RU2270686C2 - Method for preparing hepatoprotective and hypocholesterolemic agent - Google Patents

Abstract

FIELD: medicine, pharmaceutical industry, phytotherapy.

SUBSTANCE: invention relates to a method for preparing the hepatoprotective and hypocholesterolemic agent. Method for preparing hepatoprotective and hypocholesterolemic agent involves using common wormwood-leaved ragweed (Ambrosia) as the parent raw gathered in the blooming phase. Raw is milled and extracted with ethanol three times under the definite conditions, extracts are combined, filtered and evaporated under vacuum. An aqueous-alcoholic vat residue is mixed with hot water and heated to the complete removing ethanol and then purified from lipophilic substances. The prepared purified aqueous extract comprising the sum of polyphenolic compounds is divided for two equal parts. One part is evaporated to dryness and dry sum of polyphenolic compounds is prepared - fraction 1; the second part is treated sequentially with solvents being with ether firstly to isolated fraction 2 and then this part is treated with ethyl acetate to isolate fraction 3 followed by treatment with butanol to isolated fraction 4. For each extraction solvents are taken in the definite amount of the aqueous extract volume. Extraction time is 5 min, multiplicity is 5, settling is 5 min followed by removing solvents under vacuum up to dry residue. Method provides preparing an agent from common wormwood-leaved ragweed (Ambrosia) possessing effective hepatoprotective and hypocholesterolemic effect.

EFFECT: improved preparing method.

4 tbl, 4 ex

Description

The invention relates to the pharmaceutical industry, and in particular to a method for producing biologically active substances from ragweed ragweed with hepatoprotective and hypocholesterolemic action, as well as the solution of the quarantine problem for the majority of the population.

Wormwood (Ambrosia artemisiifolia L.), being a malicious quarantine plant, easily adapts to any climatic conditions and during the flowering period creates serious problems for the majority of the population, causing allergic diseases (Ado V.A., Astafieva N.G. Pollinoza. - M .: Knowledge, 1991 .-- 224 p.).

The plant is practically not used in the national economy, with the exception of obtaining anti-allergic drugs from flower pollen (Studying the antigenic properties of an allergoid from ragweed pollen. B.N. Raikis, S.A. Ugrimov, F.L. Makhlinovskaya and others. Bull. Experimental biology and medicine - 1980. - T. 90, No. 8. - S.197-198).

The invention raises a serious environmental problem, as it allows to destroy the thickets of malicious quarantine plants during the growth period to the flowering phase. On the other hand, the invention relates to the pharmaceutical industry and relates to the production of ragweed from ragweed sum of polyphenolic compounds with hepatoprotective and hypocholesterolemic effects.

In medicine, the use of total polyphenolic phytopreparations and biologically active additives with these types of activity is widely practiced. Thus, a number of preparations from milk thistle are known: “Legal”, “Karsil”, “Silibor” (Quantitative determination of silybin and the amount of flavolignans in Silibum Marianum (L.) fruits. V.A. Kurkin, G.G. Zapesochnaya, E. V. Avdeeva et al. Plant resources - 1996. - T.32, issue 3. - S.80-86), as well as combined dry plant extracts: "Sibektan" - the amount containing dry extract of tansy, pulp of milk thistle, hypericum and birch; "Hepatofalk plant" - an extract of milk thistle, large celandine and Javanese turmeric. From leaves of Amur velvet receive "Flacoside"; pumpkin is the raw material for the well-known total preparations “Tykveol” and “Tykveinol” (Russian Medicines. Radar - Encyclopedia of Medicines. - 9th ed. revised and revised. Edited by G.L. Vyshkovsky. - M .: Radar, 2002 .-- 1504 p.).

Closest to the claimed invention is a method for producing a dry purified extract from spotted milk thistle - Silymar (Sokolskaya T.A. Complex processing of spotted milk thistle fruits and the creation of the preparation Silimar based on it. Chemical and pharmaceutical journal. - 2000. - T. 34, No. 9. - S.27-30). The essence of this method is that the crushed fruit is extracted three times at a temperature of 55-60 ° C with 80% ethyl alcohol. The time of the first extraction is 3 hours, the second is 2 hours, the third is 1 hour. The ratio of raw material to extractant at the first contact of the phases is 1: 8, the extractant is fed to the second and third extraction in the amount drained. The first and second extracts are evaporated in vacuo to 20-25% of the initial volume (concentration), the third extract is fed to fresh raw materials. The water-alcohol bottoms (after cooling) are treated with stirring 2 times with petroleum ether, and 20% of the volume of the bottoms are taken for each extraction solvent. The time of each extraction is 20 minutes, sedimentation 30 minutes The VAT residue purified from fatty substances is poured with stirring into a 0.05% hydrochloric acid solution 23: 100); the reaction mass is stirred for 15 min, while the precipitate is precipitated - silymar. Silymar is filtered from the mother liquor, then washed with water to neutral washings, weathered under draft for 8-10 hours, and then dried in a air dryer at a temperature of 45-50 ° C for 8-10 hours. The dried product is crushed and sieved through a nylon sieve.

