KR20110053590A - A composition comprising the fermented rhizoma of curcuma aromatica salisb for treating and preventing alcoholic liver disease - Google Patents

Abstract

PURPOSE: A composition containing fermented Curcuma aromatica Salisb. extract is provided to remove DPPH radical and ABTS radical and to suppress liver damage. CONSTITUTION: A pharmacetucialc composition for preventing and treating alcoholic liver diseases contains fermented Curcuma aromatica Salisb. extract as an active ingredient. The Curcuma aromatica Salisb. is fermented by Aspergillus sp. fungi at 150°C and 2090% of relative humidity for 1 hour to 1 week. The fermented Curcuma aromatica Salisb. extract is obtained by isolating with water including purified water, methanol, ethanol, or mixture solvent thereof. A health food for preventing and treating alcoholic liver diseases contains the ffermented Curcuma aromatica Salisb. extract as an active ingredient.

Description

A composition comprising the fermented rhizoma of curcuma aromatica salisb for treating and preventing alcoholic liver disease

The present invention relates to a composition for the treatment and prevention of alcoholic liver disease containing fermented turmeric extract.

[Document 1] Kim Chang-min, Shin Min-kyo et al., Jungdam Book Publishing, Chinese Medicinal Dictionary, Vol . 7 , pp3282-3287, 1997

[Document 2] Korean Patent Registration No. 10-0842022

[Reference 3] Yu, L., Perret, J., Davy, D., Wilson, J., & Melby, CL. Antioxidant properties of cereal products. Journal of Food Science, 67 , pp 2600.2603 (2002)

 Drapper HH, Hadley M. Malondialdehyde determination as index of lipid peroxidation. Methods Enzymol. 186: pp421-431 (1990)

Akerboom TP, Sies H. Assay of glutathione, glutathione disulfide, and glutathione mixed disulfides in biological samples. Methods Enzymol. 77: pp 373-382 (1981)

Document 6 Dai, Y. and AI Cederbaum. 1995. Cytotoxicity of acetaminophen in human cytochrome P4502E1-transfected HepG2 cells. J. Pharmacol. Exp. Ther. 273: pp1497-1505)

Document 7 Jamenez-Lpez, J. M. and A. I. Cederbaum. 2003. Green tea polyphenol epigallocatechin-3-gallate protects HepG2 cells against CYP2E1-dependent toxicity. Free radic. Biol. & Med. 36: pp359-370

The liver is a very important organ that is responsible for various metabolism, detoxification, decomposition, synthesis and secretion in our body. First, the liver has the ability to manage energy metabolism, which metabolizes all the nutrients absorbed by food into energy-producing substances that are supplied or stored throughout the body. Second, the liver has the function of synthesizing, storing, and distributing fats such as serum proteins, bile acids, phospholipids and cholesterol of about 2,000 enzymes, albumin and coagulation factors. Third, the liver has the ability to excrete various metabolites into the duodenum through the bile ducts, and the immune function plays an important role in maintaining our lives. Finally, the liver detoxifies and breaks down drugs, toxicants, and alcohol. However, the liver detoxification function is easy to damage the liver cells can cause drug, toxic, alcoholic liver diseases and the like.

Alcoholic liver disease can be divided into alcoholic fatty liver, alcoholic hepatitis, and alcoholic cirrhosis according to the clinical symptoms and usually occur when drinking 60-80 g of alcohol per day for 10 years. Alcoholic fatty liver is caused by the accumulation of cholesterol and triglycerides in the liver cells due to excessive alcohol intake, which can be recovered soon after drinking, but will continue to develop hepatitis if continued drinking. Alcoholic hepatitis is a condition of necrosis and inflammation of hepatocytes and shows various symptoms such as fatigue, loss of appetite, weight loss, jaundice, fever, and right upper abdominal pain. About 40% of patients with alcoholic liver cirrhosis develop alcoholic cirrhosis. . Alcoholic cirrhosis is a condition that is impossible to recover from normal liver, and has various symptoms such as general fatigue, loss of appetite, ascites, esophageal varices, bleeding, hepatic encephalopathy and lethargy. In Esau, 50% of deaths from end-stage liver disease are known to be caused by alcohol.

