RU2716161C1 - Method for preparing a medicament having adaptogenic activity - Google Patents

Abstract

FIELD: chemical-pharmaceutical industry.

SUBSTANCE: invention refers to chemical-pharmaceutical industry, namely to a method for preparing a drug exhibiting adaptogenic activity. Medicinal agent represents a dry extract produced from the following vegetal raw materials in ratio: 178.57 g of Chinese liquorice roots and rhizomes; Caragana jubata wood 321.42 g; Polygonatum odoratum roots 53.57 g; Polygonatum humile roots 53.57 g; Orchis mascula root tuber 178.57 g; rhubarb roots 107.14 g; roots and rhizomes of common madder 107.14 g, which are milled to particle size of 1 mm diameter and extracted 50 % ethyl alcohol in ratio raw material : extractant, equal to 1:16, at temperature 60 °C and constant stirring. Process is repeated three times and the combined extractions are boiled off to 1/3 of the initial volume and purified by separation. After that, the purified extract is additionally boiled off till 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: invention 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, 2 dwg, 11 tbl, 1 ex

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 over 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 [5].

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”.

Based on the prescription “licorice-7”, in indications for use: “for pain in the region of the kidneys, lower back, stiffness of the extremities, sudden hot flashes in different parts of the body” [14], a dry plant extract (RES) from licorice roots and rhizomes was developed Ural (Glyzyrrhiza uralensis Fisch), wood of caragana mane (Caragana jubata (Pall.) Poir, roots bought fragrant (Polygonatum odoratum (Mill) Druse, roots bought squat (Polygonatum humile Fisch ex Maxim), masculine tuber (L. maschula) ), the roots of common rhubarb (Rheum rhabarbarum L.), the roots and rhizomes of madder madder (Rubi a tinctorum (L.).

The components of this tool contain a wide range of biologically active substances belonging to different classes of chemical compounds: terpenes (glycyrrhizic acid, diosgenin, saponin RO-2, PO-3), flavonoids (liquiquirithin, quercetin, rutin, vitexin, myricitin, chrysoriol, apigenin and their glycosides), phenolcarboxylic acids (caffeic, chlorogenic, neochlorogenic, gallic, coumaric, cinnamon), anthocyanins (chresatemin, seranin, cyanine), anthraquinones (chrysofanol, emodin, alizarin, purpurin, lucidin primerverozidium, rubium, rubri acid, 10, 11, 12].

The pharmacological activity of these species is known, so licorice extracts exhibit anti-inflammatory and immunomodulating activity [10]; Karagany - hypoglycemic, anti-inflammatory [10]; bought fragrant - immunomodulatory [12], bought squat - coagulant [12]; orchis antioxidant [12]; rhubarb - anti-inflammatory [9]; madder dye - choleretic and antibacterial [11].

Thus, according to folk and scientific medicine, preparations from the roots and rhizomes of licorice of the Urals, carafes of mane, roots bought fragrant, rhizomes bought squat, tubers of male Orchis, roots of common rhubarb, roots and rhizomes of madder 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, hydroxycinnamic acids, anthocyanins).

To achieve this goal, plant material (raw materials) - 178.57 g of roots and rhizomes of Ural licorice; 321.42 g of wood caragana mane; 53.57 g of roots bought fragrant; 53.57 g of roots bought squat; 178.57 g of male Orchis tubers; 107.14 g of the roots of rhubarb ordinary; 107.14 g of roots and rhizomes of madder madder 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 60 ° C with 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 odor 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) - 178.57 g of the roots and rhizomes of Ural licorice; 321.42 g of wood caragana mane; 53.57 g of roots bought fragrant; 53.57 g of roots bought squat; 178.57 g of male Orchis tubers; 107.14 g of the roots of rhubarb ordinary; 107.14 g of roots and rhizomes of madder madder 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. It is 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 toxicity of the extract (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 animals was conducted for 14 days. The observed signs of intoxication were recorded, an autopsy and examination of dead animals were 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 of 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 extract dry to the group of low toxic substances according to Sidorov's classification [13].

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, a generally accepted swimming technique with a load of 5% of body weight was used [6]. An aqueous solution of RES was administered intragastrically in a volume of 1.0 ml / 100 g containing 0.5; 1.0; 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. 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 [6]. 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 given in table 1, a single administration of RES in the volumes 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% compared with the data of animals of the control group. 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 obtained data indicate that RES has a stimulating effect on general 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; efficiency 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, we used the generally accepted method of running animals on a 10-track treadmill at a speed of 40 m / min [7]. 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 [8]. 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 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 in 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 data from 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 the 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 [6, 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 in animals, a complex of dystrophic changes in the internal organs characteristic of the stess reaction develops: involution of the lymphoid organs, adrenal hypertrophy, the appearance of destructive lesions of the gastric mucosa. More pronounced changes in internal organs were noted with immobilization stress, which is obviously associated with a longer stress exposure. The prophylactic administration of dry plant extract in the volume of 3.0 ml / 100 g had a pronounced anti-stress 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 similar indicators of 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 the dry plant extract showed that its preventive administration in the 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 drug, is a series of functional and metabolic changes occurring at various levels of biological organization and provided by a wide range of biologically active substances that make up its composition, such as terpene, flavonoids, anthocyanins, phenol carboxylic 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, as is known, is 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 units 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 states of various origins.

Thus, the data obtained 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 intended to increase the nonspecific resistance of the body, prevent fatigue during physical and mental stress, as well as increase health.

