The way to obtain 1-hydroxy-2,3,4,5-tetramethoxybenzene with choleretic and hepatoprotective action

 

(57) Abstract:

The invention relates to medicine. Plant material the grass Galeni horned extracted with 70% ethanol three times with respect to raw material: solvent 1: 10. The extractant contacting with hexane. Chromatographic hexane extract on a column of silica gel. Then spend evaporation, dissolution hexane by heating, filtration and recrystallization from ethanol. The invention improves the product yield. 3 table.

The invention relates to the field of medicine and relates to a method of obtaining biologically active compounds from plant material, has choleretic and hepatoprotective effect.

A method of obtaining 1-hydroxy-2,3,4,5 - tetramethoxybenzene of all plant species Halenia asclepidea (HBK) Acting Don. by extraction of dried crushed material (422 g) hexane, dissolving the precipitate in CH2Cl2, filtration and extraction with alkali Claisen's, followed by evaporation, thin-film chromatographytandem main soluble material (0,222 g) and continuous elution on Merk silica gel with petroleum ether (Et OAc (8: 1)), recrystallization from methanol. The output is to 0.011% by weight of the feedstock [Stout, G. H. , Fries J. L is XI-2,3,4,5-tetramethoxybenzene by extraction of the crushed air-dry raw material (550 g), acetone (three times), liquid-liquid extraction with CH2Cl2/ pentane United filtrate, filtration, evaporation CH2Cl2/ pentane, thin-layer chromatography on silica gel coarse thick yellow-brown extract (29 g) followed by crystallization from methanol. The output is 1% by weight of the feedstock. [Stout, G. H. , Balkenhol W. J. Xanthones of the Gentianaceae. - I Frasera caroliniensis Walt // Tetrahedron. 1969. -Vol. 25 (9), pp. 1947-1960] .

The disadvantages of these methods are low yield of the target product, the duration and the complexity of the process.

The closest technological essence and the achieved result is a way to obtain 1-hydroxy-2,3,4,5-tetramethoxybenzene from Halenia asclepidea (HBK) G. Don. consists in the fact that air-dry raw material is extracted with hexane, the residue is evaporated and chromatographic followed by recrystallization from methanol [Stout, G. H. , Fries J. L. The xanthones of Halenia species. Short communication // Phytochemistry, 1970, vol. 9, N 1. - pp. 235 - 236] . The disadvantage of this method is the low yield of the target product (0,011%).

The objective of the invention is to increase the yield of the target product from the aerial parts of Halenia corniculata (L. ) Cornaz. with choleretic and hepatoprotective action is edusim way: plant material is extracted with 70% ethanol three times in the process of evaporation, concentration of the combined extracts in vacuo to an aqueous residue (0.5 l), followed by treatment with hexane. Hexane extract after chromatography, evaporation, dilution with hexane by heating, filtration, recrystallization (twice) from ethanol allows you to get the finished product. The output is 0.17% of the weight of raw material (PL. 1).

The following examples illustrate the inventive method.

Example 1: 100 g powdered air-dried raw herbs Halenia corniculata (L. ) Cornaz. was extracted with 60% ethanol three times in the ratio of raw material and solvent of 1: 8 (18-20oC, 72 hours). The combined extracts were concentrated in vacuo to an aqueous residue (0.5 l), which were processed in a separating funnel with hexane (8 x 150 ml). Chromatographic analysis of the hexane extract of grass Halenia corniculata (L. ) Cornaz. showed the presence of Mironovich aglycones (system I, system II; paper Fn - 16), which are localized in one spot. Hexane extract was evaporated on a rotary evaporator to dryness and the obtained dry residue in the number of 25.6 g (2,56%). The obtained hexane fraction was dissolved by heating in 100 ml of hexane and left to cool to room temperature. The precipitated crystals were filtered and tx2">

Example 2: 100 g powdered air-dried raw herbs Halenia corniculata (L. ) Cornaz was extracted with 70% ethanol three times in the ratio of raw material and solvent of 1: 10 (18-20oC, 72 hours). The combined extracts were concentrated in vacuo to an aqueous residue (0.5 l), which were processed in a separating funnel with hexane (8 x 150 ml). Chromatographic analysis of the hexane extract of grass Halenia corniculata (L. ) Cornaz showed the presence of Mironovich aglycones (system I, system II; paper Fn - 16), which are localized in one spot. Hexane extract was evaporated on a rotary evaporator to dryness and the obtained dry residue in the number of 25.6 g (2,56%). The obtained hexane fraction was dissolved by heating in 100 ml of hexane and left to cool to room temperature. The precipitated crystals were filtered and recrystallized twice from ethanol. It was obtained 0.17 g of 1-hydroxy-2,3,4,5-tetramethoxybenzene.

