The method of obtaining biologically active substances with antiaggregatory and hypotensive action

 

The invention relates to medicine. As raw materials take the dried aerial part with the roots of the unique meadow. It is pulverized and extracted with 50% ethanol three times for 60 min, at a ratio of 1:10 (raw - extractant), in the presence of calcium carbonate in a water bath. The alcoholic extract combine, the alcohol is distilled off, the aqueous residue is evaporated, mixed with polyamide sorbent and contribute to the chromatographic column. The column is sequentially washed with water and 50% ethyl alcohol. Of the alcohol fraction after removal of the solvent receive a complex of flavonoids, and water complex iridoids and polysaccharides. The invention allows to realize the specified destination. 3 table.

The invention relates to medical and pharmaceutical industry and can be used in medicine for drug development with antiaggregatory and hypotensive activity.

One of the urgent problems of medical science and practical health care is the prevention and treatment of diseases of the blood and cardiovascular system. The social significance of this problem is due to a wide spread and severity of disease. To tie the th toxicity, is the actual problem.

Among the wild plants that affect the cardiovascular system, are species of the family Nalichnaya - Foxglove (Digitalis grandiflora Mill. ), ochanka of Rostkoviana (Euphrasia rostkoviana Hayne), Linaria vulgaris Mill.), unique meadow (Melampyrum pratense L. ). They are widely used in both scientific and folk medicine, homeopathy, veterinary medicine [1]. Despite the wide distribution of these species on the territory of Russia the majority of them does not form dense thickets, suitable for industrial use, and only unique meadow has a sufficient resource base. It is widely distributed in the Central and Northern regions of Russia in the zone of coniferous and mixed forests, forms extensive thickets. The aboveground part of the unique meadow has long been used as an effective sedative, hypotensive, neuroleptic drugs [2, 3]. It prepared water extract (infusions and decoctions), alcohol extract (tincture), but they contain relatively low amounts of biologically active substances [table 1].

Phytochemical analysis of aerial parts roots of the unique meadow has allowed to establish the existence of such biologically active visa), alkaloids, polysaccharides [4, 5], which are involved in complex pharmacological action.

Way to complex natural products, which antiaggregatory activity against platelet-known method of obtaining a dry extract lnjanki ordinary, which also belongs to the Figwort family. He is a long extraction of biologically active substances with a water-alcohol mixture (30 days), the freezing of the extract in cryogenic freeze apparatus KC-30 at a temperature (-25^o(C)-(-30^oC) drying in two stages under vacuum 40-60 mm RT.article when the temperature of 36-38^o[6].

This way it is advisable to take for the prototype due to the presence of common operations, similar biological activity Botanical affinity raw sources.

The prototype has the following disadvantages: labor intensive method of production, associated with the need to use special equipment for cryogenic freeze-drying; the duration of the method of obtaining (about 30 days); no parameters standardization and quality control of the product; the source of raw material preparation Linaria vulgaris L. ATA does not form; the lack of anti-hypertensive drug activity; biologically active substance lnjanki ordinary possess vasoconstrictor action, increases blood pressure, stimulates polyreactive system [7], which does not allow to use them for the development of antiaggregatory means of hypotensive effect.

The aim of the invention is a method of obtaining biologically active substances with antiaggregatory and hypotensive action from vegetable raw materials with a sufficient resource base, which is simple, high content of biologically active substances in the product, the absence of stage cryogenic drying.

The objective is achieved by the fact that as a source of raw materials used aerial part with roots (grass) are unique arc - Melampyrum pratense L.

Air-dry grass (aerial part with roots) is crushed to particle size of 10 mm and extracted 3 times in 60 minutes 50% ethyl alcohol in the ratio of raw materials-extractant 1: 10 in round bottom flask with reflux in a boiling water bath in the presence of calcium carbonate (0.5 g per 100 g raw material), neutralizing free organic acids that destroy natural glycosides in the extraction process. Spirtos vacuum to a volume of 200 ml, mixed with polyamide sorbent and contribute to the chromatographic column (3,580 cm). The column is sequentially washed with water and 50% ethyl alcohol, separately collecting alcohol and aqueous fractions. The alcohol fraction is evaporated in a vacuum to obtain a dry residue (a complex of flavonoids). The aqueous fraction is evaporated in a vacuum until a dry residue, which was dissolved in 70% ethyl alcohol, when standing from the resulting solution falls amorphous precipitate cream color. The precipitate is filtered off, dissolved in water and acetone is added precipitation of white (complex polysaccharides). An alcoholic solution after separation of the precipitate polysaccharides dilute with water to 1 liter and precipitated impurities ballast substances with excess acetone, filtered. The filtrate is successively passed through the layer of aluminum oxide (3,5/4 cm) and polyamide (3,5/4 cm) is distilled off in a vacuum until a dry residue (a complex of iridoids).

