KR20020082121A - Pharmaceutical composition useful for prevention or treatment of hepatitis containing flavonol derivatives as active ingredients - Google Patents

Abstract

PURPOSE: A therapeutic or preventive pharmaceutical composition for hepatitis containing as an effective component flavonol derivatives which strongly inhibit the formation of an e antigen and are low in cytotoxicity is provided which can be effectively used for treatment of chronic hepatitis B. CONSTITUTION: The pharmaceutical composition for the prevention and treatment of hepatitis contains flavonol derivatives represented by the formula 1 or a pharmaceutically acceptable salt thereof, wherein the flavonol derivatives contain myricetin represented by the formula 2, quercetin represented by the formula 3 and morin represented by the formula 4. In formula, R1, R2, R3 and R4 are independent, H, OH or 2. For example, a mixture of 1g myricetin, quercetin or morin, 0.6g sodium chloride, 0.1g ascorbic acid is dissolved in distilled water to produce 100ml solution, which is heated at 20deg.C for 30mim.

Description

Pharmaceutical composition useful for prevention or treatment of hepatitis containing flavonol derivatives as active ingredients}

The present invention relates to the use of a flavonol derivative having a structure of Formula 1 in the prevention or treatment of hepatitis B.

<Formula 1>

In Formula 1, R ₁ , R ₂ , R ₃ and R ₄ are independent of each other, and are H, OH or an alkoxy group of C _{1 to} C ₃ .

<Formula 2>

<Formula 3>

<Formula 4>

Human hepatitis B virus (HBV) causes acute and chronic hepatitis, and can develop into diseases such as self-controlling acute hepatitis, blunt hepatitis or chronic active hepatitis. Chronic active hepatitis develops, causes cirrhosis and liver cancer, and can be acutely acute with liver failure (Brechot, C., J. Hepatol. , 4, 269-279, 1987). When infected with chronic hepatitis, most patients show persistent or active hepatitis, while some do not. The pathogenesis or causative factors of the viral hepatitis are not clear yet. Since some evidence suggests that liver damage caused by the hepatitis B virus is mediated by the host's immune cells (Milich, DR et al., J. Immunol. , 143, 3141-3147, 1989), Intensive studies have been conducted on the immune response of specific B and T cells and the direct or pathogenic mechanisms of viral proteins (R. Zschke, O. et al., Nature , 348, 252-254, 1990). . However, the causes of the clinical pathways that still vary from patient to patient are not clear.

Hepatitis B has been studied with great interest in its treatment because of its severity. Currently, several drugs and vaccines have been developed and have received good reviews in preclinical and clinical trials, but few drugs have been applied as actual therapeutics. To date, the only treatment for hepatitis B virus (HBV) hepatitis patients is human immunity. It depends on the system. Human immune action against HBV removes HBV particles, but on the other hand destroys hepatocytes, so side effects of these immune actions can lead to fatal diseases. In addition, since these problems make it difficult to predict disease progression, it is desirable to use a combination of antiviral therapy and immunotherapy for fast and efficient hepatitis treatment. In other words, during the treatment of viral hepatitis, infected hepatocytes are destroyed by cytotoxic T lymphocytes (CTLs), and virus particles released from the destroyed hepatocytes remove the neutralizing antibodies against the virus to prevent reinfection into the hepatocytes. Proliferation of should also be suppressed.

Several methods have been tried to treat the hepatitis B virus, including interferon, nucleic acid derivatives or immunomodulators, but only interferon-α has been shown to be the only therapeutic effect. have. Interferon-α was first tested by Sculled et al in the mid-1970s as a treatment for chronic hepatitis B. It has been shown to be effective in inhibiting HBV replication. However, interferon-α, when administered three times a week for at least three months, shows a therapeutic effect that inhibits virus growth in an average of 20% of patients, but does not show sustained inhibitory effects. Patients or children due to infection are resistant to interferon-α and therefore have a low therapeutic effect. In addition, it has recently been reported that only 50% or less of acute and chronic patients infected with the hepatitis B virus are suitable for the treatment of interferon-α, among which various enzymes (AST, Only if the amount of SGOT, ALT, SGPT, etc. is increased, the interferon-α treatment can be expected to be effective. Therefore, there is an urgent need to develop a hepatitis B virus therapeutic agent that can treat patients with resistance to interferon-α. In particular, as a method of inhibiting the proliferation of hepatitis B virus, research has been conducted to find a substance that inhibits the attachment of a virus to a receptor or specifically inhibits an active protein or a polymerase of a virus.

