GLUTATHIONE-LOADED NANOEMULSION 263 no phase separation or inversion, cracking, creaming, or coalescence after these stress tests were selected for further storage stability studies. Physical characteristics based on visual observations confi rm that whether the formula- tion is physically stable or not (16). The color of the nanoemulsion was milky white, this was because of preparation temperature change, i.e., emulsifi cation at 25°C. With the increase of preparation temperature from 20 to 70°C, the nanoemulsion tends to develop transparency because of the emulsion droplet diameter decreasing from 10.3 μm to 51 nm, proving the formation of nanoemulsions (19). There was no change in the milky white color, no liquefaction, and no phase separation in NE-19 and B-19 on storage at 4, 25, 40, and 40°C + 75% RH over the 90-d testing period. This shows the stability of NE-19. The stability against Ostwald ripening is outstanding because of the extremely low solu- bility of the paraffi n oil in the continuous (19,20), whereas absence of liquefaction pro- vides strong evidence of nanoemulsion stability (21). The absence of liquefaction might be because of the adopted method of high-pressure homogenization. Homogenization decreases liquefaction (22). There was no phase separation in any NE-19 and B-19 samples Figure 4. Rheogra m of B-19 and NE-19 at 25°C. Figure 3. Drople t size distribution and intensity distribution data of freshly prepared NE-19.

JOURNAL OF COSMETIC SCIENCE 264 at intervals 0 h, 24 h, 48 h, and 72 h, and on days 7, 14, 21, 28, 45, 60, and 90. There was no phase separation in any NE-19 and B-19. However, phase separation occurred in the NE-19 and base stored at 40 and 40°C + 75% RH at the 90th day. This may have occurred because of Ostwald ripening in which molecules move as a monomer, and the formation of larger droplets occurred because of the coalescence of small droplets by dif- fusion processes driven by the gain in surface free energy (6). In most colloidal dispersions, the sizes of dispersed structures and the density difference between the continuous and dispersed phase are small enough that thermal energy can keep the colloids from sedimentation under gravity for an extended period of time (21). Turbidity is haziness or cloudiness or disturbance of a liquid caused by a large number of factors (droplet size, etc.) and the measurement of turbidity is a key test for stability (23). The turbidity of NE-19 and B-19 was checked visually at 4, 25, 40, and 40°C + 75% RH and there was no turbidity seen in them. The variation of the turbidity of a sample as a function of time depends on the fl occulation rate. The turbidity of a nanoemulsion results from the contributions of conventional aggregates, bigger drops, and mixed aggregates (24). pH is the count of total ions present in a formulation. Stable formulations afford very little change in pH. The pH of the skin is normally considered 3–7 (25). The pH of NE-19 tallies with skin pH and there is a little decrease in the pH of NE-19. At the start of the stability study, the pH of fresh samples of NE-19 and B-19 were 5.79 and 5.92. At the end of 90 days of the stability studies, the pH were 4.53, 4.47, 4.43, and 4.37 and 4.41, 4.35, 4.48, and 4.31 at 4, 25, 40, and 40°C + 75% RH, respectively. By applying two-way ANOVA, the change in pH of NE-19 and B-19 was found to be insignifi cant with respect to time. The pH values of both nanoemulsions, which were unchanged could be because of the stability of the ingredients in the formulation. Thus, this indicated that there was no degradation or ionization of chemicals in the formulation at storage conditions during the testing period. However, because the particle size and the pH value did not signifi - cantly change across different conditions, we considered our nanoemulsion to be stable. Changes in the electrical conductivity can imply nanoemulsion variability and may fl uc- tuate the nanoemulsion droplet size (26). In these studies, the increase in electrical conduc- tivity was minor and prediction of emulsion stability in this way was diffi cult because the relationship between an increase in electrical conductivity and emulsion instability is not linear. Thus, we could not conclusively determine the nanoemulsion’s stability by this parameter. Similar results are reported by Bernardi et al. (27). Physical stability of nanoemulsions throughout their life is very important, and no or min- imal changes in the particle size distribution are necessary. The nanoemulsion stability is strictly related to the nanodroplet size distribution. A large droplet size may enhance Ostwald ripening which causes the increase in droplet size and can lead to creaming and coalescence. A fast droplet size increase indicates low system stability (24). The mean droplet sizes recorded for fresh NE-19, after 30, 60, and 90 days were 96.05 nm, 155, 181.3, and 190, respectively. The range of the droplet size of all formulations should be between 30 and 500 nm (28). There was no large increase in the droplet size of NE-19. The nanoemulsions had polydispersity index values less than 0.3 throughout the 90-d testing period, indicating the high fi delity of the system (low polydispersity), which may refl ect the overall stability of this formulation and synthesis method. Polydispersity val- ues near 1.0 are indicative of a polydisperse system (27). The long-term stability of nano- emulsions was evaluated and was also verifi ed by stability studies conducted over 3 mo.