The content of extractives, the dry residue and the humidity determined by the method GFHS [8].

Analysis on the content of flavonoids 1. Qualitative detection of 0.05 Obtained substance was dissolved in 10 ml of 40% ethyl alcohol. To 5 ml of the resulting solution add 3 ml of 2% aqueous solution aluminas hydrochloric acid and 10 mg of crystalline magnesium, heated in a boiling water bath for 3 min, appears orange-red staining (candinavia reaction).

2. Quantitative determination of flavonoids About 0.05 g (accurately weighed) of the complex of flavonoids is placed in a volumetric flask with a capacity of 25 ml, dissolved in 10 ml of 50% ethanol and bring the volume of the solution up to the mark with 50% ethanol, and mix (solution A). 5 ml (the exact amount) solution And quantitatively transferred into a volumetric flask of 50 ml, add 3 ml of 2% aqueous solution of aluminum chloride, bring the volume up to the mark with 50% ethanol, mix. After 30 minutes, measure the optical density of the solution in the spectrophotometer at a wavelength of 385 nm in a cell with a layer thickness of 10 mm as the reference solution using solution consisting of 5 ml And brought to 50% ethanol and up to the mark in a volumetric flask with a capacity of 50 ml, the total Content of flavonoids in terms of cynaroside and completely dry the product in % (X%) is calculated by the formula:

where E₁^1%=3612,2 - specific absorption complex cynaroside with aluminium chloride at a wavelength of 385 nm; D is the optical density of the investigated solution; m is the mass of raw material in grams; W - humidity in %.

Analisando water. To 5 ml of the resulting solution add 5 drops of reagent Trim-hill (mixture consisting of 5 ml of concentrated chlorostoma portly acid, 10 ml of 0.2% solution of copper sulphate and 100 ml glacial acetic acid), heated in a boiling water bath for 10 min, appears blue staining.

2. Quantitative determination of iridoid
Carried out by the reaction with the reagent Trim-hill method Gregor and Simchen [9].

Analysis of polysaccharides
Qualitative detection
To 0.05 g of the drug was added 1 ml of 10% NaOH solution, heated in a boiling water bath for 5 minutes, appears yellow staining.

To 0.05 g of the drug add 1-2 ml of 10% hydrochloric acid, boil for 1-2 minutes and add 5 ml of Fehling's reagent (a mixture of equal volumes of solutions a and B. Solution A: 34,66 g recrystallized copper sulfate is dissolved in water, acidified with 2-3 drops of diluted sulfuric acid, and dilute with water to 500 ml Solution b: 173 g of sodium potassium tartrate and 50 g of sodium hydroxide dissolved in 400 ml of water, after cooling, dilute with water to 500 ml), heated to boiling, appears orange-red precipitate.

Example: 100 g air-dry grass (aerial part with the roots) are unique meadow crush the g of calcium carbonate, heated in a boiling water bath for one hour under reflux, cooled alcohol removing the merge, and the extraction process was repeated twice under the same conditions. Alcoholic extract combine (3 l), filtered through cotton gauze swab, alcohol is distilled off, the aqueous residue is evaporated in vacuum to a volume of 200 ml, mixed with polyamide sorbent (50 g) obtained by the method of Litvinenko Century. And. [10], dried at a temperature of 40^oWith and contribute to the chromatographic column (3.580 cm), filled with 1/3 volume of polyamide sorbent. The column is sequentially washed with water (3 l) to negative reactions to iridoid (control - reaction with the reagent Trim-hill) and 50% ethanol (3 l) to negative reactions to the flavonoids ( control - canadanova reaction), separately collecting alcohol and aqueous fractions.

The alcohol fraction is evaporated in a vacuum until a dry residue (a complex of flavonoids). The yield of the target product 0.8 g, i.e. 0.8% of the original number of dry raw material. The complex of flavonoids is a dark-brown powder, no odor, weak bitter taste, gives a qualitative response to flavonoids. With the, the which is dissolved in 70% ethanol, cooled, when standing falls amorphous precipitate cream color. The precipitate is filtered off, dissolved in 50 ml of water and add 100 ml of acetone precipitation of white. The precipitate is filtered off, washed with acetone, dried (complex polysaccharides). Complex polysaccharides is a white powder, odorless, sweet taste, gives a qualitative response to polysaccharides. The yield of the target product, 3.0 g, i.e. 3% of the original dry matter. The moisture content of 0.32%.

