JP2003531861A - Composition for treating hepatitis B and cirrhosis - Google Patents

Abstract

(57) [Summary] The present invention provides a mixed extract of a plant of yellow cabbage (Phellendron amurense Rupr. Cortex) and a plant of Patrina (Patrinia scabiosaefolia FISCH.) Showing a therapeutic effect on hepatitis B and cirrhosis.

Description

Detailed Description of the Invention

[0001]

【Technical field】

The present invention relates to a pharmaceutical composition which is effective for treating hepatitis B and cirrhosis, which comprises a mixed extract of yellow pelt peel and Ominaeshi (soy sauce).
edron amurense RUPRCHT cortex) and a mixture extract of P. scutellatum (Patrinia scabiosaaefllia FISCH.) as an active ingredient, which is an active ingredient for hepatitis B virus activity and liver cirrhosis treatment, and has antiviral effect, immunopotentiating effect, and fibrotic hepatocyte TECHNICAL FIELD The present invention relates to a pharmaceutical composition having the effect of regenerating and a method for producing the same.

[0002]

[Background technology]

Although hepatitis B virus (HBV) is a DNA virus, it is a virus that easily mutates. The HBV genome is a double-stranded circular (doubl of about 3.2 kb).
e strand circular) DNA, which has an extremely compressed structure. Each open reading frame (ORF) has one OR
F is cleverly made so as to hold different genetic information in whole or in part with other ORFs. Unlike other DNA viruses, HBV undergoes a reverse transcription process when it is replicated in infected cells. That is, a replication intermediate called a pregenome of 3.5 kb, which is longer than the length of the original genome, is produced, and double-stranded DNA is replicated by the reverse transcriptase / DNA activity of the virus itself.

[0003] Unlike such a normal DNA virus that replicates a DNA chain as it is, such a replication mode is first transcribed from a DNA chain to an RNA chain by a reverse transcriptase of a host, and the reaction proceeds by an RNA polymerase specific to the virus. To be Therefore, HBV is a DNA virus, but exhibits high variability comparable to that of a general RNA virus. Mutations in the HBV gene occur arbitrarily, but have a constant growth rate and frequency.

On the other hand, the host side also shows humoral and cell-mediated immune reactions to HBV-derived proteins, and thus the residual viral mutations occur continuously from such reactions. HBV DNA polymerase having reverse transcriptase activity reverse transcribes pregenomic RNA to replicate viral DNA. The series of replications is independent of the host chromosome. However, it has been revealed that a part of the viral DNA enters the host chromosome. When HBV DNA enters the chromosome of the host, the gene becomes unstable, and the X protein expressed from the HBV DNA promotes transcription of the host cell, and P53 gene function is likely to decrease. If this situation continues, hepatocellular carcinoma may develop.

Most primary hepatocellular carcinomas begin with viral hepatitis. Hepatitis virus species are
There were 5 types from A type to E type, but recently the viral gene was cloned and
Six types are known in total, but among them, the ones that have been epidemiologically pointed out to be related to liver carcinogenesis are hepatitis B virus (HBV) and hepatitis C virus (HC
V). The outer coat of HBV is exposed on the surface of virus particles and is a part directly related to the immune system of the host.

[0006] HBV is therefore altering the structure of surface antigens in order to escape and survive the host's immune response. Changes in the antigen structure occur in various ways and are important factors that determine the progress of disease and persistent infection. The host's immune response to the virus is not appropriate, or the acute hepatitis develops into chronic hepatitis due to factors such as lack of T cell function, endocrine function like steroids, and drugs.

If the symptoms of hepatitis persist for 6 months or longer and there is no improvement in biochemical and serological tests, it is called chronic hepatitis. Chronic hepatitis is further divided into chronic persistent hepatitis and chronic active hepatitis. Among them, chronic active hepatitis causes progressive damage of liver tissue, leading to cirrhosis, liver dysfunction, and death. Chronic hepatitis due to hepatitis B and C viruses has a high rate of liver cirrhosis, and if it progresses further from here, it will develop into hepatocellular carcinoma. There are various causes of cirrhosis, including viral infections, parasites, acquired syphilis, alcohol drinking, drug and chemical toxicity, bile duct obstruction, congestion, hemochromatosis, and Wilson's disease.

