KR20040018730A - Composition comprising the extracts of Cucumis melon Linn. var. ma-gua, Ixeris dentata and Youngia sonchifolia for therapy against chronic viral hepatitis diseases - Google Patents

Abstract

PURPOSE: Provided is a composition comprising extracts of Cucumis melon Linn. var. ma-gua, Lxeris dentata and Youngia sonchifolia for the treatment of chronic viral hepatitis diseases. It increases liver function, inhibits HBV DNA proliferation to show antiviral effect, and is thus useful for prevention and treatment of chronic viral hepatitis diseases. CONSTITUTION: A pharmaceutical composition for prevention and treatment of chronic viral hepatitis diseases is characterized by containing, based on the total weight of the composition, 0.5-50% of crude extracts of Cucumis melon Linn. var. ma-gua, Lxeris dentata and Youngia sonchifolia having effective inhibition effect on HBV DNA proliferation. The crude extract is obtained by using a polar solvent selected from water, methanol, ethanol, a mixture thereof, and a buffer solution.

Description

Composition containing the extracts of quince, cauliflower and horsetail extract for treating viral liver disease {Composition comprising the extracts of Cucumis melon Linn. var. ma-gua, Ixeris dentata and Youngia sonchifolia for therapy against chronic viral hepatitis diseases}

The present invention relates to a composition for the prevention and treatment of viral liver disease, and more particularly, hepatitis and cirrhosis, etc., using the effect of inhibiting hepatitis B virus transcriptase activity of the extracts of apples, bitter gourd, and perilla extract and complex drugs comprising them. The present invention relates to pharmaceutical compositions and dietary supplements useful for the prevention and treatment of viral liver diseases.

More than 300 million people worldwide are infected with hepatitis B virus and are estimated to be chronic carriers, and hepatitis C virus carriers are estimated to be about 100 million, and their numbers continue to increase. Each year, more than 2 million people have hepatitis due to chronic viral infections and develop cirrhosis and liver cancer. Types of viral hepatitis-inducing viruses are known as type A, type B, type C, delta type, GBV, etc., but type B and type C have the highest risk, and their pathological mechanisms are actively studied. It is becoming.

Hepatitis B virus (HBV), a representative virus that causes hepatitis, is a virus that is susceptible to mutation despite being a DNA virus. The HBV gene is about 3.2 Kb of incomplete double strand cyclic DNA and has an extremely compact structure. So far it is known that the HBV DNA has a transcriptase gene (pol), a viral shell gene (capsid), a viral surface antigen gene (HBsAg), and an X gene that is involved in many functions and cancers. Unlike ordinary DNA viruses that replicate intact, 3.5 Kb of pregenomic RNA chains are transcribed from the DNA chain by host transcriptases, and double-stranded DNA is reversed by virus-specific reverse transcriptase (pol). Is replicated. This replication process occurs independently of the host chromosome. However, it has been found that some of the viral DNA gets into the host's chromosome. When HBV DNA gets into the host's chromosome, the gene becomes unstable and the X protein expressed therein promotes the transcription of the host cell and the p53 gene function. It is likely to be inhibited. If this situation continues, it will develop into HCC.

As described, HBV exhibits a high degree of variability comparable to that of normal RNA viruses because of its replication pattern similar to that of RNA viruses. HBV gene mutations occur randomly but the frequency is known to be constant. HBV gene mutations result in structural changes in surface antigens in the HBV envelope. Changes in the structure of various surface antigens are factors that can escape the humoral and cellular immune responses of the host, and are important factors in determining disease progression or persistent infection.

Hepatitis B has a latent period of 1-4 months, which is more susceptible to infection through blood than oral infections, fatigue and loss of appetite after infection, and acute hepatitis symptoms such as discomfort or digestive problems in the right upper abdomen. Acute hepatitis develops into a chronic form due to inadequate host immune responses to viruses, lack of T cell function, endocrine functions such as steroids, and drugs.

Currently, only a prophylactic vaccine has been developed for the treatment of hepatitis, and no satisfactory treatment has been developed. Recently, a nucleoside analog called lamivudine has been clinically prescribed under the approval of the US FDA. have. On the other hand, hepatitis C has been shown to be somewhat effective because ribavirin, alpha interferon, etc. is a complex prescription. However, these synthetic drugs and recombinant medicines are still required to develop new drugs because they have physical problems such as side effects and recurrence caused by resistant viruses due to long-term use.

