RU2160599C1 - Method for producing preparation possessing anti- inflammation activity - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: method involves extracting the above- ground portion of fleecy Panceria plant with 50- 55% ethyl alcohol taken in 1: (14-16) raw material - extragent proportion, with water absorption coefficient at 55-56 C taken into account. The process is repeated three times. Extraction time is 60 min in the first, the second and the third phase contact. The extracted stuff is filtered, mixed, boiled down and cleaned by separation. The cleaned product is dried in vacuum-type drying installation and comminuted in propeller-type mill. Finished product output is 28.9 ± 0.5 % of initial mass of the vegetable raw material. EFFECT: higher concentration of active substances in the finished product. 10 tbl

Description

 The invention relates to the field of pharmacy and relates to a method for producing funds from plant materials with anti-inflammatory activity.

 Currently, much attention is paid to the study, development and implementation of drugs for the treatment and prevention of inflammatory diseases, since inflammatory diseases are widespread, people of different ages suffer from them, they are often accompanied by severe clinical manifestations and cause partial or complete disability of large contingents population.

 Despite the fact that the current arsenal of anti-inflammatory drugs includes a significant number, the problem of finding new highly effective anti-inflammatory drugs that do not have side effects remains very relevant. This is due to the fact that there are no “ideal” drugs, not all anti-inflammatory drugs meet the high requirements for effectiveness and safety. The object of study - wool shellfish extract is prepared from local plant materials and therefore is more affordable and cheaper. A known method of obtaining an infusion of wooly pantherium, which is used for hepatitis and intoxications (prototype) / Krylov G.V. The herbs of life and their seekers. - Tomsk, - 1992. - 217 p. // V. Minaeva. Medicinal plants of Siberia. - 5th ed., Revised. and augmented. - Novosibirsk: Science. - 1991 .-- 431 p. / However, its pharmacotherapeutic effectiveness does not provide the desired result. This is due to the fact that when using as an extractant water 40 - 50% of biologically active substances remains in the meal / Muravyov I.A. Technology of drugs. - M., - 1971, - 751 s./. In addition, the disadvantage of this method of obtaining a medicinal product is a lower content of flavonoids - the main active substances.

 The objective of the present invention is to increase the anti-inflammatory activity of the drug by increasing the content of active substances in it.

To achieve this goal, the plant material is crushed to a particle size of not more than 5 mm, extracted with 50 - 55% ethyl alcohol in the ratio of raw materials: extractant 1: 14 - 16 for 60 minutes at a temperature of 60 ± 2.5 ^o C with constant stirring. The process is repeated three times. The extraction time at the 1st, 2nd and 3rd phase contacts is 60 minutes. The combined extracts are filtered through sulphurous cloth, evaporated to 1/3 of the original volume and purified by separation. The purified extract is concentrated with a double park to 1/5 of the original volume, then dried in a vacuum dryer and crushed in a propeller-type mill. The yield of dry product is 28.9 ± 0.5%.

 The proposed method allows to obtain the final product, which is a brown powder with a pleasant odor and a bitter astringent taste with a moisture content of not more than 5%.

 The method is illustrated by the following examples.

 Example 1

1 kg of grass of the wool shell is crushed in a mill to a particle size of 0.5 mm in diameter (sieve No. 50 GOST 214-83). The crushed raw materials are loaded into an extraction apparatus with a stirrer and external steam heating. Pour 13 liters of 50% ethyl alcohol in the ratio of raw materials: extractant 1: 14, taking into account the coefficient of water absorption. Extracted at a temperature of 60 ^o C and constant stirring for 60 minutes. The extract is filtered through a tin cloth in the collection. Pour 50% ethyl alcohol in an amount equal to the drained, and again extracted 60 minutes. A third extraction is carried out in a similar manner within 60 minutes. The first discharge is 10.5 liters, the second discharge is 9 liters, the third is 9 liters. The combined extracts are evaporated to 1/5 of the original volume, then dried in a vacuum dryer at a temperature of 60 ^o C for 8 hours and ground in a propeller-type mill. Get 0.287 kg of the finished product, which is 28.7% of extractive substances by weight of the feedstock. The dry extract is a brown powder with a pleasant smell and a bitter astringent taste; lumps, moisture content - 5%.

