RU2654709C1 - Anti-inflammatory agent - Google Patents

Abstract

FIELD: pharmaceutics.

SUBSTANCE: invention relates to the pharmaceutical industry, namely to an anti-inflammatory agent. Anti-inflammatory agent, which is a tincture of the dried and crushed herb of Hedysarum fruticosum Pallas, obtained by percolation using 70 % ethyl alcohol as an extractant, at the ratio of raw materials : extractant 1:10, while in the soaking stage 11.29 ml of 70 % ethyl alcohol are poured into 10 g of raw materials.

EFFECT: obtained product withstands acute toxicity tests and has a high anti-inflammatory effect.

1 cl, 6 dwg

Description

The invention relates to medicine, namely to the treatment of inflammatory diseases, and can be used as an anti-inflammatory agent. An anti-inflammatory agent is proposed, which is a 70% tincture of the dried and crushed aerial parts of shrubby hedgehog (Hedysarum fruticosum) obtained by percolation in the ratio of raw material: extractant 1:10.

Pharmacological regulation of inflammatory processes is an urgent problem of medical science and practice. Currently, synthetic anti-inflammatory drugs are known, for example, acetylsalicylic acid (ASA), indomethacin, and some others (Mashkovsky M.D. Medicines: T. 2. - M., 1998. - 592 p.). However, in some cases, their use is limited due to the high possibility of developing undesirable drug reactions, poor tolerance. In addition to synthetic drugs, phytopreparations from medicinal plants are known to have anti-inflammatory effects. They have several advantages: better tolerance, less toxicity, polyfunctionality.

The objective of the invention is to obtain tinctures based on natural plant materials with high anti-inflammatory activity and low toxicity.

The essence of the invention lies in the fact that the proposed tincture of the dried and crushed above-ground parts of shrubby penny obtained by percolation using 70% ethyl alcohol as extractant in a ratio in the weight parts of the raw material: extractant 1:10. A method of manufacturing an anti-inflammatory agent includes obtaining tinctures by the method of percolation, characterized in that in stage 1 of percolation, 10 g of dried and ground aerial parts of shrubby penny are poured with 11.29 ml of 70% ethyl alcohol.

Percolation - filtering the extractant through the plant material in order to extract soluble substances in the extractant. The percolation method includes three successive stages: soaking the raw material (swelling), infusion and percolation itself.

Soaking (swelling) is carried out outside the percolator. For soaking use from 50 to 100% extractant relative to the weight of the raw material. After stirring, the raw materials are left for 4-5 hours in a closed container. During this time, the extractant penetrates between the particles of plant material and into the cells, the raw material swells, increasing in volume. In this case, the active substances dissolve inside the cell.

Infusion is the second stage of the percolation process. Swollen or dry material is loaded into the percolator and filled with extractant until a “mirror” is formed, the layer height above the raw material should be about 30-40 mm, and infusion is carried out for 24-48 hours, during which an equilibrium concentration is reached. For many types of raw materials, the infusion time can be reduced.

Actually percolation - the continuous passage of the extractant through a layer of raw materials and the collection of percolate. At the same time, the percolate is drained and the extractant is simultaneously fed from above at a speed not exceeding 1/24 of the used percolator volume for 1 hour. In this case, the saturated extract is displaced from the plant material by the current of fresh extractant and a difference in the concentrations of extracted substances in the raw material and the extractant is created. The percolation rate should be such that diffusion of the extracted substances into the extract has time to occur. When preparing tinctures, percolation is completed by obtaining five or ten volumes (depending on the properties of the raw material) of the extract in relation to the mass of the loaded raw material (Industrial Technology of Medicines: Textbook. In 2 volumes, Volume 2 / V.I. Chueshov, M.Yu. Chernov, L.M. Khokhlova et al .; Edited by V.I. Chueshov. - Kharkov: MTK-Kniga; NFAA Publishing House, 2002. - 716 p.).

An anti-inflammatory agent is proposed, which is a tincture of the dried and crushed aboveground parts of shrubby penny in the ratio of raw materials: extractant 1:10, obtained by percolation using 70% ethyl alcohol as an extractant.

The proposed tool is manufactured as follows: the collected aerial part of the shrubby penny is dried and crushed using a mill. Sifted through a sieve with a hole diameter of 3 mm. 10 g of crushed raw material is poured into 11.29 ml of 70% ethanol (100% extractant is used for soaking in relation to the mass of the raw material, taking into account the density of the alcohol at 70% concentration equal to 0.8854 g / ml), mix and leave for 4 hours in a closed container. The swollen raw material is placed in a percolator, poured with an extractant to form a “mirror” and insisted for 24 hours. After the time, percolation itself is carried out. Drain percolate and simultaneous supply of extractant is carried out at a speed not exceeding 1/24 of the used percolator volume for 1 hour. Store the resulting tincture in a dark, cool place.