Therefore, in order to reduce the mortality rate of alcoholic liver disease, alcoholic fatty liver, which is the initial alcoholic liver disease, should be treated appropriately. However, to date, no appropriate therapeutic agent has been developed to treat the disease.

Tuber of Curcuma aromatica Salisb, the ginger family, is dried or dried after removing the bark. In Japan, it is root stem. In China, Cucuma kwangsiensis SG Lee & CF Liang (廣西 莪 朮), Curcuma wenyujin YH Chen & C. Ling (溫 郁金) and Curcuma phaeocaulis Valeton (蓬 莪 朮) Roots of the roots (Curcuma longa Linn), also known as fall turmerics. It has a similar appearance to Curcuma longa Linn, but has smooth features on both sides of the leaf. It is grown and cultivated in South China, India, Okinawa, and Southeast Asia. When it is mixed with alcohol, it is named as yellow gold, and its shape is similar to Azul (莪), and it is called magic because it treats diseases of horses. (Document 1: Kim Chang-min, Shin Min-gyo et al., Jungdam Book Publishing, Chinese Medicine Dictionary, Vol . 7 , pp3282-3287, 1997)

       Turmeric has been recorded since BC and has been used as a dye and food coloring material and is the source of Indian curry. In Japan, turmeric is used as a coloring material for radish, and it is grown as a special crop in Okinawa, Japan, which is known as a longevity village in the world, and is used as a health food.

     On the other hand, Korean Patent Registration No. 10-0842022 (document 2), after cutting the dried turmeric into ethanol-containing extraction tank and extracted it, concentrated under reduced pressure to proceed fermentation sequentially containing a large amount of organic acids beneficial to the human body The turmeric fermentation method for obtaining the turmeric fermentation extract has been disclosed.

    However, none of the above documents discloses or mentions the medical effect of fermented turmeric extract as a therapeutic agent for alcoholic liver disease.

    Therefore, the present inventors fermented the fermented turmeric extract with a bacterium, and the fermented golden extract obtained through a conventional extraction process shows a relatively high scavenging activity in terms of DPPH radical, ABTS radical scavenging ability, and inhibits lipid peracid activity. It has been shown that the alcoholic liver injury model has shown strong potency to inhibit liver damage, and thus, the fermented turmeric extract of the present invention was found to be useful for the treatment and prevention of alcoholic liver disease, thereby completing the present invention.

In order to accomplish the above object, the present invention provides a pharmaceutical composition for the prevention and treatment of alcoholic liver disease containing fermented turmeric extract as an active ingredient.

The fermented turmeric as defined herein is dried Rhesus bacteria (Aspergillus genus fungus), preferably, Rhesus bacillus Aspergillus oryzae , sulfuric acid strains in turmeric samples that have been dried and sterilized 0.120%, preferably 110%, More preferably, it is inoculated at 15% (v / v%), and the relative humidity of the water is 2090%, preferably 4080% at a fermentation temperature of 150 ° C, preferably 1040 ° C, more preferably 2030 ° C. Fermentation obtained through a process comprising the step of fermenting at a fermentation time of 1 hour to 1 week, preferably 2 days to 5 days, and more preferably about 30 to 40 hours at 5070%. Contains turmeric.

Extracts as defined herein can be used in C ₁ to C ₄ lower alcohols such as water, methanol, and ethanol, including purified water, or mixed solvents thereof, preferably water, methanol, 50-100% water and ethanol mixed solvents. Contains extracts.

       Alcoholic liver disease as defined herein includes alcoholic fatty liver, alcoholic hepatitis, alcoholic cirrhosis, preferably alcoholic fatty liver.

Hereinafter, the present invention will be described in detail.

The extract of the present invention can be obtained as follows.

