KR20180085204A - Composition for prevention and treatment of liver disease or obesity comprising Rheum undulatum Linne extract and Glycyrriza uralensis Fischer extract as active ingredients - Google Patents

Abstract

The present invention relates to a composition for prevention and treatment of liver disease or obesity comprising extracts of Rheum rhabarbarum and Glycyrrhiza uralensis as active ingredients. Since it was confirmed that the composition of the present invention has excellent cytoprotective effects against oxidative damage and liver protection and obesity inhibiting effects, the composition can be used as a pharmaceutical composition or a functional food composition for prevention and treatment of the liver disease or obesity.

Description

[0001] The present invention relates to a composition for prevention and treatment of liver disease or obesity comprising an extract of rhubarb and licorice as an active ingredient,

The present invention relates to a composition for preventing and treating liver disease or obesity, which comprises rhubarb and licorice extract as an active ingredient.

Reactive Oxygen Species (ROS) is a reductive metabolite of oxygen that is produced by the normal metabolism of cells. When the balance of the intracellular metabolism is broken due to factors such as stress, drugs, environmental pollutants, and bacterial infection, . Oxidative stress is an imbalance between the production of reactive oxygen species and the antioxidant reaction that removes it, increasing reactive oxygen species in the cell and damaging it by reacting with DNA, protein, and lipid. It causes cancer, arteriosclerosis, Diabetes, and neurodegeneration. Therefore, various antioxidants that can treat these diseases by increasing intracellular antioxidant function have been actively searched for and investigated for its action.

Excessively increased reactive oxygen species in the cell liberate arachidonic acid (AA) by phosphorylating phospholipids and fatty acids in the cell membrane through the activation of phospholipase. The released AA is a proinflammatory mediator, inducing apoptosis by liberating cytochrome-c from cytoplasmic Ca2 + by sphingomyelinase and mitochondrial membrane potential through mitochondrial membrane potential, and accelerating cellular damage when iron acts as a catalyst. When hepatocyte apoptosis continues, liver fibrosis causes hepatic cirrhosis. Although damaged liver tissue is normally restored by glue fibers, abnormal restoration of the extra cellular matrix (ECM) protein accumulates and fibrosis may occur. Continuous hepatic tissue damage and fibrosis cause cirrhosis. Therefore, the oxidative stress model using AA and iron in hepatocytes can be a model of good cytological studies for screening antioxidants that can treat liver disease.

Rheum undulatum Linne (Rheum undulatum Linne) is a medicinal herb widely distributed in Asia. It is a perennial plant belonging to Polygonaceae. It has been used in oriental medicine for the treatment of peritonitis, enteritis, gallstone disease, chronic diarrhea, jaundice and gallstone disease. Thrombosis, and the like have been reported. However, there is no research on the hepatoprotective effect.

Licorice (Glycyrrhiza uralensis Fischer) is a perennial plant belonging to the genus Fabeceae. It lives in various places such as Korea, China and Japan. It has an effect of clearing the action of other drugs and is used as an important medicinal herb in oriental medicine. Licorice has been reported to reduce blood sugar and abdominal fat, and antidepressant and antimicrobial efficacy.

Korean Patent No. 10-0310979

 Ko HL, Jegal KH, Song SY, Kim NE, Kang J, Byun SH, et al., Water Extract of Rosa laevigata Michx. Protects Hepatocytes from Arachidonic Acid and Iron-mediated Oxidative Stress. The Korean Journal of Herbology, 2015; 30 (6): 7-15.  Bats R, Brenner, Liver fibrosis. The Journal of Clinical Investigation, 2005; 115 (2): 209-18.  Park S-M, Lee G-W, Cho Y-H, Effect of Rheum undulatum extract on antioxidant activity and matrix metalloproteinase-1 in human skin fibroblasts. Journal of Life Science, 2008; 18 (12): 1700-4.  Song J-H, Yang T-C, Chang KW, Han SK, Yi HK, Jeon J-G, In vitro anti-cariogenic activity of dichloromethane fraction from Rheum undulatum L. root. Archives of pharmacal research, 2006; 29 (6): 490-6.

It is an object of the present invention to provide a pharmaceutical composition for protecting and preventing liver damage containing rhubarb and licorice extract as an active ingredient.

Another object of the present invention is to provide a functional food composition for liver protection containing rhubarb and licorice extract as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for the prevention and treatment of fatty liver or obesity containing rhubarb and licorice extract as an active ingredient.

Still another object of the present invention is to provide a functional food composition for prevention and improvement of fatty liver or obesity containing rhubarb and licorice extract as an active ingredient.

In order to achieve the above object, the present invention provides a pharmaceutical composition for protecting and preventing liver damage, which comprises an extract of Rheum undulatum Linne and a licorice extract (Glycyrrhiza uralensis Fischer) as an active ingredient.

In one embodiment of the present invention, the extract may be a hot-water extract of rhubarb and licorice, respectively.

In one embodiment of the present invention, the rhubarb extract and licorice extract may be mixed at a weight ratio of 1:10.

In one embodiment of the present invention, the composition exhibits liver protective effects against liver damage by oxidative stress. In one embodiment of the present invention, the oxidative stress may be induced by AA (arachidonic acid) or iron (ferric nitrilotriacetic acid), but the present invention is not limited thereto.

In one embodiment of the present invention, the composition exhibits a protective effect on the liver acute liver damage induced by CCl _4.

In one embodiment of the present invention, the composition of the present invention exhibits inhibition of apoptosis, protection of mitochondria, phosphorylation of LKB1 in hepatocytes, activation of AMPK pathway, reduction of serum ALT levels and / or inhibition of hepatic tissue damage and inflammation .

