臺灣博碩士論文加值系統

本研究以奇美醫院所開發出之具美白效能中藥複方－五白散分別以濕式研磨法將藥材顆粒微小化及以索式萃取法分別以甲醇、95 %、50 %及30 %乙醇水溶液為溶劑萃得其有效成分，將上述所取得進行美白及抗氧化之體外試驗。最後再進行美白效能評估之人體試驗，以凝膠及乳液二種不同賦形劑加以比較其對人體皮膚吸收的效果。
五白散以濕式研磨法研磨24 hr可有效將藥材尺寸由最大75μm縮小至平均粒徑為600 nm。體外試驗發現五白散抑制酪胺酸酶能力及抗氧化能力均隨濃度的提高而增加，經濕研後之五白散因粒徑變小、比表面積變大，質傳阻力小，其溶解出的有效成分會比原始粉末多，試驗效果也比原始粉末好。而經索式萃取後粗萃物因去除大部分纖維雜質，單位克數的有效成分增多，故效果比前者為佳。
濃度10 mg/mL中，總酚類化合物測定以每克五白散50 %乙醇粗萃物之含量33.46±0.05 mg最高，其次依高低順序為95 %乙醇>甲醇>30 %乙醇；在濃度60 mg/mL時，只有50 %乙醇粗萃物之酪胺酸酶活性抑制率可達50 %以上，其IC50為55.3 mg/mL；從多巴醌自氧化抑制試驗中，五白散50 %乙醇粗萃物抑制效果最佳，在濃度70 mg/mL時其抑制率達75.2 %。五白散之30 %乙醇粗萃物在總抗氧化能力之延遲時間以135秒表現最好，也無呈色衰退之現象，但於總抗氧化能力之總還原試驗中，以50 %乙醇粗萃物之效果最佳，IC50為3.99 mg/mL，顯示可能為內含有效成分不同所造成。五白散對於DPPH自由基清除、過氧化氫清除及螯合亞鐵離子能力，在所有試驗中呈現最佳的效果，其中皆顯示50 %乙醇水溶液粗萃物其效果最佳。而於10名受試者的手臂美白效果評估為期15天的試驗中，黑色素指數有下降的趨勢，其中五白散50 %乙醇粗萃物之效果較為明顯，且乳液的美白效果比凝膠為佳，最多差了6個百分點。
再將白芷、白附子、白茯苓、白蘞、藥材A等五種單方藥材的50 %乙醇粗萃物分別進行美白及抗氧化分析，試驗結果此五種藥材之性質相近，其中白芷之50 %乙醇粗萃物的效果最好。且可由結果推測五白散總抗氧化延遲時間試驗會造成所呈顏色衰減的現象可能是由白附子及藥材A所造成。

In this study, we conducted in vitro test in which the abilities of whitening and anti-oxidation of Wu-Bai Chinese herbs composite prepared by wet-mill method and soxhlet-extraction method were defined. Wu-Bai Chinese herbs composite is found to have whitening function by Chi-Mai Medical Center. The purpose of wet-mill is to reduce the particle size of this Chinese herb and increase its surface area. As in soxhlet-extraction method, the Wu-Bai herb composite were extracted with methanol as well as 95 %, 50 % and 30 % aqueous solutions of Ethanol to obtain a highly concentrated effective ingredients. Both the wet mill-ed sample and the extracted ingredients were then formulated as gel form or lotion and tested for its whitening function in human skin.
The particle size of Wu-Bai herb composite was reduced from 75 μm to 600 nm by 24 hours wet-mill procedure. From the test in vitro, we found that the abilities of tyrosinase inhibition and anti-oxidation of Wu-Bai Chinese herb increase as its concentration increases. And milled Wu-Bai Chinese herb powder is more effective than original Wu-Bai herb powder due to its smaller particle size, which should provide larger specific surface area and smaller resistance of mass transfer. As for the extract of Soxhlet extraction, since solvent extraction removes most of impurities, the active ingredients per gram of extract is increased, therefore the results of tests were better than that of original powder and milled powder.
