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系統識別號 U0007-2807201118460200
論文名稱(中文) 肉豆蔻抗痤瘡之活性成分研究
論文名稱(英文) Anti-acne Principle Constituents of Myristica fragrans Houtt.
校院名稱 臺北醫學大學
系所名稱(中) 生藥學研究所
系所名稱(英) Graduate Institute of Pharmacognosy
學年度 99
學期 2
出版年 100
研究生(中文) 黃俊瑋
研究生(英文) Chun-Wei Huang
學號 M303098002
學位類別 碩士
語文別 中文
口試日期 2011-07-20
論文頁數 100頁
口試委員 指導教授-王靜瓊
委員-呂彥禮
委員-鄭幼文
中文關鍵字 肉豆蔻  痤瘡  抗菌  抗發炎 
英文關鍵字 Myristica fragrans Houtt.  acne  anti-microbial activity  anti-inflammation 
學科別分類
中文摘要 痤瘡又稱為青春痘,是一種在青春期常見的皮膟病症並在皮膟上留下疤痕,往往引起自尊心下降以及憂鬱等不好的影響,且目前尚無較安全及有效的治療方法,因此本研究以抗痤瘡菌為主要活性追蹤,篩選27種中草藥,繼而分離純化其活性成分,結果顯示肉豆蔻50%甲醇萃取物抑制作用較其他中草藥顯著,對金黃色葡萄球菌在10 mg/紙錠下,其抑菌圈直徑為13.5 mm。
肉豆蔻種仁富含揮發油,其中黃樟素及肉豆蔻醚是主要有毒成分,因此利用水蒸氣蒸餾法,得到精油後(essential oils),藥渣再以50%甲醇溶液萃取(MFE-M),兩種萃取物進行抗菌試驗,結果顯示:精油對金黃色葡萄球菌以及痤瘡桿菌的最小抑菌濃度分別為80 % (v/v)和20 % (v/v);而MFE-M萃取物對兩株菌最小抑菌濃度(MIC)皆為0.64 mg/紙錠下。利用抗菌活性追蹤分離具活性之劃分部,以矽膞管柱分離到無抗菌活性的myristicin (1) 及3個有效新木質素類天然物,分別為(+)-erythro-(7S,8R)-Δ8’,3,3’,5’ -trimethoxy -8-O-4’-neolignan (2), (+)-erythro-(7S,8R)-Δ8’,3,3’,4,5’- tetramethoxy -8-O-4’-neolignan (3) 和erythro-Δ8’-7-acetoxy-3,4,3’,5’- tetramethoxy -8-O-4’- neolignan (4)。結果顯示: (2)抑菌活性最強,且對金黃色葡萄球菌及痤瘡桿菌MIC分別為3.12 μg/ml及6.25 μg/ml。進而利用LPS以及熱處理的P. acnes (PA) 誘導RAW 264.7細胞發炎反應,評估(2)-(4)抑制NO、PGE2產生及COX-2、iNOS蛋白表現之作用,結果發現:(2)具有最強的抑制NO生成作用,其IC50分別為11.07 μg/ml(誘導劑:LPS)以及11.53 μg/ml(誘導劑:PA,)。
據結果,MFE-M萃取物具有抗痤瘡之作用,且含有抗痤瘡致病菌及抗發炎活性成分(2),因而分析其內有毒成分黃樟素以及肉豆蔻醚以評估其毒性。結果MFE-M萃取物中肉豆蔻醚為2.47 %、黃樟素0.01 %,而精油中則為2.62 %及4.46 %,表示去精油後之肉豆蔻萃取物,其黃樟素明顯降低。繼而將兩萃取物再進行皮膟單一劑量毒性檢測,將其塗抹於大鼠皮膟,24及48小時後觀察,幾乎不會引起刺激反應。因此,我們發現MFE-M其內黃樟素含量顯著下降、含有抗痤瘡致病菌、抗發炎之活性成分(2),且不具皮膟刺激性,因而推測此萃取物適合開發為抗痤瘡之醫藥用品。
英文摘要 Acne is a common skin condition in adolescence and usually results in scarring. It also leads to severe psychological side effects, such as self-esteem reduction and depression. So far, there are not safty and effective drugs for acne therapy. Therefore, 27 kinds of herbs were screened by anti-acne bacterial assay. Among them, 50% methanol extract of Myristica fragrans Houtt. (MF) most inhibited acne bacteria and in the dose of 10mg/disc, the inhibition zone diameter was 13.5 mm. Then we explored the anti-acne bacterial components in MF. The major toxic components of MF were safrole and myristicin. The essential oils of MF were extracted by hydrodistillation and the dregs were extracted by 50% methanol (MFE-M), again. And then the essential oil and MFE-M were evaluated the anti-bacterial activity. Results showed the minimum inhibitory concentration (MIC) of the essential oil against S. aureus and P. acnes were 80 % (v/v) and 20 % (v/v), respectively;and the MIC of MFE-M against both S. aureus and P. acnes was 0.64mg/disc. The active fractions (MFE-M) were isolated with silica gel column and combined with anti-microbial assay. Four compounds were obtained from MFE-M, myristicin (1) which was non-active and 3 effective neolignans: (+)-erythro-(7S,8R)-Δ8’,3,3’,5’-trimethoxy-8-O-4’-neolignan (2)、(+)-erythro-(7S,8R)-Δ8’,3,3’,4,5’-tetramethoxy-8-O-4’-neolignan (3) and erythro-Δ8’-7-acetoxy-3,4,3’,5’-tetramethoxy-8-O-4’-neolignan (4). However, (2) was the strongest anti-microbial activities than the others, the MICs against S. aureus and P. acnes were 3.12 μg/ml and 6.25 μg/ml, respectively. Moreover, (2) could inhibited NO, PGE2 production and COX-2, iNOS eapression in LPS or heat killed-P. acnes induced RAW 264.7 cells and the 50% inhibitory concentrations (IC50) were 11.07μg/ml (by LPS inducer ) and 11.53 μg/ml ( by heat killed- P. acnes inducer).
