KR100321445B1 - Purification method of 5-hydroxymethyl-2-furfural from pollen - Google Patents

Abstract

PURPOSE: A purification method of 5-hydroxymethyl-2-furfural from pollen is provided, which 5-hydroxymethyl-2-furfural can be useful for the treatment of hepatitis B virus. CONSTITUTION: A purification method of 5-hydroxymethyl-2-furfural from pollen comprises the steps of: extracting pollen with water; extracting the water extract of pollen with at least one organic solvent selected from the group consisting of ether, chloroform, ethylacetate and n-butanol; and subjecting the solvent-philic extract to reverse silica-chromatography, wherein the skin of the pollen is removed prior to the water extraction; the water extraction is carried out in 60 to 90 deg. C of water for 12 to 48 hours; the water extract is centrifuged and ultrafiltrated; and the reverse silica-chromatography uses 0.05% trifluoroacetate containing pH 7.0 distilled water as a buffer solution.

Description

How to purify 5-hydroxymethyl-2-furfural from pollen

The present invention relates to a method for purifying 5-hydroxymethyl-2-furfural useful as a therapeutic agent for hepatitis B from pollen.

Hepatitis B virus ("HBV") is a virus of the Hepadnaviridae strain that specifically infects the human body, and it is known that about 250 million people worldwide are infected with HBV. The hepatitis B virus is distributed in various parts of the world, but the incidence rate varies greatly from country to country, with the US at 0.5%, Japan at 1.5%, Europe at 1%, 8% in Korea, 15% in China and Africa. The incidence rate is as high as 7%. To date, the Korean population has about 8% of the hepatitis B virus, 15 to 20% of which is known to develop cirrhosis, and about 20% of patients with cirrhosis develop liver cancer. It is estimated that 700,000 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 60 to 110 days, and 90 to 95% is fully recovered through various clinical stages.In patients who have not recovered from infection, HBV DNA is assimilated into genomic DNA of human hepatocytes, resulting in chronic activity. Hepatitis, cirrhosis, liver cancer, etc. will develop. Chronic infection with HBV, like other diseases, causes chronic viral infections, lymphoma diseases and chronic kidney disorders. Thus, chronic infection is a highly fatal disease that develops into a more powerful form of the 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. of Medicine 305, 1067 (1981); SN Zaman et al., Lancet 1, 1357 (1985)).

To date, several drugs have been developed as treatments for hepatitis B, but few have satisfactory efficacy. Ara-A (Vidaravine) is by far the only drug approved by the US FDA. Ara-A has shown significant therapeutic effects in clinical trials, but its use is extremely limited due to the toxicity of the drug itself and is used in combination with interferon. Side effects include nausea, loss of appetite, diarrhea, vomiting and reversible bone marrow suppression due to thrombocytopenia (J.A. Payne et al., Disease a Month, March, pp. 117-159 (1988)). Acyclovir (9- (2-hydroxy-ethoxymethyl guanine)) or interferon, on the other hand, shows some therapeutic effects on hepatitis B due to administration, but returns to its pre-dose state if the drug is discontinued. Because long-term administration should be, because of this there is a problem causing severe side effects. Other phosphonoformates (Phosphonoformate (Foscar-net)), Suramin, Thymosin, Prosta-glandin, 2-CDG, Famciclovir and others are known to be promising But more verification is needed. FIAU (Fialuridine), one of the most promising drugs in terms of efficacy and toxicity, has been discontinued due to unexpected death in Phase II clinical trials.

Until now, studies on hepatitis B treatment have been predominantly related to nucleoside analogs, but severe side effects of these analogs are pointed out as the biggest disadvantage. On the other hand, research has been reported to develop a hepatitis B therapeutic agent from natural drugs that are not nucleoside analogs. Extracts of medicinal plants, Phyllthus niruri and Phyllthus amarus, widely known as Indian jaundice treatments, have been reported to be very effective in the inhibition of hepatitis B virus in animal experiments. In clinical trials (PSVenkateswaran et al., Proc. Natl. Acad. Sci. 84, 274 (1987); BS Blumberg et al., Vaccine 8, 86 (1990)).

