Evaluation of green tea extracts for UV protection and inhibition of Melanin synthesis in cells and tissue cultures

Abstract

Green tea, a popularly consumed beverage worldwide, contains polyphenols that have been shown to provide many attributes to health. Among green tea polyphenols, EGCG (Epigallocatechin gallate) is reported to be the most active antioxidant. In this research green tea leaves were extracted using distilled water, and citrate buffer of various pH: 3, 3.5, 4 and 4.5. It was found that the EGCG yield with citrate buffer was 3 folds greater than with distilled water; however, the pH of the buffer had little effect. This experiment chose green tea extract with citrate buffer pH 4.5 which close to skin pH. Keratinocytes, melanocytes and fibroblasts were prepared from newborn foreskins and used to examine the cytotoxicity of green tea extract and EGCG with the MTT assay method. The results show concentrations below 90 and 60 µg/ml respectively, were not toxic. Inhibition of melanin synthesis was studied on melanocyte/keratinocyte co-cultures. Green tea extract and kojic acid reduced melanin content by 14.47 and 33%, respectively. Keratinocytes were cultured in medium containing test sample, irradiated with ultraviolet light B (UVB) at 400 mj and assayed for cell viability by MTT method. It was found that survival of cultured cells irradiated in medium or medium containing green tea, EGCG or Ectoin were 73.34, 98.15, 96.32 and 101.05%, respectively. A 3D skin model was derived from skin cells resulting in a skin equivalent consisting of fibroblasts in collagen raft supporting differentiated keratinocytes. The skin model and excised human skin were cultured in medium containing test sample and were irradiated with UVB. The skins were assayed for cell viability or evaluated for sunburn cells by preparing paraffin cross sections stained with Haematoxylin and Eosin. Supernatant was assessed by measuring interleukin 1 alpha (ILlα) content using ELISA kit. The results show that after irradiation, survival of cells in the cultured skin model with medium or medium containing green tea extracts or Ectoin was 59.29, 94.08 and 97.29 %, respectively, and ILlα content was 15.13, 14.52 and 14.05 pg/ml, respectively. While the survival of cells in excised human skin cultured in medium or medium containing green tea extracts or Ectoin, was 68.81, 85.24 and 92.02%, respectively; number of sunburn cells was 2000, 1000 and 900 cells/mm of skin, respectively; and ILlα content was 105.36, 79.28 and 77.04 pg/ml, respectively. Green tea extract could reduce damage from UVB irradiation in cells and tissue cultures including inhibited melanin synthesis in cell cultures.