Ultraviolet-B Protective Effect of Flavonoids from Eugenia caryophylata on Human Dermal Fibroblast Cells

Background: The exposure of skin to ultraviolet-B (UV-B) radiations leads to deoxyribonucleic acid (DNA) damage and can induce production of free radicals which imbalance the redox status of the cell and lead to increased oxidative stress. Clove has been traditionally used for its analgesic, anti-inflammatory, anti-microbial, anti-viral, and antiseptic effects. Objective: To evaluate the UV-B protective activity of flavonoids from Eugenia caryophylata (clove) buds on human dermal fibroblast cells. Materials and Methods: Protective ability of flavonoid-enriched (FE) fraction of clove was studied against UV-B induced cytotoxicity, anti-oxidant regulation, oxidative DNA damage, intracellular reactive oxygen species (ROS) generation, apoptotic morphological changes, and regulation of heme oxygenase-1 (HO-1) gene through nuclear factor E2-related factor 2 antioxidant response element (Nrf2 ARE) pathway. Results: FE fraction showed a significant antioxidant potential. Pretreatment of cells with FE fraction (10–40 μg/ml) reversed the effects of UV-B induced cytotoxicity, depletion of endogenous enzymatic antioxidants, oxidative DNA damage, intracellular ROS production, apoptotic changes, and overexpression of Nrf2 and HO-1. Conclusion: The present study demonstrated for the first time that the FE fraction from clove could confer UV-B protection probably through the Nrf2-ARE pathway, which included the down-regulation of Nrf2 and HO-1. These findings suggested that the flavonoids from clove could potentially be considered as UV-B protectants and can be explored further for its topical application to the area of the skin requiring protection. SUMMARY Pretreatment of human dermal fibroblast with flavonoid-enriched fraction of Eugenia caryophylata attenuated effects of ultraviolet-B radiations It also conferred protection through nuclear factor E2-related factor 2-antioxidant response pathway and increased tolerance of cells against oxidative stress Flavonoid-enriched fraction can be explored further for topical application to the skin as a ultraviolet-B protectant. Abbreviations used: ABTS: 2,2’-azino-bis-(3-ethylbenzothiazoline- 6-sulphonic acid), AO: Acridine orange, ANOVA: Analysis of variance, ARE: Antioxidant response elements, BSA: Bovine serum albumin, CAPE: Caffeic acid phenethyl ester, CAT: Catalase, DCFH-DA: 2’,7’-dichlorofluorescein diacetate, DMEM: Dulbecco's Modified Eagle's Medium, DMSO: Dimethyl sulfoxide, DNA: Deoxyribonucleic acid, DPBS: Dulbecco's phosphate buffered saline, DPPH: 2,2-diphenyl-1-picrylhydrazyl, ECL: Enhanced chemiluminescence, EDTA: Ethylenediaminetetraacetic acid, ELISA: Enzyme-linked immunesorbent assay, EtBr: Ethidium bromide, FBS: Fetal bovine serum, FE fraction: Flavonoid-enriched fraction, FRAP: Ferric reducing antioxidant power, GPx: Glutathione peroxidase, GR: Glutathione reductase, GST: Glutathione-S-transferase, GSH: Reduced glutathione, GSSG: Oxidized glutathione, HDF: Human dermal fibroblast, HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid, HRP: Horseradish peroxidase, HO-1: Heme oxygenase-1, HPTLC: High-performance thin layer chromatography, Keap-1: Kelch-like ECH-associated protein-1, MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, NaCl: Sodium chloride, NFDM: Nonfat dry milk, Nrf2: Nuclear factor E2-related factor 2, NQO1: NAD (P) H: Quinine oxidoreductase 1, OH: Hydroxyl ions, PBST: Phosphate buffered saline with 0.1% tween 20, PCR: Polymerase chain reaction, PMSF: Phenylmethanesulfonyl fluoride, Rf: Retention factor, ROS: Reactive oxygen species, rRNA: Ribosomal ribonucleic acid, SDS: Sodium dodecyl sulfate, SOD: Superoxide dismutase, TLC: Thin layer chromatography, TLC-DPPH: Thin layer chromatography-2,2-diphenyl-1-picrylhydrazyl, UV: Ultraviolet, UV-A: Ultraviolet-A, UV-B: Ultraviolet-B, UV-C: Ultraviolet-C, and qPCR: Quantitative polymerase chain reaction.