Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
  • A61K36/18—Magnoliophyta (angiosperms)
  • A61K36/185—Magnoliopsida (dicotyledons)
  • A61K36/72—Rhamnaceae (Buckthorn family), e.g. buckthorn, chewstick or umbrella-tree
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics

Definitions

• the present invention relates to a composition for the prevention and treatment of hepatitis B containing extracts or fractions of Genus Hovenia and functional food for the prevention and improvement of hepatitis B containing the same. More precisely, the present invention relates to a composition for the prevention and treatment of hepatitis B containing extracts or fractions of Genus Hovenia extracted with water, alcohol, an organic solvent or a mixture thereof and a functional food for the prevention and improvement of hepatitis B containing the same.
• Hepatitis B virus causes acute and chronic hepatitis in human which might be developed into self- limited acute hepatitis, fulminant hepatitis or chronic active hepatitis, etc.
• Chronic active hepatitis is developed into liver cirrhosis and hepatoma and serious acute disease accompanying liver failure (Brechot . , J. Hepatol., 4, 269-279, 1987).
• Most of patients with chronic hepatitis have consistent or active hepatitis symptoms, but some of those patients do not exhibit any symptoms. The break out mechanism of viral hepatitis and causative factors have not been disclosed, yet.
• liver damage caused by hepatitis B virus is mediated by immune cells of a host (Milich, D. R. et al., J. Immunol., 143, 3141-3147, 1989)
• studies have been focused on viral antigen causing liver cell necrosis, immune response of specific B cells and T cells, cellular pathogenesis or oncogenesis directly caused by virus protein (R. Zschke, O. et al . , Nature, 348, 252-254, 1990).
• virus protein R. Zschke, O. et al .
• HBV mediated diseases including hepatitis are difficult to treat because the development of effective anti-HBV medicines has not been so successful.
• virology made a great advance, so that the process of virus proliferation that has been a cause of many human diseases including HBV has been well acknowledged and based on that the development of a therapeutic agent for pathogenic virus mediated disease is competitively going on.
• the antiviral agents developed so far are mostly the drugs using nucleoside analogues.
• HBV is the factor highly responsible for the development of cancer. Liver cancer takes the third place in death rate by cancer in Korea. Among liver cancer patients in Korea, 70% are caused by hepatitis B virus and 10% are caused by hepatitis C virus.
• liver cancer it is the best way to reduce risk of liver cancer by preventing the viral infection and inhibiting the proliferation of such virus.
• human immune system has been the only way to treat hepatitis patients infected with HBV so far.
• HBV can be eliminated but at the same time liver cells are also destroyed, suggesting that such side effect can cause another serious disease.
• Such side effect is difficult to predict, so it is required to execute antiviral therapy and immune therapy altogether for the fast and efficient treatment of hepatitis.
• Liver cells recovered from viral hepatitis are destroyed by cytotoxic T lymphocytes (CTL) and the released viral particles from those destroyed liver cells destroy neutralizing antibodies working for the virus.
• CTL cytotoxic T lymphocytes
• core is a structural epitope
• antigen s surface is the epitope produced by the surface protein of the virus.
• Antigen e is a marker in blood showing the infection by hepatitis B virus. Antigen e is not always necessary for the proliferation of HBV and its role in the life cycle of HBV has not been disclosed, yet. But, antigen e is presumed to play an important role considering that a gene of the antigen is well preserved in a similar animal virus. So, antigen e is an immunological target to eliminate the virus, in particular for a host to eliminate the virus (Pignatelli, M. et al., J. Hepal. 4, 15-16. 1987).
• Hepatitis B virus has been treated by interferons, nucleoside analogues or immune regulators. As a result, only interferon- ⁇ has shown a treatment effect, so that it has been approved as a drug treating hepatitis B in USA.
• Nucleoside analogue the first drug developed for the treatment of hepatitis, had inhibited the virus in the early stage but had carried serious side effects such as generation of varieties and resistance as time went on.
• Interferon- ⁇ was first tried as a drug for chronic hepatitis B in the mid 1970s by Sculled et al. Since then, it has been known to have HBV inhibiting effect.
