The Studies on Purification of Total Flavonoids and Anti-oxidative Damage on Cadiocytes of Bauhinia Championii Flavones
|School||Guilin Medical College,|
|Course||Cardiovascular within science|
|Keywords||Bauhinia championii flavones purification cradiomyocyte oxidative damage protective effect|
Objective:1.To study the technology on abstraction and purification of totalflavonoids from Bauhinia championii（Benth.）Benth.（BCF）2.To observe inhibitory the effects of Bauhinia championii flavones oncadiocytes apoptosis induced by H2O2..3.To investigate the effects of Bauhinia championii flavones onhypoxia-reoxygenation induced injury in myocardial cells.Methods:1. To extract and purify total flavonoids from Bauhinia championi（iBenth.）Benth.1.1Bauhinia championii flavones were identified by stronger ammoniawater reaction, AlCl3reaction, concentrated sulfuric acid reaction, potassiumborohydride reaction and HCl-Mg reaction.1.2Quantitative determination of Bauhinia championii flavones: baicalinwas used as control article, Bauhinia championii flavones was determined at theOD278.5nmand substed to the standard curvilinear equation.The contents of totalflavonoids were calculated with baicalin reference substance by the followingformulas:Content（%）＝Ctotal flavonoids×Vmeasuring liquid／Mcrude extract×100% Extraction rate（%）＝Content×WTextrac（g）/Crude drug （g）×100%1.3To extracted the Bauhinia championii flavones:Using cold diffusionmethod, total flavonoids were extacted by different polar liquid.1.4To optimize the purification of the Bauhinia championii flavones:Macroporous resin model was determined by static absorption-deabsorptionexperiment. To optimize the purification process, with total flavone purity andrecovery as targets.2.To study the effects of Bauhinia championii flavones on cadiocytesoxidative damagePeroxidative damage model was induced by adding100μmol·L-1H2O2andthe hypoxia/reoxygenation injury on neonatal rat cardiomyocytes.Pretreated bySXN (100mg·L-1) or Vit.C (100mg·L-1), BCF (50,100or200mg·L-1). Cellviabilities were assay by MTT，Annex v-FITC/PI staining was used to detectapoptosis rate, ELISA was used to evaluate tumor necrosis factor alpha （TNF-α）,and total antioxidant activity （T-AOC）, total of superoxide dismutase （T-SOD）,lactate dehydrogenase （LDH）, malondialdehyde （MDA）, glutathione （GSH）,nitric oxide synthase （NOS） were determined. while the protein expression ofBax, NF-κβp65and Bcl-2was observed by immunohistochemistry.Results:1. To optimize the purification of the Bauhinia championii flavones: Bothabsorption ability and deabsorption ability of D101were higher than AB-8orpolyamide.The optimal purification process was: the maximum sample volumewas less than16.85mg·g-1（flavones/wet resin）,15.5mg·mL-1（pH3-4） sampleliquid, pillar diameter ratio1:6,4times of column volume （BV）, and1BV·h-1 speed of60%ethanol elution. Using this process, the average flavone purity was48.95%, while the recovery rate was96.09%.the extraction rate of totalflavonoids was2.6%（vs. crude drug）.2. To study the anti-oxidative damage on cadiocytes of Bauhiniachampionii flavones2.1Effects of Bauhinia championii flavones on cadiocytes apoptosisinduced by H2O2: Compared with model group, BCF pretreatment couldincrease cell viability, T-AOC、T-SOD、GSH and the expression of Bcl-2，whiledecrease cadiocyte apoptosis, LDH、MDA、TNF-α and the expression of Bax（P<0.01or P<0.05）.2.2Effects of Bauhinia championii flavones on hypoxia-reoxygenationinduced injury in myocardial cells:In morphology of myocardial cells,Toobserve the Myocardial primitive cells when culture3～5d later.The fusion ofcell develop radially with pseudopod coupling into network, andsynchronization pulsation.In the group of H/R model,The endochylemavacuolization, cell fusion contracted or disappeared, as well as cell peeled off,suspension and necrolysis, cell beat extentand frequency inhibition could beseen by morphological observation.These case were attenuated significantly inBauhinia championii flavones group.Compared with model group, flavonesfrom Bauhinia championii pretreatment could decrease cadiocyte apoptosis,TNF-α、iNOS and the expression of Bax and NF-κβp65，while kept the level ofeNOS,increase the expression of Bcl-2（P<0.01or P<0.05）.Conclusions:1. Extraction and purification of total flavonoids from Bauhinia championii（Benth.）Benth, D101type of resin is a better material for purifyingBauhinia championii. It is feasible to apply the optimized process intopurification total flavones from Bauhinia Championii.2. BCF has inhibitive effects on cadiocytes oxidative injury induced byH2O2and H/R, which the mechanisms are associated with scavenging freeradicals by increasing GSH and T-SOD, inhibiting lipid peroxidation withdecreasing MDA, improving myocardial metabolism by reducing LDH,reducing myocardial injury with decreasing iNOS and increasing eNOS,inhibiting myocardial cell apoptosis by up-regulating Bcl-2, downregulating Bax,NF-kappa beta protein expression, and decreasing the content of TNF-α.