In one embodiment of the present invention, the composition comprises an indicator component of a rhubarb extract and / or a licorice extract as an active ingredient, wherein the indicator component is selected from the group consisting of Sennoside A, Emodin, Chrysophanol, Aloe-emodin, Rhein, and Glycyrrhizin acid, Liquiritigenin and Isoliquiritigenin which are indicator components of licorice extract, preferably Isoliquiritigenin, Emodin and / or Rhein.

The present invention also provides a functional food composition for liver protection comprising an extract of Rheum undulatum Linne and Glycyrrhiza uralensis Fischer as an active ingredient.

In one embodiment of the present invention, the rhubarb extract and licorice extract may be mixed at a weight ratio of 1:10.

In one embodiment of the present invention, the composition exhibits antioxidant activity.

In one embodiment of the present invention, the food composition of the present invention inhibits apoptosis, mitochondrial protection, phosphorylation of LKB1 in hepatocytes, activation of AMPK pathway, reduction of serum ALT levels and / or inhibition of hepatic tissue damage and inflammation .

In one embodiment of the present invention, the composition comprises an indicator component of a rhubarb extract and / or a licorice extract as an active ingredient, wherein the indicator component is selected from the group consisting of Sennoside A, Emodin, Chrysophanol, Aloe-emodin, Rhein, and Glycyrrhizin acid, Liquiritigenin and Isoliquiritigenin which are indicator components of licorice extract, preferably Isoliquiritigenin, Emodin and / or Rhein.

The present invention also provides a pharmaceutical composition for prevention and treatment of fatty liver or obesity comprising the extract of Rheum undulatum Linne and Glycyrrhiza uralensis Fischer as an active ingredient.

In addition, the present invention provides a functional food composition for preventing or alleviating fatty liver or obesity containing the extract of Rheum undulatum Linne and Glycyrrhiza uralensis Fischer as an active ingredient.

Compositions containing rhubarb and licorice extract of the present invention exhibited a oxidative stress-induced HepG2 cells apoptosis in the in vitro model utilizing inhibitory effects and mitochondrial protection by AA with iron, between acute by CCl ₄ In the in vivo model of the damaged mice, the ALT levels in the serum were decreased, and the effect of inhibiting the damage and inflammation in the liver was exhibited. In addition, high fat diet-induced fatty liver models did not affect feed intake but showed weight loss and serum triglyceride reduction.

These results show that the present invention, in which rhubarb and licorice extracts are mixed, shows that the composition has cytoprotective effect, hepatoprotective effect and obesity inhibitory effect against oxidative damage, so that the composition of the present invention is useful for preventing or treating liver disease or obesity A pharmaceutical composition or a functional food composition.

FIG. 1 shows the results of experiments on the protective effects of AA and iron-induced cell death of R. undulatum extract (RUE) and G. uralensis extract (GUE) in HepG2 cells. (A) Results of cell viability measurement. (Data represent the mean ± SE of three replicates). (B) Cell morphology. Optical microscopy observations show changes in cell morphology (magnification 200x). (C) Immunoblot analysis of proteins involved in apoptosis. Immunoblot analysis of procaspase 3, poly ADP-ribose polymerase (PARP), B-cell lymphoma-extra large (Bcl-xL) and β-actin was performed on HepG2 cell lysates. HepG2 cells were incubated with 10 μg / ml RUE and 100 μg / ml GUE for 1 hour, followed by 30 μM AA for 12 hours and then 5 μM iron for 2 hours. The statistical significance of the deviation between the experimental group and the control (vehicle treated group) (** ρ <0.01) or between the RUE and GUE treated groups (# ρ <0.05 or ## ρ <0.01) was determined.
Figure 2 shows the results of evaluating the effects of RUE and GUE on mitochondrial dysfunction and oxidative stress.
(A) Mitochondrial membrane permeability (MMP). 1C, HepG2 cells were treated with 0.05 μg / ml of rhodamine 123 for 1 hour, and the intensity of rhodamine 123 was measured by flow cytometry. The RN1 fraction represents the cell population.
(B) RN1 fraction. Data represent the mean ± SE of three replicate experiments. The statistical significance of the deviation between the experimental group and the control (vehicle treated group) (** ρ <0.01) or between the AA and iron treated groups (## ρ <0.01) was determined.
(C) Cellular ROS production. Cellular ROS production was measured by dichlorofluorescin (DCF) fluorescence. RUE and GUE treatment reduced AA and iron-induced ROS production.
FIG. 3 shows the results of experiments on the effect of RUE and GUE on phosphorylation of LKB1. Immunoblot analysis of p-LKB1 and β-actin in lysates of HepG2 (A), AML 12 (B) and SKHep 1 (C) cells treated with 10 μg / ml RUE and 100 μg / Respectively.
Figure 4 shows the results of an experiment on the effects of RUE and GUE on CCl4-induced liver toxicity in mice.
(A) Serum levels of ALT. Serum levels of ALT were measured with a semi-automated blood chemistry analyzer. Data represent the mean ± SE of three replicate experiments. The statistical significance of the deviation between the experimental group and the control (vehicle treated group) (** ρ <0.01) or the CCl4 treated group (## ρ <0.01) was determined.
(B) Histological analysis. Hepatic tissue sections were stained with Harris hematoxylin and eosin (magnification 200x).
FIG. 5 shows the results of measuring weight gain and serum triglyceride levels.
(A) Effect of RUE and GUE on mouse weight gain. Male C57BL / 6 mice were fed either a normal diet (ND) or a high fat diet (HFD) for 9 weeks. During the last 4 weeks, 10 mg / kg RUE and 100 mg / kg GUE were orally administered three times per week.
(B) Effect of RUE and GUE on serum triglyceride levels. Each treatment group and ND (** p <0.01) or HFD alone (# p <0.05, ## p <0.01).
FIG. 6 shows the results of experiments on the effect of RUE and GUE on fatty liver.
(A) Oil red O staining of the mouse liver section. (B) Oil red O Positive area.
FIG. 7 is a real-time RT PCR assay showing the inhibitory effect of RUE and GUE on fatty liver-related genes. HepG2 cells were treated with 10 [mu] g / ml RUE and 100 [mu] g / ml GUE for 1 hour and treated successively with 10 uM T090 for 12 hours. GAPDH was used as a normalizing reference. The statistical significance of the deviation between the treated and vehicle-treated controls (** ρ <0.01) or between RUE and GUE treated cells (# ρ <0.05 or ## ρ <0.01) was established.
FIG. 8 shows the result of an experiment in which the hepatoprotective effect against liver damage of RUE and GUE was tested with HFD. (A) Harris' hematoxylin and eosin staining of the mouse liver section. (B) Serum AST levels.
9 is a UPLC chromatogram of five marker components of RUE.
(A) UPLC chromatogram of commercial standard compounds. Chromatograms were obtained at 340 nm (a) and 254 nm (b, c, d, e). Sennoside (a), Emodin (b), Chrysophanol (c), Aloe-emodin (d), Rhein (e).
(B) UPLC chromatogram of five marker components of RUE.
FIG. 10 shows UPLC chromatograms of the three marker components of GUE.
(A) UPLC chromatogram of commercial standard compounds. Chromatograms were obtained at 254 nm (A) and 280 nm (B). Glycyrrhizine (a), Liquiritigenin (b), Isoliquiritigenin (c).
FIG. 11 shows the results of experiments on hepatocyte protective effects of Glycyrrhizin acid, Liquiritigenin, Isoliquiritigenin, Sennoside A, Emodin and Rhein among the index components identified from RUE and GUE.