Fermented turmeric extract as defined herein is a step 1: preparing dried turmeric by sterilizing turmeric powder obtained through washing, cutting, drying and pulverizing with water flowing through the turmeric, and is preferably dried (fungus of the genus Aspergillus). Preferably, the coarse turmeric sample obtained in the first step of Rhodiphyte bacterium Aspergillus oryzae is inoculated with 0.120%, preferably, 110%, more preferably 15% (v / v%) of the yellow rice strain, Fermentation temperature 150 ° C, preferably 1040 ° C, more preferably at 2030 ° C 2090% relative humidity with water, preferably 4080%, more preferably 5070%, fermentation time 1 hour to 1 week Preferably, a second step of fermentation for 2 to 5 days, more preferably for about 30 to 40 hours; About 1 to 30 times the weight of the fermented turmeric sample obtained in step 2, preferably about 5 to 15 times the water, C ₁ to C ₄ lower alcohol or a mixed solvent thereof, preferably water or ethanol More preferably, water or 50 to 100% ethanol as the extraction solvent, about 1 to 24 hours, preferably about 2 to 5 hours at an extraction temperature of 0 ℃ to 350 ℃, preferably 50 ℃ to 300 ℃ Fermentation of the present invention through a third step process of filtration, concentration under reduced pressure and drying the extract obtained after hot water extraction, heat extraction, preferably 1 to 10 times, preferably 2 to 7 times by repeated extraction by hot water extraction method Turmeric extract can be obtained.

Accordingly, the present invention provides a pharmaceutical composition for the prevention and treatment of alcoholic liver disease containing the extract obtained by the above production method as an active ingredient.

The composition for the prevention and treatment of alcoholic liver disease of the present invention comprises 0.1 to 50% by weight of the extract based on the total weight of the composition.

However, the composition is not limited thereto, and may vary depending on the condition of the patient, the type of disease, and the progress of the disease.

The composition comprising the extract of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

Compositions comprising extracts according to the invention are formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterile injectable solutions, respectively, according to conventional methods. The carriers, excipients and diluents that may be used in the composition comprising the extract of the present invention may also include 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 have. 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 in the extract. ) Or lactose, gelatin and the like are mixed. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . 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.

Preferred dosages of the extracts of the present invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the desired effect, the extract of the present invention is preferably administered at 0.01 mg / kg to 10 g / kg, preferably 1 mg / kg to 1 g / kg per day. The administration may be carried out once a day or divided into several doses. Accordingly, the dosage is not limited in any way to the scope of the present invention.

The composition of the present invention may be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration can be expected, for example by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

The present invention also provides a health functional food for the prevention and improvement of alcoholic liver disease containing the fermented turmeric extract as an active ingredient.

      Accordingly, the present invention also provides a food and food additive containing the fermented turmeric extract as an active ingredient having an effect of preventing and improving alcoholic liver disease.

      The composition comprising the extract of the present invention may be used in various ways, such as drugs, foods and drinks for the prevention and improvement of fatty liver disease. Foods to which the extract of the present invention may be added include, for example, various foods, beverages, gums, teas, vitamin complexes, health supplements, and the like, in the form of powders, granules, tablets, capsules, or beverages. Can be used.

Since the extract of the present invention has little toxicity and side effects, it is a drug that can be used safely even when taken for a long time for the purpose of prevention.

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

In addition to containing the extract as an essential ingredient in the indicated ratio, the health beverage composition of the present invention is not particularly limited in the liquid component and may contain various flavors or natural carbohydrates as additional ingredients, as in general beverages. Examples of the above-mentioned natural carbohydrates include 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 Sugar, 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 its Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, 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 in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

As described above, the fermented turmeric extract of the present invention not only exhibited relatively high scavenging activity in terms of DPPH radical and ABTS radical scavenging ability, but also showed lipid peracid inhibitory activity, and exhibited potent liver damage inhibiting effect in alcoholic liver damage model. In addition, the fermented turmeric extract of the present invention can be usefully used as a composition for the treatment and prevention of alcoholic liver damage.

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples.

However, the following Examples and Experimental Examples are only illustrative of the present invention, and the content of the present invention is not limited to the following Examples and Experimental Examples.

Example 1 Preparation of Fermented Turmeric Extract

1-1. Preparation of Turmeric Ferment

2006 was used for cutting and dried turmeric dry powder 100kg ground by pulverization after the turmeric purchased from the magnitude turmeric Farming Association based in Jindo jisanmyeon as a raw material, the fermentation strain is a hwanggukgyun (hwanggukgyun Aspergillus oryzae purchased from Chungmu fermentation Purified by Chungmu fermentation, the extract was prepared under four different conditions using different extraction solvents.

