RU2160598C1 - Method for producing preparation possessing cholagogue and anti-inflammation activity - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: method involves extracting vegetable material like Odontites vulgaris herb with 35-45% ethyl alcohol in 1:(12-14) raw material - extractant proportion taking into account water absorption coefficient. The process takes 60 min at 60-65 C and constant stirring. The process is repeated two times. The third extraction is carried out in the same mode during 30 min. Mixed water-alcohol extraction stuff is filtered, boiled down to 1/3 of the initial volume. The rest in cube is processed with separation. The obtained product is boiled down to 1/3 of the initial volume and dried in vacuum-type drying box. EFFECT: higher cholagogue and anti-inflammation activity. 13 tbl

Description

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

 There is a method of obtaining a decoction of ordinary dentate (Nikolaev S.M., Sambueva Z. G., Batorova S.M., Hingeyeva S.L. Promising plant species with choleretic action from the arsenal of Tibetan medicine // Proceedings of the Siberian Academy of Medical Sciences of the USSR, Novosibirsk, 1987 , pp. 48-51), used as a choleretic agent. To prepare the broth, the crushed medicinal plant material - the grass of the dentate ordinary, is filled with room temperature water, taken into account the coefficient of water absorption, in an appropriate container and insisted on a boiling water bath with frequent stirring for 30 minutes, then cooled for 10 minutes, filtered (squeezing the remainder of the plant material ) and add water to the required volume of extraction. The disadvantage of this method is the relatively low choleretic activity compared with the amount obtained by the claimed method.

 The objective of the invention is to increase the yield of active substances, increase choleretic activity and the establishment of anti-inflammatory effects.

To solve this problem, the crushed plant material (ordinary grass) is extracted with 35-45% ethyl alcohol in the ratio of raw materials: extractant 1: (12-14) taking into account the coefficient of water absorption for 60 minutes at a temperature of 60-65 ^o C and stirring constantly. The process is repeated twice. In a similar manner, a third extraction is carried out for 30 minutes. Water-alcohol extracts are combined and evaporated to 1/3 of the original volume.

 The resulting product is purified by separation and dried in a vacuum oven. The yield of the finished product is 20.58% by weight of the plant material. Identified distinguishing features allow us to conclude that the proposed technical solution meets the criterion of "novelty."

 One of the pressing problems of medical science and practical health care is the prevention and treatment of diseases of the liver and biliary tract. The social significance of this problem is due to the widespread, diverse etiological factors and the special severity of diseases that lead to disability of the population. In this regard, the search and development of new effective medicines from plant materials that have low toxicity and do not have side effects with prolonged use is an urgent task.

 Given the needs of practical health care in herbal medicines intended for the treatment of diseases of the liver and biliary tract, a method has been developed to obtain a dry extract from the grass of ordinary dentate.

 Further research and the data presented in the text allow us to conclude that the proposed solution meets the criterion of "inventive step" and the proposed in the claims allow to achieve the goal.

 The final product is an amorphous powder of a brownish-black color, a loss in mass upon drying of 3.5%.

 We carried out a chemical analysis of the dry extract.

1. Based on the qualitative phytochemical analysis of the dry extract, the presence of:
1) flavonoids
0.1 g of the obtained extract is dissolved in 50 ml of 40% alcohol.

 To 3 ml of the resulting solution add 3 ml of a 2% solution of aluminum chloride, a yellow color appears.

 Thin-layer chromatography on Silufol plates in a solvent system of chloroform-acetic acid (5: 2): when viewing the chromatogram in UV light before and after treatment with ammonia vapors, the following were detected: apigenin - Rf = 0.69, luteolin - Rf = 0, eight; luteolin-7-glucoside - Rf = 0.6. Their identification was carried out in comparison with authentic samples.

 2) tannins.

 0.05 g of the obtained extract is dissolved in 5 ml of purified water.

 To 2 ml of the resulting solution add 2-3 drops of a solution of iron-ammonium alum, a black-green color appears.

