WO2008010620A1

WO2008010620A1 - Composition comprising the crude drug extracts for improving liver function

Info

Publication number: WO2008010620A1
Application number: PCT/KR2006/003817
Authority: WO
Inventors: Jong Moon Jung; Eu Gene Lee; Seung Sook Lee; Ji Hoon Kim; Kyung Bum Kim
Original assignee: BEN'S LAB Co Ltd
Current assignee: BEN'S LAB Co Ltd
Priority date: 2006-07-21
Filing date: 2006-09-26
Publication date: 2008-01-24
Anticipated expiration: 2009-01-21

Abstract

A composition of the present invention is effective in liver protection and safe to human, so that this composition may be available for a pharmaceutical composition and a health functional food for protection of liver cells and prevention or remedy of liver injury.

Description

COMPOSITION COMPRISING THE CRUDE DRUG EXTRACTS FOR IMPROVING LIVER FUNCTION

Technical Field

[1] The present invention relates to a composition for improving liver function and, more particularly, to a composition including an extract of aloe vera, an extract of green tea leaves, an extract of wormwood, an extract of guava leaves, an extract of asparagus, vitamin C, and vitamin B complex. Background Art

[2] The liver is an organ present in the human body. It functions for storage and circulation of blood, regulation of blood quantity, and detoxification, and is closely related to spirit activity. Since the human body is always exposed to harmful pollutants and toxic substances, the liver is continuously burdened with the process of detoxifying. Additionally, an injury of the human liver caused by a mental stress is a serious problem. A mental relaxation may restore an injured liver, but a busy present- day life does not allow much room for mental relaxation. Furthermore, overdrinking and smoking increase a liver injury, which may often result in a decline in a detoxifying function, a malfunction of a human immunity system, and an outbreak of many diseases.

[3] Carbon tetrachloride, D-galactosamine, etc. are known as substances for inducing liver toxicity. Carbon tetrachloride, which is a kind of aliphatic halogen hydrocarbon, is a xenobiotic chemical to injure a biological membrane and to invite a toxic operation of the liver and the kidney (Bruckner, J. V., Fund. Appl. Toxicology, 6, ppl6-34; Butler, T. C, /. Pharmacol. Exp. Ther., 134, pp311-319).

[4] Recently, in order to prevent or remedy liver toxicity, many experiments on inhibitory action of free radical generation through esculent plants or medicinal plants have been reported (Caragy, A. B., Food Technology, 46, pp65-68, 1992; Liang jun, Y., et al., Biochem. and Biophy. Res. com., 212, pp360-366, 1995; Kim, H. K., et al., Korean J. Food ScL Technol., 27, pp80-85, 1995; Middleton, E., Int. J. Pharmacognosy, 34, pp344-348, 1996). Especially, silymarin (SLM) and biphenyl dimethyl dicarboxylate (BDD) have been used as naturally originated material for medical treatment of liver diseases. SLM is a substance obtained from a fruit of silybum marianum (carduus marianus) which belongs to the composite family. BDD is an artificial compound which is similar with schizandrin which is an ingredient of fruits of schisandra chinensis.

[5] Inventors of the present invention desired to observe the efficacy of liver function improvements of herb composite medicine which is expected to be clinically effective in prevention or remedy against liver toxicity, and thus desired to evaluate a possibility of an effective remedy for liver toxicity.

[6] Aloe vera (Aloe barbadensis Miller) is used in the form of a gel with an aloe peel stripped off. Gel-form aloe vera contains a lot of active ingredients such as polysaccharids which has a high moisturizing capacity, an immunity reinforcing effect, and a curative property, and glycoprotein which decreases blood sugar (Ahn, D. G., the Korean pictorial book of medical herbs, pp280, Kyohak Publishing Company, 2000; Kim, C. M., the colored pictorial book of the herb remedy, pp306, Academy Book Company, 2001).

[7] Green tea leaves are leaves of a tea tree (camellia sinensis), also named tea plant.

Green tea, which is a tea made of non-fermented leaves, is on the increase of consumption according to a recently rising interest in health and is validated with providing a wide variety of health benefits by scientific evidence.

[8] Polyphenol of green tea is known as catechin. The followings have been reported as the staple catechins of green tea: epicatechin(EC), epicatechin-3-gallate, epigal- locatechin (EGC), epigallocatechin-3-gallate (EGCG) (Zhao Wenhua, Chen Junshi. /. Clin. Nutr. 10(2), ppl38~142, 2001).

