2.2. Solution preparation2.2.1. Curcumin–zein solution1.7 g zein was added to 100.0 ml 85% (v/v) aqueous ethanolsolution with magnetic stirring at 500 rpm (IKA R05) for 1 h, thencurcumin powder was added and the mixture was stirred continuouslyfor another hour.2.2.2. Pectin solutionPectin powder was dispersed in double distilled water at aconcentration of 0.1% (w/v), and stirred for 3 h, then filteredthrough filter paper (Fisher Science, P5) to remove any insolublecomponents.2.3. Preparation of curcumin loaded zein–pectin nanoparticles5.88 ml of curcumin–zein ethanol solution was rapidly droppedinto 25.00 ml of pH 4.0 adjusted water using a syringe with constantstirring at 900 rpm (IKA R05, USA). The resulting dispersionwas then stirred for another 5 min, and then the ethanol wasevaporated using a rotary evaporator (Rotavapor R110, BüchiCrop., Switzerland). Water (adjusted to pH 4.0) was added to compensatefor the lost ethanol, so that the final volume of the dispersionwas 25.00 ml. The dispersion was then filtered and pouredinto a 31.25 ml pectin solution with continuous stirring at600 rpm for 30 min.2.4. Nanoparticle characterization2.4.1. Particle size and zeta potential measurementsThe particle size distribution of the colloidal dispersions formedwas measured by dynamic light scattering (DLS), Nano-ZS (MalvernInstruments, Worcestershire, UK). The Z-average diameter andpolydispersity index (PDI) were calculated from the light scatteringmeasurements. The electrical characteristics (f-potential) of the particles in the colloidal dispersions were determined using amicro-electrophoresis device (Nano-ZS, Malvern Instruments,Worcestershire, UK). Samples were diluted with water (adjustedto pH 4.0) prior to measurements to avoid multiple scatteringeffects.2.4.2. Particle yield and curcumin loading efficiency (LE)Freshly prepared colloidal dispersions were centrifuged at3000 rpm for 10 min (Sorvall RC-6 Plus, Thermo ElectronCorporation, USA) to separate any large particles, and then thenanoparticles in the serum phase were freeze dried and weighed,and the curcumin content in the dried nanoparticles was determined.The particle yield and the curcumin loading efficiency werecalculated as follows:Particle yield ð%Þ ¼the weight of the freeze dried particlestotal weight of curcumin; zein and pectin 100%ð1ÞLoading efficiency ð%Þ ¼curcumin in nanoparticlestotal curcumin input 100% ð2Þ2.4.3. Curcumin loading determination10 mg of freeze-dried nanoparticles were solubilized in 10 mlDMSO and then stirred overnight in the dark. The resultingsolution was then centrifuged at 15,000 rpm for 30 min. Thesupernatant was diluted 10 times with DMSO, and the absorbanceat k435 nm was measured using a UV-spectrophotometer(Ultrospec 3000 Pro, Biochrom Ltd, England) to determine thecurcumin concentration on the basis of a calibration curve whichwas previously established using standard solutions (0–10 lg/mlfree curcumin in DMSO).