KR100331174B1 - Pharmaceutical composition for hepatitis B comprising the extract of Eugenia caryophyllata as a pharmaceutically active ingredient - Google Patents

Abstract

The present invention relates to a new medical use of the clove extract, and more particularly, to a hepatitis B therapeutic agent comprising the water extract of the clove, its dry powder or a water-soluble fraction obtained by dissolving it in water and centrifugation. Clove extract of the present invention shows an excellent effect in the field of treatment of HBV infection that does not yet have a stable and effective therapeutic agent, the toxicity is also significantly lower than the known compounds, the manufacturing process is also very simple and economical and effective hepatitis B virus infection It can be used as a therapeutic agent.

Description

Pharmaceutical composition for hepatitis B comprising the extract of Eugenia caryophyllata as a pharmaceutical active ingredient

The present invention relates to a new medical use of the clove extract, and more particularly, to a hepatitis B therapeutic agent comprising the water extract of the clove, its dry powder or a water-soluble fraction obtained by dissolving it in water and centrifugation.

Hepatitis is a disease in which liver function is lowered due to inflammation or rapid necrosis of liver cells, and is classified into three types of viral hepatitis, alcoholic hepatitis, and toxic hepatitis depending on the cause. About 30 hepatitis viruses are known, of which the A, B, C, D and E viruses have a great effect on the human body. Chronic hepatitis, which lasts for more than six months, is mainly caused by viruses of type B, C, and D. Most acute hepatitis can be cured by conservative therapy. Can be. Alcoholic hepatitis is common in the United States and Europe, but in Korea, viral hepatitis accounts for more than 90% of all liver diseases. In the United States alone, about 200,000 people are infected each year, and at least 10% of the population in the Far East and Africa, including Korea, are carriers, many of which are known as hepatocellular carcinoma (HCC) (Tiollas et al ., Nature 317). 487-495, 1985). It has also been reported that only about 5-10% develop HBV in adults infected with HBV, but 90% develop in HBV-infected infants (Chisari et al., Proc. Natl. Acad. Sci. USA 84: 6909) . -6913, 1989).

The hepatitis B virus was first discovered by Bloomberg in the United States in 1964, and is spread by blood, saliva, semen, and breast milk from patients. In most cases, acute hepatitis B recovers and antibodies form and become a semi-permanent immune condition, but it becomes chronic in about 5 to 10% of cases. About 20% of chronic hepatitis B develops into cirrhosis, some of which develop into liver cancer.

Although much is known about the structure of HBV, little is known about the pathological mechanism of hepatocellular damage and malignant transformation by viral infection. This is because the hepatitis B virus has a limited host range and the experimental approach was difficult because the cell culture system was not established in vitro (Chisari et al., Proc. Natl.Aca.Sci. USA 84: 6909-6913, 1987). In the early 1990s, Korba et al. Developed a HepG2 2.2.15 cell line in which HBV is propagated by introducing the HBV gene into human liver cancer cell line HepG2, and developed and reported a cell culture anti-HBV drug discovery system using the same. (Korba and Milman, Antiviral research 15: 217-228, 1991; Korba and Gerin, Antiviral research 19: 55-70, 1992). HepG2 2.2.15 cell line is useful because it can search many samples in a short time (Jansen, Antimicrob. Agents Chemother . 37: 441-447, 1993), and is easier to culture than the HB 611 cell line developed in Japan for the same purpose. The high yield of virus can be applied to cell culture plate systems (Kruining, J. of Hepatology 22: 263, 1995).

Α-interferon, lamivudine, recombinant HBV surface antigen protein, and antigen-antibody complex have been developed as hepatitis B therapeutics or vaccines.However, α-interferon has many side effects, is expensive, and the treatment period is long. Its use is less and less because it is long and less effective. In addition, traditional medicines such as Astragalus, licorice, red ginseng, baekhwasachocho, Cordyceps sinensis, and aging are widely used as an adjunct to the treatment of hepatitis and liver disease.

