RU2068269C1 - Method of preparing agent showing antisclerotic and cholagogue activity - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: red clover herb (Trifolium sativum Crome) is extracted with 75-80% ethanol at ratio raw : extractant = 1:(8-12) by eddy extraction method at stirring rate 8000 rev/min for 10 min followed by filtration. Then grist is placed into laboratory reactor with mixer and extracted with 75-80% ethanol three times for 30 min at 75-85 C. Ratio raw : extractant = 1:(8-12). Extracts were combined, filtered and evaporated under vacuum up to complete alcohol removing and cooled. Precipitate of lipophylic substances is separated, filtered and dried. Yield of the end product is 14.5-15.3%. Procedure duration is 5-6 hr. Yield is increased by 4-5-fold as compared with that in prototype. Antisclerotic activity of product is increased by 15-20% and that of cholagogue - by 1.2-fold as compared with standard "Flamin". EFFECT: simplified method, increased yield, enhanced activity.

Description

 The invention relates to the pharmaceutical industry and relates to the production of medicines from plant materials.

 Herbal products with pronounced antisclerotic and choleretic activity, as a rule, contain a complex of polyphenolic compounds (flavones, flavonols, isoflavonols, hydroxycinnamic acids, etc.).

A known method of producing a complex of polyphenolic compounds based on the collection of six types of medicinal plant materials using hot water (80 90 ^o ). However, in the described method, water is used as an extractant, which extracts a significant amount of concomitant substances (lipids, proteins, carbohydrates, etc.) from plant materials that do not have antisclerotic and choleretic activity.

 A method is described for producing flavonoids from the seeds of Milk Thistle (Salybum marianum), namely, in order to increase the purity of the target product, the raw material is boiled with carbon tetrachloride, then it is treated with a polar solvent, for example, formamide, the mixture is extracted with acetone or ethyl acetate, followed by the addition of aqueous sodium chloride solution, the target product is precipitated in water from the biphasic solvent system treated after repeated extraction with ethylene glycol containing calcium chloride and, if necessary gence, chromatographed on a polyamide column. The output of flavonoids is 1.97% of the feedstock. The resulting total flavonoid preparation from milk thistle seeds exhibits a pronounced hepatoprotective and choleretic effect. The duration of the method is 45 hours (3). The specified method was chosen by us as an analogue.

 The disadvantages of this method are the long duration (45 hours), the complexity and multi-stage, low yield of the target product (1.97%) and the use of highly toxic and expensive solvents and reagents (carbon tetrachloride, formamide, ethyl acetate, acetone, ethylene glycol, etc.), limited stocks of raw materials (only seeds are used).

 A known method of obtaining the sum of flavonoids from grass of red clover (Trifolium satiwum Crome) (4) by extraction of plant materials 70 75% ethanol, with a ratio of 1: (3 5). Purification is carried out sequentially with chloroform and ethyl acetate, followed by precipitation with dry chloroform of the target product. The yield is 3.83% in terms of air-dry feedstock. The duration of the method is 25 hours. The resulting total polyphenolic preparation from red clover grass exhibits pronounced antisclerotic activity (4). The specified method is chosen by us for the prototype.

 The disadvantages of this method are: long duration (25 hours) and the complexity, the use of toxic reagents (chloroform, ethyl acetate), significant losses of biologically active active substances in the production process and low yield of the target product (3.83%).

The aim of the invention is:
1. Increasing the yield of the target product;
2. Simplification and reduction of the duration of the method;
3. Exclusion of highly toxic reagents;
4. The expansion of the spectrum of pharmacological action;
5. An increase in antisclerotic activity.

 In the inventive method, we propose to use red clover grass, collected and dried by the shadow method during flowering, as a source of raw materials. Red clover (sowing) is universally introduced into the culture and its raw material base is practically unlimited. There is an agreement with the MGO "YUGAGROKONT" (Krasnodar Territory) on the procurement and supply of raw materials, and with the Krasnodar pharmaceutical factory on the production of the proposed method for the substance of the total polyphenol preparation from red clover grass, which under the name "Trizoflan" is allowed for clinical trials by the pharmacological committee of the Ministry of Health of the USSR as an antisclerotic and lipid-lowering agent.

