巴西蘑菇粗多醣體萃取及其對人體肝癌細胞生存力之影響

Extraction of crude polysaccharide extracts from Agricus brasiliensis and its effect on cellular viability in human hepatoma cells

呂嘉均(jia jun Lu)

指導教授 ： 林俊義 ； 李明明 ； 石信德 ； 范宗宸

亞洲大學/健康學院/生物科技學系碩士班/碩士(2008年)

摘要

巴西蘑菇(Agaraicus brasiliensis)，原產於南美巴西，產期為春秋兩季，是一種藥食兼用的珍稀菇類，含豐富的營養物質和生物活性物質，具有增強免疫力、抗腫瘤、抗氧化、抗病毒、抗致突變等功用，近來科學研究發現巴西磨菇的多醣體有抑制腫瘤細胞的效果。本研究將新鮮巴西蘑菇子實體冷凍乾燥成粉末，分別以熱水萃取(Hot wather extracts，60℃、70℃、80℃、90℃、100℃)以及磷酸緩衝液(Phosphate Buffer Saline extracts)合併超音波破碎(0 min、5 min、10 min、15 min)萃取，利用上述兩種萃取方法所得之萃取液，經酚硫酸法(Phenol-Sulfuric Acid Assay)測定發現，以10 公克巴西磨菇粉末在100℃熱水萃取，可得粗多醣體含量為5.68mg/ml；10 公克巴西磨菇粉末加磷酸緩衝液合併超音波破碎15 分鐘之萃取物含粗多醣含量為8.08 mg/ml。結果並發現巴西磨菇加磷酸緩衝液合併超音波振盪之萃取，菇傘粗多醣體含量8.37 mg/ml 顯著高於菇柄含量之7.63 mg/ml。 進一步研究其粗多醣體對人類肝癌細胞株（HepG2､Hep3B）與正常肝細胞細胞株（Chang Liver cell）之抑制及誘導分化作用，並利用乳酸脫氫酶 (LDH assay)測定、琥珀酸脫氫酶 (MTT assay)分析細胞生存力；肝臟生化酵素指標酵素丙胺酸轉胺酶 (ALT assay) 及天門冬胺酸轉胺酶 (AST assay)作為肝細胞代謝功能活性分析，結果發現正常Chang Liver 肝細胞及肝癌細胞與巴西蘑菇粗多醣體0.32 mg/ml 培養48 小時後，對正常肝細胞株有顯著增進細胞生長及降低細胞外液乳酸脫氫酶(Lactate Denydrogenase, LDH)作用。對肝癌細胞株 (HepG2，Hep3B)則有抑制生長效果，亦即顯著增加細胞外液LDH 活性，HepG2 之LDH 活性增加達40%及Hep3B 之LDH 活性增加達60%。從顯微鏡觀察肝細胞生長狀況，發現HepG2 及Hep3B 經巴西蘑菇粗多體處理後有細胞裂解及漂浮現象，部分細胞呈現空泡化，或圓形皺縮狀，且其外型較小。在培養48 小時候，粗多醣體有效劑量在0.32m/ml 濃度時，對正常肝細胞(Chang Liver)增加AST活性達50%及ALT活性達60%，對肝癌HepG2 細胞則減少AST 活性45%及ALT 活性40%；對肝癌細胞Hep3B AST 活性減少40%及ALT 活性減少55%。研究結果顯示巴西蘑菇粗多醣體對肝癌細胞株的抑制作用較正常肝細胞顯著，對正常肝細胞可能傷害較小。

關鍵字

巴西蘑菇 ； 肝癌

並列摘要

Agaricus brasiliensis is a native mushroom of Brazil, South America. It is considered as an culinary-medical and functional food, a rare specie of mushroom rich in nutritious substance and living organism which enhance the immune system, antitumor, antioxidant,antivirus,and antimutagenic, abilities. Lately, scientific researchers have found that the polysaccharide of this mushroom is effective for the control of tumor cells. For this research, the fresh fruiting body of A. brasiliensis was refrigerated, dried, and ground into powder. With various temperatures of hot water or various times of Phosphate buffer saline combined with ultrasonic oscillation, 5.68 mg/ml of crude polysaccharides were extracted from 10g powders of Agaricus brasiliensis fruiting body by 100℃ hot wather, and 8.08 mg/ml polysaccharides by 15 min ultrasonic oscillation. The result also revealed that the quantity of polysaccharide contained in the cap of mushroom was 8.37mg/ml, which was higher than the 7.63mg/ml of polysaccharide contained in the stalk. The effects of crude polysaccharide on human liver cancer cell（HepG2､Hep3B）and normal liver cell（Chang liver cell）were studied using LDH assay and MTT assay to analyze the viability of liver cells. And using ALT assay and AST assay were used to analyze the metabolism of the liver cell function activity. The result showed that the Chang liver cell or liver cancer cell(HepG2, Hep3B) cultured with 0.32 mg/ml crude polysaccharide for 48 hours could increase Chang liver cells growth and reduce to LDH activity.On the other hand, the liver cancer cells (Hep G2，Hep3B) growth was restrained, yet their LDH activity was increased: HepG2 to 40% and Hep3B to 45%. The observation of microscope showed that cells of HepG2 and Hep3B were lysed after treating with A. brasiliensis crude polysaccharides. Some of cells appeared to be shrunk, empty and became smaller. The best concentration of crude polysaccharide among four treatments was 0.32 mg/ml. After 48 hour incubation, the AST and ALT activities of normal Chang liver cell were increased 5% and 60% indiviedually, where the AST and ALT of HepG2 were decreased 38% and 45%, and those of Hep3B were decreased 35% and 55% respectively.

並列關鍵字

Agricus brasiliensis ； hepatoma

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