Chronic hepatitis virus is a causative virus that ultimately progresses from hepatitis to cirrhosis, which causes frequent liver failure or liver cancer. Analysis of serological data shows that 10-15% of those infected are infected with hepatitis B and C virus at the same time. These double infected people cause even more serious liver disease. In the United States alone, more than 10,000 people die each year from viral chronic liver disease, and the number of people infected is increasing every year. There are more than 100 million hepatitis B carriers in China, which account for 10-12% of the total population. Especially in Korea, there are more than 3 million chronic carriers as well as acute hepatitis patients. Moreover, in Korea, the proportion of early hepatitis B viral hepatitis virus carriers is known to be significantly higher for serious liver diseases such as chronic hepatitis, cirrhosis and liver cancer. Therefore, it is urgently needed to develop a therapeutic agent with lower side effects and lower cost than lamivudine, which can cure chronic viral hepatitis patients.

Drugs currently being used for the treatment of viral hepatitis and cirrhosis are alpha-interferon, lamivudine, and the like, which are known as immunoactive agents and anti-viral agents, and are commercially available as clinically prescribed antiviral agents, but have many disadvantages. Lamivudine, an organosynthesized nucleoside analogue, is the biggest problem of drug-resistant virus in long-term use, and even if a mutation occurs in the YMDD motif of the site having reverse transcriptase activity of HBV transcriptase protein Adefovir, another nucleoside analogue that exhibits inhibitory activity, causes long-term doses of resistant viruses and renal failure.Also, alpha-interferon, a protein expressed by genetic recombination, is used in chronic C-type infected patients. Only about 30% of patients have a significant response and other side effects. Moreover, the long-term administration of alpha-interferon is a frequent and problematic problem for the emergence of resistant type C viruses against this drug.

In recent years, the combination prescription has become the best to suppress the emergence of drug-resistant mutant virus, so the combination administration of ribavirin and alpha-interferon has become common. Nevertheless, new forms of more complete therapies need to be released, which may be better than organic synthetic antiviral agents found in plant-derived natural products. However, although it is necessary to develop a therapeutic agent to replace the existing antiviral agent as a treatment for liver disease caused by chronic infection of hepatitis virus, it is considered that the time gap is too long until new drug development from these plant natural products is clinically applied.

Therefore, the purpose of prevention or treatment of hepatitis is to prevent the virus proliferation by prescribing a vaccine or to stop the progression of hepatitis by using drugs that inhibit the virus proliferation and to prevent the progression of cirrhosis and liver failure. I'm trying to stop. Therefore, the development of recombinant vaccines and new antiviral agents such as lamivudine and acyclovir, such as synthetic drugs, are being developed, but as a complete treatment for hepatitis, it is still in question, and its use is limited due to its side effects and high cost. It is true.

Therefore, researches are being actively made to create new therapeutic agents with low toxicity and side effects from folk remedies or natural products, especially herbal medicines that have been applied since ancient times. For example, a composition consisting of a mixed extract of Matari and Hwangbaekpi has been tried as a therapeutic agent for hepatitis B and liver cirrhosis (Korean Patent Application No. 2000-23564), tanshinol B, tanshinone IIB, prochebaquinone A, salvia extract and It has been suggested that the composition consisting of the saliva fraction is effective in the treatment of hepatitis B (Korean Patent Application No. 2000-27306).

Cucumis melon Linn. Var. Ma-gua used in the present invention is a plant widely distributed in the Heilongjiang region of China, a hybrid of melons. The seeds of the melon in Korea also came down as a variant of the melon, and this is also a hybrid, which is grown in northern China. The taste is sweet and has a slightly soft texture. The mature fruit has a blue surface, has a dark blue pattern, and usually weighs about 1kg. It is about 30-40cm in size and texture. see. Usually, it is harvested when it is medium-aged in August-September, and it does not grow well when it is planted in the following year. Its effect is known to be good for liver function and has been edible as fruit in northeastern China for a long time.

Ixeris dentata is a perennial herb of the Compositae, which is used as a decoction or crushed externally using starch. It is effective in stopping diarrhea, reducing fever, regenerating and detoxifying skin. It is used to treat serpentine, urinary stones, eczema, pneumonia, bruises, and fractures.

