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Evaluation of mucosal permeation of stigmasterol from the glutaraldehyde cross linked chitosan microspheres at increasing experimental temperatures was performed. The activation energy of permeation, partition, and diffusion were estimated to understand the permeation kinetic with respect to the temperature. The formulation depicting least activation energy possessed the increased permeation thresholds of drug at the site of application. The encapsulation efficacy and mucoadhesive strength were found to be directly proportional to the polymer‐emulsifier ratio. Decreased intensity in crystallography directed the molecular dispersion of microencapsulated drug. The depleted enthalpic phase transition in thermogram affirmed the stigmasterol encapsulation. The sphericity and the size of microspheres were determined by scanning electron photo micrograph. The in vivo quantification of oral Candida infection with different statistical approach and histopathological observation of infected tongue of mice on treatment with the stigmasterol encapsulated microspheres showed significant anti oral candidiasis activity by reduction of fungal colony count and recovery of papillae, reorganization of basal cell layer and newly formed papillae during 21–28 days of treatment.

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