On the other hand, Eugenia caryophyllata Thunberg is a dried clove bud of the family Clove (Myrtaceae), and has been used as a dry stomach, antibacterial, and antifungal drug since it is a potent medicinal plant. Cloves are dark brown or dark red and have burns of 10 to 18 mm in length with four thick calyxes and four membranous stools on top, with a peculiar smell, taste and intensity, and later numb the tongue slightly. The active ingredient is eugenol, acetyleugenol, and the like, which are essential oils, and flavonoids contain rhamnetin, kaempferol, and alkaloids such as hygenamine. In addition, galotanic acid (galotanic acid), β-caryophyllene, 2-hydroxy 4,6-dimethoxy 5-methylacetophenone and the like. Clove pharmacological action, in addition to the above-mentioned stomach, antibacterial, antifungal activity, uterine contraction, animal harvesting tube contraction, analgesia by local anesthesia, adrenaline-antagonism, mucosal stimulation, expectorant action, promoting biosynthesis of collagen fibers, etc. This is known.

Specifically, inhibition of acetylcholine esterase activity on the pharmacological effects of cloves (Akinrimisi et al., West Afr. J. Pharmacol. Drug Res ., 1975 2 (2): 127-131), antimicrobial activity (Ramanoelina Et al . , Arch. Inst. Pasteur Madagascar, 1987, 53 (1): 217-226), anticancer activity through induction of glutathione S-transferase (Zheng et al . , J. Nat. Prod. , 1992, 55 (7): 999-1003), reverse transcriptase inhibitory activity of moloney murine leukemia virus (MMLV) (Suthienkul et al., Southeast Asian J. Trop. Med, Public Health , 1993, 24 (4): 751-755), anti Convulsive action (Pourgholami et al., J. Ethnopharmacol ., 1999, 64 (2): 167-171). However, the efficacy of cloves for the treatment of HBV infection is unknown.

There are efforts to recent search for compositions having a pharmacological activity from plant extracts continues, research reports on the anti-HBV effects of saengyakjae is required Lantus (Phyllanthus) in plants of pearl seconds in vivo (in vivo) and in vitro (in vitro (Venkateswaren et al., 1987) reported significant anti-HBV effects in clinical trials (Wang et al. , J. Lab. Clin. Med . 126: 350-352, 1995). It is actively underway. In addition, recently, in vitro, the inhibitory activity of HBV DNA polymerase isolated from the serum of hepatitis patients and the binding ability of surface antigens were examined in 105 Korean herbal medicines, and 15 herbal extracts were reported to have anti-HBV effects. (Chung et al., Phytotherapy research 9: 429-434, 1995).

As such, herbal medicines do not contain only one active ingredient, and most of the ingredients are competitive (competitive). This competitive action causes the components to exhibit complementary, synergistic or inhibitory effects on each other. Therefore, herbal medicines may be used in the form of extracts rather than separating specific components, and this can be said to effectively exhibit the unique function of the herbal medicines.

Therefore, the present inventors conducted a study to find a substance having anti-HBV activity against various kinds of natural products by using a drug screening system using HepG2 2.2.15 cultured cells. The present invention was completed by confirming that it exhibits HBV activity.

It is an object of the present invention to provide a plant extract having HBV inhibitory activity.

Specifically, an object of the present invention is to provide a therapeutic agent for hepatitis B virus infection prepared from plant extracts, which is simple to manufacture, high in stability and efficacy, and low in toxicity.

FIG. 1 shows the extract of clove at concentrations of 64 (lane 1), 128 (lane 2), 256 (lane 3) and 512 (lane 4) μg / ml, respectively, to determine whether the water extract of cloves inhibits the replication of HBV DNA. After 8 days of incubation, the gel photograph shows the result of Southern blot using a 3.2 kb HBV gene probe by electrophoresis of HepG2 2.2.15 cell line culture.

Lane C: negative control untreated sample

Lane PC: positive control treated with 10 μM ddC

M: size marker (λ / Hin d III)

RC: relaxed circular HBV DNA

In order to achieve the above object, the present invention provides a hepatitis B therapeutic agent comprising the extract of Eugenia cryophyllanta Thunberg as an active ingredient.

Specifically, the clove extract is obtained from the buds of the clove ( Eugenia cryophyllanta Thunberg) belonging to the Myrtaceae, and is usually collected when the bud turns from blue to bright red between September and March of the following year. The vase is dried in the sun and extracted. Clove extract in the present invention may be used in all of the water extract, saline extract or alcohol extracts such as ethanol, water extract is particularly excellent in the HBV growth inhibitory effect.

