| Purpose:1.The automatic analysis method of thin layer chromatography was established,and the results of thin layer chromatography of Polygalae Radix from different sources were analyzed by computer image analysis technology,which provided reference for the accuracy of the identification and the automatic analysis and evaluation of the quality grade of Polygalae Radix.2.The extraction methods of Polygalae Radix were screened and the technological conditions of DISS parts prepared by macroporous resin method were determined,which provided support for the study of active factions of Polygalae Radix.3.Screen the protective effect of different fractions of Polygalae Radix on SH-SY5Y cells damaged by CORT,and explore the effect of DISS part of Polygalae Radix on SH-SY5Y cells and other indexes such as LDH,ROS and MMP.To study the partial protective mechanism of DIISS,and to provide support for the pharmacodynamic mechanism of Polygalae Radix in the treatment of depression.4.To investigate the protective effect of DISS on SH-SY5Y cells damaged by CORT in hypoxic culture environment,and the effect on ROS,MMP and other indexes.To study the partial protective mechanism of DISS under hypoxia,and to provide support for the pharmacodynamic mechanism of Polygalae Radix in the treatment of depression.Material and method:1.Using thin layer chromatography to identify 18 different Polygalae Radix samples,using automatic analysis method to evahuate the similarity and Polygalae Radix control medicinal materials from different sources,and make grade classification.2.75%ethanol was used to extract Polygalae Radix hrough the enrichment of macroporous adsorption resin and the gradient elution of different concentrations of ethanol,HPLC method was used to detect different eluents with DISS as the index to determine the elution conditions.3.The SH-SY5Y cells were damaged by CORT,and the cell viability was determined by MTT method LDH levels were detected by LDH release assay,and levels of intracellular reactive oxygen species and mitochondrial membrane potential were measured by kit.Hoechst/PI double staining method was used to detect cell apoptosis,intracellular calcium content was determined by caldum ion kit,and intracellular ATP,ADP,AMP and NAD contents were determined by HPLC.The neuroprotective effect and mechanism of DISS on CORT damaged cells were comprehensively evaluated and analyzed.4.The model of SH-SY5Y cells damaged by hypoxic CORT was established,and the cell viability was determined by MTT method.The levels of intracellular reactive oxygen species and mitochondrial membrane potential were determined by fluorescence probe kit.Apoptosis was detected by Hoechst33342/PI double staining method.With ROT as control,the mitochondrial membrane potential of cells was measured by IC-1 kit,and the mechanism of action of DISS on mitochondrial function of cells damaged by CORT unnder hypoxia was analyzed.Results:1.The automatic TLC analysis method was used to screen and evaluate 18 Polygalae Radix samples from different sources,and the Herba Polygalae telephioidis and 17 batches of Polygalae Radx were identified.The similarity between the 17 batches of Polygalae Radix and the control was higher than 0.85,indicating that the homogeneity of the 17 batches of Polygalae Radix was good.The 17 batches of Polygalae Radix were classified into four batches of first grade,seven batches of second grade and six batches of third grade with similarity greater than 0.85 or 0.93.2.75%ethanol was used to extract Polygalae Radix,and the extract was separated by AB-8 macroporous adsorption resin.The mass concentration of the loading solution was 0.6g/ml,the maximum loading volume was 1.5BV,and the eluent was eluted by 10%and 40%ethanol(6BV),and by 75%and 95%ethanol(4BV).Using DISS as the index,HPLC was used to detect the different parts of Polygalae Radix.3.In the study of the effect of DISS on the viability of SH-SY5Y cells induced by CORT,the minimum dose of CORT significantly inhibited the viability of SH-SY5Y cells was 50 μmol/L,and the best dose was 600 μmol/L.he effect of DISS on the growth of cell vitality was dose-dependent at 10,20,40mg/ml.It had obvious protective effect on CORT damaged cells,reduced LDH level and improved cell damage.According to ROS and mitochondrial membrane potential kit,the green fluorescence increased significantly and the red fluorescence decreased significantly after CORT injury.After partial administration of DISS,the green fluorescence changed from strong to weak,and the red fluorescence changed from weak to strong,indicating that DISS can significantly improve the ROS level of CORT injury cells and enhance the expression of mitochondrial membrane potential.Hoechst33342/PI double staining showed that DISS could improve the apoptosis and necrosis of Cort-induced cells The results showed that DISS could partially reduce the calcium influx induced by CORT.The contents of ATP,ADP,AMP and NAD in the cells were detected by HPLC.After CORT injury,the contents of the four components in the cells decreased significantly.After partial treatment with DISS,the intracellular components were improved to varying degrees.DISS could significantly improve the activity of CORT damaged cells,but had no effect on CORT and ROT damaged cells.Accordng to JC-1 fluorescent probe kit,DISS can significantly improve the mitochondrial membrane potential of CORT-damaged cells,but has no significant effect on the mitochondrial membrane potential of CORt-damaged cells with ROT.4.In the study of hypoxia damage to SH-SY5Y cell vitality,the time of hypoxia significantly reduced cell vitality was 8h.According to ROS and MMP kits,after partial administration of DISS,the green fluorescence changed from strong to weak,and the red fluorescence changed from weak to strong,indicating that DISS can significantly improve the increase of ROS level and the decrease of mitochondrial membrane potential caused by CORT under hypoxia condition.By Hoechst/PI double staining,DISS can mprove the apoptosis and necrosis of Cort-damaged cells under hypoxia condition.5.Under hypoxia condition,the mitochondrial membrane potential of SH-SY5Y cells damaged by CORT and ROT was measured.According to the detection of JC-1 mitochondrial membrane potential kit,DISS could significantly improve the mitochondrial membrane potential of SH-SY5Y cells damaged by CORT under hypoxia condition.The mitochondrial membrane potential of SH-SY5Y cells damaged by CORT and ROT had no significant effect.Conclusion:1.A new automatic analysis method of thin layer chromatography was established and the results of thin layer chromatography were identified.18 batches of Polygalae Radix from different sources were compared and analyzed and classified into three grades,which provided research support for the identification and evaluation of Polygalae Radix.2.AB-8 macroporous adsorption resin was used to separate the extracts of Radix polygata,and eluted with 10%,40%,75%and 95%ethanol,respectively.The DISS part had a protective effect on SH-SY5Y cells damaged by CORT.he mechanism of action is that DISS partially inhibits CORT-induced cell vitality decline,oxidative stress injury,cell apoptosis,calcium ion influx and the reduction of intracellular ATP,ADP,AMP,NAD content.DISS fraction protects Cort-damaged cells partly through mitochondrial pathway.3.In the hypoxia state,DISS fraction has a protective effect on the damage of CORT SH-SY5Y cells,inhibiting the decrease of cell vitality,oxidative stress damage and cell apoptosis caused by the common damage of hypoxia and CORT.DISS can protect cells from hypoxia and CORT damage through mitochondrial pathway. |
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