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Antimutagenicity of water extracts from Cassia tora L. prepared under different degrees of roasting and their protective effects on DNA damage
|關鍵字:||Cassia tora L.;決明子;roasting;Ames test;COMET assay;antimutagenicity;DNA damage;mechanism;anthraquinones;焙炒;安氏試驗;彗星分析法;抗致突變性;DNA傷害;作用機制;類化合物||出版社:||食品科學系||摘要:||
本研究主要以安氏試驗及單細胞電泳法(或稱彗星分析法, COMET assay)探討不同焙炒程度決明子之水萃取物(未焙炒、焙炒150℃、焙炒200℃與焙炒250℃)對多種致癌物之抗致突變性及抗DNA損傷之保護效果，並就其可能之作用機制作一探討。在所選取的濃度範圍(0.25-5 mg/plate)內，不論有無S9 mix，四種樣品對Salmonella typhimurium TA98與TA100均不具毒性及致突變性。對需肝臟酵素活化之benzo[a]pyrene (B[a]P)、2-amino-6-methyldipyrido(1,2-a:3':2'-d)imidazole (Glu-P-1)、2-amino-3-methylimidazo[4,5-f]quinoline (IQ)、及3-amino-1,4-dimethyl-5H-pyrido(4,3-b)indole (Trp-P-1)等致突變劑，均具有顯著之抑制效果(p<0.05)。其中以未焙炒的效果最好，在TA98的系統，對B[a]P、Glu-P-1、IQ及Trp-P-1之IC50分別為1.07、0.57、0.15及0.15 mg/mL；於TA100則為0.14、0.17、0.48及0.2 mg/mL。整體而言，決明子水萃取物之抗致突變性隨其焙炒程度增加而漸趨減弱(未焙炒>150℃>200℃>250℃)。至於對不需酵素活化之直接致突變劑MNNG及NQNO則無任何抗致突變效果。不同焙炒程度決明子之水萃取物對IQ及B[a]P引起之回復突變不具生物抗致突變性。以光譜分析致突變物-抑制劑形成分子複合體的結果顯示，未焙炒決明子水萃取物為一干擾分子，可直接與致突變物質發生交互作用，降低其生物作用性。未焙炒決明子之水萃取物對於S9 mix中之ethoxy- (代表CYP-450 1A1)及methoxyresorufin O-deethylase (代表CYP-450 1A2) 酵素活性分別具有98.0 %及89.3 %之抑制率；同時對NADPH cytochrome P-450 reductase活性亦有類似之抑制效果。推測其抑制CYP-450的反應途徑可能有部分出自於干擾NADPH至細胞色素間之電子傳遞。此外，在電子順磁共振光譜(EPR)的檢測系統下，未焙炒決明子之水萃取物可清除83.7 % 由IQ於活化過程產生之超氧陰離子。綜合上述，不同焙炒程度決明子之水萃取物其抗致突變性乃是屬於去致突變作用，而非生物抗致突變作用。
利用彗星分析系統，樣品在選取之濃度範圍(0.1-2 mg/mL)內，對人類淋巴球細胞不具細胞毒性(細胞存活率>95 %)。然而，當作用劑量高於0.5 mg/mL，決明子水萃取物對人類淋巴球細胞卻具有不同程度之促DNA損傷效應，三者中以焙炒250℃之樣品引起的傷害較為顯著(tail moment≒15)。以Trp-P-1為傷害誘導劑的測試系統，不同焙炒程度之決明子其抗基因毒性與添加濃度呈劑量與反應之相關性(p<0.05)，未焙炒、150℃及250℃焙炒決明子之水萃取物於2 mg/mL可分別達到75 %、62 %與45 %抑制DNA損傷的效果。決明子水萃取物不論其抗致突變性或抗DNA損傷的活性，皆呈未焙炒>150℃焙炒>250℃焙炒的趨勢，保護作用隨焙炒程度增加而逐漸減弱。針對抗基因毒性機制之探討，未焙炒決明子之水萃取物能直接與Trp-P-1、Glu-P-1發生交互作用形成分子複合體；對於Trp-P-1之代謝活化物具有38.7 % 之清除效應。與人類淋巴球細胞預培養30分鐘後，對於之後Trp-P-1誘導的DNA損傷可提昇30 %的保護效果；然而對已經Trp-P-1造成的DNA損傷並不具後續促進修復的能力。將決明子水萃取物經酸水解，以HPLC分析可得到三種已知之類化合物(AQ)︰chrysophanol、emodin及rhein，其中以未焙炒之決明子含量最高，每克水萃取物約含有rhein (10.42 mg)>chrysophanol (0.61 mg)> emodin (0.28 mg)。AQ含量隨焙炒程度增加而降低，焙炒250℃之樣品甚至偵測不到上述三種AQ。Emodin (-S9 mix)與rhein (+S9 mix)在COMET assay中具有輕微促DNA斷裂的反應。對於Trp-P-1誘導之DNA損傷，chrysophanol、emodin及rhein則分別有79.0 %、63.7 %與37.9 %之抑制效果。
此外，本研究另探討不同焙炒程度(未焙炒、150℃、250℃)決明子之水萃取物在無外生性酵素(S9 mix)存在下，對多環芳香化合物benzo[a]pyrene (B[a]P)誘導人類肝癌Hep G2細胞株DNA損傷之影響。結果顯示，以不同焙炒程度決明子之水萃取物(0.1-2 mg/mL)處理Hep G2細胞24小時，均不構成該細胞之細胞毒性(細胞存活率>90 %)與基因毒性。抗基因毒性方面，樣品對於B[a]P誘導之DNA損傷，可隨其作用劑量呈線性之抑制關係(p<0.05)。未焙炒、150℃焙炒及250℃焙炒之決明子於添加濃度1 mg/mL下，可分別減輕72 %、60 %及23 %之DNA傷害，保護效果亦隨著焙炒程度增加而顯著減弱。探討其作用機制則發現，決明子之水萃取物可作用於Hep G2胞內活化及解毒B[a]P之主要代謝酵素。對於ethoxyresorufin O-deethylase (EROD)，同樣呈現未焙炒(63.9 %)>150℃焙炒(41.6 %)>250℃焙炒(17.5 %)之抑制趨勢。對NADPH cytochrome P-450 reductase之活性，未焙炒及焙炒150℃之樣品亦分別有49.5 %及38.4 %之抑制率，顯示其抑制EROD之作用途徑可能部分出自於干擾NADPH至細胞色素間之電子傳遞。此外，未焙炒及焙炒150℃決明子之水萃取物對glutathione S-transferase (GST) 之活性可分別提昇1.28及1.21倍，促進glutathione與B[a]P之親電性活化物鍵結，迫使其代謝成更具水溶性之形式而排出細胞外。決明子水萃取物中具有抗致突變性之作用成分anthraquinones類化合物(AQ)，其含量隨焙炒程度增加而減低。對於B[a]P誘導Hep G2之DNA損傷，chrysophanol、emodin及rhein則分別有89.4 %、85.9 %及71.2 %之極佳抑制效果，此顯示決明子經不同焙炒處理後其抗基因毒性之表現，可能與其AQ的含量變化有關。
