Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/50815
標題: The effects of processing conditions on the content of isoflavones in soy products and the effects of isoflavones on DNA damage in mammal cell by single cell gel electrophoresis
加工條件對大豆製品異黃酮含量之影響及以單細胞電泳法探討其對哺乳類 細胞DNA損傷影響之研究
作者: 高嘉鴻
Kao, Chia-Hung
關鍵字: 大豆製品
異黃酮
單細胞電泳法(彗星分析法)
哺乳類動物細胞
Daidzein genistein
DNA損傷
soybean products
isoflavone
daidzein
genistein
single cell gel electrophoresis (Comet assay)
mammal cell
DNA damage
出版社: 食品科學系
摘要: The objectives of this study were to determine the contents of isoflavone glucosides (daidzin, genistin) and their aglycones (daidzein, genistein) in commercial soybean and related bean products by HPLC; and to investigate the effects of processing conditions on changes in the isoflavones content of black soybean milk, yuba and chaw-tofu. The contents of isoflavones (daidzin, genistin, daidzein and genistein) in black soybean were higher than those in soybean. The contents of isoflavones in soy sprouts were also higher than those in soybean. The contents of isoflavone glucosides (daidzin, genistin) in unfermented soybean products followed the order of yuba (3196 mg/g dry wt) > hard bean curd (2164mg/g dry wt)> tofu (1971 mg/g dry wt)> soy sprout (1487 mg/g dry wt)> soymilk (851 mg/g dry wt). The contents of isoflavone aglycones (daidzein, genistein) in unfermented soybean products followed the order of yuba (410mg/g dry wt) ≒ soy sprout (401 mg/g dry wt)≒ tofu (377mg/g dry wt)> hard bean curd (175 mg/g dry wt)> soymilk. The contents of daidzein and genistein in fermented soy products followed the order of soybean paste (935 mg/g dry wt)≒chaw-tofu (855 mg/g dry wt)> toshi (653 mg/g dry wt)> sufu (302 mg/g dry wt)> miso (267 mg/ g dry wt)> soy sauce (18 mg/g dry wt). The levels of daidzein and genistein in fermented soybean products were higher than those in unfermented products, except soy sauce. When black soybeans were soaked in water at 30 and 50℃ for various periods of time, the contents of daidzein and genistein increased with an increase of the soaking time while daidzin and genistin decreased. There was no significant difference (p<0.05) in the contents of isoflavones in black soybean under soaking at 30℃ for 12h and at 50℃ for 2h. The amounts of isoflavones in black soybean changed markedly under soaking at 20-60℃ for 8h. The content of isoflavone glycosides and their aglycones in black soybean ranged from 226~227and 360~547 mg/g dry wt, respectively, under soaking at 50℃ for 8h. The change in the contents of isoflavones in black soybean during soaking might be related to its b-glucosidases activity. The effect of soaking temperature in b-glucosidases activity of black soybean was in the order of 50> 40> 60> 30> 20℃. No significant difference (p>0.05) was found in the sensory evaluation of black soy milk, prepared under different soaking conditions, which contained different amounts of daaidzein and genistein. The contents of daidzein and genistein in Yuba (B) prepared by soaking soybeans at 50℃for 6 h was 389 and 505 mg/g dry wt, respectively. The contents of these two compounds in Yuba (B) were about 2.5 times those in Yuba (A) that prepared by soaking soybeans at 30℃for 12 h. Yuba (B) contained higher ash but lower protein and fat content than did Yuba (A). The contents of daidzein and genistein in chaw-tofu increased during the fermentation period while the contents of daidzin and genistin decreased. In addition, 9-25% of the contents of isoflavones in chaw-tofu were removed after frying treatment. The effects of isoflavone aglycones on DNA damage in mammals cells, human blood lymphocyte, human promyelocytic leukemia (HL-60) cells and Chinese hamster ovary (CHO) were investigated by means of single cell gel electrophoresis (Comet assay). Moreover, the effects of UV and H2O2 mediated DNA damage in human lymphocyte were also investigated. After incubation with cells for 30 min, daidzein induced DNA damage in CHO and HL-60 cells only at a higher concentration of 40 mg/ml but did not induce DNA damage in human lymphocyte at the same concentration. DNA damage in CHO and HL-60 cells increased significantly(p<0.05) under treatment with genistein at a concentration of 5 mg/ml, especially the HL-60 cells. The mean values of tail DNA % increased to 28.65 in HL-60 cells. DNA damage in human blood lymphocyte exhibited a significant increase only at a concentration greater than 40 mg/ml, and the mean values of tail DNA % increased to 21.89. Genistein caused greater DNA damage than did daidzein in these three kinds of cells at a concentration of 0-40 mg/ml. In addition, genistein caused more DNA damage in CHO and HL-60 cells than in human lymphocytes. The reason might be that genistein can inhibit topoisomerase and then induce protein-linked DNA damage. UV-C induced DNA damage in human blood lymphocyte was inhibited by daidzein and genistein in a concentration dependent manner(0-40 mg/ml). When the cells were preincubated with daidzein or genistein (40 mg/ml) before expose to UV-C for 5 min, the mean values of tail DNA % values decreased from 69.19 to 21.69 and 34.66, respectively, compared to the control group. No protective effect was found when cells did not undergo preincubation with daidzein and genistein before exposure to UV-C. The protective effects of these two compounds on UV-C mediated DNA damage might be due to the absorption of UV-C or induction antioxidant enzymes in cells by these two compounds. Furthermore, daidzein and genistein exhibited no protective effect on H2O2 induced DNA damage in human blood lymphocytes. In conclusion, the protective effects of daidzein and genistein on DNA damage induced by UV light irradiation were greater than those of by H2O2.
