KR20140018706A - Composition comprising extract of fermented curcuma longa by natural fermented soybean and curcuminoid derivatives isolated therefrom for treating or preventing liver disease - Google Patents

Abstract

The present invention relates to a composition for preventing or treating liver disease containing a fermented chrysanthemum fermented curd extract or a curcuminoid derivative compound isolated therefrom. The composition inhibits the expression of? -SMA and TGF-? 1 in liver tissues or hepatocytes and has an effect of reducing the amount of TNF- ?, and is useful as a therapeutic agent or a health food for liver diseases such as hepatitis, liver fibrosis and liver cirrhosis Lt; / RTI >

Description

TECHNICAL FIELD The present invention relates to a composition for preventing or treating liver disease comprising fermented kojunil extract or fermented kojunilide derivatives isolated therefrom.

The present invention relates to a composition for the prevention or treatment of liver disease containing fermented Chungkookjang fermented koji extract or a glucuronide derivative isolated therefrom.

The liver is a living organ that plays a pivotal role in the metabolism of substances in the body and in the body, and continuously produces enzymatic reactions and energy metabolism. Hepatitis, hepatic fibrosis, liver cirrhosis, and hepatocellular carcinoma account for the highest proportion of chronic diseases in Korea, together with circulatory diseases, and account for a large proportion of deaths caused by diseases. In particular, it has a high interest in drinking as it is relatively high in developed countries and high risk of liver damage due to heavy drinking. Continuous hepatic tissue damage due to viral infection or drinking is characterized by the development of hepatitis, hepatic fibrosis in hepatitis, liver cirrhosis in liver fibrosis and liver cancer. Considering the physiological characteristics and importance of liver tissue, the treatment or prevention of liver disease is very important, and it is required to develop a therapeutic or prophylactic composition which can reduce liver tissue damage and ultimately be applied to treatment.

Liver fibrosis refers to a condition in which damaged liver tissue is transformed into fibrous tissue such as collagen, rather than restoring normal hepatocytes, as part of a biochemical adaptation reaction accompanying chronic liver disease such as hepatitis. The production of ECM (extracellular matrix) is increased due to hepatic fibrosis, but decomposition is relatively reduced, and when it becomes worse, it progresses to liver cirrhosis, liver failure or liver cancer. Liver cirrhosis is a chronic inflammation of the normal liver tissue regeneration nodules (regenerative nodules (phenomenon of making small lumps), such as a fibrotic tissue, which means liver function is reduced.

Liver fibrosis is a biocompatible reaction that occurs in the repair process of tissue damage, but it is a fibrous tissue that can not perform any intrinsic functions such as metabolism of the substance in the body and secretion of bile. Liver function is inevitably lowered . However, the mechanism of hepatic fibrosis has not been clarified so far, so proper therapeutic drugs have not been developed. The overexpression of ECM in hepatic fibrosis or cirrhosis is caused by reactive oxygen species, growth factors, profibrogenic cytokines secreted by the activation of Kupffer cell, which is an intrahepatic macrophage, (HSC) is promoted by the hepatic stellate cells (HSCs) and the like, thereby increasing the production of connective tissues in the activated hepatic stellate cells.

Hepatic stellate cells were discovered by Kupffer in 1876, described by Ito, and established by Wake in 1971. The hepatic stellate cells are located in the space of Disse between the endothelial cells of the vascular endothelial cells and the hepatocytes, and they are star-shaped and called ito cells. In addition, it has a lipid droplet containing vitamin A, which can be used as a vitamin A storing cell, a fat storing cell, or a perisinusoidal hepatic lipocyte. (HSC) in the late 1990s and has been used up to now. Cooper cells (macrophages) are activated by phagocytosis of hepatocytes damaged by various toxic substances or directly activated by toxic substances to produce transforming growth factor-β (TGF-β), platelet-derived growth factor Of the cytokine secreted by these cytokines, the Cooper itself is autocrine. In addition, the cytokines secreted by Cooper cells activate the sinusoidal endothelial cells and hepatic epithelial cells in the liver. The collagen type IV collagen secreted by activated hepatic stellate cells and increased collagen production break down the normal constitutive ratio of ECM (extracellular matrix), leading to basement membrane-like matrix (eg, 4 Type collagen) is degraded. At this time, the intercellular ECM (interstitial type matrix, for example, type I and type III collagen) are twisted together to form a fibril-forming collagen, accumulate in the space of Disse, The basement membrane ECM and the accumulated intercellular ECM activate the hepatic cell. Activated hepatic stellate cells promote the production of macroglobulin and tissue inhibitor of metalloproteinases (TIMP), inhibitors of collagenase (metalloproteinase (MMP)) activity, and inhibit the degradation of overexpressed intercellular ECM . The intercellular ECM accumulated in the deep seas inhibits the exchange of substances between the blood and the hepatocyte, making it difficult for the hepatocyte to supply nutrients and to release toxic substances, and the hepatocytes are also continuously damaged. As a series of these reactions are repeated, connective tissues accumulate in liver tissue, resulting in liver fibrosis or cirrhosis. (J. Clin. Invest., 2005, 115 (2), 209-218; Gastroenterology, 2008, J. Gastroenterol., 2000, 35, (9), 16, pp. 1655-1669, Arch Pathol. , 665-672).

Recently, TGF-beta (transforming growth factor-beta), a cytokine released in macrophages and hepatic stellate cells, has been found to be an important mediator of hepatic fibrosis. In addition, it has been reported that when the action of TGF-β is blocked through the modification of TGF-β antibody, antisense RNA, and cellular TGF-β receptor, liver fibrosis process is remarkably suppressed. However, these studies were performed only at the experimental level and there are no drugs that are actually used clinically until now.

Hepatitis B, one of the causes of viral hepatitis, has been on the decline in the number of carriers and patients due to the development of preventive vaccines, but infections are still frequent as they are still classified as Type 1 infectious diseases. Furthermore, in the case of hepatitis A and hepatitis C, the development of a suitable therapeutic agent or vaccine is not performed, and the onset is relatively increased. The risk of hepatitis is more likely to develop into hepatic fibrosis, liver cirrhosis and liver cancer if appropriate rest and treatment are not performed than hepatitis itself, suggesting the importance of treatment or prevention of hepatitis. Despite these risks, however, there are few hepatitis treatments available to date.

Curcuma longa ( Curcuma longa ) is a perennial herb that is cultivated widely in tropical and subtropical regions, mainly from Southeast Asia, from India. Ulgum is also known as turmeric and is grown in the southern part of Korea recently and is used as a raw material for curry. Ulyum contains 1 ~ 5% of essential oil and contains 0.3% of curcumine, a yellow pigment, and is used as a directional nutrient for hepatic salts, cholangitis, gallstone, and quadratic jaundice. As an example of the use of Ulgum in liver disease, when an extract of Ulgum was administered to hepatitis mice induced with D-galactosamine (400 mg / kg), Tc-methabrophenine labeled with radioactive substance Tc-mebrofenin has been reported to be efficacious in the excretion of ganoderma lucidum extract (in order to confirm the effect indicator before interleukin) (Indian J. Physiol. Pharmacol., 2003, 47, 332-336). In addition, it has been reported that the rumen extract inhibits hepatotoxicity in rats that inhibit replication of the B virus, which is known to be the cause of hepatitis, and overexpress the HBV-related X protein (HBx) (Intergr. Cancer Ther., 2011, 10 , 168-177).

On the other hand, most studies on compounds derived from the gilt-infested compounds that inhibit hepatitis, hepatic fibrosis or cirrhosis have been reported in curcumin (Br. J. Pharmacol., 2010, 161, 1137-1149; J. Nat Most of them are against bis-demethoxy curcumin and demethoxy curcumin, and most of them are based on antioxidants, It has been confirmed that studies on the effect of inhibiting the hepatitis, hepatic fibrosis, or cirrhosis of other Chinese cabbage compounds other than the above compounds are hardly carried out.

In the meantime, it is not easy to develop Ulgum as a medicine or functional food. It is difficult to develop Ulgum as a medicinal product and functional food, because curcuminoids such as curcumin, bisdimethoxy curcumin and dimethoxy curcumin, And secretion (ADME) are difficult. In particular, curcumin has been reported to be poorly soluble in water because it exhibits properties that are hardly soluble in water, and various attempts have been made to increase the solubility of such curcuminoids (J. Med. Chem., 2007 , 50, 6284-6288; Biochem. Pharmacol., 2006, 72, 928-940).

