Research On Mechanism And Rules Of Antioxidant Interactions Among Four Daily Fruits/Vegetables Extracts And Four Phytochemicals

Vegetables and fruits from daily diet are thought to protect against many chronic diseases caused by oxidative stress,such as tumors,inflammation,diabetes,atherosclerosis,and among others.Different combinations of phytochemicals have been shown to produce antioxidant interactions（e.g.,synergistic,antagonistic,additive effects）.According to different molecular structures and solubility properties,phytochemicals can be divided into hydrophilic phytochemicals which soluble in polar solvents（e.g.,water,methanol,ethanol,etc.）and lipophilic phytochemicals which soluble in non-polar solvents（e.g.,chloroform,n-hexane,dichloromethane,etc.）.Up to now,studies on phytochemical antioxidant interactions based on chemical models or only limited to the same type of phytochemicals.There is a lack of researches on the antioxidant interaction rules and mechanisms among phytochemicals based on different types,ratios,and concentrations.Previous researches also lack consideration of cellular absorption and cell membrane transporters on phytochemical antioxidant interactions.Few study focus on target proteins of phytochemical antioxidant interactions.This research explored antioxidant interactions rules and mechanisms among four fruits（mulberries,blueberry,mango,watermelon）and four vegetables（eggplant,purple potato,carrot,tomato）based on chemical and cells models through cytotoxicity,intracellular ROS level,expression of antioxidant enzymes,expression of inflammatory factors.After identifying the main phytochemicals of fruits and vegetables by high-performance liquid chromatography-mass spectrometry,four phytochemicals（caffeic acid,p-coumaric acid,β-carotene,lycopene）which were the main hydrophilic or lipophilic phytochemicals were chosen to explore antioxidant interaction rules on different concentration and ratios.The role of cell membrane transporter and the possible target of antioxidant interaction were also explored.Research results are as follows:1.Antioxidant activities（DPPH and ABTS）and combination index were used as indcators and evaluation methods,respectively.The antioxidant synergistic effect was observed when the hydrophilic extracts were in the majority(e.g.,mulberry:mango=9:1 in ABTS model,CI₂₅=0.66±0.12,CI<1).When lipophilic phytochemicals were in the majority,combination groups often show antioxidant antagonism(e.g.,blueberry:mango=1:9 in ABTS mode,CI₂₅=1.11±0.09).2.This research established H₂O₂-induced H9c2 cell model,using cytotoxicity（determined by MTT）,intracellular reactive oxygen species（ROS）level,cell total antioxidant capacity（CAA）,expression of intracellular antioxidant enzymes（SOD,CAT,GPx）,intracellular lipid peroxide（MDA）level,expression of inflammatory factors（ICAM-1,VCAM-1,IL-6,IL-1β,IL-8,TNF-α）in cells as indicators.It found that when the hydrophilic extracts were in the majority,combination groups showed synergistic effects（e.g.,eggplant:carrot=9:1,intracellular ROS level was 30.37±0.25,which was significantly lower than that of eggplant only:34.34±0.36 and carrot only:46.23±0.51）.On the other hand,when the lipophilic extracts were in the majority,the combination groups showed antagonistic effects（e.g.,tomato:purple sweet potato=7:3,the intracellular ROS level was 68.21±0.95,significantly higher than that of tomato only:34.51±0.76 and purple sweet potato only:34.21±0.68）.3.The main phytochemical components in fruit and vegetable extracts were identified by UPLC and HPLC-MS.The relationship between the phytochemicals and antioxidant interaction was analyzed by principal component analysis（PCA）.It was found that the main phytochemicals of hydrophilic extracts were anthocyanins,phenolic acids and flavonoids.The main phytochemicals of lipophilic extracts wereβ-carotene and lycopene.According to PCA analysis,delphinin,cyanidin,caffeic acid,p-coumaric acid,gallic acid may play a key role in the synergistic effect.β-Carotene and lycopene may have a key effect on antagonistic effect.4.This study established H₂O₂-induced H9c2 cell model,using cytotoxicity（determined by MTT）,intracellular reactive oxygen species（ROS）level,cell total antioxidant capacity（CAA）,expression of intracellular antioxidant enzymes（SOD,CAT,GPx）,intracellular lipid peroxide（MDA）level,intracellular lactate dehydrogenase（LDH）leakage rate as indicators to explore antioxidant interaction among four phytochemicals（caffeic acid,p-coumaric acid,lycopene,β-carotene）at different concentrations and ratios.When the total concentration was the same,combination groups showed synergistic effects as the hydrophilic phytochemicals were in the majority,while combination groups showed antagonistic effects as the lipophilic phytochemicals in the majority.When the ratio of combination groups was the same,the antioxidant interaction changed as increase of concentration.For example,group（p-coumaric acid:β-carotene=1:9）showed antagonistic effect（synergistic rate SR:-11.68±1.25,SR<0）at 3.0μM,while this group showed antioxidant synergistic effect（synergistic rate SR:12.27±3.24,SR>0）at 0.5μM.5.H₂O₂-induced H9C2 cell model was established to investigate the effects of caffeic acid/p-coumaric acid on the cell absorption ofβ-carotene and lycopene.Intracellular cell membrane transporter expression,intracellular ROS level,and Nrf2/HO-1 signaling pathway after addition of cell membrane transporter（SR-BI and CD36）inhibitors were also analyzed.It was found that caffeic acid/p-coumaric acid could increase the protein expression of SR-BI and CD36,increase the absorption rate ofβ-carotene and lycopene,reduce the intracellular ROS level,up-regulate the accumulation of Nrf2 protein in the nucleus,and promote the expression of its downstream antioxidant proteins（HO-1,GCLC and NQO1）with inhibitor of cell membrane transporters.This phenomenon may be one of the mechanisms of antioxidant synergism among these four phytochemicals.6.Molecular docking technology was used to explore the interaction and possible spatial conformation between the oxidation products ofβ-carotene/lycopene,caffeic acid,p-coumaric acid,and the BTB/Kelch domains of Keap1 protein.It was found that the oxidative products of carotenoids（OPC）may preferentially bind to the BTB domain.In this case,the complex binding ability of caffeic acid/p-coumaric acid to the BTB-OPC complexes is lower than that of the BTB domain only,which may be one of the mechanisms of antioxidant antagonism among four phytochemicals.Caffeic acid/p-coumaric acid may preferably bind to the Kelch domain.In this case,the ability of the Kelch-phenolic acid complexs is higher than that to the Kelch domain only.It suggested that the Kelch or BTB domain of Keap1 proteins may selectively bind to phytochemicals with different polarities,which may be possible mechanisms of antioxidant synergistic effect among four phytochemicals.