KR0149986B1 - Anti-hbv agent containing phyllanthus - Google Patents

Abstract

The present invention relates to a hepatitis B therapeutic composition comprising an extract of Pillanthus urinaria (Phyllanthus urinaria) or Pilanthus ussuriensis (Phyllanthus ussuriensis), obtained by extracting them with an aqueous solvent The extract has the ability to bind to the hepatitis B surface antigen, and not only inhibits HBV-DNA polymerase activity, but also removes HBV in serum, and thus the pharmaceutical composition containing it as an active ingredient is very useful as a therapeutic agent for hepatitis B. Can be used.

Description

Hepatitis B Therapeutics Containing Extracts of Phyllanthus Species

FIG. 1 is a graph showing the degree of inhibition of HBV DNA polymerase activity according to the concentration of Phyllanthus ussuriensis and Phyllanthus urinaria extract.

FIG. 2 shows electron micrographs of HBV (magnification: 30,000 times), and FIGS. 2a and 2b show HBV states before and after administration of 0.1% aqueous solution of P. urinaria extract, respectively.

The present invention relates to a hepatitis B treatment agent comprising Korean Pilandus ussuriensis or Phyllanthus urinaria, and more particularly, to P. Usientiensis, which is a weed native to Sanya in Korea and P. Oura aqueous extract is an effective drug for the treatment of hepatitis B and to use it as a treatment for hepatitis B.

Hepatitis B virus (hereinafter abbreviated as HBV) is a virus of the Hepadnaviridae strain that is specifically infected with the human body. The incubation period is about 60 to 110 days and 90 to 95% Complete recovery from infection In patients who have not recovered from infection, HBV DNA is assimilated into genomic DNA of human hepatocytes, leading to chronic active hepatitis, cirrhosis, liver cancer, and the like. Chronic hepatitis caused by HBV is a disease with a high mortality rate, leading to chronic viral infections, lymphomas and chronic kidney disorders, and eventually death.

Humans have begun to become infected with HBV. It is estimated to be around 2000, and there is already a description of jaundice in BC. The hepatitis B virus was discovered by Bloomberg (B.S.) in 1964, and it is also known that hepatitis B can cause liver cirrhosis and liver cancer. In Korea, Chung (W.K.) has studied the pathology and clinical aspects of hepatitis B viral hepatitis, and especially found a viral hepatitis that does not express jaundice.

Currently, about 200 million people worldwide are carriers of HBV, and 80% of them are concentrated in the Asia-Pacific region. In Korea, hepatitis B virus surface antigen (HBsAg) is reported in 7% of the population. (Lee Se-hoon et al., A Study on the Relationship between the Positive Rate of HBsAg and HBs and Liver Function Tests in Patients with Comprehensive Health Examination, Journal of the Korean Society of Immunology, 7: 265-273, 1965)

Despite the development of HBV vaccines since 1981 and immunizations for those who may be exposed to newborns and hepatitis, the number of carriers has not been significantly reduced to date. This is due to the vertical reduction of HBV (infection from the mother to the fetus). It is the main cause of this carrier (Chung, DK et al., Vertical transmission of hepatitis B antigen, J. Catholic Med. Coll., 27: 256-267, 1976; Chung, WK et al., Preventin of perinatal transmission of hepatitis B virus: A comparison between the efficacy of passive and passive-active immunization in Korea, J. Infec. Dis., 151: 280-286, 1985). In the case of children born to HBeAg-positive persons, especially those who are born to HBeAg-positive, although infection through the placenta is not common, they may be immune to later HBV if exposed to HBV during birth or placenta damage or sensitized to HBeAg through placenta. It can cause defects and, as a result, it is more likely that newborns will become chronic carriers. Therefore, women who are already carriers of hepatitis B should be cured before their age of 20 at the latest, or at least HBeAg lost in their blood.

Recently, PCR (Polymerase Chain Reaction) technology has been introduced into this field, and it is now possible to measure HBV DNA in carriers' blood, so the time when HBeAg was the only indicator of hepatitis B has passed. In order to reduce the absolute number of hepatitis B carriers and to prevent the occurrence of hepatitis, cirrhosis and liver cancer, not only in Korea but also in Asia-Pacific, newborns are vaccinated and HBV is more effective than HBeAg in blood. There is a demand for developing a method for significantly reducing the amount of DNA itself.