Hepatitis B virus replication produces three major antigens (c, s, e antigens), the c (core) antigen is a structural antigen crystal and the s (surface) antigen is an antigenic crystal that is represented by viral surface proteins. The e antigen is a marker in the blood to detect hepatitis B virus infection. The e antigen is not a prerequisite for the propagation of hepatitis B and its role in the life history of the hepatitis B virus is not yet clear. However, it is speculated that the antigen plays an important role in view of the conserved gene of the e antigen. Thus, the e antigen is an immunological target for virus removal, in particular the host plays an important role in virus removal (Pignatelli, M. et al., J. Hepal. 4, 15-16, 1987).

Soluble e antigens circulate and secrete in the blood. e antigens are present in the serum and may be transmitted from mother to newborn through the placenta, but not to s antigens. When e antigens reach the thymus through the circulatory system, it is expected that T helper cells will lose their function. Therefore, newborns born from positive mothers have an immunological defect called tolerance of c antigen / e antigen-specific T-helper cells (Milich, DR, et al., PNAS USA 87, 6599-6603, 1990). . Newborns born from e-positive mothers due to the non-response of T helper cells are more likely to go chronic when infected with HBV.

Meanwhile, various vegetables and phenolic compounds are anti-cancer, anti-viral and lipids has been reported that the anti-function and cardiovascular disease prevention such beneficial pharmacological activity oxidizing bar (johujong, yichunja, ohseongchan after seoyeon me, food in the fruit around Testimonies , Hyoil Cultural History, 1998).

Therefore, the present inventors have selected a material showing anti-hepatitis B virus activity among natural products and researched to develop it as a hepatitis treatment, flavonol derivatives including mycetin, quercetin and moline, which are widely present in plants, hepatitis B The present invention has been completed by confirming that the cytotoxicity is low while strongly inhibiting the production of the viral e antigen, which can be usefully used for the prevention or treatment of hepatitis B.

It is an object of the present invention to provide a use of a flavonol derivative having a structure of formula 1 for the prevention and treatment of hepatitis B.

1 is a graph showing that myricetin, quercetin and morin inhibit the secretion of hepatitis B virus e antigen from extracellular to hepatocytes infected with hepatitis B virus,

FIG. 2 is a graph showing that myricetin, quercetin and morin do not affect the production of hepatitis B virus e antigen in intracellular infected hepatitis B virus,

3 is a graph showing that myricetin, quercetin and morin do not affect the production of intracellular hepatitis B virus s antigen in hepatocytes infected with hepatitis B virus,

4 is a graph showing that myricetin, quercetin and morin do not affect the production of extracellular hepatitis B virus s antigen in hepatocytes infected with hepatitis B virus,

Figure 5 shows that myricetin, quercetin, and morine do not affect DNA replication of hepatitis B virus,

1: DMSO; 2: 1 μM DDC; 3: 0.1 μM (0.023 μg / mL) 3TC;

4: 1 μM (0.23 μg / mL) 3TC; 5: 10 μM (2.3 μg / mL) 3TC;

6: 0.1 μM (0.03 μg / mL) mycetin; 7: 1 μM (0.32 μg / mL) myricetin;

8: 10 μM (3.2 μg / mL) mycetin; 9: 0.1 μM (0.03 μg / mL) quercetin;