Youngia sonchifolia or Ixeris sonchifolia is also a perennial herb of Compositae. Pain (鎭 痛), jingyeong (鎭 痙) action, and anti-inflammatory action to eliminate pus, is effective in detoxification. Appendicitis, enteritis, dysentery, various purulent inflammations, hemostasis, nasal bleeding, headache, toothache, abdominal pain, yellow spear, calculus

However, in the above-mentioned documents and research reports, the biological activity of the herbal medicine using the extracts of the cabbage, cabbage, and horseradish against viral liver disease has not been reported to date.

The present invention relates to a composition for the prevention and treatment of hepatitis, hepatic cirrhosis, especially by hepatitis B virus, which contains extracts of quince, nirbis and perilla as main herbal ingredients.

The present inventors pharmacologically observed the therapeutic effect of the combined hepatitis against viral hepatitis and cirrhosis, and focused on the possibility of preventing and treating hepatitis cirrhosis with respect to the herbal medicine that has traditionally accumulated many clinical data.

As a result, the present invention was completed by confirming that the complex herbal medicine according to the present invention inhibits the activity of hepatitis B virus transcriptase, thereby inhibiting virus proliferation and having prophylactic and therapeutic activity of viral liver disease.

Therefore, an object of the present invention is a pharmaceutical composition showing the effect of preventing and treating viral liver disease containing a complex herbal medicine containing extracts of the quince, mule, and perilla seed having an inhibitory activity of hepatitis B virus replication enzyme as an active ingredient. And to provide a dietary supplement.

1 is a photograph showing the effect of inhibiting the HBV replication enzyme activity of the complex drug KYH-1 by treatment with the concentration of the complex drug KYH-1 upon expression of activated HBV copy enzyme in the insect cell line,

FIG. 2A shows the pregenomic in cells on day 4 and day 8 after treatment with 0.15% KYH-1 extract to cell lines infected with Woodchuck Hepatitis B virus (WHBV) to observe the inhibition of viral replication activity of KYH-1. A table comparing RNA and WHBV surface antigen mRNA amounts,

FIG. 2B is a Southern view of nuclear DNA, virion DNA, and DNA replication intermediates treated with 0.15% KYH-1 extract in a cell line infected with Woodchuck Hepatitis B virus to observe the inhibition of viral replication activity of KYH-1. The result of blot analysis.

In accordance with the above object, the present invention is a viral liver disease containing a crude extract of a complex herbal medicine, including Cucumis melon Linn.var.ma-gua, Ixeris dentata and Youngia sonchifolia . In particular, it provides a composition for the prevention and treatment of viral hepatitis B disease.

The crude extract refers to water, lower alcohols such as methanol ethanol, and extracts soluble in a mixed solvent thereof.

In addition, the present invention is an essential complex herbal, that is, sesame, bitter, walnuts, in addition to kelp ( Laminaria japonica ), er ( Pueraria thunbergiana BENTH.), King walrus ( Lactuea indica var. Dracoglossa ), mulberry ( Morus alba L.), etc. May contain herbal medicines.

Hereinafter, the present invention will be described in detail.

The crude herbal crude extract of the present invention, after washing or peeling the plants and cut them, at a temperature of 10 to 40 ℃, preferably 18 to 25 ℃ about 1 to 30 days, preferably about 5 to 20 days It was naturally dried for a period of time, powdered and then dissolved in an extraction solvent such as water, lower alcohol and buffer solution at a concentration of 1 to 40%, preferably 10 to 20%, at 50 to 100 ° C, preferably 70 to 80 ° C. After about 1 hour to 1 day, preferably about 1 hour to 5 hours of hot water, centrifugation may be performed to obtain a crude extract as a supernatant. In the present invention, as a preferred embodiment, crude extract is obtained by bathing the compound herbal powder using phosphate buffer.

The compositional ratio of the main herbal medicine of the complex herbal medicine of the present invention, ie, bitter bark: bark, is 30: 1: 1 to 5: 1: 1, preferably 15: 2: 1 by weight. When these active ingredients are mixed at this composition ratio, the prophylactic and therapeutic effects of the viral liver disease of the present invention can be maximized.

