Investigation On The Therapeutic Activity Of Total Flavones From Humulus Scandens On Ulcerative Colon And Simultaneous Determination Of Luteolin And Vitexin In Rat Plasma By RP-HPLC

ObjectiveTo investigate the therapeutic activity of Total Flavones From Humulusscandens and the possible mechanisms.A specific and accuratehigh-performance liquid chromatographic method has been developed andvalidated for the simultaneous determination of luteolin and vitexin in the plasmaof rat.Methods①Mouse animal model of colitis was established by TNBS/ethanol enema.The experimental animals were divided into6groups: contort group, modelgroup, SASP group (300mg/Kg), HTF group(150，300，450mg/Kg). The animalswere administrated SASP.HTF respectively (from2nd day after the experimentof ulcerative colitis model to the end of experiment,21days in total).At the end ofthe experiment, the activity of myeloperoxidase (MPO) and the content ofmalondiadehyde(MDA),superoxide dismutase(SOD) and the mucosapathophysiology was measured and thymus and spleen of rats were weightedrespectively.②The sample was treated with6.0%perchloric acid to precipitatethe protein. Luteolin and vitexin were extracted with ethyl acetate. The organiclayer separated was dried and reconstituted in the mobile phase. The mobilephase was methanol-20mol/L potassium dihydrogen phosphate buffer (58:42,pH=4.0), The HPLC separation was performed on C18column and the UV detector was set at350nm detection wavelength350nm, flow rate of1.0mL/min, column temperature of30℃.③Reference to the2010edition of the"Chinese Pharmacopoeia" requirements on the quality standards of thecontrolled release formulations, combined with colon targeting drug deliverysystem is characterized by the establishment of appropriate quality standardsassessment. Preparation of dosage forms, e critical relative humidity (CRH), thecapsule in vitro intestinal experiments to be evaluated.Results①The obvious effects of HTF at dosage of450mg/Kg on TNBS-inducedcolitis were observed, MPO activity of colonic tissues, meanwhile, the content ofMDA reduced, and the activity of SOD increased. The results showed that HTFcould alleviate the pathophysiological lesion of colon of rat.②By the druggranulation, the process of filling the colon capsule line is relatively simple, andgreat maneuverability. Capsules prepared in artificial gastric juice, artificialintestinal fluid, almost no release, the release of sensitive artificial colon fluid.③The standard curve for luteolin and Vitexin in plasma were linear over therange of38.5~4350and16.5~1860ng/mL, with the correlation coefficients0.9996and0.9999, respectively. The assay recoveries for luteolin and Vitexinranged from102.3to104.7%and93.82~101.4%, respectively. The intra-andinter-day precisions (RSD) for luteolin and Vitexin were all less than7.9%and5.4%. The sample was stable within24h at4℃storage,30days at-20℃storage, and undergoing four freeze–thawassay cycles.ConclusionsA potent therapeutic activity exerted by Humulus total flavonoids onulcerative colon. The method developed was applied successfully to studypharmacokinetics of effective composition (luteolin) of total flavones fromHumulus scandens extract in rat after A single dose of capsule administration.