Methods: The nanoemulsion was prepared by using high and low energy emulsification technique. D-optimal mixture experimental design was generated as a tool for optimizing the composition of nanoemulsions suitable for topical delivery systems. Effects of formulation variables including KMO (2.0%-10.0% w/w), mixture of castor oil (CO):lemon essential oil (LO; 9:1) (1.0%-5.0% w/w), Tween 80 (1.0%-4.0% w/w), xanthan gum (0.5%-1.5% w/w), and deionized water (78.8%-94.8% w/w), on droplet size as a response were determined.

Results: Analysis of variance showed that the fitness of the quadratic polynomial fits the experimental data with F-value (2,479.87), a low P-value (P<0.0001), and a nonsignificant lack of fit. The optimized formulation of KMO-enriched nanoemulsion with desirable criteria was KMO (10.0% w/w), Tween 80 (3.19% w/w), CO:LO (3.74% w/w), xanthan gum (0.70% w/w), and deionized water (81.68% w/w). This optimum formulation showed good agreement between the actual droplet size (110.01 nm) and the predicted droplet size (111.73 nm) with a residual standard error <2.0%. The optimized formulation with pH values (6.28) showed high conductivity (1,492.00 µScm-1) and remained stable under accelerated stability study during storage at 4°C, 25°C, and 45°C for 90 days, centrifugal force as well as freeze-thaw cycles. Rheology measurement justified that the optimized formulation was more elastic (shear thinning and pseudo-plastic properties) rather than demonstrating viscous characteristics. In vitro cytotoxicity of the optimized KMO formulation and KMO oil showed that IC50 (50% inhibition of cell viability) value was >100 µg/mL.

METHOD: A prospective, randomized, double-blind and placebo-controlled study was performed in RTI-hospitalized children. Patients were randomized to either the probiotic (n = 60, mean age 13.81 ± 0.90 months) or placebo (n = 60, mean age 12.11 ± 0.73 months) which were administered upon admission, continued during hospitalization and 4-week post-discharged. RTI and gut health parameters were assessed at these time points using validated questionnaires while concentrations of inflammatory cytokines were assessed via oral swabs.