RU2769666C1 - Liquid extract of jiaogulan, having adaptogenic activity - Google Patents

Abstract

FIELD: pharmaceutics.

SUBSTANCE: invention relates to pharmaceutical industry, namely to use of an herbal preparation as an agent with adaptogenic activity. Use of a liquid extract of jiaogulan herb, obtained by percolation with 70 % ethyl alcohol in ratio of 1:1, as an agent with adaptogenic activity.

EFFECT: above-described use of a liquid extract of jiaogulan herb, obtained by percolation with 70 % ethyl alcohol in ratio of 1:1, provides a pronounced antihypoxic and antioxidant effect, increased physical endurance, increased activity of enzymes of antioxidant protection — catalase.

1 cl, 4 tbl

Description

The present invention relates to pharmacy, namely to herbal medicines used as therapeutic and prophylactic adaptogenic agents.

Currently, the basis of adaptogenic, general tonic drugs are drugs from such medicinal plants as: real ginseng, Chinese magnolia vine, prickly eleutherococcus, leuzea safflower, Rhodiola rosea, growing in the Far East, Siberia [Shabanov P.D. Adaptogens and antihypoxants // Reviews of clinical pharmacology and drug therapy. - 2003. - Vol. 2. - No. 3. - S. 50-81]. In this regard, there is a question of expanding the range of adaptogenic drugs and the introduction into practice of previously little-studied types of medicinal plant materials through the search for new introduced species. One of such promising crops adapted to the conditions of the Republic of Bashkortostan is Gynostemma pentaphyllum (Thunb.) from the Cucurbitaceae family.

A widespread phytoadaptogen is real ginseng. As of 2021, the State Register of Medicines of Russia includes herbal remedies based on real ginseng, which have a general tonic effect: Ginseng tincture, Herbion ginseng, Doppelherz Ginseng, Ginseng with vitamin C [State Register of Medicines [Electronic resource]. Access mode: http://grls.rosminzdrav.ru/default.aspx. Access date: 05/10/2021].

The prototype of the invention is the well-known drug "Ginseng tincture" from the roots of this ginseng, which has a number of disadvantages in terms of procurement of raw materials: the average mass of raw materials must be at least 10.0 g; the age of the plant in the wild is 25-30 years, in the cultivated one - 5-8 years.

The objective of the invention is to expand the range of adaptogenic herbal products.

The technical result when using the invention is to obtain an agent that provides an increase in physical endurance, antihypoxic and antioxidant effects.

The essence of the invention: the use of a liquid extract of the herb Gynostemma pentaphyllum, obtained by percolation with 70% ethyl alcohol in a ratio of 1:1, as a means with adaptogenic activity.

Receiving technology. The liquid extract was obtained by percolation. To do this, an accurate sample of plant material was crushed to a particle size of 3.0 mm and filled with an equal volume in relation to the sample of 70% ethyl alcohol and left to wet the raw material for a day. Then the raw materials, tightly tamping, were loaded into a percolator, filled with 70% ethyl alcohol to a “mirror” and left for extraction for 48 hours to achieve an equilibrium concentration. Further percolated at a rate of 1/48 of the working volume per hour. In this case, the first portion of the product was collected in a separate container in the amount of 85% relative to the mass of the raw material. Then the percolation was carried out into another container until the raw material was completely depleted, until the outflowing solution became colorless. The second portion of the resulting product was evaporated under vacuum at a temperature of 50-60°C to 15% relative to the mass of raw materials. Both products were combined and an extract was obtained in a ratio of 1:1 [Alekseev, K.V. Pharmaceutical technology / K.V. Alekseev, S.A. Kedik. - M.: AO IFT, 2019. - 570 p.].

The resulting liquid extract was a greenish-brown liquid with a spicy-herbaceous odor. The finished extract was placed in tightly sealed dark glass bottles and stored in a place protected from light at a temperature of 15 to 25°C [State Pharmacopoeia of the Russian Federation. - XIV ed. - M., 2018. - T. 2 - 3262 p.].

