RU2238317C2 - Method for preparing active complex based on cultured cells of plants of araliaceae family - Google Patents

Abstract

FIELD: biotechnology, medicinal, cosmetic and food industry.

SUBSTANCE: method involves additional thermal treatment of air-dried biomass of cultured cells of plants of Araliaceae family at temperature 60-150^oC for 15-90 min. method involves mutual independence for real-time in culturing biomass cycle, the moment for preparing active complex and, if necessary, products based on thereof. The prepared active complex shows enhanced adaptogenous activity as compared with the parent raw and retains quality of sorbent. The complex, its extracts and products based on thereof can be used for satisfying vital requirements of humans in accordance with conventional designation of biomass of Araliaceae family.

EFFECT: improved preparing method, valuable properties of complex.

4 cl, 4 tbl, 3 ex

Description

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

It is known that the biomass of cultured aralia cells contains biologically active substances (BAS).

Known methods for producing biologically active substances by extracting them. Various technological methods and modes have a significant impact on the content and ratio of components extracted from plant materials, as well as their pharmacological activity [I. Muravyov Technology of drugs: 2 vol. 3rd ed. M .: Medicine, 1980].

However, it is known that the main advantages of plant products are determined by the combined effect on the body of a complex of cell substances in general.

For pharmacology and practical medicine, the urgent task of increasing the activity of drugs is both newly created and already used in practice. With the increase in the number of chronic diseases requiring long-term medication and often accompanied by various metabolic disorders in the body, it is of interest to expand the range of mild drugs by obtaining modified plant complexes. Partial conversion of substances of the native complex of already known plants can lead to the appearance of new synergistic effects in its pharmacological activity or to an increase in the bioavailability of the complex [Lesiovskaya EE, Pivovarova AS, Frolova N.Yu., Marchenko NV, Drozhzhina E.V. A study of the interaction of adaptogen plants // V International Congress “Actual problems of creating new drugs of natural origin”: Sat. ref. - St. Petersburg, 2001. S.242-246].

Known with an indication of its inherent pharmacological activity, ginseng root, approved for medical use and suitable for storage. It is dried at a temperature not exceeding 60 ° C and is called “white” ginseng [State Pharmacopoeia of the USSR. 11th ed. Issue 2: General methods of analysis. Medicinal plant material. -M .: Medicine, 1990. S. 348-349].

Korean "red" ginseng is allowed for medical use; the color on the outside and at the fracture is reddish-brown. The method of obtaining it is not shown, the composition of the biologically active substances of the complex is not specified, and the difference in its activity from that of the "white" root is not indicated [State Pharmacopoeia of the USSR. 11th ed. Issue 2: General methods of analysis. Medicinal plant material. -M .: Medicine, 1990. S.349].

Recently, data on wide therapeutic (anti-carcinogenic, immunotropic, etc.) effects of the “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 intestinal bacterial Metabolite (IH901) // International Ginseng Conference'99 "Ginseng: Its Sciences and its Markets. Advances in Biotechnology, Medicinal Applications @ Marketing": Program @ 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. O.Shin, Y. K, Cho, M.K. Ki, J.S. Lee, J. G. Nam, M. H. Chei, 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. P.52]. It was found, in particular, that “red” ginseng shows a 7-fold higher antioxidant effect and a 30-fold stronger vasodilating effect than the usual “white” root [Jeong Hill Park, Jong Moon Kirn, Sang Beom Han, Na Young Kim . A new processed Ginseng with Fortified Activity // The 7th International Symposium on Ginseng: Program @ Abstract. Seoul Korea, 1998. P.42].

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

A known method of obtaining “scarlet” ginseng by periodically spraying hot water or immersing the root in hot water [Application 59-38931 of Japan, MKI A 61 K 35/78. The method of obtaining “scarlet ginseng” // ISM. 1985. A 61 K. No. 9]. With these methods, biologically active substances remain in the composition of the whole root.

The above processes for obtaining "red-scarlet" ginseng are multi-operational, time-consuming (at least 12 hours). They provide for the use of raw root, which leads to the need to combine the moments of root processing and collection of raw materials.

The known biomass of ginseng is dry, it is dried at a temperature of no higher than 55 ° C, has a color from light yellow to light brown, humidity is not more than 8%, the content of the total glycosides is not less than 1.5%, it has adaptogenic (including antioxidant and antihypoxic activity) and sorption properties [VFS 42-1891-89 / USSR Ministry of Health. Pharmacopoeia Committee; Pat. 2058784 of the Russian Federation, IPC A 61 K 35/78. Absorbent agent].

Closest to the proposed one is a method when the raw biomass of cultured cells is subjected to mechanical destruction and heat treatment in an autoclave at a temperature of 115 ° C for 15 minutes to increase the yield of glycosides. Then the liquid fraction is separated, concentrated, dried, obtaining up to 1.2% of a dry water-soluble powder with a glycoside content of 7.08% [US Pat. RF 2039817, IPC C 12 N 5/04. A method of obtaining biologically active substances from biomass of cultivated plant cells].

