KR100332485B1 - Pharmaceutical composition for hepatitis B comprising the extract of Ephedra sinica as a pharmaceutically active ingredient - Google Patents

Abstract

The present invention relates to a new medical use of ephedra extract, and more particularly, to a hepatitis B therapeutic agent comprising the water extract of ephedra, its dried powder or water-soluble fraction obtained by dissolving it in water and centrifuged. The ephedra 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, 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 Ephedra sinica as a pharmaceutically active ingredient

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

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. About 200,000 people are infected each year in the United States alone, and at least 10% of the population in the Far East and Africa, including Korea. Are carriers and many of them progress to hepatocellular carcinoma (HCC) (Tiollas et al ., Nature 317: 487-495, 1985). 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, but 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.

Ephedra sinica STAPF is a perennial herb, the main components of ephedra are ephedrine, pseudo-ephedrine, 1-N-methylephedrine, 1-norephedrine, dN- pseudomethylephedrine, d -Demethyl-pseudo-ephedrine, ephedine and the like nonacosanol, nonacosane, triacontaol and the like. Ephedra essential oil has been reported to have an inhibitory effect against influenza virus in vitro and to be cured by subcutaneous injection in mice infected with type A or PR type ₈ influenza virus. Jeo Jung-seop, Dohae Hyangdae Dictionary (Plants), Younglimsa, 1998). In addition, ephedrine, a representative substance of ephedra alkaloid, is known to have antipyretic, anti-inflammatory, bile secretion, as well as cardiovascular system, and has been used for sweating, asthma, and cold. However, it has a blood pressure raising action, so the use of hypertension patients should be careful. As such, various pharmacological effects of ephedra are known, but there is no known effect of ephedra on the treatment of HBV infection.

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 . , Proc. Natl. Acad. Sci . USA 84: 274-278, 1987), and reported clinically significant anti-HBV effects (Wang et al. , J. Lab. Clin. med 126:. 350-352, 1995) has been actively studied for commercialization. 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).

These herbal medicines do not contain only one active ingredient, and in most cases, the components act competitively with each other to exhibit a complementary to synergistic or inhibitory effect. Therefore, the herbal medicine may be used in the form of an extract instead of using a specific component separately, and this can be said to exhibit the inherent efficacy of the herbal medicine.

Therefore, the present inventors conducted a study to find a substance having anti-HBV activity in 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 the 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 ephedra extract at concentrations of 64 (lane 1), 128 (lane 2), 256 (lane 3) and 512 (lane 4) μg / ml, respectively, to confirm whether the water extract of ephedra 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 with Ephedra sinica STAPF extract as an active ingredient.

Specifically, the ephedra extract is obtained from the ultra plantain of Ephedra sinica STAPF, which belongs to Ephedraceae. That is, green thin branches from August to October. After picking up and removing the roots, the mud and the roots are removed and dried in a well-ventilated place or dried about 60% and then dried again in the sun. In the present invention, ephedra extract may be any water extract, saline extract or alcohol extracts such as ethanol, water extract is most preferred in terms of HBV inhibitory activity.

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

The ephedra extract in the hepatitis B treatment of the present invention may be in the form of a dry powder prepared by decompression drying and lyophilization of the water extract prepared above. At this time, the yield of the dry powder of the ephedra water extract prepared in a preferred embodiment of the present invention was 16.4% 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 ephedra 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 ephedra water extract dry 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, and then the total solvent. An aqueous fraction having an average yield of 72.7% was obtained.

The term 'water extract of ephedra' used without particular limitation herein refers to both the water extract of ephedra mentioned above, its dry powder, or a water-soluble fraction obtained by dissolving it in water and centrifuging it.

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

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 ephedra extract is treated to final treatment concentrations of 64, 128, 256 and 512 μg / ml, respectively. Specifically, the cells are treated for 8 days while replacing 2 ml of the cell culture solution containing ephedra extract 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. The extracellular HBV DNA is then isolated by treating the digestion solution with the addition of proteinase K. 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 ephedra extract of the present invention significantly inhibited HBV proliferation at the concentration of 128 ㎍ / ㎖, completely inhibited the virus proliferation at a concentration of 256 ㎍ / ㎖, positive control It showed a better effect than didideoxycytidine (ddC) used as a drug. EC ₅₀ of the ephedra extract of the present invention for inhibition of HBV DAN growth 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. Experimental results showed that ephedra extract had an inhibition of HBV surface antigen production of less than 30% at 64-128 ㎍ / ml, but an inhibitory effect of nearly 80% at 256 ㎍ / ml or higher.

On the other hand, the cytotoxicity test of ephedra extract showed that 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.

As described above, the ephedra extract of the present invention 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 therapeutic agent while having high safety due to low cytotoxicity.

Hepatitis B therapeutic agent using the ephedra 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 a general pharmaceutical preparation.

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 may comprise at least one excipient such as starch, calcium carbonate, sucrose (Sucrose) in ephedra extract. 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.

The effective dose of ephedra extract in the pharmaceutical composition for treating hepatitis B of the present invention is 10 to 2,000 mg / kg, can be administered once to several times a day, which is the age, sex, degree of disease, etc. Can be increased or decreased accordingly. The ephedra 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 Ephedra Extract 1

The ephedra used for the experiment was 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 164 g of dry powder.

Example 2 Preparation of Ephedra Extract 2

Using 1 kg of dried dry herbal medicine, divided into 100 g each in an electronic shaker, added 1 L of water, extracted only once for 3 hours at 100 ° C, combined the extracts, concentrated under reduced pressure, and lyophilized to obtain dry powder 183 g.