[9] Wormwood (A. princeps var. orientalis (PAMPAN.) HARA) is a perennial herb and its leaves contain oil in which cineole is included 25 to 30% by weight. Besides, contained in wormwood are terpinen-4-ol, β-carophyllene, linalool, artemisia alcohol, camphor, borneol, etc. The leaves contain tetracosanol, β-sitosterol, 1-chebulachitol, and 1-inositol. The root and the stem contain artemose being similar with inulin. The root also contains a variety of polyin compounds such as heptadec-1, 7, 9,-trien-l l, 13, 15-triyne, tetradeca-8, 10, 12,-trine-6-ene-3-one, and methyl 2-decen-4, 6, 8-triynate. The small branch contains substances acting like oxytoxin. Furthermore, ridentin of sesquiterpene lactone is extracted.

[10] Guava (psidium guajava L.) is a small tree native to America and belonging to the

Myrtaceae family and the Psidium genus. The leaves, the bark of a tree, and the fruit are used for health food and medicinal purposes. With a high propagation power, guava can be cultivated without agricultural chemicals. It is generally known that guava is effective in dysentery and diarrhea.

[11] The most highly contained ingredient of guava is tannin, which is effective in reinforcing a gastrointestinal function, inhibiting aging, and treating cancer. As other ingredients, vitamins and minerals such as oils, gasoline, insulin, vitamin B complex, vitamin C, magnesium, and potassium are contained. Also, medicinal actions such as sterilization, anti-diarrhea, anti-inflammatory, stoppage of bleeding, inhibition of histamine release, decrease in blood sugar, anti-oxidation, and inhibition of vascular permeability are known (Ahn, D. G., the Korean pictorial book of medical herbs, Kyohak Publishing Company, 1999).

[12] Asparagus (Asparagus officinalis L.) is a perennial herb and all of its root, stem, leaf, flower, seedcase and seed contain a kind of flavone. The root and stem contain asparagine, steroidsaponin glycoside, coumarin, carotene, and oil. Among them, steroidsaponin is also referred to as sarsasapogenin. The leaf contains asparagine, carotene, glutathione, rutin (This is contained 100mg% in end parts of a new branch and 30mg% in the stem.), vitamin C (This is contained 25mg% in a new stem and 252.5mg% in a trunk.), carbohydrate (almost glucose), and peptidase. The leaf contains rutin and four kinds of flavone compounds, which are hydrolyzed to quercetin, glucose, and rhamnose. The ripe fruit contains saccharide, fat, capsanthin, and alkaloid. The seed contains glucomannan in which mannose and glucose are 1: 1 in molecular weight ratio. The herb contains asparagin, coniferin, saponin, and helidonin (Jeong, B. S., et al., the Korean medicinal stuff dictionary, YoungLim Book Center, ppl65-166, 1998).

[13] As discussed above, aloe vera, green tea leaves, wormwood, guava leaves, and asparagus have been scientifically studied on their benefits and toxicities. However, no research paper has been yet reported regarding a composition containing extracts from the above for improving liver function.

[14] Therefore, inventors of the present invention have focused their efforts on studying foodstuffs available for improving liver function, so have discovered that a composition containing extracts from aloe vera, green tea leaves, wormwood, guava leaves, and asparagus, vitamin C, and vitamin B complex had a good effect on the improvement in liver function.

[15]

Disclosure of Invention Technical Problem

[16] It is therefore an object of the present invention to provide a composition being effective in protecting liver cells and remedying liver injury. Technical Solution

[17] In order to produce a medicine effective in protecting liver cells and remedying liver injury, the present invention provides a use of a composition including an extract of aloe vera, an extract of green tea leaves, an extract of wormwood, an extract of guava leaves, an extract of asparagus, vitamin C, and vitamin B complex.

[18] The present invention further provides a pharmaceutical composition including an extract of aloe vera, an extract of green tea leaves, an extract of wormwood, an extract of guava leaves, an extract of asparagus, vitamin C, and vitamin B complex, and also including a pharmaceutically permissible carrier, excipient, or diluent.

[19] The present invention still further provides a method for preventing or remedying liver injury by giving a medicine to a human or a mammal, the medicine including an extract of aloe vera, an extract of green tea leaves, an extract of wormwood, an extract of guava leaves, an extract of asparagus, vitamin C, and vitamin B complex, and also including a pharmaceutically permissible carrier or excipient.

[20] In this specification, an extract is dissolved in water, lower alcohol Cl to C4 such as ethanol, methanol, and butanol, or their mixture, and preferably in water.

[21] Additionally, vitamin B complex includes vitamin Bl (thiamin), vitamin B2

(riboflavin), vitamin B3 (niacin), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxine), vitamin B 12 (cobalmin), vitamin B7 (biotin; vitamin H), and vitamin B9 (folic acid; vitamin M).