In the present invention, in addition to the essential macaw, nibbarri, and dulcidae as a supplemental herbal quincu: citrus: deciduous: Dashima: 칡: wangdeulsu: mulberry combination ratio of 30: 1: 1: 1: 2: 1: 1 to 5: 1: 1 The weight ratio (g) is 1: 1: 3: 1: 1, preferably 15: 2: 1: 2: 5: 2: 1.

The present invention provides a pharmaceutical composition for the prevention and treatment of viral liver disease, including complex herbal extracts obtained in the extraction process.

The composition for the prevention and treatment of viral liver disease of the present invention comprises 0.5 to 50% by weight of the combined herbal based on the total weight of the composition.

It was confirmed that the HBV transcriptase activity was significantly reduced when the suspension solution of the compound herbal powder obtained by the above method was treated in different concentrations in the insect cell line expressing the HBV mutant gene.

In addition, HBV DNA replication intermediate production and HBV virion DNA production were inhibited when the HepG2.2.15 cell line was treated with the composite drug powder and the suspension solution.

And, in an experiment measuring the antiviral effect of the compound drug powder in the liver cell line of Woodchuck HBV-infected Woodchuck experimental animals, the compound drug of the present invention is the level of pre-chamber RNA and Woodchuck virus surface antigen mRNA of Woodchuck It was confirmed that the hepatitis B virus proliferation process was inhibited by inhibiting the production of Woodchuck HBV DNA replication intermediates and Woodchuck HBV virion DNA production.

Compositions comprising the multi-drug of the invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

Compositions comprising the multi-drug of the invention may further comprise a suitable carrier, excipient and diluent according to conventional methods.

Carriers, excipients and diluents that may be included in the composition comprising the complex herbal of the present invention include 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 oil.

Compositions comprising the complex herbal medicine according to the present invention, respectively, in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, oral formulations, external preparations, suppositories, and sterile injectable solutions according to a conventional method. Can be formulated and used.

The pharmaceutical preparations thus prepared may be administered by a route selected from oral, parenteral (including transdermal, intradermal, intramuscular and intravenous), rectal and inhalation.

The amount of the combined herbal medicine of the present invention may vary depending on the age, sex, and weight of the patient, but in general, an amount of 0.1 to 1000 mg / kg, preferably 10 to 100 mg / kg, is given to an adult. , May be administered once to several times daily. In addition, the dosage of the combination herbal may be increased or decreased depending on the route of administration, the severity of the disease, sex, weight, age, and the like. Therefore, the above dosage does not limit the scope of the present invention in any aspect.

The present invention also provides a dietary supplement containing the complex herbal medicine.

The composition comprising the complex herbal of the present invention can be used in various ways, such as drugs, foods and drinks for the prevention and treatment of viral liver disease. Examples of the foods to which the compound herbal medicines including the main apple, the horsetail, and the bark may be added include various foods, beverages, gums, teas, vitamin complexes, and health supplements.

The medicinal plants of the present invention, algae, bitter herbs are almost no toxic and no side effects, and therefore are drugs that can be used with confidence even for prolonged administration.

The combination herbal of the present invention may be added to food or beverage for the purpose of preventing viral liver disease. At this time, the amount of the extract or fraction in the food or beverage is generally added to the health food composition of the present invention 0.01 to 30% by weight of the total food weight, the health beverage composition is 0.02 to 30 g based on 100 ml, Preferably it can be added in the ratio of 0.3-1 g.

The health beverage composition of the present invention has no particular limitation on the liquid component except for containing the herbal medicine as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates, etc. as additional ingredients, like ordinary drinks. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, 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 said 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 salts thereof. , Organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, 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 from the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, the present invention will be described in more detail based on the following Examples and Experimental Examples, but the content of the present invention is not limited by the following Examples and Experimental Examples.

Example 1 Preparation of Compound Herbal Extract 1

The apples (cultivated at the Lightweight Yangjong Development Co., Ltd., China, Suwon University Laboratory) were washed, 2.5cm and the shells were removed from the nipples, chopped, and naturally dried at a temperature of 25 ° C. for about 20 days to be powdered with a blender.

The moths (grown in the Suwon University lab) were washed with roots, air dried at a temperature of 20 ° C, and then powdered with a blender.

The seedlings (cultivated in the Suwon University laboratory) were removed from the roots and leaf parts, washed, cut and sprinkled, and then naturally dried at a temperature of 25 ° C. for about 15 days and then powdered with a blender.

