WO2001082941A1

WO2001082941A1 - Composition for treatment of b-type hepatitis and liver cirrhosis

Info

Publication number: WO2001082941A1
Application number: PCT/KR2001/000723
Authority: WO
Inventors: Eun Kyung Hong; Young Shin Chung
Original assignee: Medvill Co., Ltd.
Priority date: 2000-05-02
Filing date: 2001-05-02
Publication date: 2001-11-08
Also published as: CN100341537C; CN1427723A; AU2001256809A1; KR100389131B1; KR20010104497A; JP2003531861A; JP4087114B2

Abstract

The invention provides a mixed extract of Phellodendri cortex and Patrinia that has a therapeutic effect on hepatitis B and liver cirrhosis.

Description

COMPOSITION FOR TREATMENT OF B-TYPE HEPATITIS AND LIVER CIRRHOSIS

Technical Field The present invention relates to a composition useful in the treatment of

Hepatitis B and liver cirrhosis. More particularly, the present invention relates to pharmaceutical compositions comprising as an active ingredient a mixed extract of Phellodendri cortex and Patrinia scabiosaefolia FISCH., which compositions are useful against Hepatitis B virus activity and liver cirrhosis and having antiviral effect, immunoadjuvant activity, and effectiveness in the regeneration of fibrotic hepatocytes, and to a process for the preparation of such compositions.

Background Art

Hepatitis B virus (HBV) is, although it is a DNA virus, prone to mutation. The HBV genome is consisted of a double stranded circular DNA of about 3.2 kb and has an extremely compact structure. Open reading frames (ORFs) in the gene are arranged in a very complicated way such that one ORF overlaps with another ORF partially or wholly to support different pieces of genetic information. PIBV, unlike other DNA viruses, undergoes reverse transcription in the course of the replication of its genome in an infected cell. That is, a full-length RNA called a progenomic RNA(pgRNA) to denote its role in genomic replication is initially produced.

In the replication, the DNA strands are transcribed into RNA strands by a host RNA polymerase II and then used as a template for the synthesis of genomic DNA by a viral reverse transciϊptase, different from the case of ordinary DNA viruses which directly replicate their DNA strands. As a result, HBV shows a high frequency of mutation comparable to general RNA viruses. While mutation in the HBV genome occurs randomly, the rate and the frequency of proliferation are constant. Because hosts produce humoral and cellular immune responses against HBV-derived proteins, mutations occur continuously in viruses survived such responses. While HBV replication cascade runs independent of the host chromosomes, a part of the viral DNA could be integrated into the host genome. When HBV DNA has been integrated into the host genome, the viral genome becomes unstable- and -the X protein expressed therefrom promotes transcriptions in the host cell, causing the inhibition in the function of the p53 gene. If these conditions persist, hepatocellular carcinoma would occur.

Most of primary hepatocellular carcinoma originates from viral hepatitis. Hepatitis viruses had been classified into 5 types, A through E. Recently, a new type of hepatitis virus, Type G, has been cloned. Thus 6 types of hepatitis viruses are known. Among these, hepatitis B virus (HBV) and hepatitis C virus (HCV) have been epidemiologically associated with the carcinogenesis in the liver. The external coat of HBV directly relates to the immune systems of hosts because that part of virus is exposed on the surface. Thus, HBV keeps changing the structure of its surface antigens so as to escape from the immune system of a host and survives. Such changes in the structures of antigens show a broad spectrum and constitute important factors in the progress of the disease or persistent infection. Acute hepatitis progresses into the chronic form due to such factors as ineffective immune response of the host against the virus, functional deficiency of T cells, endocrine function including steroids, drugs, etc.

When symptoms of hepatitis persist for at least 6 months and no improvement is found in biochemical and serological investigations, the hepatitis is labeled chronic. Chronic hepatitis is in turn classified into chronic persistent hepatitis and chronic active hepatitis, where the latter develops into cirrhosis, liver failure, and even death due to the gradual progress of tissue damage in the liver. Chronic hepatitis induced by FIBV or HCV shows a high tendency to progress to cirrhosis and, if advanced further, to hepatocellular carcinoma. Liver cirrhosis can result from various causes including viral infection, parasites, acquired syphilis, alcohol, drug or chemical toxicity, biliary obstruction, congestion, hemochromatosis, Wilson's disease, etc.

