KR20140043176A - Pharmacological composition for prevention and treatment of liver injury, and functional food including thereof - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing and treating liver injury and health functional food including thereof, and more specifically, to a pharmaceutical composition for preventing and treating liver injury which contains extracts of medical herbs including Havenia dulcis, arrowroot, and Chinese dioscorea having excellent prevention and treatment effects against liver injury as effective components; and a food composition for preventing and treating liver injury. The composition of the present invention reduces the activity of ALT and AST in plasma and the death rate of mice regarding APAP-induced liver injury, increases the content of GSH and GSH-px in liver cell crushed liquid, concentration dependently reduces the necrosis of liver tissue by APAP and the infiltration of white blood cells, and controls the activity of COX-2 and TNF-α, thereby being effectively used for preventing and treating liver injury.

Description

Pharmacological Composition for Prevention and Treatment of Liver Injury, and Functional Food including including

The present invention relates to a pharmaceutical composition for preventing and treating liver damage and a health functional food comprising the same, and more particularly, to a pharmaceutical composition including hawthorn, reeds and powder which exhibits an excellent prophylactic or therapeutic effect on liver damage. It relates to a pharmaceutical composition for the prevention and treatment of liver damage containing the extract as an active ingredient and a food composition for the prevention and improvement of liver damage.

The liver is a major organ in the Chinese medicine and Western medicine that is responsible for the blood metabolism and detoxification of the human body.

The earth is the fruit or seed of the fruit of the walnut tree Hovenia dulcis Thunb., A plant of the Rhamnaceae family. ㆍ Herbology Editorial Committee of Korean Medicine schools.Herology [Boncho-hak] .Seoul: Young-Lim Press. 2007 ： 184, 580, 734) It has been reported that the medicinal herb has been reported to protect liver against liver damage and ethanol poisoning (Dae-Hwan Youn et. Al., The Korea journal of herbology. 2006; 21: 7-19; Jong Ho Kim et. ., Yakhak Hoeji. 2008; 52: 56-61). Earth tree, on the other hand, refers to the bark of the bark tree.

Herbal medicine is a root stem (dough) of the Dioscorea batatas Decne. Or Yam ( Dioscorea japonica Thunb.), Which is a plant of the family Dioscoreaceae, and is dried or dried. With the efficacy of 정, 益肺, 益肺 보 보, 신 H (Herbology Editorial Committee of Korean Medicine schools.Herology [Boncho-hak]. Seoul ： Young-Lim Press. 2007 ： 184, 580, 734) As a result, it has been used mainly for the purpose of 津 氣 최근 최근, and recent studies have reported antioxidant and anti-inflammatory effects on oxidative damage.

Brown root is the root of the legumes (Pueraria lobata Ohwi), the legumes, as it is or it has been removed from the skin. It has been used as a main cause of alcohol in 醫 寶 鑑 으로 in the East (Kyung-Hye Jeon and Yoon-Bum Kook, The Korean journal of oriental medical prescription. 2009; 17: 29-43). It has been reported to have a significant effect on liver damage or other liver damage caused by alcohol metabolism (Dong Hwan Hyun et. Al., Korean journal of oriental physiology & pathology. 2003; 17: 297-307; Heyong-Gyoo Park et. al., Journal of Korean oriental medical society. 2000; 21: 186-98).

However, there is no report on the effect of the extract of the drug consisting of these drugs to prevent or treat acute liver damage.

Acetaminophen (N-acety1-p-aminophenol, paracetamol, APAP) is a generic drug that is used worldwide for antipyretic and analgesia and is a relatively safe drug when used in therapeutic concentrations. However, it is well known that excessive doses of APAP not only cause liver necrosis, neurotoxins, liver cirrhosis, but also have side effects of death (Ray SD et. Al., J.). Pharmacol Exp Ther. 1996; 279: 1472-83; Webster PA et. Al., J Clin Pharmacol. 1996; 36: 397-402). In experimental animals, 90% of APAP concentrations are released into bile or plasma after binding to glucoronic acid or sulfate in hepatocytes, and 5-10% of APAP is metabolized by P450 (CYP), especially CYP2E1 (Ray SD et al., J Pharmacol Exp Ther. 1996; 279: 1470-83). Toxic substances such as N-acetylp-benzoquinoneimine (NAPQI) generated during the metabolism of APAP cause glutathione (GSH) depletion, resulting in fatal damage to the cell membrane, leading to liver necrosis and inability to recover, leading to death. (Webster PA et. Al., J Clin Pharmacol. 1996; 36: 397-402; Albano E et. Al., Mol Pharmacology. 1985; 28: 306-11; Kyle ME et. Al., Biochem Biophys Res Commun 1987; 149: 889-94; Mahadevan SB et.al., Arch Dis Child. 2006: 91: 598-603). In particular, it is known that a large amount of leukocytes is introduced to cause acute inflammation, especially when an excessive amount of APAP is administered to a living body. That is, proinflammatory cytokines such as interferon-γ (IFN-γ), tumor necrosis fator-α (TNF-α), interleukin-1 (IL-1) and IL-6, which are well known as cytokines for inflammatory reactions. (proinflammatory cytokines) are mass produced within 10 hours to accelerate the inflammatory response (Ishida Y et al., J Leukoc Biol. 2004; 75: 59-67; Hogaboam CM et. al., Am J Pathol. 2000; 156; : 1245-52). Moreover, inflammatory mediators such as cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) are induced by APAP and cause fatal damage in vivo (Bhave VS et. Al., Toxicol Appl Pharmacol. 2011; 251: 173). -80). As a result, about 10% of patients with liver disease caused by APAP have serious toxicity in children rather than adults, and have been reported to be more serious in alcohol users than in non-alcoholic users (Prescott LF. Br J Clin Pharmacol). 2000; 49: 291-301). Recently, in order to discover drugs that can remove the toxicity of APAP, researches are actively conducted on herbal medicines that minimize side effects to the human body, including various foods that we ingest daily.

