WO2004019963A1

WO2004019963A1 - Composition comprising crude drug extract having treating and preventing activity of hepatic disease caused by viral infection

Info

Publication number: WO2004019963A1
Application number: PCT/KR2003/001727
Authority: WO
Inventors: Jin-Ho Lee; Young-Ho Kim; Ki-Su Lee; Chi-Young Ahn
Original assignee: Bioone Co., Ltd.
Priority date: 2002-08-27
Filing date: 2003-08-26
Publication date: 2004-03-11
Also published as: AU2003253470A1

Abstract

The present invention relates to a composition comprising crude drug extract, specifically, the extract of the Cucumis melon Linn. var. ma-gua (Jing feng seed), Ixeris dentata, Youngia sonchifolia and the combination thereof showing inhibiting effect on the polymerase enzyme activity of hepatitis B virus and hepatitis C virus for the prevention and treatment of hepatic disease caused by viral infection.

Description

COMPOSITION COMPRISING CRUDE DRUG EXTRACT HAVING TREATING AND PREVENTING ACTIVITY OF HEPATIC DISEASE CAUSED BY VIRAL INFECTION

BACKGROUND OF THE INVENTION Technical Field

The present invention relates to a composition comprising crude drug extract having antiviral activity for the prevention and treatment of hepatic disease caused by viral infection.

Background Art

The present invention relates to a composition comprising crude drug extract, specifically, the extract of the Cucumis melon Linn. var. ma-gua (Jing feng seed), Ixeris dentata, Youngia sonchifolia and the combination thereof showing inhibiting effect on the polymerase enzyme activity of hepatitis B and C virus for the prevention and treatment of hepatic disease caused by viral infection.

Over 300 million peopie are chronic carriers of hepatitis B virus (HBN) and over

100 million are chronic carriers of hepatitis C virus (HCN). Among them, over about 2 million peoples suffered from initial hepatitis syndrome and died with progressed hepatic diseases such as hepatic cirrhosis and liver cancer, which cause to death. It has been known that the viral types to cause viral hepatitis are A type, B type, C type, delta type, GBN and so on, and among them, B type and C type viruses are most risky, the mechanism of which has been studied till now (Alter, M. J.; Hepatology, 26(suppl. 1), p62S-65S, 1997).

Although HBN (Hepatitis B virus), a representative virus causing hepatitis among those is DΝA virus, it is a virus vulnerable to mutation. The gene of HBN is about 3.2Kb of incomplete double strand cyclic DΝA having extremely condensed structure being consisted of polymerase gene (pol), viral capsid gene, viral surface antigen gene (HbsAg) and X gene involved with various function including carcinogenic function. HBN is different from common DΝA virus replicating DΝA chain in that 3.5 Kb of pregenomic RΝA chain of HBN is transcribed from DΝA chain by host transcription enzyme and then double stranded DΝA is replicated by HBN specific reverse transcriptase (pol), of which replication process occurs independently with host chromosome. However, it has been discovered that the part of viral DΝA is inserted into host chromosome, which causes host DNA to be unstable, and host cell transcription is promoted by X protein expressed from the DNA, which cause host cell to develop to HCC (Hepatocellular carcinoma) in the end (Di. Bisceglie, A. M., and Bacon, B. R; Scientific American, Oct. p80-85, 1999). As described above, since HBN has similar replication mechanism to RΝA virus although it is DΝA virus, it shows high mutational character corresponding to that of common RΝA virus, particularly in structural change of surface antigen present in HBN envelope, which is a factor that makes virus deviate from humoral and cellular immunologic response and becomes a factor determining the progress of disease or incessant infection.

Hepatitis B having about one to four months of the latent period can be contaminated through blood rather than oral infection and shows acute hepatitis syndrome such as severe fatigue, weak appetites, discomfort at upper right sided abdomen, digestion disorder and so on. Acute hepatitis develops to chronic hepatitis caused by the abnormal immmunologic response of host cell against the virus, the functional deficiency of T cells, endocrinal function such as steroid and various drugs.

Although there are antiviral drugs on the market approved for clinical use, those have several drawbacks. Lamivudine, the nucleoside analog approved by FDA, has been prescribed combined with alpha interferon to treat hepatitis C, however, there are problems such as the occurrence of drug resistant HBN mutants after prolonged treatment, high cost and adverse effect on human body etc (Jaris, B, and Faulds, D.; Drugs, 58(1), plOl-141, 1999). Adefovir, one of other nucleoside analog, shows inhibiting activity against above YMDD motif mutants through its long-term use, however, it has also problems such as the occurrence of drug resistant mutants and renal toxicity due to long term use (Tsiang M et al; Hepatology, 29, pl863-1869, 1999). Alpha interferon (IFΝ-α), expressed protein prepared by genetic recombination method, is frequently used to treat chronic hepatitis C infected patient, however, it has also problems such that a beneficial response to IFΝ-α treatment is observed in only 30% of patients with other adverse side effect and further, there occurs drug resistant hepatitis C mutants through long term use (McHutchison et al. ; New England J. Med., 339, pl485- 1492, 1998). Recently, combination therapies using IFΝ-α and other antiviral drug such as ribavirin are currently in use and have increased the response rate to therapy, however, drug resistant HCN mutant is still a problem with long-term IFΝ-α therapy (Schvarcz, R et al; J. Med. Virol, 46, p43-47, 1995). In the rational drug design for such therapies, one ideally would like to utilize an antiviral drug that targets different components of the viral life cycle. There has been needed to identify new modes of action in therapy that can complement current antiviral drugs in clinical use and the investigation studies have been concentrated to find effective therapeutic drugs from natural product exhibiting antiviral potential toward an agent of viral hepatitis recently. (Schuppan, D. et al, Hepatology, 30(4), pl099-1104, 1999; Seff, L. B. et al, Hepatology, 34(3). p595-603 2001)

