초록 ▼

1. 제 1 단계(1차 연도) 가) 연구자료 및 연구기자재 확보 나) HSV 질환 치료의 처방에 관한 자료의 1차 수집 다) 시료 및 기존 항바이러스성 화학물질의 수집 라) 한약 단방 및 복방의 수집 및 추출 연구 마) 항바이러스제의 일차 검색 바) 1차 검색에서 항바이러스 활성을 나타낸 물질의 유기용매의 용 해도 차이를 이용한 분획실험 및 분석실험 사) 1차 검색에서 항바이러스 활성을 나타낸 물질의 Column chromatography, TLC (Thin Layer Chromatography) 및 HPLC (High Perfor

1. 제 1 단계(1차 연도) 가) 연구자료 및 연구기자재 확보 나) HSV 질환 치료의 처방에 관한 자료의 1차 수집 다) 시료 및 기존 항바이러스성 화학물질의 수집 라) 한약 단방 및 복방의 수집 및 추출 연구 마) 항바이러스제의 일차 검색 바) 1차 검색에서 항바이러스 활성을 나타낸 물질의 유기용매의 용 해도 차이를 이용한 분획실험 및 분석실험 사) 1차 검색에서 항바이러스 활성을 나타낸 물질의 Column chromatography, TLC (Thin Layer Chromatography) 및 HPLC (High Performance Liquid Chromatography)를 이용한 분획실험 및 분석실험 아) 항바이러스성 물질의 2차 검색 자) 2차 검색에서 항바이러스 활성을 나타낸 물질의 유기용매의 용 해도 차이를 이용한 분획실험, 분석실험 및 항바이러스 활성 검 색 차) 2차 검색에서 항바이러스 활성을 나타낸 물질의 TLC(Thin Layer Chromatography) 및 HPLC(High Performance Liquid Chromatography) 를 이용한 분획실험, 분석실험 및 항바이러 스 활성 검색 카) 한약탕제를 이용한 신규 항바이러스제의 탐색법 확립 및 탐색 타) HSV의 계대배양조건 확립 및 전자현미경에 의한 관찰 2. 제 2 단계(2차 연도) 가) 1차년도에 검색된 항바이러스 분획의 상승, 병용, 저해, 길항효 과에 관한 실험 및 항바이러스 활성 검색 나) 2차년도에 검색된 항바이러스 분획의 상승, 병용, 저해, 길항효 과에 관한 실험 및 항바이러스 활성 검색 다) 검색된 항바이러스 물질의 정성, 정량분석 실험, 분리정제 실험 및 항바이러스 활성 검색 라) 검색된 항바이러스 물질의 1D, 2D-NMR(Nuclear Magnetic Resonance)등의 기기분석을 통한 구조결정 실험 및 항바이러 스 활성 검색 마) 검색된 항바이러스 신물질의 활성 평가법 확립

Abstract ▼

In order to search for anti-Herpes simplex virus(HSV) type-1 agents from Korean medicinal herbs and Korean traditional prescriptions(herb complexes), we selected 57 medicinal herbs and 13 prescriptions, based on a review of the Korean traditional medicine books. Both methanol extract and boiling-wat

