臺灣金線連水溶性多醣分子結構特徵

Abstract

臺灣金線連 (Anoectochilus formosanus) 的水溶性多醣, 已被證實具刺激人類週邊單核球細胞 (PBMCs) 分泌細胞激素的能力, 因此本論文針對此水溶性多醣進行純化分離與結構分析｡ 金線連多醣由全草之熱水萃取物中, 以蛋白質酵素與澱粉酵素處理, 去除大部份的蛋白質與α-D-glucan (或澱粉) 後, 再以4倍乙醇沉降收集, 收率為全草乾重之3.03 %｡ 中性與酸性多醣以DEAE-650M的陰離子交換樹脂區分, 其收率分別為可萃取多醣的47.6 與30.9 %｡ 中性多醣區分再使用斐林試劑進行Cu++選擇性錯合, 可沉澱中性多醣中的甘露聚醣, 並得到不產生錯合反應的半乳聚醣, 其化學鍵結位置依13C-NMR碳譜分析, 結果皆為β-(1→4)的鍵結, 且為直線型之同質多醣, 其視分子量分別為52.7與21.2 kDa｡ 酸性多醣裡, 高離子強度的區分為一含有78.6 %的半乳糖醛酸, 且具有果膠質多醣rhamnogalactan II區塊與(1→3,1→6) -β-D-galactan的特徵｡ 而低離子強度的區分, 以一重量平均分子量為51.5 kDa的醣蛋白所構成, 其單醣組成含有阿拉伯糖､半乳糖､葡萄糖與甘露糖, 相對莫耳百分比為29.8 : 35.1 : 10.3 : 23.8｡

Cytokine stimulating activity of Anoectochilus formosanus on human peripheral blood nuclear cells has been associated with its water-soluble polysaccharides. To study the molecular characteristics of these polysaccharides, the polysaccharide enriched fractions were prepared from hot-water extracts of whole plant through protease and amylase digestion and collected with ethanol precipitation. The yield of water-soluble polysaccharide fraction (AFP) accounted for 3.03% of dried mass. The AFP was further separated into neutral (AFPN) and acidic (AFPA) fraction by DEAE-anion-exchange chromatography with yields of 47.6% and 30.9% of AFP, respectively. The AFPN was composed of two linear homopolysaccharides, a galactan and a mannan, which can be separated by selecting precipitation of Fehling’s solution through copper (II) ion and mannan complex formation. The glycosidic linkage of both polysaccharide were found to be β-(1→4) revealed by 13C-NMR analyses. The apparent molecular weight of the galactan and the mannan were 21.2 and 52.7 kDa, respectively, according size-exclusion chromatographic analyses. The acidic polysaccharide fraction (AFPA) was found to be a complicate polysaccharide mixture. The major components of acidic fraction were pectic polysaccharides which were compose of 78.6% of galacturonic acid and showed characteristic of rhamnogalactan II and (1→3, 1→6)-β-D-galactan. One proteoglycan was collected in acidic fraction from low anionic strength eluting portion of DEAE-chromatography. The proteoglycan contents 58.4% protein and 41.6% glycan with molecular weight of 51.5 kDa. The sugar compositions of the glycan were arabinose, galactose, glucose and mannose in a molar ratio of 29.8: 35.1: 10.3: 23.8.