Example 3: 100 g powdered air-dried raw herbs Halenia corniculata (L. ) Cornaz. was extracted with 80% ethanol three times in the ratio of raw material and solvent of 1: 12 (18-20oC, 72 hours). The combined extracts were concentrated in vacuo to an aqueous residue (0.5 l), which were processed in a separating funnel with hexane (8 x 150 m is Konov (I; system II; paper Fn - 16), which are localized in one spot. Hexane extract was evaporated on a rotary evaporator to dryness and the obtained dry residue in the number of 25.6 g (2,56%). The obtained hexane fraction was dissolved by heating in 100 ml of hexane and left to cool to room temperature. The precipitated crystals were filtered and recrystallized twice from ethanol. There was obtained 0.16 g of 1-hydroxy-2,3,4,5-tetramethoxybenzene.

PHARMACOLOGICAL STUDY OF SELECTED 1-HYDROXY-2,3,4,5-TETRAMETHOXYBENZENE

Choleretic activity of 1-hydroxy-2.3.4.5-tetramethoxysilane.

Definition choleretic activity 1 - hydroxy-2,3,4,5-tetramethoxybenzene conducted on 50 white rats of both sexes weighing 180-200 g according to the method of N. P. Steed, A. N. Oleinik [Racer N. P. , Olejnik A. N. Comparative effects of atropine and metacin on the exocrine function of the liver // Pharmacology and toxicology. - 1967. -So 30. -N 3. -S. 334 - 337] . The animals were housed for 5 hours under General anesthesia (1% aqueous solution of Barbara intraperitoneally in a volume of 1 ml per 100 g of animal) at room temperature. Bile was collected using a special cannula that is inserted in the common bile duct, through each PCB intestine of rats in the form of an aqueous solution of 1 ml. Rats of the control group was injected distilled water in equiano number. The degree choleretic action of the investigated tools to be judged by the rate of secretion of bile, the total volume and the number of allocated each time a portion of bile cholesterol [drogbas S. M. Comparative study and action features choleretic drugs on zheleobrazovatel the function of the liver in norm and pathology. Abstract. thesis Dr. med. Sciences. - Kharkov. - 1972. - 34 S. ), bilirubin [Racer N. P. Neurohumoral mechanism choleretic action of insulin // problems of endocrinology and hormonal therapy. - 1956. -N-6. -S. 75-78] , total bile acids [Karbach J. I. Quantitative determination of bile acids in bile and blood using chromatographic method // Biochemistry. - 1961. -So 26. -N 2. -S. 305 - 309). The obtained data statistically processed using t-test, Student [Montevista-öhringen E. C. Simplified mathematical-statistical methods in medical research // Pathological physiology and experimental therapy. 1964, N 4. -S. 71-78). The definition of ED501-hydroxy-2,3,4,5-tetramethoxybenzene conducted according to methods described Ivanov, Y. I. , Pogorelec O. N. [Ivanov Y. I. , Pogorelec O. N. am. - M. : Medicine, 1990. -224 C. ).

As a result of experiments it was determined that the xanthone-1 in a dose of 5 mg/kg exerts a moderate increase choleretic response in rats. Thus, the rate of secretion of bile within an hour after its introduction was increased by 19% after 2 hours by 14% and remained high during the experience. The total number of selected bile exceeded the control by 14% (table. 2). Against this background, in secureremote bile of rats increased the concentration of total of Khalatov 21%, bilirubin by 23%. With increasing doses of xanthone increased choleretic response in rats. In particular, at the dose of 10 mg/kg the rate of secretion of bile through after its introduction was increased by 44%, after 2 hours at 33%, and after 3-4 hours 16-20% compared with the control (table. 2). At the same time increased the total number of selected bile 28%, increased the concentration of total of Khalatov 11% and excretion of cholesterol. As the dose increases, the xanthone 1 to 30 and 50 mg/kg was noted further increase choleretic response in rats and an increase in bile concentrations of total bile acids. Thus, the rate of bile secretion in rats was increased one hour after the introduction of the xanthone 1 at a dose of 30 mg/kg of 47% and remained at approximately the same level during the whole experiment omniremote bile by 18%. The xanthone-1 at a dose of 50 mg/kg increased the rate of bile secretion in rats one hour after its introduction by 32% after 2 hours at 59%, after 3 hours at 79% after 4 hours by 43% (table. 2). At the same time increased the total number of selected bile more than 1.5 times compared with the control. Against this background, the total concentration of Khalatov exceeded the control by 30% and increased secretion of bilirubin and excretion of cholesterol compared to similar indicators in rats of the control group.

On the basis of the received data, 1-hydroxy-2,3,4,5 - tetramethoxybenzene shows a marked choleretic action.

It is established that ED501-hydroxy-2,3,4,5-tetramethoxybenzene 5 corresponds to 0.6 mg/kg of body weight.

Hepatoprotective effect of 1-hydroxy-2,3,4,5-tetramethoxybenzene.

