The method of obtaining the active complex based on a cultivated plant cells collection kalievyh

 

(57) Abstract:

The invention relates to biotechnology and can be used in medical, cosmetic and food industries. The method consists in the fact that air dry biomass cell culture of plants of family kalievyh subjected to additional heat treatment at a temperature of 60-150C within 15-90 minutes Method provides for mutual independence in cycle time cultivating biomass of receipt of the active complex and, if necessary, products based on it. Received active complex is greater than the original, adaptogenic activity and preserves the quality of the sorbent. The complex, extract and products on their basis can be used to satisfy vital human needs in accordance with the traditional purpose of biomass kalievyh. 3 C.p. f-crystals, 4 PL.

The invention relates to biotechnology and can be used in medical, cosmetic and food industries.

It is known that the biomass is cultivated cells kalievyh contains biologically active substances (BAS).

Known methods for producing biologically active substances by their removal. Various technologies the raw material components, as well as their pharmacological activity [Ant I. A., the Technology of medicines: 2 T. 3rd ed. M.: Medicine, 1980].

However, it is known that the main advantages of herbal products are determined by the cumulative effects of complex substances cells in General.

For pharmacology and practical medicine urgent task of increasing the activity of medicines as newly created and already applied in practice. With the increasing number of chronic diseases requiring long-term medication and is often accompanied by various disorders of metabolism in the body, it is of interest to expand the range of drugs soft impact due to the production of modified vegetation complexes. Partial transformation of substances native complex is already known plants can lead to new synergies in its pharmacological activity or to increase bioavailability of the complex [Lesiovskaja E. E., Pivovarov A. S., Frolova N. Y., Marchenko N. In., Draggin E. C. a Study of the interaction of plant adaptogens // V international Congress “Actual problems of creation of new medicinal preparations of natural origin”: Sa. I approved for medical use and is suitable for storage. It is dried at a temperature not above 60°C and is called “white” ginseng [State Pharmacopoeia of the USSR. 11th ed. Vol.2: General methods of analysis. Medicinal plant raw materials. -M.: Medicine, 1990. S. 348-349].

Approved for medical use Korean red ginseng, the color of the outside and on the turn reddish-brown. The method of obtaining it is not indicated, the composition of BAS complex are not clear and distinct activity from that "white" root not specified [State Pharmacopoeia of the USSR. 11th ed. Vol.2: General methods of analysis. Medicinal plant raw materials. -M.: Medicine, 1990. S. 349].

Recently abroad receive data on a wide therapeutic (anti-carcinogenic, immunotropic and other) action red ginseng root [H. Hasegava, R. Suzuki, J.-H. Sung, K.-S. Lee, S. Matsumiya, S.-K. Lee and M. Uchiyama. Antimetastatic Activity of Ginseng saponin called intestinal bacterial Metabolite (IH901) // International Ginseng Conference'99 "Ginseng: Its Sciences and its Markets. Advances in Biotechnology, Medicinal Applications @ Marketing: Programme @ Abstracts. P. 30; A. Kumagai. Recent Advances of Korean Red Ginseng studies in Japan // Proceeding of the 6th International Symposium. Seoul Korea, 1993. P. 14; Y. A. Shin, Y. K,Cho, M. K. Ki, J. S. Lee, J. G. Nam, M. H. Choi, Y. B. Kim, K. W. Choi. Effect of Korean Red Ginseng on immunological Markers of Persons with Human Immunodeficiency Virus // Proceeding of the 6th International Symposium. Seoul Korea, 1993. 30 times more powerful vasodilator action, than the usual “white” root [Jeong Hill Park, Jong Moon Kirn, Sang Beom Han, Na Young Kim. A new processed Ginseng Fortified with Activity // The 7th International Symposium on Ginseng: Program @ Abstract. Seoul Korea, 1998. P. 42].

A method of obtaining “red” ginseng root, when the raw root is placed in a sealed bag, heated to 60-100°C to soften, then frozen, place in an atmosphere of inert gas and freeze dried [Application 62-5406 Japan, MKI a 61 K 35/78, similar to: Pat. 4446130 USA, MCI 35/78. The method of obtaining dried ginseng for medicinal drug // ISM. 1985. 61 K. No. 2 (II)].

A method of obtaining “red” ginseng by periodically spraying hot water or dip the root in hot water [Application 59-38931 Japan, MKI a 61 K 35/78. A method of obtaining a “red ginseng” // ISM. 1985. 61 K. No. 9]. In these ways BAS remain part of the whole root.

The above processes produce "red-red" ginseng of mnogooperatsionny, long time (at least 12 hours). They involve the use of raw root that leads to the necessity of pairing moments processing of root and collection of raw materials.

Known biomass ginseng dry, it is dried at a temperature not exceeding 55°C, has a color ranging from light yellow to light brown, and antihypoxic activity and sorption properties [VFS 42-1891-89 / Ministry of health of the USSR. Pharmacopoeia Committee; U.S. Pat. 2058784 OF THE RUSSIAN FEDERATION, IPC A 61 K 35/78. Means of adsorption].