In the present study, antimutagenicity and the protective effects on DNA damage of water extracts from Cassia tora L. (WECT) treated with different degrees of roasting (unroasted and roasted at 150, 200, and 250℃) were evaluated by Ames test and the Single cell gel electrophoresis assay. No toxicity or mutagenicity to Salmonella typhimurium TA98 and TA100 was found in the WECT at a dose of 0.25-5 mg per plate with and without S9 mix. WECT had a very marked and dose-dependent inhibition effects on the Aroclor 1254-hepatic S9-mediated mutagenicity of benzo[a]pyrene (B[a]P), 2-amino-6-methyldipyrido(1,2-a: 3': 2'-d) imidazole (Glu-P-1), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 3-amino-1,4-dimethyl-5H-pyrido(4,3-b)indole (Trp-P-1). The antimutagenicity of WECT decreased with an increasing roasting temperature in the following order: unroasted>150℃>200℃>250℃. For strain TA98, the IC50 of water extracts of unroasted Cassia tora L. (WEUCT) toward B[a]P, Glu-P-1, IQ and Trp-P-1 were 1.07, 0.57, 0.15 and 0.15 mg/mL; whereas for strain TA100, the IC50 were 0.14, 0.17, 0.48 and 0.2 mg/mL, respectively. Neither the mutagenicity of MNNG nor that of NQNO was suppressed by WECT in TA98 and TA100. WECT had no inhibitory effects toward IQ and B[a]P in the bio-antimutagenic assay. Mutagen-inhibitor interaction (molecular complex formation) could be illustrated by a change in the UV spectrum, suggesting that WEUCT may act as an "interceptor molecule", interacting with mutagens directly and limiting their bioavailability. Methoxy- and ethoxyresorufin-O-dealkylase activities of rat microsomes, linked to cytochrome CYP-450 1A1 and 1A2 monooxygenases catalyzing N-hydroxylation of mutagens, were effectively inhibited by WEUCT (98.0 % and 89.3 %). The NADPH-dependent reduction of cytochrome P-450 activity was similarly inhibited, implying the inhibitory effect on the CYP-450 activity may be, at least in part, due to an impairment of the electron transfer from NADPH to the cytochrome. In addition, WEUCT showed 84.7 % scavenging effect on superoxide anion generated in the activation process of IQ by S9 mix in electron paramagentic resonance (EPR) system. The results presented herein suggest that the antimutagenicity of WECT were due to a desmutagenic action, but not a bioantimutagenic action.
