臺灣金線連水溶性多醣特徵與生物活性

Characteristics and bioactivities of water soluble polysaccharide of Anoectochilus formosanus

楊麗嬋(Li-Chan Yang)

指導教授 ： 呂廷璋

共同指導教授 ： 林文川(Wen-Chuan Lin)

國立臺灣大學/生物資源暨農學院/食品科技研究所/博士(2012年)

https://doi.org/10.6342/NTU.2012.10475

摘要

臺灣金線連冷水萃取第二型阿拉伯半乳聚醣在本研究中被證實其鍵結主幹為β-(1→3; 1→6)連結的半乳糖，其單醣組成有阿拉伯醣、半乳醣、葡萄醣與甘露醣。經高效能分子排阻層析分析確認其分子量為29 kDa。
本研究中，阿拉伯半乳聚醣經體內與體外試驗，被確認其具有益菌生效應。就體內試驗方面，阿拉伯半乳聚醣能增進小鼠糞便中雙歧桿菌的數目以及降低盲腸內容物的酸鹼值。體外試驗則分別採用糞便混合菌株與純菌株發酵二種方式，來進行益菌生效應的評估。糞便混合菌株發酵試驗，證實該阿拉伯半乳聚醣主要能增進雙歧桿菌的增長，純菌株發酵試驗中，透過逆轉錄聚合酶鏈式反應測試阿拉伯半乳聚醣對雙岐桿菌的作用，結果發現該多醣不僅刺激碳水化合物水解酶的表現，同時也會增進與養份攝取有關的ABC轉運蛋白表現。影響該蛋白的表現被認為有可能是阿拉伯半乳聚醣其益菌生效應的機轉。
因為該阿拉伯半乳聚醣能夠增進腸道中短鏈脂肪酸的產生，所以推測其可能具有抗骨質疏鬆的效果。因此本論文著手評估阿拉伯半乳聚醣其益菌生效益與抗骨質疏鬆兩種生理活性間的關係。本論文使用去卵巢ICR小鼠模擬骨質疏鬆的動物模式，並投與阿拉伯半乳聚醣 (15 mg/kg, 口服)或合併使用鏈黴素歷時三周，評估其改善骨質疏鬆的效果與益菌生效應間的關係。該試驗結果顯示，阿拉伯半乳聚醣能夠增進小鼠糞便中雙歧桿菌的數目。飲水中加入鏈黴素不僅會破壞阿拉伯半乳聚醣的益菌生效果，同時與去卵巢組和偽手術組比較，鏈黴素更使小鼠盲腸酸鹼值顯著的增高。研究中證實，投與阿拉伯半乳聚醣可以改善去卵巢小鼠血中骨鈣素濃度、降低血清骨膠蛋白碳末端肽鏈、預防骨質流失。但合併鏈黴素處理後，卻會抑制阿拉伯半乳聚醣的抗骨質疏鬆效果。骨骼組織型態分析顯示阿拉伯半乳聚醣可以改善骨體積百分比。本研究證實阿拉伯半乳聚醣確實具有抗骨質疏鬆的效果，而且此活性與其益菌生效應有關。
另一部分，阿拉伯半乳聚醣以體內與體外試驗來評估其免疫調控的活性。該阿拉伯半乳聚醣對巨噬細胞具有刺激的效果。結果顯示該多醣能刺激巨噬細胞增生與增進其吞噬活性。同時也能增進小鼠巨噬細胞株RAW264.7 對一氧化氮和細胞激素如腫瘤壞死因子α、白細胞介素1素、白細胞介素10、顆粒球群落刺激生長因子與顆粒單核球群落刺激生長因子等的分泌。體內評估方面，以腹腔注射投與阿拉伯半乳聚醣，會使小鼠脾臟重量增加，促進脾細胞的增生以及提高血清中顆粒球群落刺激生長因子的濃度。
該阿拉伯半乳聚醣更進一步被研究其對分泌顆粒球群落刺激生長因子的機轉探討。結果顯示該機轉可能與MAPK與NFκB的調控路徑有關，另一方面欲了解阿拉伯半乳聚醣對嗜中性白血球缺乏症的影響，以接種結腸癌CT26小鼠合併使用5-氟尿嘧啶為動物試驗模式，投與阿拉伯半乳聚醣(15, 45 mg/kg口服)，發現阿拉伯半乳聚醣能改善其嗜中性白血球缺乏症的副作用。
結論，臺灣金線連阿拉伯半乳聚醣在本研究中被證實具有益菌生效應、抗骨質疏鬆與調節免疫等生理活性。

