Screening And Determining Anti-breast Cancer Compounds Of Phenolic Acid And Flavonoid In Traditional Chinese Medicines

Objective:In the study, hollow fiber cell fishing with high performance liquid chromatography(HFCF-HPLC) and its application of active screening in traditional Chinese medicine(TCMs) were extended and verified further. A two-phase/three-phase hollow fiber liquid phase synergistic microextraction(2p/3p-HF-LPSME) was developed and introduced to quantify the trace level of active ingredients in TCMs. Methods:Hollow fiber seeded MCF-7 cells was inserted into water decoctions of Lonicera japonica, Herba Taraxaci and Cortex Eucommiae to screen and fish simultaneously phenolic acids and flavonoids active compound. Some of the structures of active compounds screened and fished were identified by comparing with the retention time of the reference substances. And the hollow fibers filled with the receptors(estrogen receptor, epidermal growth factor receptor, cyclooxygenase-2) associated with cancer cells as well as the cell membrane and cell organelle separated from live MCF-7 cells were used respectively in preliminary researching the binding sites and the targets of potential phenolic acids and flavonoids on MCF-7 cells. The active compound’s abilities to interact with their targets were compared and discussed. On the basis of active ingredients screening and their structure identified, two hollow fibres filled with water-soluble and fat-soluble acceptor phases were introduced into the sample vial for the extraction and preconcentration of phenolic acids and flavonoids active compounds. After extraction, the two acceptor phases were withdrawn and transferred into an Eppendorf tube with a clean microsyringe and dissolved or diluted with the moderate amount of methanol. The combining solution was mixed evenly and injected into the HPLC system for analysis. Results:For HFCF-HPLC, the surface properties of the hollow fiber seeded human breast cancer cell MCF-7, the survival rate of cell seeded, the cell death rate after screening active compounds, the non-specific binding between active centres on the fiber and the target compounds, the positive and negative controls, the repeatability and reliability of HFCF-HPLC were investigated in detail. Cell/target fishing factor of active compound was redefined and improved. For 2p/3p-HF-LPSME, several factors influencing performance, including extraction solvent, p Hs of the sample and acceptor phases, extraction time, stirring rate, salt concentration in the sample solution, and volume of sample phase, were investigated and optimized. Under optimized conditions, the enrichment factors of 2p/3p-HF-LPSME for analytes ranged from 9 to 171, the linear ranges were 0.0064-32.0, 0.0016-1.6, 0.0038-7.5, 0.0009-1.8, 0.0200-4.0, 0.0032-6.4, 0.0036-3.6 μg m L-1 for chlorogenic acid, caffeic acid, p-hydroxycinnamic acid, ferulic acid, rutin, galuteolin and quercetin, good linearities were obtained for all analytes with regression coefficients of between 0.9939 and 0.9996, the precisions(RSD 2.3% to 10.4%), the satisfactory recoveries(90.0% to 106.3%) and the limits of detection(0.3 to 4.0 ng m L-1) were also achieved. Conclusions:HFCF-HPLC is a simple, quick, stable and reliable method to screen and analyze bioactive compounds as well as research the possible targets of active compound, and 2p/3p-HF-LPSME is a trace level of active ingredients assay technique with high concentration and high sensitivity. HFCF-HPLC, combined with 2p/3p-HF-LPSME-HPLC, has been successfully applied in screening, identification and determination of phenolic acids and flavonoids in Lonicera japonica, Herba Taraxaci and Cortex Eucommiae. As a result, HFCF-HPLC, combined with 2p/3p-HF-LPSME-HPLC or other high sensitive modern testing technology, provides us new idea and method to indentify active ingredients, clarify TCM effect characteristic, and quantify the trace level of active ingredients in TCM.