Therefore, there have several reports on the studies of natural product having been used as medicinal materials in Oriental countries: for example, KR patent publication No. 2001-104497 published on Nov. 26, 2001, discloses a pharmaceutical composition comprising the mixture extract of Patrinia scabiosaefolia FlSCH and Phellodendron amurense Rupp. for the treatment of hepatitis B and hepatic-cirrhosis disease, and KR patent publication No. 2000-0027306 published on May. 15, 2000, discloses a pharmaceutical composition comprising the extract of Savia miltiorrhiza Bunge, tanshinol B, tanshinone II B, prazewaquinone A isolated therefrom for the treatment of hepatitis B disease.

Cucumis melon Linn. var. ma-gua, a variant of melon belonged to Cucurbitaceae, is distributed in Heilung river region in China and cultivated in northern China. Matured fruit weighing about 1kg has blue color striped surface and about 30 to 40cm of length. It tastes sweet and therefore, has been on sale at vegetable and fruit market as an edible fruit in China from long years ago.

Ixeris dentata, a perennial herb belonged to Compositae, is distributed in sunny area in Korea, Japan, China etc. It has been used as an indigenous therapy useful to treat diarrhea, pneumonia, bone fracture and a bruise, and therefore, has been on sale at vegetable and fruit market as an edible vegetable in Korea from long years ago (Chung B. S. and Shin M. K, HyangyakDaesacheon, pl056-1057, Youngrimsa, 1998).

Youngia sonchifolia, a perennial herb belonged to Compositae, is distributed in humid area in Korea, Japan, China etc. Its young sprout has been used as a indigenous therapy useful to treat pain, convulsion, various inflammatory diseases and has been on sale at vegetable and fruit market as a edible vegetable in Korea from long years ago. Its main components are known to flavonoid, arnino acid, phytosterol, and saccharide etc (Chung B. S. and Shin M. K, HyangyakDaesacheon, pl056-1057, Youngrimsa, 1998).

However, there has been not reported or disclosed that the extract of above plants has treating or preventing activity of viral liver disease in any of above cited literatures, the disclosure of which is incorporated herein as reference. To investigate a therapeutic effect of various plant extract on viral hepatitis and hepato-cirrhosis, inventors oi the present invention have intensively carried out several molecular biological experiments and finally completed present invention by confirming that the extract of Cucumis melon Linn. var. ma-gua, Ixeris dentata, and Youngia sonchifolia, inhibit the polymerase enzyme activity and the proliferation of hepatitis B virus, and have treating and preventing activities of viral liver disease caused by viral infection.

SUMMARY OF THE INVENTION

The present invention provides a pharmaceutical composition comprising a crude extract of at least one crude drug or their combination selected from the group consisting of Cucumis melon Linn. var. ma-gua (Jing feng seed), Ixeris dentata and Youngia sonchifolia, as an active ingredient in an effective amount to treat and prevent hepatic diseases caused by viral infection.

The present invention also provides a use of above extract for the preparation of pharmaceutical composition to treat and prevent hepatic diseases caused by viral infection in human or mammal.

The present invention also provides a health food or food additives comprising above extract for the prevention or alleviation of hepatic diseases caused by viral infection.

Disclosure of the invention

Accordingly, it is an object of the present invention to provide a pharmaceutical composition comprising a crude extract of at least one crude drug or their combination selected from the group consisting of Cucumis melon Linn. var. ma-gua (Jing feng seed), Ixeris dentata and Youngia sonchifolia, as an active ingredient in an effective amount to treat and prevent hepatic diseases caused by viral infection.

Above described crude extract comprises the extract prepared by extracting plant material with water, lower alcohols such as methanol, ethanol, preferably methanol and the like, or the mixtures thereof.

Further, it is another object of the present invention to provide a pharmaceutical composition containing the extracts of Cucumis melon Linn. var. ma-gua, Ixeris dentata and Youngia sonchifolia, preferably with a ratio of 5-30: 1~3: 1, most preferably 15: 2: 1 by weight.

It is another object of the present invention to provide a pharmaceutical composition additionally comprising crude extract of the crude drug selected from the group consisting of Laminaria japonica, Pueraria thunbergiana BENTH., Lactuca indica var. dracoglossa and Morus alba L., besides above described essential crude extract and a pharmaceutically acceptable carrier thereof as an active ingredient for preventing and treating hepatic diseases caused by viral infection.

Further, it is another object of the present invention to provide a pharmaceutical composition containing the crude extract of Cucumis melon Linn. var. ma-gua, Ixeris dentata, Youngia sonchifolia, Laminaria japonica, Pueraria thunbergiana BENTH., Lactuca indica var. dracoglossa and Morus alba L., preferably with a ratio of 5~30:

1-3: 1: 1-3: 1-10:1-3:1, most preferably 15: 2: 1: 2: 5: 2: 1 by weight.

It is an object of the present invention to provide a use of crude extract of at least one crude drug or their combination selected from the group consisting of Cucumis melon Linn. var. ma-gua (Jing feng seed), Ixeris dentata and Youngia sonchifolia, for the preparation of therapeutic agent for treating and preventing hepatic diseases caused by viral infection in human or mammal.