The toxic components of MFE-M and essential oil were analyzed by HPLC system. Results showed that MFE-M was in 2.47% myristicin and 0.01% safrole and essential oil was 2.62% and 4.46%. And then, the MFE-M and essential oil were smeared on rats for 24 and 48 h, the skin of rats was not caused irritation. According to the above results, MFE-M showed anti-acne effect which contained anti-microbial and anti-inflammatory compound, (2). However, MFE-M contained a few safrole and myristicin and did not cause skin irritation. Therefore, we suggested this extract has the potential to develope to anti-acne drugs and supplements of cosmetic.
論文目次 目錄...................................................... I
圖目錄 ................................................. IV 表目錄 .................................................. VI
縮寫表 ................................................. VII
摘要 ..................................................... 1
Abstract ................................................. 3
壹、 緒論 ................................................ 5
一、 痤瘡概論 ............................................ 5
(一) 痤瘡病因 .......................................... 6
(二) 痤瘡症狀與分級 .................................... 9
(三) 西方醫學治療痤瘡之方法 ........................... 12
(四) 傳統醫學治療痤瘡之方法 ........................... 16
二、 肉豆蔻文獻回顧 ..................................... 17
(一) 肉豆蔻基原 ....................................... 17
(二) 植物性狀 ......................................... 18
(三) 藥材性狀 ......................................... 20
(四) 肉豆蔻典籍考察 ................................... 21
(五) 肉豆蔻藥理 ....................................... 28
三、 實驗方法介紹 ....................................... 31
(一) 抗菌實驗模式 ..................................... 31
(二) 抗發炎實驗模式 ................................... 31
四、研究目的 ............................................ 32
五、 研究流程 ........................................... 33
貳、 實驗材料與方法 ..................................... 34
一、 實驗材料 ........................................... 34
二、 實驗方法 ........................................... 38
(一) 肉豆蔻成分含量分析及分離純化 ..................... 38
(二) 肉豆蔻抑制痤瘡致病菌生長之評估 ................... 42
(三) 肉豆蔻抑制發炎活性評估 ........................... 46
(四) 肉豆蔻單一劑量皮膟毒性試驗 ....................... 53
參、 實驗結果 ........................................... 55
一、 27種中草藥萃取物抗菌試驗 ........................... 55
二、 肉豆蔻成分分離純化 ................................. 56
(一) 抗菌活性追蹤分離肉豆蔻及結構解析 ................. 56
三、 肉豆蔻之天然物抗痤瘡之活性 ......................... 65
(一) 肉豆蔻之天然物抗痤瘡致病菌活性 ................... 65
(二) LPS誘導RAW 264.7細胞發炎反應及MTT試驗 ............ 67
(三) 熱處理P. acnes誘導RAW 264.7細胞發炎反應 .......... 70
四、 肉豆蔻萃取物中myristicin及safrole之含量 ............ 73
(一) Myristicin及Safrole檢量線 ........................ 73
(二) 分析方法之確效性 ................................. 73
(三) Essential oil及MFE-M成分含量 ..................... 74
五、 肉豆蔻單一劑量皮膟毒性試驗 ......................... 76
肆、 討論 ............................................... 77
伍、 結論 ............................................... 82
陸、 參考文獻 ........................................... 83
附錄一 .................................................. 90
附錄二 .................................................. 92
附錄三 .................................................. 95
附錄四 .................................................. 98
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