The method of obtaining funds that have anti-inflammatory activity

 

(57) Abstract:

The invention relates to the field of pharmacy and relates to a method of receiving funds from vegetable raw materials, which possess anti-inflammatory activity. The aboveground part panzeria woolly extracted with 50% to 55% ethyl alcohol in the ratio of raw material : solvent 1 : 14 - 16 taking into account the coefficient of water absorption at 55 - 65oC. the Process was repeated three times. The extraction time at the 1st, 2nd and 3rd contacts phases 60 minutes Extraction filter, combine, evaporated and purified by separation. The purified extract was dried in a vacuum drier and pulverized in the mill propeller type. The output of the finished product is of 28.9 0.5% of the initial mass of plant material. The invention allows to increase the content of active substances in the product. 10 table.

The invention relates to the field of pharmacy and relates to a method of receiving funds from vegetable raw materials, which possess anti-inflammatory activity.

At present great attention is paid to the study, development and implementation in practice of medicinal products for the treatment and prevention of inflammatory diseases as inflammatory diseases most widely the cause partial or complete disablement of large populations.

Despite the fact that the modern Arsenal of anti-inflammatory drugs has a significant number of problem investigations of new high-performance anti-inflammatory drugs that do not have side effects, is very important. This is due to the fact that there is no "perfect" drugs, not all drugs are anti-inflammatory activities meet the highest standards of efficiency and safety. The object of the study - extract panzeria woolly prepared from local plant materials and is therefore more accessible and affordable. A method of obtaining an infusion of panzeria woolly, which is used in hepatitis and intoxications (prototype) /Krylov, C. Herbs of life and their seekers. - Tomsk, - 1992. - 217 C. // Minaeva Century, Medicinal plants of Siberia. - 5th ed., Rev. and expanded. - Novosibirsk: Nauka. - 1991. - 431 S. / However pharmacotherapeutic efficiency it does not provide the desired result. This is because when using as extractant water 40 - 50% of the biologically active substances remains in the meal / Ant I. A. Technology of drugs. - M, - 1971, - 751 S./. In addition, the disadvantage of this method of obtaining medicines is mintsa increase anti-inflammatory activity at the expense of increasing the content of active substances.

To achieve the above objectives, the plant material is crushed to particle size of not more than 5 mm, extracted with 50% to 55% ethyl alcohol in the ratio of raw material : solvent 1 : 14 - 16 for 60 min at a temperature of 60 2,5oC and constant stirring. The process was repeated three times. The extraction time at the 1st, 2nd and 3rd contacts phase - 60 minutes. The combined extract was filtered through serosianno cloth, evaporated to 1/3 of the initial volume and purified by separation. The purified extract concentrate Domarkas to 1/5 of the initial volume, then dried in a vacuum drying apparatus and crushed in the mill propeller type. The yield of dry product is 28,90,5%.

The proposed method allows to obtain the final product, a powder brown color 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 panzeria woolly ground in a mill to a particle size diameter of 0.5 mm (sieve No. 50 GOST 214-83). The crushed raw material is loaded into estrazioni apparatus with stirrer and external steam heating. Fill in 13 l of 50% ethanol in aspect] constant stirring for 60 minutes. The extract is filtered through serosianno cloth in the collection. Fill with 50% ethanol in a quantity equal to the slit, and again extracted with 60 minutes. Similarly, conduct and third extraction for 60 minutes. The first drain - 10.5 l, the second drain - 9 l, the third 9 sheets combined extract evaporated to 1/5 of the initial volume, then dried in a vacuum dryer at a temperature of 60oC for 8 hours and pulverized in the mill propeller type. Get 0,287 kg of the finished product, which is 28.7% of extractives by weight of the feedstock. The dry extract is a brown powder with a pleasant smell and a bitter astringent taste; crumpled, the moisture content is 5%.