3) iridoids
0.05 g of the obtained extract is dissolved in 5 ml of purified water.

 A few drops of Stahl's reagent are added to 2 ml of the resulting solution (1 g of p-dimethylaminobenzaldehyde, 5 g of phosphoric acid and 50 g of acetic acid, diluted with water to 100 ml), brought to a boil in a water bath, a blue color appears.

4) phenol carboxylic acids
two-dimensional ascending chromatography on paper brand FN-12 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).

 A method for the quantitative determination of the sum of flavonoids has been developed.

 2. Quantitative determination of the amount of flavonoids by spectrophotometric method.

 An analytical sample of raw materials is crushed to the size of the particles passing through a sieve with holes with a diameter of 1 mm

 About 1.0 g (accurately weighed) of the crushed raw material is placed in a conical flask with a thin section with a capacity of 100 ml, 50 ml of 70% alcohol are added, weighed. The flask is attached to the reflux condenser and heated in a boiling water bath for 60 minutes, periodically shaking to close the particles from the walls. After cooling to room temperature, the flask is weighed, adjusted with 70% alcohol to the original mass. The extract is filtered through a funnel with a cotton swab into a dry flask with a capacity of 100 ml, discarding the first 10 ml of the filtrate (solution A).

 In a volumetric flask with a capacity of 25 ml, 2 ml of solution A and 2 ml of solution of aluminum chloride in 70% alcohol are added and the volume of the solution of 70% alcohol is adjusted to the mark and stirred. After 30 minutes, the optical density of the solution was measured on a spectrophotometer at a wavelength of 395 nm in a cuvette with a layer thickness of 10 mm.

 As a comparison solution, use a solution consisting of 2 ml of extraction, 1 drop of diluted acetic acid and brought with 70% 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 luteodine, prepared similarly to the test solution, is measured.

где D - оптическая плотность испытуемого раствора;
D_о - оптическая плотность раствора ГСО лютеолина;
m - масса сырья в граммах;
m_о - масса ГСО лютеолина в граммах;
W - потеря в массе при высушивании сырья в граммах.The content of the sum of flavonoids in terms of luteolin for 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 GSO solution of luteolin;
m is the mass of raw materials in grams;
m _o is the mass of GSO luteolin in grams;
W is the loss in mass upon drying of the raw material in grams.

 Found 0.64 ± 0.02% flavonoids.

 The proposed method of obtaining is quite simple, does not require a complex cleaning system, allows you to get a product of constant composition. The technology can be implemented in enterprises producing drugs.

 To prove the materiality of the proposed features, the influence of the composition and nature of the extractant, its relationship with the raw material, temperature, degree of grinding of the raw material, duration and multiplicity of the number of extractions on the yield of biologically active substances from the studied medicinal plants was studied.

 A quantitative assessment was carried out according to the yield of the amount of extraactive substances (GF XI, issue 1, p. 295) and the content of the sum of flavonoids in terms of the luteolin standard.

 The metrological characteristic of the determination of the sum of extractive substances and the sum of flavonoids was carried out on a sample of ordinary dentate collected in Ivolginsky district in 1998 (table 1).

 The selection of the optimal extractant is one of the main points in obtaining extracts. To select the type of extractant to study, the processes of extraction of raw materials with water, ethyl alcohol of various concentrations: 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 95.

 The results are presented in table 2.

 As can be seen from the table, 35-45% alcohol is the optimal extractant, because it allows you to get the largest amount of the sum of flavonoids, taking into account the output of the sum of extractive substances.

 It is known that the particle size of the raw materials determines the completeness and rate of transition of extractable substances into the solution. The dependence of the yield of the sum of flavonoids and the sum of extractive substances on the degree of grinding of raw materials is established. The raw material was subjected to grinding to a particle diameter of 0.5; 1; 2; 3; 5; 7; 10 mm.

 The analysis data are presented in table 3.