 The acute toxicity of the dry extract 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 the animals was carried out 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 the dry extract of the panzeria of the woolly white mice, signs of intoxication are observed, which are expressed in a slowdown in the orientational reaction, sudden movements, the appearance of tonic and then tonic clonic seizures. Animals died with respiratory arrest phenomena mainly during the first days from the beginning of the introduction of the extract. Kerber LD50 with intraperitoneal administration of dry extract of woolly panzeria corresponded to 4625.0 ± 390.0 mg / kg. With the intragastric administration of this extract at the maximum possible dose, no death of animals occurred and no obvious signs of intoxication were observed. This data allows us to attribute the test dry extract to the group of low toxic substances according to the classification of Sidorov / Sidorova K.K. On the classification of toxicity of poisons with parenteral methods of administration // Toxicology of new industrial chemicals. - M. - 1973. - Issue. 13. - S. 47-51./.

 Subsequently, the effect of woolly panzeria extract on the main phases of the inflammatory process was investigated. Experiments on the effect of the obtained extract on the alteration processes were carried out on 30 white outbred male rats with an initial mass of 160.0 ± 10.0 g. Damage to the tissue in the back of the rats was caused by subcutaneous injection of 0.5 ml of 9% acetic acid with simultaneous intraperitoneal administration of 0.4 ml 10% rheopolyglucin solution (300 mg / kg). Planimetry of the damaged area was performed on days 7, 14, and 21. The extract at a dose of 100 mg / kg and paste at a dose of 100 mg / kg (in terms of absolutely dry raw materials) of the woolly panzeria were administered to two groups of animals once a day orally one hour before the injection of acetic acid, and then daily for 21 days. The control group of animals received distilled water in an appropriate volume according to a similar scheme. The research results are presented in table 1.

 From the data given in table 1, it follows that the extract of the panzerium has an antialterative effect at all periods of observation, and the pharmacotherapeutic efficacy of the infusion according to this criterion is inferior to the action of the extract.

 Example 2

1 kg of the aboveground part of the woolly shell is crushed in a mill to a particle size of 0.5 mm in diameter (sieve No. 50, GOST 214 - 83). The crushed raw materials are loaded into an extraction apparatus with a stirrer and external steam heating. Pour 15 liters of 53% ethyl alcohol in the ratio of raw materials: extractant 1: 15, taking into account the coefficient of water absorption. Extracted at a temperature of 65 ^o C and constant stirring for 60 minutes. The extract is filtered through a tin cloth in the collection. Pour 50% ethyl alcohol in an amount equal to the drained, and again extracted 60 minutes. A third extraction is carried out in a similar manner within 60 minutes. The first discharge is 11.25 liters, the second discharge is 9.6 liters, the third is 9.5 liters. The combined extracts are evaporated to 1/5 of the original volume, then dried in a vacuum dryer at a temperature of 60 ^o C for 8 hours and ground in a propeller-type mill. Get 0.283 kg of the finished product, which is 28.3% of extractive substances by weight of the feedstock. The resulting dry extract is a brown powder with a pleasant odor and a bitter astringent taste; lumps, moisture content - 5.93%.

 Experiments to evaluate the effect of the obtained extract on exudation processes were carried out on gray mice of the F1 line (CBA x C57B1 / 6) with an initial weight of 20.0 ± 1.0 g. Gray mice were injected 0 hours before subplanar injection into the right hind limb. 2 ml of a 3% solution of formalin, and then 5 hours and 18 hours after this, a panzerium extract was introduced into the stomach of animals with doses of 100 mg / kg and 300 mg / kg. For comparison, in another group of rats, under the similar conditions, the panzerium infusion was administered at an optimal therapeutic dose of 100 mg / kg (in terms of absolutely dry raw materials). The control group was injected with distilled water in an equal volume. 24 hours after the introduction of formalin, the antiexudative effect of these agents was evaluated by the oncometric method. The research data are presented in table 2. From the data given in table 2, it follows that the extract of panzeria at a dose of 100 mg / kg has a pronounced antiexudative effect. An increase in the dose of this extract does not lead to a significant increase in the recorded effect.

 Example 3

1 kg of the aboveground part of the woolly shell is crushed in a mill to a particle size of 0.5 mm in diameter (sieve No. 50, 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 the ratio of raw materials: extractant 1: 16, taking into account the coefficient of water absorption. Extracted at a temperature of 55 ^o C and constant stirring for 60 minutes. The extract is filtered through a tin cloth in the collection. Pour 55% ethyl alcohol in an amount equal to the drained, and again extracted 60 minutes. A third extraction is carried out in a similar manner within 60 minutes. The first discharge is 12 liters, the second discharge is 10.28 liters, the third is 10.15 liters. The combined extracts are evaporated to 1/5 of the original volume, then dried in a vacuum dryer at a temperature of 60 ^o C for 8 hours and ground in a propeller-type mill. Get 0.281 kg of the finished product, which is 28.1% of extractive substances by weight of the feedstock. The dry extract is a brown powder with a pleasant smell and a bitter astringent taste; lumps, moisture content 5%.

