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    • 专利摘要:
    PURPOSE: Provided is a composition comprising an extract of Cucumis melon linn. var. ma-gua for the prevention and treatment of chronic viral hepatitis disease. The extract of Cucumis melon linn. var. ma-gua inhibits DNA proliferation of HBV to show antiviral activity, and is thus used for remedies or health foods for the prevention and treatment of chronic viral hepatitis disease. CONSTITUTION: A pharmaceutical composition for the prevention and treatment of chronic viral hepatitis disease is characterized by containing a crude extract of Cucumis melon linn. var. ma-gua 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.
    公开号:KR20040018731A
    申请号:KR1020020050689
    申请日:2002-08-27
    公开日:2004-03-04
    发明作者:이기수；김영호；이진호；안치영
    申请人:주식회사 바이오원；이기수；김영호；이진호；안치영；
    IPC主号:
    专利说明:

    Composition containing the extract of Cucumis melon Linn. var. ma-gua for therapy against chronic viral hepatitis disease}
    [6] The present invention relates to a composition for the prevention and treatment of viral liver disease, and more particularly, the pharmaceutical composition useful for preventing and treating viral liver diseases such as hepatitis and cirrhosis, using the inhibitory effect of hepatitis B virus transcriptase activity of the extracts. A composition and a dietary supplement.
    [7] 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 at 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.
    [8] 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. To date, the HBV DNA has a transcriptase gene (pol), a virus shell gene (capsid), a viral surface antigen gene (HBsAg), and several X genes involved in cancer function. Unlike ordinary DNA viruses that replicate DNA chains intact, HBV is first transcribed with 3.5 Kb of pregenomic RNA chains in the DNA chain by host transcriptase, and then reversed to virus-specific reverse transcriptase (pol). The double chain DNA is replicated by this. This replication process occurs independently of the host chromosome. However, some of the viral DNA has been found to enter the host's chromosome. When HBV DNA gets into the host's chromosome, the gene becomes unstable and the X protein expressed therein promotes transcription of the host cell and inhibits p53 gene function. Is likely to be. If this situation continues, it will develop into HCC.
    [9] As described, HBV exhibits a high degree of variability comparable to that of a normal RNA virus, due to 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.
    [10] 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.
    [11] 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, and alpha-interferon is combined with lamivudine. It is becoming. On the other hand, hepatitis C has been shown to some extent 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.
    [12] 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. The number of hepatitis B virus carriers in China is more than 100 million, which corresponds to 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.
    [13] 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. These drugs are commercialized 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.
    [14] 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 for new drug development from these plant natural products to be clinically applied.
    [15] 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, which are synthetic drugs, have been developed, but as a complete treatment for hepatitis, it is still in question, and its use is extremely limited due to side effects and high cost. It is true.
    [16] Therefore, researches are actively underway to create new therapeutic agents with low toxicity and side effects from folk remedies or natural products that have been applied for a long time. 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).
    [17] 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. It is usually harvested in mid-August between September and September, and it doesn't grow well when the seeds are planted the next year. Its effect is known to be good for liver function and has been edible as fruit in northeastern China for a long time.
    [18] However, in fact, the above-mentioned documents and research reports have not been reported so far the physiological activity of the herbal medicine for the viral liver disease using the extract of the apple.
    [19] The present invention relates to a composition for the prevention and treatment of hepatitis, hepatic cirrhosis, especially caused by hepatitis B virus, which contains extracts of the apple.
    [20] The present inventors have pharmacologically observed the therapeutic effect of hepatic extracts and complex herbal medicines containing the same against viral hepatitis and cirrhosis, and whether there is a possibility to prevent and treat hepatitis and cirrhosis of the herbal medicine which has accumulated a large amount of clinical data. The main focus was on.
    [21] As a result of the study, the extract of the causal extract according to the present invention inhibits the activity of hepatitis B virus transcriptase, thereby inhibiting the virus growth and confirming the fact that it has the prophylactic and therapeutic activity of viral liver disease.