Therefore, in order to overcome the problems of absorption, distribution, metabolism and secretion (ADME) of the herbaceous compounds, the present inventors prepared fermented fermented chongkukjang by fermenting chungkukjang in Ulgum, , It was confirmed that the curcumin, bisdimethoxy curcumin, dimethoxy curcumin and the like were converted into other kinds of cucuminoid compounds which are easily absorbed into the human body. That is, the bioavailability may be improved since the process of absorbing and metabolizing the liver after absorption of the carcuminoid-based compound into the body proceeds through the microorganism (Biochem. Pharmacol., 2006, 72, 928-940; Biotechnol Lett., 2005, 27 (18), 1411-1425). In addition, when fermented, the contents of curcumin, bisdimethoxy curcumin, dimethoxy curcumin, and the like are reduced compared with non-fermented products, because the hydroxyl group is increased in the aliphatic chain of curcumin or metabolized in the form of a sugar attached to the hydroxyl group.

On the other hand, the inventors of the present invention have completed the present invention by confirming that the extract of Chungkukjang fermented beef curd or the curcuminoid compounds isolated therefrom have excellent therapeutic effects of hepatitis, liver fibrosis or cirrhosis.

Prior arts relating to the extracts of Ulgum or Fermented Ulgum include "a composition for the treatment and prevention of alcoholic liver diseases containing fermented Ulgum extract, Korean Patent No. 1117301", "food composition for improving hangover and liver function, Korean Patent No. 743861 ", "Herbal Medicine Extract for the Prevention and Treatment of Liver Fibrothermia, Korean Patent No. 682045 ", and the like, Or the composition is extracted and prepared by fermenting the mixture, or the composition is different in application. Therefore, the composition of the present invention is different from that of the present invention.

Korean Patent No. 1117301 (Composition for the Treatment and Prevention of Alcoholic Liver Diseases Containing Fermented Curd Extract, Registered on Mar. 2, 2012.) Korean Registered Patent No. 574097 (registered food composition for hangover and liver function improvement, registered on April 19, 2006) Korean Registered Patent No. 743861 (registered herbal composition for liver disease prevention and treatment, registered on July 24, 2007) Korean Registered Patent No. 682045 (registered herbal medicine extract for prevention and treatment of liver fibrosis, registered on Mar. 2, 2007)

Bataller, R., Brenner, D. A., Liver fibrosis., J. Clin. Invest., 2005, 115 (2), 209-218. Friedman, S. L., Mechanisms of hepatic fibrogenesis., Gastroenterology, 2008, 134 (6), 1655-1669. Moreira, R. K., Hepatic stellate cells and liver fibrosis., Arch. Pathol. Lab. Med., 2007, 131 (11), 1728-1734. Popov, Y., Schuppan, D., Targeting liver fibrosis: strategies for development and validation of antifibrotic therapies., Hepatology, 2009, 50 (4), 1294-1306. Deshpande, U.R. et al., Hepatobiliary clearance of labeled mebrofenin in normal and D-galactosamine HCl-induced hepatitis rats and the protective effect of turmeric extract., Indian J. Physiol. Pharmacol., 2003, 47, 332-336. Stoker., J. et al., Prospective comparative study of spiral computer tomography and magnetic resonance imaging for detection of hepatocellular carcinoma., J. Ethanopharmacol., 2009, 124, 189-196. Kim, J. et al., Chemopreventive effect of Curcuma longa Linn on liver pathology in HBx transgenic mice., Intergr. Cancer Ther., 2011, 10, 168-177. R Vijayakumar, G., Divakar, S., Synthesis of guaiacol-alpha-D: -glucoside and curcumin-bis-alpha-D: -glucoside by an amyloglucosidase from Rhizopus. Biotechnol Lett., 2005, 27 (18), 1411-1415. Tang, Y., Chen, A., Curcumin prevent leptin raising glucose levels in hepatic stellate cells by blocking translocation of glucose transporter-4 and increasing glucokinase. J. Pharmacol., 2010, 161, 1137-1149. Shu, J. C., Curcumin prevents liver fibrosis by inducing apoptosis and suppressing activation of hepatic stellate cells, J. Nat. Med., 2009, 63, 415-420. Strimpakos, A. S., Sharma, R.A., Curcumin: preventive and therapeutic properties in laboratory studies and clinical trials., Antioxid. Redox Signal, 2008, 10, 511-545. Safavy, A., Design and development of water-soluble curcumin conjugates as potential anticancer agents, J. Med. Chem., 2007, 50, 6284-6288. Weber, W. M., TPA-induced up-regulation of activator protein-1 can be inhibited or enhanced by analogues of the natural product curcumin., Biochem. Pharmacol., 2006, 72, 928-940.

It is an object of the present invention to provide a composition for treating or preventing liver disease containing a fermented cucumber fermented Curcuma longa extract or a curcuminoid derivative isolated therefrom.

The present invention relates to a composition comprising curcumalogin A (compound 1 ), curcumalogin B (compound 2 ), curcumalogin C (compound 3 ), and curcumarogine D (compound 1 ) curcumalongin D, compound 4 ), 1- (4-hydroxy-3-methoxyphenyl) -7- (3,4-dihydroxyphenyl) -1,6- heptadiene- (4-hydroxy-3-methoxyphenyl ) -7- (3,4-dihydroxyphenyl) -1,6-heptadiene-3,5-dione, compound 5), 5-hydroxy-1,7-bis (4-hydroxy Heptadien-3-one, compound 6 ), 1,7-bis (4-hydroxyphenyl) Dihydroxyphenyl) -1,6-heptadiene-3,5-dione, compound 7 ) And 1- (4-hydroxyphenyl) -7- (3,4-dihydroxyphenyl) -1,6-heptadiene- , 4-dihydroxyphenyl) -1,6-heptadiene-3,5-dione, compound 8 ), wherein the curcuminoid derivative Including water Soybean Fermented turmeric (fermented Curcuma longa by natural fermented soybean extract of the present invention.

[Formula 1]

(2)

(3)

[Chemical Formula 4]

[Chemical Formula 5]

The chungkukjang fermented beef extract can be an extract prepared by extracting fermented chonggukjang fermented beef with water, lower alcohol having 1 to 4 carbon atoms, acetone, ethyl acetate, chloroform or a mixed solvent thereof.

The cheonggukjang fermented beef can be fermented fermented beef chongkukjang. Preferably, the fermented corn may be obtained by fermenting a mixture of 0.1 to 10 parts by weight of chrysanthemum powder and 40 to 100 parts by weight of water in 100 parts by weight of corn.

The liver disease may be a disease selected from the group consisting of hepatitis, liver fibrosis and cirrhosis.

The present invention also relates to a composition comprising curcumalogin A (compound 1 ), curcumalogin B (compound 2 ), curcumalogin C (compound 3 ), curcumarogin D (compound 3 ) (curcumalongin D, compound 4 ), 1- (4-hydroxy-3-methoxyphenyl) -7- (3,4-dihydroxyphenyl) -1,6- heptadiene- - (4-hydroxy-3- methoxyphenyl) -7- (3,4-dihydroxyphenyl) -1,6-heptadiene-3,5-dione, compound 5), 5-hydroxy-1,7-bis (4- Hydroxyphenyl) -4,6-heptadiene-3-one (Compound 6 ), 1,7-bis (4-hydroxyphenyl) (3,4-dihydroxyphenyl) -1,6-heptadiene-3,5-dione, compound 7 ) and 1- (4-hydroxyphenyl) -7- (3,4-dihydroxyphenyl) -1,6-heptadiene- 3,4-dihydroxyphenyl) -1,6-heptadiene-3,5-dione, compound 8 ) is used as a cocuminoid derivative compound. And to a health functional food for prevention or improvement of liver disease containing fermented kojangguk extract.

The present invention also relates to a composition comprising curcumalogin A (compound 1 ), curcumalogin B (compound 2 ), curcumalogin C (compound 3 ), curcumarogin C (compound 3 ) D (curcumalongin D, compound 4 ), 1- (4-hydroxy-3-methoxyphenyl) -7- (3,4-dihydroxyphenyl) -1,6-heptadiene- 1- (4-hydroxy-3- methoxyphenyl) -7- (3,4-dihydroxyphenyl) -1,6-heptadiene-3,5-dione, compound 5), 5-hydroxy-1,7-bis (4 4-hydroxyphenyl) -4,6-heptadiene-3-one (Compound 6 ), 1,7- Bis (3,4-dihydroxyphenyl) -1,6-heptadiene-3,5-dione, Compound 7 ) and 1- (4-hydroxyphenyl) -7- (3,4-dihydroxyphenyl) -1,6-heptadiene- a (3,4-dihydroxyphenyl) -1,6-heptadiene -3,5-dione, 1 or more large kyumi cannabinoid derivative compound selected from the group consisting of compound 8) It relates to the prevention and treatment of liver disease, which pharmaceutical composition for oil.

