| ObjectiveZhuang-medicine "Gocaekmbaw",Gyhostemma pentaphyllum(Thunb.)Makino,mainly contains saponins,flavonoids,polysaccharides,amino acids and other components,with anti-inflammatory,anti-oxidation,anti-tumor,anti-virus and other effects.The objective of this study was to isolate and identify the flavonoids from G.pentaphyllum,to evaluate the ability of scavenging free radicals and antioxidant activity in vitro,and to analyze the structure-activity relationship.It is expected to provide potential candidate drugs for renal diseases caused by oxidative injury and theoretical basis for clinical rational drug use.Methods1.G.pentaphyllum was exhaustively extracted with 80%ethanol by refluxing.Total flavonoids were isolated and purified by macroporous resin SP825L,silica gel,sephadex LH-20,semipreparative HPLC.The chemical structures were identified with UV,MS and NMR data.2.Antioxidant activity of flavonoids of G.pentaphyllum in vitro was evaluated by DPPH,O2-,· OH assay3.The oxidative damage model was established using different concentrations APPH to induce LLC-PK1 cell for different hours,and then treated with the flavonoids for 10 h.The effects of flavonoids from G.pentaphyllum on cell viability of LLC-PK1 cell damaged by AAPH were detected by MTT assay.The contents of ROS were detected by DCFH-DA fluorescent probe method via flow cytometer.The contents of SOD and GSH were detected by NBT and DTNB-linked colorimetry assay,respectively.Results1.Ten flavonoids were isolated from G.pentaphyllunm.They were identified by the mass and NMR data,combined with the reported references.Compounds ?,?,?,?,?,? and ? were identified as quercetin-3-O-(2",6"-di-?-L-rhamnosy)-?-D-galactopyranoside(?),quercetin-3-O-(2",6"-di-?-L-rhamnosy)-?-D-glucopyranoside(?),quercetin-3-O-(2'-?-L-rhamnosy)-?-D-galactopyranoside(?),quercetin-3-O-(2'-a-L-rhamnosy)-?-D-glucopyranoside(?),kaempferol 3-neohesperidoside(?),hyperin(?),isoquercitrin(X),respectivly.Owing to the low quality of compounds ?,? and ?,they were identified as kaempferol-rhamno-hexoside,kaempferol-hexoside,kaempferol-hexoside.by MS data,respectively.Compound ? and ?,? and ?,? and ?,? and ?,? and X are isomers each other,respectively.Compounds ? to ? were isolated from Cucurbitaceae family for the first time.2.Flavonoids ? to ? from G.pentaphyllum showed strong activity scavenging DPPH free radical,with IC50 values of 71.4,29.5,48.3,79.2 ?M,respectively,especially compound ? showed the strongest activity.In the scavenging · OH free radical,the IC50 values of compounds ? to ? were 403.6,396.8,358.1,481.9 ?M,respectily.Compounds ? and ? showed stronger activity than that of compounds? and ?.They all showed weak activity scavenging O2-free radical.3.The LLC-PK1 cell viability were 69.8%after treated with AAPH at 30 mM for 10 hours.So they were chosen to be as an oxidant stress model.Compared with AAPH group,LLC-PK1 cells treated with compounds ? to ? 50 ?M for 10 hours showed a dose-dependent increase in cell viability,from 69.8%to 97.7%?102.8%?107.7%?105.9%,respectively(p<0.05).4.In the model group,the expressions of ROS were increased(p<0.05),whereas the expressions of SOD and GSH were decreased(p<0.05).Compared with model group,the expressions of ROS in LLC-PK1 cells treated with flavonoids ? to ? the were decreased from117 to 53.6?40.5?49.2?56.7(p<0.05),and the expressions of SOD and GSH in the experimental groups were increased from 5.62 U/g to 6.39?10.73?8.09?7.46 U/g(p<0.05),4.25 mmol/g to 5.53?6.33?8.48?7.12 mmol/g,respectively(p<0.05).ConclusionTen flavonoids were isolated from G.pentaphyllum,and they were 5 pairs of isomers,among which seven structures were identified.They all possess very strong ability to remove DPPH free radicals in vitro,They reduced oxidative damage and repaired effects by reducing the content of ROS and increasing the content of SOD and GSH in LLC-PK1 cells damaged by AAPH oxidation.It provides evidence for reward diseases caused by oxidative damage. |
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