| 摘要: | 本研究為尋求抗流行性感冒病毒(H1N1)藥物，進行訶子成分之分離及抗病毒活性測試，追蹤其有效活性成分。研究內容包括：一、化學探討- 由含水丙酮萃取物，經由大孔吸附樹脂、多葡糖聚脂及逆相層析等管柱，分離出 [1] Phenolcarboxylic acid: gallic acid (1), protocatechuic acid (2), chebulic acid (3)。 [2] Glloylglucoses: 1,6-di-O-galloyl-β-D-glucopyranose (4), 1,3,6-tri-O-galloyl-β-D-glucopyranose (5), 1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose (6)。 [3] Galloylshikimic acids: 4-O-galloylshikimic acid (7), 5-O-galloyl shikimic acid (8)。 [4] Ellagitannins: casuariin (9), chebulanin (10), corilagin (11), chebulagic acid (12), chebulinic acid (13), castalagin (14) 等十四個化合物。 二、抗H1N1流感病毒活性MTT測試- [ I ] 藥物及流感病毒同時加入細胞培養測試，以1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose(6)抗病毒活性最優，其細胞存活率為144.5 %，高於ribavirin (105.0 %)的效果，MIC值與正對照組ribavirin同為12.5 μg/ml，其次為chebulinic acid (13), 131.8 % ，MIC值為25 μg/ml。 [ II ] 加入藥物1小時後，再加入流感病毒。 在沒有藥物的保護作用之下，其細胞受病毒感染後，細胞存活率為8.5 %，而1,2,3,4,6-penta-O-galloyl-?nβ-D-glucopyranose (6), 135.9 % 和chebulinic acid (13), 134.1 %之抗病毒活性最好，其細胞存活率比正對照組ribavirin, 112.6 %高。在細胞存活率的數據顯示，本組實驗之MIC以1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose效果最好為12.5 μg/ml，和ribavirin一樣。[ III ] 加入病毒一小時後，再加入藥物，評估治療效果。在沒有任何藥物的保護作用之下，其細胞受病毒感染後，細胞之存活率為7.9 %，正對照組ribavirin為111.6 %， 而所試驗之化合物中以chebulagic acid (12), 126.9 % 和chebulinic acid (13), 120.7 % 治療性抗病毒作用最好，高於ribavirin的作用，其次1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose(6)和castalagin (14)的細胞存活率分別為109.2 % , 110.8 % ，和ribavirin的效果一致。正對照組ribavirin之MIC為12.5 μg/ml，而以上四個有效化合物之MIC均為25 μg/ml。 結論：(1) 由化學結構與活性相互關係之探討得知，多酚性化合物對流行性感冒病毒(H1N1)之抑制、預防及治療作用，以1,2,3,4,6-penta-O-galloyl-?nβ-D-glucopyranose (6) 及chebulinic acid (13)呈現最顯著之抗病毒作用。同時，取代之galloyl或chebuloyl等acyl group，總數目之多寡將影響其活性之大小。 (2) 依化學結構分類，小分子之Phenolcarboxylic acid類化合物僅以protocatechuic acid (2)之作用較佳；Glloylglucoses類化合物則以接五個galloyl基之1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose (6) 抗H1N1病毒效果最好；Galloylshikimic acids類之抑制H1N1病毒作用不明顯；Ellagitannins類化合物呈現最佳之抗H1N1作用，但糖基上若有兩個以上之OH基未接取代基時，其作用顯著下降。
This study aimed to find out anti-H1N1 virus drugs from Terminaria chebula approached by constituents isolation of and anti-H1N1assay. The methods and results include: (1) Chemical investigation- From aqueous acetone extract, by high porous adsorbents resin, polydextran, and reverse column chromatography and fourteen compounds were isolated as following: [1] Phenolcarboxylic acid: gallic acid (1), protocatechuic acid (2), chebulic acid (3). [2] Glloylglucoses: 1,6-di-O-galloyl-β-D-glucopyranose (4), 1,3,6-tri-O-galloyl -β-D-glucopyranose (5), 1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose (6). [3] Galloylshikimic acids: 4-O-galloyl shikimic acid (7), 5-O-galloyl shikimic acid (8). [4] Ellagitannins: casuariin (9), chebulanin (10), corilagin (11), chebulagic acid (12), chebulinic acid (13), castalagin (14). (2) Antivirus activity tests against influenza H1N1 by MTT assay- [ I ] Drugs and virus were added to cell culture plate at the same time. The 1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose (6) showed the highest anti-H1N1 activity and the cell viability was 144.5 %, higher than the effect of ribavirin (105.0 %). The second is chebulinic acid(13), 131.8 % and 1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose (6), had equivalent activity as the positive control, ribavirin and the MIC was 12.5 μg/ml. [ II ] When treatments with or without tested drugs were added 1 hour pre-incubated with the cells before adding virus, the cells without any treatments were infected and the viability was 8.5 %. However, 1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose (6), 135.9 % and chebulinic acid (13), 134.1 % showed excellent protective activity and the cell viability was 144.5 %, which is higher than ribavirin (112.6 %), the positive control. The MIC of 1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose (6) was 12.5 μg/ml and similar to the one of ribavirin. [ III ] Add drugs to cell culture plate at 1 hr after virus added. After virus infection, if without any drug’s treatment, the cell viability is 7.9 %. Using ribavirin as positive control, the cell viability was 111.6 %. chebulagic acid (12), 126.9 % and chebulinic acid (13), 120.7 % possess the best therapeutic activity and higher activity than ribavirin for H1N1 virus infection. The second and the third are 1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose (6), castalagin(14) and their cell viabilities were 109.2 % and 110.8 %, respectively, the same as ribavirin. The MIC of ribavirin was 12.5 μg/ml, the MIC of four active polypenolics were 25 μg/ml. In conclusion: (1) Based on SAR discussion, the inhibition, prevention, and therapeutic effects of polyphenolics for H1N1 virus infection, both 1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose (6) and chebulinic acid (13) showed the most significant activity. The number of substituted galloyl or chebuloyl acyl group has impact on the activity. (2) According to chemical classification, small molecule’s phenolcarboxylic acid, protocatechuic acid (2) is the only one which show good activity; in glloylglucoses, five galloyl substituted 1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose (6) exhibited excellently anti-H1N1 virus activity; the activity of galloylshikimic acids were not significant; ellagitannins showed the best, but if sugar moiety remains more than two free OH residues, the anti-H1N1 virus activity would decreased significantly. |
