| Affinity chromatography is a kind of liquid chromatography which uses the specific affinity between the fixed phase and the target component to realize the separation and purification of the target component.Cell membrane chromatography is a rapidly developing bio affinity chromatography technique,which can simulate the interaction between drug and cell membrane receptor in vitro by using cell membrane and chromatographic carrier as the stationary phase.Lipid raft affinity chromatography was developed based on the principle and construction of cell membrane chromatography.Lipid rafts are the microregions in the membrane lipid bilayer,which are rich in special lipids and proteins.By using the interaction between receptor and drug which is rich in lipid raft,the active sites of Chinese traditional medicine can be screened online quickly and effectively.P-glycoprotein(P-gp),as an important transporter related to drug transport on the cell membrane,is located in the lipid raft region.P-gp excreted the chemotherapeutic drugs out of the cell through dependent method,thus affecting the accumulation of chemotherapeutic drugs in the cell.Therefore,more and more studies have focused on the development of P-gp inhibitors in order to improve the chemotherapy effect of drugs on tumors by reversing multidrug resistance.Over the past few decades,P-gp inhibitors have undergone three generations of development,but have not met expectations due to limited efficacy,drug interactions,and severe adverse reactions.In the process of searching for a new generation of chemotherapeutic sensitizers,P-gp inhibitors derived from natural drugs are attracting more and more attention from the research community.The traditional screening model for P-gp inhibitors is the Caco-2 monolayer model,but this model has some shortcomings such as long experimental cycle and inability to conduct high-throughput screening.Based on the previous work of the research group,the lipid raft in Caco-2 cells was extracted and characterized in this paper,and a screening model of lipid raft chromatography rich in P-gp was constructed to investigate the retention characteristics of lipid raft chromatography.The anthraquinone active components in rhubarb were extracted and separated by pH gradient combined with solvent flotation method.The active constituents of ginsenoside were extracted and separated by two-phase extraction method,and the extraction process was optimized by response surface method.The crude extract of bayberry leaves was obtained by ultrasonic assisted solvent extraction.In this paper,a lipid raft affinity chromatographic screening model rich in P-gp was successfully constructed to screen the active sites of P-gp substrates or inhibitors in natural drugs,and provides useful thinking for the modern development of natural drugs.Chapter ? ReviewIn this chapter,P-gp and affinity chromatography are summarized.Firstly,the structure,distribution and physiological function of P-gp are summarized.At the same time,the research progress of P-gp inhibitors was described.The classification,screening methods and natural product inhibitors of P-gp were introduced.Then the mechanism and classification of affinity chromatography are summarized.At last,the research status of affinity chromatography in the screening of active components of traditional Chinese medicine was summarized,which provided the basis for the establishment of lipid raft chromatography screening model.Chapter ? Establishment of lipid raft chromatographic screening modelIn this chapter,the culture and amplification of Caco-2 cells were carried out,and the cells were extracted by the method of non-ionic scale remoter.The lipid rafts rich in P-gp were collected after sucrose density gradient centrifugation.The characteristic proteins in lipid rafts were characterized by Western blot and immunofluorescence labeling.The lipid raft rich in P-gp and-NHS modified silica gel were combined as the fixed phase of the lipid raft column,and the column was packed by low pressure wet method.The retention characteristics of lipid raft chromatography were investigated with verapamil as a positive drug and pyrazinamide as a negative drug.The results showed that the lipid raft could be successfully extracted from Caco-2 cells by non-ionic scale remover.The constructed fixed phase of silica gel chromatography modified with P-gp lipid raft can specifically bind to the drugs targeted by P-gp and produce obvious retention behavior.The lipid-raft chromatographic screening model based on P-gp regulation established in this chapter lays a foundation for on-line and rapid highthroughput screening of P-gp substrates or inhibitors from natural drugs.Chapter ? On-line screening of active components of rhubarb based on lipid raft chromatographyIn this chapter,the active components of anthraquinones in rhubarb were separated and enriched by pH gradient combined with solvent flotation.The obtained flotation site of rhubarb was screened out potential active components of rhubarb by the lipid raft chromatographic model constructed earlier,and the flotation process was optimized by single factor experiment.The MTT method and rhodamine-123(rho-123)accumulation experiment were used to evaluate P-gp function in vitro.Lipid rafts chromatography screening results show that the flotation parts ? on lipid raft column retention behavior,has the obvious inference substrates or inhibitors containing P-gp.Using single factor experiment of flotation parts ? main ingredient emodin in flotation conditions optimization,get the best technological conditions for ethyl acetate as solvent flotation,flotation time 50 min,ion concentration 0.4 mol/L,system of pH 7.8,and the gas velocity of 20 mL/min.P-gp function in vitro preliminary evaluation results show that the lipid rafts flotation column of rhubarb parts ? can obviously increase the volume of rho-123 in cells,has the function of inhibiting P-gp further validated experiment preliminary build P-gp lipid rafts chromatographic high-throughput screening model is practical and feasible.Chapter ? On-line screening of total ginsenoside and its lipid raft by double water phase extractionIn this chapter,total ginsenosides from ginseng were extracted by ethanolammonium sulfate two-phase aqueous extraction method,and the extraction process of ginsenosides was optimized by Box-Behnken Design response surface method,and the obtained ginsenosides were extracted by lipid raft chromatography constructed in the earlier stage for P-gp functional screening.The optimal extraction process was: 22.82% ammonium sulfate,31.64% ethanol,the pH value of the system was 6.9,and the temperature was room temperature.The results of lipid raft chromatography showed that the extraction site of ginsenoside contained P-gp substrates or inhibitors.The extraction part contained ginsenoside Rb1,ginsenoside Rg1,ginsenoside Re,etc.,and it was found that ginsenoside Rb1 was obviously retained in the lipid raft by lipid raft chromatography screening,so ginsenoside Rb1 may be a P-gp substrate or inhibitor.Chapter ? On-line screening of flavonoids from bayberry leaves by lipid raft chromatographyIn this chapter,on the basis of the successful P-gp lipid raft chromatographic screening model,the crude extract and flavonoids from bayberry leaves obtained by ultrasound-assisted solvent extraction were separated by lipid raft chromatographic column.At the same time,a DPPH-HPLC method for online screening of antioxidant active ingredients was established.The results of lipid raft chromatography showed that the crude extract of bayberry leaves contained P-gp substrates or inhibitors.Flavonoids such as myricetin,quercetin,myricitrin and kaempferol were mainly contained in the crude extract of bayberry leaves.Quercetin was found to be significantly retained in lipid raft,so quercetin may be a P-gp substrate or inhibitor.The results of online screening by DPPH-HPLC showed that the antioxidant activity of flavonoids in the crude extracts of bayberry leaves was the strongest of quercetin,followed by myricetin,and the antioxidant capacity of both was stronger than that of L-ascorbic acid,while the antioxidant capacity of myricitrin and kaempferol was weaker.It is suggested that the components screened by lipid raft chromatography constructed by Caco-2 cells not only act on P-gp,but also have other activities such as antioxidant.The realization of such multi-activity screening may be related to the rich variety of receptors,enzymes and other signaling pathways on lipid raft. |
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