or antitumor alkylating agent such as chlorambucil or melphalan. Other options may include such agents as a cytokine, growth factor, bacterial endotoxin or a lipid component of bacterial endotoxin. In any case, it is believed that such agents, as listed above, if this is desirable, together with the triterpene compounds of the present invention with success to contact the referring agent, preferably an antibody, so as to ensure their targeting, internalization, secretion or presentation of the blood components in the localization of target cells (see, e.g., Ghose et al., 1983; Ghose et al., 1987).

A number of chemotherapeutic and other pharmacological agents currently successfully kongugiruut with antibodies confirming their pharmacological functionality (see, for example, Vaickus et al., 1991). Examples of anticancer agents that were tested include doxorubicin, daunomycin, methotrexate, vinblastine, and others (Dillman et al., 1988; Pietersz et al., 1988). Moreover, it was described the accession of other agents, such as neocarzinostatin (Kimura et al., 1983), macromill (Manabe et al., 1984), Treiman (Ghose, 1982) and α-amanitin (Davis & Preston, 1981). Specific methods of obtaining conjugates triterpene compounds of the present invention with suitable guides molecules specifically described above.

VII. Other uses of the compounds according to the present invention

Applicants specifically discusses the use of the compounds of the present invention for a number of other applications in addition to the treatment or prevention of cancer. In particular, the applicants consider the use of the triterpene compounds of the present invention as solvents, antifungal and antiviral agents, ichthyocides or molluscicides, contraceptives, protivogelmintny equipment, protection against UV-radiation, which means salt, diuretics, anti-inflammatory the agents, regulators of cholesterol metabolism, effectors of the cardiovascular system, antiulcer funds, painkillers, sedatives, immunomodulators, antipyretics, regulators of angiogenesis, as agents for reducing capillary fragility, as agents for combating signs of aging and as agents for improving cognition and memory.

Compounds of the present invention play a role in the regulation of angiogenesis. Angiogenesis or neovascularization is defined as the formation of new blood vessels. Tumors and cancer induce angiogenesis to ensure the activity of the tumor due to supply them with oxygen and nutrients. The development of new blood vessels also provides cancer cells with an exit in other parts of the body. Therefore, suppression of angiogenesis should have a positive effect on cancer patients. On the other hand, sometimes angiogenesis is necessary, for example, in the healing of wounds. Similar lesions can be external wounds or wounds to internal organs, causes are accidents, burns, trauma and surgery. Thus, agents stimulating angiogenesis, are important for wound healing.

Considered also note the persistence of the compounds of the present invention for the modulation of cholesterol metabolism. In particular, connections, and nutraceutical compositions of the present invention can be used to reduce levels of serum cholesterol in patients. Therefore, when treating patients with the use of triterpene compositions of the present invention either oral or intravenous hopefully mortality associated with elevated cholesterol and related cardiovascular diseases will be reduced.

It is believed that treatment of cardiovascular diseases, the compounds of the present invention can be used for the treatment of arrhythmia and also as vascular relaxants causing hypotensive activity.

Another particularly important application of the compounds of the present invention is their use as anti-inflammatory agent. It was shown that the active triterpene compounds of the present invention are powerful inhibitors of the transcription factor NF-KB, which plays an important role in the formation of the inflammatory response. This fact is particularly important, given the increasing amount of evidence of the Central role of the inflammatory response in the processes of carcinogenesis. Treatment of patients with the use of triterpene compounds, announced aamah here it may, therefore, facilitate a wide range of painful conditions associated with inflammation, including the formation of tumors and tissue damage.

The initial phase of the inflammatory response is characterized by increased permeability of blood vessels and the release (exudation) of histamine, serotonin and basic polypeptides and proteins. This is accompanied by redness and formation of edema. After that there is cellular infiltration and the formation of new connective tissue. It is believed that the use of compounds of the present invention may limit these early phase of inflammation, and thereby to reduce the negative effects associated with the inflammatory condition.

Type of plant from which were selected compounds of the present invention, Acacia victoriae, was chosen in particular because of the natural region of origin are the arid zone. An important feature of the metabolism of plants in these areas is the production of compounds that protect cells from UV exposure. The applicants, in particular, believe that the triterpene compounds of the present invention can serve as protection from UV radiation. Consequently, it can be considered that the compounds of the present invention will find wide application in cases when the desired assetsassets from ultraviolet radiation. For example, such application involves the use of triterpene compounds of the present invention as components of anti-tan and other similar lotions, intended for application on human skin.

The potential advantage of such compositions is defined chemoprotective effects due to the compounds of the present invention. Therefore, lotions and remedies against sunburn, containing triterpene compounds of the present invention, in particular, will be suitable for use by individuals, characterized by a predisposition to skin cancer. Examples of such persons are people with sensitive skin or individuals with a genetic predisposition to skin cancer. The form of such predispositions include inherited mutations in oncogenes and violations of the cellular mechanisms mediating the DNA repair after UV-induced damage. In particular, significant mutations in genes that regulate the repair processes, for example, cutting of UV-induced thymine-siminovich dimers. Similarly, the compounds of the present invention can be added in the composition is any composition which is desirable superior protection against UV radiation, and these compounds can be applied to any living or inanimate object, for which predusmatriva what is protection from UV.

Other possible applications triterpene compounds of the present invention are the protection from damage of the Central nervous system, such as loss of memory, or to improve cognitive abilities, use as an antioxidant (to control the levels of oxidants in the blood) or increasing the amount of nitric oxide (NO), necessary for the treatment of hypertension or atherosclerosis. In addition, the applicants, in particular, consider the local application of the triterpene compounds of the present invention to enhance sexual function. Also discusses topical application of the compounds of the present invention to improve the content of dermal collagen, which is associated with overcoming the effects of aging.

IX. The tests and methods for screening active compounds

Specialists in this field of technology is known for a number of tests that can be used for further characterization of triterpene compounds of the present invention. They include tests for biological activity, as well as tests for chemical properties. The results of these tests allow us to make important conclusions about the properties of compounds and their possible application for the treatment of humans and other mammals. Tests, presumably applicable for these purposes, include the screening of biological active is ti in vivo and in vitro immunological tests.

(i) the in vivo Tests

The present invention involves the use of various animal models of the disease. In this case, the identity between mouse and man, is a great opportunity to test the function of the potential therapeutic agent, for example, triterpene compounds of the present invention. You can use murine tumor models, which are characterized by good predskazatelei for cancer in humans and other mammals. In these models can be performed orthotopic or systemic injection of tumor cells to simulate the primary and/or metastatic cancer. Alternatively, the cancer can be induced in animals by affecting them agents known that they are involved in the process of malignant transformation and/or tumor development.

Treatment of animals with the use of the test compounds provides for the introduction of this connection to this animal in an appropriate form. The introduction should be carried out in any of the ways that can be used for clinical or sub clinical purposes, including, but not limited to, oral, intranasal, transbukkalno, rectal, vaginal or local introduction. Alternatively, the introduction can be carried out by nutritarian is or bronchial installation (backfilling), intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. In particular, intravenous injection system, introduction to a specific site through the blood or lymph and intratumoral injection.

To determine the effectiveness of compounds in vivo may be based on different criteria. Such criteria include, but are not limited to, survival, reduced susceptibility to tumor or mass, stopping or slowing tumor growth, resorption of tumors, inhibition or prevention of metastasis, increased activity, improved immunological effector function and improve digestion.

One of the specifically applicable tests in vivo for antitumor activity is the use of a mouse skin model. The mouse skin model, which represents one of the most well-studied experimental models of multistage carcinogenesis, allows to identify three distinct phases of cancer: initiation, activation and development (progression). Currently, it is known that cellular evolution malignancy includes sequential alteration of many proto-oncogenes and/or tumor suppressor genes whose products are involved in the major mechanisms of signaling and/or gene expression regulation. Phase activate and development of skin tumors characterized by selective and permanent hyperplasia, changes in differentiation and genetic instability, leading to specific proliferation of initiated cells with the formation of papillomas and carcinomas. It was shown that the induction of persistent hyperplasia is clearly correlated with the activity of different agents stimulating the development of skin tumors, such as forblue esters, some peroxide and chrysarobin. It was shown that in the mouse skin model, all known carcinogens and tumor promoters induce persistent hyperplasia of the epidermis. In General, it is foreshadowed in the development of the inflammatory response.

Extensive data indicate a clear correlation between Carcinogenicity and mutagenicity. Many tumor-initiating agents either generate or metabolically converted in electrophilia agents capable of covalently to communicate with the cellular DNA. Some free radicals and modified DNA bases, which are free radicals involved in tumor initiation and/or activation of tumor stages in carcinogenesis. It was proved that the activation of a gene Ha-ras occurs in the early stages of skin carcinogenesis in mice, which, apparently, is equivalent to the event initiation. For example, it was shown that the presence of activated gene c-Ha-ras in skin papillomas mouse, caused by the action of 7,12-dimethylbenz[a]anthracene, associated with you is a CMOS frequency transverse And→ T in the 61st codon. Subsequent studies showed that the type of mutation depends on the chemical nature of the initiator and does not depend on the activator, which allows you to assume the availability of a direct effect of the inductor on a gene-By-ras. Moreover, infection of murine skin On genome-rs, activated virus (v-Ha-ras), may serve as a triggering event in the two-stage carcinogenesis. It should be noted that all skin chemical carcinogens and inducers of skin tumors have been shown to produce mutations in the oncogene H-ras. However, activators of skin tumors do not cause gene mutation On-rs.

(ii) Confirming the In vivo tests and clinical analysis

To a person skilled in the art it should be clear that chemotherapeutic agents, including triterpene compounds of the present invention, or their combination with additional agents should be tested in models in vivo prior to administration to humans. Such preclinical test animal is the standard method in this field. For such confirmatory tests, all that is required is appropriate from a scientific point of view, the animal model of the analyzed diseases, such as animal having a solid tumor. In this context, can be used any animal, such as, for example, mouse, rat, sea is Kai mumps, hamster, rabbit, dog, chimpanzee, etc. From the point of view of cancer treatment, research using small animals such as mice, are widely distributed from the point of view of predictability of clinical efficacy in relation to man, and, therefore, such animal models are preferred from the point of view of the present invention as they are readily available and relatively inexpensive, at least compared to other experimental animals.

The way to test an experimental animal can be designed by the person skilled in the art. All that is necessary to conduct such a test, is to create the equivalent of the treated group and enter the test connection to one group of parallel conducting various forms of control on the equivalent animals in the other group (the group). In the course of research carried out continuous monitoring of animals, then animals killed for analysis of effect of treatment.

One of the main applications of the present invention is the treatment of cancer. Therefore, the antitumor tests can be conducted to determine specific effects on the vascular system of the tumor and the overall antitumor effects. As part of such research, you must also Prov is employed to monitor the specificity of such effects, including the General condition of these animals.

With regard to the treatment of solid tumors, it is believed that an effective amount of triterpene compounds of the present invention should be such that they provide death or apoptosis, at least 10% of the cells in the tumor. Preferably should be destroyed, at least about 20%, about 30%, about 40% or about 50% of the cells in the specific area of the tumor localization. Most preferably must be destroyed 100% of the cells in the area of tumor localization.

The level of cell death in tumors evaluated in relation to the level of healthy tissues in all parts of the body. Preferred should be using doses of compounds of the present invention, is able to induce tumor necrosis, at least about 60%, about 79%, about 80%, about 85%, about 90%, about 95% to and including 100%, and used the dose should not cause significant side effects or other adverse effects on the animal. All these definitions can be easily implemented and accurately evaluated by an expert in the field of technology. For example, nurses, scientists and doctors can use such data obtained in experimental animals for optimization of dose, appropriate for the treatment of humans. In patients with launched the first disease is the occurrence of side effects at a certain level can not be taken into account. However, for patients in the early stages of the disease can be applied more moderate doses in order to achieve a significant therapeutic effect in the absence of side effects. The results of these studies on experimental animals should be statistically significant compared to control levels, and should be reproducible in different tests.

In addition, specialists in the art should be understood that combinations and doses of the compounds of the present invention, which cause specific tumor necrosis, in accordance with the present invention can be used, starting from the lower limit of effective range. For example, in embodiments which provide for the continuous use of active agents, the initial dose, which results in only about 10%increase level of necrosis, however, is applicable, in particular, because often there is a property such initial dose to stimulate the destructive processes in the tumor, amplified in the subsequent resumption of treatment. In any case, even if ultimately not achieved approximately 40%tumor suppression, however, it is obvious that can be used with any induction of thrombosis and necrosis, unless it provides for the improvement of patients before treatment. Bol is e, it is assumed that the dose of the compounds of the present invention, which prevents or reduces the probability of metastasis, or de novo carcinogenesis, should also have a therapeutic beneficial effect on the patient undergoing treatment.

As discussed above in connection with the test system in vitro, it is obvious that combinations of agents for the joint application should be tested and optimized. Compounds of the present invention can be directly analysed in combination with one or more chemotherapeutic agents, immunotoxins, coagulants, etc., Should be the analysis of the combined action of these agents, and its results should be evaluated in accordance with the above descriptions.

(iii) in vitro Tests

In one embodiment of the present invention, screening of plant extracts carried out in vitro to identify those compounds that can inhibit the growth of or to destroy tumor cells. The destruction of tumor cells or cytotoxicity, usually occurs by necrosis or apoptosis. Necrosis is common enough mechanism “triggered” external signals. In this process we lose the integrity of cell membranes and cellular compartments. On the other hand, as optos, or genetically programmed cell death, is a complex process involving morphological events that are synchronized activation and deactivation of specific genes (Thompson et al., 1992; Wyllie, 1985).

An effective way to test the cytotoxicity in vitro involves the systematic effects of selected plant extracts on the panel of tumor cells. Such tests and lines of tumor cells suitable for carrying out such tests are well known to specialists in this field of technology. Specifically preferred lines of human tumor cells for use in tests in vitro antitumor activity, is a cancer cell line of human ovarian SKOV-3, HEY, OCC1 and OVCAR-3; T-leukemia cells, Jurkat; line tumor human breast MDA-468; cancer cells of the prostate gland human LNCaP; line melanoma cells A-M and Hs294t; and cancer cells of human kidney 769-P, 786-0, A. The preferred type of normal cells for use as control cell lines are foreskin fibroblasts human FS or Hs27.

Analysis of the in vitro efficacy of compounds for the destruction of tumor cells can be performed, for example, by means of tests on the expression and the induction of various genes involved in blocking cell cycle (P21; P27; inhibitors of cyclin-chief of the independent kinases) and apoptosis (bcl-2, bcl-XLand bax). To conduct this test, cells are treated with test compound, are lysed secrete proteins and then analyzed by electrophoresis in LTO-SDS page, and related proteins in the gel is transferred onto nitrocellulose membranes. Initially, these membranes being probed using primary antibodies (such as antibodies to proteins P21, P27, bax, bcl-2 and bcl-XLand so on) and then detects diluted with secondary antibodies conjugated with horseradish peroxidase, and these membranes exhibit chemoluminescence (ADI) method detectionin reagent, with subsequent visualization of ECL-film. Through analysis of the relative content of these proteins can be estimated percentage of cells present at this stage of the cell cycle, for example, stage G0/G1stage S stage or G2/M.

Cytotoxicity of compounds against cancer cells can be effectively evaluated in vitro using MTT staining or crystal violet. In this method, the cells were seeded, treated with various concentrations of test compounds and incubated paint or MTT, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium, Sigma Chemical Co.), or crystal violet. The treated MTT Cup put lyse buffer (20% sodium dodecyl sulphate in 50% DMF) and conduct additional encubierta the s, followed by reading the optical density (OD) at 570 nm. Colored crystal violet cups washed to remove dye of Sorenson's buffer (0.1 M sodium citrate, pH 4.2; 50% ethanol by volume) and read at 570-600 nm (Mujoo et al., 1996). The levels of relative values of optical density are a total of cytotoxicity.

(iv) Immunological tests

Immunological tests can be used in connection with the present invention, for example, for screening of extracts of other species that are not acacia Victoria Acacia victoriae, the presence of triterpene compounds of the present invention. Covered by the present invention immunological tests include, but are not limited to, those tests that have been described in U.S. patent No. 4367110 (“sandwich”test with two monoclonal antibodies) and in U.S. patent No. 4452901 (Western blotting). Other tests are immunoprecipitate labeled ligands and immunocytochemical methods, both in vitro and in vivo.

In the most General and direct sense, immunological tests are tests on the binding. Some preferred immunological tests are different ways enzyme-linked immunosorbent assays (ELISA) and radioimmunoassay analysis (RIA), known in the art. Particularly effective are the immunocytochemical detection methods using histo is logicheskih drugs.

In one typical embodiment, ELISA antireticulin antibodies immobilized on the selected substrate with affinity in relation to proteins, such as the well polystyrene microtiter plate. Then well add a test composition suspected of containing triterpene compounds of the present invention, such as the extract of the herb, related to Acacia victoriae. After binding and washing to remove non-specific immune complexes associated antigen can be detected. In principle, the detection is carried out by adding another antibody specific to the desired antigen and attached to a detectable label. This option ELISA is a simple “sandwich ELISA”. Detection can also be implemented by adding a second antibody specific for the desired antigen with subsequent addition of a third antibody which has affinity for binding to the second antibody and a third antibody linked with a detectable label.

Modification of the ELISA methods known to experts in this field of technology. In one such embodiment, the sample to be tested for the content of the desired antigen, immobilized on the surface of the hole and then brought into contact with the preparation of antibodies. After binding and suitable washing detects education is avchina immune complexes. If the original antigen-specific antibodies is connected with a detectable label, the immune complexes may be detected by the direct method. In this case, the immune complexes may be detected using a second antibody which has affinity to the first antigen-specific antibody, and this second antibody linked with a detectable label.

Can also be used in a competitive ELISA: test sample compete for binding with a known quantity of labeled antigens or antibodies. This is the number of reactive molecules in an unknown sample is determined by mixing of the sample with a known amount of labeled molecules prior to or during incubation in sensitized holes. The presence of the molecules of the reactive species in the sample leads to a decrease in the number of labeled molecules associated with the hole, which ultimately leads to a weakening of the final signal.

Regardless of the format used for ELISA characterized by a number of common stages, such as sensitization, incubation or binding, washing to remove not specifically related molecules and detection associated immune complexes. These stages are described below.

The antigen or antibodies can also be attached to a solid substrate, such as a tablet, Shari and, coli, membrane, or column media, and can be used to analyze the sample applied to the immobilized antigen or antibody. For sensitization tablet either antigen or antibody, typically, the wells of the tablet is incubated with a solution of the antigen or antibody either overnight or within a certain period of time. Then wells should be rinsed in order to remove not fully absorbirowawrzegosa material. Then any remaining after that available surface in the hole “cover” non-specific protein, which is antigenically neutral with respect to the analyzed antisera. These are bovine serum albumin (BSA), casein or solutions of milk powder. This coating provides blocking of nonspecific binding sites immobilized on the surface and thus reduces the background caused by nonspecific binding of the antisera to the surface.

When conducting ELISA, apparently more common ways to use secondary or tertiary detection in contrast to the direct method. Thus, after binding of the antigen or antibody to the well, coating non-reactive material to reduce background, and washing to remove unbound material, immobilizers surface is subjected to contact with the analyzed clinical or bio is ogicheskom sample under conditions effective to provide education of the immune complex (antigen/antibody). Then, for detecting the immune complex, you must use labeled voicesvoices ligand or antibody, or a secondary binding ligand or antibody conjugate labeled “tertiary” antibody or third binding ligand.

The phrase “under conditions effective for the formation of the immune complex (antigen/antibody)” means that the preferred conditions which include dilution of antigens and antibodies such solutions as solutions of BSA, bovine gamma globulin (BGG) or phosphate buffered saline (SFR)/twin room. Such added agents also help to reduce the level of nonspecific background.

Suitable conditions also mean that the incubation is carried out at such temperature and for such a period of time sufficient to ensure efficient binding. Stage of incubation generally last approximately 1-2 to 4 hours, the temperature preferably is about 25-27°or they can be carried out during the night at about 4°With or similar

After all stages of incubation in ELISA, prokontaktirovat surface is washed to remove not formed a complex material. Usually for washing use a solution SFR/twin room or oralny buffer. After the formation of specific immune complexes between the analyzed sample and source related material and subsequent washing can be identified even a very small amount of immune complexes.

For carrying out the detection by the second or third antibody must be attached label. Preferably this should be an enzyme that contributes to the development of painting as a result of incubation with an appropriate chromogenic substrate. For example, the first or second immune complexes may be subjected to contact and incubation with the antibody, conjugated to urease, glucoseoxidase, alkaline phosphatase or hydroperoxidase within a certain period of time and under conditions that favor the formation of the following immune complex, for example, by incubation for 2 hours at room temperature in SR-containing solution, such as SFR/twin room.

After incubation with the labeled antibody and washing to remove unbound material perform quantitative evaluation of labels, for example, by incubation with a chromogenic substrate such as urea and bromocresol purple or 2,2’-Azino-di-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and H2About2if an enzyme label is horseradish peroxidase. Quantitative assessment is then carried out according to the degree of okra is the air traffic management, for example, using a spectrophotometer in the visible spectrum. Alternatively, the label can be chemoluminescence. The use of such labels is described in U.S. patent No. 5310687, 5238808 and 5221605.

Methods of analysis in vitro and in situ are well known and include an assessment of binding of antigen-specific antibodies with tissues, cells or cell extracts. Specialists in the art-known standard methods of this type. For example, antibodies that are specific against tumor antigens can be used in conjunction with both fresh-frozen and fixed in formalin, embedded in paraffin tissue blocks prepared for immunocytochemical analysis (IZA). Each tissue block may include 50 mg residual diffuse tumors. The method of preparing tissue blocks obtained from samples consisting of particles was previously successfully applied in ICHA for prognostic estimates, for example, in cases of tumors of the breast, i.e. it is well known to specialists in this field of technology.

Briefly, frozen sections can be prepared by soaking 50 ng of frozen diffuse tumors at room temperature in SFR in small plastic capsules; deposition of particles by centrifugation; their resuspendable in a viscous caulking environment; inversion caps the crystals and re-precipitation by centrifugation; instant freezing in isopentane at -70°; cutting the plastic capsule and selection of frozen tissue in the form of a cylinder; a review of this cylinder in the Chuck Cristallago of the microtome; and manufacturing 25-50 serial sections, each of which on average gets about 500 clearly intact tumor cells.

Continuous slices can be prepared in a similar manner, including re-hydrating the 50 mg sample in a plastic microcentrifuge tube; centrifugation; re-suspension in 10%formalin with 4-hour fixation; washing/centrifugation; re-suspension in the warmth of 2.5%agar; centrifugation; cooling in ice water for solidification of the agar; the allocation of the tissue-agar block from the tube; the straining unit and fill it in paraffin; and preparation prior to 50 series-permanent slices.

In light of this statement can be used screening tests for the identification of compounds with essentially the same chemical characteristics and biological activity compared to those described in this text. In particular, in this application, you can use the tests on the biological activity of triterpene glycosides those plants that are closely related Acacia victoriae, for example, representatives of the genus acacia, Acacia. the label of these tests can have different formats, what can be determined by the type of activity, the screening which is done. Preferred are those tests that investigate the antitumor activity, such as has been described here for extracts from Acacia victoriae. Used in the present description, the term “antitumor activity” means the suppression of intercellular signaling, growth, metastasis, cell division, cell migration, formation of colonies in soft agar, suppression of contacts, invasionist, angiogenesis, development of a tumor or other malignant phenotype and induction of apoptosis. In particular, are considered functional tests, which perform analysis on the use of the compounds of the present invention as antifungal and antiviral agents, ichthyocides or molluscicides, contraceptives, anthelminthic, means for protection against UV-radiation, means salt, diuretics, anti-inflammatory agents, regulators of cholesterol metabolism, cardiovascular effectors, antiulcer funds, painkillers, sedatives, immunomodulators, antipyretics, regulators of angiogenesis and means reducing capillary fragility. In the light of the present description, such tests are well known to specialists in this field of technology. As with the direct t of the CTE on activity in vitro and in vivo, these tests may include analysis of the suppression of binding to a substrate, ligand, receptor or other binding partners with the compound of the present invention.

X. Growth and tissue culture Acacia victoriae

An important aspect of preparing compounds of the present invention is the availability of tissue Acacia victoriae. Since the applicants have shown that the compounds of the present invention are concentrated in the roots and beans Acacia victoriae, the availability of these tissues is the most important.

Also applicants have shown that another source for the selection of the compounds of the present invention are young seedlings. Acacia victoriae is growing in the South-Western USA and Australia, i.e. plant material available to people. In addition, approximately 2500 seeds of Acacia victoriae was may 7, 1998, made by the applicants in the American type culture collection (ATSS: 10801 University blvd., Manassas, VA 20110-2209, USA). Those deposited seeds assigned to the access number in ATSC No. 209835. They were deposited in accordance with the terms and conditions defined by the Budapest agreement on the Deposit of microorganisms for a period of at least thirty (30) years and for a period of at least five (05) years after the most recent request for the provision of the deposited sample obtained by the Depositary, or on the validity of this paten is a, regardless of its duration, and this material must be replaced if they lose their viability within a specified period.

Thus, in the light of this application specialist in the art can plant the seeds from the specified Depository, to grow from them plants and allocate tissue obtained from plants with the aim of obtaining triterpene compounds and nutraceutical compositions of the present invention. You can also select the fabric from plants in natural populations of Acacia victoriae. However, obtaining tissue for isolation of the compounds of the present invention can be achieved more easily, if you want to play the tissues of Acacia victoriae will apply appropriate technology of cultivation. One of the conditions for obtaining such tissue must be large-scale cultivation of this species. However, the preferred choices are tissue culture Acacia victoriae and use aeroponic for growing.

(i) Cultivation methods aeroponic

Several advantages can be achieved when using methods aeroponic for growing Acacia victoriae. First, in this case, the growth rate of plants is approximately two times higher than the growth rate achieved by standard methods of cultivation. Secondly, if necessary, the roots can be easily collected bezposrednio plants. Moreover, the cutting of roots leads to intense lateral growth of the fibrous part of the root. Consequently, the roots can be collected several times during the year. In populations of Acacia victoriae wild-type picking beans is limited to a few weeks a year, and the collection of roots without damage or destruction of plants is very limited.

Aeroponically the rearing system is a closed system in which the plant roots are in the air and sprayed a full nutrient medium. Roots close in a waterproof box and through certain periods of time are sprayed with a nutrient medium. The nutrient solution contains all the necessary components for these plants complete their life cycle. Despite the fact that different plants require different levels and compositions for optimal growth, in General, once a balanced nutrient solution gives satisfactory results.

(ii) Tissue culture Acacia victoriae

Obtaining tissue cultures represent a different way of growing Acacia victoriae. For the formation of tissue culture, seeds of Acacia victoriae washed thoroughly with tap water with antimicrobial soap and water and treated with 20%solution of household bleach for 15 minutes. After repeated washing with deionized water, the seeds treated with boiling water in a is x stimulation of germination and incubated over night. The next morning the seeds again disinfected with household bleach and washed 2-3 times with sterile deionized water. Then the treated seeds are cultivated in the medium of Murashige-Skoog (MS) (Murashige et al., 1962), supplemented with MS vitamins and 2% sucrose (for cultures of explants used add 3% sucrose) and Wednesday utverjdayut by adding 0.7% agar, or 0.2% gelrite.

Used for culturing the explants represent almost any fabric, including the tips of the shoots, segments, nodes, hypocotyl and segments of roots. Usually the explants cultivated in pure MS or MS supplemented with growth regulators such as IAA, NAA, IBA, 2,4-D and VAR (separately or in combination). Usually culture is supported at 25±2°s setperiod 16 hours light intensity of 1000 Lux lighting used white fluorescent lamp cold light effect. The resulting seedlings incubated in the greenhouse when spraying for 1 month to seal tissue with subsequent planting in the greenhouse, in the open ground or changes made in the system aeroponic.

In the present invention were formed culture of hairy roots of Acacia victoriae. The development of hairy roots is due to the contamination of plants and nodule bacteria Agrobacterium rhizogenes strain R-1000, which leads to integration into the genome of plants is tion and expression of T-DNA. Culture of hairy roots are characterized by rapid growth, pleiotropy and intensive branching roots in hormone-free medium, and also exhibit a high level of genetic stability (Aird et al., 1988). Genetic transformation and induction of hairy roots of Acacia victoriae and optimal conditions for growth are described in detail in one of the sections in the examples. Culture of hairy roots provide a rapid, large-scale tissue growth, which can be used to highlight the triterpene compounds of the present invention.