 Experiments on the effect of the extract of panzeria on proliferation processes were carried out on 30 white outbred male rats with an initial weight of 200.0 ± 10.0 g with subcutaneous implantation of sterile cotton swabs on the recommendation of Trinus et al. / Trinus F.P., Mohort N.A. ., Klebanov V.N. Non-steroidal anti-inflammatory drugs. - Kiev, - 1975. - 239 p. /.

15 mg sterile cotton balls were implanted under the skin of rats in the back region under aseptic conditions. Panzeria extract at a dose of 100 mg / kg and panzeria infusion at a dose of 100 mg / kg (in terms of absolutely dry raw materials) were administered to animals orally 1 time per day for 7 days. Animals of the control group received distilled water in similar volumes and mode. On the 8th day, granulomas were removed from animals and they were weighed in their raw form and after drying at a temperature of 70 ^o C for 24 hours. The difference in granuloma masses in rats receiving extract and infusion and animals receiving distilled water was used to judge the effect of the test substances on the proliferation phase. The data obtained are shown in table 3. From these data, it can be concluded that the extract of panzeria at a dose of 100 mg / kg does not have a statistically significant effect on the formation of fibro-granulation tissue.

 Thus, the data of a series of studies indicate that the extract of panzeria at a dose of 100 mg / kg has a pronounced anti-inflammatory effect, optimizing the course of the inflammatory reaction, which is obviously due to the presence of a complex of biologically active substances, in particular flavonoids and other natural compounds.

Based on a qualitative phytochemical analysis of the sum of flavonoids and extractive substances, the presence of:
1) Flavonoids:
two-dimensional ascending chromatography on paper brand "Filtrak" FN - 12 in solvent systems:
1 - butanol - acetic acid - water (4: 1: 5)
2 - acetic acid 15%.

 When viewing the chromatogram in UV light before and after treatment with a 2% aluminum chloride solution, the following were found: rutin — Rf = 0.38 (1), Rf = 0.4 (2); quercetin = Rf = 0.73 (1), Rf = 0.04 (2). Identification was carried out with authentic images.

2) Phenolcarboxylic acids:
two-dimensional ascending chromatography on FN-12 grade paper in a solvent system: 1-n-butanol - acetic acid - water (4: 1: 5), 2 - 15% acetic acid.

After processing the chromatogram in ammonia vapors and viewing in UV light, they were identified with authentic samples:
caffeic acid Rf = 0.79 (1), Rf = 0.53 (2).

 chlorogenic acid Rf = 0.75 (1), Rf = 0.63 (2).

3) Tannins:
0.05 g of the obtained extract is dissolved in 5 ml of purified water. To 2 ml of the resulting solution add 2 to 3 drops of a solution of iron-ammonium alum, a black and blue color appears.

 2. Quantification.

 Sum of flavonoids.

 An analytical sample is crushed to the size of particles passing through a sieve with an opening diameter of 1 mm.

 About 1 g (accurately weighed) of the crushed raw material is placed in a flask with a thin section with a capacity of 150 ml, 100 ml of 50% alcohol are added. The flask is attached to a reflux condenser and heated in a boiling water bath for 60 minutes, periodically shaking to flush the particles of raw materials from the walls. The hot extract is filtered through a filter into a volumetric flask with a capacity of 200 ml, similarly carry out the second and third extraction of 50 ml of 50% ethanol. After cooling, the combined extracts were adjusted with 50% alcohol to the mark and stirred (solution A).

 In a volumetric flask with a capacity of 25 ml, add 1 ml of solution A and 1 ml of 2% solution of aluminum chloride in 95% ethanol and adjust the volume of the solution with 95% alcohol to the mark. After 40 minutes, the optical density of the solution was measured on a spectrophotometer at a wavelength of 415 nm in a cell with a layer thickness of 10 mm.

 As a comparison solution, use a solution consisting of 1 ml of extraction, 1 drop of diluted acetic acid and brought 95% alcohol to the mark in a 25 ml volumetric flask.

 At the same time, the optical density of the solution of the State standard sample (GSO) of rutin, prepared similarly to the test solution, is measured.

где
D - оптическая плотность испытуемого раствора;
D_о - оптическая плотность раствора ГСО рутина;
m - масса сырья в граммах;
m_о - масса ГСО рутина в граммах;
W - потеря в массе при высушивании сырья в процентах.The content of the sum of flavonoids in terms of rutin on absolutely dry raw materials in percent (X) is calculated by the formula

Where
D is the optical density of the test solution;
D _about - the optical density of the solution of GSO rutin;
m is the mass of raw materials in grams;
m _o - mass GSO routine in grams;
W is the loss in mass upon drying of the raw material as a percentage.

