KR20010076077A - Novel extract from Agrimonia pilosa L. inhibiting synthesis of surface antigen of hepaptitis B virus, process for preparation the same and the use thereof - Google Patents
Novel extract from Agrimonia pilosa L. inhibiting synthesis of surface antigen of hepaptitis B virus, process for preparation the same and the use thereof Download PDFInfo
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- KR20010076077A KR20010076077A KR1020000003489A KR20000003489A KR20010076077A KR 20010076077 A KR20010076077 A KR 20010076077A KR 1020000003489 A KR1020000003489 A KR 1020000003489A KR 20000003489 A KR20000003489 A KR 20000003489A KR 20010076077 A KR20010076077 A KR 20010076077A
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- 239000000427 antigen Substances 0.000 title claims abstract description 37
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- 102000036639 antigens Human genes 0.000 title claims abstract description 37
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- 241000700721 Hepatitis B virus Species 0.000 claims abstract description 40
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims abstract description 21
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- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
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- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
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Abstract
Description
본 발명은 천연식물로부터 분리한 신규한 B형 간염바이러스의 표면항원 생성 억제물질과 그 억제물질 추출방법 및 용도에 관한 것이다. 더욱 상세하게는, 본 발명은 짚신나물(Agrimonia pilosa L.)로부터 분리한 B형 간염바이러스 표면항원(HBsAg) 생성을 억제하는 추출물과 상기 추출물의 간염 및 간암치료제로서의 용도에 관한 것이다.The present invention relates to a surface antigen production inhibitor of novel hepatitis B virus isolated from natural plants, and a method and use for extracting the inhibitor. More specifically, the present invention relates to an extract for inhibiting hepatitis B virus surface antigen (HBsAg) production isolated from Agrimonia pilosa L. and its use as a therapeutic agent for hepatitis and liver cancer.
B형 간염바이러스는 사람의 간세포에 감염하여 간세포의 DNA에 B형 간염바이러스의 유전체가 삽입되어 바이러스에 감염된 간세포를 암세포화하는 것으로 알려져 있다[J Gen Virol. 79(3):591-600, 1998]. 간조직에서 유래된 세포주는 정상적인 간조직에서 유래된 Chang 세포주가 있고 간암조직에서 유래된 HepG2, Hep3B, PLC/PRF-5 등의세포주가 있는데 HepG2 세포는 B형 간염바이러스 유전체를 갖고 있지 않은데 비해 Hep3B와 PLC/PRF-5 세포는 자체의 DNA유전체에 B형 간염바이러스 유전체를 포함하고 있다[DNA cell Biol., 17(5):415-425,1998; Cancer Res. 57(22):5137-5242, 1997; Virology 191(1)31-41, 1992]. 이들 B형 바이러스 유전체를 포함하고 있는 세포주들의 배양액에서 B형 간염 바이러스의 표면항원이 검출된다. 따라서 이러한 세포주들은 간염치료제 개발의 대상으로 사용되고 있다.Hepatitis B virus is known to infect human hepatocytes and insert the genome of hepatitis B virus into the DNA of the hepatocytes to cancer cellize the hepatocytes infected with the virus [J Gen Virol. 79 (3): 591-600, 1998]. Hepatocyte-derived cell lines include normal hepatocyte-derived Chang cell lines, and hepatocarcinoma-derived hepG2, Hep3B, and PLC / PRF-5 cell lines. HepG2 cells do not have the hepatitis B virus genome. And PLC / PRF-5 cells contain the hepatitis B virus genome in their DNA genome [DNA cell Biol., 17 (5): 415-425,1998; Cancer Res. 57 (22): 5137-5242, 1997; Virology 191 (1) 31-41, 1992]. Surface antigens of hepatitis B virus are detected in the cultures of cell lines containing these B virus genomes. Therefore, these cell lines are used for the development of hepatitis therapeutics.
