Studies On Synthesis And Antioxidant Activity Of Sapogenin Thiosemicarbazone And Its Zinc Complex

Camellia Oleifera saponin,a pentacyclic triterpenoid saponin isolated from Camellia Oleifera seeds,is composed of three parts:organic acid,glycosyl and sapogenin.However,due to its large molecular weight,complex structure and difficulty in separation and purification,the application of pharmacologic activity of Camellia Oleifera saponin has been greatly limited.In this study,sapogenin with small molecular weight,simple structure,easy separation and purification and similar pharmacological activity with Camellia Oleifera saponin was obtained by hydrolysis.Then the structure of sapogenin was modified by introducing thiosemicarbazone group with extensive biological activity to obtain sapogenin thiosemicarbazone and sapogenin thiosemicarbazone-zinc complex,their antioxidant activity was investigated by in vitro antioxidant experiment and their ability to inhibit Keap1-Nrf2 interactions in oxidative stress defense signaling pathway and bind with epidermal growth factor receptor?EGFR?was investigated by molecular docking simulation.The main research methods and conclusions are as follows:?1?The crude camellia oleifera saponin was purified by macroporous adsorption resin,and the crude sapogenin was obtained after alkali hydrolysis and acid hydrolysis to remove organic acid and glycosyl.The pure sapogenin was separated by extraction,recrystallization and column chromatography,and its structure was characterized by IR,UV,NMR and elemental analysis.?2?The sapogenin thiosamicarbazone was obtained by dehydration condensation of sapogenin and thiosemicarbazide and then coordinated with zinc ion to obtain sapogenin thiosemicarbazone-zinc complex,their structures were characterized by IR,UV,NMR and elemental analysis.The single factor experiment was used to optimize the reaction conditions,the optimal synthesis conditions of sapogenin thiosemicarbazone were as follows:the reaction temperature was 75?,the reaction time was 8 h,the catalyst was glacial acetic acid,the mole ratio of sapogenin and thiosemicarbazide was 1:1.2,the yield of the product was 79.6%.The optimal synthesis conditions of the sapogenin thiosemicarbazone-zinc complex were as follows:the reaction temperature was 65?,the reaction time was 6 h,the reaction solvent was methanol,the mole ratio of ligands and zinc acetate dihydrate was 1:0.6,the yield of the product was72.1%.?3?The IC₅₀ values of the scavenging DPPH,ABTS⁺and hydroxyl radicals by sapogenin measured by in vitro antioxidant experiments were 2.36 mg/m L,3.36 mg/m L,2.08 mg/m L,respectively.The IC₅₀ values of the scavenging three radicals by sapogenin thiosemicarbazone were 0.66 mg/m L,0.46 mg/m L,0.68 mg/m L,respectively.The IC₅₀ values of the scavenging three radicals by sapogenin thiosemicarbazone-zinc complex were 1.19 mg/m L,0.9 mg/m L,0.57 mg/m L,respectively.The results showed that the scavenging ability of the sapogenin thiosemicarbazone and its zinc complex was significantly improved while compared with that of the sapogenin,the scavenging activity of sapogenin thiosemicarbazone on DPPH and ABTS⁺radical was stronger than that of its zinc complex and their scavenging activity on hydroxyl radical had a small difference.?4?The interactions between sapogenin,sapogenin thiosemicarbazone,sapogenin thiosemicarbazone-zinc complex and Keap1 in the Keap1-Nrf2 binding domain were investigated by molecular docking simulation to explore their ability to inhibit Keap1-Nrf2interactions,thus to investigate their effect on the body's antioxidant capacity.The results showed that the negative interaction energy of sapogenin,sapogenin thiosemicarbazone and its zinc complex docked with Keap1 in the Keap1-Nrf2 binding domain were 18.53 kcal/mol,21.06 kcal/mol,39.40 kcal/mol,respectively.All of them could occupy the binding sites of Nrf2,thus inducing the release of Nrf2 and enhancing the antioxidant capacity of the body.?5?Because the thiosemicarbazone compounds have extensive antitumor activity,the interactions between sapogenin,sapogenin thiosemicarbazone,sapogenin thiosemicarbazone-zinc complex and EGFR were investigated by molecular docking simulation,thus to explore its antitumor activity as EGFR inhibitors.The results showed that the negative interaction energy of sapogenin,sapogenin thiosemicarbazone and its zinc complex docked with EGFR were 42.92 kcal/mol,46.77 kcal/mol,102.7 kcal/mol,respectively.All of them had good binding effect with EGFR and could be expected to be used as EGFR inhibitors to achieve antitumor efficacy.Moreover,the interaction between the sapogenin thiosemicarbazone-zinc complex and EGFR was the strongest,followed by the sapogenin thiosemicarbazone and sapogenin.