Polysaccharide eliciting immunostimulating activity, method for its preparing and using

FIELD: organic chemistry, polysaccharides.

SUBSTANCE: invention describes polysaccharide of the formula (I):

wherein n = 7-8; Araf means arabinofuranose; Galp means galactopyranose, and Rhap means rhamnopyranose and designated for its using as a medicinal agent in treatment of immunosuppressive diseases. Also, invention relates to a method for preparing this polysaccharide that involves gathering flowers of plant Calendula officinalis (pot-marigold), their treatment by extraction method involving cleansing flowers, milling flowers, treatment of milled flowers by laser radiation, suspending the prepared mixture in water, maceration of suspension and separating the prepared liquid. Then method involves treatment of prepared liquid by the isolating method including precipitation with methanol, centrifugation and chromatography separation under the bioanalysis control.

EFFECT: improved preparing method.

16 cl, 26 dwg, 2 tbl, 4 ex

The technical field to which the invention relates.

The present invention relates to a polysaccharide of formula I, and detecting immunostimulirutuyu activity, the method of its production, to its use in the treatment of immunosuppressive diseases and to its containing pharmaceutical compositions.

The level of technology

The immune system can be defined as a collection of molecules, cells and organs, whose complex interactions form an emergent system, usually capable of protecting the individual from external attacks, and from their own modified cells.

The immune system can be divided into two functionally different parts: part, the elements of which are innate, and part of whose elements are acquired. Innate immunity attributed to immune elements that are non-specific and non-adaptive. They are able to distinguish between foreign tissues/organisms, but is not able to recognize certain pocahotas. Acquired immunity is attributed to a group of elements that are specific and adaptive. They are able to distinguish foreign cells from their own and can distinguish one foreign antigen from another. Acquired immunity has memory. This makes it possible to immunization and resistance to re-infection with the same microorganism. The cells responsible C the immunity, are lymphocytes, in which there are two sub-classes - In - and T-cells.

Acquired immunity can be achieved either by natural infection or vaccination (active through), or by injection of immunocytes (passive way). While the first path is detected by the prolonged action, which may even become permanent, the last step is not long.

Patients with immunosuppressive diseases treated with Immunostimulants in order to activate their immune system. Various Immunostimulants known from US 4801578, US 5417979, WO 9851319. Immunostimulants on the basis of polysaccharides known from DE 18917177 and EP 0225496. In these patents describes polysaccharides with immunostimulatory activity and receive them from cultures of plant cells. As plants use Echinacea purpurea, Echinacea angustifolia and Calendula officinalis.

However, none of these stimulants does not find a satisfactory activity in the case of immunosuppressive diseases. Thus, there remains a need in other Immunostimulants, improved detecting activity in a wide range.

The invention

The present invention relates to a polysaccharide of formula I, and detecting immunostimulirutuyu activity, the method of its production, its use in the treatment of immunosuppress the situations diseases and to its containing pharmaceutical compositions.

In one aspect the invention relates to polysaccharides of the formula I:

Scheme I

where n=7-8.

In the second aspect of the invention relates to a method of producing polysaccharide of formula I. this method comprises, first, obtaining an aqueous extract of the plant Calendula officinalis according to the method described in the process of simultaneous consideration of the request of the authors of the present invention, and, secondly, the selection of the polysaccharide using a combination of separation methods, directed by bioanalysis.

In another aspect the invention relates to the use of polysaccharide of formula I of the present invention as a therapeutic agent in the treatment of immunosuppressive diseases, such as cancer, tuberculosis, influenza, the common cold, allergies, lupus, psoriasis and AIDS.

In another aspect the invention relates to pharmaceutical compositions containing a polysaccharide of formula I.

This application includes presents figures and tables.

Figure 1 represents a scheme of allocation PF2.

Figure 2 is a circuit isolation PF2R.

Figure 3 represents a range of¹H-NMR PF2RS8A.

Figure 4 represents a range of¹³C-NMR PF2RS8A.

Figure 5 is a TLC of the products of hydrolysis PF2RS8A TPA and other monosaccharides.

Figure 6 submitted is a selection scheme PF2RS8B2, i.e. polysaccharide of formula I.

Figure 7 is a HPLC profile PF2RS8B2 with evaporative light scattering detector.

