| Objective:Based on the specific binding of drug molecules to cell membrane rectors,a screening method was proposed and established by combining phospholipase C（PLC）sensitized hollow fiber microscreening by solvent seal（SS-PLC-HFMS）with high performance liquid chromatography（HPLC）technology.The active compounds in Magnolia officinalis cortex,Sesami semen nigrum and Asari radix et rhizoma were screened,and the active groups that could interact with PLC were discussed,and the activities of PLC were determined,SS-PLC-HFMS was verified by molecular docking.A simple and efficient solvent film liquid-phase microextraction（SF-LPME）was established by using a new solvent carrier for the enrichment and concentration of the screened active compounds.Methods:（1）The PLC sensitized hollow fiber microscreening by solvent seal was established.Taking PLC as the active target,the organic solvent was filled in the fiber wall pore,so that PLC solution was sealed in the fiber lumen and placed into the sample phase solution containing honokiol and magnolol standards.The magnetic agitator was used for constant temperature water bath screening.The screening process maintained appropriate water bath temperature and p H environment to ensure PLC activity.In this study,the factors that may affect the screening results were optimized,the methodological investigation was carried out under the best screening conditions,and the active compounds in the extract of Magnolia officinalis cortex were screened and analyzed by combining HPLC.By means of molecular docking,the binding mode of PLC and small molecule compounds was discussed.（2）The SS-PLC-HFMS method was used to screen sesamin,asarinin and sesamolin.The screening conditions that might affect the screening results were optimized.At the same time,phillygenin and sesamol,which are similar in structure to the active compounds screened above,were also screened.Compared with the structural characteristics of five compounds,the active groups that can be combined with PLC were discussed by comparing the screening results and the results of PLC activity determination.In this study,the active constituents of Sesami semen nigrum and Asari radix et rhizoma were screened by combining HPLC under the optimal screening conditions.（3）In this study,the extractant was loaded on the micro non-woven fabric to make microsolvent film,which were placed in the sample phase solution containing active compounds,stirred and extracted on the magnetic stirrer,and the extractant was quickly collected by squeezing the microsolvent film for HPLC analysis.The conditions that might affect the extraction results were optimized,the methodological investigation was carried out under the best screening conditions,and the target analytes in Magnolia officinalis cortex,Sesami semen nigrum and Asari radix et rhizoma were enriched and determined.Results:（1）PartⅠ:The best experimental conditions for SS-PLC-HFMS were as follows:types of hollow fiber:PVDF（inner diameter:0.5 mm）;types of organic phase in fiber wall pore:heptanol;screening temperature:45°C;screening time:60 min;PLC solution concentration:1 mg/m L;sample phase concentration:15μg/m L;PLC and sample phase solution p H:6.0.The chromatographic peak area of active compounds after PLC screening was higher than that after phosphate buffer solution screening.By comparing retention time,honokiol and magnolol were determined to be effective with PLC,and SFPLC was 61.0 and 48.5,respectively.The molecular docking results were consistent with the screening results of SS-PLC-HFMS,indicating that both honokiol and magnolol could be combined with PLC.（2）PartⅡ:The best experimental conditions for SS-PLC-HFMS were as follows:types of hollow fiber:PP（inner diameter:0.6 mm）;types of organic phase in fiber wall pore:octanol;screening temperature:45°C;screening time:60 min;PLC solution concentration:1 mg/m L;sample phase concentration:5μg/m L;PLC and sample phase solution p H:6.0.The compounds that acted with PLC were sesamin,asarinin and sesamolin,with SFPLC of8.2,15.7 and 18.7,respectively.The SFPLC of phillygenin and sesamol were 0.By comparing the structural characteristics of the five compounds,it was found that the lignans containing methylene dioxy showed stronger activity.（3）PartⅢ:The optimal extraction conditions for SF-LPME were as follows:extractant types:undecanol;quantity of solvent film:10 pieces;extraction time:30 min;p H of sample phase solution:7.0;salt concentration of sample phase solution:0%.Under the optimal extraction conditions,the enrichment factors of five target analytes ranged from 124.3 to274.3.The linear ranges were 2-800 ng/m L（sesamin,asarinin,sesamolin and magnolol）and1-400 ng/m L（honokiol）,respectively.The correlation coefficients were all greater than0.9900.The limits of detection and quantification were 0.6-1.8 ng/m L and 1.9-6.0 ng/m L,respectively.The RSDS of intra-day and inter-day precision were 0.4%-9.5%and 5.4%-9.6%,respectively.The average recoveries were 86.6%-100.7%.The average content of sesamin and sesamolin in Sesami semen nigrum were 0.34 mg/g and 0.14 mg/g,respectively.The average content of sesamin and asarinin in Asari radix et rhizoma were 0.08 mg/g and 0.23mg/g,respectively.The average content of magnolol and honokiol in Magnolia officinalis cortex were 2.19 mg/g and 4.29 mg/g,respectively.Conclusions:In this paper,SS-PLC-HFMS was established to screen the active compounds in Magnolia officinalis cortex,Sesami semen nigrum and Asari radix et rhizoma.The binding mode of PLC to active compounds was discussed,and the active groups that could be combined with PLC were studied.At the same time,the PLC activity before and after screening was determined to ensure the stability of PLC activity during the in vitro.The method was verified by molecular docking method.The active compounds screened were enriched and determined by SF-LPME.This method is simple,rapid,economical and high enrichment efficiency.It can screen active compounds in complex samples for specific targets,and integrates separation,purification and screening,which has a wide application prospect. |
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