• interferon- ⁇ Since the production of interferon- ⁇ was possible by gene recombination, the use of interferon has been in great increase (J. Infect. Dis., 143: 772-783, 1981). However, the treatment effect of interferon is not satisfactory. When interferon- ⁇ was administered for at least three months three times a week, the virus proliferation inhibition effect was confirmed in only 20% of patients, and that effect was not even consistent. Particularly, patients by vertical transmission (mother-child) , largely in Asian had resistance against interferon- ⁇ , resulting in very low treatment effect. It has recently been reported that only up to 50% of acute and chronic HBV patients are fitted for the treatment with interferon- ⁇ .
• interferon- ⁇ The treatment effect of interferon- ⁇ is only expected in those patients showing increased levels of enzymes (AST, SGOT, ALT. SGPT, etc) produced in liver caused by the virus. Therefore, it is an urgent request to develop a new drug to treat HBV to treat those patients particularly having resistance against interferon- ⁇ and nucleoside analogues. It is considered as a better approach to inhibit the adhesion of the virus onto a receptor or to find a material capable of inhibiting specifically the protein or enzyme activating the virus.
• enzymes AST, SGOT, ALT. SGPT, etc
• Genus Hovenia is a deciduous broad-leaved tree belonging to Rhamnaceae, which is distributed in south Kangwon and Kyunggi province at the altitude of 50-800 m and is also found in Japan and China. Korea, Japan and China have different varieties on their own except the basic species (Hovenia dulcis Thunb. ) characterized by light green flowers and hairless leaves.
• the endemic species in Korea is Hovenia dulcis var. Koreana Nakai blooming white flowers.
• the endemic species in Japan are Hovenia dulcis v ar. Latifolia Nakai having larger leaves than the basic species and Hovenia dulcis var. tomentella Makino having hairy leaves.
• the present invention provides a composition for the prevention and treatment of hepatitis B containing extracts of Genus Hovenia as an active ingredient.
• the present invention also provides a composition for the prevention and treatment of hepatitis B containing fractions of Genus Hovenia extracts each obtained by extracting with hexane, ethyl acetate, chloroform and butanol stepwise as an active ingredient.
• the present invention further provides a treatment method for hepatitis B containing the step of administering a pharmaceutically effective dose of extracts of Genus Hovenia or fractions thereof to a subject with hepatitis B.
• the present invention also provides a prevention method of hepatitis B containing the step of administering a pharmaceutically effective dose of extracts of Genus Hovenia or fractions thereof to a subject with hepatitis B.
• the present invention also provides a use of extracts of Genus Hovenia or fractions thereof for the production of the composition for the prevention and treatment of hepatitis B.
• the present invention also provides a functional food for the prevention and improvement of hepatitis B containing extracts of Genus Hovenia as an active ingredient .
• the present invention provides a functional food for the prevention and improvement of hepatitis B containing fractions of Genus Hovenia extracts each obtained by extracting with hexane, ethyl acetate, chloroform and butanol stepwise as an active ingredient.
• the present invention provides a composition for the prevention and treatment of hepatitis B containing extracts of Genus Hovenia as an active ingredient.
• the extract of Genus Hovenia of the present invention is preferably prepared by the method comprising the following steps:
• step 2) concentrating the extract of step 1) under reduced pressure
• step 2) drying the concentrate of step 2) to give dried powder.
• the extraction method of the invention is not always limited thereto.
• Genus Hovenia is preferably selected from the group consisting of Hovenia dulcis Thunberg, Hovenia dulcis var Koreana Nakai, Hovenia dulcis var Latifolia Nakai and Hovenia dulcis var tomentella Makino, and it can be cultivated or purchased in the market.
• step 1) of the method seeds or fruits of Genus Hovenia are used.
• the alcohol is preferably Ci - C 4 lower alcohol, and this lower alcohol is preferably ethanol or methanol.
• the preferable concentration of ethanol is 50% and the preferable concentration of methanol is 100%, but not always limited thereto.
