Nanoformulations Of Triacylglycerol-bound Punicic Acid And Cuminaldehyde For Improved Bioavailability And Increased Antihepatotoxic Activities

Liver diseases are one of the major causes of morbidity and mortality in the world,and currently,hepatoprotective drugs obtained from natural products continue to provide an effective means of treating such disorders.The disease is predominantly caused by toxic chemicals,excessive alcohol consumption,infection and autoimmune disorders.Most of these hepatotoxic agents destroy liver cells mainly by inducing lipid peroxidation and excessive oxidative stress.Therefore,bioactive compounds with antioxidative and antiinflammatory properties are likely to prevent and treat the drug-induced liver injury(DILI).However,the antihepatotoxic drugs in the system are woefully inadequate in meeting the therapeutic demands of patients because of low efficacy,severe adverse effect and high cost associated with their use.Consequently,there is the urgent need to investigate other promising antihepatotoxic agents to improve the situation so that more lives can be saved.There are also several hepatoprotective compounds that have been isolated from natural products,but their therapeutic efficacy have been limited by poor aqueous solubility,low oral bioavailability and increased enzymatic degradation.In this regard,developing an appropriate nanoformulation for antihepatotoxic compounds with improved bioavailability and increased biological activities could be one of the innovative ways to increase the number of such drugs in clinical use.Herein,the model compounds used in the present study,viz.,macromolecule triacylglycerol-bound punicic acid(TPA)and small-molecule cuminaldehyde(CuA)are respectively obtained from pomegranate and cumin oils with remarkable pharmacological activities such as anticancer,antiinflammatory and antidiabetes.However,poor in vivo absorption of TPA and CuA,as well as low oral bioavailability due to rapid metabolism,chemical instability and high lipophilicity has adversely affected their wide clinical applications.Thus,the development of novel drug delivery systems to potentiate the bioactivity of these oils could be a step in the right direction.The present study therefore developed nanoformulations of TPA and CuA for improved bioavailability improvement and increased antihepatotoxic activities.This thesis comprises four chapters with three contributing areas as detailed below:Chapter 1 Review of natural-based antihepatotoxic components and appropriatedrug delivery systemsThis chapter provides a comprehensive perceptivity of natural-based antihepatotoxic components,videlicet TPA and CuA,as well as appropriate delivery systems of these components.The section also highlighted bioactivities of TPA and CuA including antioxidation and antiinflammatory properties with prospective management of drug-induced liver diseases.In order to justify the design of this study,the current research status of TPA and CuA in terms of source,extraction techniques,pharmacokinetic and the health-promoting properties,as well as limitations and future perspectives were discussed appropriately.Additionally,the prospects of enhancing the activities of the bioactive natural conpounds via improved bioavailability systems were also evaluated.The review of the literature thus provides a firm background to the present study.Chapter 2 Triacylglycerol-bound Puncic acid-loaded Lipid-based Formulations:Isolation,preparation,optimization and characterizationThis section aimed at optimizing the niosomal and liposomal formulation of TPA,viz.,TPA-loaded niosome(TPA-NS)and TPA nanoliposome(TPA-NL)using Box-Behnken design(BBD),central composite design(CCD)and response surface methodology(RSM)respectively for in-vitro evaluation.Through RSM-BBD tool,masses of span 80(70 mg),cholesterol(19.99 mg)and TPA(6.48 mg)were optimized via quadratic model to achieve desired characteristics of TPA-NS.Likewise,the RSM-CCD tool was also applied to optimize TPA-NL preparation with phospholipid:cholesterol ratio(w/w)of 6:1 and mass of TPA(7mg)selected as the optimal independent factors after the observed data were fitted to quadratic polynomial equation(Z = 83.35 + 2.30N + 2.14M-0.71NM + 3.016N2 + 0.92M2).The optimized TPA-NS formulation demonstrated discretely spherical morphology with larger z-average vesicle size(491.90+46.30 nm),reasonable polydispersity index(PDI,0.324±0.05)and higher zeta potential(z-potential,-38.58±0.21),as well as enhanced entrapment efficiency(EE,87.48±.4.79).However,the optimal TPA-NL was relatively spherical in shape and homogenously dispersed with smaller mean droplet size(123.54±1.92 nm),narrowed PDI(0.21±0.046),comparatively stable z-potential(-39.09±0.66 mV)and appreciable EE(85.77±1.05%).FTIR characterization of the two formulations showed that TPA was successfully entrapped in the niosomal and nanoliposomal vesicles.Additionally,TPA-NS and TPA-NL were relatively stable for the 1-month storage period and demonstrated remarkable in-vitro dissolution compared with the free TPA.Besides,the TPA-NS followed Weibull function with combination of anomalous and Fickian diffusion release mechanisms in the dissolution media.Collectively,the preliminary evidence suggested that conventional vesicle liposome and its look-alike niosome can be applied to incorporate large molecule TPA with improved in-vitro release.Chapter 3 Formulation optimization,characterization and in-vitro evaluation ofcumimaldehyde-loaded Lipid-derived nanocarriersIn this chapter,the development,optimization,characterization and in-vitro release studies of two novel formulations of CuA,namely CuA self-emulsified nanoemulsion(CuA-SEN)and Cremophor-EL-based CuA micelles(C-CuA-M)were investigated.Through BBD tool,the nanocarrmers(CuA-SEN and C-CuA-M)were successfully designed and developed.Homogenously CuA-SEN with acceptable characteristics(mean droplet size-48.83±1.06 nm;PDI-0.232 ±0.140;ZP--29.92 ± 1.66 mV;EE-91.51±0.44%;and drug-loading capacity(DL)-9.77±0.75%)was formulated using quadratic model.CuA-SEN was relatively stable with remarkable increased in-vitro drug release(IVDR)of CuA via Fickian diffusion mechanism.Meanwhile,experimental conditions of mass of CuA(0.41 g),mass of Cremophor-EL(0.60 g)and volume of water(5.0 mL)which translated into optimized C-CuA-M with relatively larger size dimension(55.14±3.79 nm),fairly broad PDI(0.366±0.015)and acceptable z-potential(-15.17±2.19 mV)were observed for C-CuA-M at a quadratic desirability function of 0.917.Besides,C-CuA-M had appropriate%EE(88.45±1.42%)and DL(8.43±0.86%)which was reasonably stable with slightly lower in-vitro release rate of CuA compared with CuA-SEN.Generally,the two developed formulations demonstrated promising physico-chemical characteristics coupled with improved stability and in-vitro drug release.Chapter 4:Improved oral bioavailability and increased antihepatotoxic activitiesof Triacylglycerol-bound Punicic acid and cuminaldehyde nanoformulationsThis section reports the successful improvement of oral bioavailabilities of TPA and CuA nanoformulations,viz..TPA-NS,TPA-NL,CuA-SEN and C-CuA-M respectively.The formulations with better physico-chemical characteristics and oral bioavailabilities were selected for antihepatotoxic activities evaluation in CCl4 treated mice.Comparatively,TPA-NL(164.14%)exhibited improved oral bioavailability in comparison with free TPA and TPA-NS(124.22%).More importantly,in vivo studies provided substantial evidence for the enhanced antihepatotoxic effects of TPA-NL(2 mg/kg/body weight)in CC14 induced hepatotoxicity via suppression of inflammation,reduction of oxidation-dernved stress,and restoration of liver functions.Taken together,the liposomal nanoformulation of TPA significantly improved the antihepatotoxic effects of TPA.Likewise,CuA-SEN showed marked increase in the plasma pharmacokinetics with enhanced relative oral bioavailability(171.45%)compared with the free CuA and C-CuA-M(132.48%).CuA-SEN(200 mg/kg/body weight)exhibited remarkable higher serum levels of GSH,SOD,CAT and TNF-?plus IL-6(serum and liver tissues)with reduced AST,ALT and MDA levels.Thus,improving bioavailability of CuA via SEN could be a promising tool for enhancing the antioxidation,antiinflammation and antihepatotoxicity of the drug.Generally,through appropriate formulations,the bioavailabilities of macromolecule TPA and small-molecule CuA could be improved for increased antihepatotoxic activities.