**8. Formulation of nanoparticles**

An o/w nanoemulsion of gallic acid and quercetin was prepared by using a GMO/chitosan framework as reported by with slight modifications. Briefly, isolated gallic acid (100 mg) and quercetin (100 mg) were dissolved in molten GMO (2 g), then add 12.5 ml of 0.1% poloxamer 407 sonicated at 18 W for 3 min in probe sonicator. To this emulsion, dropwise 12.5 ml of 2.4% chitosan solution was added again using probe sonicator at 16 W for 4 min [40, 41]. Finally this phase was subjected to twelve cycles of HPH at 15,000 psi to give the nanoemulsion. Then, lyophilized with 2% mannitol as a cryoprotectant for 48 hr. Central composite design [42] was applied to examine the combined effect two variables, each at 2 levels and the possible 9 combinations of CS nanoparticles.