In the above water extract is obtained by any one of the method of cold soaking, ultrasonic, reflux, in a preferred embodiment of the present invention by adding water at a rate of 1 L per 100g dried clove by heating to reflux at 100 ℃ for 2 to 3 hours only By extract, clove water extract was prepared.

Clove extract in the hepatitis B treatment of the present invention may be in the form of a dry powder prepared by drying under reduced pressure and lyophilized water extract prepared above. In this case, the yield of the dried powder of the clove water extract prepared in one preferred embodiment of the present invention was 16.7% on average.

In addition, the water-soluble fraction obtained by dissolving the dried powder in water and centrifugal separation may also be used as an active ingredient of the hepatitis B therapeutic agent of the present invention. In this case, the centrifugation is carried out by dissolving the dry powder in water at a concentration of 40 to 60 mg / ml, more preferably 51 to 52 mg / ml, and performing it for 25 to 35 minutes, preferably 30 minutes at about 10,000 g. It is preferable. After centrifugation and sterilization with a membrane filter can be obtained an aqueous fraction of the clove extract having the HBV inhibitory activity. Preferably, the membrane filter for sterilization has a pore size of about 0.2 micrometer.

In a preferred embodiment of the present invention, 51.2 mg of dried clove water extract powder was dissolved in 1 ml of water for 20 minutes, centrifuged at 15 ° C. and 10,000 g for 30 minutes, and then the supernatant was sterilized with a membrane filter. An aqueous fraction having an average yield of 74.3% was obtained.

Hereinafter, the term 'clove water extract' used without particular limitation means all of the water extract of clove mentioned above, the dried powder thereof, or the water-soluble fraction obtained by centrifugation by dissolving it in water.

In the present invention, the anti-HBV activity of the clove extract is examined by measuring the inhibition of extracellular HBV DNA release and the inhibition of HBV surface antigen production in the cell culture.

First, HepG2 2.2.15 in which HBV gene was introduced into HepG2, a human liver cancer cell line, was used as a cell line for measuring the release level of extracellular HBV DNA.The cell line cultured in RPMI-1640 medium was dispensed into a cell culture plate. After sufficient incubation, the clove extract is treated to a final treatment concentration of 64, 128, 256, 512 μg / ml, respectively. Specifically, 2 ml of the cell culture solution containing the clove extract is treated for 8 days while exchanging daily. The HepG2 2.2.15 cell line used in the above was developed by Korba et al. For the anti-HBV drug search, and has the advantage of being able to search a large number of samples in a short time, easy to cultivate, and high virus yield. The treatment period of the sample is 8 days in which HBV DNA excretion increases rapidly and a constant amount of virus is continuously detected.

On day 8 after sample treatment, cell cultures are collected and treated with polyethylene glycol (PEG) solution to precipitate virions. Subsequently, extracellular HBV DNA is isolated by treating the digestion solution to which proteinase K is added. It is electrophoresed, transferred to a nylon membrane, and HBV DNA is detected by Southern blot. In this case, a well-known HBV gene may be used as a probe. In the experimental example of the present invention, a 3.2 kb 'adr' subtype HBV gene obtained by cleaving pHBV315 with a restriction enzyme BamH I was used as a probe.

As shown in the experimental example of the present invention, Southern blot analysis showed that the clove extract of the present invention significantly inhibited HBV proliferation at the concentration of 128 ㎍ / ㎖, completely inhibited the virus proliferation at the concentration of 256 ㎍ / ㎖, positive control It showed a better effect than didideoxycytidine (ddC) used as a drug. The EC ₅₀ for inhibition of HBV DAN proliferation of the clove extract of the present invention was found to be 126 μg / ml.

Another anti-HBV activity measurement method is to investigate the degree of inhibition of HBs antigen production by radioimmunoassay.

To this end, the collected cell culture solution is reacted with HBs antibody-labeled beads, followed by reaction with HBs antibody-labeled [ ¹²⁵ I] and measuring radioactivity. As a result, clove extract showed less than 30% of HBV surface antigen production inhibition in 64 ~ 128 ㎍ / ml, but showed more than 80% inhibition in more than 256 ㎍ / ml.