Acute toxicity of dry extract was determined on outbred mice-males with initial mass 20,01,0 g intraperitoneal and intragastric administration dissolved in water extract. Each dose of the extract was tested on 10 animals. Observation of the animals were within 14 days. Registered observable signs of intoxication, conducted an autopsy and examination of the dead animals. The results showed that intraperitoneal injection of large doses of dry extract panzeria wooly be eniah, the appearance of the tonic, and then tonicclonic seizures. Animals were killed with symptoms of respiratory failure mainly during the first day started with an introduction of the extract. LD50 for Cerberus intraperitoneal injection of dry extract panzeria woolly corresponded 4625,0390,0 mg/kg after intragastric administration of the extract at the highest possible dose of death did not come and did not observe obvious signs of intoxicatie. This data allows us to include the subject dry extract to a group of low-toxic substances according to the classification Sidorova / Sidorov, K. K. On the classification of the toxicity of poisons parenteral ways of doing // the Toxicology of new industrial chemical substances. - M - 1973. - Vol. 13. - S. 47-51./.

Further it was investigated the effect of the extract panzeria woolly on the main phases of the inflammatory process. Experiments on the influence of the obtained extract on the processes of alteration were held on 30 outbred rats-males with initial mass 160.010.0, tissue Damage in the area back in rats was induced by the subcutaneous administration of 0.5 ml of 9% acetic acid, with simultaneous intraperitoneal introduction of 0.4 ml of 10% solution reopoligliukina (300 mg/kg). The layout of the damaged zone is Ancaria woolly was administered to two groups of animals 1 time per day orally one hour before the injection of acetic acid, and then every day for 21 days. Control group animals received distilled water in an appropriate amount according to the same scheme. The results are shown in table 1.

From the data presented in table 1, it follows that the extract panzeria has antiulcerative effect on all periods of observation, and pharmacotherapeutic efficiency of the extract on this criterion yields to the action of the extract.

Example 2.

1 kg of the above-ground part panzeria woolly ground in a mill to a particle size diameter of 0.5 mm (sieve No. 50, GOST 214 - 83). The crushed raw material is loaded into the extraction apparatus with stirrer and external steam heating. Pour 15 l 53% of ethyl alcohol in the ratio of raw material : solvent 1 : 15 taking into account the coefficient of water absorption. Extracted at a temperature of 65oC and continuous stirring for 60 minutes. The extract is filtered through serosianno cloth in the collection. Fill with 50% ethanol in a quantity equal to the slit, and again extracted with 60 minutes. Similarly, conduct and third extraction for 60 minutes. The first drain - 11,25 l, the second drain - 9,6 l, third - 9,5 HP combined extract evaporated to 1/5 of the initial volume, sateo type. Get 0,283 kg of the finished product, which is 28.3% of extractives by weight of the feedstock. The obtained dry extract is a brown powder with a pleasant smell and a bitter astringent taste; crumpled, moisture content 5,93%.

Experiments to assess the impact of the obtained extract by the process of exudation were conducted on gray mice F1(CBA x C57B1/6) with initial mass 20,01,0, Gray mice 2 hours before subplantar injection in the right hind limb was injected with 0.2 ml of 3% formalin solution, and then after 5 hours and 18 hours after that in the animals ' stomach probe was introduced extract panzeria in doses of 100 mg/kg and 300 mg/kg Other group of rats for comparison in similar conditions was administered infusion panzeria in optimal therapeutic dose of 100 mg/kg (in terms of absolutely dry raw material). The control group was administered distilled water in equal volume. 24 hours after injection of formalin assessed antiexudative action of these funds ecometrics method. The research data presented in table 2. From the data presented in table 2, it follows that the extract panzeria at a dose of 100 mg/kg has expressed antiexudative action. The dose d is

