KR20180083146A - Composition for preventing or treating liver disease comprising pericarpium zanthoxyli extracts - Google Patents

Abstract

The present invention relates to a composition for preventing, treating, or improving liver diseases, containing an akane extract as an active ingredient. The akane extract of the present invention is excellent in effects of protecting hepatocytes through protection of mitochondria and activation of an Nrf2 pathway while showing hepatoprotective functions without causing cytoxicity to normal cells, thereby being used as an active ingredient for functional health foods to prevent liver disease or treat agents for liver disease.

Description

TECHNICAL FIELD The present invention relates to a composition for prevention or treatment of liver disease,

The present invention relates to a composition for preventing or treating liver disease, which comprises an extract of Leucovie chinensis as an active ingredient.

The liver is an important organ responsible for metabolism, detoxification, degradation, synthesis and secretion in the human body. It is located between the digestive system and the systemic circulatory system in the body and functions to defend the whole body from exogenous substances have. Since the in vivo substance entering the living body passes through the liver once, the liver has a higher risk of being exposed to many toxic substances than the other organs in addition to the nutrients. However, when the liver is regenerated, it is recovered to a normal state when there is some damage, but if the damage is sustained, the liver is completely destroyed and the hepatic function is lowered. do. If such liver damage is chronicized, liver fibrosis, cirrhosis, and liver cancer occur regardless of the cause.

The causes of liver damage include stressful chronic fatigue and excessive intake of food or alcohol containing fat, harmful substances such as virus infection, various medicines, and malnutrition.

As a method for treating liver diseases caused by liver damage, dietary therapy and pharmaceutical therapy are widely used, and these two methods are used in combination. In addition, there are silymarin and biphenyl dimethyl dicarboxylate (BDD) as representative treatment for liver disease, but these drugs are also not a fundamental therapeutic agent, Can induce various side effects in the body, and thus it can not be commercialized.

Therefore, it is urgently required to develop a new therapeutic agent for liver disease using natural products which have excellent therapeutic effects to replace conventional therapeutic agents and have no side effects even when administered in large amounts or for a long period of time.

On the other hand, pericarpium zanthoxyli is a species belonging to Z. rutaceae Zanthoxylum, Z. piperitum De Candolle, Z. schinifolium Siebold et Zuccarini or Z. bungeanum Maximowicz ) Refers to ripe fruit bark. It affects menopause, parenchyma, and nerves (肾经). It has the efficacy of Onshan-zhan (cold spring), news (deodorization), dehumidifying tongue (痛 止痛), insecticide, and sea urchin poison. It stimulates appetite and increases gastrointestinal motility Vomiting, diarrhea, and toothache, and it is also used as an insect repellent for roundworms (Korean Medical Dictionary Compilation Committee, Dictionary of Oriental Medicine, Jungdam, pp.1534, 1851 ~ 1852, 1998).

There are several kinds of essential components such as alpha-sanshool, beta-sanshool, sanshoamide, limonin, tannin, hyperin, xanthoxylin and citronellal, phellandren, dipentene and geranial. The pharmacological studies on chunghwa have been reported on the anticancer, anti - obesity and antioxidant effects of Chuncheon extract. Local sagittal paralysis of one of the chunks of sanshool, promotion of gastrointestinal motility and promoting circulation of intestinal blood circulation have been reported. However, the clinic is used mainly for the digestive system prescription such as Dagoon bath, and the research on the liver disease is very insufficient.

Accordingly, the inventors of the present invention evaluated the hepatocyte protective effect against oxidative stress in vitro in vitro and evaluated the hepatoprotective effect in vivo to be used as a material for the treatment and prevention of liver disease, .

Korean Patent No. 10-0864455 Korean Patent Publication No. 10-1383146

It is an object of the present invention to provide a pharmaceutical composition for the prevention or treatment of liver diseases containing an extract of Chenopodium ambrosioides as an active ingredient.

It is another object of the present invention to provide a health functional food for preventing or ameliorating liver disease, which contains an extract of Chuncheoncho as an active ingredient.

In order to achieve the object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating liver diseases, which comprises an extract of Chuncheon mushroom as an active ingredient.

In one embodiment of the invention, the extract is selected from the group consisting of water, C1-C4 alcohol, hexane, ethyl acetate, butylene glycol, propylene glycol, glycerin, acetic acid, ether, chloroform, Can be obtained by using a solvent to be selected.

In one embodiment of the present invention, the extract may be contained in an amount of 0.1 to 50% by weight based on the total weight% of the composition.

In one embodiment of the present invention, the extract exhibits hepatocyte protective activity through Nrf2 pathway activation and mitochondrial protection.

In one embodiment of the present invention, the liver disease may be liver cirrhosis, hepatic fibrosis, acute hepatitis, chronic hepatitis or liver cancer.

Further, the present invention provides a health functional food for preventing or ameliorating liver disease, which comprises an extract of mulberry root as an active ingredient.

In one embodiment of the invention, the extract is selected from the group consisting of water, C1-C4 alcohol, hexane, ethyl acetate, butylene glycol, propylene glycol, glycerin, acetic acid, ether, chloroform, Lt; / RTI > may be obtained using a solvent of choice.

In one embodiment of the present invention, the liver disease may be liver cirrhosis, hepatic fibrosis, acute hepatitis, chronic hepatitis or liver cancer.

In one embodiment of the present invention, the health functional food may be in the form of tablets, capsules, pills, or liquids.

The present invention relates to a composition for preventing or treating hepatopathy comprising an extract of Chenopodiumposis as an active ingredient. The extract of Chenopodium albumolum of the present invention is excellent in hepatocyte protective effect through Nrf2 pathway activation and mitochondrial protecting action, By confirming that it does not cause toxicity to normal cells, there is an effect that it can be used as an effective ingredient of a therapeutic agent for liver disease or health functional food for prevention of liver disease.