On the other hand, as a result of cytotoxicity test of clove extract, the cytotoxicity when the sample was treated at 512 ㎍ / ml concentration was 9.3%, which is very low compared to 4.4% cytotoxicity at 10 μM of ddC, a positive control drug. I could confirm it.

Clove extract of the present invention as described above has the effect of inhibiting HBV DNA replication, as well as the inhibitory effect of HBV surface antigen production, and can be used as an effective hepatitis B treatment while having high safety due to low cytotoxicity.

Hepatitis B therapeutic agent using the cloves extract of the present invention as an active ingredient can be administered orally or parenterally during clinical administration and can be used in the form of general pharmaceutical preparations.

That is, the pharmaceutical composition of the present invention may be administered in various oral and parenteral dosage forms in actual clinical administration, and when formulated, diluents such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants that are commonly used Or using excipients. Solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, which solid preparations contain at least one excipient such as starch, calcium carbonate, sucrose in sucrose extracts. Or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As a suppository base, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

In addition, the extract of the clove of the present invention can significantly increase the efficacy of the traditional herbal medicine by adding to the existing herbal medicine prescription.

In the pharmaceutical composition for treating hepatitis B of the present invention, the effective dose of the clove extract is 10 to 2,000 mg / kg, and can be administered once a day to several times, which is the age, sex, degree of disease, etc. Can be increased or decreased accordingly. Clove extract of the present invention was found to be several orders of magnitude safer than dideoxycytidine used as a positive control in cytotoxicity experiments.

Hereinafter, the present invention will be described in detail with reference to Examples.

However, the following examples are illustrative of the present invention, and the scope of the present invention is not limited thereto.

Example 1 Preparation of Clove Extract 1

The cloves used in the experiments were stored and stored in the herbal medicine reservoir of the Korea Food and Drug Administration. Using 1 kg of dried dry herbal medicine, divided by 100 g into an electronic shaker, 1 L of water was added and extracted only once at 100 ° C. for 3 hours. Each extract was combined, concentrated under reduced pressure, and lyophilized to give 167 g of dry powder.

Example 2 Preparation of Clove Extract 2

Using 1 kg of chopped dried herbal medicine, divided into 100 g each, put in an electronic shaker, 1 L of water was added and extracted only once at 100 ° C. for 3 hours. The extracts were combined, concentrated under reduced pressure, and lyophilized to obtain 180 g of dry powder.

Example 3 Preparation of Clove Extract 3

Using 1 kg of chopped dried herbal medicine, divided into 100 g each in an electronic shaker, added 1 L of water, extracted only once at 100 ° C. for 3 hours, combined the extracts, concentrated under reduced pressure, and lyophilized to obtain 154 g of dry powder.

Using 1 kg of dried clove according to the manufacturing method according to the present invention as described above, dividing each 100g into an electronic medicinal water bath and add 1 L of water and extract only once for 2 to 3 hours to combine each extract solution The mixture was concentrated under reduced pressure, lyophilized to a dry powder, and used as a test substance. Each yield averaged 16.7%.

Example 4 Preparation of a Clove Extract Water-Soluble Fraction 1

51.2 mg of the dry powder obtained in Example 1 was dissolved in 1 ml of water for 20 minutes, centrifuged at 15 ° C. and 10,000 g for 30 minutes, and then the supernatant as a soluble powder was filtered through a water-soluble membrane filter (membrane filter, cut off 0.2 μm). Sterilization yielded 0.72 ml

Example 5 Preparation of Clove Extract Water Soluble Fraction 2

51.2 mg of the dry powder obtained in Example 2 was dissolved in 1 ml of water for 20 minutes, centrifuged at 15 ° C. and 10,000 g for 30 minutes, and the supernatant as a soluble powder was filtered and sterilized with a water-soluble membrane filter to obtain 0.76 ml.

Example 6 Preparation of Clove Extract Water Soluble Fraction 3

51.2 mg of the dry powder obtained in Example 3 was dissolved in 1 ml of water for 20 minutes, centrifuged at 15 ° C. and 10,000 g for 30 minutes, and the supernatant as a soluble powder was filtered and sterilized with a water-soluble membrane filter to obtain 0.75 ml.