Fermented turmeric extract as defined herein is prepared in the dried state of Kookgi ( Aspergillus oryzae purchased from Chungmu fermentation, CF1001), and the turmeric is cut by drying the surface after washing three times with running water through Jinyang cultivation 5 mm or less). The turmeric which passed through 100mesh sieve after drying for 24 hours (less than 5% of water content) with a heat dryer (instead of pores), followed by the process of 1st grinding (adjustment), 2nd grinding (milling), and 3rd grinding (dusting). The powder was airtightly packed and stored at room temperature to prepare.

   The prepared turmeric powder was sterilized using a solid fermenter (3ton, manufactured by Innobio) owned by Gyeongbuk Bio Industrial Research Institute located in Andong, Gyeongbuk. (Sterilization conditions 121 ℃, 1.0 atm, 20min).

   When the sterilization of the turmeric powder is completed, the fermentation tank was inoculated with sulfuric acid strain 2% (v / v%?) Compared to the powder and the temperature was 25 ° C, 60% moisture, fermentation time 36 hours.

1-2. Preparation of Turmeric Fermented Water Extract (FCH)

  0.5 L of purified water was added to 25 g of the fermented turmeric obtained in step 1-1, and the extract was heated and extracted with hot water at 250 ° C. for 3 hours, and the filtrate was concentrated with a vacuum concentrator (Hanshin, HS-2000), and then dried. 23.8 g of fermented water heated extract dried product were obtained and used in the following experiment. (Hereinafter referred to as FCH).

1-3. Preparation of Turmeric Fermented Water Extract (FCC)

  0.5 g of purified water was added to 25 g of the fermented turmeric obtained in step 1-1, and the mixture was extracted for 3 hours at room temperature. The filtrate was concentrated with a vacuum concentrator (Hanshin, HS-2000), and dried to fermented turmeric. 17.4 g of room temperature extraction dried product was obtained and used in the following experiment. (Hereinafter referred to as "FCC").

1-4. Preparation of Turmeric Fermented 80% Ethanol Extract (FCE)

  0.5 g of EtOH 80% was added to 25 g of the fermented turmeric obtained in step 1-1, and the mixture was heated and extracted at 250 ° C. for 3 hours, and the filtrate was concentrated with a vacuum concentrator (Hanshin, HS-2000) and dried. 10.0 g of turmeric fermented water extract dried product was obtained and used in the following experiment. (Hereinafter referred to as "FCE").

1-5. Preparation of Turmeric Fermented Methanol Extract (FCM)

  0.5 g of MeOH was added to 25 g of the fermented turmeric obtained in step 1-1, followed by heat extraction at 250 ° C. for 3 hours, and the filtrate was concentrated in a vacuum condenser (Hanshin, HS-2000) and dried to ferment turmeric. 6.2 g of water extract dried material was obtained and used in the following experiment (hereinafter referred to as 'FCM').

Experimental Example 1. Experiment of radical scavenging activity using DPPH (2,2-diphenyl-1-picrylhydrazyl) reagent

    In order to confirm the radical scavenging ability using the DPPH (2,2-diphenyl-1-picrylhydrazyl) reagent of the fermented turmeric extracts of the present invention obtained in the above examples, the experiments described in the literature were applied as follows (Document 3 Yu, L., Perret, J., Davy, D., Wilson, J., & Melby, CL (2002) .Antioxidant properties of cereal products.Journal of Food Science, 67, 2600.2603.)

    In order to measure the electron donating transfer capacity of the sample according to the turmeric extract obtained in the above example, radical scavenging ability experiment using DPPH (2,2-diphenyl-1-picrylhydrazyl) reagent was performed as follows.

    In 20 μl of freeze-dried fermented turmeric extract (FCH and FCC dissolved in distilled water, FCM, FCE dissolved in ethanol, 0, 1000, 2000, 3000, 4000, 5000, 10000, 15000, 20,000 μg / ml Each prepared at a concentration) and then adding 180 μl of a 0.25 mM DPPH (2,2-diphenyl-1-picrylhydrazyl) solution (manufactured by Sigma, D9132) and reacting for 30 minutes at 37 ° C. (BIO-TEK manufacturer, 1010-1) and DPPH radical scavenging ability was investigated by measuring the degree of absorbance reduction with the control group.

    DPPH is itself a very stable free radical, a purple compound that exhibits a characteristic light absorption at 517 nm, and it is stable in alcohol and the like and decolorized by a protein-radical scavenger during the antioxidant mechanism. It can also be easily observed.

