Study On Metabolism Of Amentoflavone And Isoginkgetin In Vitro And In Vivo,Preparation And Evaluation Of Their Nanomicelles

Objective: Biflavonoids are a special kind of flavonoids isolated from gymnosperms such as Selaginella biloba,Ginkgo biloba,and Shizuku cypress.In recent years,diflavonoids have received more and more attention.Amentoflavone and isoginkgetin are two types of biflavonoids extracted from Ginkgo biloba leaves.Due to its low content in nature,there are currently few studies on the two biflavonoids,and no studies have been conducted on its metabolites and metabolic pathways in vivo and in vitro.In addition,the two compounds have poor solubility and low bioavailability.Therefore,this experiment was aimed at the study of the metabolites and metabolic pathways of the two biflavonoids in vivo and in vitro.And the two kinds of biflavones were to prepared into mixed nanomicelles to improve their solubility,stability,cytotoxicity and compare the similarities and differences of its metabolism with the prototype drug.Methods: UHPLC-Q/TOF-MS method was used to identify the metabolites of amentoflavone and isoginkgetin in rats,rat liver microsomes,and rat intestinal flora,and the metabolic pathways were analyzed and compared.In this experiment,TPGS and soluplus were used as carriers,and nanomicelles were prepared by thin film hydration method.After condition optimization,the particle size,Zeta potential,encapsulation efficiency,drug loading,stability,cytotoxicity,and metabolites in rats were studied.Furthermore,cytotoxicity and metabolites in rats of drug-loaded TPGS/soluplus mixed micelles were compared with those of diflavone monomers.Results: In this experiment,a total of 40 metabolites of amentoflavone were identified,34 in vivo,20 in liver microsomes,and 16 in intestinal flora.They were mainly distributed in rat feces,but not detected in bile or plasma.A total of 38 metabolites of isoginkgetin were found,of which 32 were in vivo and 18 were in vitro.Metabolites in vivo were only distributed in the feces of rats.In vitro metabolism,there were 17 metabolites in intestinal flora and 7 metabolites in liver microsomes.The mixed nanomicelles of amentoflavone and isoginkgetin were successfully prepared with particle sizes of 67.33±2.01 nm and 62.34±1.10 nm,and Zeta potentials of-0.84±0.04 m V and-0.35±0.18 m V,respectively.Encapsulation efficiencies were(99.18±0.76)% and(96.92±0.66)%,and the drug loading was(2.47±0.01)% and(2.42±0.02)%,respectively.The physical and chemical properties of the mixed nanomicelles could remain stable within 60 days,and the cytotoxicity was much greater than that of the two biflavones.Furthermore,the metabolites of nanomicelles in rats were significantly reduced,but metabolites can be detected in plasma,bile and urine,which indirectly indicated that the internal absorption of the two diflavonoids has increased.Conclusions: All the results indicated that amentoflavone and isoginkgetin were mainly excreted by gastrointestinal metabolism after oral administration,and they were difficult to be absorbed into the blood to exert pharmacological effects and had low bioavailability.When the two biflavones were prepared into mixed nanomicelles,their stability,solubility,cytotoxicity and internal absorption were increased,which provided a new choice for the development of new drugs.