植物萃取物對痤瘡致病菌之生長、菌膜形成與脂解酶活性的抑制作用

Inhibitory effects of botanical extracts on the growth, biofilm and lipase activity of acne-causing bacteria

嶺瑤蓉(Yau Rong Liing)

指導教授 ： 蔡帛蓉

國立臺灣師範大學/教育學院/人類發展與家庭學系/碩士(2011年)

摘要

尋常性痤瘡(acne vulgaris)，是一種常見的皮膚疾病。痤瘡桿菌(Propionibacterium acnes)和表皮葡萄球菌(Staphylococcus epidermidis)存在於人類皮膚含皮脂腺的毛囊(pilosebaceous follicle)，在尋常性痤瘡的致病過程中扮演著重要角色。這些痤瘡致病菌(acne-causing bacteria)會形成菌膜(biofilm)抵抗外在不良的環境條件，也是產生抗藥性的因子之ㄧ，另外其所分泌的胞外脂解酶，水解皮脂中三酸甘油酯供給菌體所需養份，同時誘發後續的發炎反應。P. acnes感染角質細胞時，會刺激細胞產生超氧陰離子(superoxide anions)，雖抑制P. acnes生長但會引發角質細胞分解，誘導發炎反應。
我們篩選34種市售植物乙醇萃取物的抗菌活性，結果發現迷迭香(Rosmarinus officinalis )、鼠尾草(Salvia officinalis L. )、紅茶、綠茶(Camellia sinensis )及馬鞭草(Verbena officinalis Linn)可有效抑制P. acnes生長。所以在後續實驗，我們製備了上述五種植物的乙醇、甲醇、乙酸乙酯(ethyl acetate)及水萃取物作為試驗材料，分別評估其抗菌能力以及對P. acnes菌膜(biofilm)，脂解酶活性(lipase activity)和對活性氧屬的抑制效果。最後，使用HPLC分析迷迭香萃取物之有效成份並加以定量。
本研究結果發現，在乙醇萃取物中，抑制P. acnes生長效果最好的是紅茶及綠茶，而抑制S. epidermidis生長效果最好的則是馬鞭草。在甲醇萃取物中，抑制P. acnes生長效果最好的是紅茶及綠茶，而抑制S. epidermidis生長效果最好的則為迷迭香萃取物。在乙酸乙脂萃取物中，抑制P. acnes生長效果最好的是紅茶及綠茶，而抑制S. epidermidis生長效果最好的則為迷迭香。在水萃取物中，抑制P. acnes和S. epidermidis生長效果最好的皆為紅茶及綠茶，迷迭香於本實驗的條件下，不具有抑菌的效果，馬鞭草萃取物不具有抑制S. epidermidis生長的效果。
植物萃取物對P. acnes菌膜形成(biofilm formation)之影響的實驗結果，在菌膜生成過程中，觀察植物萃取物於培養第16 hr和48 hr時對於菌膜biomass的影響，這兩個時間點分別用來代表萃取物抑制菌膜形成(prevention of biofilm formation；簡寫為PBF)和移除菌膜的能力(removal of established biofilm；簡寫為REB)。結果發現，迷迭香、鼠尾草、紅茶、綠茶及馬鞭草的乙醇、甲醇、EA及水萃取物具有PBF之能力，而鼠尾草和綠茶的乙醇、甲醇及水萃取物具REB之效果。
植物萃取物抑制P. acnes及S. epidermidis脂解酶活性的實驗結果中，馬鞭草、迷迭香、鼠尾草、綠茶以及紅茶萃取物皆顯著抑制脂解酶活性。植物萃取物清除自由基之能力的實驗結果中，(a)清除DPPH及一氧化氮自由基能力：綠茶及紅茶萃取物清除DPPH及一氧化氮自由基能力最好，迷迭香及馬鞭草萃取物次之，鼠尾草萃取物最差。(b)清除超氧陰離子自由基能力：綠茶及紅茶萃取物清除超氧陰離子自由基能力最好，迷迭香及鼠尾草萃取物次之，馬鞭草萃取物最差。(c)清除羥基自由基能力：綠茶及紅茶萃取物清除羥基自由基能力最好，鼠尾草及馬鞭草萃取物次之，迷迭香萃取物最差。(d)總多酚含量：綠茶及紅茶萃取物總酚含量最高，馬鞭草萃取物次之，迷迭香及鼠尾草萃取物最少。HPLC分析迷迭香萃取物之有效成份的實驗結果中，除了迷迭香水萃物外，迷迭香乙醇、甲醇及EA萃取物皆含有迷迭香、鼠尾草酚及鼠尾草酸等有效成份，並且含量皆是迷迭香甲醇萃取物最高、乙醇萃取物次之，EA萃取物含量最少。
綜合上述結果，迷迭香、鼠尾草、紅茶、綠茶及馬鞭草萃取物均具有抑制痤瘡致病菌生長、菌膜形成及脂解酶活性之能力，並且具有優異的抗氧化能力，故以上五種植物萃取物具有治療痤瘡及抗氧化的潛力。未來在臨床上可應用於預防或是緩解痤瘡，也能減少使用抗生素所產生之抗藥性。