Figure 8 is an HPLC profile PF2RS8B2 with UV photodiode detector.

Figure 9 represents a range of¹H-NMR PF2RS8B2.

Table 1 reflects the effect of samples PF2RS8A and PF2RS8B2 on the transformation of lymphocytes.

Figure 10 represents a range of¹³C-NMR PF2RS8B2, i.e. polysaccharide of formula I.

Figure 11 represents a range of DEPT-135 PF2RS8B2.

Figure 12 is a spectrum DEPT-90 PF2RS8B2.

Figure 13 is a range HMQC PF2RS8B2.

Figure 14 represents a range of¹H¹H-COSY PF2RS8B2.

Figure 15 is a range of¹H¹H-COSY PF2RS8B2.

Figure 16 represents a range HMQC PF2RS8B2.

Table 2 reflects the range of¹³C-NMR PF2RS8B2.

Figure 17 is a range NMS PF2RS8B2.

Figure 18 is a range NMS PF2RS8B2.

Figure 19 is a TLC of the products of hydrolysis PF2RS8B2 TPA and other monosaccharides.

Figure 20 is a graph of measurement of molecular weight.

Detailed description of the invention

As mentioned above, in the first aspect of the invention relates to a polysaccharide of formula I:

Scheme I

where n=7-8.

The configuration structure of the compounds of formula I are reflected in the W scheme II:

Scheme II

where n=7-8.

The polysaccharide has a molecular weight of about 10,000.

In the second aspect of the invention relates to a method of producing polysaccharide of formula I. This method comprises, first, obtaining an aqueous extract of the plant Calendula officinalis according to the method described in the process of simultaneous consideration of the request of the authors of the present invention "Method of production of aqueous plant extracts and the extracts obtained in this way", and registered simultaneously with the present application, and, secondly, the selection of the polysaccharide using a combination of separation methods, directed by bioanalysis.

Aqueous extract of Calendula officinalis get, putting the flowers of the specified plants described later process.

a) Purification of flowers.

b) Grinding the flowers.

c) Processing the crushed flowers laser radiation.

d) the Suspension in the water of the mixture obtained in stage c).

e) Maceration of the suspension obtained in stage d).

f) Separating the resulting liquid.

Clearance (stage a) is carried out, washing the flowers of Calendula officinalis water. The amount of water used at this stage, is not defined and may vary depending on the degree of contamination of plants. Although higher and lower temperatures are not excluded, the temperature in the s must be between 10 and 40° C, preferably between 20 and 35°and most preferably -28°C. you Can use the washing chamber to facilitate this stage. As the amount of water and the residence time of plant materials in the washing chamber is not defined and can therefore vary depending on the degree of contamination of plants. Stage leaching can be performed several times with the stage of drying in between. Such a stage of drying preferably takes place by placing plant material in the sun.

As soon as the flowers are thoroughly cleaned, crushed (stage b) in the usual ways, such as with the use of the machine for grinding or even manually. Although higher and lower temperatures are not excluded, the temperature at which the flowers are crushed, should be from 10 to 40°C.

Then the crushed flowers processed by laser radiation (stage c). As the laser light source is used preferably red line laser diode capable of generating radiation at a wavelength in the range 150-810 nm. Preferably the wavelength of the laser radiation is 200-400 nm, and most preferably 250 nm. The power of the laser radiation is preferably 1-60 wt, preferably 10-30 wt and most preferably 20 watts. The spot has a diameter of preferably 1 to 6 mm, pre is reverent - 2-5 nm, and most preferably 4 mm crushed plant material exposed to laser radiation, so that was exposed to the mixture or her most. This reach or manually moving the laser generator in the plant raw materials are crushed or missing material on a belt conveyor through a set of multiple laser generators. Preferably each kilogram of crushed material is treated with laser radiation within 3-10 minutes, preferably within 5 minutes, Although higher and lower temperatures are not excluded, the temperature at which the crushed plant material is treated with laser radiation should be from 10 to 40°C.

Then processed with laser radiation with the material suspended in water (stage d). At this stage you can use any commercial mineral water. The suspension carried out in such a way as to present 50-300, preferably 100-250 g of the treated material in 1 liter of water. Although higher and lower temperatures are not excluded, the temperature at which the suspended powdered plant material should be from 10 to 40°C.

