KR20080092167A - Anti-hepatitis b virus agent from paeonia lactiflora - Google Patents

Abstract

An extract of Paeonia lactiflora or 1,2,3,4,6-penta-O-galoyl-beta-D-glucose compound, a main ingredient of the Paeonia lactiflora is provided to show excellent hepatitis B virus inhibitory effect, thereby being used as a therapeutic agent of hepatitis B. A composition for treating or preventing hepatitis B comprises an ethanol, methanol. ethyl acetate or butanol extract of Paeonia lactiflora or 1,2,3,4,6-penta-O-galoyl-beta-D-glucose compound isolated therefrom as an effective ingredient.

Description

Hepatitis B therapeutic composition comprising peony extract as an active ingredient {Anti-hepatitis B virus agent from Paeonia lactiflora}

Figure 1 is a hepatitis B treatment, lamivudine (3TC) to determine whether the peony 1,2,3,4,6-penta-O-galloyl-beta-D-glucose (PGG) inhibits hepatitis B virus ) Is treated with HepG2 2.2.15 cell line by concentration (0.08-20 μg / ml), and the result of slot blot of the culture using 677 bp HBV gene probe.

2 is a graph showing the inhibitory effect of hepatitis B virus surface antigen (HBsAg) on 1,2,3,4,6-penta-O-galloyl-beta-D-glucose.

The present invention relates to a peony extract having a hepatitis B virus (HBV) inhibitory effect and 1,2,3,4,6-penta-O-galloyl-beta-D-glucose.

Hepatitis B virus (HBV) is a major cause of acute or chronic hepatitis or liver cancer, and has become an important problem not only in Korea but also in the world. It is estimated that more than 300 million people are infected worldwide, especially in the Far East, including Korea, and in Africa, where more than 10% of the population is prevalent. Therefore, the development of new treatments will lead to rapid market expansion not only in the domestic market but also in many parts of the overseas market. In 1995, according to the World Health Statistics Yearbook of WHO, liver cancer accounted for 33.4 per 100,000 people among the leading causes of death among men in their 40s. This is the second largest member of the OECD, demonstrating the serious health consequences of about 40 people dying from liver cancer per day.

  Treatment for chronic hepatitis is interferon alpha, which is approved by the FDA. It is known to be effective for 40% of patients with acquired hepatitis in adults and hepatitis C in 20-25%, but it is known to cause side effects similar to flu symptoms. Lamivudine (3TC), recently patented by Glaxo-Welcome Pharmaceuticals, is the most representative nucleoside-based compound synthesized with ddC as a parent, and acts on hepatitis B virus as well as reverse transcriptase of AIDS virus. The compound blocks DNA synthesis from the RNA necessary for the virus to multiply. However, since DNA polymerase of host cell can recognize lamivudine of abnormal structure, it can be synthesized into complete DNA by replacing with normal nucleoside. Therefore, the host cell is less affected and can act effectively on the virus to effectively block the proliferation. However, when the compound is administered for a long time, the virus becomes resistant by modifying the structure of reverse transcriptase in order to survive itself and having the ability to recognize lamivudine, such as the DNA polymerase of the host cell. The drug is also known to cause vomiting as a side effect. Therefore, the global trend is at the stage of synthesizing and developing other nucleoside antiviral agents that are in the same class as lamivudine, and also simultaneously administering two nucleoside compounds at the same time to reduce resistance to this class of compounds. I am using a prescription.

According to the development status of hepatitis B treatment drug in Korea, on October 26, 1996, Sino-Foreign Pharmaceuticals developed NP-77A extracted from plantain. It is the first developed and released in the world. The company also announced the next generation of hepatitis B treatment L-FMAU, developed jointly by US researchers on February 27, 1998. Korean wild grass, Phyllanthus urinaria; Dr. Kyungpook National University School of Medicine Institute of Liver jeongtaeho was announced shows an excellent effect in the treatment of hepatitis B, extracts of a fox beads).

