KR20040043255A - Liver disease treating agent containing the extract and flavonoid compounds isolated from Rhus verniciflua Stokes - Google Patents

Abstract

PURPOSE: Provided is a pharmaceutical composition containing, as an active ingredient, the polar solvent or non-polar solvent soluble extract of Rhus verniciflua Stokes, and sulfuretin and fustin compounds isolated therefrom. The pharmaceutical composition is useful for prevention and treatment of liver diseases. CONSTITUTION: A pharmaceutical composition for prevention and treatment of hepatocirrhosis caused by hepatic fibrosis, as well as amelioration of liver function is characterized by containing the polar solvent or non-polar solvent soluble extract of Rhus verniciflua Stokes, fustin of the formula(1) and sulfuretin of the formula(2) isolated therefrom, as an active ingredient, and pharmaceutically acceptable carrier or excipient thereof.

Description

Liver disease treating agent containing the extract and flavonoid compounds isolated from Rhus verniciflua Stokes}

The present invention is intended to be used as a therapeutic agent or health food effective for improving liver function and preventing and treating liver cirrhosis caused by hepatocellular fibrosis, which contains fustine and sulferetine compounds isolated from polar solvent-soluble extracts, non-polar solvent-soluble extracts, and fractions thereof. It is.

While active oxygen is responsible for the host's defense function of killing intracellular microorganisms that are phagocytosed by electrophilic substances caused by endogenous and exogenous factors, the excess free radicals are liberated in the surrounding tissues to peroxidize unsaturated fatty acids in the cell membrane. It promotes permeability and induces destruction, which induces cytotoxicity, which causes in vivo poisoning, aging, carcinogenesis and immunosuppression, and is the most important cause of tissue destruction observed during inflammatory reactions (Lee , KS et al., J. Clin, Invest ., 96, pp2461-2468, 1995: Milan, L. et al., Free Radicals in Chemistry and Biology, pp29-31, 283-284, CRC press. 1989).

Induction of liver damage by oxidative stress has been reported to not only promote the synthesis of lipid peroxides in the serum of animals, but also stimulate collagen accumulation in tissues and increase collagen production (French, SW et al., Exp. Mol. Pathol., 58, pp61-75, 1993). Recently, the link between lipid peroxidation and hepatic fibrosis has been extensively studied (Nordman, R., C. et al., Free Radic. Biol). Med., 12. pp219-240, 1992; Parola, M., G. et al., Leonarduzzi, G. et al., Free Radic. Biol. Med ., 20, pp351-359, 1996; Sokol, RJ. et al., Gastroenterology , 99 , pp1061-1071, 1990), The quantitative measurement of lipid peroxides and the indicators of hepatic fibrosis are significant in that they can simultaneously detect antioxidant and antifibrotic effects (Parola, M., G. et al., Hepatology , 16, pp 1014-1021, 1992).

Hepatic fibrosis The production of MDA occurs in the course of hepatic fibrosis (Muriel, P. and OR Suarez . ; J. Appl. Toxicol., 14, pp423-426, 1995). Has also been reported to stimulate collagen synthesis and the activity of hepatic stellate cells (Freeman, BA and JD Crapo., Lab. Invest ., 47, pp412-426, 1982).

Cytotoxicity by free radicals is known to be detoxified by the action of the body's detoxification mechanism. In general, hepatic detoxification mechanism is detoxified or inactivated by metabolic enzyme system present in the smooth endoplasmic reticulum of hepatocytes. It can be divided into phase I phase and phase II phase which are discharged to the body (Routledge, P, A. and DG Shand., Res. Pharmacol. Toxicol ., 19, pp 447-468, 1979). Cytochrome P-450, which is involved in phase I phase, is a mixed function oxidase, and exhibits different spectra depending on the form and tissues in which the drug binds. It is classified into Form I and Form II which forms formaldehyde and Form II which forms p -aminophenol based on aniline, which is known to be involved in the generation of active oxygen in microsomal system (Leonard). , V. and JB Schenkman Biochem Biophys Res Commun, 142, pp623-630, 1987:....... Past, MR and DE Cook Biochem, Pharmacol, 31, pp3329-3334, 1982).

Xanthine oxidase and aldehyde oxidase, which are involved in other phase I reactions, are non-microsomal oxidation, especially xanthine oxidase in viruses, bacteria and the activity is increased when the parasite infection or the like (Tubaro, E. et al, Arzneim Forsch (Drug Res), 26, pp2185-2186, 1976;....... Tubaro, E. et al, Biochem Pharmacol. , 29 , pp 1939-1942, 1980). Xanthine oxidase and aldehyde oxidase are enzymes that generate free radicals and are molybdenum-containing oxidases present in the cytosol fraction. Oxygen molecules in the reaction solution are used as electron acceptors in catalyzing biochemical reactions. And superoxide anion, hydrogen peroxide and finally hydroxy radicals (Granger, DN and DA Parks., The Pharmacologist, 26 , pp159-164. 1983). Induction of hepatic fibrosis resulted in a marked increase in the activity of the enzyme, suggesting that the increase in production of bioactive oxygen activates the enzyme system involved in non-microsomal oxidation. It is thought to inhibit the production of lipid peroxidation.

