Synergistic Antioxidant Effects And Mechanisms Of Flavonoids(Quercetin,Luteolin) And Carotenoids(Lycopene,Lutein)

Plant foods play an important role in maintaining human health.Their health effects result from bioactive ingredients,which exhibit antioxidant activity and the prevention of chronic diseases,such as diabetes and cardiovascular diseases.Also,the complex interactions between bioactive ingredients contribute to the health effects of plant foods,by forming complexes or interacting with each other to achieve antioxidant,anti-inflammatory,and neuroprotective effects.Among them,the prevention of oxidative stress is critical for bioactive ingredients to prevent the development of chronic diseases.However,there remain some issues unresolved with regard to the antioxidant interactions among phytochemicals.For example,it is not clear how they regulate the endogenous reactive oxygen species（ROS）production.In addition,previous researches are based on in vitro models,such as chemical or cellular models,which lack biological relevance.Especially,hydrophilic antioxidants and lipophilic antioxidants usually co-exist in foods,but their effects and mechanism of preventing oxidative stress remain unclear.In this study,two flavonoids（quercetin and luteolin）and two carotenoids（lycopene and lutein）,which are commonly found in fruits and vegetables,were combined in different ratios（10:1,5:1,1:1,1:5,1:10）to study the antioxidant interaction and mechanism using both in vitro and in vivo models.The findings are as follows:（1）The in vitro antioxidant interactions of flavonoids and carotenoids were investigated using ABTS and DPPH radical scavenging assays,and H₂O₂-induced oxidative damage cell models.The results showed that flavonoids and carotenoids in different ratios exhibited significant synergistic or antagonistic effects on radical scavenging capacity,increasing cellular antioxidant capacity,and antioxidant enzyme activities（p<0.05）.The synergistic effect was stronger when flavonoids were in higher proportion in the combination.Especially,the lycopene and quercetin combination showed stronger synergistic effects.In addition,compared to Caco-2,L-02 cells,the flavonoids and carotenoids combination showed stronger synergistic effects in HUVEC cells.（2）The effects of flavonoid and carotenoid combinations on their cellular uptake were analyzed by high performance liquid chromatography（HPLC）,and the effects of the combination on carotenoid transporter SRBI and NPC1L1 were investigated using Western blotting.In HUVEC cells,synergistic combinations promoted carotenoid uptake,such as lycopene:quercetin=1:5 combination（M2）,increased 271%of lycopene uptake（p<0.05）,and synergistically increased SOD,GSH-Px,and CAT activity（p<0.05）.While antagonistic combinations inhibited carotenoid uptake,such as lutein:quercetin=5:1 combination（M6）,decreased 17%lutein uptake,and exhibited significant antagonistic effects on CAT activity（p<0.05）.SRBI and NPC1L1 were involved in the transport of carotenoids as their expression was increased by lycopene or lutein.The changes in the expression of SRBI and NPC1L1 were consistent with the effect of flavonoids on carotenoid uptake.Therefore,flavonoids may regulate the transport SRBI and NPC1L1,thus affecting carotenoid uptake and their antioxidant activity.（3）The H₂O₂-induced HUVEC oxidative damage cell model was established to study the effects of flavonoid and carotenoid combinations on the regulation of the redox signaling pathway.Results showed that the combinations increased the m RNA and protein expression levels of Nrf2 and detoxification enzymes HO-1 and NQO1,and increased the translocation of Nrf2 to the nucleus,which promoted the activation of Nrf2.Among them,M2 and lutein:luteolin=1:5（M3）showed the strongest synergistic effects（p<0.05）.Especially M2 increased by around 3-fold of nucleus Nrf2expression.They may mitigate oxidative damage in HUVEC cells through Nrf2/HO-1/NQO1 signaling pathway and prevent cardiovascular diseases.