현재까지 알려진 간염치료제는 면역활성을 유발하는 면역조절제와 항바이러스제제로 구분할 수 있다. 면역조절제는 인체내에 면역활성을 증가시켜서 간염바이러스의 활성을 억제하는 효과를 가진 특성을 가지는데, 여러 면역조절제중에 알파 인터페론이 간염치료에 가장 큰 효과를 나타내는 것으로 알려져 있다. 항바이러스 치료제는 주로 올리고뉴클레오타이드(oligonucleotide)의 유사형체을 이용하는데, Glaxo-Wellcome사에서 발매하는 라미뷰딘[Lamivudine,ara-arabinoside monophosphate, 2',3', dideoxy-cytidine, PNAS 88(19): 8495-8499,1995], L-FMAU [Biochem Pharmacol 55(8):1181-1187, 1998] 등이 알려져 있는데이들은 DNA의 구조와 유사한 형체를 가진 물질로서 바이러스가 증식할 때 바이러스유전자와 경쟁적인 관계를 가짐으로써 바이러스의 증식효과를 억제하는 특성을 가지고 있다. 그러나 이들 간염바이러스제제의 바이러스퇴치률은 매우 낮다. 라미뷰딘과 비슷한 DNA 유사형인 안티센스 올리고뉴클레오타이드[antisense oligonucleotide, 15-S-asON: Yao Z.Q. et al., 1995]를 사용하여 HBsAg의 발현 억제율은 81%으로 나타나 있으나 64.789cpm에서 12.143cpm으로 떨어진 값으로 일반적인 자연 방사선 측정치가 500cpm이상이므로 50cpm의 변화는 B형 간염바이러스 표면항원 생성 억제에 대한 효과가 있다고 할 수 없다(Yao Z.Q. et al., 1995).Hepatitis therapies known to date can be divided into immunomodulators and antiviral agents that induce immune activity. Immunomodulators have the property of inhibiting the activity of hepatitis virus by increasing immune activity in the human body. Among the immunomodulators, alpha interferon is known to have the greatest effect in the treatment of hepatitis. Antiviral drugs mainly use analogues of oligonucleotides, such as lamivudine, ara-arabinoside monophosphate, 2 ', 3', dideoxy-cytidine, PNAS 88 (19): 8495- 8499,1995], L-FMAU [Biochem Pharmacol 55 (8): 1181-1187, 1998], etc., which have a shape similar to that of DNA, and have a competitive relationship with the viral gene when the virus proliferates. It has the property of suppressing the proliferative effect of viruses. However, the virus eradication rate of these hepatitis virus preparations is very low. Antisense oligonucleotide, 15-S-asON: Yao Z.Q., a DNA analogue similar to lamivudine. et al., 1995], the inhibition rate of HBsAg was 81%, but the value from 50.789cpm to 12.143cpm, which is more than 500cpm. It is not effective (Yao ZQ et al., 1995).
한편, Interferon를 이용한 HBsAg의 발현 억제실험(Berthillon et al., 1996)에서는 10,000unit를 처리했을 때 본 발명에서 실험에 이용한 PLC/PRF/5세포에서 B형 간염바이러스 표면항원의 생성 억제률이 10,000 단위를 처리했를때에 40%의 생성억제률을 나타내었다.On the other hand, in the experiment of inhibiting the expression of HBsAg using Interferon (Berthillon et al., 1996), the inhibition rate of hepatitis B virus surface antigen production was 10,000 in PLC / PRF / 5 cells used in the experiment when the 10,000 units were treated. When the unit was processed, the production inhibition rate was 40%.
Lamivudine은 oligonucleotide analog로서 많은 부작용이 노출되고 있으며 interferone도 낮은 치료효과로 상당히 높은 dose(일백만 unit/회)를 투여해야만 치료효과를 나타내나 이에 따른 많은 부작용을 나타내고 있다.Lamivudine is exposed to many side effects as an oligonucleotide analog, and interferone also has a low therapeutic effect and only a very high dose (one million units / time) is required for treatment.
본 발명은 천연식물로부터 얻은 추출물로서 추출방법이 간단하고 그 재배가 매우 손쉬워서 다량의 제제를 기존 간염치료제보다 낮은 가격으로 생산, 판매할 수 있는 장점이 있다. 천연의약품은 개발효과가 높아 개발비용이 적게 소요된다는 점이다. 실제로 화학합성법에 비해 천연의약품 개발비용은 약 40% 수준에 불과하다(생물산업협회, 바이오인더스트리, 1995).The present invention is an extract obtained from a natural plant, the extraction method is simple and its cultivation is very easy, there is an advantage that can be produced and sold a large amount of the formulation at a lower price than conventional hepatitis treatment. Natural drugs have high developmental effects and therefore require low development costs. Indeed, compared with chemical synthesis, the cost of developing natural drugs is only about 40% (Bioindustry Association, Bioindustry, 1995).
또, 천연 의약품의 상품화 성공률은 임상 Phase Ⅰ에서 합성의약품의 25%보다 훨씬 높은 67%를 보이고 있으며, 임상 Phase Ⅲ에서도 천연 의약품의 성공률은 합성의약품의 성공률 66%를 훨씬 앞지르고 있다.In addition, the commercialization rate of natural drugs is 67%, which is much higher than 25% of synthetic drugs in clinical phase I, and the success rate of natural drugs far exceeds that of synthetic drugs in clinical phase III.
