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The antimicrobial and anti-inflammatory effects of spice extracts against Propionibacterium acnes
Toll-like receptor 2
|Abstract:||尋常性痤瘡(acne vulgaris)俗稱青春痘是一個非常常見的皮膚疾病，好發年紀除了青春期的青少年外，在成年人也常見。痤瘡的致病機轉牽涉到非常多的因素，包括不正常的過度角質化、皮脂分泌增加、荷爾蒙、免疫反應、皮膚內微生物特別是痤瘡桿菌。常見的治療方式是使用抗生素，但是現行資料顯示人體皮膚內的痤瘡桿菌對於常用抗生素有越來越多抗藥性的情形出現，因此開發非抗生素類的治療藥物是一個漸受重視的議題。本研究的目的為篩選出具有抗菌作用能夠抑制痤瘡桿菌(Propionibacterium acnes)的生長，或具有抗發炎作用的天然食材能夠抑制由P. acnes 所誘發的發炎反應以緩解尋常性痤瘡。
首先進行預實驗，以抗菌能力作為篩選標的，得知4種香料(culinary spices)的乙醇萃取物具有抑制痤瘡桿菌生長的作用。這4種香料為迷迭香、百里香、丁香和奧勒岡，其最低抑菌濃度(MIC)分別為4 , 8, 8, 16 mg/mL。接著進行本研究，
將這4種香料以不同溶劑(乙醇、乙酸乙酯、水)萃取，分析項目有(1)抑制P. acnes與表皮葡萄球菌(S. epidermidis)生長的能力，(2)抗發炎活性：抑制P. acnes刺激單核球細胞THP-1細胞產生促發炎細胞激素的能力，(3)探討其抗發炎作用的可能機制。
本研究結果發現，在乙醇萃取物中，抑制P. acnes 和S. epidermidis生長效果最好的是迷迭香，百里香與奧勒岡次之，丁香最差；乙酸乙酯萃取物抑制P. acnes生長的效果以奧勒岡最好，抑制S. epidermidis生長的效果以迷迭香效果最好，而丁香乙酸乙酯萃取物於本實驗條件下，對於兩株菌皆無抗菌的效果；在水萃取物的部份，抑制P. acnes和S. epidermidis生長的效果以丁香效果最好，迷迭香和百里香於本實驗的條件下，不具有抑菌的效果。在抗發炎活性方面，以不影響細胞存活率的萃取物濃度與THP-1細胞共培養，發現迷迭香、丁香與奧勒岡的乙醇萃取物均能夠抑制P. acnes刺激THP-1細胞產生促發炎細胞激素 (TNF-α, IL-8 and IL-1β)的分泌與mRNA基因表現量。然而這些香料的乙酸乙酯與水萃取物卻有促發炎的現象。由於已知P. acnes活化toll-like receptor 2(TLR2)以及下游的訊息傳遞路徑，活化轉錄因子NFκB，進而調控促發炎細胞激素的表現。所以我們進一步探討其抗發炎機制是否經由TLR2和NFκB路徑。結果發現P. acnes顯著的誘導THP-1細胞的NFκB活化情形以及TLR2的mRNA表現，而這些活化路徑均能被迷迭香、丁香與奧勒岡乙醇萃取物所抑制。所以綜合上述實驗結果，我們推測這3種香料乙醇萃取物的抗發炎機制是能透過減弱單核球細胞膜上的TLR2的表現以及轉錄因子NFκB的活化，進而降低促發炎細胞激素的分泌與基因表現。
Acne vulgaris is a skin disorder of the pilosebaceous follicles that commonly occurs in adolescence and in young adulthood. The pathogenesis of acne is complex, but this process occurs in the pilosebaceous unit and there is strong evidence to suggest that it involves sebaceous hyperplasia, follicular hyperkeratinization, bacterial hypercolonization, together with the immune reaction and inflammation. Several lines of evidence suggest that the anaerobic bacterium Propionibacterium acnes is the etiological agent of inflammatory acne. For many years, antibiotics have been used to treat acne vulgaris, however, antibiotic resistance has been increasing in prevalence within the dermatologic setting. Bacterial resistance is an ongoing problem in the treatment of acne vulgaris, therefore, an agent which can inhibit P. acnes growth and suppress the inflammatory response will provide promising benefits to patients with acne vulgaris. The objective of this study was to screen and evaluate the antimicrobial and anti-inflammatory effects of culinary spice extracts against P. acnes. In the preliminary study, the inhibitory effects of various ethanolic spice extracts on P. acnes growth were determined by broth dilution method. We found that 4 ethanolic spice extracts possessed antibacterial activity against P. acnes. The minimum inhibition concentrations (MICs) of rosemary, clove, thyme, and oregano were 4, 8, 8, and 16 mg/mL, respectively. We examined the antimicrobial and anti-inflammatory effects of the above spice extracts and addressed possible mechanism of anti-inflammation in the subsequent study. In this study, ethanol, ethyl acetate and aqueous extracts of rosemary, clove, thyme, and oregano were prepared. Results showed that, rosemary showed the greatest antimicrobial activity against P. acnes and S. epidermidis than thyme, oregano and clove in ethanolic extracts. For ethyl acetate extracts, oregano showed the greatest antimicrobial activity against P. acnes, and rosemary showed the greatest antimicrobial activity against S. epidermidis, however, clove has no inhibitory effect on growth of both P. acnes and S. epidermidis. For aqueous extracts, clove showed the greatest antimicrobial activity against P. acnes and S. epidermidis, rosemary and thyme has no bactericidal effect. It is widely accepted that inflammatory acne may be induced by host immune reactions to P. acnes and production of pro-inflammatory cytokines such as IL-1β, IL-8, and TNF-α. We also found that P. acnes lead to increased production and gene expression of TNF-α, IL-8, and IL-1β in THP-1 cells. Ethanolic extracts of rosemary, clove and oregano strongly suppressed the secretion of P. acnes-induced pro-inflammatory cytokines by THP-1 cells, including TNF-α, IL-8, and IL-1β, without inhibition of cell viability. Whereas, ethyl acetate and aqueous extracts of rosemary, clove and oregano raised production of P. acnes-induced pro-inflammatory cytokines. P. acnes, by acting on TLR2, may stimulate the secretion of cytokines by macrophages. TLR2 was also proved to be sufficient for NF-B activation in response to P acnes. We found that ethanolic extracts of rosemary, clove and oregano suppressed P. acnes-induced TLR2 mRNA expression and NF-κB activation. Taken together, these spice extracts possess the anti-inflammatory activity against P. acnes, which may be through down-regulating TLR2 expression and NF-κB activation. In conclusion, these results suggest that ethanolic extracts of rosemary, clove and oregano, or their components, exhibit antimicrobiral and anti-inflammatory activities and may be useful in the treatment of acne vulgaris.
|Appears in Collections:||學位論文|
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