    [22] Accordingly, it is an object of the present invention to provide a pharmaceutical composition and a dietary supplement showing a prophylactic and therapeutic effect of a viral hepatic disease containing an extract of an apple having an inhibitory effect of hepatitis B virus replication enzyme as an active ingredient.
    [1] 1 is a purchase place and instructions of the seed of the apple used in the present invention,
    [2] FIG. 2 is a photograph showing the effect of inhibiting HBV transcriptase activity through priming reaction after treating an asterisk extract and five kinds of herbal extracts when HBV transcriptase expression is activated in an insect cell line.
    [3] Figure 3 is a photograph showing the effect of inhibiting the HBV copy enzyme activity of the complex drug through the priming reaction after treating the complex drug containing the apocarcinoma at the concentration of activated HBV copy enzyme in the insect cell line,
    [4] FIG. 4A shows intracellular intracellular treatments on day 4 and day 8 after treatment with 0.15% of herbal extracts treated with Woodchuck hepatitis B virus (WHBV) -infected cell lines in order to observe the inhibition of viral replication activity of the complex containing herbals. Table comparing the amounts of pregenonomic RNA and WHBV surface antigen mRNA,
    [5] Figure 4b is treated with 0.15% complex drug in the cell line infected with Woodchuck hepatitis B virus in order to observe the inhibition of the virus replication activity of the extract containing the herbal medicine, the viral DNA in the nucleus, intracellular virion DNA and DNA replication intermediates Southern blot analysis of the photographs.
    [23] In accordance with the above object, the present invention provides a crude extract of Cucumis melon Linn.var.ma-gua effective in inhibiting hepatitis B virus proliferation.
    [24] The crude extract means water, methanol, ethanol, lower alcohols, mixed solvents thereof and extracts soluble in buffers.
    [25] Hereinafter, the present invention will be described in detail.
    [26] In order to prepare a crude extract of the apple effective in inhibiting the hepatitis B virus proliferation of the present invention,
    [27] The crude crude extract is washed and peeled after cutting off the cabbage, and naturally dried at a temperature of 10 to 40 ℃, preferably 20 to 30 ℃ for about 1 to 50 days, preferably about 15 to 25 days After pulverization, the solution was dissolved in an extraction solvent such as water, lower alcohol, buffer, etc. at a concentration of 1 to 70%, preferably 40 to 60%, and about 1 hour at 50 to 100 ° C, preferably 70 to 80 ° C. To 1 day, preferably about 1 to 5 hours, and then centrifuged to obtain the supernatant extract. In the present invention, crude extract is obtained by bathing the powder of cabbage using phosphate buffer as a preferred embodiment.
    [28] The present invention provides a pharmaceutical composition effective for the prevention and treatment of viral liver disease containing the extract of the apple obtained in the extraction process.
    [29] The composition for the prevention and treatment of viral liver disease of the present invention comprises 0.5 to 50% by weight of the extract based on the total weight of the composition.
    [30] It was confirmed that the HBV transcriptase activity was significantly reduced when the complex containing the herbal medicine obtained by the above method was treated in each concentration of the insect cell line expressing the HBV mutant gene.
    [31] The composition comprising the extract of the apple of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.
    [32] The composition comprising the extract of the present invention may further comprise a suitable carrier, excipient and diluent according to conventional methods.
    [33] Carriers, excipients and diluents that may be included in the composition comprising the extract 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.
    [34] Compositions comprising extracts according to the invention are formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterile injectable solutions, respectively, according to conventional methods. Can be used.
    [35] The pharmaceutical preparations thus prepared may be administered by a route selected from oral, parenteral (including transdermal, intradermal, intramuscular and intravenous), rectal and inhalation.
    [36] The amount of the extract of the present invention may vary depending on the age, sex, and weight of the patient, but generally in adults, the amount of 0.1 to 1000 mg / kg per day, preferably 10 to 100 mg / kg, It may be administered once to several times daily. In addition, the dosage of the extract can be increased or decreased depending on the route of administration, the degree of disease, sex, weight, age and the like. Therefore, the above dosage does not limit the scope of the present invention in any aspect.
    [37] In addition, the present invention provides a health supplement containing the apple extract.