Kelp (cultivated in Suwon University laboratory) was washed in fresh water to completely remove the salty taste, and then only the root portion was removed, dried for about 5 days at a temperature of 20 to 25 ℃ and then pulverized with a blender.

The algae and the algae are the algae. Among them, the algae are collected (cultivated in the Suwon University lab), peeled, shredded, dried naturally at a temperature of 25 ° C. for 20 days, and then powdered by mixing.

The king eel (cultivated in the Suwon University laboratory) was washed, chopped, dried for about 20 days at a temperature of 18 to 25 ℃, and then powdered by a mixer.

Mulberry (cultivated in the Suwon University laboratory) was washed and mulberry leaves, then dried for about 20 days at a temperature of 18 to 25 ℃ and then powdered with a blender.

45 g of quince, 6 g of bitter gourd, 3 g of dried persimmon, 6 g of kelp, 6 g of 칡, 15 g of king radish, 6 g of mulberry leaf and 3 g of mulberry leaf were mixed to prepare a compound herbal powder and named KYH-1. The compound herbal powder KYH-1 was prepared as a 10% solution in PBS buffer, heated at 80 ° C for 2 hours, centrifuged at 10,000 rpm for 5 minutes using a centrifuge (Centrifuge 5471R, Eppendorf), and then The solution was taken and used as a sample for cell experiments.

Example 2. Preparation of Complex Herbal Extract 2

100 g of the mixed herbal powder of Example 1 was added with 1 L of an aqueous 80% ethanol solution and refluxed four times at 50 ° C. for 1 hour, and all were combined and filtered through a filter paper (Watman, USA), followed by a reduced pressure concentrator (Eyela). , N-1000, Japan) was concentrated under reduced pressure to obtain a 25g extract was used as a sample.

Reference example. Hepatitis B Virus Enzyme Expression

To express hepatitis B virus polase (pol) protein in insect cell lines, cloning HBV cloning gene (HBV pol, Invitrogen) to baculovirus (FLAG-pol-stem-loop: FPL-pol) It was then transformed by infecting the insect cell line Sf9 cell line. Transformed FPL-pol insect cell lines were cultured in T-75 flasks, with 5% FCS (fetal calf serum, Gibco), 0.1% Pluronic F-68 in TNM-FH medium. And Grace medium (Grace'smedium, Gibco Co., Ltd.) containing 50 μg / ml gentamicin were used and incubated at 26 ° C. for 48 hours.

Experimental Example 1. Measurement of HBV DNA replication inhibition ability by the priming reaction method

In order to examine the effect of inhibiting the hepatitis B virus (HBV) proliferation that causes viral hepatitis of the complex herbal composition (KYH-1) of the present invention, the degree of DNA replication enzyme activity inhibition of hepatitis B virus was measured.

When expressing the hepatitis B virus transcriptase of the reference example, 10 ml of SF9 cells in the amount of 50 μl, 100 μl, and 500 μl of the 10% compound KYH-1 extract (supernatant) of Example 2 from the beginning of culture, respectively. In addition to the culture medium, replication enzyme activity was measured after 48 hours.

1st group: Control group which added only saline solution.

2nd group: 50 microliters of KYH-1 10% extracts (final concentration 50 micrograms / ml).

3rd group: 100 microliters of KYH-1 10% extracts (final concentration 500 microgram / ml).

4th group: 500 microliters of KYH-1 10% extracts (final concentration 1000microgram / ml).

HBV transcriptase protein activity expressed in Sf9 cell line infected with FPL-pol recombinant baculovirus was measured by the priming reaction method (Lanford et al., J. Virology , 69 , 4331, 1995). The expressed transcriptase protein was purified by immunoaffinity chromatography by binding to M2 beads (Sigma) to which anti-FLAG monoclonal antibody was bound, followed by HBV transcriptase-bound free. HBV transcriptase-primer complexes were allowed to form on genomic RNA. When the reaction primer ^{32 P-TTP (3,000Ci / m㏖} , NEN Co.) that were so stuck into determine the activity of HBV replication enzyme, ³² P-TTP is the radiation amount of the cloned enzyme labeled protein loaded on SDS-PAGE gels It was confirmed. At this time, if the composite herbal composition inhibited the synthesis of the primer by inhibiting the synthesis of the primer, it was determined that the herbal composition had the effect of inhibiting the HBV DNA synthesis ability of the composition.

