ANTIOXIDANT ACTIVITY OF EGT 23 (a) (b) (c) 326 328 330 332 334 Field (mT) Figure 4. Singlet oxygen quenching effect of EGT by ESR study. (a) Control (without EGT). (b) EGT 10 mg/ml. (c) EGT 20 mg/mJ. Table I Inhibition of EGT on Lipid Peroxidation Initiated by Singlet Oxygen Liposomes alone Li posomes + rose bengal Lip + rose bengal + 20 µM EGT nmol LPO/ml 23.81 91.84 26.53 As a source of singlet oxygen, the photo-irradiated rose bengal system was used. Liposomes prepared from phosphatidyl choline with 10 mM rose bengal were irradiated by using a Sylvania 150 W slide projector. Oxidation products in the liposomes were assayed with K-Assay1M LPO-CC from Kamiya Biomedical Company (Seattle, WA). Data are expressed as mean value from dependent examinations in duplicate. address the contribution of 1 O 2 to new MMP-1 production, the effect of NaN 3, a typical quencher against 1 0 2 , on UV A-induced MMP-1 production was examined. UV A ex­ posure to fibroblasts at 40 J/cm2 dramatically increased MMP-1 secretion by 70-fold in cultured medium (data not shown). Fibroblasts produced a small amount of MMP-1 (0.3 ± 2.3 units/cells) in sham­ irradiated cells. On the other hand, UV A-exposed fibroblasts irradiated with 20 J/cm2

24 Treatment UV-B (100 J/m2 ) UVB + 20 µM EGT UVB + 100 µM EGT JOURNAL OF COSMETIC SCIENCE Table II Induction of TNF-a by UV-B in Fibroblasts Net CAT activity (nmol/mg/h) 39.87 15.97 22.07 Fibroblast line XPTNF2, which is an SV40 fibroblast line deficient in DNA repair and carrying the TNF-a promoter chloramphenicol acetyltransferase (CAT) reporter gene. Assay of TNF-a promoter activity as described in text. Assays in duplicate, background subtracted, and results averaged. UVA showed a 100-fold increase in MMP-1 secretion (30.7 ± 4.8 units/cells). The addition of EGT at 1 mg/ml to the system reduced MMP-1 secretion to 1 7 .8 ± 6.2 units/cell, a 42% reduction, and at a concentration of 2 mg/ml, MMP-1 secretion was reduced to 14.9 ± 3.0 units/cell, a 52% reduction (Figure 5). MMP-1 mRNA EXPRESSION Fibroblasts exposed to UV A enhance MMP-1 production with up-regulation of MMP-1 50 .!!l. 40 0 ..... ;2 30 i::: ::s - ..... ri. 10 0 EGT(mg/ml) UVA (J/cm2) 0 0 0.3 0.5 1.0 2.0 0 20 20 20 20 20 Figure 5. EGT-suppressed MMP-1 production induced by UV A irradiation. Human fibroblasts were exposed to UVA at a dose of 20 J/cm2 in the presence of various concentrations of EGT in HBS. MMP-1 activity and cell numbers were measured after UVA irradiation for 24 h. n = 4. Significance: *p 0.05 **p 0.01.