1 kg of the above-ground part panzeria woolly ground in a mill to a particle size diameter of 0.5 mm (sieve No. 50, GOST 214 - 83). The crushed raw material is loaded into the extraction apparatus with stirrer and external steam heating. Pour 16 l of 50% ethanol in the ratio of raw material : solvent 1 : 16 with regard to the rate of water absorption. Extracted at a temperature of 55oC and continuous stirring for 60 minutes. The extract is filtered through serosianno cloth in the collection. Fill in 55% ethanol in the amount equal to the slit, and again extracted with 60 minutes. Similarly, conduct and third extraction for 60 minutes. The first drain - 12 l, the second drain - 10,28 L., third - 10,15 L. combined extract evaporated to 1/5 of the initial volume, then dried in a vacuum dryer at a temperature of 60oC for 8 hours and pulverized in the mill propeller type. Get 0,281 kg of finished product that 28.1% of extractives by weight of the feedstock. The dry extract is a brown powder with a pleasant smell and a bitter astringent taste; crumpled, the moisture content of 5%.

Experiments on the effect of the extract panzeria on the processes of proliferation were carried out on 30 white besporodnye of Trinos with co / Trinus F. P., Mokhort N. A., Klebanov Century. N. Nesteroidnyi anti-inflammatory drugs. - Kiev, - 1975. - 239 S./.

Sterile cotton balls weighing 15 mg implanted under the skin of rats in the back under aseptic conditions. Extract panzeria at a dose of 100 mg/kg and infusion panzeria at a dose of 100 mg/kg (in terms of absolutely dry raw material), was administered to animals orally 1 time a day for 7 days. Animals of control group received distilled water in the same volume and mode. On the 8th day the animals were removed granulomas and were weighing them in raw form and after drying at a temperature of 70oC for 24 hours. The difference in the masses of granulomas in rats treated with the extract and infusion and animals treated with distilled water, judged the influence of the subjects of funds for the phase of proliferation. The data obtained are given in table 3. From these data, it can be concluded that the extract panzeria at a dose of 100 mg/kg had no statistically significant influence on the formation of fibrous granulation tissue.

Thus, the data conducted a series of studies indicate that the extract panzeria at a dose of 100 mg/kg has a pronounced anti-inflammatory effect, optimizing for inflammatory reacts and other natural compounds.

On the basis of qualitative phytochemical analysis of the amount of flavonoids and extractives installed:

1) Flavonoids:

two-dimensional ascending chromatography on paper brand "Filtrak" FN - 12 in the solvent system:

1 - butanol - acetic acid - water (4:1:5)

2 - acetic acid 15%.

While browsing the chromatogram in ultraviolet light before and after treatment with 2% solution of aluminum chloride detected: rutin - Rf = 0,38 (1), Rf = 0.4 (of 2), quercetin = Rf = 0,73 (1), Rf = 0,04 (2). Identification was performed with authentic images.

2) phenol carbonic acids:

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

After processing the chromatogram in pairs ammonia and browsing in UV-light identified with authentic samples:

coffee acid Rf = 0,79 (1), Rf = 0,53 (2).

chlorogenic acid Rf = 0.75 In (1), Rf = 0,63 (2).

3) Tannins:

0.05 g of the obtained extract was dissolved in 5 ml of purified water. To 2 ml of the resulting solution add 2 - 3 drops of solution salesonlinevuh alum, appears black and blue colouring.

2. Quantitative determination.OSI a sieve with the hole diameter 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, add 100 ml of 50% alcohol. The flask is attached to the back of the fridge and heated in a boiling water bath for 60 min, shaking occasionally to wash particles of raw materials from the walls. Hot extract is filtered through a filter into a volumetric flask with a capacity of 200 ml, similar to holding a second and third extraction with 50 ml of 50% ethanol. After cooling, the combined extract was adjusted to 50% alcohol up to the mark and mix (R-RA).

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

As the reference solution using solution consisting of 1 ml of extract, 1 drops of diluted acetic acid and brought to 95% alcohol to the mark in a volumetric flask of 25 ml

Simultaneously measure the optical density of the solution State standard (GSO) routine, prepared similarly to the subject an solution.