The use of 70% ethyl alcohol as an extractant is due to the fact that the maximum amount of active substances is extracted from the dried and ground aerial parts of shrubby penny with ethyl alcohol of 70% concentration.

Phytochemical screening of tinctures showed the presence of the following groups of biologically active substances (BAS): flavonoids (1.6%), phenolic compounds ((gallic acid (14.6%), protocatechuic acid (11.2%), caffeic acid (7.9% ), chlorogenic acid (24.4%)), organic acids, calculated as malic acid (3.5%), tannins (about 8.5%), ascorbic acid (0.0096%), free sugars (galactose , glucose, fructose, rhamnose), polysaccharides (12.5%), triterpene saponins (2.6% in terms of astragaloside), carotenoids (in terms of β-carotene 0.00428%), amino acid you (3.2% in terms of glutamic acid). The pronounced anti-inflammatory effect of the proposed tincture is due to the presence of phenolic compounds in plant raw materials (Yordanov D., Nikolov P., Boychinov Asp. Phytotherapy. Herbal treatment. - Sofia, 1970. - 344 pp.) The presence of ascorbic acid and tannins as concomitant compounds also justifies the use of tincture of shrubbery as an anti-inflammatory agent.

To determine acute toxicity used the method of B.M. Shtabsky (Shtabsky BM On the methodology for determining the medium-lethal doses and concentrations of chemicals / BM Shtabsky et al. // Hygiene and sanitation. - 1980. - No. 10. P. 49-51.). The studies were carried out on outbred white mice of both sexes weighing 18.0-26.0 g. The proposed tincture was administered once intragastrically at doses of 1 to 5 g / kg (in terms of dry raw materials) in a volume of 0.5 ml. After administration, each group of animals (at least 6 animals) was placed in an isolated cage at standard temperature and food conditions and monitored for 24 hours (LN Sernov Elements of experimental pharmacology / LN Sernov, V.V. Gatsura. - M., 2000 .-- 352 p.).

It was established in the experiment that a single administration of tincture of Lyceum shrub at doses of 2000-5000 mg / kg does not affect the general condition of the animals, symptoms of acute poisoning were not recorded, and there was no death of animals. Since the investigated dose infusions did not lead to the death of any animal, it was concluded that the LD _50> 2000 mg / kg and, in accordance smodifitsirovannoy Classification for Economic Cooperation and Development (OECD), analyzed tincture based on the dried and crushed aerial parts Hedysarum shrub belongs to hazard class IV (low toxic substances), like almost all total phytopreparations (I. Lobanova. Study of acute toxicity and antioxidant activity of dry aspen leaf extract / I.Yu. Lobanova, V.F. Tur Getskova, Ya.F. Zverev, O.S. Talalaeva // Fundamental Research. - 2012. - No. 9. - S. 308-312).

During the entire observation period, the behavioral reactions of the animals of the experimental group were within the physiological norm: normal drinking and eating behavior, normal coordination of movements, normal frequency and depth of respiratory movements, normal consistency of fecal masses, frequency of urination and color of urine were noted. During this time, the mice gained weight (table in Fig. 1).

To prove the anti-inflammatory activity of a 70% tincture from the dried and ground aerial parts of shrubby penny, a study was conducted on a model of formalin edema (Khabriev R.U. Guide to experimental (preclinical) study of new pharmacological substances. - M .: Medicine, 2005. - 832 p.). The experimental work was performed on white male Wistar rats weighing 183.0-226.0 g. The experiment involved 42 animals, which were kept under standard vivarium conditions of the Federal State Budget Educational Institution of Higher Medical Education of the State Medical University of the Russian Ministry of Health under a 12-hour lighting regimen and free access to water and poop. Each group consisted of animals of approximately the same mass in the amount of 6 individuals.

The results of statistical processing of chemical and pharmacological experiments were conducted according to the State Pharmacopoeia (GF) of the XIII edition. Data processing was carried out using Microsoft Excel 2016 packages. Independent groups were compared using the non-parametric Mann-Whitney U-test, the differences were considered significant at p≤0.01 and p≤0.05 (Gubler E.V. Computational methods for the analysis and recognition of pathological processes. - L., 1978 - S. 72).