10: 1 μM (0.34 μg / ml) quercetin; 11: 10 μΜ (3.4 μg / ml) quercetin;

12: 0.1 μM (0.03 μg / mL) morine; 13: 1 μM (0.3 μg / ml) morine;

14: 10 μM (3.0 μg / ml) moline;

I: cellular DNA;

D2: double strand linear DNA;

D1: double strand circular DNA;

S: single strand DNA

6 is a graph showing that quercetin, morphine, and myricetin do not affect the polymerase activity of hepatitis B virus,

FIG. 7 is a graph showing that quercetin, maurine and myricetin activate the production of T-helper cells in e-antigen producing transgenic mice.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention or treatment of hepatitis B containing a flavonol derivative of formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention also provides a pharmaceutical composition for preventing or treating hepatitis B, which contains as an active ingredient a combination of two or more selected from the group consisting of myricetin, quercetin, moline and pharmaceutically acceptable salts thereof.

The present invention also provides a food additive for preventing or treating hepatitis B, which contains a flavonol derivative having a structure of Formula 1 or a salt thereof as an active ingredient. The food additives include beverage compositions and the like.

The present invention also provides an immunostimulator containing the flavonol derivative as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for preventing or treating hepatitis B, which contains a flavonol derivative of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

In Formula 1, R ₁ , R ₂ , R ₃ and R ₄ are independent of each other, and are H, OH or an alkoxy group of C _{1 to} C ₃ .

As the flavonol derivative of the present invention, mycetin of formula (2), quercetin of formula (3), moline of formula (4), and the like are preferable. The flavonol derivatives are mycetin of Formula 2 wherein R ₁ = H, R ₂ = OH, R ₃ = OH and R ₄ = OH in Formula 1; Quercetin of Formula 3 wherein R ₁ = H, R ₂ = OH, R ₃ = OH and R ₄ = H; And moline of Formula 4 wherein R ₁ = OH, R ₂ = H, R ₃ = OH and R ₄ = H.

The present invention also provides a pharmaceutical composition for preventing or treating hepatitis B, which contains as an active ingredient a combination of two or more selected from the group consisting of myricetin, quercetin, moline and pharmaceutically acceptable salts thereof.

Flavonol derivatives of the present invention represented by the formula (1) can be used in the form of a pharmaceutically acceptable salt, the salt is an acid addition salt formed by a pharmaceutically acceptable free acid is useful. Inorganic acids and organic acids can be used as the free acid. Hydrochloric acid, bromic acid, sulfuric acid, phosphoric acid, etc. may be used as the inorganic acid, and citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid, gluconic acid, methanesulfonic acid, glyconic acid, succinic acid, 4-toluenesulfonic acid , Gluturonic acid, embonic acid, glutamic acid, aspartic acid and the like can be used. The flavonol derivatives of formula (1) may also be pharmaceutically acceptable metal salts, in particular alkali metal salts, formed from bases. Examples of these are sodium salts and potassium salts.

In the present invention, the antiviral activity of the flavonol derivative against hepatitis B virus is measured by enzyme-linked immunoassay (EIA) using a human liver tissue-derived cell line.

Specifically, the present invention uses a 2.2.15 cell line that quantitatively secretes HBeAg, which is a representative marker of hepatitis B virus, and treats the flavonol derivative for a predetermined time while increasing the concentration of the flavonol derivatives in the culture medium. Measure the amount of HBeAg.

As a result, the antiviral activity of the flavonol derivative against the hepatitis B virus was analyzed by enzyme immunoassay (EIA) using HBeAg, which is a representative marker of hepatitis B virus, depending on the concentration of the flavonol derivative. The amount was significantly reduced. In addition, it was confirmed that the secreted e antigen in cells outside does not affect greatly suppressed, but e antigen produced in the cell (see FIGS. 1 and 2). On the other hand, the flavonol derivatives of the present invention did not affect the intracellular production and secretion of hepatitis B virus s antigen to the outside of the cells (see Fig . 3 and 4 ).