The active oxygen species generated during the metabolism of cells act as mediators such as gene expression and cell proliferation regulation. However, excessive production of reactive oxygen species causes oxidative stress and causes cell damage. Clinically, oxidative stress is associated with diseases such as inflammation, aging, tumors, and apoptosis. The liver, which is the most active metabolite in the human body, is affected by oxidative stress due to metabolites, and excessive oxidative stress in the liver interferes with the expression of enzymes, signal molecules and genes in the liver, And is known to be a major cause of acute interstitial disease. Therefore, researches are being actively conducted to find candidates for liver protection through the discovery of substances that can inhibit toxicity due to oxidative stress.

Rhubarb has been proven in various effects such as antibacterial, anti-inflammation, anti-thrombosis. It is known that chrysophanol, emodin, aloe-emodin, rhein, physcion, sennoside A, B, C, D and E and stilbene compounds rhaponticin, piceid and deoxyrhaponticin are known as components of rhubarb. In particular, piceatannol, a component of rhubarb, has been reported to exhibit antioxidative effects by increased expression of HO-1 through the signal of Nrf2. Licorice has been reported to have various effects such as blood sugar, abdominal fat loss, anti-inflammation, anti-allergic effect. However, the combined effect of rhubarb and licorice on the liver damage induced by oxidative stress has not been reported yet. Therefore, the inventors of the present invention have confirmed the effect of the combination treatment of rhubarb and licorice on AA and iron-induced oxidative stress and CCl4-induced cirrhosis model experimental animals in HepG2 cells.

The protective effects of RUE and GUE on cytotoxicity induced by oxidative stress were investigated by MTT assay. As a result, it was confirmed that RUE and GUE inhibited AA and iron induced cytotoxicity in a dose - And GUE combination treatments showed higher cell viability than the individual treatments. In addition, the cytoprotective effects of RUE and GUE were confirmed by immunoblotting analysis of the expression of procaspase 3, PARP, and Bcl-xL, apoptosis-related proteins. In the AA and iron treatment groups, the expression of procaspase 3, PARP, and Bcl-xL protein was decreased, and RUE and GUE pretreatment effectively inhibited the expression. Caspase 3 is present in the cell as an inactivated form, procaspase 3, but is activated by degradation in response to stimuli such as reactive oxygen species. PARP is also activated by degradation by stimulation of reactive oxygen species. In addition, Bcl-xL, one of the apoptotic defense factors, is present in the outer membrane of mitochondria and is known to control apoptosis by controlling the release of cytochrome-c. Therefore, the combined treatment of RUE and GUE seems to have a cytoprotective effect on apoptosis induced by AA and iron. In addition, the combined treatment of RUE and GUE effectively inhibited the production of reactive oxygen species induced by AA and iron. These results suggest that the combined treatment of RUE and GUE shows cytoprotective effect on hepatocyte apoptosis induced by oxidative stress by inhibiting reactive oxygen species formation and antioxidative effects.

Mitochondria are major subcellular organelles involved in cell survival that produce ATP through oxidative phosphorylation of energy stored in various organic materials in response to external stimuli. Thus, excessive oxidative stress leads to the accumulation of active oxygen species in the cell, resulting in dysfunction through changes in the membrane potential of the mitochondria, thereby activating mitochondrial apoptosis. In the present invention, the measurement of mitochondrial membrane potential by flow cytometry analysis showed that the treatment of AA and iron resulted in a decrease in the mitochondrial membrane potential, and that the RUE and GUE pretreatment decreased AA and iron-induced mitochondrial membrane potential Were statistically significantly inhibited. The protective effect of AA and iron induced mitochondrial dysfunction of RUE and GUE was confirmed by the present invention.

