KR19990047905A - Hepatitis C Therapeutics with Ogapi Extract - Google Patents

Abstract

The present invention relates to a hepatitis C therapeutic agent comprising an extract of the genus Acanthopanacis cortex .

More specifically, the present invention relates to a hepatitis C therapeutic agent containing an extract obtained by boiling the bark of the roots, stems and branches of the genus Ogalpi with distilled water or cooling with an organic solvent as an active ingredient. Since it exhibits a strong inhibitory activity against hepatitis protease, it can be usefully used as a therapeutic agent for hepatitis C and can be used in various food and beverages.

Description

Hepatitis C Therapeutics with Ogapi Extract

The present invention relates to a hepatitis C therapeutic agent comprising an extract of the Acanthopanacis cortex tree, and more particularly, to extracts obtained by boiling the bark of roots, stems and branches of the genus Agalpi in distilled water or cooling them with an organic solvent. It relates to a hepatitis C treatment including.

Hepatitis C Virus (abbreviated as "HCV") is a virus that is a major cause of hepatitis known to cause non-A or non-B hepatitis. Not only does it cause hepatitis, but it is also involved in cirrhosis or transition to liver cancer (Alter, HJ et al., N. Eng. J. Med , 321 , p. 1494-1500, 1989 ). HCV has been reported to be infected with 1% of the world's population, with an estimated 1 million new cases of HCV infected each year, with 1.5-2.0% of the total population in Africa, Japan and Korea. Is known to be a chronic carrier (Purcell, FEMS Microbial. Rev, 14 , p . 181-192, 1994 ; van der Poel, Hepatitis C Virus, 1994 : HW Reesink ed., Current Studies in Hematology and Blood Transfusion, p. 137-193; Alter, HJ, AJ Zuckerman ed., Viral Hepatitis and Liver Disease , p.537-542, 1988 ).

The main feature that distinguishes hepatitis from HCV infection from hepatitis B caused by infection with Hepatitis B virus (hereinafter referred to as HBV) is the possibility of progression from hepatitis C to chronic hepatitis HBV infection. It is much higher than in the case of. That is, about 40-50% of HCV-infected patients develop hepatitis and become chronic hepatitis patients, and about 20% become more advanced and become cirrhosis or liver cancer patients (Dienstag, JL, Semin. Liver Dis, 6, p. 67-81, 1986 ).

Transfusion causes more than half of hepatitis patients infected with non-A or non-B hepatitis virus to become chronic hepatitis, and about 10-20% of patients with non-A or non-B hepatitis develop cirrhosis .

Anti-HCV antibody formation rates in patients without HBV infection and with cirrhosis or liver cancer are known to be much higher than controls (for HBV infection) (Ikeda et al., Hepatology , 1993 , vol. 18 , 47-53). According to the survey, 75% of people with HCV will develop liver cancer about 15 years after the disease. This is much higher than the 27% hepatocarcinoma progression seen with HBV infection, suggesting that HCV infection is more closely correlated with liver cancer than any carcinogen known to date.

Currently, diagnostic reagents have been developed for HCV to prevent some of the infections caused by blood transfusions, but there is still a risk of infection through routes other than blood transfusions since vaccines have not been developed. In order to know whether a patient is infected with HCV, antibodies can be detected with a diagnostic reagent only 6 months or more after the infection. Therefore, blood within 6 months of infection may be mistaken for normal blood and used for transfusion. In addition, about 10% of people with HCV have a risk that no HCV antibody can be found and thus blood from such patients may be mistaken for normal blood and used for transfusion.

Therapies for effectively treating chronic infection of HCV have not been developed yet, and the only administration of α-interferon as a therapeutic agent for HCV has been carried out. However, these therapies have recently been shown to be ineffective against HCV having an Interferon resistance sequence, requiring the development of new types of therapeutics. The new form of HCV therapy would be most effective with inhibitors of proteins such as proteases or RNA polymerases for the treatment of RNA viruses such as the AIDS virus.