1 is a graph showing the results of measurement of the survival rate of hepatic parenchymal cells (HepG2 cells) treated with Pericarpium zanthoxyli water extracts (PZW) of the present invention; (A) Results of MTT assay for cell viability of HepG2 cells treated with PZW alone, (B) Results of cell viability MTT assay of HepG2 cells treated with arachidonic acid (AA) and iron after PZW pretreatment.
FIG. 2 is a graph showing the results of measuring the protective effect of HepG2 cells on methanol extracts of Chuncheon (PZM) and Chenopodium albumalum; (A) Results of cell viability MTT assay of HepG2 cells treated with AA + iron after PZM pretreatment. (B) Results of cell viability MTT assay of HepG2 cells treated with AA + iron after pretreatment of Celsius fruit extract.
FIG. 3 is a graph showing the results of measurement of AA + iron-induced apoptosis inhibitory effect of PZW of the present invention; FIG. (A) Western blot analysis for the expression of PARP and procaspase-3 in HepG2 cells, (B) a graph quantifying the level of PARP-related protein expression by AA + iron or PZW treatment, (C) And the expression level of procaspase-3-related protein according to the present invention.
FIG. 4 is a graph showing the inhibitory effect of AA + iron-induced H ₂ O ₂ production of Chrysanthemum morifolium extract (PZW) of the present invention.
FIG. 5 is a graph showing the ability of the present invention to recover the AA + iron-induced GSH content of PZW.
FIG. 6 is a graph showing the mitochondrial dysfuncion-inhibiting effect induced by AA + iron of Chrysanthemum morifolium extract (PZW) of the present invention.
FIG. 7 is a graph showing the results of measuring the degree of Nrf2 activation by water-soluble extract (PZW) of the present invention; (B) PZW treatment concentration (3, 10, 30, 100 μg / ml) in the western blot analysis of the degree of Nrf2 activation according to (A) PZW 100 μg / (C) Western blot analysis of Nrf2-related antioxidant protein GCLC and NQO1 expression according to PZW 100 μg / ml treatment time (12, 24 h).
FIG. 8 is a graph showing the effect of chrysanthemum water extract (PZW) of the present invention on CCl ₄ -induced liver damage; (A) liver damage index ALT measurement result, and (B) liver damage index AST measurement result.
9 is a diagram showing an H & E staining for histological changes analysis between the damage induced by CCl ₄ according to one thousand seconds water extract (PZW) processing of the present invention; (A) Normal liver tissues pictures, (B) 300 mg / kg of only administering a cross PZW tissue picture, (C) cross-induced liver damage in CCl ₄ tissue picture, (D) CCl ₄ + silymarin 100 mg / kg (E) liver tissue photographs with CCl ₄ + PZW 100 mg / kg, (F) liver tissue photographs with CCl ₄ + PZW 300 mg / kg.
FIG. 10 shows the results of analysis of cleaved caspase-3 and cleaved PARP-positive cells in order to examine the effect of the present invention on the immunological changes of CCl ₄ -induced impairment of PZW; (A) Normal liver tissues pictures, (B) 300 mg / kg of only administering a cross PZW tissue picture, (C) cross-induced liver damage in CCl ₄ tissue picture, (D) CCl ₄ + silymarin 100 mg / kg (E) liver tissue photographs with CCl ₄ + PZW 100 mg / kg, (F) liver tissue photographs with CCl ₄ + PZW 300 mg / kg.
11 is a graph showing the results of analysis of NT and 4-HNE-positive cells in order to examine the effect of the PZW of the present invention on the immunological changes of CCl ₄ induced damage; (A) Normal liver tissues pictures, (B) 300 mg / kg of only administering a cross PZW tissue picture, (C) cross-induced liver damage in CCl ₄ tissue picture, (D) CCl ₄ + silymarin 100 mg / kg (E) liver tissue photographs with CCl ₄ + PZW 100 mg / kg, (F) liver tissue photographs with CCl ₄ + PZW 300 mg / kg.

The present invention is characterized in that it is possible to use chrysanthemum extract as an effective ingredient capable of preventing or treating liver disease.

As described in the prior art, the current therapeutic agents for liver diseases are not fundamentally treated, resulting in insufficient pharmacological efficacy and cause side effects in the body. Therefore, efforts have been made to discover novel liver disease drugs based on natural materials , The present inventors confirmed through experiments that it is possible to use the extract of Chenopodiumposis.

According to one embodiment of the present invention, the herringbone extract of the present invention has no cytotoxicity and is excellent in hepatocyte coagulation effect against oxidative stress (see Examples 2 to 6).

The human body is continuously exposed to reactive oxygen species (ROS), and when the ROS is excessively generated or when the oxidant and the antioxidant are out of balance, the cells or tissues are damaged and oxidative stress is generated do. In response to this oxidative stress, non-enzymatic antioxidants such as glutathione (GSH), carotenoids, flavonoids, and intrinsic antioxidant enzymes such as catalase, superoxidedismutase and the like protect the body from oxidative stress, Lt; / RTI >

According to one embodiment of the present invention, the herringbone extract of the present invention has excellent hepatocyte protective effect through Nrf2 pathway activation and mitochondrial protecting action, and also shows significant hepatoprotective action in animal models (see Examples 7 to 11) ).

Oxidation of fatty acids and lipids stimulates the release of AA and inflammatory mediators, impairing phosphorylation activity and cell signaling. The increase of intracellular AA leads to apoptosis by opening mitochondrial permeability transition pore (mPTP), thereby increasing cytochrome-c release in cytoplasm and increasing the concentration of ceramide to increase cytotoxicity of AA It is known that it happens. After AA treatment, treatment of iron catalyzes oxidation to increase the oxidative stress of the cells, causing malfunction of the mitochondria.

It was confirmed in Example 8 that the activation of the Nrf2 pathway was involved before the cell protection period for apoptosis induced by such oxidative stress.

Modulators that modulate the Nrf2 pathway may be used as therapeutic agents for the treatment of liver disease. Therefore, the inventors of the present invention have experimentally proved that the herb extract of the present invention is excellent in protecting the hepatocyte through Nrf2 pathway activation and mitochondrial protecting action, and thus it has been confirmed that the herb extract can be used as a main material for the treatment of liver disease, Completed.

Accordingly, the present invention is characterized by providing a pharmaceutical composition for preventing or treating liver disease containing an extract of Chenopodiumposis as an active ingredient.

The herb extract according to the present invention may be obtained by extracting and extracting the herb extract for one thousand seconds using the extraction and separation methods known in the art. The 'extract' defined in the present invention may be prepared by using an appropriate solvent It can be extracted from a thousand seconds. Any suitable solvent that can be used to obtain the extract may be any of those accepted in the art, and water or an organic solvent may be used. Examples of the solvent include alcohols having 1 to 4 carbon atoms, acetone, ether, and the like, including purified water, methanol, ethanol, propanol, isopropanol, butanol, Various solvents such as benzene, chloroform, ethyl acetate, methylene chloride, hexane and cyclohexane may be used alone or in combination. But is not limited to.