When cirrhosis progresses due to various causes, hepatic parenchymal tissue cells are lost, and the collapse of the reticular tissue accompanied by vascular curvature and the proliferation of fibrous tissue, and the proliferation of residual hepatocytes and the proliferation of widely distributed connective tissue are caused. It occurs and gradually shrinks. While the liver fluke proliferates, necrosis and regeneration occur in the parenchyma. The liver loses its normal structure, and the lobule is divided by the proliferation of connective tissue or reconstructed by merging. Since this reconstruction obstructs the blood flow path of the central vein in the portal vein, it causes a circulatory disorder and induces portal hypertension resulting in ascites, collateral circulation (dilation of the esophageal varices or umbilical veins, so-called Mezusa). Form the head of)
, Splenomegaly, etc. HBV-positive cirrhotic nodules are composed of cells with a close proliferation pattern, and it is seen that cell groups such as clones already exist at the stage when they can be regarded as a precancerous state. However, in this state, the onset of cancer
on) there is no abnormality in the gene.

Drugs used for the treatment of chronic hepatitis B with HBV include antiviral agents and immunopotentiators depending on the mechanism of action. As an antiviral drug, Ara
-A, Ara-Amp, acyclovir, suramin and the like. Recently, as a new treatment for chronic hepatitis B, lamivudine (TC)
Has been used in clinical trials. Lamivudine is an oral antiviral agent as a hexane derivative, specifically inhibits the activity of reverse transcriptase of HIV (acquired immunodeficiency virus), and has been used as a therapeutic agent for AIDS in Europe and America since 1995. Lamivudine was first tested experimentally in patients with chronic hepatitis B by Dienstas, etc., and HBV DNA became negative during administration, resulting in 19%.
The patient had normal ALT levels and persistent negative blood HBV DNA was observed. However, lamivudine has the disadvantage that it is difficult to use for a long time because of the toxicity of the drug itself, and when the drug is stopped, the suppressed virus continues to grow,
It will return to its original state. Recently, it has become clear from patients who have been resistant to long-term administration of lamivudine of 6 to 12 months that HBV mutants resistant to this drug emerge.

Interferon, an immunopotentiator, has also been used to treat hepatitis B, but its effectiveness is very low. However, administration for 3 to 6 months is the standard, and more useful results are expected when administered over a long period of time, but the side effects are large, and when the drug administration is discontinued, the virus proliferation increases again. It will return to its original state. In the treatment of chronic hepatitis B, improvement of the ALT value and negative conversion of HBe antigen serve as an index for treatment. However, in some cases, the state of active hepatitis may continue despite the negative HBe antigen in the blood.

However, no actual medical treatment method for liver cirrhosis is known in any of the medical treatment methods known to date.

DETAILED DESCRIPTION OF THE INVENTION Therefore, an object of the present invention is to provide a composition having a therapeutic effect on chronic hepatitis B.

[0013]   Another object of the present invention is to provide a composition having a therapeutic effect on liver cirrhosis.

[0014] Yet another object of the present invention is to provide a composition effective for increasing the antibody titer and T cell proliferation.

A further object of the present invention is to provide a method for producing a composition for treating hepatitis B.

The object of the present invention as described above can be achieved by a composition for treating hepatitis B containing a therapeutically effective amount of a mixed extract of yellow scallions and Prunus chinensis. Further, a composition containing a therapeutically effective amount of a mixed extract of yellow knot skin and Prunus chinensis according to the present invention is useful as a composition for treating liver cirrhosis, and shows an increase in titer and T cell proliferation effect during antibody production. It is useful as a pharmaceutical composition having

The name Ominaeshi used in the present invention, which is also called Rokusho, is called Ominaeshi (Patrinia scabiosaefolia FISCH.), Otokoeshi (Shirohana Roshisho; sibilica JUSS.). Ominaeshi is a perennial herbaceous plant that grows naturally in the mountains of temperate regions around the world including Korea. The whole plant is used, and in Kampo, it is used for eye diseases, Streptococcus pyogens, edema, and inflammation of zoster.

[0018] In addition, there is a report that the heat extract of Pleurotus cornucopiae has an antitumor effect (for example, uterine cancer, esophageal cancer, gastric cancer, intestinal cancer, lung cancer, etc.),
Strongly suppresses the growth of Staphylococcus aureus, Streptococcus, etc. in an in-vitro test. Since it has the ability to regenerate damaged hepatocytes, it is known to prevent degeneration of the damaged hepatocytes, improve circulation in the portal vein, and promote regeneration of hepatocytes. It is known to act on the central nervous system to stabilize nerves, exhibit a strong analgesic effect, and have a blood pressure lowering effect and an antidiuretic effect.