In addition, the present invention as a result of measuring the effect of flavonol derivatives on DNA replication and DNA polymerase activity of hepatitis B virus to investigate the antiviral activity against hepatitis B virus, DNA replication and polymerase activity of hepatitis virus did not appear to affect (see Figs . 5 and 6 ).

Therefore, the pharmaceutical composition containing the flavonol derivative of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient inhibits the secretion of hepatitis b virus e antigen, which keeps viral hepatitis in a chronic state, thereby preventing chronic and acute hepatitis. It can be usefully used for the prevention and treatment of.

According to the present invention, by using myricetin, quercetin, and moline as an active ingredient for the treatment of hepatitis B virus, and mixing it with a pharmaceutically acceptable carrier, a pharmaceutical composition for the prevention and treatment of these diseases can be prepared.

The pharmaceutical composition for preventing and treating hepatitis B of the present invention may further include conventionally used excipients, disintegrants, sweeteners, lubricants, flavoring agents, etc., tablets, capsules, powders, It may be formulated into granules, suspensions, emulsions, syrups, and other liquids.

In particular, the pharmaceutical compositions of the present invention may be formulated for oral administration such as tablets, troches, lozenges, water-soluble or oily suspensions, prepared powders or granules, emulsions, hard or soft capsules, syrups or It is formulated with elixirs. Binders such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin for preparation in formulations such as tablets and capsules; Excipients such as dicalcium phosphate; Disintegrants such as corn starch or sweet potato starch; Lubricants such as magnesium stearate, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax. Capsules contain liquid carriers, such as fatty oils, in addition to the substances mentioned above.

In addition, the pharmaceutical composition of the present invention can be administered parenterally, and parenteral administration is by subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection. To formulate into a parenteral formulation, the compound of Formula 1 is mixed in water with a stabilizer or buffer to prepare a solution or suspension, which is formulated in unit dosage forms of ampoules or vials.

Toxicity test results showed that these flavonol derivatives showed little toxicity when administered orally to rats at a dose of 5 g / kg and showed no adverse effects on the function of organs, including liver.

In addition, the flavonol derivatives are known to show no adverse effects on the function of organs, including the liver ( Merck Index Vol. 11th edition. 1989). In the present invention, through the cytotoxicity and oral administration acute toxicity experiments, it was confirmed that these flavonol derivatives including mycetin, quercetin, and moline are safe.

The dosage of the active ingredient according to the present invention is appropriately selected depending on the absorption rate, inactivation rate and excretion rate of the active ingredient in the body, the age, sex and condition of the patient, and the severity of the disease to be treated, but generally in adults one day The compound of formula 1 may be administered once to several times in an amount of 0.1 to 500 mg per kg of body weight, preferably 0.1 to 100 mg.

The present invention also provides a food additive or beverage composition for hepatitis B prophylaxis or treatment comprising a flavonol derivative having a structure of Formula 1 or a salt thereof as an active ingredient.

When the flavonol derivative of the present invention is used as a food additive, the flavonol derivative may be added as it is or used with other food or food ingredients, and may be appropriately used according to a conventional method. The mixed amount of the active ingredient can be suitably determined according to the purpose of use (prevention, health or therapeutic treatment). In general, the flavonol derivatives may be added in the amount of 0.0001 to 10% by weight, preferably 0.1 to 5% by weight based on the raw materials in the manufacture of the food or beverage. However, in the case of long-term intake for health and hygiene or health control, the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety. Is sure. Acute toxicity testing in rats demonstrated no death was observed at the oral dose of 5 g / kg / day.

There is no particular limitation on the kind of food. Examples of the food to which the substance can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, ice cream, various soups, drinks, tea, drinks, Alcoholic beverages and vitamin complexes.

The present invention also provides an immunostimulating agent having the flavonol derivatives or their pharmaceutically acceptable salts as an active ingredient.