This method of obtaining the total preparation is very time-consuming, combined with the consumption of large volumes of solvents, and the use of hydrochloric acid, which can have an undesirable effect on glycosides, namely, initiate the hydrolysis of phenolic glycosides, is required at the stage of precipitation of silymar.

The aim of the invention is to develop a method for producing a drug with hepatoprotective and hypocholesterolemic action from a malignant quarantine plant - ragweed ragweed (Ambrosia artemisiifolia L.), which makes it possible to create a new therapeutic agent and also purposefully destroy a harmful weed plant before the flowering phase.

An essential distinguishing feature of this method is its availability, low cost of feedstock, as well as the implementation of the most important quarantine task.

An example of a specific implementation. Raw materials - the aerial part of ragweed are collected during the period of growth and the beginning of budding (before flowering!). The crushed raw materials are exhaustively extracted with 96% ethanol, at a temperature of 60 ° C and constant stirring (extraction ratio is 3; the ratio of raw materials to extractant is 1: 7, 1: 5, 1: 5; the time of each operation is 1 h). The extracts are combined, filtered and evaporated in vacuo to 10-15% of the original volume (concentration). The water-alcohol bottoms are poured into hot water with stirring (1:10); the reaction mass is stirred for 1 h at a temperature of 80 ° C until complete removal of ethanol residues. The aqueous extract after cooling is filtered through a fine-pore paper filter. A purified aqueous extraction of the sum of polyphenolic compounds is obtained, which is divided into two equal portions, one of them is evaporated to dryness and the dry sum of polyphenolic compounds is obtained (fraction I). The allocation scheme of polyphenolic compounds from ragweed is presented in figure 1.

The second portion is transferred to a separatory funnel and sequentially treated first with diethyl ether, then with ethyl acetate and n-butanol, with a solvent in the amount of 3-5% of the volume of aqueous extraction taken for each extraction. The time of each extraction is 5 minutes, multiplicity 5, sedimentation 5 minutes The solvents — diethyl ether, ethyl acetate and n-butanol — are evaporated in vacuo to a dry residue and fractions II, III, IV are obtained, respectively.

Polyphenolic compounds contained in the obtained fractions are phenolcarboxylic acids, flavonoids and coumarins. Table 1 shows the differences in the qualitative composition of individual fractions.

As for the quantitative composition, in the total recovery (fraction I), the flavonoid content is maximum (4.45 ± 0.09%). Fraction II (ether extraction) contains mainly aglycons of flavonoids (1.04 ± 0.05%), fraction IV (butanol extraction) glycosides (2.52 ± 0.08%), and fraction III (ethyl acetate extraction) both (1.06 ± 0.05%). The results are presented in table 2.

From the presented data it is seen that coumarins are the main substances of the total fractions. Fraction II contains mainly coumarin aglycones (30.47 ± 1.5%), fraction IV glycosides (20.02 ± 0.88%), and fraction III both (25.08 ± 1.15%).

There is a small amount of sesquiterpene lactones, of which cumanin and peruvine are dominant.

The hepatoprotective and hypolipidemic effect of the studied substances was studied on white outbred male rats weighing 220-250 g in 2 series of experiments.

The hepatoprotective effect was studied on a model of acute toxic liver damage with carbon tetrachloride. The total substances in doses of 200 mg / kg were started orally 6 days before the start of the reproduction of the model of acute liver damage and then every day for another 6 days (12 days in total) against the background of the reproduction of CCl ₄ lesions of the liver, 3 times every other day 50 % solution of CCl ₄ in liquid paraffin in a dose of 0.15 ml / 100 g body weight. Slaughter of animals was performed 24 hours after the last injection of CCl ₄ . The official hepatoprotector - “Karsil” in a therapeutic dose (100 mg / kg) was used as a comparison drug (Mashkovsky M.D. Medicines: In 2 vols. - 14th ed. Revised and supplemented. - M .: New Wave , 2000 .-- 2 vol.). At the same time, slaughter of intact animals starving for 12-14 hours.