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 herringbone extract of the present invention, and any known method can be used.

Preferably, water is used as a solvent, and a fresh water extract can be obtained by hot water extraction.

In addition, the pharmaceutical composition of the present invention may be prepared by using pharmaceutically acceptable and physiologically acceptable adjuvants in addition to the above-mentioned effective ingredients. Examples of the adjuvants include excipients, disintegrants, sweeteners, binders, , A lubricant, a lubricant or a flavoring agent.

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 composition may be in the form of 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 solutions 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 can be additionally added and formulated into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Further, it can be suitably formulated according to each disease or ingredient, using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA as an appropriate method in the field.

The appropriate dosage of the pharmaceutical composition of the present invention may be determined by various methods depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate and responsiveness of the patient It can be prescribed.

The pharmaceutical composition of the present invention can be administered orally or parenterally, and when administered parenterally, it can be administered by intravenous injection, subcutaneous injection, muscle injection, intraperitoneal injection, transdermal administration, or the like.

The concentration of the active ingredient contained in the composition of the present invention is determined in consideration of the purpose of treatment, the condition of the patient, the period of time required, the severity of disease, and the like, and is not limited to a specific range of concentration.

In one embodiment of the present invention, the herb extract of the present invention may be contained in an amount of 0.1 to 50% by weight based on the total weight of the composition.

The hepatic diseases which can be treated, prevented or ameliorated by the extract of the present invention include, but are not limited to, liver cirrhosis, liver fibrosis, acute hepatitis, chronic hepatitis or liver cancer.

In particular, the herb extract of the present invention has an effect of protecting liver damage through activation of the Nrf2 pathway.

Among the liver diseases described above, cirrhosis is characterized by morphological features of fibrous tissue proliferation and regenerative nodule formation as a result of extensive destruction of normal liver tissue due to chronic inflammation, and secondly, deformation of liver blood vessels and liver function (Bataller B, Brenner DA, Hepatic Fibrosis. In: Arias IM et al., The liver biology and pathobiology, 5th ed., Wiley-Blackwell, 2009, p.433-452). Symptoms of cirrhosis can be manifested in a wide variety of symptoms, such as spider blood vessels appearing on the skin, abnormal metabolism of the hormones, redness of the palms more than normal, men do not destroy female hormones, resulting in bigger chest and lower sexual function. As the spleen grows larger, it can be touched on the left side, revenge can be swollen, both legs swollen, and extended blood vessels can protrude to the outside of the skin. In addition, jaundice can occur due to liver function deterioration, and personality changes or loss of consciousness due to hepatic coma. If the esophageal variceal hemorrhage is present, the blood may be ejaculated or blackened or stained. (Goldmann DR, Complete home medical guide, DK publishing, 2003, p.643-651).

These liver cirrhosis is a disease that manifests the proliferation and regeneration nodule of the fibrous tissue. It is impossible to return to the normal state, and the treatment for cirrhosis is not to cure but to make full use of the present liver function. In the case of viral cirrhosis, drugs such as peginterferon or antiviral agent can be used to suppress the cause, but there is no therapeutic agent for actively treating cirrhosis.

In addition, hepatic fibrosis (hepatic fibrosis) is a condition in which the balance of synthesis and degradation of connective tissues is lost, and connective tissues accumulate in the liver tissues and are accompanied by necrosis or inflammation. In particular, hepatic stellate cells (HSCs), which play a role in the storage of vitamin A under normal liver function, are rapidly transformed into myofibroblasts by acute chronic liver damage and rapidly proliferate to form collagen, It is known to promote the fibrotic process of the liver by increasing and transferring the production of extracellular matrix such as proteoglycan, hyaluronan and the like to produce excessive connective tissue [Friedman et al., Proc. Natl. Acad. Sci. USA, 82: 8681 (1985) Gressner et al., Biochem. Biophys. Res. Commun., 151: 222 (1988) Gressner et al., J. Hepatol., 22: 28 (1995)].

The liver cancer may also be caused by symptoms such as liver cirrhosis and hepatic fibrosis which are the liver damage diseases described above and may be caused by the continuous causative action of liver damage, and thus the herbal extract of the present invention may be effective for the treatment of liver cancer.

Furthermore, the composition of the present invention can be used as a food composition in addition to a pharmaceutical composition for the treatment and prevention of liver diseases. In addition to containing an effective component of the herb extract, the food composition may contain various flavors or natural Carbohydrates and the like as additional components.

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 can be formulated in the same manner as the above pharmaceutical composition and used as a functional food or 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 above food composition may contain flavoring agents such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, colorants and aging agents (cheese, chocolate, etc.), pectic acid, Salts of alginic acid and its salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages 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.

As an active ingredient of the present invention, it is a natural substance and has little toxicity and side effects, so that it can be safely used even when taken for a long time.

The health functional food of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, and circles for the purpose of improving and preventing liver damage.

In the present invention, the term " health functional food " 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 obtained by mixing the herbal extract of the present invention as an excipient, binder, disintegrant and other additives in a conventional manner, Alternatively, the mixture can be directly compression molded. 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 herbal extract of the present invention and an additive such as an excipient. The soft capsule is prepared by adding a herb extract And filling the mixture with 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 the extract of Chenopodium ambrosioides, an effective ingredient of the present invention, excipient, binder, disintegrant, etc., by a conventionally known method and, if necessary, Or the surface may be coated with a material such as starch, talc.

The granular health functional food may be prepared by granulating a mixture of the extract of Angelica keiskei koidzaiensis extract, an active ingredient of the present invention, an excipient, a binder and a disintegrant into a granular form by a known method, and if necessary, adding a flavoring agent, ≪ / RTI >

The health functional food may be in the form of tablets, capsules, pills or liquids, and examples thereof include beverages, meat, chocolates, foods, confectionery, pizza, ramen, other noodles, gums, candy, ice cream, alcoholic beverages, And health supplement foods.

Hereinafter, the present invention will be described in more detail by way of examples. It will be apparent to those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited to these embodiments.