Liver is a major organ that manages blood metabolism and detoxifies the human body in oriental medicine and western medicine. When the liver is damaged, it causes inflammatory reactions and various diseases in the human body. It is important. Accordingly, the present inventors subtracted that most liver diseases occurring in the human body are caused by oxidative stress along with the inflammatory response while searching for substances that are effective in preventing and treating liver damage. Various studies were conducted on the liver protection effects of extracts combining specific herbs against APAP-induced liver damage. As a result, the extracts can be used as pharmaceutical compositions for the prevention and treatment of liver damage. Was completed.

An object of the present invention is to provide a pharmaceutical composition for the prevention and treatment of acute liver damage and a food composition for the prevention and improvement of liver damage.

In order to achieve the above object, acute liver damage containing as an active ingredient an extract of a medicinal herb comprising at least one medicinal herb, Puerariae Radix and Dioscoreae Rhizoma, selected from the group consisting of earth and earth It provides a preventive and therapeutic pharmaceutical composition for.

In addition, the present invention is an acute liver injury containing as an active ingredient extracts of medicinal herbs, including hawthorn medicinal herbs, reeds and pesticides selected from the group consisting of earth and earth tree containing food acceptable food supplement additives as an active ingredient It provides a preventive and improved food composition for.

Hereinafter, the present invention will be described in detail.

In the present invention, the medicinal herb wood is preferably earth.

In the prophylactic and therapeutic pharmaceutical composition for liver damage of the present invention, the extract preferably further comprises a pharmaceutically acceptable carrier, wherein 50 ~ 100 weight of the roots based on 100 parts by weight of the medicinal herbs It is preferable that it is 48-97 weight part and acid.

In addition, in the pharmaceutical composition for preventing and treating liver damage of the present invention, the extract iii) 5 to 20 times the weight of the drug weight and extracting for 1 to 4 hours at 80 to 150 ℃; Ii) removing the impurities precipitated in the extraction solution by centrifugation at 1000 to 5000 rpm and filtering the filtered solution through a 0.3 to 0.6 mu m filter; And iii) concentrating the filtrate with a rotary vacuum concentrator.

The present invention confirmed the protective effect of these extracts against APAP-induced liver damage. Specifically, the control group administered with APAP significantly increased liver tissue damage indicator enzymes ALT and AST, and significantly depleted GSH and GSH-px known as antioxidants and enzymes in vivo, and COX-2 and TNF-α. Inflammatory mediators were mass produced. However, the experimental group administered with the extract of the present invention significantly inhibited ALT and AST increased by APAP, and increased production of GSH and GSH-px depleted by APAP as well as mass production by APAP. It has an effect of significantly inhibiting COX-2 and TNF-α. In particular, the 100 mg / kg and 200 mg / kg administration group of the extract of the present invention showed a degree similar to the normal liver damage protection effect induced by APAP. Therefore, these results suggest the liver protection effect of the extract of the present invention against APAP-induced liver damage, the composition of the present invention can be used as a useful material for liver disease care.

The present invention suggests that the combination consisting of medicinal herbs, Puerariae Radix and Dioscoreae Rhizoma is a composition for the protection of liver function. On the other hand, medicinal herbs means earth and earth tree, preferably earth. In the present invention, the effects of these extracts on acetaminophen (APAP) -induced liver damage in mice were confirmed.

Specifically, the present invention was prepared by extracting with hot distilled water. On the other hand, it can be extracted using other organic materials including ethanol and the like. The extract was lyophilized after filtration through a vacuum distillation system. The mice fasted overnight were orally administered with or without the extract at various doses (25-200 mg / kg / day). After 30 minutes, APAP was applied to the oral cavity at a single dose (400 mg / kg). The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mouse plasma were measured. Glutathione (GSH), glutathione peroxidase (GSH-px), cyclooxygenase-2 (COX-2) activity and tumor necrosis factor-α in hepatic cell lysate; TNF-α) levels were studied. Liver sections were stained with haematoxylin & eosin, anti-TNF-α and anti-mouse COX-2 antibodies.

APAP treatment increased plasma AST and ALT activity but inhibited GSH and GSH-px levels in hepatic cell lysate. In addition, liver damage was significantly accelerated by APAP treatment. In addition, APAP surprisingly raised COX-2 activity and TNF-α levels in hepatic cell lysate. However, administration of the extract of the herbal combination of the present invention could counteract this effect. Histological studies provided supporting evidence for biochemical and molecular analysis.

These results suggest that the extract of the medicinal combination of the present invention has a strong protective effect on liver function against APAP-induced liver damage, and this property may contribute to the treatment of liver disease.

The extract of the combination of the present invention can be obtained by extracting the medicine of the present invention with water or an organic solvent, the organic solvent may be exemplified by lower alcohol, acetone, chloroform, methylene chloride, ether, ethyl acetate, hexane and the like. . As the lower alcohol, methanol, ethanol, propanol and butanol can be mentioned, and ethanol is most preferable.

Specifically, 1 to 20 times, preferably 5 to 15 times, more preferably 10 times of water is added to the medicinal product dried or powder, and 1 to 24 at a temperature of 80 to 150 ℃, preferably 90 to 100 ℃ Extraction for hours, preferably 2 to 6 hours, more preferably 2 hours and then filtered to produce a hydrothermal extract of the medicinal herb. The organic solvent extract of the medicinal herb can be prepared by adding 1-5 times, preferably 3 times, the organic solvent to the medicinal herb dry matter or powder, extracting at room temperature, and concentrating the filtrate obtained by filtration under reduced pressure. In the above extraction methods, the extraction process may be repeated twice or more as necessary, and the extract obtained after filtration may be lyophilized or dried under reduced pressure to a powder form.

The "pharmaceutically acceptable carrier" is a pharmaceutically acceptable substance such as a liquid or solid filler, diluent, excipient or solvent which serves to transport the active ingredient from one organ or part of the body to another organ or part of the body. , Composition or vehicle.