Closest to the present invention is a method, when raw biomass of cultured cells to increase the output of glycosides is subjected to mechanical destruction and heat treatment in an autoclave at a temperature of 115°C for 15 minutes After which the liquid fraction is separated, concentrated, dried, getting up to 1.2% of dry water-soluble powder containing glycosides 7,08% [U.S. Pat. RF 2039817, IPC 12 N 5/04. The method of obtaining biologically active compounds from the biomass of cultured plant cells].

A significant drawback of this method is the use of raw biomass, which inextricably links the process with the cultivation cycle. Sprat biomass, which is up to 20% of the raw material remains unused. The overall composition of the obtained biologically active substances not described, its activity is not characterized. The final product has an increased content of glycosides, however, it is known that in addition glycosides tissue kalievyh contain a number of other BAS [Hiromichi Okuda, Yinan Zheng, Yukinaga Matsuura, Takeshi Takaku. Studies on biologically active substances in non-saponin called fraction of Korean Red ginseng // Proceeding of the 6th International Symposium. Seoul Korea, 1993. P. 110-112].

Task izobretalem (including as part of the meal remaining after extraction of the complex of biologically active substances from air-dry biomass), active complex with enhanced adaptogenic activity, to expand the range of such tools. The invention also includes the preservation of the sorption properties of biomass, the achievement of mutual independence in time of cultivation cycle and moments produce an activated complex in the composition of raw materials or taking out BAS, as well as obtain products with activated biomass and extracted with BAS.

The problem is solved as follows.

Air-dry biomass cell culture kalievyh, dried at a temperature not exceeding 60°C directly before processing or dry in the stage of storage, is subjected to additional heat treatment. As a result, the biomass becomes a characteristic reddish-brown color (“red” biomass). The process temperature varies in the range of 60 to 150°C., preferably from 100 to 135°C, time is 15-90 minutes Received active complex biomass in General is higher than the original adaptogenic (including antioxidant and antihypoxic activity and preserves the quality of the sorbent. Vzaimozavisimosti in wreathed fact, what is pre-dried to air-dry biomass and its activation is possible at any time, and the moisture content of the biomass before and after the additional heat treatment corresponds necessary for storage.

Given the patterns of subject as biomass cell culture collection kalievyh different strains, and its meal, which is the waste of traditional technologies while receiving infusions of air-dry biomass type Biosensing, tincture” [VFS 42-1890-89 / Ministry of health of the USSR. Pharmacopoeial Committee].

The invention is illustrated by the following examples.

Example 1.

Air-dry biomass, cell culture of plants of family kalievyh different strains or her meal is subjected to additional heat treatment at a temperature of 60-150°C. for 15-90 minutes

Biomass acquires a characteristic hue (the experience visually determine when the optimum results of the process occurring within the stated formula. The original experience is the property of the applicant, contains its know-how and is not the subject of this application).

The results are presented in table.1.

Example 2.

About 1.0 g (accurately weighed) ismalic the 200-250 ml, add 50 ml of purified water, close the tube and leave for 1 hour. Then the flask connected with a reflux condenser, heated, maintaining a low boil for 2 hours. After cooling the flask with the contents thoroughly shaken and filtered through a dry filter paper into a dry flask with a capacity of 150-200 ml.

25 ml of the filtrate pipette is transferred into a pre-dried at a temperature of 100-105°With a Cup diameter of 7-9 cm and evaporated on a water bath to dryness. The Cup and the residue is dried at a temperature of 100-105°C to constant mass, then cooled for 30 min in a desiccator over anhydrous calcium chloride and weighed, counting the percentage of extractives [State Pharmacopoeia of the USSR. 11th ed. Vol.1: General methods of analysis. - M.: Medicine, 1987. S. 295].

The amount of extractives extracted 40-70% ethyl alcohol, is determined by a similar scheme, in which purified water is replaced with the corresponding alcohol concentration.

The results are presented in table.2.

Example 3.

Shredded biomass, sifted through a sieve with holes of 1 mm diameter, is subjected to successive extraction with different solvents.

A portion of about 5.0 g (textlayout petroleum ether. Petroleum ether is poured into a previously dried and weighed round bottom flask, the solvent is distilled off and weigh the flask with the remainder, determining the mass of petroleum extract (%).

Remaining after extraction with petroleum ether pour out a portion of chloroform. After 3 hours, the chloroform fraction is poured into pre-weighed round bottom flask and distilled under vacuum on a rotary evaporator to dryness, determining the mass of a chloroform extract (%).

The remaining sample within 6 hours extracted with a mixture of 1:2 ethyl alcohol with chloroform (V/V), the extractant is poured into pre-weighed flask and distilled under vacuum on a rotary evaporator, determining the mass of total glycoside fraction (GFS %).