It is an object of the present invention to provide a method of treating or preventing hepatic diseases caused by viral infection in a mammal comprising administering to said mammal an effective amount of crude extract of at least one crude drug or their combination selected from the group consisting of Cucumis melon Linn, var. ma-gua (Jing feng seed), Ixeris dentata and Youngia sonchifolia, together with a pharmaceutically acceptable carrier thereof.

It is another object of the present invention to provide a health food or food additives comprising above extract, together with a sitologically acceptable additive for the prevention and improvement of hepatic diseases caused by viral infection.

Above described hepatic diseases comprise the diseases caused by hepatitis B virus or hepatitis C virus infection and the like.

The pharmaceutical composition of the present invention can contain about 0.01 ~ 50 % by weight of the above extract based on the total weight of the composition.

The health food of the present invention comprises above extracts as 0.01 to 80 %, preferably 1 to 50 % by weight based on the total weight of the composition. Hereinafter, the present invention is described in detail.

An inventive extract of Cucumis melon Linn. var. ma-gua(commoxι name: Ma- gua), Ixeris dentata and Youngia sonchifolia can be prepared in detail by following procedures,

For example, the inventive crude extract of Ma-gua can be prepared by following procedure; Ma-gua is washed, peeled, cut into small pieces, dried at 10 to 40°C, preferably 18 to 25°C, for 1 to 30 days, preferably 5 to 20 days and pulverized; The powder was mixed with 2 to 25-fold, preferably, approximately 5 to 10 fold volume of distilled water, buffer, saline, lower alcohols such as methanol, ethanol, butanol and the like, or the mixtures thereof, preferably PBS buffer; the mixture is subjected to the extraction with hot water, cold water, reflux extraction, double boiling extraction or ultra-sonication extraction at the temperature ranging from 20 to 100°C, preferably from 60 to 100°C, for the period ranging from 1 to 24 hours with 1 to 5 times, preferably 2 to 3 times, consecutively; the residue thereof is filtered and the supernatant is concentrated with rotary evaporator, at the temperature ranging from 20 to 100°C, preferably from 50 to 70 °C and then dried by vacuum freeze-drying, hot air-drying or spray drying to obtain dried crude extract powder of Ma-gua which can be soluble in water, lower alcohols, buffers or the mixtures thereof.

Other crude extracts than aforementioned Ma-gua extract, i.e. the crude extract of Ixeris dentata, Youngia sonchifolia or combination thereof can be prepared by the identical or modified method with above described procedure.

Additionally, crude extract of present invention can be subjected to following procedure; the above prepared crude extract is suspended in water, and then is mixed with 1 to 100-fold, preferably, 1 to 5 -fold volume of non polar solvent such as ethyl acetate, chloroform, hexane and the like; the non-polar solvent soluble layer is collected to obtain non-polar solvent soluble extract of the present invention and remaining polar solvent soluble layer is collected to obtain polar solvent soluble extract of the present invention soluble in water, lower alcohols, or the mixtures thereof. Also, above described procedures may be modified or subjected to further step to fractionate or isolate more potent fractions or compounds by conventional procedure well- known in the art, for example, the procedure disclosed in the literature (Harborne J. B. Phytochemical methods: A guide to modern techniques of plant analysis, 3^rd Ed. pp6-7, 1998). The combination extract of Cucumis melon Linn. var. ma-gua, Ixeris dentata and Youngia sonchifolia is designated as KYH-1 hereinafter.

In an embodiment of the present invention to investigate the effect of the extract of Cucumis melon Linn. var. ma-gua, Ixeris dentata, Youngia sonchifolia or KYH-1 on antiviral activity, HBV DNA pol activity was measured in HBV pol gene-transfected insect cell line and it is confirmed that the inventive crude extracts inhibit HBV DNA replication by reducing HBV pol activity. Through above experiment, it is found that inventive KYH-1 powder treated to

HepG2.2.15 cell line inhibited the production of HBV DNA replicative intermediate and HBV virion DNA and shows the higher inhibiting activity comparing with that of KYH-1 extract prepared by double boiling method. hi another embodiment of the present invention, KYH-1 decreases the woodchuck pregenomic RNA level and the mRNA level of woodchuck virus surface antigen and suppresses the production of woodchuck HBV DNA replicative intermediates and woodchuck HBV virion DNA in KYH-1 treated- woodchuck HBV infected liver cell line, thereby it is verified that inventive KYH-1 inhibits hepatitis B virus proliferation. In another embodiment of the present invention, KYH-1 exhibits the antiviral activity against BVDV as a surrogate model of HCV in BVDV-infected uterine cell.

In accordance with an aspect of the present invention, there is provided a pharmaceutical composition comprising the crude extract of Cucumis melon Linn. var. ma-gua, Ixeris dentata, Youngia sonchifolia or combination thereof prepared by above described preparation method as active ingredients for the treatment and prevention of hepatic diseases caused by viral infection.

The combination of crude extract, KYH-1, comprises the extracts of Cucumis melon Linn. var. ma-gua, Ixeris dentata and Youngia sonchifolia, preferably with a ratio of 5-30: 1-3: 1, most preferably 15: 2: 1 by weight to maximize their efficacy.

It is another object of the present invention to provide a treating method comprising administering a pharmaceutical composition comprising said extract prepared by above described preparation method to mammals including human suffering from viral hepatic diseases. The inventive composition for treating and preventing chronic viral hepatitis diseases by inhibiting HBV DNA replication may comprises above extracts as 0.01 - 50 % by weight based on the total weight of the composition.

