KR100309699B1 - Agent for treating b type hepatitis containing pollen extract - Google Patents

Abstract

PURPOSE: A water soluble pollen extract obtained by heating pollen and then subjecting to centrifugal separation and ultrafiltration to remove water insoluble materials is provided which has an effect on treatment of B type hepatitis. CONSTITUTION: Epidermis removed pollen is extracted with water, and the extract is mixed with a pharmaceutically acceptable carrier, an excipient or a diluent to produce the titled agent. For example, 90g pollen is extracted with 450ml distilled water at 80deg.C for 24 hr. The extract is subjected to centrifugal separation for 30 min and then ultrafiltration.

Description

Non-B hepatitis treatment containing pollen extract

1 shows the results of quantitative analysis of the inhibitory effect of pollen extract on the production of hepatitis B virus DNA by comparative quantitative PCR method.

The present invention relates to a hepatitis B treatment comprising a pollen extract. More specifically, it is obtained by heating the pollen, which is known to be very effective for the recovery of liver damage as a dietary supplement, in a range not to be boiled with a water bath and then removing insoluble substances by known methods such as centrifugation and ultrafiltration. A hepatitis B treatment comprising a water-soluble pollen extract.

Hepatitis B virus ("HBV") is a virus of the Hepadnaviridae strain that specifically infects the human body. Around 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% hepatitis B virus, of which 15 to 20% is known to progress to cirrhosis and 20% of cirrhosis patients develop liver cancer. Of cirrhosis patients and 140,000 liver cancer patients (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 is completely recovered through various clinical stages. In patients who have not recovered from infection, HBV DNA is assimilated into the genomic DNA of human hepatocytes, leading to chronic active hepatitis, cirrhosis, and liver cancer. Chronic infection with HBV, like other diseases, causes chronic viral infections, lymphoma diseases and chronic kidney disorders. Thus, chronic infection is a disease with a very 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. of Medicine 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 type B surface antigens (HBs Ag) as immunogens. Hepatitis B surface antigen can be obtained from the plasma of carriers infected with hepatitis B virus or through genetic engineering techniques. Such surface antigenic vaccines are generally effective in preventing infection of individuals, 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 injection site as well as the age of the individual to whom the vaccine is administered and the degree of inhibition of the immune system. In addition, all people who receive the vaccine have some risk of developing hypersensitivity.

Once infected, adequate treatment is required. To date, several drugs have been developed as treatments for hepatitis B, but few have satisfactory efficacy. Ara-A (Vidarabine) 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 inhibition of bone marrow caused by thrombocytopenia. (J.A. Payne et al., Disease a Month, March, pp. 117-159 (1988)). Acyclovir (9- (2-hydroxyethoxymethyl guanine)) or interferon, on the other hand, shows some therapeutic effects on hepatitis B due to administration, but when the drug is stopped Long-term dosing because it returns, there is a problem that causes severe side effects. Other promising phosphonoformates (Phosphonoformate, Foscarnet), Suramin, Thymosin, Prostaglandin, 2-carbocyclic analogue of 2-deoxyguanosine, Famciclovir, etc. Although it is known that this method requires more verification. FIAU (Fialuridine), which is the most promising drug in terms of efficacy and toxicity, has been discontinued due to unexpected death in clinical stage II.

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, studies have been reported to develop a hepatitis B therapeutic agent from natural drugs that are not nucleoside analogs. 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 experiments. In clinical trials (PS Venkateswaran et al., Proc. Natl. Acad. Sci. 84, 274 (1987); BS Blumberg et al., Vaccine 8, 86 (1990)).

On the other hand, pollen, a germ cell of a plant, is widely used as a health food in many countries as a surgical pollen corresponding to sperm. However, the epidermis of pollen is so hard that only the epidermis of the pollen can remove the various active ingredients, and the epidermis of pollen must be removed because it causes allergic skin disease in humans. Several animal experiments have been reported using the pollen from Sweden AB Chanel, which has been the only one that has successfully removed the epidermis with enzymes. In particular, Cernitin T60, a water-soluble fraction of pollen extract, has been reported to be very effective in the recovery of liver damage in several animal experiments (J. Wojcicki et al., Acta. Pharmacol. Toxicol. Suppl. 59, 233 (1986) Samochowiec et al., Arch. Exp. Veterinaermed 43, 521 (1989); J. Wojcicki et al., Herba Pol. 30, 213 (1984)). Of course, these results are not directly related to the hepatitis B virus and are only known to be effective in repairing liver damage.

