RU2836177C1 - Method of producing phytosubstance with adaptogenic and antioxidant activity (versions) - Google Patents

Abstract

FIELD: pharmaceutics.

SUBSTANCE: group of inventions relates to pharmaceutical industry, namely to methods of producing a phytosubstance in form of a dry extract with adaptogenic and antioxidant activity. Method of producing a phytosubstance in the form of a dry extract with adaptogenic and antioxidant activity, involving extraction of crushed Schisandra chinensis raw material with 70% ethyl alcohol, wherein raw material used is Schisandra chinensis leaves, which are crushed to particles with size of 3.0 mm, extraction is carried out at ratio of raw material and extractant 1:30 by percolation, then obtained extract is purified with ethyl alcohol 95%, purified extract is condensed under vacuum and dried till moisture content is not more than 5.0%. Method of producing a phytosubstance in the form of a dry extract with adaptogenic and antioxidant activity, involving extraction of crushed Schisandra chinensis raw material with 70% ethyl alcohol, wherein raw material used is Schisandra chinensis shoots, which are crushed to particles with size of 5.0 mm, extraction is carried out at ratio of raw material and extractant of 1:20 by percolation, then the obtained extract is purified with ethyl alcohol 95%, the purified extract is condensed under vacuum and additionally dried till moisture content is not more than 5.0%.

EFFECT: above described methods enable to obtain a phytosubstance with adaptogenic and antioxidant action in the form of a dry extract.

2 cl, 3 tbl, 2 ex

Description

The group of inventions relates to the pharmaceutical industry and is aimed at the development and use of pharmaceutical substances with adaptogenic and antioxidant activity.

The growing demand for natural medicines leads to the depletion of natural reserves, so the introduction of medicinal plants into cultivation, especially valuable, rare, endangered species, as well as the rational use of raw materials is particularly important [Hamilton A.S. Medicinal plants, conservation and livelihoods. Biodivers Conserv. 2004; 13: 1477-517]. The approach to the use of cultivated valuable medicinal plants in pharmacy has a number of major advantages, the main one of which is a sufficient raw material base [Orlovskaya T.V. Pharmacognostic study of some cultivated plants in order to expand their use in pharmacy: author's abstract. diss. doctor of pharmaceutical sciences: Orlovskaya T.V. - Pyatigorsk, 2011: 50]. Expanding the range of drugs with adaptogenic action remains relevant today, the solution to this issue is achieved through the use of new types of medicinal plants or the use of other morphological groups of raw materials that were not previously used.

The following preparations are obtained from Schisandra chinensis: “Schisandra seed tincture”, “Schisandra fruit tincture” with a general tonic effect (ICD-10), which are included in the State Register of Medicines of Russia (2024) [State Register of Medicines [Electronic resource]. Access mode: https://grls.pharm-portal.ru. Access date: 06.07.2024].

There is a well-known “Lemongrass seed tincture” (FS.3.4.0029.22) from the seeds of a wild vine with a lignan content in terms of schizandrin of at least 0.3%, obtained by maceration in 95% ethyl alcohol [https://pharmacopoeiaTegmed.ra/pharmacopoeia/izdanie-14/1/1-1/statisticheskaya-obrabotka-rezultatov-khimicheskogo-eksperimenta/?ysclid=lua6jxinli93 9937061 [Electronic resource] (date of access: 07/08/24)].

The closest analogue of the invention is the method for obtaining the product "Chinese magnolia vine tincture", which consists in the fact that Chinese magnolia vine seeds are extracted with 70-90%) ethyl alcohol first at room temperature, and then by heating in the temperature range of 65-70°C, i.e. extraction is used in three extractors. The method ensures obtaining a tincture with a content of the sum of lignans in the preparation in terms of γ-schisandrin of 2.12% [patent RU 2368388, 2009].

The disadvantage of analogues is the use of seeds as medicinal raw materials and the irregularity of fruiting of wild magnolia vine, which can affect the yield. Chinese magnolia vine is a Japanese-Manchurian endemic with an East Asian type of range [Plant resources of Russia: Wild flowering plants, their component composition and biological activity. Family Magnoliaceae - Juglandaceae, Ulmaceae, Moraceae, Cannabaceae, Urticaceae / Ed. A.L. Budantsev. - St. Petersburg; Moscow: Partnership of scientific publications KMK. 2008; 2: 421]. In conditions of cultivation of the vine in a temperate climate, due to weather conditions, the fruits (seeds) do not always ripen. But at the same time, the vine has natural vitality and a high shoot-forming capacity [Bilalova R.A. Experience of introduction of Schisandra chinensis in the Southern Urals / Bulletin of RUDN. Series: Agronomics and Animal Husbandry. 2022; 17 (4): 448-454].

