RU2695335C1 - Method of producing an agent possessing adaptogenic activity - Google Patents

Abstract

FIELD: pharmaceutical industry.

SUBSTANCE: invention refers to pharmaceutical industry, namely to a method for preparing an agent possessing adaptogenic activity. A method for preparing a medicinal agent having adaptogenic activity, which is a dry extract obtained from the following herbal raw materials in ratio: high-grade rhizomes root 333 g, buckthorn sea-buckthorn fruits 333 g, coriander seeds of inoculation 333 g, which is ground in a mill to particle size of 1 mm in diameter and extracted with 50 % ethyl alcohol in ratio of raw material: extractant equal to 1:16, at temperature of 60 °C and constant stirring, the process is repeated three times, after which the combined extracts are evaporated to 1/3 of the initial volume and purified by separation, the obtained purified extract is additionally added to 1/5 of the initial volume, dried in a vacuum drier at temperature of 60 °C for 8 hours and ground in a mill of a propeller type.

EFFECT: agent possesses pronounced adaptogenic activity, is intended for increase of nonspecific body resistance, prevention of fatigue at physical and mental loads, and also for increase of working capacity.

1 cl, 9 tbl

Description

The invention relates to the field of pharmacy and relates to a method for producing funds from plant materials with adaptogenic activity.

In the last decade, due to increasing environmental and social pressure, there has been a significant decrease in overall resistance associated with depression of adaptive-compensatory mechanisms. Under these conditions, preventive measures aimed at increasing the nonspecific resistance of the body are of particular importance. One of the methods to achieve this condition is the use of pharmacological agents with adaptogenic activity and increasing resistance to a wide range of stress factors. The most promising direction is the development of new drugs based on raw materials of natural origin, a growing interest in which is noted throughout the world.

This is due to the fact that preparations obtained from raw materials of plant and animal origin, seafood and beekeeping, have several advantages compared to synthetic products: they contain a wide range of biologically active substances; have several types of pharmacological activity; characterized by a smooth increase in the pharmacological effect. In addition, drugs of natural origin are characterized by low toxicity and the absence of adverse reactions with prolonged use [3].

A promising direction in the search and development of new adaptogenic drugs of natural origin is the study of nonspecific drugs from the arsenal of Tibetan medicine, recommended for the weakened, as well as people of advanced age, as tonic, “giving longevity and health” [11, 12].

Based on the “three friends” prescription [12], a dry plant extract (RES) from roots and rhizomes of Elecampane (Inula helenium L.), fruits of buckthorn buckthorn (Hippophae rhamnoides L.), coriander fruits (Coriandrum sativum L.) was developed.

The components of this tool contain a wide range of biologically active substances belonging to different classes of chemical compounds: terpenes (α-pinene, limonene, lipalool, camphor, geraniol, myrcene, azulene), flavonoids (quercetin, rutin, vitexin, orientin, apigenin, cynaroside, apigenin and their glycosides), phenol carboxylic acids (caffeic, salicylic, gallic, coumaric, cinnamon, chlorogenic), carotenoids (β-carotene, lutein, cryptoxanthin), anthocyanins (peonin), etc. [5, 8, 9].

The pharmacological activity of these species is known, so extracts of elecampane exhibit antioxidant and immunomodelling activity; Buckthorn buckthorn - wound healing, antioxidant and immunomodulating activity; coriander fruits - antioxidant [5, 8, 9].

Thus, according to folk and scientific medicine, preparations of elecampane, sea buckthorn and coriander have a pronounced antioxidant and immunomodulating effect, which indicates the advisability of using this composition as an adaptogenic agent.

The aim of the invention is to increase the adaptogenic activity of the drug due to the higher content of active substances (terpenoids, flavonoids, carotenoids, hydroxycinnamic acids, anthocyanins).

To achieve this goal, plant material (raw materials) - the root of elecampane high (333 g), buckthorn buckthorn fruits (333 g), seed coriander fruits (333 g) are ground in a mill to a particle size of 1 mm in diameter (sieve No. 10 GOST 214-83 ) and extracted with 50% ethyl alcohol in the ratio of raw materials: extractant equal to 1:16 (taking into account the coefficient of water absorption), at a temperature of 60 ° C and constant stirring. The process is repeated three times. The extraction time at the 1st and 2nd phase contacts is 60 minutes, at the 3rd phase contact - 30 minutes. The combined extracts are evaporated to 1/3 of the original volume and purified by separation. The purified extract is doubled to 1/5 of the original volume, dried in a vacuum dryer at a temperature of 60 ° C for 8 hours and ground in a propeller-type mill. Get 270.0 g of the finished product, which is 27.0 ± 0.5% by weight of air-dry raw materials.