Accordingly, the present inventors, while trying to find a hepatitis B therapeutic agent from natural medicine, confirmed that the water-soluble extract of pollen and 5-hydroxymethyl-2-furfural, a single component compound separated therefrom, are active ingredients (the same patent application) 94-1240), thus completing the process for purifying this active ingredient from pollen to reach the present invention.

5-hydroxymethyl-2-furfural is present in fruit juices, foods, liquor and the like and is known to be easily made from polysaccharides such as sugar (Chemical Abstract 77, 85191a; 82, 168916v; 85, 44835k; 81, 39339q; 82, 113463q; 76, 12684p). However, there are no reports on the presence of this substance in the pollen or how to isolate it from the pollen.

Accordingly, it is an object of the present invention to provide a method for purifying 5-hydroxymethyl-2-furfural from pollen extracts.

According to the present invention, a method of extracting pollen with an aqueous solvent, extracting the obtained water-soluble extract with an extracting solvent again, and purifying 5-hydroxymethyl-2-furfural, wherein the obtained solvent affinity extract is reversed-phase silica-chromatography. This is provided.

In the process of the invention, the pollen is preferably removed from the epidermis before extraction with an aqueous solvent. In order to increase the yield of tablets, it is necessary to remove the hard epidermis, and furthermore, it is desirable to remove the epidermis of pollen because it may cause allergic diseases. Thus, the epidermis may be removed mechanically or chemically, but commercially available products from which the epidermis has been completely removed may be used (eg from AB Chanel, Sweden).

The epidermis has been removed with an aqueous solvent, preferably water, to obtain a water-soluble extract. At this time, the mixture is extracted by mixing at a ratio of 10 to 40 g of pollen with respect to 100 ml of water. When extracting the pot with water, the temperature of the water bath is maintained for 12 to 48 hours at 60 to 90 ℃ to extract the water-soluble components. It is particularly preferable to extract the water-soluble component by hot water bath at 80 ° C for 24 hours.

In order to separate a single component from the obtained water-soluble extract, it is preferable to centrifuge and ultrafilter before applying to the next process. In addition to these methods, any method capable of removing impurities from the extract may be used as necessary.

The water soluble extract is extracted again with a solvent, preferably an organic solvent. Examples of organic solvents that can be used include ether, chloroform, ethyl acetate or n-butanol, and these solvents may be used alone or in a mixture of two or more, or sequentially. Ethyl acetate is suitable as a single solvent because of its low toxicity and high extractability. Most preferably, the ratio of extract to solvent is 1: 1 by volume.

The extract obtained by extraction with an organic solvent is purified by reversed phase silica-chromatography. The buffer used for chromatography is preferably a distilled water frame of pH 7.0 containing 0.05% trifluoroacetate.

As a result of reverse phase HPLC and NMR structure analysis, the separated components were identified as 5-hydroxymethyl-2-furfural and their effects on hepatitis B. The degree of inhibition of hepatitis B surface antigen was analyzed using a commercialized hepatitis B surface antigen detection kit (Auszyme ®, Monoclonal Abbott lab., # 1980-24). The degree of inhibition against hepatitis B virus DNA is analyzed by comparative quantitative PCR method (R.K. Saiki et al., Science 239, 487 (1988)).

The following provides examples of the present invention, but the scope of the present invention is not limited to these examples.

Example

Step 1: Preparation of Water Soluble Extract

90 g of an enzymatically cuticle pollen, a product of Sweden AB Chanel, was heated at 80 ° C. for 24 hours while stirring with 450 ml of distilled water to 20% (w / v) by weight. The pollen extract thus obtained was centrifuged at 8500 rpm for 30 minutes to obtain a supernatant from which an insoluble substance was removed. The supernatant was purified using ultrafiltration membrane (Whatman, # 1) to obtain a water-soluble pollen extract from which residual insoluble matter was completely removed.