  In spite of many studies on hepatitis B virus, only a few diseased animals infected with the virus due to HBV's narrow host range, which can grow only in the liver of humans, chimpanzees, woodchuck, ducks, etc. This study was used to search for the efficacy of treatment for hepatitis, and this suggests that it is not suitable as the first screening method because of the high cost. In 1991, Dr. Korba of Georgetown University, USA, developed and reported an anti-HBV drug screening system using HepG2.2.15 cell line, which has the characteristics of HBV proliferation and production by introducing HBV gene into human liver cancer cells (Korba and Milman, Antiviral research 15: 217-228, 1991; Korba and Gerin, Antiviral research 19: 55-70, 1992). The HepG2.2.15 cell line is useful because it can search many samples in a short time, and it is reported that the cell reproducibility and viral yield are higher than that of the HB 611 cell line developed in Japan for the same purpose. J. of Hepatology 22: 263, 1995). Using this screening method, we tried to search the efficacy of many natural products easily and to find anti-HBV bioactive substances derived from natural products more efficiently.

Peeonia lactiflora Pall.) Is a plant belonging to the genus Peony, perennial, thickening of root, but growing long, fusiform, long, about 30cm long. This plant is an alleviation, analgesic, astringent, and alleviator for muscle spasms, headaches, abdominal pain, dysentery, bacterial infections, cold, and landscaping. Mainly acting in the liver, and softens the liver energy and blood replenishment function is irregular menstrual cycles and stress is an excellent effect when you are irritated. However, there is no known effect of peony on the treatment of HBV infection.

Therefore, the present inventors prepared extracts for dozens of herbal medicines for the purpose of developing inhibitors of hepatitis B virus and measured the inhibitory effect of hepatitis B virus by using a drug screening system using HepG2 2.2.15 cultured cells. And 1,2,3,4,6-penta-O-galloyl-beta-D-glucose have been found to have an excellent effect on hepatitis B and have completed the present invention. Pharm. Bull. 29, 2131-2134 (October 2006).

 It is an object of the present invention to provide a composition comprising a peony extract having an inhibitory effect on hepatitis B virus and 1,2,3,4,6-penta-O-galloyl-beta-D-glucose as an active ingredient. .

EMBODIMENT OF THE INVENTION Hereinafter, the structure of this invention is demonstrated concretely.

In order to achieve the above object, the present invention provides a hepatitis B therapeutic agent comprising a peony extract and 1,2,3,4,6-penta-O-galloyl-beta-D-glucose as an active ingredient.

 As a method for producing a peony extract of the present invention, it is possible to use known methods. For example, a peony may be taken and dried or dried, and ethanol, methanol, ethyl acetate, butanol, and the like may be added, followed by immersion extraction or warm extraction at room temperature.

Peony extract is designated as a usable food ingredient of the Korea Food and Drug Administration, and it can be used as a clinical treatment for hepatitis B or as an active ingredient of functional foods because it has no side effects or toxicity. Judging.

The peony extract according to the present invention is considered to be a drug that can be safely administered without substantially any side effects or toxicity, and when the pharmaceutical composition is clinically used, it is formulated as a conventional pharmaceutical preparation according to a conventional method in the pharmaceutical field. It is available.

Hereinafter, the configuration and operation of the present invention will be described in more detail with reference to Examples. However, the following examples are only for the purpose of illustrating the present invention is not limited to the scope of the present invention.

Example 1 Preparation of Peony Extract

Peony was purchased in Kyungdong Yangnyeong Market as a dry herb and used as an extracting material. Peony dry medicinal herbs and ethanol (ethanol) were mixed at a ratio of 1: 3 (W / V), respectively, extracted by 7 days of cold soaking, filtered using a filter paper filter, and the filtrate was concentrated at 40 ° C. using a vacuum concentrator. The concentrate was re-dissolved in 70% methanol, and then extracted sequentially using four solvents (hexane, ethyl acetate, butanol, and water) according to the polarity, and concentrated at 40 ° C. using a vacuum condenser, and the water layer was freeze-dried. Lyophilization using was used as an extract sample.

