ANTIOXIDANT ACTIVITY OF EGT 19 EXPERIMENT PROCEDURES REAGENT AND CELL CULTURE Dulbecco's modified Eagles medium (DMEM) was purchased from Nikken Bio Medical Laboratory (Kyoto, Japan). Fetal calf serum (FCS), TRizol reagent, and the Super Script first-strand system for RT-PCR were purchased from Invitrogen (Carlsbad, California). Alloxan, hematoporphyrin, hypoxanthine, nitroblue tetrazolium, rose bengal, tyrpsin, tyrpsin inhibitor, 2,2,6,6-tetramaethyl-piperidone hydrochloride (TMPD), and xanthine oxidase, were purchased from Sigma-Aldrich Chemical Company (St. Louis, MO). Takara Tag was purchased from Takara shuzou (Shiga, Japan). FITC-labeled collagen type I was purchased from Yagai Co. Ltd. (Yamagata, Japan). All other chemicals used were of the purest grade commercially available. The normal human fibroblasts were purchased from Kurabo Industries Ltd. (Osaka, Japan), and these cells were maintained with DMEM with 5% FCS. Cells were grown in a humidified incubator at 37°C under a 5% CO 2 atmosphere. SCAVENGING ABILITY AGAINST •02 - Superoxide was generated using a hypoxanthine/xanthine oxidase system. EGT or a blank was added to 1 ml of a superoxide buffer consisting of 50 mM KH2PO4 (pH 7.4), 1 mM NaEDTA, 1 mM hypoxanthine, and 100 mM nitroblue tetrazolium. Xanthine oxidase (0.33 units) was added, the sample was held at 25°C, and the OD 5600m was read at 10 min. The scavenging ability was calculated as the percent difference between the antioxidant sample and control. Alloxan was also used to generate superoxide. Alloxan forms a redox cycle with its reduced form, dialuric acid, and generates superoxides (23). It was prepared fresh at 100 mM in phosphate-buffered saline with 1 % sodium citrate, diluted, and used immedi­ ately. Phosphatidylcholine liposomes in phosphate-buffered saline with 1 % sodium citrate were mixed with the 50-mM alloxan solution in the presence of various antioxi­ dants, all at 20 µM. Oxidation products in the liposomes were assayed after 60 minutes using the K-Assay LPO-CC lipid peroxide kit from Kamiya Biochemicals (Seattle, WA). QUENCHING ACTIVITY AGAINST 1 02 The 1 0 2 quenching activity of EGT was estimated both by ESR-spin trapping (24) and lipid peroxidation using liposomes. TMPD was used as a 1 O 2 -trapping reagent. Various concentrations of the compounds were added to a solution containing 0.05 mM hema­ toporphyrin and 50 mM TMPD in 100 mM Tris-HCI buffer (pH 8.0). The resulting solutions were transferred into an ESR quartz flat cell and set in the cavity of the ESR. After UV A irradiation (0.65 J/cm2 , 1 Kw Xenon lamp USHIO Inc.) filtered with a YV-3 3 filter (Toshiba Glass Co.), the ESR spectra were recorded with a field modulation frequency of 100 kHz and a modulation amplitude of 0.1 mT at an output power of 5 mW. Mn2 + doped in MnO was used as a standard. All experiments were carried out at 21 °C. Singlet oxygen was also generated by mixing phosphatidylcholine liposomes in a 10-mM sodium phosphate buffer (pH 7.4) with 10 µM rose bengal, with and without antioxi-

20 JOURNAL OF COSMETIC SCIENCE dant, in a cuvette, and irradiating it on ice with a Sylvania 150 W slide projector spot tungsten filament two inches from the lens for 60 minutes. Oxidation products in the liposomes were assayed using the K-Assay™ LPO-CC lipid peroxide kit from Kamiya Biochemicals (Seattle, WA). TNF-a EXPRESSION OF FIBROBLASTS AFTER UVB IRRADIATION The fibroblast line XPTNF2 is an SV 40-transformed fibroblast line deficient in DNA repair and carrying the TNF-a promoter chloramphenicol acetyltransferase (CAT) re­ porter gene (25). The assay of TNF-a promoter activity using fluorescent chloramphen­ icol was as described. The assays were in duplicate, and the background was subtracted and the results averaged. MMP-1 OF HUMAN FIBROBLASTS AFTER UV A IRRADIATION MMP-1 activity. Fibroblasts were seeded at a density of 2 x 104 cells per well in a 96-well plate. After one day, cells were exposed to 20 mJ/cm2 UVA in Hank's buffer (Ca, Mg free) containing various concentrations of EGT. Following UVA irradiation, cells were cultured with DMEM-supplemented 0.1 % BSA for 24 h, and the culture medium was assayed for MMP-1 activity. MMP-1 is secreted into the medium in inactive from, and so it is necessary to convert it to the active form using a proteinase such as trypsin. The medium (0.05 ml) was collected, and 0.01 ml of trypsin was added, followed by 15-min incubation at 3 7 ° C. The resulting solution was incubated with FITC-labeled collagen type I, which is a substrate for MMP-1, for 2 h at 37°C. A 40-mM o-phenanthrolin was added and incubated for 30 min at 37°C. The degraded collagen was extracted with 0.05 ml of 0.17 M Tris-HCI (pH 9.5) containing 70% EtOH, and centrifuged for 15 min at 12000 rpm. The MMP-1 activity was estimated by measuring the fluorescence intensity of the FITC-peptide cleaved by MMP-1 (excitation: 495 nm emission: 520 nm). MMP-1 activity was expressed as type I collagen degraded per minute (unit/min/µg collagen). RT-PCR. Fibroblasts were seeded at a density of 1 x 108 cells per 35-cm dish. After 1-day culture, cells were applied to Hank's buffer (Ca, Mg free) containing various concentrations of EGT and then exposed to 20 mJ/cm2 UV A light (Toshiba FL-20S BLB). Following UVA irradiation, cells were cultured in DMEM-supplemented 0.1 % BSA for 24 h, and then cells were submitted for the determination of the MMP-1 mRN A expression level. Total RNA from the cells was extracted with TRizol reagent according to the manu­ facturer's protocol. The first-strand cDNA synthesis containing 1.5 µg of total RNA was primed with oligo(dT) and M-MLV reverse transcriptase. Primers used for PCR ampli­ fication of MMP-1 and the glycerolaldehyde-3-phosphate dehydrogenase (G3PDH) housekeeping gene were designed as follows: for MMP-1 forward primer, 5'-CGACTCTAGAAACACAAGAGCAAGA-3' and reverse primer, 5'-AAGGTT­ AGCTTACTGTCACACGCTT-3' for G3PDH forward primer, 5 '-ACCAC­ AGTCCATGCCATCAC-3' and reverse primer, 5'-TCCACCACCCTGTTGCTGTA-3'. The cycling conditions in the first-strand cDNA system consisted of denaturation at 94°C for 1 min, annealing at 60°C for 2 min, and extension at 72°C for 2 min. PCR was