LKB1 is known as a tumor suppressor and has been shown to protect against apoptosis due to cell depletion. LKB1 promotes the activation of AMPK, an energy metabolism regulator, as one of the AMPK upstream kinases, and acetylation of LKB1 in the liver is involved in the intracellular location and activity of LKB1. Therefore, we examined the effect of RUE and GUE on the activation of LKB1 in HepG2 cells, AML12 cells and SKHep1 cells by hepatocyte immunoblotting analysis. Activation of LKB1 was observed in AML12 cells and SKHep1 cells for 10 min in HepG2 cells. Therefore, RUE and GUE are thought to have hepatocyte protective efficacy through activation of LKB1. In HepG2 cells on the basis of efficacy due to the oxidative stress of RUE and GUE in mouse cirrhosis model induced by CCl ₄ it was tested the efficacy of RUE and GUE. CCl ₄ , a typical hepatotoxicity inducer used in the in vivo model, reacts with oxygen in the body to produce CCl ₃ , which causes hepatotoxicity, and attacks the phospholipid of the cell membrane to induce lipid peroxidation. This lipid peroxidation has a toxic effect on major cellular structures including mitochondria, DNA, and cell membrane, resulting in necrosis of hepatocytes. These reactive oxygen species production and apoptosis contribute to various liver diseases such as alcoholic and non-alcoholic fatty liver disease, hemochromatosis and hepatotoxicity. RUE and GUE improved the serologic index of ALT in the CCl ₄ - induced hepatotoxicity model and inhibited the transformation of hepatocytes and infiltration of inflammatory cells, which are histological indicators. This effect was more effective than the single treatment of each extract with the combined treatment of RUE and GUE.

Therefore, we confirmed that RUE and GUE have an effective hepatoprotective effect in the in vivo model through inhibition of hepatic tissue damage and inflammation caused by oxidative stress induced by CCl ₄ .

In addition, RUE and GUE did not affect the feed intake in the high fat diet - induced fatty liver model and showed decreased body weight and serum triglyceride levels in the combined treatment group compared to the single treatment group. In addition, liver tissue showed a decrease in adipocytes in Harris' hematoxylin and eosin and Oil red O staining.

These results demonstrate that the combined treatment of rhubarb and licorice not only has cytoprotective and liver protective effects against oxidative damage in vitro and in vivo models, but also has the effect of preventing or treating obesity. May be an effective drug candidate to protect or prevent or treat obesity.

Accordingly, the present invention can provide a composition for preventing or treating liver disease or obesity comprising rhubarb and licorice extract as an active ingredient.

The rhubarb extract and the licorice extract according to the present invention can be obtained by extracting and isolating from nature using an extraction and separation method known in the art. The 'extract' defined in the present invention can be prepared by extracting rhubarb Or licorice, for example, crude extracts of rhubarb or licorice, polar solvent-soluble extracts or non-polar solvent-soluble extracts. As an appropriate solvent for extracting the extract from the rhubarb or licorice, any organic solvent which is pharmaceutically acceptable may be used, and water or an organic solvent may be used, and for example, purified water, An alcohol having 1 to 4 carbon atoms, acetone, ether, benzene, and the like, including methanol, ethanol, propanol, isopropanol, butanol, Various solvents such as chloroform, ethyl acetate, methylene chloride, hexane and cyclohexane may be used alone or in combination. As the extraction method, any one of the methods such as hot water extraction method, cold extraction method, reflux cooling extraction method, solvent extraction method, steam distillation method, ultrasonic extraction method, elution method and compression method can be selected and used. In addition, the desired extract may be further subjected to a conventional fractionation process or may be purified using a conventional purification method.

There is no limitation on the method for producing the rhubarb or licorice extract of the present invention, and any known method can be used. For example, the rhubarb or licorice extract contained in the composition of the present invention may be prepared in powder form by an additional process such as vacuum distillation or freeze-drying or spray drying, . Further, the primary extract can be further purified by using various chromatographies such as silica gel column chromatography, thin layer chromatography, high performance liquid chromatography and the like, You can get it. Therefore, in the present invention, the rhubarb or licorice extract is a concept including all the extracts, fractions and tablets obtained in each step of extraction, fractionation or purification, their diluted solutions, concentrates or dried products.

The pharmaceutical composition of the present invention may be prepared by using pharmaceutically acceptable and physiologically acceptable adjuvants in addition to the above-mentioned active ingredients. Examples of the adjuvants include excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, A lubricant or a flavoring agent can be used. The pharmaceutical composition may be formulated into a pharmaceutical composition containing at least one pharmaceutically acceptable carrier in addition to the above-described active ingredients for administration. The pharmaceutical form of the pharmaceutical composition may be granules, powders, tablets, coated tablets, capsules, suppositories, liquids, syrups, juices, suspensions, emulsions, drops or injectable solutions. For example, for formulation into tablets or capsules, the active ingredient may be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Also, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included as a mixture. Suitable binders include, but are not limited to, natural sugars such as starch, gelatin, glucose or beta-lactose, natural and synthetic gums such as corn sweeteners, acacia, tracker candles or sodium oleate, sodium stearate, magnesium stearate, sodium Benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum and the like. Acceptable pharmaceutical carriers for compositions that are formulated into a liquid solution include sterile water and sterile water suitable for the living body such as saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, One or more of these components may be mixed and used. If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.