When extracellularly secreted e-antigens, which are infected by viral infection, reach the thymus through the circulatory system, they lose the function of T-helper cells, resulting in tolerance of c antigen / e antigen-specific T-helper cells. do. Viral immunity by activating T-helper cells in transgenic mice designed to initiate e antigen production by administration of zinc (Zn) with quercetin (see FIG. 7A ), maline (see FIG. 7B ) and myricetin (see FIG. 7C ) It is expected to release no response.

Hereinafter, the present invention will be described in detail by way of examples.

The following examples illustrate the present invention in detail, and the content of the present invention is not limited by the examples.

Pharmaceutical compositions comprising the compound of formula 1 as an active ingredient of the present invention may be administered parenterally or orally, and are prepared in the following parenteral formulations by injection, oral formulations by syrups and tablets.

Example 1 Preparation of Injection Solution

Injection solution containing 10 mg of the active ingredient was prepared by the following method.

1 g of mycetin, quercetin or moline, 0.6 g of sodium chloride and 0.1 g of ascorbic acid were dissolved in distilled water to make 100 ml. The solution was bottled and sterilized by heating at 20 ° C. for 30 minutes.

The components of the injection solution are as follows.

Mycetin, quercetin or maline ... 1 g

Sodium Chloride ・ ・ ・ ・ 0.6 g

0.1 g of ascorbic acid

Distilled water ··················

Example 2 Preparation of Syrup

Syrup containing acid addition salt of the flavonol derivative of Formula 1 of the present invention and a pharmaceutically acceptable salt thereof as an active ingredient of 2% (weight / volume) is prepared by the following method.

Acid addition salts, saccharin and sugars of the flavonol derivatives were dissolved in 80 g of warm water. After the solution was cooled, a solution consisting of glycerin, saccharin, spices, ethanol, sorbic acid and distilled water was prepared and mixed thereto. Water was added to this mixture to make 100 ml.

The components of the syrup are as follows.

Acid addition salt of myricetin, quercetin or maline ... 2 g

Saccharin: 0.8 g ················

25.4 g of sugar

Glycerin ... 8.0 g

Spices ··················· 0.04 g

Ethanol 4.0 g

0.4 g of sorbic acid

Distilled water ·····················

Example 3 Preparation of Tablet

A tablet containing 15 mg of active ingredient is prepared by the following method.

250 g of mycetin, quercetin or maurine, 175.9 g of lactose, 180 g of potato starch and 32 g of colloidal silicic acid were mixed. 10% gelatin solution was added to the mixture, which was then ground and passed through a 14 mesh sieve. It was dried and the mixture obtained by adding 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate was made into a tablet.

The components of the tablet are as follows.

Myricetin, quercetin or maline 250 g

Lactose ········ 175.9 g

Potato starch ········· 180 g

Colloidal silicic acid 32 g

10% gelatin solution

Potato starch · 160 g

Talc · 50 g

Magnesium stearate 5 g

Example 4 Preparation of Food and Beverage

The present inventors prepared a food or beverage composition containing a flavonol derivative as an active ingredient as follows.

<4-1> Preparation of Chewing Gum

Gum base 20%

Sugar 76.36-76.76%

Mycetin, quercetin, or maurine 0.24-0.64%

1% fruit flavor

Water 2%

Chewing gum was prepared using conventional methods using the above composition and content.

<4-2> Preparation of Candy

50 to 60% sugar

Starch syrup 39.26-49.66%

Mycetin, quercetin, or maurine 0.24-0.64%

Orange flavor 0.1%

Candy was prepared using conventional methods with the above composition and content.

<4-3> Preparation of Biscuits

Force Class 1 88 kg

Gravity First Class 76.4 ㎏

16.5 kg per white

2.5 kg of salt

2.7 kg of glucose

Palm shortening 40.5 kg

5.3 kg of ammo

Medium kg 0.6 kg

0.55 kg sodium bisulfite

Rice flour 5.0 kg

Vitamin B1 0.003 kg

0.003 kg of vitamin B2

Milk Flavor 0.16 ㎏

71.1 kg of water

Whole milk powder 4 kg

Substitute powder 1 kg

0.1 kg of calcium phosphate

Spray salt 1 kg

25 kg of spray oil

0.1-0.5 kg of myricetin, quercetin or maline

Biscuits were prepared using conventional methods with the above composition and content.

