蛹蟲草子實體生物指標物與非消化水溶性多醣特徵

蛹蟲草 (Cordyceps militaris) 為傳統中草藥，因成分與冬蟲夏草相似 (蟲草素、蟲草酸、腺苷)，可經人工栽培而受重視，蛹蟲草多醣亦為具功效成分之一，因種類繁多無法單一且明確地表示；目前對於蛹蟲草組成當中指標成分的統整及對非消化水溶性蛹蟲草多醣的研究較少，故本研究除就白米培養蛹蟲草子實體組成當中生物指標物分析，亦針對水溶性非消化多醣，進行分子結構特徵分析。以乾基表示，灰分與粗脂含量約 4.0 %，粗蛋白含量約 18.2 % ，總膳食纖維達45.5%。粉末組成當中指標化合物麥角固醇及麥角硫鹼含量則皆低於0.8 mg/g，蟲草素含量 (11.8 mg/g ) 高於結構相似的腺嘌呤核苷酸 (0.2 mg/g)，菇類當中常見的醣類化合物包括蟲草酸、海藻糖及β-(1,3)-D-葡聚醣，含量各為2.8% (28.6 mg/g) 、22.6% (226.9 mg/g) 及9.1% (91.2 mg/g)。熱水萃取蛹蟲草多醣 (CMCP) 當中約48% 為可消化的α-D-葡聚醣，由此可知，CMCP如未經純化，具功能性的非消化多醣於水萃多醣的比例並不高。SDF(1,3) 為蛹蟲草水溶性非消化多醣 (SDF) 去除β-(1,3)-D-葡聚醣，進一步以陰離子交換樹脂劃分SDF(1,3)，觀察SDF(1,3)各區分多醣特徵，皆以半乳甘露聚醣為主。且皆具有刺激RAW 264.7 巨噬細胞株釋放細胞激素 NF-κB 及iNOS活性；進一步觀察多醣特徵，SDF(1,3)-F1無果膠相關結構 ; SDF(1,3)-F2及SDF(1,3)-F3 除甘露糖及半乳糖之外，亦有發現酸性醣存在，其中以F3酸性醣比例最高，並發現F3具有(1,4)-β-D-galactan (LM5) 及 (1,4)-α-D-galacturonan 相關結構；SDF(1,3)-F4雖無酸性醣但是具有明顯帶電荷蛋白，發現F4具有(1,4)-β-D-galactan 結構存在。關於結構與細胞活性的關係，F3及F4刺激巨噬細胞釋放細胞激素因素之一，推測在於其具有β- (1,4)-半乳糖糖苷鍵鍵結的半乳聚醣。

關鍵字

蛹蟲草子實體；蟲草素；水溶性非消化多醣；單株抗體；促發炎活性

並列摘要

In recent years, market demand of Cordyceps militaris has increased because of its similar biological activities to the well-known traditional medicine C. sinensis. Cordyceps militaris was found to been riched in cordycepin, cordyceps acid polysaccharides and other natural biological active ingredients. Furthermore, the industrial production of Cordyceps militaris has been achieved using artificial cultivation technology. However, the profile of components of C. militaris in the form of fruiting bodies which contain compositions, marker components and its indigestible polysaccharide is not available. The molecular characteristics of polysaccharides may be associated to the biological activities. Therefore, the objective of this thesis is to know the biological activities compounds content and to explore the molecular characteristics, immunomodulatory activity of indigestible polysaccharide in C. militaris. Based on dry mass, fruiting bodies of C. militaris was high in contents of crude fiber (45.5%) and crude protein (18.2%), crude ash and crude fat were in the range of 4%. Both the content of ergosterol and ergothioneine are less then 0.8 mg/g. Cordycepin content (11.8 mg/g) is much higher than adenosine (0.2 mg/g). The content of mannitol, trehalose and β-(1,3)-D-glucan which were major mushroom components were 2.3%, 22.6% and 9.1% respectively. Water soluble cordyceps militaris crude polysaccharide (CMCP) contain approximately 50% of digestible -α-D-glucan, which means that there a need for further purified of CMCP. In order to explore the functional polysaccharide, SDF(1,3) is the soluble dietary fiber of C. militaris treated with exo- and endo-beta-(1,3)-glucanase. According to the monosaccharide composition, SDF(1,3) is galactoglcomannan which can be separated to four fractions by charge density. The results show that all of fractions significantly stimulated the releasing of NF-κB and iNOS in RAW 264.7 macrophage. According to the ELISA response of the monoclonal antibody LM5, LM19 and LM20 affinity result, the stimulating activity of RAW 264.7 macrophage of fraction III and IV are related to the β- (1,4)- galactan.