The effectiveness of the hepatoprotective effect was evaluated by normalizing the functional and biochemical parameters of the liver, which are the most informative tests proving the effectiveness of the compounds tested as hepatoprotectors: the activity of alanine aminotransferase (AlAt), alkaline phosphatase (ALP) and total bilirubin in blood serum, protein, nucleic acid acids (NK), triglycerides (TRH) and TBA-active products in the liver homogenate (Kolb V.G., Kamyshnikov BC Handbook of clinical chemistry - Minsk: Be Larus, 1982.- 336 p.). The results are presented in table 3.

The hypolipidemic effect of these fractions was studied on a model of vitamin hyperlipidemia caused by the oral administration of vitamin D ₂ . at a dose of 320,000 units together with cholesterol at a dose of 200 mg / kg daily for 4 days. The studied fractions in the form of an aqueous suspension were administered per os in doses of 100 mg / kg 6 days before the introduction of vitamin D ₂ and cholesterol, and then against the background of the reproduction of the hyperlipidemia model. As a comparison drug, Lipanor was used, which is used orally in animal experiments at a dose of 50 mg / kg (Mashkovsky M.D. Medicines: In 2 vols. - 14th ed. Revised and additional - M .: New Wave, 2000 .-- 2 vol.). Slaughter was done by decapitation 24 hours after the last exposure. In the blood serum, total cholesterol was determined by the method of Ilk and triglycerides by the method of Gottfried S.P., Rosenberg V. (Kolb V.G., Kamyshnikov BC Handbook of clinical chemistry - Minsk: Belarus, 1982. - 336 p.) And expressed in mmol / l. Statistical processing of the results was carried out using t-student test. The results are presented in table 4.

The therapeutic and prophylactic administration of total polyphenolic substances in doses of 200 mg / kg according to the studied biochemical parameters had a protective effect in the conditions of intoxication with carbon tetrachloride.

The most active were fractions III (ethyl acetate extraction) and IV (butanol extraction), under the influence of which normalization of all studied biochemical parameters of blood serum occurs: AlAt activity (cytolysis index) and alkaline phosphatase (cholestasis index) decreased by 57, 51% and 67, respectively. 38% (P _to <0.001), the total bilirubin content decreased by 48 and 34% (P _to <0.001, P _to <0.05). Moreover, a decrease of 12% (P _to > 0.05) and 39% (P _to <0.001) of the content of TBA-active products, respectively, was observed in the liver homogenate. The content of TEG decreased by 47% (P _to <0.001) and 30% (P _to <0.05) compared with the control, and there was an increase in the content of protein and nucleic acids by 32% (P _to <0.05), 23 % (P _to > 0.05) and 61, 43% (P _to <0.001), respectively (Fig. 2, 3).

It should be noted that the hepatoprotective effect of the sum of polyphenols at a dose of 200 mg / kg did not significantly differ from that of carzil in the following indicators: the content of TBA-active products, triglycerides, protein and nucleic acids in the liver.

In a number of other indicators, fractions III and IV exceeded the efficiency of the action of carcil, namely, in the degree of change in the activity of AlAt and alkaline phosphatase, which decreased with the introduction of carcil by 31% (P _to <0.001) and 47% (P _to <0.005), respectively. The bilirubin content in the blood serum, which under the action of Karsil practically did not normalize 4% (P _to > 0.1).

It should be noted that under the influence of these fractions there is a complete normalization of indicators such as AlAt, alkaline phosphatase, total bilirubin, TBA-active products, protein and nucleic acids in the liver, because they did not significantly differ from the level of intact animals (P _and > 0.1).

The effectiveness of lipid-lowering effects was evaluated by normalizing the content of cholesterol and triglycerides in blood serum, which are the most informative tests proving the effectiveness of compounds tested as lipid-normalizing agents.

Therapeutic and prophylactic administration of various fractions (Fig. 4) at doses of 100 mg / kg had a lipid-lowering effect. The most pronounced decrease in cholesterol and triglycerides (by 70% and 53%, respectively, P _to <0.001) in the blood was observed under the influence of fraction I. The introduction of ethyl acetate fraction III leads to a decrease in cholesterol by 52% (P _to <0.001), and triglycerides 36% (P _to <0.005). The ether fraction II reduces blood cholesterol and triglycerides by 30% (P _to <0.005) and 21% (P _to <0.05), respectively. At the same time, the activity of fraction IV was not significantly different from the activity of fraction I: cholesterol and blood triglycerides decreased by 68% (P _to <0.001) and 45% (P _to <0.001), respectively. Under the influence of fractions I and IV, a complete normalization of the studied parameters of lipid metabolism is observed, since they did not significantly differ from those in intact rats (P _and > 0.1).