< Example  1> Materials and methods

<1-1> Preparation of materials and reagents

(NQO1), anti-laminase (IL-1), and anti-IL-2 / C, anti-cleaved caspase-3 (Asp175) and horseradish peroxidase-conjugated goat anti-mouse IgG were obtained from Cell Signaling Technology (Beverly, MA, USA) Anti-Nrf2 and anti-cleaved PARP (h215) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The horseradish peroxidase-conjugated anti-rabbit IgG was purchased from Thermo (Rockford, Ill., USA), and anti-glutamate-cysteine ligase catalytic subunit (GCLC) and anti -4-Hydroxynonenal (4-HNE) was purchased from Abcam (Cambridge, MA, USA). Arachidonic acid (AA) and rhodamine 123 (Rh 123) were purchased from Calbiochem (San Diego, CA, USA) and anti-Nitrotyrosine (NT) was purchased from Millipore Corporation (Temecula, ) And other reagents such as 2 ', 7'-dichlorodihydrofluorescine diacetate (HDCF-DA), ferric nitrilotriacetic acid (Fe-NTA), anti-β- actin antibody, silymarin and other reagents were purchased from Sigma , USA).

&Lt; 1-2 >

The chrysanthemum used in the present invention was purchased from Daegu, Korea, washed with water and shaken, and 100 g of dry weight was transferred in 1.7 L of water for 3 hours. The extract thus obtained was filtered through a filter paper (No. 2 filter paper, Nalgene, New York, NY, USA) and then concentrated using a vacuum rotary condenser (EYELA CCA-1110, SER.NO.10406050, Tokyo, Japan) 27.33 g was obtained. The yield of the water extract was 3.38%. The extract of Pericarpium zanthoxyli water extracts (PZW) was stored at -20 ° C. until use. The extract was dissolved in water to give a 0.22 μm filter (Millipore, USA) and used in the following examples.

<1-3> Cell culture

Human hepatic parenchymal hepatocytes (HepG2 cells) were purchased from the American Type Culture Collection (ATCC, Rockville, Md., USA). Using a Dulbecco's modified Eagle's medium (DMEM) medium containing 10% heat-treated fetal bovine serum (FBS) and 50 units / ml penicillin and 50 μg / ml streptomycin, HepG2 cells were cultured in an incubator maintained at 5% CO ₂ . Cells were cultured in a 100 mm dish to have confluence of 80% or more, and only cells not exceeding 20 passages were used.

<1-4> Breeding of experimental animals

The experimental animals were fed with ICR mice (22-24 g) at 6 weeks of age from Orient (Seongnam, Korea) and adapted to the laboratory environment for 1 week. The animals were used at the temperature of 20-23 ℃, (Nestle Purina Petcare Korea, Seoul, Korea) and drinking water were allowed to eat freely, while keeping the light / dark cycle at 12 hour intervals. This study was carried out after approval of DHU IACUC (Approval No. DHU2016-073) by the Experimental Animal Ethics Committee of Daegu Haany University.

<1-5> Treatment of experimental animals

The control group was the control group and the PZW group was administered 300 mg / kg of the extract. CCl ₄ diluted with corn oil (0.5 ml / kg) was injected peritoneal (ip). CCl ₄ group was used to induce hepatotoxicity. Liver was used for disease treatment agent of silymarin (ilymarn) as a positive control CCl ₄ treatment before silymarin 100 mg / kg, one thousand seconds the water extract of 100 or 300 mg / kg administered 4 days and the group treated for CCl ₄ each silymarin 100 group, PZW 100 group and PZW 300 group. A total of 30 animals were used in each group (see Table 1).

The experimental animal groups used in the experiments of the present invention Experimental group
(Groups) Corn oil Corn oil
+ CCl ₄ water Silymarin
100 mg / kg Chrysanthemum water extract
100 mg / kg Chrysanthemum water extract
300 mg / kg Control - - - - - - PZW + - - - - + CCl ₄ - + + - - - Silymarin 100 - + - + - - PZW 100 - + - - + - PZW 300 - + - - - +

<1-6> Statistical processing

The analytical values of the evaluation are expressed as mean (SD). Multiple comparisons were made between different concentration groups, and equi - dispersion tests were performed with the Levene test. In the absence of significant differences in the equilibrium distribution of the Levene test results, the results were analyzed by one-way ANOVA test, followed by post test with LSD test. Non-parametric test and Kruskal-Wallis H test were performed when there was a difference in iso-dispersion through Levene test. When significant differences were observed in the Kruskal-Wallis H test, Mann-Whitney U (MW) test was performed to compare the differences between the experimental groups. Statistical analysis was performed using SPSS (Release 14.0K, SPSS Inc., USA). P values less than 0.05 were considered significant.

< Comparative Example  1> Thousand  Production of methanol extract and fruit extract

&Lt; 1-1 > Preparation of methanol extract

Pericarpium zanthoxyli methanol extracts (PZM) were obtained and stored in the same manner as in Example <1-2>, except that 2.0 L of 100% methanol was used as an extraction solvent instead of 1.7 L of water. For example.

<1-2> Preparation of extracts

Example 1-2 < RTI ID = 0.0 > 1-2 < / RTI > except for using 100 g dry weight of the mixture of chestnut fruit purchased from Seo Hyun Herbal Medicinal Farming Association (Chungcheongnam- &Lt; / RTI &gt; were obtained and stored in the following examples.

< Example  2> Thousand  When the extract is a liver parenchyma cell ( HepG2  cell) on the survival rate in vitro  exam)

The cell survival rate was measured by MTT assay in order to examine the effect of the water extract obtained from Example <1-2> on the survival rate of hepatic parenchymal cells (HepG2 cells). The MTT assay was carried out by adding 0.5 mg of 3- (4,5- (dimethylthiazol) -2-yl) -2,5-diphenyltetrazolium (MTT, Sigma, St. Louis, Mo., USA) / ml was added to the wells and incubated for 4 hours. The medium was carefully removed and the formazan crystals were dissolved in DMSO and incubated at 570 nm using a microplate reader (Infinite 200 Pro, Tecan, Mannedorf, Switzerland) Absorbance was measured. Cell viability was expressed as a percentage of the control cell using the formula shown in Equation 1 below.

[Equation 1]

Viability (% of control) = (absorbance of treated sample) / (absorbance of control) x 100

Hepatocytes were treated with HepG2 cells at concentrations of 3, 10, 30, 100 and 300 μg / ml for 24 hours. The results were 105.77 ± 2.64 (%), 106.03 ± 2.88 (%) and 107.72 ± Cell viability was found to be 2.84 (%), 109.55 ± 1.10 (%) and 110.30 ± 3.48 (%), indicating that 1000 sec did not show any cytotoxicity (see FIG. 1A).