In liver cirrhosis, loss of liver parenchyma cells, collapse of reticulin architecture accompanied by vascular bending, fibrosis, proliferation of residual hepatocytes and of widespread connective tissue are observed and the liver gradually contracts Proliferation of extracellular matrix with, concomitant necrosis and regeneration in the parenchyma leads to a distortion of normal liver architecture, and lobules are altered by division or merge due to the proliferation of connective tissue. This alteration obstructs portal vein blood flow, causing portal hypertension, which in turn leads to ascites, collateral circulation (formation of so- called medusa head due to esophageal varix or umbilical vein dilatation), splenomegaly, etc. Proliferation pattern of nodules found in HBV positive cirrhosis comprises intimate cells, and the presence of colonies similar to clones is observed in pre-carcinoma phase. However, genetic abnormalities associated to the initiation of carcinoma are not observed in this phase. Therapeutic agents which have been used for the treatment of chronic hepatitis B are classified into antiviral agents and immuno adjuvants, on the basis of their mechanism of action. For example, antiviral agents include Ara-A, Ara-Amp, acyclovir, and suramin. As a new treatment for chronic hepatitis B, lamivudine, 3TC, is under clinical trials. Lamivudine is a nucleic acid derivative useful as an oral antiviral agent and has been used as an AIDS treatment since 1995 in western countries because it specifically inhibits the reverse transcriptase activity of HIV. Lamivudine was originally tested by Dienstas et al. on patients with chronic hepatitis B. During this administration, the patients turned to HBV DNA negative and 19% of the patients showed recovery to normal ALT range, which indicated the prolonged negative of HBV DNA in serum. However, lamivudine has a disadvantage that it cannot be administered for a long period because of toxicity, and the proliferation of virus is rebounded and returns to the initial state if the administration of the drug is ceased. Recently, it has been found that an HBV variant resistant to lamivudine appears in patients who exhibit resistance to the administration of the drug for an extended period of 6 to 12 months. Interferon, an immunoadjuvant, has also been used in the treatment of hepatitis B, only with a very limited effectiveness. However, standard administration lasts for 3 to 6 months. Although a better result is expected from an extended administration, it is accompanied by substantial adverse effect, and the proliferation of virus is resumed and eturns to the initial state if the administration of the drug is ceased. In the treatment of chronic hepatitis B, improvement in the ALT level concomitant with negative HBe antigen can be the therapeutic index. However, some patients remain in the active hepatitis state even though they are shown to be negative for the HBe antigen. After all, there has not been any practical method of curing liver cirrhosis up to now.

Disclosure of Invention

Therefore, an object of the present invention is to provide a pharmaceutical composition useful in the treatment of chronic hepatitis B.

Another object of the present invention is to provide a pharmaceutical composition useful in the treatment of liver cirrhosis.

A further object of the present invention is to provide a pharmaceutical composition useful in the activities of antibodies and the proliferation of T cells. A still further object of the present invention is to provide a process for the preparation of a pharmaceutical composition for the treatment of hepatitis B.

The objects of the present invention can be achieved by a pharmaceutical composition for the treatment of hepatitis B, comprising a mixed extract of Phellodendri cortex and Patrinia scabiosaefolia FISCH in a therapeutically effective amount. In addition, the composition according to the present invention which comprises a mixed extract of Phellodendron and Patrinia is also useful in the treatment of liver cirrhosis, and in the activities of antibodies and the proliferation of T cells.

Patrinia, including Patrinia scabiosaefolia FISCH., Patrinia villosa JUSS., Patrinia sibirica JUSS., and the like, is a species of perennial plant wild in hills and mountains all over the temperate zone including Korea. It is used in the form of whole plant and, in oriental medicine, for anti-inflammatory effect in ocular diseases, Streptococcus pyogens, edema, leukorrhea and the like.

A thermal extract of Patrinia has been reported to have anti-tumor effect (e.g., against uterine cancer, esophageal cancer, intestinal cancer, and lung cancer) and strongly inhibits the growth of Staphyloccus aureus, Streptococci, etc. in vitro. The extract is also known as having the ability to regenerate injured hepatocytes, prevent their degeneration, and improve the circulation in the portal vein, resulting in the stimulation of the regeneration of hepatocytes. It also acts on the central nervous system to sedate the nerve, exhibit a strong analgesic effect as well as a hypotensive effect and an antidiuretic effect.