The overall objective of the invention is to obtain new medicinal pharmaceutical substances from the leaves and shoots of Schisandra chinensis (Turcz.) Baill., and to expand the range of agents with adaptogenic and antioxidant activity.

The single technical result of using the invention is the production of a phytosubstance with adaptogenic and antioxidant action in the form of a dry extract.

The proposed method for obtaining a phytosubstance with adaptogenic and antioxidant activity (option 1) is carried out as follows. The leaves of Chinese magnolia vine - S. chinensis (Turcz.) Baill. are crushed to particles of 3.0 mm in size, the ratio of raw materials to extractant is 1:30, for soaking the leaves are poured with 70% ethyl alcohol in a volume equal to the mass of the raw materials, and left for 5 hours in a closed container. Then the raw materials are loaded into a percolator, tightly tamped, poured with the same extractant to a "mirror" and infused for 24 hours. Then percolation is carried out at a rate of 1/24 of the working volume per hour. The resulting percolates are collected separately: the first portion (concentrated) is obtained from the percolator, the second portion - until the raw materials are completely depleted, which is evaporated under vacuum at a T of 50-60 ° C, a vacuum of 600-650 mm Hg. st. until the calculated volume is obtained. The extracts are combined, concentrated, purified with 95% ethyl alcohol. The purified extract is then concentrated under vacuum and dried in a drying cabinet at a temperature of no more than 60°C to a humidity of no more than 5.0%.

The proposed method for obtaining a phytosubstance with adaptogenic and antioxidant activity (option 2) is carried out as follows. The shoots of Chinese magnolia vine - S. chinensis (Turcz.) Baill. are crushed to particles of 5.0 mm in size, the ratio of raw materials to extractant is 1:20, for soaking the shoots are poured with 70% ethyl alcohol in a volume equal to the mass of the raw materials, and left for 5 hours in a closed container. Then the raw materials are loaded into a percolator, tightly tamped, poured with the same extractant to a "mirror" and infused for 48 hours. Then percolation is carried out at a rate of 1/24 of the working volume per hour. The resulting percolates are collected separately: the first portion (concentrated) is obtained from the percolator, the second portion - until the raw materials are completely depleted, which is evaporated under vacuum at a T of 50-60 ° C, a vacuum of 600-650 mm Hg. st. until the calculated volume is obtained. The extracts are combined, concentrated, purified with 95% ethyl alcohol. The purified extract is then concentrated under vacuum and dried in a drying cabinet at a temperature of no more than 60°C to a humidity of no more than 5.0%.

Experimental studies have shown that the highest yield of extractive substances was observed from extracts on 70% ethyl alcohol under the following conditions: for leaves of S. chinensis (Turcz.) Baill. - at a ratio of raw material to extractant of 1:30 with a grinding of 3.0 mm; for shoots of S. chinensis (Turcz.) Baill. - at a ratio of raw material to extractant of 1:20 with a grinding of 5.0 mm. Of the traditional extraction methods used, it turned out that the completeness of extraction was observed when using the percolation method. For this purpose, a weighed portion of plant material with an exact mass was crushed to a particle size of 3.0 mm for leaves (the ratio of raw material to extractant was 1:30) or 5.0 mm for shoots (the ratio of raw material to extractant was 1:20), filled with 70% ethyl alcohol in a volume equal to the mass of the raw material, and left for 5 hours (raw material wetting stage). Then the raw material was loaded into a percolator, tightly tamped and filled with the same extractant to a "mirror". Extraction of biologically active substances in leaves was carried out for 24 hours and 48 hours in shoots. Then percolation was carried out at a rate of 1/24 of the working volume per hour. The obtained percolates were collected separately: the first portion (concentrated) was obtained from the percolator, the second portion - until the raw material was completely depleted, which was evaporated under vacuum (T 50-60 °C, vacuum 600-650 mm Hg) until the calculated volume was obtained. The extracts were combined, concentrated, purified with 95% ethyl alcohol. The purified extract was further concentrated and dried in a drying cabinet at T no more than 60 °C to a humidity of no more than 5.0%) [Chueshov V.I., Chernov N.E., Khokhlova L.N., Boguslavskaya L.I., Pashnev P.D., Lyapunova O.A. et al. Industrial technology of drugs. - X.: MTK-Book; NFAU Publishing House. 2002; 2: 716].

The proposed invention is illustrated by the following examples.

Example 1. Leaves of S. chinensis (Turcz.) Baill. weighing 100.0 are crushed to 3.0 mm, the ratio of raw material to extractant is 1:30, poured with 70% ethyl alcohol in a volume of about 100 ml and left for 5 hours to soak the raw material in a closed container. Then the raw material is loaded into a percolator, tightly tamped, poured with the same extractant to a "mirror" and infused for 24 hours. Then percolation is carried out at a rate of 1/24 of the working volume per hour. The resulting percolates are collected separately: the first portion (concentrated) is obtained from the percolator, the second portion - until the raw material is completely depleted, which is evaporated under vacuum: T 50 ° C, vacuum 600 mm Hg. When combining percolates, the resulting volume is 3000 ml. The extract is then concentrated and purified with 95% ethyl alcohol. The purified extract is then concentrated under vacuum and dried in a drying cabinet at a temperature of no more than 60°C to a humidity of no more than 5.0%).