Hereinafter, the present invention will be described in detail.

The fermented chongkukjang fermented beef extract of the present invention can be prepared by adding 2 to 50 times (w / v) water, 1 to 4 carbon atoms lower alcohol, acetone, ethyl acetate, chloroform or a mixed solvent thereof And then extracting it. The lower alcohol may be selected from ethanol, methanol, propanol, isopropanol and butanol.

The fermented chongkukjang fermented beef is preferably fermented with a mixture of 0.1 to 10 parts by weight of chrysanthemum powder and 40 to 100 parts by weight of water, based on 100 parts by weight of corn. The mixture of the koji, chungkukjang powder and water can be fermented at a temperature of 20 to 30 DEG C for 30 to 45 days. The fermentation conditions can be performed under conditions of relative humidity of 35 to 65%. In addition, the mixture of urogen, chungkukjang powder and water can be prepared by pre-fermenting at 70 to 90 캜 for 1 to 5 hours before fermentation. In addition, the mixture of the koji, chungkukjang powder and water can be post-fermented at a temperature of 70 to 90 ° C for 1 to 5 hours after fermentation.

On the other hand, the chungkukjang can be prepared by drying the chungkukjang prepared by a conventional method and then preparing it as a powder.

Preferably the said chungkukjang contains boiled beans in straw or Bacillus sp.) strain.

The fermentation temperature is preferably 20 to 37 占 폚. The fermentation time is preferably 12 hours to 10 days.

Thus, preferably the said chungkukjang contains boiled beans in straw or Bacillus sp.) strain at 20 ~ 37 ℃ for 12 ~ 10 days.

The soybean for manufacturing Cheonggukjang may be any kind of soybean, but preferably soybean or black soybean may be used.

The chungkukjang can be prepared by adding 0.01 to 10 parts by weight of straw to the boiled soybean when the boiled soybean is 100 parts by weight. At this time, the Bacillus genus ( Bacillus sp.) can be used to ferment Chongkukjang.

The chungkukjang contains 0.001 to 1 part by weight of Bacillus sp. sp.) can be prepared by mixing the fermented soybeans with boiled soybeans. As the Bacillus sp. Strain, Bacillus subtilis is preferably used.

In addition, when boiled beans and the above-mentioned Bacillus subtilis are mixed, when 100 parts by weight of boiled beans is used, the ratio of Aspergillus and 0.001 to 1 part by weight of at least one strain selected from yeast can be mixed.

To the genus Aspergillus strain is Aspergillus the duck (Aspergillus oryzae , Aspergillus sojae, Aspergillus niger niger). Among the yeasts, Saccharomyces cerevisiae , Saccharomyces veanans ( Saccharomyces cerevisiae , a bayanus), jjayi Kosaka My process ruksi (Zygosaccharomyces rouxii ) and the like may be used.

Therefore, the chungkukjang fermented corn oil extract of the present invention is preferably a fermented,

Preparing a mixture by mixing 0.1 to 10 parts by weight of chrysanthemum powder and 40 to 100 parts by weight of water in 100 parts by weight of corn;

Fermenting the mixture at a temperature of 20 to 30 DEG C for 30 to 45 days to prepare fermented chongkukjang fermented koi; And

Adding and adding water, 2 to 50 times (w / v) water, a lower alcohol having 1 to 4 carbon atoms, acetone, ethyl acetate, chloroform or a mixed solvent thereof to the fermented chungkukjang fermented cherries, .

Compounds isolated from the fermented Cheonggukjang fermented koji extract of the present invention can be obtained by extracting the fermented Cheonggukjang fermented koji extract with an organic solvent (alcohol, aqueous alcohol solution, acetone, chloroform, etc.), sequential fractionation with hexane, ethyl acetate and butanol, The active compound of the present invention can be easily obtained by a known method used for separation and extraction of plant components, such as a method of using an adsorbent resin such as -20 resin, a method by column chromatography, or the like.

Preferably, hexane, ethyl acetate, butanol, etc., of 2 to 50 times (w / v) the weight of the extract are sequentially added to the extract of Chungkookjang fermented beef cattle, and the fraction is purified and fractionated by chromatography. The active compounds can be isolated.

Chromatography used in the present invention includes silica gel column chromatography, LH-20 column chromatography, thin layer chromatography (TLC) and high performance liquid chromatography high performance liquid chromatography, etc.) can be used.

In addition, the activity of the fermented Cheonggukjang fermented beef extract of the present invention or the glucuronide derivative compound isolated therefrom can be measured by fermenting the fermented beef with the fermented bean curd; Extracting the fermented chongkukjang fermented beef with water, a lower alcohol having 1 to 4 carbon atoms, acetone, ethyl acetate, chloroform or a mixed solvent thereof, and then fractionating the mixture with hexane and ethyl acetate; Purifying and isolating the glucuronide derivative compound by chromatography using pure water; Identifying the cucuminoid derivative compound through the chemical structure of the separated active compound; And measuring the inhibitory activity of the glucuronide derivative compound against TNF-α, TGF-β and α-SMA.

In addition, the present invention provides a pharmaceutical composition comprising a fermented chrysanthemum fermented curd extract or a curcuminoid derivative compound isolated therefrom. The pharmaceutical composition comprising the fermented Cheonggukjang fermented kojang extract according to the present invention or the glucuronide derivative compound isolated therefrom may be administered orally or parenterally in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, And may be formulated in the form of a tablet, an external preparation, a suppository, and a sterile injection solution. Examples of carriers, excipients and diluents that can be contained in the composition comprising the fermented Cheonggukjang fermented corn oil extract or the glucuronide derivative compound isolated therefrom include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, But are not limited to, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, Mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. The solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like. These solid preparations can be prepared by adding to the above fermented chrysanthemum fermented curd extract or a glucuronide derivative compound separated therefrom at least one excipient, Starch, calcium carbonate, sucrose or lactose, and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of suppository bases include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like.

The dose of the fermented Cheonggukjang fermented koji extract or the glucuronide derivative compound separated therefrom depends on the age, sex, and weight of the subject to be treated, the specific disease or pathological condition to be treated, the severity of the disease or pathological condition, . Dosage determinations based on these factors are within the level of ordinary skill in the art and generally the dosage ranges from 0.01 mg / kg / day to approximately 2000 mg / kg / day. A more preferable dosage is 0.1 mg / kg / day to 500 mg / kg / day. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way. The chungkukjang fermented kojang extract of the present invention or a glucuronide derivative compound isolated therefrom can be administered to mammals such as livestock and human in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine dural or intracerebral injection. The cheonggukjang fermented kojang extract of the present invention or a glucuronid derivative compound isolated therefrom has little toxicity and side effects, and thus can be used safely even when taken for a long time for the purpose of prevention.

The present invention also provides a health functional food for prevention or improvement of liver disease comprising the above fermented chongkukjang fermented bean curd extract and a pharmaceutically acceptable food supplementary additive. The health functional food of the present invention includes forms such as tablets, capsules, pills, and liquids. Examples of the foods to which the extract of the present invention can be added include various foods, beverages, gums, tea, vitamins , And health functional foods.

The present invention relates to a composition for preventing or treating liver disease containing a fermented chrysanthemum fermented curd extract or a curcuminoid derivative compound isolated therefrom. The composition inhibits the expression of [alpha] -SMA and TGF- [beta] 1 in liver tissue or hepatocytes and is excellent in reducing the amount of TNF- [alpha], and is useful as a therapeutic agent for hepatic diseases such as hepatitis, liver fibrosis and liver cirrhosis Can be usefully used.

FIG. 1 shows HPLC (high performance liquid chromatography) spectral results showing changes in compound contents before fermentation and after fermentation.
FIG. 2 shows the result of TLC (thin layer chromatography) for the change of the compound content of the fermented koji extract of Cheonggukjang.
FIG. 3 is a diagram showing the results of confirming the structures of Compounds 1 to 4 isolated from Cheonggukjang fermented koji extract through HMBC and NOESY.
4 is a graph showing the results of confirming the activity of? -SMMA (? -Smooth muscle actin) using Western blotting after treating the compounds of the present invention with MEF cells.
FIG. 5 shows TGF-β1 mRNA expression and transcriptional activity in the case of treatment with curymarogin A compared with the control without compound treatment in MEF cells by real-time PCR (FIG. 6A) and reporter gene assay (reporter gene assay, Fig. 6B).

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein but may be embodied in other forms. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the concept of the invention to those skilled in the art.