Then the suspension was incubated for 5-25 days, preferably 7-15 days at a temperature of from 2 to 10°C, preferably from 4 to 8°so that what s happened maceration (stage e).

Finally, after the stage of maceration carry out the separation of the liquid phase from the solid phase (stage f). Separation can be achieved one by decantation or preferably by decantation followed by filtration. The filtering is preferably carried out under pressure. It is most preferable to carry out three consecutive filtration under pressure through a filter of 5 μm, 1 μm and 0.22 μm. Although higher and lower temperatures are not excluded, the temperature at which the branch should be from 10 to 40°C.

Finally, get a water extract brownish-yellow color.

Then the extract obtained is subjected to the process of selection, which includes precipitation with methanol, centrifugation and chromatographic separation, napravlenie the bioanalysis. Applied biennales carried out in vitro by adding samples of lymphocytes isolated from the blood of mice, according to the method described in the literature: Max W. et al., Journal of Natural Products, vol.54, no.6, pp.1531-1542 (1991). Control the inclusion of thymidine, which means the replication of DNA. This inclusion reflects the increase in the number of lymphocytes, and increase the activity of lymphocytes. These used selection methods include repeated ethanol precipitation, centrifugation, dialysis and/or column chromatography.

Thus, lyophilized 112 l received the CSO pre-aqueous extract and obtain 800 g of a yellowish-brown powder ("PF2"). The resulting powder was fractionary on the fraction soluble in Meon, and the residue, insoluble in MeOH (PF2R, 350 g), which detects the activity of transformation of cells. Part (80 g) of such material is subjected to deposition of MeOH, centrifugation, dialysis and/or column chromatography on Sephadex DEAE that leads to the selection of multiple crystalline substances, which are defined as inactive inorganic salts (figure 1).

Part (270 g) PF2R, which is subjected to the deposition of MeOH to a final concentration of 25, 50 and 67%, leads to active precipitates. The most active material is the precipitate obtained from a 50% solution of MeOH (PF2RS8, of 51.2 g, LT=+1059%). The processing part 5 g PF2RS8 by dissolution in water followed by centrifugation and chromatographic separation of the supernatant on Sephadex G-25 leads to active polysaccharide-enriched fraction, called PF2RS8A (0,13 g, LT=+1074%). Then from the second part (6 g) precipitate 50% MeOH get PF2RS8A' (0.3 g). As shown in figure 2 identified a number of other factions and crystalline substances, but they do not show the level of activity PF2RS8A.

PF2RS8A characterize spectroscopy (spectra¹H,¹³C-NMR and DEPT (undistorted enhancement by polarization transfer (Distortionless Enhancement by Polarization Transfer)and chemical analysis (hydrolysis with TFA and analyzed by TLC on silica gel). Cm. figures 3, 4 and .

More active mixture of polysaccharides PF2RS8A (1.4 g) was isolated from the rest of the precipitate 50% MeOH (PF2RS8). At the same time, the processing portion (2.0 g) insoluble fraction 67% solution of MeOH (PF2RS9A; LT=+735%) leads to the separation of a mixture of polysaccharides (PF2RS9A, 0.3 g), which analysis method¹H-NMR identify as PF2RS8A. So PF2RS9A (0.2 g) are combined with PF2RS8A (1.4 g)and the mixture is called "PF2RS8B", is subjected to further separation on Sephadex G-50 (20-80 mm), as shown in figure 6. Elution with water to give six fractions (PF2RS8B 1, 2, 3, 4, 5 and 6), analysis by HPLC which shows that only the primary isolate PF2RS8B2 is homogeneous for detection as with evaporative light scattering detector and a UV detector (figures 7 and 8). Spectral analysis methods¹The h and¹³C-NMR (figures 9 and 10) gives the spectra of the specified protein isolate, which is very similar to the spectra obtained for PF2RS8A (figures 2 and 4), suggesting that it is the main polysaccharide in the mixture. Therefore, the fraction PF2RS8B2 consists of a polysaccharide of formula I and water. The latter can be removed by methods known in the art.

The bioanalysis of homogeneous isolate PF2RS8B2 and the source for it mixture PF2RS8A when the same analysis shows the activity of transformation of leukocytes (LT) 6203% 3532%, respectively (table 1). A higher level of activity, shown in isolation, suggests that this polishuri which is the main active component of the biological effect of the extract of the present invention.