In another aspect, the present invention provides a functional food composition for liver protection or obesity inhibition, which comprises rhubarb and licorice extract as an active ingredient. The functional food composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient as well as ordinary food compositions, in addition to containing the active ingredient, rhubarb and licorice extract. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. The above-described flavors can be advantageously used as natural flavorings (tau martin), stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.). The food composition of the present invention may be formulated in the same manner as the above pharmaceutical composition and used as a functional food or may be added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, meat, chocolates, foods, confectionery, pizza, ram noodles, other noodles, gums, candy, ice cream, alcoholic beverages, vitamin complexes, .

In addition, the food composition may contain various additives such as various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and heavies (cheese, chocolate, etc.), pectic acid, Alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the food composition of the present invention may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks.

The functional food composition of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, and circles for the purpose of preventing and protecting liver damage. In the present invention, the term 'functional food composition' refers to foods manufactured and processed using raw materials or ingredients having useful functions in accordance with Law No. 6727 on Health Functional Foods, Or to obtain a beneficial effect in health use such as physiological action. The health functional foods of the present invention may contain conventional food additives and, unless otherwise specified, whether or not they are suitable as food additives are classified according to the General Rules for Food Additives approved by the Food and Drug Administration, Standards and standards. Examples of the items listed in the above-mentioned 'food additives' include chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; Natural additives such as persimmon extract, licorice extract, crystalline cellulose, high color pigment and guar gum; L-glutamic acid sodium preparations, noodle-added alkalis, preservative preparations, tar coloring preparations and the like. For example, the health functional food in the form of tablets may be prepared by granulating a mixture of an active ingredient of the present invention, such as a rhubarb or licorice root extract, with an excipient, a binder, a disintegrant and other additives in a usual manner, Compression molding, or direct compression molding of the mixture. In addition, the health functional food of the tablet form may contain a mating agent or the like if necessary. The hard capsule of the capsule type health functional food can be prepared by filling a normal hard capsule with a mixture of the active ingredients of the present invention, such as rhubarb or licorice extract, with an excipient such as an excipient, and the soft capsule is prepared by adding rhubarb or licorice extract An excipient and the like may be filled in a capsule base such as gelatin. The soft capsule may contain a plasticizer such as glycerin or sorbitol, a coloring agent, a preservative and the like, if necessary. The ring-shaped health functional food can be prepared by molding a mixture of an effective ingredient of the present invention, such as rhubarb or licorice root extract, an excipient, a binder and a disintegrant, by a conventionally known method and, if necessary, The surface can be coated with a material such as starch, talc or the like. The granular health functional food may be prepared by granulating a mixture of the active ingredient of the present invention, such as rhubarb or licorice root extract, excipient, binder, disintegrant, etc., into granules by a known method. If necessary, And the like.

Hereinafter, the present invention will be described in more detail with reference to Examples. It should be understood, however, that these examples are for illustrative purposes only and are not to be construed as limiting the scope of the present invention.

Experimental Method

1. Reagents

Anti-caspase 3 antibody, anti-poly (ADP-ribose) polymerase (PARP) antibody, anti-B-cell lymphoma extra large (Bcl-xL) antibody, anti-phospho-liver kinase B1 (LKB1) conjugated anti-rabbit IgG antibody and HRP-conjugated anti-mouse IgG antibody were purchased from Cell Signaling Technology (Beverly, Mass., USA) and arachidonic acid (AA), ferric nitrilotriacetic acid (iron) -2-yl) -2,5-diphenyl- tetrazolium bromide (MTT), Dimethyl sulfoxide (DMSO), T0901317 (T090), rhodamine 123 (Rh123), 2 ', 7'-Dichlorodihydrofluorescin diacetate (DCFH 2 -DA), anti-β-actin antibody, carbon tetrachloride (CCl ₄ ), and T0901317 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Liquid ALT reagent sets were purchased from Pointe Scientific (Canton, MI, USA). Cholesterol assay kit and triglyceride colorimetric assay kit were purchased from Cayman (Ann Arbor, MI, USA).

2. Preparation of extract

200 g of rhubarb and 200 g of licorice were purchased from Daewon Pharmacy (Daegu, Korea), filtered, and lyophilized. The recovery rate of rhubarb was 11.33% and the recovery rate of licorice was 15.61%.

3. Cell culture and treatment

HepG2 cells were cultured in 10% Fetal Bovine Serum (FBS, Gibco, USA) supplemented with 10% fetal bovine serum (HepG2 cells, SKHep1 cells, mouse hepatocyte AML12 cells and cervical cancer cells, Hela cells were purchased from the American Type Culture Collection AML12 and Hela cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Welgene, Daegu, Korea) containing 1% normocin (InvivoGen, San Diego, Dulbecco's modified Eagle's medium (DMEM) (Welgene, Daegu, Korea) containing 1% Penicillin / Streptomycin (InvivoGen, San Diego, Calif., USA) And cultured at 37 ° C and 5% CO ₂ . Cells were depleted of FBS for 12 hours, then treated with RUE and GUE for 1 hour before the experiment, 30 μM AA for 12 hours, and 5 μM iron for 2 hours.