Although several drugs have been developed as a treatment for hepatitis B, few have satisfactory efficacy, and the fact that currently used drugs such as Ara-A and Acyclovir are also highly toxic. It is becoming. Phosphonoformate (Foscarnet), Suramin, Prostaglandin, carbocyclic analogue of 2-deoxyguanosine (FCD), and Famciclovir (Famciclovir) are known to be promising. Much verification is required. To date, very few drugs have been used in humans to improve HBsAg, HBeAg, and HBV-DNA, which are indicators of hepatitis B without side effects, and, moreover, drugs with fewer side effects that can be used by mothers or women of childbearing age. It's hard to see.

Benkateswaran et al. (1987), Bloomberg et al. (1988), and Yanagi (1989) used the Phyllanthus plant (Euphorbiaceae), which was used early in India, to treat hepatitis B. Was intended. In 1991, Unander, DW et al., Journal of Ethnopharmacology, 34 (1991) 97-133) summarized data from around the world on the use and biochemical analysis of Pilandus plants. Among the 550 species of Pilandus, P. niruri, P. airy-shawii (amarus (?)), P. amarus, P. fraternus webster, P. urinaria, P. gasstroemi Muell- It is composed of seven species including Arg and P. thymoides. P. amarus and P. niruri are described in Venkateswaran, PS et al., Effect of an extract from Phyllanthus niuri on hepatitis B and woodchuck hepatitis virus: in vitro and in vivo studies, Proc. Natl. Acad. Sci., 84: 274, 1987) and hepatitis B virus in ducks (Nis, J., et al., Effect of Phyllanthus amarus on duck hepatitis B virus replification in vitro, J. of Med. Virol., 32: 212 -218, 1990), indicating its effectiveness but failing in actual clinical use (Leelarasamee, A., et al., Failure of Phyllanthus amarus to eradicate hepatitis B surface antigen from symptomless carriers, Lancet, 1: 335, 1990). In addition, P. thymoides had the most effective binding ability with HBsAg in vitro, but it was due to the specific lectin binding to HBsAg. (Shead, A. et al., Neutralization but not cure of duck hepatitis B by Australian Phyllanthus extracts.Abstract 602 In: Scientific Program and Abstracts volume, the 1990 International Symposium on Viral Hepatitis and Liver Disease, April 4-8, 1990 , Houston, Texas). In view of the above, some examples of P. amarus, P. nururi, etc. among the plants of Pilandus have been used as folk remedies for jaundice or hepatitis, and they have suggested the effectiveness of hepatitis B in vitro. Indeed, when applied to humans, the effectiveness of hepatitis B is not found.

As mentioned above, the inventors of the present invention, while developing a hepatitis B therapeutic agent that can be safely used without side effects even for mothers or childbearing women and children, has been focused on plants of the genus Pilandus as a natural drug with low toxicity. Two kinds of Pilandus, Phyllanthus urinaria (Phyllanthus urinaria) and Phyllanthus ussuriensis (Physanthus spp.), Which are native to Korean wild fields, are found in Gyeongsangbuk-do and Jeju Island, respectively. As a result of the extraction and research, it was found that these aqueous extracts had a clinically remarkable effect on hepatitis B in humans and led to the present invention.

That is, an object of the present invention is to find out that the aqueous extract of Phyllanthus urinaria or Phyllanthus ussuriensis is clinically effective against hepatitis B in humans, and hepatitis B comprising the same It is to provide a pharmaceutical composition useful for treatment.

In order to achieve the above object, the present invention provides a hepatitis B therapeutic composition comprising an extract of Pillanthus urinaria (Phyllanthus urinaria) or Pilandus ussuriensis (Phyllanthus ussuriensis).

Hereinafter, the present invention will be described in more detail.

[P. Collection of Ussuriensis and P. Koreana]

P. urinaria and P. ursuliansis used in the present invention are annual herb belonging to Euphorbiaceae, P. urinaria is from Okpo-myeon and Yuseong-myeon, Oksan-myeon, Dalseong-gun, Gyeongsangbuk-do, and P. Each of them was collected from the wild, and their seeds were obtained and cultivated in Osan-ri, Jicheon-myeon, Chilgok-gun, Gyeongbuk. Their botanical properties and scanning electron microscopic comparison are shown in Table 1 below.