Most of the active oxygen are oxidizing materials such as superoxide anion, hydroxy radical (OH) and hydrogen peroxide (H).₂O₂Hydroxy radicals are known to have the strongest activity. Superoxide dismutase is an O₂H₂O₂To Free (J.A.).Superoxide, superoxide dismutase and oxygen toxicity in "metal ion activation of dioxygen",J. G. Spiro ed., John Willy & Sons, Inc., p209, 1980), catalase is H₂O₂H₂O and O₂Glutathione peroxidase also generates the free radicals₂Detoxification enzymes that are converted to O and excreted in vitro (Lawrence, R.A. and R.F.Burk.,Biochem. Biophys. Res. Commun.,71,pp952-958, 1976).

The free radical-mediated process in the cytoplasm is associated with an oxidation reaction known as oxidative stress as a change in the oxidation-reduction balance, with an increase in lipid peroxide in the plasma of patients with liver disease. It has been reported that this increased lipid peroxide in plasma can be very harmful in hepatic tissues as well as kidney, heart and other tissues (Kose, K., C. Yazici and O. Assioglu., Clin) . Biochem., 34 (2), pp 125-129, 2001: Pancewicz, SA et al., Med. Sci. Monit., 7 (6) , pp 1230-1235, 2001). In recent years, there has been an increase in lipid peroxides due to oxidative stress exposure in cholestatic liver disease, and oxidative stress protective effects have been decreased in experimental cholestatic rats. (Oetting-Deems, R. et al., J. Nutr., 123, pp1414-1420, 1993; Ono, M., C. et al., J. Lab. Clin. Med., 118) . , pp476-483, 1991;... Tubaro, E., B. et al, Biochem Pharmacol, 29, pp1939-1942, 1980). Recently, antioxidant effect, a function that inhibits or prevents lipid peroxidation, has been reported to have hepatoprotective and anti-inflammatory effects (Das, D. et al., Biochem. Biophys. Acta., 1502 (3), pp351-362 , 2000; Lin, CC, et al., Am. J. Clin. Med., 28 (1) , pp87-96, 2000), Antioxidants are intended to protect liver and hepatocytes against attack by intermediates of reactive oxygen. It is being used for research, and the possibility of application in liver disease is being studied. To date, antioxidants such as flavonoids, silymarin or vitamin E extracted from natural products have been reported to be effective for lipid peroxidation and liver fibrosis (Mourelle, MP et al., Clin. Pharmacol ., 3, pp183-187, 1989; Past, MR and DE Cook., Biochem, Pharmacol., 31, pp3329-3334, 1982).

As used herein, Rhus verniciflua Stokes is a deciduous broad-leaved sub-tree belonging to the family Anacardiaceae. Widely distributed, deciduous or evergreen, mostly arborescent or shrub, but partly vine-grown (Barkley Fred Alexander., Ann. Of the Missouri Bot. Garden., 24 (3) , pp265-500, 1937)

The sap of the lacquer tree is called lacquer. In oriental medicine, dryness removes blood and promotes blood circulation. Insufficiency, abdominal pain, excessive stomach acid, Jinhae, pulmonary tuberculosis, tuberculosis, constipation, diabetes, malaria, anti-inflammatory , Arthritis, embalming, dry stomach, women's meridian pain, etc., has been reported recently (Namba, T., Colored Illusteations of Wakan Yaku.p215, Hoikusha Publishing Co. Ltd., Osaka, 1980) ). Also, in folk medicine, lacquer is said to be more effective than wild ginseng, and it is said that it is effective for digestive medicine, liver blood, and blood in blood, to control heart disease, to eliminate tuberculosis bacteria in lung, and to be excellent medicine for neuralgia, arthritis, skin disease, etc. As components of the lacquer tree, there are known uricol and flavonoid strains and phenolic compounds, such as gallic acid.

In addition, Korean Patent Laid-Open Publication No. 2002-2096 (January 9, 2002) discloses a method for removing allergens from lacquer trees and the antioxidant and hangover-relieving effects of the lacquer extract, and Korean Patent Publication No. 2001-111159 (2001). 12.17), extracts from the lacquer tree, allergen-inducing compounds, hexane fraction, ethyl acetate fraction, butanol and water fraction, anticancer, antimicrobial, antioxidant, antilipid peroxidation activity, hepatitis virus inhibitory activity, liver function The technical contents have been disclosed for the improvement and hangover effect, and Korean Patent Registration No. 251526 (January 12, 2000) discloses the efficacy of anti-cancer, anti-viral, anti-fungal and antioxidant agents of urushiol-based compounds and lacquer extracts including the same. In addition, Korean Patent Publication No. 1996-7000738 (February 24, 1996) describes the antiviral efficacy of young extracts of young lacquer and elm flower parts. There is disclosure.