 The optimum degree of grinding of raw materials is a particle size of 5 mm.

 In detail, the optimal ratio of raw materials-extractant for a single gulf of raw materials, see table 4.

 The maximum yield of extractives and flavonoids is observed when the ratio of raw materials and extractant 1:12. A further increase in the volume of the ratio of raw materials: extractant to (1:12) is inappropriate, since the consumption of extractant increases.

Based on experimental data, it can be concluded that an increase in the temperature of the extractant positively affects the course of the extraction process. It was found that at 60 ^o C there is a maximum yield of extractive substances and flavonoids (table 5).

 In order to determine the duration and frequency of the extraction number, we studied the dependence of the time to reach equilibrium concentration in the raw material-extractant system with a three-time extraction of raw materials.

To do this, we extracted the crushed raw materials with 40% alcohol in a ratio of 1:12 in a water bath at a temperature of 60 ^o C with constant stirring under reflux. At predetermined time intervals (15, 30, 45, 60, 75, 90 minutes), the extracts were filtered (1 phase contact), determining the content of the sum of extractive substances and the sum of flavonoids. Two subsequent extraction of the squeezed raw material was carried out in the same way for the same period of time, each time supplying 40% alcohol in an amount equal to the volume of the fused extraction (II and III contact phases), were also analyzed for the content of the sum of extractive substances and the sum of flavonoids. Experimental data indicate that the equilibrium state occurs at I, II phase contacts after 60 minutes, with III phase contact after 30 minutes (table 6).

 During phase I contact, about 60-70% of extractive substances and the sum of flavonoids are extracted. II contact provides a yield of 20-30% and III contact - 10-20%, i.e. triple extraction is sufficient for complete depletion of raw materials.

 Determination of the yield of extractives in all these experiments was carried out according to the following methodology of the same type given in State Pharmacopoeia XI (State Pharmacology of the USSR, issue 1. General methods of analysis, Ministry of Health of the USSR, Nth ed. Supplementary M., 1987 - 336 s).

 Example 1

1 kg of grass of the common serratus is ground in a mill to a particle size of 5 mm in diameter (sieve N 20 GOST 214-83). The crushed raw materials are loaded into an extraction apparatus with a stirrer and external steam heating. Pour 12 liters of 35% alcohol in the ratio of raw materials: extractant 1:12 taking into account the coefficient of water absorption. It is extracted at a temperature of 55 ^° C. with constant stirring for 60 minutes. The extract is filtered through a tin cloth in the collection. A further 2 extractions are carried out for 60 minutes and 30 minutes, each time supplying 35% alcohol to the extractor in an amount equal to the volume drained, 1st discharge - 9.9 L, 2nd discharge - 9.8 L, 3rd plum - 10 l. The combined aqueous-alcoholic extracts are evaporated to about 1/3 to 1/4 of the original volume. The bottom residue from the three extracts is subjected to purification by separation and dried on stainless baking sheets in a vacuum dryer at 65-70 ^o C for 8 hours. Get 195.0 g of the finished product, which is 19.50% of the dry extract by weight of the feedstock.

 Dry extract is an amorphous powder of brownish-black color with a pleasant smell, crumbles, with an astringent taste. The loss in mass upon drying is 3.5%.

The acute toxicity of the dry extract of the common cinquefoil grass was determined on white mice of both sexes (weight 20.0 ± 2.0 g) with intraperitoneal administration. The obtained data were processed statistically using the Kerbury method. The test substance was administered in doses of 500-6000 mg / kg with an interval between doses of 200 mg / kg. Observation of animals was carried out for 14 days. It was found that LD _{50 of} dry extract of grass of ordinary dentinata when administered intraperitoneally to white mice is 5234 mg / kg, which makes it possible to attribute the test agent according to the classification of K.K. Sidorova (On the classification of toxic poisons with parenteral routes of administration // Toxicology of new industrial chemicals, M. 1973, issue 13, p. 47-51) to relatively harmless substances.