≪ Example 1: Preparation of fermented cornstarch of Cheonggukjang &

1 kg of Ulgum (purchased from Korea, Seoul, Ilshin Sangyo) was dried and powdered, and 10 g of chungkookjang powder (Product No. 157, Tinvas scoria, Muan) was added to the powdered koji, and 400 g of purified water And then fermented at 82.5 ± 2.5 ° C for 4 hours. The fermentation was then continued for 40 days at a temperature of 25 ° C and a humidity of 60%. Dried at 82.5 ± 2.5 ° C for 2 hours, and then dried with hot air at 87.5 ± 2.5 ° C for 2 hours to prepare a fermented broth having a moisture content of 3% or less.

Example 2: Confirmation of Solvent Extraction Conditions of Active Material from Cheonggukjang Fermented Curd

In order to compare the extracting conditions of the fermented chongkukjang fermented beef curd prepared in Example 1 and the degree of extracting the active substance from the active substance, the extracts were prepared by adding distilled water, a 30 to 100% aqueous ethanol solution, a solvent such as methanol, acetone, ethyl acetate, Was inhibited against? -SMA. To this end, 100 g each of 100 g of the fermented koi or chongkukjang fermented koi were heated and extracted with 300 ml of the solvent for 3 hours using a reflux condenser under the conditions shown in Table 1 below, and the extract was concentrated under reduced pressure. Thereafter, the inhibitory activity of each extract (50 占 퐂 / ml) on? -SMA was confirmed using the method described in Example 5.

As shown in Table 1, it was confirmed that the chungkukjang fermented koji extract extracted with 30% ethanol aqueous solution had a relatively high inhibitory activity against? -SMA in the extracts of the respective solvents. As the content of alcohol was increased, the extraction of 30% ethanol aqueous solution was set as the optimal extraction condition, considering the relatively low extractable amount of curcumin and the relatively low level of ADME and high cytotoxicity of curcumin. In addition, it was also confirmed that the chungkukjang fermented beef extract of the present invention had remarkably high inhibitory activity against? -SMA, rather than the extract of 30% ethanol aqueous solution.

Subsequently, the extract of Chungkukjang fermented koji extracted with the aqueous 30% ethanol solution was sequentially fractionated with hexane, ethyl acetate and butanol to obtain concentrated fractions, and then the inhibitory activity against each of? -SMA was determined by the same method As a result, it was found that the inhibitory activity against α-SMA was similarly strong in the ethyl acetate and butanol fractions of fermented Cheonggukjang fermented Ulgum (see Table 2).

Solvent condition Inhibitory activity against? -SMA (%)



Extract of Chungkookjang fermented Ulgum
(50 mu g / ml) 30% aqueous ethanol solution 34.3 50% aqueous ethanol solution 35.3 70% ethanol aqueous solution 39.3 90% aqueous ethanol solution 39.7 100% ethanol 45.8 100% methanol 46.2 100% acetone 29.9 100% ethyl acetate 32.7 100% chloroform 28.3 Distilled water 18.02 Extract of non-fermented turmeric
(50 mu g / ml)
30% aqueous ethanol solution
10.2

Solvent condition Inhibitory activity against? -SMA (%)
Extract of Chungkookjang fermented Ulgum
(50 mu g / ml) A hexane fraction of a 30% aqueous ethanol solution 15.3 Of a 30% aqueous ethanol solution
Ethyl acetate fraction
42.4 Butanol fraction of 30% aqueous ethanol solution 45.8

< Example  3: fermentation of Cheonggukjang Turmeric  The preparation of the extract and Diarylheptanoids  Isolation and Purification of Compounds>

3 kg of the fermented chongkukjang fermented beef of Example 1 was heated and extracted with 15 liters of 30% aqueous ethanol solution for 3 hours over a period of 4 hours using a reflux condenser. The 30% aqueous ethanol solution (hereinafter, referred to as "A fraction") was concentrated under reduced pressure to obtain 182 g of an extract. The concentrated extract was suspended in water (5 L), and then subjected to solvent fractionation successively with n-hexane (5 L), ethyl acetate (5 L) and butanol (2 L), followed by α-SMA inhibition As a result of the activity, strong activity was confirmed in ethyl acetate fraction and butanol fraction. Thereafter, the ethyl acetate fraction and the butanol fraction were used in combination as the progress of the experiment.

The fractions (130 g) of ethyl acetate and butanol combined were subjected to silica gel column chromatography (10 × 30 cm; 63-200 μm particle size) under the solvent gradient condition of chloroform-acetone (19: 1 → 1:19) F7: 10.5 g; F6: 12.6 g; F7: 22.4 mg) was obtained in the same manner as in Example 1 (F1: 17.6 g; F2: 8.5 g; F3: 5.6 g; F4: 9.8 g; The fraction F3 was recrystallized in hexane-ethyl acetate solvent to obtain pure curcumine (4.2 g). Five small fractions were obtained by performing silica gel column chromatography (5 x 40 cm; 40 to 63 μm particle size) under chloroform-acetonitrile (9: 1 to 1: 9) solvent gradient conditions of fraction F4 (F4.1- F4.5). Demethoxycurcumin (1.3 g) and bisdemethoxycurmin (2.5 g) were obtained by recrystallization of the fractions F4.1 and F4.2 using a chloroform solvent. The small fraction F4.4 (90 mg) was dissolved in acetonitrile (MeCN) -water (0-40 min: 45% acetonitrile, 40-45 min: 100% acetonitrile, 45-55 min: 100% acetonitrile) Optima Pak C ₁₈ column 10 x 250 mm; 10 mu M particle size 2 ml / min; UV detection: 205, 254nm] the embodiment to obtain the _{5 (t R = 30.0min, 5.5㎎} ) and compound _{6 (t R = 34.0min, 7.0㎎} ) compound.

Fraction F5 was subjected to Sephadex eletch-20 column chromatography (7 x 40 cm) using methanol as a developing solvent to obtain three small fractions (F5.1-F5.3). The small fraction F5.2 (3.1 g) was subjected to silica gel column chromatography (5 × 40 cm; 40 to 63 μm particle size) under the solvent gradient condition of chloroform-acetonitrile (9: 1 → 1: 9) (F5.2.1F5.5.5). The small fraction, F5.2.3 (110 mg), was purified on HPLC (Optima Pak C ₁₈ column 10x250 mm) using acetonitrile-water (065 min: 57% acetonitrile, 6570 min: 100% acetonitrile). 10 mu M particle size 2 ml / min; UV detection: 205, 254nm] the embodiment to obtain the title compound _{7 (t R = 42.0min, 13.5㎎} ), compound _{8 (t R = 58.0min, 7.5㎎} ) and compound _{3 (t R = 61.0min, 8.0㎎} ) . A small fraction F5.2.4 (95㎎) HPLC (060 min : 35% acetonitrile, 6065min: 100% acetonitrile), the compound was obtained as a condition of the same column as the _{1 (t R = 46.0min, 4.5㎎} ) and compound _{2 (t R = 48.5min, 3.5㎎} ) was obtained. Final compound _{4 (t R = 31.0min, 11.5㎎} ) is a small fraction F5.2.5 (150㎎) HPLC to obtain the (035min: 100% acetonitrile: 32% acetonitrile, 40min).

In addition, as a result of comparing the effective compounds of the extracts of Chongkukjang fermented koi (40 days fermentation) extracted with a 30% ethanol aqueous solution and the non-fermented koji extracts by HPLC, it was found that there was a therapeutic effect of hepatitis, liver fibrosis or cirrhosis It was confirmed that the contents of the known curcumin, dimethoxy curcumin and bisdimethoxy curcumin were decreased, and the compounds 1 to 8 of the present invention were increased (see FIG. 1).

Similarly, the fermented fermented Chungkookjang fermented beef extract (fermented product on the 40th day) and the fermented fermented fermented cherries were fermented by TLC (fermentation on days 0, 10, 20 and 30) (See FIG. 2), it was confirmed that the distribution of the compounds was also different in each of the extracts of the same amount. That is, the content of curcumin, dimethoxy curcumin and bisdimethoxy curcumin was reduced, and it was expected that the compounds 1 to 8 of the present invention were relatively increased.

Thus, curcumin, dimethoxy curcumin and bisdimethoxy curcumin, which are present in large amounts in the ganoderma extract but not easily absorbed, distributed, metabolized and secreted (ADME) in the intestinal tract, are converted into the compounds of the present invention through fermentation, The inventive Chungkookjang fermented Ulgum extract was thought to improve absorption, distribution, metabolism and secretion (ADME) in the intestinal tract than the Ulguk extract.