The inventive composition may additionally comprise conventional carrier, adjuvants or diluents in accordance with a using method well known in the art. It is preferable that said carrier is used as appropriate substance according to the usage and application method, but it is not limited. Appropriate diluents are listed in the written text of Remington's Pharmaceutical Science (Mack Publishing co, Easton PA).

Hereinafter, the following formulation methods and recipients are merely exemplary and in no way limit the invention.

The composition according to the present invention can be provided as a pharmaceutical composition containing pharmaceutically acceptable carriers, adjuvants or diluents, e.g., lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starches, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrofidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil. The formulations may additionally include fillers, anti-agglutinating agents, lubricating agents, wetting agents, flavoring agents, emulsifiers, preservatives and the like. The compositions of the invention may be formulated so as to provide quick, sustained or delayed release of the active ingredient after their administration to a patient by employing any of the procedures well known in the art.

For example, the compositions of the present invention can be dissolved in oils, propylene glycol or other solvents that are commonly used to produce an injection. Suitable examples of the carriers include physiological saline, polyethylene glycol, ethanol, vegetable oils, isopropyl myristate, etc., but are not limited to them. For topical administration, the extract of the present invention can be formulated in the form of ointments and creams.

Pharmaceutical formulations containing present composition may be prepared in any form, such as oral dosage form (powder, tablet, capsule, soft capsule, aqueous medicine, syrup, elixirs pill, powder, sachet, granule), or topical preparation (cream, ointment, lotion, gel, balm, patch, paste, spray solution, aerosol and the like), or injectable preparation (solution, suspension, emulsion). The composition of the present invention in pharmaceutical dosage forms may be used in the form of their pharmaceutically acceptable salts, and also may be used alone or in appropriate association, as well as in combination with other pharmaceutically active compounds.

The desirable dose of the inventive extract or composition varies depending on the condition and the weight of the subject, severity, drag form, route and period of administration, and may be chosen by those skilled in the art. However, in order to obtain desirable effects, it is generally recommended to administer at the amount ranging from 0.1 to 1000 mg/kg, preferably, 10 to 100 mg/kg by weight/day of the inventive extract or compounds of the present invention. The dose may be administered in single or divided into several times per day. hi terms of composition, the amount of inventive extract should be present between 0.01 to 50% by weight, preferably 0.5 to 40% by weight based on the total weight of the composition.

The pharmaceutical composition of present invention can be administered to a subject animal such as mammals (rat, mouse, domestic animals or human) via various routes. All modes of administration are contemplated, for example, administration can be made orally, rectally or by intravenous, intramuscular, subcutaneous, intracutaneous, intrathecal, epidural or intracerebro ventricular injection.

Also, the present invention provide a composition of the health food beverage for the prevention and improvement of hepatic disease caused by viral infection adding above described extracts 0.01 to 80 % by weight, amino acids 0.001 to 5 % by weight, vitamins 0.001 to 2 % by weight, sugars 0.001 to 20 % by weight, organic acids 0.001 to 10 % by weight, sweetener and flavors of proper amount.

Above described extract of Cucumis melon Linn. var. ma-gua Ixeris dentata, Youngia sonchifolia or combination thereof can be added to food and beverage for the prevention and improvement of hepatic diseases caused by viral infection.

To develop for health food, examples of addable food comprising above extracts of the present invention are various food, beverage, gum, vitamin complex, health improving food and the like, and can be used as power, granule, tablet, chewing tablet, capsule or beverage etc. Above described composition therein can be added to food, additive or beverage, wherein, the amount of above described extract in food or beverage may generally range from about 0.1 to 80w/w %, preferably 1 to 50 w/w % of total weight of food for the health food composition and 1 to 30 g, preferably 3 to 10 g on the ratio of 100m£ of the health beverage composition.

Providing that the health beverage composition of present invention contains above described extract as an essential component in the indicated ratio, there is no particular limitation on the other liquid component, wherein the other component can be various deodorant or natural carbohydrate etc. such as conventional beverage. Examples of aforementioned natural carbohydrate are monosaccharide such as glucose, fructose etc; disaccharide such as maltose, sucrose etc; conventional sugar such as dextrin, cyclodextrin; and sugar alcohol such as xylitol, and erythritol etc. As the other deodorant than aforementioned ones, natural deodorant such as taumatin, stevia extract such as levaudioside A, glycyrrhizin et al., and synthetic deodorant such as saccharin, aspartam et al., may be useful favorably. The amount of above described natural carbohydrate is generally ranges from about 1 to 20 g, preferably 5 to 12 g in the ratio of 100 mi of present beverage composition.

The other components than aforementioned composition are various nutrients, a vitamin, a mineral or an electrolyte, synthetic flavoring agent, a coloring agent and improving agent in case of cheese chocolate et al., pectic acid and the salt thereof, alginic acid and the salt thereof, organic acid, protective colloidal adhesive, pH controlling agent, stabilizer, a preservative, glycerin, alcohol, carbonizing agent used in carbonate beverage et al. The other component than aforementioned ones may be fruit juice for preparing natural fruit juice, fruit juice beverage and vegetable beverage, wherein the component can be used independently or in combination. The ratio of the components is not so important but is generally range from about 0 to 20 w/w % per 100 w/w % present composition. Examples of addable food comprising aforementioned extract therein are various food, beverage, gum, vitamin complex, health improving food and the like.

