DLS,ZETA,FT-IR

Citrus lemons purchased in autumn, winter, spring, and summer were manually juiced. The juice was filtered through filter paper and a 100 μm nylon mesh. The sample underwent sequential centrifugation at 4°C: 2000 × g for 10 min, 6000 × g for 20 min, and 10000 × g for 40 min. The supernatant was filtered through a 0.45 μm syringe filter. PEG6000 was added to a final concentration of 10% (w/v), and the mixture was incubated overnight at 4°C. The next day, samples were centrifuged at 10000 × g for 30 min. The precipitate was resuspended in PBS and stored at -80°C.Samples of nanovesicles were diluted 1:7 in deionized water to measure the zeta potential. A zeta compact analyzer (CAD Instruments, France) was then used to measure the samples' surface charge at 25°C. Three separate measurements were made for each sample, and the average value was recorded. This method ensures accurate assessment of the nanovesicles' surface charge and colloidal stability . To analyze the nanovesicle size, the DLS technique was employed. Initially, the nanovesicle containing solution was diluted with physiological saline (NaCl) in a 1:2 ratio. Subsequently, a small volume of the diluted sample was deposited onto the particle size analyzer (VASCO, Cordouan Technologies, France). The analysis was performed at room temperature (25°C) and with three repetitions. Particle size distribution and average hydrodynamic diameter of nanovesicles were calculated and reported by the device software. This method allows precise measurement of particles in the nanometer range.FT-IR spectroscopy was conducted using a Thermo nicolet series 112 FT-IR spectrometers (USA) to identify the functional groups present in the extracted nanovesicles. The spectra were recorded in the wavenumber range of 400 to 4000 cm⁻¹ . Potassium bromide (KBr) pellets containing the dried nanovesicles were prepared and placed in the sample holder of the FT-IR spectrometer. Infrared light was passed through the pellets, and the absorption spectra corresponding to the characteristic functional groups in the nanovesicles were obtained and analyzed.