Referring to the results of FIG. 1, lane 1 is a control group treated with saline only, and the activity of basic replication enzymes can be observed, and lanes 2, 3, and 4 were treated with concentrations of the 10% extract of the compound herbal KYH-1 for each concentration. As the treatment concentration was increased, the replication enzyme activity was suppressed, and it was confirmed that the activity was significantly reduced when the KYH-1 in lane 4 was treated with 500 µg / ml.

Therefore, it was confirmed that the complex herbal composition of the present invention can be usefully used for screening the activity of HBV virus transcriptase protein expressed in insect cell lines according to the difference in concentration.

Experimental Example 2. Determination of anti-viral activity of KYH-1 combination drug against hepatitis B virus expressed in HepG2.2.15 cell line.

HepG2.2.15 cell line (Gorgetown University, Dr. Korba, USA) is a cell line in which HBV is propagated by transforming the liver cancer cell line HepG2 with HBV DNA and using this cell line to observe the proliferative inhibitory effect of KYH-1 complex drug against HBV. It was.

Efficient inhibitory concentrations of KYH-1 complexes for the production of intracellular HBV DNA replication intermediates (EC ₅₀ ) and inhibitory concentrations of KYH-1 complexes for HBV virion DNA secreted into cell line cultures (EC ₉₀ ). Decided. The inhibition of HBV production by the KYH-1 complex herbal powder and the bath solution was measured in comparison with lamivudine (3TC, Glaxo Wellcome) used as a positive control.

Cell lines were incubated in each of them using a T-96 well plate, and KYH-1 solution was added thereto at a ratio, and the lamivudine solution was diluted and added as a control. HBV virus particles and HBV DNA produced by each cell line were measured during 7 days of incubation, and cell death was measured in dilutions showing cell characteristics.

As shown in the results of Table 1, the concentrations showing the same level of activity of EC ₅₀ and EC ₉₀ were observed at about one-third of the concentrations of the compound herbal powder compared to the bath solution. EC ₅₀ and EC ₉₀ of the KYH-1 complex herbal powder was found to be 2.8 times higher than the activity of the KYH-1 bath solution. In addition, there was almost no cytotoxicity by this combination drug, and the selectivity index (SI) was calculated using the highest concentrations of the tested compound drugs and drugs (SI = CC ₅₀ / EC ₉₀ ).

EC ₅₀ EC ₉₀ CC ₅₀ S.I. KYH-1 Complex Herb Powder 90 µg / ml 254 µg / ml > 3700 µg / ml 14.6 KYH-1 Complex Herb Heat Bath Liquid 259 µg / ml 697 µg / ml > 1000 µg / ml 1.4 Lamivudine (3TC) 0.06 μM 0.166 μM 2165 μM 13042

Experimental Example 3. Measurement of effect of KYH-1 complex herbal medicine on WHBV virus proliferating cell line

KYH-1 in primary hepatocytes (Dr. Jacobs, Cornell University, Cornell University, USA) selected from Woodchuck experimental animals infected with Woodchuck HBV virus (WHBV), whose genetic function is similar to that of human HBV virus. Anti-viral activity by the combined herbal powders was measured by comparing RNA amounts. Alpha-interferon is a drug currently used to inhibit the hepatitis virus proliferation, used as a positive control to see the efficacy of KYH-1.

A KYH-1 complex herbal powder 0.15% and a primary hepatocyte control treatment the concentration in the culture medium is 10 ⁶ IU of alpha-unit - the primary liver cells treated with interferon (Roche Co.) and cultured for two weeks. After 4 days of incubation, the virus-related DNA and RNA products were extracted in the cell line, and after 8 days, the same amount of DNA and RNA extracts were obtained. Southern blot hybridization was performed to measure DNA and RNA levels through phosphoimage analysis. Samples were used based on the amount of 18S ribosomal RNA of the hepatocyte cell line at the time of hybridization, and the analysis result was expressed as a percentage of 18S RNA.

In the results of Figure 2a, WHBV RNA was reduced by about 20% in the cell line treated with KYH-1 multidrug powder for 4 days, which means a reduction of infectious virus particles in the subsequent replication cycle. In the cell line treated with KYH-1 complex herbal powder for 8 days, the WHBV pregenonomic RNA was reduced by 25% compared to the untreated group and the amount of RNA corresponding to the WHBV surface antigen was also reduced (see FIG. 2A).