[22] Preferably, the above composition has the following mixed rates of a 20-40 weight

% aloe vera extract, a 10-30 weight % green tea leaves extract, a 5-20 weight % wormwood extract, a 5-15 weight % guava leaves extract, a 5-15 weight % asparagus extract, a 0.5-5 weight % vitamin C, and a 0.5-5 weight % vitamin B complex.

[23] Extracts of the present invention may be obtained as follows.

[24] First of all, aloe vera, green tea leaves, wormwood, guava leaves, and asparagus are respectively dried and then ground to powder. Dried powder is dissolved in a solvent which is water or lower alcohol such as ethanol, methanol, and butanol. The volume of such a solvent is about 1 to 20 times, preferably about 2 to 7 times, of the weight of dried powder. Alternatively, a solvent may be a mixture of water and lower alcohol, which have a ratio of about 1 :0.1 or 1 : 10. Water is preferable as a solvent. Extraction temperature and time are 20 to 100⁰C, preferably 60 to 8O⁰C, and about 0.5 hours to 2 days, preferably 1 hour to 1 day. A water-hot extraction, an enfleurage extraction, a reflux condensing extraction, or an ultrasonic extraction is carried out once to five times, preferably three times. An extract is then filtrated with a reduced pressure. A filtrated extract is concentrated using a vacuum rotating concentrator with a reduced pressure at 20 to 100⁰C, preferably 20 to 7O⁰C.

[25] The present invention provides a pharmaceutical composition that includes extracts of aloe vera, green tea leaves, wormwood, guava leaves, and asparagus, which are obtained by the above-described method. This composition also includes vitamin C and vitamin B complex.

[26] To investigate the medical benefits of the above composition in the improvement of liver function and the protection of liver cells, several experiments on cells and animals were conducted.

[27] A pharmaceutical composition of the present invention may further include carrier, excipient, and diluent, which are normally used in the manufacture of pharmaceutical compositions.

[28] A pharmaceutical medication of a composition according to the present invention may be given in the form of a pharmaceutically permissible salt, independently, or in combination with other pharmaceutical active compounds.

[29] A pharmaceutical composition of the present invention may be formed into oral types such as powder, granule, pill, capsule, suspension, emulsion, syrup, and aerosol, external remedy types, suppository types, and injection types. Carrier, excipient, and diluent included in a composition may be lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral. Additionally, filler, diluent, binder, humectant, disintegrator, or surfactant may be used to make a medicine. A solid medicine for oral medication may be pill, powder, granule, or capsule, which may be made of the above composition and at least one excipient, e.g., starch, calcium carbonate, sucrose, lactose, and gelatin. Additionally, lubricant such as magnesium stearate or talc may be used. A liquid medicine for oral medication may be suspension, solution, emulsion, or syrup, which may be mixed in wafer or liquid paraffin, and may include humectant, sweetener, aromatic, or preservative. A medicine for non-oral medication may be a sterilized aqueous solution, a non-aqueous solvent, suspension, emulsion, freeze drying medicine, or suppository. As non-aqueous solvent and suspension, vegetable oil such as propylene glycol, polyethylene glycol, and olive oil, or injectable ester such as ethylolate may be used. Witepsol, macrogol, tween 61, cacao oil, laurin oil, or glycerogelatin may be used for suppository.

[30] Preferable dosage of a composition of the present invention may be suitably selectable and depend on a condition and a weight of patient, a degree of disease, a form of medicine, and a path and a term of medication. For good efficacy, dosage of a composition of the invention may be 0.0001 to 100 mg/kg per day, preferably 0.001 to 100 mg/kg per day. Medication may be once a day or several times a day.

[31] A composition of the present invention may be medicated through several paths to mammal such as rat, mouse, livestock, and human. A medication may be conducted by means of oral administration, rectal administration, intravenous injection, intramuscular injection, subcutaneous injection, or intracerebroventricular injection.

[32] With little toxicity and side effect, a composition of the present invention may be safely used even in a long-term use for prevention.

[33] The present invention provides a functional food for protecting liver cells and preventing liver injury, the functional food including an extract of aloe vera, an extract of green tea leaves, an extract of wormwood, an extract of guava leaves, an extract of asparagus, vitamin C, and vitamin B complex, and also including additive with permissible in sitology.

[34] A functional food of the present invention includes the aforesaid composition in the form of powder or extract.