The composition for treating liver damage of the present invention may be prepared as a medicament by adding one or more pharmaceutically acceptable carriers together with the active ingredient. The carrier may include, but is not limited to, saline, buffered saline, water, glycerol and ethanol, and any suitable agent known in the art (Remingtons's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA) may be used. .

Formulations for pharmaceutical preparation of the extract of the present invention can be administered orally during clinical administration and can be used in the form of general pharmaceutical formulations, when formulated, commonly used fillers, extenders, binders, wetting agents, disintegrating agents, interfacial It is prepared using diluents or excipients such as active agents. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and liquid preparations for oral use include suspensions, solvents, emulsions, and syrups. In addition to liquids and paraffins, various excipients may be included, such as wetting sweeteners, fragrances, preservatives and the like.

In addition, the herbal medicine that may be added to the composition of the present invention may be any pharmaceutically acceptable herbal medicine, for example, Angelica tenuissimae Radix, Gastrodiae Rhizoma, Bapleuri Radix, Angelica ( Angelicae gigantis Radix, Persicae Semen, Cinnamomi Ramulus, Rhubarb (Rhei Rhizoma), Licorice (Glycyrrhizae Radix), Cnidii Rhizoma, Aurantii nobilis Pericarpium, Taxa (Alismatis Rhizoma) Coptidis Rhizoma, Scutellariae Radix, Hoelen, Peeoniae Radix, Atractylodis Rhizoma alba, Phellodendri Cortex, Gardeniae Fructus, Pinelliae Tuber, Ramulu Set (Uncaria) Uncus, Ponciri Fructus, Ginseng (Gingseng), Liriopis Tuber, Polygalae Radix, Acori graminei Rhizoma, Atractylodis Rhizoma alba, Chrysanthemi Flos, Windproof (Ledebouri) ), Ginger (Zingiberis Rhizoma crudus), forget-me-not (Natrii sulfas), control (Ziz) yphi Fructus, Salviae Radix, Mautan Radicis Cortex, Rehmanniae Radix, Minthae Herba, Polyporus, Polygonimultiflori Radix, Allii tuberosi Semen, Cassia Semen, Goji (Lycii Fructus), Araliae cordatae Radix, Eucommiae Cortex, Hedyotis Herba, Saururus Herba, Artemisiaecapillaris Herba, Anemarrhenae Rhizomath, Safflower Flos, Astragali Radix, Lycopodium, Ginkgonis Folium, Polygonati Rhizoma, Nelumbinis Semen, Fossilia ossis Mastodi, Lycoii radicis Cortex, Radix this ), Rehmanniae Radix preparata, Perillae Semen, Thujae Semen, Malt (Hordei Fructus germinatus), Cuscutae Semen, Mordaedae Radix, Pini koraiensis Radix Or it can mix and use.

The composition of the present invention may be administered in various parenteral formulations during actual clinical administration, and solid preparations include tablets, pills, powders, granules, capsules, and the like. In addition to water, liquid, and paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. Specifically, preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. Propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used as the non-aqueous solvent and suspension agent. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like. In addition, calcium or vitamin D ₃ may be added to enhance the efficacy of the treatment. Such compositions may be presented in unit-dose (single) or multi-dose (several) containers, such as sealed ampoules and vials, and immediately before use, sterile liquid carriers such as injectable water. Can be stored under freeze-drying conditions requiring only the addition of. Immediate injection solutions and suspensions can be prepared from sterile powders, granules and tablets.

The formulations of the present invention can be applied differently depending on the age, sex, condition of the subject, the absorption of the active ingredient in the body, the inactivation rate and excretion rate, the drug used in combination. The invention also includes formulations of dosage units. The formulations are present in individual dosage forms, such as tablets, coated tablets, capsules, pills, suppositories, and ampoules, wherein the amount of active compound in the drug corresponds to the fraction or multiple of the individual dosage. Dosage units may contain, for example, one, two, three or four times the individual dosage, or 1/2, 1/3 or 1/4 times. The individual dosages preferably contain an amount in which the active compound is administered at one time, which usually corresponds to all, 1/2, 1/3 or 1/4 times the daily dosage.

As used herein, the term "extract" refers to an active ingredient isolated from natural products. The extract may be obtained by an extraction process using water, an organic solvent, or a mixed solvent thereof, and includes an extract, a dry powder thereof, or any form formulated using the same.

As used herein, the term "prevention" means any action that inhibits or delays the development of liver injury by administration of the composition.

As used herein, the term "treatment" means any action that improves or advantageously alters the symptoms of liver injury by administration of the composition.

Extract in the present invention can be used to extract using water, an organic solvent, or a mixed solvent thereof. Preferably it is extracted using an organic solvent, in particular ethanol. The extracted liquid can be used directly or by concentrating and / or drying. When extracted with an organic solvent, methanol, ethanol, isopropanol, butanol, ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, dichloromethane, N, N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,3-butylene glycol, propylene glycol, or a mixed solvent thereof may be used and extracted by room temperature or warming under conditions where the active ingredient of the herbal medicine is not destroyed or minimized. Depending on the organic solvent to be extracted, the degree of extraction and loss of the active ingredient of the drug may vary, so select an appropriate organic solvent. The extraction method is not particularly limited, and examples thereof include cold extraction, ultrasonic extraction, and reflux cooling extraction. Filtration is a process of removing the suspended solid particles from the extract, it may be used to filter the particles using cotton, nylon or the like, or may be used, such as ultrafiltration, cryofiltration, centrifugal separation, but is not limited thereto.

Concentration of the extract may be used, such as concentrated under reduced pressure, reverse osmosis concentration. The post-concentration drying step includes, but is not limited to, freeze drying, vacuum drying, hot air drying, spray drying, vacuum drying, foam drying, high frequency drying, infrared drying and the like. If desired, a process of grinding the final dried extract may be added.

In addition, the extract can perform an additional fractionation process. Preferably, the extract is suspended in distilled water to obtain a nonpolar solvent soluble layer by extraction and separation with a nonpolar organic solvent such as hexane, ether, dichloromethane, chloroform, ethyl acetate, or a mixed solvent thereof. It can be used by concentrating and / or drying it.

