人工培養烏苓參之基源、多醣物化性質及免疫調節活性研究

Abstract

烏苓參 (Xylaria nigripes, XN)是屬於炭角菌科下的一種高價值藥用真菌，在中藥的使用上具有利尿、補心腎、增強免疫、改善失眠及改善手術後失血等功效。本研究目的為確認兩種不同來源人工培養烏苓參TXN與CXN之基源，並進一步了解其子實體中多醣體之物化性質及免疫調節活性。由ITS定序的基源分析結果確認TXN與CXN皆為烏苓參，因此將兩者的ITS序列登錄於NCBI的GenBank資料庫中，登錄碼分別為KJ637786及KJ627787。經過熱水萃取與酵素純化步驟，所得烏苓參純化水溶性多醣TXNP及CXNP之產率分別為8.3%與7.8%，在紅外線吸收光譜中兩者圖譜的曲線呈現相近趨勢，為典型的多醣圖譜，具有羥基、醣苷鍵結及醣單元等特徵吸收峰，其中TXNP為白色粉末，是一種分子量853.8 kDa、具有三螺旋構型的大分子葡聚醣；而CXNP為深棕色粉末，是一種分子量14.7 kDa、不具有三螺旋構型的小分子異質多醣，由木糖、甘露糖及葡萄糖以41.5%、41.4%及17.1%之莫耳比例所組成，由此可知TXNP及CXNP為不相同的烏苓參多醣。在LPS誘發RAW264.7細胞發炎的模式中，TXNP及CXNP呈劑量效應抑制IL-1、TNF-α、NO及PGE2之分泌、iNOS及COX-2之表現及轉錄因子NF-κB之活化，但對IL-6分泌量則無明顯影響，然而TXNP與CXNP則不會影響細胞的存活率，表示以上抑制效果確實來自於TXNP及CXNP的活性，而非多醣處理對RAW264.7細胞造成毒性所引起。這些結果證明烏苓參多醣對發炎相關因子具有良好的調節作用；其中以TXNP的活性較CXNP佳，說明多醣物化性質之差異對活性功效具有不同程度的影響。

Xylaria nigripes (XN), also known as Wu Ling Shen, is a high value medicinal fungus belonging to the family Xylariaceae. In traditional Chinese medicine, it possesses the properties of diuretic, invigorating kidney, enhancing immune function, improving insomnia and post-surgery bleeding, and others. This study aimed to confirm the phylogenetic relationship of two cultivated XN that derived from different origins, namely TXN and CXN, as well as examining the physicochemical properties and immunomodulatory activities of polysaccharides prepared from their fruiting bodies. Based on the ITS phylogenetic analysis, TXN and CXN were confirmed to be XN and their ITS sequences have been registered in the NCBI Genbank with accession numbers KJ627786 and KJ627787, respectively. By hot water extraction and enzyme purification, the yields of purified water soluble polysaccharides of TXNP and CXNP were 8.3% and 7.8%, respectively. The FT-IR spectra of these two polysaccharides showed a relatively similar profile, which possessed the characteristic peaks of the hydroxyl group, pyranose ring and glycosidic bond. TXNP was white powders and identified to be a glucan, which had a molecular weight of 853.8 kDa coupled with triple helix conformation. While CXNP was dark brown powders and identified to be a hetropolysaccharide that had a molecular weight of 14.7 kD. The monosaccharide composition of CXNP contained xylose, mannose and glucose in a molar ratio of 41.5%, 41.4% and 17.1%, respectively. Results of these physicochemical properties suggested that TXNP and CXNP were two different kinds of polysaccharides. In the LPS-stimulated RAW 264.7 cell inflammation study, results showed that both TXNP and CXNP dose-dependently suppressed the production of IL-1, TNF-α, NO and PGE2, with the exception of IL-6. The expression of iNOS, COX-2 and NF-κB were also down-regulated by these XN polysaccharides. The MTT assay showed that TXNP and CXNP had no effect on RAW264.7 cells viability. Therefore, it implied that these inhibiting activities were actually mediated by TXNP and CXNP, not due to their cytotoxicity. In conclusion, this study indicated that hot water extracted polysaccharides from the fruiting bodies of the cultivated XN were effective in modulating the inflammation related factors, in which TXNP was more potent than CXNP; suggesting that the differences in the physicochemical properties of polysaccharides may affect their biological activities.