關鍵字

尋常性痤瘡 ； 痤瘡桿菌 ； 表皮葡萄球菌 ； 脂解酶 ； 菌膜 ； 抗菌 ； 抗氧化 ； HPLC

並列摘要

Acne vulgaris is a chronic disorder of the pilosebaceous follicles of the skin. Acne-causing bacteria, such as Propionibacterium acnes and Staphylococcus epidermidis, play an important role in the pathogenesis of acne vulgaris. Both bacteria resided within the follicles grow as a biofilm and hydrolyzed triglyceride to free fatty acids by bacterial lipase has been proposed as a major factor in the pathogenesis of acne vulgaris. ROS, and especially superoxide anions, were rapidly produced by keratinocytes upon stimulation by P. acnes surface proteins. In addition, superoxide anions were generated by P. acnes stimulated keratinocytes and involved in the development of acne inflammatory lesions. The aim of this study was to investigate inhibitory effects of various botanical extracts on growth, biofilm formation, and extracellular lipase activity of acne-causing bacteria and their anti-oxidation properties. Aqueous, methanolic, ethanolic and EA extract of rosemary were analyzed and quantified by HPLC.
The antimicrobial activity of extracts was assessed by determining of minimum inhibitory concentration (MIC) values obtained by a modified microdilution broth method. Results showed that ethanolic, methanolic, ethyl-acetate and aqueous extracts of rosemary, sage, verbena, green tea and black tea significantly inhibited the growth of acne-causing bacteria, including P. acnes and S. epidermidis. Aqueous extract of verbena significantly inhibited the growth of P. acnes.
We also tested the inhibitory activity of botanical extracts on the biofilm-forming ability of P. acnes in a microtiter plate model. In a preliminary experiment, we determined the optimal conditions for P. acnes biofilm formation. A mature biofilm (i.e. a biofilm in which the biomass does not significantly increase any further) was established following 48 hr incubation. Following 16 hr incubation, ethanolic, methanolic, ethyl-acetate and aqueous extracts of rosemary, sage, verbena, green tea, and black tea significantly prevented biofilm formation of P. acnes. In addition, ethanolic, methanolic and aqueous extracts of sage and green tea could remove established mature biofilm of P. acne.
Furthermore, we conducted experiments to investigate the inhibitory effect of botanical extracts on crude lipase activity of acne-causing bacteria. Results showed that ethanolic, methanolic, ethyl-acetate and aqueous extracts of rosemary, sage, verbena, green tea and black tea significantly inhibited lipolytic activity of crude lipase purified from P. acnes and S. epidermidis.
In the present study, the antioxidant activity of botanical extracts was determined by the scavenging activities of 2, 2-diphenyl-1-picrylhydrazyl (DPPH), nitric oxide, superoxide and hydroxyl radical . We also evaluated the total phenolic contents of botanical extracts. The potent radical scavenging effects were observed in ethanolic, methanolic, ethyl-acetate and aqueous extracts of rosemary, sage, verbena, green tea, and black tea. Besides aqueous extract of rosemary, rosmarinic acid, carnosol and carnosic acid were determined and quantified by HPLC in the methanolic, ethanolic and EA extract of rosemary.
In conclusion, extracts of rosemary, sage, verbena, green tea, and black tea had potent inhibitory effects on the growth, lipolytic activity and biofilm formation of acne-causing bacteria and possessed excellent anti-oxidative properties.These data suggested that extracts of rosemary, sage, verbena, green tea, and black tea may be potentially therapeutic agents for the treatment of acne vulgaris.

並列關鍵字

Propionibacterium acnes ； Staphylococcus epidermidis ； herbs ； anti-acne ； lipase ； biofilm formation ； anti-oxidation ； HPLC

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