[0019] The roots of Prunus domestica include essential oils, various saponins, carbohydrates and coumarins (c
a small amount of alkaloids such as umarin). The dermis layer of the root includes acetic acid, formic acid, valeric acid and the like, and contains alkaloids such as catinine and valerianine. Among them, valeric acid is known to have a very strong analgesic effect. The dried seeds contain 19.4 to 19.9% protein and 30 to 34.4% fat. However, no specific research has been conducted on the active ingredient of Prunus chinensis and its pharmacological effect.

[0020] The yellow-skinned bark, which is a further different plant used in the present invention, is the bark of the yellow-spotted or the yellow-spotted tree that grows naturally in Japan, South Korea, China and the like. Phellodendron amurense RUPRECH
T). As its varieties, Latihorio latomunakakawamoto, japonicum oy, ferro dendron insular nakai, ferro dendron morenakai,
There are Ferrodendron Sacarinens Sargent. Yellow-yellow skin contains 1.5 to 4.5% of a yellow or yellowish brown pigment substance and several alkaloid components. The main component of alkaloids is berberine. In addition, palmatine and magnoflorin
rine), guanidine, jateorigin (jateo)
rrizine, ferrodendrine, candicine, menisperine and the like are included, and as bitterness, obakunone and obaculactone, β-sitosterol. si
Tosterol) and the like are known. These components have strong antibacterial action, hypotensive action, central nervous system depressant action, acetylcholine enhancing action and anti-inflammatory action, and were used for bone diseases and jaundice in the herbal order and pharmacy. Further, it is effective against typhoid fever and cholera, and is also effective against healthy stomach and intestine. On the other hand, these bark have been used as gastroenteritis, abdominal pain, jaundice, etc. as a bitter stomachic agent, an anti-inflammatory agent for intestinal regulation, and an astringent agent.

The present invention is a galenic drug, and has an important characteristic that it not only reduces hepatitis B virus but also shows an effect on liver cirrhosis. The composition of the present invention, which uses a mixed extract of Prunus chinensis and T. chinensis, has a direct antiviral effect against hepatitis due to infection with hepatitis B virus, and at the same time treats inflammation and immunizes the host immune system. Among them, it has an effect of strengthening T helper cells. In addition, by recovering the central veins in hepatocytes through the regeneration of hepatocytes in fibrotic liver tissues, a therapeutic effect on liver cirrhosis is shown.

The composition according to the present invention comprises a therapeutically effective amount of a mixed extract of Pelium japonicus and P. chinensis. Here, the mixed extract can also be obtained by mixing the yellow tangerine peel and the Prunus chinensis and extracting them, and the extract of the yellow prickle bark and the extract of Ominaeshi may be mixed.

Since the present invention exhibits an excellent therapeutic effect due to the synergistic effect in the state where the extracts of these two types of plants are mixed, the composition ratio of the yellow algal skin and the stamens of these two types of plants is 2: It is preferable to set the weight ratio in the range of 5 to 5: 2. When the extracts of the active ingredients of these two kinds of plants are mixed within this range, the effects of the antivirus, cirrhosis and immunity enhancement of the present invention can be maximized.

The composition of the present invention has a direct anti-HBV effect and, at the same time, enhances the immune system involving T cells, and thus has a high therapeutic effect on hepatitis B,
Although it has the effect of regenerating fibrotic hepatocytes, it has extremely low toxicity and is evaluated as the most ideal drug.

According to another feature of the present invention, the pericarp and stamen are mixed and extracted with an aqueous solvent and filtered; the filtrate is saturated under high pressure and the precipitate of coagulated protein produced is removed by centrifugation. A step of adding an organic solvent to the filtrate to remove an organic solvent-soluble substance, and then separating and drying an aqueous layer, the method for producing a pharmaceutical composition is provided.

[0026] The total extract of yellow pearl oyster bark and Prunus chinensis used as an active ingredient in the composition of the present invention is prepared by mixing Omina pie and yellow pearl bark in a ratio of 1: 1 (weight ratio), and then After heat-extracting with water or distilled water under high pressure, the extract is centrifuged to remove the precipitate, and the extract is boiled under high pressure again to coagulate the remaining protein and then filtered. And remove. The filtrate is saturated again under vapor pressure to coagulate residual protein, centrifuged to remove the precipitate and then filtered. A solvent, for example, chloroform and hexane, is added to the filtrate, and the remaining resin dissolved in the filtrate is removed.After removing all the extracts that can be extracted with a solvent such as fiber, the aqueous layer is purified using talc or the like, Lyophilize to obtain a powder of total extract.

