Screening The Active Components Of Moutan Cortex By Cell Membrane Immobilized Chromatography

Cell Membrane Mobilized Chromatography (CMC) is a method which uses cell membrane as the immobile phase toscreen the active components from drugs. This method reflects the binding of active components and biotargets in cellular membranes directly, and combines the screening and separation of active ingredient from drugs together immediately and effectively.There are numerous biotargets such as receptors on cell membrane. Drugs can bind with the targets on cell membrane and have pharmacological effects. A variety of compounds are found in traditional Chinese herbs, the active compounds and the mechanisms of many herbs are still not clearly now. If the Chinese herb extract can protect the cells injuries, its effect should be formed by binding to the receptor and other targets on the membrane. Based on this theory, the method of Cell Membrane mmobilized Chromatography (CMC) is established. The method mimickes the binding of drug and biotargets on cell membtane, washing off unbound of compounds, and then releases the bound compounds from the cell membrane. The released compounds are analysised by Chromatography-MS. At last, the pharmacological effect of the screened compounds is verified. The convenient and efficient method---Cell Membrane Mobilized Chromatography overcomes the disadvantages of traditional screening methods, and is accepted as a new approach to study material foundation of traditional Chinese medicine. It is especially suitable for the active compounds screening from natural herbs..Moutan cortex is the dried root cortex of Paeonia suffruitcosa Andrews, which belongs to the paeoniaceae family. It can cool blood, promot blood circulation and move stasia without bleeding. It is a commonly used herb in traditional Chinese herbal formulae in treating renal disease. However, the nephroprotective active components in Moutan cortex are still unknown. Mesangial cells and renal proximal tubule cells are important constitutive cells of the kidney, and have been verified to be involved in the pathological processes of many renal diseases. In the present study, the membrane of mouse renal glomerular mesangial (MES) cell and human proximal tubular epithelial (RPT) cell are treated as immobile phases, and the active compounds in Moutan cortex are screened by CMC.The first part of the experiment, Moutan cortex was extracted by water. The UPLC fingerprint of Moutan cortex extraction was eatablished, A C18chromatographic column was used at25℃. The flow rate was0.49mL/min and the sample injection volume was10μL. The binary gradient consisted of0.1% formic acid/water (A) and acetonitrile (B). For separation, the gradient was0-0.14min,5% B;0.14-2.04min,5-16%B;2.04-6.0min,16-33% B;6.0-8.5min,33-80% B. Peaks were detected at254nm. Ten batches of Moutan cortex were analysised and nineteen common peaks were obtained and were identified by UPLC/MS and standard refrences.In the second part of experiment, the components in Moutan cortex were screened by MES cell membrane immobilized chromatography. The MES cells were incubated with Moutan cortex extraction in a physiological condition. After incubation, the supernatant was discarded, and the remaining cells were washed with PBS until no compound was detected by UPLC in the washing eluate. Finally, the remaining cells were dissociated in a acid condition. Compounds released from target sites were concentrated by an SPE column and analyzed by UPLC/TOF-MS. The elution times, incubation time were investigated and optimized. Three compounds that interacted with MES cells were identified:paeoniflorin, pentagalloylglucose (PGG) and paeonol.In the third part of experiment, the components in Moutan cortex were also screened by RPT cell membrane immobilized chromatography. The components in Moutan cortex were combined with receptors, channels and enzymes of RPT under physiological conditions. Unbound substances were washed off, and bound compounds were dissociated by specific phosphate buffered solution (PBS). Compounds released from target sites were concentrated by an SPE column and analyzed by UPLC/TOF-MS. The elution times, incubation time were also investigated and optimized. Three compounds that interacted with RPT cells were identified as paeoniflorin, pentagalloylglucose (PGG) and paeonol. The fourth part of the experiment, the pharmacological effect of the three screened compounds were tested. The effect of PGG, paeonol and paeoniflorin on hydrogen peroxide induced superoxide anion production and NADPH oxidase activity were studied. The results showed that paeoniflorin, PGG and paeonol decreased hydrogen peroxide-induced superoxide anion and NADPH oxidase activity in MES and RPT cells. It was indicated that paeoniflorin, PGG and paeonol might be the bioactive compounds in Moutan cortex that were protective against renal injury and could be as parameters for evaluating the quality of Moutan cortex. MES and RPT cell membrane ommobilized chromatography are useful methods for screening nephroprotective compounds from Chinese herbs.