The sample is extracted from the to conventional Soxhlet extractions, dried in air, and weighed with an accuracy of 0.001 g, transferred to a conical flask with a capacity of 200-250 ml, poured 100 ml of purified water, attach a reflux and 30 min boil on a sand bath. Then the extract is decanted into a volumetric flask with a capacity of 250 ml through 5 layers of gauze, nested in a glass funnel with a diameter of 55 mm, the Extraction was repeated 3 more times for 30 min, Prilepa 100 ml of water each time. The filter is washed in fougnie tubes with a capacity of 150 ml, add 25 ml of 95% alcohol, stirring heated in a water bath at 30°C for 5 minutes, the test Tube is cooled, the contents centrifuged for 30 min with a frequency of 5000 rpm

The supernatant liquid is filtered under vacuum at a residual pressure of 13-16 kPa through dried to constant weight glass filter FOR diameter 40 mm Precipitate quantitatively transferred on the same filter with 10 ml of a mixture of water and 95% alcohol (1:1) and washed with 10 ml of 95% alcohol (acetone). The filter residue is dried in air, is brought to constant weight at a temperature of 102-105°C and weighed.

The content of water-soluble polysaccharides (GRP) calculate the GF formula [State Pharmacopoeia of the USSR. 11th ed. Vol.2: General methods of analysis. Medicinal plant raw materials. - M.: Medicine, 1987. S. 379].

Conventional technologies [Ant I. A., the Technology of medicines: 2 T. 3rd ed. - M.: Medicine, 1980] received infusions, tinctures, extracts, based on the activated biomass. Defined qualitative and quantitative composition active substances: water, vodopidgotovki substances, glycosides (GFS), water-soluble polysaccharides (GRP), as well as lipophilic substances before and after the additional heat treatment.

Compare the exist, extracted water or (40-70) % odoperovali solutions, “red” biomass above 1.5-2.0 times. The content of the glycosidic fraction in all samples more than 2.5-3.0 times, the content of water-soluble polysaccharides (GRP) increased 3 times, they are 2 times more soluble proteins. The amount of lipophilic substances above 1.5-2.0 times.

To evaluate the pharmacological activity of the obtained complex as a complex index adaptogenic qualities were studied comparative antihypoxic and antioxidant activity of complexes “red” and “white” biomass of different strains kalievyh.

Evaluation of acute hypobaric hypoxia tinctures(1:10; 1:15; 1:20), obtained by the method of percolation 40% ethyl alcohol [Ant I. A., the Technology of medicines: 2 T. 3rd ed. - M.: Medicine, 1980. - So 3. S. 175]. The drug was administered intraperitoneally, 30 min spent the rise of the mice in the chamber with a speed of 50 m/s at a height of 11 km Exposition at the critical height is 20 minutes, the Evaluation was performed by the percentage of surviving animals at this height (PL.3).

The data presented indicate that not only the lifetime of the animals at the critical height (11 km) during hypobaric hypoxia was increased 3-4 times in toy activity of all investigated drugs.

To assess pharmacological activity was also conducted comparative research antihypoxic activity of GRP complexes “red” and “white” biomass of different strains kalievyh (PL.4).

The data obtained in all cases show that not only the lifetime of the critical heights (11 km) animals treated GRP from “red” biomass, was in 2-2,5 times higher, but the percentage of such animals 4 times was higher than that treated with GRP from “white” biomass.

All of the above can serve as a basis for obtaining the target product, where as an effective component can be used active complex “red” cell biomass of the plant tissue culture collection kalievyh or extract from it.

Research in this direction continues.

It was established experimentally that the proposed method obtained in the implementation of the bioactive complex and products based on it, are optimal from the standpoint of the combination of simplicity and the achieved result.

Thus, a technological method of production of the active complex “red” biomass based on cultured cells of plants of the family kalievyh through the net, take up to 1.5 hours of time stage. The method allows to obtain not only extractable by solvents BAS and products based on them, but also bioactive complex in the composition of the biomass. Last not least, because many SUPPLEMENTS and medicinal compositions is the raw material itself or its meal, which, in addition to pharmacological and sorption activity. Achieved mutual independence in cycle time cultivation of biomass, production of the active complex and products based on it.

Received the claimed method is complex, extracting, and their products can be used to satisfy vital human needs, at least according to the traditional purpose of the biomass kalievyh.

1. The method of obtaining the active complex in the composition of raw materials on the basis of cultivated cells of plants of the family kalievyh with power adaptogenic action and absorption capacity, providing for use as feedstock air-dry biomass, additional heat treatment at a temperature of 60-150C within 15-90 minutes

2. The method according to p. 1, characterized in that the additional Ter is omnitele carry out the extraction of the soluble complex of biologically active substances in terms defined for storage of the active complex in the composition of raw materials.

4. The method according to p. 3, characterized in that the extraction of soluble complex of biologically active substances (wt.% from biomass) is carried out until obtaining: extractives extracted water, 30-60, including GRP - from 8.0 to 20.0, and/or extractives extracted 40-70% water-alcohol solutions, - 26,0-57,0, and/or the amount of glycosides from 6.0 to 10.5, and/or lipophilic substances from 0.1 to 2.

 

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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.

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