石榴抗痤瘡之活性成分研究

The Anti-acne Principle Constituents of Punica granatum Linne

李佳蓉(Chia-Jung Lee)

指導教授 ： 王靜瓊

臺北醫學大學/藥學院/藥學系(博士班)/博士(2011年)

https://doi.org/10.6831/TMU.2011.00046

摘要

痤瘡(acne)俗稱青春痘，是一種常見的皮膚疾病，好發於青春期或荷爾蒙失調的病人，也常見於成年人。痤瘡產生包含許多複雜因素如：毛囊角化異常、皮脂分泌旺盛、荷爾蒙失調、毛囊內細菌增生(痤瘡桿菌、金黃色葡萄球菌)，或由細菌所誘發之發炎反應。常見的治療方式是使用角質溶解劑與抗生素，但部份藥物皮膚刺激性過高，或痤瘡桿菌與金黃色葡萄球菌產生抗藥性，因此開發具有更安全有效的治療藥物，是一個很重要的議題。
根據古代典籍與健保資料庫進行痤瘡常用藥材之探勘，配合市售抗痘產品調查，以抑制金黃葡萄球菌 (Staphylococcus aureus) 與痤瘡桿菌(Propionibacterium acnes)生長之抗菌能力、抑制脂質分解酵素活性、抗發炎與抗氧化做為活性篩選標的，並分析植物化學成分，找尋中草藥之抗痤瘡活性與植物成分關聯性，做為日後開發抗痤瘡藥物開發之研究依歸。結果顯示，固澀藥中石榴、訶子，同時具有良好的抗菌與抗發炎活性，及抑制脂質分解酵素的活性。植物化學成分分析中也發現，石榴與訶子含有最高的總多酚類成分。再以唇形科植物所建立抗痤瘡活性與植物化學成分含量之關聯性探討，結果發現：植物具有抗發炎作用則抗菌活性也較強，利用SPSS軟體以無母數分析法進行回歸分析，其相關係數(R = 0.767，p<0.01)。而清除DPPH自由基之抗氧化活性與總多酚及總黃酮類含量之關係也明顯成正比(R = 0.542及0.613，p<0.01)。
34種常用於治療青春痘之中藥中，以石榴50%甲醇萃取物具有好的抗發炎及抗菌效果，比其他藥材明顯，且具有抗氧化與抑制脂質分解酵素活性，又富含多酚類成分，因此本論文將針對石榴，進行抗痤瘡活性成分分離鑑定，與抗痤瘡活性機制探討。首先，利用不同溶劑萃取石榴，其中以70%丙酮萃取物抗菌活性最強，且總多酚類成分含量最多，繼而利用抗痤瘡桿菌與金黃色葡萄球菌進行活性追蹤，配合Diaion、TSK與ODS等管柱分離，結果得到4個具有抗菌活性成分，punicalagin (1)、punicalin (2)、strictinin A (3)與granatin B (4)。將4個活性多酚成分進行抗痤瘡活性評估，結果顯示：
(1). 1與2對P. acnes之最小抑菌濃度(MIC)為6.25 μg/disc，S. aureus之MIC為12.5 μg/disc。
(2). 1、3及4於200 μg/ml時，抑制超過30%脂質分解酵素活性。
(3). 1-4與角質細胞(HaCaT cell) 作用24小時後，具有明顯的劑量依存性抑制角質細胞增生，在無細胞毒性之濃度下 (25 μg/ml) ，1與2可抑制由testosterone所誘導之角質細胞增生。
(4). 以不影響細胞存活率的濃度下與巨噬細胞(RAW264.7)共同培養，1-4皆具有抑制LPS或P. acnes誘導細胞產生nitric oxide (NO) 作用，及抑制iNOS蛋白質表現。4亦具有抑制COX-2蛋白質表現，而降低前列腺素(PGE2)產生。另外1-4也具有抑制P. acnes刺激單核球細胞(THP-1)細胞產生促發炎細胞激素 (TNF-α與IL-8)的分泌。
綜合而論，利用古代典籍考究與現代科學的驗證，我們發現清熱藥與固澀藥中，富含多酚類成分，具有抗菌與抗發炎活性，且70%丙酮萃取多酚類含量最高。根據抗痤瘡活性追蹤分離石榴得到4個多酚類成分，皆具有抗菌、抑制脂質分解酵素、抑制角質細胞增生與抗發炎作用，又以1與2抗菌與抑制角質細胞增生活性最強，而4對LPS或P. acnes誘導之RAW264.7與THP-1細胞，抑制發炎介質(NO、PGE2、TNF-α與IL-8)活性最明顯。所以不同的多酚成分，對不同的痤瘡病因有不同效果，因此推測石榴70% 丙酮萃取物具有抗痤瘡作用，故將其進行單一劑量皮膚毒性評估，結果顯示石榴萃取物於10 mg/site之大鼠皮膚上，無明顯毒性。綜合結果，石榴萃取物中的主成分同時抑制多種痤瘡發生的病因，且萃取物無皮膚刺激，若加以定量確保品質，推測石榴萃取物式轉譯成抗痤瘡醫藥品的好原料。