 Evaluation of the choleretic activity of the extract of common dentate was carried out on outbred rats of both sexes with an initial weight of 170-200 g in acute experiments according to the standard technique. Rats were anesthetized by intraperitoneal administration of a 1% solution of barbamil in a volume of 0.8 ml / 100 g weight. Bile was obtained using a polyethylene cannula inserted into the common bile duct every hour for 5 consecutive hours. Dry extract (SE) in the form of an aqueous solution was injected into the duodenum with a syringe at doses of 100 and 300 mg / kg (doses at which pronounced choleretic activity was established). The rats of the control group were injected with an equivolume amount of distilled water. As a comparison drug used a decoction of ordinary dentate, which was introduced according to a similar scheme. The degree of choleretic activity was judged by the rate of secretion and the total amount of bile excreted, by the content of bile acids in bile (Y. I. Karbach. Quantitative determination of bile acids in bile and blood using the chromatographic method // Biochemistry, 1961, v. 21, No. 2 , pp. 305-309), as well as the number of bilirubin and cholesterol excreted with bile. The total amount of cholesterol was determined by the method of S.M. Drogovoz (Violation of the intensity of bile secretion and the chemical composition of bile during liver dystrophy caused by carbon tetrachloride. // Questions of Chemistry, 1971 - issue 4, p. 397-400), and bilirubin - according to the van der Berg method in the modification of N.P. Horse (The neurohumoral mechanism of the choleretic action of insulin // Problems of Endocrinology. 1956, N 6, p. 75-78). Statistical processing of the results presented in table 7, carried out by E.V. Monceviciute-Ehringen (simplified mathematical and statistical methods in medical research work // Pathological physiology and experimental therapy, 1964, N 4, pp. 71-78).

 As follows from table 7, the extract of dentate ordinary has a pronounced choleretic effect. At the same time, the rate of bile secretion increased by 34% after 1 hour after the introduction of the extract of dentinum ordinary, and in the following hours, in particular, at a dose of 300 mg / kg, it was almost 1.5 times higher than the control. In addition, the serum extract stimulated the synthesis and secretion of bile acids, promoted the secretion of bilirubin and the excretion of cholesterol with bile. At the same time, the choleretic activity of the proposed agent was higher than that of a decoction of ordinary dentate.

 We studied the effect of the serratus extract on the exudative phase of inflammation according to the generally accepted method for 30 outbred white rats of both sexes weighing 180-200 g. Aseptic inflammation of the extremity was caused by subplant implantation of 0.1 ml of a 3% formalin solution into the right hind paw of the rat (Strelnikov Yu. E. Comparative characterization of the anti-inflammatory action of certain pyrimidine derivatives // Pharmacology and Toxicology. 1960, No. 6, pp. 526-531). An aqueous solution of the serratus extract was administered intragastrically at doses of 50, 100 and 200 mg / kg in a volume of 1 ml / 100 g 3 hours before the administration of the phlogogenic agent, and then 5 and 18 hours after that. The animals in the control group were injected with an equal volume of distilled water according to a similar scheme. Caleflon at a dose of 100 mg / kg was used as a comparison drug. 24 hours after the introduction of formalin, the edema was determined by the oncometric method according to the difference between the volumes of the inflamed and uninflamed paws of animals. The data obtained are shown in table 8.

 As follows from the data given in table 8, the extract of dentate in all the studied doses had an antiexudative effect. At the same time, the severity of its effect depended on the dose used, if when introducing the extract of ordinary dentate at a dose of 50 mg / kg, only a tendency to decrease the volume of edema was detected (the difference is not significant), then an increase in the dose of the tested phytoextract was accompanied by an increase in its activity as follows: its administration in doses of 100 and 200 mg / kg inhibited the development of edema by 34 and 29%, respectively, compared with the data of animals of the control group. In this case, the comparison drug had a similar antiexudative effect.