Active polysaccharide PF2RS8B2 (polysaccharide of formula (I) characterized by spectroscopy (spectra¹H,¹³C-NMR, HMQC (heteronuclear multiple quantum coherence Heteronuclear Multiple Quantum Coherence)and 2D¹N¹H COSY (two-dimensional proton-proton correlation spectroscopy nuclear magnetic resonance (2-Dimensional proton-proton NMR correlation spectroscopy)and chemical analysis (hydrolysis with TFA and analyzed by TLC on silica gel). Thus, the signals¹H-NMR (figure 9) δ 5,3 and 3.2 ppm indicate the polysaccharide. Signals¹³C-NMR (figures 10-12) at δ 111,9 (d), 110,1 (d) ppm are attributed to the anomeric carbons 1→3, associated with α-L-arabinofuranose & end α-L-arabinofuranose (indicated by Araf and Araf^', respectively). Signals δ 106,1 (d) and 105,9 (d) are attributed to the anomeric carbons 1→6 associated with β-D-galactopyranose, and 1→3, 1→6 associated with β-D-galactopyranose (indicated by Galp' and Galp, respectively), while the signal at δ 100,2 (d) ppm is attributed to the anomeric carbon α-L-rhamnopyranose (indicated by Rhap). The signals of the anomeric proton (H-1) is also easily recognized because of their relatively low field shift in the spectra of¹H-NMR. A direct correlation between the signals of the proton and carbon-13 observed in the HMQC spectrum (figure 13), determines the signals¹H-NMR δ 5,08 (Ushs), 5,3 (Ushs), 4,47 (d, J=7.9 Hz), a 4.53 (d, J=7,3 Hz) and 5.1 (Ushs) ppm corresponding to the anomeric protons α-L-arabinofuranose (Araf^'), α-L-arabinofuranose (Araf), β-D-galactopyranose (Galp') and α-L-rhamnopyranose (Rhap). Using these signals as standard, you can find other proton signals, analyzing the spectra 2D¹N¹H COSY (figure 14, 15). In this way HMQC spectra determine the appropriate signals of carbon (figures 13, 16 and table 2). The sequence of sugars is determined as follows. Low field shift of the signal at C-3 Araf (δ 79,4) and Galp (δ 82,8) and signals-6 Galp Galp and' (δ 69,2) suggest that the carbon associated with other carbohydrate chains. The observation of a broad correlation between C-1 Araf and C-6 Galp Galp and' in the spectrum NMS (heteronuclear multiple correlation relations (Heteronuclear Multiple Bond Correlation)) (figure 17) implies 1→6, associated with the main chain β-D-galactopyranose. From observations of the broad correlation between C-1 Araf and C-3 Galp (figure 18) Araf is 1→3 associated with Galp. From the spectrum¹H-NMR (figure 9) calculate the ratio of sugars according to the integration values of the peaks in the anomeric protons, corresponding Araf:Araf':Galp:Galp':Rhap / 3:1:2:2:moemomu PF2RS8B2 considered as a polysaccharide with large side chains with the primary structure shown by the formula I (scheme I). Scheme II is the same is the structure, showing stereochemical configuration of the individual sugars.

To confirm the identity of its constituent sugars, PF2RS8B2 hydrolyzing with TFA (0.5 M, 100-120°C), thus making the analysis by TLC (figure 19). The presence of sugars α-L-arabinofuranose and β-D-galactopyranose easy to install, while the presence of additional sugar α-L-rhamnopyranose determined harder, probably because of the amount of the hydrolyzate applied to the plate for TLC.

The molecular weight of PF2RS8B2 evaluated by the method of gel (size) chromatography. The average molecular weight PF2RS8B2 estimated therefore is 10000 (see figure 20).

The polysaccharide of formula I suddenly finds very high activity as an immunostimulant, which is reflected in the examples below. The third aspect of the invention thus relates to the use of polysaccharide of formula I in the treatment of immunosuppressive diseases, such as cancer, tuberculosis, influenza, the common cold, allergies, lupus, psoriasis and AIDS. Non-limiting examples of cancer is liver cancer, lung cancer, kidney cancer, colon cancer, breast cancer, prostate cancer or adenocarcinoma of the prostate; cancer of the brain, such as astrocytoma and glioblastoma is mA; cervical cancer and bladder cancer.