However, none of the literature teaches or mentions the efficacy effect of the family of flavonoid compounds isolated from the lacquer tree.

In this study, flavonoid-based compounds, fustin and sulfetretin, were isolated from the lacquer tree as a part of screening for bioactive substances. Serum biochemical tests and hydroxyproline in liver tissues were performed to investigate the antifibrotic and antioxidant effects of these compounds. The present invention was completed by measuring the concentration of and lipid peroxide, searching the effect on the production system of lipid peroxidation, and confirming the effect on cirrhosis of the liver.

SUMMARY OF THE INVENTION An object of the present invention is to provide a pharmaceutical composition containing lacquer polar solvent soluble extract, non-polar solvent soluble extract and fustine and sulfetretin compounds isolated therefrom useful for the prevention and treatment of liver disease.

1 is a diagram showing the effect on the production of hydroxyproline in the liver tissue, which is an indicator of liver fibrosis of the polar solvent or nonpolar solvent soluble extract of lacquer and the fustine and sulferetine compounds isolated therefrom.

Figure 2 is a diagram showing the effect on the lipid peroxidation content in liver tissue produced by induction of hepatic fibrosis of the polar solvent or nonpolar solvent soluble extract of the sumac, fustin and sulferretin compound,

Figure 3 is a diagram showing the effect of xanthine oxidase and aldehyde oxidase, hepatic cytosolic free radical forming enzymes of the polar solvent or nonpolar solvent soluble extract of the sumac, fustin and sulferetine compounds.

In accordance with the above object, the present invention provides a pharmaceutical composition useful for the prevention and treatment of cirrhosis of the lacquer nonpolar solvent soluble extract or polar solvent soluble extract as an active ingredient and containing a pharmaceutically acceptable carrier or excipient.

The nonpolar solvent soluble extract means an extract that can be extracted in a nonpolar solvent such as hexane, chloroform, ethyl acetate, etc., and the polar solvent soluble extract means an extract that can be extracted in a polar solvent such as acetone, ethanol, methanol, and the like.

The present invention provides a pharmaceutical composition useful for the prevention and treatment of liver disease, which comprises a fustin or sulfretin compound as an active ingredient and contains a pharmaceutically acceptable carrier or excipient.

The liver disease includes liver diseases such as hepatitis, liver fibrosis, fatty liver, liver failure, liver cirrhosis and the like.

The method of separating the lacquer extract and fustin or sulfetretine compound of the present invention is as follows.

Hereinafter, the present invention will be described in detail.

The sumac extract of the present invention is about 5 to 20 times, preferably about 10 to 15 times, water, lower alcohol or about 1: 0.1 to 1:10, preferably 1: 1 weight of dried sumac (kg). These mixed solvents having a mixing ratio (kg / L) of 0.2 to 1: 5 are used in extraction methods such as ultrasonic extraction and reflux extraction for about 1 hour to 1 day at an extraction temperature of 10 to 50 ° C, preferably 20 to 30 ° C. To obtain crude extract which is a soluble extract according to water, lower alcohol or a mixed solvent thereof; After the crude extract is suspended in a polar solvent such as water or methanol, it is dissolved in a polar solvent by adding a nonpolar solvent such as hexane, ethyl acetate or chloroform in a volume of about 1 to 100 times, preferably about 1 to 5 times the suspension. Fractionating the nonpolar solvent soluble layer once to 10 times, preferably 2 to 5 times, to obtain a nonpolar solvent soluble portion and a polar solvent soluble portion; The non-polar solvent soluble layer can be separated using silica gel column chromatography and thin layer column chromatography (TLC).

Hereinafter, a process of separating the polar solvent or nonpolar solvent soluble extract, fustine or sulfetretin compound from lacquer wood,

For example, a first step of pulverizing the dried lacquer tree, reflux extraction with a polar solvent such as water, methanol or butanol, followed by filtration, concentration under reduced pressure and lyophilization to obtain a crude extract soluble in a polar solvent;

A second step of suspending the crude extract in water to sequentially dissolve it in a nonpolar solvent such as chloroform and ethyl acetate, and then fractionating and filtering with a separatory funnel to obtain a nonpolar solvent soluble extract;

A third step of sequentially fractionating and filtering the aqueous layer using a polar solvent such as butanol, water and the like, and obtaining a polar solvent soluble extract;

Subsequently, column chromatography and TLC are performed on the two-phase nonpolar solvent soluble extract, which is preferably a developing solvent having a ratio of chloroform-methanol-water (73:27:10, lower layer; v / v) in the silica gel column. Was developed on a column to obtain fustine and sulfuretine compounds having _Rf values of 0.53 and 0.65 (developing solvent: chloroform-methanol-water (73:27:10, lower layer; v / v) on TLC, respectively). It consists of four steps.

