Application Of Hollow Fiber Target Fishing And Liquid-phase Microextraction For Q-marker Study Of Salvia Miltiorrhiza

Objective:In order to effectively control the quality of Salvia Miltiorrhiza,the study is intended to establish a hollow fiber target fishing method based on acetylcholinesterase(AchE)for the screening of active components in the treatment of cardiovascular and cerebrovascular diseases in Salvia Miltiorrhiza combined with HPLC.From the perspective of new solvents and materials,a new extraction solvent is planned to be synthesized and adopted as extraction phase for overcoming the disadvantages of high toxicity and limited extraction efficiency of traditional organic solvents in liquid-phase microextraction,such as high toxicity and limited extraction efficiency.The selected active components were enriched,purified and quantitatively analyzed.Methods:In the sample phase obtained by dilution of Salvia Miltiorrhiza extract,the target fiber prepared in advance was added to screen by stirring under constant temperature simulating the physiological environment in vivo.The main factors affecting screening were optimized via changing the single variable method.Then the methodology study was investigated under the screening conditions unchanged.The active components of Salvia Miltiorrhiza in the treatment of cardiovascular and cerebrovascular diseases was preliminarily identified by HPLC-UVD analysis.After preliminary determination of the active components of Salvia Miltiorrhiza in the treatment of cardiovascular and cerebrovascular diseases,the switchable hydrophilic deep eutectic solvent prepared by diethanolamine and hexanoic acid was added to the sample phase solution as the extraction phase.After extraction,mineral acid was added to change the solubility of the extraction solvent.After centrifugation,the hydrophobic upper solution was collected for sample analysis.The factors affecting the extraction performance were investigated and optimized.The structures of hydrophilic deep eutectic solvent and the recovered hydrophobic super were characterized by Fourier transform infrared spectroscopy.The established sample pretreatment method was compared with other published methods to further confirm the feasibility of the method.Results:The optimal screening conditions based on target acetylcholinesterase were as follows:the type of hollow fibers was polypropylene(inner diameter:0.6 mm,aperture:0.2μm),the wall hole solvent was octanol,sample phase volume was 6 mL,pH 5.7,AchE concentration was 1 mg/mL,pH 6.5,screening temperature was 37℃,screening time was 90 min.The screening concentrations of the four active compounds in the sample solution(based on dihydrotanshinone I with the minimum retention time)were 5μg/mL,20μg/mL,11μg/mL and 22μg/mL,respectively.respectively.Under the optimal experimental conditions(270 nm and 286 nm),the screened active compounds were dihydrotanshinone I(t_R17.467),cryptotanshinone(t_R20.285),tanshinone I(t_R20.927),tanshinone IIA(t_R24.902),respectively.The binding ability of one active ingredient in the same herb from different localities of growth is similar.The conditions of LPME based on switchable deep eutectic solvent after optimization were as follows:the extractant was 180μL diethanolamine-hexanoic acid with molar ratio 1:1,the phase conversion solvent was 5 mol/L hydrochloric acid200μL,and the extraction time was 20 s.Under the optimal conditions,the enrichment factors of dihydrotanshinone I,cryptotanshinone,tanshinone I and tanshinone IIA were 274,261,59 and 236,respectively.The linear range of tanshinone IIA was 0.003-0.6μg/mL,and the linear range of the other three was0.005-0.9μg/mL.The limits of detection and quantitation were 0.5-0.7 ng/mL and1.5-2.1 ng/mL,respectively.The RSD of precisions were ranged from 0.5%to 8.6%.The average recoveries were 94.6%-104.6%.The average contents of four active components in Salvia Miltiorrhiza were 0.58,1.52,1.42 and 2.02 mg/g,respectively.Conclusions:In this study,the hollow fiber target fishing method based on AchE was established,combined with HPLC-UVD to screen trace active ingredients in Salvia Miltiorrhiza for the treatment of cardiovascular and cerebrovascular diseases.Four active ingredients were preliminarily identified.A novel switchable hydrophilic DES was synthesized and successfully applied to the separation and enrichment of four active components of Salvia Miltiorrhiza in microextraction,which expanded the variety of switchable solvents and provided a more convenient,efficient and green pretreatment method for complex sample analysis.