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

The objective of the invention is to obtain an active complex with enhanced adaptogenic activity in the composition of raw materials of cultured cells of plants of the aralian family of plants (including as a part of the meal remaining after extraction of the biologically active substances complex from air-dried biomass), to expand the range of such agents. Moreover, the objective of the invention also includes preserving the sorption qualities of biomass, achieving mutual independence in the time of the cultivation cycle and the moments of obtaining the activated complex as a part of raw materials or extracting biologically active substances from it, as well as obtaining products with both activated biomass and extracted biologically active substances.

The problem is solved as follows.

The air-dried biomass of the Aralian cell culture, dried at a temperature not exceeding 60 ° C immediately before processing or dry in the storage stage, is subjected to additional heat treatment. As a result, the biomass acquires a characteristic reddish-brown hue (“red” biomass). The process temperature ranges from 60 to 150 ° C, preferably from 100 to 135 ° C, the time is 15-90 minutes The resulting active biomass complex as a whole has a higher adaptogenic (including antioxidant and antihypoxic) activity than the original one and retains the quality of the sorbent. The time independence of the biomass cultivation cycle, the moment of activation of the complex and the preparation of products based on it, is achieved by the fact that pre-dried to air-dry biomass and its activation are possible at any time, and the moisture content of the biomass before and after additional heat treatment is necessary for storage.

The above regularity obeys both the biomass of the cell culture of the Aralian family of various strains and its meal, which is a waste of traditional technologies for obtaining tinctures from air-dried biomass of the type “Bio-Ginseng, tincture” [VFS 42-1890-89 / USSR Ministry of Health. Pharmacopoeia Committee].

The invention is illustrated by the following examples.

Example 1

The air-dried biomass of a cell culture of plants of the Aralian family of various strains or its meal is subjected to additional heat treatment at a temperature of 60-150 ° C for 15-90 minutes.

Biomass acquires a characteristic hue (Experience has been gained in visually determining the moment when optimal results of the process are achieved within the framework of the claimed formula. The original experience is the property of the applicant, contains his 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) of the crushed biomass sifted through a sieve with holes with a diameter of 1 mm is placed in a conical flask with a capacity of 200-250 ml, 50 ml of purified water is added, closed with a stopper and left for 1 hour. Then the flask is combined with a reflux condenser, heated, maintaining a weak boil for 2 hours. After cooling, the flask with the contents is thoroughly shaken and filtered through a dry paper filter into a dry flask with a capacity of 150-200 ml.

25 ml of the filtrate is pipetted into a cup with a diameter of 7-9 cm previously dried at a temperature of 100-105 ° C and evaporated to dryness in a water bath. The cup with the residue is dried at a temperature of 100-105 ° C to constant weight, then cooled for 30 minutes in an desiccator over anhydrous calcium chloride and weighed, calculating the percentage of extractives [State Pharmacopoeia of the USSR. 11th ed. Issue 1: General methods of analysis. - M .: Medicine, 1987. P.295].

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

The results are presented in table.2.

Example 3

The crushed biomass sifted through a sieve with holes with a diameter of 1 mm is subjected to sequential extraction with various solvents.

A portion of about 5.0 g (accurately weighed) is wrapped in a filter paper cartridge, placed in a Soxhlet apparatus and extracted with petroleum ether for 3 hours. Petroleum ether is poured into a pre-dried and weighed round bottom flask, the solvent is distilled off and the flask is weighed with the residue, determining the mass of the petroleum extract (%).

The sample remaining after extraction with petroleum ether is filled in with chloroform. After 3 hours, the chloroform fraction was poured into a pre-weighed round bottom flask and distilled to dryness under vacuum on a rotary evaporator, determining the mass of the chloroform extract (%).

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

The sample is removed from the Soxhlet apparatus, dried in air, weighed to the nearest 0.001 g, transferred to a conical flask with a capacity of 200-250 ml, poured 100 ml of purified water, the reflux condenser is attached and boiled in a sand bath for 30 minutes. Then, the extract is decanted into a 250 ml volumetric flask through 5 layers of gauze embedded in a 55 mm diameter glass funnel. The extraction is repeated 3 more times for 30 minutes, pouring 100 ml of water each time. The filter is washed with water over a volumetric flask, the contents of the flask are adjusted to the mark (solution A).

25 ml of solution A is placed in centrifuge tubes with a capacity of 150 ml, 25 ml of 95% alcohol are added, while stirring, they are heated in a water bath at 30 ° C for 5 minutes. The tubes are cooled, their contents are centrifuged for 30 minutes at a frequency of 5000 rpm.