The advantage of tissue culture is that can be formed clonal cultures that Express a connection according to the present invention. Such crops can be grown in large volumes and can be multiplied up to industrial scale plant tissue in order to emphasize triterpene compounds. In addition, the regenerated from these cultures plants are typically characterized by significant variability. Therefore, using these crops can be derived clonal cell lines or regenerated from such crop plants, which will be “elite” from the point of view of obtaining from them the triterpene compounds of the present invention. The resulting plants can be reproduced by snoopie the Oia and selected in each generation of selection with obtaining these onselectionchanged elite lines.

Elite does not necessarily have to be formed on the material tissue cultures, however, due to the significant genetic variability, they do exist in wild populations of Acacia victoriae. Therefore, applicants believe that found in natural populations of Acacia victoriae genetic variation includes variation of genes controlling the development of the endogenous triterpene compounds. If so, then it is possible to identify in populations of Acacia victoriae individuals, which are characterized by elevated levels develop triterpenes compared with other individuals of these natural populations, and possible selection of such lines for use in cropping systems aimed at obtaining tissue for isolation of triterpene compounds of the present invention. The rearing system may be, for example, standard agricultural cultivation, aeroponic, tissue culture or any suitable method of reproduction fabric Acacia victoriae. In addition, these plants can be selected for use in breeding activities for obtaining varieties, which are more elite and also reproducible as homozygotes.

XI. Definition

The article “a” means “one or more”. Thus, the word “component” (“a'm moiety”) may refer to one, two, three or the great number of components.

The “active ingredients” refers to the most pure extract, remain active. In connection with the present invention, the terms “active ingredient” or “active compound” refers to active triterpene compounds identified by the authors of the present invention. These compounds were purified and identified, for example, in the fraction of UA-BRF-004-DELEP-F094.

“Beans” refers to the fruits of Acacia victoriae type of beans.

The term “cytotoxic” determines cell death, while the term “cytotoxic” defines the suppression of growth and/or proliferation of cells.

“Apoptosis” is defined as a normal physiological process of programmed cell death that occurs during embryogenesis and during tissue homeostasis. The process of apoptosis can be divided into a number of metabolic changes in apoptotic cells. Separate enzymatic cascade reactions in various regulatory mechanisms or mechanisms of signal transmission can be determined in connection with detection of apoptosis in a cell or cell population or identified in connection with the loss of the ability to apoptosis in cancer cells. Also apoptotic program is reflected in the morphology of cells that includes the changes of the plasma membrane (for example, the loss of its asymmetry), condensation cytoplas the s and kernel and mineclearance splitting the DNA (the so-called “spraying” of chromatin). The result is cell death, is transformed into the so-called “apoptotic calf.”

Assessment methods a variety of enzymatic processes and signaling, involved in apoptosis, designed in the form of standard protocols multifactor study of apoptosis. An example of one of the markers of early stages of apoptosis is the release of mitochondrial cytochrome-C with subsequent activation kasperskogo-3 way (PharMingen, San Diego, CA). Induction of caspases (group cytoplasmic proteases) is one of the most significant properties of apoptosis. In particular, caspase-3 plays a key role in this process. After the activation of the caspase break down proteins-target:

one of the most important of these is RARP, poly-ADP-ribosomally localized in the nucleus. Therefore, effective detection of apoptosis can be carried out in tests aimed at the detection of the release of cytochrome C, the detection of caspase-3 and the detection destruction RARP.

Moreover, the agents that cause the release of cytochrome C from the mitochondria of malignant cells, can be identified as potential drugs aimed, at least some aspects of cellular control of programmed cell death.

Another test for apoptosis is the detection of annexin-V BioWhitaker, Walkerville, MD). Usually phosphatidylserin (FS) is located on the inner surface of the plasma membrane. However, in the early stages of apoptosis is the externalization of PS. Annexin-V is a calcium-binding protein that binds to the FC and can be detected after staining complex annexin-U-FITZ by flow cytometry (Martin et al., 1995). The ability of cells treated as described in the present invention are compounds of Acacia victoriae, to bind annexin-V, is taken as an indicator of the entry of cells into apoptosis.

In other examples, the applicants had used the test kinase PI-3 and to identify apoptotic activity of cells treated with compounds having antitumor activity of compounds isolated from Acacia victoriae. Phosphoinositide-3-cinea (PI3K), which is associated with the cellular membrane enzyme capable of fosforilirovanii inosine ring phosphatidylinositol the atom at the 3-position, which thereby defines a new lipid signaling pathway in those cells in which the active PI3K. In case the activity of P13-kinase, another kinase, denoted by the ACT, the mate was recruited to the cell membrane. The ACT is encoded by the oncogene and catalytically activated after joining the membrane. Fully-activated kinase ACT plays a key role in the survival of cells. Interaction PI3KAKT is a mechanism by which cells escape apoptosis. Thus, the method of suppressing PI3K in cells of malignant tumors causes a possible therapeutic tool to restore at least some elements of the cellular control of apoptosis.

The expression “abnormal proliferation” refers to the number of genetically determined changes that occur in cells during pathological condition known as cancer. Eventually, this process leads to loss of control of apoptosis in cancer cells. This can happen Paladino and is usually referred to as: (1) “initiation”, which determines the phase in which an external agent or stimulus causes a genetic change in one or more cells; and (2) activation, defined as stage, leading to further genetic and metabolic changes that can cause inflammation. In the stage of activation of the cells ' metabolic transition to the stage of cell growth, in which apoptosis is blocked.

“Malignant cells” are defined as cancer cells, devoid of the normal mechanisms of control of cell growth through a series of metabolic changes that occur during the initiation and activation of the beginning stages of malignancy. These changes are the result of genetic changes in the cells (either activating mutations and/or increased expression of proto-oncogene either inactivating mutations and/or the attenuation of the expression of one or more tumor-suppressor genes). The products of most of oncogenes and tumor-suppressor genes are components of the cascade reactions of the transmission signal, which controls entry into the cell division or exit, differentiation, damage to the coding DNA and the initiation of reparative mechanisms, and/or regulate program cell death. In cells operate multiple parallel mechanisms of growth regulation, cell differentiation, control of DNA damage and apoptosis. Almost all of the tumor and cancer cells have mutations in multiple oncogenes and tumor suppressor genes.

“Extract” or “fraction” refers to a sequential samples obtained in various ways from the tissues. These “extracts” or “faction” can be analyzed on the desired antitumor activity, and additionally “extracted” or “fractionated” for more pure components corresponding to the active component.

“Triterpen or triterpene glycoside” refers to new and/or biologically active saponine compounds identified here from Acacia victoriae. Triterpenes or triterpene glycosides do not have to be isolated from Acacia victoriae, because Liu is my specialist in the art based on the data of the present invention may allocate a connection from related species or chemically synthesize analogues triterpenes and triterpene glycosides, as described here. “Triterpenes” of the present invention include saponine compounds described herein that include at least triterpene(s) link(rd) and, in the case of triterpene glycosides, sugar or saccharide. Also, these terms encompass compounds, including additional components or chemical functional group including, but thereby not limited to, the monoterpenoid links that will be specified in another part of this application. Thus, triterpenes of the present invention also include the aglycones formed by hydrolysis of the sugar components, but also include other modifications of the triterpene compounds, unless such modifications do not violate the biological activity of these compounds.

XII. Examples

The following examples are provided to illustrate preferred variants of the present invention. Specialist in the art should be understood that the methods described in the following examples are ways to successfully implement the present invention, and therefore, they can be considered as a preferred model for its implementation. However, based on the present invention, each specialist in the art it should be clear that a particular option may be made various changes, which will give the same or similar results, not beyond ideas, creatures and scope of the present invention. More specifically, it should be clear that various chemically and physiologically related agents can be used to replace the agents described here, if it will achieve the same or similar results. All such substitutions and modifications, obvious to every expert, are seen as responsible entity, the scope and idea of the present invention, formulated in the accompanying claims.

EXAMPLE 1

Preliminary screening and purification of active anticancer component of Acacia victoriae

Sixty species were collected during the implementation of the research program DELEP (the study of leguminous plants of the desert zone) in order to identify new compounds with attractive biological properties. The program DELEP (University of Arizona in Tucson) includes a collection of desert species of leguminous plants, created with the cooperation of the University of Arizona and southwestern arboretum them. Bthompson. Experimental field samples were collected from each plant species, air-dried for 3-4 days, crushed to a particle size of 3 mm in a mill Willie (the screen with apertures of 3 mm) and extracted 2 or 3 the Aza method of percolation with a mixture of 1:1 dichloromethane (DCM) and methanol (Meon). Each percolation extraction was carried out for at least 5 hours and often continued throughout the night. The bulk of the extract was obtained after the first two stages of percolation cleaning. Then the biomass was washed with a volume of methanol equal to half of the unoccupied volume with the separation of the crude extract contained in methanol aliquot. Usually the samples were isolated and prepared for biotests removing methanol under vacuum, passing the aqueous phase through particles RP-C18, the return of the active components in the Meon and then rotary evaporation Meon with obtaining the extract in the form of solids. The crude extract is then resuspendable in water, DMSO or mixtures (less polar compounds were resuspendable in DMSO, while the more polar compounds were resuspendable in water or in a mixture of water and DMSO; aglycones were resuspendable in DMSO).

Each of the extracts were then subjected to screening against a panel of tumor and non-neoplastic human cells, including lines of cancer cells human T-leukemia cells, epidermoid cells of human breast cancer cells human cancer cells of the prostate gland human foreskin fibroblasts human endothelial cells and cancer cells of human kidney. Initially, cells were planted in 96-well plates at 18-24 at 37° C. Then the cells were treated with various concentrations of plant extracts and incubated for 72 hours at 37°and stained With either 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) (Sigma Chemical Co.) within 4 hours, or crystal violet (Sigma Chemical Co.) within 20 minutes at room temperature. On MTT-coated tablets inflicted lytic buffer (20% sodium dodecyl sulfate in 50%DMF) and incubated for 6 hours followed by optical density reading at 570 nm. Colored crystal violet tablets washed, the dye was extracted within 3-4 hours of Sorenson's buffer (0.1 M sodium citrate, pH 4.2; 50% ethanol by volume) and tablets read at 570-600 nm (Mujoo et al., 1996). The cytotoxicity of the tested extracts was determined by comparing the data read OP between “pure” processed medium and cells treated with plant extract. The level of cytotoxicity in percent was determined by subtracting from 100 percent control, where the percent of control = [((OP cells treated with plant extract (treated sample)/(OD of cells treated with empty environment (untreated sample)))×100].

During the initial screening for one plant extract has been demonstrated strong growth inhibition of cancer cells with low toxicity in about the wearing of normal human fibroblasts. This extract indicated UA-BRF-004-DELEP-F001, was isolated from bean plants Acacia victoriae. This extract exhibited IC50at the level of approximately 12 µg/ml (SKOV-3), 26 mg/ml (OVCAR-3) and 13 µg/ml (HEY) when testing cell lines of ovarian cancer man; more than 50 µg/ml (A-M) and about 38 μg/ml (HS294T) against melanoma cells; approximately 15 µg/ml for epidermoid human cells (A431); and more than 50 µg/ml for the line of breast cancer cells MDA-468 (figure 1) (see example 13, in describing these cell lines). In the normal foreskin fibroblasts human (FS) and mouse fibroblasts (L929), handled by the same extract, cytotoxicity was not revealed.

According to TLC this extract contained a mixture of a large number of components. Therefore, preliminary efforts were concentrated on the purification of the extract with the aim of identifying the active components responsible for the selective cytotoxicity. Chromatographic fractions, enriched with active ingredients that have been selected from the original extract in accordance with the basic circuit shown in Fig.

The original extract, UA-BRF-004-DELEP-F001, was prepared from 538 g of plant material Acacia victoriae method percolation (twice), as described above. Then the extract was dried in vacuum to obtain about 52,0 g of powder. After that 51,5 g dry m the material three times was treated with 1 l of ethyl acetate (EtOAc). Approximately of 15.75 g dissolved in EtOAc material was subjected to column chromatography on silica gel (1.5 kg). 54 subtractio 670 ml was suirable using mixtures of increasing polarity of hexane, EtOAc and Meon. 54 subfraction were selected in 13 separate fractions, indicated UA-BRF-004-DFELEP-F006 to UA-BRF-004-DELEP-F018. Then these fractions were tested for antitumor activity using the procedure described above. None of the tested fractions did not show antitumor activity, identified for UA-BRF-004-DELEP-F001.

Insoluble in EtOAc material (approximately to 34.7 g) was also chromatographically on silica gel (1.7 kg). 51 subtractio 670 ml and 3 additional subtractio a total volume of 21 l was suirable mixtures with increasing polarity DCM, Meon and water. These subtractio collected in 8 separate fractions, indicated UA-BRF-004-DELEP-F019 to UA-BRF-004-DELEP-F026, in accordance with the table. 6.

Then, each fraction was subjected to screening for anticancer activity against a panel of tumor cells as described above for the crude extract. One of these factions, UA-BRF-004-DELEP-F023, showed antitumor activity, higher compared to UA-BRF-004-DELEP-F001. The results showed that 6 ág/ml fraction of UA-BRF-004-DELEP-F023 showed cytotoxicity at 50% (OSS), 6% (SKOV-3), 85% (HEY) and 48% (OVCAR-3) cells of ovarian cancer man; cytotoxicity approximately 60% in prostate cancer cells human (LNCaP); cytotoxicity approximately 92% against leukemia cells (Jurkat) and cytoxicity approximately 73% in freshly isolated cells of ovarian cancer man from samples of ascitic fluid in patients with (FTC). In biotests with respect to the normal cells were determined IC50: to 10.6 ág/ml for cells FS and 23 µg/ml for HUVEC cells (figure 2).

Further, the purification of the biologically active components of UA-BRF-004-DELEP-F023 was carried out by separation by liquid chromatography with an average pressure (GHSD) repeatedly reversed phase order selection and characterization of the active(s) component(s). Samples were suirable of degassed mixtures with increasing concentrations of acetonitrile (ACN) in water in a 4 l 10% increments in the following stages: 0%, 10%, 20%, 30%, 40% acetonitrile in water. Then 2-4-liter fractions were suirable methanol. 10 fractions were selected after repeated passes and marked from UA-BRF-004-DELEP-F027 to UA-BRF-004-DELEP-F036 in accordance with table. 7.

Table 7

Elution fractions from UA-BRF-004-DELEP-F027 to UA-BRF-004-DELEP-F036
The number of fractionsTotal weight (g)Eluent
F027 6,950-20% acetonitrile in water
F0280,9930-40% acetonitrile in water
F0291,4630-40% acetonitrile in water
F0300,8630-40% acetonitrile in water
F0310,1530-40% acetonitrile in water
F0321,0130-40% acetonitrile in water
F0330,5430-40% acetonitrile in water
F0340,5030-40% acetonitrile in water
F0352,1930-40% acetonitrile in water
F0361/1730-40% acetonitrile in water

Some of these fractions were similarly highlighted by TLC. A strong manifestation of antitumor activity was found for faction (35), UA-BRF-004-DELEP-F035, characterized by one of the most high outputs.

Screening of UA-BRF-004-DELEP-F035 on the antitumor activity was determined IC50level, respectively, 3,0, 1,2, 2.0 and 3.5 g/ml against cell lines of ovarian tumors HEY, SKOV-3, OVCAR-3 and C-1 (resistant to this drug called cisplatin subline OVCAR-3); IC50=2.4 µg/ml against tumor cells of the pancreas (Raps-1); IC50=1.2 µg/ml, and 3.0 μg/ml and 3.7 μg/ml, respectively, for cell lines p. the targeted tumor 769-P, 786-0 and A; IC50=130 ng/ml for T-leukemic Jurkat cells; and ICso in the range of 1-3 µg/ml against In-leukemic cell lines KG1, REH and NALM-6 (3, 4). As shown in table 8, the purification of the crude plant extract leads to a sharp increase biological activity.

Table 8

The cytotoxicity of the crude extract in comparison with UA-BRF-004-DELEP-F035
Human cancer cellsIC50(ág/ml)
The crude extractUA-BRF-004-DELEP-F035
HEY123,0
SKOV-3251,2
OVCAR-3252,0
MDA-468509/0

Fraction 35 detects the IC50at the level of approximately 4.7 µg/MP against normal fibroblasts and the IC50approximately 13.3 µg/ml against normal cells s27 person. When the effect of fraction 35 (F035) was evaluated with respect to the normal populations of erythroid and myeloid lines (cells isolated from the bone marrow) at a concentration of 3.0 μg/ml, the level of inhibition was 12-18% (table).

Table 9

Influence fracc and 35 on erythroid and myeloid colony of cells
 Erythrocytes (number of colonies)The percentage of inhibitionMyeloid cells (number of colonies)The percentage of inhibition
No processing261-111-
F035 (30 µg/ml)16945352
F035 (3 µg/ml)212189712
F035 (0.3 ág/ml)2485,01197 (stimulation)

From the above data a strong antitumor activity fraction 35 was the Biotest corresponding to that described above, using the concentration of fraction 35 until 0,095 µg/ml In this assay, various concentrations of fraction 35 were applied to the extended panel lines of tumor cells. Obtained in this test, the results show that the fraction of 35 even at a concentration of 1.56 mg/ml exhibits antitumor activity against many cell lines (table 10).

Table 10

The cytotoxicity of different concentrations of UA-BRF-004-DELEP-F035 for different lines of tumor cells
UA-BRF-004-DELEP-F03550 mg/ml 25 mg/ml12.5 ág/mlat 6.25 µg/mlof 3.12 µg/mlof 1.56 µg/ml0.78 µg/ml0.39 µg/mlof € 0.195 mg/ml0,095 mg/ml
SKOV-394%83,40%78,50%71%54%27%0%0%0% 
OVCAR-395%92,80%91%87%79%46%9%21,40%18% 
S-1 (OVCAR-3 variant)97%71%87%77%59%29%0%0%0% 
HEY97%79,10%53,90%43,30%19,20%0%0%0%0% 
C-2(HEY variant)96%93,20%90,50%88,90%86,80%82,50%73,4050%36,10% 
A-8((HEY (option)97,20%95,70%94,80%89%75,00%59,30%18%0%0% 
MCF-7 79,70%23%5%3,10%8,10%17,20at 8.60%17,4019,20 
W-2083%90%0%4%12,50%15,50%21,30%24%34% 
MDA-MB-45398,40%97,20%94,60%89,90%85,40%the point 81.60%65,70%54,90%38,50% 
MDA-46896,50%93,80%82%65%39%8%8%8%8% 
SKBR-383,90%62,70%51,70%47,80%45,20%39,60%35,90%28,60%21,90% 

Table 10 (continued)
PANC-197,30%90,20%66,80%30,40%0%0%0%0%0% 
769-P96,80%97%90%95%94,00%91,70%63% 18%10%17,80
786-097,90%89%80,30%75%66%32%0%0%0% 
A-49899%97%95,50%80%47%19%16%15%14% 
LLC-1to 84.70%42,70%17,80%6,60%10,70%10,90%3,80%6,80%0% 
A96,60%91,10%59,70%34,80%21,20%15,60%0%0%0% 
JURKAT88,40%88,70%88,80%89,60%88,60%88,50%80%69,30%46,60%0%
Hs2788%83%47%0%6%11%11%13%18% 
FS FIBRO78,30%74,70%73,70%66,50%42,30%0%16%23,20% 
HL-6063,00%22,00%30,00%25,00%0,00%0,00%0,00%0,00%0%0%
MDA-MB-43596,50%96,40%97%96%94%to 84.70%40,60%15%14,70% 
DOV-1395,80%92,30%86,70%77,80%57,50%14,20%17,50%11,10%12,20%17,20%
MCF-10A97,50%11,70%0%1%0%0%0%0%0% 
MCF-10F97,70%5,80%0%0%0%0%0%0%0% 
KG-177%75%72%67%59%44%35%13%0%0%
OCI-246,40%21%12%12,30%9%12%% 0%0%0%
OCI-371%60%45%41%30%the ceiling of 5.60%0%0%0%0%

Example 2

Methods of isolation of active components from Acacia victoriae

Was developed a method of directly obtaining fractions containing the active components of UA-BRF-004-DELEP-F035 allocated during the pre-treatment described in example 1. Approximately 9665 g fresh tissue beans Acacia victoriae were crushed in a hammer mill with a 3 mm sieve and then was extracted with 80%Meon in water (three times) followed by filtration. 8200 g husks discarded. The results of the three washes were collected separately, labeling them as the following fraction: F068 21.5 l (first washing); F069 24 l (second wash); and F070 in 34,3 l (third washing). Fraction F068 further purified by separation on a 1-liter aliquots, adding 400 ml of water to each aliquot and washing with chloroform (2×250 ml). The joint polar phase (28,5 l) was defined as the fraction F078, and the combined organic phase was defined as the fraction F079 (yield 42 g after removal of the organic solvent on a rotary evaporator).

Meon removed from F078 in vacuum and then F078 was fractionally method OF GHSD using column size 29 on; 460 mm, loaded 530 g restored Bakerbond RP-C18, particle size 40 μm. 500 ml of an aqueous solution aspirated into the column and fractions were collected in accordance with table.

Table 11

Elution fractions from F091 to F094
The number of fractionsThe collected volume (l)Total weight (g)EluentComments
-4 100% waterSugar and strong RBC (erythrocyte)-lytic component
F0914~4010% acetonitrile in water19,6 g fractions from 16 to 29
F09248920% acetonitrile in waterFlavonoids
F093435130% acetonitrile in waterWhite fluffy solid, slightly irritant breathing
F0941,3577100% methanolFine-grained powder, irritant breathing

Then, each fraction was dried by removal of methanol, passing through the particle-18 returned in methanol and was isolated in vacuum in the form of solids. Solid resuspendable in water and tested for antitumor AK is Yunosti (less polar fractions in water was added DMSO; the aglycones resuspendable in DMSO). The results showed that the biological activity of methanol (100%) as eluent, designated as fraction F094 essentially equivalent activity defined for a fraction of UA-BRF-004-DELEP-F035 (table). The active components are also contained in the faction F093. Chemical similarity fractions F094 and F035 confirmed using methods TLC and HPLC, although F094, apparently, contains additional components.

Table 12

The cytotoxicity of different concentrations F094 with respect to the various lines of tumor cells
UA-BRF-004-DELEP(beans)-F035769-PPANC-1HEYMDA-MB-453Jurkat
50 mg/ml96,60%97%95%94,20%89,60%
25 mg/ml93,30%93,50%66,50%92,80%89,80%
12.5 ág/ml92,80%74,60%50,10%87,10%89,40%
at 6.25 µg/ml92,40%50,60%17,90%85%89,30%
of 3.12 µg/ml88,30%21,90%0%77,30%89%
of 1.56 µg/ml63,20% 1,10%0%58,50%88%
0.78 µg/ml80%0%0%47%73,80%
0.39 µg/ml4,50%0%0%47%65,70%
0,19 mg/ml2,10%0%0%26,80%0%

F094 was further fractionated in accordance with the table. 13 and analyzed by TLC and biotests with the aim of identifying the purified(s) active(s) component(s). The results of biotests conducted on various quantities obtained fractions (F138-F147)shown in table 14.

Table 13

Elution fractions F138-F147
The number of fractionsCollected subfraction (ml)Total weight (g)Eluent
-1-5(160)160% Meon in the water
F1386(65);1360% Meon in water (6)
7-8(50)70% Meon in water (7-8)
F1399(25)3970% Meon in the water
F14010(20)9370% Meon in the water
F14111(35)57 70% Meon in the water
F14212(50)5470% Meon in the water
F14313(55)6270% Meon in the water
F14414(70)2970% Meon in the water
F14515(65)1770% Meon in the water
F14616(80)5480% of the Meon in the water
F14717(80);780% of the Meon in water (17);
18(100)100% Meon in water (18)

Table 14

Biological testing of the fractions F137-F147
 50 mg/ml25 mg/ml12.5 ág/ml
F137   
769-P81,5045,5018,10
Raps-174110
HEY6,200
MDA-MB-45376,7038,8026,80
JURKAT67,7067,5067,80
F138   
769-P 96,5095,6095,30
Raps-195,5093,4573,50
HEY65,3058,3021,50
MDA-MB-45396,1094,2092,5
JURKAT87,508887,50
F139   
769-P97,3094,2094,20
Raps-196,6094,1086
HEY89,7065,80of 60.50
MDA-MB-453959591,90
JURKAT88,5088,5088,50
F140   
769-P91,7088,9087,50
Raps-19594,6092,50
HEY95,4072,1062,80
MDA-MB-45386,2080,2075,20
JURKAT68,4067,8068,10
F141   
769-P97,8095,1095
Raps-196, 8095or roughly 85.60
HEY9668,8060,6

Panc-1
MDA-MB-4539594,5094
JURKAT88,5088,4088
F142   
769-P92,5090,2088,20
Panc-19693,6088,60
HEY9874,8066
MDA-MB-45386,1075,4072,90
JURKAT67,9067,10to 66.30
F143   
769-P98,3096,8098,30
Panc-196,7094,70or roughly 85.60
HEY98,507364
MDA-MB-45396,709594,10
JURKAT88,008888
F144   
769-P89,8088,6089,50
96,6093,8090,90
HEY98,5075,3062,20
MDA-MB-45386,7078,5075,80
JURKAT65,7065,7065
F145   
769-P9290,2086,30
Panc-196,7091,4084,80
HEY97,5082.30 level58,60
MDA-MB-45385,4074,4048,90
JURKAT67,9068,4068,60
F146   
769-P97,3097,3063,30
Panc-19788,9043,40
HEY97,6070,5022
MDA-MB-4539594,8078
JURKAT88,6088,2088,10

F147   
769-P44,3023,405
Panc-140110
HEY000
MDA-MB-453705057
JURKAT86,308478,70

The percentage inhibition of growth

Although the above procedure was aimed at highlighting the active components of the beans Acacia victoriae, active components can be extracted from the roots. In this case, the roots of the ground within half an hour and fill in 100% methanol. Then the mixture is filtered and diluted to 80%Meon in the water. If you want to spend the extraction of a large number of roots, preferred can then be carrying out the extraction by percolation, as described above. The rationale for this distinction extraction procedures is that the roots are usually freshly extracted, and beans, as a rule, before extraction dried.

Example 3

Step preparative procedure for obtaining the active components of the fraction of UA-BRF-004-DELEP-F094

A modified procedure of extraction/separation was used for stepwise (stepwise) obtain mixtures of active components of the composition fraction of UA-BRF-004-DELEP(beans)-F094 (F094). This procedure was repeated several times, gradually receiving highly active fraction. Usually 20-25 g F094 or the e equivalent was dissolved in 150-175 ml of 50% Meon in water, then aspirated in column (26 mm × 460 mm)+(70 mm × 460 mm), RP-C18, 40 μm, 1200 g, balanced 60% Meon/N2O. Fractions were suirable stages 8 l 60% Meon/N2About; 7.5 l 70% Meon/N2O; and 2 liters of clean Meon and the fractions obtained were identified in accordance with table. 15. Fraction F035-B2 contains a mixture of active ingredients available in F094, F133-136 (isolated from F093) and F138-147 (isolated from F094), as shown in figa-F. F094 is an acceptable substitute F035 at 1-2-fold decrease in the efficiency, and the fraction F035-B2 has a lower efficiency than F094.

Table 15

Selection F035-B2
The number of fractionsThe collected volume (l)Total weight (g)Eluent
F23781,860% Meon in the water
F238 this1870% Meon in the water
F035-B23,58070% Meon in the water
F23931970% Meon in the water
F240220100% Meon

To further purify the active components faction F035-B2, applicants have developed a procedure for obtaining fractions with analytical HPLC profiles of the fraction of UA-BRF-004DELEP-F035. This procedure consisted of the following: preparative HPLC was carried out on the material fractions F035-B2 using columns for reversed-phase chromatography with particles 10 microns in order to gradient elution with mixtures of acetonitrile and water, ranging from 26% acetonitrile in water to 40% acetonitrile in water with a step gradient of 1-2%, followed by rinsing with 100% acetonitrile and washing 100% methanol, which results in a unique division F035-B2 into several fractions containing 0-20 minute peaks (based on the standard 6-µm analytical method HPLC with RP-C18), which were not found in the original faction F035 (figa). The rest of the fractions obtained were consistent with 1-3-component fractionation F035. As shown in the above analysis, the fraction F139-F147 similar to these fractions, showing somewhat higher antitumor activity against one of the lines of tumor cells in comparison with others. A unique blend of active ingredients present in the fraction F035 may be obtained, in General, by modifying the original song in the front row of solvents from 16% to 26% acetonitrile in water followed by purification method GHSD obtaining multiharmonic quantities equivalent of UA-BRF-004-DELEP-F035.