< Example  4 : Cucuminoids  Physicochemical Properties and Chemical Structure Analysis of Derivatives>

Example  4-1: Kekyumarojin  A (Compound 1)

Curcumalongine

Yellow amorphous powder

UV (MeOH)? _Max nm (log?) 295 (3.53), 398 (4.06)

IR (KBr) v _max 3415 (OH), 1665 (C = O), 1598, 1547, 1511, 1489, 1273 cm ^&lt;

¹ H-NMR and ¹³ C-NMR (see Tables 3 and 4)

EIMS m / z (rel.int.): 352 ([M] ⁺ , 100), 322 (59), 278 (22), 267 (20), 191 (52), 180 (28)

HRFABMS m / z 353.1022 [M + H] + (calcd for C 20 H 17 O 6, 353.1025)

Compound 1 was obtained as a yellow powder. Compound ¹ 1 H NMR spectrum is 3,4,5-tetra substituted benzene ring the _{[δ H 7.23 (1H, d} , J = 1.8 Hz, H-2 ') and 7.21 (1H, d, J = 1.8 Hz , the in H-6 ')], a 2 B 2 of the aromatic hydrogen _{[δ H 7.63 (2H, d} , J = 8.4 Hz, H-2 ", H-6") and 6.94 (2H, d, J = 8.4 Hz , H-3 ", H- 5")], a trans -olefinic hydrogen _{[δ H 7.67 (1H, d} , J = 15.6 Hz, H-7 ") , and 7.08 (1H, d, J = 15.6 Hz, H -8 "), a methoxy group _{[δ H 3.99 (3H, s} , 3'-OCH 3)] was confirmed in the other olefinic hydrogen in _{[δ H 6.52 (1H, s} , H-7 ')] , And β- diketone hydrogen was confirmed in δ _H 5.92 (1H, s, H-4). The results consistent with these ¹ H NMR results were obtained from the ¹³ C NMR spectrum. Methoxy groups at δ _C 56.5, four olefinic carbons at δ _C 146.7, 139.3, 113.3 and 112.2, conjugated ketone carbon at δ _C 186.6, hydroxylated carbons at δ _C 176.1, and β- diketone forms The carbon was identified at δ _C 105.4 and 12 aromatic carbons were identified.

Additional HSQC experiments were performed to determine the number of carbons. The final number of carbon atoms was determined to be 20. Δ _C 146.7 (C-2) and 176.1 (C-5), which are the deshielding carbon of two different olefinic carbons, were determined by carbon substitution with hydroxyl groups. These observations were confirmed in the characteristic IR absorption bands (1665, 1598 cm ^-1 ) and UV (398, 295 nm) spectra. From these results, it can be seen that compound 1 has a (3 H ) CH-CO-CH = C (OH) -CH = CH-). An HMBC experiment was conducted to confirm the precise connection of compound 1 . C-2, C-3, C-2 ', C-6' and H-2 'from H-7''/ C-7 linkage confirmed that the 1,3,4,5-tetrasubstituted aromatic ring is linked to C-2 of the furanone ring via olefinic carbon C-7'. This result was confirmed by further confirmation of HSQC and HMBC correlations to C-7 'and C-7' of the two aromatic rings. Thus, compound 1 is 2- (4,5-dihydroxy-3- methoxyphenyl) 4-hydroxyphenyl) ethenyl-3 (2 H ) -furanone was named curcumalongin A.

Example  4-2: Kekyumarojin  B (Compound 2)

Curcumalongin B

Yellow amorphous powder

UV (MeOH)? _Max nm (log?) 290 (3.76), 410 (4.12)

IR (KBr) v _max 3416 (OH), 1665 (C = O), 1588, 1547, 1512, 1490, 1284 cm ^&lt;

¹ H-NMR and ¹³ C-NMR (see Tables 3 and 4)

EIMS m / z (rel.int.): 382 ([M] ⁺ , 100), 352 (20), 322 (8), 308 (10), 278 (6), 267 (9) , 191 (10), 180 (24)

HREIMS m / z 382.1051 [M] + (calcd for C 21 H 18 O 7, 382.1053)

Compound 2 was obtained as a yellow powder and the molecular formula of Compound 2 was determined as C ₂₁ H ₁₈ O ₇ by observing the molecular ion peak at m / z 382.1051 [M] ⁺ . From the IR spectrum of Compound 2 Hydroxyl 3416cm ^-1, respectively make the sharing group the carbonyl group the functions attributed to the aromatic ring at 1665cm ^-1 at 1588, 1547, 1512, and 1490cm ^-1. Compound 2 Although ¹ H NMR and ¹³ C NMR spectrum is similar to compound 1 confirmed the additional-methoxy peak at _{δ H 3.99 (3H, s,} 3 "-OCH 3). This result A ₂ B ₂ present in the aromatic compounds (1) ABX spin system than in a substituted form _{[δ H 7.43 (1H, d} , J = 1.8 Hz, H-2 "), 7.20 (1H, dd, J = 8.4, 1.8 Hz, H-6") and 6.91 (1H, d, J = 8.4 Hz, H-5 "). Compound 2 was a compound of the form in which methoxy was added to Compound 1 . Through the HMBC, it was finally confirmed that the methoxy group was present at the C-3 "by connecting the methoxyproton with the proton peak present at H-2" and 5 "to C-3" (δ _C 149.2) there was. Thus, compound 2 is a 2- (4,5-dihydroxy-3- methoxyphenyl) methenyl-5- (4-hydroxy-3-methoxyphenyl) ethenyl-3 (2 H) -furanone, was named curcumalongin B.

Example  4-3: Kekyumarojin  D (compound 3)

Curcumalongin D

Yellow amorphous powder

UV (MeOH)? _Max nm (log?) 290 (3.76), 418 (4.12)

IR (KBr)? _Max 3416 (OH), 1588, 1547, 1512, 1490, 1284 cm ^&lt;

¹ H-NMR and ¹³ C-NMR (see Tables 3 and 4)

HREIMS m / z 408.1782 [M] + (calcd for C 21 H 28 O 8, 408.1784)

Compound 4 was obtained as a yellow powder and from its IR spectrum Hydroxyl 3315 cm &lt; ^{-1 &} gt ;. ¹ H NMR spectrum of compound 3 is 1,3,4-trisubstituted benzene ring of _{[δ H 6.77 (1H, d} , J = 2.0 Hz, H-2 "), 6.67 (1H, d, J = 8.5 Hz, H -5 "), and 6.62 (1H, dd, J = 8.5, 2.0 Hz, H-6"] in, the _{1,3,4,5-tetrasubstituted benzene ring [δ H} 6.54 (1H, s, H-6 ' ) and 6.50 (1H, s, H- 2 ')] and two methoxy groups _{[δ H 3.77 (3H, s} , 3'-OCH 3) and 3.75 (3H, s, 3 " -OCH 3)] These observations suggest that 4-hydroxy-3-methoxyphenyl and 4,5-dihydroxy-3-methoxy phenyl groups are present in EIMS at m / z 137 and 153 peaks. a peak attributed to the group _{[δ H 2.63 (1H, m} , H-7a) /2.56 (1H, m, H-7b), 1.81 (1H, d, J = 10.5 Hz, H-2a) /1.61 (1H m, H-2b), 1.74 (1H, m, H-6a) /1.60 (1H, m, H- ) And three oxymethine groups were confirmed in [δ _H 4.66 (1H, d, J = 10.5 Hz, H-1), 4.19 (1H, m, H-5) From the HMQC and ¹³ C NMR results, it was found that (-CH (OH) -CH ₂ -CH (OH) -CH ₂ -CH (OH) H ₂ -CH ₂ -.) Skeleton was confirmed that the ¹ H NMR and ¹³ C NMR spectrum of compound 3 has two additional hydroxyl groups, except for the C-5 ', and the compound (3 S, 5 S) -3 , 5-dihydroxy-1,7-bis (4-hydroxy-3-methoxyphenyl) found that similar to the heptane. the results also, by observing the molecular ion peak of [m] ⁺ at m / z 408.1782 C ₂₁ H2 ₈ O ₈ (calculated C ₂₁ H ₂₈ O ₈ , 408.1784). HMBC experiments were conducted to ascertain the precise structure of the novel compound 3 . H-2 "and H-5" / C-3 ", H-2 '/ C-3' and C-4"',H-6' / C -4 ' and C-5', 3'-OCH 3 / C-3 ' and _{3 "-OCH 3 / C-3} " was confirmed by the observation of compound 3 these results are (1 R , 3 R , 5 S ) -trihydroxy-1- (4,5-dihydroxy-3-methoxyphenyl) -7- (4-hydroxy-3-methoxyphenyl) heptane was named curcumalongin D.

