Antioxidant Effects Of Daidzein Or Coenzyme Q10 Alone Or In Combination With Micro-nutrients In In Vitro Systems And In Animal Models

Objectives:1.Using in vitro chemical and biological model systems to study the antioxidative mechanisms of daidzein（Da）,coenzyme Q10（CoQ10,CQ）,lycopene（Lyc） and lutein（Lut）,and to study the antioxidant activities of them and synergistic effects of different antioxidants.2.Using an animal model to evaluate the in vivo antioxidant defense effects of daidzein and coenzyme Q10 alone and to evaluate the combined effects of them with mixed carotenoids（lycopene and lutein,LL） or/and mineral nutrients（znic and selenium,ZS）.Methods:1.In vitro antioxidant activities of Da,CQ,Lyc and Lut.The reducing power,Fe（Ⅱ） and Cu（Ⅱ） chelating activity,scavenging activities of DPPH and O₂^- radicals of the antioxidants were determined.The inhibition effects of antioxidants on Fe（Ⅱ）-VC-induced lipid peroxidation in liver microsome and on Cu（Ⅱ）-induced oxidation in LDL were evaluated.2.Synergistic in vitro antioxidative effects of different antioxidants.Synergistic antioxidative effects of each antioxidant with VE and synergistic effects among Da,Lyc and Lut and among CQ,Lyc and Lut were determined in Fe（Ⅱ）-VC-induced lipid peroxidation in liver microsome.Synergistic antioxidative effects of each antioxidant with VC were determined in Cu（Ⅱ）-induced oxidation in LDL.3.Antioxidant effects of daidzein alone or in combination with micro-nutrients on antioxidant status in rats.The rats were fed with a diet based on AIN-76 rodent diet but containing 4% lard as well as 4%corn oil.They were randomized into 9 groups:control,D（H） （daidzein with the purity of 80%）,D（H）+ZS,D（H）+LL,D（H）+ZS+LL,D（L） （daidzein with the purity of 40%）、D（L）+ZS,D（L）+LL,D（L）+ZS+LL.The dosages of Da,Lyc,Lut,Zn and Se were 5 mg/kg bw·d、2 mg/kg bw·d、1 mg/kg bw·d、1 mg/kg bw·d and 4μg/kg bw·d respectively.The plasma and liver antioxidant enzymes （SOD、GSH-px、CAT） and MDA levels,lymphocyte DNA damage,plasma NO concentration,plasma lipid profile and indexes of bone health in each group were determined.4.Antioxidant effects of coenzyme Q10 alone or in combination with micro-nutrients on antioxidant status in rats.The rats were randomized into 6 groups:control,CQ、CQ+ZS,CQ+LL, CQ+ZS+LL and VE。The dosages of CQ,Lyc,Lut,Zn,Se and VE were 10 mg/kg bw·d、2 mg/kg bw·d、1 mg/kg bw·d、1 mg/kg bw·d、4μg/kg bw·d and 2 mg/kg bw·d respectively.The plasma and liver antioxidant enzymes（SOD、GSH-px、CAT） and MDA levels,lymphocyte DNA damage,plasma NO concentration,plasma lipid profile in each group were determined.Results:1.In vitro antioxidant activities of Da,CQ,Lyc and Lut and the synergy of different antioxidants.（1） Da had low reducing power,chelating activity and DPPH-scavenging activity, while had relatively high O₂^--scavenging activity.Although Da inhibited lipid peroxidation in microsome initiated by Fe（Ⅱ）-VC,the concentration required for this effect were relatively high compared to those of other antioxidants.However,Da inhibited Cu（Ⅱ）-induced oxidation in LDL with a physiological concentration.（2） CQ had no reducing power,chelating activity and radical-scavenging activity, while it effectively inhibited lipid peroxidation in microsome.It also exerted weak antioxidant activity in LDL oxidation induced by Cu（Ⅱ）.（3） Lyc had relatively high reducing power and high radical-scavenging activity. Its inhibition effect on Fe（Ⅱ）-VC-induced lipid peroxidation in microsome were even greater than that of VE.However,it had no effect on Cu（Ⅱ）-induced oxidative modification of LDL.（4） Lut had relatively high reducing power,while it is a weak chelator and weak radical-scavenger.Its effects on lipid peroxidation in microsome and oxidative modification of LDL were both minor.