Protective Effects of a Topical Antioxidant Complex Containing Vitamins C and E and Ferulic Acid Against Ultraviolet Irradiation-Induced Photodamage in Chinese Women

It is well-known that continuous exposure to ultraviolet irradiation (UVR) leads to various of biological effects, including cutaneous erythema, edema, sunburn, immunosuppression, photoaging, as well as skin carcinogenesis.¹ One of the mechanisms is that UVR increases the cellular levels of reactive oxygen species (ROS), which damage lipids, proteins, and nucleic acids in both epidermal and dermal cells, and stimulates the inflammatory process in the skin.²

Ultraviolet A (UVA), in a wavelength of 320 to 400 nm, contributes up to 95% of total UV exposure and is a significant source of oxidative stress in human skin.^3,4 Experimental findings show that cumulative UVA doses are capable of inducing cellular DNA damage, which may lead to photoaging of the skin, altered expression of oncogenes and tumor suppressor genes, and skin carcinoma.^5-7 Ultraviolet B (UVB), in a wavelength of 290 to 320 nm, critically damages cellular macromolecules and induces the formation of ROS. Newly formed ROS, such as hydroxyl radical and superoxide anion, can activate genes, damage DNA, oxidize cell lipids and proteins, and ultimately lead to apoptotic or necrotic cell death.^8-10 To protect cells from UVR-induced damage, the skin has an elaborate antioxidant defense system consisting of enzymatic and nonenzymatic components to quench reactive oxygen intermediates.¹¹ However, with the ongoing exposure to UVR, the body's endogenous antioxidative system will be overwhelmed and the production of ROS increases. Supporting the skin's antioxidant defense system is a necessary strategy to combat oxidative damage induced by UVR.

Vitamin C (ascorbic acid) and vitamin E (mainly α-tocopherol) are the 2 elemental nutrients that could quench free radicals and prevent ROS-induced cellular damage. Vitamin C can directly scavenge free radicals in the water phase and can protect cellular membranes.¹² In contrast, vitamin E is mainly a free radical scavenger in the lipid phase by means of inhibiting lipid peroxidation.¹³ When vitamin C and vitamin E coexist, they demonstrate synergistic effects.¹⁴ Ferulic acid is a potent phenolic antioxidant found ubiquitously and at high concentrations in plants.¹⁵ It has been demonstrated that ferulic acid can protect membranes from lipid peroxidation, neutralize alkoxyl radicals, and prevent cells from damage induced by hydroxyl radicals, nitric oxide, and superoxide radical.¹⁶ When ferulic acid was incorporated into a formulation of vitamin C and/or vitamin E, the topical delivery of

the vitamins was improved, the chemical stability improved, and the photoprotection against ssUVR doubled.¹²

Antioxidants have been suggested as a useful supplement to prevent UVR-induced cutaneous damages. They are designed to exert protective effects from inside skin and remain active for several days.^17,18 In recent years, a variety of naturally occurring antioxidant compounds from plant origin have been demonstrated to be beneficial by quenching free radical generation and preventing photodamage in skin. The present study was elaborated to investigate the protective effects of a topical antioxidant complex containing Vitamin C/E and ferulic acid on photodamage induced by solar-stimulated UVR (ssUVR).

Subjects Twelve healthy female Chinese subjects aged between 18 and 60 years with Fitzpatrick skin type III or IV were enrolled in this study. Informed consent and photography consent were obtained from each subject before enrollment. The study protocol complied with the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the Institutional Medical Ethic and Human Research Committee of China Medical University. The clinical assessment was conducted in winter to avoid the confounding effect of environmental sun exposure.

Test Materials Test materials were provided by Skinceuticals Laboratory of L'Oreal Research and Innovation. The vehicle control product was a hydroglycolic base containing water, dipropylene glycol, glycerin, laureth-23, phenoxyethanol, sodium hyaluronate, and sodium hydroxide. The antioxidant complex product contained ascorbic acid, tocopherol, ferulic acid, and the same hydroglycolic base as the vehicle control.