In order to observe the protective effect of HepG2 cells at 1000 sec, HepG2 cells were treated with 10, 30, 100 and 300 μg / ml of water extracts one hour before, and AA (10 μM, 12 hours) and iron (5 μM, 1 hour). The survival rate of AA + iron was 33.82 ± 2.34% as compared with the control group. However, cell survival rate of 10, 30, 100, and 300 μg / The results showed that the water-extracts of Chuncheon mushrooms had a hepatocyte protective effect in a concentration-dependent manner by increasing the cell survival rate in a concentration-dependent manner by 43.37 ± 3.10, 68.96 ± 4.63, 104.15 ± 3.42 (%) and 98.89 ± 2.75 1B).

As a result, cytotoxicity did not appear at 24 hours treatment at a concentration of 3 to 300 μg / ml. Thus, the inventors of the present invention found that the water extract of Chrysanthemum japonica is stable in the body and toxic to normal hepatocytes Therefore, it has been confirmed that it can be used safely as an active ingredient of a therapeutic agent or health functional food for the treatment and improvement of liver disease. In addition, it was confirmed that hepatocyte protective effect was effected by concentration -

< Example  3> A thousand years  The liver parenchyma cells ( HepG2  cell protection effect

In order to compare the protective effect of hepatic parenchymal cells (HepG2 cells) according to the extraction solvent or site of use, the chrysanthemum methanol extracts (10, 30, 100, 300 (10 μM, 12 h) and iron (5 μM, 1 h) were incubated in HepG2 cells one hour before treatment with the extracts (3, 10, 30, 100 and 300 μg / And the cell viability was measured using the same method as in Example 2 above.

The cell viability after oxidative stress induced pretreatment with methanol extract (PZM) was compared with the pretreatment with PZW pretreatment in Example 2, The water extracts of chrysanthemum were recovered to almost the same level as control (100%), but the methanol extracts of chrysanthemum recovered only up to 90% It was confirmed that the protective effect was excellent (see FIG. 1B and FIG. 2A).

On the other hand, the hepatocyte protective effects of extracts of Chuncheon water extract (PZW) extracted from only the skin of the example <1-2> and the extracts of Chenchus fruit extract and the skin of the comparative example <1-2> (100%) at 100 μg / ml, but only 42% at 100 μg / ml concentration, and 300 The cell survival rate at the concentration of μg / ml was also 84% level, and it was confirmed that the water extract of Chuncheon showed a remarkable effect of protecting the liver from the chunghwa seed extract (see FIGS. 1B and 2B).

< Example  4> Thousand  AA + iron-induced inhibition of cell death

Treatment of iron after treatment with arachidonic acid (AA) (AA + iron) induces apoptosis, and evaluation of whether or not the water extract of the present invention can inhibit apoptosis induced by AA + iron The following experiment was performed by Western blotting.

Western blot analysis was performed by incubating HepG2 cell with 30 μg / ml of water extract (100 μg / ml) for 1 hour before induction of apoptosis with AA + iron and lysing buffer [20 mM Tris Cl pH 7.5), 1% Triton X-100, 137 mM sodium chloride, 10% glycerol, 2 mM EDTA, 1 mM sodium orthovanadate, 25 mM beta 100 μl of b-glycerophosphate, 2 mM sodium pyrophosphate, 1 mM phenylmethylsulfonylfluoride, and 1 mg / ml leupeptin) was added to the wells, And scraped and centrifuged at 15,000 x g for 20 minutes at 4 占 폚. 30 μg of the quantified protein was electrophoresed on 10% SDS-PAGE, transferred to Nitrocellulose paper and bound to PARP and procaspase-3 antibody, and then the secondary antibody, horseradish peroxidase conjugated anti- And reacted with rabbit IgG (horseradish peroxidase-conjugated anti-rabbit IgG) antibody. The degree of protein expression was determined using ECL detection reagents (Amersham Biosciences Corp., Piscataway, NJ, USA) and analyzed using an image analysis system (Image J, rsb.info.nih.) For densitometric analysis. gov / ij) were used.

As a result of the above experiment, it was confirmed that the protein expression level of PARP and procaspase-3 decreased by AA + iron was increased in a concentration-dependent manner by the water extract of Chrysanthemum indicum, Reference).

< Example  5> Thousand  Antioxidative effect of extract

The treatment of arachidonic acid (AA) and iron catalyzes oxidation, increasing oxidative stress and causing dysfunction of mitochondria. Therefore, H2DCF-DA was used to measure the amount of active oxygen in the cells in order to confirm the antioxidative effect of the water extract of Chuncheon. H2DCF-DA is a cell-permeable non-fluorescent probe, which is converted to 2 ', 7'-dichlorodihydrofluorescin (DCF) by intracellular esterases and H ₂ O ₂ , and the amount of H ₂ O ₂ is relatively measured. (PZW) (30, 100 μg / ml) was treated with AA + iron for 1 hour before HepG2 cells were treated with a microplate reader (Infinite 200 Pro, Tecan, Mannedorf, Switzerland) Fluorescence intensity was measured at 485 nm and emission 535 nm wavelength.

As a result of the above experiment, it was observed that the amount of H ₂ O _{2 in the} cells treated with AA + iron was significantly increased by 2.02 ± 0.17 times as compared with that of the control (Control). However, when PZW 30 and 100 μg / ml were treated, the amount of H ₂ O ₂ increased 1.52 ± 0.10 times and 0.84 ± 0.10 times, respectively. Especially, at 100 μg / ml, (See FIG. 4). These results indicate that PZW suppresses the production of AA + iron - induced free radicals (ROS) and inhibits apoptosis of HepG2 hepatocytes. Therefore, it was confirmed that H ₂ O ₂ induced by AA + iron was reduced by water extract of Chuncheon, and the antioxidative effect of Chuncheon water extract was confirmed.

< Example  6> Thousand  Depending on the treatment of the extract GSH  Content change

GSH plays an important role in protecting cells from reactive oxygen species (ROS) in living cells. GSH has antioxidant functions by directly binding to active oxygen or by participating in antioxidant recycling such as vitamin C. High concentrations of GSH in cells can protect cells from oxidative stress primarily by protecting ROS, but when GSH is inhibited, it increases ROS, increases cell death, and is sensitive to oxidative stimuli do. In addition, the increase of intracellular reactive oxygen species induces cell death by inducing GSH deficiency, and many antioxidant drugs have cytoprotective action by increasing expression and activity of enzyme to produce intracellular deficient GSH .