Further improvement of the above-described procedure, extrac the AI, as well as other extraction procedures described in this text can be effected by the use of mixtures of the three solvents, acetonitrile, methanol and water. Their percentages can dynamically grow and can be optimized by a specialist in the field of standard chromatographic separation. In the same way related on silica gel phase can vary depending on a combination of combined reversed-phase systems, including, but not limited to, C-8, CN, dimethyldi and C-18. In the final stages even silica gel normal phase can be used for final cleanup procedures.

Example 4

Alternative procedures for isolating active ingredients from Acacia victoriae

Faction F094 (250 g), F035 (50 mg) and powdered beans Acacia victoriae (1 kg) (i.e., “fruit-seed” powder) were obtained as described above. This example describes an analytical procedure for the analysis of fractions F094 and subsequent fractionation F094.

4.1. Analytical procedure

A number of methods, including a variety of C8 and C18 column gradient and isocratic conditions were used for splitting fraction F094. The monitoring involved the analysis of UV absorption at 220 nm and detection of light scattering by evaporation (ELSD). The best resolution of the peaks occurred when the moveable the e phase triperoxonane acid (TFU). The following technique, which received the name “acacia-257”. This method provides a good solution level in a short time fractionation.

Washroomcare was equipped with a detector with diode array (DAD) or a detector with variable wavelength and column 4,6×150 mm Inertsil C18, 3 MK (MetaChem). The detector was set at a wavelength of 220 nm.

Used the following gradients.

Time (min)Acetonitrile, %Water with 0.1% TFU,%
03070
363664
424258
42,13070
473070

On Fig shows the chromatogram F094 obtained by this method. F094 consists of three groups or families with a presence in each of them several peaks. Group-1 (8-20 minutes: peaks A-D), group-2 (22-35 minutes; peaks E-N) and group-3 (36-47 minutes; the peaks I-L). Fraction F035 was also analyzed in this way: the corresponding chromatogram is shown in Fig. The peaks of the second group is more numerous in the case of F035 compared to F094, where the most numerous peaks of the 1st group.

4.2. Fractionation

4.2.1. Initial fractionation

Machineprecision was concentrated at the peaks of the group-1. Prepreparation column Symmetry C8 (Waters) (7,8×300 mm, 7 MK) used for this purpose with the parameters of gradient elution is shown below. Seven sub-fractions were isolated in accordance with Fig. The last fraction (No. 2160-007-31) includes all peaks group-2 and group-3. These fractions, as well as original material (F094) were included in the biological testing.

Time (min)Acetonitrile, %Water with 0.1% TFU,%
0,02773
38,03070
42,19010
48,09010
49,02773
65,02773

4.2.2. The second fractionation

The purpose of this stage fractionation was the separation of the peaks of the compounds of group-2. For this I used the same prepreparation column C8. The mobile phase was isocratic: 32% acetonitrile in water + 0.1% of TFU. Chromatographic mark pointing to division 7 of the fractions shown in Fig. The first separated fraction in this case covers all the peaks of the group-1.

4.2.3. Biological tests

The results of biological testing of sub-fractions obtained with the first and boromrajchonnanee, shown, respectively, in table 16 and 17.

Table 16

Cytotoxicity of sub-fractions of the first fractionation against Jurkat cells
No. factionWeight (mg)*Cytotoxicity; IC50(ág/ml)
2160-007-032,7inactive
2160-007-071,9inactive
2160-007-111,3inactive
2160-007-151,6inactive

2160-007-19 (peak D1)1,71,2
2160-007-25 (peak D2)2,9the 5.7
2160-007-313,21,3
2160-007-34 (F094)9,30,17
* The precision of the estimate weight of about ± 20%.
Table 17

Cytotoxicity of sub-fractions of the second fractionation against Jurkat cells
No. factionWeight (mg)*Cytotoxicity: IC50(ág/ml)
2160-025-017,241,2
2160-025-024,742,8
2160-025-033,63 1,0
2160-025-04 (G1 peak)1,370,64
2160-025-05 (peak G2)2,071,56
2160-025-06of 3.640,33
2160-007-34 (F094)12,090,17
* The precision of the estimate weight of about ± 20%.

Two purified triterpene glycoside, designated as D1 and G1 were obtained from fractions F094 Acacia. In the acid hydrolysis of D1 is formed aglycon.

4.4. Step preparative fractionation to obtain peak areas D and G/H

F094 (2.3 g) was fractionally on the column for HPLC Prep PFP (pentafluorophenyl) (50×250 mm, 10 μm). Mobile phase: acetonitrile / water with 0.1% triperoxonane acid (TFU) with a gradient of acetonitrile from 27% to 32%, while 38 minutes. As shown in Fig, in this way were separated fractions containing the peaks D and G/h is Selected when this preparative separation of the fractions were subjected to biological testing. The results of the analytical tests D and G/H is shown in Fig and 31. In this case, applied the method of “acacia-257”described earlier in this section.

Faction G/H was further purified on a column of PFP Prep order to obtain G1 with chromatographic purity 68%: this material was further purified on prepreparation C18 column to obtain pure G1.

Approximately 100 mg of Masi G/H were loaded onto the same column PFP, which is described above. Used the following gradient.

Time (min)Acetonitrile, %Water % 0.1% TFU
02773
12971
403466

Received five fractions (G1, G2, G3, H1 and H2). Analytical data for G1 (Fig) indicated that 68%of chromatographic purity. This fraction was further purified on prepreparation column.

Used column YMC C18-Aq (10×250 mm, 5 μm). Mobile phase: 31% acetonitrile in water with 0.1% TFU. The end product of G1 were characterized by chromatographic purity 100% (Fig).

Fraction D (45% D1) with PFP column Prep was first fractionated using column Waters C-18 (25×100 mm). Mobile phase: 61% methanol in water with 0.1% TFU. HPLC analysis showed that D1 is characterized by a purity of 78% (Fig) and contains an additional peak (marked D1.5). Sample D1 with chromatographic purity 100% (Fig) was obtained by further fractionation of the crude D1 at column YMC C18-Aq a mixture of 33% acetonitrile - water with 0.1% TFU as mobile phase. It has been shown that D1 is more stable in dilute acid solutions than in water at 40°C. With this in mind, the composition of the solvents in PTS during the harsh conditions of D1 was included TFU (0,1%).

4.4.1. Biological tests

Biotests were performed using cell lines (Jurkat, and the influence of sub-fractions and pure D1 and G1 are shown, respectively, in table. 18, 19 and 20. D1 and G1 were tested at two different pH values. The obtained data indicate a slightly higher activity at pH 6.5 than at pH 7.5. However, cell growth was suppressed by about 40% at lower values of pH.

Table 18

Cytotoxicity of fractions with PFP column Prep
No. factionDescriptionWeight (mg)*Cytotoxicity: IC50(ág/ml)
2160-035-22peak D1,70,52
2160-047-01peaks G/N1,240,12
2160-047-03peaks I/J/K1,660,19
2160-047-05The plot of peak L1,170,18
2160-047-07peak M1,720,24
2160-007-34F094 0,21

Table 19

Cytotoxicity of fractions obtained by fractionation G/H
No. factionThe weight (the g)* Cytotoxicity: IC50(ág/ml)
2160-53-8-G11,871,23
2160-53-11-G20,762,2
2160-53-14-G30,674,35
2160-53-17-N10,296,25
2160-53-20-H20,4512,8
2160-007-34(F094) 0,38
Table 20

Cytotoxicity D1 and G1 at pH=6.5 and 7.5
No. factionWeight (mg)*Cytotoxicity: IC50(ág/ml)
  pH=6.5pH=7,5
2160-69-29(D1)1,0361,010,98
2160-083-30(G1)1,9510,30,49
2160-007-34(F094) 0,150,22

4.4.2. Acid hydrolysis D1

Example 5

The structure of D1, G1 and B1

5.1. Structure D1

D1 is the main component beans Acacia victoriae. When testing the connection was established, significant biological activity.

5.1.1. Full molecule D1

D1 was isolated as a colorless amorphous solid from a partially purified extract F094 obtained using preparative HPLC-separation in accordance with the above-described examples. When mass spectroscopy MALDI its molecular mass was 2104 Amu and minus ion sodium real molecular mass was 2081. In the high-resolution method of mass spectroscopy FAB was confirmed this molecular weight and defined the molecular formula C98H155NO46. This molecule is too large to determine its structure only by the spectroscopic method; hence used of the separation shown in figure 1 Fig. D1 on Fig marked as structure "(1)".

Proton and carbon NMR of molecules D1 showed the presence of triterpene, two monoterpenes and approximately eight sugars (see table 21 the figures obtained13C-NMR in column (1)).

Table 21

Parameters13C-NMR for D1 (1), G1 (14), B1 (21), aglycone (2) and acacia acid (3): numbers in parentheses 1, 14, 21, 2, and 3 correspond to the structures of D1, G1, B1, aglycone and acacia acid depicted in Fig, Fig and pig, respectively.
The carbon atom No.(1)(14)(21)(2) in DMSO-d6(3)
Triterpen. part     
136,1336,1336,1336,0738,90
227,1527,1527,1529,2828,03
389,8689,84 76,7877,94
4 40,0939,8539,7139,28
5 57,08 54,8455,78
6 19,54 18,0318,71
734,5934,5934,5834,2733,51
8 40,8240,0940,8239,79
9 48,08 46,1147,15
10 37,9437,9436,5937,31
G24,2924,5424,4926,9723,77
12124,04124,04124,09122,04122,61
13143,70143,7143,68142,61144,29
14 42,6442,63 42,01
1536,2036,3936,51 35,74
16 74,26 72,4174,22
17 52,29 49,7051,67
18 41,6441,60 40,97
1948,6748.3 46,8548,42
20 35,8835,95 36,64
21 78,61 76,7873,32
2239,8641,741,9438,0741,97
2328,6228,6128,6526,6028,65
2417,1217.11 per bbl17.11 per bbl16,0615,55
2516,2216,2216,2515,1916,47
2617,7317,7218,0716.78 in17,43
2727,4027,3227,4028,2427,11
28173,39175,34175,39176,64179,14
2929,4129,4329,4128,7729,97
3019,4219,4219,5318,6518,26
External     
1168,69168,68168,74  
2132,92132,92132,82  
3148,48148,02    
424,4924,5824,56  
541,9541,3340,83  

22,86
681,0181,0   
7145,93144,01   
8112,53112,44112,53  
916,7516,716,74  
1056,5112,49   
Internal monoterpene     
1168,17169,01168,19164,0 
2132,49128,52132,49135,20 
3148,03of 145.95 137,05 
424,2924,2924,30 
541,3339,8639,7376,03 
6 73,61 129,41 
7144,03144,43 119,80 
8116,0116,0115,33up 11,86 
923,7623,724,2112,81 
1056,6256,61 64,28 

5.1.2. Strong acid hydrolysis D1

As a result of hydrolysis of Dl 3 n model HC1 at 100°C for 2 hours was obtained aglycon D1”shown as (2) Fig;

and as shown by mass spectroscopy, had a molecular weight of 652. According to NMR data in the aglycone Dl has triterpen and modified monoterpene, and no sugar. By saponification (1,3 n NaOH at 100°C for 30 minutes in methanol) this product was digested additionally, highlighting the following :

A. Triterpen. Data13C-NMR spectrum is identical to the data obtained previously for acacia acid (see Fig, structure (3)and table. 21, data13C-NMR in column (3)), and set the molecular weight of 488 agree is raised with its structure.

5.1.2.b. Cyklinowanie monoterpene. Molecular weight and NMR of this compound indicated the presence of carboxylic acids, the two methyl groups attached to the double bond, and two vinyl protons of determining existence of paranavai patterns. Although this structural unit indicated at Fig as (4), is also present in the aglycone D1”, it does not appear in the original D1. This D1 includes acyclic monoterpene marked on Fig as (5), and this structure cyclists during acid hydrolysis in accordance with the below:

These patterns along with the parameters of the molecular mass and spectral properties of D1, in good agreement with the structure of the aglycone D1 shown in Fig under the label (2). Selected parameters13C-NMR are shown in table 21 in column (2).

5.1.3. Weak saponification D1

When processing D1 0.5 n NH4OH at room temperature for 1 h complete conversion in two new connections.

A. Monoterpene. This molecule has a molecular weight of 200, and the NMR parameters indicate the presence of patterns acyclic monoterpene, which confirms the expected splitting. This structure is shown in Fig as (5).

5.1.3.b. Triterpene/monoterpenoid oligosaccharide. This compound is more polar than D1, and NMR data are consistent with prisutstvie the m acacia acid, one monoterpene and several monosaccharides. This structure 36 as shown in (6).

5.1.4. Analysis of sugars D1

Using strong acid hydrolysis D1 (2 n Hcl at 100°C for 2 hours) followed by derivatization (getting trimethylsilyl esters) and the method of GC/MS confirmed the presence of eight sugar residues in the parent molecule: arabinose, ramnose, fucose, xylose, 6-deoxyglucose (= Hinojosa), N-acetylglucosamine and two molecules of glucose.

5.1.5. More active saponification triterpene/monoterpenoid oligosaccharide

As a result of processing triterpene/monoterpenoid oligosaccharide of 0.3 n NaOH for 1 hour at 60°were obtained With three connections.

A. Oligosaccharide. Selection and analysis of this highly polar fragment confirmed that it is an oligosaccharide. Analysis of sugar by the method of acid hydrolysis (2 n Hcl for 2 hours at 100° (C) and data analysis GC/MS in relation trimethylsilyl esters of monosaccharides confirmed that this oligosaccharide is tetrasaccharide composed of two molecules of glucose and one each of the molecules of arabinose and ramnose.

5.1.5.b. Monoterpenoid glycoside.

According to NMR data, this component corresponds to the structure of (8), shown in Fig. The results of acid hydrolysis (2 n Hcl for 2 hours PR is 100° (C) identified sugar as 6-deoxyglucose. Processing monoterpenoid glycoside β-glucosidase resulted in the receipt of monoterpene with the structure (9), shown in Fig, NMR data which correspond to the structure of TRANS-2-hydroxymethyl-6-hydroxy-6-methyl-2,7-octadiene acid. Hydrolysis of this bond under the action β-glucosidase indicates that the relationship between these two groups is β-Suazo.

S Triterpene glycoside.

This compound has a molecular weight of 951 and according to NMR data, corresponds to the lactone acacia acid containing trisaccharide position C-3, which is shown as (10b) on Fig. Acid hydrolysis (2 n Hcl for 2 hours at 100° (C) of this compound, its constituent sugars identified as N-acetylglucosamine, fucose and xylose in the form of their trimethylsilyl derivative method GC/MS. This molecule was identified in the open form of the acid/alcohol, which is marked on Fig as (10A), and in the closed lactone ring form, as shown in Fig as (10b).

Analysis of sugars and molecular weight of the fragments compared to the intact molecule D1 confirmed that all parts of D1 correspond to fragments, patterns which Fig designated as(5), (7), (8) and (10A).

5.1.6. Weak acid hydrolysis D1

Weak acid hydrolysis D1 (1 n Hcl for 16 hours at 25° (C) let p in order to obtain two new molecules.

A. Monoterpenoid sugar.

Molecular weight, NMR spectra and the analysis of sugars correspond to the structure of monoterpene-6-deoxyglucose. The structure of this molecule is shown as (11) on Fig.

5.1.6.b. Triterpen-monoterpene-glycoside.

The second molecule was identified as tridecanolactone glycoside, and the structure of this molecule is shown in Fig as (12).

5.1.7. Connection subgroups in D1

According to NMR data, carboxylic acid external monoterpene forms an ester according to atom C-4 ring 6-deoxyglucose (Hinojosa). Data of NMR and hydrolysis showed that the anomeric carbon Hinojosa attached to a hydroxyl group when the atom C-6 internal monoterpene. Stereochemical parameters of the anomeric carbon in Hinojosa indicate that it βconnection.

Conducting hydrolysis (2 n Hcl for 2 hours at 100° (C) and analysis of isomerization of sugars show that the monomers of this tetrasaccharide are two molecules of glucose and one molecule of arabinose and ramnose. Tetrasaccharide directly protelevision on atom C-28 carboxylic acid triterpene, as shown in Fig. The analysis of mass spectroscopy with ion trap indicates that tetrasaccharide structure has two remaining glucose and arabinose attached to occupying a Central position residue ramnose, as this show is about to Fig. The types of relationships these sugars are not yet known.

According to NMR data established that N-acetylglucosamine (NAG) is attached directly to the atom C-3 triterpene. Other elements of the sequence of sugars fucose are in the middle and xylose in the end, what the data indicates LC/MS products of partial hydrolysis (1 n Hcl for 1 hour at 60°With 50% methanol). The type of relationship between these sugars are not yet known.

5.1.8. Ellipsoid-E

As part D1 are triterpen and two monoterpene, usually found in the saponins described for other plant species, including other members of the genus Acacia. Although the structure D1 is similar to ellipsoidal-E (Fig)described for Archidendron ellipticum (Beutler et al., 1997), in the present invention, the specific rotation D1 was [α]D=-30,0°that does not match the value -24,3°provided for ellipsoid-E.

The previously described ellipsoid-E (Beutler et al., 1997) and D1 are characterized by different retention time in HPLC (15.2 minutes for D1 and 12.5 minutes for ellipsoid-E). Therefore, these two molecules must have some differences, such as the specific place of joining their subunits or the presence of optical or structural isomers.

Applicants found that the specific rotation of the inner monoterpene shown in Fig as (9)is +11,2° in methanol and +16° PI is reforme. For a similar fragment of ellipsoid-E was established -9,1° in chloroform. Moreover, only one chiral center in the inner monoterpene D1 was set configuration "S", which is the opposite configuration found in ellipsoid-that is, Specific rotation external monoterpene D1 is under study. Moreover, according to proton NMR, the double bond in monoterpene D1 have the TRANS-configuration, while the double bond of monoterpenes ellipsoid-E are in the “CIS”configuration (Beutler et al., 1997). These two characteristics define the first structural difference found between D1 and ellipsoidal-that is, the Enzymatic catalytic hydrolysis of individual sugars showed that the location of sugars is the same as in ellipsoide-E.

5.2. Structure G1

Biological tests of this material showed that G1 biologically more active than D1.

5.2.1. The intact molecule G1 (14)

The second structure established in the present invention, is G1. It also was isolated from fraction F094 by preparative HPLC, but with less output connections. G1 characterized by a lower polarity than D1. Method of mass spectrometry laser desorption (MALDI) was determined by the molecular weight 2065, which is 16 Amu less than D1. Specific rotation G1 was -26,9° (in methanol). According to proton NMR G1 is a saponin, very similar to D1, but differs from D1 by the absence of one atom of oxygen in the external monoterpene, which, accordingly, is a TRANS-2,6-dimethyl-6-hydroxy-2,7-octadiene acid: see (14) on Fig and separate spectral parameters13C-NMR in table. 21 in column (14). The destruction of G1 shown in diagram 2 (Fig).

5.2.2. Weak saponification G1

When processing a G1 with a solution of 0.5 n NH4OH at room temperature for even a few minutes is a complete conversion to a more polar product weak saponification and monoterpene.

A. Monoterpene. Molecular mass and NMR spectra of this material indicate the presence of a methyl group at atom C-2, which has hydroxymethyl. This is shown under the label (15) on Fig.

5.2.2.b. Triterpen-monoterpenoid oligosaccharide.

According to NMR data of this compound is identical in retention time in HPLC and proton spectra the NMR structure (16) on Fig similar to the structure (6) on Fig originating from D1, and it includes acacia acid, one monoterpene and eight monosaccharides, which was shown for D1. The similarity in structure (16) and (6) indicates the similarity of the stereochemical parameters obtained for internal monoterpene D1.

5.2.3. Analysis of sugars G1

When strong acid hydrolysis of G1 (2 n Hcl for 2 hours at 100° (C) receive the same monosaccharide residues, CTO in D1: arabinose, rhamnose, fucose, xylose, 6-deoxyglucose, N-acetylglucosamine and two molecules of glucose.

5.2.4. Acid hydrolysis G1

Acid hydrolysis product of a weak saponification identified three molecules, as in the case of D1. For each of them was carried out by NMR and analysis of sugar (2 n Hcl for 2 hours at 100°). The result is shown in Fig like structure (16).

A. The oligosaccharide includes two molecules of glucose and one molecule of arabinose and ramnose, as shown in Fig under the label (17).

5.2.4.b. Monoterpenoid glycoside includes acyclic monoterpene shown in Fig as (15), and 6-deoxyglucose, and its full structure is marked on Fig as (18).

S Triterpene glycoside includes acacia acid and one molecule of N-acetylglucosamine, fucose and xylose. The order of these sugars is the same as in D1. This structure is in Fig marked as (19).

5.2.5. Ellipsoid-And

The composition of terpenes and sugars in G1 is the same as ellipsoid (see Fig and Beutler, 1997). However, as has been established, ellipsoid-And markedly different retention time in HPLC (G1 29,09 minutes; ellipsoid-And to 26.04 minutes): this indicates that these two molecules must to some extent be different, for example, a particular order of connection parts or the presence of optical isomers, or both at the same time. We have also identified RA the differences in the values of chemical shifts in the spectra of 1H and13C-NMR G1 and ellipsoid-A. it is Assumed that the specific rotation of the internal and external monoterpenes in the composition of these compounds may also vary. On Fig structure (14) is G1.

5.3. Structure B1

According to the biological tests B1 significantly less active compared to D1 or G1.

5.3.1. The intact molecule B1 (21)

Selection B1 was carried out by obtaining a plant extract and flash chromatography with a C-18 preparative and subsequent prepreparation chromatography with C-18. According to NMR data B1 is typical for a family of saponins with triterpen-monoterpene-Hinojosa-monoterpene structure. Also NMR data indicate the presence of four deoxysaccharides and one N-acetyl group: i.e. this molecule must be different from D1 by their sugar components. Specific options13C-NMR are shown in table 21 in the column (21). Cleavage of this molecule displayed on Fig.

5.3.2. Analysis of sugars B1

These NMR indicate the presence of more than one copy of one residue 6-desoximetasone (i.e. fucose, ramnose, 6-deoxyglucose). Analysis of the sugar composition of the full molecule after hydrolysis (2 n Hcl for 2 hours at 100° (C) showed the presence of nine sugars: one molecule of fucose, arabinose, xylose, Hinojosa and glucosamine and two molecules of glucose and ramnose. In the similar molecule is glucosamine, which is the “rest” of the N-acetylglucosamine. Structure B1 shown in Fig under the label (21).

5.3.3. Weak saponification B1

When processing B1 solution of 0.5 n NH4HE at room temperature for even a few minutes is a complete conversion to a more polar compound, the product is weak saponification and monoterpene.

A. Monoterpene. Molecular weight and NMR data this material has the same structure as monoterpene of the composition of D1 shown in Fig under the label (5). On Fig it is shown under the label (22).

5.3.3.b. Triterpen/monoterpenoid oligosaccharide. These NMR of this compound indicates that it includes acacia acid, one monoterpene and several monosaccharides. This is shown in Fig as (23).

5.3.4. More active saponification triterpen/monoterpenoid oligosaccharide

More active saponification (0.3 n NaOH for 1 hour at 6° (C) weak product of saponification identified three molecules, as previously obtained for D1 and G1. Data were obtained NMR and analysis of sugars for each of them.

A. The oligosaccharide includes glucose, arabinose and two molecules ramnose. This is shown in Fig under the label (24).

5.3.4.b. Monoterpenoid glycoside includes 6-deoxyglucose and monoterpene. This is shown in Fig under the label (25).

S Triterpene glycoside includes acacia acid attached is trasferito in the C-3 position. This tetrasaccharide includes one molecule of N-acetylglucosamine, fucose, glucose and xylose. This is shown in Fig like structure (26).

Example 6

De-esterification of triterpene compounds of the present invention

Conducted deesterification F094 and products deesterification tested to search for active components. 1,00 g F094 was dissolved in 100 ml of water and then adding 1 g of KOH and heated under reflux for one and a half hours. The solution was left to cool to room temperature and its pH was brought to 7 with 1 n Hcl and then washed with hexane (2×50 ml). Then the aqueous solution was subjected to further stepwise extraction with obtaining fractions 159-162. For example, the first solution was extracted with n-butanol (2×50 ml) to give to 0.127 g soluble in organic solvents solids (fraction F159) after drying in vacuum. The aqueous phase was acidified to pH 5 using 1 n Hcl and was extracted with EtOAc (2×50 ml) to obtain the 0,397 g dissolved in EtOAc solids (F160) and then n-butanol (2×50 l) to obtain the 0,338 g solids (F161) after removal of organic solvents. Finally, the aqueous layer was neutralized to pH 7 1 n NaOH. From the final aqueous layer was allocated 1,808 g solids (F162).

Biological tests showed that the products deesterification have the weak activity or forgo it altogether. Was tested cytotoxicity F159-162 against cell lines 769-P, Panc-1, HEY, MDA-MB-453 and Jurkat. Activity revealed only F161, which showed cytotoxicity against MDA-MB-453 at 1.6% at 50 μg/ml, and for F159 against Jurkat cells at the level 15,50%, 6,60% 3,80%, respectively, at 50 μg/ml, 25 μg/ml and 12.5 μg/ml, These data indicate that for the manifestation of biological activity in the presence of the ester side chain is necessary. It is considered that the ester side chain in compounds of the present invention provides antitumor activity and/or consistently worked with the triterpene skeleton of these compounds of the present invention with the receipt showing them a strong antitumor effect.

Example 7

Hydrolysis of sugars in the compounds of the present invention

Included in the F094 sugar were also obtained for the purpose of characterization of active components. 12 g F094 was dissolved in 400 ml of 2 n n2SO4and heated under reflux for 75 minutes, during this time was the formation of insoluble material. The solution was cooled and was filtered through a sintered porous glass. The residue was washed with water, receiving 4.8 g aglycone (F191), as evidenced by TLC analysis. The filtrate dark amber color neutralized KOH or NaOH. Collected is razuysya white precipitate. The solvent was removed in vacuum and the product resuspendable in methanol, resulting in the obtained white precipitate, which, apparently, corresponds to the salts of the sulfate of what the data indicates test flame coloration. Almost transparent filtrate was dried in vacuum, and the residue was azetilirovanie and analyzed by HPLC, according to which it includes a mixture of sugars, which is shown in figa and 17C. This mixture is believed to contain at least five sugars. These sugars can be further characterized by the selection tetramethylsilane derivatives for characterization by GC/MS; chromatography on paper; the allocation of benzyl derivatives by HPLC separation or NMR DEPT; or13C-NMR that in full described above. Using the standard one - and two-dimensional pulp, paper and normalnofocus thin-layer chromatography, the major sugars were identified as glucose, xylose, rhamnose and arabinose, along with some minor additional sugar, which is probably the source of the aminoglycoside in particular, acetamidophenol sugar.

The number of different sugars, apparently, explains the formation of complex HPLC spectra, which indicate the presence of dozens of closely related compounds. In particular, some of the assets the major components putatively glycosylated at two different sites (site alcohol and customers carboxylic acid). Various combinations of six sugars attached on these two sites, apparently, give a large number of closely related compounds, which separation is very difficult.

Alternatively, when a weaker hydrolysis in distilled with 100% ethanol azeotropic mixture with 5% water) and from 0.1 to 2.0 n H2SO4(despite the fact that further processing does not change) at low heat to the point of reflux distilled, but without hard reflux distilled, forms a mixture of aglycones. Some components are lost; this indicates a certain degree of isomerization under more severe conditions.