As shown in Table 1, P. urinaria has a relatively large amount of red pigment and is characterized by rhabdom in seeds, and P. amirus from the United States is a plant close to P. Ussuriensis, with a few rows of seeds. see.

[P. Preparation of Ussuriensis and P. Koreanis Extract]

Each plant is washed with water, air-dried at a constant temperature, and then chopped. Then, about 50 times of water is added and heat-extracted at about 60 to 90 ° C. for about 3 to 5 hours. The extract is centrifuged, filtered and lyophilized to yield an extract powder (yield 8-9%), used at 4 ° C.

[P. Effects of Ussuriensis and P. urianna Extracts]

In the aqueous extracts of P. urinaria and P. ursuliansis obtained above, a substance binding to HBsAg is present, showing the ability to bind HBsAg in serum and hepatitis B vaccine in vitro. Due to this binding capacity, these plant extracts inhibit the binding of HBsAg and HBsAb, and each has an antagonistic inhibitory effect at a concentration of 1.25mg / ml.

In addition, P. Usualiensis and P. urinaria extracts inhibit HBV DNA polymerase activity in vitro, which inhibits HBV DNA polymerase activity in proportion to concentration at about 34.1 μg / ml or more. In particular, P. urinaria extract causes severe degeneration of the virus at 0.1% concentration.

Despite the above strong effects, P. Ussienriensis and P. urinaria extracts show no toxicity or mutagenicity in toxicity and mutagenicity tests in mice and dogs. In other words, P. urinaria extract was not toxic even when administered intraperitoneally to mice at an amount of 70 mg / kg, which is 3,500 mg intraperitoneally administered to a person weighing 50 kg. In addition, the oral administration of these plant extracts to normal mice does not alter the concentrations of SGOT, SGPT, ALP and bilirubin, suggesting that P. usuriensis and P. urinaria extracts do not affect liver function. . In the Ames test using Salmonella typhimurium TA100 and TA98, P. Usualiensis and P. urianna extracts were negative as in the control group, indicating that they are not mutagenic.

In addition, P. Usorientis and P. urianna extracts showed no abnormalities in liver function test even when administered to dogs for 120 days. HBsAb-positive reactions were observed when P. urirea was administered. It can be inferred that the substance is formed in serum.

On the other hand, woodchuck hepatitis virus (WHV), a virus of the same type as human HBV, is found in North American Woodchurch (Marmota monax), and the infection of WHV causes human-like chronic hepatitis. Experimentally reproduced (Summers, J. Smolec, JM and Snyder.R., A virus similar to human hepatitis B virus associated with hepatitis and hepatoma in woodchuck, Proc. Natl. Acad. Sci. USA, 75: 4533-4537, 1978) Woodchurch was used as an experimental animal, and the WHV DNA concentration of Woodchurch was significantly decreased by the administration of P. urianna extract.

In several clinical trials in humans, P. urinaria and P. superiorriensis extracts showed remarkable effects. After 3 months of P. urinary tract extract administration to healthy HBV carriers, HBV-DNA in the serum became negative and HBsAb in the serum became positive (ELISA method). Even in cases where mild chronic hepatitis was suspected as a carrier of HBV, HBsAb was positive after 3 months of P. urinary administration. However, HBsAb was negative when radioisotope was used, probably due to the presence of substances in the blood that specifically bind to HBsAg when absorbed in P. urinaria extract. In the case of liver cirrhosis combined with chronic active hepatitis due to HBV infection, HBV-DNA became negative only 3 months after P. urinary administration, and the activity of GOT and GPT in serum was significantly improved.

Based on the various test results, it can be seen that the P. uranusiensis and P. urinary aqueous extract can be used as an effective hepatitis B treatment with little toxicity or side effects.