<4-4> Preparation of Ice Cream

Milkfat 10.0%

Non-fat solids 10.8%

Sugar 12.0%

Starch syrup 3.0%

Emulsifying stabilizer (span) 0.5%

Spices (Strawberries) 0.15%

Water 63.31-62.91%

Mycetin, quercetin, or maurine 0.24-0.64%

Ice cream was prepared using conventional methods using the above composition and content.

<4-5> Preparation of Beverage

522 mg of honey

Chioctosanamide 5 mg

Nicotinamide 10 mg

Riboflavin Sodium Hydrochloride 3 mg

Pyridoxine hydrochloride 2 mg

Inositol 30 mg

Orthoic acid 50 mg

Myricetin, quercetin, or murine 0.48-1.28 mg

200 ml of water

With the above composition and content, drinks were prepared using conventional methods.

<4-6> Preparation of Sausage

Pork 63.6%

Chicken 27.5%

Starch 3.5%

Soy Protein 1.7%

Saline 1.62%

Glucose 0.5%

Other additives (glycerine) 0.94-1.34%

Mycetin, quercetin, or maurine 0.24-0.64%

Sausages were prepared using conventional methods using the above composition and content.

<4-7> Preparation of Chocolate

Sugar 34.36-34.76%

Cocoa Butter 34%

Cocoa Mass 15%

Cocoa Powder 15%

Lecithin 0.5%

0.5% vanilla

Mycetin, quercetin, or maurine 0.24-0.64%

With the above composition and content, chocolate was prepared using conventional methods.

Experimental Example 1 Determination of Anti-Hepatitis Virus Activity of Flavonol Derivatives

In order to investigate the effects of flavonol derivatives on the anti-hepatitis virus activity against hepatitis B virus, the treatment of flavonol derivatives with concentrations in animal cells derived from human liver tissues was examined.

<1-1> 2.2.15. Cell culture

In order to investigate the activity, the hep B2 gene (human hepatoblastoma cell) derived from human liver tissue is inserted into the hepatitis B virus gene and 2.2.15 which discharges the hepatitis B virus e antigen (HBeAg) into the medium. . Cell line (Keui Ueda et al., Virology, 169, 213-21, 1989) was used. 2.2.15. Cells were cultured using MEM medium (Gibco) supplemented with 4 μg / ml gentamycin (Gm; Sigma) and 10% heat treated fetal bovine serum (FBS; Gibco) in 10 cm diameter Petri dishes. , 37 ° C, 5% CO ₂ incubator was grown to form a cell monolayer, trypsin treatment at 3-4 days intervals were passaged.

<1-2> Flavonol Derivative Determination of Hepatitis B Virus e Antigen Production

In the case of hepatitis B virus e antigen, enzyme immunoassay was performed using a reagent for diagnosing hepatitis B antigen (Enzygnost HBeAg monoclonal; Behring). 2.2.15 cultured in Experimental Example <1-1>. Cells are added to a 24-well plate with 1 ml of DMEM medium culture medium containing FBS at a concentration of 2 × 10 ^{4 per} well and incubated for 24 hours in a 37 ° C., 5% CO ₂ incubator, followed by DMEM medium without FBS. After 4 hours of changing to 1 ml of the culture solution, the flavonol derivatives myricetin, quercetin, and morine were respectively treated. After 24 hours of treatment with the extract, only the culture was taken and the culture was centrifuged at 1,500 rpm for 5 minutes to remove the cell isolate. Only the supernatant obtained at this time was dispensed in a predetermined amount onto a diagnostic reagent plate coated with a monoclonal antibody to HBeAg (HBeAb), and then left in a 37 ° C. incubator for 1 hour. The diagnostic reagent plate was washed three times with phosphate buffer solution, and then a predetermined amount of HBeAb labeled with peroxidase was dispensed into each well and left in a 37 ° C. incubator for 1 hour to prevent secondary binding with HBeAg. It was done. Unbound excess antibody was removed by washing three times with phosphate buffer solution, and the color reaction was induced by dispensing a certain amount of a solution containing TMB (tetramethyl benzidine dihydrochloride) substrate which causes color reaction by peroxidase. The diagnostic reagent plate that completed the reaction was evaluated for the change of e antigen by reading the degree of absorption at 450 nm.