An example of a specific experiment # 1 (formalin edema model).

The experiments were carried out on white male rats of the Wistar strain weighing 196-219 g. Animals were selected so that the initial average volume of the paws in all experimental groups was approximately the same.

During the experiment, 70% tincture of the dried and ground aerial parts of shrubby penny (25 ml / kg), acetylsalicylic acid (300 mg / kg), 70% ethyl alcohol and distilled water are introduced into the stomach through a probe in a volume of calculation of 5 ml per 200 g of animal mass, observing the time interval before the introduction of formaldehyde for 2 hours and 5 hours and 18 hours after its introduction, and then daily for 7 days.

An acute inflammatory reaction is reproduced by subplant implantation (under the paw aponeurosis) of 0.1 ml of a 2% formalin solution in the form of an aqueous solution in the rat left paw. This causes paw swelling that develops over time. The degree of edema reflects the intensity of inflammation. The magnitude of the edema is measured by the amount of water displaced when the paws are immersed in it.

Experimental animals are divided into 7 groups of 6 animals each. Animals of the first group are intact, the second group is orally treated with purified water, the third - 70% ethyl alcohol. The fourth and fifth groups create inflammation, against the background of which appropriate solvents are introduced: purified water and ethyl alcohol of 70% concentration. Animals of the sixth group on the background of the introduction of formalin give tincture of penny shrub. Animals of the seventh group under similar conditions together with formalin receive a comparison drug, which serves as acetylsalicylic acid at a dose of 300 mg / kg. The paw volume is measured 3, 24, 72, 120, 168 hours after the introduction of formalin into the paw.

Antiexudative effect is studied by the method of oncometry (Trinus F.P., Mohort N.A., Klebanov B.M. Nonsteroidal anti-inflammatory drugs. - Kiev: Health, 1975. - P. 205). The development of edema is evaluated by the formula 1:

where P is the increase in edema;

About - the volume of the paw after the introduction of the inducer of inflammation;

And - the volume of the paw before the introduction of the inducer of inflammation.

The effect of the therapeutic effect (inhibition of inflammation) was evaluated by the degree of suppression of the inflammatory reaction compared to the control and calculated by the formula 2:

where And - the volume of the paw before the introduction of the inducer of inflammation;

About - the volume of the paw after the introduction of the inflammation inducer;

o - a group of animals that received a pharmacological preparation;

to - a group of animals that received an inducer of inflammation.

The results of a comparative study of the effect of prophylactic administration of tincture on the development of formalin-induced edema of rat paw are presented in the table in FIG. 2.

The initial limb volume of intact rats was 1.01 ± 0.01 cm ³ (purified water). In the group of control animals, 1.00 ± 0.01 cm ³ (70% ethyl alcohol). As can be seen, the initial volume of rat paws in all groups was approximately the same and there were no statistically significant differences (p> 0.05), this is consistent with literature data (Mosesova N.S., Kovaleva V.L., Larencova L.I. Experimental study of the anti-inflammatory properties of Mexidol // Proceedings of the III All-Russian scientific-practical conference "Education, science and practice in dentistry" - Moscow. - 2006. - P. 108-110.). With the induction of inflammation with formalin after 3 hours, an increase in limb volume by 0.88 ± 0.04 cm ³ was observed (p <0.01). Against the background of administration of ASA, the limb volume in rats increased compared to the control to 1.66 ± 0.05 cm ³ (p <0.01), the inhibition of inflammation was 35.63%).

With the introduction of the analyzed tincture, the volume of the limb increased compared to the control, respectively, to 1.43 ± 0.03 cm ³ (p <0.01), and the inhibition of inflammation was 50.83%.

Next, the effect of tincture on the dynamics of formalin-induced inflammation was studied. The data from these studies are presented in FIG. 3 and FIG. four.

With the induction of inflammation with formalin, it was found that the inflammatory reaction formed within 3 hours (Fig. 2). This effect reached a maximum on the first day (1.85 ± 0.03 cm ³ (p <0.01), 1.77 ± 0.04 cm ³ (p <0.01)) and persisted for 7 days.

When using ASA in experimental animals, the limb volume after a day significantly increased to 1.69 ± 0.05 cm ³ (p <0.01), with a tendency to decrease on the 3rd day. A return to the original value is noted after 7 days. In the experimental group where tincture was used, the limb volume 1 day after formalin administration was 1.47 ± 0.02 cm ³ (p <0.01), the dynamics of limb volume decrease was observed on the 3rd day, by the 7th day the volume limbs decreased to 1.38 ± 0.02 cm ³ (p <0.01).

