Development and stability studies of sunscreen cream formulations containing three photo-protective filters

2.3.1 Centrifugation tests

Centrifugal tests were performed for emulsions directly after preparation. Those tests were repeated after 1 day, 7 days, 14 days, 21 days, and 28 days of storage. They were performed at 5000 rpm and 25 °C for 10 min by placing 10 g of each sample in centrifugal tubes.

2.3.2 pH determination

The pH value of various emulsions stored at different conditions was determined using a digital pH Meter. The pH tests were repeated for multiple emulsions after 1 day, 3 days, 7 days, 14 days, 21 days, and 28 days of storage.

2.5.1 Aerobic plate count

Aerobic plate counts were determined by inoculating 0.1 ml of the homogenate sample onto triplicate sterile plates of prepoured and dried Standard Method Agar. Then, plates were incubated for 48 h at 35 °C (ISO NF- 21149, 2006). Duplicates of each dilution (1 ml) of neutralized and non neutralized samples were pour-plated using Standard Method Agar (Oxoid, Basingstoke, Hampshire, England) and incubated at 30 °C for 48 h. Plates containing 25–250 colonies were counted and the average number of CFU/g was calculated.

2.5.2 P. aeruginosa count

Pseudomonas aeruginosa were enumerated on Pseudomonas Agar Base (CM 559, Oxoid) supplemented with fucidin, cephaloridine and cetrimide, providing a selective medium for P. aeruginosa. Colonies were counted after two days of incubation at 25 °C (ISO NF- 22717, 2006).

2.5.3 S. aureus

Population of S. aureus was determined by standard plating methods (ISO NF- 22718, 2008). Colonies of Staphylococcus were selected, gram-stained, and observed for oxidase and catalase reactions to confirm their presence. All microbial counts were transformed into logarithms of the number of colony-forming units (log 10 CFU/g).

2.5.4 Yeast and mold counts

The method involved enumeration of colonies on the Sabouraud dextrose chloramphenicol agar medium. Enumeration was carried out as a pour plate, surface spread, or membrane filtration method (ISO NF- 16212, 2008). Microbiological tests were repeated for formulations at 25 °C after 0, 7, 14, 21 and 28 days of preparation.

3.1.1 Color

The findings revealed that the freshly prepared emulsions were pale yellow, soft yellowish white, yellowish white and white in color for F₁, F₂, F₃ and F₄. Little changes in color were observed for emulsions F₁, F₂ and F₃, as well as the end of storage period is characterized by the following colors: soft yellowish white, white and yellowish white (Tables 3 and 4). For example, for F₁, the change in color was observed from the 21st day. This change was presumably due to the oily phase separation promoted at higher temperature. Interestingly, no change in color was observed for F₄ at the different storage conditions: 8, 25 and 40 °C ± 2 °C, up to 28 days of observation.

3.1.2 Liquefaction

The viscosity of emulsion is often reported to play a vital role in its flow properties (Nasirideen et al., 1998). Starting from the emulsion preparation, the temperature and time processes begin to contribute to its separation, leading to a decrease in viscosity which results in liquefaction increase (Herbert et al., 1988). As far as the findings of the present study, no liquefaction was observed for the emulsions in any of the storage conditions under investigation, i.e., 8, 25 and 40 ± 2 °C during the 28 days of observation. The absence of liquefaction provided strong evidence for the stability of the emulsions under investigation.

3.1.3 Phase separation test

Creaming leads to phase separation and is often attributed to density differences between the two phases under the influence of gravity (Derick, 2000). The findings of this present work revealed that all the formulation samples were stable in all storage conditions, i.e., 8, 25 and 40 ± 2 °C during the 28 days of the observation period.

3.1.4 Centrifugation test

No phase separation was observed after centrifugation in any of the samples stored at different conditions up to 21 days. A weak phase separation was, however, recorded on the 21st day and up to the 28th day for preparation of F₁ and F₃ kept at 40 °C. No other phase separation was observed till the end of the experimental period. This was presumably due to the proper homogenization speed during emulsion formulation which might have prevented the breakage of the formulations during testing (Abdurahman and Rosli, 2006).

3.1.5 pH value determination

Monitoring the pH value is crucial for determining the emulsions’ stability. In fact, pH changes indicate the occurrence of chemical reactions that can give an idea on the quality of the final product. Furthermore, the most important parts of chemical stability are performances on accelerated testing and kinetics of pH profiles (Issa et al., 2000).

The pH of human skin normally ranges from 4.5 to 6.0. Therefore, in order for a formulation to possibly gain admission for industrial application, it should have a pH that is included into this range (Matousek et al., 2003). Emulsions formulated in this work had a pH value of 6.1, which is close to the neutral pH. Moreover, the pH of the various emulsion samples stored at various storage conditions, i.e. 8, 25 and 40 °C, were noted to undergo a continuous decrease up to one month of observation (data not shown). The emulsions had stable pH values for almost all conditions tested (data not shown). In the end of storage, at 40 °C, a statistically significant decrease in the pH of the emulsion was observed. The high temperature contributes to the destabilization of the emulsion by hydrolysis, but it did not affect the overall quality of emulsions because the pH values remained around pH 6.0, which is an acceptable and non skin irritating pH value.

3.3.1 Aerobic plate count

The log mean count recorded for the Aerobic plate count of samples on day 0 was about 2.01 log10 CFU/ g. On day 28 of storage, the log mean count of Aerobic plate count reached 4.33, 4.3, 3.7 and 3.33 for F₁, F₂, F₃ and F₄, respectively, which did not approximate the maximum limit of 6,9 log10 CFU/g for Aerobic plate count recommended by ISO NF- 21149 (2006) in processed cosmetics (Table 5).

3.3.2 P. aeruginosa and S. aureus counts

The results from the Pseudomonas and S. aureus detection tests were negative, thus confirming that all formulated emulsions met the conventional standards specified with regard to fitness for human consumption (ISO NF- 22717, 2006; ISO NF- 22718, 2008). (Table 5).

3.3.3 Yeast and mold counts

Yeast and molds have been tested in cosmetic products to assess microbiological safety and product quality during processing and storage (ISO NF- 16212, 2008). The levels of these microorganisms were noted to remain under the standard limit. In fact, the initial yeast and mold counts recorded for all treatments were under the detection limit (ISO NF- 16212, 2008).

Moreover, the yeast and mold count values recorded for the formulated sample F₄ were noted to show delayed proliferation when compared to F₁, F₂ and F₃ (Table 5).

In conclusion, the combination of 7% benzophenone-3, 7% ethylhexyl methoxycinnamate and 6% titanium dioxide seems to be very interesting since it preserved physicochemical properties of the product and was efficient against the growth of different spoilage microorganisms. It should be noted that the maximum authorized levels are 7.5% for -Ethylhexyl 4-methoxycinnamate are 7% for benzophenone-3 according to FDA (FDA, 1999) and 25% for titanium dioxide (Salvador and Chisvert, 2005) in the F₄ formulation.