    [38] The composition comprising the extract of the apple of the present invention can be used in a variety of drugs, food and beverages for the prevention and treatment of viral liver disease. Examples of the food to which the compound herbal containing the extract may be added include various foods, beverages, gums, teas, vitamin complexes, and health supplements.
    [39] Since the extract of the apple itself of the present invention has little toxicity and side effects, it is a drug that can be used safely even for long-term administration for the purpose of prevention.
    [40] The extract 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 in the food or beverage may generally be added at 0.01 to 30% by weight of the total food weight, the health beverage composition is 0.02 to 30 g, preferably 0.3 to 3 g based on 100 ml Can be added.
    [41] The health beverage composition of the present invention has no particular limitations on the liquid components other than the above-mentioned extracts as essential ingredients in the indicated proportions, and may contain various flavors or natural carbohydrates as additional ingredients, as in general beverages. Examples of the above-mentioned natural carbohydrates include monosaccharides such as 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.
    [42] 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.
    [43] 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.
    [44] Example 1. Preparation of Crude Crude Extract
    [45] Seed seeds (Lightweight Yangjong Development Co., Ltd., China, see Fig. 1) were imported and grown in the Suwon University laboratory. After washing the fruit of the cabbage, remove the 2.5cm and the peel from the stem, and then chopped, and then dried for about 20 days at a temperature of 25 ℃ and powdered with a blender.
    [46] Example 2. Preparation of a compound herbal extract containing an apple
    [47] 84 g of the complex herbal powder containing 45 g (about 53.6%) of the apple powder of Example 1 was prepared in a phosphate buffered saline at a concentration of 10%, followed by bathing at 80 ° C. for 2 hours, and then 10,000 Centrifuged for 5 minutes at rpm, the supernatant was taken and used as a sample in the cell experiment.
    [48] Example 3. Preparation of a compound herbal extract containing an apple
    [49] 100 g of the composite herbal powder containing 53.6 g of the powder of Example 1 in 1 liter of 80% ethanol aqueous solution was extracted under reflux four times at 50 ° C. for 1 hour, and all were combined and filtered through a filter paper (Watman, USA). This was concentrated under reduced pressure with a reduced pressure concentrator (Eyela, N-1000, Japan) to obtain a 25g extract was used as a sample.
    [50] Reference example. Hepatitis B Virus Enzyme Expression
    [51] In order to express the hepatitis B virus polase (pol) protein in insect cell lines, an HBV cloning gene (Invitrogen) was cloned into baculovirus (FLAG-pol-stem-loop: FPL-pol). The Sf9 cell line was infected and transformed. 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's medium, Gibco Co., Ltd.) containing 50 μg / ml gentamicin were used and incubated at 26 ° C. for 48 hours.
    [52] Experimental Example 1. Determination of HBV DNA replication inhibitory ability
    [53] In order to examine the hepatitis B virus (HBV) proliferation inhibitory effect of the hepatitis B extract of the present invention, hepatitis B virus DNA inhibitor activity was measured.
    [54] When expressing the hepatitis B virus transcriptase of the reference example, from the beginning of culture, 10% complex herbal extract (supernatant) containing the quince of Example 2 was added to the Sf9 cell culture medium at a final concentration of 500 µg / ml, respectively. After hours the transcriptase activity was measured.
    [55] 1st group: control group which added only saline solution
    [56] Group 2: Add 10% apple extract (final concentration 500 µg / ml)
    [57] Group 3: Add 10% bitter extract (final concentration 500㎍ / ㎖)
    [58] 4th group: 10% of the extract of Algae extract (final concentration 500µg / ml)
    [59] Group 5: 10% kelp extract added group (final concentration 500 μg / ml)
    [60] Group 6: 10% 칡 extract addition group (final concentration 500 µg / ml)
    [61] The seventh group: 10% royal broth extract added group (final concentration 500 ㎍ / ㎖)
    [62] Group 8: alpha-interferon addition group (10 6 IU, Roche)
    [63] 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 Bead (Sigma) to which an anti-FLAG monoclonal antibody was bound, and then pre-prepared with HBV transcriptase. HBV transcriptase-primer complexes were allowed to form on genomic RNA. The activity of HBV transcriptase was measured to the extent that 32 P-TTP (3,000 Ci / mmol, NEN Co., Inc.) was inserted during the primer reaction, and the amount of 32 P-TTP-labeled transcriptase protein was measured on the SDS-PAGE gel. It was confirmed. At this time, if the 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.
