JOURNAL OF COSMETIC SCIENCE 176 promote healthy vision. M. cochinchinensis is a good source of carotenoid, particularly beta- carotene and lycopene, as well as vitamins A and E (2,3). Vitamins A, C, and E are acceptable cosmetic components because of their antioxi- dant effi cacies. The oil in Gac aril is a valuable source of carotenoids (4,5) as well as signifi cant amounts of vitamin E and omega-3 fatty acids (6). The antioxidant con- tent of Gac aril is 5- to 10-fold higher than that of other sources such as grapefruit, guava, and tomato (7). The carotenoid compounds in Gac exhibit substantial free radical scavenging ability. The antioxidant activity of alpha tocopherol in Gac may play an important role in ameliorating antioxidative DNA damage (8) induced by H2O2 and Ultraviolet light in the C spectrum (UVC) in TK6 and L929 cells, as de- termined by cytotoxic activity measurements via 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT assay) (9). Moreover, Gac extract maintains antioxidant activity after transformation processes such as powdering, drying, and freezing (10). Gac extract is safe for human and animal con- sumption as applied in the food, beverage, pharmaceutical, and cosmetics industries (11). This study aimed to determine the antioxidant activity and tyrosinase inhibition activity of Gac extract and the effi cacy of Gac cream in 22 subjects after 28 and 56 days of use. Whitening, moisturizing, and antiwrinkle effects were evaluated. The skin tolerance and safety of this cream were also evaluated. MATERIALS AND METHODS MATERIALS Gac extract was a supplied by Specialty Natural Products Co. Ltd. (Bangkok, Thailand). The Gac extract, which is extracted by ethanol and water, contained 825.30 mg/100 g lycopene, 385.0 mg/kg beta-carotene, and 61.1 mg/kg alpha-tocopherol. Ethyl acetate, n-hexane, ethanol, methanol, and trichloroacetic acid were supplied by Merck (Bangkok, Thailand) ascorbic acid was supplied by Finechem (Bangkok, Thailand) linoleic acid was supplied by Sigma-Aldrich (Nonthaburi, Thailand) and 2,2’-diphenyl-1-picrylhydrazyl (DPPH) was supplied by Sigma. Ferric chloride (FeCl3), potassium ferricyanide (K3[Fe(CN)6]), ammonium thiocyanate (NH4SCN), and ferrous chloride tetrahydrate (FeCl2·4H2O) were supplied by Fisher Scientifi c (Bangkok, Thailand). Sodium dihydrogen phosphate (NaH2PO4·2H2O) and disodium hydrogen phosphate (Na2HPO4·12H2O) were supplied by Bdh-Prolabo (Bangkok, Thailand). The measurement was conducted by supplier provided Gac extract. EXTRACT PREPARATION Using 80% ethanol and 20% water to absorb plant materials, and a drying process using a rotary evaporator in a vacuum can enhance greater effi cacy of plants (12). The red mem- brane surrounding the seeds (seed membrane) of the Gac fruit were also extracted in ethanol and water measured by supplier properly mixing the solvent with the Gac fruit using a blender. This extract was evaporated to a cream paste consistency using the rotary evaporator in a vacuum at 37–40oC. The extract was diluted in propylene glycol to obtain Gac extract.

CLINICAL EVALUATION OF GAC EXTRACT 177 METHODS IN VITRO BIOLOGICAL TESTS DPPH radical scavenging assay. The DPPH assay was performed according to the method developed by Prior et al. (13) using solutions of 1 mM L -ascorbic acid or 1 mM Trolox to supply vitamin C or E, respectively. Ten milliliters each of Gac extract and vitamin C or E solution were mixed with 190 ml of DPPH solution. The solution was mixed by stir- ring, covered with aluminum foil and incubated in the dark for 30 min at room tem- perature. The light absorbance of the solution was measured at 515 nm using a microplate reader and the results were expressed as ascorbic acid equivalents (mg AAE/mg sample) or Trolox equivalents (mg TEAC/mg sample). 2, 2’-Azinobis 3-ethylbenzothialine-6-sulfonic acid cation radical scavenging assay. The 2, 2’-azinobis 3-ethylbenzothialine-6-sulfonic acid (ABTS) scavenging assay and DPPH scavenging assay have been used to determine free radical scavenging activity (14). The method used in this study was based on that of Van den Berg et al. (15) as slightly modi- fi ed by Thaipong et al. (16) using ABTS and solutions of 1 mM L -ascorbic acid and 1 mM Trolox. Ten milliliters each of Gac extract and vitamin C or E solution were mixed with 190 mL of ABTS solution. The solution was mixed by stirring, covered with aluminum foil and incubated in the dark for 30 min at room temperature. The light absorbance of the solution at 734 nm was measured using a microplate reader and the results were ex- pressed as ascorbic acid equivalents (mg AAE/mg sample) or Trolox equivalents (mg TEAC/mg sample). Ferric reducing antioxidant power assay. The ferric reducing antioxidant power (FRAP) assay involves the reduction of a ferric tripyridyltriazine complex to its fer- rous form to produce color in the presence of antioxidants (16). The procedure was developed by Thaipong et al. (17) using an FRAP reagent. In the assay, 300 mM acetate buffer was mixed with 20 mM ferric chloride solution and 10 mM 2,4,6-tris(2-pyridyl)-1,3,5-triazine (TPTZ) solution. Gac extract, vitamin C, and vitamin E was individually mixed with FRAP reagent, covered with aluminum foil, and incubated for 15 min in the dark. The light absorbance of the solution at 593 nm was measured using a microplate reader, and the results were expressed as ascorbic acid equivalents (mg AAE/mg sample) and Trolox equivalents (mg TEAC/mg sample). Tyrosinase inhibition activity. A total of 40 ml of Gac extract dissolved in 0.1 M etha- nol was added to 80 ml of phosphate buffer (pH 6.8) and 40 ml of L -dopa. A 40-ml aliquot of 0.02 mg/ml tyrosinase enzyme solution was added, and the mixture was in- cubated for 20 min. The light absorbance of the resolution was measured at 475 nm, and the percentage of inhibition activity of the tyrosinase enzymes was calculated as inhibition (%) = [(Acontrol–Bsample)/Acontrol] × 100% (18), where A is the change in the absorbance of the control sample between incubation times of 0.5 and 1.0 min and B is the change in the absorbance of the test sample after the same incubation time (19). Each result is the mean of three concurrent readings. Kojic acid was used as a positive control. Cream preparation. In this study, 5% Gac extract was incorporated into an O/W emul- sion and glycerin as excipient and continuous stirring until dispersion was complete.