The composition for preventing and treating liver disease of the present invention comprises 0.1 to 20% by weight of the compound based on the total weight of the composition.

Compositions comprising a compound of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

The compositions comprising the compounds according to the invention are each formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, external preparations, suppositories, and sterile injectable solutions according to conventional methods. Carriers, excipients and diluents that may be included in the composition comprising the extract include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, Calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations may include at least one excipient such as starch, calcium carbonate, sucrose ( It is prepared by mixing sucrose or lactose and gelatin. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Liquid preparations for oral use may include various excipients, such as wetting agents, sweeteners, fragrances, preservatives, etc., in addition to water and liquid paraffin, which are commonly used to include suspensions, solutions, emulsions, and syrups. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The amount of the extract or compound of the present invention may vary depending on the age, sex, and weight of the patient, but in general, the amount of 5 to 500 mg / kg, preferably 100 to 250 mg / kg, once or three times a day. It can be administered separately. In addition, the dosage of the extract can be increased or decreased depending on the route of administration, the degree of disease, sex, weight, age and the like. Therefore, the above dosage does not limit the scope of the present invention in any aspect.

The present invention provides a dietary supplement comprising a polar solvent or a non-polar solvent soluble extract of lacquer tree showing a preventive effect of cirrhosis of the liver and a food acceptable additive.

Compositions comprising the extracts of the present invention can be used in a variety of drugs, food and beverages for the prevention of inflammatory diseases. Examples of the food to which the polar solvent or the non-polar solvent soluble extract of the lacquer can be added include various foods, beverages, gums, teas, vitamin complexes, health supplements, and the like, and powders, granules, tablets, capsules, or the like. It can be used in the form of a drink.

The polar solvent or non-polar solvent soluble extract itself of the lacquer of the present invention has little toxicity and side effects, so it is a drug that can be used safely even when taken for long periods of time.

The extract of the present invention may be added to food or beverage for the purpose of preventing inflammatory diseases. At this time, the amount of the extract in the food or beverage is generally added to the health food composition of the present invention 0.01 to 15% by weight of the total food weight, the health beverage composition is 0.02 to 10 g, preferably based on 100 ml Can be added in a ratio of 0.3 to 1 g.

The health beverage composition of the present invention has no particular limitation on the liquid component except for containing the extract as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. Examples of the above-mentioned natural carbohydrates are conventional monosaccharides such as disaccharides such as glucose and fructose, such as maltose, sucrose and the like, and polysaccharides such as dextrin, cyclodextrin and the like. Sugars and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of natural carbohydrates is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and salts thereof. , Organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. The compositions of the present invention may also contain pulp for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components can be used independently or in combination. The proportion of such additives is not so critical but is generally selected from the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

The present invention is described in more detail based on the following examples and experimental examples, but the present invention is not limited thereto.

Reference Example 1. Laboratory Equipment and Reagents

^{The 1} H-NMR and ¹³ C-NMR instruments were 600 MHz (Bruker, Advance-600, 6630-205) and the mass spectrometer (MS spectrum) was used with FAB-MS (HP, JEOL, JMS-AX505WA). , All solvents used A-grade reagent.

Example 1 Preparation of Sumac Extract

The lacquer tree used in the present invention was collected in Gangwon-do, Korea in the fall of 1998, and was verified by Prof. Se-Young Yoon of the Department of Plant Resources, Sangji University. The tested specimen (# NATCHEM-18) was deposited in the Botanical Sample Room of Sangji University.

2 kg of the dried lacquer was selected and chopped, and 6 liters of methanol was added thereto, followed by reflux extraction three times, filtered through a filter paper, and distilled under reduced pressure to obtain a viscous lacquer crude extract (280 g).

Example 2 Preparation of Succulent Polar Solvent and Nonpolar Solvent Soluble Extract

Sucrose crude extract of Example 1 was suspended in 3 L of water, and then, 3 L of chloroform was added to the separatory funnel, and the resultant was repeatedly fractionated five times with a chloroform layer and a water layer. The same volume of ethyl acetate solvent as the water layer was repeated five times in the same manner as above to obtain a substance that was soluble in the ethyl acetate layer as much as possible until the color of the solution became light. The chloroform layer and ethyl acetate layer were dried in a vacuum concentrator, respectively, to obtain a chloroform soluble extract (69 g) and an ethyl acetate soluble extract (95 g) and used as a sample.