The root of Patrinia contains essential oil, various saponins, carbohydrates, and trace amounts of alkaloids such as cumarin. In the root dermis, there can be found acetic acid, formic acid, valeric acid as well as alkaloids including chatinine and valerianine. Among these, valeric acid is known to have a very strong analgesic effect. Dried seeds contain 19.4-19.9% of proteins and 30-34.4% of fats. However, studies on the particulars of active ingredients or pharmacological properties oϊ Patrinia have hardly been carried out yet.

The cortex Phellodendron, the other plant of the present invention, is the bark oϊ Phellodendron amurense RUPRECHT, which grows wild in East Asia and is the best known of Phellodendron species. Other variants of corktree include P. latifoliolatum Nakai, KAWAMOTO, P. japonicum OHWI, P. insular e NAKAI, P. molle NAKAI, and P. s argent. Phellodendri cortex contains yellow or yellowish brown pigments and several kinds of alkaloids of 1.5-4.5%. The major component of the alkaloids is berberine. Also contained are palmatine, magnoflorine, guanidine, jateorrhizine, phellodendrine, candicine, menispermine, and as bitter substances, obakunone, obakulactone, β-sitosterol, etc. These substances have strong antibacterial activity, hypotensive activity, inhibiting effect on central nerve system, acetyl cholinergic effect and anti-inflammatory effect, and are known to be traditionally used in bone disorders and jaundice. Phellodendri cortex is also useful in the treatment of typhoid fever, cholera and in nourishing stomach and normalizing intestinal flora. Thus, Phellodendri cortex has been used in the treatment of gastritis, abdominal pain, jaundice, and the like, as bitter tonic agents, probiotics, or anti-inflammatory astringents. The plant extract of the present . invention has an important characteristic that it shows a therapeutic effect on liver cirrhosis in addition to inhibiting the proliferation of HBV. Thus, the pharmaceutical composition of the present invention using a mixed extract of Patrinia and Phellodendri cortex exhibits a direct antiviral effect against hepatitis caused by a hepatitis B virus infection and at the same time has the effect of healing inflammation and enhancing T helper cells in the immune system of the host. It also exhibits the effect of treating liver cirrhosis by restoring the central vein in the liver through regenerating hepatocytes in the fibrotic liver tissue.

The composition according to the present invention comprises a mixed extract of Phellodendri cortex and Patrinia in a therapeutically effective amount. The mixed extract may be obtained by extracting a mixture of Phellodendri cortex and patrinia or by combining a Phellodendri cortex extract and a Patrinia extract.

In the present invention, the extracts of the two plants in the mixed state show an excellent therapeutic effect due to a synergistic effect. Thus, the ratio of the two plants may preferably be in the range of 2:5 to 5:2 by weight. It is believed that the antiviral effect, cirrhosis treating effect, and the immunoadjuvant effect of the present invention are optimized when the extracts of the two plants are mixed within the above weight ratio.

The composition of the present invention may be appreciated as one of the most optimized therapeutic agents, because it achieves a great effectiveness in the treatment of hepatitis B by means of enhancing the immune system associated to T cells in addition to having a direct anti-HBV effect and exhibits the effect of regenerating fibrotic hepatocytes with a very low toxicity.

According to another aspect of the invention, there is provided a process for the preparation of a pharmaceutical composition, which process comprises the steps of extracting a mixture oϊ Phellodendri cortex and Patrinia with an aqueous solvent and filtering the extract; saturating the filtrate under an elevated pressure and removing the resulting precipitates of coagulated proteins by centrifugation; and adding an organic solvent to the filtrate to remove substances soluble in the organic - - solvent, and then separating and drying the aqueous layer.

In a preferred embodiment of the present invention, the combined extract of Patrinia and Phellodendri cortex is obtained by mixing and milling Patrinia and Phellodendri cortex with a ratio of 1 : 1 by weight, thermally extracting the mixture under un elevated pressure using tab water or distilled water, removing precipitates by centrifuging the extract, boiling the extract again under an elevated pressure to coagulate the residual proteins which is then removed by filtration. The filtrate is again saturated with steam to coagulate the residual proteins, centrifuged to remove precipitates, and then filtered again. A solvent such as chloroform or hexane is added to this filtrate so as to remove any extract components which can be extracted by the solvent, including resins, or fibrous substances solubilized in the filtrate. Thereafter, the aqueous layer is purified using talc or the like and lyophilized to give the mixed extract powders.

The mixed extract of the present invention prepared as described above can be formulated into an appropriate pharmaceutical composition, alone or together with pharmaceutically acceptable carriers, and then used as a therapeutic agent for hepatitis B and liver cirrhosis by oral administration or injection.