Therefore, the inventors of the present invention, while trying to develop a hepatitis B treatment from natural drugs based on the fact that more than 70% of hepatitis B is known to be caused by the virus, the water-soluble extract of pollen removed epidermis B The present invention was completed by finding that it strongly inhibits the production of hepatitis virus.

It is an object of the present invention to find out that the water-soluble extract of the pollen from which the epidermis has been removed has a strong inhibitory effect on the production of hepatitis B virus and to provide a hepatitis B therapeutic agent comprising the same.

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

First, distilled water is put in a pollen from which the epidermis has been removed, followed by water bath to obtain a pollen extract. At this time, the concentration of pollen in distilled water is 10 to 40% (w / v), preferably 20% (w / v), the heating of the hot water bath is not boiling, that is, 60 to 90 ℃, preferably At 80 ° C. for 10 to 48 hours, preferably 24 hours. The pollen extract thus obtained can be used as a therapeutic agent, but since it contains many impurities, centrifugation and ultrafiltration are performed to make it more pure. Centrifugation and ultrafiltration are just one example of a method for obtaining pure pollen extract, but the method is not limited thereto. In addition, the rotational speed (rpm) during centrifugation or the type of membrane during ultrafiltration may also be arbitrarily selected and used.

The pure water-soluble pollen extract obtained above was treated with a cell line producing hepatitis B virus, and cultured for a predetermined time, and then the cell cultures were collected to quantitatively analyze the degree of inhibition of the production of hepatitis B surface antigen and hepatitis B virus DNA. .

The degree of inhibition on surface antigens can be analyzed using, for example, a commercialized hepatitis B surface antigen detection kit (Auszyme R, Monoclonal, Abbott lab., # 1980-24).

The degree of inhibition against hepatitis B virus DNA is described, for example, by comparative quantitative PCR methods (RK Saiki et al., Science 239, 487 (1988); M. Escarceller et al., Anal. Biochem. 206, 36 (1992) S. Diviacco et al., Gene 122, 313 (1992). As primers used for PCR, the base sequence between the nuclear antigen gene and the polymerase gene of hepatitis B virus was used as a 3-terminal primer, and the base sequence located 320 bp above the 3-terminal primer was used as the 5-terminal primer. As an internal indicator of the PCR reaction, Trp R (RP Gunsalus and C. Yanofsky, Proc. Natl, Acad. Sci. 77, No. 22, 7117 (1980)), which is an E. coli gene, is used. As a result of the PCR reaction, 320bp fragment of the hepatitis B virus DNA and 450bp gene of the Trp R gene are amplified. DNA amplified by the quantitative PCR method is confirmed by electrophoresis on 7% polyacrylamide gel and then silver stained.

As a result of the above experiments, it was found that the water-soluble pollen extract showed a strong inhibitory effect on the production of hepatitis B surface antigen and hepatitis B virus DNA. Thus, water-soluble pollen extract can be used as an effective hepatitis B treatment.

The hepatitis B therapeutic agent of the present invention includes a water-soluble pollen extract as an active ingredient, and may also include a pharmaceutically acceptable carrier, excipient, diluent, and the like. The hepatitis B treatment agent can be administered orally or parenterally, and the daily dosage can be used in 5 ~ 25mg / kg body weight, preferably 10mg / kg body weight, but various conditions such as the patient's symptoms, age and sex Can be adjusted according to. In addition, the hepatitis B therapeutic agent of the present invention may be combined with conventional excipients such as fillers, anti-agglomerating agents, lubricants, flavoring agents, etc. according to known methods in the form of any preparation such as oral, subcutaneous, and intravenous injections suitable for the selected route of administration. Can also be prepared.

Hereinafter, the present invention will be described in more detail with reference to Examples. The following examples are given for illustrative purposes only and in no way limit the scope of the invention.