The hepatitis B therapeutic composition of the present invention contains an aqueous extract of P. ursihensis or P. urinia as an active ingredient, and is also pharmaceutically acceptable such as a carrier, diluent, filler, anti-agglomerating agent, lubricant, flavoring agent, and the like. Ordinary excipients can be added to prepare pharmaceutical preparations such as oral, subcutaneous, and intravenous in accordance with known methods. The hepatitis B therapeutic agent may be administered orally or parenterally, oral dosage may be used as an aqueous extract in an amount of 100 to 1,000 mg, preferably 200 to 600 mg per kg body weight per day. It can be adjusted according to various conditions such as patient's symptoms, age, sex, and weight.

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

The following examples are given for illustrative purposes only and are not intended to limit the scope of the invention.

Production Example

[P. Preparation and Storage of Ussuriensis and P. Koreanis Extract]

Each plant was washed with water, air-dried at room temperature, and chopped, and then, 1000 ml of distilled water was added per 20 g of the plant, and extracted at 80 ° C. for 4 hours. The extract was centrifuged at 3000 rpm and Whatman No. After filtration with 2 filter paper, it was filtered again with 0.45 탆 Seitz filter paper. The filtrate was lyophilized to obtain 1.6 g (8% aqueous) of extract powder, which was used for the next experiment while storing at 4 ° C.

The extract powder has high water solubility (20% or more), and when orally administered to humans, the amount of 200mg per kg of body weight per day was dissolved in 50 to 100ml of water divided by 2 to 3 times, and in the case of animals, normal human administration About three times the amount was used by dissolving in an appropriate amount of water according to the type, size and weight of the animal.

[Effective test 1- in vitro]

[Sedimentation of HBsAg-positive Serum, P. Ussienhensis, and P. urianna Extracts]

0.5 ml of HBsAg positive serum was dispensed into 5 ml test tubes, and a 2% solution of the extract was gradually layered thereon to observe the formation of sedimentation rings.

As a result, the extracts of both plants reacted with HBsAg positive serum to form white sedimentation rings (positive sedimentation reaction).

[Effective test 2-in virto]

[Test of binding capacity of HBsAg and P. Ussienhensis and P. urianna extract]

The binding capacity of the extract and HBsAg was determined by measuring the extent to which the extract inhibits the binding of HBsAg and HBsAb, and the HBsAg titer was measured as follows using an enzyme immunoassay device (ELISA kit; Behring).

Into each hole of a 96-hole microplate coated with an antibody against HBsAg, 100 μl of the sample was reacted at 40 ° C. for 1.5 hours, and then, each ball was washed three times with a washing solution, and HRP (Horse Radish Peroxidase) conjugated to HSsAg. 100 μl of the antibody was added and reacted again at 40 ° C. for 1 hour, and washed four times. Then, color was developed for 30 minutes at the place where o-phenylenediamine dihydrochloride solution was added, followed by adding 100 μl of 0.5 N sulfuric acid solution at 492 nm. Absorbance was measured to quantify HBsAg.

(1) Test of binding capacity using HBsAg positive patient serum

The extract was dissolved in distilled water, and 400 μl of HBsAg positive serum was mixed with 400 μl of solution prepared at final concentrations of 10, 5, 2.5, 1.25 and 0.63 mg / ml, and reacted at 20 ° C. for 1 hour and centrifuged at 3000 rpm for 15 minutes. After separation, the titer of HBsAg in the upper layer was measured to evaluate the degree of reduction of HBsAg. Table 2 shows the extent to which P. Usorriensis and P. urinaria extract inhibit the binding of HBsAg and HBsAb in serum.

(2) Test of binding capacity using HBsAg in hepatitis B vaccine

The extract was dissolved in distilled water and mixed with 400 μl of the final concentrations of 0.63, 0.31, and 0.16 mg / ml, respectively, and 400 μl of HBsAg solution diluted 50-fold with Hepavax (20 μg) of Green Cross. After reacting for a period of time, the titer of HBsAg was measured to investigate the decrease in HBsAg. Table 3 shows the extent to which the P. Usualiensis and P. urianna extracts inhibit the binding of HBsAg and HBsAb in the hepatitis B vaccine.