[35] A composition of the present invention may be variously used in medicine, food, beverage, etc. for an improvement in the liver. A composition of the invention may be added to a great variety of food such as beverage, chewing gum, tea, vitamin compound, and additive food, in the form of pill, powder, granule, infusion, capsule, or beverage. A composition may occupy 0.01 to 15 weight % of food or beverage. In case of beverage, a composition may be added with 0.02 to 5g, preferably 0.3 to Ig, in 100ml.

[36] A composition in beverage may contain any other conventional ingredients such as flavor or natural saccharide. As examples of natural saccharide, there are monosaccharide such as glucose and fructose, disaccharide such as maltose and sucrose, polysaccharide such as dextrin, cyclodextrin, xylitol, sorbitol, and erythritol. As examples of flavor, there are natural flavor such as thaumatin and stevia extract (e.g., rebaudioside A and glycyrrhizin), and synthetic flavor such as saccharin and aspartame. A natural saccharide may be generally about 1 to 2Og, preferably about 5 to 12g, in a composition of 100ml.

[37] Furthermore, a composition of the present invention may contain nutrient, vitamin, mineral (electrolyte), natural or synthetic flavor, stain (cheese or chocolate), pectic acid and salt, alginic acid and salt, organic acid, protective colloid, pH conditioner, stabilizer, preservative, glycerin, alcohol, and carbonation reagent. Besides, a composition of the invention may contain flesh of fruit used for juice or beverage. These additives may be added independently or in combination with 0 to 20 weight parts per 100 weight of a composition according to the present invention.

Advantageous Effects

[38] A composition of the present invention is effective in liver protection and safe to human, so that this composition may be available for a pharmaceutical composition and a health functional food for protection of liver cells and prevention or remedy of liver injury.

[39]

Brief Description of the Drawings

[40] FIG. 1 is a diagram showing an experimental result of liver cell toxicity in various densities of a composition according to the present invention.

[41] FIG. 2 is a diagram showing an effect of liver cell protection against t-BHP-induced toxicity of a composition according to the present invention. [42] FIG. 3 is a diagram showing a measurement data of GOT and LDH in a culture medium using a HepG2 cell in which toxicity is induced by t-BHP to verify an effect of liver cell protection of a composition according to the present invention.

[43]

Best Mode for Carrying Out the Invention

[44] The present invention will now be described more fully hereinafter with reference to the embodiments below.

[45] This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, the disclosed embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[46] The principles and features of this invention may be employed in varied and numerous embodiments without departing from the scope of the invention. Mode for the Invention

[47] 1st Embodiment. Manufacture of Extracts

[48] Aloe vera, green tea leaves, wormwood, guava leaves, and asparagus were acquired in a store and a market. Aloe vera, green tea leaves, wormwood, guava leaves, and asparagus, each of which is lkg, were dried and ground. Resultant powders each of which is 300 to 600g were put in a bottle, and distilled water of 2 liters was put in too. Then water-hot extraction was carried out three times at regular intervals (12h, 6h, 3h) with a temperature of 7O⁰C. An extract was filtrated using a filter paper (Whatman, U.S.) with a reduced pressure. Distilled water was removed from a filtrated extract at a temperature of 4O⁰C by using a vacuum rotating concentrator. As a result, aloe vera, green tea leaves, wormwood, guava leaves, and asparagus, which are 15g, 5 Ig, 25g, 24g, and 1Og, respectively, were obtained as water-soluble extracts. By repeating five times the above process, aloe vera, green tea leaves, wormwood, guava leaves, and asparagus were obtained to 75g, 255g, 125g, 12Og, and 50g, respectively.

[49] Respective extracts were prepared in the form of powder by using a spray dryer or a freezing dryer, and kept with absorption of moisture restrained at a temperature of 4⁰C.

[50]

[51] 2nd Embodiment. Manufacture of a Composition Including Extracts

[52] 2-1. Manufacturing Example (I) of a Composition

[53] An aloe vera extract of 35g, a green tea leaves extract of 15g, a wormwood extract of 15g, a guava leaves extract of 1Og, and an asparagus extract of 15g, which were obtained according to the 1st embodiment, were mixed with vitamin C of 5g, vitamin B l of 1.5g, vitamin B2 of Ig, and vitamin B3 of 2.5g. Therefore, this composition (hereinafter, referred to as BS-I) was lOOg in total. [54]

[55] 2-2. Manufacturing Example (2) of a Composition

[56] An aloe vera extract of 2Og, a green tea leaves extract of 2Og, a wormwood extract of 1Og, a guava leaves extract of 2Og, and an asparagus extract of 15g, which were obtained according to the 1st embodiment, were mixed with vitamin C of 5g, vitamin B 1 of 3g, vitamin B2 of 2g, and vitamin B3 of 5g. Therefore, this composition (hereinafter, referred to as BS-2) was also lOOg in total.