The study of hepatoprotective activity of 1-hydroxy-2,3,4,5 - tetramethoxybenzene conducted on 82 white rats of both sexes weighing 180-200 g method Levshin B. I. [Levshin B. I. Experimental pharmacotherapy drugs selenium and thiazolidine hepatic toxicity: author. thesis Dr. med. Sciences. - Kharkov, 1973. - 29 c. ] . Experimental toxic hepatitis caused by subcutaneous administration to rats 50% oil (V 1 was administered intragastrically at a dose of 10 mg/kg, starting from the second day of the experience, once a day during the whole experiment. Animals of the control group were injected in equiano amount of distilled water. Evaluation of the functional state of rat liver with toxic hepatitis was performed at 7, 14, 21 and 28 days of experience on biochemical parameters of blood serum. In addition, evaluated the intensity of the processes of lipid peroxidation (LPO) (the content of malondialdehyde MDA in the blood) and antitoxic function of liver bromsulphalein test. The determination of the activity of alanine aminotransferase (Alat) and aspartate aminotransferase (AST) was performed on Reitman and Frankel, the content of total protein by biuret reaction, cholesterol, lipoproteins by Burstein and Samae thymol test - Huergo, Popper [Kolb Century BC , Kamyshnikov B. C. - Handbook of clinical chemistry. - Minsk. - 1982. - 366 c. ] . Peroxidation was monitored by determining the content of MDA in serum [Gamerbots R. A. Seleznev , I. E. a Method of increasing the intensity of free radical oxidation oil containing blood components and diagnostic value // Laboratory work. 1981. - #4. -S. 209 - 211] . Bromsulphalein test was carried out according to the method Solovyov Century and of the liver in albino rats by the method of bromsulphalein samples // Laboratory work. - 1976. -N 9. -S. 538 -542. ).

Assessment of liver functions in rats with toxic hepatitis showed that the xanthone-1 has a strong pharmacological effect. In the early stages of the experiment, animals treated with xanthone-1, was observed a moderate decrease of the main manifestations of syndromes pathogenesis of hepatitis cytolysis and cholestasis, as well as slowed down the accumulation of MDA in the serum. So, on the 7th day experience was significantly decreased level of activity of Alat, indicating the decrease in the severity of cytolysis syndrome (PL. 3). On the 14th day of the experience of the activity of Alt and AST decreased by 22% and 23%. Then throughout the experiment continued reduction in the activity of AST and Alt. This showed a significant reduction in the activity of Alat almost 1.5 times. From table. 3 shows that the animals in this group was significantly decreased accumulation in the blood products of peroxidation of lipids. The content of MDA in animals treated with the xanthone-1, was reduced by 16%. Clearly slowed the accumulation of MDA in serum 14 days of the experiment. About the severity of cholestasis judged on the basis of a comprehensive assessment of cholesterol and lipoproteins in the serum. On day 7 was observed a decline in cholesta is the control. There was observed a marked decrease of these indices, respectively, on 23 and 26%.

Changes thymol samples indicating a reduction of dysproteinemia, has been noted on day 7 of the experiment, and the subsequent timing observations of the effect of using a xanthone 1 was increased (table. 3). Indirect confirmation of the reduction dysproteinemia is a tendency to increase the total protein content in the blood 14 days. The use of xanthone 1 contributed to significant increase since 7 days excretory functions of the liver according to bromsulphalein samples. Factor retention of bromsulphalein decreased by 18% from 7 days (table. 3). While this effect was observed in the subsequent observation period.

On the basis of the foregoing that when the experimental (Tetrachloromethane) liver damage, 1-hydroxy-2,3,4,5-tetramethoxybenzene exhibits hepatoprotective effect. Pharmacological effect it is implemented primarily at the level of stabilization of membrane structures of hepatocytes and suppression of free radical oxidation of the phospholipids of cell membranes. As a consequence, restores the lipid and protein metabolism, detoxifying function of the PECs is xeaxanthin has hepatoprotective action.

Determination of the acute toxicity of 1-hydroxy-2.3.4.5-tetramethoxysilane.

The acute toxicity of 1-hydroxy-2,3,4,5-tetramethoxybenzene intraperitoneal single dose according to the method of Cerberus was determined on white mice with weighing 18-20 g of both sexes. Within 15 days of recorded appearance, behavior, and death of animals. The value of LD50was calculated by the method of C. B. Prozorovsky [Prozorovsky Century B. Using the method of least squares for probit analysis of the mortality curves // Pharmacology and toxicology. - 1962. -N 1. - S. 115 - 120). Toxicity was determined according to the classification of toxic substances K. K. Sidorov [Sidorov, K. K., classification of toxic poisons when parenteral routes of administration. - In the book. : Toxicology of new industrial chemical substances. - M. : Medicine, 1973. vol. 13. -S. 47 - 51] .

Found that LD501-hydroxy-2,3,4,5-tetramethoxybenzene corresponds 8368 162 mg/kg of body weight. According to the classification of toxic substances K. K. Sidorov [1973] the xanthone is a relatively harmless substance.

The way to obtain 1-hydroxy-2,3,4,5-tetramethoxybenzene with choleretic and hepatoprotective effect by hexane extraction, chromatography and UPA is Galeni horned 70% ethanol at the ratio of raw material: solvent 1: 10, followed by contacting the extract with hexane, chromatographytandem on a column of silica gel and evaporation, dissolution in hexane, filtration and recrystallization from ethanol.

 

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