(3) Antagonistic binding of anti-HBs antibodies to HBsAg

To determine whether the anti-HBs antibody against HBsAg and the extract of the plant were antagonisticly bound, 400 μl of anti-HBs antibody-positive serum and the extract were dissolved in distilled water, respectively, to final concentrations of 10, 5, 2.5 and 1.25 mg. After mixing 400 μl of the solution made of / ml, the titer of the anti-HBs antibody in the mixed solution was measured. Titers of anti-HBs antibodies were quantified using an HBsAg coated anti-HBs antibody test kit, and the method is the same as the HBsAg titer measurement method. The antagonistic inhibition of the binding reaction of HBsAg and HBsAb in serum by P. Ussaliens and P. urianna extract is shown in Table 4 below.

As shown in Table 4, each plant showed an antagonistic effect even at a concentration of 1.25 mg / ml.

[Efficacy test 3- in vitro]

[P. Determination of HBV DNA Polymerase Activity of Usurianiensis and P. Koreanis Extracts]

The extract was dissolved in distilled water to final concentrations of 340.6, 68.1, 34.1 and 6.8 ㎍ / ml and reacted with HBV particles (Dane particles) to measure the inhibitory activity of DNA polymerase activity.

0.2 ml of serum containing HBV particles was mixed with 2.3 ml of 0.01 M tris-hydrochloric acid (pH 7.4), 0.15 M NaCl solution and centrifuged at 10,000 rpm for 10 minutes at 4 ° C. The upper layer was layered in 2.5 ml of 30% (w / v) sucrose, 0.01 M tris-hydrochloric acid (pH 7.4), 0.15 M NaCl, 0.001 M EDTA, 0.1% 2-ME, 1 mg BSA solution and 50,000 rpm at 4 ° C. Centrifuged for 4 hours. After removing the upper layer, the pellet was dissolved in 25 µl of 0.01 M tris-hydrochloric acid (pH 7.4), 0.15 M NaCl, 1% NP-40, 0.1% 2-mercaptoethanol solution, and 25 µl of the extract dilution solution and polymerase. Mixed solution [0.2 M Tris-hydrochloric acid (pH 7.4), 0.08 M MgCl, 0.24 M NHCl, 1 mM dATP, 1 mM dTTP, 0.0025 nM H-dCTP, 0.0025 nM 25 μl H-dGTP (both inactive 21 Ci / mM) was added and mixed. The mixed solution was reacted at 37 ° C. for 3 hours, and then dropped onto Whatman 3 mm filter paper. Filter paper was washed with 15% trichloroacetic acid and 90% ethanol, dried in air, and radioactivity was measured to evaluate the degree of inhibition of DNA polymerase activity.

1 is a graph showing the degree of inhibition of HBV DNA polymerase activity according to the concentrations of P. Ussurianissis and P. urianna extract. As shown here, the plant extract was found to inhibit HBV DNA polymerase activity in proportion to concentration at about 34.1 μl / ml or more.

[Efficacy test 4-in vitro]

[P. Electron Microscopic Observation of HBV-induced Activity of Ouraceae Extracts]

The HBV particles were obtained by layering the serum of HBsAg and HBeAg positive patients on a 20% sucrose cushion and ultrafast centrifugation (Sorvall) at 4 ° C. and 100,000 g for 4 hours, and then pelleting the first with phosphate buffer (PBS). Suspended to 1/20 of the amount. 2% aqueous solution of P. quinaria extract was diluted 10-fold and 20-fold with distilled water (0.2 and 0.1% solution)

Determination of the inactivation of P. urinia extract against HBV was performed by distilled water as a control and mixing the diluted P. urinia 0.2 and 0.1% solution with the HBV particle solution in the same amount and 30 minutes at room temperature. After being left for a while, morphological changes of HBV particles were observed by electron microscopy.

Electron microscopic observation showed that a drop of the prepared sample solution was dropped on the grid, negatively stained with 1% phosphotungstic acid, washed twice with PBS, and the excess moisture remaining on the grid was measured in Wattman No. After one complete removal, the result was observed by an electron microscope (Zeiss) and photographed.

2a and 2b is a result of observing the HBV state before and after administration of 0.1% aqueous solution of P. urianna extract by electron microscopy. As shown here, the P. urinaria extract was found to cause severe degeneration of the virus at 0.1% concentration.

[Toxicity Test 1- in vivo]

[P. Acute Toxicity Test of Mice of Ouraria Extracts]

Mice were administered intraperitoneally at doses of 70, 84, 100, 120 and 144 mg / kg of P. urinaria extract, respectively, for a total of five groups. Observations were made until 7 days after administration, and dissection was performed to observe abnormalities of internal organs. The results are shown in Table 5.