A fourth aspect of the invention relates to pharmaceutical compositions containing a polysaccharide of formula I.

The polysaccharide of the present invention can be entered or separately in the form of a pure substance or in the form of pharmaceutical preparations, although preferably the compound of the invention is administered in combination form. The combination medicinal product preferably has the form of a composition which (1) contains one polysaccharide according to the invention; (2) contains one or more corresponding binders, carriers and/or excipients, and (3) may also contain additional therapeutically active substances.

Carriers, binders and/or auxiliary substances should be pharmacologically portable so that they can be combined with other components of the composition or preparation, and not to have a harmful effect on the body that is treated.

Such compositions include compositions suitable for oral and parenteral (including subcutaneous, intradermal, intramuscular and intravenous) administration, although the best method of administration depends on the condition of the patient.

The composition can be in the form of single doses. The compositions have according to methods known in the field of pharmacology. Soo the relevant number of active substances suitable for injection, can change as a function of specific areas of therapy. In General, the concentration of the active substance in the composition in the form of a single dose ranges from 5% to 95% of the entire composition.

The application of the invention is illustrated below by examples.

Example 1. Obtaining an aqueous extract of the flowers of Calendula oficinalis according to the method of the invention.

The flowers of Calendula oficinalis (500 g) was placed in the washing chamber and is subjected to washing with water at about 28°C. Then the flowers are crushed using a machine for grinding. Received 500 g of crushed material is subjected to processing by the radiation of the red line laser diode, capable of emitting radiation with a wavelength of 250 nm, a power of 20 W and a spot diameter of 4 mm Treatment is carried out manually by moving the laser generator according to the shredded material within 2.5 min, so that the entire mixture is irradiated or her most. The material is then processed by the laser, are suspended in 2 l of water at a temperature of about 20°C. Then the suspension was incubated for 12 days at a temperature of 4°C. Finally, carry out the separation of liquid and solid phases, first by decantation of the liquid (solid pressed to facilitate separation) and then three consecutive filtration under pressure through the filters 5, 1, and 0.22 μm at a temperature of about 20°C. the manual gives approximately 1.7 l of solution (aqueous extract) brownish-yellow color.

Example 2. The selection of the polysaccharide of formula I is carried out as described previously in the description.

Example 3. The polysaccharide of formula I have to determine its activity as an immunostimulant by quantitative evaluation of the activity of transformation of cells (LTA). Under the activity of transformation of cells refers to the fact that lymphocytes are transformed from the passive to the active state, which is necessary to combat disease through an immunological mechanism, or to restore the immune system, which could weaken various factors. Tests are carried out according to the literature method (Max W. et al., Journal of Natural Products, vol.54, no.6, pp.1531-1542 (1991)), adding an in vitro solution of the polysaccharide of the invention to the lymphocytes are isolated from the body of the mouse. Control the inclusion of thymidine, which means the replication of DNA. This inclusion reflects the increase in the number of lymphocytes, and increase the activity of lymphocytes.

The polysaccharide of formula I gives LTA +6203% relative to unstimulated lymphocytes.

Example 4. The test of the polysaccharide according to the invention toxicity

Materials and methods

The test Protocol security in vivo

For testing biological safety, animals were divided into three groups of 10 animals each. Animals were injected orally, by nasogastric what about the probe with varying doses of the polysaccharide of the present invention (EU) every 48 h for 30 days. After this period, the remaining animals were kept for 1 month to assess long-term toxicity after administration of the test compounds.

Laboratory animals

Mouse

The experiments were conducted in three strains of mice BALB/c, CBA and C57/BL6. Animals of each strain were divided into 5 groups of 10 mice each, and after the introduction of each of the groups were defined as follows:

Controls: injected with 200 µl of water

EU: injected dose of the EU 2750 mg/kg of body weight in a volume of 200 ál (5x solution)

EU-In: each animal was administered a dose of the EU 550 mg/kg in a volume of 200 ál (1x solution)

EU: each animal was administered a dose of the EU 55 mg/kg of body weight in a volume of 200 ál (0,1x solution)

EC-D: each animal was administered a dose of the EU 11 mg/kg of body weight in a volume of 200 ál (0,h solution).

