Preparation,Characterization And The Therapeutic Efficacy Against Ulcerative Colitis Of Baicalein-Cyclodextrin Inclusion

Objective:Baicalein(Ba)is a flavonoid glycoside extracted from Scutellaria baicalensis with antioxidant and anti-inflammatory activities.However,the disadvantages of poor water solubility and low bioavailability limit its effects.In order to improve the solubility,absorption and bioavailability of baicalein in vivo,β-cyclodextrin(β-CD)and hydroxypropyl-β-cyclodextrin(HPβ-CD)are selected as inclusion subsidiary materialin this study.Baicalein-cyclodextrin inclusion complexes are prepared by freeze-drying method,and their structures are characterized and verified.The pharmacokinetics and therapeutic effect on ulcerative colitis(UC)of inclusion complexes are discussed.Method:(1)Firstly,the phase solubility method is used to study the effects of β-CD and HP-β-CD on the solubility of Ba.The preparation conditions of baicalein-cyclodextrin inclusion complexes are optimized by single factor experiment and star point design-response surface method.The inclusion rate is taken as the investigation index.(2)Differential scanning calorimetry(DSC),infrared spectroscopy(IR),X-ray diffraction(XRD),scanning electron microscopy(SEM)and the molecular simulation are used to characterize and verify the structures of Ba-β-CD and Ba-HP-β-CD inclusion complexes.The dissolving properties of Ba and two inclusion complexes are assessed by the experiment of dissolution and solubility in vitro.(3)The concentration of baicalin in rat plasma is determined by HPLC after oral administration of Ba and two inclusion complexes.The pharmacokinetic parameters are calculated by DAS 2.0 software,and the absorption degree and oral bioavailability in vivo are compared.(4)A Lipopolysaccharide(LPS)-stimulated inflammatory RAW264.7 cell model and a dextran sodium sulfate(DSS)-induced UC mouse model are established.Inflammatory RAW264.7 cells and UC mice are intervened with baicalein and inclusion complexes.The levels of inflammatory cytokines and biochemical indexes in the cell supernatant,serum and colon of mice are detected by ELISA kits.The antioxidant and anti-inflammatory activities of baicalein and inclusion complexes in vitro and in UC mice are evaluated.(5)The distributions of baicalein and inclusion complexes in liver,colon and intestine of normal mice and DSS-induced UC mice are determined by UPLC-Xevo TQ-MS/MS.Result:(1)HP-β-CD and β-CD both have good effects on the solubility of Ba and the effect of the former is better than that of the latter.The optimum preparation process of Ba-β-CD inclusion complex is that the inclusion temperature is 60℃,the stirring time is 95 min,the molar ratio of Ba and β-CD is 1:2.The optimum preparation process of Ba-HP-β-CD inclusion complex is that the molar ratio of Ba and HP-β-CD is 1:2,the inclusion temperature is 70℃,stirring time is 120 min,and the volume ratio of water to ethanol is 2:3.The inclusion complexes prepared by the optimum processes are fluffy and have good solubility.(2)The characteristic peak of Ba is not detected in Ba-β-CD and Ba-HP-β-CD inclusion complexes via DSC,XRD and IR.The morphology of inclusion complexes are irregular clastic in SEM.After being encapsulated,Ba is located in the center of β-CD and HP-β-CD,separatly forming 3 and 2 intermolecular hydrogen bonds.Inclusion complexes exert well in the molecular simulation,which are new phases.The solubility and dissolution of Ba are greatly improved via inclusion.Compared with Ba,the solubility of Ba-β-CD and Ba-HP-β-CD inclusion complexes are increased by 29.7 times and 37.6 times respectively,and the dissolution in 180 minutes are increased by 11.86 times and 12.53 times respectively.(3)After complexed with β-CD and HP-β-CD,t1/2,Cmax and AUC0-∞ of Ba are improved(p<0.05),the degree and rate of absorption are increased,and the oral bioavailability are increased by 3.95 times and 5.80 times respectively.The bioavailability of Ba-HP-β-CD inclusion complex is the better than that of Ba-β-CD inclusion complex.Tmax has no difference.(4)At a dose range of 12.5～100 μmol/L,Ba,Ba-β-CD inclusion complex and Ba-HP-β-CD inclusion complex are non-toxic on RAW264.7 cells and effectively reduce the levels of nitric oxide(NO),tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)in LPS-stimulated RAW264.7 cells(p<0.05).At a dose of 40 mg/kg,compared with Ba,Ba-β-CD inclusion complex and Ba-HP-β-CD inclusion complex can also improve the symptoms of mice weight loss,diarrhea and hematochezia,reduce the spleen index,improve the shortening of colon,restore goblet cells and crypts,alleviate the damaged colonic mucosa and the infiltration of inflammatory cells,reduce the levels of diamine oxidase(DAO),IL-6,TNF-α and interleukin-1β(IL-1β)in serum,and the levels of myeloperoxidase(MPO)and malondialdehyde(MDA)in colon,and increase the content of interleukin-10(IL-10)in serum(p<0.05).In summary,they have anti-inflammatory effect in RAW264.7 inflammatory cells and anti-UC activity in DSS-induced UC mice,and two inclusion complexes have better antioxidant and anti-inflammatory effects in vitro and in vivo.(5)The tissue distribution of Ba and inclusion complexes after oral administration found that:baicalein and baicalin are mainly distributed in the intestine of normal and UC mice,but less in the liver and colon.The amount of baicalein in these tissues is higher than that of baicalin.Compared with Ba,the drug peak concentrations of Ba-β-CD and Ba-HP-β-CD inclusion complex in the colon,intestine and liver of mice are higher.The peak concentrations of baicalin and baicalein in the colon of DSS-induced UC mice are generally higher than those of normal mice.Conclusion:Ba-β-CD inclusion complex and Ba-HP-β-CD inclusion complex are successfully prepared.They can improve the solubility,oral bioavailability and anti-inflammatory activity in RAW264.7 inflammatory cells and DSS-induced UC mice of Ba.The distributions of baicalein and baicalin in organs under normal and UC pathological conditions are definite,and the drug concentrations in ulcerative colon are improved.The study provides a new way to improve the solubility and bioavailability of insoluble compounds,and provides an experimental basis for the industrialized research and development of baicalein preparation.