KR101059075B1 - Composition for the treatment and prevention of alcoholic liver disease containing non-leaflet extract - Google Patents

Abstract

The present invention relates to a composition containing a non-leaflet extract, wherein the non-leaflet extract of the present invention exhibits a relatively high scavenging activity in terms of DPPH radical scavenging ability as well as a lipid peroxidation inhibitory ability. In the alcoholic liver injury model, showing strong potency to inhibit liver damage, the non-leaflet extract of the present invention can be usefully used as a composition for the treatment and prevention of alcoholic liver disease.

Non-leaflets, DPPH, lipid peroxidation inhibitory, alcoholic liver damage

Description

A composition comprising the extract of Eriobotryae Folium for treating and Preventing alcoholic liver disease}

The present invention relates to a composition for the treatment and prevention of alcoholic liver disease containing non-leaflet extract.

Nishioka et al., Biological and Pharmaceutical Bulletin, 25: 1053-1057 (2002)

Taniguchi et al., Phytochemistry, 59: 315-323 (2002)

Shimizu et al., Chemical and Pharmaceutical Bulletin,

34: 2614-2617 (1986)

De Tommaasi et al., Planta Medica, 57: 414-416 (1991)

Jung et al., Archives of Pharmacal Research, 22: 213-218.

(1999)

Taniguchi et al., Phytochemistry, 59: 315-323 (2002)

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

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

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

Loquat leaf ( Eriobotryae Folium ) is the leaf part of loquat ( Eriobotrya japonica ), an evergreen tree belonging to the Rosaceae family. In oriental medicine, fruits, roots, stem bark and leaves are used for medicinal purposes. It is known that non-leaflets have the effect of clearing the lungs, harmonizing the stomach, strengthening the group, and cutting down the wall. The major components of the non-leaflets contain many triterpenoid compounds including ursolic acid, oleanolic acid, maslinic acid, and tometic acid (Taniguchi et al., Phytochemistry, 59: pp315-323 (2002)). Others have been reported to contain polyphenols such as sesqui terpene glycosides and flavonoid glycosides (Shimizu et al., Chem. Pharm. Bull., 34: pp2614-2617 (1986)); De Tommaasi et al., Planta Medica, 57: pp 414-416 (1991); Jung et al., Archives of Pharmacal Research, 22: pp213-218 (1999); Taniguchi et al., Phytochemistry, 59: pp315-323 (2002)).

Accordingly, the inventors showed that the non-leaflet extract exhibited relatively high scavenging activity and high lipid peroxidation inhibitory activity as compared with BHT, which is a synthetic antioxidant, as well as high level of liver damage suppressing effect in alcoholic liver damage model. The present invention has been found to be useful for the treatment and prevention of non-lobe alcoholic liver disease.

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

Extracts as defined herein are C ₁ to C ₄ lower alcohols, such as water, including purified water, methanol, ethanol, butanol, or mixed solvents thereof, preferably water, 1-100% water and ethanol mixed solvents, more preferably Preferably 1 to 30% water and extracts soluble in a mixed solvent of ethanol.

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

Hereinafter, the present invention will be described in detail.

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

The non-leaflet extract as defined herein is about 1 to 30 times the sample weight of water, preferably about 5 to 15 times the water, C ₁ to C ₄ lower alcohols or mixed solvents thereof, preferably water and ethanol Mixed solvent, more preferably water, 1 to 100% water and ethanol mixed solvent, still more preferably 1 to 30% water and ethanol mixed solvent as extraction solvent, 0 ℃ to 350 ℃, preferably 50 ℃ 1 to 10 times by hot water extraction, heat extraction, preferably hot water extraction for about 1 hour to 24 hours, preferably about 2 hours to 5 hours at a temperature of about 300 ° C., more preferably 70 ° C. to 100 ° C. Preferably, the extract obtained after repeated extraction 2 to 7 times may be filtered, concentrated under reduced pressure and dried to obtain the non-foliar extract of the present invention.

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

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

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

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

Compositions comprising extracts according to the invention are formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterile injectable solutions, respectively, according to conventional methods. The carriers, excipients and diluents that may be used in the composition comprising the extract of the present invention may also include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber. Alginate, gelatin, calcium phosphate, calcium silicate, cellulose rose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil Can be. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations may include at least one excipient such as starch, calcium carbonate, sucrose in the extract. ) Or lactose, gelatin and the like are mixed. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

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

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

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

Therefore, the present invention also provides a food and food additive containing the non-leaflet extract as an active ingredient having an effect of preventing and improving alcoholic liver disease.

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

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

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

In addition to containing the extract as an essential ingredient in the indicated ratio, the health beverage composition of the present invention is not particularly limited in the liquid component and may contain various flavors or natural carbohydrates as additional ingredients, as in general beverages. Examples of the above-mentioned natural carbohydrates include monosaccharides such as disaccharides such as glucose and fructose such as maltose, sucrose and the like and polysaccharides such as dextrin, cyclodextrin and the like Sugar, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of natural carbohydrates is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. The compositions of the present invention may also contain pulp for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components can be used independently or in combination. The proportion of such additives is not so critical, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

The non-leafy 5% ethanol extract of the present invention not only showed high scavenging activity in terms of DPPH radical scavenging activity but also inhibited lipid peroxidation in comparison with BHT, a synthetic antioxidant. In addition, the non-leaflet extract of the present invention can be usefully used as a composition for the treatment and prevention of alcoholic liver damage.