    [64] The results of Figure 2 shows that the addition of the apple extract, compared with the control group certainly inhibits the virus replication enzyme, showing the same degree of suppression as the alpha-interferon treatment, effective in inhibiting the proliferation of hepatitis B virus It could be confirmed.
    [65] Experimental Example 2. Determination of HBV DNA replication inhibitory ability of complex herbal extracts containing horses
    [66] In order to examine the hepatitis B virus (HBV) proliferation inhibitory effect of the hepatitis B virus that has a hepatitis B extract containing 53.6% of the extract of the present invention, the degree of DNA replication enzyme activity of hepatitis B virus was measured.
    [67] When expressing the hepatitis B virus transcriptase of Example 3, the 10% complex herbal extract (supernatant) of Example 2 was added to 10 ml cell culture medium in the amounts of 50 µl, 100 µl and 500 µl, respectively, from the beginning of culture. After 48 hours, the transcriptase activity was measured according to the method of Experimental Example 1 above.
    [68] 1st group: Control group which added only saline solution.
    [69] 2nd group: 50 microliters of 10% multiple herbal extracts (final concentration 50 micrograms / ml).
    [70] 3rd group: 100 microliters of 10% multiple herbal extracts (final concentration 100 micrograms / ml).
    [71] 4th group: 500 microliters of 10% multiple herbal extracts (final concentration 500 microgram / ml).
    [72] Referring to the results of FIG. 3, lane 1 is a control group treated only with saline, and the activity of basic replication enzymes can be observed, and lanes 2, 3, and 4 were treated with concentrations of 10% complex herbal extracts by concentration. As the concentration was increased, it was confirmed that the transcriptase activity of hepatitis B virus was suppressed and the transcriptase activity was significantly decreased when the concentration of lane 4 was treated at 500 μg / ml.
    [73] Therefore, it was confirmed that the complex herbal composition containing the apple 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.
    [74] Experimental Example 3. Determination of anti-viral activity of complex herbal containing apoplex against hepatitis B virus expressed in HepG2.2.15 cell line.
    [75] HepG2.2.15 cell line (Georgetown University, Dr. Korba, USA) is a cell line in which HBV is propagated by transforming the liver cancer cell line HepG2 with HBV DNA. Observed.
    [76] Efficacy inhibitory concentrations (EC 50 ) for intracellular HBV DNA replication intermediate production of the medicinal herb complexes and the inhibitory concentrations for HBV virion DNA secreted into the cell line culture (EC 90 ) were determined. The inhibition of HBV production by the combined herbal powders and the bath solution was measured in comparison with lamivudine (3TC, Glaxo Wellcome) used as a positive control.
    [77] Cell lines were incubated in each of them using a T-96 well plate, to which a compound containing aquatic extracts was added at a predetermined rate, 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.
    [78] 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 combined herbal powder was found to be 2.8 times higher than the activity of the bath liquid of the combined herbal medicine. 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 ).
    [79] EC 50 EC 90 CC 50 S.I. Complex Herb Powder90 µg / ml254 µg / ml> 3700 µg / ml14.6 Complex Herbal Thermal Solution259 µg / ml697 µg / ml> 1000 µg / ml1.4 Lamivudine (3TC)0.06 μM0.166 μM2165 μM13042
    [80] Experimental Example 4 Determination of the Complex Herbal Effect Containing Apple in WHBV Virus Proliferating Cell Line
    [81] Contains horses from primary hepatocytes (Dr. Jacobs, Cornell University, Cornell University, USA) selected from Woodchuck laboratory animals with hepatitis caused by infection with the Woodchuck HBV virus (WHBV), which is genetically similar to human HBV virus. Anti-viral activity by one combination drug powder was measured by comparing RNA amounts. Alpha-interferon is a drug currently used to inhibit the hepatitis virus proliferation, and used as a positive control group to see the efficacy of the compound herbal.