• Methanol extraction is preferably performed at room temperature and 50% ethanol extraction and hot water extraction are preferably performed at 70-80 ° C, but not always limited thereto. Distilled water, ethanol and methanol are added to the dried Genus Hovenia fruits 1-10 times the fruit amount, followed by extraction. The extraction is preferably performed 1 - 5 times. It is more preferred to perform extraction two times repeatedly, but not always limited thereto .
• the organic solvent is preferably selected from the group consisting of hexane, ethyl acetate, chloroform and butanol.
• step 2) of the above method and the drying process of step 3) are performed by the conventional methods used in this field.
• the extract of Genus Hovenia can inhibit the generation of HBV e antigen and s antigen and inhibit the binding between the surface antigen of HBV and the HBV receptor of liver cell, suggesting that the extract of the present invention can be effectively used as a composition for the prevention and treatment of hepatitis B.
• the present invention also provides a composition for the prevention and treatment of hepatitis B containing fractions of Genus Hovenia extracts each obtained by extracting with hexane, ethyl acetate, chloroform and butanol stepwise as an active ingredient.
• the present invention also provides a composition for the prevention and treatment of hepatitis B containing butanol fraction of Genus Hovenia extracts as an active ingredient .
• the fractions of Genus Hovenia extracts are preferably prepared by the method comprising the following steps :
• step 1) preparing extracts from the dried Genus Hovenia plant using water, alcohol or a mixture thereof; 2) dissolving the extract of step 1) with an organic solvent;
• step 5 drying the concentrate obtained in step 4) under reduced pressure to give dried powder.
• step 1) of the above method the extract of Genus Hovenia plant can be prepared by the extraction method mentioned above.
• step 2) the organic solvent is added in the order of hexane, ethyl acetate, chloroform and butanol.
• step 3 the fraction is preferably separated using a separatory funnel, but not always limited thereto. All of the fractions obtained from each stage can be used and the fraction of butanol layer is most preferred.
• the methanol extract of Genus Hovenia fruits was added with organic solvents such as hexane, ethyl acetate, chloroform and butanol stepwise to give fractions.
• organic solvents such as hexane, ethyl acetate, chloroform and butanol stepwise to give fractions.
• the effect of the butanol fraction on the generation of HBV e antigen was investigated.
• the butanol fraction of the methanol extract of Genus Hovenia fruits reduced the activity of HBV e antigen and interrupted the binding of HBV onto its receptor on the liver cell surface (see Figures 4 and 6) .
• composition of the present invention can include, in addition to the extract of Genus Hovenia or the fraction thereof, one or more effective ingredients having the same or similar function to the extract or fraction.
• the extract of Genus Hovenia of the present invention can be administered orally or parenterally and be used in general forms of pharmaceutical formulation.
• the extract of the present invention can be prepared for oral or parenteral administration by mixing with generally used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactant.
• Solid formulations for oral administration are tablets, pills, powders, granules and capsules. These solid formulations are prepared by mixing the pharmaceutical composition of the present invention with one or more suitable excipients such as starch, calcium carbonate, sucrose or lactose, gelatin, etc.
• Liquid formulations for oral administrations are suspensions, solutions, emulsions and syrups, and the above-mentioned formulations can contain various excipients such as wetting agents, sweeteners, aromatics and preservatives in addition to generally used simple diluents such as water and liquid paraffin.
• Formulations for parenteral administration are sterilized aqueous solutions, water-insoluble excipients, suspensions, emulsions, lyophilized preparations, suppositories and injections.
• Water insoluble excipients and suspensions can contain, in addition to the active compound or compounds, propylene glycol, polyethylene glycol, vegetable oil like olive oil, injectable ester like ethylolate, etc.
• Suppositories can contain, in addition to the active compound or compounds, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerol, gelatin, etc.
• the composition of the present invention can be administered by parenterally and the parenteral administration includes subcutaneous injection, intravenous injection or intramuscular injection.
• the dosage unit can contain, for example, 1, 2, 3 or 4 individual doses or 1/2, 1/3 or 1/4 of an individual dose.
