| 摘要: | 本論文是以微波(Microwave)方式萃取中草藥，並探討中草藥萃取液對紫外線所誘導細胞死亡現象之抑制活性探討。本實驗利用UVB照射老鼠黑色素腫瘤細胞(B16 melanoma cells)，後添加不同中草藥萃取液(甘草、桑枝、桑白皮、銀杏葉、白芷、牡丹皮)，結果顯示甘草對照射後細胞，仍有99.5%之存活率。因此，進一步挑選四種甘草內之主要成分進行實驗，分別有Glycyrrhizic acid（GZA）、18α-Glycyrrhetinic acid、18β-Glycyrrhetinic acid（GTA）、Dibenzoylmethane(DBM)等，三種成分在不同的濃度作用下，細胞均有50~70%以上之存活率。另以膠體電泳 (gel electrophoresis）分析紫外線照射後DNA變化，加上DAPI螢光染劑看細胞核的螢光反應，結果顯示DNA有斷裂情形及細胞核有螢光反應，因此，經紫外線照射後之細胞死亡現象與細胞凋亡(apoptosis)有關。另外探討UVB照射經甘草萃取液作用後之存活細胞是否和蛋白質合成有關，以Cycloheximide (蛋白質新合成抑制劑)進行分析，實驗結果顯示甘草萃取液對抑制細胞死亡的現象，與蛋白質的合成有關。接著探討中草藥萃取液之抑制活化作用，是否與自由基清除作用有關，以L-(+)-Ascorbic acid、(-)-epigallocatechin gallate (EGCG)等二種抗氧化劑實驗，結果顯示此兩種物質對受UVB照後之受傷細胞，皆不具有明顯影響作用，因此推判甘草萃取液的抑制活性機制與抗氧化劑的清除效果，沒有很明顯的關聯性。在應用方面，將甘草萃取液與防曬霜標準品(SPF4)一起應用，實驗結果顯示有添加甘草萃取液比單獨只用防曬霜的防禦效果增加。此研究結果或許能夠進一步應用於皮膚的療效上，使已受紫外線傷害的皮膚得到很好的改善。
The object of this research is use microwave to extract Chinese herbs, and investigate activity inhibition of Chinese herbs extraction on UVB induced cell death. B16 melanoma cell is introduced for UVB expose with several Chinese herbs extractions, such as licorice root, mulberry, white mulberry, ginkgo, angelica root, Paeony root bark. Especially “licorice root” shows better inhibition result. Thus, we chose the follow four major compounds of licorice root to do the next experiment, such as Glycyrrhizic acid (GZA), 18α-Glycyrrhetinic acid, 18β-Glycyrrhetinic acid (GTA), dibenzoylmethane(DBM). Except DBM, the other shows over 50~70% cell viability in different concentrations.
In additional, using gel electrophoresis to observe the change of DNA with Chinese herbs extraction. And DAPI is also observed fluorescence due to change of nuclei. Another side, to investigate relation between cell activity and protein synthesize, we use cycloheximide to analysis, which is a kind of protein neosynthesis inhibitor. From the experiment result, we know that cell death inhibition of Chinese herbs is relative to protein synthesize. Besides, for study inhibition of Chinese herbs extraction whether is relative to scavenge of free radical. Using L-(+)-Ascorbic acid, (-)-epigallocatechin gallate (EGCG) with damaged cell by UVB. Both of them are no influence to the cell, so infer that inhibition is not relative to scavenge of free radical.
At the final, apply Chinese herbs with sunscreen (SPF4), within Chinese herbs extraction will gets more protection result than without. This result could do advanced application on therapy of skin, and make UVB damaged skin get improve. |