關鍵字

臺灣金線連 ； 多醣 ； 第二型阿拉伯半乳聚醣 ； 益菌生 ； 骨質疏鬆 ； 免疫調節 ； 顆粒球群落刺激因子

並列摘要

A type II arabinogalactan extracted with cold water from Anoectochilus formosanus was demonstrated that has β-(1→3; 1→6) linked Galactose as major structure with branches containing arabinose, galactose, glucose and mannose residues. The molecular weight was 29 kDa determined by high performance size-exclusion chromatography.
This study demonstrated the prebiotic effects of arabinogalactan (AFP) of A. formosanus in vivo and in vitro. In vivo study, AFP could enhance the number of fecal bifidobacteria and reduced the pH values in cecums in mice as inulin acted. In vitro studies used commercial strains and fecal strains for fermentation. AFP influenced the bifidobacteria majorly and in RT-PCR analysis, AFP not only stimulated the carbohydrate enzymes but also induced the expression of ABC transporter associated to nutrient intake. It was suggested as the prebiotic mechanism of AFP.
Since AFP could enhance the production of intestinal short chain fatty acids that were regarded asanti-osteoporosis compounds. But the correlation of AFP between prebiotic and osteoporosis haven’t been demonstrated. It is the first time that evaluated AFP could prevent bone loss in ovariectomized mice through its prebiotic effect. In this experiment female ICR mice were treated with sham-operated or ovariectomized (OVX) that could lead to bone loss while remaining OVX mice were allocated into groups that were AFP (15 mg/kg p.o. daily) with/without streptomycin sulfate (SM) treatment or SM treatment only for 3 weeks.
Results showed AFP could enhance the number of bifidobacteia in feces. SM treatment could reduce prebiotic effect of AFP and let cecum pH value higher than OVX and sham groups. The experiment demonstrated that AFP improved the level of serum osteocalcin, decreased serum C-terminal cross-linked telopeptides of type I collagen (CTx) and prevented bone calcium loss resulting from OVX but in AFP with SM treatment and SM treatment groups exhibited no differences in above bone marker to OVX group. Bone histomorphometry showed AFP improved bone volume /total volume (BV/TV) % and the number of trabecular in femur. Through SM treatment, this study demonstrated that AFP prevented bone loss in OVX mice via its prebiotic effect.
AFP was evaluated the immunomodulatory effects in vivo and in vitro. AFP exhibited the stimulatory effects on the macrophage system. It shows the strong proliferation and phagocytic activity in vitro. Also, the production of NO and cytokines as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-10, granulocyte-colony stimulating factor (G-CSF) and granulocyte macrophage-colony stimulating factor (GM-CSF) appears to be significantly induced by AFP in the murine macrophages RAW264.7. In vivo evaluation showed that intraperitoneal injection of AFP increased the spleen weight, splenocytes proliferation and level of serum G-CSF in mice.
AFP was investigated the signaling pathway of G-CSF secretion in RAW264.7 cells. The results suggested that the intracellular signaling through the activation of MAPK and NF-κB signaling pathways. Oral administration of AFP (15, 45 mg/kg) in mice bearing CT26 carcinoma could reduce the neutropenia caused by 5-fluorouracil (5-FU) injection in CT26 carcinoma inoculated mice.
In conclusion, the prebiotic, anti-osteoporosis and immunomodulatory effects were involved in the bioactivities of a type II arabinogalactan from A. formosanus.

並列關鍵字

Anoectochilus formosanus ； polysaccharide ； type II arabinogalactan ； prebiotic ； osteoporosis ； immunomodulatory ； granulocyte-colony stimulating factor

參考文獻

1. Kan, W. S., Pharmaceutical Botany. 1 ed.; National Research Institute of Chinese medicine: 1969.

2. Lin, C. C.; Huang, P. C.; Lin, J. M., Antioxidant and hepatoprotective effects of Anoectochilus formosanus and Gynostemma pentaphyllum. Am J Chin Med 2000, 28, (1), 87-96.

3. Wang, S. Y.; Kuo, Y. H.; Chang, H. N.; Kang, P. L.; Tsay, H. S.; Lin, K. F.; Yang, N. S.; Shyur, L. F., Profiling and characterization antioxidant activities in Anoectochilus formosanus hayata. J Agric Food Chem 2002, 50, (7), 1859-65.

4. Shih, C. C.; Wu, Y. W.; Lin, W. C., Scavenging of reactive oxygen species and inhibition of the oxidation of low density lipoprotein by the aqueous extraction of Anoectochilus formosanus. Am J Chin Med 2003, 31, (1), 25-36.

5. Wang, L. F.; Lin, C. M.; Shih, C. M.; Chen, H. J.; Su, B.; Tseng, C. C.; Gau, B. B.; Cheng, K. T., Prevention of cellular oxidative damage by an aqueous extract of Anoectochilus formosanus. Ann N Y Acad Sci 2005, 1042, 379-86.