    [82] Were cultured for 2 weeks the stem cells treated with interferon (Roche Co.) week-second embodiment of the composite herbal powder containing alpha magwa of liver state, and control the processing unit 10 6 IU to the culture to a concentration of 0.15%. After 4 days of incubation, virus-related DNA and RNA products were extracted from the cell line, and after 8 days, the same amount of DNA and RNA extracts were obtained and Southern blot hive with hepatitis B virus surface antigen (HBsAg) DNA probe. Southern blot hybridization was performed to measure DNA and RNA levels through phosphoimage analysis.
    [83] 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.
    [84] In the results of Figure 4a, WHBV RNA was reduced by about 20% in the cell line treated with a four-day treatment of the complex containing herbal powder powder, which means a reduction of infectious virus particles in the subsequent replication cycle. In the cell line treated with the compound 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. 4A).
    [85] Compared with the amount of intranuclear WHBV DNA, out-secreted virion DNA and replication intermediate DNA produced in the culture of the cell line without the multi-drug powder, the multi-drug-containing multi-drug powder and the alpha-interferon-treated cell line, respectively In the cell line treated with the multi-drug powder, almost all of the viral DNA in the nucleus disappeared, and the amount of intermediate DNA and virion DNA also decreased significantly (see FIG. 4b). Through this result, it was confirmed that the complex herbal powder containing the devil had an effect in inhibiting the replication of Woodchuck hepatitis B virus.
    [86] Clinical Example 1. Verification of therapeutic effect by administration of complex herbal medicine containing hepatic to patients (volunteers) with liver disease of hepatitis and cirrhosis.
    [87] 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.
    [88] The compound containing the medicinal herbs of Example 2 were administered orally at least 1.0 days per adult for 25 days or more for 25 days. 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.
    [89] Table 2 shows the results of clinical treatment by the combination drug administration containing horses. 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.
    [90] SymptomTreatment period (days)Diagnosis Before afterGOTGPTHBV DNAHBsAgAnti-HBsAgHBeAgAnti-HBeAg Cirrhosis and ascites35I'm×××+-+ - after2815--+-+ Hepatitis and Cirrhosis90I'm5244×+ -×- after6285++--+ Hepatitis B25I'm××++ -+ - after2331-+- -+ Fatty liver25I'm8197++-- - after3767-+- -+ Hepatitis B155I'm3451++- -+ after3553-+- - - Hepatitis B60I'm213290++-+ - after3234-++++
    [91] 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.
    [92] Formulation Example 1 Preparation of Injection
    [93] Example 1 Dry Extract ............... 100 mg
    [94] Sodium metabisulfite ......................................... 3.0 mg
    [95] Methylparaben ...... 0.8 mg
    [96] Propylparaben ...................................... 0.1 mg
    [97] Sterile Distilled Water for Injection ...
    [98] The above ingredients were mixed and adjusted to 2 ml by a conventional method, followed by filling into a 2 ml ampoule and sterilizing to prepare an injection.
    [99] Formulation Example 2 Preparation of Tablet
    [100] Example 1 Dry Extract ............... 200 mg
    [101] Lactose 100 mg
    [102] Starch ......................................... 100 mg
    [103] Magnesium Stearate ...............
    [104] The tablets were prepared by mixing the above components and tableting according to a conventional method for producing tablets.
    [105] Formulation Example 3 Preparation of Capsule
    [106] Example 1 Dry Extract ............... 100 mg
    [107] Lactose ...... 50 mg
    [108] Starch ............................ 50 mg
    [109] Talc ........................................ 2 mg
    [110] Magnesium Stearate ...............
    [111] The capsules were prepared by mixing the above components and filling the gelatin capsules according to a conventional method for preparing capsules.
    [112] Formulation Example 4 Preparation of Liquid
    [113] Example 1 Dry Extract ............... 1000 mg
    [114] Sugar ........................... 20 g
    [115] Isomerized sugar ......................................... 20 g
    [116] Lemon scent .........................