HCV can be recovered by inoculating the chimpanzees with plasma from non-Hepatitis A or non-B hepatitis patients (Bradley, DW et al., Gatroenterology, 88 , p.773-779, 1988 ). The HCV thus obtained has a gene in the form of a positive strand ribonucleic acid consisting of 9400 bases, from which a polyprotein consisting of 3010-3033 amino acids is produced (Choo, QL et al., S cience, 244 , p). 359-362, 1989 ; Choo, QL et al., PNAS, 88, p.2451-2455, 1991 ). Given this genetic structure, HCV is similar to flaviviruses or pestiviruses, so HCV has been classified as a new genus belonging to Flaviviridae (van Doorn, Review). J. Med. Virol, 43, p.345-356, 1994 ).

The polyprotein of HCV consists of the core-E1-E2-NS2-NS3-NS4A-NS4B-NS5A-NS5B from the amino terminal. That is, at the amino terminus of the polyprotein, there are structural proteins (cores, E1 and E2), which are components of the nucleocapsid and envelope of the virus, and at the carboxy terminus thereof, the non-structural essential for HCV proliferation. Proteins (NS2, NS3, NS4A, NS4B, NS5A, NS5B) are located. The polyprotein is a protein peptide peptidase and NS2-3 protein and NS3 protein, which is present in the virus, acts to cleave specific sites of the precursor polyprotein, thereby removing nine mature viral proteins. Make it up Specifically, the NS3 protein has been found to act on four cleavage sites existing between NS3 protein and NS4A protein, between NS4A protein and NS4B protein, between NS4B protein and NS5A protein, and between NS5A protein and NS5B protein (Hijikata et al. , PNAS , 88, p.5547-5551, 1991; Bartenschlarger et al., J. Virol, 68, p.5045-5055, 1994 ; Grakoui et al., J. Virol, 67 , p.1385-1395, 1993 Tomei et al., J. Virol, 67 , p. 4017-4026, 1993 ). In addition, these NS3 proteins have been reported to increase their enzymatic activity by binding to the coenzyme and the coenzyme bound to the coenzyme NS4A protein (Shimizu, Y. et al., J. Virol, 70, p. 127-132, 1996 ; Love RA et al., Cell. 87 , p . 331-342, 1996 ).

NS3 protein has proteolytic activity up to one-third of its amino terminus and helicase activity in the remainder, so that each activity occurs when NS3 protein is expressed separately in vitro . (D, Souza et al, J. Gen. Virol. 76, p . 1729-1736, 1993 ). NS3 protein with 57th amino acid histidine, 81th amino acid aspartate, and 139th amino acid serine is the activity found in typical Trypsin-like serine proteinase. It has a site (catalytic motif), and this property has been found to appear consistently by analyzing the amino acid sequence in a number of variants of HCV discovered so far.

However, NS3 protein is 1) linked to helicase protein, 2) divalent metal ions such as zinc (Zn), and 3) NS4A peptide consisting of 54 amino acids is involved as a major factor in degrading enzyme activity. And trypsin-like serine protease in that it interacts with the NS3 protein to help form a protein structure with degrading enzyme activity (Hijikata et al., J. Virol. 67 , p.4665-4675, 1993 ). Is different.

Among NS3 polypeptides, proteins composed of about 180 amino acids at the amino terminus were also found to have degrading enzyme activity (Raffaele DF et al., Biochem. 35, p.13282-13287, 1996 ), depending on the presence or absence of the NS4A peptide. It is known that there is a large difference in activity. In addition, when the protein purified by expressing only about 180 amino-terminal amino acids of NS3 is present at a low concentration, the maximum activity (V _max ) appears only when the NS4A peptide is present at a concentration of about 1000-2000 times higher. That is, when NS3 protein and NS4A are present in low concentration, the overall reaction rate is slowed because the active structure of the complex protein of NS3 and NS4A is also present in low concentration. When the NS4A peptide is present in high concentration, the complex protease is present in most active structures. Because the reaction rate is faster.

