黃芩、芍藥、梔子及其水解物對黑色素之影響曁大黃、首烏及決明子蒽醌類配醣體之定量;I.The effects of extracts of Scutellaria radix, Paeoniae lactiflorae radix, and Gardenia fructus and their hydrolysates on melanin II.Determination of anthraquinone glycosides in Rhei rhizome, Polygoni multiflori radix, and Cassiae torae semen

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Title:	黃芩、芍藥、梔子及其水解物對黑色素之影響曁大黃、首烏及決明子蒽醌類配醣體之定量;I.The effects of extracts of Scutellaria radix, Paeoniae lactiflorae radix, and Gardenia fructus and their hydrolysates on melanin II.Determination of anthraquinone glycosides in Rhei rhizome, Polygoni multiflori radix, and Cassiae torae semen
Authors:	曹惠婷;Hui-Ting Tsao
Contributors:	中國醫藥大學：中國藥學研究所碩士班
Keywords:	黑色素;蒽;醌;類;melanin;anthraquinone
Date:	2007-07-13
Issue Date:	2009-08-11 10:25:29 (UTC+8)
Abstract:	第一部份自古以來，不同人種對於膚色各有偏好，而透過不同的方式修飾膚色。膚色與黑色素之數量、種類及分佈有關，而黑色素之形成來自表皮基底層之黑色素細胞，其生合成過程中以酪胺酸酶為關鍵步驟。目前常使用之美白成分如熊果素及對苯二酚，有不盡完善之處，因而尋求具美白作用之天然物，天然物成分多以配醣體形式存在於植物中，有文獻報導苷元之活性較配醣體佳，故本研究擬比較黃芩、芍藥及梔子水煎劑在水解前後對於黑色素生成之影響。本研究以蘑菇酪胺酸酶之抑制平板圈試驗及抑制率試驗進行篩選，再利用小鼠黑色素瘤細胞探討此三者對於黑色素形成及細胞存活率之影響，並以活性阻斷及合成抑制試驗評估其對細胞酪胺酸酶活性之作用，進一步以西方墨點法測定對酪胺酸酶表現之影響。結果顯示，黃芩水煎劑及梔子水解物對於蘑菇酪胺酸酶之抑制效果不佳，而黃芩水解物 (IC50 18.6 mg/mL)、芍藥水煎劑 (IC50 17.0 mg/mL) 與水解物 (IC50 7.3 mg/mL) 及梔子水煎劑 (IC50 21.8 mg/mL) 皆有良好之抑制作用。另外，此三者之水煎劑對於黑色素之生成皆無顯著影響，而黃芩水解物反而促進黑色素之生成，芍藥水解物則具抑制作用。對於細胞酪胺酸酶活性，黃芩水煎劑及水解物皆有促進作用，芍藥及梔子水煎劑無顯著影響，而芍藥水解物之抑制作用增強。由以上結果顯示，此三種中藥以芍藥水解物抑制黑色素效果較強，其作用機轉可能經由抑制酪胺酸酶活性；而黃芩水解物可促進黑色素生成，其機轉可能與酪胺酸酶活性及表現有關。第二部份中藥所含之多酚類成分主要以配醣體之形式存在，而許多配醣體需經過腸道內之酵素或菌叢水解成極性較低之苷元，方可被人體吸收，因此在品管上若儘量能掌握各具活性苷元之總配醣體含量，則更貼近實際可為人體吸收之量，以再提升中藥之品質管制。但目前各國藥典對於中藥之品質管制只限於單一配醣體或苷元之定量，然而，這些尚不足以反應出中藥之實際效價，因此本研究以酸水解方式，定量大黃、生首烏、製首烏及決明子四種中藥酸水解前後之苷元總量，以進一步了解中藥可為人體吸收成分之含量。將四種藥材水煎劑，加酸及加熱水浴處理後，以高效液相層析儀定量大黃之aloe-emodin、rhein、emodin、chrysophanol及physcion，生首烏、製首烏之emodin及physcion，以及決明子之chrysophanol及physcion之酸水解前後含量變化，並藉此回推其總配醣體含量。結果顯示，大黃水煎劑之aloe-emodin、emodin、chrysophanol、及physcion之含量在水解後分別增加154%、145%、127% 及95%，生首烏水煎劑之emodin及physcion分別增加1174 % 及800%，主要以配醣體形式存在水煎劑中；而決明子水煎劑之chrysophanol及physcion則幾乎以配醣體存在；至於製首烏之emodin及physcion含量在水解前後無明顯差異。本研究此建立之方法，可應用於大黃、生首烏及決明子之總配醣體含量測定，而製首烏建議直接測定其苷元含量。 I. From the ancient times, humans prefer different skin colors and try diverse methods to modify them. Skin colors are related to the numbers, types and distribution of melanins, which are produced by the melanocyte on the basal layer of epidermal. In the process of melanins formation, tyrosinase dominates the rate limiting steps to transform tyrosine into dopaquinone. Nowadays, whitening agents as arbutin and hydroquinone are not satisfied, and scientists have been making an efforts to find whitening agents from natural products. According to the reports, aglycones have better bioactivity than glycosides. In this study, the decoctions and hydrolysates of Scutellaria radix (SR), Paeoniae lactiflorae radix (PLR), and Gardenia fructus (GF) on melanin synthesis were investigated. The inhibition activities were screened with mushroom tyrosinase, and then the effects on melanin formation and cell proliferation were