As shown in Table 1, fermented turmeric methanol extract (FCM) and ethanol extract ( FCE) showed relatively high scavenging activity in the radical scavenging activity using DPPH (2,2-diphenyl-1-picrylhydrazyl) reagent. Indicated.

Extract SC ₅₀ (μg / ml) FCH 301.3 ± 17.1 ^b FCC 400.9 ± 21.4 ^c FCE 114.6 ± 14.7 ^a FCM 121.7 ± 3.0 ^a DPPH radicals (SC ₅₀ values) are expressed as the amount of individual extract (μg) that eliminates DPPH radicals by 50%. Data are expressed as mean ± SD. Different letters (a, b ,, c) in the column mean statistically different by Ducan's multiple range test ( p <0.05).

Experimental Example 2 Radical Scavenging Activity Using ABTS Reagent

       Experiments disclosed in the literature as follows to determine the radical scavenging ability of the fermented turmeric extracts of the present invention obtained in the above Example using ABTS (2,2'-azinbis (3-ethyl-benzothiazoline-6-sulfonic acid)) reagent The experiment was conducted by applying the method (Document 4: Drapper HH, Hadley M. Malondialdehyde determination as index of lipid peroxidation.Methods Enzymol. 186: 421-431 (1990))

     Radical scavenging activity was performed using ABTS (2,2'-azinbis (3-ethyl-benzothiazoline-6-sulfonic acid)) reagent to determine the electron donating transfer capacity of each sample of turmeric extract obtained in the above example.

     When ABTS and potassium persulfate are mixed and placed in the dark, ABTS + is produced. The cation is removed by reacting with the antioxidants of the extract, so that the characteristic blue-green color is discolored and its absorbance can be measured by measuring its antioxidant capacity.

     Dissolve in 7 mM ABTS solution (phosphate buffer; purchased from GIBCO, 563383), BIO BASIC manufacturer, NBPJ17C) and 100 mM potassium persulphate (potassium buffer (GIBCO manufacturer, 563383), Aldrich manufacturer, 216224)). After mixing and reacting in the dark for about 24 hours, the absorbance was diluted to 1.0 to 1.2 at 734 nm.

      10 μl of the sample was added to 390 μl of the diluted solution and mixed well, and the absorbance (BIO-TEK 1010-1) was measured at 734 nm.

     ABTS is a relatively stable free radical and is widely used to detect antioxidant activity in conjunction with the DPPH method. In addition, the antioxidant activity by this method is applicable to the measurement of lipophilic or hydrophilic antioxidants by inhibiting or scavenging the ABTS radicals.

As shown in Table 2, fermented turmeric methanol extract (FCM) and ethanol extract ( FCE) showed relatively high ABTS radical scavenging ability.

Extract SC ₅₀ (μg / ml) FCH 240.2 ± 11.3 ^c FCC 363.9 ± 5.6 ^d FCE 83.1 ± 21.7 ^b FCM 42.76 ± 37.1 ^a ABTS cationic radicals SC ₅₀ radicals (SC ₅₀ values) are expressed as the amount of individual extract (μg) that eliminates ABTS radicals by 50%. Data are expressed as mean ± SD. Different letters (a, b ,, c) in the column mean statistically different by Ducan's multiple range test ( p <0.05).

Experimental Example 3. Lipid Peroxidation Inhibitory Activity

       In order to confirm the lipid peroxidation inhibitory activity of the fermented turmeric extracts of the present invention obtained in the above example, the experiments were modified as follows. (Reference 5: Akerboom TP, Sies H. Assay of glutathione, glutathione disulfide, and glutathione mixed disulfides in biological samples.Methods Enzymol. 77: 373-382 (1981))

       Linolenic acid emulsion by mixing 0.1 ml of linoleic acid (Linoleic acid, Sigma, L1376), 0.2 ml of Tween (JUNSEI, 8L4006) 80, 19.7 ml of distilled water to measure the electron donating transfer capacity of the sample for each turmeric extract obtained in the above example Using linoleic acid emulsion, Lin's method was performed to confirm the lipid peroxidation inhibitory ability.