GSH analysis was carried out as follows to confirm the protective effect of GSH-induced ROS production induced by AA + iron in the hepatic injury state of the water extract obtained in Example <1-2>. GSH was treated with AA + iron and MPA buffer and scraped with a scrapper to obtain a supernatant. The GSH determination kit (Oxis International) was used to treat hepatic parenchymal cells (HepG2 cells) 30, 100 μg / The fluorescence intensity was measured at excitation 405 nm wavelength using a microplate reader (Infinite 200 Pro, Tecan, Mannedorf, Switzerland).

As a result of the above experiment, it was evaluated whether GSH mediated the protective effect of chrysanthemum water extract on AA + iron-induced cytotoxicity. AA + iron treatment resulted in 16.54 ± 1.26 GSH compared to control. The GSH decreased significantly to 53.05 ± 11.98 and 76.32 ± 4.57 by pretreatment with PZW 30 and 100 μg / ml, respectively . From these results, it was confirmed that the decrease of GSH due to the increase of AA + iron-induced free radicals was increased by the water extract of Chuncheon (see FIG. 5).

Therefore, it was confirmed that the water extract of Chuncheon mackerel has an effect on the increase of GSH, and the cell death inhibition, cytoprotective effect and antioxidant ability thereof.

< Example  7> Thousand  Mitochondrial disorders of extracts ( mitochondrial dysfuncion ) Inhibitory effect

Mitochondrial membrane permeability (MMP) was analyzed by flow cytometry in order to confirm mitochondrial protection efficacy of the mulberry extract obtained in Example <1-2>.

The changes of MMP were analyzed by flow cytometry after staining HepG2 cells with Rh 123, a membrane osmotic cationic fluorescent dye. In detail, HepG2 cells were stained with 0.05 μg / ml of Rh 123 for 30 minutes, treated with trypsin, resuspended in PBS supplemented with 1% FBS, and analyzed on a Partec FACS flow cytometer (Goerlitz, DE) , Respectively. Each analysis recorded fluorescence of 10,000 cells per sample.

As a result of the above experiment, the control group was 7.93 ± 4.93, but AA + iron was 63.61 ± 5.41. The increase of RN1 fraction was 16.25 ± 13.59 and 4.13 ± 0.04 by PZW 30 and 100 μg / (Fig. 6). As a result, it was confirmed that the increase of RN1 fraction due to AA + iron was decreased by the water extract of Chuncheon. These results indicate that mitochondrial protective effects are mediated by PZW to exhibit hepatocyte protective action.

< Example  8> Thousand  By extract Nrf2  Activation

The following experiment was carried out to investigate the activation of Nrf2, an antioxidant transcription factor, as a mechanism of PZW protection against AA + iron-induced cytotoxicity.

The activation of Nrf2 and the related antioxidant genes were examined by western blotting in the following manner to confirm activation of Nrf2 by water extract of the present invention obtained in the above Example <1-2>.

Western blots were treated with AA + iron for 1 h before treatment with HepG2 cell extracts (30, 100 μg / ml), and nuclear fractions were collected from NE-PER ™ Nuclear and Cytoplasmic Extraction Reagents (Thermo, Rockford, IL The cells were extracted with lysis buffer (20 mM Tris Cl pH 7.5, 1% Triton X-100, 137 mM sodium chloride, Glycerol, 2 mM EDTA, 1 mM sodium orthovanadate, 25 mM beta-glycerophosphate, 2 mM sodium pyrophosphate, 1 mM phenylmethylsulfonyl 100 μl of 1 mg / ml leupeptin) was scraped with a scraper and centrifuged at 15,000 × g for 20 minutes at 4 ° C. to remove debris. 30 μg of the quantified protein was electrophoresed on 10% SDS-PAGE, transferred to Nitrocellulose paper, and bound to Nrf2, GCLC and NQO1 antibody. Then, the secondary antibody, horseradish peroxidase conjugate anti- (Horseradish peroxidase-conjugated anti-rabbit IgG) or horseradish peroxidase-conjugated anti-mouse IgG antibody. The degree of protein expression was determined using ECL detection reagents (Amersham Biosciences Corp., Piscataway, NJ, USA) and analyzed using an image analysis system (Image J, rsb.info.nih.) For densitometric analysis. gov / ij) were used.

As a result of the above experiment, PZW increased the activity of Nrf2 at 3 hours and increased the expression of GCLC and NQO1, which are antioxidative proteins, at 24 hours after treatment with PZW of 100 μg / ml for 24 hours in HepG2 cells (See FIG. 7). Therefore, it was confirmed that the water extract of Chuncheon extract activates both antioxidant genes GCLC and NQO1 related to Nrf2, an antioxidant transcription factor.

< Example  9> Thousand  The extract CCl ₄ in  Induced To liver damage  Impact on in vivo )

The ALT and AST measurements, which are used as an ideal index for evaluating liver damage, and the effect on the histological changes were examined to determine the liver protection effect of CCl ₄ damage on the water extracts obtained in Example <1-2> Hematoxylin-eosin (H & E) staining was performed as follows.

The animals for the experiment were experimented with the groups shown in the above Example <1-5>, and blood was collected from these animals (see Table 1). Blood was collected from 1 ml of the abdominal vein of the experimental animals and centrifuged at 3000 × g for 15 minutes at 4 ° C to obtain serum. Analysis of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum was performed using an automated kits (Pointe Scientific Inc., Canton, MI, USA) and an automated blood chemistry analyzer Riele GmbH & Co KG, Berlin, Germany).

ALT results showed 6.6 ± 2.1 U / L in the control and 744.4 ± 233.1 U / L in the CCl _4- administered group, resulting in significant hepatotoxicity. However, the increase in ALT was 634.0 ± 142.2 and 327.6 ± 181.7 U / L in the PZW 100 and PZW 300 groups, respectively, especially in the PZW 300 group (see FIG. 8A).

As a result of AST measurement, the control (control) showed 4.8 ± 0.4 U / L, and CCl ₄ And 715.2 ± 111.1 U / L in the administration group, resulting in significant hepatotoxicity. However, the increase in AST was reduced to 529.8 ± 142.7 and 462.2 ± 110.5 U / L in the PZW 100 and PZW 300 groups, respectively, and a significant decrease in AST was observed in dependence on the PZW concentration (see FIG. 8B).

