Development Of Three Dihydromyricetin/ Cyclodextrin's Derivatives Inclusion Complexes:Preparation,Characterization,and Evaluation

Dihydromyricetin?DMY?,a bioactive component extracted from vine tea,which has various of biological and pharmacological activities.However,it follows that the clinical application of DMY would be severely hindered by its pharmacodynamic properties including low water solubility and bioavailability.In this study,the three DMY/CDs IC were prepared respectively in order to enhance its solubility and bioavailability.It has been demonstrated that DMY form complexes with cyclodextrin's derivatives?CDs?of 1:1 stoichiometry via analysis of phase solubility,in which three DMY/CDs IC was formatted spontaneously and exothermically,and the preparation process of three DMY/CDs IC was optimized by Box-Behnken mehtod.Then the three DMY/CDs IC was investigated through FT-IR,PXRD,DSC,NMR,and SEM,which was further confirmed by molecular modeling study.Besides,the preliminary pharmaceutical properties of three DMY/CDs IC,including solubility,dissolution rate,radical scavenging ability,pH stability and metal ion stability,the cell viability of HepG₂cells treated with various concentration of DMY and DMY/CDs IC for 24h and48h,and the pharmacokinetics in mice after oral administration of DMY and three DMY/CDs IC,were also investigated and compared.?1?In the section of three DMY/CDs IC preparation,the concentration of DMY in aqeous solutions was determined via HPLC,in which the regression equation Y=28.641X-18.453,R²=0.9999,indicating that it has a good linear relationship between DMY and the peak area at a concentration of ranging from 10?g/mL to 60?g/mL.The precision,repeatability and recovery experiments all show that the established analylase method is feasible and accurate.Moreover,it has been demonstrated that DMY form complexes with cyclodextrin's derivatives?CDs?of 1:1 stoichiometry via analysis of phase solubility at a concentration of CDs ranging from 0 mM to 10mM,and the thermodynamic studies were also performed,indicating that three DMY/CDs IC was formatted spontaneously and exothermically,and a relatively low temperature was preferred.Finally,the inclusion process of three DMY/CDs IC was optimized by Box-Behnken Design-response surface method?BBD-RSM?,showing that the opimized temperature of DMY/HP-?-CD IC,DMY/DM-?-CD IC and DMY/SBE-?-CD IC was 28°C,40°and 30°C respectively,and other opimized conditions including time,molar tatio and rotating speed were 2h,1:1 and 200rpm respectively.?2?In the section of characterization of three DMY/CDs IC,complexes of DMY with cyclodextrin's derivatives?HP-?-CD,DM-?-CD,SBE-?-CD?were prepared successfully by freeze drying method,and further investigated through FT-IR,PXRD,DSC,SEM,and NMR analysis.Meantime,two possible inclusion model of three DMY/CDs IC was predicted,one is the B ring of DMY and part of ring C were included in the cavity of HP-?-CD in the struture of DMY/HP-?-CD IC,the other is the A ring and C ring as well as part of B ring was included in DM-?-CD and SBE-?-CD in the structure of DMY/DM-?-CD IC and DMY/SBE-?-CD IC.Besides,two possible different inclusion model were further confirmed by molecular modeling study,predicting that DMY/SBE-?-CD IC was the most stable among the three inclusion complexes,and the Hydrogen bonding interactions and vander Waals interactions are the predominant forces that facilitate formation of the inclusion complexes.?3?In the section of initial evaluation of pharmacological properties.The solubility of three DMY/CDs IC was obviously enhanced by forming inclusion complex between DMY and CDs.Meantime,the results of disslution study was consistent with both phase solubility study and solubilization test,indicating that the solubility in water and dissolution rate was improved significantly compared than pure DMY.The results of stability study show that three DMY/CDs IC should be preserved in dry enviroment,and the pH stability and metal stability of three inclusion complexes was also obviouly enhanced.Besides,the antioxidant study show that the antioxidant capacity of DMY was superior to TBHQ,and the reducing power of three inclusion complex was higher than DMY.Overmore,the scavenging capability and antioxidant stability with time prolonged of the three DMY/CDs IC on DPPH free radicals,OH free radicals,O^2-free radicals and ABTs⁺free radicals was significantly enhanced,and the scavenging capability on OH free radicals and ABTs⁺free radicals was appearently enhanced.?4?Furthermore,The cell viability of HepG₂ cells treated with various concentrations of DMY and DMY/CDs IC for 24h and 48h was investigated by MTT method,indicating that the antitumor effect was not eliminated by forming three inclusion complexes between DMY and CDs,and the three inlclusion complexes has moderate sustained release effect.Finally,the concentration of DMY in rat was determined via HPLC,and there is a good linear relationship between the concentration of DMY and the peak area ratio of DMY and TFN by methodological verification.Meantime,the bioavailability of DMY in three DMY/CDs IC in mice were greatly significantly improved compared than raw DMY via the pharmacokinetics study in mice,providing a alternatively promising vehicle to enlarge the bioavailability of DMY.All experiments on the above show the prepared three DMY/CDs IC all significantly improve both the solubility and the bioavailability of DMY,and the other pharmaceutical properties including dissolution rate;stability;antioxidant ability and antitumor effect were also enhancd.Therefore,the obtained three DMY/CDs IC can be used as a potential approach to expand its clinical application.