PREPARATION AND EVALUATION OF CREAM MASK 455 that of glycerin. Glycerin exists in the skin’s outer layer, the stratum corneum, as a natural endogenous humectant and it is also a cosmetic ingredient regularly used as a hygroscopic and humectant agent (36). In this study, glycerin at 10% showed greater than 50% of moisture retention activities (Figure 1B). Similar but lower trends were observed in sea- weed extracts, and these values were signifi cantly different across species. Among tested seaweed extracts, U. reticulata showed the lowest moisture retention activity (Figure 1B). S. crassifolium, K. alvarezii, and a mixture of four seaweeds exhibited the strongest moisture retention capacities as compared with water. Ten percent solution of S. crassifolium, K. alvarezii, and a mixture of four seaweeds had values of moisture retention activities at 39.8%, 43.7%, and 40.2%, respectively (Figure 1B). It is clears that S. crassifolium, K. alvarezii, and the mixture of four seaweeds can be used for dry skin masks to deliver moisturizers to the skin. Inhibitory effects of seaweed extracts on mushroom tyrosinase activity. Melanin is a major factor to determine skin color and plays important roles in the prevention of sun-induced skin injury (37). In this study, the inhibition of tyrosinase activity with various concentrations of seaweed extracts was quantifi ed using arbutin as a control. As shown in Figure 1C, arbutin exhibited the best inhibitory effect on mushroom tyrosinase activity compared with control. Among tested seaweed extracts, S. crassifolium, K. alvarezii, and the mixture of extracts inhibited mushroom tyrosinase activity in dose-dependent manner. The extract of S. crassifolium, K. alvarezii, and the mixture showed stronger inhibitory effect on tyrosinase activity than those of U. reticulata and C. lentillifera. S. crassifolium and K. alvarezii at con- centrations from 0.5 to 1 mg/mL reduced DOPA oxidase activity by 9.1–13.7% and 12.6–20.1%, respectively. At concentrations of 0.5 and 1 mg/mL, the mixture of seaweed extracts reduced DOPA oxidase activity by 4.7% and 9.6%. Our data showed similar results with Chang and Teo (38) and Chan et al. (39). Antibacterial activity of seaweed extracts. Preservative-free cosmetics and antimicrobial plant/ seaweed extracts have attracted attention because of its ability to reduce the risk of allergies connected to synthetic preservatives. In this study, four selected seaweed extracts were examined for their antibacterial activity against three Gram-positive bacteria (E. faecalis, S. aureus, and B. cereus), three Gram-negative bacteria (E. coli, P. aeruginosa, and S. enterica), and one fungus (Candida albicans). As shown in Table III, the MIC of seaweed extracts was varied according to species. The MIC values ranged from 2.5 to 10 mg/mL. The U. reticulata extract was the most effective against E. faecalis, S. aureus, B. cereus, P. aeruginosa, and S. enterica (MIC: 2.5–3 mg/mL), whereas it inhibited E. coli and C. albicans growths at MIC of 10 mg/mL. K. alvarezii was strongly inhibited E. faecalis, B. cereus, and C. albicans (MIC: Table III MIC (mg/mL) of Seaweed Extracts Species Gram-positive bacteria Gram-negative bacteria Yeast E. faecalis S. aureus B. cereus E. coli P. aeruginosa S. enterica C. albicans U. reticulata 2.5 3 3 10 3 2.5 10 K. alvarezii 3 5 3 10 10 10 3 S. crassifolium 3 2.5 2.5 2.5 5 5 2.5 C. lentillifera 10 10 10 10 10 10 10 Mixture 10 5 10 5 10 5 10 Streptomycin (μg/mL) 256 256 128 32 256 128 — Cycloheximide (μg/mL) — — — — — — 32

JOURNAL OF COSMETIC SCIENCE 456 3 mg/mL), whereas S. aureus was highly inhibited by K. alvarezii with a MIC of 5 mg/mL. This result concurred with a previous report described by Prasad et al. (40) that Gram-positive bacteria were more susceptible to crude Kappaphycus sp. extract than Gram-negative bacteria. Both Gram-positive and Gram-negative bacteria were more in- hibited with S. crassifolium extract, and the MIC values ranged from 2.5 to 5 mg/mL. This result was similar to the study of Tajbakhsh et al. (41), in which MIC value of Sargassum oligocystum extracts was ranging from 3 to 10 mg/mL. C. lentillifera and a mixture of sea- weed extracts in our study also showed the inhibition of bacteria and fungus growths, and the MIC values were 5–10 mg/mL. Taken together, it can be concluded that the extracts of all tested seaweed species and mixture of extracts showed antibacterial activity against bacteria and fungus and can be used as ingredients for the development of cosmetic products. Biological active components in seaweed extracts. It is reported that seaweed is widely used in numerous cosmetic products in treatment of skin problems, such as aging, dryness, tanning, and pigment disorders. Steroids, polysaccharides, polyphenols, carotenoids, and vitamins were considered as the components that contribute to the health benefi cial properties of seaweeds (1). We, therefore, identifi ed active compounds in the seaweed extracts. Results showed that water extracts of selected seaweeds were rich in polysaccharide including carrageenan in K. alvarezii (46% of extract), aginate in S. crassifolium (53% of extract), and ulvan in U. reticulata and C. lentillifera (46% and 18% of extracts). In addition, we identi- fi ed higher amount of fucoxanthin in S. crassifolium (25 μg/g extract). Because selected seaweed extracts had antibacterial, cell proliferation, moisture retention, and tyrosinase inhibitory activities, we further identifi ed active compounds responsible for these activa- tions. Here, we demonstrated that carrageenan and alginate at concentration of 100 μg/mL had cell proliferation, moisture retention, and tyrosinase inhibitory activities, ulvan at a concentration of 100 μg/mL had moisture retention activity, and fucoxanthin at a concen- tration of 50 μM had tyrosinase inhibitory activities (Figure 2). Taken together, we suggested that carrageenan, aginate, ulvan, and fucoxanthin are responsible for seaweed extract bioactivities. EVALUATION OF CREAM MASK FORMULATION CONTAINING MIXTURE OF U. RETICULATA, S. CRASSIFOLIUM, K. ALVAREZII, AND C. LENTILLIFERA EXTRACTS It is reported that a mixture of ingredients in cosmetics usually increases benefi cial ac- tivities, and together with bioactivity results aforementioned, we formulated seaweed cream mask (SCM) containing 5 mg/mL of mixture of four seaweed extracts, U. reticulata, S. crassifolium, K. alvarezii, and C. lentillifera. According to Asian guidelines for the safety assessment of a cosmetic product, the deter- minant step in the development of a cosmetic formulation involves stability study, with the objective of predicting physicochemical and microbiological alterations that may occur since its manufacturing, until the end of its expiration date. Besides, this study al- lows the evaluation of the cosmetic product performance, safety, and effi cacy and contrib- utes for its development time reduction which is highly required by the market and the consumers. For the purpose of commercial applications, the physicochemical and micro- biological properties of the cream mask product containing the mixture of seaweed ex- tracts were evaluated (Table IV). As shown in Table IV, the color of SCM changed from white to yellowish brown with a specifi c odor of seaweed. All cream masks were stable and homogeneous for up to 12 mo of storage. With the addition of mixture of seaweed