In order to search for anti-Herpes simplex virus(HSV) type-1 agents from Korean medicinal herbs and Korean traditional prescriptions(herb complexes), we selected 57 medicinal herbs and 13 prescriptions, based on a review of the Korean traditional medicine books. Both methanol extract and boiling-water extract were tested by means of the MTT assay(tetrazolium-based colorimetric assay). Seven of the 70 methanol extracts (from three single herbs and four herb complexes): and six of the water extracts (four individual herbs and two complexes) showed efficacy against HSV-1. Prescription #11 was also interesting because both methanol and water extracts were active. On the whole the prescriptions (herb complexes) were more potent than the individual herbs. Korean traditional prescriptions showing anti- HSV activities as methanol extracts were Paekyopsan, Chesupwilyungtang, Yongdamsagantang, and prescription 11. Four methanol extracts showing anti-HSV activities were fractionated by hexane and their efficacies were tested. Hexane fractionations of Paekyopsan, Chesupwilyungtang, and prescription 11 showed in anti-HSV activities in tissue culture and their selectivity indexes (SI) were 19.50, 3.32 and 42.90, respectively. Methanol fractionation of Paekyopsan showed anti-HSV activities and its SI was 1.40. Especially Paekyopsan showed anti-HSV activities both haxane and methanol fractionation. In order to search for anti-Herpes simplex virus (HSV) type-1 agents from Korean medicinal herbs and Korean traditional prescriptions (herb complexes), we selected 80 medicinal herbs and 45 prescriptions, based on a review of the Korean traditional medicine books. Both methanol extracts and boiling-water extracts were tested by means of the MTT assay (tetrazolium based colorimetric assay). Ten of the 125 methanol extracts: CM-11, CM-18, CM-19, CM-21, CM-22, CM-39, MM-3, MM-18, MM-29, MM-73 (see explanation of nomenclature below), showed efficacy against HSV-1. Twelve of the water extracts: CW-2, CW-3-I, CW-3-II, CW-18, MW-3, MW-5, MW-6, MW-12, MW-47, MW-69, MW-73 and MW-79 were active. #3 (individual herb) and #73 (individual herb) were interesting because both water and methanol extracts were active. Especially, #3 is a part of composition of Hongillaksamdungbang and Hojanghaedokt'ang which have anti-HSV-1 activities. The SI value of MW-69 and CW-18 was relative high as 10.2±0.7 and 11.8±2.2. The cytotoxic effect on Vero cells of Panch'onch'ongbang, Taraxacum platycarpum H. Dahlst. and acycloguanosine was determined by MTT assay. Water extracts of Panch'onch'ong bang(prescription) and Taraxacum platycarpum H. Dahlst. showed very weak cytotoxic effects on Vero cells at > 100㎍/㎖ but acycloguanosine showed strong cytotoxic effects on Vero cells at > 100㎍/㎖. As a result, #3, #73, MW-69 and CW-18 are considered as potentially useful for anti-HSV-1 agent and will be the focus of further research. The combination effect of Korean traditional prescriptions was evaluated by the median effect principle after Chou et al. In HSV-1 infected Vero cell line, combination of Chonghyangulgumbang and Taehwangobaejago showed a synergistic effect (combination index, CI<1). The effect of Korean prescriptions was tested in cutaneous model infection of shaved Balb/c mice with HSV-1. Chonghyangulgumbang showed about 60 % of increased life span in comparison with that of control group mouse. In order to search for anti-Herpes simplex virus(HSV) agents from Korean traditional medicine, we selected 200 specimens of Korean traditional medicines. And then methanol extracts of these 200 specimens were screened to detect anti-HSV activity by MTT assay, seven specimens of these 200 specimens showed efficacy. We isolated known Compounds from M-1, M-2 and M-3, these Compounds had activity against HSV. β-sitosterol isolated from M-1 and M-2 is common sterol in plants, and isorhamnetin 3-O-β-D-glycopyranoside isolated from M-3 is flavonoid glycoside in numerous plant. Previously, anti-HSV activity for the two compounds were not reported. Methanol extract of M-1 had efficacy in 0.8mg/ml concentration. The water and butanol layer of M-1 showed anti-HSV activity (0.41-1.23, 0.13-0.41 mg/ml concentration), but EtOAc fraction didn't. After the butanol layer was fractionated by Sephadex LH-20 column chromatography, 100% MeOH fraction showed efficacy in active experiment (0.045mg/ml concentration) but the others didn't. Compouns A was separated from the 100% MeOH fraction by Prep. TLC (Hexane:EtOAc:EtOH=1.5:8:1) and showed anti-HSV activity in 1.23-3.3mg/ml concentration. Methanol extract of M-2 had efficacy in 0.8mg/ml concentration. After methanol extract of M-2 was fractionated by chromatographic separation using Amberlite XAD-4 and Silica gel (n-Hexane:EtOAc:$CHCl_3$=5:1:2), Compouns B was isolated. Compounds B showed anti-HSV activity in 1.23-3.3mg/ml concentration. Methanol extract of M-3 had efficacy in 0.8mg/ml concentration. After methanol extract of M-3 was fractionated by chromatographic separation using Amberlite XAD-4, the 100% MeOH layer showed anti-HSV activity (0.045-0.13mg/ml) but the others didn't. Compound C was isolated from the 100% MeOH fraction by Sephadex LH-20 column chromatography (70% MeOH) and showed anti-HSV activity in concentration. Chemical structures of these compounds were elucidated on the basis of physico-chemical and spectral data of IR, EI-MS, \FAB-MS, $^{1}H-NMR$, $^{13}C-NMR$, $^{1}H-^{1}H$, COSY, $^{13}C-^{1}H$, COSY, HMBC. Structures of compounds A, B and C were identified as; Compound A m.p. : 140°, $[α]_D^25$ : -37° (c=2, $CHCl_3$) β-sitosterol ($C_{29}H_{50}O$) Compound B m.p. : 140°, $[α]_D^25$ : -37° (c=2, $CHCl_3$), β-sitosterol ($C_{29}H_{50}O$), Compound C m.p. : 215-218°, $[α]_D^23$ : -21.7° (c=0.06, MeOH), Isorhamnetin 3-O-β-D-glucopyranoside ($C_{22}H_{22}O{12}$ Compound A and Compound B are common β-sitosterol, Compound C is known flavonoid glycoside. Anti-HSV activity of Compound A, Compound B, Compound B-1 and Compound C was newly elucidated in this report. The determination of structure of Compound B-1 is in progress.

목차 Contents

• 목 차...18
• 제1장 서론...19
• 제2장 국내$\cdot$외 기술개발 현황...24
• 제3장 제1세부 연구개발수행 내용 및 결과...28
• 제1절 연구내용 및 방법...28
• 제2절 연구결과...39
• 제4장 제2세부 연구개발수행 내용 및 결과...71
• 제1절 서론...71
• 제2절 연구 방법...73
• 제3절 연구개발수행 내용 및 결과...78
• 제4절 생리활성실험...114
• 제5절 결론...119
• 제5장 연구개발목표 달성도 및 대외기여도...122
• 제1절 단계별 연구개발 목표 및 달성도...122
• 제2절 관련분야의 대회 기여도...123
• 제6장 연구개발결과의 활용계획...125
• 제7장 참고문헌...128