RU2396973C2 - Anti-inflammatory and analgesic agent and method for preparing thereof - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine and pharmaceutics, namely to creation of an anti-inflammatory and analgesic agent. The agent contains Sphagnum fuscum extract including polysaccharides and phenol compounds (phenol acids, flavonoids, coumarins). The method for preparing the agent involves 20% water-ethanol extraction in a four-repercolation battery.

EFFECT: agent prepared by the presented method is low-toxic.

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Description

The invention relates to the field of medicine, pharmacy, pharmacognosy, to drugs with anti-inflammatory and analgesic effects, and methods for their preparation.

Rational therapy of wound lesions includes the mandatory antimicrobial component. Today, clinical medicine has a wide range of antibacterial and antifungal agents (methyluracil ointment, nystatin, sanguirytrin, etc.), however, many of them have side effects in the form of dispersions, hepato-, nephrotoxic and teratogenic effects, allergic reactions, dysbacterioses, immunopathies. One of the ways to solve this problem is the use of antimicrobial preparations of plant origin, which have significant advantages over various synthetic and semi-synthetic antimicrobial agents: low toxicity, the absence of side effects, the effect on multiresistant strains of gram-positive and gram-negative bacteria, a beneficial effect on non-specific resistance of the body, the presence of anti-inflammatory and regenerative action.

Known drugs that have a similar effect, which are represented by the following groups: wound healing; anti-inflammatory; painkillers.

Sphagnum fuscum extract is characterized by a combination of these properties. The use of sphagnum moss in folk medicine is known. Its wide distribution and low cost, combined with the presence of bactericidal, wound healing and hemostatic properties have provided him with universal recognition especially during the Second World War. It is also important that for the entire time of its use, not a single case of contraindications was recorded [1].

Closest to the proposed one is an anti-inflammatory agent obtained in the Biochemical Laboratory of the Plant Resources Department of the BIN of the USSR Academy of Sciences, which represents concentrated aqueous extracts from Sphagnum magellanicum. To obtain it, crushed moss was poured with hot purified water and put in a thermos for a day. Sphagnum infusion was aspirated on a Buchner funnel and boiled under vacuum in a water bath at a temperature not exceeding 70 ° C. The extract obtained after distillation of water was treated with heated alcohol, the precipitated precipitate was filtered off, and the filtrate was distilled off to a concentration of thick syrup [2]. The method is characterized by sufficient complexity and is quite expensive. In addition, the previously used Sphagnum magellanicum has less biological activity and resource base [3].

A new technical task is the development of a drug of sphagnum moss, which has a comprehensive anti-inflammatory and analgesic effect, and a more rational way to obtain it.

To solve this problem, a drug with anti-inflammatory and analgesic effects based on sphagnum moss contains polysaccharides, phenolic compounds: phenol acids, flavonoids, coumarins in the following ratio of components, wt.%:

Polysaccharides 40 ± 14.65 Phenolic compounds 1.6 ± 0.24 Phenolic acids 1.04 ± 0.24 Flavonoids 0.454 ± 0.25 Coumarins 0.055 ± 0.03

In the method for producing an anti-inflammatory and analgesic agent by extracting sphagnum moss raw materials, extraction is carried out by re-method according to N.A. Chulkov, S. fuscum raw materials are extracted, extraction is carried out with a 20% water-ethanol mixture in a battery of 4 re-colitors.

To refine the parameters of the method, first extracts were obtained from S. fuscum raw materials in various ways: water-ethanol 20% extract (maceration, 70 ° C and percolation, 23 ° C), water extract (ultrasound extraction with IR drying and maceration, 95%). They showed varying degrees of activity against dermatophytosis pathogens: Trychophyton rubrum, T. mentagrophytes and M. Canis. The best results were obtained with a 20% aqueous ethanol extract of S. fuscum (Table 5).

The method of preparation of the extract is as follows.

The tool is obtained by repercolation according to N.A. Chulkov. According to this technique, a raw material of S. fuscum, crushed to 3-5 mm, is loaded into a battery of 4 percolators, which is extracted with 20% ethanol. The essence of this technique is that in the extraction process, two periods are distinguished: starting and working. In the starting period, one percolator is loaded daily. Raw materials are divided into equal parts. The first portion is soaked with an equal volume of 20% ethanol, the swollen feed is loaded into the head percolator, poured with a double volume of extractant and left for a day. The next day, two holidays are collected: one for soaking a second portion of the raw material in an amount equal to its mass, the other in double quantity (for insisting the raw material in the second percolator). At this time, pure extractant is poured into the first percolator in an amount equal to the volumes of the vacations taken. On the third day, two holidays are collected from the second percolator to work with a third portion of the raw material intended for loading into the third percolator.