Among the 54 amino acid NS4A peptides, the 21st to 34th amino acids are known to play the most important role in forming the active structure of protease. Attempts have been made to produce complex proteases with active structures by fusion and expression of all parts in sequence. In addition, a heterologous protein, such as glutathione S-transferase (GST) or maltose binding protein, is fused to the amino terminus of the NS3 protein (Akiko Mori et. al., FEBS Letter 378 , p. 37-42, 1996 ), and a histidine tag was attached at the amino or carboxy terminus. In either case, however, the arrangement of NS3 protein and NS4A peptides were fused in the order in which they appear on the HCV genome.

Kim et al. Revealed the crystal structure of a complex protein in which an NS3 protease and an NS4A peptide form an active structure, and showed that the NS4A peptide contributes to the formation of an active structure when the position approaches on the NS3 protein (JL). Kim et al., Cell 87 , p. 343-355, 1996 ), the active structure of the NS4A peptide is located closer to the amino terminal region of the NS3 protein. From this fact, the inventors have found that a novel fusion that can have some of the activity that occurs when a portion of the NS4A peptide is added to the NS3 protease consisting of 181 amino acids in contrast to the original HCV genome sequence is about 1000-2000 times the NS4A peptide. GST has been synthesized and patented (Korean Patent Application No. 97-59161), and GST is also used as a substrate of the protease which is purified by the above-described embodiment of the invention. -NS5AB-UB protein was synthesized, and a method for measuring proteolytic enzyme activity in a short time by measuring the detachment of about 20 C-terminal amino acids by reacting under appropriate buffer conditions was developed.

On the other hand, the plants of the genus of the genus used in the present invention are Agalthopanax ( Acanthopanax sessiliflorus Seeman) grown in Korea or cultivated in Korea ( Acanthopanax chiisanensis Nakai), the island of the Agalthophyte ( Acanthopanax koreanum Nakai), the hairy Ogalpi ( Acanthopanax rufinerve Nakai), Acanthopanax seoulense Nakai, Acanthopanax senticosus Harms, and Acanthopanax sieboldianum Makino, and Acanthopanax gracilistylus Smith, which are native or grown in China. Ogapi refers to the root and bark of the above-mentioned trees, and has been used as a tonic in Korean medicine for a long time. As well as tonic, it has the effects of anti-inflammatory, analgesic, swelling and blood loss, active paralysis, It is used to treat diseases such as back pain, edema, changjong, respectively, and a placebo on musculoskeletal medicine (gimjaegil, primary natural drug Dictionary, commercial, p.261, 1984; Oishi one, pharmacognosy, p.121, 1989; Encyclopedia Chinese medicine , Shanghai Science and Technology Publishing House, Vol . 2, p.793).

Algae's components and its physiological activity have been actively studied, and lignans, terpenoids, coumarins, flavonoids, and phenylpropanoid compounds have been separated, and pharmacological effects on the respective components have been reported. In addition, studies have been reported to introduce medicinal effects such as tonic and gangjeong appearing in Ogapi (Korean Patent Publication No. 91-7606; Korean Patent Publication No. 94-618). No therapeutic effect has been reported for hepatitis C.

Therefore, the present inventors have been trying to obtain a HCV therapeutic agent from natural medicines, while distilled water extracts and organic extracts of Ogapi, which have been widely used for treating diseases such as numb pain, low back pain, swelling, swelling, and musculoskeletal The present invention was completed by finding that the solvent extract exhibits strong inhibitory activity against the protease of HCV.

An object of the present invention is to provide a hepatitis C therapeutic agent containing the extract of the genus Agpipi as an active ingredient.

Figure 1 shows the protease inhibitory activity of the distilled distilled water extract of the present invention by performing SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

Lane 1: Substrate

Lane 2: Substrate + Protease

Lane 3: standard molecular weight protein

Lanes 4 to 8: Substrate + Protease + Organic Distilled Water Extract

Final concentrations: 333 (μg / mL), 166 (μg / mL), 83 (μg / mL), 42 (μg / mL), 21 (μg / mL).

Lanes 9-13: Substrate + Protease + Declarator Distilled Water Extract

Final concentrations: 333 (μg / mL), 166 (μg / mL), 83 (μg / mL), 42 (μg / mL), 21 (μg / mL).