The mixed extract according to the present invention obtained by the method as described above is formulated into a suitable pharmaceutical composition alone or together with a pharmaceutically acceptable carrier in a therapeutically effective amount, It can be used orally or by injection as a therapeutic agent for hepatitis B and cirrhosis.

[0028] The effective amount range of the mixed extract according to the present invention may vary depending on what the intended effect is, but in general, when it is orally administered to an adult, it is generally 5 per kg body weight per day.
A dose of -50 mg, preferably 10-40 mg can be administered. Furthermore, the extract according to the present invention can be blended or used in combination with agents such as hepatitis therapeutic agents, anti-inflammatory agents, antiviral agents, etc., if necessary. Examples of agents that can be used in combination with the composition according to the present invention include DDB (diphenyldimethyldicarboxylate), garlic oil, glycyrrhizin, silymarin and the like.

[0029]

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described more specifically based on Examples and Experimental Examples, but these are merely provided for explaining the present invention, and the present invention is not limited to these. .

Example 1: Preparation of a mixed extract Dried yellow husks (Phellodendron amurense RUPR)
Echt) and oak (Patrinia scabiosaefolia)
FISCH. ) And 1) were mixed in a weight ratio of 1: 1 and pulverized with a pulverizer. From this mixed powder, 100 g was collected, 3,000 ml of distilled water was added thereto, and saturated extraction was performed under high pressure for 50 minutes. Then, the extract was separated and the residue was removed. The extract was centrifuged to remove the precipitate, and the aqueous solution was filtered and then concentrated to a total volume of 1,500 ml and filtered. Further, the precipitate containing the coagulated substance, particularly the protein, which was produced by saturating for 15 minutes under high pressure (121 ° C, 15 pound / in a2) was removed by centrifugation and then filtered. The filtrate was put in a separatory funnel, and 400 ml of chloroform was added to elute the resin and fibers, and the chloroform layer was separated and removed. After repeating this method twice, 200 ml of n-hexane was added again to the aqueous layer to elute the remaining protein, resin, fiber and n-hexane soluble substance. The aqueous layer was recovered and heated at 60 to 80 ° C., and then 500 g of talc was added and stirred. This was filtered again under reduced pressure to remove talc, the filtrate was again slowly filtered, and the filtrate was freeze-dried to give a powder. By this method, about 15 g of a mixed extract was obtained with a yield of about 15% (on a dry weight basis) from a mixture of yellow scab and Minamiea.

Example 2: Preparation of Extract with Alcohol [0031] A mixture of dried yellow malt bark and Pleurotus sinensis 1: 1 on a weight basis was ground with a grinder to obtain 100 g of powder, and 70% methanol was added thereto. After 1,500 ml was added and left for 60 hours with frequent stirring, this was filtered under reduced pressure and the residue was removed to obtain an alcohol extract. The alcohol was recovered using a reflux cooling device, 1,000 ml of distilled water was added to and mixed with the remaining alcohol extract, and the mixture was heated at 100 ° C. for 5 minutes to be solubilized. A suitable amount of talc was added to the cloudy extract, and the mixture was stirred, filtered under reduced pressure and subjected to primary purification. The purified filtrate was put in a separatory funnel, 300 ml of chloroform was added to elute the resin and the fibrous material, and the chloroform layer was separated and removed, and then 200 ml of n-hexane was added again to the aqueous layer to remove the remaining protein, resin, Fibrous substances and n-hexane soluble substances were eluted. 60-80 to collect water layer
After heating at 0 ° C., an appropriate amount of talc was added again, and the mixture was stirred, filtered under reduced pressure to remove talc, and the filtrate was gradually filtered again, and then freeze-dried to give a powder. According to this method, about 10 g of the mixed extract of yellow scalp and Oenashi was obtained with a yield of about 10% (dry weight basis).

Composition Example 1: Tablet 250 mg of the freeze-dried powdered mixed extract of the present invention prepared in Example 1
260 mg of lactose for direct hitting of excipient and 35 m of Avicel (microcrystalline cellulose)
g, disintegration aid sodium starch glycolate (glyconate) 15 m
g, L-HPC (Low-hydroxypropylc) for direct hitting which is a binder
80 mg of ellulose) was stirred and put in a U-type mixer and mixed for 20 minutes. After mixing, add 10 mg of magnesium stearate as a lubricant and add 3
Mix for minutes. After quantitative test and moisture resistance test, tableting and film coating,
Tablets were prepared containing 250 mg of the mixed extract per tablet.