Then the aglycone(s) F191 (1 g) was metilirovanie by heating under reflux for 5-7 hours with methyliodide (1 ml) and anhydrous K2CO3(1 g) in anhydrous acetone (10 ml). The result has been 0,315 g of insoluble material and 0.54 g of methyl esters identified F197. 500 mg F197 was then divided by the method GHSD using columns 15×460 mm (45 g SiO2, 15-25 μm), in which the sample was previously adsorbing 1.5 g of SiO2(15-25 μm). The connection was suirable 790 ml of 7%aqueous isopropyl alcohol (IPA) in hexano (subtractio 1-10), 470 ml of 10%isopropyl alcohol in hexano (subtractio 1-14), 275 ml of a mixture of 20%IPS - hexane (subtractio 14-15), 200 ml of dichloromethane and 100 ml of a mixture of DCM/MeOH (1:1), in accordance with table 22.

Table 22

Fractionation F197 on fractions from F198 to F208
The number of fractionsThe number of collected sub-fractions (volume, ml)Total weight (mg)Notes
F1981(100)14 
F1992-3(120)126Additionally fractionated on F209-214
F2004(40)8 
F2015-6(110)86Additionally fractionated on F215-219
F2027-8(170)37 
F2039-10(250)17 
F20411(100)5 
F20512(150)38 
F20613(150)10 
F20714-15(345)86 
F20816-18(300)105 

Biological testing F191 and F197 showed cytotoxicity against tumor cells of the ovary (line HEY), renal cell cancer (line 769-P), cancer cells of the pancreas (Raps-1), T-leukemic Jurkat cells and breast cancer cells MDA-MB-453 at stake is intratech, the corresponding shown in tabl.

Table 23

Biological testing of the fractions F191 and F197
 50 mg/ml25 mg/ml12.5 ág/ml
F191   
769-P82,356,333,7
Raps-19064of 40.3
HEY94,571,60
MDA-MB-45353,522,37,3
Jurkat69,668,645,3
F197   
769-P84,361,140,5
Raps-193,584,453,8
HEY94,494,762,1
MDA-MB-453of 76.879,268,8
Jurkatto 70.270,656,9

F199 (116 mg) was additionally fractionally using the same column, which was used for fractionation F191, and suirable 100 ml of a mixture of 2% IPS - hexane, 525 ml of a mixture of 4% IPS - hexane and 250 ml of a mixture of 1% IPS - hexane in accordance with table.

Table 24

Fractionation F199 on fractions from F209 to F214
The number of fractionsThe number of collected sub-fractions (volume, ml)Total weight (mg)Notes
F2091-7(225)5 
F2108(20)1 
F2119(20)2 
F21210-14(140)90Additionally fractionated on F220-228
F21315-17(220)17 
F21418(250)10 

F212 (85 mg) was additionally fractionally by HPLC on a Waters chromatograph Prep LC-4000 column (Alltech Econosil C18 (22×500 mm; 10 μm; balanced blend of 75% acetonitrile - water), was suirable a mixture of 80% acetonitrile - water and washed with acetonitrile at a flow rate of 40 ml/min with the installation of the detector at 220 nm and selection of sub-fractions every 30 seconds (20 ml) according to table.

Table 25

Fractionation F212 on fractions from F220 to F228
The number of fractionsThe number SOBR is the R of the sub-fractions (volume, ml)Total weight (mg)Notes
F220(940)12 
F2211-241 
F22225-273 
F22328-3855S characterized as its acetylated derivative, according to the13C and1H-NMR DEPT, HPLC, TLC RP18 and MS
F22439-413 
F22542-544 
F22655-741 
F22775-1025 
F2281032Rinsing with acetonitrile

F223 was first purified in the form of a methyl ester derivative with getting S-191. C-191 subjected to the classical procedure acetylation. In particular, 191 (47 mg) was stirred overnight at room temperature with a mixture of acetic anhydride and pyridine in the ratio 2:1. The reaction is extinguished by water and the solution was distributed between diethyl ether and 5 n hydrochloric acid. Then the organic phase is washed to neutrality, evaporated on a rotary evaporator and the residue was subjected to PTSH: drugs the top plate 20× 20 cm was suirable a mixture of hexane:isopropanol (90:10) followed PTSH-elution with a mixture of dichloromethane:methanol (98:2) to obtain S-acetate (F229, which later was identified as S-194).

F201 (85 mg) also has fractionally method GHSD on the same column, which was divided F199, suirable and collected in a mixture of 2% IPS-hexane (120 ml)in a mixture of 4% IPS-hexane (330 ml), a mixture of 7% IPS-hexane (460 ml)in a mixture of 20% IPS-hexane (150 ml)in dichloromethane (50 ml) and the mixture dichloromethane/Meon (1:1) (70 ml) in accordance with table.

Table 26

Fractionation F201 on fractions from F215 to F219
The number of fractionsThe number of collected sub-fractions (volume, ml)Total weight (mg)Notes
F2151-5(510)3 
F2166-10(175)54Methyl ester aglycone II is also characterized
F21711-14(225)4 
F21815(150)14 
F21916(120)10 

Example 8

Biological characteristics of active triterpenes according to the present invention

Angiogenesis, or the image is of new blood vessels is a process in which cells are recruited factor(s), by(mi) tumor to ensure itself of the supply of the vascular system. Suppression of angiogenesis slows the growth of tumors by limiting blood flow to the tumor. This function was analyzed using the test on the proliferation of endothelial cells of capillaries of a bull, in which cells were treated with fraction 35 (UA-BRF-004-DELEP-F035). The test was carried out as follows:

the endothelial cells of the capillaries of the bull were obtained and cultured in a standard way (Folkman et al., 1979). These cells were washed SFR and suspended in a solution of trypsin (0.05 per cent). Cell suspension (25,000 cells per 1 ml) were formed in culture medium DMEM + 10% BSA + 1% GPS, were sown on gelatinisation 24-hole culture plates (0.5 ml per well) and the suspension is incubated for 24 hours at 37°C. the Culture medium was replaced with 0.25 ml of DMEM + 5% BSA + 1% GPS and used different concentrations of UA-BRF-004-DELEP-F035. After 20-minute incubation was added to the culture medium and FGF-βreaching a final volume of 0.5 ml of DMEM + 5% BSA + 1% GPS + 1 ng/ml FGF-β. After 72 hours the cells were dispersible in trypsin, resuspendable in Hematall (Fischer Scientific, Pittsburgh, PA) and counted on colter-meter, measure the electrical resistance of the non-conductive particles, suspendiruemye in the electrolyte (the so-called “Coulter principle”) (O eilly et al., 1997).

The results of this test have shown significant inhibition of proliferation of endothelial cells in the presence of basic fibroblast growth factor, and without it (figure 5). These data show that the active components of the herbal extract are strong inhibitors of proliferation of endothelial cells and are often a predictor of in vivo suppression of angiogenesis. In addition, this fraction does not affect the migration of capillary endothelial cells, indicating the absence of toxicity to normal cells (6).

Common problem associated with steroid saponins (e.g., digitonin and Genin-diosgenin isolated from Yam), is the lysis of red blood cells. Using a simple blood test in vitro culture has been shown that after treatment of 1 mg/ml of UA-BRF-004-DELEP-F035, was observed only a very weak lysis. In contrast, treatment of 10-25 µg/ml digitonin gives 100%lysis of blood in vitro.

In addition, to further explore the mechanism by which inhibition of tumor cells active components induced TNF-α the activation of transcription factor NF-KB was analyzed in Jurkat cells (3·106), which was treated with 1-2 μg/ml of UA-BRF-004-DELEP-F035 and UA-BRF-004-(Bob)DELEP-F094. This analysis was carried out as follows: cells Jurkat will precede the flax was treated with 1-2 μg/ml or F035 F094 for 15 hours at 37° C. the Cells were collected and resuspendable in 1 ml of culture medium RPMI and treated 100 PKM TNF-α for 30 minutes at 37°C. After treatment of TNF-α, nuclear extracts were prepared according to Schreiber et al. (1989). Briefly, cells were washed in chilled on ice SFR and suspended in 0.4 ml of lysis buffer (10 mm HEPES, pH of 7.9; 10 mm KCl, 0.1 mm EDTA, 0.1 Mm EGTA, 1 mm dithiothreitol, 0.5 mm PMSF, 2 μg/ml leupeptin, 2 μg/ml Aprotinin, and 0.5 mg/ml of benzamidine). The cells were left on ice for 15 minutes, and thereto was added 25 μl of 10% Nonidet-40. The tubes were mixed on a mixer and microcentrifuge within 30 seconds. Nuclear sludge resuspendable in 25 µl chilled on ice nuclear extraction buffer (20 mm HEPES, pH of 7.9; 0.4 M Nad, 1 mm EDTA, 1 mm EGTA, 1 mm dithiothreitol, 1 mm PMSF, 2 μg/ml leupeptin, 2 μg/ml Aprotinin, and 0.5 mg/ml of benzamidine) and the tubes were incubated on ice with intermittent stirring. Nuclear extract was microcentrifuged in 5 stages with 4°and received supernatant kept at -70°C.

Test for change in electrophoretic mobility (TIP) was carried out by incubation of nuclear extracts (7 μg of protein) with32P-labeled oligonucleotide gene NF-KB (SEQ ID NO 1; consensus oligonucleotide NF-kV; Santa Cruz Biotechnology) in the presence of 0.5 μg of poly (dI-dC in binding buffer (25 mm HEPES, pH 7,9; 0.5 mm EDTA, 0.5 mm dithiothreitol, 1% Nonidet P-40, 5% glaze the ins and 50 mm NaCl) for 20 minutes at 37° (Nabel & Baltimore, 1987; Collart et al., 1990; Hassanain et al., 1993). Formed DNA-protein complex was separated from free oligonucleotide in 5%native polyacrylamide gel using a buffer containing 50 mm Tris, 200 mm glycine and 1 mm EDTA. This gel was fixed in 10%acetic acid and dried, and the stripes were visualizable autoradiographically using intensifying screen at -70°C.

Results TIEP show that in untreated cells low basal level of factor NF-KB, which is activated TNF (Fig: tracks 1 and 2). Pre-treating the cells with 1 μg/ml or F035 F094 with subsequent activation of TNF (Fig: tracks 4 and 8) resulted in the absence of suppressing the activation of NF-KB. When cells were treated with 2 µg/ml of UA-BRF-004-DELEP-F035 or UA-BRF-004(beans)-DELEP-F094 (Fig: tracks 6 and 10), found a clear suppression of the activation of NF-KB under the influence of TNF. The results of this analysis show that, and F035, and F094 able to induce potent anti-inflammatory response. In addition to confirming the potential anti-inflammatory activity of triterpene compounds, the results obtained, in particular, are significant additional argument indicating a key role of inflammation in carcinogenesis (Sieweke et al., 1990; Prehn, 1997; Schuh et al., 1990).

Example 9

The analysis of the mechanism of signal transmission F035

To further clarify the molecules of the situations targets for active compounds of the extract of the plant Acacia victoriae was the analysis of the effect of F035 on the activity of phosphatidylinositol-3-kinase (PI3K), as well as on the activity of the INSTRUMENT (protein kinase-B, which is the serine ser / thr kinase), a negative effector of PI3 kinase. The PI3 kinase is an enzyme that participates in signaling pathways of growth factors by binding to the receptor and preceptoring tyrosine kinases. Two known inhibitor of PI3 kinase: wortmannin, which is a fungal metabolite, and LY294002, which is a synthetic compound that is structurally similar to plant bioflavonoid quercetin.

This test was carried out as follows: Jurkat cells (1x107), starving during the night, and treated with various concentrations (1-8 mg/ml, depending on cell line) fractions F035 for different time (2-16 hours) at 37°C. After different time intervals, the cells were collected and washed SFR at 2000 rpm for 10 minutes. Cells were literally in lysis buffer with 1% NP-40 for 30 minutes at 4°and the resulting lysates were isolated by centrifugation for 5 minutes at 15,000 rpm at 4°C. To conduct thus PI3 kinase, 5 μl of rabbit anti-R antibodies (R - adapter protein tyrosinekinase receptor: Upstate Biotechnology Inc.) incubated with 1 ml of cell lysate for 90 minutes at 4°C. the Immune complexes were isolated using 100 µl of the beads with 20% protein-And/sepharose for a further 90 minutes at 4°C. Obtained immunodeficiency is precipitate sequentially washed with: (a) SFR, 100 mm PA3VO4, 1% Triton X-100; (b) 100 mm Tris, pH of 7.6, 0.5 mm LiCl, 100 mm Na3VO4; (C) 100 mm Tris, pH of 7.6, 100 mm NaCl, 1 mm EDTA, 100 mm PA3VO4; and (d) 20 mm HEPES, pH 7.5, 50 mm NaCl, 5 mm EDTA, 30 mm NaPPi, 200 mm PA3VO4, 1 mm PMSF, 0,03% Triton X-100. Then immunoprecipitate resuspendable in 30 μl of kinase-reactive buffer (33 mm Tris, pH of 7.6, 125 mm NaCl, 15 mm MgCl2, 200 mm of adenosine, 20 mm ATP, 30 µci γ-32P-ATP). Phosphatidylinositol (PI: 50 μl) was dried in nitrogen atmosphere and resuspendable in 20 mm HEPES, pH 7.5, 2 mg/ml, and were treated by ultrasound on ice for 10 minutes. P13-kinase reaction was initiated by adding 10 μl of the suspension PI and 10 ál γ-labeled ATP. The reaction mixture was left for half an hour at room temperature followed by stopping the reaction by adding 100 ál of 1 n Hcl. Lipids were extracted in 600 µl of a mixture of chloroform:methanol (1:1) and separated on silica gel (G60) by thin layer chromatography (TLC) with elution with a mixture of chloroform:methanol:NH4OH:H2O(60:47:2:11,3). Radioactively labeled phosphatidylinositol was visualizable autoradiographically and the level of inhibition was quantified using the Storm device (Okada et al., 1994; Vlahos et al., 1994). The results obtained (Fig) show that after 2 and 6 hours after treatment F035 (4 µg/ml) occurred suppression of the activity of PI3 kinase. Similarly, when EDI was treated with 2 µg/ml F035 for 15 hours, the level of the observed suppression was 95%, which is similar to the action wortmannin (a fungal metabolite, which is a known inhibitor of PI3 kinase) in Jurkat cells.

Further, it was studied the effect of F035 on the ACT, which is a negative effector of PI3 kinase. The ACT, also known as protein kinase, is the cellular homologue of the viral oncogene v-akt, activated next growth factor and operates as part of the mechanism element of which is the activation of PI3K, sensitive to the action of wortmannin. Gene ACT encodes a serine-threonine the protein kinase: for this gene was demonstrated amplificatori 12.1% of cases of ovarian carcinomas and 2.8% of cases of cancer of the breast. The ACT of participating in anti-apoptotic mechanism through phosphorylation of Bad - antiapilepticescoy molecules (factor of overcoming apoptosis). Patients with tumor of the ovary with amendments ACT differ poor prognosis (Bellacosa et al., 1995). As shown, the ACT of actively blocking apoptosis, in particular, due to the activation of p70S6 kinase (Kennedy et al., 1997). P70S6 kinase is a mitogen-activated serine ser / thr protein kinase, required for cell growth and development in the phase of G1cell cycle (LUT & Blenis, 1996). The activity of p70S6 kinase is controlled by multiple phosphorylation events on the site, located in the same polypeptide within the catalytic and pseudoobstruction domains (Cheatham et al., 1995; Weng et al., 1995).

The effect of F035 on phosphorylation of the ACT were analyzed as follows. The Jurkat cells (5·106) were in serum-free medium and treated them F035 for 15 hours and 2 hours with wortmannin at 37°C. the Cells were induced cd3XL (cross stitched CD3) for 10 minutes at 37°or not induced at all and literally in the ACT-lysis buffer, and proteins were separated by electrophoresis in 8%of the LTO-page and analyzed by the method of Western blotting using antibodies to phosphorylated by serine-473 ACT (New England Biolabs) and to the General ACT. Test the effect of F035 on kinase 70S6 can be done the same way, but using a set of antibodies to the p70S6 kinase Phosphoplus (New England Biolabs), designed to analyze the phosphorylation of p70S6 kinase (serine-411, threonine 421/serine-424). The results of the study ACT (Fig) showed that cross-stitched cd3 induces a weak phosphorylation of the ACT. Subsequent treatment of the cells 1 and 2 µg/ml F035 caused a marked suppression of phosphorylation ACT (activation ACT), which is similar to 2-hour treatment of cells 1 µm wortmannin. However, differences in the overall level of expression of the ACT was not. A similar suppression of phosphorylation ACT also been demonstrated for cell tumor ovarian OVCAR-3 and C-2 (option line HEY) and Jurkat cells treated with 2-4 mg/ml F094. The obtained data is e show what F035 inhibits the phosphorylation of kinases ACT in Jurkat cells and ovarian cancer cells. This is strong evidence that the signaling mechanism involving the P13 kinase/AKT, as shown, passes anti-apoptotic signal (Kennedy et al., 1997). The results confirm that F035 and F094 mediate apoptosis of tumor cells by suppressing the signaling mechanism of PI3 kinase.

Example 10

Detection of apoptosis in cell cycle analysis and test for binding of annexin-V

To further characterize the mechanism of inhibition of growth and cytotoxicity shown by the active compounds of this plant extract, about 1×106tumor cell lines OVCAR-3 were sown on 60-mm3Cup, was treated with various concentrations of UA-BRF-004-DELEP-F035, and incubated for 18-24 hours at 37°C. the Cells were collected, washed twice SFR and resuspendable concentration of 1×106cells in 1 ml Paraformaldehyde (final concentration 1%) was added dropwise to gently mix the cells. Cells were again washed SFR after 15-minute incubation on ice and the precipitate resuspendable in 70%ethanol, cooled on ice, and incubated over night at -20°C. Finally, the ethanol once laundered SFR and cells resuspendable 10 μg/ml of propidium iodide (Sigma Chemical Co.) with 0.1% RNase (RNse). Cells once again incubated at room temperature for 30 minutes and then was transferred to a 4°and analyzed after 18 hours by flow cytometry (Pallavicini, 1987). The results show that before treating the cells with UA-BRF-004-DELEP-F035, 48% of the cells were in the phase of G0/G1, 36% of the cells were in the S phase and 7% of the cells were in the phase of G2/M. However, at 48 hours after treatment of cells OVCAR-3 fraction F035 approximately 58% of the cells were in the phase of G1and only 27% of the cells were in the S phase of the cell cycle: an increase of 8% increases the proportion of cells in the G1and about 10% fewer cells in the S phase of the cell cycle (figa, In). The results suggest explicit blocking of tumor cells of human ovarian OVCAR-3 in the phase of G1.

It was also studied the effect of F035 on the profile of the cell cycle of breast cancer cells human. The breast cancer cells MDA-MB-435 and MDA-MB-453 was treated with various concentrations of F035 and analyzed after 72 hours, as described above. The results show that F035 induces apoptosis of cells MDA-MB-435, as evidenced by the appearance of a peak corresponding to the phase Sub.G0(table). In addition, regulation of the cell cycle was also observed by the reduction in the percentage of cells in the cell cycle phases S and G2/M.

Table 27

Analysis of cell cycle in tumor cells of breast cancer MDA-MB-435 after processing F035
Stage of the cell cycleControlF035 (6 µg/ml)F035 (3 µg/ml)F035 (1 µg/ml)
Sub. G00,82%16,0%12,7%0,90%
G152,0%50,0%50,3%51,0%
S35,0%26,0%26,0%36,0%
G2/M16,0%10,0%2,0%14,0%

Using cells MDA-MB-435 results obtained show that F035 induces blocking the cell cycle with increase of approximately 10% of the number of cells in the phase of G1and a decrease of 4% to 10% of cells in the S phase of the cell cycle at 72 hours after treatment F035 (table). These data indicate that blocking cell cycle and apoptosis of tumor cells induced by the given plant extract.

Table 28

Analysis of cell cycle in tumor cells of breast cancer MDA-MB-453
Stage of the cell cycleControlF035 (6 µg/ml)F035 (3) - Rev. kg/ml) F035 (1 µg/ml)
Sub. G00,96%2,2%1,8%1,5%
G162,0%72,0%71,0%69,0%
S26,0%19,0%16,3%21,0%
G2/M12,5%8,5%10,4%10,0%

The Jurkat cells (1×106) was treated with various concentrations of UA-BRF-004-DELEP-F035 (50-1000 ng/ml) for 18 hours at 37°C. the Cells once washed SFR, resuspendable in binding buffer (10 mm HEPES/NaOH, 140 mm NaCl, 2 mm CaCl2)containing 5 ál conjugate annexin-V-FITZ (Biowhittaker, Walkersville, MD), and incubated for 10 minutes in the dark. Cells were washed and resuspendable in binding buffer containing 10 μl of 20 μg/ml of propidium iodide (Sigma Chemical Co.), and analyzed using cell sorting device with induced fluorescence (FACS) (Martin et al., 1995).

The results show that purified active components were able to induce apoptosis of Jurkat cells. This observation is further confirmed by the ability of treated cells to bind annexin-V, which is an indicator of the entry of cells in apoptosis (table. 29). Usually, phosphatidylserin (FS) is localized on the inner surface of p is akmaticheskoy membrane. However, in the early stages of apoptosis is the output of PS on the outer surface of the plasma membrane. Annexin-V is a calcium-binding protein that binds to FS and can be detected by flow cytometry by staining with annexin-V-FITZ (Martin et al., 1995).

Table 29

Analysis of apoptosis on binding parameters with annexin-V in Jurkat cells, treated with various concentrations of UA-BRF-004-DELEP-F035
UA-BRF-004-DELEP-F035 (µg/ml)Positive for annexin-V cells, %
Raw6
anti-Fas (positive control)20,0
1 mcg/ml16,0
2 mcg/ml18,0

Example 11

UA-BRF-004-DELEP-F035 as chemoprotective agent

The impact of UA-BRF-004-DELEP-F035 was studied on the model of multistage skin carcinogenesis in SENCAR mice. The skin of these animals were treated (“painted”) with acetone, carcinogen DMBA (7,12-dimethylbenz[a]anthracene), DMBA + UA-BRF-004-DELEP-F035 and DMBA + fraction 60 (negative control) at low (100 ug UA-BRF-004-DELEP-F035 or faction 60 1 animal) and high (500 ug UA-BRF-004-DELEP-F035 or faction 60 1 animal) doses of plant extract used twice a week for 4 weeks. UA-BRF-004-DELEP-F035 Il is the control applied to the skin of mice for 5 minutes before application of DMBA. Animals observed the formation of papillomas and then they were killed and tissues were analyzed histologically (figa-F). The results of the systematic analysis on figa, figw, figa, figw, Fig and Fig.

After 8 weeks of carrying out these experiments in the group of mice treated with DMBA noted in 8 moles per 1 animal, while in the groups with joint exposure to DMBA and UA-BRF-004-DELEP-F035 was 0.66 moles per 1 animal; in the group with DMBA treatment and faction 60 (negative control) were available for 6.9 moles per 1 animal. These results indicate a significant protective effect of UA-BRF-004-DELEP-F035 against tumors, while fraction 60, basically, had no protective effect.

Further studies in mice in vivo, it was shown that UA-BRF-004-DELEP-F035 is chemoprotective agent in cases with tumors induced by carcinogens, by preventing mutations in the ras oncogene. The stage of initiation of carcinogenesis in the skin of mice were provided by direct exposure to the carcinogen (e.g., DMBA) and, mainly, it was irreversible. Suppression of carcinogenesis was determined in 8 weeks to reduce the intensity of formation of papillomas induced by DMBA. Molecular analysis of the treated skin showed that UA-BRF-004-DELEP-F035 prevents the ability of DMBA to induce mutations in the ras oncogene (see section shall emery 14, 15 and 16 below).

Example 12

The procedure for identifying active triterpenoids from Acacia victoriae

Procedure was to effectively identify active triterpenes in the tissue sample plants. This procedure was carried out as follows. Approximately 5 g of leaves and twigs cut into small pieces with scissors or, alternatively, samples of roots were cut with a knife into small pieces. The plant material was processed in a small mixer, combined with approximately 25 ml 80% (V/V) methanol and kept at least for 2 hours with shaking every hour. Insoluble material was removed by centrifugation at 10000 g. Then the extract was used for carrying out thin-layer chromatography on reversed-phase plates (aluminum TLC plates, RP-C18 F254swith 40% acetonitrile (V/V). After spraying the TLC plates with a mixture of 0.1% vanillin (4-hydroxy-3-methoxybenzaldehyde/N2SO4and drying at 70°C for 15-30 minutes, active triterpenoids connection visualizeus in the form of brownish-red spots (Rf=0.2 and 0.3).

Example 13

Localization triterpene compounds in plants Acacia

victoriae

In the initial stages of research, the dried aerial parts of the plants were collected in early summer to obtain extracts. Re charges in the autumn appeared to be inactive. P the following was carried out a systematic analysis to determine the relative levels of the presence or absence of active triterpene compounds in different parts of the plant Acacia victoriae. After monitoring the chemistry of plants, it was found that substantially all of the active components of the aerial parts of this plant are concentrated in beans and leaves, while they are mainly or entirely absent in branches, stem bark, leaves and seeds. Therefore, the active collection of material is limited to approximately three weeks from the beginning of the formation of the beans prior to their disclosure. It was also found that the roots of plants also produce the same active material in the event of fluctuating ratio of sugars and active components. The last parameter indicates that Aeroponics, in which there is strong root growth, while preserving the normal development of plants may be suitable for Acacia victoriae.

Example 14

Lines of tumor cells and their growth

The following line human tumor cells were obtained from American type culture collection (ATSS: Rockville, MD)cell line SK-OV-3 and OVCAR-3 (ovary), Jurkat (T cell leukemia), U-937 (histiocytoma lymphoma), MDA-MB-468, MDA-MB-453, MDA-MB-435, SK-BP-3, MCF-7, MDA-MB-231, BT-20 (breast), LNCaP, PC-3, DU145 (the prostate gland), 769-P, 786-0, A-498 (kidney) and PANC-1 (pancreas). Cell lines and HEY Dov-13 (ovaries) were obtained from M.D.Anderson Cancer center. Following the untransformed cell line MCF-10A and 10F (the epithelium of the mammary gland) were obtained from Cancer C is NTRA M.D.Anderson. Line Hs-27 (foreskin fibroblasts) and L929 (mouse fibroblasts) were obtained from ATS. Line SK-OV-3, MDA-MB-468, Hs-27, L929 were cultured in minimal supportive environment. Line OVCAR-3, Jurkat, U-937, LNCaP, DU-145, PC-3, HEY, Dov-13, PANC-1, MCF-10A, MCF-10F and other cell lines breast cancer were cultured in medium RPMI-1640, and the culture medium F-12 was used for cell growth 769-P, 786-0 and a-498. All the used culture medium was supplemented with 10% fetal calf serum, 200 mm glutamine and 0.05% gentamicin.

Example 15

Amplification of mutant 61-th codon CAA→one HUNDRED and gene Ha-ras mice using specific for this mutation primers (MSP)

This Protocol is used according to Nelson et al., (1992). Reverse primer, designated as 3MSP61mut, designed so that the 3’-terminal nucleotide (A) mated with an average nucleotide (underlined) in codonsrepresenting transverse 61st codon of the gene Ha-ras, and provide selective amplification of mutant DNA in the conditions described below. The test is based on the fact that Taq polymerase lacks 3’-ectonucleoside activity and therefore is not able to correct the error in the 3'-end origimage primer. Test conditions depend on reverse primer, not capable of any substantial extent otjihase on the sequence of the wild type, consequently the those which of completion of a circuit does not occur. Using the same direct primer one reaction takes place with reverse includes an error primer (3MSP61mut), and the other reaction takes place with a reverse primer corresponding to the wild type (3MP61wt). This Protocol revealed only transversehowever, these mutations are mostly prevalent among mutations affecting the 61st codon. The result of this transversely formed RFLP-site cut by the restriction enzyme Xbal. Mutations can be detected by processing the amplified DNA with the restriction enzyme Xbal or by direct DNA sequencing using32R-terminal tagging primer 5MSP61. Reactions, including products with a mutation, can be subjected to electrophoresis in 2%low-melting agarose for subsequent purification and sequencing. Sequences of the used primers, 5MSP61, 3MSP61mut and 3MSP61wt below: and, respectively, in SEQ ID NO 2, SEQ ID NO 3 and SEQ ID NO 4.