（5） Each of the Da,CQ,Lyc and Lut synergistically inhibited Fe（Ⅱ）-VC-induced lipid peroxidation in microsome with VE（P=0.000）.The synergism of CQ and Lyc with VE were especially greater.There were also synergistic antioxidant effects among Da,Lyc and Lut（P＜0.01） and among CQ,Lyc and Lut in this model（P=0.000）.（6） Each of the Da,CQ and Lut synergistically delayed Cu（Ⅱ）-induced lipid oxidative in LDL with VC（P=0.000）.2.Antioxidant effects of daidzein alone or in combination with micro-nutrients on antioxidant status in rats.（1） High-purity daidzein alone enhanced plasma SOD and TAOC levels and liver GSH-px activity,and decreased lymphocyte DNA damage in rats（P＜0.05）. Low-purity daidzein alone enhanced plasma SOD and TAOC levels and liver SOD and CAT activities in rats（P＜0.05）.（2） When daidzein was combined with mixed carotenoids and/or minerals, antioxidant enzymes activities in plasma and liver and TAOC level in plasma and brain were raised more frequently and largely,and levels of lipid peroxidation in plasma and brain and lymphocyte DNA damage were decreased further,compared with daidzein supplemented alone.As the number of antioxidants supplemented increased,change of biomarkers of antioxidant status became greater and more frequent.（3） All treatments significantly increased plasma NO concentration（P＜0.05）, while there was no difference among all supplementations.（4） All supplementations have no effects on plasma lipid profile and indexes of bone health in rats,compared with the control.（5） Though low-purity daidzein treatments enhanced biomarkers of antioxidant status a little more frequently than high-purity daidzein treatments,there was no difference between the corresponding high-and low-purity daidzein treatments.3.Antioxidant effects of coenzyme Q10 alone or in combination with micro-nutrients on antioxidant status in rats.（1） Rats treated with CQ had higher plasma SOD and TAOC levels and liver SOD activity than rats in the control group（P＜0.05）,while CQ alone had no significant effect on lymphocyte DNA damage. （2） Combined treatments of CQ with micro-nutrients further enhanced antioxidant enzymes activity and reduced MDA levels & DNA damage,especially combined treatments of CQ with mixed carotenoids and with mixed carotenoids and minerals.（3） All supplementations have no effects on plasma lipid profile and plasma NO concentration in rats.Conclusions:1.Though daidzein had mild inhibition effects on Fe（Ⅱ）-VC-induced lipid peroxidation in microsome,it effectively inhitited Cu（Ⅱ）-induced oxidation in LDL with a physiological concentration,which may be due to its interaction with Apo B in LDL.2.Liver microsome could reduce CQ to CQH₂,which protected microsome against lipid peroxidation.LDL reduced CQ in much lesser extent.3.Lycopene is a good radical-scavenger and it largely inhibited microsome lipid peroxidation,while had no effect on oxidation modification of LDL.Lutein had minor effects on both of the two models.4.There were synergistic effects between different antioxidants in in vitro,systems, which may be related to the regeneration of one antioxidant by another,different location and orientation in biological systems owing to their structural features and complementation of antioxidative mechanisms.5.In our animal model,daidzein and coenzyme Q10 exerted moderate antioxidative protective effects.When they were supplemented in combination with other antioxidants,especially with mixed carotenoids or with mixed carotenoids and mineral,the effects were raised to some extent.The purity of daidzein had insufficient effects on its antioxidant effects.6.Daidzein enhanced plasma NO concentration in rats.The results of our study suggested that daidzein or coenzyme Q10 supplementation in combination with nutrients may have beneficial effects on human health.