Thus, aglycones and glycosides of phenolic acids, coumarins and flavonoids contained in total polyphenolic extracts from ragweed ragweed have the ability to lower blood cholesterol and triglycerides in hyperlipidemia. Moreover, the lipid-lowering activity of glycosides in vivo is higher than that of aglycones, which, apparently, is associated with their better solubility in water, which results in their better bioavailability.

It should be noted that the lipid-lowering activity of fractions I and IV from ragweed is not inferior in its effectiveness to the action of the standard preparation Lipanora.

Table 1
The chemical composition of total polyphenolic fractions from ragweed Biologically active fraction Connection class Hydroxycinnamic acids Coumarins Flavonoids Fraction I Contains fractions II, III, IV Fraction II ferulic ◆
isoferulic ◆
coffee *
chlorogenic * esculletin * ◆
scopoletin * ◆
umbelliferon * ◆ yaceidin * ◆ (5,7,4'-trihydroxy-3,6,3'-trimethoxyflavone) quercetin Faction III ferulic ◆
isoferulic ◆
coffee *
chlorogenic * esculletin * ◆
esculin * ◆
scopoletin * ◆
scopolin * ◆
umbelliferon * ◆
skimmin * ◆ isoramnetin *
quercetin * Fraction IV coffee chlorogenic glucoside * esculin * ◆
scopolin * ◆
skimmin * ◆ isoramnetin-3-rutinoside * ◆, quercimeritrin * ◆, isocvercitrin *, xanthomicrol glycosides * ◆ (4 ', 5-dihydroxy-6,7,8-trimethoxyflavone) and 4', 5-dihydroxy-3,6,7,8 -tetramethoxyflavone * ◆ * - predominant compounds;
◆ - identified for ragweed for the first time

table 2
The content of the main biologically active compounds in the total polyphenolic fractions of ragweed Biologically active fraction Sesquiterpene lactones,% Flavonoids,% Coumarins,% Fraction I 2.45 ± 0.05; ε = 2.10% 4.45 ± 0.09; ε = 2.06% 16.03 ± 0.50; ε = 3.16% Fraction II - 1.04 ± 0.05; ε = 4.96% 30.47 ± 1.50; ε = 4.91% Faction III 1.01 ± 0.05; ε = 4.61% 1.06 ± 0.05; ε = 4.80% 25.08 ± 1.15; ε = 4.59% Fraction IV 1.51 + 0.06; ε = 4.0% 2.52 ± 0.08; ε = 3.07% 20.02 ± 0.88; ε = 4.42%

Table 4
Hypolipidemic effect of ragweed extracts in rats with hyperlipidemia caused by the administration of vitamin D ₂ together with cholesterol Indicators Intact n = 4 Control n = 5 Fraction I, 100 mg / kg, n = 5 Fraction II, 100 mg / kg, n = 5 Fraction III, 100 mg / kg, n = 5 Fraction IV, 100 mg / kg, n = 5 Lipanor, 50 mg / kg, n = 4 Cholesterol, mmol / L 1.71 ± 0.088 6.57 ± 0.363
P _and <0.001
+ 284% 2.05 ± 0.220
P _to <0.001
-69%
P _and > 0.1 4.62 ± 0.256
P _to <0.005
-30% P _and <0.001 3.16 ± 0.281
P _to <0.001
-52% P _and <0.005 2.08 ± 0.306
P _to <0.001
-68%
P _and > 0.1 1.96 ± 0.079
P _to <0.001
-70%
P _and > 0.1 TRG syv. mmol / l 0.747 ± 0.089 1,768 ± 0,093
P _and <0.001
+ 136.7% 0.830 ± 0.092
P _to <0.001
-53%
P _and > 0.1 1,388 ± 0,091
P _to <0.05
-36%
P _and <0.005 1.127 ± 0.105 Pk <0.005 -37% Pb <0.05 0.975 ± 0.133
P _to <0.005
-45%
P _and > 0.1 0.905 ± 0.207
P _to <0.001
-49%
P _and > 0.1