WECT exhibited no cytotoxicity to human lymphocytes at a concentration of 0.1-2 mg/mL, the cell viability was greater than 95 %. However, in the COMET assay, WECT caused a different extents of DNA damage in human lymphocytes at a concentration over 0.5 mg/mL, especially the sample of roasted at 250℃ (Tail moment=15). All three types of WECT (unroasted and roasted at 150, and 250℃) presented antigenotoxic effects on DNA damage in human lymphocytes induced by Trp-P-1 and in a dose-dependent manner (P<0.05). At a concentration of 2 mg/mL, the inhibitory effects were observed in the order of unroasted (75 %)>roasted at 150℃ (62 %)>roasted at 250℃ (45 %). It revealed that increasing roasting temperature of the seeds of Cassia tora L. might decrease their antigenotoxic activity both in the Ames test and the COMET assay. Mutagen-inhibitor interaction was identified in spectrophotometry studied, suggesting that WEUCT may produce complexes with Glu-P-1 and Trp-P-1. Using a modified COMET assay procedure, WEUCT exhibited 38.7 % scavenging effect on reactive intermediates of Trp-P-1 generated from metabolism system. Pre-treatment of the human lymphocytes with WEUCT for 30 min resulted in a modest repression of DNA damage (30 %). In contrast, no promotive effect of excision-repair was found during DNA damage expression time in post-treatment scheme. Further, three anthraquinones (AQ): chrysophanol, emodin and rhein were determined from acid hydrolyzed WECT by HPLC. It was found that the amounts of these AQ in WECT all decreased with an increasing roasting temperature. The contents of chrysophanol, emodin and rhein in WEUCT were 0.61, 0.28 and 10.42 mg/g, respectively. Emodin (-S9 mix) and rhein (+S9 mix) exhibited slight DNA damage in human lymphocytes in the COMET assay. However, chrysophanol, emodin and rhein shown 79.0, 63.7 and 37.9 %, respectively, protective effects on DNA damage induced by Trp-P-1.
The effects of WECT on B[a]P-induced DNA damage in human hepatoma cell line Hep G2 were investigated in the COMET assay without exogenous activation mixtures (S9 mix). WECT alone shown neither cytotoxic nor genotoxic toward Hep G2 cells under a concentration of 0.1-2 mg/mL. B[a]P-induced DNA damage in Hep G2 cells was reduced by WECT in a dose-dependent manner (P<0.05). At a concentration of 1 mg/mL, the inhibitory effects on DNA damage were in the order of unroasted (72 %)>roasted at 150℃ (60 %)>roasted at 250℃ (23 %). Ethoxyresorufin-O-dealkylase activity of Hep G2 cells, linked to cytochrome CYP-450 1A1 monooxygenases, were effectively inhibited by WECT, and a similar inhibition was observed in the following order: unroasted (63.9 %)>roasted at 150℃ (41.6 %)>roasted at 250℃ (17.5 %). The activity of NADPH cytochrome P-450 was also decreased by unroasted and roasted at 150℃ samples (49.5 % and 38.4 %), implying the inhibitory effects on the CYP-450 activity may be, at least in part, due to an impairment of the electron transfer from NADPH to the cytochrome. Furthermore, glutathione S-transferase activity was slightly increased by the treatment with unroasted (1.28-fold) and roasted at 150℃ (1.21-fold) samples at a concentration of 1 mg/mL, compared to the control group. In addition, the contents of antimutagenic AQ in WECT, including chrysophanol, emodin and rhein were decreased with an increasing roasting temperature in the following order: unroasted>150℃>250℃. Each of these AQ also demonstrated significant antigenotoxic activity in the COMET assay. The inhibitory effects of chrysophanol, emodin and rhein on B[a]P-mediated DNA damage in Hep G2 cells were 89.4, 85.9 and 71.2 %, respectively. These findings suggested that the decrease in the antigenotoxic activity of the roasted samples might be due to the reduction in its anthraquinones content.
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