Leaves from the first percolator are fed to the second percolator, and pure extractant, etc., is again fed into it. A day after loading the last percolator, the working period begins with the receipt of the first portion of the finished product in an amount equal to the weight of the loaded raw materials. At the same time, the entire vacation is collected from the first percolator and fed to the second percolator, into which then a clean extractant begins to flow. The first percolator is loaded with a new portion of raw material, which is treated with holidays from the last percolator. The resulting water-alcohol extracts are combined and filtered through a paper filter, and then dried at a temperature not exceeding 30 ° C. The yield of dry extract is 6.92%. Quality assessment is carried out according to organoleptic characteristics, solubility, the content of polysaccharides, phenolic compounds (phenol acids, flavonoids, coumarins) and heavy metals [4, 5, 6].

The extracts were tested for toxicity, antimicrobial (antibacterial and antifungal) activity and analgesic effect [7].

According to the test results, it was possible to conclude that the studied extract from S. fuscum is low toxic. When introduced into the stomach to white outbred male mice at a dose of 10,000 mg / kg, there were no symptoms of acute poisoning, there was no death of the animals and changes in their motor activity and behavioral reactions within two weeks of observation. Thus, according to the existing classification (GOST 12.1.007-76), it corresponds to hazard class IV “Low-hazard substances”.

It was found that in promising concentrations (31.7-62.5 μg / ml), the extract acts on pathogens of dermatophytosis. At the same time, test mushrooms Trichophyton rubrum and Microsporum canis show higher sensitivity to the studied drugs (Table 1).

According to the experiment, the extract exhibits pronounced bacteriostatic and bactericidal effects. The action of the extract is comparable, and sometimes even exceeds the effect on bacteria of water extracts of St. John's wort, perforated and sage, which are pharmacopeia antimicrobial agents. At a dilution of 1/80, it inhibits the multiplication of microorganisms of the strains of Escherichia coli H-304, Staphylococcus aureus P-209 and Klebsiella pneumoniae, slightly affecting the strain Pseudomonas aeruginosa ATCC 9027 (b / c - 1/20). The bactericidal effect is manifested in higher concentrations: 1 / 40-1 / 20, with the exception of the effect on Staphylococcus aureus P-209 (b / c - 1/80). Table 2 shows the antimicrobial activity of a 20% aqueous ethanol extract of S. fuscum.

The analgesic effect was investigated, revealing the antiexudative effect and pain sensitivity of mice. With subplanetary administration of agar solution, intensive formation of inflammatory edema of the hind limb was observed in mice. Under these experimental conditions, a preliminary 5-fold intragastric (once a day) administration of the extract in the dose range of 100-500 mg / kg led to a significant decrease in the formation of inflammatory swelling of the paws of mice (Table 3).

The most pronounced antiexudative effect of the extract was observed for doses of 250 and 500 mg / kg - inhibition of edema was 30.1% and 31.1%, respectively. The preliminary administration of the comparison drug indomethacin (10 mg / kg) to mice caused a more effective inhibition of the development of edema - 47.2% (Table 1, which presents data on the effect of preliminary administration of the extract from (50-500 mg / kg) and indomethacin ( 10 mg / kg) for the formation of agar edema of the extremities in white outbred male mice, * differences with control are significant (p≤0.05).

It is known that the analgesic effect of phlogolitics consists of peripheral and central effects. The peripheral components involved in analgesia include decongestion. The weakening of exudation under the influence of the studied extract allowed us to suggest the analgesic properties of the studied extract on the model of "vinegar cramps" in mice.

Judging by the experimental data obtained (Table 4), the extract also has a pronounced analgesic effect. Preliminary 5-fold intragastric administration (once a day) of this extract in the dose range of 250-500 mg / kg leads to a reliable, almost identical to the similar action of indomethacin in reducing the average number of writhing - by 36.9% and 42.9%, respectively (Table 2, which shows the effect of preliminary administration of a 20% aqueous ethanol extract from S. fuscum (50-500 mg / kg) and indomethacin (10 mg / kg) on the pain sensitivity of white outbred male mice in the model " vinegar cramps ", * differences with control are significant (p≤0.05 )

Thus, experimental studies of the biological activity of the extract of S. fuscum showed that it is low toxic and at the same time has a pronounced analgesic and antimicrobial effect, comparable to the similar effects of pharmacopeia drugs selected as standards. S. fuscum extract differs from its analogues in its mild and complex action. The combination of the properties of the proposed tool will allow you to use it in the treatment of wound and inflammatory processes, burn skin lesions, infected wounds.

Information sources

1. Savich-Lyubitskaya S.I. The use of sphagnum (peat) moss in medicine / S.I. Savich-Lyubitskaya // Nature. - 1943. - N4. - S.41-50.

2. Savich-Lyubitskaya L.I. Flora of spore plants of the USSR. - M .: AN USSR, 1952. - S.97-109 (prototype).

3. Babeshina L.G. Sphagnum mosses of the Tomsk region and the prospects for their use in medicine. - Abstract. Cand. biol. sciences. - Tomsk, 2002, 19 p.