For concentration of 10 mg/ml, every gram of Wu-Bai herb gave 33.46±0.05 mg phenolic compounds while extracted with 50 % aqueous solution of ethanol, which rank the highest in yield. The rest of yield was as following: 95 % aqueous solution of ethanol > methanol > 30 % aqueous solution of ethanol. When concentration reaches 60 mg/mL, only the extract of 50 % aqueous solution of ethanol exhibits more than 50 % of inhibition in tyrosinase activity and the IC50 value is 55.3 mg/mL; In the inhibition test of dopachrome autoxidation, the extract from aqueous solution of 50 % ethanol gave the best performance: when concentration is 70 mg/mL, the inhibition percentage is 75.2 %; The best lag time in total anti-oxidation activity test is 135 seconds, which was given by extract from aqueous solution of 30 % ethanol and no discoloration was observed either. However, extract from 50% aqueous solution of ethanol ranked the best in the ability test of total reduction where IC50 is 3.99 mg/mL. This result may suggest the difference in active ingredients. Wu-Bai Chinese herbs composite is effective in terms of DPPH free radical scavenging test, hydrogen peroxide scavenge test and ferrous iron chelated test. Among all samples, the extract of 50 % aqueous solution of ethanol showed the best result. Results from the whitening test performed by applying above-mentioned formulations on the arms of 10 human subjects for 15 days indicated the trend of reduction in the melanin index. Again, the extract from 50 % aqueous solution of ethanol is the most effective one. Besides, formulated in lotion form showed at least 6% better result than that of gel form.
Furthermore, extraction were performed for each constituents of the Wu-Bai Chinese herbs composite, Angelicaedahuricae radix, Aconiticoreani tuber, Proia cocos, Ampelopsis radix and Herbal A, with 50 % aqueous solution of ethanol and the whitening and antioxidation test were conducted respectively. All samples showed very similar results while the extract of Angelicaedahuricae radix was the best among all. In addition, the results also suggest that Aconiticoreani tuber and Herbal A may contribute to the discoloration in lag time of total anti-oxidation test.

摘要．．．．．．．．．．．．．．．．．．．．．．．．．．．I
Abstract．．．．．．．．．．．．．．．．．．．．．．．．．III
目錄．．．．．．．．．．．．．．．．．．．．．．．．．．． VI
圖目錄 ．．．．．．．．．．．．．．．．．．．．．．．．．XI
表目錄．．．．．．．．．．．．．．．．．．．．．．．．．XIV
第一章　前言．．．．．．．．．．．．．．．．．．．．．．．1
第二章　原理及文獻回顧．．．．．．．．．．．．．．．．．．4