（4）The H₂O₂-induced HUVEC oxidative damage cell model was used to study the effect of flavonoid and carotenoid interaction on the pathway of cellular ROS production employing flow cytometry,western blotting,and RT-q PCR.It was found that the ROS level and protein expression of NADPH oxidase Nox2 and Nox4 were significantly inhibited by the flavonoid and carotenoid combination（p<0.05）.Especially,the combinations such as lycopene:luteolin=1:5（M1）,M2,and M3synergistically reduced the expression of Nox4（p<0.05）.Especially M2 decreased by around 3.5-fold of ROS level compared to the H₂O₂-treated group.Flavonoid and carotenoid combinations may regulate cellular ROS levels by regulating Nox4expression.Combinations such as M2 and M3 synergistically promoted the m RNA level and protein expression of SIRT1,an upstream regulator of oxidative stress,which could be critical to the prevention of oxidative stress.Further,the H₂O₂-induced HUVEC cell model and synergistic group M2 was used to study the signaling pathway of ROS generation.The RNA sequencing study revealed that the gene expression of M2 in HUVEC cells was significantly different from that of the H₂O₂-treated group（p<0.05）,and the differential expressed genes were mainly enriched in inflammatory response pathways such as NF-κB,IL-17,and TNF signaling pathway,which significantly downregulated on the expression of inflammation-related genes（p<0.05）.M2 synergistically reduced the m RNA levels of inflammatory genes such as COX-2 and BCL-6.Further,it was found that the activation of NF-κB,a transcription factor of the inflammatory signaling pathway,was significantly reduced by M2（p<0.05）.Compared to the H₂O₂-treated group,the p-p65 expression decreased by around 1.8-fold.Lycopene and quercetin could inhibit ROS production through SIRT1/NF-κB/Nox4 signaling pathway to ameliorate oxidative stress and inflammation,thus preventing cardiovascular disfunctions.（5）The analysis of CESTA and fluorescence quenching assay revealed the effect of M2 on improving the thermostability of SIRT1 protein and increasing the SIRT1deacetylation activity,suggesting that they can act as SIRT1 activators.Molecular docking analysis revealed that quercetin and lycopene docked to the N-terminal domain of the small molecule activator binding region of SIRT1 protein and interacted with SIRT1 through hydrogen bonding and other forces,with binding energy at-6.16kcal·mol^-1 and-8.7 kcal·mol^-1,respectively.Lycopene and quercetin could bind to the N-terminal STAC domain at the same time,and the site of interaction between lycopene and SIRT1 is close to the docking site of quercetin,indicating that they may work together in the STAC domain.Thus,quercetin and lycopene synergistically reduced the ROS level by altering the SIRT1 signaling pathway to prevent oxidative stress.（6）D-galactose-induced oxidative stress model in ICR mice was established,and M2 was used to study the antioxidant interaction in vivo.It was found that D-galactose-induced tissue damage,while M2 ameliorated the heart and hippocampus damage,increased the activities of serum SOD and CAT activities,inhibited the serum MDA content,reduced the heart 4-HNE and Nox4 expression,restored the m RNA levels of BDNF and PSD95.M2 also mitigated the inflammatory response in mice heart,reduced the m RNA levels of COX-2 and IL1-β,and significantly decreased the m RNA levels of IL-6,IL1-β,and TNFαin mouse hippocampus compared to D-galactose-treated mice（p<0.05）.Further,we found that M2 increased the m RNA levels of SIRT1,Nrf2,HO-1,and NQO1 in mice and synergistically increased the m RNA levels of SIRT1 and Nrf2.Compared to the D-galactose-treated group,the m RNA level of SIRT1and Nrf2 were increased by around 3-fold and 2.4-fold,respectively.Therefore,M2may activate the SIRT1/Nrf2/HO-1/NQO1 signaling pathway to prevent oxidative stress.These results further confirmed the synergistic effects of lycopene and quercetin to regulate the SIRT1 signaling pathway to prevent oxidative stress.In summary,the antioxidant interactions between flavonoids and carotenoids were systematically investigated in this study.The results showed that the flavonoid and carotenoid combinations exhibited significant antioxidant interaction in vitro（p<0.05）.In addition,the expression of cell membrane protein SRBI and NPC1L1 was affected by the flavonoids,which affected the carotenoid uptake and their cellular antioxidant activities.Flavonoids in combination with carotenoids significantly altering Nrf2/HO-1 signaling pathway to prevent oxidative damage（p<0.05）.Among them,the combination of quercetin and lycopene exhibited significant antioxidant synergistic effects（p<0.05）.The lycopene and quercetin combination significantly prevented intracellular ROS accumulation and oxidative stress by regulating the SIRT1/NF-κB/Nox4 pathway.Simultaneously,they ameliorated cardiac and hippocampal damage in D-galactose-treated ICR mice,and synergistically prevent oxidative stress and inflammatory response by SIRT1/Nrf2/HO-1/NQO1 signaling pathway.This research elucidates the mechanism of antioxidant interactions among phytochemicals and provides a theoretical basis for the development of balanced dietary patterns,which is important for promoting human health.