investigated by mouse melanoma cells (B16-F0). The effects on the expression level of tyrosinase in B16-F0 cells were also carried out by western blot assay. The results showed that the hydrolysate of SR (IC50 18.6 mg/mL), decoction (IC50 17.0 mg/mL) and hydrolysate (IC50 7.3 mg/mL) of PLR, and decoction of GF (IC50 21.8 mg/mL) inhibited mushroom tyrosinase whereas the decoctions of SR and GF had little effects. However, all of these three decoctions had no effect on melanin formation. The hydrolysate of PLR inhibited melanogenesis whereas the hydrolysate of SR promoted it. As for tyrosinase in B16-F0 cells, the decoction and the hydrolysate of SR showed promoted effects, the hydrolysate of PLR showed inhibited effects, and the decoction of PLR and GF had little effects on it. In this study, the hydrolysate of PLR presents the strongest whitening effect by the inhibition of tyrosinase activity, and the hydrolysate of SR enhanced melanogenesis by promoting the activity and expression level of tyrosinase. II. Polyphenols are predominantly present as glycosides in Chinese herbs, and many of them are hydrolyzed to less polar aglycones by enzymes or bacteria in intestine and then became absorbable. Therefore, for quality control of Chinese herbs, the amount of total glycosides is more appropriate to serve as a standard since most glycosides are assimilated with aglycone form after oral administration. Currently there are limited requirements for the quality control of crude drugs in pharmacopoeias of many countries and only assay of one glycoside or aglycone are required. However, these requirements cannot ensure the efficacy of Chinese herbs. This study attempted to determine the contents of algycones in Rhei rhizome (RR), crude Polygoni multiflori radix (cPM), processed Polygoni multiflori radix (pPM), and Cassiae torae semen (CS) after acid hydrolysis to investigate the total amount of absorbable components. The decoctions of these four Chinese herbs were hydrolyzed by adding hydrochloric acid and heat. The contents of aloe-emodin, rhein, emodin, chrysophanol and physcion from RR, emodin and physcion from cPM and pPM, and chrysophanol and physcion from CS were determined by HPLC before and after hydrolysis. The total glycoside contents were calculated by subtracting the amounts of aglycones in decoction from those in hydrolysate. The results showed that the contents of aloe-emodin, emodin, and chrysophanol in RR after acid hydrolysis were increased by 154%, 145%, 127% and 95%, respectively. Emodin and physcion in cPM were increased by 1176% and 800%, respectively. Chrysophanol and physcion in CS existed solely as glycoside forms. However, emodin and physcion in pPM had no significant difference between decoction and hydrolysate. The methods developed in this study are applicable for the determination of total glycosides in RR, cPM, and CS, whereas pPM was suggested to determine the contents of aglycones directly.
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