The therapeutically effective amount of the mixed extract of the present

.invention may vary depending on the desired effect. In general, for an oral administration to an adult, the effective dose may range from 5 to 50 mg, and preferably, from 10 to 40 mg per day per kg bodyweight. The mixed extract of the present invention can also be used, if appropriate, in combination with other pharmaceuticals such as therapeutics for hepatitis, anti-inflammatory agents, and antiviral agents. Pharmaceuticals that can be used in combination with the composition of the present invention include diphenyl dimethyl dicarboxylate, garlic oil, glycyrrhizin, and silymarin. Brief Description of Drawings

FIG. la is a graph showing the therapeutic effect of Clinical Example 1 (HBe antigen and antibody).

FIG. lb is a graph showing the therapeutic effect of Clinical Example 1 (HBs antigen and antibody). . .

FIG. 2a is a graph showing the therapeutic effect of Clinical Example 2 (HBe antigen and antibody).

FIG. 2b is a graph showing the therapeutic effect of Clinical Example 2 (HBs antigen and antibody). FIG. 3a is a graph showing the therapeutic effect of Clinical Example 3

(HBe antigen and antibody).

FIG. 3b is a graph showing the therapeutic effect of Clinical Example 3 (HBs antigen and antibody).

^• FIGS. 4a to 4d are pictures showing the therapeutic effect against liver cirrhosis for groups that received a control, DMN, DMN + 150 mg/kg of the composition of the present invention, and DMN + 500 mg/kg of the composition of the present invention, respectively.

FIG. 5 shows a UV spectrum obtained from a Patrinia extract. FIG. 6 shows a UV spectrum obtained from a Phellodendri cortex extract. FIG. 7 shows a UV spectrum obtained from a mixed extract oϊ Patrinia and

Phellodendri cortex according to the present invention.

FIG. 8 shows an HPLC chromatogram obtained from a Patrinia extract. FIG. 9 shows an HPLC chromatogram obtained from a mixed extract of Patrinia and Phellodendri cortex according to the present invention. FIG. 10 shows an HPLC chromatogram obtained from a Phellodendri cortex extract.

FIG. 11 shows an HPLC chromatogram obtained from a mixed extract of Patrinia and Phellodendri cortex according to the present invention. Best Mode for Carrying Out the Invention

The present invention will now be described in more detail, based on the preparation and clinical examples. However, it should be appreciated that these examples are presented for the purpose of illustration only and cannot be construed as limiting the scope-of protection sought in the present applicatio which.is defined in the attached claims.

Example 1 : Preparation of a mixed extract

A 100 g aliquot of a powder obtained by mixing and milling equal amounts of dried Phellodendri cortex and Patrinia was taken and 3,000 ml of distilled water was added thereto. Extraction was conducted with saturation at elevated pressure for 50 minutes. The resulting extract was collected and the residue was removed. The chloroform extract was then subjected to centrifugation to remove precipitates, and the aqueous extract was filtered. The resulting filtrate was concentrated to a total amount of 1500 ml and then filtered again. Thereafter, the filtrate was saturated under elevated pressure (121°C, 15 psia) for 15 minutes, and the resulting coagulates, especially precipitates containing proteins, were removed by centrifugation followed by filtration. The filtrate was transferred to a separatory funnel and 400 ml of chloroform was added to abstract resins and fibrous substances, etc. Then the chloroform layer was separated and disposed. This extraction was repeated two times, and 200 ml of n-hexane was added to the aqueous layer to abstract residual proteins, resins, fibrous substances, and other n- hexane soluble substances. The aqueous layer was then collected, heated to 60 to 80°C, and then stirred with the addition of 500 g of talc. The talc was removed by suction filtering and the filtrate was once again filtered slowly, and then lyophilized to powder. This gave about 15 g of extract from the mixture of Phellodendri cortex and Patrinia with a yield (dry weight basis) of about 15%>.