Rats

Animals were divided into 4 groups:

Control: introduced 1 ml of water

EU: injected dose of the EU 2750 mg/kg of body weight in a volume of 1 ml (5x solution)

EU-In: each animal was administered a dose of the EU 550 mg/kg in a volume of 1 ml (1x solution)

EU: each animal was administered a dose of the EU 55 mg/kg of body weight in a volume of 1 ml (0,1x solution).

The test on the model of lymphocyte proliferation in human peripheral blood in vitro.

To establish the effect on the peripheral blood lymphocytes (PBL) of the person built the dependence of dose-response.

Lymphocytes were isolated from whole peripheral Kravis using density gradient Ficoll Isopaque (Histopaque 1077, Sigma Diagnostics). The blood was diluted with PBS (phosphate buffered physiological saline solution) and added to a test tube with 3 ml of Ficoll. The tube was centrifuged for 30 min at 400 g at room temperature. After centrifugation opaque interphase between Ficoll and plasma containing mononuclear cells was aspirated using a Pasteur pipette (Fig. 21).

LPK were treated for 72 h in different concentrations of EU (from 2 mg to 15 mg per ml) or other currently available agents with potent immunomodulatory effects in vitro, but also showing a strong toxicity in vivo such as concanavalin a and phytohemagglutinin (PHA) in concentrations ranging from 10 mg to 1.25 μg per 1 ml

For this analysis used the kit for colorimetric measurement of cell proliferation (Anti-Brdu colorimetric kit. Cell Proliferation ELISA (Roche Diagnostics)), which is an alternative to the analysis of the incorporation of radioactive [³H]-thymidine into newly synthesized DNA.

Cells were cultured in 96-well-plate at 37°C for 48 h with density 5×10⁴cells per well. The experiment was performed in at least triplicate for each hole. As the culture medium used RPMI 1640 (Sigma).

Then to each well was made on 15 μl solution of 5-bromo-2-dose irradiation on neurogenesis (BrdU) and left in the incubator is an additional 24 hours

After 72 h from the start of incubation, the cells were centrifuged at 300 g for 10 min, fixed by heating (60°C for 1 h, and DNA was denaturiruet add Fix Denat (200 ál per well). The denaturation of DNA needed to improve binding of the antibody against BrdU.

In each well was added 100 μl of a solution of AntiBrdU antibodies and left incubated for 90 min at 15-25°C. When this occurs, the binding of the antibody antiBrdU with BrdU, involved in the composition of DNA.

- Formed immune complexes detected after adding 100 μl of substrate solution to each well. As a stopping solution using 1 M sulphuric acid in the amount of 25 μl per well.

- Quantitative evaluation of the reaction is carried out with the use of advance spectrophotometer (tablet reader for ELISA, mounted on a wavelength of 450 nm). The intensity of staining (i.e. optical density) correlates with the amount of synthesized DNA, i.e. cell proliferation in microculture.

The test on the model of cytotoxicity of natural killer cells (NK)

To assess the induction of the cytotoxic activity of NK cells in mice of BALB/C daily for 7 days was administered orally daily dose of the EU in the range of 1.1-0.22 mg per animal.

Cells from the spleen of these animals were used as effector cells, and cell lines lymphoma YAC-ispolzovali as target cells. Quantification was performed using the standard method of analysis of NK cytotoxicity using⁵¹Cr. As a positive control was used connection Celoron, which is known as an inducer of cytotoxic activity of NK cells. He was introduced as the EU.

Test results

Safety testing in vivo in mice

In tests on biological safety in mice BALB/C (Fig) neither the control group nor in the groups treated with the EU in the dose of 55 mg/kg of body weight (0,1x concentration) and 11 mg/kg of body weight (0,h concentration), within 30 days of the study and during the next 30 days follow-up after the end of the study among the animals death were noted. At a dose of 550 mg/kg (1x concentration) 15 days after the start of the study 50% of the animals died (LD₅₀), while the other 50% remained alive. At the dose of 2750 mg/kg (5x concentration) after 20 days from the start of the study 80% of the animals died, while the remaining 20% remained alive until the end of the study. This experiment was repeated three times and the results were in all cases similar that indicates the representativeness of the analysis.