The proposed method allows to obtain the final product, a dry extract, which is a fine brown powder with a pleasant smell and sweet taste, with a moisture content of not more than 5%.

The method is illustrated by the following examples.

Example

Plant material (raw materials) - Elecampane rhizomes high (333 g), buckthorn buckthorn fruits (333 g), seeds of coriander seed (333 g) are ground in a mill to a particle size of 1 mm in diameter (sieve No. 10 GOST 214-83). The crushed raw materials are loaded into an extraction apparatus with a stirrer and external steam heating. Pour 16 liters of 50% ethyl alcohol in a ratio of raw materials-extractant equal to 1:16, taking into account the coefficient of water absorption of the raw materials. Extracted at a temperature of 60 ° C with constant stirring for 60 minutes The extract is filtered through a tin cloth in the collection. Two more extractions are carried out for 60 and 30 minutes, feeding 50% alcohol each time to the extractor in an amount equal to the volume of the drained extract (1st discharge - 10.85 L, 2nd discharge - 9.80 L, 3rd discharge - 9.60 L) . The combined extracts are evaporated to 1/3 of the original volume and purified by separation. The purified extract is doubled to 1/5 of the original volume, dried in a vacuum dryer at a temperature of 60 ° C for 8 hours and ground in a propeller-type mill. Get 270.3 g of the finished product, which is 27.06% by weight of air-dried raw materials. Dry extract is a fine powder of brown color with a pleasant smell and sweet taste; crumbles, the moisture content is 4.05%.

Acute extract toxicity (RES) was determined on white outbred male mice with an initial weight of 20.0 ± 1.0 g with intraperitoneal and intragastric administrations of the extract dissolved in water. Each dose of the extract was tested in 10 animals. Observation of the animals was carried out for 14 days. Observed signs of intoxication were recorded, autopsy and examination of dead animals was performed. The results of the studies showed that with the intraperitoneal administration of large doses of dry extract to white mice, signs of intoxication are observed, expressed in a slowdown in the orientational reaction, sudden movements, the appearance of tonic, and then tonic-clonic seizures. Animals died in cases of cardiovascular failure mainly during the day from the introduction of dry extract. Kerber LD50 with intraperitoneal administration of dry 1960 extract ± 60.76 mg / kg. With the intragastric administration of this drug at the maximum possible dose, the death of animals was not recorded, and there were no obvious signs of intoxication. These data allow us to attribute the test dry extract to the group of low toxic substances according to Sidorov's classification [10].

Subsequently, the effect of RES on physical endurance and resistance to hypoxia was investigated.

The effect of dry extract on physical endurance and resistance to hypoxia.

Studies were conducted on 55 white Wistar rats of both sexes with an initial weight of 160-180 g contained in the standard diet of vivarium.

To determine the total physical endurance, the generally accepted swimming technique with a load of 5% of body weight was used [4]. An aqueous solution of RES was administered intragastrically in a volume of 1.0 ml / 100 g containing 0.5; 1.0; A 3.0% extract solution once 1 hour before testing, and also repeatedly within 7 days in a volume of 1.0 ml / 100 g containing 3.0 ml of water infusion 1 time per day before meals. The animals of the control group were intragastrically injected with a similar volume of distilled water. As a comparison drug, a dealcalated solution of liquid Eleutherococcus extract was used in a volume of 1 ml / 100 g [3]. The duration of swimming of the animals to complete fatigue was evaluated, the criterion of which was a 10-second immersion in water. The data obtained are shown in table 1

As follows from the data. Listed in table 1, a single introduction of RES in the volume of 0.5 and 1.0 ml / kg did not significantly affect the duration of swimming of animals, while when using it in the volume of 3.0 ml / kg, an increase in overall physical endurance by 20% was noted compared with the data of the control animals groups. Based on this, in further experiments, the volume of RES corresponding to 3.0 ml / kg was used as an experimental therapeutic.

Seven-day preventive administration of RES in the volume of 3.0 ml / kg was accompanied by a more significant increase in overall physical endurance, as evidenced by an increase in the duration of swimming of animals of the experimental group by an average of 30% compared with the control group.

Thus, the data obtained indicate that RES has a stimulating effect on overall physical endurance, characterized by the predominance of aerobic processes of tissue energy supply. It was found that the test agent possesses the most pronounced actoprotective activity when administered in a volume of 3.0 ml / kg containing 3.0 ml of aqueous infusion; effectiveness increases with repeated administration.