Example 2: Preparation of an alcohol extract To 100 g of a powder obtained by mixing and milling equal amounts of dried Phellodendri cortex and Patrinia, 1,500 ml of 70% methanol was added and allowed to stand 60 hours with intermittent stirring. The mixture was then subjected to suction filtering to give an alcohol extract. Subsequently, to the alcohol extract remaining after the removal of alcohol in a reflux condenser, 1,000 ml of distilled water was admixed and the mixture was heated to 100°C for..5 minutes to allow solubilization of water-soluble substances. This turbid extract was primarily purified by the addition of an appropriate amount of talc, followed by stirring and suction filtering. The purified filtrate was transferred to a separatory funnel and 300 ml of chloroform was added to abstract resins and fibrous substances, etc. Then the chloroform layer was separated and disposed. 200 ml of n-hexane was added to the aqueous layer to abstract residual proteins, resins, fibrous substances, and other n-hexane soluble substances. The aqueous layer was then collected, heated to 60 to 80°C, and then stirred with the addition of a suitable amount of talc. The talc was removed by suction filtering and the filtrate was once again filtered slowly, and then lyophilized to powder. This gave about 10 g of extract from the mixture of Phellodendri cortex and Patrinia with a yield (dry weight basis) of about 10%.

Formulation Example 1 : Tablets 250 mg of the mixed extract in the form of lyophilized powder, which was prepared in above Example 1, was admixed in a U shaped mixer for 20 minutes with 260 mg of lactose, an excipient, 35 mg of Avicell (microcrystalline cellulose),15 mg of sodium starch glyconate, a disintegrant, and 80 mg of Low-hydroxypropyl cellulose, a binder. After the mixing was completed, 10 mg of magnesium stearate, a lubricant, was added followed by additional mixing for 3 minutes. The mixture was tab letted and film coated after quantitative test and moisture resistance test, producing tablets containing 250 mg of the mixed extract per tablet.

Formulation Example 2: Syrups An appropriate amount of sucrose was dissolved in a predetermined amount of water, and 80 mg of para-oxymethyl benzoate and 16 mg of para-oxypropyl benzoate were added thereto as preservative. 4.5 g of the mixed extract in the form of lyophilized powder, which was prepared in above Example 1, was then added and completely dissolved with the temperature maintained at 60°C, after which the mixture was cooled and made to 150 ml by adding distilled water, giving a syrup formulation.

Formulation Example 3 : Capsules

500 mg of the mixed extract in the form of lyophilized powder, which was prepared in above Example 1, was filled in a hard gelatin capsule to produce a capsule formulation.

Formulation Example 4: Beverages

500 mg of the mixed extract in the form of lyophilized powder, which was prepared in above Example 1, was dissolved in an appropriate amount of water, and additives like vitamin C, a flavoring agent like citric acid, sodium citrate, a high fructose syrup were added and an amount of sodium benzoate was added as a preservative. Then the mixture was made to a total volume of 100 ml by adding water to produce a beverage formulation.

Formulation Example 5: Injections

200 mg of the mixed extract in the form of lyophilized powder, which was prepared in above Example 1, was dissolved in 200 mg of physiological saline containing 1% polyoxyethylene hydrogenated castor oil by heating such that an injection formulation containing the mixed extract in an amount of 0.1% was prepared.

Clinical Example 1 : Administration of the composition of the present invention to a patient with chronic hepatitis B A capsule formulation containing the composition of the present invention which was prepared in above Example 1, was orally administered to a male patient of age 38 having a chronic hepatitis B, at a dosage of 2 capsules twice a day (2000 mg/day). The results are shown in FIG. 1. FIG. 1 illustrates the results of an examination of anti-HBV antibodies with the RIA (radioisotope analysis) method, and indicates the therapeutic effect of the- composition of the present invention on hepatitis B (FIG. la: HBe antigen, FIG. lb: HBs antigen).

Clinical Example 2: Administration of the inventive composition to a patient with chronic hepatitis B A capsule formulation containing the composition of the present invention was orally administered for 11 months to a male patient of age 36 diagnosed as having chronic hepatitis B, at a dosage of 2 capsules twice a day (2000 mg per day). The result was that antibodies against hepatitis B virus were produced, and thereby both HBe antigen and HBs antigen were almost cleared such that the relevant antigen titers are lowered to about 0. In the present Example (FIG. 2a and FIG. 2b), antigens were detected at high levels before the start of the therapy because the hepatitis was chronic. Administration of a composition of the present invention led to the production of antibodies against the HBe antigen and a consequent reduction of the antigen, as well as the reduction of HBs antigen for a similar reason. Thus, the antigen titers were found to have been lowered to about 0 after 6 months of the administration initiation.

Clinical Example 3 : Administration of the inventive composition to a patient with chronic hepatitis B A capsule formulation containing the composition of the present invention was orally administered for 8 months to a male patient of age 34 having chronic hepatitis associated to HBV, at a dosage of 2 capsules twice a day (2000 mg per day). The results are shown in FIG. 3a and FIG. 3b. As can be seen from the figures, the antigen titers for HBV were lowered to about 0. The above clinical examples demonstrate that the composition of the present invention has an excellent effectiveness for the treatment of hepatitis B.

