KR100342936B1 - Method for extracting scopoletin from oenanthe javanica - Google Patents

Abstract

PURPOSE: Provided is a method for extracting scopoletin from Oenanthe javanica. The scopoletin inhibits the generation of hepatitis B surface antigen. Therefore, a pharmaceutical composition containing it as an active ingredient is used as a therapeutic for hepatitis B. CONSTITUTION: A method for extracting scopoletin from Oenanthe javanica is characterized by extracting Oenanthe javanica with aqueous solvent, such as water then organic solvent, such as methanol or acetonitrile, sequentially; and performing reversed phase HPLC with the extract to obtain scopoletin.

Description

How to extract scopoline from buttercup

The present invention relates to a method for extracting scopoletin from buttercups, and more particularly, to a method for extracting and purifying buttercups with an aqueous solvent and an organic solvent to obtain scopoletin useful as a hepatitis B therapeutic agent.

Hepatitis B virus (HBV) is a virus of the Hepadnaviridae strain that specifically infects the human body, and an estimated 250 million people worldwide are infected with HBV. The hepatitis B virus is distributed in various parts of the world, but the incidence rate differs by country, with the US 0.5%, Japan 1.5%, Europe 1%, etc., 8% Korea, 15% China, Africa The incidence of about 7% is high. To date, the Korean population has about 8% hepatitis B virus, of which 15-20% progress to cirrhosis and 20% of liver cirrhosis patients develop liver cancer, even if no further carriers occur. It is estimated that 10,000 patients with cirrhosis and 140,000 patients with liver cancer will develop (IH Hu et al., Monthly Pharmacy April, pp 54-59 (1992)).

The incubation period of the hepatitis B virus is about 60 to 110 days, and 90 to 95% of the hepatitis B virus recovers completely after various degrees of clinical trial. In patients who do not recover 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, like other diseases, causes chronic viral infections, lymphoma diseases and chronic kidney disorders. Therefore, chronic hepatitis is a disease with a high mortality rate that develops into a more powerful form of disease and eventually leads to death (Don Ganem et al., Ann. Rev. Biochem. 56, 651 (1987); RP Beasley et al., Lancet 2, 1129 (1981); DA Shafritz et al., New England J. Med. 305, 1067 (1981); SN Zaman et al., Lancet 1, 1357 (1985)).

To date, many vaccines have been developed to prevent HBV infection. These vaccines use hepatitis B surface antigen (HBsAg) as an immunogen, which can be obtained from the plasma of carriers infected with hepatitis B virus or through genetic engineering techniques. Such surface antigen-based vaccines are generally effective in preventing individual infections, but not all levels of protective antibodies are formed in all individuals to which the vaccine is administered. The degree of formation of these antibodies is influenced by factors such as the age of the group administered and the degree of suppression of the immune system, as well as the injection site, and all people who receive the vaccine also have some risk of hypersensitivity.

Once a patient is infected with HBV, adequate treatment is required. To date, several drugs have been developed for the treatment of hepatitis B, but few have satisfactory efficacy. Ara-A is the only drug that has been approved by the US FDA so far, and clinical trials have shown significant results, but its use is extremely limited due to the toxicity of the drug itself, and is used in combination with interferon. Side effects of Ara-A include nausea, anorexia, diarrhea, vomiting, and reversible myelosuppression due to platelet reduction (J. A. Payne et al., Disease a Month, March, pp. 117-159 (1988)). Acyclovir (9- (2-hydroxy guanine) or interferon, on the other hand, shows some therapeutic effects on hepatitis B at the time of administration, but returns to the pre-administration state after discontinuation. Long-term administration should be performed, which causes severe side effects. Meanwhile, phosphonoformate (Foscarnet), suramin, prostaglandin, 2-CDG (carbocyclic analogue of Although 2-deoxyguanosine, Famciclovir, etc. are known to be promising, much validation is still needed .. In case of FIAU (Fialuridine), which is known to be the most promising in terms of efficacy and toxicity among recently developed drugs, clinical II In some cases, trials were discontinued due to unexpected death at the stage.

Until now, studies on nucleoside analogues have been mainly used as a treatment for hepatitis B, but the serious side effects are pointed out as the biggest disadvantage. On the other hand, studies have been reported to develop a hepatitis B therapeutic agent from natural medicine that is not a nucleoside analog. Extracts of medicinal plants, Phyllanthus niruri and Phyllanthus amarus, widely known as Indian jaundice treatments, have been reported to be very effective in the inhibition of hepatitis B virus in animal studies. In clinical trials (PS Venkateswaren et al., Proc. Natl. Acad. Sci. 84. 274 (1987): Blumberg et al., Vaccine 8, 274 (1990)).