Composition Example 2: Syrup A suitable amount of sucrose is dissolved in a fixed amount of water, and 80 mg of paraoxymethylbenzoate and 16 mg of para-oxypropylbenzoate are added thereto as a preservative,
4.5 g of the freeze-dried powdered mixed extract of the present invention prepared in Example 1 was added and completely dissolved while maintaining the temperature at 60 ° C. Then, the mixture was cooled, and distilled water was added to give 15
A syrup was prepared by making up to 0 ml.

Composition Example 3: Capsule 500 mg of the freeze-dried powdered mixed extract of the present invention prepared in Example 1
Was filled in a hard gelatin capsule to produce a capsule.

Composition Example 4: Beverage 500 mg of the freeze-dried powdered mixed extract of the present invention prepared in Example 1
Is dissolved in an appropriate amount of water, vitamin C as an auxiliary component, citric acid as a flavoring agent, sodium citrate, and high fructose are added in appropriate amounts, and an appropriate amount of sodium benzoate is added as a preservative, and then water is added, The whole amount was made up to 100 ml to prepare a beverage composition.

Composition Example 5: Injectable solution 200 mg of the freeze-dried powdered mixed extract of the present invention produced in Example 1
Was dissolved in 200 mg of physiological saline containing 1% polyoxyethylene hydrogenated castor oil by heating to prepare an injection preparation containing the mixed extract at a concentration of 0.1%.

Clinical Example 1: Administration of the composition of the present invention to a hepatitis B patient A capsule containing the composition of the present invention prepared in Example 1 was administered to a 38 year old male patient with chronic hepatitis B for 1 day. 2 capsules were orally administered twice per day. The result is shown in FIG. FIG. 1 shows the results of investigation of the anti-HBV antibody by the RIA (radioisotope analysis) method, showing the therapeutic effect on hepatitis B of the present invention (FIG. 1a: HB).
e antigen, Figure 1b: HBs antigen).

Clinical Example 2: Administration to Patients with Chronic Hepatitis B A 36-year-old male patient diagnosed with chronic hepatitis B is treated with two capsules of the composition of the present invention twice a day (2,000 mg / day). ) As a result of oral administration for 11 months,
It can be seen that antibodies to hepatitis B virus are generated, both HBe and HBs antigens are almost absent, and the antigen titer drops to near zero or decreases. In the case of clinical example 2 (Figs. 2a and 2b), B
The patient had chronic hepatitis B, and many antigens were detected before the treatment, but as a result of administration of the composition of the present invention, antibodies against HBe antigens were produced, HBe antigens were decreased, and HBs antigens were also decreased. After 6 months, it was observed that it had dropped to near zero.

Clinical Example 3 A patient with chronic hepatitis due to HBV (34 years old, male) was orally administered with 2 capsules (2,000 mg / day) of the composition capsule of the present invention twice a day for 8 months. The results are shown in Figures 3a and 3b. From this figure, it can be seen that the antigen level for HBV dropped to near zero.

From the results of these clinical cases, it can be seen that the composition of the present invention has an excellent effect on the treatment of hepatitis B.

Experimental Example 1: Therapeutic effect of the product of the present invention on cirrhosis In order to investigate the effect of the composition of the present invention on cirrhosis, DMN (N-nitrosodimethylamine) 40 mg / kg was intraperitoneally administered to white rats. A single injection induced cirrhosis. DMN is a substance that causes tissue changes most similar to human cirrhosis and is a model often used for cirrhosis experiments.

Twenty-four white rats were divided into four groups of six mice each, one group was a control group, and two groups were DMN4.
Only 0 mg / kg was administered, DMN 40 mg / kg was administered to 3 groups, and the composition of the present invention was further administered at a dose of 150 mg / kg over 2 weeks.
N40 mg / kg was administered, and the product of the present invention was further administered at a dose of 500 mg / kg for 2 weeks. Saline was administered to the control group and the group to which only DMN was administered for the same period.

After 2 weeks, all animals were sacrificed to collect blood, and the liver was removed and fixed in 4% formalin solution. From blood, indexes such as ALT, AST, bilirubin, and albumin are investigated, and in liver tissue, in order to observe the morphology of the tissue according to the progressing process of lesions, hyalline degeneration, fibrin, etc. AZAN stain that stains pathological products such as

In order to perform AZAN (azocarmine) dyeing, a mordant (modant: 10)
% Potassium dichromate (K2Cr2O7) aqueous solution 50 ml + 10% trichloroacetate (CCl2COOH) aqueous solution 50 ml), and then treated with an azocarmine solution at high temperature. After dyeing, the mixture was cooled, fractionated with an aniline-alcohol solution until the nuclei were clearly distinguished, and stopped with acetic acid-alcohol. Aniline Blue-
After treatment with the Orange G solution, the slides were completed by fractionating and dehydrating with 100% ethanol and encapsulating.