Experimental Example 1 : Therapeutic effects on liver cirrhosis

In order to examine the effect of the composition of the present invention on - the-liver cirrhosis, liver cirrhosis was induced in rats by single injection of N-nitroso dimethyl amine (DMN) (40 mg/kg, i.p.). DMN causes alterations in tissue that are most similar to those found in liver cirrhosis in human and, hence, frequently used in liver cirrhosis animal experiments.

24 rats were allocated to 4 groups of 6 animals, where Group 1 served as control, Group 2 received 40 mg/kg of DMN only, Group 3 received 40 mg/kg of DMN followed by 2 a week period which the composition of the present invention was administered at a dosage of 150 mg/kg, and Group 4 received 40 mg/kg of DMN followed by 2 weeks of the period which the composition of the present invention was administered at a dosage of 500 mg/kg. The Groups 1 and _.2 received physiological saline for the period where Groups 3 and 4 received the composition of the present invention.

After 2 weeks, all the animals were sacrificed in order to take blood samples and to excise liver tissues for the fixation in a 4% formaldehyde solution. Such indices as ALT, AST, bilirubin, albumin, and the like were determined from blood samples. In order to observe the morphology of tissues depending on the development of lesions, liver tissues were stained with AZAN dye, which stains pathological products such as hyaline degeneration and fibrin, and examined under an optical microscope.

AZAN (azocarmine) staining was carried out by dipping the tissue in a mordant (10% aqueous potassium dichromate (K₂Cr₂0₇) 50 ml + 10% aqueous trichloroacetic acid (CCl₃COOH) 5 ml) and then in an azocarmine solution at an elevated temperature. After staining, differentiation in aniline-alcohol was continued until the nuclei appear satisfactorily while the rest of the tissue decolorized, and acetic acid-alcohol was used to fix the stain. Then, the tissue was treated with an Aniline blue-Orange G solution and differentiated with 100% ethanol, drained, and sealed to give the completed slide.

FIG. 4 shows the result of the examination under microscope. In FIG. 4a, normal liver tissue is displayed where the central vein is clearly seen. In FIG. 4b, the blue regions can be identified as fibrotic tissue in the DNA induced-liver cirrhosis. In FIG. 4c, cirrhosis was ameliorated when 150 mg/kg of the composition of the present invention was administered, which can be seen from the reduction of the blue staining region. In FIG. 4d, it can be seen that the fibrotic region recovered to almost normal level when the composition of the present invention was administered to a level of 500 mg/kg. The central vein also showed a pattern indicating gradual recovery.

The above results demonstrate that the composition according to the present invention has an ability to regenerate fibrotic liver and a therapeutic effect on liver cirrhosis.

Experimental Example 2: Comparison of immunoadjuvant effect (the effect of enhancing T cell dependent antibody production in vivo) between the composition of the present invention and individual extracts oϊ Phellodendri cortex and Patrinia

25 Balb/c mice (body weight 17-20 g) were allocated to 5 groups of 5 animals. Sheep red blood cells (SRBC) were intra peritoneally transplanted i.p. Then, each of the individual extracts oϊ Phellodendri cortex and Patrinia, which are components of the composition of the present invention, and the composition of the present invention dissolved in water were orally administered to the mice at a dosage of 3-100 mg/kg body weight for 3 days starting from the day of transplantation (Day 0, 1, 2).

For the control group, physiological saline was given by the same route of administration. Four days after, the animals were sacrificed and their spleens were isolated aseptically. The tissue was chopped, and spleen cells were separated using a mesh and isolated using the plunger of a syringe in a petri dish containing 3 ml of HBSS (Hank's balanced salt solution). The resulting cell suspension was transferred to a 15 ml conical tube and left standing for 5 minutes. 2 ml of the supernatant was removed and centrifuged at 1200 rpm for 10 minutes. The supernatant was disposed and cells were resuspended in 2 ml of EBSS, diluted 30-fold in EBSS, and used for the plaque forming cell analysis in 100 (μl) aliquots. SRBC washed with EBSS (25 μl, target cell), guinea pig complement diluted 2-fold in EBSS (25 μl) and agarose (0.85% in EBSS, 350 μϊ) were mixed and poured into a petri dish to set, and then allowed to stand in a C0₂ incubator at 37°C for about 1 hour. The number of antibody forming cells was determined in a modified Jerne plaque assay. Plaques and cells were counted and reported in the number of antibody forming cells/10 cells.