The supernatant is filtered under vacuum at a residual pressure of 13-16 kPa through a 40 mm diameter POR016 glass filter dried to constant weight. The precipitate is quantitatively transferred to the same filter using 10 ml of a mixture of water and 95% alcohol (1: 1) and washed with 10 ml of 95% alcohol (acetone). The filter cake is dried in air, adjusted to constant weight at a temperature of 102-105 ° C and weighed.

The content of water-soluble polysaccharides (GRP) is calculated according to the formula given in the State Pharmacopoeia [State Pharmacopoeia of the USSR. 11th ed. Issue 2: General methods of analysis. Medicinal plant material. - M .: Medicine, 1987. P.379].

Methods of traditional technology [Muravyov I.A. Technology of drugs: 2 vol. 3rd ed. - M .: Medicine, 1980] obtained infusions, tinctures, extracts based on activated biomass. The qualitative and quantitative composition of biologically active substances was determined: water-, water-alcohol-soluble substances, glycosides (SGF), water-soluble polysaccharides (GRP), as well as lipophilic substances before and after additional heat treatment.

Comparative results are given in table.2.

The analysis shows that the quantitative content of substances recovered by water or (40-70)% water-alcohol solutions in the “red” biomass is 1.5-2.0 times higher. The content of the glycoside fraction in all samples is 2.5-3.0 times greater, the content of water-soluble polysaccharides (GRP) is increased 3 times, they contain 2 times more water-soluble proteins. The amount of lipophilic substances is 1.5-2.0 times higher.

To assess the pharmacological activity of the obtained complex as a complex indicator of adaptogenic qualities, the comparative antihypoxic and antioxidant activities of the complexes of “red” and “white” biomass of different aralia strains were studied.

An assessment of acute hypobaric hypoxia of tinctures (1:10; 1:15; 1:20) obtained by percolation on 40% ethyl alcohol [Muravyov I.A. Technology of drugs: 2 vol. 3rd ed. - M .: Medicine, 1980. - V.3. S.175]. The drug was administered intraperitoneally, after 30 min the mice were lifted in a pressure chamber at a speed of 50 m / s. to a height of 11 km. Exposure at a critical height - 20 minutes. The assessment was carried out according to the percentage of surviving animals at a given height (Table 3).

The data presented indicate that not only the life time of animals at a critical height (11 km) during hypobaric hypoxia was increased by 3-4 times compared with the control, but the percentage of surviving animals was from 30 to 50%, which indicates an increased antioxidant activity of all investigated drugs.

To assess the pharmacological activity, a comparative study of the antihypoxic activity of GRP from the complexes of “red” and “white” biomass of different strains of aralia was also performed (Table 4).

The data obtained in all cases indicate that not only the life time at a critical height (11 km) of animals receiving GRP from the “red” biomass was 2–2.5 times higher, but the percentage of such animals was 4 times higher, than those receiving GRP from “white” biomass.

All of the above can serve as a basis for obtaining the target products, in which an active complex of “red” biomass of cells from tissue culture plants of the Aralian family plants or extracts from it can be used as an effective component.

Research in this direction is ongoing.

It was experimentally established that the claimed method parameters obtained during its implementation of the bioactive complex and products based on it are optimal from the standpoint of combining simplicity and the achieved result.

Thus, a technological method has been proposed for producing the active complex of “red” biomass based on cultured cells of plants of the Aralian family by additional heat treatment of air-dried biomass. The heat treatment of dry raw materials is carried out in one simple stage, which takes up to 1.5 hours. The method allows to obtain not only biologically active substances extracted with solvents and products based on them, but also a bioactive complex in the composition of biomass itself. The latter is no less important, since the composition of many dietary supplements and medicinal compositions includes the raw material itself or its meal, which in addition to pharmacological and sorption activity. Mutual independence is achieved in the time of the biomass cultivation cycle, the moments of obtaining the active complex and products based on it.

The complex, extracts and their products obtained by the claimed method can be used to meet human vital needs, at least in accordance with the traditional purpose of the Aralia biomass.

Claims (4)

1. A method of obtaining an active complex in the composition of raw materials based on cultured cells of plants of the Aralian family, with enhanced adaptogenic action and sorption ability, involving the use of air-dried biomass as a feedstock, its additional heat treatment at a temperature of 60-150 ° C for 15- 90 min 2. The method according to claim 1, characterized in that the additional heat treatment is preferably carried out at a temperature of 100-135 ° C. 3. The method according to claim 1, characterized in that it further carries out the extraction of a complex of soluble biologically active substances in the time period determined for storage of the obtained active complex in the composition of the raw material. 4. The method according to claim 3, characterized in that the extraction of a complex of soluble biologically active substances (wt.% Of biomass) is carried out to obtain: extractives extracted with water, 30-60, incl. GRP - from 8.0 to 20.0, and / or extractives extracted with 40-70% aqueous-alcoholic 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.