Example  4-4: Kekyumarojin  C (compound 4)

Curcumalongin C

Yellow amorphous powder

UV (MeOH)? _Max nm (log?) 264 (3.74), 422 (4.27) IR (KBr) ? _Max 3415 (OH), 2917, 1737 (C = O), 1626, 1599, 1513, 1467, 1272 cm ^&lt;

^& Lt; ¹ &gt; H NMR and &lt; ¹³ &gt; C NMR (see Tables 3 and 4)

EIMS m / z (rel.int.): 384 ([M] ⁺ , 100), 366 (47), 284 (51), 267 (55), 208 (32), 192 (53), 177 HREIMS m / z 384.1213 [m] + (calcd for C 21 H 20 O 7, 384.1209)

Table 3 below shows ¹ H NMR (600 MHz) of the compounds 1 to 4 of the present invention and Table ¹³ shows ¹³ C NMR (150 MHz) data.

¹ H NMR location Compound 1 Compound 2 Compound 3 Compound 4 δ _H mult.
( J in Hz) δ _H mult.
( J in Hz) δ _H mult.
( J in Hz) δ _H mult.
( J in Hz) One
7.53 d (15.6) 4.66 d (10.5)
2
6.67 d (15.6) 1.81 d (10.5)
1.61 m 3 3.87 m
4 5.92 s 5.91 s 5.98 s 1.64 m
1.46 m 5 4.19 m
6 6.72 d (15.6) 1.74 m
1.60 m 7 7.60 d (15.6) 2.63 m
2.56 m One' 2' 7.23 d (1.8) 7.23 d (1.8) 6.90 h 6.50 s 3 ' 4' 5 ' 6 ' 7.21 d (1.8) 7.21 d (1.8) 6.87 s 6.54 s 7 ' 6.52 s 6.52 s One" 2" 7.63 d (8.4) 7.43 d (1.8) 7.34 h 6.77 d 3 " 6.94 d (8.4) 4" 5 " 6.94 d (8.4) 6.91 d (8.4) 6.88 d (8.4) 6.67 d (8.5) 6 " 7.63 d (8.4) 7.20 dd (8.4, 1.8) 7.18 d (8.4) 6.62 dd (8.5, 2.0) 7 " 7.67 d (15.6) 7.67 d (15.6) 8" 7.08 d (15.6) 7.14 d (15.6) 3'-OCH ₃ 3.99 s 3.94 s 3.89 s 3.77 s ₃ "-OCH 3 3.99 s 3.90 s 3.75 s

¹³ C NMR location Compound 1 Compound 2 Compound 3 Compound 4 δ _C δ _C δ _C δ _C One 141.8 74.6 2 146.7 146.7 122.5 41.9 3 186.6 186.6 184.4 71.6 4 105.4 105.3 101.8 39.5 5 176.1 176.1 184.4 64.8 6 122.4 39.4 7 141.5 32.0 One' 124.4 124.4 127.3 133.5 2' 107.9 107.9 104.7 102.1 3 ' 149.2 149.0 149.2 148.7 4' 137.5 137.4 137.5 136.0 5 ' 146.5 146.5 146.5 146.0 6 ' 113.6 113.6 110.3 107.4 7 ' 112.2 112.2 One" 127.7 128.2 128.2 134.7 2" 131.2 111.0 111.5 112.9 3 " 117.0 149.2 148.8 148.1 4" 160.8 150.4 150.0 145.4 5 " 117.0 116.4 116.3 115.6 6 " 131.2 124.6 123.9 121.6 7 " 139.3 139.7 8" 113.3 113.5 3'-OCH ₃ 56.5 56.4 56.6 56.1 ₃ "-OCH 3 56.5 56.3 56.4

Example  4-5: 1- (4- Hydroxy -3- Methoxyphenyl ) -7- (3,4- Dihydroxyphenyl ) -1,6- Heptadiene -3,5- Dion  (Compound 5)

1- (4-hydroxy-3-methoxyphenyl) -7- (3,4-dihydroxyphenyl) -1,6-heptadiene-3,5-dione

Yellow needle

EI-MS m / z : 354 [M] ^&lt; ⁺ & gt ^;

^{1 H-NMR (500 MHz,} acetone- d 6): 7.59 (1H, d, J = 16.0 Hz, H-7), 7.54 (1H, d, J = 16.0 Hz, H-1), 7.18 (2H, d, J = 8.0 Hz, H -6 ', 6 "), 7.07 (1H, s, H-2'), 7.05 (1H, s, H-2"), 6.88 (1H, d, J = 8.0 Hz H-5 ', 5''), 6.72 (1H, d, J = 16.0 Hz, H-6), 6.60 (1H, d, J = 16.0 Hz, H- 4), 3.92 (3H, s , 3 "-OCH 3)

^{13 C-NMR (125 MHz,} acetone- d 6): 184.9 (C-3, 5), 150.0 (C-4 "), 148.8 (C-4 ', 3"), 146.3 (C-3'), C-1 '), 128.3 (C-1'), 128.3 (C-1 ''), 123.9 6), 122.2 (C-2 ), 116.2 (C-5 '), 115.2 (C-3'), 111.4 (C-2 "), 101.8 (C-4), 56.0 (3" -OCH 3)

Example  4-6: 5- Hydroxy -1,7- Vis (4- Hydroxyphenyl ) -4,6- Heptadiene -3-one (Compound 6)

5-hydroxy-1,7-bis (4-hydroxyphenyl) -4,6-heptadien-3-one

Yellow needle

EI-MS m / z : 310 [M] &lt; ⁺ & gt ^;

¹ H-NMR (500 MHz, acetone- d ₆ ): 7.54 (1H, d, J = 16.0 Hz, H-7), 7.53 (2H, d, J = 8.0 Hz, H- 7.07 (2H, d, J = 8.0 Hz, H-2 ', 6'), 6.88 (2H, d, J = 8.0 Hz, H-3 ", 5"), 6.74 (2H, d, J = 8.0 Hz H-3 ', 5'), 6.53 (1H, d, J = 16.0 Hz, H-6), 5.79 2H, m, H-1)

^{13 C-NMR (125 MHz,} acetone- d 6) 200.4 (C-3), 168.0 (C-5), 160.4 (C-4 "), 156.6 (C-4 '), 140.5 (C-7), C-1 '), 130.8 (C-2 ", 6"), 130.2 (C-2', 6 '), 127.8 (C-1), 116.1 (C-3 ', 5'), 101.0

Example  4-7: 1,7- Vis (3,4- Dihydroxyphenyl ) -1,6- Heptadiene -3,5- Dion  (Compound 7)

1,7-bis (3,4-dihydroxyphenyl) -1,6-heptadien-3,5-dione

Yellow needle

EI-MS m / z : 340 [M] ^&lt; ⁺ & gt ^;

^{1 H-NMR (500 MHz,} acetone- d 6): 7.50 (2H, d, J = 16.0 Hz, H-1,7), 7.03 (2H, d, J = 8.0 Hz, H-6 ', 6 " ), 7.15 (2H, s, H-2 ', 2 "), 6.85 (2H, d, J = 8.0 Hz, H-5', 5"), 6.54 (2H, d, J = 16.0 Hz, H- 2,6), 5.92 (2H, s, H-4)

^{13 C-NMR (125 MHz,} acetone- d 6) 184.3 (C-3, 5), 148.5 (C-4 ', 4 "), 146.6 (C-3', 3"), 141.3 (C-1, 6), 122.0 (C-2, 6), 116.4 (C-5 ', 5 "), 115.2 ', 2''), 101.8 (C-4)

Example  4-8: 1- (4- Hydroxyphenyl ) -7- (3,4- Dihydroxyphenyl ) -1,6- Heptadiene -3,5- Dion  (Compound 8)

1- (4-hydroxyphenyl) -7- (3,4-dihydroxyphenyl) -1,6-heptadiene-3,5-dione

Yellow needle

EI-MS m / z : 324 [M] ^&lt; ⁺ & gt ^;

¹ H-NMR (500 MHz, acetone- d ₆ ): 7.59 (1H, d, J = 16.0 Hz, H-7), 7.58 (1H, d, J = 16.0 Hz, H- (1H, d, J = 8.0 Hz, H-2 &quot;, 6 "), 7.20 (1H, d, J = 8.0 Hz, H-6 '), 7.07 , J = 8.0 Hz, H- 3 ", 5"), 6.90 (1H, d, J = 8.0 Hz, H-5 '), 6.67 (1H, d, J = 16.0 Hz, H-6), 6.66 ( 1H, d, J = 16.0Hz, H-2), 5.95 (2H, s,

^{13 C-NMR (125 MHz,} acetone- d 6) 183.9 (C-3, 5), 160.4 (C-4 "), 148.7 (C-4 '), 146.4 (C-3'), 141.4 (C- (C-1), 122.9 (C-6), 122.2 (C-6) ), 122.1 (C-2), 116.8 (C-3 ", 5"), 116.5 (C-5 '), 115.3

< Example  5: Anti-fibrosis  Measuring action>

In order to confirm the anti-fibrosis effect of the compounds of the present invention, the amount of expression of α-SMA, which is a typical protein which is increased when fibrosis due to tissue damage of hepatocytes occurs using western blot, was confirmed.