• An individual dose preferably contains the amount of active compound which is administered in one application and which usually corresponds to a whole, 1/2, 1/3 or 1/4 of a daily dose.
• the effective dosage of the composition of the present invention is 0.0001 ⁇ 10 g/kg per day and preferably 0.0001 ⁇ 5 g/kg per day, and administration frequency is 1 ⁇ 6 per day.
• the composition for the prevention and treatment of hepatitis B of the present invention is evaluated to be a safe substance since its estimated LD 5O value is much greater than 5 g/kg in rats, which is confirmed by acute toxicity assay with rats tested via oral administration.
• the composition for the prevention and treatment of hepatitis B of the present invention can be administered alone or together with surgical operation, hormone therapy, chemo-therapy and biological regulators to prevent and treat hepatitis B.
• the present invention further provides a treatment method for hepatitis B containing the step of administering a pharmaceutically effective dose of extracts of Genus Hovenia or fractions thereof to a subject with hepatitis B.
• the present invention also provides a prevention method of hepatitis B containing the step of administering a pharmaceutically effective dose of extracts of Genus
• the present invention also provides a use of extracts of Genus Hovenia or fractions thereof for the production of the composition for the prevention and treatment of hepatitis B.
• the extract of Genus Hovenia or fraction thereof can inhibit the generation of HBV e antigen and s antigen and interrupt the binding of HBV surface antigen onto its receptor, but has no cytotoxicity, so that it can be effectively used for the prevention and treatment of hepatitis B.
• the present invention also provides a functional food for the prevention and improvement of hepatitis B containing extracts of Genus Hovenia as an active ingredient .
• the present invention provides a functional food for the prevention and improvement of hepatitis B containing fractions of Genus Hovenia extracts as an active ingredient.
• the Genus Hovenia extract of the present invention can be used as food additive.
• the Genus Hovenia extract can be added as it is or as mixed with other food components according to the conventional method.
• the Genus Hovenia extract can be obtained by extraction using hot water and ethanol, and the concentration of ethanol is preferably 50%.
• the mixing ratio of active ingredients can be regulated according to the purpose of use (prevention, health enhancement or treatment) .
• the composition of the present invention is added preferably by up to 15 weight part and more preferably by up to 10 weight part.
• the content can be lower than the above but higher content can be accepted as well since the composition has been proved to be very safe.
• the food herein is not limited.
• the Genus Hovenia extract of the present invention can be added to meats, sausages, breads, chocolates, candies, snacks, cookies, pizza, ramyuns, flour products, gums, dairy products including ice cream, soups, beverages, tea, drinks, alcohol drinks and vitamin complex, etc, and in wide sense, almost every food applicable in the production of health food can be included.
• the composition for health beverages of the present invention can additionally include various flavors or natural carbohydrates, etc, like other beverages.
• the natural carbohydrates above can be one of monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xilytole, sorbitol and erythritol.
• natural sweetening agents such as thaumatin and stevia extract, and synthetic sweetening agents such as saccharin and aspartame can be included as a sweetening agent.
• the content of the natural carbohydrate is preferably 0.01-0.04 g and more preferably 0.02-0.03 g in 100 mt of the pharmaceutical composition of the present invention.
• the Genus Hovenia extract of the present invention can include in variety of nutrients, vitamins, minerals, flavors, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acid, protective colloidal viscosifiers, pH regulators, stabilizers, antiseptics, glycerin, alcohols, carbonators which used to be added to soda, etc.
• the Genus Hovenia extract of the present invention can also include natural fruit juice, fruit beverages and/or fruit flesh addable to vegetable beverages. All the mentioned ingredients can be added singly or together.
• the extract of Genus Hovenia or fraction thereof can inhibit the generation of HBV e antigen and s antigen and interrupt the binding of HBV surface antigen onto its receptor but has no cytotoxicity, it can be effectively used for the production of a composition for the prevention and treatment of hepatitis B or a functional food for the prevention and improvement of hepatitis B.