2-1 皮膚的構造　．．．．．．．．．．．．．．．．．．．．．4
　　2-1-1皮膚的層次(Layers of the skin) ．．．．．．．．．．．4
2-1-2皮膚的生理功能 ．．．．．．．．．．．．．．．．．6
2-2黑色素細胞(Melanocytes) ．．．．．．．．．．．．．．．11
2-3黑色素的生成 ．．．．．．．．．．．．．．．．．．．．11
2-4影響黑色素生成的因素．．．．．．．．．．．．．．．．．12
2-4-1酪胺酸酶(Tyrosinase)活性抑制 ．．．．．．．．．．．12
2-4-2還原劑對黑色素生成的影響 ．．．．．．．．．．．．14
2-4-3促進表皮內黑色素的排除 ．．．．．．．．．．．．．14
2-5自由基、抗氧化與美白 ．．．．．．．．．．．．．．．．14
2-5-1皮膚老化之相關機制 ．．．．．．．．．．．．．．14
2-5-2皮膚老化的自由基學說 ．．．．．．．．．．．．． 18
2-5-3自由基．．．．．．．．．．．．．．．．．．．． ．19
2-5-4自由基對人體的影響 ．．．．．．．．．．．． ．．20
2-5-5人體對抗自由基系統 ．．．．．．．．．．．．．．．22
2-6文獻回顧 ．．．．．．．．．．．．．．．．．．．．． 27
2-6-1市面上添加之美白成份．．．．．．．．．．．． ．．27
2-6-2天然中草藥介紹 ．．．．．．．．．．．．．．．．31
2-7 天然抗氧化成分－酚類化合物(phenolic compound) ．．．．37
2-8實驗方法介紹 ．．．．．．．．．．．．．．．．．．．38
2-8-1濕式研磨法 ．．．．．．．．．．．．．．．．．．38
2-8-2索式萃取法(Soxhlet extraction) ．．．．．．．．．．39
2-8-3 酚類化合物含量測定．．．．．．．．．．．．．．．41
2-8-4美白效能評估 ．．．．．．．．．．．．．．．．．41
2-8-5總抗氧化力測定 ．．．．．．．．．．．．．．．． 42
2-8-6 DPPH自由基清除試驗．．．．．．．．．．．．．．． 43
2-8-7清除過氧化氫(H2O2)能力測試 ．．．．．．．．．．．44
2-8-8 螯合亞鐵離子能力測定．．．．．．．．．．．．．．44
2-8-9 美白效果評估之人體試驗．．．．．．．．．．．．．45
第三章　實驗 ．．．．．．．．．．．．．．．．．．．．．47
3-1實驗設備 ．．．．．．．．．．．．．．．．．．．．．．47
3-1-1實驗藥品 ．．．．．．．．．．．．．．．．．．． 47
3-1-2實驗儀器．．．．．．．．．．．．．．．．．．．． 48
3-2 實驗方法．．．．．．．．．．．．．．．．．．．．．． 48
3-2-1濕式研磨法 ．．．．．．．．．．．．．．．．．． 48
3-2-2索式萃取(Soxhlet extraction)法 ．．．．．．．．．． 49
3-2-3酚類化合物含量測定．．．．．．．．．．．．．．． 49
3-2-4 酪胺酸酶(Tyrosinase)活性抑制法 ．．．．．．．．．50
3-2-5 多巴醌(Dopachrome)自氧化作用抑制試驗．．．．．． 51
3-2-6總抗氧化測試法 ．．．．．．．．．．．．．．．．．52
3-2-7 DPPH自由基清除試驗 ．．．．．．．．．．．．． 53
3-2-8清除過氧化氫(H2O2)能力測試 ．．．．．．．．．．．54
3-2-9螯合亞鐵離子能力測定．．．．．．．．．．．．．． 55
3-2-10美白效果評估之人體試驗．．．．．．．．．．．．．55
第四章　結果與討論 ．．．．．．．．．．．．．．．．．．．58
4-1最佳研磨時間 ．．．．．．．．．．．．．．．．．．．．58
4-2索式萃取五白散 ．．．．．．．．．．．．．．．．．．．58
4-3 總酚類化合物含量測定．．．．．．．．．．．．．．．． 62
4-4 酪胺酸酶 (Tyrosinase)活性抑制試驗 ．．．．．．．．．．64
4-5 多巴醌(Dopachrome)自氧化抑制試驗．．．．．．．．．．． 67
4-6總抗氧化能力試驗 ．．．．．．．．．．．．．．．．．．70
4-6-1校正曲線之製作 ．．．．．．．．．．．．．．．．．70
4-6-2五白散之總抗氧化能力 ．．．．．．．．．．．．．．70
4-7 DPPH自由基清除試驗 ．．．． ．．．．．．．．．．．．81
4-8清除過氧化氫(H2O2)能力測試 ．．．．．．．．．．．．．85
4-9 螯合亞鐵離子能力之測定．．． ．．．．．．．．．．．．88
4-10 美白效能評估之人體試驗．． ．．．．．．．．．．．． 92
第五章　單方中草藥粗萃物性質分析．．．．．．．．．．．．．96
5-1 單方中藥材之索式萃取．．．．．．．．．．．．．．．． 96
5-2 單方中藥材之總酚類化合物含量測定．．．．．．．．．． 97
5-3 單方中藥材之酪胺酸酶活性抑制試驗．．．．．．．．．． 99
5-4 單方中藥材之多巴醌自氧化抑制試驗．．．．．．．．．． 101
5-5 單方中藥材之總抗氧化能力試驗．．．．．．．．．．．．101
5-6 單方中藥材之DPPH自由基清除試驗．．．．．．．．．．108
5-7 單方中藥材之清除過氧化氫(H2O2)試驗．．．．．．．．．108
5-8 單方中藥材之螯合亞鐵離子能力試驗．．．．．．．．．．111
第六章　結論 ．．．．．．．．．．．．．．．．．．．．．．117
第七章　參考文獻 ．．．．．．．．．．．．．．．．．．．．120
附錄一．．．．．．．．．．．．．．．．．．．．．．．．．．129

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