The inventive composition may additionally comprise one or more than one of organic acid, such as citric acid, fumaric acid, adipic acid, lactic acid, malic acid; phosphate, such as phosphate, sodium phosphate, potassium phosphate, acid pyrophosphate, polyphosphate; natural anti-oxidants, such as polyphenol, catechin, α- tocopherol, rosemary extract, vitamin C, green tea extract, licorice root extract, chitosan, tannic acid, phytic acid etc. The above extract of Cucumis melon Linn. var. ma-gua Ixeris dentata, Youngia sonchifolia or the combination thereof may be 20 to 90 % high concentrated liquid, power, or granule type.

Similarly, the above extract of Cucumis melon Linn. var. ma-gua Ixeris dentata, Youngia sonchifolia or the combination thereof can comprise additionally one or more than one of lactose, casein, dextrose, glucose, sucrose and sorbitol.

Inventive extract of the present invention have no toxicity and adverse effect and, therefore, they can be used with safe.

It will be apparent to those skilled in the art that various modifications and variations can be made in the compositions, use and preparations of the present invention without departing from the spirit or scope of the invention.

The present invention is more specifically explained by the following examples. However, it should be understood that the present invention is not limited to these examples in any manner.

Brief Description of the Drawings

The above and other objects, features and other advantages of the present invention will more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which;

Fig. 1 is the description of Ma-gua on sale;

Fig. 2 shows the inhibiting effect of inventive extracts on HBV pol activity in priming reaction;

Fig. 3 shows the inhibiting effect of complex herbal extract, KYH-1 on HBV pol activity in priming reaction;

Fig. 4 represents the anti-HBV activity of complex herbal extract after treatment of

KYH-1 to WHBV-infected primary hepatocyte cell;

Fig. 5 presents the southern blot analysis for anti-HBN activity of complex herbal extract after treatment of KYH-1 to WHBN-infected primary hepatocyte cell; Fig. 6 shows antiviral effect of KYH-1 on BNDN replication;

Fig. 7 shows antiviral effect of ribavirin on BNDV replication;

Fig. 8 shows antiviral effect of IFΝ-α onBVDV replication. BEST MODE FOR CARRING OUT THE INVENTION

EXAMPLES

The following Reference Example, Examples and Experimental Examples are intended to further illustrate the present invention without limiting its scope.

Example 1. Preparation of the crude extract of Ma-gua Cucumis melon Linn. var. ma-gua, (Ma-gua, Jing Feng seed, Kyoungryang-

Yangjong Inc., China, see Fig. 1) cultivated in Suwon University located in Kyonggi-do, South Korea was washed, peeled and the 2.5cm top of Ma-gua fruit was removed. And then it was cut into small pieces and dried at 25 °C for 20 day to be pulverized.

And the pulverized powder was suspended at 10%(w/v) in PBS buffer and was heated in a double boiler at 80°C for 2 hours. The suspension was centrifuged at the speed of 10,000rpm for 5 mins (Centrifuge 547 IR, Eppendorf) and the supernatant thereof was used as a sample in following Experiment.

Example 2. Preparation of the crude extract of Ixeris dentαtα The radix of Ixeris dentαtα cultivated in Suwon University located in Kyonggi- do, South Korea was washed and dried at 25 °C for 20 day to obtain pulverized powder.

And the powder was suspended in PBS with a ratio l:10(w:v) and heated in a double boiler at 80°C for 2 hours. The suspension was centrifuged at the speed of 10,000 rpm for 5 mins (Centrifuge 547 IR, Eppendorf) and the supernatant thereof was used as a sample in following Experiment.

Example 3. Preparation of the crude extract of Youngia sonchifolia

The radix of Youngia sonchifolia was cultivated in Suwon University located in Kyonggi-do, South Korea was washed and dried at 20-25 °C for 15 day to obtain pulverized powder.

And the powder was suspended in PBS with a ratio l:10(w:v) and heated in a double boiler at 80°C for 2 hours. The suspension was centrifuged at the speed of 10,000rpm for 5 mins (Centrifuge 5471R, Eppendorf) and the supernatant thereof was used as a sample in following Experiment.

Example 4-7. Preparation of the crude extract of various herbs

Each crude extract of Laminaria japonica, Pueraria thunbergiana, Lactuca indica var. dracoglossa and Morus alba L. was prepared by the modified method disclosed in Example 1-3.

Example 8. Preparation of complex herbal extract, KYH-1

45g of Ma-gua powder, 6g of Ixeris dentata, 3g of Youngia sonchifolia, 6g of Laminaria japonica, 15g of Pueraria thunbergiana BENTH., 6g of Lactuca indica var. dracoglossa, 3g of Morus alba L were mixed to obtain their complex mixture designated as KYH-1. KYH-1 was suspended in PBS with a ratio l:10(w:v) and heated in a double boiler at 80°C for 2 hours. The suspension was centrifuged at the speed of 10,000rpm for 5 mins (Centrifuge 5471R, Eppendorf) and the supernatant thereof was used as a sample in following Experiment.

Example 9. Preparation of alcohol extract of KYH-1. lOOg of complex herbal mixture KYH-1 prepared in Example 8 was added to 1 H of 80% ethanol, stirred, refluxed 4 times at 50-60 °C for 1 hour and filtered. The filtrate was concentrated under reduced pressure and freeze-dried to obtain 25 g of complex herbal extract powder.