Treatment of KYH-1 complex powder and alpha-interferon, respectively, compared to the amount of intranuclear WHBV DNA, virion DNA and intermediate intermediate DNA produced in cell line culture without KYH-1 complex powder In one cell line, the amount of DNA was significantly reduced, especially in the cell lines treated with KYH-1 complex powder, almost all of the viral DNA in the nucleus was lost, and the amount of intermediate DNA and virion DNA was also greatly reduced (see Figure 2b). Through this result, it was confirmed that the KYH-1 complex herbal powder has an effect in inhibiting the replication of Woodchuck hepatitis B virus.

Clinical Example 1. Verification of therapeutic effect by administration of KYH-1 combination herbal medicine to patients with hepatitis and liver cirrhosis (volunteer).

Changes in biochemical and molecular biological values of humans related to viral hepatitis and cirrhosis (tested for the presence of HBV DNA in the body) were examined.

The KYH-1 complex herbal of Example 2 was administered orally by 1.0-1.5 g per adult per day for 25 days or longer to determine the effect. These indicators include changes in GPT and GOT levels in the blood, and indicator changes such as HBs and anti-HBs antibodies and HBe and anti-HBe antigens to measure signs of liver disease and the presence of HBV DNA in serum. Was measured by the PCR method. GOT and GPT refer to the levels of aspartic acid and alanine degrading enzymes, respectively, and when liver problems occur and hepatocytes are destroyed, this component enters the blood and is detected and sensitively reflects hepatocellular necrosis. -40 UI.

Table 2 shows the results of clinical treatment by KYH-1 combination herbal administration. Remaining HBV DNA may be a problem for recurrence in hepatitis patients. The results of Table 2 show that after the combination drug administration, HBV DNA disappeared and was not detected in 5 of 6 patients. It was confirmed that the herbal medicine can be effectively used for the treatment and prevention of hepatitis B by inhibiting HBV DNA transcriptase activity and inhibiting HBV DNA proliferation. In particular, in hepatitis B patients who received 60 days of combined herbal medicine, it was confirmed that GOT and GPT levels were significantly reduced, which could be effectively used for the treatment of hepatitis B.

Symptom Treatment period (days) Diagnosis Before after GOT GPT HBV DNA HBsAg Anti-HBsAg HBeAg Anti-HBeAg Cirrhosis and ascites 35 I'm × × × + - + - after 28 15 - - + - + Hepatitis and Cirrhosis 90 I'm 52 44 × + - × - after 62 85 + + - - + Hepatitis B 25 I'm × × + + - + - after 23 31 - + - - + Fatty liver 25 I'm 81 97 + + - - - after 37 67 - + - - + Hepatitis B 155 I'm 34 51 + + - - + after 35 53 - + - - - Hepatitis B 60 I'm 213 290 + + - + - after 32 34 - + + + +

Hereinafter, an example of the preparation of the pharmaceutical composition will be described, but it is not intended to limit the present invention but merely to explain in detail.

Formulation Example 1 Preparation of Injection

Example 1 Dry Extract ............... 100 mg

Sodium metabisulfite ......................................... 3.0 mg

Methylparaben ...... 0.8 mg

Propylparaben ...................................... 0.1 mg

Sterile Distilled Water for Injection ...

The above ingredients were mixed and adjusted to 2 ml by a conventional method, and then filled into 2 ml ampoules and sterilized to prepare an injection.

Formulation Example 2 Preparation of Tablet

Example 1 Dry Extract ............... 200 mg

Lactose 100 mg

Starch ......................................... 100 mg

Magnesium Stearate ...............

The tablets were prepared by mixing the above components and tableting according to a conventional method for producing tablets.

Formulation Example 3 Preparation of Capsule

Example 1 Dry Extract ............... 100 mg

Lactose ...... 50 mg

Starch ............................ 50 mg

Talc ........................................ 2 mg

Magnesium Stearate ...............

The capsules were prepared by mixing the above components and filling the gelatin capsules according to a conventional method for preparing capsules.

Formulation Example 4 Preparation of Liquid

Example 1 Dry Extract ............... 1000 mg

Sugar ........................... 20 g

Isomerized sugar ......................................... 20 g

Lemon scent .........................

Purified water was added to adjust the total volume to 100 ml.