In the present invention, the term "pharmaceutically acceptable salts" means salts derived from pharmacologically or physiologically acceptable inorganic acids, organic acids and bases. Examples of suitable acid include hydrochloric acid, bromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, Formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, and the like. Salts derived from suitable bases may include alkali metals such as sodium, alkaline earth metals such as magnesium, ammonium and the like.

The pharmaceutical composition for preventing and treating liver injury diseases of the present invention comprises 0.1 to 50% by weight of the extract or compound based on the total weight of the composition. In addition, the composition does not increase the efficacy, but may include additional ingredients that are commonly used in the pharmaceutical composition to improve the smell, taste, time and the like. In addition, the composition adds inorganic and organic additives such as vitamins B1, B2, B6, C, E, niacin, carnitine, betaine, folate pantothenic acid, biotin, zinc, iron, calcium, chromium, magnesium, and mixtures thereof. It can be included as. In addition, the composition may include a substance having a therapeutic activity against liver damage, used alone or in the past.

As used herein, the term "patient" refers to humans and horses, sheep, pigs, goats, and camels that have diseases caused by liver damage and their immediate and indirect causes, and whose symptoms may be improved by administering the compositions of the present invention. Means animals such as nutrition, dogs. By administering to a patient a composition comprising an extract of the present invention, the liver damage mentioned above can be effectively prevented and treated. The composition of the present invention can be administered in parallel with existing therapeutic agents for liver injury.

As used herein, the term "administration" means introducing a predetermined substance into a patient by any suitable method, and the route of administration of the composition of the present invention is oral or parenteral via any general route as long as the target tissue can be reached. May be administered. In addition, the composition may be administered by any device in which the active agent may migrate to the target cell.

The composition of the present invention is administered in a pharmaceutically effective amount. As used herein, the term “pharmaceutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dose level refers to a patient's sexually transmitted disease, age, severity, and drug activity. , Drug sensitivity, time of administration, route of administration and rate of release, duration of treatment, factors including concurrently used drugs, and other factors well known in the medical arts. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. It may be single or multiple doses. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without adverse effect, and can be easily determined by those skilled in the art. The method of administering the composition comprising the extract or compound prepared according to the preparation method of the present invention is preferably oral administration or intravenous administration. In general, when the effective dose is oral administration, it is usually 1 to 1 time per adult. 500 mg / kg is preferred, and in the case of intravenous administration, 1 to 100 mg / kg is preferred, and may be administered 2-3 times a day. Dosage levels for a particular patient may vary depending on sex, age, health condition, diet, time of administration, method of administration, drug mixture, the condition of the patient, and the extent of the onset of neurological disease.

In addition, the present invention provides a food composition for preventing and improving liver damage, including as an active ingredient extracts of medicinal herbs, including earthworms, roots and pesticides, including food acceptable additives.

In addition, the extract according to the present invention may be provided in the form of a food composition for the purpose of preventing and improving liver damage. The food composition of the present invention includes all forms such as functional food, nutritional supplement, health food and food additives. Food compositions of this type may be prepared in a variety of forms according to conventional methods known in the art.

For example, as a health food, the extract of the present invention may be prepared in the form of tea, juice and drink for drinking, or may be ingested by granulation, encapsulation and powdering. In addition, the extract of the present invention can be prepared in the form of a composition by mixing with a known substance or active ingredient known to have the effect of preventing and improving liver damage.

Functional foods also include beverages (including alcoholic beverages), fruits and their processed foods (e.g., canned fruits, bottled, jam, maalmalade, etc.), fish, meat and processed foods such as ham, Etc.), breads and noodles (eg udon, buckwheat noodles, ramen noodles, spaghetti, macaroni, etc.), fruit juice, various drinks, cookies, Retort food, frozen food, various kinds of seasoning (for example, soybean paste, soy sauce, sauce, etc.) by adding the extract of the present invention.

In addition, in order to use the extract of the present invention in the form of a food additive, it may be prepared and used in powder or concentrate form.

In the present invention, "functional food" means a food manufactured and processed using raw materials or ingredients having functional properties useful for the human body according to Act No. 6767 of the Health Functional Food Act, and "functional" refers to the structure of the human body And ingestion for the purpose of obtaining nutrients for function or for obtaining useful effects in health uses such as physiological actions.