Reference Example 1. Expression of hepatitis B virus polymerase

For the expression of hepatitis B virus (HBN) polymerase (pol) in insect cell line, HBN pol gene (Invitrogen Co.) was cloned in baculo virus system (FLAG-pol-stem- loop:FPL-pol) and transfected into Sf9 cell line. Transfected FPL-pol insect cell line was grown at 26°C for 48 hours in T-75 flask containing cultivation medium of TΝM- FH supplemented with 5% fetal calf serum (GIBCO BRL), 0.1% pluronic F-68 and 50 μg/mβ gentamicin and Grace's medium(GIBCO BRL).

Experimental Example 1. Inhibitory effect of KYH-1 on HBV DΝA polymerization To assess the antiviral activity of the inventive extracts and KYH-1, the inventive herbal extract of Example 1 to 8 were treated to Sf9 cells to determine their inhibition activity on HBN DΝA polymerase activity. When HBV pol of Reference Example 1 was expressed, each crude extract of

Exmple 1 to 7 was treated to be the final concentration of 500/zgM. And 50 U, 100 t and 500 μi of 10% KYH-1 extract (supernatant) of Example 8 were also added into 10 ml of Sf9 insect cell culture medium and their polymerase activity was assayed after 48 hours.

Group 1 : Saline treated Group 2: 10% ma-gua extract treated Group 3: 10% Ixeris dmtata extract treated Group 4: 10% Youngia sonchifolia extract treated

Group 5: 10% Laminaria japonica extract treated Group 6: 10% Pueraria thunbergiana extract treated Group 7: 10% Lactuca indica var. dracoglossa extract treated

Group 8: 10⁶I.U. IFN-α treated

Group 1 : Saline treated

Group 2: 50μl of 10% KYH-1 extract treated

Group 3: 100 ≠ of 10% KYH-1 extract treated

Group 4: 500 € of 10% KYH-1 extract treated The HBV pol activity FPL-pol expressed recombinant baculovirus-infected Sf9 cell line was monitored by the priming reaction method disclosed in the literature (Lanford et al J. Virology, 69, p4331, 1995).

Expressed HBV pol protein was mixed with anti-FLAG monoclonal antibody conjugated M2 bead (Sigma Co.) and subjected to immunoaffinity chromatography to obtain purified HBV pol. HBV pol-primer complex formation on pregenomic RNA bound HBV pol was performed on the M2 bead bound with anti-Flag mAb against Flag- pol protein at 30C for 30 min.

Polymerase assay was performed in the presence of [α- P]TTP(3,000Ci/mmol,

NEN and its incorporation rate was regarded as the HBV pol activity which was determined by counting the amount of labeled protein on SDS-PAGE and autoradiography. h case that the inventive extracts inhibited polymerase activity and the primer synthesis, thereby it was judged that the inventive extracts had the inhibitory activity of

HBV DNA synthesis. As shown in Fig. 2, the crude extracts of Ma-gua (lane 2), I. dentata (lane 3), Y. sonchifolia (lane 4), Pueraria thunbergiana (lane 6), Lactuca indica var. dracoglossa

(lane 7) inhibit the HBV pol activity as much as IFN-α (lane 8). As shown in Fig. 3, lane 1 is the control group treated with saline solely, which shows fundamental polymerase activity, lane 2, 3, and 4 are the KYH-1 -treated groups at various concentrations.

Based upon the intensity of autoradiographic exposure, KYH-1 showed 60-70% inhibition of polymerase primer activity at 50 and 500 zg with an increase inhibitory effect at 1000/tg/mt

Finally, it was confirmed that HBV pol activity was inhibited with dose dependent manner and HBV pol activity in the presence of 500μJl of 10% KYH-1 complex herbal extract was dramatically decreased.

And also inventive complex herbal composition can be used to screen the HBV pol activity in insect cell line in accordance with the concentration difference.

Experimental Example 2. Anti-viral effect of KYH-1 complex herbal extract on HBV in HepG2.2.15 cell HepG2.2.15 cell line (Gorgetown Univ., Korba Ph.D, U.S.A.) is a HBV DNA- transfected HepG2 cell line, which produces HBV. It was used for the observation on the antiviral activity against HBV of KYH-1 complex herbal extract in this experiment.

Effective concentrations of KYH-1 against fntracellular HBV DNA replicative intermediates (EC₅₀) and against HBV virion DNA secreted in the culture media (EC₉₀) were determined. Both of the activities of KYH-1 powder and other extract were compared to that of lamivudine (3TC), used as a positive control.

HepG2.2.15 cells were cultured in 96-well plate and treated with various concentrations of KYH-1 for 7 days. The amount of HBV particles and produced HBV DNA amount were measured and cellular apoptosis was assayed with medium diluents showing cellular characteristics.

As shown in Table 1, the EC₅₀ and EC₉₀ of KYH-1 powder were 2.8 fold greater than the activity of heat extracted KYH-1. No toxicity was observed with the extracts at the dosages, therefore, the highest concentration of tested drug was used in calculating a selectivity index (S.I.=CC₅o/EC₉₀).

[Table 1]

Experimental Example 3. Antiviral effect of KYH-1 complex herbal extract on

WHBV producing cell line

In woodchuck primary hepatocyte cell (Cornell Univ., Jacob Ph. D., U.S.A.) established from woodchuck animal infected with woodchuck hepatitis B virus (WHBV) having similar genetic function to human HBN anti-viral activity of KYH-1 powder extract was measured by RΝA expression assay.

The antiviral activity of KYH-1 powder was compared to that of IFΝ-α, which is currently used to inhibit hepatitis virus proliferation as a positive control drug.