In the above method, the flavonol derivative of the present invention was measured at various concentrations to reduce the hepatitis e antigen.

As a result, all of the flavonol derivatives including myricetin, quercetin, and morphine showed anti-hepatitis virus activity by rapidly lowering the production of e antigen secreted outside the cell even at a concentration of 5 μg / ml ( FIG. 1 ). In this case, the drug concentration (IC ₅₀ ) that inhibits e antigen generation by 50% in HepG2 2.2.15 cells is determined and shown in Table 1 below. However, the derivative did not appear to affect the hepatitis B virus e antigen production in the cell ( Fig. 2 ).

Flavonol derivatives IC ₅₀ (μg / ml) Mycetin 0.89 Quercetin 1.50 Maureen 1.41

<1-3> Effect of Flavonol Derivatives on Hepatitis B Virus s Antigen

The present inventors examined whether the flavonol derivative influences the production of hepatitis B virus s antigen in the same manner as in Experimental Example <1-2>.

As a result, the flavonol derivatives including myricetin, quercetin, and moline did not affect the production of hepatitis B virus s antigen in cells and the production of s antigens secreted out of cells ( FIGS. 3 and 4 ).

Experimental Example 2 Determination of Hepatitis B Virus DNA Polymerase Activity Inhibition Effect of Flavonol Derivatives

Hepatitis B virus was dissolved in polymerase buffer (10 mM Tris HCl [pH 7.5], 200 mM dATP, dCTP and dGTP, 0.1% Triton X-100, 50 mM MgCl ₂ ), and α- ³² P-dCTP The reaction was carried out with the bonol derivatives for 16 hours. The reaction was stopped with 6 μl of 0.5 M EDTA, spotted on Whatman DE81 filter paper, washed four times with 0.5 M Na ₂ HPO ₄ , twice with distilled water and then with 95% ethanol to determine the amount of combined radiation. It measured by (image analyzer). 3TC, an existing polymerase activity blocker, was used as a positive control.

As a result, the flavonol derivatives including quercetin, morphine, and myricetin of the present invention had no effect on DNA replication of hepatitis B virus ( FIG. 5 ), and did not show polymerase activity ( FIG. 6 ). Treatment with 3TC as a control showed decreased polymerase activity at 10 μM 3TC (32%) and 20 μM 3TC (22%).

Experimental Example 3 Analysis of Immune Boosting Effect of Flavonol Derivatives

In order to determine whether the flavonol derivatives have an immunopromoting effect, the present inventors have administered quercetin, morphine, and myricetin to transgenic mice prepared to initiate e antigen production by administration of zinc (Zn) with normal mice. Cell and T-helper cell production activities were measured.

As a result, quercetin (see FIG. 7A ), morphine (see FIG. 7B ) and myricetin (see FIG. 7C ) induced T-helper cell production in transgenic mice constructed to initiate e antigen production by zinc (Zn) administration. It was found that the flavonol derivatives show an immunostimulating effect.

Experimental Example 4 Cytotoxicity Test of Flavonol Derivatives

CC ₅₀ was measured as follows to verify the toxicity to the cells by verifying the activity of the flavonol derivatives from the experimental examples.