The results of the examination under the microscope are shown in FIG. FIG. 4-1 shows normal liver tissue in which the central vein is clearly visible. FIG. 4-2 shows that cirrhosis was induced by feeding DMN, and fibrotic liver tissue showed a blue color. In FIG. 4-3, when the composition of the present invention was administered at 150 mg / kg, the degree of liver cirrhosis was alleviated and the area stained blue was reduced. In FIG. 4-4, it can be observed that the fibrotic portion returned to a normal state when 500 mg / kg of the product of the present invention was administered. The central vein also showed a gradual recovery.

From the above results, it was clearly demonstrated that the composition of the present invention has the ability to regenerate fibrotic hepatocytes and has a therapeutic effect on liver cirrhosis.

Experimental Example 2: Comparison of immunity-enhancing effect of a composition of the present invention with a single extract of Pelium chinensis and Scutellaria baicalensis, which are constituents of the composition of the present invention (T cell-dependent antibody formation reaction in vivo) 25 Balb / c mice having a body weight of about 17 to 20 g were divided into 5 groups, 5 mice per each group, and SRBC (sheep red blood cell) 2.4 × 10 8 cells were intraperitoneally transplanted to obtain the present invention. Each of the extracts of yellow husks and Prunus chinensis, which are constituents of the composition, and the composition powder of the present invention are dissolved in water, and 3 to 100 m / kg of body weight for 3 days from the first day of transplantation.
An amount of g was administered orally (0, 1, 2 days).

A physiological saline was orally administered to the control group. After 4 days, the animals were sacrificed and the spleens were aseptically removed, the tissues were minced, spleen cells were separated using a mesh, and HBSS (Hank's balanced saline (Hank's balance) was isolated.
Cells were released using a syringe plunger in a Petri dish containing 3 ml of d salt solution).

The cell suspension was transferred to a 15 ml conical tube and left for 5 minutes, then 2 ml
The supernatant was collected and centrifuged at 1200 rpm for 10 minutes. The supernatant was discarded and the residue was resuspended in 2 ml of EBSS and diluted 30-fold with EBSS. 100 μl of this spleen cell dilution was used for plaque forming cell analysis at one time. 100 μl of the thus-obtained spleen cell dilution was washed with EBSS for SRBC (25 μl, target cells), and guinea pig complement was diluted 2-fold with EBSS.
25 μl) and agarose (0.85% in EBSS, 350
μl) and then poured into a Petri dish to solidify and left in a CO 2 incubator at 37 ° C. for about 1 hour. Then, antibody-forming cells (antibody forming ce)
lls) is the modified jani plaque analysis method (modified je)
It was measured using a lane plaque assay. The number of plaques and the number of cells were counted, and the result was shown as the number of antibody-producing cells per 10 6 cells.

As a result, as shown in Table 1, it can be seen that the number of antibody-producing cells when the composition of the present invention was administered was much higher than when each of the components was administered.

[0051]

[Table 1]

Experimental Example 3: Effect of composition of the present invention on T cell activation reaction The effect of the composition of the present invention on T cell activation reaction was measured by the mixed immune cell reaction method. Major histocompatibility gene complex (MHC: major histocom)
When immune cells are separated and mixed from two kinds of mice having mutually different antigens, T cells that recognize the antigen of the other cell proliferate.

B6C3F1 (H-2k) and BD differing in major histocompatibility complex antigens
The spleens were aseptically removed from two F1 (H-2d) mice to release spleen cells. The separated immune cells were adjusted to 2.5 × 10 6 cells / ml, and 100 μl was added to each well in a 96-well plate to make 200 μl.

The composition of the present invention was treated at a concentration of 0.01 to 100 μg / ml, and then cultured at 37 ° C. in a CO 2 incubator for 3 days. Eighteen hours before the end of the culture, 1 μCi of [3H] thymidine was added to each well. Automatic cell harvester (automa
After harvesting the cells using a tic cell harvester, [3H
] The degree of proliferation of immune cells was measured by measuring the degree of thymidine absorption.

As a result, as shown in Table 2, the proliferation of T cells increased depending on the concentration of the product of the present invention.

