Therapeutic Effect Of Brusatol Self-microemulsifying Drug Delivery System Against Ulcerative Colitis

ObjectiveBrucea javanica?L.?Merr,a plant species of the family Simaroubaceae,the fruits are used in traditional folk medicine for treating various inflammatory disorders including dysentery,which is consistent with UC nowadays on the clinical symptoms.Modern medicine for Brucea javanica was mainly focused on its anti-tumor effect,but neglected its traditional application for dysentery.The quassinoid compounds are the bioactive components in Brucea javanica.Brusatol,one of the major active quassinoid of Brucea javanica,which was reported to be a potent anti-inflammatory agent.Nevertheless,the low aqueous solubility and rapid first-pass metabolism after oral administration had severely limited its potential medicinal application.In order to improve the dissolution and bioavailability of brusatol,we focused our attention on a nanoformulation named self-microemulsifying drug delivery system.The aim of the present study was to develop a nanoformulation of SMEDDS containing brusatol to increase its solubility and bioavailability,and enhance its anti-colitis activity.In the research,we isolated brusatol from Brucea Javanica dregs,and then evaluated the anti-inflammatory effects of brusatol on LPS-stimulated RAW 264.7 cells in vitro and explore its underlying mechanism.It is proposed to develop brusatol self-microemulsion delivery system?BR-SMEDDS?and the formulation was characterized by evaluating its physicochemical characterizations.A HPLC-MS/MS method was developed for the determination of brusatol in rats plasma.Pharmacokinetics of BR-SMEDDS and BR-suspension in rats after oral administration were investigated.The pharmacokinetics parameters were calculated and yielded a relative bioavailability of BR-SMEDDS.The anti-colitis activity of BR-SMEDDS was evaluated on DSS-induced colitis mice model and TNBS induced colitis rat model.From the regulation of anti-oxidative and anti-inflammatory status to elucidate the potential underlying mechanism which could be scientifically elaborating the material basis of Brucae javanica for the treatment of“liji”.Methods1.The isolation and identification of brusatolThe Brucea javanica dregs were extracted with 95%EtOH under heating.The solvent was concentrated to give a crude extract,followed by suspending in H2O.The aqueous layer was further extracted with Petroleum ether and EtOAc.The EtOAc layer was evaporated under vacuum to afford extracts and subjected to silica gel column chromatography and Sephadex LH-20 eluted with CH2Cl2-MeOH which was obtained a compound.2.The in vitro anti-inflammatory effects of brusatol in LPS-stimulated RAW 264.7 cellsThe in vitro anti-inflammatory effects of brusatol was investigated on LPS-stimulated RAW 264.7 cells.The effect of brusatol on the production of TNF-?,IL-1?,NO and PGE2in LPS-stimulated RAW 264.7 macrophage was measured by kits,the expression of NF-?B p65 proteins in RAW264.7 cells were detected by western blot.3.Preparation of brusatol self-emulsifying drug delivery system and characterization its physicochemical propertiesThe solubility of brusatol in Ethyl Polyenoate,MCT,Solutol HS15,Cremophor RH40,Propylene Glycol,PEG 400,Glycerol was measured.Pseudo-ternary phase diagram was adopted to determine the ratio of surfactant and cosurfactant?Km?.The prescription of BR-SMEDDS was optimized by mean particle size and drug loading.The developed BR-SMEDDS formulation was characterized by evaluating its physicochemical characterizations?including droplet size,polydispersity index,morphology,zeta potential,drug encapsulation efficiency?and stability.4.Pharmacokinetics studies of BR-SMEDDS in ratsThe analysis was performed with an Agilent 1260 Infinity HPLC system interfaced with an Agilent 6460 Triple Quad LC-MS/MS system with an electrospray ionization source.A HPLC-MS/MS method was developed for the determination of brusatol in rats plasma,BR-suspension was performed as a comparsion.Pharmacokinetics of BR-SMEDDS and BR-suspension in rats after oral administration were investigated.Plasma concentration-time curve was fitted by using the Drug and Statistics software?version 3.0?.Pharmacokinetics parameters and relative bioavailability of BR-SMEDDS was calculated.5.Therapeutic effect of BR-SMEDDS against DSS-induced ulcerative colitis in miceA total of 160 adult male Balb/c mice were randomly sorted into 8 different experimental groups?20 mice per group?.Acute ulcerative colitis was induced by adding DSS?dissolved in fresh distilled water?to the drinking water to a final concentration of 3%?wt/vol?for 7consecutive days.Control group and DSS group received equal volume of blank-SMEDDS,DSS-treated group were oral received with BR-SMEDDS?0.25,0.5 and 1 mg/kg?,BR-suspension?1mg/kg?,SASP?200 mg/kg?,AZA?13 mg/kg?once daily for 7 days.Disease activity index?DAI?and the degree of colonic injuries?colon length and histologic assessment of colons?were measured to evaluate the therapeutic effect of BR-SMEDDS on DSS-induced UC in mice.Meanwhile,colon oxidative status?MPO,MDA,GSH-Px,SOD?,colon inflammatory cytokines?TNF-?,IFN-?,IL-1?,IL-4,IL-6 and IL-10?were analyzed,the expression of TLR4,MyD88 and NF-?B p65 proteins were detected by western blot,to explain the potential mechanisms of BR-SMEDDS.on DSS-induced UC in mice.6.Therapeutic effect of BR-SMEDDS against TNBS-induced ulcerative colitis in rats48 male SD