4. Belikov V.V. Unified method for determining flavonoids for standardization of phytochemicals / V.V. Belikov // New drugs from plants of Siberia and the Far East. Theses of the All-Union Conference. - Tomsk, 1989 .-- S.21-22.

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

6. The State Pharmacopoeia of the USSR. Issue 1. 11th ed. M., 1987 .-- 335 s.

7. Guidance on the experimental (preclinical) study of new pharmacological substances / Ed. Corresponding Member RAMS prof. RU. Khabrieva. - 2nd ed., Revised. and add. - M .: OJSC "Publishing House" Medicine ", 2005. - 832 p.: Ill.

Table 1 BAV Group The content in the extract Polysaccharides 40 ± 14.65 Phenolic compounds: 1.6 ± 0.24 Phenolic acids 1.04 ± 0.24 Flavonoids 0.454 ± 0.25 Coumarins 0.055 ± 0.03

table 2 Test mushrooms Selection source Test drug Trichoplyton rubrum Tricholyton mentagrophytes Microsporum canis S. fuscum Water-ethanol 20% extract (maceration, 70 ° C) 15.7 15.7 15.7 Water-ethanol 20% extract (percolation, 23 ° C) 7.9 15.7 15.7 Water extract (maceration, 95%) 31.7 62.5 62.5 Water extract (ultrasound extraction with IR drying) 125 125 62.5 Individual substances Coffee acid 62.5 250 31.7 Coumarin 125 62.5 62.5 Comparison Products Nitrofungin 7.9 31.7 15.7 Sanguirythrin 15.7 15.7 15.7

Table 3 Research drugs Act The strain of microorganisms (sowing dose ≈100000 microbial bodies) Escherichia coli H-304 Staphylococcus aureus P-209 Pseudomonas Aeruginosa ATCC 9027 Klebsiella pneumoniae 20% water-ethanol extract from S.fuscum b / c * 1/80 1/80 1/80 1/20 b / c ** 1/40 1/80 1/40 1/20 Water extract from sage officinalis b / c 1/40 1/80 1/40 1/80 b / c 1/40 1/80 1/40 1/80 Water extraction from Hypericum perforatum b / c 1/80 1/80 1/40 1/40 b / c 1/40 1/40 1/20 1/20

Table 4 Monitoring group, dose (mg / kg) The average increase in limb mass in the group (M ± m), mg Inhibition of edema at the peak of inflammation (%) Control (n = 20) 118.20 ± 11.62 - Indomethacin, 10 (n = 20) 62.40 ± 5.46 * 47.2 20% water-ethanol extract, 50 (n = 20) 109.2 ± 9.8 9.2 20% water-ethanol extract, 100 (n = 20) 95.6 ± 9.24 * 19.1 20% water-ethanol extract, 250 (n = 20) 82.6 ± 8.82 * 30.1 20% water-ethanol extract, 500 (n = 20) 81.4 ± 7.64 * 31.1

Table 5 Monitoring group, dose (mg / kg) The number of cramps in 20 minutes (M ± m) Inhibition of pain sensitivity (%) Control (n = 20) 50.4 ± 4.84 - Indomethacin, 10 (n = 20) 22.6 ± 2.44 * 55,2 20% water-ethanol extract, 50 (n = 20) 52.8 ± 4.50 - 20% water-ethanol extract, 100 (n = 20) 44.2 ± 3.46 12,4 20% water-ethanol extract, 250 (n = 20) 31.8 ± 2.64 * 36.9 20% water-ethanol extract, 500 (n = 20) 28.8 ± 2.58 * 42.9

application

Table 1. The quantitative content of biologically active substances in the extract of S. fuscum, wt.%.

Table 2. Antifungal activity of 20% aqueous ethanol extract from S. fuscum (μg / ml).

Table 3. Antimicrobial activity of 20% water-ethanol extract of S. fuscum * - bacteriostatic effect;

** - bactericidal effect.

Table 4. The effect of preliminary administration of the extract from (50-500 mg / kg) and indomethacin (10 mg / kg) on the formation of agar edema of the extremities in outbred male mice, the differences with the control are significant (p≤0.05).

Table 5. The effect of preliminary administration of a 20% water-ethanol extract from S. fuscum (50-500 mg / kg) and indomethacin (10 mg / kg) on the pain sensitivity of white outbred male mice on the model of “vinegar cramps”, * differences with reliable control (p≤0.05).

Claims (1)

A drug with anti-inflammatory and analgesic effects, characterized in that it contains Sphagnum fuscum extract obtained by extraction with a 20% water-ethanol mixture in a battery of four repercolators, while the extract contains polysaccharides and phenolic compounds (phenol acids, flavonoids, coumarins) in the following ratio of components, wt.% of the total mass of the extract:
Polysaccharides 40 ± 14.65 Phenolic compounds 1.549 ± 0.24