Acute inflammation is manifested by characteristic changes in blood leukogram and erythrocyte sedimentation rate (ESR). These parameters were studied in control and experimental animals 3 hours after the introduction of formalin.

To study blood leukograms, the total number of leukocytes was calculated by the standard method in the Goryaev chamber. Blood smears were fixed with methanol for 10 minutes and stained according to Romanovsky-Giemsa. Blood cells were counted under an immersion lens × 90, eyepiece × 5. The blood sampling of animals was performed by partial cutting of the tip of the tail, 18 hours after the induction of inflammation.

According to the leukogram indicators, the agranulocyte / granulocyte index was calculated (anti-inflammatory activity index - I), which is the ratio of the sum of agranulocytes (lymphocytes and monocytes) to the sum of granulocytes (neutrophils and eosinophils):

As can be seen from the table in FIG. 4, in all control groups of animals, the introduction of phlogogen caused an increase in the number of neutrophils, which is characteristic of an acute inflammatory process. The number of mature forms of neutrophils and eosinophils in the blood of animals that were injected with formalin increased to 53.33 ± 2.39% and 2.67 ± 0.84%, respectively; 52.50 ± 2.91% and 2.17 ± 0.40% (p> 0.05), compared with intact groups. The number of monocytes and lymphocytes decreased, respectively, to 4.83 ± 1.14%, 4.33 ± 0.84% (p> 0.05) and 49.00 ± 2.32%, 49.17 ± 3.57% ( p <0.01).

In groups of animals treated with drugs after formalin administration, opposite changes were observed, for example, under the influence of ASA and tincture, 70% of the number of neutrophils decreased to 38.5 ± 2.11%, 33.83 ± 2.8% (p < 0.01), lymphocytes decreased respectively to 65.50 ± 2.28%, 49.33 ± 4.31% (p> 0.05). The number of monocytes and eosinophils with the introduction of the same drugs increased to 7.00 ± 1.29%, 7.33 ± 1.23% (p> 0.05) and 0.67 ± 0.33%, 1.17 ± 0.48% (p <0.05), respectively.

To evaluate the anti-inflammatory activity of tincture and ASA on a model of formalin-induced inflammation, the index of anti-inflammatory activity was calculated by the formula 3.

The highest value of the index of anti-inflammatory activity was noted in ASA - 1.82 ± 0.05, and the lowest when modeling inflammation in water - 0.92 ± 0.03. In the presence of inflammation, the proposed index decreases, and its value depends on the severity of the pathological process (table in Fig. 5).

Based on the magnitude of the agranulocyte / granulocyte index value, the drugs were ranked according to their anti-inflammatory activity (in decreasing order of activity) on a model of formalin-induced inflammation (Fig. 6). Thus, the greatest anti-inflammatory activity in the model of formalin-induced inflammation was detected in the proposed tincture, and the smallest in water, which has practically no anti-inflammatory effect.

A sign of an inflammatory reaction is also an increase in the erythrocyte sedimentation rate (ESR). This is due to an increase in the content of fibrinogen (one of the proteins of the acute phase of inflammation), which leads to agglutination of red blood cells. ESR was determined immediately after blood collection (no later than 2 hours) using the Panchenkov micromethod.

ESR in the group of animals where formalin was used was 12.53 ± 0.43 mm / h, which is 8.5 times more than in the intact group. Against the background of tincture of 70% and ASA, ESR significantly decreased to 7.60 ± 0.16 mm / h and 5.47 ± 0.18 mm / h (p <0.01), respectively (Fig. 5).

An analysis of the results of our studies shows that our tincture has a more pronounced effect than the comparison drug (acetylsalicylic acid) and reliable in comparison with the results obtained in the control group of animals.

Thus, the possibility of using a 70% tincture of the dried and crushed aboveground parts of shrubby penny in the ratio of raw materials: extractant 1:10 as an anti-inflammatory agent in medicine has been experimentally established. Our tincture has a pronounced anti-inflammatory activity. When compared with acetylsalicylic acid, tincture of penny shrub 70% is not inferior in effectiveness, and in some cases exceeds it.

Claims (1)

An anti-inflammatory agent, which is a tincture of the dried and crushed aboveground part of the shrubby penny, obtained by percolation using 70% ethyl alcohol as an extractant, with a ratio of raw materials: extractant 1:10, while 11.29 ml is poured at the stage of soaking 10 g of raw material 70% ethyl alcohol.

Images