EXAMPLE

1. Preparation of Pollen Extract

90 ml of distilled water was added to 90 g of an epidermis-free pollen (Sweden AB Chanel) and heated at 80 ° C. for 24 hours while stirring. The pollen extract thus obtained was centrifuged at 8500 rpm for 30 minutes to remove insoluble matter and to obtain a supernatant. The supernatant was filtered using an ultrafiltration membrane (Whatman, # 1) to obtain a water-soluble pollen extract from which residual insoluble matter was completely removed.

2. Cell culture and treatment of pollen extract

Cells producing hepatitis B virus, 2.2.15 (MA Shells et al., Proc. Natl. Acad. Sci. USA 84, 1005 (1987)), were cultured in a T75 flask with 10% FBS (Fetus Bovine Serum: GIBCO). , # 16000-028), IMDM medium with 1% ABAM (Antibiotic-Antimyoctic, GIBCO BRL, # 15240-013) and Geneticin (Geneticin, Sigma, # G-9516) at a final concentration of 400 μg / ml After incubation with (GIBCO Cat No. 430-2200) for 5 days, 1.0 to 1.5 × 10 ⁵ cells / ml were dispensed into each ball of the 24-hole microplate and incubated for about 1 to 2 hours in a 5% CO ₂ incubator. Cells were treated with a serial dilution pollen extract by 5 ⁿ after adhering to the bottom with the same medium by each gongdang 0.1㎖. At this time, the standard Ara-AMP (Sigma, # A-5392) was used in the same dilution. Ara-AMP is a substance that increases the solubility by attaching one phosphate group to Ara-A and is almost equivalent to Ara-A in terms of its efficacy (IVD weller et al., Gut 26, 745 (1985)). We tried to reduce the experimental error to the maximum by maintaining the number of repetitions of three specimens. Cultures and cultured cells were collected 6 to 8 days after treatment to analyze the degree of inhibition against hepatitis B virus.

3. Analysis of Inhibition of Hepatitis B Surface Antigen

Hepatitis B surface antigen detection kit (Auszyme) commercialized using cell culture solution treated with pollen extract obtained from the above , Monoclonal, Abbott lab., # 1980-24) quantitatively analyzed the inhibition of hepatitis B surface antigen production from pollen extracts.

The cell culture was diluted with phosphate buffered saline (ph 7.0) so that the final absorbance was in the range of 1.0 to 1.5 (approximately 20 to 50-fold dilution), so that 0.2 ml of the diluent and 0.2 ml of the control group without treatment of pollen extract were applied to the surface antigen. After reacting the primary monoclonal antibody, HRP (Horse Radish Peroxidase) -linked secondary monoclonal antibody with the beads already bound to each other at 40 ° C. for 2 hours, the beads were washed three times with distilled water to remove unbound substances. Thereafter, 300 µl of a 0-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 group and the experimental group was measured with a spectrophotometer equipped with 492 nm, and the degree of inhibition of hepatitis B surface antigen production according to the concentration of pollen extract was calculated using the following equation:

The absorbance of the control group was not treated with pollen extract was 0.889, the absorbance according to the concentration of the pollen extract and the calculated hepatitis B surface antigen generation inhibition calculated from it is shown in Table 1 below.

The water-soluble pollen extract exhibited an inhibitory effect of about 50% at a concentration of 1.8 mg / ml and a 20% inhibitory effect at a concentration of 0.015 mg / ml. It can be seen that.

4. Analysis of Hepatitis B Virus DNA Suppression by Comparative Quantitative PCR Method

A comparative quantitative PCR method was used to analyze the degree of inhibition of hepatitis B virus DNA of pollen extract.

Cultured cells were collected by centrifugation from the culture obtained in Example 2, and the collected cultured cells were treated with 0.5 ml of crushed solution (0.1N NaOH, 0.1% SDS) and incubated at 65 ° C for 30 minutes. After the culture was extracted by a conventional method using phenol / chloroform, three volumes of 100% ethanol were added and centrifuged at 14000 rpm at 4 ° C. Precipitated DNA (hereinafter referred to as “purified DNA”) was dried and dissolved in 100 μl of TE buffer (10 mM Tris, pH 8.0, 1 mM EDTA).

Meanwhile, the following primers were synthesized by a gene synthesizer (Applied Biosystem, model 380A, USA) using an automated solid phase phosphoamidite chemistry to amplify the purified DNA by PCR. It was.