In the analysis of toxicity in mice CBA (Fig) in the control group and the groups treated with the EU at a dose of 55 mg/kg of body weight (0,1x concentration) and at a dose of 11 mg/kg weight of the (0,h concentration), mortality was not observed after 30 days of the study. On the contrary, in the group treated with 550 mg/kg of body weight, on the 20th day was observed in 50% mortality, whereas the remaining 50% of mice remained alive until the end of the study.

The highest mortality was observed in the group treated with the EU at a dose of 2750 mg/kg (5x concentration), in which 7 mice were killed 20 days, and the other 3 remained alive until the end of the study.

In groups of mice S/B16 that oral was given water (control), and the EU at a dose of 55 mg/kg of body weight (0,1x concentration) or 11 mg/kg of body weight (0,h concentration), mortality was not observed.

On the contrary, in the group of mice S/B16, which has introduced a number of EU, equivalent to 550 mg/kg (1x concentration)was observed the death of 60% of the animals to 20 days, whereas the rest of the animals remained alive until the end of the study.

The highest mortality was observed in the group treated with the EU at a dose of 2750 mg/kg (5x concentration). As can be seen from Fig, mortality in this group was 80% to 20 days, while the remaining 20% of the animals remained alive until the end of the study.

Safety tests in vivo on rats

In safety tests on rats Wistar mortality was not observed in the control group and the groups treated with the EU at a dose of 55 mg/kg of body weight (0,1x concentration) and 550 mg/kg (1x concentration). However, in the group of rats receiving EU dose mg/kg (5x concentration), was marked by the death of 6 animals (60% of the whole group) to 15 days (LD₅₀), while the remaining 4 animals remained alive until the end of the study (Fig). This experiment was repeated three times and the results were in all cases similar that indicates the representativeness of the analysis.

As you can see, the results of studies of biological safety were very similar in the case of three different strains of mice (BALB/C, CBA, C57/BL6), was not observed any significant differences, and for all three lines, the 50% lethal dose (LD₅₀) was the same and amounted to 55 mg/kg (1x concentration). Therefore, the dose corresponding to 0,1 and 0,h concentrations can be safely used in the subsequent experiments, because they are non-toxic.

In contrast, in experiments on acute toxicity in rats 50% lethal dose (LD₅₀) was 2750 mg/kg (5x concentration), instead of 550 mg/kg (1x concentration)at which mortality was not revealed.

Thus, it is possible to conclude that any significant differences in lethal dose between lines of the same species of laboratory animals (mice) were observed, whereas between different species of animals observed significant differences in the 50% lethal doses, with higher lethal doses were observed in rats.

Should the tmetal, all mice and rats that survived during acute toxicity study within 30 days (the introduction of the EU through the day), watched by an additional 30 days to assess the long-term effects after administration of the compounds. Death or any physical changes during this time period were noted.

On dead animals performed the autopsy, and as shown by a preliminary conclusion, the most affected organ was the liver (necrosis).

These experiments on the acute toxicity of the polysaccharide of the invention were conducted in mice and rats in at least triplicate to obtain very close results. Presents data obtained in one experiment.

The study of the cytotoxicity of natural killer cells

In Fig. 26 shows the lysis of cells YAC-1 (in percentage terms), as measured by release of⁵¹Cr, when used as effector cells in the spleen of mice BALB/c mice, which were administered orally for 7 days EU at a dose of 55 mg/kg body weight, or 11 mg/kg of body weight.

As you can see, the lysis of cells (in percent) does not exceed 10%, which allows to conclude that the investigational compound does not induce cytotoxicity of natural killer cells.

These experiments were conducted in triplicate with obtaining very b is izkuyu results. Presents the data in one of the experiments (see table 1, 2).

The concentration of connections	Sample
Without Con A	Without Con A
PF2RS8A	PF2RS8B2
	%		%		%		%
0	165±31	0	197±51	0
0.01	311±41	+91	205±49	+24
0.1	371±51	+127	361±76	+83
1	556±26	+237	649±48	+229
10	3829±380	+2220	2348±305	+1092
100	5993±1760	+3532	12417±3153	+6203
Kohn And 8 µg/ml	1762±349		1507±250
The influence of sample PF2RS8A and PF2RS8B2 on the transformation of lymphocytes (female mouse SMC).
Table 1.