Example 2. Isolation of 1,2,3,4,6-penta-O-galloyl-beta-D-glucose

      77 mg of 14.4 g of ethyl acetate extract was injected into a silica column (23 × 250 mm, DAISO) of high performance liquid chromatography (AKTA Explorer 10, Amersham Pharmacia) as a mobile phase solvent (cyclohexane- Isopropanol (0:20-> 20:80)) was taken in a test tube with 17 ml, 36 fractions were obtained, concentrated under reduced pressure using a speed vac, and used as a fraction sample. The 18th fraction having the best effect among the separated fractions was repeatedly obtained under the same conditions, and then concentrated under reduced pressure using a concentrator. The collected 18th fraction was injected into 100 mg of silica gel (23 × 250 mm, DAISO) of high-speed liquid chromatography (Waters Delta 600), and then the concentrations of cyclohexane and isopropyl alcohol, which are mobile phase solvents, were changed. -> 0: 100) was collected in a test tube and five fractions were obtained by concentration under reduced pressure using a speed vac and collected fraction samples. In the experiment, 100 mg of the most active fraction was injected into a Prefect column (Prepak C18 cartridge, 19 × 300 mm, Waters) of High-Speed Liquid Chromatography (Waters Delta 600), followed by water and mobile phase solvent. The concentration of acetonitrile (acetonitrile) (100: 0-> 0: 100) was collected in a test tube, and the second most active fraction of the three fractions was collected by concentration under reduced pressure using a speed vac and isolated. 53 mg of purified pure material was obtained and analyzed by nuclear magnetic resonance spectra and mass spectrometry. The isolated material was obtained as 1,2,3,4,6-penta-O-galloyl-beta-D as shown in Chemical Formula 1. It was confirmed that it is glucose.

1,2,3,4,6-penta-O-galloyl-beta-D-glucose isolated from the peony was dissolved in DMSO at 20 mg / ml and stored at -20 ° C. In order to determine the hepatitis B virus inhibitory ability of the composition of the present invention was carried out as follows.

Example 3. Hepatitis B Virus Inhibitory Activity Screening

2.2.15 Cell line at 37 ° C, 5% CO2, 100% humidity in RPMI-1640 medium containing 5% fetal bovine serum and 350 μg / ml gentamicin (G-418). Passaging. Insert 10x104 cells / well of cells into a 24-well plate and incubate under the above conditions in a CO2 incubator. The sample and medium to be tested are replaced with new ones on days 2, 4 and 6, and only incubated for 48 hours after changing only the medium on day 8. 0.4 ml of medium is used for viral DNA quantification and the remaining cells are tested for viability using crystal violet. At this time, cytotoxicity was expressed as CC50 (50% cytotoxicity) using the DMSO value as a control and% of control. The measurement of viral DNA uses slot blot hybridization technology. The denatured medium is placed in a slot blot apparatus and blotted onto a nylon membrane. A 677-bp probe made by amplifying a portion of HBV DNA by PCR was hybridized at 42 ° C. for 20 hours and analyzed by an easy analyzer. The antiviral effect on the sample is expressed by looking at the value of DMSO as a control group and calculating the relative value.

 HBs antigen was measured by using an immunokit to collect the cell culture solution on the 8th day after treatment with natural products to the cells, and the value was compared with the DMSO treatment group to determine the antigen production inhibitory ability of the control group.

Hepatitis B virus inhibitory activity (100 μg / ml) extract Hepatitis B virus inhibition rate (%) Cytotoxicity (%) Methanol extract 25.7 2.5 Hexane extract 7.5 3.8 Ethyl acetate extract 72.5 4.5 Butanol extract 32.2 3.1 Water extract 1.4 0.3

As shown in Table 1, the methanol, ethyl acetate, butanol extract of peony shows the effect of inhibiting the proliferation of hepatitis B virus, and it can be seen that there is little cytotoxicity and can be used safely.

In addition, the hepatitis B virus inhibitory activity inhibitory activity of 1,2,3,4,6-penta-O-galloyl-beta-D-glucose obtained by separating the most effective ethyl acetate through activity tracking is shown in FIG. As shown in FIG. 1, the concentration-dependent inhibition was shown, and the 50% inhibition concentration was 1.0 μg / ml.

In addition, as shown in FIG. 2, HBs antigens are suppressed in a concentration-dependent manner, and are about 25 at 4 μg / ml, the highest concentration of 1,2,3,4,6-penta-O-galloyl-beta-D-glucose. Inhibition of HBs antigen was also shown to be excellent.

As described above, the present invention provides peony extract and 1,2,3,4,6-penta-O-galloyl-beta-D-glucose, which are one of the main components of the peony, are excellent in inhibiting hepatitis B virus. It is a useful invention that can be used as.

Claims (2)

Hepatitis B treatment or prevention composition comprising the peony ethanol, methanol, ethyl acetate or butanol extract as an active ingredient. A composition for treating hepatitis B comprising 1,2,3,4,6-penta-O-galloyl-beta-D-glucose compound as an active ingredient.

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