30 JOURNAL OF COSMETIC SCIENCE SKIN HYDRATION TEST Skin surface hydration state was assessed by a skin capacitance-based instrument (cor- neometer CM 820, Courage & Khazaka GmbH). The measurements by the interdigital electrode indicate changes in skin capacitance due to variations in the moisture content of the stratum corneum (SC). Twenty female volunteers, aged between 22 and 63, applied the tested compositions to three different places on the lower arm. The mean value of the three obtained measurements was calculated. The measurements were taken immediately after application, after 8 hours, and after 12 hours. Untreated skin of the contralateral lower arm was used as a control. Subjects were acclimatized to an ambient temperature of 22øC and relative humidity of 60% for 45 minutes prior to the mea- surements. The recorded capacitance values were converted into arbitrary hydration units varying from 0-120 rcu (relative corneometer units) (16,17). BIOSORPTION EXPERIMENTS A series of incubations with 1 g of dry, crushed Dunaliella powder and 25 ml of Dead Sea concentrated brine (Mineral Skin OsmoterZM) was carried out in various combina- tions of brine pH, temperature, and exposure times. Algal biomass and brine were mixed and incubated in Erlenmeyer flasks, and mixed with an orbital shaker (EnvironShaker 3328 type, Lab-Line) or with a magnetic lab stirrer. The ratio of mineral biosorption to algal biomass was evaluated in correlation with the pH of the brine, temperature of the solution, and exposure time. After incubation, the brine was separated from the biomass by centrifugation (Sorval RC-58 centrifuge, 10 min, 24,000 g). Non-sorbed salt residues were removed from the algae by two consecutive washing cycles, each of 150 ml distilled water. The algal precipitate was lyophilized, and the dried biomass was digested overnight in 4% nitric acid. The concentrations of acid-soluble calcium, magnesium, and potassium in the biomass were measured by atomic absorption (Varian, type Spectra 10 AA). DESORPTION EXPERIMENTS Dunaliella salina biomass was saturated with minerals from Dead Sea Mineral Skin OsmoterZM brine under the optimal conditions found previously: pH 5-5.5, 15-minute exposure time, 33øC. The saturated biomass was washed twice with distilled water, and the slurry was kept at 4øC. Portions of the biomass were introduced into distilled water in a ratio of 0.5% dry weight to water, and exposed to an experimental design matrix of parameters. Design-Ease TM software was used for the experimental design and analysis (Stat-Ease Ltd. 1992, Minneapolis). Residual levels of minerals in the dry algal biomass were evaluated by atomic abosption. RESULTS SKIN ROUGHNESS EVALUATION The surface roughness of the skin was evaluated quantitatively by mean, standard

MINERAL-ALGAL-BOTANICAL COMPLEX 31 deviation, and minimum and maximum values of the Rz parameter, as observed in the laser profilometric test. The results are shown in Table II. The improvement in the skin roughness was defined as the difference in the Rz value between two periodic measurements over four weeks, compared to the initial Rz value. In both treatments, the observed improvements were found to be significant (p 0.001). Wilcoxon matched pairs, signed rank, tests were used, comparing Rz values before and after each treatment. It was observed that the mean Rz value was reduced by 43.2% after treatment with the cream enriched with 5 % Triple D Complex TM, while after treatment with the control cream, the mean Rz value was reduced by only 24.9%. The difference between the two treatments was significant, as analyzed by Kruskal-Wallis one-way non-parametric ANOVA (p 0.001). This significant improvement in the roughness of the skin after four weeks of applications is illustrated in Figure 1. SKIN HYDRATION ANALYSIS The SC hydration state was determined by the results of corneometeric analysis of 20 female volunteers treated with cream enriched with 5% Triple D Complex TM and compared to the results of the vehicle cream control. The results are shown in Table III. Small non-significant changes were observed between measurements of untreated skin control areas (data not shown). Wilcoxon matched pairs, signed rank, tests were used to examine changes in values, before and after each treatment. Application of cream with 5 % Triple D Complex TM had shown a significant improvement of 10.5% after 8 hours and 6.4% after 12 hours (p 0.001). The control treatment had shown a significant improvement of 7.9% after 8 hours and 5.1% after 12 hours (p 0.001). The difference between the two treatments was analyzed by Kruskal-Wallis one-way non-parametric ANOVA and was found to be significant (p 0.001 for 8-hour mea- surement, p 0.05 for 12-hour measurement). The results are presented in Figure 2. THE INFLUENCE OF VARIOUS PARAMETERS ON THE PASSIVE MINERAL BIOSORPTION TO THE DUNALIELLA SALINA BIOMASS The ratio of mineral biosorption to algal biomass was evaluated according to changes in the pH of the Mineral Skin Osmoter TM, temperature of the solution, and exposure time. When exposed to Dead Sea concentrated brine (Mineral Skin OsmoterTM), Dunaliella sali,a dry biomass became enriched with calcium and magnesium, with a preference for Table II Rz Values of Skin Treatments With Basic Cream (control) and the Same Cream Enriched With 5 % Triple D Complex TM, as Observed by Computer-Aided Laser Profilometric Analysis, Lasting 4 Weeks Variable Mean SD Minimum Maximum N Control Rz before 235.22 57.10 166.20 393.10 20 Rz after 175.81 40.65 122.70 278.00 20 Cream + 5% Triple D Complex TM Rz before 231.53 43.12 137.60 339.20 20 Rz after 131.18 25.14 75.90 174.10 20