본 발명 짚신나물 추출물은 천연제제로서 기존 간염치료제로 사용되고 있는 lamivudine이나 interferone보다 저렴한 가격에 생산할 수 있고 치료효과 역시 위의 치료제보다 월등하다고 볼 수 있다. 결국, 본 발명으로 국내 간염치료제 연간 3000억 시장에서 최소 300억 이상의 기대효과를 올릴 수 있을 것으로 추정된다.The present invention can be produced at low price than lamivudine or interferone, which is used as a conventional hepatitis treatment as a natural formulation, and the therapeutic effect is also superior to the above treatment. As a result, the present invention is expected to achieve at least 30 billion or more expected effect in the annual hepatitis treatment drug market 300 billion.
따라서, 본 발명의 목적은 짚신나물(Agrimonia pilosa L.)로부터 B형 간염바이러스 표면항원 생성을 억제하는 물질을 추출 분리하는 방법을 제공함에 있다. 본 발명의 다른 목적은 짚신나물로부터 상기 B형 간염바이러스 표면항원 생성을 억제하는 물질을 추출 분리하여 제공함에 있다. 본 발명의 또 다른 목적은 짚신나물로부터 상기 B형 간염바이러스 표면항원 생성을 억제하는 물질들의 간염 및 간암치료제로서의 용도를 제공함에 있다.Accordingly, an object of the present invention is to provide a method for extracting and separating a substance that inhibits the hepatitis B virus surface antigen generation from Agrimonia pilosa L. Another object of the present invention is to extract and isolate a substance that inhibits the hepatitis B virus surface antigen generation from the straw herb. Still another object of the present invention is to provide a method of treating the hepatitis B virus surface antigen production from hay fever as a therapeutic agent for hepatitis and liver cancer.
본 발명의 상기 목적은 천연식물 짚신나물(Agrimonia pilosa L.)로부터 추출물을 분리한 후 이 추출물이 PLC/PRF/5 세포에서 B형 간염바이러스 표면항원 생성 억제능을 조사하고 세포에 대한 독성을 조사한 다음 짚신나물 추출물 현탁액에서 비수용성 유기용매로 추출했을 경우 수용성 분획으로 추출되며 이 수용성 분획에서 수용성 유기용매로 추출하여 침전물로 분리되는 물질들임을 확인하고 임상실험을 실시하므로써 달성하였다.The object of the present invention is to isolate the extract from natural plant straw ( Agrimonia pilosa L. ) and then the extract to investigate the hepatitis B virus surface antigen production inhibitory ability in PLC / PRF / 5 cells and to investigate the toxicity to the cells When extracted with a non-aqueous organic solvent from the extract of the straw herb extract, it was extracted into a water-soluble fraction, which was achieved by conducting a clinical experiment after confirming that the water-soluble organic solvents were separated into precipitates.
이하, 본 발명의 구체적인 구성 및 작용을 설명한다.Hereinafter, the specific configuration and operation of the present invention.
도 1은 본 발명 B형 간염바이러스의 표면항원 생성을 억제하는 물질의 바람직한 추출방법을 나타낸 것이다.Figure 1 shows a preferred extraction method of a substance for inhibiting the surface antigen production of hepatitis B virus of the present invention.
도 2은 본 발명 추출물을 포함한 용액이 PLC/PRF/5 세포주에서 B형 간염바이러스의 표면항원 생성을 억제하는 것을 나타낸 결과이다.Figure 2 is a result showing that the solution containing the extract of the present invention inhibits the surface antigen production of hepatitis B virus in PLC / PRF / 5 cell line.
도 3은 본 발명 추출물의 건조 무게당 PLC/PRF/5 세포주에서 B형 간염바이러스의 표면항원 생성을 억제하는 것을 나타낸 결과이다.Figure 3 shows the results of inhibiting the surface antigen production of hepatitis B virus in the PLC / PRF / 5 cell line per dry weight of the extract of the present invention.
도 4은 본 발명 추출물의 농도에 따라 PLC/PRF/5 세포주에서 B형 간염바이러스의 표면항원 생성을 억제하는 것을 나타낸 결과이다.Figure 4 is a result showing the inhibition of the surface antigen production of hepatitis B virus in PLC / PRF / 5 cell line according to the concentration of the extract of the present invention.
도 5은 본 발명 추출물을 포함한 용액이 세포의 증식에는 영향을 주지 않음을 밝힌 결과이다.5 is a result that the solution containing the extract of the present invention does not affect the proliferation of cells.