Example 3 Preparation of Ephedra Extract 3

Using 1 kg of dried dry herbal medicine, divided into 100 g each in an electronic shaker, added 1 L of water, extracted only once for 3 hours at 100 ° C, combined the extracts, concentrated under reduced pressure, and lyophilized to obtain dry powder 157 g.

As described above, using 1 kg of dried ephedra according to the manufacturing method according to the present invention, dividing each 100 g into an electronic medicinal water bath and adding 1 L of water and extracting only once for 2 to 3 hours at 100 ℃ 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 16.4%.

Example 4 Preparation of Ephedra 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, and 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 0.73 ml.

Example 5 Preparation of Ephedra Extract Water-Soluble Fraction 2

51.2 mg of the dried 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 then the supernatant as a soluble component was filtered and sterilized with a water-soluble membrane filter to obtain 0.68 ml.

Example 6 Preparation of Ephedra Extract Water-Soluble Fraction 3

51.2 mg of the dried 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 then the supernatant as a soluble component was filtered and sterilized with a water-soluble membrane filter to obtain 0.77 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 72.7%.

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

In order to investigate the effect of ephedra extract inhibiting the growth of HBV in HepG2 2.2.15 cells and inhibiting the release of virion extracellularly, the sample was treated with cultured cells, and the culture medium was collected to quantify HBV DNA.

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. The ephedra extract was diluted to 4 concentrations and dispensed in 20 wells into a 24 well cell culture plate, followed by 2 ml of 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. 2 ml of the cell culture solution containing each sample was treated for 8 days while the cells were exchanged daily, and cell division and ephedra 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 immobilized using an 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).

Eotneunde The results are shown in Figure 1, ephedra extract inhibited HBV proliferation in a concentration dependent manner, as shown in FIG. E. sinica immersion extract significantly inhibited HBV proliferation at the concentration of 128 ㎍ / ml, and completely inhibited virus proliferation at concentrations above 256 ㎍ / ml, which was released into the cell culture medium. ) DNA band was not observed and showed better effect than the positive control 10 μM 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 determined to be 126 μg / ml or less.

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

1) Measurement of HBs Antigen

Cell cultures were collected on day 8 after cells were treated with samples 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 reaction degree was expressed in cpm on a gamma-meter (gamma counter, PACKARD, USA), and the antigen-producing ability 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)

The results of measuring the amount of HBV antigen in the cell culture after the sample treatment are shown in Table 1 below.

Inhibition Rate of HBs Antigen Production by Ephedra Extract (%) Treatment concentration % Inhibition Ephedra extract 64 (μg / ml) 28.4 ± 2.0 128 (μg / ml) 39.1 ± 1.7 256 (μg / ml) 52.4 ± 2.3 512 (µg / mL) 88.5 ± 1.8 ddC 10 (μM) 14.4 ± 4.1

As a result, ephedra immersion extract inhibited HBV antigen production in a concentration-dependent manner at all concentrations. Ephedra immersion extract showed less than 30% HBV inhibition of surface antigen production from 64 to 128 ㎍ / ml, but showed an inhibitory effect of nearly 80% above 256 ㎍ / ml. 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

Cytotoxicity of ephedra 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, followed by incubation 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, the cytotoxicity was 4.4%, and the ephedra sinica STAPF. Of the present invention was compared with ddC when the CC ₅₀ (50% cytotoxic concentration) was 168 μM. It was found that the cytotoxicity of the water-soluble fraction obtained after centrifugation by evaporation of ephedra extract prepared by extraction, concentrated drying, and then dissolved in water.

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

Ephedra Extract (Dry Powder) 100 mg

100 mg lactose

10 mg talc

2) In 1 package

100 mg water soluble fraction of ephedra

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

Ephedra Extract (Dry Powder) 100 mg

100 mg corn starch

100 mg lactose

2 mg magnesium stearate

2) In 1 tablet

100 mg water soluble fraction of ephedra

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

Ephedra Extract (Dry Powder) 100 mg

100 mg corn starch

100 mg lactose

2 mg magnesium stearate

2) 1 capsule

100 mg water soluble fraction of ephedra

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)

Ephedra Extract (Dry Powder) 50 mg

Appropriate sterile distilled water for injection

pH adjuster

2) in 1 ampoule (2 ml)

50 mg water soluble fraction of ephedra

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

Ephedra Extract (Dry Powder) 1g

10 grams of sugar

5 grams of mannitol

Purified water

2) in 100 ml

100 mg water soluble fraction of ephedra

10 grams of sugar

5 grams of mannitol

Purified water

The water extract of Ephedra sinica STAPF. Prepared according to the present invention, its dry powder or water-soluble fraction obtained after centrifugation by dissolving it in water has excellent effects in the field of HBV therapeutics, which do not yet have a safe and effective therapeutic agent. Its toxicity is also much lower than that of known compounds, and it 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, which comprises a water-soluble fraction obtained by dissolving water extract of Ephedra sinica STAPF. Or its dry powder 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 lyophilization of the evaporated water extract of ephedra 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 water extract of ephedra in a concentration of 40 to 60 mg / ml, and centrifuging it for about 25 to 35 minutes at 15 ° C and 1000 g. Or prophylactic pharmaceutical compositions. The hepatitis B treatment or prevention method according to claim 4, wherein the water-soluble fraction is obtained by dissolving the water extract of ephedra in 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.