乳酸菌發酵蕎麥萃取物抑制大腸癌細胞HT-29增生及對人類腸道細胞株Int-407抗細胞毒性之研究

Abstract

蕎麥 (Fagopyrum esculentum Moench) 含有豐富的營養成分。研究指出蕎麥含有多種抗氧化成分，如芸香苷 (rutin)、槲皮素 (quercetin)等酚類化合物與類黃酮物質，具有抗氧化、抗腫瘤等作用。益生菌 (probiotics) 已被證實，具有加強免疫調節，降低腸道中有毒物質、致突變物的反應及降低腸癌發生率等有益人體健康之功效。因此，本實驗以不同乳酸菌發酵蕎麥種子與發芽蕎麥，評估其甲醇萃取物是否能抑制大腸癌細胞株增生及抑制4NQO誘導人類腸道細胞株Int-407細胞毒性之能力。本實驗以L. acidophilus BCRC 14079、L. casei 01、L. plantarum BCRC11697、L. rhamnosus GG BCRC 16000、B. infantis BCRC14602、B. longum BCRC14634做為發酵菌酛，結果顯示，六株乳酸菌發酵不同發芽天數之蕎麥芽，在乳酸菌菌數、pH值和可滴定酸含量方面，發芽五天之蕎麥芽為其最適利用之發酵基質。以發芽五天之蕎麥與未發芽蕎麥種實作發酵基質，結果顯示發酵蕎麥芽萃取物在總酚、總類黃酮含量及抗氧化能力均顯著高於未發芽之發酵蕎麥，並且在抑制大腸癌細胞株HT-29增生方面，以L. casei 01發酵蕎麥芽萃取物 (600 μg/mL) 在24及48小時處理下，最低可顯著使HT-29 之存活率分別65.8% 及 48.6% 。之後，利用抗致突變劑4NQO誘導人類腸道細胞株Int-407產生細胞損傷，結果發現L. casei 01發酵蕎麥與蕎麥芽萃取物可顯著提升細胞株之存活率，其中以200 μg/mL之發酵蕎麥芽萃取物效果最佳。利用彗星電泳法評估L. casei 01發酵蕎麥與蕎麥芽萃取物降低4NQO誘導Int-407產生細胞損傷及基因毒性之效果。進一步以分段式預反應，探討萃取物抗細胞毒性機制。以L. casei 01發酵蕎麥與蕎麥芽之萃取物可降低4NQO誘導Int-407細胞之基因毒性。發現L. casei 01發酵蕎麥與蕎麥芽萃取物之抗細胞毒性機制主要為阻斷作用 (Blocking effects) 與生物抗細胞毒性作用 (Bioanticytotoxic effect)。

Buckwheat (Fagopyrum esculentum Moench) is abundant in nutrients, and contains antioxidants, such as rutin, quercetin, polyphenols and flavonoids. Buckwheat has a lot of functional activities of such as antioxidative and antitumor activities. Study has shown that probiotics has many beneficial effects on human health such as strengthening immunity regulation, decreasing the toxigenic/mutagenic reactions in the gut and lower the risk of colon cancer. The aim of this study was to evaluate the anticytotoxic and antigentoxic effects of the extracts of fermented buckwheat and buckwheat sprouts. Results showed that the fermented 5-day germinated buckwheat sprouts were the chioce fermented material for the starters (L. acidophilus BCRC 14079、L. casei 01、L. plantarum BCRC11697、L. rhamnosus GG BCRC 16000、B. infantis BCRC14602、B. longum BCRC14634). The extracts of fermented buckwheat sprouts, its total phenolic contents, total flavonoid content, DPPH-radical scavenging activity and metal chelating activity were significantly higher (p>0.05) than the extracts of fermented buckwheat. The extracts of fermented buckwheat sprouts of L. casei 01 could reduce the cell viability in HT-29. The lowest in HT-29 for 24, 48h and the viability were 65.8% and 48.6%, respectively. Furthermore, the prevention of 4NQO-induced DNA damage on Int-407 cell line was tested by comet assay. The extracts of fermented buckwheat and buckwheat sprouts of L. casei 01 could reduce the genotoxicity of 4NQO-induced DNA damage on Int-407. The possible anticytotoxic mechanisms of extracts were investigated by section-preincubation test. The major mechanisms of the extracts of fermented buckwheat and buckwheat sprouts of L. casei were blocking effects and bioantitoxic effects.