Preparation,Bioactivity And Anti-aging Mechanism Of Dihydroquercetin And Its Derivative

In this study,dihydroquercetin(DHQ)was prepared by flash-assisted extraction by using wooden stakes of Larix gmelinii as materials,and dihydroquercetin derivative(DHQA)was prepared through aminomethylation.This study focused on the extration,preparation,structure identification,activity identification,pharmacokinetics,enzyme kinetics and molelular mechanism of anti-aging of DHQ and DHQA.The results are as follows:1.The technological conditions for flash-assisted extraction of DHQ from L.gmelinii were optimized by single factor experiments and RSM.Results showed that the relative degrees of factors influenced DHQ extration yield was:ethanol concentration>solid-to-liquid(g/mL)ratio>flash-assisted extraction time>extraction time.The optimal extraction conditions were flash-assisted extraction time(3min),extraction time(3h),solid-to-liquid(g/mL)ratio(1:15),and ethanol concentration(75%).The highest DHQ extration yield rate was 6.15%under the optimal conditions.The relative error between this rate and theoretical rate of model optimalization was 0.99%.2.DHQ samples prepared by flash-assisted extraction were qualitatively,quantitatively and structurally analysed by HPLC,scanning electron microscopy,infrared spectrum and XRD diffraction.Results showed that the DHQ purity was 96%,and the surface structure,stretching vibration of functional group and absorption peak of diffraction of samples had nearly the same characteristics as the DHQ standard.3.DHQA was prepared using aminomethylation,which had overcome the low water solubility and bioavailability of DHQ.The structural and physical properties of DHQA were characterized through HPLC,nuclear magnetic resonance,scanning electron microscopy,X-ray diffraction,and thermogravimetry.Results showed that DHQA was converted into the spherical,loose,porous and amorphous form,but the major structure of DHQ remained unchanged.Solubilization and dissolution tests were also performed.Results showed that the solubility and dissolution rates of DHQA were approximately 16.28 and 6.31 times higher than those of DHQ,respectively.The MTT assay for cytotoxicity showed non-toxic effect of DHQA against non-cancerous HEK-293T cells(EC50=820.00 ?M),but toxic effect against cancerous Hela cell(EC50=138.17 ?M).The antioxidant capacity in vitro showed the DPPH radical scavenging activity and reducing Fe3+power of DHQA were stronger than those of Vc,but the effect of them on ABTS was the same as that of Vc.Meanwhile,the ABTS radical scavenging activity and reducing Fe3+power of DHQA were stronger than those of DHQ.Results from pharmacokinetics showed that DHQA could reach the highest concentration(22.03 ?g/mL)within 30min,which was 2.5 times that of DHQ.4.The anti-aging mechanism of DHQ and DHQA on normal Drosophila melanogaster were evaluated by survival test,biochemical criterion determination and the expression of antioxidant enzyme genes.Results showed that DHQ and DHQA treatments could prolong the life span,increase antioxidant enzyme activity and reduce MDA content.For female D.melanogaster,0.5mM DHQ and 0.25mM DHQA had the best effects for prolonging the life span,which could also significantly increase T-SOD,CuZn-SOD,Mn-SOD and CAT activities and decrease MDA content(P<0.05),but the effects were significant among groups only for CuZn-SOD,Mn-SOD and MDA content(P<0.05).The effects were significant among groups for the expression of genes encoding CuZn-SOD and Mn-SOD.For male D.melanogaster,1.0 mM DHQ and 0.5mM DHQA had the best effects for prolonging the life span,which could also significantly increase the above four kinds of enzyme activities and decrease MDA content(P<0.05),but the effects were significant among groups only for CAT activity and MDA content(P<0.05).The effects were significant among groups for the expression of genes encoding Mn-SOD and CAT.DHQ and DHQA doses of more than optimal had effects of decreasing life span,decreasing enzyme activities and increasing MDA content as compared with the optimal doses,meaning that excessive DHQ and DHQA might have negative effects on organisms.5.The anti-aging mechanism of DHQ and DHQA on high fat injured Drosophila melanogaster were evaluated by survival test,biochemical criterion determination and the expression of antioxidant enzyme genes.Results showed that DHQ and DHQA treatments could significantly alleviate the injury caused by high fat in both female and male D.melanogaster(P<0.05).1mM DHQ and 0.5mM DHQA were the optimal doses for female and male D.melanogaster.Compared with control(CK),the activities of 4 enzymes were significantly increased and MDA content was significantly decreased under DHQ and DHQA treatments(P<0.05).However,the effects of DHQ and DHQA treatments on SOD increase were less than on CAT and MDA changes,which has the same trend of relative gene expression of these three enzymes,implying that DHQ and DHQA treatments might alleviate the injury caused by high fat in D.melanogaster by increasing CAT activity and decreasing the levels of lipid peroxidation in cell membrane.However,the mechanism of such roles needs further study.6.The roles of DHQ and DHQA in protecting oxidative damage of D.melanogaster caused by H2O2 and paraquat were studied.Results showed that DHQ and DHQA treatments could effectively decrease the oxidative damage caused by these reagents.The optimal dose group had the same role in increasing the life span of D.melanogaster(P<0.05).Under optimal dose treatment,the roles of DHQ and DHQA in oxidative damage protection caused by H2O2 and paraquat were significantly between female and male D.melanogaster(P<0.05),and there were significant roles between groups(P<0.05).Meanwhile,the protection ability of D.melanogaster to oxidative damage caused by paraquat was stronger than that caused by H2O2,demonstrating that DHQ and DHQA could effectively decrease the damage of superoxide anion free radical to organism.7.Results from enzyme kinetics of DHQ and DHQA,and synergistic antioxidant effect of them with Vc showed that:?DDHQ and DHQA could inhibit the activity of tyrosinase to a certain degree.High concentrations of DHQA had nearly the same inhibition effect on tyrosinase as ?-arbutin.?ADH and ALDH could be strongly activated by DHQ and DHQA.The activation rates of DHQ and DHQA,under 4mM treatment,to ADH were 71.83%and 68.09%,respectively.The activation rates of them to ALDH were 100%under 1mM treatment.?DHQ and DHQA had a certain degrees of synergistic antioxidant capacity with Vc,but the dose of Vc was different for different free radicals.