The content of total flavonoids rutin and mortar;

Dabout- the optical density of the solution of the state security service routine;

m is the mass of raw material in grams;

mabout- weight GSO routine in grams;

W is the loss in weight on drying of raw materials in percentage.

Note. Preparation of the solution of the state security service routine.

About 0.5 g (accurately weighed) of the state security service routine, previously dried at T - 130-135oC for 3 h, dissolved in 85 ml of 95% ethanol in a volumetric flask with a capacity of 100 ml by heating on a water bath, cooled, quantitatively transferred into a volumetric flask 100 ml capacity, bring the volume of solution in the same spirit, up to the mark and mix.

Found 5,19% 0,048.

The proposed method of obtaining relatively simple, does not require complicated purification, allows to obtain a product of constant composition. The technology can be implemented at the enterprises for the production of medicines and Wellness resources.

To establish the optimum process conditions were studied the main factors influencing the processes of extraction of plant material: the nature of the extractant, the ratio of raw materials, temperature, degree of crushing of raw materials, duration and frequency of extraction.

Quantitative is to support the amount of flavonoids rutin standard. Metrological characteristics determine the amount of extractives and the amount of flavonoids was carried out on samples panzeria woolly collected in the vicinity of the village of Sokol, Buryatia in 1998 (table 4).

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

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

On the basis of experimental data it was established that the optimal extractant according to the amount of extractives and flavonoids selected 50% ethanol (table 5).

We conducted a study of the dependence of the yield amounts of flavonoids and extractives, depending on the temperature of extraction in the temperature range from 20 to 100oC. the Results are presented in table 6.

Based on the data analysis revealed that the optimum temperature extraction 60 2,5oC. Further increase in temperature is impractical, because the possibility is about material, which leads to an increase of the surface of the extracted material due to rupture of the cells, which increases the contact between the solvent and the material, resulting in accelerated extraction process.

To study the effect of the degree of grinding of plant material at the exit of the amount of flavonoids and extractives raw material was subjected to grinding to particle diameter 0,5; 1,0; 2,0; 3,0; 5,0; 7,0; 10,0. The results are shown in table 7.

The optimal degree of grinding of the raw material is a particle size of 0.5 mm

We conducted a study of the dependence of the yield amounts of flavonoids and extractives on the ratio of raw materials extractant. The results of the study are shown in table 8.

To determine the duration and the ratio of the number of extraction the dependence of time to achieve equilibrium concentration in the system raw - extractant with triple extraction of raw materials. For this purpose, carried out the extraction of the crushed material with 50% ethanol in the ratio of 1 : 14 in a water bath at a temperature of 60oC with constant stirring under reflux.

At set time intervals (30, 60, 90, 120 min) extraction Phil is Similarly conducted two subsequent extraction of pressed raw materials at the same time, adding each time 50% ethanol in a quantity equal to the amount drained extraction (2nd and 3rd contact phases), is also analyzed for the content of total flavonoids and extractives. Experimental data indicate that the equilibrium state occurs when the first, second and third contact phase after 60 minutes (table 9).

During the 1st contact phase is extracted about 60 - 70% of the amount of flavonoids and amount of extractives. The second contact provides an output of about 25-30%, and the third is about 5-10%, i.e. for a complete depletion enough to triple extraction.

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

To calculate the economic efficiency of the proposed method is currently not possible, however, the foregoing advantages, in combination with a simple scheme of obtaining promotes the rational use of medicinal plants and offer the prospect of implementation of this method in the pharmaceutical industry.

The method of obtaining funds, Allam, as plant material used medicinal plant - panzeri woolly, which is extracted with 50% to 55% ethyl alcohol in the ratio of plant material : the extractant is 1 : 14 - 16, three times for 60 minutes at a temperature of 55 - 65oC.

 

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