5MSP61 (23-d): 5’-CTA AGC CTG TTG TTT TGC AGG AC-3’ (SEQ ID NO 2);

3MSP61mut (20-d): 5’-CAT GGC ACT ATA CTC TTC TA-3’ (SEQ ID NO 3);

3MSP61wt (20-d): 5’-CAT GGC ACT ATA CTC TTC TT-3’ (SEQ ID NO 4).

The sequence 3MSP61wt includes 2 or 3 errors compared with sequences, respectively, N-ras and K-ras fragment size is 110 base pairs. DNA-matrix and reagents for amplification the following:

DNA (positive is positive control) OR 1.0 microgram

DNA (negative control, i.e. wild type) OR 1.0 microgram

DNA (sample, i.e. profilizirovannye block) OR 5,0 ál

Without DNA (i.e. H2O) 5,0 ál

The Rxn buffer (10x) (10x = 500 mm KCl,

100 mm Tris, pH of 8.3; 15 mm MgCl2) 5,0 ál

A mixture of dNTP, 500 μm of each

(final concentration =20 μm) of 2.0 ál

[32P]dCTP, 3000 CI/mmol, 5 mcmi,

1.7 picomol, 0,034 μm to 0.50 ál

5’-primer (10 mcmol/µl), 7.5 pcmall

(the final concentration of 0.15 μm) of 0.75 ál

3’-primer (10 mcmol/µl), 7.5 pcmall

(the final concentration of 0.15 μm) of 0.75 ál

Taq polymerase (5 units/µl of 3.0 units) of 0.60 ál

H2O to 50.0 ál 50 ál

Mineral oil 2 drops

Conditions cycle amplification using thermoacetica company Perkin-Elmer were as follows:

Preheating thermoacetica to 95°
No. 512-2195°1 min 30 sec.1 cycle
No. 512-2295°60 sec.30 cycles
57°60 sec.
72°60 sec.
No. 512-10wetting at 4°

The verification test was carried out by the processing of the following controls: wild type (WT), mutation of the wild type (MUT) and negative control (H2). As a positive control was used mutant (ILO) DNA from plasmids pHras61mut. Plasmid pHras61 includes a cloned DNA sequence of the 2nd exon of the gene Ha-ras cells from mouse tumor Sencar. The correctness of the cloned mutations were confirmed by DNA sequencing. This mutation is transverse CAA→a HUNDRED in codon 61 (located in the 2nd exon) gene Ha-ras mouse, which is a DNA sample from the tumor adenocarcinoma containing the gene mutation of Ha-ras in the 61st codon (see Fig).

Example 16

Test with PCR™ “hot start” on RFLP-mutations in codons 12/13 gene H-ras mouse

This test is based on the destruction of native MnlI restriction site covering all 3 nucleotide of the 12th codon and the first nucleotide of the 13th codon (GGA GGC, nucleotides 34-37 in the coding sequence of genes Ha-ras, rat, and mouse). Recognition site MnlI is N7GGAG. Mutations in any of these four sites will lead to the inability Mnll cut PCR fragment, which contains this area. The disadvantage of this test is that sometimes incomplete digestion with MnlI. This fact complicates the distinction between a very small percentage resistant to splitting the DNA of wild-type and characterized by a low level of true mutations. It is sometimes observed when the source of DNA consists of a mixture of DNA on the who type and DNA mutation and when to increase the sensitivity used 32P-labeled fragment. The primer set for PCR™used in this test are given below and in the sequence SEQ ID NO 5 and SEQ ID NO 6. The size of the amplified gene product Ha-ras 12 214 base pairs.

Primer#3 (5’): 5’-CCT TGG HUNDRED AGT GTG CTT CTC ATT GG-3’ (SEQ ID NO: 5);

Primer#6 (3’): 5’-ACA GCC CAC CTC TGG CAG GTA GG-3’ (SEQ ID NO: 6).

Primer #6 is used for sequencing with 55°under the following reaction conditions:

The Rxn buffer (10x) (10x = 500 mm KCl,

100 mm Tris, pH of 8.3; 15 mm MgCl2) 1,0 ál

A mixture of dNTP, 0.5 mm of each nucleotide 1,0 ál

Primer (5’) 6 pcma

Primer (3’) 6 pcma

32P-dCTP, 3000 CI/mmol to 0.5 ál

Taq polymerase (5 units/μl, 0,65%) of 0.13 ál

H2About 10 ál

DNA (positive control) >200,0 ng

DNA (negative control, i.e. wild type) >200,0 ng

DNA (sample, i.e. profilizirovannye block) 5,0 ál

Without DNA (i.e. H2O) 5,0 ál

Mineral oil 2 drops

Conditions of amplification using a set of reagents

for PCR Perkin-Elmer PCR™ were as follows:

Preheating of the amplifier 94°
No. 512-8794°2 minutes1 cycle
No. 512-8894°30 sec.8 cycles
68°30 sec.
72°1 minutes
No. 512-8994°30 sec.32 cycle
60°30 sec.
72°1 minutes
No. 512-10wetting at 4°

Example 17

Test results: detection of mutations in a gene called c-Ha-ras

4 days after the last injection of DMBA, plant extract and control, isolated from fresh frozen tissue DNA (5 mice from each group) were analyzed by PCR™ in the presence of mutations in codons 12 and 13 and in codon 61 of the gene c-Ha-ras. The applicants were used samples of 4-day exposure, as some 2-day samples of DNA were destroyed and were not suitable for analysis of Ha-ras. In codons 12 and 13 is MnlI restriction site covering 3 nucleotide of the 12th codon and the first nucleotide of the 13th codon in the sequence of the wild type. Mutation of any of them has resulted in the loss of MnlI site. On the material of genomic DNA was amplified 1st exon (which are the 12th and 13th codons) of the gene c-Ha-ras, for which we used thermoacetica company Perkin-Elmer. The reaction mixture was extracted with a mixture of phenol-l3and the DNA precipitated with ethanol. This DNA is then resuspendable in enzyme buffer and PCR is the product had restrictively MnlI with subsequent electrophoresis of the obtained product in 8%not denaturing polyacrylamide gel. In the tested material was not identified losses MnlI restriction site and it was concluded that in codons 12 and 13 mutations are absent. DNA for analysis of Ha-ras was isolated from paraffin histological sections with a thickness of 8 μm, the obtained 2 days after the last treatment. 25 slices from each waxed block was placed in a microcentrifuge tube was deparaffinization with xylene and ethanol, centrifuged and resuspendable in 5% Chelex® with proteinase-K.

The first procedure associated with the 61-th codon gene Ha-ras, developed by Nelson et al., 1992 (see example 15). Using the same direct primer, a reaction takes place with the reverse “wrong” primer (3MSP61mut), a different reaction with a reverse primer corresponding to the wild type (3MSP61wt). In this method detected only mutation type transversewhich is the most dominant point mutation in the 61st codon. The result of this transverse created by RFLP XbaI site. Reactions, including a product with a mutation, subjected to electrophoresis in 2%low-melting agarose for subsequent purification and sequencing. The ratio cut (wild type DNA) and uncut (mutant DNA) is determined quantitatively by assessing the intensity of staining of acid what bromidum or using the 32R-tagging. As a positive control using DNA plasmids pHras61mut. Plasmid rs61 includes the cloned DNA of the 2nd exon of the gene Ha-ras tumours in Sencar mice. The correctness of the cloned mutations were confirmed by DNA sequencing. Mutation is transverse CAA→a HUNDRED in codon 61 (found in the sequence of the 2nd exon) gene Ha-ras mice. Reaction conditions described in example 15.

Example 18

The effect of F035 on the occurrence of abnormal crypts in the F344 rats exposed to azoxymethane

Male rats of the Fischer-344 (F344) were obtained from the lab Charles River (Raleigh, NC) at the age of 6 weeks. Rats were fed without restriction diet AIN-76A purchased from Dyets Inc. (Bethlehem, PA). In the composition of the diet is casein (20%), DL-methionine (0,3%), corn starch (15%), sucrose (50%), corn oil (5%), cellulose (5%), salt mixture of AIN-76 (3,5%), vitamin mix AIN-76 (1%) and choline bitartrate (0,2%). Animals no restrictions were given drinking water. Azoxymethane (AOM), which is the inducer of abnormal crypts of the intestine of rats was purchased from Sigma Chemical Co. (St.Louis, MO). Animals maintained on a 3-day quarantine with “rat food mix, and then fed a diet AIN-76A until the age of 7 weeks. After this animal for treatment was randomly divided into 3 groups (10 animals/group). Animals of the 1st group fed only a diet AIN-76A, 2nd the group received the diet AIN-76A with the addition of 5 mg/mg F035, and the 3rd group - AIN-76A and 10 mg/kg F035 (table).

Table 30

The experimental group studied effect of F035 on the induction of abnormal crypts in the F344 rats under the action of azoxymethane
Group # The number of animalsDose F035 (mg per 1 kg of feed)
1100
2105
31010

After such feeding during the week, all animals were injected intraperitoneally AOM (15 mg/kg body weight). Another injection of AOM was performed one week after the first. Throughout the experiment, the animals weighed weekly. Feeding the animals was performed during 4 weeks. After 31 days the animals were killed with carbon dioxide asphyxia. Then delete the colon, washed her cold SFR, cut along the middle longitudinal axis, was placed on filter paper and fixed with 70%alcohol for at least 24 hours. Tissue of the colon were stained with methylene blue (0.25% in SFR) for about 1 minute. Abnormal crypts were counted under a microscope with magnification of 20x. Abnormal crypts differ from surrounding normal crypts by increased size, dramatically increased the distance from the apex of the folds (clearance) before the Foundation of the gaps cells and increased pericryptal area. All animals received a conditional tag and count conducted two “blind”. The statistical reliability of the results when comparing between groups was determined using univariate analysis of variance ANOVA. Upon detection of differences used student test (t) with the amendment of Bonferroni to assess multiple comparisons both doses F035 with the control group. Counting samples of the colon was performed together and “blind”, i.e. without knowing the processing of any of the experimental group performed. Between the data obtained by two “counters”, there was a good match.

It was found that F035 significantly reduces the total number of foci of abnormal crypts when included in the diet in the amount of 10 mg per 1 kg of feed, which is approximately equivalent to a daily dose of 1 mg F035 per 1 kg of body weight (table, table). The same dose also significantly reduces the number of abnormal crypts in the singlet and doublet (1 and 2 abnormal crypts per focus) categories (table, table). The lower dose F035, 5 mg per 1 kg of feed (which is roughly equivalent to a daily dose of 500 mg F035 per 1 kg of body weight)reduces the total number of abnormal crypts, the number of their singlet and doublet lesions, however, the level differences compared to control statistically unreliable (table, table). Throughout the experiment, significant differences in weight gain between the ex is erimentale and control groups were absent (table, table, table).

The number of abnormal crypts in the hearth (averaged over the results of the two “counter”)
Table 31

The effect of F035 on number of abnormal crypts in the colon of rats exposed to AOM
Dose F035, g per 1 kg of feedThe number of abnormal crypts in the colon: averaged results of two “counters”
Mean ± S.O.% of controlTotalNote
086±5100  
0,00573+585-
0,0143±450+
“-” is not significantly compared with control;

“+” - significantly compared to controls (P<0,05);

With the final research
Table 32

The effect of F035 on number of abnormal crypts in the hearth in rats exposed to AOM
Agent; intake (g per 1 kg of feed)
12≥3
Mean ± S.O.%Average ± S.O.%Mean ± S.O.%
Fraction 35; 066±510017±11003±1100
Fraction 35; 0,00556±48515±1883±0100
Fraction 35; 0,0134±3*528±1*471+033
* - significantly compared to controls (P<0,05)

Table 33

The compilation of preliminary data analysis of the effect of F035 on the average number of abnormal crypts in the colon of rats exposed to AOM
NAOMAgentDose (g/kg feed)The number of anomalous creep is on the colon: mean ± S.O.
1 “counter”2nd “counter”Two “counters”
10+Only carcinogen (4 weeks)and076±495±986±5
10+F0350,00567±480±873±5
10+F0350,0134±3c51±7c43±4c
a - rats, which were injected with AOM without test agent;

b - average in AOM-injected rats (N=10) for 4 weeks;

with significantly compared to controls (P<0,05)
Table 34

The compilation of preliminary data on the effect of F035 on number of abnormal crypts in the hearth in rats exposed to AOM
AgentDose“Count”The number of abnormal crypts in the hearth: mean ± S.O.
123Only
F0355 mg1st49±415±22±067±4
  2nd63±715±13±180±8
 two “counters”56±415±13±073±5
10 mg1st27±3*7±1*0±0*34+3*
 2nd41±5*8±1*3±151±7*
 two “counters”34±3*8±1*1±043±4*
Controlb/a1st57±417±13±176±4
 2nd75±818±23±195±9
 two “counters”66±517±13±186±5
* - significantly compared to controls (P<0,05)

Table 35

The body weight of the rats, which were administered AOM and fed F035 rate of 5 mg per 1 kg of body weight
Animal No.1 weekWeek 24th week5th week
1154,4198,2215,0RUR 219.7235,8
2149,8195,1210,6210,6232,9
3154,1200,7to 228.1to 228.1248,0
4154,1199,8to 216.2220,8242,9
5158,0208,4228,4231,5256,8
6154,8196,0of 208.3213,4230,2
7164,2210,1224,4225,5246,8
8of 161.7202,3218,8220,4237,8
9153,0199,7217,0218,1238,1
10158,8198,5212,8212,3231,6
Average156, 3mm200,9218,0220,0240,1
stocks.1,41,62,22,22,7
Table 36

The weight of the body cover is, which was introduced AOM and fed F035 at the rate of 10 mg per 1 kg of body weight
Animal No.1 weekWeek 2Week 34th week5th week
1of 148.6187,1reach 201.9205,6of 224.8
2148,3189,9196,0199,0220,8
3of 149.0197,7211,2to 216.2of 237.2
4146,2189,1206,1209,2230,0
5151,9197,2214,9of 218.6241,2
6152,2190,0205,2208,1226,6
7136,1187,8211,8to 216.2241,2
8157,4207,1224,1of 224.8246,0
9141,9187,8207,7211,1235,6
10155,7185,9196,4194,9213,9
Average148, 7mm192,0207,5210,4231,7
stocks.2,02,12,72,93,2

Table 37

The body weight of the rats, which were administered AOM
Animal No.1 weekWeek 2Week 34th week5th week
1149,3195,2of 203.2214,4240,6
2166,6213,8229,8231,8250,7
3158,6195,8209,0211,2226,8
4156,4to 200.3214,3216,9231,2
5151,2194,7205,2207,4228,5
6157,3the amount of 203.9217,2217,8237,0
7146,7USD 192.1216,6217,1235,2
8145,5190,3203,8204,7to 220.3
9158,1197,4212,3211,3231,2
10157,72018 217,9219,2240,8
Average154,7198,5212,9215,2of 234.2
stocks.2,02,22,62,42,7

Example 19

Antitumor activity of aglycones

Studies have confirmed the importance of sugars for the manifestation of biological activity, because removing sugars from the kernel triterpene molecules results in a significant loss of biological activity. As shown in the table. 38, the fraction of UA-BRF-004(beans)-DELEP-F164 (generated by hydrolysis of the Sugars from the UA-BRF-004(beans)-DELEP-F094 with attached esters) and UA-BRF-004(beans)-DELE-F245 (a mixture of methyl esters of the products of hydrolysis of UA-BRF-004(beans)-DELEP-F094) reveals a distinct loss of antitumor activity against a panel of lines of tumor cells. Similarly, UA-BRF-004(beans)-DELEP-C194 (purified acetate aglycone 1) does not show significant antitumor activity against a panel of lines of tumor cells compared with data obtained for triterpene glycoside fraction 35. Therefore, there is a significant loss of biological activity after hydrolysis of the sugar component of the claimed here triterpene glycoside.

td align="left"> 93
Table 38

Biological testing of the fractions F164, F245 and S
 50 mg/ml25 mg/ml12.5 ág/mlat 6.25 µg/mlof 3.12 µg/ml
F164     
769-P4520000
Raps-157271300
Dov-138056161210
MDA-MB-45366301300
Jurkat9386553916,5
F245     
769-P26141470
Raps-14926400
Dov-139190252813
MDA-MB-4539075880
Jurkat8964230
S     
769-P136000
Raps-169000
Dov-1330000
MDA-MB-453160000
Jurkat3418920

Example 20

Analysis of the effect of F035 on cholesterol metabolism

The purpose of this analysis is to study the influence of biologically active triterpene glycosides of the present invention for the prevention of cardiovascular diseases. The immediate object of this analysis is to establish the fact that the triterpene compounds introduced into the diet of mammals, including humans, will reduce the level of serum cholesterol. Selected for the research model system line of hamsters with hyperlipidemia is a rodent model, which, unlike rat model very accurately simulate changes in the levels of plasma LDL receptor and lipop theine in humans depending on the level of cholesterol (Spady et al., 1993).

Triterpene glycoside is administered in two different concentrations in cleaned food for hamsters without any changes in the contents of calcium, potassium, phosphorus and other essential elements of the diet. In order to demonstrate the dependence on the dose used two different levels of added triterpene glycoside. The animals were provided free access to a purified diet to hamsters Dyets, prepared in accordance with the recommendations of NRC (Reeves et al., 1993) with the addition of 1% cholesterol or without it (Davis et al., 1989). Purified food for hamsters Dyets, containing cholesterol, modified by the introduction of the triterpene glycoside concentrations indicated below. Experimental pelleted feed and the control diet were prepared by the firm Dyets Inc. (Bethlehem, PA) without changes in the content of calcium, phosphorus or any other important food microcomponent. Animals were controlled on the digestibility of the feed and the increase of body weight weekly. Used animals were Golden (Syrian) hamsters outbred lines at the age of 4 weeks, not containing viral infections (Charles River Lab., Wilmington, MA). Animals were divided into groups, putting on 3 individuals on Sadok randomly using a random number generator program Statview: cages placed in a room with lighting 12 hours a day and maintaining the temperature of 22±1,0° C.

After 0, 4 and 8 weeks of feeding on the respective diets, including or not including triterpene glycoside, 12 animals in each group are selected randomly and killed in 9-11 a.m. Then remove the liver and kidneys, weighed, processed and stored at -70°for further analysis. At the time of the killing take blood by puncture of the heart before removing the liver and kidneys, and lipid testing blood picture. Conduct a comparison of the lipid profile of blood serum in the experimental and control groups of animals. Was investigated with two control groups (groups 1 and 2) and two experimental groups (groups 3 and 4). All of these groups feed on the NRC diet during the 2-week quarantine. Group 1 continues to receive NRC diet during the whole period of the experiment. Groups 2-4 fed the NRC diet with the addition of 1% cholesterol for 2 more weeks with the intention of inducing hypercholesterolemia. Then group 2 continues to receive this ration until the end of the experiment, while groups 3 and 4 are fed the same diet supplemented by triterpene glycoside (e.g., F035 or F094). The synthesis parameters of the experimental groups is given below in table.

Table 39

Modification scheme diets
Group # the Initial number of hamsters LabelThe concentration of the additive in the diet
124+12ano-
224+12aHolb1% Hol
324Holb+TGc1% Hol + 0,003% TG
424Holb+TGc1% Hol + 0,075% TG
and is designed to destroy before feeding triterpene glycoside;

b - Hod = cholesterol;

with TG = triterpene glycoside

Through 0 (the control group), 4 and 8 weeks of feeding an appropriate diet, including or not including triterpene glycoside, 12 individuals from each group are selected randomly and killed in 9-11 a.m. The liver and kidneys of hamsters were removed, weighed and analyzed for possible anomalies. Portion of each body, characterized by any of the anomalies were prepared for histological study, i.e. frozen paraffin sections, which were stained with hematoxylin and eosin. Blood was taken at the time of the killing by puncture of the heart before surgical removal of the liver and kidneys. Then prepared serum and kept her at -20°for analysis of lipid profile blood. Predumisljajem hamsters were starving during the night. The data obtained are shown in table.

Blood samples collected during this experiment are used to determine the levels of total cholesterol, triglyceride, HDL-cholesterol and LDL-cholesterol and VLDL-cholesterol (Mackness & Durrington, 1992) in the laboratory Roche Biomedical Lab., Burlington, NC. Statistical data processing was carried out on the computer “Mac-9600” software for analysis of variance, to determine levels of significance and linear regression (Armitage, 1971). In particular, the data analysis of lipid profiles in each of the experimental groups was performed by analysis of variance using the criterion Newman-Kalsa (Steel &Torrie, 1980).

Table 40

The effect of continuous feeding hamsters triterpene glycoside (TG) within 6 weeks
DietControlCholesterol0,015% TG0,03% TG
Total cholesterol (mg/DL)average141341329303
% change--is 3.5-11,1
Triglycerides (mg/DL)average133247260236
% var is the link --5,3-4,4
Cholesterol HDL (mg/DL)average141281250246
% change---11by 12,4
The LDL cholesterol (mg/DL)average0313615
% change--16,1-51,6

12 hamsters in the group has received cleared homyachkovym diet with 1% cholesterol

Example 21

Analysis of the prevention of UVB-induced carcinogenesis using fractions 35

This analysis was focused on prevention of carcinogenesis induced by UV-B, in the model of the skin of mice using a triterpene glycosides of the present invention. The immediate object of this study is to identify the fact that in the model the skin of mice triterpene glycosides will prevent damage caused by UV-Century Experimental mouse model was used because it most closely corresponds to a similar situation in humans. In this analysis it is planned to demonstrate that topical application of the active triterpene compounds of the present invention in acetone at which the auger back of hairless mice SKH-1, irradiated UV-b, to prevent skin damage caused by such exposure.

In this analysis, hairless mice SKH-1 is irradiated with ultraviolet light In a dose of 1.8 kJ/m2within 15 minutes. Previously, mice exposed to two different doses F035 (2 mg and 4 mg in one dose), and also put a negative control (F060 or acetone). It is believed that to obtain statistically significant results, each group must have at least 10 mice. Each test compound is applied topically for 10 minutes before irradiation three times a week for 6-10 weeks. Studies conducted over a short period of time to assess the preventive effect of these compounds. To see formed, visually distinct tumor did not expect, even when exposed only to UV-b, given in the data time frame only damage to the skin. Can be identified weak erythema (slight reddening of the skin), which disappeared the next day after exposure.

As the UV source used device with 8 UV lamps Westinghouse FS40, radiometer/photometer IL-1400A and attached to phototherapy UVB-radiometer IL-1403 detector SEL-240/UVB-1/TD. In the middle part of this device has several chambers, each of which was placed in 1 mouse. On the sides of the chambers there are holes for ventilation in process the e-irradiation of mice. During irradiation, the camera rotates in the horizontal plane to provide uniform exposure to UV-b in mice. The device has doors that must be closed during start-up, UVB lamp, and therefore, the ultraviolet light remains inside the device. To measure the level of UV-b is used UVB-radiometer. The mouse should not be in the cells for more than 10-15 minutes.

The purpose of this analysis was to demonstrate the light shielding effect against induced by UV-b damage to the skin of mice. UV-b is absorbed directly cellular DNA and cause damage that can induce mutations in genes targets, ultimately leading to cancer. Early detection of damage and prevention of such injuries may indicate the presence chemoprotective effects (Berton et al., 1997; Chatterjee et al., 1996; Youn et al., 1997; Shirazi et al., 1996; Baba et al., 1996; Takema et al., 1996).

Table 41

Modes of exposure to UV-b
Only UV-b10 mice in group
Acetone + UV-b10 mice in group
F035 (2 mg dose), UV-b in 5-10 minutes10 mice in group
F035 (4 mg dose), UV-b in 5-10 minutes10 mice in group
F060 (2 mg dose), UV-b in 5-10 minutesMA in
F060 (4 mg dose), UV-b in 5-10 minutes10 mice in group

The experimental group for the study are described in table. 41. It is assumed that the size of these groups is sufficient to control the variability of hyperplasia of the skin and skin inflammation in this group, evidence of the variability of the thickness of the epidermis and inflammation of the skin in animals of the same age and stage of development. The main parameters that register in this analysis are hyperplasia and cutaneous inflammation. The remaining skin retain for the measurement of other biological markers, such as modified nucleotides in DNA (8-OH-dG) and the expression of the oncogene (oncogene Ha-ras).

Animals have free access to granular feed and drinking water throughout the experiment. Animal control on digestibility of food and the increase of body weight weekly. Using hairless mice lines SKH-1 outbred females aged 7 weeks, not containing viral infections (Charles River Lab., Wilmington, MA). Animals are divided into groups, putting on 5 individuals per Sadok randomly using a random number generator program Statview: cages placed in a room with lighting 12 hours a day and maintaining a temperature of 22±1,0°C.

Statistical processing of the obtained data hold n is the computer “Mac-G3” with the original program for analysis of variance, definition of levels of significance and linear regression (Armitage, 1971). In particular, data on the thickness of the epidermis in each group of medicinal substances evaluated using analysis of variance (Armitage, 1971).

Example 22

Influence of biologically active triterpenes on the expression of proteins involved in blocking the cell cycle and apoptosis

Apoptosis is defined as a normal physiological process of programmed cell death that occurs during embryonic development and in maintaining tissue homeostasis. The process of apoptosis can be divided into a number of metabolic changes in the apoptotic cell. Separate enzymatic stage several regulatory or signaling mechanisms can be analyzed in order to demonstrate the progress of apoptosis in the cell or cell population, or to detect the disruption of cell death in cancer cells. Apoptotic program is also observed by morphological features, which include changes in the plasma membrane (such as loss of skewness), condensation of cytoplasm and nucleus and mineclearance DNA cleavage. It all ends with death of cells, which, breaking down, turns into “apoptotic calf.”

Methods of analysis of some enzymatic reactions and processes PE is Adachi signals, involved in apoptosis, were developed as the standard protocols used in multifactor study of apoptosis. One of the examples of markers of early stages of apoptosis is the release of cytochrome C from mitochondria and subsequent activation kasperskogo-3 way (PharMingen, San Diego, CA). Induction of caspase (family of cytoplasmic proteases) is one of the most frequently observed signs of apoptosis. In particular, caspase-3 plays a key role in this process. When activated caspase split the target proteins, and one of the main proteins such is the PARP protein (nuclear protein). Therefore, effective from the point of view of identification of apoptosis are the tests for detection of the release of cytochrome C, the detection of the activity of caspase-3 and identification of the cleavage of PARP protein.

Moreover, the agents that cause the release of cytochrome C from the mitochondria of tumor cells, can be considered as a possible therapeutic agent for reducing at least one aspect of the mechanism of cellular control of programmed cell death.

Another test for apoptosis based on the detection of annexin-V (BioWhitaker, Walkerville, MD). Usually, phosphatidylserin (FS) is located on the inner surface of the cell membrane.

However, in the early stages of apoptosis is the externalization (peremeshany is on the external surface) FS. Annexin-V is a calcium-binding protein that binds to the FC and can be identified by flow cytometry with FITZ-staining of annexin-V (Martin et al., 1995). The ability of cells treated with compounds Acacia victoriae described in the present invention, to bind annexin-V is considered as an indication that these cells are susceptible to apoptosis.

In other examples, the applicants was used to test the PI3 kinase aimed at evaluating apoptotic activity in cells treated with anticancer compounds isolated from Acacia victoriae. Phosphoinositide-3-kinase (PI3K), an enzyme associated with cell membrane that can fosforilirovanii on the 3rd position Inositol ring phosphatidylinositol, thereby defining a new lipid signaling mechanism in those cells in which the PI3 kinase active. If PI3K is active at the cell membrane mate was recruited kinase, denoted by the ACT. The ACT is the product of an oncogene, which is catalytically activated upon recruitment to the membrane. Fully activated the ACT plays a key role in the survival of cells. A mechanism involving PI3K/AKT, corresponds to the mechanism of avoidance cell apoptosis. Thus, the method of suppressing PI3K in cancer cells, obviously, can be a therapeutic method for restoring at least some is, some aspects of the mechanism of cellular control of apoptosis.