Example 2. Shoots of S. chinensis (Turcz.) Baill. weighing 100.0 are crushed to 5.0 mm, the ratio of raw material to extractant is 1:20, poured with ethyl alcohol (70%) in a volume of about 100 ml and left for 5 hours to soak the raw material in a closed container. Then the raw material is loaded into a percolator, tightly tamped, poured with the same extractant to a "mirror" and infused for 48 hours. Then percolation is carried out at a rate of 1/24 of the working volume per hour. The resulting percolates are collected separately: the first portion (concentrated) is obtained from the percolator, the second portion - until the raw material is completely depleted, which is evaporated under vacuum: T 60 ° C, vacuum 650 mm Hg. When combining percolates, the resulting volume is 2000 ml. The extract is then concentrated and purified with 95% ethyl alcohol. The purified extract is then concentrated under vacuum and dried in a drying oven at a temperature of no more than 60°C to a humidity of no more than 5.0%.

The obtained phytosubstances: dry extracts of leaves and shoots of S. chinensis (Turcz.) Baill. were powdery loose masses of dark brown color with a specific odor and bitter taste. Dry extracts of leaves and shoots of S. chinensis (Turcz.) Baill. are well soluble in water and ethyl alcohol, forming cloudy solutions, and are insoluble in organic solvents. The moisture content of dry extracts from leaves and shoots of S. chinensis was 3.67±0.06% and 4.63±0.11%, respectively (no more than 5.0%).

The obtained results of the quantitative content of marker components in dry extracts of leaves and shoots of S. chinensis were: flavonoids in terms of rutin 6.22%±0.14%) and 8.96%)±0.21% (should be at least 6.0%) and 8.0%, respectively), lignans in terms of schizandrin A - 5.40±0.11% and 15.56%±0.32% (should be at least 5.0% and 15.0%), respectively), saponins in terms of β-escin - 19.37%±0.57% and 15.43%±0.40% (should be at least 19.0% and 15.0%, respectively).

It is known to use "Schisandra seeds tincture" as a stimulant that affects the central nervous system, cardiovascular and respiratory systems, increases blood pressure, mental and physical performance. Indications for use: mental and physical overstrain, convalescence after somatic and infectious diseases, to increase the body's performance, in complex therapy for asthenic syndrome [State Register of Medicines [Electronic resource]. Access mode: https://grls.pharm-portal.ru. Access date: 06.07.2024. 2) Zilfikarov I.N. Natural drugs: chemical composition and standardization. Reference and scientific-practical publication. - M: Publishing house "SLON PO". 2021: 712]. "Lemongrass fruit tincture" has hepatoprotective, immunomodulatory, hypoglycemic, antiallergic, stress-regulating properties, and also increases hematopoiesis and the body's resistance to intoxication, including alcohol [Plant resources of Russia: Wild flowering plants, their component composition and biological activity. Supplements to volume 1 / Ed. A. L. Budantsev. - St. Petersburg; M: KMK Scientific Publications Partnership. 2018: 409].

In the scientific, medical and patent literature, no information was found about the known production of phytosubstances from the leaves and shoots of S. chinensis (Turcz.) Baill. (Turcz.) Baill. (Turcz.) Baill. and their use as agents with adaptogenic and antioxidant activity. The obtained dry extracts, used in the below-listed methods for determining biological activity, were dissolved in purified water to obtain aqueous solutions in a ratio of 1:30 for dry extracts from leaves and 1:20 from shoots.

The adaptogenic effect of phytosubstances from S. chinensis leaves and shoots was studied on white male mice (n=56, m 18.0-25.0 g) in 2 reproduction systems. In 1 system, normobaric hypoxia with hypercapnia was reproduced, in which laboratory animals were placed in containers (V=250 ^cm3 ) and the reserve time under hypoxic conditions was recorded. After the experiment, the animals were decapitated, the brain was removed and biochemical studies were immediately carried out. The antioxidant status in homogenates was determined by measuring the level of malondialdehyde (nmol/g raw tissue) and catalase (mkat/g raw tissue).

In the 2nd system, physical performance was assessed by the duration of swimming of mice with a load (~ 7% of the mass) on the 15th day of the experiment until complete fatigue (immersion of the animal under water and refusal to swim). After swimming, the mice were dried and the experiment was repeated an hour later. To reduce the impact of stress, the mice were previously accustomed to water: swimming without a load for 1 min (3 times, with an interval of 3 days, water T 26-28 ° C).