Step 2: Preparation of Solvent Affinity Extract

1.5 L of the water-soluble pollen extract prepared in step 1 was extracted by adding ether in a ratio of 1: 1 by volume, and then the solvent layer and the water layer were separated. The water layer was extracted by adding chloroform in a ratio of 1: 1 by volume, and then the solvent layer and the water layer were separated. The water layer was extracted by adding ethyl acetate in a ratio of 1: 1 by volume, and then the solvent layer and the water layer were separated. The solvent layer was separated by adding n-butanol at a ratio of 1: 1 based on volume again. The solvent of the solvent layer thus obtained was evaporated, and then all were collected and dissolved in 200 ml of distilled water.

Step 3: Reversed Phase Silica-MC Chromatography

15 ml of the extract obtained in step 2 were adsorbed onto a silica-MC column (mid-chain, Amicon diameter 1.5 cm, length 20 cm) previously equilibrated with distilled water (PH 7.0) containing 0.05% trifluoro acetate. Elution was performed for 150 minutes at a rate of 7 ml per minute with buffer.

Thereafter, the same buffer containing 10%, 20% and 30% ethanol was eluted in a step gradient at the same rate.

The elution airfoil at this time is shown in FIG. Only peak WIII in the eluted fractions was collected to give 5-hydroxymethyl-2-furfural.

Analysis by reverse phase HPLC

Extraction before reverse phase silica-MC-chromatography and peak WIII after chromatography and commercially available 5-hydroxymethyl-2-furfural (Aldrich, # H4080-7) were run on reverse phase HPLC columns (C18: Bondapack, 30 mm × 300 mm). ). The composition of the buffer solution with time was as follows, and the dissolution rate at this time was 1 ml / min. A and B refer to HPLC's A pump (distilled water) and B pump (solvent such as acetonitrile), respectively.

Table 1: Composition of time and buffer

The experimental results are shown in FIGS. 2 and 3. Figure 2 shows the results of reversed phase HPLC analysis of the extract before reversed phase silica-MC chromatography. There were three peaks peaking at 6, 8 and 11 minutes. FIG. 3A shows the result of purity analysis of the peak WIII of FIG. 1 by reversed phase HPLC, confirming that it is a single peak of 11 components with high purity, and it can be seen that it is the same as the peak of 11 components among the 3 peaks of FIG. . Figure 3B shows the results of reverse phase HPLC analysis of commercial 5-hydroxymethyl-2-furfural. Comparing this result with the 3A diagram, it can be seen that both materials show the same peak pattern.

NMR Structure Analysis

The peak of FIG. 3 is shown in FIG. 4 by HMR structural analysis. As can be seen from this result, it was confirmed that the component separated according to the method of the present invention is 5-hydroxymethyl-2-furfural as a single component.

Example 2: Analysis of the degree of inhibition against hepatitis B surface antigen

In Example 1, the degree of inhibition of hepatitis B surface antigen was analyzed as follows for each fraction of the chromatography.

Cells producing hepatitis B virus, 2.2.15 (MA Shells et al., PNA, S. 84, 1005 (1987)), were harvested in 10% FBS (Fetus Bovine Serum: GIBCO, # 16000-028) in T75 flasks. 5 days with IMDM medium (GIBCO Cat No. 430-2200) with 1% ABAM (GIBCO BRL, # 15240-013), Geneticin (Sigma, # G-9516) to a final 400 μg / ml concentration After culturing for 24 hours, the cells were aliquoted to 1.0-1.5x10 ⁵ cells / ml / well in 24 co-microplates and incubated for 1-2 hours in a 5% CO ₂ incubator. After the cells adhered to the bottom, the fraction of each elution step of the chromatography in Example 1 was lyophilized and dissolved in 1 ml of distilled water, and treated with 0.1 ml per each pore. Three replicates per sample were maintained to minimize experimental errors. Cultures were collected 6-8 days after sample treatment.