The water-soluble fraction is dissolved in water at a concentration of 40 to 60, preferably 51 to 52 mg / ml, and centrifuged at 15 ° C. and 10,000 g for 25 to 35 minutes, preferably 30 minutes, followed by filtration with an aqueous membrane filter. It could be obtained by sterilization. The average yield of the water soluble fraction relative to the total solvent was 74.3%.

Experimental Example 1 Anti-HBV Activity Experiment of Clove Extract 1

In order to investigate the effect of the extract of Clove on inhibiting the proliferation of HBV in HepG2 2.2.15 cells and inhibiting the release of virion extracellularly, HBV DNA was quantified by treating the sample with culture cells and collecting the culture solution.

1) Cell line acquisition and culture

The HepG2 2.2.15 cell line used in the experiment was purchased from Professor Korba of the College of Medicine, Georgetown University, USA. Cells were thawed immediately upon arrival and placed in RPMI-1640 (Gibco BRL, USA) medium containing 5 mM HEPES, 5% fetal bovine serum (FBS), 330 mg / ml penicillin, 100 μg / ml streptomycin 37 C was incubated in a 5% CO ₂ incubator. Cells proliferated through passages 4-5 were stored in liquid nitrogen, and thawed and used for screening for anti-HBV efficacy.

2) HBV gene probe

The pHBV315 cloned with the 'adr' subtype HBV gene was distributed from Professor Kim Gil-hyun, Ewha Womans University College of Pharmacy, and the plasmid was digested with restriction enzyme BamH I and the 3.2 kb HBV gene was quenched by Qiagen gel extraction kit. Hilden, Germany) and then used as a probe (probe) in the Southern blot analysis experiments.

3) Treatment of Extract

Screening of anti-HBV effects in samples using HepG2 2.2.15 cell line was performed based on Korba's method (Korba and Gerin, Antiviral research 19: 55-70, 1992).

After propagating the HepG2 2.2.15 cell line, the concentration was adjusted to 2 × 10 ⁵ cells / ml, and 1 ml per well was dispensed into a 24-well cell culture plate. This was incubated in a CO ₂ incubator until the cells were fully grown and used for anti-HBV efficacy screening. Dilute the clove extract to 4 concentrations and dispense 20 μl / well into the 24 well cell culture plate, and add 2 ml of the cell culture solution (2% FBS) preheated to 37 ° C. The treatment was performed at 64, 128, 256 and 512 µg / ml. In addition, the positive control drug dideoxycytidine (ddC) (Sigma) was treated to a final concentration of 10μM. The cells were treated for 8 days with 2 ml of the cell culture solution containing each sample, and the aliquots of the cells and the clove extract were treated three times in duplicate.

4) Southern blot analysis

Southern blot analysis to determine the extent to which the sample inhibits the release of extracellular virion DNA was performed according to Kim et al. (Kim et al., J. of Applied Pharmacol . 6: 139-144, 1998). . The treatment period of the sample was 8 days after HBV DNA excretion was rapidly increased after the cells had grown sufficiently to continuously detect a certain amount of virus. Cell cultures were collected on the 8th day from the day the cells were treated with the sample, and then treated with PEG solution to precipitate virions and the digestion solution added with proteinase K to treat extracellular HBV DNA. Was separated. After electrophoresis on a 0.8% agarose gel, HBV DNA was transferred to a Hybond ^™ -N ⁺ nylon membrane filter (Amersham, UK) and fixed using an ultraviolet light fixture (Ultraviolet Crosslinker). Hybridization was performed using a 3.2 kb HBV gene obtained by cleaving pHBV315 with the restriction enzyme BamH I as a probe. The labeling of the probe and the detection of HBV DNA were performed by Southern blot method using ECL direct ^™ labeling and detection kit (Amersham).

The results are shown in FIG . 1 , as shown in FIG. 1 , the clove extract inhibited HBV proliferation in a concentration-dependent manner. C. ( E. caryophyllata ) immersion extract significantly inhibited the proliferation of HBV at the concentration of 128 ㎍ / ml, and completely inhibited the growth of virus at concentrations of 256 ㎍ / ml. The effect was better than 10 μM of ddC. Inhibition of HBV proliferation of ddC in HepG2 2.2.15 cells has been reported to have an EC ₉₀ (90% effective concentration) of 6 μM (Korba 1992), and the concentration of 10 μM of ddC used in this experiment completely inhibited HBV proliferation. ( FIG. 1 , lane PC).

