Extraction Of Flavones With Microwave Radiation, Benzensulfonate Modification And Pharmacokinetics Experiments

Flavonoids,quercetin,daidzein and genistein,are a class of naturally occurring polyphenolic compounds that have been isolated from various vascular plants such as hippophae rhamnoides L.,kudzuvine root and soybean et al.Despite their in vitro biological activity,such as antioxidant,antiinflammatory,antiviral,antiproliferative, and anticarcinogenic effects,quercetin,daidzein and genistein have not been employed widely in therapeutic medicine as drugs because their poor lipophilicity, hydrophilicity and their first pass effect,which result from their polar hydroxyls in molecules,will result in weak bioavailability(quercetin 3.6%,daidzein 12%,genistein 30%).In this paper,we focus on investigating the microwave radiation extraction method and extraction mechanism of three flavones,design and synthesis of prodrugs of flavones and their pharmacokinetics,some derivatives' activities.The microwave radiation extraction method of three flavones(quercetin,daidzein and genistein) from three plants,hippophae rhamnoides L.,kudzuvine root and soybean respectively were investigated,and microwave radiation extraction mechanism were also studied systematically with IR,FM etc.The sample was comminuted to powder of 0.022～0.056mm,9.4%DMF were added into sample and put it into microwave oven.The sample was heated up with microwave and light wave combination(55:45) by 6min or 8 min durativly.Flavones were extracted twice by 200mL ethanol.After recycling ethanol,flavones were obtained.To optimize pharmacokinetics of three flavones which resulted in increasing their oral bioavailability,the seventeen new sulfonic acid esters of three flavones were designed according to principles of prodrugs and synthesised in high yield with excellent regioselectivity.Among them there are 3 quercetin derivatives,7 daidzein derivatives and 7 genistein derivatives respectively.Their structures were characterized by IR,MS,elemental analysis and ¹H NMR spectra.The crystal structures of compound 1,11and13 were reported respectively.As expected,each S atom locates at the center of the tetrahedral geometry.In the crystal structure of 11 and 13,molecules are linked through intermolecular hydrogen bonds.In addition, relatively shorter centroid distances among the rings are observed,implying the existence ofÏ€-Ï€stacking interactions in the compounds 1 and 11.The prodrug studies method of benzene sulfonic acid esters was set up and reaction pathway of hydrolysis was also investigated.It contains mensuration of solubility,partition coefficients and hydrolysis constants of quercetin,daidzein and their analogs 1,12 and 13.The solubility of the prodrugs increased in all examined solvents compared with their technicalmaterial.The apparent lipid/water partition coefficients of compound 1,12 and 13 are 2.04,3.57 and 1.97 respectively.All prodrugs hydrolysis are modeled as a pseudo first-order reaction under constant conditions of pH and temperature,and their hydrolysis constants are 5×10^-5s^-1, 9×10^-5s^-1and 1×10^-5s^-1 respectively.Their half lives T_1/2 are 3.85,2.14 and 19.25h, respectively.The 4'-OH in compound 12 was hydrolysised firistly to obtain compound 13,and farther to get daidzein.The experiment results and counts of ChemAxon of these compounds indicate the oral bioavailabilities are improved comparing with their technicalmaterials.An HPLC procedure was developed and validated for the determination of free daidzein(genistein) and its prodrugs in biological samples with genistein(daidzein) as the internal standard.The analyte and internal standard (genistein or daidzein) were extracted from plasma samples by CH₃COOC₂H₅,and chromatographed on a Diamonsil Cog column(200×4.6 mm,I.D.,5μm) with the mobile phase consisting of water:formic acid(1:1,v/v) at a flow rate of 0.5 mL/min. The intra-and inter-day precision in terms of RSD were both under 14%,and the accuracy in terms of RE ranged from-10%to 13%.The extraction recoveries of daidzein,genistein and their prodrugs all exceed 66.1%.Prodrug screening and prodrug pharmacokinetics experiments were operated using Wistar rats.Among 17 sulfonic acid esters there are 7 prodrugs of these flavones.The pharmacokinetics of these prodrugs were systematically investigated. The relative bioavailabilities of prodrug12,13 and 15 compared with daidzein at the same molar dose were calculated to be 42.9%,21.5%and 26.8%,respectively;and the relative bioavailabilities of prodrug6,7,9 and 10 compared with genistein at the same molar dose were calculated to be 198.6%,110.9%,159.2%and 253.8%, respectively.Higher relative bioavailabilities of prodrug10 and prodrug 7 compared with genistein indicated great improvements of their pharmacokinetic behaviors after structural modifications.Prodrug 10 and 7 were new drug candidates of genistein with better oral bioavailability.The anticancer and inhibiting the viability of vascular smooth muscle cells activities of some sulfonic acid ester derivatives of daidzein were screened in vitro, their structure-activity relationship were investigated by results and counts of computer-assisted drug design software ChemAxon.Compounds 12 and 13 showed better anti-HL-60 activities than daidzein(P＜0.01).Conpound 12 and 13 showed better inhibiting the viability of vascular smooth muscle cells activities than daidzein. The compound 12 shows about 100 times inhibiting activity than daidzein and compound 13 is 10³ times.Inhibitory rate of the compound 13 is 56.06%at 10^-7mol/L in vitro.