As a result, the number of antibody forming cells was remarkably greater in the case of the composition of the present invention than in the case of the individual component extracts, indicating the stronger immunoadjuvant effect obtained in the present invention. This is demonstrated in Table 1 below.

Table 1. Effect of the composition of the present invention on the T cell dependent antibody production

Experimental Example 3 : Effect of the composition of the present invention on T cell activation

Effects of the composition of the present invention on T cell activation were determined using the mixed immune cell response method. As immune cells isolated from two kinds of mice with different MHC class antigens are mixed, T cells recognizing antigens on the cells of the other kind of mouse proliferates.

From two strains of mice, B6C3F1 (H-2k) and BDF1 (H-2d) which have mutually different MHC class antigens, the spleens were isolated aseptically and spleen cells were liberated. The resulting isolated immune cells were adjusted to 2.5 x 10⁶ cells/ml and allocated to a 96 well plate in 100 μl/well and made to 200 μl.

To this, the composition of the present invention was added to a concentration of 0.01 to 100 μg/ml, and the plate was incubated in a C0₂ incubator at 37°C for 3 days. 18 hours before the termination of the incubation, 1 μCi/well of [Η]-thymidine was added. Cells were harvested using an automatic cell harvester, and the extent of immune cell proliferation was determined by measuring the [^JFI]-thymidine uptake.

The results listed in Table 2 below indicate that the proliferation of T cells was enhanced in a manner dependent on the content of the composition of the present invention.

Table 2. Effect of the composition of the present invention on the T cell proliferation

Experimental Example 4: Acute toxicity test

30 normal female ICR mice (bodyweight 19 ± 1 g) were allocated to 5 groups of 6 animals. The LD₅₀ p.o. of the mixed extract of the present invention was determined in mice orally administered the mixed extract in an amount of 3 g /kg body weight to Group A, 6 g/kg to Group B, 9 g/kg to Group C, 12 g/kg to Group D and 15 g/kg to Group E using the Behrens-Karber method. LD₅₀ is a significant value representing an index of acute toxicity of drugs. It indicates the safety of the composition of the present invention.

The results listed in Table 3 below show that no mouse was brought to death even in the group which received 15 g/kg bodyweight of the mixed extract, thereby making it unable to determine the LD₅o value. Therefore, LD₅₀ p.o. of the mixed extract of the present invention is higher than 15 g/kg bodyweight, which demonstrates that the mixed extract of the present invention is very safe to the human body. In other words, it is evident that the mixed extract of the present invention can be administered safely without significant toxicity.

Autopsy and histo-pathological analysis were carried out as described below. At the end of experiment, all the surviving animals were anesthetized with ether and sacrificed by bloodletting. Organs were separated and examined for abnormalities by naked eyes. For histo-pathological analysis, organs were fixed in a 10% neutral formaldehyde solution for at least 10 days, dehydrated, and then embedded in a paraffin embedding center (Fisher, Histomatic Tissue Processor, 166A) and cut into 5 μl slices using a rotary microtome (AO Rotary Microtome), which were then examined after staining with Hematoxylin-Eosin.

Histo-pathological findings from the dissection and microscopic examination of all animals in each group were as follows. When the mixed extract of the present invention was administered to a mouse up to 15 g/kg bodyweight, there were no abnormal findings due to the drug administration in the liver tissue. Abnormal findings were not observed in kidney, myocardial cells, gastrointestinal tract, pancreas, lung, spleen, adrenal, brain, testis, ovary, bone marrow, and the like either. It thus can be concluded that the mixed extract of the present invention did not show an adverse effect due to its acute toxicity on any of the organs when administered to a mouse in the maximum dose of 15 g/kg body weight, which means that the extract would be a safe drug which do not induce any toxicity.

Table 3. Lethal Dose (LD₅₀) of the mixed extract by oral administration

Notes)

*Z: half value of the number of dead animals from two consecutive doses

**d: Difference between two consecutive doses

Experimental Example 5: Characterization of the mixed extract by UV absorption spectrum

The mixed extract of Patrinia and Phellodendri cortex according to the present invention was examined using a UV spectrophotometer (Pharmacia, Ultrospec 2000) and HPLC (HP 1090). FIG. 5 is the result of a UV absorption spectrum for a Patrinia extract (0.5 mg/ml), showing a rise in the absorption at 282 nm. FIG. 6 is the result of a UV absorption spectrum for a Phellodendri cortex extract (0.25 mg/ml), where the absorption increases at 278 nm and 325 nm. FIG. 7 is the result of a UV absorption spectrum for the mixed extract of patrinia and Phellodendri cortex according to the present invention (0.5 mg/ml), showing rises in the absorption at 281 nm and 316 nm.