Three groups of animals were used for the study: 1 - the experimental group, which was given an aqueous solution of dry extracts of Schisandra leaves and shoots to drink; 2 - the comparison group, which was given an aqueous solution of pre-dealcoholized "Schisandra seed tincture", included in the state register of medicines; 3 - the control group, which was given distilled water to drink. Aqueous solutions of the extracts were administered orally to male mice for 14 days, 3 drops at a time. The volume of aqueous solutions of extracts was calculated based on the recommended daily dose for an adult (m=70 kg) - 30 drops 3 times a day: 30 drops * 3 = 90 drops / day, for a weight of 1 kg there is approximately 1.3 drops / kg. The daily dose for mice (m=20.0 g) is 0.026 drops. For the possibility and convenience of dosing, the aqueous solution of dry extracts was diluted 100 times. Therefore, mice weighing 20.0 g were administered 3 drops.

According to the obtained results, under normobaric hypoxia, S. chinensis leaf extracts increased the lifespan of mice by 2.4 times (p<0.044108), and S. chinensis shoot extracts - by 2.5 times (p<0.040745) relative to the control group. The experimental groups were practically not inferior in activity to the comparison drug (p<0.0134 relative to the control). Studies of the antioxidant status in the experimental groups showed that the level of malondialdehyde was higher than in the control, which is probably explained by a longer lifespan under hypoxia. An increase in destructive changes in cells leads to an increase in the activity of antioxidant defense enzymes. Thus, it can be observed that the catalase level in animals of the experimental group and the comparison drug is higher than in the control group. At the same time, the experimental group from leaf extracts showed almost the same results as the comparison drug group, and the group from shoot extracts - more (Table 1).

The physical endurance test showed that with repeated swimming with a load, the duration of swimming increased in all groups: in the comparison drug and in the experimental groups, almost 2 times relative to the time of the first swim, and in the control group - 1.2 times (Table 2).

The antioxidant activity (AOA) of the phytosubstances was determined by the chemiluminescent method in vitro . A 0.05% solution of ascorbic acid and a decoction of rose hips (1:10) were used as a comparison drug. Aqueous extracts (0.01-1 ml) were introduced into model systems in which the formation of active oxygen species (ROS) was formed and lipid peroxidation (LPO) reactions occurred.

1 model system consisted of a phosphate buffer with a solution of sodium citrate and luminol (pH 7.5), into which 1 ml of 50 mM iron sulfate solution was introduced as an initiator of the oxidation process. The luminescence was recorded for 5 minutes.

The 2 model system included lipoprotein complexes prepared from chicken yolk homogenized with phosphate buffer (1:5). The initiator of the oxidation process was also a 50 mM solution of iron sulfate.

Model systems without adding the studied extracts served as controls. Luminescence was recorded using the “KhLM-003” device. AOA was assessed by the decrease in luminescence intensity (Table 3).

It was revealed that aqueous extracts of phytosubstances have a pronounced AOA and affect both the formation of ROS and the LPO process.

Thus, the use of the invention ensures:

1. The obtained phytosubstances from the leaves and shoots of Schisandra chinensis have an adaptogenic effect due to increased physical endurance, antihypoxic and antioxidant effects, which allows expanding the range of herbal medicines on the Russian pharmaceutical market.

2. The proposed phytosubstances (dry extracts) from the leaves and shoots of Schisandra, obtained by percolation with 70% ethyl alcohol, allow the extraction of a complex of substances of phenolic (flavonoids and lignans) and triterpene nature.

3. Pharmaceutical substances can be produced in industrial conditions at pharmaceutical enterprises as independent medicinal products, and can also be used to obtain other medicinal forms.

Claims (2)

1. A method for obtaining a phytosubstance in the form of a dry extract with adaptogenic and antioxidant activity, including the extraction of crushed raw materials of Chinese magnolia vine with 70% ethyl alcohol, characterized in that the raw materials used are magnolia vine leaves, which are crushed to particles of 3.0 mm in size, the extraction is carried out at a ratio of raw materials and extractant of 1:30 using the percolation method, then the resulting extract is purified with 95% ethyl alcohol, the purified extract is concentrated under vacuum and dried to a moisture content of no more than 5.0%. 2. A method for obtaining a phytosubstance in the form of a dry extract with adaptogenic and antioxidant activity, including the extraction of crushed raw materials of Chinese magnolia vine with 70% ethyl alcohol, characterized in that the raw materials used are Schisandra shoots, which are crushed to particles of 5.0 mm in size, the extraction is carried out at a ratio of raw materials and extractant of 1:20 using the percolation method, then the resulting extract is purified with 95% ethyl alcohol, the purified extract is concentrated under vacuum and dried to a moisture content of no more than 5.0%.