This goal is achieved in that the method of obtaining red clover from the aerial part (Trifolium sativum Crome) is carried out by extraction of raw materials with 75 80% ethanol at a ratio of raw materials extractant 1: 8 1:12 by vortex extraction at a stirring speed of 8000 rpm for 10 min subsequent filtration of the extract. Next, the meal is placed in a laboratory reactor with a stirrer, pour 75 80% ethanol at a temperature of 75 85 ^o at a ratio of raw material extractant 1: 8 12 and extracted for 30 minutes The operation is repeated three times. Next, the extracts are combined, filtered and evaporated in vacuo until the alcohol is completely removed. The aqueous residue is cooled in the refrigerator (up to 5 hours at a temperature of 4 ^o C). The precipitated lipophilic substances are separated, filtered through a porous cloth on a suction filter. The purified aqueous extract is sent to a vacuum oven to obtain a dry purified extract. The yield of the target product 14.5 15.3%. The duration of the method 5 6 hours

In the inventive method, vortex extraction of raw materials is carried out in the device "Melextractor", where two processes occur simultaneously: grinding and extraction; moreover, due to the presence of two pairs of knives located horizontally and at an angle of 45 ^o to the working axis of the device, the grinding of raw materials goes in all directions with tearing of the cell walls, which contributes to the additional extraction of polyphenols not only from intercellular, but also intracellular juice, and therefore, helps to increase the yield of the target product. Due to the high mixing speed in the shortest possible time, equilibrium in the raw material extractant system occurs, which significantly reduces the duration of technological processes.

 Further extraction of the raw materials in a laboratory extractor with a hot ethanol mixer thrice contributes to the maximum extraction of polyphenols from the raw materials.

 The exception used in the prototype stage of the extraction of polyphenolic compounds with ethyl acetate and the subsequent precipitation of polyphenols with dry chloroform, significantly reduces the loss of the target product (3-4 times).

Example 1. 500 g of plant material (the aerial part of red clover grass) is poured into 4.0 L of 75% ethanol (room temperature) and mixed in a Melextractor at a speed of grinding knives at 6000 rpm for 8 minutes. Then the feed was filtered. After filtering, the meal is transferred to a laboratory reactor with a stirrer, it is poured with 75% ethanol at a temperature of 75 ^o with a feed-extractant ratio of 1: 8 and extracted for 20 minutes. For a more complete depletion, meal in a laboratory reactor with a stirrer is poured twice more. The extracts are combined, filtered and evaporated in vacuo until the alcohol is completely removed. The aqueous precipitate was placed in a refrigerator (4 ^° C) for up to 5 hours. Then, the supernatant was decanted, filtered through a porous cloth on a suction filter and sent to a vacuum oven to obtain a dry, purified extract. The yield of the target product is 14.5% i.e. 72.5 per 500 g of raw materials. The total duration of the method ≈7 hours

 The target product is the sum of polyphenols from red clover grass - is a dark brown powder with a specific smell and taste, soluble in aqueous ethanol.

Example 2. 500 g of the aerial part of red clover grass is poured with 5 l of 80% ethanol and mixed in a Melextractor device at a knife rotation speed of 8000 rpm for 10 minutes. Then the extract is filtered. The meal is transferred to a laboratory reactor with a stirrer and three times filled with 80% ethanol at a temperature of 80 ^o C at a ratio of raw material extractant 1:10 and extracted three times for 30 minutes. Then proceed as described in example 1.

 The yield of the target product was 15.3% i.e. 76.5 g per 500 g of raw materials.

Example 3. 500 g of the aerial part of red clover is filled with 6.0 L of 80% ethanol and mixed in a Melextractor device at a blade rotation speed of 10,000 rpm for 15 minutes. Then the extractor is filtered. The meal is transferred to a laboratory reactor with a stirrer and three times filled with 80% ethanol at a temperature of 85 ^o at a ratio of raw material extractant 1:12 and extracted three times for 40 minutes. The extracts are combined, filtered and evaporated until the alcohol is completely removed. Then proceed as described in example 1.

 The yield of the target product was 15.1% i.e. 75.5 g per 500 g of raw materials.

 Examples of specific performance are confirmed by table. 1 7.

 From the table. 1 it follows that the optimal ratio of raw material extractant is a limit of 1: 8 1:12, while ensuring the highest yield of the target product.

 From the table. 2 it follows that the optimal concentration of the extractant of ethyl alcohol is 75 to 80% which provides the greatest yield of polyphenols.

 From the table. 3 it follows that the maximum yield of polyphenols is provided at a speed of rotation of grinding knives 8000 rpm

 From the table. 4 shows that the optimal duration of mixing, providing a maximum yield of polyphenols of 10 minutes

From the table. 5 shows that the maximum yield of polyphenols occurs when extracting meal with ethanol at a temperature of 75 85 ^o C.

 From the table. Figure 6 shows that for the complete extraction of polyphenols from raw materials, meal in the laboratory reactor is poured three times with hot ethanol and the extracts are collected three times, which are then combined.

 As can be seen from the table. 7, in a laboratory reactor, raw materials must be extracted for 30 minutes, which ensures the highest polyphenol yield.

