人工培養大蟬花之多醣物化性質及免疫調節活性研究

Abstract

大蟬花 (Cordyceps cicadae Shing, CC) 為高經濟價值之傳統藥材，其具有清熱解毒、補肝腎、明目安神及增強免疫等功效。本研究目的首先確認採集之本土大蟬花子實體 (CCF) 之基源，進而探討其人工栽培子實體之多醣物化特性及免疫調節活性。根據ITS定序的基源分析結果確認CCF為大蟬花物種，其ITS序列已登錄於NCBI的GenBank資料庫中，登錄碼為KP771879。大蟬花子實體之水溶性粗多醣CP及純化多醣CPP產率分別為3.4%及1.2%。在物化性質方面，CPP總糖含量為75.1%，蛋白質含量為3.92%，而CP總糖含量為53.4%，蛋白質含量為6.29%，兩者在FT-IR的圖譜曲線呈現相近趨勢，皆為典型的多醣圖譜，具有各種如C-OH、C-H及C-O-C等多醣特徵吸收峰。CP與CPP皆是以甘露糖為主，其次為葡萄糖及半乳糖所組成的異質性多醣，然而兩者的差異主要在於分子量的分布情形，CP包含三個分子量大小的分布，分別是21.5 kDa、3.1 kDa 及少量678 kDa，而CPP則是由24.3 kDa所組成的單一分子量多醣。在LPS誘發RAW264.7細胞發炎模式中，CP與CPP在不影響細胞存活率情況下，對於促發炎因子的分泌具有不同程度的抑制效果，其中CPP呈劑量效應抑制nitric oxide (NO)、IL-1β及TNF-α之分泌，但對IL-6分泌抑制則不明顯；CP呈劑量效應抑制IL-1β及TNF-α的分泌，但對NO及IL-6的抑制僅在高濃度CP (80及160 μg/mL) 處理下才有效果。在以BALB/c小鼠為實驗動物下，CP各劑量的處理 (75、150及300 mg/kg) 相較於控制組，對小鼠脾臟細胞受LPS刺激所產生的IL-1β及IL-6均呈劑量效應的抑制，且不影響脾臟細胞存活率及細胞族群分布比例。從上述結果證明，大蟬花水溶性粗多醣CP及水溶性純化多醣CPP的物化性質雖有差異，但在離體或活體活性試驗中皆表現良好抗發炎活性。

Cordyceps cicadae (CC) is a high economic value ingredient of traditional medicine. It is believed to possess the properties of detoxifying, invigorating the liver and kidney, improving eye sight, enhancing immune function, and others. This study aimed to validate the authenticity of locally collected CCF, and examine the physicochemical properties and immunomodulatory activities of polysaccharides from cultivated CC fruiting bodies. According to the result of ITS phylogenetic analysis, CCF was confirmed to be CC and its ITS sequences has been registered in the NCBI GenBank as an accession number KP771879. The yields of water-soluble crude polysaccharides (CP) and purified polysaccharides (CPP) from CC fruiting bodies were 3.4% and 1.2%, respectively. Results of the physicochemical properties showed that the contents of total carbohydrate and protein in CPP were 75.1% and 3.92%, respectively, whereas CP were 53.4% and 6.29%, respectively. The FT-IR spectra of these two polysaccharides showed a relatively similar profile, which possessed the characteristic peak of the C-OH, C-H, and C-O-C stretching vibration. CP and CPP showed a similar monosaccharide composition, of which the predominant monosaccharide was mannose, followed by glucose and galactose. However, there were apparent differences in molecular weight distribution between CP and CPP; two major (21.5 kDa and 3.1 kDa) and one minor (678 kDa) macromolecular populations were found in CP, whereas CPP contained only one macromolecular population (24.3 kDa). In anti-inflammatory study, results showed that CP and CPP had different degrees of inhibition on the inflammatory factors in the LPS-induced RAW264.7 microphages. CPP dose-dependently suppressed the production of nitric oxide (NO), IL-1β, and TNF-α, with the exception of IL-6. Although CP had similar effects on the IL-1β and TNF-α production, its inhibitory effects on NO and IL-6 were only noted in high-doses (80 and 160 μg/mL). In in-vivo study, results showed that compared with the control group, animals treated with different doses of CP (75, 150, and 300 mg/kg) showed no effect on the cell viability and cellularity of splenocytes but dose-dependently suppressed the LPS-induced IL-1β and IL-6 production from mice splenocytes. In conclusion, this study indicates that although CP and CPP possess differences in the physicochemical properties, they are effective in inhibiting the production of inflammatory mediators both in-vitro and in-vivo.