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

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

Example 1 Preparation of 5% Ethanol Extract of Non-leafed

36 kg of 5% ethanol mixed solvent was added to 3.6 kg of non-leaflets (collected from Jangheung Arboretum, Jangheung-gun, Jeonnam), and extracted by heating at 100 ° C for 4 hours. The filtrate was concentrated under reduced pressure and freeze-dried at -40 ° C for 48 hours. 720 g (yield: 20.0%) of non-leaflets 5% ethanol extract of the present invention was obtained and used as a sample of the following experimental example (hereinafter referred to as 'EFE-5%').

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

In order to confirm the radical scavenging ability using the DPPH (2,2-diphenyl-1-picrylhydrazyl) reagent of the non-leaflet extract of the present invention obtained in the above example, the experiments described in the literature were applied as follows (Antioxidant properties of cereal products.Journal of Food Science, 67, pp 2600.2603.)

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

In 20 μl of freeze-dried non-leaflets extract (EFH, EFE-5% is dissolved in distilled water, concentrations are all 0, 20 μg / ml) 0.25 mM DPPH (2,2-diphenyl-1-picrylhydrazyl) solution (Sigma Manufacturer, D9132) after the addition of 180 μl was reacted for 30 minutes at 37 ℃ and measured at 517 nm (BIO-TEK manufacturer, 1010-1) and the DPPH radical scavenging ability by measuring the degree of absorbance decrease with the control.

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

As a result, as shown in FIG. 1, non-leafy 5% ethanol extract (EFE-5%) was compared with BHT, a synthetic antioxidant of the same concentration, in the radical scavenging activity using DPPH (2,2-diphenyl-1-picrylhydrazyl) reagent. It showed relatively high radical scavenging activity.

Experimental Example 2 Lipid Peroxidation Inhibitory Activity

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

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

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

As a result, as shown in Figure 3, non-leafy 5% ethanol extract (EFE-5%) showed a relatively higher lipid peroxidation inhibitory activity than BHT, a synthetic antioxidant of the same concentration.

Experimental Example 3. Experiment of inhibiting alcoholic liver damage

The inhibitory effect of alcoholic liver damage of the non-leaflet 5% ethanol extract of the present invention obtained in the above example was measured by applying the method described in the literature as follows (Jamenez-Lpez, JM and AI Cederbaum. 2003. Green tea polyphenol epigallocatechin- 3-gallate protects HepG2 cells against CYP2E1-dependent toxicity.Free radic. Biol. & Med., 36: pp 359-370).

The hepatoprotective activity of the sample was measured in 1 ml of MEM [Minimum Essential Medium (MEM), Hyclone, 01486] was dispensed in 24 well plates at 5 × 10 ⁴ cells / well and incubated for 16 hours.

After 16 hours of incubation, the medium (Minimum Essential Medium; MEM) was removed and each well contained a sample and 200 mM EtOH. Medium containing 3% serum (Minimum Essential Medium (MEM), Hyclone, 01486) was added and reacted in an incubator at 37 ° C. for 24 hours. This process was repeated for 5 days, and on the 7th day, the medium containing the sample and ethanol was removed, and the XTT-PMS solution [1 mg XTT (XTT Sodium Cell Culture Test, manufactured by Sigma, X4626) and 10 g PMS ( phenazine methosulfate, manufactured by Sigma, P9625) / mL] and reacted for 2 hours. The viability of the cells was measured for absorbance at 450 nm using a microplate reader (BIO-TEK, 1010-1) to determine the degree of formation of the generated formazan (BIO-TEK, 1010-1). ) Was measured. As a negative control, only culture medium was used instead of the sample, and only 200 mM ethanol was treated as a positive control.

       As a result, as shown in Figure 3, the non-leaflet 5% ethanol extract showed a strong protective effect against hepatocellular damage caused by alcohol.

Figure 1 shows the results of a comparison experiment of DPPH radical scavenging ability between the non-leaflet 5% ethanol extract and BHT (data is expressed as mean ± SD. The alphabet at the top of the bar is statistically (student t-test); p <0.05) different;

Figure 2 shows the results of a comparative experiment of inhibiting lipid peroxidation between non-lobe 5% ethanol extract and BHT (data is expressed as mean ± SD. The alphabet at the top of the bar is statistically (student t-test); p <0.05) different;

Figure 3 shows the results of a liver protective effect of HepG2 / 2E1 cells of non-lobe 5% ethanol extract (expressed as 100% cell survival in the control group. Data is expressed as mean ± SD. The alphabet of is statistically different from each group (student t-test; p <0.05).

Claims (8)

Pharmaceutical composition for the prevention and treatment of alcoholic liver disease containing non-leaflet extract as an active ingredient. The pharmaceutical composition of claim 1, wherein the extract is an extract available in water including purified water, C ₁ to C ₄ lower alcohols, or a mixed solvent thereof. The pharmaceutical composition of claim 2, wherein the extract is an extract available in a mixed solvent of water and ethanol. The pharmaceutical composition of claim 1, wherein the alcoholic liver disease is alcoholic fatty liver, alcoholic hepatitis, or alcoholic cirrhosis. Health functional food for the prevention and improvement of alcoholic liver disease containing non-leaflet extract as an active ingredient. 6. The dietary supplement of claim 5 in the form of a powder, granule, tablet, capsule or beverage. Food and food additives containing a non-leaflet extract having an effect of preventing and improving alcoholic liver disease as an active ingredient. The food according to claim 7, which is a variety of foods, beverages, gums, teas, vitamin complexes, or dietary supplements of candies.

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