	C-1	C-2	C-3	C-4	C-5	S-6
Araf-(1→	110.1 d	85.0 d	74.5 d	86.7 d	63.2 t	-
		84.7 d
→3)-Araf-(1→	111.9 d	84.0 d	79.4 d	86.5 d	63.9 t	-
				63.7 t
		83.7 d			64.8 t
→6)-Galp-(1→	106.1 d	72.9 d	75.3 d	72.2 d	76.4 d	69.2 t
→3)	105.9 d	73.5 d	82.8 d	73.5 d	76.1 d	69.2 t
	105.8 d
→6)
Rhap-(1→	100.2 d	72.2 d	73.5 d	72.5 d	71.1 d	19.2
Data13C-NMR PF2RS8B-2 (125 MHz, D2O, relative to the DSS)
Table 2.

1. The polysaccharide of formula I

where n=7-8 and where Araf means arabinofuranose, Galp means galactopyranose and Rhap means rhamnopyranose.

2. The polysaccharide according to claim 1 for use as lekarstvennoj the funds for the treatment of immunosuppressive diseases.

3. The polysaccharide according to claim 1 for use in the treatment of immunosuppressive diseases.

4. The polysaccharide according to claim 1 for use in the treatment of immunosuppressive diseases, such as cancer, tuberculosis, influenza, the common cold, allergies, lupus, psoriasis and AIDS.

5. The polysaccharide according to claim 1 for use in the treatment of liver cancer, lung cancer, kidney cancer, colon cancer, breast cancer, prostate cancer or adenocarcinoma of the prostate; cancer of the brain, such as astrocytoma and glioblastoma; cancer of the cervix and cancer of the urinary bladder.

6. A method of obtaining a compound according to claim 1, including 1) the Department of owers of the plant Calendula officinalis, 2) processing them according to the method of extraction, including

a) purification of flowers;

b) grinding the flowers;

c) processing the crushed flowers laser radiation;

d) the suspension in the water of the mixture obtained in stage C);

e) the maceration of the suspension obtained in stage d);

f) separating the resulting liquid;

and 3) the processing liquid obtained in stage f), the allocation method, including precipitation with methanol, centrifugation and chromatographic separation, directed by bioanalysis.

7. The method according to claim 6, where stage 3) includes

g) the lyophilization liquid obtained in stage f),

h) OS is the establishment of methanol liofilizirovannogo substances, obtained in stage g),

i) separating the solid phase from the liquid phase,

j) depositing a solid phase obtained in stage i), with methanol to a final concentration of 25%, 50% and 67%,

k) dissolving in water precipitates obtained at stage (j) at 50% and 67%, centrifugation and implementation of the chromatographic separation of the supernatant;

l) identification of active fractions of bioanalysis;

m) the implementation of the second chromatographic separation of active fractions;

n) identification of active fractions of bioanalysis;

o) removing the solvent.

8. The method according to PP and 7, where the process of laser radiation with the red line laser diode capable of generating radiation with a wavelength in the range 150-810 nm, power 1-60 W and a spot diameter of 1-6 mm

9. The method of claim 8, where the wavelength is in the range of 200-400 nm, preferably 250 nm, at a power of 20 W and a spot diameter 4 mm

10. The method according to any of the preceding items, where each kilogram of crushed material is treated with laser radiation within 3-10 minutes, preferably within 5 minutes

11. The use of polysaccharide according to claim 1 to obtain a therapeutic agent for the treatment of immunosuppressive diseases.

12. The application of item 11 to obtain a therapeutic agent for the treatment of cancer, tuberculosis is uleta, flu, cold, allergies, lupus erythematosus, psoriasis and AIDS.

13. The application of section 12 to obtain a therapeutic agent for treating liver cancer, lung cancer, kidney cancer, colon cancer, breast cancer, prostate cancer or adenocarcinoma of the prostate; cancer of the brain, such as astrocytoma and glioblastoma; cancer of the cervix and cancer of the urinary bladder.

14. Pharmaceutical preparation for the treatment of immunosuppressive diseases, containing the polysaccharide according to claim 1.

15. The pharmaceutical preparation according to 14, further comprising a pharmaceutically acceptable carrier.

16. Pharmaceutical drugs are 14 or 15, further comprising at least one pharmaceutically active compound.