도 6은 본 발명에 따른 짚신나물의 전초를 건조 분쇄하여 증류수에 혼합한 용액을 동량의 여러 비수용성 유기용매로 추출하여 얻은 수용성 분획들이PLC/PRF/5 세포주에서 B형 간염바이러스의 표면항원 생성을 억제한 효과를 나타낸 결과이다Figure 6 shows the surface antigen generation of hepatitis B virus in the PLCC / PRF / 5 cell line is water-soluble fractions obtained by drying and grinding the straw shoots of straw according to the present invention in a distilled water and a solution of the same mixed with distilled water It is the result which showed the effect that suppressed
도 7은 본 발명에 따른 짚신나물의 전초를 건조 분쇄하여 증류수에 혼합한 용액을 동량의 여러 수용성 유기용매로 추출하여 얻은 침전물들이 PLC/PRF/5 세포주에서 B형 간염바이러스의 표면항원 생성을 억제한 효과를 나타낸 결과이다.Fig. 7 shows that the precipitates obtained by dry grinding and crushing the outposts of straw thin herbs according to the present invention in distilled water are extracted with several water-soluble organic solvents in the same amount to inhibit the surface antigen generation of hepatitis B virus in PLC / PRF / 5 cell line. The result is an effect.
도 8은 본 발명에 따른 짚신나물에 다닥냉이, 쐐기풀을 첨가하여 혼합 분쇄하여 얻은 추출물이 PLC/PRF/5 세포주에서 B형 간염바이러스의 표면항원 생성을 억제하는 것을 나타낸 결과이다.8 is a result showing that the extract obtained by mixing and grinding by adding the horseradish and nettle to the straw shin according to the present invention inhibits the surface antigen production of hepatitis B virus in PLC / PRF / 5 cell line.
도 9은 본 발명 추출물을 복용한 사람의 임상결과(GOT, GPT)를 나타낸 그림이다.9 is a diagram showing the clinical results (GOT, GPT) of the person taking the extract of the present invention.
본 발명은 건조시킨 짚신나물(Agrimonia pilosa L.)의 전초를 분쇄한 후 증류수에 현탁시킨 추출물이 B형 간염 바이러스에 감염되어 있는 PLC/PRF/5 세포에서 B형 간염바이러스 표면항원 생성의 억제를 조사하는 단계; 이 추출물을 비수용성 유기용매로 추출하여 수용성 분획으로 얻어지는 단계; 수용성 분획을 수요성 유기용매로 처리하여 침전물로 분리되는 물질들이 B형 간염바이러스 표면항원 생성의 억제능이 있음을 조사하는 단계로 구성된다.The present invention is directed to the inhibition of hepatitis B virus surface antigen production in PLC / PRF / 5 cells infected with hepatitis B virus by crushing dried outposts of Agrimonia pilosa L. and then suspended in distilled water. Investigating; Extracting the extract with a water-insoluble organic solvent to obtain a water-soluble fraction; The water-soluble fraction was treated with a demanding organic solvent to investigate whether the substances separated from the sediment have the ability to inhibit hepatitis B surface antigen production.
이하, 본 발명의 구체적인 구성 및 작용을 실시예를 통하여 상세히 설명하고자 하지만 본 발명의 권리범위가 이들 실시예에만 제한되는 것은 아니다.Hereinafter, the specific configuration and operation of the present invention will be described in detail with reference to the examples, but the scope of the present invention is not limited only to these examples.
실시예 1: 본 발명 짚신나물 추출물 제조Example 1 Preparation of Invention Straw Sprout Extract
짚신나물(Agrimonia pilosa L.)의 전초를 건조시킨 후에 분쇄하여 분쇄물 1g 당 증류수 40ml에 가하여 현탁시킨 후 분쇄물액을 고속원심분리하여 불용성분을 제거한 후에 추출액을 건조하여 단위 건조무게를 측정하였다. 이때 추출용액의 1ml의 건조무게는 20mg이었다.After drying the outposts of Agrimonia pilosa L. dried, ground and suspended in 40ml of distilled water per 1g of pulverized powder, the ground liquid was separated by high-speed centrifugation to remove insoluble components, and the extract was dried to measure the unit dry weight. At this time, the dry weight of 1ml of the extraction solution was 20mg.