0.2 ml of 0.01% FeSO _4, 0.2 ml of 0.56 mM H ₂ O ₂ , and 0.4 ml of the sample were mixed in 1 ml of Linoleic acid emulsion prepared above and left at 37 ° C. for 14 hours. 0.2 ml of 0.4% BHT (2, 6-Di-tert-butyl-4-methylphenol, manufactured by Sigma, B1378), 0.2 ml of 4% trichloroacetic acid (Sigma T9159), 0.8% TBA (4, 6-Dihydroxy-2 mercaptopyrimidine 98%, Wako Co., Ltd., 202-00902) 2 ml were added and mixed well and then boiled at 100 ° C. for 10 minutes. After cooling for 5 minutes, centrifugation at 3000 rpm for 20 minutes, absorbing only the supernatant, the absorbance at 532 nm was measured using an instrument (BIO-TEK, 1010-1).

As shown in Table 3, fermented turmeric methanol extract (FCM) and ethanol extract ( FCE) showed relatively higher lipid peroxidation inhibitory activity than cold and hot water extracts.

Extract Inhibition (%) FCH 13.85 ± 3.43 ^b FCC 7.93 ± 5.41 ^c FCE 71.87 ± 3.43 ^a FCM 76.46 ± 1.32 ^a Sample final concentration is 180 μg / ml. Data are expressed as mean ± SD. Different letters (a, b ,, c) in the column mean statistically different by Ducan's multiple range test ( p <0.05).

Experimental Example 4. Cytotoxicity Test

In order to confirm the cytotoxicity of the fermented turmeric extracts of the present invention obtained in the above example, it was measured by modifying the method such as Rochem. (Document 6: Dai, Y. and AI Cederbaum. 1995. Cytotoxicity of acetaminophen in human cytochrome P4502E1-transfected HepG2 cells.J. Pharmacol.Exp.Ther . 273: 1497-1505)

HepG2 / 2E1 cell line of 2 × 10 ⁵ cells / well in a 24 wells plate (293GPG cell number, San Jose CA USA) to measure the electron donation delivery capacity of the turmeric extract for each sample obtained in the above example Cells were infected with HepG2 (ATCC, purchased from Manassas VA USA), and then medium (Minimum Essential Medium (MEM), Hyclone, 01486) was used for 24 hours.

       Remove the medium after the primary culture and the medium containing 3% serum (Minimum Essential Medium (MEM), Hyclone, 01486), fetal bovine serum, where to buy Hyclone, KTJ32092 (FBS) ) 1 ml was dispensed and the reculture was repeated for 5 days. After culture, on day 7, 0.25 ml of XTT-PMS solution (1 mg XTT (XTT Sodium Cell Culture Test, Sigma, X4626) without phenol red) and 10 g PMS (phenazine methosulfate, Sigma manufacturer) in each well , P9625) / mL of MEM (Minimum Essential Medium (MEM), Hyclone, 01486) mixed solution) was added and incubated again for 2 hours. The cytotoxicity of the cells was measured for absorbance at 450 nm using a microplate reader (BIO-TEK, 1010-1) for the formation of formazan (BIO-TEK, 1010-1). It was measured by.

As a result of the experiment, as shown in Figures 1 to 2, it was confirmed that the fermented turmeric cold water extract (FCC) and hot water extract ( FCH) of the present invention is relatively cytotoxic.

Experimental Example 5. Experimental Inhibition of Alcoholic Liver Injury

       The inhibitory effect of alcoholic liver damage of the fermented turmeric extracts of the present invention obtained in the above example was measured by applying the method described in the literature as follows. (Document 7: Jamenez-Lpez, JM and AI Cederbaum. 2003. Green tea polyphenol epigallocatechin-3-gallate protects HepG2 cells against CYP2E1-dependent toxicity.Free radic. Biol. & Med. 36: 359-370)

In order to measure the electron donor delivery ability of the sample for each turmeric extract obtained in the above example, the hepatoprotective activity of the sample was determined by using HepG2 / 2E1 cell line (a cell line ((293GPG cell line number, purchased by San Jose CA USA)) HepG2 (ATCC, Manassas VA). 1 ml MEM (Minimum Essential Medium (MEM), Hyclone, 01486) was dispensed in 24 well plates at 5 × 10 ⁴ cells / well and incubated for 16 hours.