When excessive amount of APAP is administered to a living body, necrosis of liver tissue releases intracellular ALT and AST, and it enters the serum and increases serum ALT and AST, causing death at 24-96 hours (Ray SD et. Al., J Pharmacol). Exp Ther. 1996; 279: 1470-83; Webster PA et.al., J Clin Pharmacol. 1996; 36: 397-402; Albano E et. Al., Mol Pharmacology. 1985; 28: 306-11). This characteristic increases the time it takes for the clot to form, namely prothrombin time (PT), as well as increases in transaminases and bilirubin concentrations and necrosis of liver tissue (Hinson JA et. Al., Drug Metab Dispos. 1980; 8: 289-94). At this time, AST and ALT, which are well known as indicators of liver toxicity, increase and enter the serum. The mechanism of hepatotoxicity by APAP is known to be dependent on P450 cytocrome C (Webster PA et. Al., J Clin Pharmacol. 1996; 36; 397-402; Albano E et. Al., Mol Pharmacology. 1985; 28: 306-11). In the present invention, whether the extract according to APAP administration inhibits the protective effect and mortality against liver toxicity. As a result, compared to the mortality rate of the control group administered only APAP, the extract of the present invention showed a survival effect in both the 100 mg / kg and 200 mg / kg administration group. In addition, the plasma AST and ALT increased significantly compared to the control group administered with APAP only, whereas the experimental group administered with the extract of the present invention decreased depending on the concentration. It gave evidence that the organization could be restored. In addition, when APAP is administered to a living body, the drug is metabolized to produce a large amount of free radicals, thereby depleting the antioxidants and enzymes of the living body (Ray SD et. Al., J Pharmacol Exp Ther. 1996; 279: 1470-83 Webster PA et. Al., J Clin Pharmacol. 1996; 36: 397-402; Albano E et. Al., Mol Pharmacology. 1985; 28: 306-11). The present invention examined the activity of GSH and GSH-px, etc. in order to determine the antioxidant activity in vivo in mice administered APAP and the extract of the present invention. As a result, as shown in FIG. 3, the control group administered with APAP was depleted about 57% and 65% of GSH and GSH-px, respectively, compared to the normal control group, whereas the experimental group to which the extract of the present invention was administered was increased with increasing concentrations of GSH and GSH. The content of -px was increased. In particular, the 100 mg / kg and 200 mg / kg administration groups showed a similar increase in the amount of GSH and GSH-px, similar to the normal control group. In vivo, 90% of APAP doses are combined with glucoronic acid or sulfate in hepatocytes and bile is released into the plasma, and 5-10% of APAP is metabolized by P450 (CYP), especially CYP2E1 (Webster PA et. al., J Clin Pharmacol. 1996; 36: 397-402; Albano E et. al., Mol Pharmacology. 1985; 28: 306-11). Toxic substances such as NAPQI, which occur in the metabolism of APAP, result in the depletion of GSH, resulting in fatal damage to the cell membrane, leading to necrosis of the liver and incapacity for recovery (Hinson JA et. Al., Drug). Metab Dispos. 1980; 8: 289-94; Rajesh B and Parames CS.Phytother Res. 2006; l20: 595-601). In addition, inhibition of GSH-px by goldthioglucose has been shown to be sensitive to hepatocytes and thus inhibit the hepatotoxicity induced by APAP (Hinson JA et. Al., Drug Metab Dispos. 1980; 8: 289-94). In order to protect the liver from APAP, there is a need for a drug capable of inducing the improvement of the depleted antioxidant GSH and the antioxidant enzyme GSH-px. On the other hand, when an excessive amount of APAP is administered to a living body, a large amount of leukocytes are introduced to cause acute inflammation, in which IFN-γ, IL-1 and IL-6, including TNF-α, a well known cytokine causing an inflammatory response, The same proinflammatory cytokines are mass produced within 10 hours to accelerate the inflammatory response (Ishida Y et al., J Leukoc Biol. 2004; 75: 59-67; Hogaboam CM et. Al., Am J Pathol. 2000; 156: 1245-52). Moreover, inflammatory mediators such as COX-2 and PGE2 are known to be induced by APAP to further accelerate necrosis of liver tissue16. In the present study, the control group administered with APAP showed massive necrosis of liver tissue, marked infiltration of leukocytes causing inflammation around the vein, and inflammatory mediators such as TNF-α and COX. 6). However, the experimental group administered with the extract of the present invention not only significantly reduced necrosis and inflammatory leukocyte infiltration of liver tissue, but also suppressed the expression of TNF-α and COX-2. In particular, this study demonstrated that the extract 100 mg / kg and 200 mg / kg administration groups significantly inhibited the activity and production of these inflammatory mediators by inhibiting the expression of COX-2 and TNF-α similarly to the normal control group (Fig. 5 and FIG. 6).

The extract of the present invention effectively inhibited ALT and AST increased by APAP, and hepatoprotective effect was increased by increasing GSH and GSH-px known as antioxidants and enzymes of living organisms depleted by APAP. In particular, the inhibitory effect of the extract of the present invention on the activity and production of excess COX-2 and TNF-α, which are inflammation mediators induced by APAP, was confirmed by inhibiting the expression of these molecules. Therefore, the extract of the present invention from liver damage caused by drugs such as APAP is considered to be a good composition that can be utilized for the action of protecting the liver function.

Extracts of the herbaceous earth or earthwood, reeds, and powdered combinations of the present invention, the extract of the present invention reduces the mortality and plasma ALT and AST activity of mice against APAP-induced liver damage, GSH and GSH- in liver cell lysate It can be effectively used for the prevention and treatment of acute liver injury because it increases the px content, reduces the concentration of liver tissue necrosis and leukocyte infiltration by APAP, and inhibits the activity of COX-2 and TNF-α.

1 is a graph showing the effect of the combination extract of the present invention on APAP (acetaminophen) -induced mortality in mice. Normal control mice received only physiological saline and APAP control mice received a single dose (400 mg / kg body weight) of APAP dissolved in physiological saline. Extracts (25, 50, 100 or 200 mg / kg body weight) were administered orally 30 minutes prior to APAP administration. Accumulated survival was measured 48 h later. Data represent mean ± standard deviation of 11 mice. #p <0.001 normal control group treated with saline garlic only contrasted group. *** Control group treated with only AP compared to p <0.001.
2 is a graph showing the effect of the extract of the present invention on APAP-induced hepatotoxicity in mice. Normal control mice received only physiological saline and APAP control mice received a single dose (400 mg / kg body weight) of APAP dissolved in physiological saline. Extracts (25, 50, 100 or 200 mg / kg body weight) were administered orally 30 minutes prior to APAP administration. Hepatotoxicity was measured after 12 h to quantify plasma ALT and AST. Data represent mean ± standard deviation of 11 mice. #p <0.001 normal control group treated with saline garlic only contrasted group. Control group treated with only AP compared to ** p <0.01 and *** p <0.001.
3 is a graph showing the effect of the extract of the present invention on APAP-induced hepatotoxicity in mice. Normal control mice received only physiological saline and APAP control mice received a single dose (400 mg / kg body weight) of APAP dissolved in physiological saline. Extracts (25, 50, 100 or 200 mg / kg body weight) were administered orally 30 minutes prior to APAP administration. After 12 h, liver GSH and GSH-px levels were measured. Data represent mean ± standard deviation of 11 mice. #p <0.001 normal control group treated with saline garlic only contrasted group. Control group treated with only AP compared to ** p <0.01 and *** p <0.001.
Figure 4 is a photograph showing the effect of the extract of the present invention on APAP-induced hepatotoxicity in mice. Normal control mice received only physiological saline and APAP control mice received a single dose (400 mg / kg body weight) of APAP dissolved in physiological saline. Extracts (25, 50, 100 or 200 mg / kg body weight) were administered orally 30 minutes prior to APAP administration. Histological analysis was measured 12 h after H & E staining. Note the narrow area of basophilic hepatocytes and some remaining macrophages around the central vein.
Figure 5 is a photograph and graph showing the effect of the extract of the present invention on APAP-induced COX-2 expression and activity in mice. Normal control mice received only physiological saline and APAP control mice received a single dose (400 mg / kg body weight) of APAP dissolved in physiological saline. Extracts (25, 50, 100 or 200 mg / kg body weight) were administered orally 30 minutes prior to APAP administration. (A) Immunohistologic analysis was measured 12 h later by anti-mouse COX-2 staining. (B) Liver COX-2 activity was measured 12 h later by COX-2 analysis. The data represent mean ± standard deviation of five mice. #p <0.001 normal control group treated with saline garlic only contrasted group. Control group treated only AP compared to * p <0.05, ** p <0.01 and *** p <0.001.
Figure 6 is a photograph and graph showing the effect of the extract of the present invention on APAP-induced TNF-α expression and activity in mice. Normal control mice received only physiological saline and APAP control mice received a single dose (400 mg / kg body weight) of APAP dissolved in physiological saline. Extracts (25, 50, 100 or 200 mg / kg body weight) were administered orally 30 minutes prior to APAP administration. (A) Immunohistologic analysis was measured 12 h later by anti-mouse TNF-α staining. (B) Liver TNF-α was measured 12 h later by TNF-α ELISA analysis. The data represent mean ± standard deviation of five mice. #p <0.001 normal control group treated with saline garlic only contrasted group. Control group treated only AP compared to * p <0.05, ** p <0.01 and *** p <0.001.
On the other hand, the extract of the present invention was expressed in MGB on the drawing.