The cell culture fluid thus obtained was quantitatively analyzed for the inhibition of hepatitis B surface antigen production using a commercially available hepatitis B surface antigen detection kit (AuszymeR, Monoclonal, Abbott lab., # 1980-24).

Dilute 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), add 0.2 ml of the dilution and 0.2 ml of the control of the kit at 40 After the reaction time, the beads were washed three times with distilled water to remove the unbound material. The beads used here are already bound to a first single antibody against a surface antigen, a second single antibody linked to HRP (Horse Radish Peroxidase). Thereafter, 300 µl of O-phenylenediamine solution containing hydrogen peroxide was added to the kit, incubated for 30 minutes, and then 0.1N sulfuric acid solution was added to stop the reaction.

Absorbance of the control and the sample was measured by a spectrophotometer equipped with 492 nm, and the degree of inhibition of hepatitis B surface antigen production of pollen extract was calculated from the measured absorbance and is shown in Table 2 below. The formula for the degree of inhibition is as follows.

A: absorbance of control group B: absorbance of sample

Table 2: Inhibition Rates by Purification Steps

As can be seen from the results, all of the initial eluted fractions showed 23-67% of inhibitory effect, but the fractions of the ethanol eluting stage showed generally insignificant inhibitory effects.

Example 3

1.5 L of the water-soluble pollen extract obtained in step 1 of Example 1 was continuously extracted with the same volume (1: 1) of ether, chloroform, ethyl acetate and n-butanol to evaporate the solvent in the solvent layer of each step to obtain a solvent extract. The water layer was lyophilized. Each substance in the powder state was dissolved in distilled water at a concentration of 4.5 mg / ml and carried out according to the inhibition assay for hepatitis B surface antigen of Example 2.

In addition, 1.5 liter of the water-soluble pollen extract obtained in step 1 of Example 1 was extracted with ethyl acetate to obtain a solvent layer and a water layer, and the inhibition of hepatitis B surface antigen was analyzed by the same method as above. The results are shown in Table 3 below.

Table 3: Extraction Methods and Inhibition Rates

In the results of the experiment, the continuous solvent extract showed an inhibitory effect of 45 to 90%, and when extracted only with ethyl acetate it can be seen that the degree of inhibition is the same as the continuous extraction with ether, chloroform and ethyl acetate.

As described above, 5-hydroxymethyl-2-furfural extracted from pollen according to the method of the present invention is very useful as a therapeutic agent for hepatitis B.

1 shows the elution form obtained by reverse phase silica-MC chromatography of a solvent extract,

2 shows the results of reverse phase HPLC analysis of the solvent extract,

3A shows reverse phase HPLC analysis of a WIII fraction obtained by reverse phase silica-MC chromatography of a solvent extract,

3B shows the results of reversed phase HPLC of 5-hydroxymethyl-2-furfural on the market,

4 shows the results of NMR structural analysis of the fraction of FIG.

Claims (5)

Pollen was extracted using water as an aqueous solvent, and the obtained water-soluble extract was extracted again using at least one organic solvent selected from the group consisting of ether, chloroform, ethyl acetate and n-butanol as an extraction solvent, and the obtained solvent affinity extract. Purification method of 5-hydroxymethyl-2-furural characterized in that the reversed phase silica-chromatography. The method of claim 1, And removing the epidermis of the pollen in advance before extracting the pollen with an aqueous solvent. The method of claim 1, Extraction using an aqueous solvent is characterized in that the extraction of the bath in 60 to 90 ℃ 12 to 48 hours. The method of claim 1, Centrifugation and ultrafiltration of the aqueous extract extracted with an aqueous solvent. The method of claim 1, Reverse phase silica-MC chromatography using distilled water at pH 7.0 containing 0.05% trifluoroacetate as a buffer.