[0056]

[Table 2]

Experimental Example 4 Acute Toxicity Experiment 30 normal ICR mice (♀, 19 ± 1 g) were divided into 5 groups of A to E, 6 mice per group, and the first group A was administered 3 g per kg body weight. As a result, the amount is increased in an equal amount, 6 g for group B, 9 g for group C, 12 g for group D and 15 g for group E.
Behrens-Krber
Law (Reference: Keijiro Takagi, et al .: Drug Experiment, Nanzando, Japan, p131, 1960)
LD50 value by oral (po) administration of the mixed extract of the present invention was measured by. LD50 (lethal dose) is an important numerical value that serves as an index showing the acute toxicity of a drug, and indicates the safety level of the product of the present invention.

As can be seen from Table 3 below, the LD50 value could not be measured without dying even one animal from the group to which 15 g of the present invention was orally administered per kg body weight. Therefore, the LD50 value by oral administration of the mixed extract of the present invention is 15 g / k.
It is more than g body weight, which proves that the product of the present invention is very safe for living body. That is,
It can be clearly seen that the mixed extract of the present invention can be safely administered with little toxicity.

The autopsy and histopathological examination methods are as follows. At the end of the test, all surviving animals were killed by exsanguination after ether anesthesia, and the organs were excised and macroscopically examined for abnormalities in all organs. All organs autopsied for histopathological examination were fixed in 10% neutral formalin solution for 10 or more, and then dehydrated, and then paraffin-embedded machine (Fi
sher, Histomatic Tissue Processor, 166
After embedding with A), a rotary microtome (AO Rotary Micro)
a 5 μl section with hematoxylin (Hematoxylin)
And was stained with Eosin and observed.

The histopathological findings of all animals in each group dissected and examined microscopically are as follows. When the composition of the present invention was administered up to 15 g per 1 kg of mouse body weight, no abnormal findings due to administration of the drug in the liver tissue were observed. In addition, no abnormal findings due to drug administration were observed in the kidney and cardiomyocytes, gastrointestinal tract, pancreas, lung, spleen, adrenal gland, brain, testis, ovary, bone marrow, etc., and the maximum dose at which the composition according to the present invention can be administered to mice It is considered that the drug is a safe drug which has no side effect due to acute toxicity on all organs even when administered at a dose of 15 g / kg body weight, and does not induce any toxicity.

[0061]

[Table 3]

Experimental Example 5. Characteristic of the Extract of the Present Invention by UV Absorbance The mixed extract of Pleurotus spp.
Rophotometer; Pharmacia, Ultrospec 200
0) and HPLC (HP1090), the results of the investigation are as follows. FIG. 5 shows the results of UV absorbance search for P. chinensis extract (0.5 mg / ml), showing the results of increasing absorbance at 282 nm. FIG. 6 shows the yellow perilla extract (0.2
5 mg / ml) was measured by UV absorbance.
Absorbance increases at nm and 325 nm. FIG. 7 is a UV spectrum of the mixed extract (0.5 mg / ml) of Pleurotus cornucopiae of the present invention (0.5 mg / ml).
The result showed that the absorbance increased at 16 nm.

Experimental Example 6. Characterization of the Composition by HPLC (High Performance Liquid Chromatography) Analysis In order to separate the mixed extract of Pleurotus cornucopiae and P. albicans of the present invention and each extract of Pleurotus cornucopiae and P. alba It was dissolved in water at a concentration of 10 mg / ml. Take 10 μl of the dissolved extract solution and perform HPLC (HP1090)
Was analyzed using.

(Analysis conditions) Column: Luna 5u C18; 4.6 mm i. d. × 250mmL (P
mobile phase: 2% acetic acid and methanol gradient system Flow rate: 1 ml / min Detection: photodioid array detector
measured at 254 nm

Characteristic peaks in each extract were observed in repeated experiments. FIG. 8 is a HPLC profile of O. chinensis extract, which is 10.245 min, 12.772 min, 14.104 min, 17 under the above analytical conditions.
． Characteristic peaks were observed at 087 minutes and 18.769 minutes. Among them, 10.
The peak shown at 952 minutes was 3,4-dihydroxybenzoic acid, and the content thereof was 0.
The peak at 022% at 17.087 minutes was chlorogenic acid, and its content was 0.238%. The peak detected at 18.769 minutes was caffeic acid, and its content was 0.107%. As a result of analyzing the composition of the present invention by HPLC under the same conditions, as shown in FIG. 9, the above-mentioned three kinds of substances from O. chinensis each contained 0.020 of 3,4-dihydroxybenzoic acid. %, Chlorogenic acid 0.270%, caffeic acid 0.044% is contained, and in addition,
Characteristic peaks appeared at 17.299 minutes, 22.048 minutes, and 22.449 minutes.

