Theacrine Preparation And Protective Effects Against Glutamate-induced HT22 Cells Damage

Glutamate(Glu)is the most abundant and widely available excitatory amino acid in the central nervous system.However,concentration of the extracellular Glu will be excessive and can be over-combined with Glu receptors,leading to the death of neurons when the brain was in ischemic and hypoxia injury.Theacrine(TC)is a purine alkaloid mainly present in Camellia kucha which has physiological functions such as sedative hypnosis,anti-depressant anti-inflammatory and analgesia.In this paper,we studied the separation and preparation of TC from Kucha dried leaves by preparative chromatography.Taking HT22 cells as the research object,a model of Glu-induced damage was established,and the protective effects of TC on Glu-induced damage and its possible mechanisms were discussed.The results are as follows:(1)Purity,yield rate,retention time and degree of separation of TC were investigated to study the proportion of mobile phase,sample concentration,sample volume as well as the mobile phase flow rate on the separation and preparation process of TC.The optimal chromatographic condition was as follows:the balance of the proportion of mobile phase 35%B was balanced 1BV(one column volume),and the sample was injected by hand,and then the proportion of mobile phase 35%B was1.01-1.50BV following the proportion of mobile phase 45%B was 1.51-3.30BV.Sample concentration was 10mg/ml,sample volume was 500?l,and the flow rate was2ml/min.The separated substances were identified as TC by UPLC-MS/MS.(2)The HT22 cells damage model was established using 5mmol/L Glu.TC and A_2A receptor antagonist SCH58261 showed protective effects on Glu-induced HT22cell damage since they improved cell morphology and increased cell viability.(3)SOD activities decreased and MDA contents increased in Glu-induced HT22 cells.However,TC significantly inhibited the effects of Glu,which showed a certain concentration dependence.(4)Hoechst 33342 staining and detecting apoptosis rate by Annexin V-FITC/PI double staining method were used to observe the HT22 cell morphology.The results indicated that Glu induced apoptotic morphology and increased the rate of apoptosis obviously.TC could obviously alleviate apoptotic morphology and decrease the rate of apoptosis induced by Glu in a certain concentration dependence manner.In conclusion,TC can be effectively separated and prepared by preparative chromatography from Camellia kucha leaves.TC affords protection against Glu-induced HT22 cell damage may via both interacting with Glu-induced oxidation stress and attenuating cell apoptosis,or may be associated with TC as an A_2A receptor antagonist.