Hereinafter, the present invention will be described in detail with reference to examples.

However, the following examples are only for exemplifying the present invention, and the contents of the present invention are not limited to the following examples and preparation examples.

Reference Example Statistical Analysis

In the statistical treatment of the present invention, all experimental values were expressed as mean ± standard error (mean ± SD), statistical analysis was performed by Student's t-test, and the significance limit was set to p <0.05.

Example 1 Materials and Methods

<1-1> Preparation of compounds and reagents

Glutathione (GSH) assay kit and glutathione peroxidase (GSH-px) assay kit were purchased from Calbiochem (Darmstadt, Germany) and COX-2 activity assay kit was purchased from Cayman Chemicals (Michigan, USA). Anti-TNF-α, anti-COX-2, anti-mouse IgG phycoerythrin, anti-mouse IgG fluorescein and TNF-α ELISA assay kit were purchased from R & D Systems (Minneapolis, USA). Acetaminophen (N-acety1-paminophenol, paracetamol, APAP) and other reagents were purchased from Sigma-Aldrich (MO, USA) in reagent grade.

<1-2> Preparation of Animal Model

Seven-week-old female Balb / c mice raised in a sterile environment were purchased from Central Animal Laboratory (Seoul). Mice were reared by feeding 12 hours of day and night cycles for 12 hours to relieve stress for one week and feeding them with feed (central animal laboratory) and sterilized water.

<1-3> Extract Preparation

The constituent drugs used in the experiments were purchased from the drug store in Gwangsan (Gwangmyeongdang, Korea) and identified at Woosuk University College of Medicine. Drug composition consists of 84 g of earth, 60 g of root and 56 g of powdered acid, weighing 200 g in total, and making it into powder, adding 10 times the amount of purified water to boil it in a shaker (Daewoong, Korea) for 2 hours, taking the solution at 3,000 rpm. After centrifugation, only the supernatant was collected, filtered using a 0.45 μm filter, concentrated in a rotary vacuum concentrator (Eyela A-1000S, Tokyo Rikakikai Co., Tokyo, Japan), and the sample was recovered and freeze-dried (ILSHIN, Korea). 27.6 g was recovered and used for the experiment while storing at -20 ° C.

<1-4> Administration of Extract

The 8-week-old Balb / c mice, which had relieved stress, were fasted for 18 hours, left for 30 minutes with an extract (25-200 mg / kg) for each concentration, and then orally administered with APAP (400 mg / kg) for 3 hours. Was left for a while, and then sufficient food and water were supplied. As described above, mortality and physiological activity experiments were conducted by APAP for 2 days. The normal control group was orally administered saline, and the APAP-administered group was administered saline to the fasted mice for 30 minutes, and APAP was administered, and saline was supplied in the same amount as that used for extract administration.

<1-5> plasma

Mice of the control group treated with saline, the APAP alone group, and the experimental group treated with APAP and the extract of the present invention were anesthetized 12 hours after APAP administration, and then collected by using a heparin-treated syringe from the hepatic portal vein. After centrifugation at rpm, plasma was obtained, stored at −20 ° C., and used to measure ALT and AST activity.

<1-6> Preparation of liver tissue lysate

After the blood was drawn, liver tissue was quickly removed and maintained in an ice box, and the amount of cooling buffer (0.01 M Tris-HCl, 0.0001 M EDTA-2Na, 0.01 M sucrose and 0.8% physiological saline, pH 7.4) was 7 times greater than liver tissue. After injection into the ground, the liver tissue was ground and centrifuged at 10,000 rpm to obtain a supernatant. The obtained supernatant was quantitated according to the method suggested by the manufacturer using a Bio-Rad Protein Assay Kit (Bio-Rad, USA), and used in the experiments for measuring COX-2 activity and TNF-α production.

<1-7> ALT and AST Measurement

Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured from plasma according to the method suggested by Yeongdong Pharmaceutical (Gyeonggi-do, Korea).

<1-8> GSH and GSH-px Measurement

The activity of GSH and GSH-px in the prepared liver tissue lysates was measured according to the method suggested by Calbiochem. GSH activity was expressed in μmol per g protein, and GSH-px activity was converted to nmol per minute per ml.