[57]

[58] 2-3. Manufacturing Example (3) of a Composition

[59] An aloe vera extract of 1Og, a green tea leaves extract of 30g, a wormwood extract of 15g, a guava leaves extract of 22g, and an asparagus extract of 7g, which were obtained according to the 1st embodiment, were mixed with vitamin C of 1Og, vitamin B 1 of 2g, vitamin B2 of Ig, and vitamin B3 of 3g. Therefore, this composition (hereinafter, referred to as BS-3) was also lOOg in total.

[60]

[61] 1st Experimental Example. Experiment for an Effect of Liver Cell Protection of a Composition Including Extracts

[62] 1 - 1. Toxicity Experiment of Liver Cells

[63] A HepG2 cell, which is a human hepatocellular carcinoma cell line, was received from Korean Cell Line Bank and subcultured. A culture medium was MEM-α containing penicillin G of 100 IU/ml and streptomycin of 100 mg/ml, mixed with FBS which is 10% of a total amount, and regulated to pH 7.4. Then a HepG2 cell was cultivated in an incubator under 37⁰C, 5% CO for 2-3 days. Cells were separated through trypsin-EDTA treatment and regulated to 1X10 cell/ml per well. Then cells of 100 μl were put in each well and preliminarily cultivated in a CO incubator under 37°C, 5% CO for 24 hours. After a culture medium was removed, cells were treated with compositions having various densities and cultivated in a CO incubator under 37°C, 5% CO for 48 hours. Then a survival rate of cells was measured by means of MTT assay.

[64] MTT assay was conducted as follows, using a technique proposed by Sladowski D. et al., An improved MTT assay. J. Immun. Methods, 157, pp203~207, 1993. An MTT solution of 5 μg/ml with PBS was applied to 96 well plates in which HepG2 cells were cultivated. These well plates were left in a CO incubator under 37⁰C, 5% CO for 2

2 2 hours. After supernatant was removed, DMSO and EtOH (1:1 v/v) of 100 μl were put in each well and shaken for 20 minutes. Then an optical density was measured at 570nm by means of an ELISA reader to verify that MTT pigment (3-(4,5)-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) forms blue formazan by mitochondrial dehydrogenase. [65] Liver cell toxicity by density of a composition was observed as the former step of an experiment to observe a protection effect of a BS-I composition against oxidizing injury of liver cells and necrosis of cells. HepG2 cells were treated with compositions of several densities and cultivated in a CO incubator under 37⁰C, 5% CO for 48

2 2 hours. When a cell survival rate was measured through MTT assay, a survival rate of cells to which compositions having the maximum density of 5,500 μg/ml were added was 85.5% in comparison with a survival rate of cells in a control group with no treatment was defined as 100%. On the other hand, a survival rate of cells in an experimental group treated with a density not exceeding 2,750 μg/ml was 100-150%. This means that no necrosis of cells was caused by a composition. It was therefore verified that a pretreatment with a composition having a density not exceeding 2,750 μg/ml did not induce a significant toxicity in cultivated liver cells (See FIG. 1). [66]

[67] 1-2. Activity of Liver Cell Protection against t-BHP-Induced Toxicity

[68] This experiment was carried out in order to observe a protection effect of a BS-I composition against oxidizing injury and necrosis of human hepatocellular carcinoma cells caused by t-BHP (tert-butyl hydroperoxide). Liver cells were distributed in 96 well plates at 1X10 cell/ml per well and preliminarily cultivated in a CO incubator under 37⁰C, 5% CO for 24 hours. Cells were treated with compositions having various densities and cultivated in a CO incubator under 37⁰C, 5% CO for 48 hours. Then t-

2 2

BHP (final density ImM) was added, and cells were cultivated again in a CO incubator under 37⁰C, 5% CO 2 for 5 hours. And a survival rate of cells was measured by means of MTT assay.

[69] MTT assay was conducted as follows, using a technique proposed by Sladowski D. et a\., An improved MTT assay. J. Immun. Methods, 157, pp203~207, 1993. An MTT solution of 5 μg/ml with PBS was applied to 96 well plates in which HepG2 cells were cultivated. These well plates were left in a CO incubator under 37°C, 5% CO for 2 r 2 2 hours. After supernatant was removed, DMSO and EtOH (1:1 v/v) of 100 μl were put in each well and shaken for 20 minutes. Then an optical density was measured at 570nm by means of an ELISA reader to verify that MTT pigment (3-(4,5)-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) forms blue formazan by mitochondrial dehydrogenase.

