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Objective: To serine palmitoyltransferase the enzyme (SPT) [EC2.3.1.50] is a key enzyme in the synthesis of sphingolipids in vivo, plays a pivotal role in the regulation of sphingolipid metabolism, affect the of various sphingolipids composition synthesis in vivo, distribution and function, cicadas spend (Cordyceps cicadae) is a valuable medicine in China belong to the insect and the Cordyceps fungus complex, the main active ingredient myriocin (myriocin, ISP-1) is a natural inhibitor of SPT inhibitory activity and also has anti-atherosclerosis, anti-fungal and other pharmacological effects of these active may find better inhibitors of SPT by adjusting the same target-the SPT, having a broad clinical value, this paper has a strong immune create a simple, high precision and accuracy the SPT inhibitor screening platform for inhibitor screening experiments provide possible technical means, but also for further research to lay the foundation. Methods: rat lungs SPT higher activity in rat lungs as an enzyme source, sonication, centrifuged, the enzyme-containing lysate was prepared, with the substrate reaction, add the internal standard material, the enzyme reaction product of the liquid-liquid extraction, HPLC determination of the content of the enzyme reaction, thereby establishing the inhibitor screening platform, the inhibition rate was calculated by the internal standard method, and Evaluation of inhibitory effect. Experiments to optimize the experimental conditions of the various steps of the lysis, extraction, enzyme reaction, also established o-phthalaldehyde (OPA) pre-column derivatization-HPLC-UV method for measuring effects of the the derivatization reaction system buffer concentration and pH, OPA amount of 2 - mercaptoethanol amount of derivative reaction time derivative of the impact of factors such as reaction temperature long-chain base derivatives on the dihydro the sheath Saddle alcohol (Sa. enzyme product) and plants sheath saddle alcohol the (Phy. internal standard) and stability. Add in the enzyme reaction system the myriocin standard, the feasibility of the test platform, and then, by adding several different Chinese medicine extracts, a preliminary screening experiment. Results: precolumn derivatization reaction at 25 ℃ for 2h optimize the reaction conditions, the detection wavelength of 230nm, the dihydro of the sheath saddle alcohol plants sheath the saddle alcohol samples derivative product placed within a 4 ° C refrigerator 7h stable the RSD measurement results were less than 4.0 The% dihydrogen sheath saddle alcohol linear range of 3.125-100.0μg/mL (r = 0.9993), phytosphingosine the saddle alcohol derivatives the linear range 5.0-500.0μg/mL (r = 0.9971); detection limit (S / N = 3:1): Phy 0.9μg/mL, Sa to 0.5μg/mL. Sonicated rat lungs enzyme prepared Lysates, cracking uniformity, stability of the protein content, by optimizing the extraction process under different conditions, the extraction rate is maintained at between 70.0% to 85.0%, and the supernatant liquid amount of 90 μL, the high liquid injector 40μL (substrate concentration: L-serine (200 mM), palmitoyl-CoA A (5 mM)) for the optimization of the enzyme reaction conditions and the inhibitor screening test, except for the Zen flower mycelium extract inhibited the rate of 69.20%, other Chinese herb extracts significantly inhibited. Conclusions: the establishment of experimental detection the SPT reaction product the high-performance liquid chromatography, the the SPT inhibitor of in vitro screening model is established on the basis of the detection method on four kinds of Chinese herb extracts and found no significant inhibition of SPT extract can increase screening efforts, in order to find better inhibitors. In this study, based on the reference to foreign literature, the first time in the country to establish a high sensitivity, low cost, convenient general laboratory operations the SPT inhibitor screening model, compared with the usual isotopic labeling method with simple equipment, no radioactive danger and other advantages. Vigorously screening novel serine palmitoyl transferase inhibitor and has broad prospects, especially found the obvious effect of the inhibitors from traditional Chinese medicine theory and economic value.
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