The following conditions were used to analyze the alkaloids berberine and palmatine, which were contained in large amounts in the yellow-teal extract.

(Analysis conditions) Column: Luna 5u C18; 4.6 mm i. d. × 250mmL (P
mobile phase: acetonitrile: water: KH2PO4: SDS = 500: 500: 3.4
1.7 Flow rate: 1 ml / min Detection: Measured at 254 nm with a photogeoid array detector

As a result of the HPLC analysis, as shown in FIG. 10, palmatine was detected at 10.629 minutes in the yellow-belt peel extract, the content of which was 0.988% and berberine was 11.
Although it was detected at 314 minutes and its content was 1.963%, in the composition of the present invention, palmatine was detected at 11.751 minutes, its content was 0.229%, and berberine was 12.650 minutes. The content was 0.405%, and the yield tended to decrease when mixed and extracted (FIG. 11).

[0069]

[Industrial availability]

The composition according to the present invention has the effects of suppressing hepatitis B virus and regenerating hepatocytes that have become fibrotic due to cirrhosis. In particular, since it has no side effects and does not induce drug resistance, it can be administered for a long period of time, and the activity of the composition is maintained even after administration for a long period of time. Therefore, it is useful for treating hepatitis B and cirrhosis.

[Brief description of drawings]

Figure 1a   FIG. 1a is a graph showing the therapeutic effect of clinical example 1 (HBe antigen).

Figure 1b   FIG. 1b is a graph showing the therapeutic effect of clinical example 1 (HBs antigen).

Figure 2a   FIG. 2a is a graph showing the therapeutic effect of clinical example 2 (HBe antigen).

Figure 2b   FIG. 2b is a graph showing the therapeutic effect of clinical example 2 (HBs antigen).

FIG. 3a   FIG. 3a is a graph showing the therapeutic effect of clinical example 3 (HBe antigen).

FIG. 3b   FIG. 3b is a graph showing the therapeutic effect of clinical example 3 (HBs antigen).

4a to 4d are a control group, a DMN-administered group, DMN + the composition 150 of the present invention.
3 is a photograph showing the therapeutic effect on liver cirrhosis for the mg / kg administration group and DMN + 500 mg / kg of the present invention administration group.

[Figure 5]   FIG. 5 is a UV search chart of the Scutellaria barbata extract.

[Figure 6]   FIG. 6 is a UV search diagram of an extract of yellow scabbard.

[Figure 7]   FIG. 7 is a UV search diagram of the mixed extract of Pleurotus cornucopiae and P. vellum of the present invention.

[Figure 8]   FIG. 8 is a HPLC analysis chart of the extract of Prunus chinensis.

FIG. 9 is an HPLC analysis chart of a mixed extract of Prunus chinensis and T. chinensis according to the present invention.

[Figure 10]   FIG. 10 is a HPLC analysis diagram of the yellow-scallop extract.

FIG. 11 is an HPLC analysis chart of a mixed extract of Prunus chinensis and T. chinensis according to the present invention.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 1/16 A61P 1/16 29/00 29/00 31/12 31/12 (81) Designated country EP ( AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE, TR), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW) , EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA , CH CN, CO, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE , KG, KP, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW F terms (reference) 4C076 AA22 AA37 AA54 BB01 BB11 CC09 DD67L EE31H EE42H GG14 4C088 AB23 AB62 BA07 BA08 MA07 MA17 MA23 MA35 MA37 MA52 MA66 NA14 ZA75 ZB11 ZB33

Claims (6)

[Claims] 1. A composition for treating hepatitis B, which comprises a therapeutically effective amount of a mixed extract of yellow scallions and Oenashishi. 2. A composition for treating cirrhosis, which comprises a therapeutically effective amount of a mixed extract of yellow scallions and Prunus chinensis. 3. The mixed extract according to claim 1, wherein the mixed extract is dried and mixed with the yellow algal bark and P. terrestris at a weight ratio of 1: 0.2 to 1: 5. Composition. 4. The mixed extract according to claim 1, wherein the mixed extract is an extract extracted from Perilla frutescens and an extract extracted from Prunus chinensis in a weight ratio of 1: 0.2 to 1: 5. Compositions mixed in a weight ratio. 5. The composition according to claim 1 or 2, wherein the mixed extract of Pelium japonicum and P. chinensis is administered in an amount of 10 to 50 mg per kg of adult body weight. 6. The tablet according to claim 1 or 2, wherein the tablet, capsule, solution,
A composition formulated by adding a pharmaceutically acceptable excipient in the form of a suspension, syrup or edible beverage.