After 16 hours of incubation, the medium (Minimum Essential Medium (MEM)) is removed and each well contains a sample and 300 mM EtOH. Medium containing 3% serum (Minimum Essential Medium (MEM), Hyclone, 01486) was added and reacted in an incubator at 37 ° C. for 24 hours. This process was repeated for 5 days, and on the 7th day, the medium containing the sample and ethanol was removed and the XTT-PMS solution (1 mg XTT (XTT Sodium Cell Culture Test, manufactured by Sigma, X4626) and 10 g PMS ( phenazine methosulfate (manufactured by Sigma, P9625) / mL was added and reacted for 2 hours.The cytotoxicity of the cells was determined by the microplate reader (BIO-TEK, 1010-1). Absorbance at 450 nm was measured using an instrument (BIO-TEK, 1010-1.) The negative control was cultured instead of the sample and only 300 mM ethanol was treated as the positive control.

 As shown in FIG. 5, the fermented turmeric cold water extract (FCC) showed a stronger protective effect against hepatocellular damage caused by alcohol than other extracts.

1 is a diagram showing the results of cytotoxicity experiments in HepG2 / 2E1 cells of fermented turmeric hydrothermal extract (FCH) (expressing 100% cell viability in the control group. Data is expressed as mean ± SD. (*) At the top is statistically (student t-test; p <0.05); shows the results of the 0 toxicity test (100% cell survival rate in the control group. Data is mean ± SD. (*) At the top of the bar is statistically different from the control (student t-test; p <0.05);

Figure 3 is a diagram showing the results of cytotoxicity experiments in HepG2 / 2E1 cells of fermented turmeric ethanol extract (FCE) (indicated cell survival rate in the control group as 100%. Data is expressed as mean ± SD. (*) At the top is statistically different from the control (student t-test; p <0.05));

Figure 4 is a diagram showing the results of cytotoxicity experiments in HepG2 / 2E1 cells of fermented turmeric methanol extract (FCM) (control cell survival rate in 100%. Data is expressed as mean ± SD. (*) At the top is statistically different from the control (student t-test; p <0.05));

Figure 5 shows the results of the protective effect of liver fermented turmeric extracts in HepG2 / 2E1 cells (expressed as 100% cell survival in the control group. Data is expressed as mean ± SD. *) Is statistically different from the control group (Ducan's multiple range test ( p <0.05), (#) at the top of the bar is statistically different from the control group (Ducan's multiple range test ( p <0.05)) FCH: Hot water extract from F. turmeric, FCC: Cold water extract from F. turmeric, FCE: Ethanol extract from F. turmeric, FCM: Methanol extract from F. turmeric)

Claims (10)

Pharmaceutical composition for the prevention and treatment of alcoholic liver disease containing fermented turmeric extract as an active ingredient. The pharmaceutical composition according to claim 1, wherein the fermented turmeric is turmeric fermented with dried Rhesus bacteria (fungus Aspergillus). According to claim 2, The fermentation turmeric is inoculated with dried sulfur bacteria (fungus of the Aspergillus genus) in a dry turmeric sample, 0.120% (v / v%) compared to the dry powder turmeric samples, fermentation temperature A pharmaceutical composition which is a fermented turmeric obtained through a process comprising fermenting at 150 ° C. with a relative humidity of 2090% for 1 hour to 1 week. The pharmaceutical composition of claim 1, wherein the extract is an extract available in C ₁ to C ₄ lower alcohols such as water containing purified water, methanol, ethanol and the like, or a mixed solvent thereof. The pharmaceutical composition of claim 4, wherein the extract is an extract available in water, methanol, 50-100% water and ethanol mixed solvent. According to claim 1, wherein the alcoholic liver disease is alcoholic fatty liver, alcoholic hepatitis, Or a pharmaceutical composition that is alcoholic cirrhosis. Health functional food for the prevention and improvement of alcoholic liver disease containing fermented turmeric extract as an active ingredient. A dietary supplement according to claim 7 in the form of a powder, granule, tablet, capsule or beverage. Food and food additives containing the fermented turmeric extract having an effect of preventing and improving alcoholic liver disease as an active ingredient. The food according to claim 9, which is a variety of foods, beverages, gums, teas, vitamin complexes, or dietary supplements of candies.

Images