 Example 2

1 kg of grass of the common serratus was ground in a mill to a particle size of 5 mm in diameter (sieve No. 20 GOST 214-83). The crushed raw materials were loaded into an extraction apparatus with a stirrer and external steam heating. Poured 12 liters of 40% alcohol in the ratio of raw materials: extractant 1:13 taking into account the coefficient of water absorption. It is extracted at a temperature of 60 ^° C. with constant stirring for 60 minutes. The extract is filtered through a tin cloth in the collection. A further 2 extractions are carried out for 60 minutes and 30 minutes, each time supplying 40% alcohol to the extractor in an amount equal to the volume drained, 1st drain - 9.9 L, 2nd drain - 9.7 L, 3 drain - 9.8 liters The combined water-alcohol extracts are evaporated to about 1/3 to 1/4 of the original volume. The bottom residue from the three extracts is subjected to purification by separation and dried on stainless baking sheets in a vacuum dryer at 65-70 ^o C for 8 hours. Get 201.5 g of the finished product, which is 20.15% of the dry extract by weight of the feedstock.

 Dry extract is an amorphous powder of brownish-black color with a pleasant smell, crumbles, with an astringent taste. The loss in mass upon drying is 2.9%.

 Evaluation of the choleretic activity of the extract of dentinata was carried out according to the method described in example 1.

 The results are shown in table. nine.

 It has been established that 40% of the denticular extract has a pronounced choleretic effect. Under the influence of the ordinary serratus extract, the rate of bile secretion at a dose of 300 mg / kg increased from 2 to 5 hours of the experiment by 39-52% compared with the control. Toothed extract, exhibiting the property of true cholesecretics, stimulated the synthesis of bile acids. The extract also accelerated the excretion of bilirubin and cholesterol with bile. At the same time, the choleretic activity of the extract exceeded that of a decoction of ordinary dentate.

 We studied the effect of the dentate extract on the course of acute peritonitis. The experiments were carried out on white infertile rats of both sexes weighing 160-180 g. Acute peritonitis was caused by intraperitoneal injection of 1 ml of 0.2% silver nitrate solution into animals (Alexandrov P.I., Speranskaya T.V., Bobkov Yu.G. et al. The effect of rutin and esculamine on some models of aseptic inflammation // Pharmacology and Toxicology, 1986, N 1, pp. 84-86). A test phytoextract at a dose of 100 mg / kg was administered 30 minutes and 1 hour after injection of the phlogogenic agent. Animals of the control group received an equivolume amount of distilled water. Caleflon at a dose of 100 mg / kg was used as a comparison drug. After 24 hours from the start of the experiment, the volume of exudate in the abdominal cavity and the number of degranulated mast cells in the mesentery were determined. For this, the mesentery was fixed with a Carnoy mixture and stained with a 0.05% toluidine blue solution. Counting the number of degranulated mast cells was carried out in 100 fields of view with an increase of x20.

 The data obtained are shown in table 10.

 As follows from Table 10, the development of acute peritonitis was accompanied by the appearance of a reddish-brown exudate in the abdominal cavity and total mast cell degranulation. Against the background of the introduction of a dentifrice extract at a dose of 100 mg / kg, the volume of intraperitoneal fluid decreased by 2.5 times compared with the data of the rats of the control group, which indicates the presence of a pronounced antiexudative activity in the test extract. At the same time, in the animals of the experimental group, the intraperitoneal fluid had an opalescent hue, which indicates a significant decrease in the number of red blood cells in it. The introduction of the serratus extract was also accompanied by a significant decrease in mast cell degranulation: for example, their number as a percentage decreased by more than 2.5 times compared with that in animals of the control group. The data obtained indicate that a tooth extract in a dose of 100 mg / kg has an anti-inflammatory effect in acute peritonitis.