Example 23

Analysis of cell cycle

For cell cycle analysis was applied standard methods of flow cytophotometry with some modifications. Briefly, 1×106cells were sown in 60-mm plates and were treated with various concentrations of F035 for 72 hours at 37°C. the Cells were washed SFR and resuspendable concentration of 1×106cells in 1 ml of Cells were fixed first 1%paraformaldehyde and then 70%ethanol, cooled on ice. Then cells were stained with iodine propidium (10 μg/ml; Sigma Chemical Co.; St.Louis, MO), containing 0.1% RNase (Sigma)for 30 minutes at room temperature and analyzed on a flow cytometer FAC firm Beckton-Dickinson.

Example 24

Test the binding of annexin-V-fluoresceinisothiocyanate (FITZ)

The induction of apoptosis in tumor cells was studied in the test for binding of annexin-V-FITZ. The Jurkat cells (1×106) was treated with different concentrations of a mixture of triterpene glycosides (F035) and purified extracts of D1 and G1 (0.5 to 2.0 µg/ml) for 18 hours at 37°°C. After washing, cells SFR their resuspendable in the buffer for binding (10 mm HEPES + NaOH, 140 mm NaCl, 2 mm CaCl2)containing 5 ál conjugate annexin-V-FITZ (BioWhittaker, Walkersville, MD), and incubated for 10 minutes in the dark. Then cells were stained with iodine propidium (20 μg/ml) and analyzed by the method of rotocol cytometry (Martin et al., 1995).

Example 25

Test with phosphatidylinositol-3-kinase (PI3 kinase)

Cells in serum-free medium Jurkat was treated with 2 µg/ml F035 for 2-15 hours or wortmannin for 0.5 hour at 37°C. the Activity of PI3 kinase was determined as described previously (Whitman et al., 1985; Royal & Park, 1995). The PI3 kinase was immunoprecipitated of 1 mg of cellular protein using 5 μl of rabbit anti-R antisera at 4°C for 90 minutes. Immune complexes were collected on the beads sepharose with 20% protein And for 90 minutes at 4°C. Then the obtained immunoprecipitate resuspendable in 30 μl of buffer for kinase reaction (33 mm Tris, pH of 7.6, 125 mm NaCl, 15 mm MgCl2, 200 mm of adenosine, 20 mm ATP, 30 µci γ-32P-ATP). The reaction of PI3 kinase initiated by adding 10 μl of the suspension PI and 10 ál γ-ATP and left for 30 minutes at room temperature. The reaction was stopped by adding 100 μl of 1 n Hcl. Lipids were extracted from the reaction mixture with a mixture of chloroform:methanol (1:1) and separated by thin-layer chromatography (TLC) with elution with a mixture of chloroform:methanol:NH4OH: H2(60:47:2:11,3) silikagelevye plates G60. Radioactively labeled phosphatidylinositol was visualizable method autoradiography and inhibition was quantitatively evaluated using the Storm 860 (Molecular Dynamics).

Example 26

Analysis of total and phosphorylated the Orme ACT

The expression of total and phosphorylated forms of the INSTRUMENT were determined by the method of Western blotting. Cell line Jurkat were cultured in medium containing 0.5% PTS (FBS)and treated F035 and purified extracts of D1 and G1 (2.0 µg/ml) for 15 minutes at 37°C. the Cells were literally in the ACT-lysis buffer (20 mm Tris-Hcl, 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1% Triton X-100, 2.5 mm sodium pyrophosphate, 1 mm 8-glycerol, 1 mm Na3VO4, 1 ml leupeptin, 1 mm PMSF, pH=7,5). Cellular protein (40 μg) were separated by electrophoresis in 8% of the LTO-page and transferred to nitrocellulose membrane. These membranes were tested with antibodies specific against phosphorylated (serine-473), or a General ACT followed by treatment with goat anti-rabbit antibody conjugated with horseradish peroxidase. Proteins were detected chemiluminescence method (ECL, Amersham, Arlington Heights, IL).

Example 27

Test for change in electrophoretic mobility (TIP)

In accordance with the above-described test TIP was conducted to study the effects of untreated (F035) and purified extracts D1 and G1 by factor NF-KB induced TNF (Genetech Inc.). Cell line Jurkat (1×1061 ml) was treated with different concentrations of the crude and purified extracts for 15 min at 37°C. Then the cells were subjected to 100 PKM TNF for 15 min at 37°C. Nuclear extras which you were prepared according to described above. Nuclear extracts were incubated with 16 fmol 45-d of the double-stranded oligonucleotide NF-kV bearing end32R-tag and derived from the long terminal repeat of human immunodeficiency virus,

5’-TTGTTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCTGG-3’ (SEQ ID NO:9),

for 15 minutes at 37°in the presence of 2 µg of poly(dI-dC). DNA-protein complex was separated from free oligonucleotide molecules in a 7.5%polyacrylamide gel. Radioactive bands from the dried gels were visualizable and quantitatively evaluated using Phospholmager (Molecular Dynamics, Sunnyvale, CA) and computer software ImageQuant.

Example 28

Induction and analysis of the inducible nitric oxide synthase (iNOS)

Cell lines U-937 and Jurkat were used for analysis of the enzyme iNOS. Cells U-937 were differentiated into macrophages during culturing them with PMA (100 nm) for 72 hours at 37°C. the Differentiated cells were treated F035 (2 μg/ml) for 15 hours with subsequent 4-hour LPS treatment (10 µg/ml) for the induction of iNOS. In Jurkat cells iNOS induced by exposure to 0.5×106cells PHA (10 μg/ml) and PMA (10 ml) for 24 hours at 37°C. Cell lysates were prepared by repeated freeze-thawing in RIPA buffer (1% NP-40, 0.5% deoxycholate sodium, 0.1% of LTOs in SFR). Cellular protein (200 µg) were separated by electrophoresis in 7.5%of the LTO-SDS page, transferred to nitrocellulose membrane, those who sabotaged using rabbit antibodies to iNOS, and then goat anti-rabbit antibody conjugated with horseradish peroxidase. Proteins were detected chemiluminescence method (ECL, Amersham, Arlington Heights, IL).

Example 29

Induction of cytotoxicity against tumor cells mixture and purified triterpene glycosides

The effect of a mixture of triterpene glycosides (F035) on the viability of a panel of cancer and normal cells was investigated in accordance with the described methods. As shown in Fig, (T-leukemic) Jurkat cells were highly sensitive to F035 with IC50=0.2 ág/ml Similarly F035 inhibited the growth of several lines of cancer cells with inhibitory concentration IC50in the range of 1.7 to 2.8 (ovarian cancer cells), 2.0 to 3.3 (kidney cancer), 0,93 (pancreatic cancer), 1,2-6,5 (prostate cancer) and 0.72-4.0 µg/ml (some breast cancer cells). However, other lines of breast cancer cells were resistant to the cytotoxic effect of F035. As seen in the last four blocks on Fig to destroy half of the untransformed cells (human fibroblasts and mouse and an immortalized epithelial cells of the mammary gland), you need more than 25 μg/ml F035: this confirms that F035 specific cytotoxic against tumor cells.

In addition, two purified triterpene glycoside, D1 and G1 were tested for their cytotoxic the activity in respect of the 5 cell lines. On Fig shows that the value of the IC50for D1 comparable with F035 for 3 cell lines (769-P, MDA-MB-453 and MDA-MB-231). In the cells With a-2 (option line HEY) and Jurkat D1 two times more active than with F035. However, the G1 is significantly more cytotoxic compared with F035 and D1 in most of the tested cell lines: this can be attributed to the lower polarity G1 compared to extract D1 (Fig).

Example 30

Blocking cell cycle and induction of apoptosis by a mixture of triterpene glycosides

To analyze the effect of F035 on the cell cycle of the line of tumor cells MDA-MB-453 and MDA-MB-435 was treated with various concentrations of F035. In table shows the increase in the number of cells in the phase of G1(7-10%) and a corresponding reduction in the percentage of cells in S phase (6-10%): this confirms the presence of blocking the cell cycle of cells MDA-MB-453 in the phase of G1. In addition, 72 hours after exposure F035 16% of the cells MDA-MB-435 (another line of breast cancer cells) is in Phase Sub.Go cell cycle (table), suggesting that cells entered apoptosis. This observation was further confirmed by analysis of apoptosis in the test TUNEL.

Table 42

Analysis of cell cycle in cells treated F035
Cell line F035 (µg/ml)Phase of the cell cycle (cell content, %)
Sub.G0G1SG2/M
MDA-MB-45301,0622613
11,5692110
31,8711610
62,272199
MDA-MB-43501,0523516
11,0513614
313502612
616502610

Cells MDA-MB-453 and MDA-MB-435 was treated with various concentrations of F035 for 72 hours at 37°C. Analysis of cell cycles performed after staining iodine propidium as described in the Methods section.

To clarify the mechanism underlying F035-induced cell death, applicants was carried out test on the binding of annexin-V-FITZ using processing F035, D1 and G1 cell line Jurkat. In table. 43 shows the binding of annexin-V cells, clicks botaniki 1 ml F035, D1 and G1 (15-17%), which indicates that the apoptotic mechanism leading to cell death.

Table 43

Test 72-hour analysis of cytotoxicity against T-leukemic Jurkat cells using D1-control, D1-aglycone, D1 without monoterpenes and monoterpenoid carbohydrates
Dose, mg/mlD1-controlD1-aglyconeD1 without monoterpenesD1 without both monoterpenesMonoterpene-carbohydrate
25,000 1005676
12,500 1005546
6,25062865433
3,1256204352
1,562610971
0,781610142
0,391570441
of € 0.195320140
0,097150 10
0,048 0000
0,000 0000
IC50(ág/ml)0,3293,6345,787>25,000>25,000

Example 31

The suppression of activity of P13 kinase a mixture of triterpene glycosides

In order to identify molecular targets F035, applicants investigated the mechanism of signal transmission of PI3 kinase. The results thus with anti-R (adapter protein) antibody and subsequent libidinously test showed that F035 inhibits the activity of PI3 kinase in the cell line Jurkat. On figa shown that after a 2-hour pre-treatment F035 activity of PI3 kinase is inhibited by 50-70%. At a 6-hour pre-treatment was observed suppression of the activity of PI3 kinase in 92-95%, which remained until the 15th hour after treatment. A similar suppression of kinase activity in Jurkat cells was established by action wortmannin (1 ám, 30 minutes after treatment), which is a known inhibitor of PI3 kinase (figa).

Example 32

Suppression of phosphorylation ACT with a mixture of triterpene glycosides, D1 and G1

The authors of the present invention have determined the effect of F035 and cleaned up the x extracts the ACT, which serine ser / thr kinase and a negative effector P13-kinase signaling mechanism. In contrast to the rapid suppression of the activity of PI3 kinase, suppression of phosphorylation of the ACT did not occur until the 15th hour after treatment. Treatment of Jurkat cells faction F035 (2 ml) for 15 hours resulted in reduced level of phosphorylation of the ACT. However, this treatment also resulted in the reduction of total protein ACT, as can be seen on figv. The applicants have confirmed that purified triterpene glycosides inhibited the activity ACT. Purified triterpene glycosides D1 and G1 (2 μg/ml) also inhibited the phosphorylation of akt and total expression level of the protein ACT (pigv). Treatment of Jurkat cells with wortmannin and LY294002 (known inhibitors RTZ-kinase) showed suppression of phosphorylation of the ACT.

Example 33

Suppression of TNF-induced NF-kV mixture of triterpene glycosides, D1 and G1

To further establish mediators of apoptotic mechanism, applicants evaluated the effect of F035, D1 and G1 on the transcription factor NF-KB, which has been shown to be involved in apoptosis. Shown in figa the results show that in Jurkat cells F035 suppresses induced factor TNF activation of NF-KB in a dose-dependent mode. In untreated cells and in cells treated only F035, activation of NF-KB is not produced in the runs. Similar results were obtained for purified extracts D1 and G1. Pre-treatment of cells with 2 ml of G1 and D1 resulted in a reduction of 54% and 87% of the levels of NF-KB, respectively (pigv). In cells treated with D1 or G1, activation of NF-KB is not detected (pigv). Since it was recently shown that PI3 kinase is involved in regulation of NF-KB, pre-treatment of cells with wortmannin (1 μm) leads to an almost complete suppression of TNF-induced NF-KB.

Example 34

Suppression of iNOS fraction F035

Because the transcription of the iNOS gene is regulated factor NF-KB, the authors of the present invention have examined the effect of F035 on the induction of iNOS enzyme. In cells U-937, which were differentiated into macrophages, iNOS was induced in response to treatment with lipopolysaccharide (figs). Pretreatment of these cells F035 (1 μg/ml) completely blocked the induction of iNOS. Wortmannin also has a similar effect on LPS-induced iNOS in these cells.

It was also investigated the effect of F035 on the induction of iNOS in Jurkat cells. iNOS was induced using PHA and PMA, as described in the Methods section. The results showed that pre-treatment of Jurkat cells faction F035 blocked the induction of iNOS (fig.46D).

Example 35

Analysis of the destruction of PARP by the method of Western blot turns

Apoptosis induced F035 and D1, were tested on FR the political cleavage of poly-ADP-ribosomally (PARP protein). The Jurkat cells (2×1061 ml) was treated with F035 (2 μg/ml) and D1 (2 μg/ml) at different time intervals. Cell lysates were prepared in buffer containing 20 mm HEPES, 250 mm NaCl, 2 mm EDTA, 0,1% NP-40, 2 μg/ml leupeptin, 2 μg/ml Aprotinin, 0.5 μg/ml of benzamidine, 1 mm dithiothreitol and 1 mm PMSF. Cellular proteins (60 µg/ml) were separated by electrophoresis in 7.5%of the LTO - polyacrylamide gel and transferred to nitrocellulose membrane using electroblotting. This membrane was tested with first monoclonal antibodies to PARP (Pharmagen), and then antimelanoma antibody conjugated with horseradish peroxidase. Protein bands were detected chemoluminescent by (ECL, Amersham). As a marker of apoptosis used the degree of rassheplenie PARP protein with a molecular mass of 116 KD on peptide fragments of 85 KD and 41 KD (Tewari et al., 1995).

Example 36

Test for caspase-3 protease

The activity of caspase-3 was measured as described previously (Enari et al., 1995) with some modifications. Briefly, Jurkat cells (1×1061 ml) was treated with F035, D1 and G1 in different periods of time. Cytoplasmic extracts were prepared by repeated freezing-thawing in 300 μl of extraction buffer (12.5 mm Tris, pH=7,0, 1 mm dithiothreitol (DTT), 0.125 mm EDTA, 5% glycerol, 1 μm PMSF, 1 μg/ml leupeptin, 1 μg/ml of pepstatin and 1 μg/ml Aprotinin). Cellular Liz who you were diluted 1:2 ICE buffer (50 mm Tris, pH of 7.0, 0.5 mm EDTA, 4 mm dithiothreitol and 20% glycerol) and incubated at 37°With 20 μm of the substrate caspase-3 (acetyl-Asp-Glu-Val-Asp-aminoethylamino). The activity of caspase-3 was monitored by the formation of fluorescent aminomethyl-coumarin on the scanner Fluoroscan II with excitation at 355 nm and the absorption at 460 nm (Labsystems, Helsinki, Finland).

Example 37

Detection of release of cytochrome C from mitochondria

The release of cytochrome C from mitochondria in response to processing F035 was determined by the method of Western blotting. The Jurkat cells (10×106) was treated with 2 µg/ml F035 for 4 and 6 hours at 37°C. the Cell precipitates were washed in sucrose buffer (0.25 M sucrose, 30 mm Tris, pH of 7.7, 1 mm EDTA). These cellular precipitation was added 20 μl of sucrose buffer containing 1 μm PMSF, 1 μg/ml leupeptin, 1 μg/ml of pepstatin and 1 μg/ml Aprotinin. Cells were broken by 120-fold pushing through a 0.3-ml “dunser” pestle “In” (“soft”) (Kontes Glass Company). Cellular protein (60 µg) was dissolved in 15%of the LTO-polyacrylamide gel and electrotransferred on nitrocellulose membrane. This membrane was tested with first monoclonal antibody specific against cytochrome C (Pharmagen), then antimelanoma antibody conjugated with horseradish peroxidase. Protein bands were detected chemoluminescent by (ECL, Amersham).

Induction of PARP cleavage by the action of F035 and D1

F035 and D1 induced PARP cleavage in Jurkat cells, depending on the duration. Data on Fig show that for 4 hours and F035, D1 and begin to induce the cleavage of PARP, and the completion of the cleavage occurs within 6-8 hours. This indicates the effective role of caspases and thereby apoptosis, i.e. the mechanism involved in cell death induced F035 and D1.

Example 39

The effect of z-vad fmk on F035-induced cell death

To further confirm the role of caspases in F035-mediated cell death have examined the effect of z-vad fmk, which is the inhibitor of caspases, cells treated F035.

Pre-treatment of Jurkat cells with 100 μm z-vad fmk for 1 hour at 37°leads to a complete reversion F035-induced PARP cleavage (Fig).

Example 40

Induction of activated caspase-3 action F035

Received by the applicants, the results clearly confirm the role of caspases in apoptosis induced by the effect of F035. Further studies were commissioned activation of caspase-3 in cells treated F035, F094, D1 and G1, because this protease in temporal dynamics is located directly in front of PARP in caspase-3 (Fig). Activation begins 4 hours after treatment in all cases, the peak is reached on 6-8th hour and then decreases.

the example 41

The release of cytochrome C from mitochondria under the effect of F035

As is, the release of cytochrome-C in some apoptotic mechanisms is the cause of the activation of caspase-3. For the analysis of the correctness of this assumption, induction of apoptosis the effect of F035 determined the levels of cytochrome C in the cytoplasmic extracts of cells treated F035. The authors of the present invention was installed in the release of cytochrome C from the mitochondria of these cells, depending on the duration (Fig). The release of cytochrome C detected 4 hours after treatment F035, which is consistent with the time when the activation of caspase-3 and begins PARP cleavage. It is necessary to investigate earlier times, to understand precedes whether the release of cytochrome C activation of caspase-3.

Example 42

Aeroponically system of cultivation

Discovery of the fact that the triterpene compounds of the present invention are concentrated in the roots and beans plants Acacia victoriae, it is desirable to develop a way of playing required tissues, which can be allocated connection data. To solve this problem was developed aeroponically system of growing roots of Acacia victoriae. Aeroponics is a closed system in which the roots of plants which are in the air and sprayed a full nutrient medium. Mailbox size 8×4×3,5 feet was made of 3/4-inch plywood sheets, fastened by screws, and is lined with plexiglass sheet to obtain a watertight volume. The top of the box was covered with two (2×8 ft) sheets of polystyrene, which were made 12 round holes, although the new design, based on PVC-coated wire cover for bird cages, closed opaque pressed black plastic film, painted white, is considered as a camera cover for future work. For spraying the roots for 12 seconds every 4.5 minutes used electronic timer.

Vertical chamber design for aeroponic is a closed system, collected from 3/4-inch PVC sheet with six hollow conical jet polypropylene spray nozzles. At the bottom of the camera was set 720-liter reservoir with nutrient solution, which is sprayed on the roots of plants from the bottom up using an external pump. Used pump Little Giant 4-MD, 3250 rpm, power 1/12 PS

The pump was controlled by a timer Tork installed at intervals of 30 seconds to spray every 4.5 minutes. The temperature was monitored using duringthere thermometer Tork, showing the temperature inside and outside the chamber; the temperature of the re which was interaval manually. In the winter months for heating the nutrient solution used two semi-submersible aquarium heater Visi-therm 300 W power; it was enough to maintain active growth of plants without heating the surrounding air in the absence of heating and cooling under a covered canopy outdoors in conditions of Tucson, Arizona.

The nutrient solution contained all the necessary elements required for the full life cycle of plants. Despite the fact that different plants require different amounts and compositions for optimal growth, in General, satisfactory results were obtained when using once a balanced solution. The composition of this solution is shown below in table.

Table 44

Nutrient solution for aeroponic
ConnectionChemicalConcentration (Cmin)
Calcium nitrateN Sa150 146
Potassium nitrateTo200
Monophosphate potassiumP90
Magnesium sulfateMD50
S134
10% Fe-chelateFe5
The copper sulfateC0,07
The chloride of manganeseMP0,8
Molybdate sodiumMo0,03
Boric acidIn0,3
Zinc sulfateZn0,1

Then the seeds of Acacia victoriae was scarificial and soaked in not containing a soil mixture of peat moss (50%) and vermiculite (50%). The seedlings were dipped twice daily and fertilize one dose osmocote (granular fertilizer on the basis of bone meal). Upon reaching the sprout length of 15-20 cm, which is usually achieved within 3-4 months of growing, the root systems were carefully washed to remove any residual peat moss and vermiculite. Then the roots were pushed through the holes in the polystyrene caps and the upper part of the seedlings were fixed with ropes attached to the fragments of the structure of the greenhouse. Cylindrical pieces of foam the size of 7 cm was enveloped in the upper part of the seedlings to avoid contact of the spray solution on the leaves and into the surrounding space. After that, the box was filled with a nutrient solution, a layer of about 30 cm, and consisted of a pump.

After placing the seedling in place and begin spraying, monitoring was limited to securing the growth of seedlings along the garter, for h the th used plastic clothespins, as well as the replenishment of the nutrient solution after the level has been decreased to 10 cm or less. Up until the seedlings were grown in the greenhouse, the temperature control of the nutrient solution was not necessary. However, if the mailbox for aeroponic exposed to an open environment (i.e. outside), it is recommended to raise the temperature of the nutrient solution up to 21°so that plants are not “asleep” during the winter months.

To collect the roots, the root mass of individual plants washed with water directly in the box to aeroponic and then cut this root mass scissors, retreating a few inches above the spray. Excess water is removed by drying with a paper towel, followed by weighing the sample. Then the root mass is crushed with scissors into pieces 3-4 inches and subjected to chemical extraction, as described above. Alternatively, to ensure the continuous collection of roots, the pump is switched off and the roots otscheplaut from the growing root mass. Then these roots are crushed into pieces the size of 3-4 inches and extracted as described. This requires careful not to damage uncollectible roots, which continue to grow.

When growing plants in aeroponic achieved a number of advantages. First, the growth rate is roughly twice as. is the standard method of growing. Secondly, if necessary, the roots can be easily gathered without harming the plant. This cutting roots additionally leads to intensive growth of laterals on the type of fibrous roots. Consequently, the roots can be collected several times a year. In addition, grown in aeroponic plants bloom in the first year of life, and the first flowering plants of open ground occurs on the 3rd to 5th year.

Example 43

Tissue culture and germination of Acacia victoriae

Seeds/substrate. Seeds were collected from plants of the Agricultural center of the University of Arizona in Tucson. The seeds were thoroughly washed with tap water, using antimicrobial soap (Vionex, Viro Res. Intern. Inc., Durango, Colorado), then was treated with 20% (V/V) household bleach for 15 minutes. After repeated washing with deionized water, the seeds were treated with boiling water (about 200 ml per 100 seeds) and left to cool overnight. Then they were treated with 20% (V/V) household bleach, 2-3 times washed in sterile deionized water and were cultured in complete medium Murashige-Skoog (MS) (Murashige &Skoog, 1962) and in MS half strength. This culture medium was supplemented with MS vitamins, 2% (V/o) sucrose and curing used or 0.7% agar, or 0.2 percent gelrite. In one experiment, seeds were scarificial concentrated the agreement acid, washed in sterile water and cultured in the medium. All culture medium autoclaved at 121°C for 15 minutes. The cultures were maintained at 25±2°s setperiod 16 hours at 1000 Lux using fluorescent lamps cold glow. Each analysis was replicated 18 times.

Reproduction. The tips of the shoots, segments, nodes, isolated from 3-week-old seedlings were cultivated only in MS and MS medium supplemented with 0.1 mg/l of auxin (IAA, NAA or IBA) and VAR (0,1; 0,3; 0,5; 1,0 and 1,3 mg/l) alone and in combinations. To analyze the formation of roots on shoots tested IAA (0.1 mg/l), IBA (0.1 and 0.6 mg/l) and NAA (0.1 and 0.2 mg/l). To migrate into the soil, the seedlings were removed from the culture tubes, roots were washed with tap water to remove glued to the roots of fragments nutrient substrate and transferred to pots filled with soil arid type. The plants were covered Magenta boxes to maintain humidity and kept at spraying and low light for 3 weeks. After 3 weeks, Magenta boxes were removed and the plants removed from the spray zone in the illuminated areas of the greenhouse.

Induction calluses. The formation of calluses induced on the material hypocotyl and root segments, cut in 3-week-old seedlings grown in vitro. The explants were cultured in MS medium, to alsenoy 2,4-D (1 mg/l), NAA (0.5 and 1 mg/l), IAA (0.2 and 1 mg/l), thidiazuron (0.2 mg/l), dicamba (0.2 and 2 mg/l), VAR (0.3 mg/l) and KN (0.5 and 3 mg/l) alone and in combinations.

The germination of seeds. Seeds treated with hot water, germinated with the emergence of the root after 3-4 days and the formation of a complete sprout for 1 week. Seeds not treated with hot water, did not grow at all. A greater number of seeds germinated on the environment, compacted using gelrite (0,2%) compared with agar (0.7 percent). The maximum level of germination reached 88.7%of what was observed in MS medium with half concentration, compacted galerita. Data on germination in different environments are summarized in table.

Table 45

The germination of seeds of Acacia victoriae
Cultural environmentThe number of seeds in cultureThe number of germinated seedsa
MS (sealed agar)4236(85,7%)
MS (sealed agar) (scarification sulfuric acid)4124(58%)
MS-1/2 (sealed agar)6048(80%)
MS-1/2 (sealed galerita)133118(88,7%)
and the number in parentheses corresponds to the percentage of germinated seeds

Suitable d is I transplant the seedlings were obtained after 3-4 weeks. Seeds of Acacia victoriae in natural conditions characterized by poor germination due to the high frequency of being in diapause (Kaul & Ganguly, 1965; Grice & Westoby, 1987). To overcome diapause you must remove the outer coat of the seed either by scraping with a sharp tool or acid scarification, or treatment with boiling water, followed by cooling in water during the night. Applicants found that the level of germination can be increased to 88.7 per cent using treatment with boiling water and subsequent cultivation of the seed culture medium MS half the concentration, compacted galerita (0,2%). According to Larsen 1964, the germination of seeds of A. victoriae in vivo in the case of treatment with boiling water can be increased up to 36%. Without pre-treatment, germination amounts to 19.4% (Kaul & Ganguly, 1965). In addition, in this case, the root development takes 12 days, and complete plantlets are formed for 79 days. Used by applicants method, germination increased (up to 88.7 per cent), and is suitable for transplanting the seedlings can be obtained for 3-4 weeks.

Culture of shoot tips: To study the propagation of shoots, their tips (approximately 1.0 cm in length) were cultured either in pure MS medium or MS medium supplemented VA or VA in combination with IAA. In a clean environment MS power shoots was low at low growth of roots (1-3 spine on the cult of the ru). In the medium containing BA (1.3 mg/l), at the tips of shoots formed multiple shoots (average 3.94 escape on culture). Among the numerous shoots one or two escape lengthened and grew for 4 weeks length 8,6 see the Combination of BA and IAA also contributed to the formation of multiple shoots.

These results are systematized in table.

Table 46

The effect of different levels of BA and IAA (0.2 mg/l) on the formation of multiple shoots from Acacia victoriae
Wednesday* +The average number of shoots per 1 tip escapeLength of shoot (cm)
BA (mg/l)IAA (mg/l)
1,303,94±1,8468,6±3,0258
0,10,21,6±0,5996,8±3,002
0,30,21,9±0,70715,8±2,794
0,50,22,8±1,6595,1±2,501
1,00,24,9±USD 2.0753,2±1,468
* MS. Data presented are average values for 18 duplicates ± standads.

At higher concentrations of BA (1.0 and 1.3 mg/l), the number of shoots increased. It was found that the combination of BA (1 mg/l) and IA (0.2 mg/l) improves the multiplication of shoots. Calluse received on the material scraps ends when all the combinations of BA-IAA. Was confirmed (Kaur et al., 1998) synergism of BA-NAA in relation to the induction of development of buds on shoots of Acacia catechu, while at higher concentrations of NAA (1-2 mg/l) no effect was observed. It was also alleged that the IAA ineffective to intensify the formation of buds on shoots; however, the material of such explants of calluse were obtained.