An alcoholic solution after separation of the precipitate polysaccharides diluted with distilled water to a volume of 300 ml and add 1.5 l of acetone. The precipitation is filtered off. The filtrate is successively passed through the layer of aluminum oxide (3.54 cm) and polyamide sorbent (3,54 cm), evaporated in a vacuum until a dry residue (a complex of iridoids). The yield of the target product of 1.0, i.e. 1% of the original dry matter. Complex iridoids is a brown powder, odorless, bitter taste, gives qualitative reactions on iridoid. The content of iridoids in the product of 8.5%, the humidity of 7.96%.

The influence of the products obtained at a concentration of 0.2 mg/ml is). As Comparators used dry extracts of unique meadow, obtained by extraction of the raw water and 50% ethyl alcohol. Control was a solution of aspirin and dipyridamole in the concentration of 0.2 mg/ml

As follows from table 2, the highest antiaggregatory activity manifests a complex of flavonoids, the percentage of platelet aggregation is reduced by 33.3%, complex polysaccharides, slows platelet aggregation by 12%. Complex iridoids and Comparators (dry extracts are unique) practically does not possess the antiaggregatory effect. Antiaggregatory activity of the complex of flavonoids is comparable with the activity of drugs such as aspirin and dipyridamole. Dry extract lnjanki (prototype) slows platelet aggregation by 50% compared with adenozindezaminazou salt (ADP) adding 0.1 ml of the extract with 1 µmol ADP 1 ml of plasma rich in platelets [6].

Having hipaageninfo activity was performed in vivo in cats [11], as Comparators used dry extracts of unique meadow, obtained by extraction of the raw water and 50% ethyl alcohol. Control was a solution of papaverine hydrochloride in a dose of 3 mg/kg (0.1 LDI studies are shown in table 3.

It is established that the Comparators (dry extracts of unique meadow) at a dose of 50 mg/kg does not show hypotensive activity. Complexes of flavonoids and iridoids, (the same dose), after 2 min after injection reduce blood pressure and 23.3 26.0%, and after 5 min 11.0 and 8.7%, respectively. Dry extracts are unique exert a hypotensive effect in a dose of 100 mg/kg of Dry extract (extractant 50% ethanol) reduce blood pressure after 2 min 28.2%, and the dry aqueous extract of 27.7%, after 5 min 15.4 and 15.6%, respectively. The complex polysaccharides in doses of 5, 20, 50 mg/kg does not show hypotensive activity.

Sources of information
1. Plant resources of the USSR. Flowering plants, their chemical composition, use, Caprifoliaceae - plantaginaceae families. - M.: Nauka, 1990, 328 S.

2. Kucherov, E. C., S. Karimov,, Lazarev A. N. and other Unique new medicinal plant // problems of sustainable use of plant resources of the southern Urals. - Ufa, 1963, S. 24-27.

3. Rabinovich, M. I. Influence of unique meadow on cardiovascular activity // 3rd Ural conference of physiologists, biochemists and pharmacologists, dedicated to the 40th anniversary of the Udmurt ASSR. - Izhevsk, 1960, S. 192-193.

4. S. Karimov, Acting westosha and introduced plants in Bashkortostan. - Ufa, 1961, vol.1, S. 137-142.

5. Kaliszewska E. E., Petrichenko C. M. and other Learning iridoids of unique meadow growing in the Urals. // The prospects of development of natural Sciences in high school. So 1. - Perm, 2001, S. 201-205.

6. AC 1034742 from 22.05.83; publ. 15.08.83, B 30.

7. Rabinovich, M. I. Materials experimental and clinical pharmacology of drugs of some plants of the genus Linaria ill.: Abstract. thesis... Dr. vet. Sciences. Omsk, 1965, 23 S.

8. State Pharmacopoeia of the USSR. - XI ed. - M.: Medicine, so 1, 2 (1987, 1990).

9. Grger G. Simchen P. 1967. Zur Kennis iridoider pflanzensteffe / Die Pharmazie, 22, 6, s.315.

10. Litvinenko Century. And., Maksutina N. P., Kolesnikov, D., Obtaining polyamide sorbent. The honey. industry of the USSR, 1962, T. 11, 3, S. 40-43.

11. Berezin, M. P., N. Vasilevskaya.E., Auerbach, M. S. and other Large workshop on the physiology of human and animals. Under the General Ed. L. L. Vasiliev and I. A. Vetyukov. - M.: Higher school, 1961, S. 675.4

Claims

The method of obtaining funds with antiaggregatory and hypotensive activity, including the extraction of vegetable raw materials the aerial parts roots are unique meadow - 50% ethyl alcohol in the presence of calcium carbonate when heated to kipawa the x extraction, chromatographic separation of polyamide sorbent using successively water and 50% ethyl alcohol, the alcohol evaporation of the fractions in vacuo to a dry residue.