Figure 2 shows the protease inhibitory activity of the methanol extract of the present invention by performing SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

Lane 1: Substrate

Lane 2: Substrate + Protease

Lane 3: standard molecular weight protein

Lanes 4 to 8: Substrate + Protease + Ogapi Methanol Extract

Final concentrations: 333 (μg / mL), 166 (μg / mL), 83 (μg / mL), 42 (μg / mL), 21 (μg / mL).

Lanes 9-13: Substrate + Protease + Declarator Methanol Extract

Final concentrations: 333 (μg / mL), 166 (μg / mL), 83 (μg / mL), 42 (μg / mL), 21 (μg / mL).

In order to achieve the above object, the present invention provides a hepatitis C therapeutic agent comprising an extract obtained by boiling the bark of the root, stem and branch of the genus Galgalpi with distilled water or cooled by an organic solvent. Hepatitis C therapeutic agent of the present invention can be used in a variety of food and beverages.

Hereinafter, the present invention will be described in detail.

The present invention provides an extract of Ogapi that can be used for the treatment of hepatitis C. The present invention provides an organic extract using an organic solvent and an organic extract.

First, the extract of Ogapi using distilled water was first put into 5 ~ 10 times (volume / mass) of distilled water in finely sliced Ogapi and boiled for 2 ~ 3 hours, then the cell was used as a filter aid in a vacuum filter equipped with a glass filter. After filling the appropriate amount of light, distilled water extract is filtered under reduced pressure. The pure distilled water extract thus obtained is freeze-dried to obtain a concentrated extract.

Using the Ogapi extract of the present invention obtained above, the extract is dissolved in distilled water at an appropriate concentration to measure the degree of inhibition of HCV protease.

In addition, the extract of organic skin using organic solvent is first made into powder by using a grinder, and then 5-10 times (volume / mass) of organic solvent is added and then extracted for 16 to 24 hours with stirring with a stirrer or not stirred. Extract by chilling for 4 to 7 days in the absence. At this time, it is preferable to use methanol or the like as the organic solvent. After extraction, centrifugation is performed using a centrifuge to remove the organ powder, and the distilled water extract is filtered in the same manner as the filtrate to obtain a pure extract. The extract is concentrated using a vacuum concentrator. The concentrated extract was dissolved in an organic solvent such as dimethyl sulfoxide (DMSO) at an appropriate concentration, and then measured for HCV protease inhibition titer.

The isolated and purified Ogapi extract is subjected to SDS-polyacrylamide gel electrophoresis (hereinafter abbreviated as "SDS-PAGE") to measure the degree of HCV protease inhibition.

When measuring the degree of inhibition of HCV protease by the above-mentioned Ogapi extract, the amino acid is separated from the substrate by the action of the protease in the sample that does not contain the Ogapi extract, the molecular weight is analyzed by using SDS-PAGE A new band appears smaller. On the other hand, in the sample to which the Ogapi extract was added, the degree of inhibition was determined according to the concentration of the added Ogapi extract, and the degree of separation could be expected. From this, the degree of inhibition activity was visually determined using a densitometer or the like. Can be determined (see FIGS . 1 and 2 ).

The hepatitis C therapeutic agent of the present invention may be separated into pure components alone or separated into multi-component mixtures alone or contained as an active ingredient, and the pharmaceutically acceptable carrier, diluent, Excipients such as fillers, anticoagulants and flavors can be added to prepare pharmaceutical preparations such as oral preparations, subcutaneous injections or intravenous injections.

Hereinafter, the hepatitis C therapeutic agent containing the ogapi extract of the present invention will be described in detail by Examples. However, the following examples are only for exemplifying the present invention, and the present invention is not limited by the examples.

Example 1 Preparation of Ogapi Extract Using Distilled Water.

500ml of distilled water was added to an electric bath (commercially available from Daewoong Pharm.), And 50g of chopped chopped oranges, which were purchased from Chinese medicine, boiled for 2 hours and 30 minutes, left to stand, and the supernatant was removed. After filling the celite with a filter aid in a vacuum filter equipped with a glass filter about 1/3, the supernatant was poured and filtered under reduced pressure to obtain a distilled water extract from which an insoluble substance was removed. The obtained extract was dissolved in distilled water at a concentration of 10 mg / ml to measure the degree of inhibition of HCV protease.

