牛樟芝CCRC-35396在MgSO4或MnSO4處理下之 生長化學成分分析及生物活性研究

Abstract

由前人研究，牛樟芝編號CCRC 35396具有生長速度快、含量豐富的adenosine，且被證實具有預防因缺乏血清而引起細胞凋亡的功效(Lu, 2006)。因此在本實驗中，選擇CCRC 35396 牛樟芝品系，探討其在不同濃度含硫鹽類逆境下，對菌體型態、菌絲體乾重、多醣體、乙醇萃取物、甲醇萃取物、多醣體分子量分布以及含硫多醣產量的影響及其在細胞保護方面的生理活性。實驗結果發現多醣體的累積，並沒有依生長期間而呈現相關性；相反地，在乙醇萃取物產量方面，卻是與生長時間呈現負相關。MgSO4 0.025% 以及0.25% 的濃度處理下可以促進菌絲的生長；而MnSO4處理下，則是在濃度於0.025%、0.05%、0.10%與0.25%，會抑制菌絲體的生長。以MnSO4 0.25%濃度處理之下，多醣體可以得到最高的產量(8.82 ± 0.79 %)。在乙醇萃取物方面，以MnSO4 0.10%處理為最佳，可達到39.00 ± 2.24 %。三萜含量方面在各個鹽類處理的條件下皆不盡相同，但在不添加鹽類處理的生長曲線之下，三萜的含量隨著培養天數增加而累積。在細胞保護方面，選擇MTT作為分析方式，結果顯示Serum-free狀態下，MnSO4 0.50% 處理時產生的含硫多醣體在30μg/ml即具有明顯保護性。此外，MPP+(1-methyl- 4-phenylpyridine)可抑制呼吸鏈的complex I，導致細胞ATP量快速下滑，同時增加電子從complex I漏損的量，因此提高了粒線體superoxide的產量，對細胞造成毒害與破壞。在MPP+(1-methyl- 4-phenylpyridine)1.5mM處理狀態下，MnSO4 0.05%、0.10%、0.25%處理所產生的含硫多醣體，在100μg/ml皆有保護細胞的效果。

In previous study, the strains CCRC 35396 of A. cinnamomea grew fastest among all the strains in our laboratory, and rich in adenosine. Also the adenosine has been proved that it could prevent cells from serum deprivation-induced apoptosis (Lu, 2006). In this study, strain CCRC 35396 was used to investigate the effects of sulfated-salt stress on the chemical constituents of A. cinnamomea and anti-apoptosis effect. Four fractions, sulfated- polysaccharides(SPS), polysaccharides (PS), ethanolic extracts, and triterpene were analyzed. The PS accumulation was not growth-associated and there was a negative correlation between growth and the yield of NPS. Mycelia were treated with MgSO4 or MnSO4 at the concentrations of 0.025％, 0.05％, 0.1％, 0.25％ and 0.5％. From the results, treatment of MgSO4 in the concentration of 0.025％ & 0.25％ could enhance the mycelial growth. Whereas MnSO4 could inhibit the mycelial growth beyond concentration of 0.025％. The treatment of MnSO4 in the concentration of 0.25％ showed the maximal yield of PS among all the tested concentrations at the value of 8.82 ± 0.79 %. MnSO4 at 0.10％ showed the highest yield of NPS at the value of 39.00 ± 2.24 %. The contents of triterpenoids were all different by series sulfated-salt treatment. The accumulation of triterpenoids were time-dependent without sulfated-salt treatment. The cell protectivity of cell was performed by the MTT test. The result showed that by SPS (30μg/ml) isolated from 0.50% MnSO4 treated mycelia exhibited protectivity significantly in Serum-free test. Meanwhile, in the presence of MPP+1.5mM, all the MnSO4 0.05%、0.10%、0.25% treated mycelia SPS showed the protectivity.