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  • 學位論文

西洋參生物轉化物的醣質組成與其對RAW264.7巨噬細胞株之影響

Carbohydrate Composition of American Ginseng Bioconversion Product and Its Effects on RAW264.7 Macrophage Cell Line

指導教授 : 呂廷璋

摘要


近年台灣人參相關的健康食品銷售量明顯增加,並產生大量的萃取殘渣,而殘渣中仍含有大量的活性物質,如人參多醣。本研究欲利用靈芝菌絲降解西洋參渣細胞壁中的木質素與纖維素,釋放活性物質,同時靈芝菌絲生長亦伴隨活性多醣的產生。論文前段先針對西洋參多醣進行化學組成與免疫活性分析,結果顯示西洋參粗多醣中有近90%的澱粉,而不可消化多醣 (NDPS) 含量約為10%,其單醣組成以阿拉伯糖和半乳糖為主,莫耳比例為1:1.2。NDPS 區分出的F3多醣,具有刺激RAW264.7巨噬細胞株釋放ㄧ氧化氮 (NO) 與腫瘤壞死因子α (TNF-α)之免疫刺激活性,根據其醣組成與大鼠單株抗體LM2單株抗體陽性反應,推斷為第二型阿拉伯半乳聚醣 (arabinogalactan type II, AG II)。後續實驗中以F3多醣為標準品,利用免疫親和反應建立一定量人參AG II方法。第二部分研究以西洋參殘渣作為靈芝菌絲體固態培養之基質,靈芝菌絲於西洋參殘渣上生長良好,於接種後試驗第0、4、8、14與30天取樣分析,第8天時菌絲覆蓋率即可達85%以上,第14天時可達100%。發酵樣品之熱水萃取層產率、水溶性碳水化合物含量、AG II含量皆隨發酵時間增加而降低,其熱水萃取層在以 RAW264.7 巨噬細胞株為模式下,能顯著抑制endotoxin (lipopolysaccharide) 所引起之NO與TNF-α釋放,且此抑制活性隨發酵時間增加而增強。將發酵物熱水萃取層劃分為粗多醣區分 (CPSF) 與酒精上清層 (ESF),兩者皆有抑制NO與TNF-α釋放之活性。CPSF中除含有大量澱粉外,其不可消化多醣的醣組成中,甘露聚醣、海藻醣、葡萄醣隨發酵時間增加,應來自靈芝所產生之多醣體,其中靈芝所產生之多醣 (1, 3)-β-D-葡聚醣,佔30天發酵物CPSF總碳水化合物的2.75 %。而ESF含有較小分子的醣類應為西洋參多醣降解物與靈芝生成物質。藉由靈芝菌絲固態發酵西洋參殘渣,能使活性多醣以及免疫相關活性物質增加,因而提高其抑制RAW264.7巨噬細胞株釋放發炎介質 (NO) 與促發炎細胞激素 (TNF-α) 分泌的免疫活性。

並列摘要


Consumption of ginseng-related health food products has increased in Taiwan in recent years. Meanwhile, food industry procudues considerable amount of extracted residues, which still contain a great amount content of bioactive materials, such as ginseng polysaccharides. In this study, Lingzhi (Ganoderma lucidum) mycelia were applied to degrade cellulose and lignin in cell walls of American ginseng (Panax quinquefolius Linn) residue and to release bioactive ingredients. The growth of Lingzhi mycelia brought the mushroom bioactive polysaccharides into the fermentation product. In the first part of this study, we screened bioactive polysaccharides in the ginseng raw material. Our results showed that the crude polysaccharide of the ginseng contained 90% of starch and 10% of nondigestible polysaccharide (NDPS) mainly composed of arabinose and galactose with molar ratio of 1:1.2. A polysaccharide fraction designated as F3 from NDPS showed significant activity to stimulate RAW264.7 cells to release nitric oxide (NO) and tumor necrosis factor-α (TNF-α) and was tentatively determined as arabinogalactan type II (AG II) according to monosaccharide composition and positive affinity of LM2 monoclonal antibody from rat. The AG II was quantified by enzyme-linked immunosorbent assay (ELISA) using isolated F3 polysaccharide as standard in following analysis. In the second part of study, ginseng residues were used as fermentation medium of Lingzhi mycelia. The mycelia grew well and products were sampled on 4th, 8th, 14th and 30th day. The results indicated that the cover ratios of mycelia were 85% and 100% on the 8th and 14th day, respectively. The yields of hot-water-fraction (HWF), water soluble carbohydrate, and AG II decreased with fermentation time. HWF of fermentation products significantly inhibited lipopolysaccharide-induced NO and TNF-α releasing in RAW264.7 cell model. The inhibitory activity increased with fermentation time. HWF was further separated into crude polysaccharide fraction (CPSF) and ethanol supernatant fraction (ESF) and both showed significant activities to reduce NO and TNF-α releasing. The CPSF was a polysaccharide enriched fraction and contained high ratio of starch. The ratios of mannose, fucose, and glucose of NDPS increased with fermentation time, as a result of growth of Lingzhi . The (1, 3)-β-D-glucan, as a Lingzhi bioactive polysaccharide marker, increased to 2.75% of total carbohydrate of CPSF on the 30th day. The ESF was a mixture of smaller polysaccharides degraded from ginseng and those from Lingzhi mycelia. It is plausible to conclude that the enhancement of activities to reduce NO and TNF-α releasing of the fermentation product is a synergistic effect of degraded ginseng polysaccharides, Lingzhi polysaccharides and other anti-inflammatory substance produced during the growth of Lingzhi mycelia on ginseng residue.

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