<1-9> COX-2 Activity and TNF-α Measurement

COX-2 activity of the prepared liver tissue lysates was measured according to the method suggested by Cayman Chemicals. The activity of COX-2 in liver tissue was determined by the amount of enzyme (nmol / min / mg protein) of 0.1 nM N, N, N ', N'-tetramethyl-pphenylenediamine oxidized.

<1-10> liver tissue

According to the experimental procedure, the control group and the experimental group were sacrificed. About 5 x 5 mm of liver tissue was extracted and fixed with 4% paraformaldehyde (pH 7.4), and paraffin blocks were prepared through a series of processes. Was sectioned into longitudinal sections. Stained with hematoxylin & eosin for histological examination, observed at low magnification (X 40) and photographed at X 200 microscopic field with magnification, and examined at X 400 microscopic field to confirm hepatocellular necrosis (Olympus, Japan) .

<1-11> Immunochemical Staining

Skin tissue buried in paraffin was terminated at 5 μm intervals, attached to slides, paraffin was removed with xylene, and tissue samples were prepared through a series of procedures. The prepared paraffin tissues were blocked with 1% bovine serum albumin for 1 hour, and then diluted anti-COX-2 and anti-TNF-α to 1: 100, and the tissues were sufficiently immersed for 12 hours or more under 4 ° C refrigeration. It was left to stand. Thereafter, goat anti-mouse IgG-PE and goat anti-mouse IgG-FITC were diluted 1: 500, left at room temperature for 1 hour, the remaining antibody was washed well, sutured with a coverslip, and a fluorescent microscope (Olympus, Japan). ) Was examined and photographed for comparative analysis.

< Example  2> APAP  Induced survival rate ALT  And AST  Effect of Extracts of the Invention on Production

 The present invention is to investigate the protective effect of the extract of the present invention against APAP induced liver damage to the mice fasted for more than 18 hours oral administration 25-200 mg / kg body weight of the extract 30 minutes after APAP (400 mg / kg) Was administered orally. Plasma was obtained after 12 hours to measure ALT and AST, and 3 hours after APAP for survival, mice (11 animals per experimental group) were supplied with sufficient food and water, and the extract was administered orally once a day. Mortality was checked for 48 hours.

As a result, as shown in Fig. 1, about 50% of the deaths in the APAP-only control group did not differ from the APAP control in the 25 mg / kg administration group, whereas the survival rate was significantly higher as the concentration was increased. In particular, 100 mg / kg and 200 mg / kg administration group survived 100%. As a result of examining the effect of the extract on the ALT and AST of plasma, as shown in Figure 2 ALT and AST was very high as 4,050 ㅁ 550 IU / L and 4,265 wh 425 IU / L, respectively, the extract 50 mg / kg administration group ( p <0.01), 100 mg / kg and 200 mg / kg administration group (p <0.001) showed a significant decrease in ALT and AST.

< Example  3> APAP  Judo GSH Wow GSH - px  Effect of Extracts on Activity

In addition, the present invention to determine the protective effect of the extract of the present invention against APAP induced liver damage 12 hours after the mouse liver tissue was collected and dissolved and then measured the activity of GSH and GSH-px. As a result, the GSH and GSH-px contents of the liver tissue of the control group APAP-treated as shown in Figure 3 was 5.62 ± 0.51 μM and 105.2 ± 12.5 nM, respectively, significantly decreased compared to the normal control (p <0.001). However, the experimental group administered more than 50 mg / kg of extract increased concentrations of GSH and GSH-px. In particular, GSH and GSH-px contents were increased in the 100 mg / kg administration group (p <0.01) and 200 mg / kg administration group (p <0.001) to the extent similar to the normal control group.

< Example  4> APAP  Protective effect of extracts against induced liver tissue damage

In addition, the present invention in order to determine the protective effect of the extract against APAP induced liver tissue damage to the mice fasted for more than 18 hours oral administration 25-200 mg per kg body weight of the extract after 30 minutes APAP (400 mg / kg) After 12 hours after oral administration, liver tissue was collected, paraffin tissue samples were prepared, and the lesions of the liver tissues were examined by H & E staining under 40 and 200 times microscope. As a result, as shown in FIG. 4, the damage of liver tissue of the control group administered with APAP resulted in a marked increase in inflammatory leukocyte infiltration of about 80% or more of liver tissue necrosis region. Hepatic necrosis and leukocyte infiltration were not improved in the 25 mg / kg administration group compared to the APAP control group, but liver necrosis and leukocyte infiltration were significantly reduced depending on the concentration in the 50 mg / kg administration group. . In particular, the 100 mg / kg and 200 mg / kg extracts showed an effect of more than 90% improvement in liver tissue necrosis and leukocyte infiltration compared to the APAP control group.

< Example  5> APAP  Induction of liver tissue COX -2 and TNF Protective effect of the extract of the present invention on the expression of -α

The present invention also examined the effect of extracts on the expression and activity of inflammatory mediators of APAP-induced inflammatory mediators. Liver tissue specimens, such as those shown in H & E staining of liver tissues, were used to examine the expression of COX-2 and TNF-α. Immunohistochemical staining was performed using the anti-mouse TNF-α antibody attached thereto, and COX-2 activity and TNF-α contents were examined in liver tissue lysates that measured GSH and GSH-px activity. 5A and 6A, the normal control group did not express COX-2 and TNF-α. However, in the APAP control group, the expression of COX-2 and TNF-α was very high in the liver tissue necrosis site. However, the expression of COX-2 and TNF-α was significantly inhibited in the administration group of extract 50 mg / kg or more. In particular, the 100 mg / kg and 200 mg / kg administration groups significantly inhibited the expression of COX-2 and TNF-α, similar to the normal control group. In addition, the present invention examined the contents of COX-2 and TNF-α in order to determine the protective effect of the extract on the activity of COX-2 and TNF-α in APAP-induced liver tissue. As a result, the COX-2 and TNF-α activities of the control group APAP administered significantly increased (p <0.001) as shown in FIGS. 5B and 6B, but 50 mg / kg excluding the extract 25 mg / kg administration group. In the above administration group, COX-2 and TNF-α activities were significantly inhibited in a concentration-dependent manner.