Example 3. Isolation of Fustin and Sulfuretine Compounds

Soluble ethyl acetate acetate extract of Example 2 was separated by column chromatography on silica gel (600 g, 7 × 70 cm, Merck, Art 7734, Germany) using an eluent (chloroform: methanol: water = 73: 27: 10). Four fractions were obtained: A (560-640 mL), B (720-880 mL), C (1040-1200 mL) and D (1280-1600 mL). Of the four fractions, and recrystallized with methanol to the B and D thin layer chromatography (TLC) phase; the sulfur retinoic (sulfuretin) each R _f value of 0.65 in (developing solvent: chloroform: water = 73:27:10: methanol) 1.5 g of fustin, 1.1 g, 0.53, were obtained and analyzed, Yoon, BJ, Pharmacological Evaluation on Anti-inflammatory Effects of the Flavonoids Isolated from the Heartwood of Rhus verniciflua Stokes.Ph . D. Thesis, Kyungsung University, Busan, 2001) were matched with the physicochemical properties described in the identification of fustine and sulfur retintin respectively and used as a sample.

Fustin (C ₁₅ H ₁₂ O ₆ )

White acicular crystal

Melting Point: 228-229 ℃

[α] _D : + 28.3 ° (c, 0.9 in 50% acetone)

EI-MS (70 eV) m / z : 288.3 (M ⁺ , [C ₁₅ H ₁₂ O ₆ ] ⁺ ) (Harborne, JB et al., Dictionary of Natural Products Vol. 9, p5455, Chapman & Hall, New York , 1994)

¹ H-NMR (500 MHz, DMSO-d ₆ ) δ: 4.97 (1H, d, J = 11.5 Hz, H-2), 4.41 (1H, dd, J = 5.5, 11.3 Hz, H-3), 5.47 ( 1H, d, J = 5.5 Hz, 3-OH), 7.64 (1H, d, J = 8.7 Hz), 6.53 (1H, dd, J = 8.7, 2.2 Hz), 6.30 (1H, d, J = 2.1 Hz , H-8), 6.89 (1H, d, J = 1.8 Hz), 6.75 (1H, d, J = 7.0 Hz), 6.76 (1H, dd, J = 1.8, 7.0 Hz)

¹³ C-NMR (125 MHz, DMSO-d ₆ ) δ: 83.5 (C-2), 72.6 (C-3), 192.4 (C-4), 128.7 (C-5), 110.1 (C-6), 162.8 (C-7), 112.1 (C-8), 164.7 (C-9), 102.4 (C-10), 128.3 (C-1 '), 115.1 (C-2'), 144.9 (C-3 ' ), 145.6 (C-4 '), 115.3 (C-5'), 119.4 (C-6 ')

Sulfur Retin (C ₁₅ H ₁₀ O ₅ )

Orange Plate Crystal

Melting Point:> 300 ℃

EI-MS (70 eV) m / z: 270.3 (M ⁺ , [C ₁₅ H ₁₀ O ₅ ] ⁺ ) (Harborne, JB et al., Dictionary of Natural Products Vol. 10, p5716, Chapman & Hall, New York , 1994)

¹ H-NMR (500 MHz, DMSO-d ₆ ) δ: 6.64 (1H, s, H-2), 7.72 (1H, d, J = 8.5 Hz), 6.71 (1H, dd, J = 1.7, 8.5 Hz, H-5), 6.75 (1H, d, J = 1.7 Hz, H-8), 6.84 (1H, d, J = 8.2 Hz, H-2), 7.45 (1H, d, J = 1.9 Hz), 7.25 (1H, dd, J = 2. / 0, 8.2 Hz)

¹³ C-NMR (125 MHz, DMSO-d ₆ ) δ: 111.9 (C-2), 147.9 (C-3), 181.7 (C-4), 124.5 (C-5), 111.9 (C-6), 166.1 (C-7), 98.3 (C-8), 167.4 (C-9), 113.2 (C-10), 123.4 (C-1 '), 116.0 (C-2'), 145.6 (C-3 ' ), 148.0 (C-4 '), 117.0 C-5'), 125.7 (C-6 ')

Experimental Example 1. Materials and Methods

Animals & Aid

As experimental animals, male Sprague-Dawley rats weighing 200 ± 10 g, which were distributed from Daehan Biolink Co., Ltd., were adapted for 1 week in animal history of the university. (Temp: 20 ± 2 ^o C, Humidity: 50%, Contrast: 12 hours light and dark cycle cycle), and used for the polar solvent or non-polar solvent soluble extract of lacquer tree and the administration of pushtin and sulferetine separated therefrom. Pretreatment was carried out for 2 weeks, and the animals were fasted with water for 24 hours before the experiment. Induction of hepatic fibrosis was performed by biliary ligation in groups other than the normal group according to the method of Kountras et al. (Kountras, J. et al., Bri. J. Exp. Path., 65, pp305-312, 1984). Hepatic fibrosis was induced, and ursodeoxycholic acid (UCDA) (50 mg / kg) was used as a control drug.