In Table 5, it can be seen that P. urinaria extract is not toxic even when administered intraperitoneally in an amount of 70 mg / kg, which is a projection of 3,500 mg intraperitoneally to a person weighing 50 kg.

A = (case) / (number of dead rats)

B = (case) / (number of surviving rats)

[Toxicity test 2- in vivo]

[P. Effects of Ussuriensis and P. urianna Extracts on Liver Function in Normal Rats]

Three rats of about 200 g were orally administered with a 2% solution of the extract instead of drinking water for 2 days, and blood was collected to change the serum GOT (glutamic oxaloacetic transaminase), GP T (glutamic pyruvic transaminase), alkaline phosphatase and bilirubin level. The measurement results are shown in Table 6 below.

(1) Serum GOT Activity Measurement

GOT activity in serum was determined by Reitman, S. and Frankel, AS, A colorimetric method for the determination of serum glutamic oxaloacetic and glutamic pyruvic transaminases, Am. J. Clin, Pathol., 28: 56, 1957 ), The substrate solution 1-asparagine and α-ketoglutaric acid was allowed to stand at 37 ℃ for 5 minutes, and then the standard solution and test serum 0.12ml each was added and reacted at 37 ℃ 60 minutes, again 2,4- 1 ml of dinitrophenyl hydrazine solution was added and reacted at room temperature for 20 minutes. 10 ml of 0.4 N NaOH, a color developing reagent, was mixed well and left at room temperature for 10 minutes, followed by spectrophotometer (Gilford 2600) at 505 nm. Absorbance was measured and expressed in units of Karmen units / ml.

(2) measurement of serum GPT activity

Serum GPT activity was measured by the method of lateman-Frankel, and left substrate substrate dl-alanine and α-ketoglutaric acid at 37 ° C. for 5 minutes, and then 0.2 ml of standard solution and test serum were added at 30 ° C. at 37 ° C. After reacting for 1 minute, add 1 ml of 2,4-dinitrophenyl hydrazine solution, and react for 20 minutes at room temperature. Add 10 ml of 0.4 N NaOH, a coloring reagent, mix well, and leave at room temperature for 10 minutes. Absorbance at 505 nm was measured in meters (Gilford 2600) and expressed in units of Karmen units / ml.

(3) measurement of serum alkaline phosphatase activity

Alkaline phosphatase activity in serum was determined by alkaline buffer solution according to King-Armstrong's method (Kind, PRN and King, DJ, Estimation of plasma phosphatase by determination of hydrolysed phenol with antipyrine, J. Clin. Pathol., 7: 322, 1954). The substrate solution (2-sodium phenyl phosphate) contained in the solution was allowed to stand at 37 ° C. for 5 minutes, and 50 µl of standard solution and test serum were added thereto, followed by reaction at 37 ° C. for 15 minutes. After standing, absorbance was measured at 500 nm and expressed in units of King-Armstrong unit / ml.

(4) Measurement of Serum Bilirubin

Determination of serum bilirubin was carried out by the methods of Malloy and Evelyn (Malloy, HT and Evelyn, KA, The determination of bilirubin with photoelectric colorimeter, J. Biol. Chem., 119: 481, 1937). Was used. First, the diazerine reagent is added to the test serum and the standard solution to convert the indirect bilirubin into a form that can react with the diazo reagent, and the diazo reagent is added to the bilirubin to be azo-bilirubin, and then an alkaline azo produced by adding a ferring reagent. -Bilirubin was measured in absorbance at 600 nm and expressed in mg / ml.

As shown in Table 6, these plant extracts did not alter the concentrations of SGOT, SGPT, ALP and bilirubin, which are indicators of liver function, and thus did not affect the liver function of normal rats.