The above-mentioned components were mixed according to a conventional method for preparing a liquid solution, filled into a 100 ml brown bottle, and sterilized to prepare a liquid solution.

In addition, health foods and health drinks were prepared in the following manner.

Formulation Example 5 Preparation of Health Food

Example 1 Dry Powder ..................... 1000 mg

Vitamin Blend ......................... 20 g

Vitamin A Acetate ............... 70 μg

Vitamin E ......................... 1 mg

Vitamin B ₁ ................................. 0.13 mg

Vitamin B ₂ ................................. 0.15 mg

Vitamin B ₆ ....................... 0.5 mg

Vitamin B ₁₂ ................................. 0.2 μg

Vitamin C ......................................... 10 mg

Biotin .......................... 10 μg

Nicotinic Acid Amide ...................................... 1.7 mg

Folic acid ......................................... 50 μg

Calcium Pantothenate ......................................... 0.5 mg

Mineral mixture ...............

Ferrous Sulfate ................................. 1.75 mg

Zinc Oxide ........................ 0.82mg

Magnesium Carbonate ......................................... 25.3 mg

Potassium monophosphate ......................................... 15 mg

Dicalcium Phosphate Dioxide ..................... 55 mg

Potassium citrate ..... 90 mg

Calcium Carbonate ... 100 mg

Magnesium Chloride .............. 24.8 mg

Although the composition ratio of the vitamin and mineral mixture is a composition suitable for a relatively healthy food in a preferred embodiment, the composition ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional health food manufacturing method, Granules may be prepared and used to prepare health food compositions according to conventional methods.

Formulation Example 6 Preparation of Health Beverage

Example 1 Dry Powder ......................................... 1000 mg

Citric Acid ..................... 100 mg

Oligosaccharide ......................................... 100 g

Plum concentrate ..................... 2 g

Taurine ..................................... 1 g

Purified water is added.

After mixing the above components in accordance with a conventional healthy beverage production method, and stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed sterilization and then refrigerated and stored in the present invention For the preparation of healthy beverage compositions.

Although the composition ratio is a composition suitable for a preferred beverage in a preferred embodiment, the compounding ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, and use purpose.

As described above, the complex herbal composition comprising the antagonist, bitter gourd, and perilla extract according to the present invention has excellent efficacy of inhibiting the replication enzyme activity of hepatitis B virus and consequently inhibiting virus growth, such as hepatitis and cirrhosis. It can be usefully used for the prevention and treatment of viral liver disease.

Claims (12)

A pharmaceutical composition for the prevention and treatment of viral liver disease, which contains a complex herbal medicine essentially comprising Cucumis melon Linn.var.ma-gua, Ixeris dentata and Youngia sonchifolia . The composition of claim 1, wherein the crude extract is extracted with one or more polar solvents selected from lower alcohols such as water, methanol, ethanol, mixed solvents and buffers thereof. The composition of claim 3 wherein the polar solvent is a buffer. The composition of claim 1, further comprising kelp ( Laminaria japonica ), Pueraria thunbergiana BENTH., Lactuea indica var. Dracoglossa and Morus alba L .. The composition according to claim 1, wherein the composition ratio of apples: crocuses: perillas is 20-30: 4-1: 2-1. A composition according to claim 5, wherein the composition ratio of apples: crocuses: barley is 15: 2: 1. The composition according to claim 1, wherein the compound herbal mixture is mixed at a weight ratio (g) of apple 45, bitter melon 6, cornflower 3, kelp 6,, 15, handicraft 6 and mulberry 3. The composition of claim 1, wherein the dosage is administered in an amount of 10-100 mg per kg of adult body weight. The composition of claim 1 comprising 0.5-50% of said extract relative to the total weight of the composition. Washed, peeled, and shredded apple, bitter gourd, walnut, kelp, 칡, handicraft, mulberry, finely dried at 18 to 25 ℃ temperature for 5 to 20 days, powder and mixed in a weight ratio of the above 6 to extract the water bath with a solvent Method of manufacturing herbal extracts. A dietary supplement comprising a compound herbal extract and a food-acceptable food supplement additive which essentially include the medicinal extracts of the quince, pertussis and perilla extract, which have a prophylactic and therapeutic effect on viral liver disease. The dietary supplement of claim 11 in the form of a powder, granule, tablet, capsule or beverage.