關鍵字

痤瘡 ； 石榴 ； 唇形科植物 ； 痤瘡桿菌 ； 金黃色葡萄球菌 ； 脂質分解酵素 ； 角質細胞增生 ； 發炎反應

並列摘要

Acne is a common skin condition and usually appears in young adolescents with hormone imbalance. Major causes in acne formation include increased sebum production by overactive oil glands, retention hyperkeratosis to block the skin pores, activity of normal skin bacteria (Propionibacterium acnes and Staphylococcus aureus) and inflammation. Nowadays, therapeutic strategy for acne vulgaris is applying keratolytic agents and antibiotics. However, the increasing incidences of skin irritation and antibiotic resistance were found in recent acne remedies. Hence, it is urgent to explore an agent with abilities in inhibiting P. acnes and S. aureus growth, suppressing inflammatory responses and reducing skin irritation. The objective of this study was to discover an anti-acne traditional Chinese medicine (TCM) through system reviewing the ancient articles and National Health Insurance research database, surveying the commercial products, selecting the candidate TCM extracts and evaluating the anti-microbial and anti-inflammatory effects.
In this study, inhibitory abilities of herbal extracts on S. aureus and P. acnes growth and lipase activities, anti-inflammatory and anti-oxidative activities were used as evaluation platforms of the anti-acne effects. Punica granatum Linn. (pomegranate) and Terminalia chebula Retz. showed stronger anti-bacterial and anti-inflammatory activities than the others. P. granatum, T. chebula and S. baicalensis also significantly inhibited lipase activity. In the phytochemical analysis, P. granatum and T. chebula were rich in polyphenolics. Based on the commercial survey, Lamiaceae plants were the most popular source in anti-acne cosmetics. Moreover, we used 28 kinds of Lamiaceae plants with the similar genus combined with published experimental methods to establish the correlations between anti-acne abilities and phytochemical contents. Results showed that polyphenolic contents had the good correlations with anti-oxidative, anti-inflammatory and anti-bacterial activities (R = 0.767, p < 0.01). Good correlation was also found between anti-inflammatory and anti-bacterial activities (R = 0.542 and 0.613, p < 0.01).
Secondly, 50 % methanol pomegranate extracts showed more significant anti-oxidative, lipase inhibitory effects, anti-inflammatory and anti-bacterial activities than the others. Therefore, anti-acne principal constituents of pomegranate were explored for its active mechanisms. Firstly, pomegranate peel was extracted via different solvents and results showed that 70% acetone Pomegranate extracts was rich in the most polyphenolics and showed the strongest inhibitory activity against S. aureus and P. acnes. Column chromatography combined with in vitro bioassay-guided fractionation was used to isolate the anti-acne principal components from the pomegranate. Four hydrolysable tannins Punicalagin (1), Punicalin (2), Strictinin A (3) and Granatin B (4) were obtained with yields of 0.093%, 0.015%, 0.003%, and 0.013%, respectively. Among them, minimum inhibitory concentration (MIC) and minimum bacterial concentration (MBC) of 1 and 2 were 6.25 and 12.5 μg, respectively. 1, 3 and 4 (200 μg/ml) displayed more than 30% of lipase inhibitory effects. All these four components significantly inhibited HaCaT cell proliferations in a concentration-dependent manner after 24 hr treatments. Besides, 1 and 2 significantly reduced the testosterone-induced HaCaT cell proliferations at a non-toxic concentration (25 μg/ml). These pomegranate-polyphenolic components also decreased LPS- and heat-killed P. acnes-induced NO productions through down-regulating iNOS expression in RAW 246.7 cells, while 4 could reduce COX-2 expressions, resulting in decreasing PGE2 productions. Moreover, these pomegranate-polyphenolic components also reduced heat-killed P. acnes-induced TNF-α and IL-8 secretions. According to above results, different pomegranate-polyphenolic components showed different anti-acne effects. We suggested that pomegranate extract was a good anti-acne material source. Therefore, the pomegranate extrct was evaluated for the skin irritation. The results showed that extracts did not display significant toxicity on Wistar rat.
Taken together, active principal constituents of pomegranate extracts could inhibit different pathogenic factors of acne without skin irritations. We suggested that pomegranate was suitable to develop the anti-acne agent.

並列關鍵字

anti-acne ； pomegranate ； Lamiaceae plants ； Propionibacterium acnes ； Staphylococcus aureus ； lipase ； keratinocytes ； inflammrtion

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