The effect of RES on physical endurance and resistance to hypoxia.

Studies were conducted on 55 white Wistar rats of both sexes with an initial weight of 160-180 g contained in the standard diet of vivarium.

To determine the speed of physical endurance, the generally accepted method of running animals on a 10-track treadmill was used at a speed of 40 m / min [6]. Acute hypobaric hypoxia was reproduced in a pressure chamber at an atmospheric pressure of 198.8 mm Hg, a partial oxygen pressure of 50 mm Hg, which corresponds to the "rise" of animals to a height of 1000 m above sea level. To reproduce histotoxic hypoxia, the animals were intraperitoneally injected once with sodium nitroprusside at a dose of 20 mg / kg [4]. Resistance to hypoxia was evaluated by the life span of the animals.

An aqueous solution of the extract was administered intragastrically in a volume of 1.0 ml / 100 g, containing a 3.0% solution of the extract once for 1 hour before testing, and also repeatedly for 7 days in a volume of 1.0 ml / 100 g containing 3.0 ml of water infusion 1 time per day until food intake. The animals of the control group were intragastrically injected with a similar volume of distilled water. As a comparison drug, a dealcalated solution of Eleutherococcus liquid extract in a volume of 1 ml / 100 g was used. The data obtained are shown in table 2.

As follows from the table, preventive-course administration of the extract is accompanied by an increase in running duration by 26% compared with the data of animals in the control group. It was found that prophylactic administration of the extract increased the life time of rats with histotoxic and hypobaric hypoxia by 20 and 38%, respectively, in relation to the control. At the same time, the actoprotective and antihypoxic activity of the extract was similar to that of the comparison drug “Eleutherococcus extract”.

The effect of dry extract on the severity of catabolic changes in organs of white rats with immobilization and emotional stress.

Studies were conducted on 55 white Wistar rats of both sexes with an initial weight of 160-180 g contained in the standard diet of vivarium. Immobilization stress was reproduced by the conventional method by fixing animals on the back for 20 hours. To reproduce emotional stress, animals were immobilized in plastic cases and immersed in water (23 ° C) along the xiphoid process of the sternum for 2 hours [7]. Statistical processing of the results was carried out using the Mann-Whitney U-test.

As can be seen from table 3, as a result of immobilization and emotional stress, animals develop a complex of degenerative changes in the internal organs characteristic of the stess reaction: involution of the lymphoid organs, adrenal hypertrophy, the appearance of destructive lesions of the gastric mucosa. More pronounced changes in the internal organs were noted during immobilization stress, which is obviously associated with a longer stress exposure. The prophylactic administration of dry plant extract in a volume of 3.0 ml / 100 g had a pronounced antistress effect, as evidenced by a decrease in the relative mass of the adrenal glands of rats of the experimental group with immobilization and emotional stress by an average of 27.9%; an increase in the relative mass of the thymus — by 30% and the spleen — by 18% compared with the corresponding indices of the animals of the control group. The antistessor activity of the test drug was comparable to that of the comparison drug - Eleutherococcus extract.

Thus, a study of the adaptogenic activity of a dry plant extract showed that its preventive administration in a volume of 3.0 ml / 100 g is accompanied by an increase in the animal organism's resistance to the action of extreme factors of various nature: intense physical activity (general and speed); hypobaric and tissue hypoxia; immobilization and emotional stress. It can be assumed that the basis of the adaptive reorganization of the body, which develops under the influence of the test agent, is a series of functional and metabolic changes that occur at various levels of biological organization and are provided by a wide range of biologically active substances that make up its composition, such as terpene, flavonoids, carotenoids, anthocyanins, phenolcarboxylic acids. It can be assumed that these biologically active substances stimulate ergotropic and trophotropic processes in cells and thereby form a state of nonspecifically increased body resistance to the action of extreme factors of various nature. In particular, during intense physical exertion, the realization of the actoprotective effect of the test drug is associated with the optimization of energy metabolism in cells, which ensures activation of ATP resynthesis and a decrease in metabolic anide, which is known to be the main limiting factor of physical performance. It was shown that the activation of ATP resynthesis under the influence of the dry plant extract is provided both by an increase in the oxygen transport function of the blood and by the optimization of the limiting links of energy metabolism in the cells, which is confirmed by the obtained data on the pronounced antihypoxic activity of the dry plant extract under oxygen-deficient conditions of various origins.