α-SMA In order to perform the Western blot, it was treated with Compound 1 ~ 8 (10㎍ / ㎖) of the present invention to an activated hepatic detail Po properties similar MEF cells. After the cells were disrupted, proteins were extracted and subjected to SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and transferred to a nitrocellulose membrane. Thereafter, the antibody was reacted with the α-SMA primary antibody and then reacted with the secondary antibody. The results were confirmed using a dedicated kit (ECL chemiluminescence kit) and the results are shown in FIG. The control group was a group without drug treatment. The extract or the solvent of the compound was dissolved in dimethyl sulfoxide (DMSO) and used in a minimum amount.

Referring to FIG. 4, it was found that the compounds 1 to 8 of the present invention strongly inhibited the expression of? -SMA, and thus it was judged that these compounds could be usefully used as a therapeutic agent for hepatic fibrosis or cirrhosis. In addition, the compound 1-8 to be determined, the effect of the compound 1-8 curcumin and bis-dimethoxy curcumin, dimethoxyethane, yet similar as curcumin, Minister of absorption, distribution, metabolism and secretion (ADME) is to be the better the compound, Or chungkukjang fermented koji extract containing the above compounds could be useful as a therapeutic agent for liver diseases.

< Example  6: Cucuminoids  Derivative TGF -β1 mRNA  Expression and transcriptional activity inhibition &gt;

TGF-β1 is a typical cytokine that is increased during fibrosis caused by tissue damage. For this, TGF-β1 mRNA expression and transcriptional activity were evaluated by real-time PCR and reporter gene assay, respectively, in MEF cells similar in nature to activated hepatic cells. In addition, the inhibitory effect of bisdimethoxy curcumin and compound 1 , curymarogin A, which had strong antifibrotic activity, on TGF-beta1 mRNA expression was evaluated by real-time PCR. The primer used in the experiment is as follows (the PCR result is expected to be 163 bp).

TGF-beta1 mRNA F: 5'-TGA ACC AAG GAG ACG GAA TAC AGG-3 '

TGF-beta1 mRNA R: 5'-GCC ATG AGG AGC AGG AAG GG-3 '

In addition, PCR conditions were 98 cycles for 30 seconds, 60 cycles for 1 minute, and 72 cycles for 1 minute. As a result, it was confirmed that the effect of curcumarogin A on TGF-β1 mRNA expression was excellent (see FIG. 5A).

Similarly, in order to confirm the activity of the human TGF-beta1 gene promoter, MEF cells were cultured in 48 well-plates, and FBS (bovine serum) and cell culture medium without antibiotics were reacted for 6 hours. Then, a luciferase reporter vector containing wild type promoter WT-TGF-beta1 (1 μg, control vector: pGL3 vector) was added to MEF cells with lipofectamine 2000 (lipofectamine 2000, ), And the activity of each promoter was confirmed after 24 hours. The promoter activity of TGF-beta1 was corrected by measuring the activity of β-galactosidase (using 0.5 μg of pCMV-β-gal) (using a luminometer, Luminoscan Ascent; Thermo Electron Co.) The results are shown in Figure 6B. The luciferase assay system was specified 24 hours after transfection and a kit manufactured by Promega was used (dual-luciferase reporter assay system, Promega, Madison, WI, USA).

5B, transcriptional activity evaluation using a TGF-beta1 reporter having a human TGF-beta1 gene promoter (promoter, 1100 bp) and a luciferase reporter gene was also carried out using the TGF-beta1 gene transcription of curcumin Inhibitory effect was observed, and it was able to demonstrate the ability of the compounds derived from fermented Chungkookjang fermented Ulgum extract to inhibit liver fibrosis.

< Example  7: Induced by administration of carbon tetrachloride Liver tissue  Chungkukjang fermentation for damage model Turmeric  By extract administration Liver index  Analysis>

This experiment was conducted at Korea Food Research Institute. Experimental animals were fed with ICR mouse (27 ± 1 g), which was 5 weeks old, from the central laboratory animal, and the temperature was 23 ± 1 ℃ and the relative humidity was 55 ± 15% ) Were used. After 1 week of purging period, the experimental group was separated by body weight and the feed and water were fed freely. Carbon tetrachloride was dissolved in corn oil at a concentration of 20% and orally administered at a dose of 1 ml / kg (body weight) twice a week. In normal control, only corn oil was orally administered twice a week, and the negative control was fed with carbon tetrachloride followed by a general diet. A positive control (manufactured by adding a 0.4% concentration of solid feed weight to a commercial product of a health functional food made from a mycelium of mycelia of mushrooms [known to have a good effect of improving liver fibrosis]) and a non-fermented cucumber extract, Fermented celery extracts were fed with carbon tetrachloride and feed containing each ingredient. After 8 weeks of carbon tetrachloride, they were fasted for 1 day and sacrificed. Blood was collected and serum was isolated. Liver tissues were washed in cold saline and stored frozen in liquid nitrogen.

Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), cholesterol and triglycerides were measured using an analytical reagent (Bayer, USA) And measured by ADIVA 1650 (Bayer, Japan). Lactate dehydrogenase (LDH) was measured by ADIVA 1650/2400 (Siemens, USA) using analytical reagent (Siemens, USA). The results were compared with those of CCl ₄ control Enzyme levels were converted to percentages and are shown in Table 5.

process ALT AST ALP LDH TC TG Normal group - - - - - - CCl ₄ control 100 100 100 100 100 100 Positive control group 78 71 76 81 98 81 Non Fermented Curd Extract
(0.4%) + CCl ₄ 72 83 99 93 92 87 Cheonggukjang fermented Ulgum extract
(0.4%) + CCl ₄ 51 42 68 61 72 76

Alanine aminotransferase (ALT) and aspartate aminotransferase (AST), which are known as indicators of liver damage, are released into the blood and become highly active as the breakdown of liver tissue progresses It is known. In particular, ALT is present in the liver and AST is abundant in the liver and skeletal muscle, and its specificity is recognized.

Therefore, as shown in Table 5, the hepatocyte-treated group showed significantly higher enzyme activity than the normal group, suggesting that liver injury was induced. On the other hand, in the group administered with Cheonggukjang fermented kojang extract, the enzyme level of ALT was reduced by 49% to 51%, which was 27% lower than that of the positive control group. In contrast, the decrease in the group treated with the non-fermented koji extract was 72%.

Alkaline phosphatase (ALP) is known to increase in bile when hepatic cells are poorly discharged or biliary obstruction. As shown in Table 5 above, the group administered with chongkukjang fermented kojang extract was administered with carbon tetrachloride ₄ control), the enzyme activity was reduced by 32%. In addition, lactate dehydrogenase (LDH) is also released into the blood when the tissue containing the enzyme is destroyed, leading to elevated levels of enzymes. In contrast to the CCl ₄ control group, 7%, while the extract group of Chungkukjang fermented with Urea extract decreased by 39%.

In addition, the content of total cholesterol (TC) and triglyceride (TG) in normal hepatocytes is in a balance between synthesis and utilization, but when liver damage is caused by carbon tetrachloride, a highly reactive CCl 3 group is produced, It has been reported that the content of cholesterol and glyceride is increased by lowering the function.

Table 5 shows that total cholesterol (TC) was not significantly influenced by CCl ₄ control as compared with the normal group, but in the case of the group administered with Cheonggukjang fermented kojang extract, it was lower than that of the group treated with carbon tetrachloride Was reduced by about 28%.

In the case of triglyceride (TG), it was confirmed that the extract of Cheonggukjang fermented kojang decreased by about 24% compared with that of CCl ₄ control.

Therefore, it was expected that the chungkukjang fermented kojang extract of the present invention was superior to the non-fermented kojang extract.

< Example  8: Toxicity test>

Example  8-1. Acute toxicity

This experiment was conducted to investigate the toxicity of the fermented Cheonggukjang fermentation extract of Example 3 to an animal body in an acute (within 24 hours) when an excessive amount was ingested in a short period and to determine the mortality rate. 30 normal mice were prepared and 10 mice were assigned to each group. In the control group, only 30% PEG-400 was administered. In the experimental group, the chongkukjang fermented kojang extract and compound 1 of Example 3 were orally administered at a concentration of 2.0 g / kg, respectively. After 24 hours of administration, the mortality rate was examined. As a result, both the control group and the experimental group administered with the Cheonggukjang fermented kojang extract and Compound 1 of Example 3 at a concentration of 2.0 g / kg survived.