The hepatocyte cells were incubated with test samples for 2 weeks. Culture media was supplemented with KYH-1 (1.5mg ) or IFΝ-α (10⁶ LU) at the interval of 2 days for 2 weeks. Nucleic acids were extracted from cells at 4 and 8 days post-treatment and hybridized with Hepatitis B virus surface antigen (HbsAg) DNA probe by Southern blot analysis followed by phosphor-image analysis. Test samples were loaded after normalizing based upon amount of 18S ribosome RNA and the results were expressed as the percentage of 18S ribosomal RNA.

As shown in Fig. 4, there was a 20% reduction of WHBV RNA after the treatment of KYH-1 for 4 days, which means the reduction of infected viral particles in continuous replication cycle 40% reduction of pregenomic RNA in KYH-1 -treated cell line after 8 days of treatment comparing with that of untreated control. WHBV surface antigen RNA was also reduced (Fig. 4).

Comparing with nuclear WHBV DNA thereof, virion DNA and replicative DNA intermediates in untreated cell, those of KYH-1 extract treated- and IFN-α treated- cell were significantly reduced. Especially in KYH-1 powder extract treated cell, nuclear

DNA was completely abolished and virion DNA and replicative DNA intermediates were significantly decreased (Fig. 5).

Through above described experiment, it is confirmed that KYH-1 has inhibitory effect on replication of WHBV

Experimental Example 4. Antiviral effects of KHY-1 on BVDV replication in vitro

BVDV is used as a surrogate in vitro assay to measure the antiviral potential of compounds against HCV (Zitzmann et al, PNAS USA, 96(21). ρpll878-11882, 1999). In this assay we can determine (1) the effective concentration of a crude extract to inhibit BVD-induced cell killing by 50% (EC₅o), (2) the effective concentration to reduce the release of progeny viral yields by 90% (EC₉o), and (3) the cytotoxic (cytotoxicity) concentration of the crude extract at which 50% of normal uninfected cells are killed (CC₅₀). Ribavirin and IFN-α, two drugs approved by FDA for the clinical management of HCN infection were served as positive controls in these assays.

The activity of herbal KYH-1 extract was analyzed in our BVDV assay and its relative activity was compared with those of ribavirin and IFΝ-α (Figure 6, 7, 8). KYH-1 exhibited antiviral activity against BVDV in this assay (Fig. 6). There was a dose dependent reduction in virus-induced cell killing, which shows a maximum 37.3% reduction at an extract concentration of 280 βg/ml (EC₅o= 530 βg/ml). Although the EC₉₀ was 630 βglml, the dramatic decrease in progeny viral yields could be explained by cytotoxic effect of the extract (CCso^ 840 βglml).

In comparison with standards, ribavirin (Fig. 7) inhibited virus-induced cell killing with a calculated EC₅0 of 9.92 μM. The reduction in viral yields was a calculated EC₉₀ of 26.3 μM. However, the concentration was greater than the midpoint of drug cytotoxicity (CC₅₀= 18.4 μM). The antiviral activity of IFΝ-α was also measured in this assay (Fig. 8). Inhibition of viras-induced cell killing was calculated EC₅₀= 0.23 ng/ml. There was a substantial reduction in viral yield with a calculated EC₉₀= 0.013 ng/ml, and IFΝ-α did not exhibit cytotoxicity up to the concentration of 1 μglm .

In reference to the inventive crude extract effective antiviral concentration relative to its cytotoxicity, a selectivity index can be calculated (S.I.= CC₅₀/EC ₀). hi this comparison the potency of IFΝ-α far exceeded that of KYH-1 and ribavirin (Table 2).

A criticism of surrogate assays lies in the relevance of the system to the interaction between infectious agent and the human host. In our case we employed BVDV as a surrogate model of HCV The two drugs approved for clinical use against HCN ribavirin and IFΝ-α, were effective in our BVDV model. By analogy, other crude extract tested in this system that exhibit similar or greater antiviral activity may also inhibit HCN

[Table 2] Antiviral activity of drugs against BVDV-infected bovine uterine ΝCL cells

Clinical Example 1. Treatment effect of KYH-1 complex herbal extract to patients with hepatic diseases

Biochemical and molecular biological tests including blood HBV DNA test on Patients suffered from viral hepatitis or cirrhosis were carried out. KYH-1 of Example 8 was orally administered to volunteers with several dosages ranging from 1.0 to 1.5g/day for 25 days. As a treatment index, GPT, GOT, HBs Ag, anti-HBsAg Ab, Hbe Ag, anti-HbeAg Ab etc. were assayed to detect the change of symptoms associated with hepatic diseases. The amount of HBV DNA in the blood was determined by polymerase chain reaction method. The GOT, the level of aspartate aminotransferase, and GPT, the level of alanine aminotransferase, blood levels are increased when liver cells are destroyed, which sensitively reflect the liver necrosis. The normal level of GOT and GPT ranges from 0 to 40 UL

Table 3 shows the clinical result after KYH-1 administration. As shown in Table 3, HBV DNA was not detected in 5 of 6 volunteers after the administration. Specifically, GOT and GPT levels were dramatically decreased in case of hepatitis B patient treated with inventive extract for 60 days.

Therefore, it is confirmed that inventive complex herbal extract having HBV DNA polymerase-inhibitory activity may be useful for treatment and prevention of liver diseases including cirrhosis, ascites, hepatitis and the like. [Table 3]

Hereinafter, the formulating methods and kinds of recipients will be described, but the present invention is not limited to them. The representative preparation examples were described as follows.