本研究是以HPLC分析市售大豆及其相關發酵、未發酵製品中, 具醣基異 黃酮(isoflavone)成分daidzin及genistin和不具醣基異黃酮 daidzein 及genistein之含量,進一步探討黑豆乳、豆皮和臭豆腐經 過不同加工 條件對異黃酮含量之影響。並以單細胞電泳法(彗星分析) 法分析 daidzein和genistein對於哺乳類動物細胞DNA損傷之影響。 實驗結果顯 示黑大豆中daidzin、genistin、daidzein和genistein 含量都高於黃大 豆,此外黃豆芽中四種異黃酮含量也都大於黃 大豆。其它未發酵大豆製 品乾重中具醣基異黃酮含量依序為豆 皮 (3196 mg/g) > 豆乾 (2164 mg/g) > 豆腐 (1971 mg/g) > 豆芽 (1487 mg/g) > 豆漿 (851 mg/g) ,不具醣基的異黃酮含量依序為 豆皮 (410 mg/g)≒豆芽 (401 mg/g)≒ 豆腐 (377 mg/g )>豆乾(175 mg/g )>豆漿。大豆發酵製品中daidzein 和genistein含量依序為 豆瓣醬 (935 mg/g) ≒臭豆腐 (855 mg/g) >豆 豉 (653 mg/g) >豆腐 乳 (302 mg/g) >味噌 (267 mg/g) >醬油 (18 mg/g)。除了醬油以外 大豆發酵製品中daidzein和genistein含量都高於 未發酵製品。 將黑大豆分別浸泡於30或50℃水溫不同時間,結果顯示隨 著浸 泡時間增加daidzein和genistein含量漸增,daidzin和genistin 漸減;50℃浸泡2小時後與30℃浸泡12小時者異黃酮含量即無 顯著性差 異。黑大豆浸泡於不同溫度(20、30、40、50、60℃)八 小時, 以50℃ 浸泡者乾重中daidzein (360 mg/g)和genistein (547 mg/g)含量最高 ,daidzin (226 mg/g)和genistin (227 mg/g)含量最 低,此現象與黑 大豆中之酵素水解daidzin和genistin有關。因 為黑大豆浸泡於50℃水 溫度中b-glucosidases酵素活性最強,其 次是40℃,再次之為60℃。以 官能品評分析不同浸泡條件製成 之高daidzein和genistein含量黑豆乳 與低含量之黑豆乳,並無顯著性差異(p>0.05)。在豆皮製做過程中改 變 黃大豆浸泡條件,結果發現Yuba B (黃大豆經50℃浸泡6小時) daidzein和genistein分別含389及505 mg/g dry wt,兩成分含量約為 Yuba A (黃大豆經30℃浸泡12小時) 2.5倍。Yuba B的基本成份相較 於 Yuba A,其中灰分含量較高而蛋白質及油脂含量較低。在臭豆腐 發酵過 程中隨著發酵時間增長,不具醣基異黃酮genistein 和daidzein 含量隨 之增加,而具醣基異黃酮daidzin和genistin則隨之減少。臭 豆腐經油 炸處理後各異黃酮成分含量卻都減少,減少比率9~25%。 以彗星分析法 檢測daidzein和genistein 濃度範圍0~40 mg/ml對 於人體靜脈血液淋巴 球細胞(Lymphocyte)、人類急性前骨髓白血 病細胞(HL-60)及中國倉鼠 卵巢細胞(CHO)之DNA損傷性,並探討 daidzein和genistein對於UV-C及 H2O2誘導人體靜脈血液淋巴球細 胞DNA損傷之影響。以Tail DNA %表示 個別細胞DNA損傷程度。 細胞與樣品反應30 min後檢測DNA損傷情形,結 果顯示daidzein 在高濃度(40 mg/ml)下才會對CHO細胞及HL-60細胞DNA 造成明 顯損傷,但對於人體血液淋巴球則不具傷害性。Genistein在低 濃度 下5 mg/ml就會造成CHO細胞及HL-60細胞DNA明顯損傷,尤其 HL-60細胞損傷最為嚴重,平均Tail DNA %增加為28.65。對於人 體血液 淋巴球DNA,低濃度genistein並不具損傷性,只有在高濃度 時40 mg/ml 才有明顯傷害,平均Tail DNA %增加為21.89。實驗結 果顯示系統中 genistein (0~40 mg/ml)各濃度下對於細胞DNA損傷性 都大於daidzein ,此外genistein對於HL-60細胞及CHO繼代細胞DNA 損傷性大於人體血液 淋巴球細胞,造成DNA斷裂的原因可能來自於 genistein抑制 topoisomerase而促使protein-linked DNA斷裂表現。 Daidzein和 genistein對於UV-C誘導之人體靜脈血液淋巴球傷 害性具有抑制效果 ,且隨著濃度(0~40 mg/ml)增加保護性增高,當系 統中分別加入 daidzein和genistein (40 mg/ml)與細胞反應30 min後照 射UV-C 5min ,細胞平均Tail DNA %由控制組的69.19降低為21.69 及34.66。若是 daidzein和genistein與細胞混和後直接照射UV-C, 兩成分則不具保護 性。此二成分抗UV-C的效果上可能來自於具有 濾光效果,或者與細胞反 應時改變細胞膜上的酵素作用及增加細胞 中抗氧化酵素含量。兩異黃酮 濃度範圍在0~40 mg/ml與細胞及H2O2 同時反應30 min後對於H2O2誘導的 DNA傷害並無抑制效果。 Daidzein和genistein對於UV-C誘導DNA傷害之 抑制效果,意謂著 可能具有抗光照引起之腫瘤的潛力。
URI: http://hdl.handle.net/11455/50815
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