Example  8-2: Experimental group  And control organ organs and tissue toxicity experiments

In order to investigate the effect of C57BL / 6J mice on each organ (tissue) of the long-term toxicity test, the experimental group administered with the concentration of 2.0 g / kg of Chungkookjang fermented kojang extract and compound 1 of Example 3 and the solvent After 8 weeks from the control animals, blood samples were collected from Glutamate-pyruvate transferase (GPT) and Blood Urea Nitrogen (BUN) using Select E (Vital Scientific NV, Netherland). As a result, GPT, which is known to be related to hepatotoxicity, and BUN, which is known to be related to renal toxicity, showed no significant difference compared to the control group. In addition, liver and kidney were cut from each animal and histological observation was carried out with an optical microscope through a conventional tissue section production process. No abnormal abnormalities were observed.

< Examples  1: Pharmaceutical Formulation example >

1-1. Manufacture of tablets

200g of Compound 1 of the present invention (curcumarogin A) was mixed with 175.9g of lactose, 180g of potato starch and 32g of colloidal silicic acid. To this mixture was added a 10% gelatin solution, which was pulverized and passed through a 14-mesh sieve. This was dried, and a mixture obtained by adding 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate was made into tablets.

1-2. Injection preparation

1 g of the compound 1 of the present invention (curcumarogin A), 0.6 g of sodium chloride and 0.1 g of ascorbic acid were dissolved in distilled water to make 100 ml. This solution was placed in a bottle and sterilized by heating at 20 DEG C for 30 minutes.

< Examples  2: Food Manufacturing example >

2-1. Manufacture of cooking seasonings

The cheonggukjang fermented kojang extract of Example 3 of the present invention was added to the cooking sauce at 0.2 to 10% by weight to prepare a cooking sauce for health promotion.

2-2. Manufacture of flour food products

The cheonggukjang fermented kojang extract of Example 3 of the present invention was added to wheat flour in an amount of 0.1-5.0 wt%, and bread, cake, cookies, crackers and noodles were prepared by using the mixture to prepare a health improving food.

2-3. soup  And juicy ( gravies )

The chungkukjang fermented beef extract of Example 3 of the present invention was added to soup and broth at 0.1 to 1.0 wt% to prepare health promotion soup and juice.

2-4. dairy product( dairy products )

The cheonggukjang fermented kojang extract of Example 3 of the present invention was added to milk in an amount of 0.1 to 1.0 wt%, and various dairy products such as butter and ice cream were prepared using the milk.

< Examples  3: Drink Manufacturing example >

3-1. Vegetable juice  Produce

The vegetable juice for health promotion was prepared by adding 0.5 g of the fermented koji fermented extract of Example 3 of the present invention to 1,000 ml of tomato or carrot juice.

3-2. Fruit juice  Produce

The fruit juice for health promotion was prepared by adding 0.1 g of the chungkukjang fermented kojang extract of Example 3 of the present invention to 1,000 ml of apple or grape juice.

Claims (13)

To keokyu Maroni Jin A (curcumalogin A, compound 1) of formula 1-5, keokyu Maroni Jean B (curcumalogin B, compound 2), keokyu Maroni binary C (curcumalogin C, Compound 3), keokyu Maroni Jin D (curcumalongin D, compound 4), 1- (4-hydroxy-3-methoxyphenyl) -7- (3,4-hydroxyphenyl) -l, 6-heptadiene-3,5-dione (1 - (4 3-methoxyphenyl) -7- (3,4-dihydroxyphenyl) -1,6-heptadiene-3,5-dione, Compound 5 ), 5-hydroxy- 4,6-heptadiene-3-one, compound 6 ), 1,7-bis (3,4 1,7-bis (3,4-dihydroxyphenyl) -1,6-heptadiene-3,5-dione, Compound 7 ) and 1 - (4-hydroxyphenyl) -7- (3,4-dihydroxyphenyl) -1,6-heptadiene- dihydroxyphenyl) -1,6-heptadiene-3,5- dione, compound 8) at least one selected from the group consisting of greater kyumi cannabinoid (curcuminoid) derivative compound is PO The soybean fermented turmeric (fermented Curcuma Longa by natural fermented soybean) composition for the treatment or prevention of liver disease, characterized in that it contains an extract.
[Chemical Formula 1]

(2)

(3)

[Chemical Formula 4]

[Chemical Formula 5]

The method of claim 1,
The Cheonggukjang fermented turmeric extract is a composition for the treatment or prevention of liver disease, characterized in that the extract is produced by extracting the Cheonggukjang fermented turmeric with water, lower alcohol having 1 to 4 carbon atoms, acetone, ethyl acetate, chloroform or a mixed solvent thereof. The method of claim 1,
The Chunggukjang fermented turmeric is a composition for the treatment or prevention of liver disease, characterized in that the fermentation is prepared by fermenting a mixture of 0.1 to 10 parts by weight of Cheonggukjang powder and 40 to 100 parts by weight of water. 4. The method according to any one of claims 1 to 3,
The liver disease is a composition for the treatment or prevention of liver disease, characterized in that the disease is selected from the group consisting of hepatitis, liver fibrosis and cirrhosis. (Curcumalogin A, compound 1 ), curcumalogin B (compound 2 ), curcumalogin C (compound 3 ), curcumalogin D (curcumalogin A, compound 4), 1- (4-hydroxy-3-methoxyphenyl) -7- (3,4-hydroxyphenyl) -l, 6-heptadiene-3,5-dione (1 - (4 3-methoxyphenyl) -7- (3,4-dihydroxyphenyl) -1,6-heptadiene-3,5-dione, Compound 5 ), 5-hydroxy- 4,6-heptadiene-3-one, compound 6 ), 1,7-bis (3,4 1,7-bis (3,4-dihydroxyphenyl) -1,6-heptadiene-3,5-dione, Compound 7 ) and 1 - (4-hydroxyphenyl) -7- (3,4-dihydroxyphenyl) -1,6-heptadiene- dihydroxyphenyl) -1,6-heptadiene-3,5- dione, one or more kinds of compounds selected from the group consisting of 8) increases kyumi cannabinoid (curcuminoid) containing the derivative Soybean Fermented turmeric (fermented Curcuma Longa by natural fermented soybean) Health functional food for the prevention or improvement of liver disease, characterized in that it contains an extract.
[Chemical Formula 1]

(2)

(3)

[Chemical Formula 4]

[Chemical Formula 5]

6. The method of claim 5,
The fermented turmeric extract is a health functional food for the prevention or improvement of liver disease, characterized in that the fermented cheonggukjang chungkukjang fermented turmeric extracted with water, C1-4 lower alcohol, acetone, ethyl acetate, chloroform or a mixture thereof . 6. The method of claim 5,
The Cheonggukjang fermented turmeric is a health functional food for the treatment or improvement of liver disease, characterized in that by producing a fermentation mixture of 0.1 to 10 parts by weight of the Cheonggukjang powder and 40 to 100 parts by weight of turmeric. 8. The method according to any one of claims 5 to 7,
The liver disease is a health functional food for the treatment or improvement of liver disease, characterized in that the disease is selected from the group consisting of hepatitis, liver fibrosis and cirrhosis. (Curcumalogin A, compound 1 ), curcumalogin B (compound 2 ), curcumalogin C (compound 3 ), curcumalogin D (curcumalogin A, compound 4), 1- (4-hydroxy-3-methoxyphenyl) -7- (3,4-hydroxyphenyl) -l, 6-heptadiene-3,5-dione (1 - (4 3-methoxyphenyl) -7- (3,4-dihydroxyphenyl) -1,6-heptadiene-3,5-dione, Compound 5 ), 5-hydroxy- 4,6-heptadiene-3-one, compound 6 ), 1,7-bis (3,4 1,7-bis (3,4-dihydroxyphenyl) -1,6-heptadiene-3,5-dione, Compound 7 ) and 1 - (4-hydroxyphenyl) -7- (3,4-dihydroxyphenyl) -1,6-heptadiene- containing dihydroxyphenyl) -1,6-heptadiene-3,5- dione, one or more kinds of compounds selected from the group consisting of 8) increases kyumi cannabinoid (curcuminoid) derivatives A pharmaceutical composition for the prevention and treatment of liver diseases, characterized in that the.
[Chemical Formula 1]

(2)

(3)

[Chemical Formula 4]

[Chemical Formula 5]

10. The method of claim 9,
The liver disease is a pharmaceutical composition for treating or preventing liver disease, characterized in that the disease is selected from the group consisting of hepatitis, liver fibrosis and cirrhosis. A novel compound of formula (1): curcumalogin A.
[Chemical Formula 1]

A novel compound of formula (2): curcumalogin B.
(2)

A novel compound of formula (4): curcumalogin D.
[Chemical Formula 4]

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