Application Of Hollow Fibre Cell Fishing And Liquid/solid Phase Microextraction In Research Of Active Curcuminoids

Objective:Hollow fibre cell fishing with high performance liquid chromatography(HFCF-HPLC),hollow fibre liquid/solid phase microextraction with HPLC(HFL/SME-HPLC)and stirrer-liquid/solid microextraction with HPLC(Stirrer-L/SME-HPLC)was developed and introduced to research the active curcuminoids in Rhizoma Curcumae Longae,Radix Curcumae and Rhizoma Curcumae.Also,the extraction mechanism of HFL/SME and Stirrer-L/SME was analysed and expounded.Methods:The cell growth state on the inner wall of the hollow fibre,which seeded with hepatoma cells(HepG-2),human ovarian cancer cells(SKOV-3)and human renal tubular cells(ACHN),the selection of cell seeding time,the affect of methanol concentration on cells survival rates,the binding between hollow fibre activity centre,bioactive compounds and nutrient solution,positive and negative control,the repeatability(retention times,relative peak areas)ofcompounds were investigated.Under the optimal conditions of HFCF-HPLC,the hollow fibre seeded with three cells,their cell membrane,organelle,and receptor(phospholipase C),coupled with HPLC,to screening potential anti-cancer group(active curcuminoids)in Rhizoma Curcumae Longae,Radix Curcumae,Rhizoma Curcumae.The binding sites and binding target of the active compounds on the cells were researched.Some effective parameters of HFL/SME or Stirrer-L/SME,coupled with HPLC,including extraction solvent,sample phase pH,NaCl concentration,extraction time,stirring rate,and sample phase volume were investigated and optimized.Also,methodology,such as linearities,were investigated.Under the optimal conditions of HFL/SME or Stirrer-L/SME,curcumin,demethoxycurcumin and bisdemethoxycurcumin as model compounds,HFL/SME or Stirrer-L/SME,coupled with HPLC,were developed for separation,enrichment and determination potential anti-cancer group(active curcuminoids)in Rhizoma Curcumae Longae,Radix Curcumae and Rhizoma Curcumae.Results:The experimental results show that:(1)according to a comparison of retention times of the reference substances,the active curcuminoids may be curcumin,demethoxycurcumin and bisdemethoxycurcumin;(2)the binding ability of active compounds with cells have nothing to do with its content in traditional Chinese medicine(TCM),the minimal levels of active curcuminoids may have stronger antitumor activity,TCMs have integration and synergistic effects of multiple components and multiple targets;(3)the active curcuminoids can affects both cell membranes and organelles,and they are phospholipase C inhibitors;(4)the extraction mechanism of HFL/SME and Stirrer-L/SME for target analyte is the combination of adsorption by the HF outer wall or the stirrer surface and organic solvent extraction;(5)HFL/SME and Stirrer-L/SME are simple,fast sample pretreatment approaches with high enrichment factor and no need for any special skill and equipment.Conclusion:HFCF-HPLC coupled with HFL/SME or Stirrer-L/SME were developed and introduced for screening potential anti-cancer curcuminoids active group,researching the binding sites,and quantifying their contents in Rhizoma Curcumae Longae,Radix Curcumae,Rhizoma Curcumae,the integration and synergistic effects of multiple components and multiple targets of TCMs are verified,Also,it provide a scientific basis and test method to clarify TCM effect characteristic,and establish quality standards of personalized active ingredients of TCM.In addition,HFL/SME and Stirrer-L/SME are sample pretreatment techniques,with simple,fast,high enrichment factor,environment friendly.