4. Measurement of cell viability

MTT assay was performed to investigate cell viability by rhubarb and licorice. HepG2 cells were plated in 48-well plates at a density of 1 × 10 ⁵ cells / well and cultured in confluency at 80-90%. The cells were treated with RUE (3, 10, 30 μg / ml) and GUE (10, 30, 100 μg / ml) for 1 h before culturing in media without FBS for 12 h MTT (0.1 mg / ml) was added for 2 hours and further cultured for 2 hours. Formazan crystals were dissolved by adding 200 μl of dimethylsulfoxide (DMSO) and the cell viability was measured at 570 nm using an ELISA microplate reader (Tecan, Research Triangle Park, NC, USA). Cell viability was calculated as a percentage of the control.

5. Measurement of intracellular reactive oxygen species

DFCH2-DA staining was performed to measure intracellular reactive oxygen species production. DFCH2-DA is a fluorescent dye that is converted to a non-fluorescent dye by intracellular esterase. This non-fluorescent dye is oxidized to DCF by reactive oxygen species and emits fluorescence (Rastogi RP, Singh SP, Hader DP, Sinha RP, Detection of reactive oxygen species (ROS) dichlorodihydrofluorescein diacetate in the cyanobacterium Anabaena variabilis PCC 7937 Biochemical and biophysical research communications, 2010; 397 (3):. 603-7). HepG2 cells were plated in 2 × 10 ⁴ cells on a Black 96-well plate and cultured in 80-90% confluency. After 12 hours of depletion, 10 μg / ml RUE and 100 μg / ml GUE were treated 1 hour before. After 12 hours of 30 μM AA treatment, the cells were exposed to 5 μM iron for 2 hours. The cells were stained with 10 μM DCFH2-DA for 30 min to measure reactive oxygen species at an excitation wavelength of 460 nm and an emission wavelength of 530 nm.

6. Western blot analysis

HepG2 cells were treated with 1 × 10 ⁵ cells in a 6-well plate, washed twice with phosphate-buffered saline (PBS), and harvested using a scapper. (Sigma, St. Louis, Mo., USA), and the supernatant was removed by centrifugation at 15,000 rpm for 30 min at 4 ° C. Protein was quantified. 30 μg of the same protein was separated by electrophoresis on sodium dodecyl sulfate polyacrylamide gel electrophoreses (SDS-PAGE) and transferred to a nitrocellulose membrane (Thermo Scientific, Rockford, Ill., USA) and reacted with the primary antibody against the target protein. After the secondary antibody treatment, expression of the target protein was confirmed using a chemiluminescent ECL (electrochemiluminescence) kit (Amersham Pharmacia Biotech, Piscataway, USA).

7. PCR analysis

Real-time RT-PCR was performed to measure the expression of FAS and ACC, the fat production-related factors. The cells were divided into 2 × 10 ⁶ cells in a 6-well plate and cultured in 80-90% confluency. Total RNA was extracted according to the protocol of Trizol (Invitrogen, Carlsbad, CA, USA) and cDNA was synthesized using oligo (dT) ₁₆ primer in 1 ug of total RNA and amplified by PCR. Real-time RT-PCR was performed using LightCycler 96 (Roche, Mannheim, Germany). Primer sequences were as follows (Table 1).

8. Mitochondrial membrane potential (MMP) measurement

To measure the changes in mitochondrial membrane potential, rh123 (Sigma-Aldrich, St. Louis, Mo., USA), a membrane permeable cationic fluorescent dye, was used. HepG2 cells were cultured on a 6-well plate and stained with 0.05 μg / ml rh123 for 30 min. Cells were treated with trypsin. 2 x 10 ⁴ cells per sample were resuspended in PBS supplemented with 1% FBS and measured with a flow cytometer (FACSCalibur; BD Bioscience Pharmingen, San Jose, CA, SA).

9. Experimental animals

Five-week-old BALB / c mice weighing 18 g and 6-week-old C57BL / 6 mice weighing 18 g were purchased from Orient Bios Co., Korea, and pre-fed for 7 days. C57BL / 6 mice were fed a 60% fat diet (D12492, Research Diets, New Brunswick, NJ, USA) in water and feed (Lab Diet 5L79, Lab Diet, USA) The medium was maintained at 25 ℃ and 50 ~ 55% humidity for 12 hours.

10. Extract treatment and hepatotoxicity

Using BALB / c mice group were divided into the control group, 10 mg / kg RUE and 100 mg / kg GUE treated, CCl ₄ group, RUE and CCl ₄ administration group, GUE and CCl ₄ administration group, RUE and GUE and CCl ₄ administration group . RUE and GUE were orally administered once a day for 3 days, and 2 hours after the last administration, CCl ₄ was intraperitoneally injected 0.5 ml per 1 kg of body weight. The same amount of saline was orally administered to the control and CCl ₄ administration groups during the entire experimental period.

11. Extract treatment and induction of fatty liver

Using the C57BL / 6 mouse, the experimental group was classified into the control group, 10 mg / kg RUE and 100 mg / kg GUE group, the high fat diet group, the RUE and high fat diet group, the GUE and high fat diet group, the RUE and GUE and the high fat diet group . Fatty liver was induced by high fat diet for 4 weeks and oral administration of 10 mg / kg RUE and 100 mg / kg GUE three times a week for 5 weeks to 4 weeks. In the control and high fat diet groups, the same amount of physiological saline was orally administered during the drug administration period.

12. Serum biochemical test

After the animal was anesthetized with ether, blood was collected from the abdominal vein, left at 37 ° C for 30 minutes, and centrifuged at 5000 x g for 20 minutes. Serum was used for biochemical tests. The ALT measurement was carried out with an absorbance of 340 nm after adding 8 μl of serum to 1.2 ml of the reagent according to the protocol of the liquid ALT reagent set. ALT values were expressed as IU / L. Serum cholesterol was measured according to the protocol of cayman cholesterol assay kit and serum triglyceride was measured according to the protocol of cayman triglyceride assay kit. Cholesterol and triglycerides were expressed in mg / dL.

