Quality Control Of Semen Oroxyli Based On The Tyrosinase Inhibitory Activity

Part 1:Optimization of ultrasound-assisted extraction of semen oroxyli flavonoids using response surface methodologyObjective:To establish an optimal extraction condition of semen oroxyli flavonoids using ultrasound-assisted extraction combined with response surface methodology.Methods:The ultrasound-assisted extraction of flavonoids with Box-Behnken design combined with the identification of the principal individual flavonoids compounds of semen oroxyli by UPLC-DAD-Q-TOF-MS/MS was proposed.Using the methanol concentration,extraction time,and liquid/solid ratio as factors,the total flavonoids content（TFC）,antioxidant activities（DPPH,ABTS,FRAP,superoxide anion and hydroxyl radicals）and tyrosinase inhibitory activity as responses,the extaction conditons were optimzed.Results:The experimental results showed that the optimal extraction conditions were as following:a solvent of 69.90%methanol,an extraction time of 30.64 min and a liquid/solid ratio of 31.30m L·g^-1,which give individual response values of 19.79 mg RE·g^-1,78.66%,99.92%,19.05 mmol·L^-1 FE·g^-1,20.49%,13.37%,and 35.13%for TFC,DPPH,ABTS,FRAP,superoxide anion,hydroxyl radicals,and tyrosinase inhibitory activity,respectively.By UPLC-DAD-Q-TOF-MS/MS analysis,the main flavonoids identified in the extracts of semen oroxyli were oroxin B,baicalin,oroxin A,baicalein and chrysin.Conclusions:Ultrasound-assisted extraction is an effective and practical method for extracting flavonoids from semen oroxyli.This optimized extraction method can effectively extract flavonoids from semen oroxyli.Part 2:Screening of potential tyrosinase inhibitors from semen oroxyli by spectrometry and in silico molecular dockingObjective:To effectively screen and identify the ligands of tyrosinase in the extract of semen oroxyli.Methods:An affinity selection assay was established to screen and identify tyrosinase inhibitors ligands from semen oroxyli extract by ultrafiltration and high-performance liquid chromatography coupled with diode array detector and time of flight mass spectrometry and in silico molecular docking.The samples were first incubated with the tyrosinase to select the optimal binding conditions.After incubation,the mixed solution was processed centrifugal ultrafiltration,and the ultrafiltrate was analyzed.By comparison of the metabolome profiles of the ultrafiltrate with activated and inactivated tyrosinase,the potential inhibitors were obtained and then identified by comparison their chemical characteristics with those of standards or references.Results:As a result,seven compounds were fished out as tyrosinase inhibitors by this assay.Among them,compared with the positive control drug resveratrol,oroxin A and baicalein showed remarkably higher tyrosinase inhibitory activity,and their binding mode with enzyme was further verified via the molecular docking analysis.Conclusions:The test results showed that the ultrafiltration and liquid chromatography coupled with mass spectrometry was an effective method for screening and analyzing bioactive compounds from traditional Chinese medicines.Part 3:Extraction of main flavonoids from semen oroxyli by deep eutectic solvents combined with tissue-smashing extractionObjective:To establish a green and fast quality control method for semen oroxyli.Methods:A green and highly efficient extraction of four bioactive flavonoids from the seeds of semen oroxyli using a combination of natural deep eutectic solvents and tissue-smashing extraction technique was applied and a UPLC method was developed for their sensitive and selective quantification.The extraction conditions were optimized by response surface methodology coupled with Box-Behnken design procedure on the basis of single factors including liquid/solid ratio,extraction speed and extraction time.Results:The experimental results showed that the composition of the deep eutectic solvents system was choline chloride/1,4-butanediol,the molar ratio was 1:3,and the water content was 40%.The optimal extraction conditions were as following:a solid/liquid ratio of 20 mg·m L^-1,an extraction speed of 5and an extraction time of 1 min.By combination of the tissue-smashing extraction and UPLC,the analysis of flavonoids was accomplished within only 6 min.Conclusions:The deep eutectic solvents combined with tissue-smashing extraction provided an ultra-rapid,environmentally friendly and promising alternation for extraction and analysis of active compounds in natural products.