Sampling and Separation of Enzyme Sources

The animals were anesthetized with carbon dioxide gas, and cut along the abdominal midline, and blood was collected from the abdominal aorta. The blood was centrifuged at 3000 rpm for 10 minutes to be used for biochemical tests. Liver tissue was perfused with saline to remove blood, and blood and other adherents were removed. Part of the liver was stored at -70 ° C for hydroxyproline and lipid peroxidation. Then, four times 0.1 M potassium phosphate buffer (pH 7.5) per gram of tissue was added and ground with a glass teflon homogenizer. Cytosolic and macrosomal fractions obtained by ultracentrifugation of this grinding solution were used as enzyme sources, and all the above operations were performed at 4 ° C or lower.

Serum biochemical test

Amino transferases (AST, ALT, Cat.No 505-OP), sorbitol dehydrogenase (SDH, Cat.No 50-UV), total bilirubin (550-50P) and γ-glutamyltransferra (Γ-GT, 545-A) was measured using a commercially available Sigma company kit (Sigma kit).

Protein Quantitation and Statistical Processing

Quantification of protein was determined by Lowry et al. (Lowry, OH, et al., J. Biol. Chem ., 193 , pp265-275, 1951). Bovine serum albumin, Siger, Fr. V ) Was measured as a standard. The results obtained in this experiment were expressed as mean ± standard deviation, and statistical significance was verified by Duncan's multiple range test.

Experimental Example 2. Measurement of total collagen in tissue

The amount of hydroxyproline in liver tissue was hydrolyzed to hydrochloric acid by hydrolysis of liver tissue according to the method of Jamall et al . (Jamall, IS and VN Finelli., Anal. Biochem ., 112, pp70-75, 1981). The solution was oxidized with chloramine-T, and then colored with Ehrlich reaction solution (p-dimethylaminobenzaldehyde) to measure absorbance at 558 nm.

As a result of administering fractions and components of lacquer to the experimental group which induced liver fibrosis by ligation of the biliary tract, and the collagen content in the liver tissue, the liver fibrosis was induced, which was about 4.5 times higher than that of the normal group. The amount of hydroxyproline in the liver tissues was suppressed by induction of liver fibrosis by administration of polar solvent soluble extract of lacquer, nonpolar solvent soluble extract, and pushtin and sulfur retinin isolated therefrom for 2 weeks. Retin and fustin were found to significantly decrease by 48% and 34%, respectively (see Figure 1).

Experimental Example 3. Determination of the effect on lipid peroxidation content

Changes of Succulent Extract, Pushtin and Sulfur Retin on Lipid Peroxidation by Altering the Method of Ohkawa et al. (Ohkawa, H., N. Ohishi et al., Anal. Biochem., 95 , pp351-358, 1979) The impact was measured.

Four milligrams of 0.1M potassium phosphate buffer (pH 7.4) was added per 1 g of liver tissue, and the same buffer was added to the ground solution for 3 hours preincubation, followed by 8.1% sodium dodecyl sulfate (SDS) and 20%. Acetate buffer (pH 3.5) and 0.8% thiobarbituric acid were added for the purpose of color development, followed by reaction at 95 ° C for 1 hour, cooling at room temperature, n -butanol: pyridine (15: 1) The red n -butanol: pyridine layer produced by centrifugation for 15 minutes was added, and the absorbance was measured at a wavelength of 532 nm.

Lactose biliary tracts were treated with hepatic fibrosis to induce hepatic fibrosis, and extracts of sumac and pushtin and sulfetretin were observed in the liver tissues. In particular, it could be seen that the sulfur-retinine and fustin, which are separated from each other, significantly reduced lipid peroxidation contents by about 60% and 47%, respectively (see FIG. 2).

Experimental Example 4. Various Enzyme Measurement Experiments

Xanthine oxidase activity was measured using the Stripe and Della methods (Stirpe, F. and CE Della., J. Biol. Chem., 244, pp3855-3863, 1969). The activity of aldehyde oxidase was measured according to the method of Rajagopalan et al. (Rajagopalan, KV, et al., J. Biol. Chem., 237 , pp922-928, 1962). .

Samples were treated daily by oral administration to the animal model of biliary cirrhosis, and rats were used for the experiment by dissecting the head 4 hours after the final sample administration.

In the experimental group that induce liver fibrosis by ligation of the biliary tract, the extracts of lacquer tree and its isolated components, pushtin and sulfetretin, were observed in the blood biochemical changes. ALT, SDH, γ-GT activity and total bilirubin were significantly increased, and the increase in blood biochemical fluctuations was statistically significant in the order of Sulfurretin, Fustine, Nonpolar Solvent Soluble Extract, and Polar Solvent Soluble Extract. It was confirmed that the inhibition (see Table 1).