On the other hand, the buttercup is also called swimming, roots, etc., belongs to the buttercup family, and the scientific name is known as Onanthe javanica, which has been widely used in folk medicine or oriental medicine for a long time. It has various pharmacological effects such as anti-fatty liver action, liver cirrhosis suppression effect, blood cholesterol lowering effect, detoxification effect, and hemostatic action. , Especially in the treatment of acute hepatitis (Chinese metabolism, Sang Kwon, pp 1044-1047; Chinese metabolism, Volume 2, pp 1334; Natural Drug Metabolism, pp 244). However, the above reports have only been found that the buttercups are effective for acute hepatitis in terms of herbal treatments, and there have been no studies on the active ingredient analysis or direct relationship with the hepatitis B virus.

Accordingly, the present inventors, while trying to find a hepatitis B therapeutic agent from natural medicine, the aqueous extract of buttercup and unipolar compound isolated from the scopoletin (scopoletin) has been identified as the active ingredient.

On the other hand, coumarin derivatives known to be widely distributed in the roots, body, fruit, etc. of various plants have the effect of inhibiting HIV virus (Totani et al., JP 03,227,923): hypotensive action (U. Zutshi et al., Indian J) Exp. Biol. 16, 836 (1978); immunosuppressive actions (HC Huanget et al., Eur. J. Pharmaco. 198, 211 (1991), etc.) have been reported.

Scopoletin is a coumarin-based compound that is present in several oleic plants, such as Olea Africana and Olea Capensis (H. Tsukamoto et al., Phytochemistry 23, 699 (1984), showing antiviral activity against Tobacco mosaic virus (MM Antonina et al., Zesz. Probl. Postepow Nack Roln. 182. 17 (1976). Although present in the skin of lemon and mandarin in small amounts, when irradiated with gamma rays and under stress, methyltransferase is converted into scoparon, a natural fungicide of plants, to induce a large amount of self-defense (IA Dubery). et al., S. Afr. I. Sci. 83, 440 (1987)), however, the relationship between scoporetin and hepatitis B virus has not been studied at all.

It is an object of the present invention to provide a method for extracting and purifying scopoletin effective in treating hepatitis B from buttercups.

The method of the present invention for achieving the above object, by first extracting the buttercups with an aqueous solvent, the second extract with an organic solvent to perform a reverse phase high-pressure liquid chromatography for collecting the partition corresponding to scopoline It is characterized by collecting.

Here, water, preferably distilled water, may be used as the aqueous solvent, and methanol, acetonitrile, or the like may be used as the organic solvent.

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

(1) Preparation of Buttercup Extract

100 ml of distilled water was added to 10 to 30 g of buttercup, and the mixture was heated at about 60 to 90 ° C for 12 to 48 hours to obtain an aqueous buttercup extract. Preferably it is kept at 80 degreeC for 24 hours. The obtained aqueous extract needs to remove various impurities by well-known methods, such as centrifugation, before entering into the next process for separating a single component.

The water-soluble buttercup extract, from which impurities have been removed, is secondaryly extracted with an organic solvent, and the extract is evaporated under reduced pressure to obtain a buttercup extract.

(2) Isolation and Purification of Buttercup Extract

The buttercup extract obtained in (1) was dissolved again in water to perform reverse phase high pressure liquid chromatography for collection. Using a C18 column, acetonitrile containing 0.1% trifluoroacetic acid as an eluting solvent: water (16: 84). (v / v) was used to elute the isocratic solution, and the collected solution of each peak separated was evaporated to dryness under reduced pressure to obtain a single component.

As a result of evaluating the degree of inhibition of hepatitis B surface antigen with respect to the collection of each peak, it was found that M1, the 24-component peak, showed a strong inhibitory effect on hepatitis B surface antigen production in reversed phase HPLC for collection. This component was confirmed to be scopoline by NMR and MASS structural analysis, and its effect on hepatitis B was also demonstrated.

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.

In the present invention, the degree of inhibition of hepatitis B surface antigen of the components extracted from buttercups is evaluated by the following method.

Analysis of the degree of inhibition of hepatitis B surface antigen

Quantitative analysis of the inhibitory effect on the production of surface B antigens using a commercially available hepatitis B surface antigen detection kit (Auszyme R, Monoclonal, Abbottlab., # 1980-24) do.

After diluting the cell culture with phosphate buffered saline (pH 7.0) so that the final absorbance is in the range of 1.0 to 1.5 (approximately 20 to 50-fold dilution), 0.2 ml of the diluent and 0.2 ml of the control solution are the primary monoantibodies to the surface antigen. After reacting HRP (Horse Radish Peroxidase) -connected secondary monoclonal antibody with beads already bound at 40 ° C. for 2 hours, the beads were washed three times with distilled water to remove unbound material. Next, 300 µl of an O-phenylenediamine solution containing hydrogen peroxide solution was added thereto, followed by incubation for 30 minutes, and then 0.1N HCl was added to stop the reaction. Absorbance of the control group and the experimental group was measured at 492 nm, and the degree of inhibition of hepatitis B surface antigen production was calculated using the following equation.

