The Protective Effect Of Herba Siegesbeckiae On Apoptosisin PC12 Cells Induced By MPP~+

Herba Siegesbeckiae has rheumatism, joint, detoxification effect,commonly used in rheumatism, muscles weakness, Yaoxisuanruan, limb numbness, hemiplegia, rubella wet sores and other diseases. Its chemical composition mainly containing terpene and glycosides, which kirenol, Herba Siegesbeckiae alcohol, Siegesbeckia glycosides in immune regulation,anti-inflammatory and anti rheumatism, cardiovascular system, disease resistance microbial, antioxidant and anti early pregnancy aspects have obvious activity. In the domestic study, Herba Siegesbeckiae extract can obviously protect cerebral ischemia again perfusion of cerebral cortex neuron injury model, and the mechanism of Herba Siegesbeckiae antioxidation. But Herba Siegesbeckiae extraction based on the material in the treatment of Parkinson’s disease and the specific mechanism have not been reported in the references.In this study, to optimize the best extracting procedure for herba siegesbeckiae, the Orthogonal test was used in the effect of alcohol concentration, volume, extraction time and times on the content of kirenol.The HPLC fingerprints of Herba Siegesbeckiae were established, and the similarity of different regions, different batches of Herba Siegesbeckiae were compared. The quantitative determination was done in each batch of herba medicine. Cell model of Parkinsonˊs disease was established to observe the protective effect of Herba Siegesbeckiae on the PC12 cell injury induced by1-methy-4-phenylpyridinium(MPP+) and study the mechanism.Part one: The Study on the fingerprint of Herba Siegesbeckiae by HPLCObjective: To establish the HPLC fingerprints of Siegesbeckia herb, and analysis similarity in fingerprints among different origin, different batches of Herba Siegesbeckiae, as well as detect the concentration of kirenol, Herba Siegesbeckiae glycosides, Herba Siegesbeckiae fine alcohol in order to provide scientific and effective method for the quality control of Herba Siegesbeckiae.Methods: HPLC was performed on a C8 column(Kromasil WQ,250mm×4.6mm, 5μm), mobile phase of the gradient elution was methanol-acetonitrile-0.05mol/L KH2PO4 solution, the column temperature was 30℃, the detection wavelength was 215 nm, flow rate was 1 m L·min-1.Results: The HPLC fingerprints of Herba Siegesbeckiae were established, and the similarity of different regions, different batches of Herba Siegesbeckiae were compared, and the quantitative determination was done in each batch of herba medicine.Conclusion: This method is simple, rapid, and accurate, It can be used in the identification and quality control of Herba Siegesbeckiae herbs.Part two: Optimizing the extraction process of Herba Siegesbeckiae by Orthogonal TestObjective: To optimize the best extracting procedure for herba siegesbeckiae.Methods: Orthogonal test was used for studying the effect of alcohol concentration, volume, extraction time and times on the content of kirenol.Results: The optimum extraction process is as follows: 12 times of 80%alcohol, extracted for 3 times, each time for 40 min.Conclusion: This extraction process is workable and reproducible, and can provide theoretic support for mass extraction of the kirenol from Herba Siegesbeckiae.Part three: The protective effect of Herba Siegesbeckiae on apoptosis in PC12 cells induced by MPP+Objective: To observe the protective effect of Herba Siegesbeckiae on the PC12 cell injury induced by 1-methy-4-phenylpyridinium(MPP+) and study its mechanism.Methods: Cell model of Parkinsonˊs disease was established by adding MPP+ into PC12 cells. The protective effects of Herba Siegesbeckiae on PC12 cells were assessed. The morphological changes of PC12 cells were observed by microscope, cell viability was analyzed by methyl thiazolyl teerazolium(MTT) method. The ROS production was measured by DCFH-DA.Results: When exposed to 400μmol·L-1 MPP+ for 48 h, the viability of PC12 cells decreased significantly, and the ROS level was increased significantly. Compared with MPP+ groups, the cell viability in treated with Herba Siegesbeckiae were significant inceased, and ROS level was significantly deceased.Conclusion: Herba Siegesbeckiae protects PC12 cells against apoptosis induced by MPP+ and the mechanism may be related to increase the ROS level. The results suggest that pretreatment of Herba Siegesbeckiae protects PC12 cells against apoptosis, its mechanism may correlate with up-regulating ROS gene.