Known methods (Korba, B. E. et al., Use of a standardized cell culture assay to assess activities of nucleoside analogs against hepatitis B virus replication,Antiviral research, 19, 55-70, 1992) using flavonol derivatives in HepG2 cells and 2.2.15. The cytotoxicity expressed against the cells was investigated.

100 μl volume of 2 × 10 ⁴ cells per well was added to a 24-well plate, incubated for 24 hours in a 37 ° C. incubator, and then treated with various concentrations of flavonol derivative samples for 3 days. The culture was removed and 20 μl of staining solution (tetrazolium compound, inner salt and phenazine ethosulfate) were aliquoted and placed in the incubator for 1 hour. The excess staining solution was removed by washing several times and 20 μl of fixation solution (50% ethanol, 1% cold acetic acid) was added and stirred for 30 minutes.The absorbance at 510 nm was read by spectrophotometer. The concentration of the extract which causes 50% absorbance reduction compared to the absorbance of the cells without the extract was determined as CC ₅₀ (50% cytotoxic concentration), and the results are shown in Table 2 below.

Flavonol derivatives CC ₅₀ (μg / ml) Mycetin 936 Quercetin 362 Maureen 398

Experimental Example 5 Oral Acute Toxicity in Rats

On the other hand, the following experiment was conducted to determine the acute toxicity of the flavonol derivatives.

Acute toxicity test was performed using 6-week-old SPF SD rats. Mycetin, quercetin, and morphine were each suspended in 0.5% methylcellulose solution and administered once orally at a dose of 5 g / kg / 15 ml. After administration of the test substance, mortality, clinical symptoms, and changes in body weight were observed. Hematological and hematological examinations were performed. Necropsy was performed to observe abdominal and thoracic organ abnormalities. As a result, no significant clinical symptoms or dead animals were noted in all animals treated with the test substance, and no toxic changes were observed in weight changes, blood tests, blood biochemical tests, and autopsy findings. As a result, the flavonol derivatives of the general formula (1) did not show a toxic change in rats up to 5 g / kg, and the minimum lethal dose (LD ₅₀ ) for oral administration was determined to be a safe substance of 5 g / kg or more.

As discussed above, flavonol derivatives containing myricetin, quercetin or moline strongly inhibit the production of e antigens that maintain hepatitis in a chronic state, and have low cytotoxicity to treat hepatitis B patients, especially chronic hepatitis patients. It can be useful to

Claims (8)

A pharmaceutical composition for preventing or treating hepatitis B containing a flavonol derivative having a structure of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient. <Formula 1> In Formula 1, R ₁ , R ₂ , R ₃ and R ₄ are independent of each other, and are H, OH or an alkoxy group of C _{1 to} C ₃ . The hepatitis B prophylaxis according to claim 1, wherein the flavonol derivative is myricetin having a structure of Formula 2 wherein R ₁ is H, and R ₂ , R ₃ and R ₄ are OH. Or therapeutic pharmaceutical compositions. <Formula 2> The method of claim 1, wherein the flavonol derivative is a quercetin (quercetin) having a structure of Formula 3 wherein R ₁ and R ₂ is H, and R ₃ and R ₄ is OH in Formula 1 or Therapeutic pharmaceutical composition. <Formula 3> [Claim _{2] The} hepatitis B prevention or hepatitis B according to claim 1, wherein the flavonol derivative is a morin having a structure of Formula 4 wherein R ₁ and R ₃ are OH, and R ₂ and R ₄ are H in Formula 1; Therapeutic pharmaceutical composition. <Formula 4> The pharmaceutical composition for preventing or treating hepatitis B according to claim 1, which comprises as an active ingredient a combination of two or more selected from the group consisting of myricetin, quercetin, moline, and pharmaceutically acceptable salts thereof. A food additive comprising the flavonol derivative of claim 1 or a salt thereof as an active ingredient. Beverage composition comprising the flavonol derivative of claim 1 or a salt thereof as an active ingredient. An immunostimulating agent comprising the flavonol derivative of claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient.