Studies On HPLC-IMS Analysis Of Chemical Constituents And Tyrosinase Inhibitory Activity In Licorice And Safflower

Tyrosinase is a key enzyme regulates the production of melanin.Tyrosinase inhibitors can be used to treat conditions caused by pigmentation,as a preservative for fruits and vegetables,as a whitening additive for cosmetics,and as a bio-insecticide.In recent years,because of the rapid growing of market demand for tyrosinase inhibitors,the pursuit of tyrosinase inhibitors with high specificity,high safety,low toxicity,and high-efficiency functions is still a hot research field.Natural tyrosinase inhibitors are recognized as easy oxidation,low abundance,and generally exist in complex plant extracts,which are very difficult separated and analyzed by conventional methods.In this study,licorice and safflower were analyzed by the liquid chromatography combined with ion mobility spectrometry?HPLC-IMS?that was developed in our laboratory.This is the first time using HPLC-IMS method to analyze flavonoids compounds presented in natural plant extracts,which extends the application of liquid chromatography-ion mobility spectromety and provides a new approach for the analysis of flavonoids.The research and results are as following:1.Investigation of the chemical composition and their antioxidant activities of glycyrrhizin extracts.Six flavonoids were isolated by preparative liquid chromatography and recycling preparative liquid chromatography from glycyrrhizin.They were characterized by Nuclear Magnetic Resonance Spectroscopy?NMR?and identified as liquiritin,isoliquiritin,liquiritigenin,isoliquiritigenin,echinatin,licochalcone B,respectively.The antioxidant activity?DPPH and ABTS⁺free radical scavenging method?and tyrosinase inhibition activity of six compounds were determined using 96-well microplate method.The results are as following:Firstly,as for DPPH method,the IC₅₀ values of liquiritigenin,isoliquiritigenin,echinatin,licochalcone B are 0.59 mg/mL,0.66 mg/mL,0.60 mg/mL,0.90 mg/mL,respectively.However,because of the low activity of liquiritin and isoliquiritin,their IC₅₀value are not available.The ABTS⁺scavenging activity IC₅₀0 for isoliquiritin,liquiritigenin,isoliquiritigenin,echinatin,licochalcone B are 0.47 mg/mL,0.02 mg/mL,0.05 mg/mL,0.54mg/mL,0.83 mg/mL,respectively,while the liquiritin activity is too low to obtain the IC_50.Secondly,the inhibitory effects against monophenol oxidase are as follows:the IC₅₀ values for liquiritigenin,isoliquiritigenin,echinatin,licochalcone B are 0.91 mg/mL,0.85 mg/mL,0.87 mg/mL,0.82 mg/mL,while the activities of liquiritin and isoliquiritin are too low to obtain.The IC₅₀ values against tyrosinase diphenolase of liquiritigenin,isoliquiritigenin,isoliquiritin and echinatin are 0.69 mg/mL,0.76 mg/mL,0.73 mg/mL,0.83 mg/mL,respectivly.And the activities of liquiritin,and licochalcone B were too lower to be determined accurately.Kinetic studies show the inhibition mechanisms of tyrosinase by liquiritin,isoliquiritin,liquiritigenin,isoliquiritigenin,echinatin and licochalcone B is a reversible mixed-type inhibition.2.Quantitative and qualitative analysis of flavonoids were performed by combined liquid chromatography with ion mobility spectroscopy and liquid chromatography-mass spectrometry.Using liquiritin,isoliquiritin,liquiritigenin,isoliquiritigenin as indicators,a liquid chromatography-ion mobility spectrometry method was used to create a calibration curve for the quantitative analysis of the fraction?of crude licorice flavonoids,and qualitative analysis was performed according to the ion mobility drift time.The contents of liquiritin,isoliquiritin,isoliquiritigenin,liquiritigenin are calculated as 4.134%,3.381%,5.831%,and 3.684%using developed HPLC-IMS method.Using liquiritin,isoliquiritin,liquiritigenin,isoliquiritigenin as standard compounds,the calibration equation for quantitative analysis of crude licorice flavonoid fraction III using HPLC-MS/MS was established,and qualitative results of liquiritin,isoliquiritin,isoliquiritigenin,liquiritigenin are 4.148%,3.378%,5.814%and 3.617%,respectively.The results of HPLC-IMS and HPLC-MS/MS are agreeing to each other.3.Column chromatography was used to separate the extracts of safflower into different fractions.The antioxidant activity?DPPH method and ABTS⁺method?and tyrosinase inhibitory activity were investigated using 96-well microtiter plates.The fractions with high tyrosinase inhibitory activity was investitated using Distillate kinetic and HPLC-IMS methods.The results are as follows:The IC₅₀ values of safflower crude extract,safflower water fraction,safflower-20%,safflower-50%,safflower IIIb and safflower IIIc against DPPH.are 0.51mg/mL,0.50 mg/mL,0.38 mg/mL,0.17 mg/mL,0.12 mg/mL and 0.13 mg/mL,seperately.As for the results of ABTS⁺scavenging activity,when the concentration of five fractions,named as safflower crude extract,safflower washed,safflower-20%,safflower-50%and safflower IIIb,higher than 0.5 mg/mL,the scavenging rate are high than 90%,and the IC₅₀value of safflower IIIc is 0.31 mg/mL,comparatively.For tyrosinase monophenolase inhibition,the inhibition activity of safflower crude extract,safflower water fraction,safflower-20%and safflower-50%inhibition activity are very similar and close to 20%,slightly lower than arbutin's activity.Safflower IIIb and safflower IIIc demostrated slightly stronger inhibitory activity,which are up to 45.29%and 69.15%,respectively.IC₅₀ value of safflower IIIc was 0.72 mg/mL,indicating that the safflower fractions can inhibit tyrosinase monophenolase effectively.The tyrosinase diphenolase inhibition activities of safflower extracts are as follows:crude extract,water fraction,safflower-20%and safflower-50%show similar activities,the inhibition rate of those four fractions are about 30%,which are lower than the arbutin's 60.16%.However,inhibitory abilities of safflower IIIb and safflower IIIc are slightly stronger compared to previous fractions,and their inhibition rate are up to 55.35%and 72.15%,with IC₅₀ values being 0.91 mg/mL and 0.69 mg/mL,seperately.Kinetic studies indicate that the tyrosinase inhibition mechanism by safflower IIIc is a reversible mixed-type inhibition.