The amount of HBV DNA band detected above was measured using a desitometer and a Bio-Profil image analysis program (Vilber Lourmat, France). EC ₅₀ for was found to be less than 126 μg / ml.

Experimental Example 2 Anti-HBV Activity Experiment 2 of Clove Extract

1) Measurement of HBs Antigen

Cells were collected on the 8th day after the samples were treated with the cells and the amount of HBs antigen was measured using a radioimmunoassay kit (Abbott, USA). Put into the the HBs antibodies labeled with the collected cell culture solution bead (bead) After 20 hours at room temperature and washing the beads with distilled water and ^[125 I] labeled HBs antibody solution was 3 hours at 45 ℃. After washing the beads again, the gamma-meter (gamma counter, PACKARD, USA) showed the degree of response in cpm, and the antigen production inhibitory activity was measured by comparing with the amount of the control antigen.

2) Statistical Processing

Experimental results were statistically analyzed using ANOVA (Kruskal-Wallis one way analysis of variance), and when comparing differences between the groups, Bonferroni's modified t-test (Ponferroni's modified t-test) was used. Significance was searched at the 0.05 or p <0.01 level.

The degree of inhibition of HBs antigen production was measured by radioimmunoassay, and the inhibition rate was calculated by the following equation.

% Inhibition of HBs antigen production = [1- (T-B) / (N-B)] × 100

* T: cpm of treatment group

* N: cpm of untreated group

* B: default cpm (background cpm)

Table 1 shows the results of measuring the amount of HBV antigen in the cell culture after the sample treatment.

Inhibition Rate of HBs Antigen Production by Clove Extract (%) Treatment concentration % Inhibition Clove Extract 64 (μg / ml) 20.5 ± 2.6 128 (μg / ml) 28.6 ± 2.0 256 (μg / ml) 77.3 ± 1.6 512 (µg / mL) 81.4 ± 1.7 ddC 10 (μM) 14.4 ± 4.1

As a result, the clove immersion extract inhibited HBV antigen production in a concentration-dependent manner at all concentrations. Clove immersion extract showed less than 30% HBV inhibition of surface antigen production from 64 to 128 ㎍ / ml, but more than 256 ㎍ / ml showed nearly 80% inhibition. The positive control ddC showed a low HBs antigen production inhibition of 14.4%, which is consistent with Kruining et al. (1995). Although ddC inhibits the replication of the HBV genome in the cytoplasm, HBs antigens generated from HBV transcripts (mRNAs) can be self-assembled together without HBV genes in the membranes of the endoplasmic reticulum, resulting in linear alignment. And spherical (sphere) through the Golgi apparatus and extracellularly released (Harrison et al., Replication of Hepatitis B Virus , 63-88, 1997). The inhibitory effect of HBV antigen production appears to be weak.

Experimental Example 2 Cytotoxicity Experiment

1) Cytotoxicity Measurement Method

The cytotoxicity of the clove extract was treated with HepG2 2.2.15 cells by mixing the extract of Example 1 or the aqueous fraction of Example 4 with the cell culture and incubating for 24 hours, and then lactate released into the cell culture by the cytotoxicity of the sample. The amount of lactate dehydrogenase (LDH) was measured using an LDH cytotoxicity kit (LDH cytotoxicity kit, Boehringer Mannheim, Germany) (Shrivastava et al . , Cell Biol. Toxicol . 8: 157-170, 1992).

2) Cytotoxicity Test Results

In order to determine the dose of the sample to detect the antiviral effect, the cytotoxicity was calculated by measuring the LDH activity released from the cells into the culture after the sample treatment. When the cell line treated with 1% Triton X-100 was used as the maximum amount of LDH produced, cytotoxicity due to the maximum concentration of the sample (512 µg / ml) was 9.3%. When ddC used as a positive control was treated with 10 μM, cytotoxicity was 4.4%, and the clove of the present invention ( Eugenia caryophyllata Thunberg) was extracted with water when compared to CC ₅₀ (50% cytotoxic concentration) of ddC with 168 μM. It was found that the cytotoxicity of the clove extract prepared by concentration and drying or the water-soluble fraction obtained after centrifugation by dissolving it in water was not significant.