An alcoholic extract of Gynostemma pentaphyllum is known to be used to treat metabolic diseases such as obesity, diabetes, or hyperlipidemia [CN 102470155, 2012].

In the available scientific, medical and patent literature, information on the popularity of the use of a liquid extract of the herb Gynostemma pentaphyllum, obtained by percolation with 70% ethyl alcohol in a ratio of 1: 1, as a means with adaptogenic activity was not found.

The preliminarily obtained liquid extract and reference drugs used in the following methods were dealcoholized, followed by dissolution of the dry residue in distilled water to obtain an aqueous extract.

The adaptogenic effect of a liquid extract of the herb Gynostemma pentaphyllum was studied on 48 outbred male mice weighing 18.0-25.0 g in two model systems.

In the first model system, normobaric hypoxia with hypercapnia was formed. Animals of the same weight were placed one at a time in hermetically sealed jars with a volume of 250 cm ³ . The survival time (reserve time) of the animals under hypoxic conditions was recorded and the percentage increase in the reserve time relative to the control was calculated. At the end of the experiment, the animals were decapitated, the brain was removed, weighed, and homogenized in 3 ml of cold Tris-HCl buffer (pH 7.4). Biochemical studies were carried out immediately. To determine the antioxidant status in the homogenates, the level of malondialdehyde (nmol/g wet tissue) and the level of catalase (mca/g wet tissue) were measured.

The second model was based on the evaluation of performance by the duration of swimming of mice with a load (7% of the animal weight). To do this, on the 15th day of the experiment, mice were forced to swim with a load attached to their backs until they were completely tired (until the animal was submerged under water and refused to swim), then the mice were dried and swimming with a load was repeated an hour later. To reduce the stress component of the test, mice were first accustomed to water in the forced swimming mode without load (3 times, with an interval of 3 days, the duration of stay in water was 1 min). The temperature of the water in the vessel was maintained at 26-28°C (thermoneutral for mice).

Animals divided or into three groups:

No. 1 - experimental group, in which the animals were fed with an aqueous extract of percolate of the herb Gynostemma pentaphyllum;

No. 2 - comparison group, in which the animals were fed with a water extract from the well-known drug "Ginseng tincture", included in the state register of medicines;

No. 3 - the control group, in which the animals were fed only with distilled water.

Aqueous extracts from the studied plant material were administered to male mice orally for 14 days, 3 drops each. The extraction volume was calculated based on the recommended daily dose for an adult weighing 70 kg - 30 drops three times a day: 30 drops * 3 = 90 drops per day, 90 drops / 70 kg = 1.29 drops / kg. The daily dose for mice weighing 20.0 g is 0.026 cap. For ease of dosing, the liquid extract (1:1) was diluted 100 times. Therefore, mice weighing 20.0 g were injected with 3 drops.

According to the obtained data, the extract of gynostemma pentaphyllum increased the lifespan of mice under normobaric hypoxia by 1.3 times (P<0.042285) relative to the control group. The experimental extract was not inferior in activity to the ginseng extract (P<0.011387 relative to the control). In the experimental groups, the level of malondialdehyde is higher than in the control, which is probably due to a longer life expectancy under hypoxic conditions. With an increase in destructive processes in the cell, the activity of antioxidant defense enzymes, in particular, catalase, increases, which can be observed in animals of the control group and the gynostemma group. At the same time, no increase in the level of catalase was noted in the ginseng group (Table 1).

The performance test showed that during repeated swimming with a load, the duration of swimming increased in all groups. Animals from the gynostemma group lasted longer, their time increased almost 2 times relative to the time of the first swimming, in the ginseng group - 1.3 times and in the control group - 1.2 times (Table 2).

In the ginseng group, the duration of swimming in both of their swims was the shortest compared to other groups: the animals very quickly acquired a vertical position, hung in the water and drowned.