실시예 2 : 짚신나물Example 2 Straw Sprouts (Agrimonia pilosa L.)(Agrimonia pilosa L.) 의 추출물이 간암세포에서 B형 바이러스 표면항원 생성억제능 효과조사Extracts Inhibit the Antigens of Hepatitis B Virus Antigen Production in Liver Cancer Cells
PLC/PRF/5 세포가 배양되어 있는 96 웰 플레이트에 배양액 100ul에 상기 실시예 1에서 얻은 추출용액 1.25ul, 4ul, 5ul, 6ul, 8ul, 10ul를 각각 48 시간 후에 배양액 100ul을 B형 간염바이러스 표면항원에 대한 항체가 부착되어 있는 마이크로플레이트에 옮기고 37℃에서 1시간 반응하고 퍼옥시데이스 효소가 결합되어 있는 표면항체 용액 25ul을 각 웰에 첨가하고 30분 반응시키고 인산완충용액으로 마이크로플레이트를 5회 세척하고 퍼옥시데이스에 대한 기질용액을 첨가하여 발색시켰다. 발색된 용액은 효소면역측정기로 450nm에서 흡광계수를 측정하였다. 대조군은 추출용액을 가하지 않은 웰의 배양액으로 하였으며 비교군으로 환자의 HBV양성 혈액과 HBV 음성혈액을 사용하였다. 실험결과, 도 2, 3, 4에서 나타낸 바와 같이 추출용액을 가하지 않은 대조군과 추출용액을 가한 실험군을 비교한 결과에서 추출용액를 증가시킴에 따라 배양액내에서 B형 간염바이러스 표면항원 생성억제률이 증가하였으며추출용액 4ul를 첨가한 경우에서 대조군에서의 B형 간염바이러스 표면항원 생성억제률이 50%로 나타났다.100 μl of the extraction solution obtained in Example 1 on 100 μl of the culture solution in a 96 well plate in which PLC / PRF / 5 cells were cultured. Transfer to a microplate to which the antibody to the antigen is attached, react for 1 hour at 37 ° C, add 25ul of the surface antibody solution bound to the peroxidase enzyme to each well, react for 30 minutes, and react the microplate with phosphate buffer solution five times. It was washed and developed by adding the substrate solution to peroxidase. The color solution was measured for the extinction coefficient at 450 nm using an enzyme immunoassay. The control group was a culture medium of the well without addition of the extract solution, and the HBV positive blood and HBV negative blood of the patient were used as a comparison group. As a result, as shown in FIGS. 2, 3, and 4, the inhibition rate of hepatitis B virus surface antigen production in the culture medium increased as the extraction solution was increased from the control group without the extraction solution and the experimental group to which the extraction solution was added. In the case of adding 4ul of extract solution, the hepatitis B virus surface antigen production inhibition rate was 50% in the control group.
실시예 3 : 추출물의 세포에 대한 안전성Example 3 Safety of Cells on Extracts
상기 실시예 2에서 분리한 추출용액 1.25ul, 4ul, 5ul, 6ul, 8ul, 10ul를 배양액에 첨가한 웰에서 배양액을 마이크로플레이트로 옮긴 웰을 인산완충요액으로 2회 세척하고 WST-1(Boeringer Mannheim Co.)을 MEM(minimal essential medium)용액에 희석시킨 용액 100ul를 각 웰에 가하고 2 시간 후에 효소면역측정기로 450nm에서 흡광계수를 측정하였다. WST-1은 Boeringer Mannheim 사에서 발매하는 세포증식을 측정하는 시약이다. 이 실험결과에서 추출물을 첨가한 웰이나 첨가하지 않은 웰의 PLC/PRF/5 세포의 세포증식은 큰 차이가 없었다. 따라서 상기 실시예 1에서 얻은 추출물은 세포의 자체 활성에는 변화없이 B형 간염바이러스의 표면항원 생성을 억제하고 있음을 도 5에 제시하였다.In the well, in which the extract solution 1.25ul, 4ul, 5ul, 6ul, 8ul, 10ul, which was separated in Example 2, was added to the culture, the wells in which the culture was transferred to the microplate were washed twice with phosphate buffered solution and WST-1 (Boeringer Mannheim). 100 μl of the solution diluted in MEM (minimal essential medium) solution was added to each well, and the absorbance coefficient was measured at 450 nm using an enzyme immunoassay device after 2 hours. WST-1 is a reagent for measuring cell proliferation released by Boeringer Mannheim. The cell proliferation of PLC / PRF / 5 cells in the wells with or without the extracts was not significantly different. Therefore, it is shown in Figure 5 that the extract obtained in Example 1 inhibits the surface antigen production of hepatitis B virus without a change in its own activity.
