While The Chinese Herbal Medicine Flavonoids And Anthraquinone-based Active Ingredient Group Screening, Separation And Determination

A novel hollow fiber cell fishing with high performance liquid chromatography(HFCF-HPLC) was extended and used to screen flavonoid and anthraquinone activecompound groups simultaneously from traditional Chinese medicines (TCMs). In thisstudy, three cells (MCF-7, SGC7901, and MADB-106) were seeded on the inner wallof the hollow fiber employed to screen bioactive components from TCM waterdecoction. The variables influencing HFCF-HPLC, such as cell seeding time,screening stirring rate and time, and active compound concentration, wereinvestigated and optimized. The surface property of the hollow fiber seeded with cells,the cell survival rate under different conditions, the nonspecific binding betweenactive centers in the fiber and the target compounds, and the repeatability andrecovery of HFCF-HPLC were analyzed and validated. Certain structures of thecompounds fished by HFCF-HPLC were identified after comparing the retentiontimes of the reference substances. To verify preliminarily the binding site between thebioactive components and cells, we separated the cell membrane and cell organellefrom live MCF-7cells. We then employed the cell membrane, and cell organelle, andthe whole cells to screen simultaneously the active compounds. The cell fishing factorof the active compound was calculated and discussed as the index of cell-drugbinding ability in HFCF-HPLC. Tamoxifen as a positive control and indomethacin asa negative control were screened by HFCF-HPLC to verify the method. Moreover,two novel techniques, one named ionic liquid-water-organic solvent three phasemicroextraction (ILWOS-3p-ME) another named multiple-solvent simultaneousmicroextraction (MSSME) have been developed and introduced for simultaneouspreconcentration and determination of flavonoids and anthraquinones activecompounds in Chinese herbal formula and its preparations followed by highperformance liquid chromatography (HPLC). In ILWOS-3p-ME, two differentdensities solvents, lighter (organic solvent) and heavier (ionic liquid) than water, wereinjected separately into a syringe, which was used as an extraction device. Shaking byhand induced the formation of a cloudy emulsion. After the mixture stood at rest for several minutes, the emulsion was readily separated into three phases: an upperorganic solvent extraction phase mainly containing the flavonoids, a middle aqueoussample phase and a lower ion liquid extraction phase mainly containing theanthraquinones. Both the upper and lower layers were transferred to a smalleppendorf (EP) tube. In MSSME, two different solvents (immiscible with each other)carried by two filter membranes (1cm×1cm), which were used as extractiondevices. Several factors affecting performance of the methods, the type and volume ofthe extraction solvent, the pH and salt concentration of sample phase, the shakingduration, the sample volume, were investigated and optimized. And we compared thetwo methods. Under the optimized conditions, the enrichment factors of the analytesby ILWOS-3p-ME and MSSME were102-221and68-132, the limits of detectionwere below0.05ng mL1and0.96ng mL1, the recoveries were80.1%-105.8%and80.1%-119.2%and precisions were5.2-7.8%å'Œ2.2%-8.8%, respectively. The resultsindicate that HFCF-HPLC is an effective and reliable method for the screening andanalysis of bioactive components. Moreover, this method can be applied to predictbioactive candidates in TCMs. ILWOS-3p-ME and MSSME are simple, rapid,practical and effective for the simultaneous extraction and preconcentration ofdifferent types of trace bioactive constituents in traditional Chinese prescription.