Acute toxicity of an aqueous extract of the herb Gynostemma pentaphyllum was carried out on 24 outbred male and female mice weighing 18.0-25.0 g by oral administration of an aqueous extract of the herb Gynostemma pentaphyllum in doses: 5000, 7500, 10000 mg/kg of animal weight in terms of by dry weight.

Previously, the animals were prepared for the study according to the scheme: fasting, marking, weighing, division into groups (4 groups of 6 animals). Then, after a single injection of the infusion, the state of laboratory animals was monitored for two weeks: appearance, behavioral response, activity, respiratory rate.

With the introduction of the studied doses of an aqueous extract of the herb Gynostemma pentaphyllum, no signs of intoxication were noted, all animals were alive, mobile, had a good appetite, and no deaths were observed. The maximum dose of the infusion is 10,000 mg/kg (Table 3). Further toxicological studies were not carried out due to the apparent non-toxicity of the studied raw materials. Thus, according to the classification of GOST 12.1.007.76, the herb Gynostemma pentaphyllum was assigned to the class of low-toxic compounds, which makes it possible to judge the safety of these types of raw materials [GOST 12.1.007-76 System of labor safety standards. Harmful substances. Classification and general safety requirements].

The antioxidant activity of the aqueous extract was determined by two methods.

The first method consisted in the study of an aqueous extract of various concentrations in vitro by the chemiluminescent method. A 0.05% solution of ascorbic acid was used as a reference preparation.

An aqueous extract (0.01–1 ml) was introduced into model systems in which the formation of reactive oxygen species (ROS) was formed and lipid peroxidation (LPO) reactions took place, which are the most common free radical oxidation processes. As a first model system, 20 ml of phosphate buffer supplemented with sodium citrate and luminol (pH 7.5) was used. Added 1 ml of 50 mm solution of iron sulfate, as the initiator of the oxidation process. Oxidation of iron salts causes the appearance of oxygen radicals and is accompanied by chemiluminescence, which is enhanced in the presence of luminol. Registration of the glow was carried out for 5 minutes.

As the second model system, lipoprotein complexes similar to blood lipids prepared from chicken yolk were used. The yolk was mixed with phosphate buffer in a ratio of 1:5 and homogenized. As an initiator of the process of oxidation of unsaturated fatty acids, 1 ml of a 50 mm solution of iron sulfate was added. Model systems without the addition of the studied extracts served as controls. Registration of the glow was carried out on the device "KhLM-003". Antioxidant activity was assessed by the decrease in luminescence intensity (Table 4).

It was found that the aqueous extract has a pronounced antioxidant activity and affects both the formation of reactive oxygen species and the rate of lipid peroxidation.

The second method was based on the ability of adrenaline to autoxidize in an alkaline medium, with the formation of a product with an absorption maximum at 347 nm. To 2 ml of bicarbonate buffer (pH=10.56) was added 0.1 ml of 0.1% adrenaline hydrochloride solution (control solution) and after 10 minutes the optical density was determined at a wavelength of 347 nm in a cuvette 10 mm thick on a ShimadzuUV1800 spectrophotometer (D ₁ ). To 2 ml of bicarbonate buffer a was added 0.03 ml of the test solution and 0.1 ml of a 0.1% solution of adrenaline hydrochloride, after 10 minutes the optical density was determined (D ₂ ).

Antioxidant activity was calculated by the formula:

AOA=(D ₁ -D ₂ )*100/D ₁

An AOA value of more than 10% indicates the presence of antioxidant activity. When determining the AOA, it was also taken into account that the studied extract has its own color, which absorbs a certain wavelength in the visible region of the spectrum. The value of antioxidant activity in the determination was 50.5%.

Thus, the use of the invention provides an adaptogenic effect by increasing physical endurance, antihypoxic and antioxidant effects, and allows you to expand the range of herbal medicines on the Russian pharmaceutical market.

Claims (1)

The use of a liquid extract of the herb Gynostemma pentaphyllum, obtained by percolation with 70% ethyl alcohol in a ratio of 1:1, as an agent with adaptogenic activity.