To study the formation of roots formed in vitro shoots were cut and transferred to culture medium containing IAA, NAA or IBA. The obtained data are systematized in table. Among the studied versions of the best options education roots occurred in MS half concentration with NAA (0.2 mg/l). Roots were formed almost 100% of the shoots. Shoots had reached a height of 9-11 cm for 4 weeks. It was reported (Kaur et al., 1998)that Acacia catechu unstable development calluses occurs in the places of articulation of shoots and roots, and to control the development of calluses the concentration of sucrose was reduced from 3% to 1.5%. Similar data were obtained for peronii lemon Feronia limonia (Purohit & Tak, 1992) and acacia Acacia auriculiformis (Das et al., 1993). In this study, the weak development of calluses was also identified in 3% sucrose, and it was reduced to a minimum of 2% sucrose. Shoots formed with roots transferred into the greenhouse. Surviving is here after the transfer was 100%. Seedlings acclimatized when spraying for 3 weeks, and then the seedlings were grown under normal greenhouse.

Culture segments of the nodes. Segments of nodes (seed nodes), cut off from couched in vitro seedlings were cultured in MS medium/ supplemented with 0.1 mg/l IAA, NAA or IBA. One Explant was developed by one or two axillary shoots. However, these shoots were characterized by weak growth. Therefore, knotting the explants were not taken for further use.

Induction calluses on the material hypocotyle and segments of roots. Calluse induced on the material segments hypocotyle, isolated from 3-week-old seedlings grown in vitro. The callus formed in the presence of 2,4-D (1 mg/l), thidiazuron (0.2 mg/l) and dicamba (0.2 mg/l), was greenish, compact and solid. The number of generated calluses was mild in most of the tested concentrations (table). Intensive education calluses found in MS medium with addition of 2,4-D (4 mg/l) + IAA (1 mg/l) + NAA (1 mg/l).

Segments of the roots, cut off from 3-week-old plantlets obtained in vitro, cultured in MS medium only with 2,4-D (1 mg/l) and 2,4-D in combination with kinesin (0.5 mg/l)and marked the formation of a light-yellowish friable calluses, and they formed a small number of roots. Education is Allyson occurred in 100% crops. Abundant formation of whitish, powdery, brittle calluses found on segments roots cultivated in the medium with the addition of BA (0.3 mg/l) and IAA (0.2 mg/l). The formation of numerous pale-yellowish calluses said segments roots cultivated in the medium with the addition of 2,4-D (4 mg/l) in combination with IAA and NAA (1 mg/l). A similar type of calluses was detected in the presence of thidiazuron (0.2 mg/l), dicamba (2 mg/l) and IAA (0.1 mg/l). On the segments of roots cultivated in the medium with dicamba (2 mg/l) and IAA (0.1 mg/l), was formed light green compact solid celluci. Attempts to regenerate plantlets on the material of these calluses was unsuccessful. For some tree species of plants, such as, for Albiizzia lebbeck (Lakshmana Rao & De, 1987) and Lonicera japonica (Georges et al., 1993), were found in the variability of the types of explants in connection with the development of calluses. Research applicants also found that developing in identical culture medium of calluses formed by hypocotyle and roots, there is a difference. Calluse formed from the tissues of the hypocotyl in combination BA-IAA were light green, compact and solid, while calluse root segments were whitish, soft (loose), brittle; it was also shown that in some embodiments, kallosh formed roots. Calluse Dalbergia latifolia when putting the x in regenerating the environment has become compact, solid and dark green, and they were differentiated buds of shoots (Pradhan et al., 1998). Research applicants was awarded a similar character development calluses, however, such calluse did not possess the ability to regenerate. In this analysis, the applicants have demonstrated that A.victoriae may be reproduced in vitro by the material of the tips of the shoots. The standardized method is applicable to microasperities elite individuals identified in the heterogeneous population of seedlings, and to maintain elite lines for further research.

Table 48

The development of calluses on the material hypocotyle and segments of the roots of Acacia victoriae
Wednesday*Nature of callus
The hypocotylRoots
1. MS + 2,4-D (1)Moderate, greenModerate, yellow
2. MS + TD (0,2)PoorPoor
3. MS + dicamba (2)Moderate, compact, greenModerate, loose, yellow
4. MS + 2,4-D (1) + KN (0.5 in)Scarce greenPoor, pale green
5. MS + KN (3) + NAA (0.5 in)Moderate, whitePoor, pale green
6 MS + TD(2) + Moderate, light greenModerate, loose, yellow
7. MS + dicamba (2) + IAA (0,2)Scarce, compact, yellowPoor, pale green
8. MS + 2,4-D (4) + IAA (1) + NAA (1)Rich, green, compact, solidModerate, yellow, friable
9. MS + BA (0,3) + IAA (0,2)Moderate, compactRich, white
* the number in parentheses indicates the quantity in mg/l

Example 44

Induction of the formation of hairy roots of Acacia victoriae to develop antitumor compounds.

Infected plant material Acacia victoriae bacterium Agrobacterium rhizogenes leads to integration into the genome of the plant and expression of T-DNA, which leads to the development of the phenotype “hairy roots” (Grant et al., 1991). Culture of hairy roots are characterized by rapid growth, associated with pleiotrophin root growth and intensively branched in hormone-free medium. Hairy roots are also characterized by a high level of genetic stability (Aird et al., 1988). Many species of dicotyledonous plants susceptible to infection with A. rhizogenes, and for many species was carried out regeneration of plants from cultures of hairy roots (Christey, 1997).

Genetic transformation and induction of hair is breaking the roots were carried out by the authors of the present invention as to generate active triterpenes of A. victoriae. The natural ability of soil bacteria Agrobacterium rhizogenes transformed genes in the genome of the recipient organism leads to the fact that parts of the roots, which have had such an infection, can be used to obtain cultures of hairy roots. Hairy roots are characterized by very rapid growth, intense branching auxiliary roots, which continue to grow in vitro on hormone-free medium.

Applicants have demonstrated induction of hairy roots of Acacia victoriae using Agrobacterium rhizogenes strain R1000 (genetically engineered strain of Agrobacterium tumefaciens, which was introduced plasmid pRiA4Agrobacterium rhizogenes - ATSC No. 43056). The production of the desired compounds of the hairy roots was confirmed by HPLC. The induction of the development of hairy roots was carried out as follows. First seeds of Acacia victoriae were collected from plants grown in the open ground in Tucson, Arizona. These seeds were treated with boiling water and left over night soaked in this water, which was cooled, and the surface of the seeds were sterilized with 15%household bleach for 30 minutes. After repeated rinsing in sterile water, the seeds were cultured in liquid MS medium (Murashige &Skoog, 1962)supplemented with MS vitamins and 2% sucrose, 250-ml conical flasks with 50 ml medium in ka the DOI of them. The cultures were maintained in a rotating shaker in the culture box at 25±2°in the dark. After 4 days of cultivation, the embryos were cut from the seedlings and used to infect bacteria.

Before using for infection, Agrobacterium rhizogenes strain R1000 were cultured overnight in medium YENB. The YENB medium was prepared by adding yeast extract (7.5 g/l) and nutrient broth (8 g/l) (Difco Lab., Detroit, MI). The embryo explants were infected using a thin needle is stainless steel, which before that had been dipped in the solution with bacteria. After infection with a drop of bacterial suspension (1:20 in MS) was applied to the surface of the Explant. Then the explants transferred to MS medium and MS medium with the addition of acetosyringone (100 μm) (3,5-dimethoxy-4-hydroxyacetophenone) (Aldrich Chem. Co., Milwaukee, WI) for culturing. Joint cultivation was carried out for 3 days in the dark. After 3 days of co-culture, the explants transferred to MS medium with the addition of Cefotaxime (500 mg/l) (Agri-Bio. North Miami, FL) to prevent excessive bacterial growth. Initiation of rooting was observed at the site of infection, mainly on the material of young developing leaves of the embryo 3-4 weeks. After 4 weeks, the explants along with the roots was transferred into a clean MS and continued what incubatio in the dark in order to achieve the development of hairy roots. The development of hairy roots was observed after 8 weeks. Educated thus the hairy roots were multiplied by standard in the MS method transfers. The transgenic nature of the hairy roots was confirmed by PCR using the primer set for amplification of the gene GOV. These primers were as follows:

1) 5’-GAGGGGATCCGATTTGCTTTTC-3’ (SEQ ID NO 7);

2) 5’-CTGATCAGGCCCCGAGAGTC-3’ (SEQ ID NO 8).

50 µl PCR reaction mixture contained primers (final concentration of each 1 μm), aq polymerase (1,0%), 125 µm of each nucleotide dNTP, 1x PCR reaction buffer, 1.5 mm MgCl2, 300 ng of isolated DNA. Used PCR conditions: initial denaturation for 5 minutes at 92°With, then spent 35 cycles of 50 seconds at 92°C, 1 minute at 55°C (annealing), 1.5 minutes at 72° (completing the circuit) and at the end of 1 minute at 72°With (final completion). Was amplified fragment length 645 base pairs.

Culture of hairy roots in liquid medium. To optimize growth conditions, hairlike roots, growing in semi-solid MS medium, cut and cultured in liquid MS medium in flasks of different capacity (125, 250, 500 and 1000 ml), filling them in, respectively, 20, 50, 100, and 400 ml of medium. The size of the original inoculum hairy roots was 6 g/L. the Growth of the hairy roots was also tested in follow what their basic environments: MS, environment N&N (Nitsch &Nitsch, 1969), environment Schenk-Hildebrandt (SH) (Schenk & Hilderbrandt, 1972) and the environment for tree species of plants WPM (Woody Plant Medium) (Lloyd &McCown, 1981). To test the effect of different carbon sources on the growth of hairy roots in MS medium was added at 2% (mass/about) the following sources: sucrose, glucose, fructose and mannose. The effect of gibberellic acid of 0.1, 0.5 and 1 mg/l) on the growth of hairy roots was tested by adding sterilized by filtration of the solution in the MS medium after autoclaving.

To initiate formation of roots at the site of infection was observed after 3-4 weeks. Four independent transformed clone of the hairy roots were formed on the material germ infected with strain R1000 in the presence of acetosyringone (100 μm). Embryos are cultivated with A. rhizogenes without acetosyringone not formed hairlike roots (table. 49). It was found that 3-day joint cultivation in the presence of acetosyringone is optimal for the induction of hairy roots. Activating effect of acetosyringone was described for sage Salvia militiorrhiza (No & Alfermann, 1993). The results showed that acetosyringone, which is the activator of the vir genes of Agrobacterium, increases the frequency of transformation. Similarly, in the study conducted here, acetosyringone is necessary is Imam for the induction of hairy roots.

Transformed the nature of these roots was confirmed by PCR amplification using the primer set, providing amplificatoare fragment of the gene GOV. Diagnostic fragment length 645 base pairs was amplified in all four tested clones of hairy roots.

Growing in liquid medium hairy roots characterized by a strong development. Among the tested source environments, the best performance due to the growth of the hairy roots were determined for MS (table). In 125-ml flasks were noted 268-fold rise in 4 weeks. When using media WPM and N&N, the rate of increase amounted to, respectively, 254 and 196. Environment B5and SH were not optimal for the growth of hairy roots. These two environments was observed slow darkening of hair roots. In one experiment, the hairy roots were grown in different capacity flasks (125, 250, 500 and 1000 ml) with 20, 50, 100, and 400 ml MS, respectively. The growth parameters are summarized in table. 50. First, the growth of the hairy roots was powerful and had reached 25,77-fold increase for 4 weeks in 125-ml flasks with the value of the original inoculum 150 mg as increasing the capacity of the flasks, the growth rate of roots was slowly dropped.

The growth of the hairy roots can be sensitive to the composition of cultural the th environment, especially to mineral ions and carbon source (Wysokinska & Chmiel, 1997). For Acacia victoriae, five different source environments (MS, N&N, SH, WPM and B5) were tested for effects on the growth of hairy roots. The best growth was the MS medium. According to the comparison of the growth of the hairy roots of Coleus forskohlii in various nutrient media (Sasaki et al., 1998) the best for the growth of the hairy roots was Wednesday WPM.

In the study, sucrose contributed to the growth of the hairy roots compared with other carbon sources (fructose, glucose and mannose). The best growth (24,52-fold increase) was detected in culture medium containing sucrose. The medium containing glucose, to some extent inhibited the growth and mannose growth was completely suppressed (table). In Catharanthus roseus develop catharanthine can be doubled by using fructose as a carbon source in the culture medium. However, it was reported that the use of fructose gave approximately 40%reduction in growth compared with sucrose (Jung et al., 1992).

For the growth of hairy roots is not necessary to add exogenous growth factors, because the genes responsible for increased susceptibility to auxins, and are in part of the Ri plasmid (Wysokinska & Chmiel, 1997). There are, however, reports that exogenous hormones stimulate the growth of the Applicants have investigated the effect of gibberellic acid (0,1, 0.5 and 1.0 mg/l) on the growth of hairy roots. The growth of the hairy roots was the best in an environment devoid GA3compared with medium containing GA3(15,77-fold increase). Different levels of GA3not significantly affect the growth (table). The wormwood genus Artemisia, GA3does not increase biomass accumulation as a whole, but facilitates more rapid achievement of a stationary phase of growth compared to cultures grown on medium without GA3(Smith et al., 1997). It was found (Rhodes et al., 1994)that the reaction of the hairy roots of white mustard Brassica Candida in GA3largely depends on the analyzed clones. However, it was revealed that GA3demonstrates a positive effect on the growth and reducing the accumulation of alkaloids along with changes in the conditions of their production. It was reported (Ohkawa et al., 1989)that GA3at concentrations of 10 ng/l and 1 mg/l accelerates growth and increases the elongation and increases lateral branching hairy roots of Datura Datura innoxia. It was confirmed (Zobel, 1989)that GA3applies to the growth of roots as similar CO2. In respect of hairy roots of Acacia victoriae, GA3does not increase growth, which may indicate different reaction to it different genotypes.

Using cultures of hairy roots of Acacia victoriae provides an effective way to obtain one is one culture plant tissue, from which can be isolated triterpene glycosides of the present invention, including the selection of mixtures or hotel purified compounds.

Different media were tested for growth in them hairlike roots. The best growth was found in MS medium containing 2% sucrose. Investigated the influence of different capacity flasks and gibberellic acid on the growth of hairy roots. Also, the hairy roots were cultured in MS liquid medium on a rotary shaker at 125-ml conical flask with application of 20 ml of medium. 4 weeks was marked 84-fold increase. The production of triterpene saponins, corresponding to those identified in the faction F035, was confirmed using HPLC using authentic standard sample.

All compositions and methods described and claimed herein can be made and used without any additional experiments in the light of this application. Although the compositions and methods of the present invention is described in preferred variants, the specialist in the art should understand that in these compositions and methods may be made of modifications, as well as in private this is s and the sequence of steps of these methods, described herein, without deviating from the concept of the essence and scope of the present invention. More specifically, it should be clear that some agents that are close and chemically, and physiologically, can be used to replace the agents described here, if it will achieve the same or similar results. All such similar substitutes, and modifications, obvious to those skilled in the art, are considered as relevant substantive scope and the idea of the present invention defined by the claims.

BIBLIOGRAPHY

The following references, to the extent that they are intended to illustrate procedures or other details supplementary to declare that this text is included here for information in the form of a bibliography.

Aerts et al., Plant J., 5:635-643, 1994.

Agrawal, "NMR spectroscopy in the structural elucidation of oligosaccharides and glycosides," Phytochemistry, 31:3307-3330, 1992.

Aird, Hamill, Rhodes, "Cytogenetic analysis of hairy root cultures from a number of species transformed by Agrobacterium rhizogenes," Plant Cell Tissue Organ Cult., 15:47-57; 1988.

Akiyama et al., J. Blol. Chem., 262:5592-5595, 1987.

Alien et al., "Leguminosae, A source book of characteristics uses and nodulation," The University of Wisconsin Press, Madison, Wisconsin, 1981.

Armitage, Jn: Statistical Methods in Medical Research, Wiley and Sons, New York, NY.p 205, 1971.

Amon, R., et al., Proc. Natl. Acad. Sci. (USA) 77: 6769-6772, 1980.

Baba, Hanada, Hashimoto, "The study of ultraviolet B-induced apoptosis in cultured mouse keratinocytes and in mouse skin," J. Dermatol. Sci., 12:18-23, 1996.

Baxter, Price Fenwick, "Sapogenin structure: analysis of the

13C and1H-NMR spectra of soyasapogenol b," J. Nat. Prod., 53:298-302, 1990.

Bellacosa, Feo, Godwin, Bell, Cheng, et al., Int. J. Cancer, 64:280-285, 1995.

Berton, Mitchell, Fischer, Locniskar, "Epidermal proliferation but not the quantity of DNA photodamage is correlated with UV-induced mouse skin carcinogenesis," Invest.

Dermatol., 109:340-347, 1997.

Beutler, Kashman, Pannell, Cardellina, Alexander, Balaschak, Prather, Shoemaker, Boyd, Bioorganic and Medicinal Chemistry, 5:1509-1517, (1997).

Boll and von Philipshom, "NMR studies and the absolute configuration of Solanum alkaloids (spiroaminoketal-alkaloids), Acta Chem. Scand., 19: 1365-1370, 1965.

Brinkmann et al., Proc. Natl. Acad. Sci., USA, 88(19):8616-8620, 1991.

Burchell et al., J. Immunol.. 131 (1): 508-513, 1983.

Campbell. in Monoclonal Antibody Technology, Laboratory

Techniques in Biochemistry and Molecular Biology Vol.13,

Burden and Von Knippenberg, Eds. pp.75-83, Amsterdam,

Elseview, 1984.

Capaidi et al., Blochem. Biophys. Res. Comm., 76:425, 1977. Capon and Thacker, "The nuclear magnetic resonance spectra of some aldofuranosides and acyclic aldose acetals," Proc.

Chem. Soc. bond., 369, 1964. Chatterj, Agarwal, Muhtar, "In Ultraviolet radiation-induced

DNA lesions in mouse epidermis," Biochem. Blophys. Res.

Commun., 229:590-595, 1996.

Cheatham et al., Proc. Natl. Acad. Sci., 92: 11696-11700, 1995.

Cheeke, Can. J. Animal Sci., 51: 621-632, 1971.

Chen and Snyder, "Diosgenin-bearing, molluscicidal in saponins from Allium vineale: an NMR approach for the structural assignment of oligosaccharide units," J. Org. Chem.,

54: 3679-3689, 1989.

Chen and Snyder, "Molluscicidal saponions form Allium vineale," Tetrahedron Lett., 28:5603-5606, 1987.

Cho, Widholm, Tanaka, Nakanishi, Murooka, "Agrobacterlum

rhizogenes-mediated transformation and regeneration of the legume Astragalus sinicus (Chinese milk vetch),"Riant Science, 138: 53-65;1998.

Chou and Blems, Cell, 85:573-583, 1996.

Christey, "Transgenic crop plants using Agrobacterium rhizogenes-mediated transformation," Doran, P.M., (ed.)

Hairy roots: Culture and applications, Harwoo,

Amsterdam, 99-111, 1997.

Colchere et al., Cancer Res., 47: 1185 and 4218, 1987.

Cohart, Baeuerle, Vassalli, Mol. Cell. Biol., 10: 1498-1506, 1990.

Creeimane et al., Proc. Natl, Acad. Sci. USA, 89: 4938-4941, 1992.

Davis & Preston Analytical Biochemistry, 116(2):402-407, 1981.

Davis, Sinensky, Junker, Pharmac. Ther., 43: 221-36, 1989.

Defago, Weg. Schweiz. Bot. Ges., 87:79-132, 1977.

Dillman et al., Antibody Immunocon. Radiopharm., 1:65-77,1988.

Doll, R. et ai., Lancet 1: 793, 1962.

Enari. Hug, Nagata, Nature, 375: 78-81, 1995.

Folkman, Haudenschild, Zetter, Proc. Natl. Acad. Sci.,

76: 5217-5221, 1979.

Franceschi et al., Proc. Nati. Acad. Sci. USA, 88: 6745-6749, 1991.

Frechet, Christ, du Sorbier, Fischer, Vuilhorgne, "Four triterpenoid in saponins from dried roots of Gypsophlla species," Phytochemistry, 30:927-931, 1991.

Gamborg, Miller, Ojima, "Nutrient requirements of suspension cultures of soybean root cells,Exp. Cell Res., 50: 151-158;

1968. Gariboldi, Verotta, Gabetta, "in saponins from Crossopteryx

febrlfuga, Phyto chemistry, 29:2629-2635, 1990.

Gefter et al., Somatic Cell Genet., 3: 231-236, 1977. Ghose et al., CRC Critical Reviews In Therapeutic Drug Carrier

Systems, 3: 262-359, 1987.

Ghose, et al., Meth. Enzymology, 93: 280-333, 1983. Coding, 1986, In: Monoclonal Antibodies: Principles and.

Practice, 2d ed., Academic Press, Orlando, Fla., pp.60-61, and 71-74, 1986.

Grant, Dommisse, Christey, Conner, "Gene transfer to plants using Agrobacterium," In: Murray, D.R., (ed.) Advanced methods in plant breeding and biotechnology, CAB

International, Wallingford, 1991:between 50 and 73. Gundalche et al., Proc. Natl. Acad. Sci. USA, 89: 2389-2393, 1992.

Hamburger, Slacanin, Hostettmann, Dyatmiko, Sutarjadi,

"Acetylated in saponins with molluscicidal activity from

Saplndus rarak: unambiguous structure determination by proton nuclear magnetic resonance and quantitative analysis," Phytochem. Anal., 3: 231-237, 1992.

Hansen, Nielsen, Berg, J. Immunologlcal Methods, 119: 203-210, 1989.

Harlow and Lane, Antibodies: A Laboratory manual. Cold Spring

Harbor Laboratory, 1988.

Harwood, Chandler, Pellarin, Bangerter, Wilkins, Long, Cosgrove, Malinow, Mazetta, Pettini, Savoy, Mayne,

"Pharmacologic consequences of cholesterol absorption inhibition: alteration in cholesterol metabolism and reduction in plasma cholesterol concentration induced by the synthetic saponin called p-tigogenin cellobioside (CP-88,818; tiqueside), J. Lipid. Res. 34:377-395, 1993.

Hassanain, Dai, Gupta, Anal. Biochem., 213:162-167, 1993.

Hostettmann et al., "Chemistry and pharmacology of natural products," In in saponins, Cambridge University Press, pp.1-548, 1995.

Hu, Alfermann, "Diterpenoid production in hairy root cultures of Salvia’ miltlorrhlza," Phy to chemistry, 32 (3): 699-703; 1993.

Huang et al., Zhongueo Yaoil XuebaOr Chemical abstract No.98100885, 3: 286-288, 1982.

Ikeda, Fujiwara, Kinjo, Nohara, Ida, Shoji, Shingu, Isobe,

Kajimoto, Bull. Chem. Soc. Jpn., 68:3483-3490 (1995).

Inoue, H., et al., Chem. Pharm. Bull. 6) 2:897-901, 1986.

Jansakul, Baumann, Kenne, Samuelsson, "Ardisiacrispin A and B, two utero-contracting in saponins from Ardlsia crispa,"

Planta Medico, 53: 405-409, 1987.

Jiang, Massiot, Lavaud, et al., "Triterpenoid glycosides from

the bark of Mimosa tenuiflora, Phytochemlstry, 30: 2357-2360, 1991.

Jung, Kwak, Kirn, Lee, Choi, Lin, "Improvement of the catharanthine productivity in hairy root cultures of Catharanthus roseus by using monosaccharides as a carbon source," Blotech. Lett., 14: 695-700; 1992.

Kamel, Ohtani, Kurokawa, et al., "Studies on Balanltes aegyptiaca fruits, an antidiabetic Egyptian folk medicine," Chem. Pharm. Bull., 39: 1229-1233, 1991.

Kasiwada et al., J. Org. Chem., 57: 6946-6953, 1992.

Kelly and Tsai, "Effect of pectin, gum arable and agar on cholesterol absorption, synthesis and turnover in rats,"

J. Nutr., 108:630-639, 1978.

Kennedy, Wagner, Conzen, Jordan, Bellacosa, Tsichlis, Nissam,

Genes and Dev., 11: 701-713, 1997.

Kimura et al., Immunogenetics., 11: 373-381, 1983. Kinjo, Araki, Fukui, Higuchi, Iceda, Nohara, Ida, Takemoto,

Miyakoshi, Shoji, Chem. Pharm. Bull. 40(12); 3269-3273 (1992).

Kizu and Tomimori, "Studies on the constituents of Clematis species. V. On the in saponins of the root of Clematis chinensis OSBECK," Chem. Pharm. Bull., 30: 3340-3346, 1982.

Kohler and Milstein, Eur. J. Immunol., 6: 511-519,1976.

Kohlr and Milstem, Nature, 256: 495-497, 1975.

Kojima and Ogura. "Configurational studies on hydroxy groups at C-2, 3 and 23 or 24 of oleanene and ursene-type triterpenes by NMR spectroscopy," Phytochemistry, 28: 1703-1710, 1989.

Kong et al., Phyto chemistry. 33: 427-430, 1993.

Konoshima and Sawada, Chem. Pharm. Bull., 30: 2747-2760, 1982.

Kutney, "Nuclear magnetic resonance (N.M.R.) study in the steroidal sapogenin series. Stereochemistry of the spiro ketal system," Steroids, 2: 225-235, 1963.

Lemieux, Kullnig, Bernstein, Schneider, "Configurational effects on the proton magnetic resonance spectra of six-membered ring compounds," J. Am. Chem. Soc., 80: 6098-6105, 1958.

Lister, P.P.., P. Holford, T. Haigh, and D.A. Morrison. Acacia in Australia: Ethnobotany and potential food crop. p.228-236. In: J. Janick (ed.). Progress in new crops. ASHS Press, Alexandria, VA, 1996.

Lloyd and McCown, "Commercially feasible micropropagation of mountain laurel, Kalmia latifolia by use of shoot tip culture," Comb. Proc. Inti. Plant Prop. Soc., 30: 421-427; 1981.

Mackness, Durrington, Converse, Skinner (Eds.), Jn:

Iiipoprotein Analysis: A Practical Approach, Oxford University Press, Oxford, p 1, 1992.

Mahato, Pal, Nandy, Tetrahedron, 48: 6717-6728 (1992).

Manabe et al, J. Lab. Clin. Med., 104 (3):445-454, 1984.

Martin et al., J. Exp. Med., 182:1545-1556, 1995.

Martin, Reueelingsperger, McGahon, Rader, van Schie, Laface, Green, J. Exp.Med., 182:1545-1556, 1995.

Massiot, Lavaud, Besson, Le Men-Olivier, van Binst, "in saponins from aerial parts of alfalfa (Medicago sativa)," J. Agric. Food Chem., 39: 78-82, 1991b.

Massiot, Lavaud, Delaude, van Binst, Miller, Fales, "in saponins from Tridesmostemon claessenssi," Phy to chemistry, 29:3291-3298, 1990.

Massiot, Lavaud, Guillaume, Le Men-Olivier, van Binst, "Identification and sequencing of sugars in in saponins using 2D1H NMR spectroscopy," J. Chem. Soc., Chem. Common., 1485-1487, 1986.

Massiot, Lavaud, Le Men-Olivier, van Binst, Miller, Fales, "Structural elucidation of alfalfa root in saponins by mass spectrometry and nuclear magnetic resonance analysis,". J. Chem. Soc., Perkin Trans., 1:3071-3079, 1988.

Massiot, Lavaud, Nuzillard. "Revision des structures des chrysantellines par resonance magnetique nucleaire" Bull. Soc. Chim. Fr., 127: 100-107, 1991a.

Miotti et al., Cancer Res., 65: 826, 1985.

Miyamoto, Togawa, Higuchi. Komori, Sasaki, "Six newly identified biologically active triterpenoid glycoside sulphates from the sea cucumber," Cucumana echinata. Annalen, 453-460, 1990.

Monk, "Variegation in epigenetic inheritance", TIG, 6: 110-114, 1990.

Mujoo, Maneval, Anderson, Gutterman, Oncogene, 12: 1617-1623, 1996.

Murashige, Skoog, "A revised medium for rapid growth and bioassay of tobacco tissue culture," Physiol. Plant, 15: 473-482; 1962.

Murashige, T. and Skoog, F. A revised medium for rapid growth and bio-assays with tobacco tissue cultures," Physlologia

Plantarum 15: 473-497, 1962.