Experimental Example 6: Characterization of the mixed extract by HPLC The mixed extract of the present invention and extracts of Patrinia or Phellodendri cortex alone were separately dissolved in water to a concentration of 10 mg/ml for separation by HPLC. 10 μl aliquots were taken from the dissolved extract solutions and used for the HPLC (HP 1090) analysis.

(Analysis Conditions) Column: Luna 5u C 18; 4.6 mm i.d. x 250 mm L (Phenomenex)

Mobile phase: A gradient system of 2% acetic acid and methanol Flow rate: 1 ml/min. Detection: at 254 nm with a photodiode array detector

In repeated experiments, peaks characteristic of respective extracts were observed. FIG. 8 shows the HPLC profile for a Patrinia extract. Under the operation parameters described above, characteristic peaks appear at 10.245, 12.772, 14.104, 17.087, 18.769 minutes. Among those, the peak at 10.952 min. represents 3,4-dihydroxybenzoic acid and showed a content of 0.022%. The peak at 17.087 represents chlorogenic acid and its content was 0.238%. The peak at 18.769 min. represents caffeine acid and its content was 0.107%. When the mixed extract of the present invention was analyzed by HPLC under the same conditions as described above, as can be seen from FIG. 9, the above three substances derived from patrinia were shown to be contained, in an amount of 0.020% for 3,4- dihydroxybenzoic acid, 0.270% for chlorogenic acid, and 0.044% for caffeine. Additional characteristic peaks appeared at 17.299, 22.048, and 22.449 minutes.,

For the analysis of berberine and palmatin, the most abundant alkaloids in Phellodendri cortex extracts, the following conditions were employed. (Analysis Conditions)

Column: Luna 5u C18; 4.6 mm i.d. x 250 mm L (Phenomenex) Mobile phase: acetonitrile: water: KH₂P0₄:SDS = 500:500:3.4: 1.7 Flow rate: 1 ml/min. Detection: at 254 nm with a photodiode array detector ..

In the HPLC analysis oϊ a Phellodendri cortex extract, as shown in FIG. 10, palmatine was detected at 10.629 min. with the content being 0.988%, and berberine at 11.314 min. with the content being 1.963%. However, in the mixed extract of the present invention, palmatine was detected at 11.751 min. with the content being 0.229%, and berberine at 12.650 min. with the content being 0.405%, which amounts to a tendency toward reduced yields in the mixed extraction (FIG. 11).

Industrial Applicability

The composition according to the present invention has the effect of inhibiting hepatitis B virus and regenerating fibrotic hepatocytes in liver cirrhosis. In particular, the composition can be administered for a long period because it does not show an adverse side effect or induce drug resistance. The composition also has the advantage that the activity of the composition is maintained during a prolonged period of administration. Therefore, the composition of the present invention can be used advantageously for the treatment of hepatitis B and liver cirrhosis.

Claims

1. A pharmaceutical composition for the treatment of hepatitis B, comprising a mixed extract of Phellodendri cortex and Patrinia in a therapeutically effective amount.

2. A pharmaceutical composition for the treatment of liver cirrhosis, comprising a mixed extract of Phellodendri cortex and Patrinia in a therapeutically effective amount.

3. The pharmaceutical composition as claimed in claim 1 or 2, wherein said mixed extract has been prepared by extracting a mixture oϊ Phellodendri cortex and Patrinia with a weight ratio of 1 :0.2 to 1 :5 on the dry weigh basis.

4. The pharmaceutical composition as claimed in claim 1 or 2, wherein said mixed extract has been prepared by mixing a Phellodendri cortex extract and a Patrinia extract with a weight ratio of 1 :0.2 to 1 :5.

5. The pharmaceutical composition as claimed in claim 1 or 2, wherein said mixed extract is administered in an amount of .10 to 50 mg/kg body weight for an adult.

6. The pharmaceutical composition as claimed in claim 1 or 2 which has been formulated in the form of tablets, capsules, solution, suspension, syrup, or beverage with the addition of pharmaceutically acceptable excipients.