 Example 3

1 kg of grass of the common serratus is ground in a mill to a particle size of 5 mm in diameter (sieve N 20 GOST 214-83). The crushed raw materials are loaded into an extraction apparatus with a stirrer and external steam heating. Pour 12 liters of 45% alcohol in the ratio of raw materials: extractant 1:14 taking into account the coefficient of water absorption. It is extracted at a temperature of 65 ^° C. with constant stirring for 60 minutes. The extract is filtered through a tin cloth in the collection. A further 2 extractions are carried out for 60 minutes and 30 minutes, each time supplying 40% alcohol to the extractor in an amount equal to the volume drained, 1st discharge - 9.8 L, 2nd discharge - 10 L, 3rd discharge - 9.8 liters The combined aqueous-alcoholic extracts are successively evaporated in portions to approximately 1/3 to 1/4 of the original volume. The bottom residue from the three extracts is subjected to purification by separation and dried on stainless baking sheets in a vacuum dryer at 65-70 ^o C for 8 hours. Get 199.5 g of the finished product, which is 19.95% of dry extract by weight of the feedstock.

 Dry extract is an amorphous powder of brownish-black color with a pleasant smell, crumbles, with an astringent taste. The loss in mass upon drying is 3.8%.

 Evaluation of the choleretic activity of the extract of common dentate was carried out according to the method similar to that in examples 1 and 2.

 The results are presented in table 11.

 From the data given in table 11, it follows that 45% of the dentinata extract also has a choleretic effect, slightly inferior to the action of 40% of the extract, and at the same time exceeds the similar effect of the dentate broth.

 We studied the effect of the dentate extract on the processes of tissue alteration and regeneration. The experiments were carried out on white outbred rats of both sexes weighing 160-180 g. The inflammatory process was modeled according to the Menkin method (Oyvin I.A., Shetel S.L. Methodology for studying local disorders of capillary permeability. Materials on the pathogenesis of inflammation and pathology of blood proteins. Dushanbe, 1961, v. 49, No. 5, pp. 167-173) by subcutaneous administration to white rats in the back region of 0.5 ml of a 9% solution of acetic acid, a solution of dextran at a dose of 300 mg / kg was simultaneously administered intraperitoneally. An aqueous solution of the serratus extract was administered intragastrically at a dose of 100 mg / kg 1 hour before the introduction of the florogenic agent, and then daily once a day throughout the experiment. Rats in the control group received an equivolume amount of distilled water according to a similar scheme. Caleflon at a dose of 100 mg / kg was used as a comparison drug. The area of necrotic tissue was evaluated on days 1, 3, 7, 14, and 21 of the experiment by applying a necrosis contour to a transparent film. The data obtained are presented in table 12.

 As follows from the data given in table 12, the course administration of the dentate extract at a dose of 100 mg / kg has an anti-inflammatory effect, reducing the degree of tissue destruction during alteration with acetic acid and contributing to the accelerated regeneration of damaged tissue. Moreover, testing on days 1 and 3 of the experiment revealed only a tendency to decrease the area of necrotic tissue (by 13 and 17%, respectively, compared to the control, the difference is not significant), then on 7, 14, and 21 days, a significant decrease in this indicator, respectively, by 28, was established. 27 and 43% compared with the same data in the control group. With the introduction of the comparison drug, a similar trend was established: a significant decrease in the degree of tissue destruction was found only on the 7th day of the experiment, however, upon further observation (on the 14th and 21st days), kaleflon had a less pronounced anti-inflammatory effect than the ordinary dentate extract.

 Comparative characteristics of the proposed method with the selected prototype are presented in table. thirteen.

Claims (1)

 The method of obtaining funds with choleretic and anti-inflammatory activity by extraction of plant materials - ordinary grass, characterized in that the extraction is carried out with 35 - 45% ethyl alcohol in the ratio of raw materials - extractant = 1: 12 - 14 twice for 60 minutes, the third extraction carried out for 30 minutes, the combined extracts are evaporated, purified by separation, concentrated with an additional park and dried in a vacuum dryer.

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