Nabel and Baltimore, Nature 326:711-713, 1987.

Nagamoto et a2., Planta Medica., 54:305-307, 1988.

Nagao, Hachiyama, Oka, Yamauchi, "Studies on the constituents of Aster tatarlcus L. f. .Structures of aster in saponins isolated from the root," Chem. Pharm. Bull., 37:1977-1983, 1989.

Nelson, Futscher, Kinsella, Wymer, Bowden, "Detection of mutant Ha-ras genes in chemically initiated mouse skin epidermis before the development of benign tumors," Proc.

Natl. Acad. Sci. USA, (14):6398-6402, 1992.

Nishino, Manabe, Enoki, Nagata, Tsushida, Hamaya, "The structure of The tetrasaccharide unit of camellidins, in saponins, possessing antifungal activity," J. Chem. Soc.,

Chem. Commun., 720-723, 1986. Nitsch, Nitsch, "Haploid plants from pollen grains," Science 163:85-87, 1969.

O'reilly, Boehm, Shing, Fukai, Vasios, Lane, Flynn, Birkhead, Olsen, Folkman, Cell 88: 277-285, 1997.

Oakenfull et al., Athe-rosc-Iero.s-LS, 48: 301 (1983).

Ohkawa, Kamada, Sudo, Harada, "Effects of gibberellic acid on hairy root growth in Datura innoxia," J. Plant Physiol., 134:633-636; 1989.

Okabe, Nagao, Hachiyama, Yamauchi, "Studies on the constituents of a Luff operculata cogn. .Isolation and structure elucidation of in saponins in the herb," Chezn.

Pharm. Bull., 37: 895-900, 1989.

Okada, Koyama, Takahashi, Okuyama. Shibata. Planta Med. 40: 185-192, (1980).

Okada, Sakuma. Fukui, Hazeki, Ui, J. Blo. Chem., 269: 3563-3567, 1994.

Pallavicini, Jn: Techniques In Cell Cycle Analysis, Gray and Parzynkiewicz (Eds.), Humana Press Inc., Clifton, NJ, pp.139, 1987./p>

Pant, Panwar, Negi, Rawat, Morris, Thompson, "Structure elucidation of a spirostanol glycoside from. Asparagus offlclnalis fruits by concerted use of two-dimensional

NMR techniques," Mag. Reson. Chem., 26: 911-918, 1988.

Fenders, Delaude, Pepermans, van Binst, "Identification and sequencing of sugars in an acetylated saponin called of Bllghia welwltschil by N.M.R. spectroscopy," Carbohyd. Res., 190: 109-120, 1989.

Pietenpol et al., Cancer Res., 55: 1206-1210, 1995.

Pieterez et al. Antibody Inmiunoconj. Radlopharm., 1: 79-103, 35, 1988.

Pisha et al., Mature Medicine, 1: 1046-1051, 1995.

Polyak et al., Genes Dev., 8: 9-22, 1994.

Potterat, Hostettmann, Stoeckli-Evans, Saadou, "in saponins with an unusual secoursene skeleton from Sesamum alatum

THONN., Helv. Chlm. Ada, 75:833-841, 1992.

Prehn, "Regeneration versus neoplastic growth,"

Carcinogenesis, 18(8): 1439-1444, 1997.

Puri, Wong, Puri, "Solasodine and diosgenin:1H and13C assignments by two-dimensional NMR spectroscopy," Mag.

Res. Chem., 31: 278-282, 1993. Reeves, Nielson, Fahey, Am. Inst. Nutr., 1939, 1993.

Reisfeld et al., Melanoma Antigens and Antibodies, p.317, 1982.

Reznicek, Jurenitsch, Kubelka, Michi, Korhammer, Haslinger, "Isolierung und Struktur der vier Hauptsaponine aus Solldago gigantea var. serotlna," Annalen, 989-994, 1990.

Reznicek, Jurenitsch, Michi, Haslinger, "The first structurally confirmed saponin called from Solldago gigantea: structure elucidation by modern NMR techniques," Tetrahedron Lett., 30: 4697-4100. 1989b.

Reznicek, Jurenitsch, Robien, Kubelka, "in saponins in Cyclamen species: configuration of cyclamiretin With and structure of isocyclamin," Phyto chemistry, 28: 825-828 .1989a.

Rhodes, et al., "Influence of exogenous hormones on the growth and secondary metabolite formation in transformed root cultures," Plant Cell Tissue Organ Culture, 3 8: 143-151; 1994.

Rodriguez, Castro, Riguera, "Holothurinosides: new antitumour non sulphated triterpenoid glycosides from the sea

cucumber Holothrula forskalii, " Tetrahedron, 47: 4753-4762, 1991.

Royal I and Park M, J. Biol. Chem. 270: 27780-27787, 1995.

Sasaki, Udagawa, Ishimaru, Hayashi, Alfermann, Nakanishi,

Shimomura, "High forskolin production in hairy rots of

Coleus forskohlll" Plant Cell Reports 17: 457-459, 1998.

Sashida, Kawashima, Mimaki, "Novel polyhydroxylated steroidal

in saponins from Allium giganteum," Chem. Pharm. Bull, 39: 698-703, 1991.

Schenk, Hilderbrandt, Medium and techniques for induction and

growth of monocotyledonous and dicotyledonous plant cell cultures," Can. J. Bot., 50: 199-204; 1972.

Schopke, Wray, Rzazewska, Hiller, "Bellissaponins BAi and BAa, acylated in saponins from Bellis perennis," Phytochemlstry, 30: 627-631, 1991.

Schreiber, Matthias, Muller, Schafmer, Nucleic Acids Res., 17: 6419, 1989.

Schuh et al., "Obligatory wounding requirement for tumorigenes is in v-jun transgenic mice," Nature, 346:756-760, 1990.

Shao, Kasai, Xu, Tanaka, "in saponins from roots of Kalopanax septemlobus. (THUNB.) KOIDZ., Ciqiu: structures of kalopanaxsaponins C, D, E and F, " CAem. Pha-nn. Bull., 37: 311-314, 1989.

Shayesteh, Lu, Kuo, Baldocchi, Godfrey, Collins, Pinkel,

Powell, Mills, Grey, Nat. Gent., 21: 99-102, 1999.

Shepard et al., J Clln. Jmmunol., 11: 117-127, 1991.

Shirazi, Liu, Trott, "Exposure to ultraviolet radiation In

increases the tolerance of mouse skin to daily X-radiation," Pad. Res., 145: 768-775, 1996.

Sieweke et al., "Mediation of wound-related rous sarcoma virus

tumorigenesis by TGF-P; Science, 248: 1656-1660, 1990.

Smith, Weathers, Cheetham, "Effects of gibberellic acid on hairy root cultures of Artemlsia annua: growth and artemisinin production," In Vitro Cell Dev. Blol., 33: 75-79; 1997.

Spady, Wollett, Dietschy, Annu. Rev. Nutr., 13:355, 1993. Steel and Torrie, In: Principals and Procedures of Statistics, 2nd Ed., McGraw-Hill, New York, p.383, 1980.

Stevenson et al., CAem. Immunol., 48:126-166, 1990.

Takema, Fujimura, Ohsu, Imokawa, "Unusual wrinkle formation after temporary skin fixation followed by UVB irradiation in hairless mouse skin." Exp. Dermatol., 5:145-149, 1996.

Tewari, Quan, 'rourke, Zeng, Beidler, Salvesan, Dixit, "Yaina/CPP32 beta, a mammalian homolog of CED-3, is a CrmA-inhibitable protease that cleaves the death substrate poly (ADP-ribose) polymerase," Cell, 81: 801, 1995.

Thompson et al., Cancer Epidemiol. Biomarker Prevent., 1: 597-602, 1992.

Thor et al., Cancer Res., 46:3118, 1986.

omas-Barbaren et al., Planta Medica., 54: 266-267 (1988).

Tori and Aono, Ann. jRept. Shionogi Res. Lab., 14: 136, 1964.

Vaickus et al., Cancer Invest., 9: 195-209, 1991. Vazquez, Quinoa, Riguera, San Martin, Darias, "Santiagoside, the first asterosaponin from an Antarctic starfish {Neosmllaster georgianus}" Tetrahedron, 48: 6739-6746, 1992.

Vlahos and Matter, FEBS Lett., 309: 242-248, 1992.

Vlahos,Matter, Hui, Brown, J. Bio. Chem., 269: 5241-5248, 1994.

Waltho, Williams, Mahato, Pal, Barna, "Structure elucidation of two triterpenoid tetrasaccharides from'androsace saxliragifolia," J. Chem. Soc., Perkin 1: 1527-1531, 1986.

Wang, He, Ling, Li, "Chemical study of Astragalus plant. .Structures of asernestioside A and B, isolated from Astragalus ernest-ii COMB. Huaxue Xuebao, 47: 583-587,

Chem. Abstr., 1989.

Wens et al., Proc. Natl. Acad. Sci., 92: 5744-5748, 1995.

White, Genes Dev., 10: 1-15, 1996. Whitman M, Kaplan D.R.. Schatthausen B, Cantley L.C. and Roberts, T.M. Nature, 315: 239-242, 1985.

Willker and Leibfritz, "Complete assignment and conformational studies of tomatine and tomatidine," Mag. Res. Chem., 30: 645-650, 1992.

Wyllie, Anticancer Res., 5: 131-136, 1985.

Wysokinska, Chmiel, "Transformed root cultures for biotechnology," Ada Blotechnol., 17: 131-159; 1997.

Yang et al., Anticancer Res., 15: 2479-2488, 1995.

Yoshikawa, Shimono, Arihara, "Antisweet substances, jujubasaponins I-III from Zizyphus jujuba,. Revised structure of ziziphin," Tetrahedron Lett., 32:7059-7062, 1991.

Yoshikawa, Suzaki, Tanaka, Arihara, Nigam, J. Wat. Prod., 60: 1269-1274 (1997).

Youn, Park, Chung, Lee, PAotodermatoi PAoto-Lmmuno-I. PAotomed., 13: 109-114, 1997.

Yukimune et al., Nature Biotech., 14: 1129-1132, 1996.

Zobel, "Study-state control and investigation of root system morphology," In: J.G. Torrey, Winship, L.J. (eds.) Applications of continuous and. steady-state methods to root biology, Kluwer, Amsterdam, 165-182, 1989.

1. Mixture for inhibiting the initiation and activation of epithelial cells mlec the supply in pre-malignant or malignant condition, stimulate apoptosis of malignant cells of a mammal, preventing abnormal proliferation of epithelial cells of a mammal, the treatment of inflammation and regulation of angiogenesis in a mammal, containing one or more selected triterpene glycosides characterized by the following properties:

a) the selectability of tissue beans or sprouts Acacia victoriae;

b) content of at least one triterpene glycoside having a molecular weight of about 1800 - 2600 Amu;

(C) the ability to induce cytotoxicity cells Jurkat and

d) the ability to induce apoptosis in Jurkat cells.

2. Mixture for inhibiting the initiation and activation of the epithelial cells of a mammal in pre-malignant or malignant condition, stimulate apoptosis of malignant cells of a mammal, preventing abnormal proliferation of epithelial cells of a mammal, the treatment of inflammation and regulation of angiogenesis in a mammal, containing one or more selected triterpene glycosides characterized by the following properties:

a) the selectability of tissue beans and seedlings of Acacia victoriae;

b) content of at least one triterpene glycoside having a molecular weight of about 1800 - 2600 Amu and

(C) the ability of the to induce the release of cytochrome C from mitochondria Jurkat cells.

3. Mixture for inhibiting the initiation and activation of the epithelial cells of a mammal in pre-malignant or malignant condition, stimulate apoptosis of malignant cells of a mammal, preventing abnormal proliferation of epithelial cells of a mammal, the treatment of inflammation and regulation of angiogenesis in a mammal, containing one or more selected triterpene glycosides characterized by the following properties: (a) the selectability of tissue beans and seedlings of Acacia victoriae; (b) content of at least one triterpene glycoside having a molecular weight of about 1800 - 2600 Amu and C) the ability to activate caspase-3 in Jurkat cell.

4. The mixture according to claim 3, where the activity specified caspase is about 0.3 to 1.6 units of fluorescence per minute on 1 mg.

5. Mixture for inhibiting the initiation and activation of the epithelial cells of a mammal in pre-malignant or malignant condition, stimulate apoptosis of malignant cells of a mammal, preventing abnormal proliferation of epithelial cells of a mammal, the treatment of inflammation and regulation of angiogenesis in a mammal, containing one or more selected triterpene glycosides characterized by the following properties: (a) the selectability of tissue beans and seedlings of Acacia victoriae; ) content, at least one triterpene glycoside having a molecular weight of about 1800 - 2600 Amu and C) the ability to induce PARP cleavage in Jurkat cell.

6. Mixture for inhibiting the initiation and activation of the epithelial cells of a mammal in pre-malignant or malignant condition, stimulate apoptosis of malignant cells of a mammal, preventing abnormal proliferation of epithelial cells of a mammal, the treatment of inflammation and regulation of angiogenesis in a mammal, containing one or more selected triterpene glycosides characterized by the following properties: (a) the selectability of tissue beans and seedlings of Acacia victoriae; (b) content of at least one triterpene glycoside having a molecular weight of about 1800 Amu - 2600 Amu; (C) the ability to inhibit the kinase activity of PI-3 in Jurkat cell.

7. Mixture for inhibiting the initiation and activation of the epithelial cells of a mammal in pre-malignant or malignant condition, stimulate apoptosis of malignant cells of a mammal, preventing abnormal proliferation of epithelial cells of a mammal, the treatment of inflammation and regulation of angiogenesis in a mammal, containing one or more selected triterpene glycosides characterized by the following properties is: (a) the selectability of tissue beans and seedlings of Acacia victoriae; b) the ability to inhibit the initiation and activation of epithelial cells of a mammal in pre-malignant or malignant condition.

8. Mixture for inhibiting the initiation and activation of the epithelial cells of a mammal in pre-malignant or malignant condition, stimulate apoptosis of malignant cells of a mammal, preventing abnormal proliferation of epithelial cells of a mammal, the treatment of inflammation and regulation of angiogenesis in a mammal, containing one or more selected triterpene glycosides characterized by the following properties: (a) the selectability of tissue beans and seedlings of Acacia victoriae; (b) the ability to induce apoptosis of malignant mammalian cells.

9. Nutraceutical composition for inhibiting the initiation and activation of the epithelial cells of a mammal in pre-malignant or malignant condition, stimulate apoptosis of malignant cells of a mammal, preventing abnormal proliferation of epithelial cells of a mammal, the treatment of inflammation and regulation of angiogenesis in a mammal containing a mixture according to any one of claims 1 to 12 in a pharmacologically acceptable medium.

10. Nutraceutical composition for inhibiting the initiation and activation of the epithelial cells of a mammal in prejlocaj the public or malignant condition, stimulate apoptosis of malignant cells of a mammal, preventing abnormal proliferation of epithelial cells of a mammal, the treatment of inflammation and regulation of angiogenesis in a mammal containing the dried and ground roots or beans Acacia victoriae in a pharmacologically acceptable medium.

11. The nutraceutical composition of claim 10, where the specified pharmacologically acceptable medium is a buffer, a solvent, a diluent, an inert carrier oil, cream or suitable for food stuff.

12. A method of obtaining a composition for inhibiting the initiation and activation of the epithelial cells of a mammal in pre-malignant or malignant condition, stimulate apoptosis of malignant cells of a mammal, preventing abnormal proliferation of epithelial cells of a mammal, the treatment of inflammation and regulation of angiogenesis in a mammal containing a mixture of one or more triterpene glycosides, including (a) obtaining tissue from the beans, roots, seedlings, plants Acacia victoriae or mixtures thereof; (b) extraction of the specified tissue solvent and (C) obtaining one or more triterpene glycosides.

13. A method of obtaining a composition selected triterpene glycosides to inhibit the initiation and activation of the epithelial cells of a mammal in preslice the public or malignant condition, stimulate apoptosis of malignant cells of a mammal, preventing abnormal proliferation of epithelial cells of a mammal, the treatment of inflammation and regulation of angiogenesis in a mammal, including:

a) preparation of tissue culture hairlike roots, including cells of the plant Acacia victoriae, which were infected with Agrobacterium rhizogenes in an appropriate culture medium, and

b) extraction of specified composition triterpene glycosides from the specified culture solvent, thereby extragere triterpene glycoside compound in which triterpene component is acacia acid, oleanolic acid or other structurally similar triterpene component having the formula:

,

where

a) R1and R2selected from the group consisting of hydrogen, C1-5-alkyl and oligosaccharide;

b) R3selected from the group consisting of hydrogen, hydroxyl, C1-5-alkyl, C1-5-alkylene,1-5-alkylsulphonyl, sugar and monoterpenoid group and

(C) the formula further includes R4where R4selected from the group consisting of hydrogen, hydroxyl, C1-5-alkyl, C1-5-alkylene,1-5-alkylsulphonyl, sugar1-5-Olkiluoto of ester is monoterpenoid group, and where R4can be attached to the triterpene component or monoterpenoid component.

14. Triterpene glycoside having a molecular weight of about 1800 - 2600 u, obtained by any method according to PP and 13.

15. Tissue culture of hairy roots, including cells of the plant Acacia victoriae, which were infected with Agrobacterium rhizogenes R1000, in the culture medium.

16. A tissue culture according to § 15, where this culture medium contains about 3% - 4% sucrose by weight.

17. Method for continuous collection of plant tissues of Acacia victoriae, comprising a) growing plants Acacia victoriae hydroponics and (b) the fee specified tissue beans and roots from the specified plants from approximately one to approximately four times per year, and the specified collection does not result in death of the specified plants.

18. The method according to 17, where this system of cultivation is Aeroponics.

19. Composition for inhibiting the initiation and activation of the epithelial cells of a mammal in pre-malignant or malignant condition, stimulate apoptosis of malignant cells of a mammal, preventing, abnormal proliferation of epithelial cells of a mammal, the treatment of inflammation and regulation of angiogenesis in a mammal containing an effective amount of purified triterpene compounds, vkluchaya what about the triterpene component, connected with monoterpenoid component, in which the triterpene component is acacia acid, oleanolic acid or other structurally similar triterpene component having the formula:

,

where

a) R1and R2selected from the group consisting of hydrogen, C1-5-alkyl and oligosaccharide;

b) R3selected from the group consisting of hydrogen, hydroxyl, C1-5-alkyl, C1-5-alkylene,1-5-alkylsulphonyl, sugar and monoterpenoid group and

(C) the formula further includes R4where R4selected from the group consisting of hydrogen, hydroxyl, C1-5-alkyl, C1-5-alkylene,1-5-alkylsulphonyl, sugar1-5-Olkiluoto of ester and monoterpenoid group, and where R4can be attached to the triterpene component or monoterpenoid component.

20. The composition according to claim 19, further comprising monoterpenoid component associated with sugar.

21. The composition according to claim 20, where R3has the formula:

R5selected from the group consisting of hydrogen, hydroxyl, C1-5-alkyl, C1-5-alkylene,1-5-alkylsulphonyl, sugar1-5-Olkiluoto of ester and monoterpenoid group.

p> 22. The composition according to claim 19, comprising a triterpene glycoside molecular formula:

where

a) R1represents oligosaccharide comprising N-acetylglucosamine, fucose and xylose, and

b) R2represents oligosaccharide comprising glucose, arabinose and rhamnose.

23. The composition according to claim 19, comprising a triterpene glycoside molecular formula:

where

a) R1represents oligosaccharide comprising N-acetylglucosamine, fucose and xylose, and

b) R2represents oligosaccharide comprising glucose, arabinose and rhamnose.

24. The composition according to claim 19, comprising a triterpene glycoside molecular formula:

where

a) R1represents oligosaccharide comprising N-acetylglucosamine, glucose, fucose and xylose, and

b) R2represents oligosaccharide comprising glucose, arabinose and rhamnose.

25. The composition according to claim 19, comprising triterpene component, oligosaccharide and three monoterpenoid link.

26. The composition according A.25, in which triterpene component is acacia acid or oleanolic acid.

27. Pharmaceutical composition for inhibiting the initiation and activation of epithelial cells is recapitalise in pre-malignant or malignant condition, stimulate apoptosis of malignant cells of a mammal, preventing abnormal proliferation of epithelial cells of a mammal, the treatment of inflammation and regulation of angiogenesis in a mammal containing composition according to any one of p-26 in a pharmaceutically acceptable medium.

28. Composition according to any one of p-26, used for the production of a drug for inhibiting the initiation and activation of the epithelial cells of a mammal in pre-malignant or malignant condition in a mammal.

29. Composition according to any one of p-26, used for the production of a drug for stimulating apoptosis of malignant cells of a mammal.

30. Composition according to any one of p-26, used to manufacture a medicinal product for preventing abnormal proliferation of epithelial cells of a mammal.

31. Composition according to any one of p-26, used for the production of a medicinal product for the treatment of inflammation in a mammal.

32. Composition according to any one of p-26, used for the production of a drug for regulating angiogenesis in a mammal.

33. Method of stimulating the release of cytochrome C from the mitochondria of mammalian cells, including the introduction of a given mammal a therapeutically effective capsules to the effective amount of the pharmaceutical composition according to item 27.

34. The method according to p, where the specified mammal is man.

35. The method according to p, where the specified cell of a mammal is a malignant cell.

36. The method according to p, where the cell is a skin cell, the cell colon cell cancer, cell, ovarian cell, a pancreatic cancer, a prostate cancer cell, a kidney cell, a cell of the lung, cell bladder epithelial cell or a cell of the mammary gland.

37. The method according to p, where specified, the introduction is carried out orally.

38. The method according to p, where specified, the introduction is carried out locally.

39. The method according to p, where specified, the introduction is carried out by intratumoral injection.

40. The method according to p, where specified, the introduction is carried out intravenously.

41. The method according to p, where specified, the introduction is carried out by inhalation of the aerosol.

42. The method according to p, optionally including the specified irradiation of epithelial cells.

43. The method according to § 42, where the specified cell is irradiated with x-rays, UV rays, γ-radiation, or microwave radiation.



 

Same patents:

FIELD: biology, genetic engineering.

SUBSTANCE: invention relates to preparing immortalized cellular lines from health human skin tissues and can be used in immunological, pharmacological, photo- and chemical-toxicological analysis of cutaneous response, for expression of heterologous genes and for construction of artificial skin. Keratinocytes are immortalized by infection of keratinocytes of health human. The human skin sample is isolated and prepared its for culturing in vitro. Keratinocytes are prepared from this prepared human skin sample and plated in serum-free medium for growing keratinocytes in cultural plates with cover alleviating attachment and growth of cells. In the process for culturing keratinocytes the serum-free medium is replaced to provide preparing the optimal confluent growth of cells in culture with continuous maintenance of cup cover. Keratinocytes are transferred in selective serum-free medium in cultural cups with cover and infected with vectors pLXSHD + SV40(#328) and pLXSHD + E6/E7. Then prepared immortalized keratinocytes are transferred in cultural cups with cover to useful medium for proliferation. Then prepared proliferated keratinocytes are transferred in medium with high calcium content for differentiation in cultural chambers with cover. Invention provides preparing the human keratinocyte cellular line that has no oncogenic property and retains capacity for differentiation and expression of proteins and enzymes expressing by normal differentiated keratinocytes being even after increased number of passages in culture. Also, this cellular line forms lamellar and polarized epithelium with keratinized layer (stratum corneum) consisting of ortho-keratinocytes in the process for culturing in organotypical culture in serum-free medium and without layer of feeding cells.

EFFECT: improved immortalizing method, valuable biological properties of cellular line.

7 cl, 2 dwg, 4 ex

The invention relates to biotechnology and Cryobiology

The invention relates to medicine, namely to cell therapy, and for the culture of cells containing precursor cells osteogenesis of the implant based on it and use it to restore the integrity of the bone
The invention relates to ophthalmology and is used for correction of degenerative changes of the retina by pathology of various origins

The invention relates to biotechnology and can be used in medical, cosmetic and food industry

The invention relates to biotechnology and medicine, specifically to the media for cryopreservation of human and animal cells

The invention relates to biotechnology and medicine, specifically to the media for cryopreservation of human and animal cells

FIELD: beauty products manufacturing.

SUBSTANCE: the present innovation deals with manufacturing cosmetic composition that contains solubilized cumic alcohol of a certain formula, glucose, ascorbic acid or compound which is split in skin up to glucose or ascorbic acid and a cosmetically acceptable carrier. Components should be taken at a certain quantitative ratio. The suggested composition improves expression of transglutaminase-1 and ceramides in skin cells and enhances absorption of glucose and ascorbic acid by cells.

EFFECT: higher efficiency.

1 cl, 5 ex, 12 tbl

FIELD: organic chemistry, chemical technology, agriculture.

SUBSTANCE: invention relates to a method for preparing the preparation comprising triterpenic acid water-soluble salts and additionally added protein-containing product and vegetable raw, the source of triterpenic acids taken in the following ratio of components, wt.-%: protein-containing product, 10-17; triterpenic acid sodium salts, 4-5, and vegetable raw, the balance. Method involves mixing triterpenic acid-containing vegetable raw with the protein-containing product taken in the ratio = (9-11):(1-2), mechanical-chemical treatment of this mixture in activator device, mixing of prepared semi-finished product with sodium carbonate or sodium hydrocarbonate taken in the ratio = (92-97):(3.5-8.3) and repeated treatment in the activator device. Method involves applying flow-type ball vibration-centrifugal or ellipse-centrifugal mills as the activator device that provide the acceleration of milling bodies up to 170-250 m/c2 and time for treatment for 1.5-3 min. Invention provides simplifying the process and the complex processing waste in lumber industry.

EFFECT: improved preparing method.

6 cl, 1 tbl, 6 ex

FIELD: medicine, hematology.

SUBSTANCE: invention relates to applying elm-leaf dropwort (Filipendula) above-ground part 40% alcoholic tincture as an agent stimulating erythroidal and granulocytic hemopoiesis stems in cytostatic myelorepressions. Agent promotes to effective stimulation of erythroidal and granulocytic hemopoiesis stems in cytostatic myelodepression. Invention is used for correction of disorders arising in blood system in administration of cytostatic preparations.

EFFECT: valuable medicinal properties of agent.

3 tbl

FIELD: medicine, cardiology, phytotherapy, pharmacy.

SUBSTANCE: invention relates to the field in development of an agent of vegetable origin used for improvement of the functional state and prophylaxis of organic alterations in cardiovascular system, in particular, atherosclerosis. The biologically active supplement used for prophylaxis of cardiovascular diseases, in particular, atherosclerosis comprises clover dry extract, haw-thorn flowers powder, ascorbic and nicotinic acids, and accessory substances taken in the definite ratio. The biologically active supplement is made in the form of tablet or capsule. No adverse responses observed in intake of the supplement. The blood cholesterol level was reduced to the normal value in 68% of patients and reducing morbidity with acute respiratory-viral infections has been noticed. In patients with ischemic heart disease with cholesterolemia the improvement of the state is noted on the background of conventional therapy: the moderate reducing the arterial pressure value in its elevation, improved in the state of health, enhanced working capacity, reduced retrosternal pains, ear noise, vertigo, headache.

EFFECT: valuable medicinal properties of supplement.

1 ex

The hemostatic tool // 2242987
The invention relates to the field of medical pharmacology, namely organic geostatical used to stop the capillary and mixed hemorrhage in traumatic lesions of the skin and muscle tissue in surgical practice
The invention relates to the field of generating plant to normalize cholesterol metabolism, prevention and treatment of atherosclerosis

The invention relates to animal husbandry, and in particular to means of stimulating the reproductive function and the natural resistance of animals, for example pigs

The invention relates to the creation of funds for treatment and prevention of obesity

The invention relates to the field of food industry, in particular confectionery industries

The invention relates to medicine, namely to the field of physiotherapy and can be used for treatment of nervous system diseases of the spine

The invention relates to new S-4 carbonate-bearing similarly to taxanes of formula 1 and their pharmaceutical salts:

where R denotes phenyl, isopropyl or tert.butyl; R1denotes-C(O)RZin which RZmeans (CH3)3CO-, (CH3)3CLO2-, CH3(CH2)3Oh, cyclobutyl, cyclohexyloxy or 2-furyl and R2denotes CH3C(O)O-, and also to pharmaceutical compositions based on them and their use as protophobic agents to treat diseases of humans and animals

Up!