Thus, the obtained data indicate that dry plant extract has a wide range of adaptogenic activity, which argues the prospect of its further research and implementation in clinical practice as a means designed to increase the nonspecific resistance of the body, prevent fatigue during physical and mental stress, as well as increase health.

Based on the qualitative phytochemical analysis [1, 2, 13], the presence of the following biologically active substances was established in the obtained dry extract.

1. Phenolcarboxylic acids. Two-dimensional upward chromatography on paper brand FN-12 in solvent systems:

- n-Butanol - acetic acid - water (4: 1: 2);

- 15% acetic acid.

When viewing the chromatogram in UV light, after treatment with ammonia vapors, identified with standard samples:

- caffeic acid - R _f = 0.80 (A), 0.42 (B);

- ferulic - R _f = 0.67 (A), 0.70 (B).

2. Flavonoids. When using the Sorbfil PTSX-AF-A-UV plates (chloroform – methanol – water solvent system, 26: 14: 3), spots of rutin (Rf about 0.6), quercetin (Rf about 0.7), and cynaroside (Rf about 0.8), apigenin (0.9).

2. Quantification

About 0.05 g (accurately weighed) of the extract is dissolved in a 25 ml volumetric flask in 10 ml of warm water (40-60 ° C), 5.2 ml of 96% alcohol are added, the solution volume is adjusted to the mark with water (solution A). 3 ml of solution A is placed in a volumetric flask with a capacity of 25 ml, 2 ml of a 2% solution of aluminum chloride in 96% alcohol are added, the volume of the solution with 20% alcohol is adjusted to the mark. After 40 minutes, the optical density of the solution was measured on a spectrophotometer at a wavelength of 405 nm in a cuvette with a layer thickness of 10 mm. As a comparison solution, use a solution consisting of 3 ml of solution A, 1 drop of diluted acetic acid and adjusted with 20% alcohol to the mark in a 25 ml volumetric flask. In parallel, the optical density of the state standard sample (GSO) of cinaroside is measured. 1 ml of the prepared GSO cinaroside solution is placed in a 25 ml volumetric flask, 1 ml of a 2% solution of aluminum chloride in 95% alcohol is added and the volume of the solution is adjusted with 20% alcohol to the mark. The comparison solution is a solution consisting of 1 ml of GSO cinaroside, 1 drop of diluted acetic acid and brought to 20% alcohol to the mark in a 25 ml volumetric flask.

The content of the sum of flavonoids in terms of cinaroside absolutely dry extract in percent (X) is calculated by the formula:

where D is the optical density of the test solution;

D _{o the} optical density of the GSO solution of cinaroside;

m is the mass of extract in grams;

m _o is the mass of GSO cinaroside in grams;

W is the mass loss upon drying of the extract in percent.

Note. Preparation of a GSO cinaroside solution: about 0.05 g (accurately weighed) GSO cinaroside, previously dried at a temperature of 130-135 ° C for 3 hours, dissolved in 85 ml of 95% alcohol in a 100 ml volumetric flask when heated in a water bath, cooled, quantitatively transferred to a volumetric flask with a capacity of 100 ml, bring the volume of the solution with the same alcohol to the mark and mix.

Found: 10.72 ± 0.18%.

The proposed production method is quite simple, does not require a complex purification scheme, allows you to get a product of constant composition. The technology can be implemented in enterprises producing drugs.

To identify the optimal technological conditions, we studied the main factors affecting the extraction process of plant materials: the nature of the extractant, its ratio with the raw materials, temperature conditions, the degree of grinding of the raw materials, duration and number of extractions.

Quantitative assessment was carried out according to the yield of extractives [1, 2] and the sum of flavonoids. Metrological characteristics are presented in table 7.

Water and ethyl alcohol of various concentrations (20, 30, 40, 50, 60, 70, 80, 90, 96%) were used as extractants.

As can be seen from table 8, 50-55% alcohol is the optimal extractant, as it allows to achieve the maximum yield of the sum of flavonoids, taking into account the yield of extractive substances.

Identification of the optimal degree of grinding of raw materials was carried out separately for each type of raw material. A quantitative assessment was carried out according to the yield of extractives. Based on the data obtained (table 9) and taking into account the fact that too fine grinding of plant material leads to contamination of the extraction with difficult-to-filter high molecular weight compounds, grinding of each type of raw material should be carried out jointly - 1 mm.

Due to the fact that the source of plant materials contains thermolabile substances (vitamins, iridoids), the influence of temperature on the extraction process was studied in the temperature range from 20 to 60 ° C. The data are presented in table 10, the optimum temperature is 60 ± 5 ° C.