13. Histopathological examination

CCl ₄ induction cirrhosis model liver tissue and high fat dietary induction fatty acid samples were immediately removed and fixed in 4% formalin. The paraffin was infiltrated through tissue processor and embedding center was used to make the block. A 4 μm-thick section was prepared with a microtome, denaturation of the nuclei in a Harris' hematoxylin solution for 10 min after deparaffinization in xylene. After washing with water, it was decolorized with 1% HCl alcohol, blue color was developed, and 10 seconds cytoplasm was stained with eosin, followed by dehydration and transparency. Frozen section of fatty liver tissue was stained with Oil red O stain for fat monitoring.

14. Analysis of RUE and GUE components

Waters ACQUITY ™ BEH C18 column (1.7 μm, 2.1 × 100) was used for the Waters ACQUITY ™ photodiode array detector (PDA) and HPLC column for high performance liquid chromatography (UPLC) Software used Empower. The sample solution was homogenously mixed with 0.1 g of precisely weighed sample, 10 ml of 30% methanol was added, and sonicated for 1 hour. The test solution was filtered with a membrane filter having a pore size of 0.2 μm or less to prepare a sample solution. The analytical wavelength of the PDA was analyzed at 340 nm for Sennoside A, Emodin, Chrysophanol, Aloe-emodin and Rhein at 254 nm, 254 nm for Glycyrrhizin acid and 280 nm for Liquiritigenin and Isoliquiritigenin. The mobile phase contained acetonitrile and water containing 0.1% Mixed solution was used and analyzed under the following conditions. 2 μl of sample was injected and the flow rate was 0.4 ml / min. As a result of analysis, qualitative confirmation was made by retention time and quantified by peak area method. Table 2 below shows the solvent gradient for the analysis of surface components in RUE and GUE.

15. Statistical processing

All experimental values were based on the results of repeated experiments more than 3 times. The mean difference between the control group and each experimental group was analyzed by ANOVA. P-value <0.05 was regarded as significant level, and it was expressed as mean ± SE value.

Experiment result

1. RUE and Of GUE  AA + iron-induced In apoptosis  Protection against

MTT assay was performed to determine the effective concentration of RUE (3, 10, 30 μg / ml) and GUE (10, 30, 100 μg / ml) in apoptosis induced by AA + iron in HepG2 cells. As a result, it showed an effective cytoprotective effect in combination treatment of 10 μg / ml RUE and 100 μg / ml guine than the single treatment group of RUE and GUE (FIG. 1A). Therefore, in the subsequent experiments, 10 μg / ml RUE and 100 μg / ml GUE were used in combination. The cytoprotective effect of RUE and GUE on oxidative stress induced by AA + iron was observed with an optical microscope. In the AA + iron-treated group, cell membrane damage was observed and cell membrane damage was protected by pretreatment with 10 μg / ml RUE and 100 μg / ml GUE (FIG. 1B). The inhibition of apoptosis induced by AA + iron was confirmed by immunoblotting analysis. In the AA + iron-treated group, the expression of procaspase 3, PARP, and Bcl-xL was decreased and the RUE and GUE-treated groups suppressed the decrease of apoptosis-related protein (Fig. 1C).

2. RUE and GUE mitochondrial  with dysfunction On active oxygen species  Impact

Oxidative stress induced by AA + iron induces apoptosis through mitochondrial membrane dysfunction. To study mitochondrial function of RUE and GUE, HepG2 cells were stained with rh123 and analyzed by flow cytometry. Due to external stimuli, the increase in mitochondrial membrane permeability causes rh123 to be absorbed into the mitochondria and fluoresce. As a result, the number of cells with low fluorescence intensity for rh123 (RN1 fraction) was significantly increased compared with the control group because the AA + iron treatment group induced decrease in mitochondrial cell membrane function and decreased membrane potential and decreased rh123 fluorescence Respectively. RUE and GUE pretreatment significantly reduced the RN1 fraction increased by AA + iron (FIG. 2A). In addition, the effects of DCFH ₂ -DA on the production of reactive oxygen species in the cells were evaluated to observe whether the inhibition of oxidative stress induced by AA + iron was mediated by the cytoprotective effect. AA + iron treatment increased the production of reactive oxygen species in the cell 6 times as compared with the control group, and production of reactive oxygen species was significantly inhibited by RUE and GUE pretreatment (FIG. 2C).

3. RUE and GUE LKB -1 on phosphorylation

AMPK pathway and activation were mediated before AA + iron induced apoptosis protection. Activated AMPK through phosphorylation regulates the activity of various enzymes in the cell through phosphorylation. It is known that LKB1, which is the upper kinase of phosphorylation of AMPK, is required. In this study, immunoblotting analysis was performed to investigate the protective effect of RUE and GUE on oxidative stress induced by AA + iron mediated LKB1 activation. The treatment of RUE 10 μg / ml and GUE 100 μg / ml for 10 minutes to 6 hours resulted in an increase in phosphorylation of LKB1 at 1 hour in HepG2 cells and at 10 minutes in AML12 cells and SKHep1 cells (FIG. 3).