Preparation of powder

Dried powder of Example 1 50mg

Lactose lOOmg

Talc lOmg Powder preparation was prepared by mixing above components and filling sealed package.

Preparation of tablet

Dried powder of Example 2 50mg Corn Starch lOOmg

Lactose lOOmg

Magnesium Stearate 2mg

Tablet preparation was prepared by mixing above components and entabletting.

Preparation of capsule

Dried powder of Example 3 50mg

Corn starch lOOmg

Lactose lOOmg

Magnesium Stearate 2mg Tablet preparation was prepared by mixing above components and filling gelatin capsule by conventional gelatin preparation method.

Preparation of in ection

Dried powder of Example 1 50mg Distilled water for injection optimum amount

PH controller optimum amount

Injection preparation was prepared by dissolving active component, controlling pH to about 7.5 and then filling all the components in 2ml ample and sterilizing by conventional injection preparation method. Preparation of liquid

Dried powder of Example 8 0.1~80g

Sugar 5-10g

Citric acid 0.05-0.3%

Caramel 0.005-0.02%

Vitamin C 0.1-1%

Distilled water 79-94%

CO₂ gas 0.5-0.82%

Liquid preparation was prepared by dissolving active component, filling all the components and sterilizing by conventional liquid preparation method.

Preparation of health food Extract of Example 8 lOOOmg Vitamin mixture optimum amount

Vitamin A acetate 70μg

Vitamin E l.Omg

Vitamin Bi 0.13mg

Vitamin B₂ 0.15ms

Vitamin B6 0.5mg

Vitamin B12 0.2μg

Vitamin C lOmg

Biotin 10μg

Amide nicotinic acid 1.7mg

Folic acid 50μg

Calcium pantothenic acid 0.5mg Mineral mixture optimum amount

Ferrous sulfate 1.75mg

Zinc oxide 0.82mg

Magnesium carbonate 25.3mg

Monopotassium phosphate 15mg Dicalcium phosphate 55mg Potassium citrate 90mg Calcium carbonate lOOmg Magnesium chloride 24.8mg

The above-mentioned vitamin and mineral mixture may be varied in may ways. Such variations are not to be regarded as a departure from the spirit and scope of the present invention. Preparation of health beverage

Extract of Example 8 lOOOmg

Citric acid lOOOmg Oligosaccharide lOOg

Apricot concentration 2g

Taurine lg

Distilled water 900m£

Health beverage preparation was prepared by dissolving active component, mixing, stirred at 85 ^°C for 1 hour, filtered and then filling all the components in lOOOmβ ample and sterilizing by conventional health beverage preparation method.

The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the present invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.

INDUSTRIAL APPLICABILITY

As described in the present invention, the extracts isolated from Cucumis melon

Linn. var. ma-gua, Ixeris dentata and Youngia sonchifolia have potent HBV and HCV inhibitory activity by inactivating viral polymerase, therefore, it can be used as the therapeutics or health food for treating and preventing hepatic diseases caused by HBN

HCV and the like.

Claims

What is claimed is;

1. A pharmaceutical composition comprising a crude extract of at least one crude drug or their combination selected from the group consisting of Cucumis melon Linn. var. ma-gua (Jing feng seed), Ixeris dentata send Youngia sonchifolia, as an active ingredient in an effective amount to treat and prevent hepatic diseases caused by viral infection.

2. The pharmaceutical composition according to claim 1 wherein said crade extract is extracted with the solvent selected from the group consisting of water, lower alcohol, buffer and the mixture thereof.

3. The pharmaceutical composition according to claim 2 wherein said buffer is PBS.

4. The pharmaceutical composition according to any of claim 1 wherein said virus is hepatitis B virus or hepatitis C viras.

5. The pharmaceutical composition according to claim 1 wherein said pharmaceutical composition comprises Cucumis melon Linn. var. ma-gua, Ixeris dentata and

Youngia sonchifolia with a mix ratio of 5-30: 1-3: 1.

6. The pharmaceutical composition according to claim 1, which further comprise the extract of complex herb selected from the group consisting of Laminaria japonica, Pueraria thunbergiana BENTH., Lactuca indica var. dracoglossa and Morus alba L. and a pharmaceutically acceptable carrier thereof as an active ingredient for preventing and treating hepatic disease caused by viral infection.

7. The pharmaceutical composition according to claim 6 wherein said pharmaceutical composition comprises Cucumis melon Linn. var. ma-gua, Ixeris dentata and

Youngia sonchifolia, Laminaria japonica, Pueraria thunbergiana BENTH., Lactuca indica var. dracoglossa and Morus alba L. with a mix ratio of 5-30: 1-3: 1: 1-3: 1-10:1-3:1.

8. A use of crade extract of at least one crude drug or their combination selected from the group consisting of Cucumis melon Linn. var. ma-gua (Jing feng seed), Ixeris dentata and Youngia sonchifolia, for the preparation of therapeutic agent for treating and preventing hepatic diseases caused by viral infection in human or mammal.

9. A health food comprising a crade extract of at least one crade drag or their combination selected from the group consisting of Cucumis melon Linn. var. ma- gua (Jing feng seed), Ixeris dentata and Youngia sonchifolia, together with a sitologically acceptable additive for the prevention and improvement of hepatic diseases caused by viral infection.

10. The health food according to claim 9 wherein said health food is provided as powder, granule, tablet, chewing tablet, capsule or beverage type.