In the PZW group treated with PZW 300 mg / kg alone, ALT and AST were 5.8 ± 0.8 and 4.0 ± 0.7 U / L, respectively. These results indicate that PZW itself does not cause significant hepatotoxicity up to a dose of 300 mg / kg, and PZW has a liver protection effect both in vitro as well as in vivo (see FIG. 8).

As a result of the above experiment, it was confirmed that CCl ₄ -induced ALT and AST were significantly decreased in the PZW 300 group, and that the group treated alone did not cause hepatotoxicity (see FIG. 8).

< Example  10> Thousand  The extract CCl ₄ in  Influence on induced histological changes in liver

In order to investigate the effect of water extracts on CCl ₄ induced hepatic histologic changes, a part of liver parenchyma was collected and fixed in 10% neutral formalin, dehydrated and paraffin embedded in the usual manner, and 3 The sections were stained with hematoxylin-eosin (H & E) and observed under an optical microscope (Nikon, Japan). The degree of liver damage was assessed using a modified histological activity index (HAI) semiquantative histopathological scoring system based on apoptosis, confluent necrosis and inflammatory cell infiltration of liver cells, (% / Mm ^{2) in the} liver parenchyma. of hepatic parenchyma and number of hepatocytes (Cells / 1000 hepatocytes) and number of infiltrating inflammatory cells (cells / mm ² of hepatic parenchyma) were measured using a video analyzer ( i Solution FL ver 9.1, IMT i- Solution Inc., Vancouver , Quebec, Canada).

The HAI scores were 0.40 ± 0.55 in the control group and 8.20 ± 1.10 in the CCl ₄ group. However, this increase by CCl ₄ treatment was significantly reduced to 4.60 ± 0.89 and 3.40 ± 0.55 by treatment with PZW 100 or 300 mg / kg, respectively (see Table 2 and FIG. 9).

The percentages of denaturation in liver parenchyma (% / mm ² of hepatic parenchyma) were 1.54 ± 0.78 in control and 83.37 ± 6.10 in CCl ₄ group. However, this increase by CCl ₄ was significantly reduced by PZW 100 and 300 mg / kg to 48.11 7.95 and 38.55 6.63, respectively (see Table 2 and FIG. 9).

The number of denatured hepatocytes (cells / 1000 hepatocytes) showed 14.80 ± 7.95 in the control and 808.80 ± 78.10 in the CCl ₄ group. However, this increase by CCl ₄ was significantly reduced to 473.60 ± 86.38 and 363.80 ± 59.82 by PZW 100 and 300 mg / kg, respectively, similar to the ratio of denatured sites in liver parenchyma (see Table 2 and Figure 9 ).

The number of infiltrating inflammatory cells (cells / mm ² of hepatic parenchyma) showed 24.20 ± 6.98 in control and 223.00 ± 76.42 in CCl ₄ group, showing a significant increase compared with control. However, this increase by CCl ₄ was significantly reduced to 71.00 ± 14.09 and 51.00 ± 15.22 by PZW 100 and 300 mg / kg, respectively (see Table 2 and Figure 9).

In the PZW 300 mg / kg single treatment (PZW group), the ratio of HAI score, percentage of denatured site in liver parenchyma, number of denatured hepatocytes, number of infiltrating inflammatory cells were 0.40 ± 0.55, 1.53 ± 0.87, 15.00 ± 7.78 , And 23.80 ± 12.64, respectively (Table 2 and Figure 9B).

As a result, it was confirmed that the fraction of centrilobular necrosis such as CCl ₄ -induced hepatocyte vacuolization, accumulation of lipid droplets in the liver and inflammatory cell infiltration was significantly decreased by 300 mg / kg water treatment , But not significantly changed in the group treated alone (see Table 2 and FIG. 9).

The effect of the water extract of the present invention on the histological changes induced by CCl ₄ was confirmed
Index (Index)


Groups General histomorphometry Histological Activity Index
(Scores;
Max = 10) Percentages of degenerative regions (% / mm ² ) Numbers of degenerative hepatocytes (cells / 1000 hepatocytes) Numbers of inflammatory cells infiltrated (cells / mm ² ) Control 0.40 + - 0.55 1.54 + 0.78 14.80 ± 7.95 24.20 ± 6.98 PZW 0.40 + - 0.55 1.53 + - 0.87 15.00 7.78 23.80 ± 12.64 CCl ₄ 8.20 ± 1.10 ^a 83.37 6.10 ^c 808.80 ± 78.10 ^c 223.00 ± 76.42 ^c Silymarin 100 4.40 ± 0.55 ^ab 47.02 ± 4.58 ^ce 453.20 ± 50.01 ^ce 71.60 ± 10.29 ^ce PZW 100 4.60 ± 0.89 ^ab 48.11 ± 7.95 ^ce 473.60 ± 86.38 ^ce 71.00 ± 14.09 ^ce PZW 300 3.40 ± 0.55 ^ab 38.55 + 6.63 ^ce 363.80 ± 59.82 ^ce 51.00 ± 15.22 ^de

^a p <0.01 as compared with control by LSD test; ^b p <0.01 as compared with CCl ₄ by LSD test; ^c p <0.01 and ^d p <0.05 compared with control by MW test; ^e p <0.01 as compared with CCl ₄ by MW test.

< Example  11> Thousand  The extract CCl ₄ in  Influence on induced immunohistochemical changes in liver

Cleaved caspase-3, cleaved PARP, nitrotrysine (NT) and 4-hydroxynonenal (4-hydroxynonenal) were used for the immunohistochemical evaluation of the effect of CCl ₄ on the water- HNE) were assessed using each antibody and avidin-biotin-peroxidase (ABC) and peroxidase substrate kit (Vector Labs, Burlingame, CA, USA). Endogenous peroxidase activity was blocked by incubation in methanol and 0.3% H ₂ O ₂ for 30 min and incubated with normalhorse serum blocking solution for 1 hour to inhibit non - specific binding of immunoglobulin. Then, the primary antibody was treated overnight with 4 ° C thermo-hygrostat, and then incubated with secondary antibody and ABC reagent for 1 hour. The tissue samples were then incubated with a peroxidase substrate kit for 3 minutes at room temperature. All tissue samples were washed three times with 0.01M PBS for each step. Cleaved caspase-3, cleaved PARP, NT, and 4-HNE positive cells were analyzed using a mobile video analyzer. Cleaved caspase-3, cleaved PARP, NT and 4-HNE positive cells were analyzed for hepatic parenchyma, centrolobular region, cells / 1000 hepatocytes in the central vein region.