< Example  3> By extract Acute toxicity  exam

The combination medicinal herbs used in the present invention were widely used as a medicinal herb, so it was judged that there would be no problem in stability.

Acute toxicity test was performed using 6-week-old SPF SD rats. Two animals per group were suspended orally administered at a dose of 5 g / kg in suspension of each extract of Example 1 of the present invention in 0.5% methylcellulose solution. After administration of the test substance, mortality, clinical symptoms, and changes in body weight were observed. Hematological and hematological examinations were performed. Necropsy was performed to observe abdominal and thoracic organ abnormalities.

As a result of the test, there were no clinically symptomatic or dead animals in all the animals to which the test substance was administered, and no toxic change was observed in weight change, blood test, blood biochemical test, and autopsy findings. All of the above extracts did not show toxic changes up to 5 g / kg in rats, and the minimum lethal dose (LD ₅₀ ) was determined to be a safe substance of 5 g / kg or more.

< Manufacturing example  1> Preparation of Therapeutic Agent for Liver Damage Containing Extract as an Active Ingredient

The inventors have confirmed that the liver damage treatment effect of the extract is excellent through the above embodiment to prepare a liver damage treatment containing the extract as an active ingredient as follows. In addition, the preparation examples of the following therapeutic agents can be applied not only to therapeutic agents but also to the production of health foods.

<1-1> soft capsule containing the extract of the present invention ( soft gelatin capsules )

20% extract of the present invention, vitamin C 4.5%, vitamin D ₃ 0.001%, manganese sulfate 0.1%, beeswax 10%, palm oil 25%, safflower seed oil 30.399%

 <1-2> Preparation of Intravenous Formulation Containing Extract of the Present Invention

Extract 0.2%, Mannitol 0.3%, Saline 9.5%

<1-3> tablet containing the extract of the present invention

Extract 35% of the present invention, vitamin C 10%, vitamin D ₃ 0.001%, manganese sulfate 0.1%, crystalline cellulose 25.0%, lactose 17.999%, magnesium stearate 2%

Preparation Example 2 Preparation of Functional Foods Containing the Extract of the Present Invention as an Active Ingredient

The present inventors have confirmed that the extract of the present invention is excellent in the liver damage treatment activity through the above embodiment to prepare a functional food containing it as an active ingredient as follows.

<2-1> Production of beverage

Honey 522 mg, thioctoamide 5 mg, nicotinamide 10 mg, riboflavin sodium 3 mg, pyridoxine hydrochloride 2 mg, inositol 30 mg, orteic acid 50 mg, extract 0.48-1.28 mg, 200 ml of water

A beverage was prepared using the above-mentioned composition and content by a conventional method.

<2-2> Of chewing gum  Produce

Gum base 20%, sugar 76.36 ~ 76.76%, extract of the present invention 0.24 ~ 0.64%

1% fruit, 2% water

Chewing gum was prepared using the above-mentioned composition and content by a conventional method.

<2-3> Manufacture of candy

Sugar 50-60%, syrup 39.26-49.66%, extract of the present invention 0.24-0.64%

Orange fragrance 0.1%

The composition and the content of the candy were prepared using a conventional method.

<2-4> Production of biscuit

Force 1st class 88 kg, gravity 1st class 76.4 kg, 16.5 kg per white spirit. 2.5 kg of salt, 2.7 kg of glucose, 40.5 kg of palm shortening, 5.3 kg of ammonia, 0.6 kg of sodium bicarbonate, 0.55 kg of sodium bisulfite, 5.0 kg of rice flour, vitamin B 10.003 kg, tamin B 20.003 kg, milk flavor 0.16 kg, water 71.1 kg, battery Milk powder 4 kg, calcium phosphate 0.1 kg, spray salt 1 kg, spray oil 25 kg, the extract of the present invention 0.2 ~ 0.5 kg

The biscuits were prepared using the above-mentioned composition and content by a conventional method.

<2-5> Production of ice cream

Milk fat 10.0%, nonfat milk solids 10.8%, sugar 12.0%, starch syrup 3.0%, emulsifying stabilizer (span, span) 0.5%, fragrance (strawberry) 0.15%, water 63.31 ~ 62.91%, extract 0.24 ~ 0.64%

Ice cream was prepared using conventional methods using the above composition and content.

<2-6> Manufacture of Chocolate

Sugar 34.36 ~ 34.76%, Cocoa Butter 34%, Cocoa Mass 15%, Cocoa Powder 15%, Lecithin 0.5%, Vanilla Flavor 0.5%, Extract of the present invention 0.24 ~ 0.64%

With the above composition and content, chocolate was prepared using conventional methods.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the invention, and are not intended to limit the scope of the invention. .

Claims (6)

Pharmaceutics for the prevention and treatment of acute liver injury, containing as an active ingredient extracts of medicinal herbs, including barley medicinal herbs, Puerariae Radix and Dioscoreae Rhizoma Composition. According to claim 1, wherein the medicinal herb is a pharmaceutical composition, characterized in that the earth. The method according to claim 1 or 2,
The extract is characterized in that it further comprises a pharmaceutically acceptable carrier. The method according to claim 1 or 2,
50 to 100 parts by weight and 48 to 97 parts by weight acid extract based on 100 parts by weight of the medicinal herbs. The method of claim 1 or 2, wherein the extract is
Iii) adding 5 to 20 times the weight of the medicine and extracting at 80 to 150 ℃ for 1 to 4 hours;
Ii) removing the impurities precipitated in the extraction solution by centrifugation at 1000 to 5000 rpm and filtering the filtered solution through a 0.3 to 0.6 mu m filter; And
Iii) concentrating the filtrate with a rotary vacuum concentrator. Prevention of acute liver damage, containing as an active ingredient an extract of a medicinal herb comprising at least one selected from the group consisting of earth and earth, including food acceptable food supplements Improvement food composition.

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