실시예 4 : 비수용성 유기 용매를 이용한 유기용해 물질의 제거Example 4 Removal of Organic Soluble Materials Using Water-Soluble Organic Solvents
상기 실시예 1에서 얻은 추출용액 500ul를 동량의 클로르포름(chloroform), 또는 헥산(hexane), 에틸아세테이트(ethyl acetate) 등과 실온에서 10분 간 잘 혼합한 다음 고속원심분리하여 수용성 분획과 클로르포름 용해 분획을 분리하고 각각의 분획을 완전히 건조시키고 MEM용액 500ul에 현탁시킨 다음 상기 실시예 2에서와 같이 실험하였다. PLC/PRF/5 세포가 배양되어 있는 플레이트웰의 배양액 100ul에 10ul의 수용성 분획과 클로르포름 용해 분획용액 10ul를 가하고 48 시간 후에 배양액 100ul을 B형 간염바이러스 표면항원에 대한 항체가 부착되어 있는 마이크로플레이트에 옮기고 37℃에서 1시간 반응하고 퍼옥시데이스 효소가 결합되어 있는 표면항체 용액 25ul을 각 웰에 첨가하고 30분 반응시키고 인산완충용액으로 마이크로플레이트를 5회 세척하고 퍼옥시데이스에 대한 기질용액을 첨가하여 발색시켰다. 발색된 용액은 효소면역측정기로 450nm에서 흡광계수를 측정하였다. 대조군은 추출용액을 가하지 않은 웰의 배양액으로 하였으며 비교군으로 환자의 HBV양성 혈액과 HBV 음성혈액을 사용하였다. 실험결과, 클로르포름을 처리했을 때 수용성 분획에서는 B형 간염바이러스의 표면항원 생성을 억제하였으나 클로르포름 용해분획에서는 B형 간염바이러스의 표면항원 생성을 억제되지 않았다(도 6 ).500ul of the extract solution obtained in Example 1 was mixed well with chloroform, hexane, ethyl acetate, and the like for 10 minutes at room temperature, followed by high-speed centrifugation to dissolve the aqueous fraction and chloroform. The fractions were separated and each fraction was completely dried and suspended in 500ul of MEM solution and then tested as in Example 2 above. 10 ul of an aqueous soluble fraction and 10 ul of chloroform soluble fraction were added to 100 ul of a plate well cultured with PLC / PRF / 5 cells. After 48 hours, 100 ul of the culture was a microplate with an antibody against hepatitis B surface antigen. After 25 hours of reaction at 37 ° C., 25 μl of the surface antibody solution bound to the peroxidase enzyme was added to each well, and reacted for 30 minutes. The microplates were washed five times with phosphate buffer solution and the substrate solution for peroxidase was removed. It was added and developed. The color solution was measured for the extinction coefficient at 450 nm using an enzyme immunoassay. The control group was a culture medium of the well without addition of the extract solution, and the HBV positive blood and HBV negative blood of the patient were used as a comparison group. As a result, when chlorform was treated, the water-soluble fraction inhibited the surface antigen production of hepatitis B virus, but the chloroform lysis fraction did not inhibit the surface antigen production of hepatitis B virus (FIG. 6).
실시예 5 : B형 간염바이러스 표면항원 억제물질의 수용성 유기 용매를 이용한 분리방법Example 5 Separation Method of Hepatitis B Virus Surface Antigen Inhibitor Using Water-Soluble Organic Solvent
상기 실시예 1에서 헥산의 수용성분획액을 동량의 아세톤(acetone)과 혼합하여 잘 섞은 다음 고속원심분리하여 상등액을 제거하고 침전물을 얻은 다음 완전건조시키고 MEM용액에 현탁시킨 다음 상기 실시예 2에서와 같이 실험하였다.In Example 1, the aqueous fraction of hexane was mixed with the same amount of acetone (acetone) and mixed well, followed by high-speed centrifugation to remove the supernatant, to obtain a precipitate, and then completely dried and suspended in MEM solution. Experimented together.
PLC/PRF/5 세포가 배양되어 있는 플레이트웰의 배양액 100ul에 10ul의 아세톤 추출침전현탁용액 10ul를 가하고 48시간 후에 배양액 100ul을 B형 간염바이러스 표면항원에 대한 항체가 부착되어 있는 마이크로플레이트에 옮기고 37℃에서 1시간 반응하고 퍼옥시데이스 효소가 결합되어 있는 표면항체 용액 25ul을 각 웰에 첨가하고 30분 반응시키고 인산완충용액으로 마이크로플레이트를 5회 세척하고 퍼옥시데이스에 대한 기질용액을 첨가하여 발색시켰다. 발색된 용액은 효소면역측정기로 450nm에서 흡광계수를 측정하였다. 대조군은 추출용액을 가하지 않은 웰의 배양액으로 하였으며 비교군으로 환자의 HBV양성 혈액과 HBV 음성혈액을 사용하였다. 실험결과, 도 3에서 아세톤 추출침전물은 세포의 자체 활성에는 변화없이 B형 간염바이러스의 표면항원 생성을 억제하고 있음을 알 수 있다.10ul of acetone extracting and precipitating suspension was added to 100ul of the plate well culture medium in which PLC / PRF / 5 cells were cultured, and after 48 hours, 100ul of the culture solution was transferred to a microplate with antibody to hepatitis B virus surface antigen. Add 25ul of the surface antibody solution which reacted at 1 ° C for 1 hour and the peroxidase enzyme is added to each well, react for 30 minutes, wash the microplate five times with phosphate buffer solution, and add the substrate solution for peroxidase. I was. The color solution was measured for the extinction coefficient at 450 nm using an enzyme immunoassay. The control group was a culture medium of the well without addition of the extract solution, and the HBV positive blood and HBV negative blood of the patient were used as a comparison group. As a result, it can be seen that the acetone extract precipitate in Figure 3 inhibits the surface antigen production of hepatitis B virus without changing the cell's own activity.