[Toxicity test 3- in vivo]

[P. Mutagenicity of Ussrianiensis and P. urianna Extracts (Ames Experiment)]

A., BN, Kammen, HO and Yamasaki, E., Hair dyes are mutagenic: Identification of a variety of mutagenic ingredients, Proc.Not.Acad Sci. (USA), 72: 2433-2427, 1975) and Nagao, M., and Suginura, T., Environmental mutagens and carcinogens, Ann. Rev. Genet., 12: 117-159, 1978). Salmonella typhimurium TA100 and TA98 are histidine demand strains originally developed in laboratories such as Ames, and are used for mutagenicity testing as a species that changes to histidine non-requirers in contact with environmental mutagenic or carcinogenic substances. do.

The extract powder was made into 2000 mg / ml aqueous solution, and 5, 10, 50 and 100 μl each were mixed with DMSO and used as 100 μl for testing. S9 fraction (10 ml of 0.15 M KCl solution in liver of about 100 g of rats) was used for the activation of mutagenic to carcinogens again by homogenizing the test tubes in which these extract solutions were dispensed and cut and homogenized (Potter-Elvejhem). Type homogenizer) Microsomal fraction obtained by centrifugation at 9000xg for 10 minutes, the protein amount of the fraction used in the experiment was adjusted to 4mg%) and the group without the S9 fraction, 0.5ml in the S9 series Add 50 μmol phosphate buffer (pH 7.4) and add 2 μM NADH, 2 μmol NADPH, 2.5 μmol glucose-6-phosphate, 0.25 unit glucose-6-phosphate dehydrogenase, 4 μM MgCl and 16.5 μM KCl This containing 0.5 ml of phosphate buffer (pH 7.4) was added to prepare about 1 ml of the fungi.

About 2 × 10 TA100 or TA98 strains for each test tube Cells were added (0.1 ml of culture) and left at 37 ° C. for 20 minutes, then mixed well with 45 ml of 2 ml agar (containing 0.7% divalent and 0.6% NaCl) solution and quickly applied thinly on a minimum of aluminose 37 ° C. Incubation for 2 days at was observed the occurrence of histidine non-recommended.

For comparative experiments, 2- (2-furyl) -3- (5-nitro-2-furyl) acrylamide (hereinafter referred to as AF-2), which has been previously defined as a carcinogen, was used at a concentration of 1 µg per 100 µl of DMSO. Each experiment was carried out twice and the average value was determined and the results are shown in Table 7 below. Mutation-positive determinations were made when two conditions were found that colonies were found to be more than twice as high as colonies cultured without extract, and that the number of colonies should also increase according to the amount of mutagen added.

As shown in Table 7, P. Usualiensis and P. urianna extract is negative in the Ames test, as in the control, it can be seen that there is no mutagenicity.

[Toxicity Test 4- in vivo]

[Toxicity Test of P. Ussienhensis and P. Koreani Extract Using Dogs]

Four Korean hybrid dogs (two males and two females; weight average 12 kg) were purchased and divided into two groups, one male and female, and orally administered a solution extracted from 600 ml of distilled water of 12 g of P. urgenthensis and P. urinaria, respectively. It was. After administration for 120 days, blood was collected and liver function test findings were measured (Dupont's Dimension 760), and the average of two animals is shown in Table 8 below.

On the other hand, the administration of about 200ml of the extract 30 minutes, 60 minutes and 120 minutes after the blood was collected and tested for the presence of HBsAb with the HBsAB measurement kit (Abbott), the results are shown in Table 9 below.

As shown in Table 8, it can be seen that the P. Usualiensis and P. urianna extract does not show abnormalities in liver function test even when administered to the dog for a long period of 120 days.

In Table 9, P. urinary administration shows an HBsAb positive reaction, even when P. urinarysis is administered to P. urinaryis, this reaction occurs even when P. urinaria is administered. It did not appear when P. Usorriensis was administered to dogs. In other words, P. urinary administration shows that substances such as HBsAb appear in serum.

In other words, P. Usorientis and P. urianna extracts showed no abnormalities in liver function test even after long-term administration to dogs, and only P. urinaria administration showed HBsAb-like responses in blood. Could.

[Effective test-in vivo]

[Effective test of P. erynia extract using woodchuck]

Serum WHV DNA concentration (pg / ml) was administered orally to the P. urinary extract in an amount of 600 mg / kg per day for six woodchuck hepatitis virus (WHV) infected human HBV strains. Was measured and the results are shown in Table 10.