4. RUE and GUE CCl ₄ on  Effect on the Liver Damage Model Induced by

In an animal study, serum ALT was measured to determine the effect of RUE and GUE on liver protection against CCl ₄ induced acute liver injury. In the CCl ₄ group, serum ALT was significantly increased to 549.90 IU / L compared to the control group (4.18 IU / L). In the RUE 10 mg / kg group alone, 347.36 IU / L and GUE 100 mg / kg alone were 146.90 IU / L, and 56.36 IU / L in RUE and GUE combination treatment group (FIG. 4A). Harris' hematoxylin and eosin staining was performed to confirm the liver protective effect of RUE and GUE. In normal control group without CCl ₄ treatment, hepatocytes were arranged well maintaining the normal lobular structure around the central vein, and other histological structures were normal. In CCl ₄ treated group, infiltration of endophytic cytoplasm and inflammatory cells was increased around the cytoplasm and many vacuoles and central veins. Compared with the RUE or GUE alone group, the RUE and GUE combination treatment groups were more relaxed than the CCl ₄ treatment group (FIG. 4B).

5. RUE and GUE  Impact on obesity

A high fat diet model was run for nine weeks to induce fatty liver in C57BL / 6 mice, and the increase in mouse body weight was confirmed (Fig. 5A). During the last 4 weeks, the combined treatment with RUE and GUE had no effect on feed intake but showed weight loss and serum triglyceride reduction. In the high fat dietary group, serum triglyceride was significantly increased to 101.22 mg / dL compared to the normal diet (66.14 mg / dL), 80 mg / dL for the RUE alone treatment and 75.97 mg / dL for the GUE single treatment high fat dietary group , And 71.24 mg / dL in the high-fat diet group administered with RUE and GUE, respectively (FIG. 5B).

6. RUE and GUE  Effect on fatty liver

The fatty liver induced by the high fat dietary model for 9 weeks was observed in the C57BL / 6 mouse for fatty liver induction. As a result of the oil red O staining, an increase in the red fat droplets in the liver tissue was confirmed (FIG. 6A). During the last 4 weeks, red fat droplets of Oil Red O staining were markedly reduced in the RUE and GUE combination treatment groups, and the results were observed to be significantly reduced as a result of quantitative observation (FIG. 6B).

In addition, genetic changes leading to fat biosynthesis were observed in hepatocytes. The expression of fatty acid synthase and acetyl Co-A carboxylase, which is involved in the biosynthesis of fat, was markedly increased when T090, a chemical inducing the biosynthesis of fat through SREBP1c activation, was administered to HepG2 hepatocytes, (Figs. 7A and 7B).

Furthermore, hepatocyte injury induced by H & E staining of hepatic tissue was significantly reduced in the RUE and GUE combination treatment groups (FIG. 8A), and the hematological index through RTA And GUE-treated group (Fig. 8B).

7. RUE and Of GUE  Component analysis

High performance liquid chromatography (UPLC) was used to analyze the index components of the rhubarb and licorice extracts used in this study. In RUE, Sennoside A, Emodin, Chrysophanol, Aloe-emodin, and Rhein were identified as the five indices of rhubarb, and Glycyrrhizin acid, Liquiritigenin and Isoliquiritigenin were identified in GUE (Table 3, 4). Table 3 shows the results of UPLC measurement of the content of five marker components of RUE, and Table 4 shows the results of UPLC measurement of the content of three marker components of GUE.

8. RUE and Of GUE  Hepatocyte protective effect of analyzed components

In RUE, Sennoside A, Emodin, Chrysophanol, Aloe-emodin, and Rhein were identified as the five indices of rhubarb, and Glycyrrhizin acid, Liquiritigenin and Isoliquiritigenin were identified in GUE. Among these components, Glycyrrhizin acid , Liquiritigenin, Isoliquiritigenin, Sennoside A, Emodin, and Rhein. Of these components, Isoliquiritigenin, Emodin, Rhein showed significant results (Fig. 11).

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (14)

A pharmaceutical composition for protecting and preventing liver damage containing an extract of Rheum undulatum Linne and Glycyrrhiza uralensis Fischer as an active ingredient. The method according to claim 1,
Wherein the extract is a hot-water extract of rhubarb and licorice, respectively. The method according to claim 1,
Wherein the rhubarb extract and the licorice extract are mixed at a weight ratio of 1:10. The method according to claim 1,
Wherein the composition exhibits a liver protective effect against liver damage by oxidative stress. 5. The method of claim 4,
Wherein the oxidative stress is induced by AA (arachidonic acid) or iron (ferric nitrilotriacetic acid). The method according to claim 1,
The composition is a composition, characterized in that indicating the protective effects against acute liver liver damage induced by CCl _4. The method according to claim 1,
Wherein said composition exhibits at least one of the effects selected from the group consisting of inhibition of apoptosis, mitochondrial protection, phosphorylation of hepatic LKB1, activation of AMPK pathway, reduction of serum ALT levels, damage to liver tissue and inhibition of inflammation . The method according to claim 1,
Wherein said composition contains isliquiritigenin, rhenin and emodin as active ingredients. A functional food composition for liver protection comprising an extract of Rheum undulatum Linne and a extract of Glycyrrhiza uralensis Fischer as an active ingredient. 10. The method of claim 9,
Wherein the rhubarb extract and the licorice extract are mixed at a weight ratio of 1:10. 10. The method of claim 9,
Wherein the composition exhibits an antioxidative activity. 10. The method of claim 9,
Wherein said composition contains isliquiritigenin, rhenin and emodin as active ingredients. A pharmaceutical composition for prevention and treatment of fatty liver or obesity comprising extract of Rheum undulatum Linne and extract of Glycyrrhiza uralensis Fischer as an active ingredient. A functional food composition for preventing or improving fatty liver or obesity containing an extract of Rheum undulatum Linne and Glycyrrhiza uralensis Fischer as an active ingredient.

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