Aid group Dose (mg / kg) ALT AST SDH γ-GT t-bilirubin Karmen IU / ℓ mU / ml Mg / dl Normal 21.9 ± 2.98 ^d 59.3 ± 7.31 ^e 17.4 ± 3.11 ^d 29.6 ± 2.90 ^d 0.53 ± 0.08 ^d Control 40.6 ± 3.29 ^a 280.7 ± 21.5 ^a 34.9 ± 4.14 ^a 65.9 ± 3.18 ^a 7.27 ± 0.98 ^a UCM 250 35.2 ± 2.98 ^{b, c} 220.4 ± 18.6 ^b 29.6 ± 2.99 ^b 55.5 ± 2.97 ^{b, c} 6.18 ± 1.17 ^{a, b} UCE 250 33.7 ± 3.11 ^c 190.8 ± 20.1 ^c 25.3 ± 3.27 ^{b, c} 48.3 ± 2.25 ^b 5.58 ± 0.67 ^b Fustin 10 30.6 ± 2.54 ^c 181.3 ± 16.2 ^c 24.7 ± 2.55 ^{b, c} 40.8 ± 3.12 ^{b, c, d} 5.14 ± 0.49 ^b Sulfur Retin 10 24.1 ± 2.16 ^d 100.4 ± 13.8 ^d 20.5 ± 1.98 ^{c, d} 36.9 ± 3.03 ^{b, c, d} 3.89 ± 0.47 ^c UDCA 50 25.3 ± 2.06 ^d 110.5 ± 15.6 ^d 18.5 ± 1.74 ^d 34.5 ± 2.45 ^{c, d} 3.31 ± 0.37 ^c Different data superscripts represent significant values from the normal group (p <0.05) by Duncan's new multi-range test.

Experimental Example 5. Related enzyme measurement experiment

Determination of superoxide dismutase activity was carried out by Marklund et al. (Marklund, S. and G. Marklund.,Eur. J. Biochem., 47, pp469-474, 1974)._,Catalase activity was measured by Aebi's method (Aebi, H., Catalase in "Method of enzymatic analysis" (H. U. vergmeter, ed.), Pp673, Academic press, New York, 1974). And measuring the activity of glutathione peroxidase by the method of Paglia and Valentine (Paglia, E. D. and W. N. Valentine, W. N.J. Lab. Clin. Med., 70,pp158-169, 1967).

Samples were treated daily with oral administration to biliary hepatic sclerosis animal models, and rats were used for the experiment by dissecting the head 4 hours after the final sample administration.

As a result of experiments confirming the effect of hepatocellular free radical scavenger enzyme on biliary tract fibrosis, the activity of superoxide dismutase was significantly reduced by inducing hepatic fibrosis compared to the contents of normal group as shown in FIG. 3 and Table 2. It was increased by the pretreatment of the extract of the sumac and the pushtin and the sulfuric retinol separated therefrom, and similar results were observed for the activity of glutathione peroxidase and catalase (see FIG. 3 and Table 2).

group Dose (mg / kg) activation SOD ^* GP^** Catalase ^*** Normal 10.2 ± 0.78 ^a 256.3 ± 18.21 ^a 2.93 ± 0.45 ^a Control 5.67 ± 0.39 ^d 155.6 ± 21.20 ^d 1.62 ± 0.15 ^d UCM 250 6.49 ± 0.54 ^{c, d} 183.7 ± 19.39 ^{c, d} 1.89 ± 0.21 ^{c, d} UCE 250 6.82 ± 0.37 ^c 197.1 ± 15.28 ^{b, c} 2.18 ± 0.25 ^{b, c} Fustin 10 7.28 ± 0.33 ^c 205.4 ± 10.42 ^{b, c} 2.24 ± 0.36 ^{b, c} Sulfur Retin 10 8.26 ± 0.43 ^b 210.6 ± 15.99 ^{b, c} 2.59 ± 0.27 ^{a, b} UDCA 50 8.87 ± 0.58 ^b 220.2 ± 13.27 ^b 2.64 ± 0.20 ^{a, b *} ; Superoxide dismutase activity: unit ^# / mg protein / min ^# ; 1 unit inhibited the rate of reducing cytochrome C by 50% in a binding system of xanthine and xanthine oxidase at pH 7.8 and 25 ° C. at a 3.0 ml reaction capacity. ^** ; Glutathione peroxidase: oxidized NADPH nmole / mg protein / min ^*** ; Reduced H ₂ O ₂ nmole / mg protein / min

Experimental Example 6. Toxicity Test

To determine the degree of toxicity, three groups of 150 ± 5 g of ICR mice (Central test animals) and 235 ± 10 g of SPF Sprague Dawley (Biogenomics) rats each Dividing the compounds of Example 2 and Example 3 of the present invention in doses of 20 mg / kg, 10 mg / kg and 1 mg / kg, respectively, was observed for toxicity for 24 hours.

As a result, no deaths were observed in all three groups, and no significant symptoms were found in weight gain and feed intake.

The compounds of the present invention can be administered in the following formulations, and the formulation examples below are merely illustrative of the present invention, whereby the content of the present invention is not limited.