[70] HepG2 cells were treated with compositions of several densities, and t-BHP was added up to ImM. Then cells were cultivated in a CO incubator under 37⁰C, 5% CO

2 2 for 5 hours, and a cell survival rate was measured through MTT assay. As the result of measurement, a survival rate of cells in an experimental group treated with t-BHP only was remarkably reduced to 32.04% in comparison with a survival rate of cells in a control group with no treatment was defined as 100%. On the other hand, a survival rate of cells in another experimental group pretreated with compositions of 2,750, 1,375, 687.5, 343.8, and 171.9 μg/ml per well and then treated with t-BHP was 149.62, 150.57, 97.34, 52.79, and 50.81%, respectively. Especially, it was verified that a survival rate of cells was remarkably increased in a jointly treated group of 2,750, 1,375, and 687.5 μg/ml than in a singly treated group of t-BHP (See FIG. 2).

[71]

[72] 1-3. Effect of Liver Protection by Serological Index

[73] This experiment was carried out in order to measure the activity of GOT and LDH in a cell culture medium of a BS-I composition. Liver cells were distributed with 3ml in 6 wells at 5X10 cell/ml and preliminarily cultivated for 24 hours. Cells were treated with compositions by density and cultivated in a CO incubator under 37⁰C, 5% CO for 48 hours. Then t-BHP (final density ImM) was added, and cells were cultivated again in a CO incubator under 37⁰C, 5% CO for 5 hours. After each cell culture

2 2 medium was obtained, a measurement of GOT and LDH activity was conducted using assay kit of Asan Pharmaceutical (Korea). As a control medicine, silymarin (S0292, Sigma, USA) having a liver protecting function was used.

[74] LDH (lactate dehydrogenase) is a kind of enzyme that transforms lactate into pyruvate. When there is an abnormal cell, LDH is drained from a cell and increased in a culture medium. GOT (glutamic oxaloacetic transaminase) is a kind of enzyme that exists in a liver cell. When the cell membrane is destroyed due to liver injury, GOT is drained from a cell and increased. That is, an injury of liver cells increases the amount of these enzymes separated to a culture medium, and medicinal substances having an effect of liver cell protection may decrease these enzymes.

[75] An effect of liver cell protection of a composition was examined by using a HepG2 cell in which toxicity is induced by t-BHP and by measuring GOT and LDH in a culture medium. In comparison with a control substance, silymarin, GOT was measured to a reduction of 10~25% in case of treatment using BS- 1 composition of 687.5-1,375 μg/ml density, and to a reduction of 73.39% at 2,750 μg/ml. LDH was measured to a reduction of 31.3-34% in case of treatment of 171.9-343.8 μg/ml density, and to a reduction of 91.28-95.76% at 687.5-2,750 μg/ml. It was therefore verified that a composition has a remarkable reduction effect in both GOT and LDH (See FIG. 3).

[76]

[77] 2nd Experimental Example. Measurement of Liver Function Improvement

Effect on Rat with Liver Toxicity Induced by Tetrachloride

[78] 2-1. Preparation of Sample and Laboratory Animal

[79] This experiment used a Sprague-Dawley rat of Orient Bio Inc. (Korea), tetrachloride of Sigma (USA), a corn oil of CJ (Korea), and a kit of Asan Phar- maceutical (Korea). This kit was used to measure the activity of enzymes such as GPT (glutamate pyruvate transaminase), GOT (glutamate oxaloacetate transaminase), and LDH (lactate dehydrogenase).

[80] A male rat of Sprague-Dawley, which was 260-28Og and 7 weeks old, was adapted to an environment for 1 week. This male rat freely took feed (Samtako, Korea) and water in an animal facility in which a brightness cycle was automatically regulated every 12 hours (07:00^s 19:00) at a temperature of 22-25°C.

[81]

[82] 2-2. Effect on Rat with Liver Toxicity Induced by Tetrachloride

[83] Ten rates formed each group, and a saline solution was used for a control group

(named normal in table). A composition of BS-I was orally administered once a day at the fixed time with 1.8g per weight kilogram for 10 days. Experimental groups were classified into three groups, namely, a control group (normal), a group with tetrachloride (CCl ) only, and a group with tetrachloride (CCl ) after treated with a composition (BS-I).

[84] In order to induce liver toxicity, tetrachloride was dissolved in a corn oil at a ratio of 1:1 (v/v) and administered by an intraperitoneal injection with 2.0 ml/kg/day. In a control group, a corn oil only was administered.