실시예 6 : 짚신나물에 다닥냉이, 쐐기풀을 첨가하여 혼합 분쇄하여 얻은 추출물에 대한 B형 간염바이러스의 표면항원 생성을 억제효과Example 6 Inhibitory Effect of Hepatitis B Virus Surface Antigen Production on Extracts obtained by Mixing and Grinding Addition of Horseradish and Nettle to Straw Sprouts
상기 실시예 1에서 얻은 짚신나물 분쇄물에 다닥냉이(Lepidium aspatum L.)과 쐐기풀(Urtica dioca L.) 분쇄물을 첨가하여 상기예 1에서처럼 추출물을 만든다음 상기 실시예와 동일한 방법으로 실험하였다. PLC/PRF/5 세포가 배양되어 있는 플레이트웰의 배양액 100ul에 10ul의 아세톤 추출침전현탁용액 10ul를 가하고 48 시간 후에 배양액 100ul을 B형 간염바이러스 표면항원에 대한 항체가 부착되어 있는 마이크로플레이트에 옮기고 37℃에서 1시간 반응하고 퍼옥시데이스 효소가 결합되어 있는 표면항체 용액 25ul을 각 웰에 첨가하고 30분 반응시키고 인산완출용액으로 마이크로플레이트를 5회 세척하고 퍼옥시데이스에 대한 기질용액을 첨가하여 발색시켰다. 발색된 용액은 효소면역측정기로 450nm에서 흡광계수를 측정하였다.An extract was prepared by adding ground horseradish ( Lepidium aspatum L. ) and nettle ( Urtica dioca L. ) ground to the powdered straw sprouts obtained in Example 1, and then experimented in the same manner as in Example 1 above. 10ul of acetone extracting and precipitating suspension was added to 100ul of the plate well cultured with PLC / PRF / 5 cells, and after 48 hours, 100ul of the culture was transferred to a microplate with antibody to hepatitis B surface antigen. Add 25ul of the surface antibody solution which is reacted for 1 hour at ℃ and peroxidase enzyme to each well, react for 30 minutes, wash the microplate 5 times with phosphate release solution, and add the substrate solution for peroxidase. I was. The color solution was measured for the extinction coefficient at 450 nm using an enzyme immunoassay.
대조군은 추출용액을 가하지 않은 웰의 배양액으로 하였으며 비교군으로 환자의 HBV양성 혈액과 HBV 음성혈액을 사용하였다. 실험결과, 도 8에서 짚신나물, 다닥냉이, 쐐기풀의 혼합 분쇄물에서 추출한 물질도 B형 간염바이러스의 표면항원 생성을 억제하고 있음을 알 수 있다.The control group was a culture medium of the well without addition of the extract solution, and the HBV positive blood and HBV negative blood of the patient were used as a comparison group. As a result, in Figure 8 it can be seen that the material extracted from the mixed grinding of straw, green horseradish, nettle also inhibits the surface antigen production of hepatitis B virus.
실시예 7 : 짚신나물 추출물의 임상시험Example 7 Clinical Trial of Straw Sprout Extract
만 38세인 성인 남자(서기돈, 주민등록번호 560915-1051125)에게 상기예 1에서 분리한 짚신나물 추출물이 포함된 용액 10ml을 2 개월간 복용한 결과, 혈중 GOT수치가 230IU/ml에서 50IU/ml 이하로, GPT수치가 388IU/ml에서 역시 50IU/ml 이하로 떨어졌으며, 처음 측정당시(1995년 3월) HBeAg이 양성에서 2개월 후에는 음성으로 바뀌었다(도 9 및 표 1 참조).A 38-year-old adult man (Seo Gi-don, Resident Registration No. 560915-1051125) was given 10 ml of the solution containing the extract of Straw Sprouts isolated from Example 1 for 2 months, and the blood GOT value was lowered from 230 IU / ml to 50 IU / ml, GPT levels dropped from 388 IU / ml to less than 50 IU / ml, and HBeAg turned negative after 2 months from positive at the time of initial measurement (March 1995) (see FIG. 9 and Table 1).