Formulation Example 1 Preparation of Injection

100 mg of fustin of Example 3

Sodium Metabisulfite3.0 mg

Methylparaben0.8 mg

Propylparaben0.1 mg

Sterile Distillation Amount for Injection

The above ingredients are mixed and prepared in a conventional manner to have a final volume of 2 ml, and then filled into 2 ml ampoules and sterilized to prepare an injection.

Formulation Example 2 Preparation of Tablet

Fustine 200 mg of Example 3

Lactose 100 mg

Starch 100 mg

Magnesium stearate proper amount

A tablet is prepared by mixing and tableting the above components according to a conventional tablet manufacturing method.

Formulation Example 3 Preparation of Capsule

Fustin 100 mg of Example 3

Lactose 50 mg

Starch 50 mg

Talc 2 mg

Magnesium stearate

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare capsules.

Formulation Example 4 Preparation of Liquid

Sulfur Retin 1000 mg of Example 3

20 g of sugar

Isomerized sugar 20 g

Lemon flavor

Purified water was added to make a total of 1000 ml. According to the conventional method for preparing a liquid, the above components are mixed, and then filled into a brown bottle and sterilized to prepare a liquid.

Although the composition ratio is a composition suitable for a preferred beverage in a preferred embodiment, the composition ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, use purpose.

Preparation Example 5 Preparation of Mixed Liquid

1000 mg of extract of Example 2

Citric Acid1000 mg

Oligosaccharides 100 g

Plum concentrate 2 g

1 g of taurine

Lemon flavor

Add 100 ml of purified water

According to the conventional method for preparing a liquid, the above components are mixed, and then filled into a brown bottle and sterilized to prepare a liquid.

Formulation Example 6 Preparation of Healthy Food

1000 mg of extract of Example 2

Vitamin mixture

Vitamin A Acetate70 μg

Vitamin E1.0 mg

Vitamin B ₁ 0.13 mg

Vitamin B ₂ 0.15 mg

Vitamin B ₆ 0.5 mg

0.2 μg of vitamin B ₁₂

Vitamin C10 mg

Biotin 10 ㎍

Nicotinic Acid Amide1.7 mg

Folic acid 50 ㎍

Calcium Pantothenate 0.5 mg

Mineral mixture

Ferrous Sulfate1.75 mg

Zinc Oxide 0.82 mg

Magnesium carbonate25.3 mg

Potassium phosphate monobasic 15 mg

Dibasic calcium phosphate55 mg

Potassium Citrate 90 mg

Calcium Carbonate 100 mg

Magnesium chloride24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixtures is mixed with a component suitable for a health food in a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional health food manufacturing method. The granules may be prepared and used for preparing a health food composition according to a conventional method.

Formulation Example 7 Preparation of Healthy Drink

1000 mg of extract of Example 2

Citric Acid1000 mg

Oligosaccharides 100 g

Plum concentrate 2 g

1 g of taurine

Add 900 ml of purified water

After mixing the above components in accordance with a conventional healthy beverage production method, and stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed sterilization and then refrigerated and stored in the present invention For the preparation of healthy beverage compositions.

Although the composition ratio is a composition suitable for a preferred beverage in a preferred embodiment, the composition ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, use purpose.

Nonpolar solvent soluble extract, polar solvent soluble extract of the lacquer of the present invention, and fustine and sulfetretin isolated therefrom are the amount of hydroxyproline in liver tissue which is an indicator of liver fibrosis, the amount of lipid peroxidation, AST, ALT, SDH By significantly reducing the amount of γ-GT activity and total bilirubin, it can be usefully used for the treatment and prevention of various liver diseases.

Claims (9)

Pharmaceutical composition useful for the prevention and treatment of cirrhosis of the lacquer non-polar solvent soluble extract or polar solvent soluble extract as an active ingredient and containing a pharmaceutically acceptable carrier or excipient. The composition of claim 1, wherein the nonpolar solvent is a nonpolar solvent selected from hexane, chloroform, ethyl acetate. The composition of claim 1, wherein the polar solvent is a polar solvent selected from acetone, ethanol and methanol. A pharmaceutical composition for treating liver disease, comprising fustin as an active ingredient and a pharmaceutically acceptable carrier. A pharmaceutical composition for the treatment of liver disease, comprising sulfuretin as an active ingredient and a pharmaceutically acceptable carrier. The pharmaceutical composition according to any one of claims 1 to 5, wherein the daily dose of the composition is from 5 mg to 500 mg per kg of body weight. The composition of any one of claims 1 to 5, wherein the composition comprises 0.1 to 20% by weight relative to the total weight of the composition. Health supplement comprising a lacquer polar solvent or a non-polar solvent soluble extract and a food supplement acceptable food additives exhibiting a liver cirrhosis prevention effect. The dietary supplement of claim 8 which is a powder, granule, tablet, capsule or beverage.