Examining all the tables, we can conclude that the maximum yield of polyphenols will occur if the feed is filled with 75 80% ethanol at a ratio of 1: 8 12, extracted by vortex extraction in a “Melextractor” at a stirring speed of 8000 rpm for 10 min And in a laboratory reactor, meal is poured with 75 80% ethanol at a temperature of 75 - 85 ^o C at a ratio of raw material extractant 1: 8 12 and extracted three times for 30 minutes.

 The isolated amount of polyphenolic compounds from red clover grass was studied for pharmacological activity (5,6,7).

Acute toxicity It was studied by the method of Kerber (5) on white mice with intraperitoneal administration of the studied compounds in a volume of 1 ml of an aqueous solution. The maximum dose possible for administration was 6000 mg / kg of the animal’s body weight; it did not cause the death of the animals, which allowed us to consider LD ₅₀ > 6000 mg / kg. According to the classification of toxicity (according to Sidorov K.K. 1973), the studied compounds should be attributed to the group of relatively harmless substances.

 The main specific antisclerotic activity was studied in comparison with the prototype. As a standard in the study of antisclerotic and lipid-lowering activity, the official preparation of this spectrum of action "Miskleron" was used.

 Biological testing of the drug was carried out on various models: intraperitoneal administration of WR-1339 triton, as well as on cholesterol and reference models. Comparison was made of intact animals and animals that received the prototype drug and the standard drug.

 Studies were conducted on rats and rabbits. As a result of the tests, it was found that the agent obtained by the claimed method has a pronounced hypolipidemic and hypocholesterolemic effect, which exceeds the action of the prototype drug by 15 20%. Moreover, the drug obtained by the claimed method has a 2.5 times higher level of cholesterol, β-lipoprotein reduction and phospholipids than that of the preparation of the standard "Minskleron" (data are given in table. 8).

 For the preparation obtained by the proposed method from red clover grass, an additional specific choleretic activity was revealed that was not described for the prototype preparation.

 Tests for choleretic activity were carried out on white rats of both sexes weighing 250 300 g. According to the method of M.D. Litvinchuk (1980) secretory bile was collected in hourly portions for 3 hours.

 The drug obtained by the proposed method from red clover grass and the reference drug "Flamin" was administered in the same dose of 50 mg / kg, as the most effective, in the form of a suspension with the addition of a few drops of Tween-80. A control group of animals under similar conditions received distilled water with the addition of an appropriate amount of Tween-80.

 In the total portion of bile, the content of bile acids and cholesterol was determined and the cholate-cholesterol coefficient was calculated (V.P. Miroshnichenko, 1978). The data are presented in table. 9.

 An analysis of the data shows that the secretion rate for 3 hours after administration of the drug obtained from red clover grass by the claimed method increased by 40% (P <0.05) compared with the control, which was 1.2 times higher than the action of the official reference drug Flamina. The drug obtained by the claimed method, reduced cholesterol in bile by 30% (P <0.05) and bile acids by 21% (P <0.05) compared with the control. The cholate-cholesterol coefficient was higher than in the control by 99% (P <0.01), which indicated an increase in the lithogenicity of bile.

So, the claimed method provides:
1. A significant simplification of the method of producing polyphenols. It does not require expensive, highly toxic reagents, easy to perform; excludes multistage. While in the known method it is proposed to use reagents toxic to the human body: chloroform, ethyl acetate.

 2. The duration of the proposed method is 5 to 6 hours, prototype up to 25 hours, i.e. the duration of the proposed method is 5 times less than the duration of the known method.

 3. Increasing the yield of the target product. In the inventive method, the yield of polyphenols is 14.5 15.3% of the feedstock, which is 4 to 5 times the yield of the product in the known method (3.83%).

4. Increasing the activity of the target product and expanding the spectrum of its pharmacological action. The total polyphenolic preparation obtained from red clover grass by the present method exceeds the antisclerotic activity of a similar preparation obtained by the known method by 15
20% and 45 50% in comparison with the Miskleron standard. In addition, for the proposed drug, the spectrum of biological action was expanded, choleretic activity was revealed, which exceeds the activity of the reference drug "Flamina" by 1.2 times.

Claims (1)

The method of obtaining funds with antisclerotic and choleretic activity, including extraction of the aerial part of the grass of red clover (Trifolium sativum Crome) 75 with 80% ethanol at a ratio of raw material extractant 1 8 12 by vortex extraction at a stirring speed of 8000 min ^-1 for 10 min, filtering the extract, extracting the meal with stirring 75 with 80% ethanol three times at 75 85 ^o C and with a ratio of raw material extractant 1 8 12 for 30 min, combining the extracts, filtering, evaporating, cooling and drying.

Images