靈芝不同培養時期的多醣成份分析與生物活性之研究

Abstract

靈芝(Ganoderma lucidum)是中國歷史悠久的中草藥，過去的研究中指出其內含的多醣體具有抗腫瘤細胞與增進免疫系統之能力。本研究的目的是系統性研究靈芝生長過程中兩個靈芝菌株J101與36123的多醣產率與藥效，期待能夠得到有效且產量高的靈芝多醣。實驗結果顯示在培養49天時，靈芝菌株J101乾重產量最高可以達到19.15 ± 0.73 g/L，而多醣產率的最大值則是出現在第14天達到470.0 ± 0.3 mg/L；靈芝菌種36123的表現與前者不同，乾重產量最大值17.30 ± 0.36 g/L出現在第35天，而多醣產率最大值出現在第49天610.0 ± 0.6 mg/L。單醣分析結果指出兩種靈芝菌株的多醣體中均以葡萄糖和半乳糖為主要成份，而兩菌種中以36123的葡萄糖含量較高，J101葡萄糖最高產量為417.66 ± 10.60 μmol/g PS。而36123葡萄糖最高產量達1496.22 ± 6.62 μmol/g PS，是J101的三倍之多。以10, 50, 100, 250, 500 μg/ml五種濃度的多醣體處理四種不同的腫瘤細胞(MCF-7、HT29、H460、CEM)以測定細胞毒殺能力，另外將多醣體以100與250 mg/ml分別處理在人類血管內皮細胞Eahy926上進行抗血管新生能力測定；結果顯示兩種菌株的任何一個培養時期的多醣均不具有細胞毒殺能力與抗血管新生能力。將J101與36123的多醣單獨處理在老鼠巨噬細胞RAW264.7上，測定腫瘤壞死因子(tumor necrosis factor-α, TNF-α)與介白素(interleukin-6, IL-6)的基因表現量，以觀察多醣體對免疫反應的影響，結果發現所有培養時期的多醣均能誘導TNF-α表現，此外，J101的第0天、第7天與第42天多醣體與36123的第0天與第21至35天多醣體能誘導IL-6表現，推測以上培養時期的多醣體能刺激細胞激素分泌，對於免疫反應有促進效果。在抗發炎能力測定方式為加入脂多醣(lipopolysaccharide, LPS)刺激RAW264.7使其發炎後測定TNF-α與IL-6的基因表現量，結果顯示，所有J101與36123的多醣體均無法抑制由LPS所誘導之TNF-α表現；J101的所有多醣對於LPS所誘導之IL-6表現均沒有抑制效果；36123第28、35、42天的多醣體預先處理後會些微抑制由LPS所誘導的IL-6基因表現，表示這三天的多醣體可能有抗發炎的能力但並不明顯。綜上所述，兩個菌種的靈芝多醣以36123產率較高，而藥效部份是所有靈芝多醣皆能促進免疫反應，在特定天數萃取的多醣可能有些微抑制發炎反應的效果，但不明顯。

Ganoderma lucidum has been reported to provide anti-tumor activity and enhance the ability of immune system, especially for its polysaccharide (PS). Thus, the aim of this study is to characterize the yield and the immune-modulating ability of polysaccharide extracted from species J101 and species 36123 in different cultural period. The maximum dry weight of J101 is 19.15 ± 0.73 g/L, which is appeared at 49th days, and that of the yield of PS is 470.0 ± 0.3 mg/L at 14th days. In contrast, the maximum dry weight of 36123 is 17.30 ± 0.36 g/L, which is obtained at 35th days, and the maximum yield of PS is 610.0 ± 0.6 mg/L at 49th days. The results of monosaccharide composition of PS after completely acid hydrolysis showed that galactose and glucose are the major component of PS. The maximum glucose of species J101 is 417.66 ± 10.60 μmol/g PS. To note that the maximum glucose content of species 36123 is 1496.22 ± 6.62 μmol/g PS, which was over three folds higher than species J101. To investigate the bioactivity of these PS with different cultural period, the cytotoxicity test was first performed on 4 tumor cell line MCF-7, HT29, H460, and CEM. The results showed that no cytotoxicity. The anti-angiogenesis ability test was performed on human endothelial cell Eahy926. The results showed that neither 100 μg/ml nor 250 μg/ml PS had the ability of anti-angiogenesis. RAW264.7 treated with PS only revealed the basal response of immunity. The results showed that all PS induce the expression of TNF-α and IL-6, which indicated these PS have ability to promote immune response. After treated with LPS to induce inflammatory response in RAW64.7, we measured the expression level of TNF-α and IL-6. The results showed that PS of species J101 couldn’t inhibit the expression of TNF-α and IL-6, which induced by LPS, indicated that these PS were not able to inhibit inflammatory response. On the contrary, the expression of IL-6 induced by LPS was slightly inhibited by 28th、35th、42th days PS of species 36123, but other days PS of species 36123 couldn’t inhibit the expression of IL-6 which was induced by LPS. To sum up, the polysaccharide yield is higher in species 36123 than species J101. All PS exhibited no cytotoxicity ability and no anti-angiogenesis ability. Besides, all PS were able to promote immune response, and the PS of species 36123 in specific cultural period might have potential to inhibit inflammatory response.