[85] After 48 hours from tetrachloride administration, an incision was conducted along peritoneum midline under ether anesthesia. Then blood was collecting from the heart, and the liver was extracted. The extracted liver was washed with a saline solution and compressed with a filter paper to remove a remaining saline solution, and then the weight was measured. Blood was left at a room temperature for 30 minutes and cen- trifugally separated at 3,000 rpm for 15 minutes. Separated blood serum was used for analysis (See Table 1).

[86] It was verified that tetrachloride destroyed cell membrane of the liver and increased permeability. It was also verified that tetrachloride caused edema and fatty degeneration and thereby the lipid level of the liver increased to cause hypertrophy. Table 1 shows the effects of tetrachloride administration on the weight of the liver. In comparison with a control group, there was an 8.7% increase of a liver weight in a tetrachloride single group. And, in comparison with a tetrachloride single group, a tetrachloride group treated with a composition exhibited an effect of 28% improvement.

[87] As shown in Table 1, the activity of enzymes GOT and GPT in blood plasma was increased because transaminase was separated into blood according to the progress of necrosis of liver cells and destruction of liver tissue. The activities of GOT and GPT in blood plasma were significantly improved to 53% and 58% in a tetrachloride group treated with a composition than a tetrachloride single group. [88] The activity of LDH in blood plasma is increased in the early stage of acute hepatitis. The activity of LDH was reduced on a level with normality in a tetrachloride group treated with a composition.

[89] [90] Table 1

Experimentalg Liver wt/g GOT(Karmen/m GPT LDH(Wroblewsk roup L) (Karmen/mL) i unit)

Normal 8.55 + 0.46 52.72 d - 8.00 18.24 = b 2.56 89.50 =! : 22.09

CC14 9.30 ± 0.52 97.68 d 1 18.39 34.40 : b 10.67 140.56 ± 15.59

BS-I + CC14 9.09 ± 0.57 73.70 d b 9.49 24.98 = b 5.23 68.37 =! : 21.52

[91] [92] 3rd Experimental Example. Experiment of Acute Toxicity [93] For safety verification of a composition of the invention, an experiment of single oral administration acute toxicity was conducted as follows. A Sprague Dawley outbred rat of 6 weeks was used as an experimental animal. Five males and five females were used for experimental groups, and one male and one female were used for a control group (normal). Enough water was supplied for 12 hours before oral administration of a BS-I composition, and no feed was supplied. A composition was orally administered with 5g per weight kilogram of a rat. After administration of a composition, feed was normally supplied. Then behavior and abnormal symptom of a rat were observed by the naked eye for 14 days.

[94] [95] Table 2

[96] [97] Table 3

[98] [99] As the result of an acute toxicity experiment, no rat died after administration of a composition as shown in Tables 2 and 3. Additionally, a health condition was good, and a weight was normal. When dissection was conducted after 14 days, it was observed by the naked eye that all organs were normal. Therefore, the safety of a composition according to the present invention was verified.

[100] [101] 1st Clinical Example. Simple Clinical Experiment [102] In order to measure the medical benefits of a BS-I composition regarding the improvement of liver function and the protection of liver cells, three hospitals M, L and J conducted an experiment on two men of late thirties and two men of late forties, who had high GOT and GPT. In this experiment, a composition of 27.5g was dissolved in distilled water of IL. Each patient took 20ml (a BS-I composition of 550mg) twice or three times a day for 2 or 3 weeks. Then GOT and GPT were measured as shown in Table 4.

[103] [104] Table 4

[105] [106] As shown in Table 4, it was verified that a composition has a distinguished effect on improvements in liver function. Also, doctors in the above hospitals gave their views on the effect of a composition.

[107] Patient Kim in hospital M took, together with a composition (three times a day), lamivudine Zefix (GlaxoSmithkline PIc, UK) and Aldactone (Pfizer Inc., USA) once a day and one tablet per once. As a result, it was verified that a composition was effective in improving liver function.

[108] Patient Hwang in hospital M took only a composition due to hardening of the arteries and fatty liver. As a result, a GOT value was dropped and it was verified that a composition was effective in improving liver function.

[109] Patient Jeong in hospital L initially had slight fatty liver and then generally rallied after administration of a composition for 2 weeks.

[HO] [111] 1st Medicine Example. Powder Medicine [112] BS-I 20mg [113] Lactose lOOmg

[114] Talc lOmg

[115] The above ingredients are mixed and then filled into airtight packing.

