Research On Anti-oxidative,Anti-proliferative And Anti-aging Activities Of Citrus Flavonoids

In many chronic diseases,such as neurodegenerative diseases and cancer,there is an important factor:reactive oxygen species?ROS?.Reasonable dietary intervention is one of the key factors to eliminate excessive ROS and prevent chronic diseases.In recent years,food and dietary supplements with antioxidant function have gradually become one of the hot spots in the field of food science,and have attracted more and more attention.Citrus fruits have a long history of planting in China,rich in nutrition and deeply loved by consumers.Modern pharmacological studies have confirmed that citrus flavonoids have strong antioxidant,antibacterial and antiviral,antitumor and other physiological activities.However,previous studies have mainly focused on crude extracts or the activity of a monomer,but little is known about the additive,synergistic or antagonistic effects existed between monomers,especially in biological activity in vivo.Therefore,in the present study,we selected four citrus flavonoids:nobiletin,hesperetin,hesperetin,naringenin,to systematically analyze its antioxidant,anti-proliferative,anti-aging activities.With PC12 nerve cell model,Hep G2 human liver cancer cell model,and Caenorhabditis elegans biological model,we explored the molecular mechanism of citrus flavonoids in vitro anti-oxidation,synergistic inhibition of human liver cancer cell proliferation and life extension,with a view to anti-cancer active drugs,anti-aging active functional foods.The specific results are as follows:Firstly,we examined the antioxidant capacity of four citrus flavonoids.NOB has the strongest oxygen free radical absorption capacity,with the highest ORAC value?52.17±1.14?mol TE/?mol?,which is significantly higher than other flavonoids,and two flavanones:ORAC values of HES and NAR followed,yet TAN has the lowest antioxidant activity.A model of oxidative damage to PC12 nerve cells treated with glutamate?Glu?was established to study the antioxidant activity of 4 monomers.In the low concentration?0.1?m?of NOB and HES,the activity of cells increased significantly?p<0.05?,while the growth rate of ROS and the release rate of LDH decreased significantly.The protective activity of TAN was the second,while that of NAR had almost no protective activity of oxidative stress.Secondly,the human hepatoma cell Hep G2 was used as a model to study the anti-tumor cell proliferation activity of 4 monomers.The combination of two monomers was used to study the joint effect,and the anti-cell proliferation pathway and its molecular mechanism were explored from the cellular level.TAN has the best anti-proliferative ability,its half-inhibitory concentration(EC₅₀)is 68.67±2.75?M,and the activity of NOB and HES is slightly weaker,78.94±2.88,92.13±2.91?M,respectively.The half cell death rate(CC₅₀)of the body to Hep G2 cells was greater than 200?M,and the selection index SI value was greater than 2,while Hep G2 cells were low sensitive to NAR,and no significant anti-proliferative activity.was detected in the concentration range of 200?M.NAR was compounded with two kinds of polymethoxyflavonoid monomers?NOB,TAN?to study the synergistic anti-proliferation effect and mechanism between the monomers.The results showed that the inhibitory effect on cancer cells of NAR was significantly increased,and the EC₅₀ decreased to 103.66±3.44 and 96.15±1.90?M,respectively.At 50%inhibition rate50%,the CI value were 0.68±0.01 and 0.748±0.02,respectively,showing a better synergistic inhibitory effect,higher than that of the monomer.It plays a very good synergistic effect within the range of 50?95%inhibition rate.Using flow cytometry to investigate the synergistic effect of two combinations?NAR+NOB,NAR+TAN?on cell apoptosis and cell cycle.When half of the compound was used in combination,the early apoptosis rate of Hep G2 cells increased significantly,and at the same time,the cells were blocked in G0/G1phase.Western blot analysis was used to analyze the effects of the two complexes on the expression levels of cell cycle regulatory proteins.Both formulations can down-regulate the expression level of CDK4 in a dose-dependent manner,but there is no dose-dependent regulation on the expression level of p21.At a high concentration of 0.5EC50×?NAR+NOB?,the expression of p21 was significantly increased about 1.48 times.It can be concluded that the two combinations downregulate the expression of CDK4 protein and simultaneously increase the expression of p21 protein to regulate the cell cycle,thereby exerting an anti-proliferative effect.Finally,the aging model of Caenorhabditis elegans was established to test the anti-aging activity of NOB.Through lifespan experiments,various health index determination experiments,anti-oxidative stress experiments,nematode active oxygen?ROS?detection,antioxidant enzyme activity determination experiments,molecular mechanism experiments,we found that NOB can be in 3.13?12.5?M It extends the life span of nematodes within a range of concentrations and has a certain concentration dependence;without affecting the body length and reproductive ability,it significantly improves mobility and reduces the content of lipofuscin in the body to delay aging.Besides,NOB can significantly extend the life of nematodes under the stimulation of high temperature,ultraviolet radiation,and oxidative stress,reduce the level of ROS and MDA contents,and increase the activity of antioxidant enzymes?SOD,CAT?,indicating the anti-aging activity of NOB is also closely related to antioxidant activity.In addition,using PCR reverse transcription technology,it was found that Chuanchensuin can up-regulate the expression of sod-3,hsp-16.2,gst-4,skn-1,sek-1 and sir-2.1,which indicates that NOB exert the effect of prolonging life and increasing stress resistance through activated DAF-16,HSF-1 and SKN-1 transcription factors to regulate the expression of downstream target genes.In short,NOB has potential application value in extending life and its related healthy life and anti-stress.