J. Cosmet. Sci., 69, 257–267 ( July/August 2018) 257 Stability Studies and Characterization of Glutathione-Loaded Nanoemulsion NAVEED ULLAH KHAN, ATIF ALI, HIRA KHAN, ZAHEER ULLAH KHAN, and ZIA AHMED, Department of Pharmaceutics, College of Pharmaceutical Sciences, Soochow University, Suzhou 215123, P. R. China (N.U.K.), Department of Pharmacy, COMSATS University Islamabad, Abbottabad Campus, Abbottabad 22060, Pakistan (A.A., Z.U.K.), Department of Pharmaceutical Sciences, Abbottabad University of Science and Technology, Havelian, Abbottabad 22500, Pakistan (H.K., Z.A.) Accepted for publication July 6, 2018. Synopsis Glutathione reduced (GSH) is the mother of all the antioxidants and has an antimelanogenic effect. It is extremely vulnerable to oxidation in the solution form which limits its use. The GSH in nano-oil droplets present a potential solution to this problem. The aim of this study was to formulate glutathione-loaded nanoemulsion and assess its stability studies over a 90-day testing period. To formulate GSH-loaded nanoemulsion pseudo- ternary phase diagram, it was built with various concentrations of water, liquid paraffi n oil, and surfactant mixture (Tween 80 and Span 80). The oily phase was prepared by dissolving the GSH (450 mg) in liquid paraffi n oil through stirring. High-energy homogenization was used to prepare the nanoemulsion. From preformulation stability studies of the 28-day testing period, nanoemulsion (NE-19) with oil and surfactant mixture ratio (1:1) of hydrophilic lipophilic balance (HLB) value 10 was selected. The samples of NE-19 and its respective base (B-19) were kept at four different storage conditions for a period of 90 days and evaluated for physical characteristics, droplet size and distribution analysis, zeta potential analysis, electrical conductivity, mobility, polydispersity, pH, phase separation, and fl ow analysis at different time intervals. Glutathione in nano-oil droplets with nonionic surfactants produced oil-in-water nanoemulsions that were thermodynamically stable over the 90-day testing period at different storage conditions. NE-19 was formulated having non-Newtonian fl ow and pseudo-plastic behavior. pH was found in the range of 5–6. Polydispersity was less than 0.3. The droplet size of fresh nanoemulsion was 96.05 nm, whereas the zeta potential was -37.1. Mobility and electrical conductivity were -2.726 μm cm/Vs and 0.0141 mS/cm, respectively. Glutathione-loaded nanoemulsions have excellent stability, promising the solution in nano-oil droplets and are suggested for in-vivo release studies for oxidative skin related diseases. INTRODUCTION In contemporary eras, extensive and continuous development in consumer claim in the fi eld of cosmetics has spurred the development of sophisticated formulations, aiming at high performance, attractive appearance, sensorial benefi t, and safety. Although with increasing Address all correspondence to Atif Ali at ajmaline2000@gmail.com or atifali@ciit.net.pk.

JOURNAL OF COSMETIC SCIENCE 258 demand from consumers, the formulators have certain problems regarding the optimum equilibrium between active compound concentration and the formulation base for skin structure regarding the ideal penetration of the active compound into the natural skin barrier (1). Glutathione is a resilient antioxidant often called the mother of all the antioxidants. It is a tripeptide antioxidant based on three amino acids cysteine, glycine, and glutamine (2,3). Glutathione transpires in both reduced (GSH) and oxidized (GSSG) states. The GSH is a biologically active sulfhydryl group which allows for interactions with a variety of biochemical systems. It is the most crucial molecule needed to stay healthy and prevents diseases. Apart from its several natural purposes, it is thought to be a contributory in generating skin lightening because of its tyrosinase-inhibitory activity (2). Many lightening compounds are melanocyte-toxic (2). These melanocyte-toxic compounds are oxidized in the cell to harvest extremely deleterious intermediates such as quinones. These quinones retract melanocytes, ultimately triggering spanking of pigment. But GSH is purported to shield the melanocytes from oxidation through its antioxidant pro- tective effects (4,5). GSH is extremely exposed to oxidation in the solution form. GSH is hydrolyzed by intestinal and hepatic gamma-glutamyl transferase, resulting in abridged bioavailability when directed orally. Most of the absorbed GSH sustains within the gut luminal cells and could be found in the general circulation (6). So, it produces pronounced stability and absorption complications (7) which restrain their persistence for new formu- lations. The submicron or nano-size of active molecules is enough to cross the skin barriers during penetration into the skin (8,9). The drug molecules sized in nanometer range offer some exclusive features which can lead to sustained circulation, upgraded drug localiza- tion, enhanced drug effi cacy, etc., and by means of the variety of dosage forms these better performances can be accomplished. Chemically unstable drugs can be supplied to the skin by means of nanosystems (10). A nanoemulsion can resolve these issues because it has the ability to protect GSH as the GSH will be in the oil globule of a O/W nanoemulsion. It has been shown that the smaller the particle size the greater the absorption into skin Stratum corneum (1). The S. corneum is the fi rst-line barrier of the skin because of its lipo- philicity and high cohesion between cells (11). Hence, good stability and penetration effi cacy could be attained. That is why a GSH-loaded nanoemulsion was formulated to achieve the desired stability. MATERIALS AND METHODS MATERIALS Glutathione reduced 98% (GSH) was purchased from Acros organics (Fair Lawn, NJ). Liquid paraffi n oil was purchased from Merck (KGaA, Darmstadt, Germany). Polyethyl- ene glycol sorbitan monooleate (Tween 80) and sorbitan monooleate (Span 80) were pur- chased from Merck. All other chemicals were of analytical grades. METHODS A modifi ed method was adopted to prepare the GSH-loaded nanoemulsion (1). Surfactant mixture (Smix) was dissolved in distilled water with continuous agitation to prepare a