草本植物萃取物之抑菌活性及抗氧化能力

微生物之研究促進了產業的興盛，但病原菌汙染卻也是無法迴避的問題，基於抗生素濫用及對環境之影響，天然抑菌素材之研究逐漸被重視。有別過往專注於單一成分之探討，未經純化之粗萃取物被認為更為貼近自然且無害，其成本及製程也更加適用於大量生產。本文中將植物之乾燥粉末以溶劑及超臨界萃取法萃取分別萃取出穿心蓮萃取液 (Andrographis paniculata extracts, APE) 、芳香萬壽菊萃取液 (Tagetes lemmonii extracts, TLE)及小花山奈萃取液 (Kaempferia parviflora extracts, KPE)，並額外以水蒸餾法萃取新鮮芳香萬壽菊之精油。實驗中先是以瓊脂擴散法確認植物萃取液對於試驗菌株之抑菌範圍，再以液態培養基稀釋法定量固定濃度之萃取液對於14株試驗菌株之抑菌活性，最後則以微量稀釋法及細胞存活率分析 (Thiazolyl Blue Tetrazolium Bromide, MTT) 檢測其最低抑制濃度。對於實驗樣品之抗氧化能力則會先定量其總酚及總類黃酮含量，再以DPPH自由基清除能力與還原力比較不同萃取條件所得之萃取液之抗氧化活性。經實驗數據顯示，在溶劑萃取法中75%與95%乙醇所萃取之APE、TLE及KPE對於Lactobacillus plantarum以外之革蘭氏陽性菌株及革蘭氏陰性菌株Vibrio cholerae有不同程度之抑菌效果，於液態培養基稀釋法實驗中可見，95%乙醇萃取之APE萃取液在10%的濃度下能夠完全抑制 (菌數自8 Log CFU/mL降至低於1 Log CFU/mL) Staphylococcus aureus、Listeria monocytogenes、及V. cholerae，並其最低抑制濃度分別為12.5 μL/mL、3.125 μL/mL及12.5 μL/mL之間。而超臨界萃取法之APE則是能夠完全抑制L. monocytogenes、Streptococcus mutans及V. cholerae等受試菌株，其最低抑制濃度分別為12.5 μL/mL、25 μL/mL及50 μL/mL之間。而芳香萬壽菊之精油則為實驗樣品中抑菌能力最廣泛者，對於L. plantarum及Pseudomonas aeruginosa以外之所有受試菌株，包含酵母菌都有著良好的抑菌活性，於實驗中1%之精油可有效將其完全抑制，且最低抑制濃度皆低於5 μL/mL。實驗所使用之三種植物中，總酚含量 (以沒食子酸當量作為總酚相對含量, mg GAE/g ) 最高者為芳香萬壽菊，其75%乙醇萃取液及其精油分別具有55.75±0.41 mg GAE/g及34.26±0.94 mg GAE/g之當量值；小花山奈則次之，75%乙醇萃取之小花山奈萃取液具有11.03±0.2 mg GAE/g之當量值；而穿心蓮萃取液則最低，僅5.57±0.16 mg GAE/g之當量值。75%乙醇萃取之芳香萬壽菊萃取液總類黃酮含量 (以槲皮素當量作為總類黃酮相對含量, mg QE/g ) 約為9.22±0.43 mg QE/g，僅為總酚含量之20%左右，其精油更是只有1.84±0.03 mg QE/g，總黃酮類僅占總酚含量之5%；而小花山奈及穿心蓮之總類黃酮含量分別為6.24±0.3 mg QE/g及2.94±0.14 mg QE/g，大約為其總酚含量的50%至60%，亦即表示小花山奈中及穿心蓮的酚類化合物多以類黃酮之形式存在。DPPH自由基清除能力則以75%乙醇萃取之穿心蓮萃取液及芳香萬壽菊之精油為最高，5%之濃度下都有96%以上之清除率，而芳香萬壽菊之75%乙醇萃取液則次之，約94%之清除率，小花山奈則相對較低，約84%之清除率，而其還原力則是與總酚之含量有相同之趨勢。

關鍵字

穿心蓮；芳香萬壽菊；小花山奈；抑菌活性；最低抑制濃度；抗氧化能力

並列摘要

Microorganism has promoted the prosperity of Primary industrial sectors, but the contamination of pathogenic bacteria is still unavoidable. Cause of the abuse of antibiotics and its negative impact on the environment, bacteriostatic material development direction began to bias the study of natural materials. In the past, we usually focused on the single component, but the purer on behalf of the more expensive. The composite crude extract was more natural and harmless, and its cost and process were more suitable for mass production. In this study, the dry powder of the plants is extracted by solvent and supercritical extraction. They are Andrographis paniculata extracts (APE), Tagetes lemmonii extracts (TLE) and Kaempferia parviflora extracts (KPE). The essential oil of T. lemmonii (TLE-EO) was extracted by water distillation. In the experiment, the agar diffusion method was used to confirm the range of antimicrobials activity of plant extracts against the test strains. The broth dilution method was used to quantify the antibicrobial activity of the extracts, and the minimum inhibitory concentration (MIC) was measured by the microdilution method with the MTT reagent. For the antioxidant capacity of the extracts, we measured the total phenolic, total flavonoid content, DPPH free radical scavenging ability and reducing power. In the experiment, it was found that APE, TLE and KPE extracted by 75% and 95% ethanol in solvent extraction inhibited Gram-negative strain Vibrio cholerae and most of Gram-positive strains. It could be seen that the 10% concentration extracts can reduce the total count number of Staphylococcus aureus, Listeria monocytogenes, and V. cholerae from 8 Log CFU/mL to 1 Log CFU/mL. Its minimum inhibitory concentration were 12.5 μL/mL, 3.125 μL/mL and 12.5 μL/mL. The APE extracted by supercritical extraction showed the antimicrobials activity against L. monocytogenes, Streptococcus mutans and V. cholerae. The minimum inhibitory concentration was 12.5 μL/mL, 25 μL/mL, and 50 μL/mL.TLE-EO showed the strongest antimicrobials activity against tested strains except Lactobacillus plantarum and Pseudomonas aeruginosa. They were be inhibited and reduced the total count number of strains to 1 Log CFU/mL or less than that by 1% of essential oil. The minimum inhibitory concentration was less than 5 μL/mL. Among the plants used in this experiment, the highest total phenol content (The gallic acid equivalent is used as the total phenol relative content, mg GAE/g) was T. lemmonii, its 75% ethanol extract and essential oils had an equivalent of 55.75±0.41 mg GAE/g and 34.26±0.94 mg GAE/g, respectively. The 75% ethanol extract of KPE and APE had an equivalent value of 11.03±0.2 mg GAE/g and 5.57±0.16 mg GAE/g. The total flavonoid content (The quercetin equivalent is used as the total phenol relative content, mg QE/g) of TLE, KPE and APE extracted by 75% ethanol are 9.22±0.43 mg QE/g, 6.24±0.3 mg QE/g and 2.94±0.14 mg QE/g, respectively. In the concentration of 5% APE and TLE-EO, that showed more than 96% DPPH free radical scavenging rate.TLE and KPE extracted by 75% ethanol got 94% and 84% scavenging rate.The result of reducing power of the extracts were the same as the total phenol content.