상기 실시예와 실험예를 통하여 설명한 바와 같이 본 발명 식물 짚신나물(Agrimonia pilosa L.)에서 분리한 짚신나물 추출물은 B형 간염 바이러스에 감염되어 있는 PLC/PRF/5 세포에서 B형 간염바이러스 표면항원의 생성을 억제하는 효과를 나타냄으로 B형 바이러스에 감염되어 유발된 간염 및 간암에 대한 치료제로서 뛰어난 효과가 있으므로 생물의약산업상 매우 유용한 발명인 것이다.As described through the above Examples and Experimental Examples, the extract of Straw Sprouts isolated from the plant Straw Sprouts of the present invention ( Agrimonia pilosa L. ) is a hepatitis B virus surface antigen in PLC / PRF / 5 cells infected with hepatitis B virus. It is a very useful invention in the biopharmaceutical industry because it has an excellent effect as a therapeutic agent for hepatitis B and liver cancer caused by infection with hepatitis B virus by showing the effect of inhibiting the production of.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003030921A1 (en) * | 2001-10-09 | 2003-04-17 | Biokorea Co., Ltd | Methods for producing agrimonia extract with improved activity against hepatitis B virus and pharmaceutical and food compositions containing said extract |
| WO2004002945A1 (en) * | 2002-06-29 | 2004-01-08 | Biokorea Co., Ltd | (e)-1-[[(2e, 4e)-1-hexyl-2,4-octadecadienyl]oxy]-2-hydroxydiazine and a pharmaceutical composition for treating hepatitis |
| WO2007100206A1 (en) * | 2006-02-28 | 2007-09-07 | Bio Korea Co., Ltd. | A pharmaceutical and food composition comprising agrimonia extracts |
| KR100763035B1 (en) * | 2006-03-02 | 2007-10-04 | 주식회사 래디안 | An extraction method of a effective component improving an acne from Agrimonia pilosa Ledebour and a composition of cosmetic including the component |
| CN110536690A (en) * | 2017-04-20 | 2019-12-03 | 株式会社杰恩森 | For preventing or treating the pharmaceutical compositions of infection with hepatitis C virus disease |
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| KR20210058164A (en) | 2019-11-13 | 2021-05-24 | 주식회사 비티씨 | Method for Producing Agrimony Extract, and Composition for Improving Liver Function or Treating Liver Desease Containing the Same |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS60255727A (en) * | 1984-05-30 | 1985-12-17 | Riyouzou Koshiura | Carcinostatic agent |
| JPS646289A (en) * | 1987-06-29 | 1989-01-10 | Lead Chem Co Ltd | Pharmaceutical composition for remedy and crisis-prevention of acquired immunodeficiency syndrome |
| JP3281458B2 (en) * | 1993-08-11 | 2002-05-13 | 日本製粉株式会社 | Glucosyltransferase inhibitors |
| KR950013517A (en) * | 1993-11-22 | 1995-06-15 | 박태성 | Tumor Therapeutic Composition |
| FR2768621B1 (en) * | 1997-09-22 | 2000-04-07 | Oreal | USE OF AN EXTRACT OF AT LEAST ONE PLANT FROM THE ROSACEA FAMILY |
| CN1197670A (en) * | 1998-05-20 | 1998-11-04 | 靳家富 | Medicine for ulcer |
-
2000
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003030921A1 (en) * | 2001-10-09 | 2003-04-17 | Biokorea Co., Ltd | Methods for producing agrimonia extract with improved activity against hepatitis B virus and pharmaceutical and food compositions containing said extract |
| KR100557015B1 (en) * | 2001-10-09 | 2006-03-03 | 바이오코리아 주식회사 | Methods for producing Agrimonia extract with improved activity against hepatitis B virus and pharmaceutical and food compositions containing said extract |
| WO2004002945A1 (en) * | 2002-06-29 | 2004-01-08 | Biokorea Co., Ltd | (e)-1-[[(2e, 4e)-1-hexyl-2,4-octadecadienyl]oxy]-2-hydroxydiazine and a pharmaceutical composition for treating hepatitis |
| WO2007100206A1 (en) * | 2006-02-28 | 2007-09-07 | Bio Korea Co., Ltd. | A pharmaceutical and food composition comprising agrimonia extracts |
| KR100763035B1 (en) * | 2006-03-02 | 2007-10-04 | 주식회사 래디안 | An extraction method of a effective component improving an acne from Agrimonia pilosa Ledebour and a composition of cosmetic including the component |
| CN110536690A (en) * | 2017-04-20 | 2019-12-03 | 株式会社杰恩森 | For preventing or treating the pharmaceutical compositions of infection with hepatitis C virus disease |
| CN110536690B (en) * | 2017-04-20 | 2022-01-11 | 株式会社杰恩森 | Pharmaceutical composition for preventing or treating hepatitis C virus infection disease |
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| Publication number | Publication date |
|---|---|
| KR100327762B1 (en) | 2002-03-15 |
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