rats were randomly divided into 6 groups,include control group,TNBS group,AZA group?AZA,9 mg/Kg?and BR-SMEDDS group?0.25,0.5,1 mg/Kg?.Except for control group,UC was induced by infra-rectal installation of TNBS?25mg/Kg?.AZA and BR-SMEDDS groups were oral administrated for 7 consecutive days,while control group and TNBS group received equal volume of blank-SMEDDS.Body weight,disease activity index?DAI?and the degree of colonic injuries?colon length,macroscopic and histologic assessment of colons?were performed to evaluate the therapeutic effect of BR-SMEDDS on TNBS-induced UC in rats.The parameters of oxidative stress?CAT,GSH,T-SOD,NOS?,the inflammatory cytokines?TGF-?,IL-4,IL-13 and IL-18?were analyzed,the expression of Keap-1 and Nrf2 mRNA in colon were measured by real time-PCR,the expression of Nrf2protein in colon was detected by immunohistochemistry,the expression of NLRP3,ASC and caspase-1 proteins were detected by western blot,to explain the potential mechanisms of BR-SMEDDS.on TNBS-induced UC in rats.Results1.The isolation and identification of brusatolThe purity of obtained compound was above 98%detected by HPLC.The structure of this compound was elucidated by NMR and determined for brusatol.2.The in vitro anti-inflammatory effects of brusatol in LPS-stimulated RAW 264.7 cellsTreatment with brusatol?at the concentrations of 25,50 or 100 nM?could dose-dependently decrease the production of TNF-?,IL-1?and PGE₂ in LPS-stimulated RAW264.7 cells,and brusatol treatment?at the concentrations of 50 and 100 nM?also decreased the production of NO in LPS-stimulated RAW 264.7 cells,furthermore,brusatol in concentrations of 25,50 or 100 nM could dose-dependently inhibit the expression of NF-?B p65 proteins in LPS-stimulated RAW 264.7 cells.In summary,brusatol has a protective effect on LPS-stimulated RAW 264.7 macrophage inflammation,and its mechanism may be related to the inhibition of the activation of NF-?B pathway and subsequent regulation the release of inflammatory cytokines and mediators.3.Preparation of brusatol self-emulsifying drug delivery system and characterization its physicochemical propertiesBased on the results of solubility,MCT,HS 15 and PEG 400 were selected as oil,surfactant and co-surfactant,respectively.The constitution of optimum formulation is as follows:Solutol HS-15/PEG 400/MCT=4:2:1,with content 2%of drug.The developed BR-SMEDDS was with smaller size,dispersed homogeneously in aqueous medium and stability was proved excellent.4.Pharmacokinetics studies of BR-SMEDDS in ratsThe calibration curve was linear over the concentration.The intra-and inter-day accuracy and precision were within 10%.The validated method was successfully applied to the requirements of biological sample analysis.The pharmacokinetic parameters of BR-SMEDDS were significantly enhanced compared with BR-suspension.The relative bioavailability of BR-SMEDDS in rats was calculated to 188.20%.5.Therapeutic effect of BR-SMEDDS against DSS-induced ulcerative colitis in miceBR-SMEDDS could significantly reduced the body weight loss,recovered colon length,decreased disease activity index and microscopic score,regulated immune-inflammatory cytokines?down-regulation the activities of TNF-?,IFN-?,IL-1?and IL-6,up-regulation the activities of IL-4 and IL-10?,diminished oxidative stress?decreased the MPO and MDA content,increased the activities of GSH-Px and SOD?,in addition,BR-SMEDDS could repress the colonic expression of MyD88,TLR4 and NF-?B p65 proteins.At the same dose,the therapeutic action of BR-SMEDDS was more pronounced than that of BR-suspension.Therefore,these results strongly indicate that brusatol could effectively attenuate colonic inflammation on DSS-induced UC in mice,and the possible mechanism might be associated with regulation of oxidative stress and anti-inflammatory status by inhibition of the TLR4-linked NF-?B signaling pathway.6.Therapeutic effect of BR-SMEDDS against TNBS-induced ulcerative colitis in ratsBR-SMEDDS could significantly reduced the body weight loss,recovered colon length,decreased disease activity index,macroscopic and microscopic score,regulated inflammatory cytokines?up-regulation the activities of TGF-?,IL-4 and IL-13,down-regulation the activities of IL-18?,diminished oxidative stress?decreased the NOS content,increased the activities of CAT,GSH and T-SOD?,regulated the expression of Keap-1 and Nrf2 mRNA and Nrf2,NLRP3,ASC and caspase-1 proteins.Taken together,these results demonstrated that brusatol could effectively attenuate colonic inflammation on TNBS-induced UC in rats,and the underlying mechanism might be associated with regulation of anti-oxidative and anti-inflammatory status by activation Nrf2 signal pathway and then inhibition of the NLRP3 inflammasome activation.ConclusionOur findings demonstrated for the first time that brusatol has a significant in vitro anti-inflammatory activity and administration of brusatol could effectively attenuate colonic inflammation in DSS and TNBS induced UC model.The BR-SMEDDS was superior to the BR-suspension,SASP and AZA in treating experimental UC,with much smaller dosage.The enhanced anti-UC effect of brusatol might be intimately associated with the improved pharmacokinetic property of brusatol by SMEDDS.Taken together,SMEDDS provides new ideas and methods for enhancing drug efficacy,the research illuminated that the developed nano-delivery system might be a promising candidate for colitis treatment.