Table 3
Hepatoprotective effect of total fractions from ragweed Indicators Intact n = 4 Control, n = 5 Fraction I, 200 mg / kg, n = 5 Fraction II, 200 mg / kg, n = 5 Fraction III, 200 mg / kg, n = 5 Fraction IV, 200 mg / kg, n = 5 Carsil, 100 mg / kg, n = 7 AlAt, mkkat / l 0.70 ± 0.046 1.85 + 0.053
P _and <0.001
+ 164% 1.27 ± 0.11
P _to <0.005,
-31%
P _and <0.005 1.37 ± 0.105
P _to <0.05,
-26%
P _and <0.01 0.80 ± 0.046
P _to <0.001,
-57%
P _and > 0.1 0.91 ± 0.044
P _to <0.001,
-51%
P _and <0.05 1.27 ± 0.046
P _to <0.001,
-31%
P _and <0.001 Total bilirubin, µmol / l 8.39 ± 1.02 20.61 ± 0.93
P _and <0.001
+ 146% 20.42 ± 1.34
P _to > 0.1
P _and <0.001 18.54 ± 1.06
P _to > 0.1
P _and <0.001 10.63 ± 1.21
P _to <0.001,
-48%
P _and > 0.1 13.65 ± 1.78
P _to <0.05,
-34%
P _and > 0.05 19.78 ± 1.99
P _to > 0.1
P _and <0.01 Liver TRH μmol / g 24.48 ± 1.67 79.76 ± 4.04
P _and <0.001
+ 226% 78.54 ± 4.52
P _to > 0.1
P _and <0.001 61.20 ± 6.97
P _to > 0.05
P _and <0.005 42.54 ± 2.92
P _to <0.001,
-47%
P _and <0.01 55.59 ± 3.67
P _to <0.005,
-thirty%
P _and <0.001 49.83 ± 4.36
P _to <0.001,
-38%
P _and <0.01 Alkaline phosphatase, units / l 176.88 ± 14.481 433.74 ± 49.945
P _and <0.001
+ 145% 254.86 ± 36.045
P _to <0.05,
-41%
P _and > 0.05 425.01 ± 5.019
P _to > 0.1
P _and <0.001 159.90 ± 19.293
P _to <0.001,
-63%
P _and > 0.1 267.51 ± 45.018
P _to <0.05,
-38%
P _and > 0.05 229.39 ± 11.526
P _to <0.005,
-47%
P _and > 0.05 Protein, mg / ml 25.5 ± 1.44 18.3 ± 0.81
P _and <0.01
-28% 20.9 ± 1.60
P _to > 0.05
P _and > 0.1 19.2 ± 0.71
P _to > 0.1
P _and <0.01 24.1 ± 1.54
P _to <0.05,
+ 32%
P _and > 0.1 22.5 ± 1.75
P _to > 0.05
+ 23%
P _and > 0.1 25.0 ± 1.32
P _to <0.005,
+ 37%
P _and > 0.1 NK, mg / g 47.75 ± 0.543 32.45 ± 1.341
P _and <0.001
-32% 42.38 ± 0.834
P _to <0.001,
+ 31%
P _and <0.005 34.65 ± 1.495
P _to > 0.1
P _and <0.05 52.34 ± 1.845
P _to <0.001,
+ 61%
P _and > 0.1 46.45 ± 0.946
P _to <0.001,
+ 43%
P _and > 0.1 45.25 ± 1.148
P _to <0.001,
+ 39%
P _and > 0.1 TBA liver
nmol / mg protein 0.1875 ± 0.0071 0.3227 ± 0.0117
P _and <0.001
+ 72% 0.2949 ± 0.0134
P _to > 0.1
P _and <0.001 0.3173 ± 0.0055
P _to > 0.1
P _and <0.001 0.2840 ± 0.0156
P _to > 0.05,
-12%
P _and <0.005 0.1980 ± 0.0169
P _to <0.001,
+ 39%
P _and > 0.1 0.1867 ± 0.0145
P _to <0.001,
+ 42%
P _and > 0.1

Claims (1)

A method of obtaining a hepatoprotective and hypocholesterolemic agent, characterized in that the raw materials used are ragweed, collected in the developmental phase before flowering, the raw materials are crushed and extracted three times with 96% ethanol at a temperature of 60 ° C with constant stirring and the ratio of the raw material extractant 1: 7 , 1: 5, 1: 5, respectively, for 1 h at each extraction, the extracts are combined, filtered and evaporated in vacuo to 10-15% of the original volume, the water-alcohol still residue is mixed with hot water and heated until ethanol is completely removed, then purified from lipophilic compounds, the resulting purified aqueous extraction of the sum of polyphenolic compounds is divided into two equal portions, one is evaporated to dryness and a dry amount of polyphenolic compounds is obtained - fraction 1, the second portion is sequentially treated with solvents, first with ether to isolate fractions 2, then treated with ethyl acetate to isolate fraction 3, then treated with butanol to isolate fraction 4, solvents are taken for each extraction in an amount of 3-5% by volume and aqueous extraction, the time of each extraction is 5 minutes, a multiplicity of 5, settling for 5 minutes, then the solvents are removed in vacuo to a dry residue.

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