EXAMPLE

Step 1: Preparation of Buttercup Extract

In 50 g of buttercup, a Chinese herb, distilled water was added 300 ml and heated at 80 ° C. for 24 hours while stirring. The buttercup extract thus obtained was centrifuged at 8,500 rpm for 30 minutes to remove insoluble precipitate and obtain a supernatant. The supernatant was filtered through an ultrafiltration membrane (whatman, # 1) to obtain an aqueous extract of buttercups from which residual insoluble matter was completely removed.

The aqueous extract of buttercup was extracted with 90% (v / v) methanol as a secondary solvent and evaporated at 70 ° C. under reduced pressure to obtain 10 g of buttercup extract.

By making the buttercup extract at various concentrations, the degree of inhibition of hepatitis B surface antigen was investigated and the results are shown in the following [Table 1].

TABLE 1

As shown in [Table 1], the buttercup extract shows a high inhibitory effect of 74% at a concentration of 9 mg / a.

Step 2. Collecting Reverse Phase High Pressure Liquid Chromatography

For collection reverse phase HPLC, separate peaks are collected by isocratic elution in the following program using μ- BondaPack C18 column (7.8 mm x 300 mm, Waters, USA). At this time, acetonitrile: distilled water (16:84) (v / v) containing 0.1% trifluoroacetic acid was used as the isocratic eluate. The elution rate was 3.5 ml / min and the absorbance was measured at 254 nm.

The result of collecting reversed phase HPLC analysis of the aqueous buttercup extract obtained in step 1 is shown in FIG.

The collected liquids of the separated peaks were evaporated to dryness under reduced pressure, and analyzed for inhibition of hepatitis B surface antigen at a concentration of 0.2m g / ml and shown in [Table 2].

TABLE 2

As shown in Table 2, the collection of reversed phase HPLC showed an inhibitory effect on hepatitis B surface antigen production in the components of several peaks. First, M1, a 24-minute peak, was identified and analyzed using HPLC, NMR, and MASS.

Analytical Reverse Phase High Pressure Liquid Chromatography

The column uses Microsorb C18 (4.6 × 250 mm, Rainin, U.S.A.). Elution buffer is isocratic eluted at a flow rate of 1.0 ml per minute using acetonitrile: distilled water (20:80) containing 0.1% trifluoroacetic acid and detected at 280 nm.

FIG. 2 shows the results of analytical reverse phase HPLC analysis of M1 component (A) and commercially available scopoletin (B) obtained by collecting reversed phase HPLC. Here, it was confirmed that the M1 peak and commercially available scopoline showed the peak shape at the same time zone, and thus the substance M1 separated and purified from the buttercup was scopoline. This is also consistent with the following NMR and MASS structure analysis results.

NMR and Mass Structure Analysis

As a result of NMR and MASS structure analysis of the 24-component peak component M1 by collecting reversed phase HPLC, it was confirmed that component M1, which was separated from the buttercup and purified, was scopoletin as a single component.

FIG. 3 shows the ¹ H NMR structural analysis of the M1 component, FIG. 4 shows the ¹³ C NMR structural analysis of the M1 component, and FIG. 5 shows the MASS structural analysis of the M1 component.

As described above, according to the method of the present invention, the scopoline extracted from the buttercup with an aqueous solvent and an organic solvent and separated and purified by reverse phase HPLC has an inhibitory effect on hepatitis B surface antigen production, and thus is included as an active ingredient. The pharmaceutical composition can be used very usefully as a hepatitis B therapeutic agent.

1 shows the results of a reversed phase HPLC analysis for the collection of an aqueous buttercup extract,

Figure 2 shows the results of analytical reverse phase HPLC analysis of M1 component (A) and commercially available scopoletin (B) obtained by collecting reversed phase HPLC,

3 shows the ¹ H NMR structural analysis of the M1 component,

4 shows the results of ¹³ C NMR structural analysis of the M1 component,

5 shows the results of MASS structural analysis of the M1 component.

Claims (4)

The extract obtained by first extracting the buttercups with an aqueous solvent and the second extraction with the organic solvent is subjected to reverse phase high pressure liquid chromatography for collection to collect the compartments corresponding to scopoline, from scopoline from buttercups. How to extract it. The method of claim 1, Wherein said aqueous solvent is water and said organic solvent is methanol or acetonitrile. The method according to claim 1 or 2, Extracting with an aqueous solvent, characterized in that the extraction in water bath for 12 to 48 hours at 60 to 90 ℃. The method according to claim 1 or 2, Eluent isocratic elution using a C18 column for collection reverse phase high pressure liquid chromatography and acetonitrile: water (16:84) (v / v) containing 0.1% trifluoroacetic acid as the eluent. How to feature.