    [117] Purified water was added to adjust the total volume to 100 ml.
    [118] 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.
    [119] In addition, health foods and health drinks were prepared in the following manner.
    [120] Formulation Example 5 Preparation of Health Food
    [121] Example 1 Dry Powder ......................................... 1000 mg
    [122] Vitamin Blend ......................... 20 g
    [123] Vitamin A Acetate ............... 70 μg
    [124] Vitamin E ......................... 1 mg
    [125] Vitamin B 1 ................................. 0.13 mg
    [126] Vitamin B 2 ................................. 0.15 mg
    [127] Vitamin B 6 ....................... 0.5 mg
    [128] Vitamin B 12 ................................. 0.2 μg
    [129] Vitamin C ......................................... 10 mg
    [130] Biotin .......................... 10 μg
    [131] Nicotinic Acid Amide ...................................... 1.7 mg
    [132] Folic acid ......................................... 50 μg
    [133] Calcium Pantothenate ......................................... 0.5 mg
    [134] Mineral mixture ...............
    [135] Ferrous Sulfate ................................. 1.75 mg
    [136] Zinc Oxide ........................ 0.82mg
    [137] Magnesium Carbonate ......................................... 25.3 mg
    [138] Potassium monophosphate ......................................... 15 mg
    [139] Dicalcium Phosphate Dioxide ..................... 55 mg
    [140] Potassium citrate ..... 90 mg
    [141] Calcium Carbonate ... 100 mg
    [142] Magnesium Chloride .............. 24.8 mg
    [143] 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.
    [144] Formulation Example 6 Preparation of Health Beverage
    [145] Example 1 Dry Powder ......................................... 1000 mg
    [146] Citric Acid ..................... 100 mg
    [147] Oligosaccharide ......................................... 100 g
    [148] Plum concentrate ..................... 2 g
    [149] Taurine ..................................... 1 g
    [150] Purified water is added.
    [151] 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.
    [152] 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.
    [153] As described above, the extract of the apple according to the present invention has an excellent effect of inhibiting the replication enzyme activity of hepatitis B virus and consequently inhibiting the growth of the virus, thereby preventing and treating viral liver diseases such as hepatitis and cirrhosis. It can be usefully used.
    权利要求:
    Claims (8)
    [1" claim-type="Currently amended] A crude extract of Cucumis melon Linn.var.ma-gua, which is effective in inhibiting the hepatitis B virus proliferation.
    [2" claim-type="Currently amended] A pharmaceutical composition for the prevention and treatment of viral liver disease, containing crude extracts of apples.
    [3" claim-type="Currently amended] The composition of claim 1 or 2, wherein the crude extract is extracted with at least one polar solvent selected from water, lower alcohols such as methanol, ethanol, mixed solvents and buffers thereof.
    [4" claim-type="Currently amended] The composition of claim 3 wherein the polar solvent is a buffer.
    [5" claim-type="Currently amended] The composition of claim 1 or 2, wherein the dosage is administered in an amount of 10 to 100 mg per kg body weight of the adult.
    [6" claim-type="Currently amended] 3. A composition according to claim 1 or 2 comprising 0.5 to 50% of said extract relative to the total weight of the composition.
    [7" claim-type="Currently amended] A dietary supplement comprising crude extracts of apples and food-acceptable food supplements that have a prophylactic and therapeutic effect on viral liver disease.
    [8" claim-type="Currently amended] 8. The dietary supplement of claim 7, in the form of a powder, granule, tablet, capsule or beverage.
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    同族专利:
    公开号 | 公开日
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    法律状态:
    2002-08-27|Application filed by 주식회사 바이오원, 이기수, 김영호, 이진호, 안치영
    2002-08-27|Priority to KR1020020050689A
    2003-08-26|Priority claimed from AU2003253470A
    2004-03-04|Publication of KR20040018731A
    优先权:
    申请号 | 申请日 | 专利标题
    KR1020020050689A|KR20040018731A|2002-08-27|2002-08-27|Composition comprising the extract of Cucumis melon Linn. var. ma-gua for therapy against chronic viral hepatitis disease|
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