Study On Anticancer Activity Of Flavonoids And Phenolic Acid In Scutellaria Barbata Based On Hollow Fibre Cell Fishing And Hollow Fibre Liquid Phase Microextraction

Objective:To establish the efficient procedure of hollow fibre cell fishing with high performance liquid chromatography(HFCF-HPLC)and hollow fibre liquid phase microextraction with HPLC(HFLPME-HPLC).Then,combining HFCF-HPLC and HFLPME-HPLC to screen and quantify the active ingredient groups of phenolic acid and flavonoids in Scutellaria barbata(S.barbata)in vitro and to preliminarily study the anticancer activity of scutellarin and its metabolite scutellarein in vivo after intraperitoneal injection of breviscapine.Methods:Hollow fibre seeded human renal tubular ACHN cells,cell membrane,cell organelle and octanol were placed into into the S.barbata extract to screen and quantify the phenolic acid(protocatechuic acid)and flavonoids(scutellarin,baicalin,luteolin,apigenin,and wogonin).Certain structures of the active ingredients,such as protocatechuic acid,scutellarin,baicalin,luteolin,apigenin and wogonin screened by HFCF were confirmed,and the contents of protocatechuic acid,scutellarin,luteolin and wogonin in S.barbata were determined by HFLPME-HPLC.Hollow fibre seeded human renal tubular ACHN or hepatoma HepG-2 cells was placed into the plasma,urine,liver/kidney homogenate to probe the interaction between scutellarin or scutellarein and the two tumor cells and to understand the potential characteristics of scutellarin,scutellarein or its breviscapine injection in the treatment or prevention of liver and kidney tumors,to extract,enrich and quantify the trace level of scutellarin and scutellarein in rat plasma,urine and target tissuesof liver or kidney,and study the PK of scutellarin and scutellarein after intraperitoneal injection of breviscapine.Results:The data from this study indicate some patterns of phenomena:(1)S.barbata could evidently induce ACHN cell apoptosis,especially early apoptosis(early apoptosis rate was12.97%).(2)Compared with the retention times of their reference substances,the six active ingredients were confirmed to be protocatechuic acid,scutellarin,baicalein,luteolin,apigenin and wogonin.The unidentified active ingredients X1,X2,X3 and X4 may exhibit certain anticancer properties.(3)The contents of protocatechuic acid,scutellarin,luteolin and wogonin in sample are 3.3、4.1、0.27、0.22 mg/g.Baicalein and apigenin were screened but not detected as their contents in S.barbata are below the LODs.(4)Scutellarin and scutellarein can be detected in just 12 min after intraperitoneal breviscapine injection in rat plasma.Scutellarin has stronger binding ability with ACHN cell and HepG-2 cell,but scutellarein has weaker binding ability with ACHN cell and HepG-2 cell.Corresponding tissue sample was screened accordingly by its tumor cells in this experiment.Scutellarin has stronger effect on ACHN cell than scutellarein,whereas both scutellarin and scutellarein have weaker effect on HepG-2 cell.(5)Double peaks were observed in mean plasma concentration profiles of scutellarin and scutellarein.The concentrations(Cmaxs)of scutellarin and scutellarein reached the peak value of 2.99 μg/mL and 3.44 μg/mL at tmaxs of 0.25 h and 0.5 h,with t1/2βs are 20.87±1.26 h and 11.60±1.28 h;the AUC0-∞of them are 31.22 mg/(L×h)and 26.25 mg/(L×h),respectively.Conclusion:In this study,the HFCF-HPLC,coupled with HFLPME-HPLC was developed and introduced to study the anticancer activity of flavonoids and phenolic acid in S.barbata,scutellarin and its metabolite scutellarein after intraperitoneal administration of breviscapine injection.From the study,we can draw the following conclusions:(1)Theminimal-level or even the trace-level of flavonoids and phenolic acid in S.barbata can have significant therapeutic effect,so the anticancer activity of the active ingredents have no relationship with their contents.(2)Scutellarin was absorbed quickly into the bloodstream and part of it was metabolized into scutellarein after intraperitoneal administration of breviscapine injection and both can reach liver or kidney,combine with ACHN or HepG-2 cells and maintain effective concentrations.However,it is worth mentioning that these researches are only for the anticancer activity of S.barbata and major ingredients of breviscapine rather than all of the active ingredients and metabolites in vivo.Therefore,the coexistence of all active ingredients and metabolites in S.barbata and breviscapine in vivo may lead to activity accumulation and multi-ingredients synergistic effects.Over all,HFCF-HPLC with HFLPME-HPLC lays experimental foundation for the anticancer research and material basis of S.barbata and breviscapine injection.The approach may provide a new method and idea for the activity research of ingredients in TCMs.