Production Example 1 Preparation of Powder

According to the preparation method of powder in the pharmacopeia formulation general formula for the following component content.

1) In 1 package

Clove Extract (Dry Powder) 100 mg

100 mg lactose

10 mg talc

2) In 1 package

100 mg of water soluble fraction of cloves

100 mg lactose

10 mg talc

Preparation Example 2 Preparation of Tablet

According to the method for preparing tablets in the general pharmacopeia formulation, it is prepared in the following component contents.

1) In 1 tablet

Clove Extract (Dry Powder) 100 mg

100 mg corn starch

100 mg lactose

2 mg magnesium stearate

2) In 1 tablet

100 mg of water soluble fraction of cloves

100 mg corn starch

100 mg lactose

2 mg magnesium stearate

Production Example 3 Preparation of Capsule

According to the method for preparing a capsule in the general pharmaceutical preparations of the Korean Pharmacopoeia according to the following component content.

1) In 1 capsule

Clove Extract (Dry Powder) 100 mg

100 mg corn starch

100 mg lactose

2 mg magnesium stearate

2) 1 capsule

100 mg of water soluble fraction of cloves

100 mg corn starch

100 mg lactose

2 mg magnesium stearate

Preparation Example 4 Preparation of Injection

It is prepared in the following component contents according to the preparation method of injectables in the general pharmacopeia formulation.

1) in 1 ampoule (2 ml)

Clove Extract (Dry Powder) 50 mg

Appropriate sterile distilled water for injection

pH adjuster

2) in 1 ampoule (2 ml)

50 mg of water soluble fraction of cloves

Appropriate sterile distilled water for injection

pH adjuster

Production Example 5 Preparation of Liquid

According to the manufacturing method of the liquid formulation in the general pharmacopeia formulation general formula to the following component content.

1) in 100 ml

1 g of cloves extract (dry powder)

10 grams of sugar

5 grams of mannitol

Purified water

2) in 100 ml

100 mg of water soluble fraction of cloves

10 grams of sugar

5 grams of mannitol

Purified water

The water extract of Clove ( Eugenia caryophyllata Thunberg) prepared according to the present invention, its dry powder or water-soluble fraction obtained after centrifugation by dissolving it in water shows an excellent effect in the field of HBV therapeutics which does not yet have a safe and effective therapeutic agent. Toxicity is also significantly lower than known compounds, which can be used as a safe and effective treatment for hepatitis B virus infection.

Claims (7)

A pharmaceutical composition for treating or preventing hepatitis B, comprising a water-soluble fraction obtained by dissolving water extract of Clove ( Eugenia caryophyllata Thunberg) or a dry powder thereof in water and centrifuging. The method of claim 1, wherein the water extract is a pharmaceutical composition for treating or preventing hepatitis B, characterized in that obtained by any one method of cold soaking, ultrasound, reflux. The method of claim 1, wherein the dry powder is a pharmaceutical composition for treating or preventing hepatitis B, characterized in that obtained by lyophilizing the concentrated water extract of the clove obtained by any one of cold needle, ultrasonic, reflux method. The hepatitis B treatment according to claim 1, wherein the water-soluble fraction is obtained by dissolving the clove water extract in water at a concentration of 40 to 60 mg / ml and centrifuging it at 15 ° C and 1000 g for about 25 to 35 minutes. Or prophylactic pharmaceutical compositions. 5. The hepatitis B treatment or prevention method according to claim 4, wherein the water-soluble fraction is obtained by dissolving the clove water extract in water at a concentration of 51 to 52 mg / ml, and centrifuging it at 15 ° C and 1000 g for about 30 minutes. Pharmaceutical composition for. The pharmaceutical composition for treating or preventing hepatitis B according to claim 1, which is mixed with a pharmaceutically acceptable carrier. The pharmaceutical composition for treating or preventing hepatitis B according to claim 1, which is formulated and used in the form of powder, granule, tablet, capsule, solution or injection.