As shown in Table 10, the blood WHV DNA concentration of the animals at the start of the test was 8,200 ~ 2,900 pg / ml in the test group, 12,000 ~ 13,000 pg / ml in the control group, and the blood WHV DNA concentration just before P. urinary administration The test group was 9,100-2,200 pg / ml, and the control group was 13,000-13,000 pg / ml. P. WHV DNA concentrations in animals 12 weeks after wearyria administration were still 11,000-1,300 pg / ml in the control group, whereas 4 out of 6 animals were 220 pg / ml and 7 pg / ml, respectively. It can be seen that it significantly decreased until.

[Clinical test]

[P. Clinical Effect of Ussuriensis and P. urianna Extracts]

Liver function test results are relatively normal, HBsAg (+) HBsAb (-) HBeAg (+) HBsAb (-) in humans (HBV carrier status), liver function test findings are slightly abnormal and serum HBsAg (+) HBsAb (-) HBeAg (+) HBeAg (-) 1 human (Hepatitis B viral chronic mild), and cirrhosis with HBsAg (+) HBsAb (-) HBeAg (+) HBsAb (-) Three patients with one human (cirrhosis + hepatitis) were tested. In one example, first, 200 mg / kg of P. Ussienhensis extract powder was divided into 3 times, dissolved in 50 ml of water, and orally administered three times a day for 11 months, and then exchanged with P. urinaria extract. In the other two cases, P. urinaria extract was continuously administered.

(1) Healthy Hepatitis B Virus Carriers

Patients who did not find any abnormalities except HBsAg positive in the liver function test were administered 200 mg / kg body weight per day of P. urianna extract.

In Table 11, the specific one becomes HBsAb (+) (which is expressed negatively by using radiotherapy) after 9 months of P. urinary administration, and HBeAb is changed to + after 3 months of P. urinary administration. will be. HBV-DNA continued to be negative from 3 months after P. urinaria administration.

(2) Hepatitis B virus carrier of hepatitis B viral chronic hepatitis state mild

Hepatic function test showed HBsAg (+) HBsAb (-) HBeAg (+) HBeAb (-) anti-HBc (+) findings and suspected chronic active hepatitis. GOT was 23.9 and GPT was 63.0. In the indicated patients, the change of serologic findings was shown in the following Table 12 while P. Ussienhensis extract was administered for 11 months and then P. urinaria was administered. P. Ussienhensis extract and P. urinaria extract were both administered 200 mg / kg body weight per day.

As shown in Table 12, the condition of HBsAg (+) HBsAb (-) HBeAg (+) HBeAb (-) and HBV-DNA (+) was maintained until 1 month after P. Usorientis administration, and GOT and GPT remained borderline. In the area, HBV-DNA continued to be negative after about 1 year after P. Usaliensis administration.

Three months after P. uriana administration, HBsAb was positive, but the radioisotope was negative. This is probably due to the presence of substances in the blood that specifically bind to HBsAg when absorbed in P. urinary extract.

HBsAb was positive and HBV-DNA continued negative throughout the entire period of P. urinary administration.

(3) Hepatitis B virus carriers of chronic active hepatitis with liver cirrhosis

Liver function tests showed GOT / GPT = 129/184, HBsAg (+) HBeAg (+) anti-HBcAb (+) anti-HBsAb (-), and there was no mass and there was a slight coarse shadow on ultrasound. . The physical findings were relatively healthy, but there were arachnoid angiomas and palmar hematomas in the chest, and hepatic cirrhosis of 2 cm below the right rib margin was found. Liver biopsy revealed that cirrhosis combined with chronic active hepatitis could not be applied to interferon.

To this patient, the serum test results were shown in Table 13 below, with 200 mg / kg body weight per day of P. urinaria extract.

As shown in Table 13, HBV-DNA became negative only 3 months after P. urinary administration and the results of transaminase were very improved. However, due to cirrhosis of the liver, continuous observation is required.

As described above, it can be seen that the extracts of P. uranussis and P. urianna can be used as an effective hepatitis B treatment with little toxicity or side effects.

Claims (2)

A composition for treating hepatitis B, comprising an extract of Pillanthus urinaria (Phyllanthus urinaria) or Pilandus ussuriensis (Phyllanthus). The composition of claim 1 wherein said extract is an aqueous extract.