When studying the influence of the ratio of raw materials to the extractant on the extraction process, it was established (table 11) that with an increase in the amount of extractant, the yield of extractives and the amount of flavonoids increase, but this increase for ratios of 1:16 and higher is insignificant, therefore, to prevent additional consumption of extractant, a ratio equal to 1:16 (taking into account the coefficient of water absorption).

To determine the duration and the required number of extractions, the time was established for reaching equilibrium concentrations of extractive substances and flavonoids in the raw material-extractant system during a three-time extraction. For this purpose, the crushed raw materials were extracted with 40% ethyl alcohol in a ratio of 1:16 in a water bath at a temperature of 60 ° C with constant stirring in a flask with the opposite. a refrigerator. The extracts obtained at certain intervals (15, 30, 45, 60, 75, 90 min) during three extractions were analyzed for the content of extractives and flavonoids. It was found that the equilibrium concentrations are established at the 1st and 2nd phase contacts after 60 minutes, at the 3rd phase contact after 30 minutes (table 12). Threefold extraction provides a fairly complete depletion of raw materials - up to 96% of active substances are extracted.

The proposed method allows to obtain a constant composition with a higher adaptogenic activity due to the higher content of active substances (flavonoids).

It is currently not possible to calculate the economic efficiency of the proposed method, however, the above advantages, combined with a simple production scheme, contribute to the rational use of medicinal plant materials and open up the prospect of introducing this method into the pharmaceutical industry.

Information sources

1. The State Pharmacopoeia of the USSR. Vol. 1. General methods of analysis / Ministry of Health of the USSR. - 11th ed., Ext. - M .: Medicine, 1987 .-- 336 p.

2. State Pharmacopoeia of the USSR: Issue. 2. General methods of analysis. Medicinal plant material / Ministry of Health of the USSR. - 11th ed., Ext. - M .: Medicine, 1989 .-- 400 p.

3. Mashkovsky M.D. Medicines - M., 2008 .-- 1200 s.

4. Methodological recommendations for the experimental study of drugs proposed for clinical study as antihypoxic drugs. - M., 1990. - 57 p. )

5. Nersesyan Z.M. Chemical investigation of seed coriander (Coriandrum sativum L.) in order to obtain pharmacologically active substances. Abstract. dis. Cand. farm. n - Pyatigorsk, 2007 .-- 17 s

6. Determination of the safety and effectiveness of biologically active food additives. Methodological guidelines. - M., 1999 .-- 87 s.

7. Pertsov S.S. Ulcerative lesions of the stomach in rats August and Wistar in acute and emotional stress // Bull. exp biol. and medicine. - 1995. - No. 11. - S. 469 - 473].

8. Plant resources of Russia: Wild flowering plants, their component composition and biological activity. In 6 t. T. 3. Families Fabaceae-Apiaceae. / comp. L.M. Belenovskaya, E.E. Lesiovskaya, N.S. Bobyleva - St. Petersburg., M, 2010 .-- 601 p.

9. Plant resources of Russia: Wild flowering plants, their component composition and biological activity. In 6 t. T. 5.2. Families Asteraceae (genera Echinops - Youngia) / comp. Belenovskaya L.M., Lesiovskaya E.E., Bobyleva N.S. - SPb., M, 2013 .-- 312 p.

10. Sidorov K.K. On the classification of toxicity of poisons with parenteral methods of administration // Toxicology of new industrial chemicals. - M. - 1973. - Issue. 13. - S. 47-51.

11. Sumati Prazdnya. Large recipe Aginsky datsan. Woodcut of Aginsky datsan. - The beginning. XX century - 151 liters Manuscript, translation by D. Dashiev

12. "Chjud-shek": Canon of Tibetan medicine. Per. from Tibetan D.B. Dashieva. - M .: "Oriental literature", 2001. - 766 p.

Claims (1)

A method of obtaining a drug having adaptogenic activity, which is a dry extract obtained from the following plant materials in the ratio: elecampane rhizomes high 333 g, buckthorn buckthorn fruits 333 g, sowing coriander fruits 333 g, which are ground in a mill to a particle size of 1 mm diameter and extracted with 50% ethyl alcohol in the ratio of raw materials: extractant equal to 1:16, at a temperature of 60 ° C and constant stirring, the process is repeated three times, after which the combined extracts are evaporated to 1/3 of the original volume and purified by separation, the resulting purified extract is added to 1/5 of the original volume, dried in a vacuum dryer at a temperature of 60 ° C for 8 hours and ground in a propeller-type mill.