<11-1> Effects of Chrysanthemum indicum extract on cleaved caspase-3 and cleaved PARP in liver tissue

The number of cleaved caspase-3 positive cells (cells / 1000 hepatocytes) showed 12.20 ± 9.18 in the control and 664.60 ± 77.89 in the CCl ₄ group, indicating a significant increase compared with the control. However, the increase of cleaved caspase-3 by CCl ₄ was significantly reduced to 433.20 ± 89.18 and 261.80 ± 50.98 in PZW 100 and 300, respectively. However, the PZW group was 12.00 ± 7.65 and showed no significant change (see Table 3 and FIG. 10).

The number of Cleaved PARP positive cells (cells / 1000 hepatocytes) showed 19.80 ± 10.94 in control and 713.40 ± 57.50 in CCl ₄ group, showing a significant increase compared with control. However, the increase of 4-HNE by CCl ₄ was 461.20 ± 77.33 and 284.00 ± 108.91 in PZW 100 and 300 groups, respectively. However, the PZW group was 19.40 ± 9.66 and showed no significant change (see Table 3 and FIG. 10).

These results indicate that PZW inhibits cleaved caspase-3, cleaved PARP associated with apoptosis and thereby inhibits CCl ₄ -induced hepatotoxicity.

<11-2> Effects of Chunsocho extract on NT and 4-HNE of liver tissue

The number of NT - positive cells (cells / 1000 hepatocytes) showed 28.40 ± 18.22 in the control and 664.20 ± 50.00 in the CCl ₄ group, showing a significant increase compared to the control. However, CCl ₄ The increase in NT by treatment was significantly reduced to 412.80 ± 133.19 and 217.20 ± 37.10 in the PZW 100 and 300 groups, respectively. However, the PZW group was 127.20 ± 20.75 and showed no significant change (see Table 3 and FIG. 11).

These results suggest that PZW inhibits nitrosative stress associated with NO and inhibits CCl ₄ - induced hepatotoxicity.

The number of 4-HNE positive cells (cells / 1000 hepatocytes) showed 62.40 ± 30.66 in the control and 795.60 ± 60.11 in the CCl ₄ group, showing a significant increase compared to the control. However, the increase of 4-HNE by CCl ₄ was 427.20 ± 83.58 and 278.60 ± 45.39 by PZW 100 and 300 mg / kg, respectively. However, the PZW alone treatment group (PZW) was 161.40 ± 37.58, indicating no significant change (see Table 3 and FIG. 11).

These results suggest that PZW can inhibit CCl ₄ -induced liver damage through the inhibition of lipid peroxidation and 4-HNE formation.

As a result of the above experiments, the expression of cleaved caspase-3 and cleaved PARP increased by CCl ₄ -induced hepatotoxicity, 4-HNE formed by NT and lipid peroxidation, which plays an important role in stimulated nitric oxide formation, (PZW 100 and 300 group) treated with 300 mg / kg, and it was confirmed that hepatotoxicity was not induced in the group treated alone (PZW group) (FIGS. 10 and 12) 11). The number of cleaved caspase-3, cleaved PARP, NT and 4-HNE positive cells was also reduced by PZW treatment, and it was confirmed that the PZW alone treatment did not induce hepatotoxicity (see Table 3).

The effect of the water extracts of the present invention on the immunological changes of CCl ₄ induced damage Index (Index)


Groups Positive cells by immunohistochemistry (cells / 1000 hepatocytes) Cleaved caspase-3 Cleaved Poly (ADP-ribose) polymerase Nitrotyrosine 4-Hydroxynonenal Control 12.20 ± 9.18 19.80 ± 10.94 28.40 ± 18.22 62.40 ± 30.66 PZW 12.00 ± 7.65 19.40 ± 9.66 27.20 ± 20.75 61.40 ± 37.58 CCl ₄ 664.60 ± 77.89 ^c 713.40 ± 57.50 ^c 664.20 ± 50.00 ^c 795.60 ± 60.11 ^a Silymarin 100 420.00 ± 33.13 ^cd 463.80 ± 57.12 ^cd 388.40 ± 69.68 ^cd 404.40 ± 51.03 ^ab PZW 100 433.20 ± 89.18 ^cd 461.20 ± 77.33 ^cd 412.80 ± 133.19 ^ce 427.20 ± 83.58 ^ab PZW 300 261.80 ± 50.98 ^cd 284.00 ± 108.91 ^cd 217.20 ± 37.10 ^cd 278.60 ± 45.39 ^ab

^a p <0.01 as compared with control by LSD test; ^b p <0.01 as compared with CCl ₄ by LSD test; ^c p <0.01 compared with control by MW test; ^d p <0.01 and ^e p <0.05 as compared with CCl ₄ by MW test.

In conclusion, the water extracts of the present invention significantly inhibited CCl ₄ -induced liver damage, indicating that 300 mg / kg of perennial water extract is superior to liver protection. These results suggest that water extracts from Chuncheon are useful for the prevention and treatment of acute chronic liver injury.

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 (8)

A pharmaceutical composition for the prevention or treatment of liver diseases comprising extracts of Chuncheon as an active ingredient. The method according to claim 1,
The extract is obtained by using a solvent selected from the group consisting of water, C1 to C4 alcohol, hexane, ethyl acetate, butylene glycol, propylene glycol, glycerin, acetic acid, ether, chloroform and a mixed solvent thereof &Lt; / RTI &gt; The method according to claim 1,
Wherein the extract is contained in an amount of 0.1 to 50% by weight based on the total weight% of the composition. The method according to claim 1,
Wherein said extract exhibits hepatocyte protective activity through Nrf2 pathway activation and mitochondrial protective action. The method according to claim 1,
Wherein the liver disease is liver cirrhosis, liver fibrosis, acute hepatitis, chronic hepatitis or liver cancer. A health functional food for preventing or ameliorating liver disease containing extract of Chuncheon as an active ingredient. The method according to claim 6,
The extract is obtained by using a solvent selected from the group consisting of water, C1 to C4 alcohol, hexane, ethyl acetate, butylene glycol, propylene glycol, glycerin, acetic acid, ether, chloroform and a mixed solvent thereof Features a healthy functional food. The method according to claim 6,
Wherein the liver disease is liver cirrhosis, liver fibrosis, acute hepatitis, chronic hepatitis or liver cancer.

Images