Example 2 Preparation of Ogapi Extract Using Organic Solvents

First, the scallop was pulverized to a fine powder by using a pulverizer. Then, 1 g of powder and 10 ml of methanol were put into a test tube, sealed with a stopper, extracted with stirring for 20 hours in a stirrer, and then the scabies were removed by centrifugation at a speed of 3000 rpm. . The obtained extract supernatant was filtered in the same manner as the method of filtering the distilled water extract in Example 1 to completely remove impurities to obtain an extract of Ogapi. The extract was concentrated using a reduced pressure rotary concentrator, and the concentrate was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mg / ml and used for measuring protease inhibitory titer of HCV.

Experimental Example Measurement of HCV Protease Inhibition by Ogapi Extract

Determination of the activity of protease by the extract of Ogapi was carried out in the following steps.

First, purified according to the process described in the Applicant issued the original method by (Republic of Korea Patent Application No. 97-59161 call reference) the protease method for measuring the gain issued by the applicant the original protease activity by purified GST -5A5B-Ubep2-2 substrate protein was obtained. GST-5A5B-Ubep2-2 substrate protein has a GST protein attached to the amino terminus of 20 amino acids 5A / 5B peptide, and contains 10 ubiquitin protein-derived amino acids at the carboxy terminus. Upon digestion, 20 amino acids are separated from the original substrate protein and can be observed by SDS-PAGE to determine their activity. PH with 50 mM Tris, 10 mM DTT, 2% chaps {CHAPS: 3-[(3-cholamidopropyl) -dimethylammonio] -1-propane-sulfonate} and 37.5% glycerol to determine the appropriate activity 7.5 buffer solution (hereinafter referred to as "assay buffer") consisting of 10 µl of substrate (concentration: 500 µg / ml) and 10 µl of type C protease (concentration: 8 µg / ml) 20 μl of solution was added and reacted at room temperature for 1 hour. To complete the reaction, 15 μl of 3X SDS-PAGE sample buffer was added thereto, and the resultant was allowed to stand at 80 ° C. for 5 minutes, followed by 20 μl of the sample, which was developed using 13.5% of SDS-PAGE. As a control to find out that the extract of Ogapi specifically inhibits the protease activity of HCV, the C. distilled water extract and C. ethanol methanol extract obtained by treating the C. locust fruit in the same manner as in Example 1 or 2 above were used.

As a result of measuring the inhibition of HCV protease by the above-mentioned organ extract, 20 amino acids are separated from the substrate by the action of the protease in the case of the control group without the inhibitor of the organ extract, that is, the protease. Although a new band of about 2000 was shown, the degree of inhibition was determined according to the concentration of the added Ogapi extract, and the higher the concentration of the Ogapi extract, that is, the higher the degree of inhibition, the less the separation. (See FIGS . 1 and 2 ).

That is, it was found that the distilled water extract and the organic solvent extract of Ogapi had a strong inhibitory effect on HCV protease, and thus the extract of Ogapi could be usefully used as an effective hepatitis therapeutic agent.

As described above, the distilled water extract and the organic solvent extract of Ogapi showed a strong inhibitory effect on HCV protease, and thus the composition comprising the Ogapi extract of the present invention as an active ingredient is useful as an effective hepatitis C therapeutic agent. Can be used. In addition, the ogapi extract of the present invention can be used in various hepatitis C therapeutic agents included in various food and beverages.

Claims (5)

Hepatitis C therapeutic agent comprising the extract. The hepatitis C therapeutic agent according to claim 1, wherein the extract is boiled with distilled water. The hepatitis C therapeutic agent according to claim 1, wherein the agapi is extracted with a methanol solvent. The hepatitis C therapeutic agent according to claim 1, which contains, as an active ingredient, one component of the extract of Ogapi alone or in a multicomponent mixture. Food and beverage comprising the extract of claim 1 as an active ingredient.