• Figure 1 is a diagram illustrating the extraction of Genus Hovenia fruits with methanol, hot-water and 50% ethanol, and the purification of active ingredients therefrom,
• Figure 2 is a graph illustrating the anti-hepatitis virus activities of the methanol, hot-water and 50% ethanol extracts of Genus Hovenia fruits determined by measuring the secretion of HBV e antigen by HBeAg ELISA,
• Figure 3 is a graph illustrating the anti-hepatitis virus activities of the methanol, hot-water and 50% ethanol extracts of Genus Hovenia fruits determined by measuring the secretion of HBV s antigen by HBsAg ELISA,
• Figure 4 is a graph illustrating the secretion of HBV e antigen, measured by HBeAg ELISA, by methanol extracts prepared by different solvents according to Figure 1
• Figure 5 is a graph illustrating the result of receptor binding assay.
• Figure 6 is a graph illustrating the result of receptor binding assay. It was investigated whether or not the butanol (BuOH) fraction obtained from the methanol extract of Genus Hovenia fruits could interrupt the binding of HBV onto its receptor on the liver cell surface by receptor binding assay by the same manner as shown in Figure 5.
• BuOH butanol
• Example 1 Preparation of water extract of Genus Hovenia ⁇ Hovenia dulcis Thunberg
• Hovenia Hovenia dulcis Thunberg 25 i of 50% ethanol (70 ⁇ 80 ° C) was added to 10 kg of dried Genus Hovenia (Hovenia dulcis Thunberg) fruits, followed by extraction two times. The extract was concentrated in a rotary evaporator, followed by drying to give 635 g of dried powder.
• Example 4 Preparation of 50% ethanol extract of Hovenia dulcis var Koreana Nakai 25 t of 50% ethanol (70 ⁇ 80 ° C) was added to 10 kg of dried Hovenia dulcis var Koreana Nakai fruits, followed by extraction two times. The extract was concentrated in a rotary evaporator, followed by drying to give 605 g of dried powder.
• Example 5 Preparation of 50% ethanol extract of Hovenia dulcis var Latifolia Nakai
• Example 6 Preparation of 50% ethanol extract of Hovenia dulcis var tomentella Making
• Example 7 Preparation of butanol fraction of methanol extract of Genus Hovenia ⁇ Hovenia dulcis Thunberg) Hexane, ethyl acetate and chloroform were added stepwise to 600 g of the methanol extract obtained in Example 3. Fractions were obtained from the dissolved mixture. Butanol was added to the remaining solution to obtain a butanol layer, which was concentrated in a rotary evaporator, followed by drying to give 52 g of dried powder.
• animal cells originated from human liver tissues where HBV is proliferating were treated with different concentrations of the Genus Hovenia extracts. Then, the inhibitory effect on the generation of e antigen and s antigen was investigated.
• HBV e antigen HBV e antigen
• HBsAg HBV e antigen
• HepG2 cells human hepatoblastoma cells
• the cells were cultured in 10 cm Petri-dishes containing
• HBeAg ELISA was performed using HBV antigen diagnostic reagent (Enzygnost HBeAg monoclonal, Behring, Germany). Particularly, 2.2.15 cells were distributed in a 96-well plate at the concentration of 2 x 10 4 cells per well. 100 ⁇ of serum-free MEM was added to each well of the plate, followed by culture in a 37 ° C, 5% CO 2 incubator for 24 hours.
• the extracts of Genus Hovenia ⁇ Hovenia dulcis Thunberg) , Hovenia dulcis var Koreana Nakai, Hovenia dulcis var Latifolia Nakai and Hovenia dulcis var tomentella Makino fruits were treated thereto at different concentrations. 24 hours after the treatment, the culture solution was centrifuged at 1,500 rpm for 5 minutes to eliminate cell debris. The obtained supernatant was distributed in the diagnostic reagent plate on which anti- HBeAg monoclonal antibody was smeared at a proper amount, which stood in a 37 ° C incubator for one hour.
