小葉葡萄抑菌成分Heyneanol A分離、鑑定及生產之研究

Abstract

本研究之目的為分離及鑑定野生葡萄中所含之抑菌物質，並探討其生產與應用。首先以濾紙片擴散法（disc diffusion method）對細本山葡萄（Vitis thunbergii Sieb. et Zucc.）、漢氏山葡萄（Ampelopsis brevipedunculata var. hancei）、厚皮葡萄（V. rotundifolia var. jumbo）和基隆葡萄（V. kelungensis）等野生葡萄之葉、枝及根萃取物進行抑菌能力篩選試驗，結果顯示葡萄屬（Vitis）葡萄之根萃取物對於枯草桿菌（Bacillus subtilis）、金黃色葡萄球菌（Staphylococcus aureus）和抗藥性金黃色葡萄球菌（methicillin-resistant Staphylococcus aureus, MRSA）等格蘭氏陽性菌（Gram-positive bacteria）具有抑制效果，最小抑菌濃度（minimum inhibitory concentration, MIC）為64-256 μg/mL。試驗進一步則將抑菌能力最佳之小葉葡萄（Vitis thunbergii var. taiwaniana）根萃取物中之主要活性成分以製備型薄層層析（preparative thin layer chromatography, PTLC）及再結晶法（recrystallization）分離純化，並將純化所得之化合物進行紫外光譜（ultraviolet spectroscopy）、質譜（mass spectroscopy）及核磁共振（nuclear magnetic resonance）分析；鑑定發現其主要活性成分為Heyneanol A。進一步試驗以液體微量稀釋法（liquid microdilution method）分析純化所得之Heyneanol A之抑菌能力，結果顯示Heyneanol A對於腸球菌（Enterococcus faecium）、枯草桿菌、金黃色葡萄球菌和抗藥性金黃色葡萄球菌具有抑制效果，最小抑菌濃度為2 μg/mL。此外其對於表皮葡萄球菌（Staphylococcus epidermidis）、無乳鏈球菌（Streptococcus agalactiae）、釀膿鏈球菌（Streptococcus pyogenes）和馬鏈球菌（Streptococcus equinus）亦具抑制效果，最小抑菌濃度為4-8 μg/mL。本研究第二部份再以高效液相層析（high performance liquid chromatoghraphy, HPLC）法分析葡萄屬六種葡萄之葉、枝及根萃取物中之Heyneanol A之含量，結果僅於根萃取物中檢出含有Heyneanol A；在試驗葡萄種類之乾燥根中，Heyneanol A含量以細本山葡萄含量的37 mg/g為最高，其次為基隆葡萄的25 mg/g、巨峰葡萄（V. vinifera X V. labrusca cv. Kyoho）的16 mg/g、5C葡萄（V. berlandieri X V. riparia cv. 5C）的14 mg/g及厚皮葡萄的5 mg/g，此結果顯示葡萄屬葡萄根中富含抑菌活性成分Heyneanol A，可作為抑菌植物材料之應用。有鑑於臺灣小葉葡萄以往栽培系統多採水平棚架，普遍存在根採收操作不便利之問題，本研究試驗以天溝系統（trough system）對小葉葡萄之植株進行整根處理，並探討處理年數對其植株根系生長分佈及抑菌活性成分Heyneanol A含量之影響，結果為整根處理植株較對照植株之主根長而生長集中、離軸根數顯著較少且採收操作時間顯著較短；整根處理植株乾燥根之Heyneanol A含量為39.9 mg/g，則與對照植株（36.5 mg/g）無顯著差異，此結果顯示整根處理有利於以收穫根為目的之葡萄栽培。

The aim of this study was to determine the antimicrobial compounds against pathogenic bacteria from grapevines and to product these active compounds from Vitis plants. We first analyzed the antimicrobial activity of leaf, vine, and root extracts from five wild grape species such as taiwan wild grape (Vitis thunbergii Sieb. et Zucc.), hancei wild grape (Ampelopsis brevipedunculata var. hancei), muscadine grape (V. rotundifolia var. jumbo), and kelung wild grape (V. kelungensis) using the disc diffusion method. The result showed that root extracts of Vitis species all inhibited Gram-positive bacteria such as Bacillus subtilis, Staphylococcus aureus and methicillin-resistant S. aureus (MRSA). The root extracts of taiwan wild grape (Vitis thunbergii var. taiwaniana), which showed markedly antimicrobial activities (minimum inhibitory concentration, MIC value of 64 μg/mL toward S. aureus), and contained three possible active compounds were further collected using preparative thin layer chromatography with recrystallization methods. One of these three purified compounds was confirmed as Heyneanol A using ultraviolet, mass spectroscopy and nuclear magnetic resonance analyses. Heyneanol A showed an MIC value of 2 μg/mL toward MRSA, S. aureus, Enterococcus faecium, and B. subtilis and that of 4 to 8 μg/mL for S. epidermidis, Streptococcus agalactiae, Str. pyogenes, and Str. equines. Moreover, the contents of Heyneanol A in root extracts were determined as 37 mg/g dry weight (DW) of taiwan wild grape, 25 mg/g DW of kelung wild grape, 16 mg/g DW of kyoho grape (V. vinifera X V. labrusca cv. Kyoho), 14 mg/g DW of 5C grape (V. berlandieri X V. riparia cv. 5C), and 5 mg/g DW of muscadine grape using high-performance liquid chromatography. Furthermore, to product roots of grapevines in a convenient way, we used the trough system to cultivate taiwan wild grape and analyzed the effects of root training on the growth, distribution, and Heyneanol A content of roots of taiwan wild grape. After three years of training, the roots of treated grapevines were significantly longer than those roots of untreated vines and the distribution of treated roots was more concentrated, which made the harvest work much easier and time-saving. In addition, the content of Heyneanol A within the roots of treated vines was 39.9 mg/g DW, and showed no significant difference with 36.5 mg/g DW of the content of untreated vines. These results suggest that the trough system could be used for root production of taiwan wild grape.