[116]

[117] 2nd Medicine Example. Pill Medicine

[118] BS-2 10mg

[ 119] Corn dextrin lOOmg

[120] Lactose lOOmg

[121] Stearic acid magnesium 2mg

[122] The above ingredients are mixed and then made according to a conventional method for manufacturing a pill medicine. [123]

[124] 3rd Medicine Example. Capsule Medicine

[125] BS-3 lOmg

[126] Crystalline cellulose 3mg

[127] Lactose 14.8mg

[128] Magnesium stearate 0.2mg

[129] According to a conventional method for manufacturing a capsule medicine, the above ingredients are mixed and then filled into a gelatin capsule. [130]

[131] 4th Medicine Example. Inj ection Medicine

[132] BS-I lOmg

[133] Mannitol 180mg

[134] Sterilized distilled water for injection 2974mg

[135] Na HPO H O 26mg

2 4,12 2 °

[136] According to a conventional method for manufacturing an injection medicine, the above ingredients are contained in an ampule (2ml). [137] [138] 5th Medicine Example. Liquid Medicine

[139] BS-1 20mg

[ 140] Isomerized sugar 1 Og

[141] Mannitol 5g

[142] Refined water suitable amount

[143] According to a conventional method for manufacturing a liquid medicine, the above ingredients are dissolved in refined water, and lemon incense is somewhat added thereto. After total volume is regulated to 100ml by adding refined water, liquid is filled in a brown bottle and sterilized.

[144] [145] 6th Medicine Example. Health Functional Food

[146] BS-2 l.OOOmg

[147] Zinc oxide 1.5mg

[148] Magnesium oxide 12mg

[149] Stearic acid magnesium 1.5mg

[150] PH color I 9mg

[151] Hydroxy propyl methylcellulose 6.8mg

[152] Glycerin fatty acid ester 0.35mg

[153] Vitamin B 30mg

[154] Vitamin C 12mg

[155] Vitamin E 23mg

[156] A mixing ratio of vitamin and mineral may be varied. According to a conventional method for manufacturing a health functional food, the above ingredients are mixed and made in the form of granule.

[157]

[158] 7th Medicine Example. Health Function Beverage

[159] BS-3 l.OOOmg

[ 160] Citric acid 1 ,000mg

[161] Oligosaccharide lOOg

[ 162] Condensed extract of apricot 2g

[163] Taurine Ig

[ 164] Total 900ml with refined water

[165] According to a conventional method for manufacturing a health functional beverage, the above ingredients are mixed and agitated with heat at a temperature of 85⁰C for about 1 hour. A resultant solution is filtered and then filled into a sterilized bottle of 2L. A mixing ratio of the above ingredients may be varied. Industrial Applicability

[166] As discussed hereinbefore, a composition of the present invention is effective in liver protection and safe to human, so that this composition may be available for a pharmaceutical composition and a health functional food for protection of liver cells and prevention or remedy of liver injury.

Claims

[1] A pharmaceutical composition for protection of liver cells and prevention or remedy of liver injury, the composition comprising an extract of aloe vera, an extract of green tea leaves, an extract of wormwood, an extract of guava leaves, an extract of asparagus, vitamin C, vitamin B complex, and at least one of a pharmaceutically permissible carrier, excipient, and diluent.

[2] The pharmaceutical composition of claim 1, wherein the extract of aloe vera has a mixed rate of a 20-40 weight %, the extract of green tea leaves has a mixed rate of 10-30 weight %, the extract of wormwood has a mixed rate of 5-20 weight %, the extract of guava leaves has a mixed rate of 5-15 weight %, the extract of asparagus has a mixed rate of 5-15 weight %, the vitamin C has a mixed rate of 0.5-5 weight %, and the vitamin B complex has a mixed rate of 0.5-5 weight %.

[3] The pharmaceutical composition of claim 1, wherein each of the extracts is dissolved in one of water, lower alcohol Cl to C4 including ethanol, methanol, and butanol, and mixture thereof.

[4] A use of a composition in order to produce a medicine effective in protecting liver cells and remedying liver injury, the composition comprising an extract of aloe vera, an extract of green tea leaves, an extract of wormwood, an extract of guava leaves, an extract of asparagus, vitamin C, and vitamin B complex pharmaceutical composition.

[5] A method for preventing or remedying liver injury by giving a medicine to a human or a mammal, the medicine comprising an extract of aloe vera, an extract of green tea leaves, an extract of wormwood, an extract of guava leaves, an extract of asparagus, vitamin C, vitamin B complex, and at least one of a pharmaceutically permissible carrier and excipient.

[6] A functional food for protecting liver cells and preventing liver injury, the functional food comprising an extract of aloe vera, an extract of green tea leaves, an extract of wormwood, an extract of guava leaves, an extract of asparagus, vitamin C, vitamin B complex, and additive with permissible in sitology.

[7] The functional food of claim 6, wherein the food is in the form of one of powder, pill, granule, capsule, and beverage.