HPLC in poliekstrakte dry established the presence of ten marker components, raw source of which is rhubarb ordinary (dezoksiratsontitsin, rapontitsin, rapontigenin, caffeic acid), licorice Ural (likviritin, glycyrrhizic acid), madder dyeing (alizarin, purpurin, ruberitrinovaya acid lutsidinprimverozid) drawing 1.

Fig. 1. Chromatogram (HPLC) of alcohol extraction from the extract "Licorice-7. The numbers indicate the compounds: 1 - caffeic acid; 2 - deoxyraponticin; 3 - liquiquirithin; 4 - raponticin; 5 - lucidin primrovezide; 6 - ruberitric acid; 7 - rapontigenin; 8 - alizarin; 9 - glycyrrhizic acid; 10 - purpurine.

The dominant compounds of the licorice 7 extract are anthraquinones, the content of which was 7.65 mg / g; stelbene content 6.13 mg / g; terpenes (glycyrrhizic acid) 1.67 mg / g, flavonoids and phenolcarboxylic acids account for 2.07 and 1.12 mg / g, respectively.

quantitation

For quantitative analysis of the polyextract, the use of glycyrrhizic acid content has been proposed [3, 4]. It is proposed to use state standard samples of glycyram (monoammonium salt of glycyrrhizic acid) since glycyrrhizic acid is an unstable substance, which prevents the use of this compound as a state standard sample.

In the quantitative determination of the glycyrrhizic acid content, the electronic spectrum of the polyextract contains one intense absorption maximum at a wavelength of 258 ± 2 nm (Fig. 2), which correlates with that of glycyrrhizic acid and glycyram [3].

Methods for the quantitative determination of glycyrrhizic acid in dry polyextract.

0.5 g (accurately weighed) of the polyextract is placed in a conical flask with a thin section with a capacity of 250 ml, 20 ml of 95% alcohol are added and mixed. Then add 50 ml of a 3% acetone solution of trichloroacetic acid and heat for 10 minutes. After cooling, the resulting solution is filtered through a paper filter, the flask is washed with two portions of a 10% trichloroacetic acid solution of 10 ml each, filtered through the same filter. Concentrated ammonia solution is added dropwise to the obtained filtrate until an abundant precipitate appears (pH from 8.3 to 8.6 according to the universal indicator). The solution with the precipitate is transferred to an ashless filter placed in a Buchner funnel. The flask and filter are washed with 50 ml of acetone in three steps. The filter cake was transferred to the flask in which the precipitation was carried out, dissolved in 50 ml of water, quantitatively transferred to a 250 ml volumetric flask and the volume of the solution was adjusted to the mark with water (solution A). 1 ml of solution A is placed in a 25 ml volumetric flask and the volume of the solution is adjusted to the mark with water (solution B). The optical density of solution B was measured on a spectrophotometer at a wavelength of 258 nm in a cuvette with a layer thickness of 10 mm, using water as a comparison solution.

In parallel, the optical density of solution B of GSO glycyram is measured. The content of glycyrrhizic acid in the polyextract in terms of glycyr in percent (X) is calculated by the formula:

where D is the optical density of the test solution (B) at a wavelength of 258 nm; D _O is the optical density of the GSO solution of glycyram (B) at a wavelength of 258 nm; m is the mass of dry extract in g; m _o - mass GSO of glycyram in grams

The error of a single determination with a confidence probability of 4.78% (Table 1).

It was determined that the total content of saponins, in terms of glycyrrhizic acid in the dry polyextract, with a confidence level of 95%, was 0.55%. The error of a single determination does not exceed 5%; the experimental results can be considered satisfactory.

The proposed method of obtaining 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 regimes, 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 terpenes in terms of glycyrrhizic acid. 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 terpenes, taking into account the yield of extractive substances.

Identification of the optimal degree of grinding of raw materials was carried out for collection as a whole. A quantitative assessment was carried out according to the yield of extractives and the sum of terpenes. 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, the influence of temperature on the extraction process was studied in the temperature range from 20 to 60 ° С. The data are presented in table 10, the optimum temperature is 60 ± 5 ° C.

When studying the influence of the ratio of raw material to 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 sum of terpenes increase, but this increase for ratios of 1:16 and above is insignificant, therefore, to prevent additional consumption of extractant, a ratio 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 terpenes in the raw material-extractant system during a three-time extraction. For this purpose, the crushed raw materials were extracted with 50% 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 a reflux condenser. The extracts obtained at regular intervals (15, 30, 45, 60, 75, 90 min) during three extractions were analyzed for the content of extractives and terpenes. 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.

It is currently not possible to calculate the economic efficiency of the proposed method, however, the above advantages in combination 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.

Sources of information

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Claims (1)

A method of obtaining a medicinal product with adaptogenic activity, which is a dry extract obtained from the following plant materials in the ratio: roots and rhizomes of Ural licorice 178.57 g; wood caraganes mane 321.42 g; roots bought fragrant 53.57 g; roots bought squat 53.57 g; male Orchis tubers 178.57 g; the roots of rhubarb ordinary 107.14 g; roots and rhizomes of madder madder 107.14 g, crushed to a particle size of 1 mm in 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 and the combined extracts are evaporated to 1 / 3 of the initial volume and purified by separation, after which the purified extract is added to 1/5 of the initial volume, dried in a vacuum dryer at a temperature of 60 ° C for 8 hours and ground in a propeller-type mill.

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