• the diagnostic reagent plate was washed with phosphate buffer three times to induce secondary binding with peroxidase labeled hepatitis B antigen. Non-conjugated remaining antibody was eliminated by washing with phosphate buffer three times. 100 ⁇ i of the solution containing tetramethyl benzidine dihydrochloride (TMB) inducing color development by peroxidase was distributed to induce color development. Upon completion of the reaction, OD 45O was measured to evaluate the changes of e antigen (HBeAg) .
• TMB tetramethyl benzidine dihydrochloride
• IC 5O in Table 1 which is the concentration of a drug that is capable of inhibiting the secretion of e antigen 50% in HepG2.2.15 cells.
• HBsAg ELISA was performed with HBV s antigen by the same sandwich method as used for HBeAg ELISA.
• IC 50 in Table 2 which is the concentration of a drug that is capable of inhibiting the secretion of s antigen 50% in HepG2.2.15 cells .
• HepG2 human hepatoblastoma cell line
• U937 human histiocytic lymphoma cell line
• Plasma membranes of HepG2 and U937 cells were separated and a plate was coated with the membranes at the concentration of 50 ⁇ g/ml. After coating for overnight, the plate was washed with PBS five times. 200 fd of PBS containing 10% BSA was added to the plate, followed by blocking at room temperature for 6 hours. Then, the plate was washed with PBST five times. The synthetic biotin- preSl 21-47 peptide was loaded on the plate at the concentration of 100 ⁇ g/ml, which was treated with the methanol extract of Genus Hovenia ⁇ Hovenia dulcis Thunberg) fruits and fractions thereof, followed by reaction at 4 ° C for overnight.
• the plate was washed with PBS-Tween (PBS-T) four times. 50 fd of biotin- preSl peptide was added thereto, followed by reaction at 37 ° C for 1 hour. One hour later, the plate was washed with PBS-T four times. 100 fd of streptavidin-HRP was added thereto, followed by reaction at 37 °C for 30 minutes. 30 minutes later, the plate was washed with PBS-T four times.
• PBS-Tween PBS-Tween
• the Genus Hovenia extract of the present invention was suspended in 0.5% methyl cellulose solution and administered orally to the rats at the dosage of 5 g/kg/15 mi. Death, clinical symptoms, and weight change in the rats were observed, hematological tests and biochemical tests of blood were performed, and any abnormal signs in the gastrointestinal organs of chest and abdomen were checked with eyes during autopsy.
• the extract orally administered in this experiment was evaluated to be a safe substance since its estimated LD 50 value was much greater than 5 g/kg in rats.
• Lactose 1 g Powders were prepared by mixing all the above components, which were filled in airtight packs according to the conventional method for preparing powders.
• Capsules were prepared by mixing all the above components, which were filled in gelatin capsules according to the conventional method for preparing capsules.
• Pills were prepared by mixing all the above components according to the conventional method for preparing pills. Each pill contained 4 g of the mixture.
• Health enhancing ground beef was prepared by mixing 10 weight part of the extract of Example 2 with ground beef according to the conventional method.
• Brown rice, barley, glutinous rice and Yulmu (Job's tears) were gelatinized according to the conventional method, dried and pulverized to obtain 60-mesh powders.
• Black soybean, black sesame and wild sesame were steamed and dried according to the conventional method and pulverized to obtain 60-mesh powders.
• Example 2 The extract of Example 2 was concentrated under reduced pressure, spray-dried and pulverized to obtain 60- mesh dry powders.
• Sun-Sik was prepared by mixing the dry powders of the grains, seeds and the extract of Example 2 according to the below ratio.
• Grains (brown rice: 30 weight part, Yulmu: 15 weight part, barley: 20 weight part) ,
• Dry powders of the extract of Example 2 (3 weight part) , Ganoderma lucidum (0.5 weight part), Rehmannia glutinosa (0.5 weight part)
• Health enhancing vegetable juice was prepared by adding 5 g of the extract of Example 1 to 1,000 mi of tomato or carrot juice according to the conventional method.
• Health enhancing fruit juice was prepared by adding 1 g of the extract of Example 1 to 1,000 iM of apple or grape juice according to the conventional method.

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• 2007
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