Radiation Source and Dosimetry A multiport solar simulator (GS-2006; Aohua Co, Beijing, China) was used in the study. This solar simulator includes a 450W xenon lamp and a dichroic mirror. Radiation was filtered to deliver a spectral output in the UV range comparable with that of the natural solar spectrum (UVA, 320-400 nm; and UVB, 290-320 nm) by a 1-mm-thick, short, cut-off filter (WG-320; Schott, Clichy, France) plus a UG 11/1-mm-thick, long, cut-off filter (Schott). The irradiance at skin level was monitored with a calibrated SUN5 digital spectroradiometer (National Institute of Measuring, Beijing, China). The relative UVA and UVB output of the solar simulator under these conditions was approximately 4.1 mW/cm², consisting of approximately 88.9% of UVA and 11.1% UVB.

Irradiation and Treatment Protocol Four unexposed sites on the dorsal skin of individual subjects were marked for the experiment. A formulation containing antioxidant complex (AOx) and a vehicle-only formulation were respectively applied onto 2 sites daily for 4 consecutive days. On day 4, 30 minutes after application of products, the AOx-treated site, the vehicle control site, as well as an untreated site (positive control) received ssUVR in a dose of 5 times the minimal erythema dose (MED). The fourth site, which received neither ssUVR nor product treatment, was defined as a negative control (blank).

Erythema and Sunburn Cell Standardized photos were taken with a digital camera (Coolpix 5700; Nikon, Tokyo, Japan), and erythema index (EI) was measured with a Mexameter (MX18; Courage+Khazaka, Koln, Germany) at baseline (day 1) and 24 hours after irradiation (day 5) for erythema evaluation. EI value was measured 3 times on each site, and those values were averaged. The EI difference value was calculated as preirradiation average value minus postirradiation average value.

Biopsy Taken As early as 24 hours after exposure to ssUVR (day 5), 4-mm punch biopsies were obtained. The biopsy sections were fixed in 10% buffered formalin and processed for hematoxylin and eosin (H&E) and immunohistochemical (IHC) staining. Routine H&E staining was performed to detect sunburn cells, which showed pyknotic nuclei and dense eosinophilic cytoplasm. The average value of positive cells per high-power field (HPF) (x400) in the epidermis was counted, respectively, for every slide, and results were expressed as the mean of 5 consecutive, different, randomly chosen views per HPF.

Immunohistochemistry and Quantification Formalin-fixed, paraffin-embedded sections were processed for IHC staining. After deparaffinization, endogenous peroxidase was quenched, and the slides were subjected to heat-induced epitope retrieval in 500 mL of citrate buffer solution (pH, 6.0) for 10 minutes using a high-pressure steamer at 121°C, then immersed in the 3% H2O2 at room temperature and incubated in the normal goat sera for 10 minutes, without being washed. After that, the slides were respectively treated with the optimized dilutions of mouse monoclonal antibodies against p53 (MAB-0142), thymine dimer (clone KTM53), CD1a (clone 010), and incubated overnight at 4°C. The following morning, the slides were treated with the appropriate biotin-labeled secondary antibody for 25 minutes at 37°C. The reaction product was developed by immersing the slides in the prepared horseradish peroxidase–labeled tertiary antibody for 25 minutes at 37°C similarly, followed by coloration with 3,3'-diaminobenzidine solution, counterstaining with hematoxylin, and dehydration with gradient ethanol and dimethylbenzene. Following immunostaining, the number of positive cells (brown-colored nuclear staining for p53 and thymine dimer; brown-colored cytomembrane and cytoplasm staining for CD1a) per HPF (x400) was calculated separately for every slide. Results were expressed as the average of 5 consecutive, different, randomly chosen views per HPF (x400).

Statistical Analysis The statistical evaluation of data was performed with SPSS software (version 16.0; SPSS, Inc, Chicago, IL). Student t test was used for normal distribution data, and Mann-Whitney U test was used for non-normal distribution data. A P value <.05 was considered significant.