 Note. Preparation of a solution of GSO rutin.

About 0.5 g (accurately weighed) of GSO rutin, previously dried at T - 130-135 ^o C for 3 hours, dissolved in 85 ml of 95% ethanol in a 100 ml volumetric flask when heated in a water bath, cooled, quantitatively transferred to a volumetric flask with a capacity of 100 ml, bring the volume of the solution with the same alcohol to the mark and mix.

 Found 5.19% ± 0.048.

 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 introduced at the enterprises for the production of pharmaceuticals and wellness products.

 To establish the optimal technological regimes, the main factors affecting the extraction of plant material were studied: the nature of the extractant, its ratio with raw materials, temperature conditions, the degree of grinding of raw materials, duration and frequency of extraction.

 A quantitative assessment was conducted on the output of the sum of extractive substances / State Pharmacopoeia. T. 11, no. 1, - 295 pp. / And the content of the sum of flavonoids in terms of the routine standard. The metrological characteristics of the determination of the sum of extractive substances and the sum of flavonoids were carried out on samples of woolly panzeria collected in the vicinity of the village of Sokol, Buryatia in 1998 (table 4).

 The selection of the optimal extractant is one of the main conditions for obtaining extracts. The extractant, acting as the active component of the mixture, affects the completeness, speed and quality of the extraction of biologically active substances from plant material.

As extractants were used hot water (90 - 100 ^o C) and ethyl alcohol of the following concentrations: 10, 20, 30, 30, 50, 60, 70, 80, 90, 95.

 Based on experimental data, it was found that 50% ethyl alcohol was selected as the optimal extractant for the yield of the sum of extractive substances and flavonoids (table 5).

We studied the dependence of the yield of the sum of flavonoids and extractive substances, depending on the temperature of extraction in the temperature range from 20 to 100 ^o C. The results are presented in table 6.

Based on the analysis data, it was found that the optimal extraction temperature is 60 ± 2.5 ^o C. A further increase in temperature is impractical, since the destruction of thermolabile substances is possible.

 It is well known that the degree of grinding of the plant material is increased, which leads to an increase in the surface of the extracted material due to cell rupture, due to which there is an increase in contact between the solvent and the material, as a result of which the extraction process is accelerated.

 To study the effect of the degree of grinding of plant material on the output of the sum of flavonoids and extractive substances, the raw material was subjected to grinding to a particle diameter of 0.5; 1.0; 2.0; 3.0; 5.0; 7.0; 10.0 The results are shown in table 7.

 The optimum degree of grinding for raw materials is a particle size of 0.5 mm.

 We studied the dependence of the yield of the sum of flavonoids and extractive substances on the ratio of raw materials - extractant. The results of the study are shown in table 8.

In order to determine the duration and multiplicity of the number of extractions, the dependence of the time to reach equilibrium concentration in the raw material – extractant system was studied for three times the raw material extraction. To do this, we extracted the crushed raw materials with 50% ethyl alcohol in a ratio of 1: 14 in a water bath at a temperature of 60 ^o C with constant stirring under reflux.

 At predetermined time intervals (30, 60, 90, 120 min), the extracts were filtered (1 contact of phases), determining the sum of flavonoids in terms of rutin and the content of extractive substances. Two subsequent extraction of the squeezed raw material was carried out in the same way for the same period of time, adding 50% ethanol each time in an amount equal to the volume of the fused extraction (2nd and 3rd phase contact), and also analyzed for the content of the sum of flavonoids and extractive substances. Experimental data indicate that the equilibrium state occurs during the first, second and third contact of the phases after 60 minutes (table 9).

 About 60 - 70% of the sum of flavonoids and the sum of extractive substances are extracted during the first phase contact. The second contact provides a yield of about 25-30%, the third - about 5-10%, that is, triple extraction is sufficient to complete depletion.

 The proposed method in comparison with the known (table 10) allows to obtain a drug of constant composition with higher anti-inflammatory activity by increasing the content of active substances.

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

Claims (1)

The method of obtaining funds with anti-inflammatory activity by extracting plant material, characterized in that the plant material is a medicinal plant - wool shell, which is extracted with 50 - 55% ethyl alcohol in the ratio of plant material: extractant, equal to 1: 14 - 16, three times for 60 minutes at a temperature of 55 - 65 ^o C.

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