發酵黑豆對低密度脂蛋白氧化作用、結腸癌細胞增生以及4NQO對人類腸道細胞基因毒性之抑制

Abstract

本研究利用Aspergillus awamori對黑豆進行固態發酵，探討不同溶劑 (水、80%甲醇、80%乙醇) 之發酵黑豆萃取物所呈現之總酚和胺基態氮含量，並分析這些萃取物對於螯合銅離子和延緩低密度脂蛋白 (low density lipoprotein, LDL) 氧化之活性，此外並檢測發酵黑豆萃取物抑制結腸癌細胞株Caco-2和HT-29增生及其抗4-nitroquinoline-N-oxide (4NQO) 誘導腸道細胞株Int-407細胞與基因毒性之效果。此外，並藉由分段式預反應探討發酵黑豆甲醇萃取物抗細胞毒性之機制。 結果顯示，無論是黑豆或發酵黑豆均具有抗氧化活性、抗癌細胞增生以及抑制4NQO誘導細胞損傷等生理活性。黑豆發酵後，以甲醇萃取物中總酚含量最高，而水萃取物具有最多之胺基態氮含量並呈現顯著最佳之LDL抗氧化效果 (p＜0.05)，在30℃發酵三天之黑豆水萃取物抑制LDL氧化之效果最強。 發酵黑豆各溶劑萃取物中，以黑豆甲醇萃取物降低結腸癌細胞增生之效果最佳。在30℃發酵三天之黑豆甲醇萃取物對4NQO誘導細胞毒性呈現最佳抑制能力。發酵黑豆保護Int-407減少4NQO對其DNA損傷之效果亦優於未發酵黑豆者佳。進一步探討發酵黑豆抗4NQO所引起細胞毒性之機制，顯示其活性來自於使4NQO不活化 (Destoxic effect)、修飾細胞之機能阻斷4NQO之傷害 (Blocking effect) 以及修補受損DNA (Bioantitoxic effect) 等作用。

In the present study, various solvent (water, 80% methanol, 80% ethanol) extracts of Aspergillus awamori-fermented black soybean were examined for total phenolic, amino nitrogen contents and the antioxidant activities including Cu2+-chelating ability and inhibition on low density lipoprotein (LDL) oxidation. In addition, the effect of fermented black soybean extracts against the proliferation of human colon cancer cells Caco-2 and HT-29 as well as on the cytotoxicity and genotoxicity induced by 4-nitroquinoline-N-oxide (4NQO) toward Int-407 were detected. Furthermore, possible anticytotoxicic mechanisms of methanol extract of fermented black soybean were investigated by section-preincubation test. The results showed that the extracts of the black soybeans, regardless of fermentation, all exhibited antioxidant activities and inhibitory effect on the proliferation of cancer cells as well as cell damage induced by 4NQO. Among the various extracts examined, methanol extract showed the highest contents of total phenolics, while water extract possessed the highest amino nitrogen contents after fermentation. It was found that the water extract of A. awamori-fermented black soybean prepared at 30℃ for 3 days exhibited the highest inhibitory effect on LDL oxidation. Of the fermented black soybean extracts examed, methanol extract was found to exhibit the highest efficiency in reducing the proliferation of cancer cells. Methanol extract of the fermented black soybean prepared at 30℃ for 3 days was noted to show the highest inhibitory effect on 4NQO-induced cytotoxicity. The extract of fermented black soybean also exhibited a higher protective effect on Int-407 against 4NQO-induced DNA damage than did the respective extract of the non-fermented black soybean. Further study on the possible anticytotoxicic mechanism, revealed that the destoxic, blocking and bio-antitoxic effect all contributed to the anticytotoxicity of A. awamori-fermented black soybean against 4-NQO.