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標題: 虎頭蜂毒與中草藥萃取物對抗臨床病原菌之研究
Studies of the microbicidal effect of Vespa venom and Chinese herbal extracts on clinical pathogens
作者: 徐慶霖
Shyu, Ching-Lin
Contributors: 邱繡河
微生物暨公共衛生學研究所
關鍵字: 黃腰虎頭蜂蜂毒;旋覆花;川烏;草烏;抗菌活性
Vespa affinis venom;Inula japonica Thunb.;Aconitum carmichaeli Debx.;Aconitum kusnezoffii Reichb.;antibacterial activity
日期: 2013
Issue Date: 2013-11-19 12:50:56 (UTC+8)
Publisher: 微生物暨公共衛生學研究所
摘要: 針對臨床上細菌之抗藥性菌株,期望以天然物蜂毒(venom)及中草藥為標的,開發出新的抗菌物質為目標來進行。因此,本研究分為黃腰虎頭蜂(Vespa affinis)毒液和中草藥萃取液兩大部分,分別進行抗菌活性研究分析。研究一為黃腰虎頭蜂工蜂毒液應用於臨床抗藥性細菌之抗菌活性研究,其毒液呈清澈淡黃色,pH值介於5到6之微酸性液體,蛋白質濃度約為16.3 mg/ml到39.4 mg/ml,且富含多種具生物活性的分子。以改良過的柯鮑二氏法(Kirby-Bauer method)測試細菌對蜂毒之感受性,結果顯示革蘭氏陽性菌和革蘭氏陰性菌對於黃腰虎頭蜂蜂毒皆具有強烈的抗菌效能。進一步利用高效液相層析管柱(Phenomenex LunaR C18(2) 5μm)純化,可將毒液分離為6個明顯區段,其中僅有區段5具有抗菌活性。再利用Phenomenex JupiterR 5 μm C4 300 A管柱,可將區段5分離出fraction 5-1 和5-2兩個區段,經敏感性測試顯示fraction 5-1具抗菌活性。Fraction 5-1經快速原子撞擊法質譜儀(fast-atom-bombardment mass spectrometer)測得其分子量為1512.96 Da,胺基酸序列定序結果顯示其為14個胺基酸分子組成之胜肽,進一步依胺基酸序列設計核酸引子,可自基因組(genome)解讀其基因序列,並證實此胜肽歸屬於肥大細胞脱粒肽(mastoparan)系列,並將此胜肽命名為肥大細胞脱粒肽-AF(mastoparan-AF),此研究結果顯示黃腰虎頭蜂毒液中短鏈胜肽mastoparan-AF具有抗菌活性。研究二鑒於市面上動物皮膚疾病之藥浴治療產品多含化學物質,恐有藥物殘留與抗藥性細菌產生之虞,故利用傳統醫學使用之抗微生物中草藥應用於動物抗藥性細菌皮膚病藥浴治療之開發。各中草藥分別以水與醇類萃取後所得之萃取物,對於動物臨床常見引起皮膚感染之細菌與黴菌,進行藥物敏感性測試試驗,篩選出旋覆花(Inula japonica Thunb.)、川烏(Aconitum carmichaeli Debx.)、草烏(A. kusnezoffii Reichb.)等中草藥萃取物具有抑菌活性。中草藥萃取物進一步輔以適當界面活性劑經小型量產製成半成品後,用於具皮膚病徵之分組試驗犬隻,觀察並予以統計其各階段之藥效。動物試驗四週後,實驗組患犬皮膚狀況分級指數分別為1.14、0.71、1.57,皆優於對照組患犬之皮膚分級指數2。研究成果顯示黃腰虎頭蜂毒液之mastoparan-AF及旋覆花、川烏、草烏等中草藥萃取物,均對臨床抗藥性細菌具有抗菌活性。未來經量產製程之調整,進一步結構蛋白之分析與詳細生物活性之探究,為下一個目標。
The study is aimed to measure the antibacterial activity of polypeptide isolated from Vespa affinis venom and Chinese herbal extracts. The venom from V. affinis is a clear and light-yellow liquid with pH range 5 to 6. The protein concentration of the venom is 16.3 to 39.4 mg/ml. A strong antibacterial activity of vespa venom for gram positive and negative bacteria is founded by the Kirby-Bauer method. This venom can be separated as six fractions which are analyzed by using reverse phase High-performance liquid chromatography (HPLC), SDS-PAGE, and fast-atom- bombardment mass spectrometer, but the fraction 5 has antibacterial activity. The fraction 5 can be divided into the fraction 5-1 and the fraction 5-2 but only the fraction 5-1 with the molecular weight of 1512.96 Dalton, showed an antibacterial activity. After forth advanced analysis, the fraction 5-1 composed of 14 amino acids is classified as mastoparan-AF, the main composed of antibacterial compound from the V. affinis venom. Based on the chemical toxicity of shampoo used for pets, the second aim of this study is to evaluate the effects of herbal extracts on bacterial pathogens of clinical cases to evaluate a potential shampoo for the pet use. The antibacterial activity of crude extracts of 22 herbs were founded to be against 7 bacteria and 4 fungi. The therapeutic effects of crude alcoholic extracts of Inula japonica Thunb., Aconitum carmichaeli Debx. and A. kusnezoffii Reichb. on canine skin diseases were studied and an excellent antibacterial activity was founded compared with the control group. In conclusion, mastoparan-AF from the V. affinis venom and alcoholic extracts of I. japonica Thunb., A. carmichaeli Debx., and A. kusnezoffii Reichb. showed strong antibacterial activity. Finally, more study is needed to determine the structural of this protein and it’s bioactivity.
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