IntCP 5 '-GAT AGG ATA GGG GCA TTT GGT GGT-3'

The primer is a 3'-terminal primer as a nucleotide sequence adjacent to a nuclear gene and a polymerase gene of hepatitis B virus.

HBV200 5 '-CGC CTC AGC TCT GTA TCG A-3'

The primer is a 5'-terminal primer as a nucleotide sequence upstream about 320bp from the primer IntCP.

TrpR5 5 '-GTA ATC TAG AGG GTA CAT ATT ATG GCC CAA CAA T -3'

The primer is a nucleotide sequence ranging from 21 bp upstream to 10 bp downstream of ATG, the translation start codon of the TrpR gene.

TrpR3 5 '-TAT TAA GCT TAC GGG TAT TTG TAG GAC GGA T -3'

The primer is a nucleotide sequence ranging from about 100 bp to 70 bp downstream of TGA, the translation stop codon of the TrpR gene.

5 μl of the above-purified DNA and 1 μl (100 pg) of TrpR gene DNA were added to the reaction tube, and 5 μl of 10-fold concentration of Taq DNA polymerase buffer solution (100 mM Tris-HC1 (pH 8.3), 500 mM KC1, 15 mM MgC1). ₂ , 0.1% (w / v) gelatin), 5 μl of dNTP's mixed solution (dGTP, dATP, dTTP, dCTP 2.0 mM each), 2 μl TrpR5, 2 μl TrpR3, 2 μl HBV200, 2 μl IntCP, 25.8 μl of distilled water and 0.2 μl of Taq DNA polymerase (5u / λ, Boehringer Mannheim, Germany) were added and mixed well.

In this case, 50 μl of mineral oil was added to the reaction solution to prevent evaporation of the solution, and polymerase chain reaction (PCR) was performed. A thermocycler (Hybaid, Thermal Reactor, England) was used and the temperature cycle program was 94 ° C., 1 minute; 50 ° C., 1 minute; 5 cycles of 72 ° C., 1 minute, 94 ° C., 1 minute; 55 ° C., 1 minute; The cycle of 72 ° C., 1 minute was repeated 30 times and finally further reacted at 72 ° C. for 10 minutes.

The two amplified genes were electrophoresed in 7% polyacrylamide gel (TBE buffer solution, 7% polyacrylamide) and then silver stained. The results are shown in FIG. 1, and in FIG. 1, the first to fifth columns sequentially dilute the water-soluble pollen extract at a concentration of 5 mg / μl by 5 ⁿ (n = 0,1,2,3,4). ) Shows the cultured cells treated with columns 7 to 11, and the cultures treated with serial dilutions (n = 0,1,2,3,4) of standard Ara-AMP at a concentration of 100 μM in 5 ⁿ increments. Cells are shown, and columns 6 and 12 show cultured cells in which the sample is not treated.

The inhibitory effect of water-soluble pollen extract against hepatitis B virus DNA was almost the same level as that of the standard Ara-AMP.

5. Measurement of Cytotoxicity

After culturing the cell line 2.2.15 as in 2 above, 2 x 10 ⁵ cells / ml were dispensed into each ball of the 24-hole microplate and further incubated for 1 hour in a 5% CO ₂ incubator. Three concentrations of 3.47 mg / ml, 0.347 mg / ml and 0.0347 mg / ml for Ara-AMP and 9 mg / ml, 1.8 mg / ml and 0.4 mg / ml for pollen extract Cells were treated with.

Three days after drug treatment, the number of viable cells were counted and compared to the number of cells in the non-drug control group (average 6.0 × 10 ⁵ cells / ball). Cytotoxicity was calculated from the measured cell number by the following formula:

Specific results of the cell numbers and the cytotoxicity calculated therefrom are shown in Table 2 below.

Ara-AMP showed strong cytotoxicity at the concentration of 0.347 mg / ml, but cytotoxicity was minimal for the water-soluble pollen extract.

Claims (3)

Hepatitis B virus production inhibiting composition comprising a pollen extract. The composition of claim 1, wherein the pollen extract is a water-soluble fraction obtained by purifying after removing the pollen from water. The composition of claim 1, wherein said pollen extract is an active ingredient and further comprises a pharmaceutically acceptable carrier, excipient or diluent.