The skin color of the human body is determined by melanin, carotenoids, hemoglobin, and bilirubin, among which melanin is the most important factor [1
]. Melanin is produced to protect the skin against damage due to UV radiation [3
]. The biosynthesis of melanin starts with the oxidation of tyrosine by tyrosinase; DOPA and dopachrome are then produced, followed by DHI-eumelanin, DHICA-eumelanin, and pheomelanin [4
]. Human skin is made of three layers: the epidermis, dermis, and subcutaneous tissue. Of these the epidermis is the outermost layer, where melanocytes that produce melanin exist in the basal lamina or the lower part of the epidermis [6
]. Skin darkening occurs when the melanin produced by the melanin-producing cell (melanocyte) is transferred to the keratinocyte and accumulates in the epidermis.
Although melanin protects the skin, the hyper-production of melanin pigment can cause melasma, freckles, and dark spots [7
]. To prevent or improve skin darkening due to melanin hyper-production, skin pigment-suppressing agents using kojic acid, arbutin, or licorice extract have been developed, but various problems such as adverse side effects and weak efficacy have been observed [9
]. Thus, developing a new skin pigment-inhibiting agent is a matter of urgency.
Corn silk is the thin, yellow- or brown-colored thread-like styles at the tip of an ear of corn (Zea mays
L.), an annual plant of the Gramineae family containing various flavonoids and terpenoids [10
]. As for its physiological activities, corn has been reported to have antioxidant [11
], anti-inflammatory [12
], anti-diabetic [13
], anti-fatigue [14
], and neuroprotective effects [15
]. In particular, its diuretic action is well known [16
]. However, the activity of corn silk related to skin pigment suppression has yet to be reported. Thus, the study was performed to investigate the inhibitory effect of corn silk on melanin production in Melan-A cells by measuring melanin production and protein expression. Melan-A cells are highly pigmented melanocytes and provide an excellent parallel non-tumorigenic cell line derived from C57Bl/6 mice [17
]. In addition, corn silk extract aqueous solutions were applied on the human face with hyperpigmentation twice a day for 8 weeks, and skin color was measured to check for any adverse reactions and examine the degree of skin pigment reduction.
L-Dopa, arbutin and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). All solvents used for isolation were analytical grade products acquired from Merck Co. (Darmstadt, Germany). FBS (Fetal bovine serum), RPMI medium, and PS (Penicillin-Streptomycin) were purchased from Gibco BRL (Grand Island, NY, USA). Tyrosianse and TRP-2 antibody were purchased from Santa Cruz Biotech (Santa Cruz, CA, USA).
3.3. Cell Culture
Melan-A cells, mouse melanocytes [17
], were cultured at 37 °C, 5% CO2
using RPMI 1640 medium containing 10% fetal bovine serum, 1% penicillin-streptomycin, and 200 nM phorbol-12 myristate 13-acetate. Cells were inoculated on a 24-well plate at a concentration of 1 × 105
cells/well and incubated for 24 h, and then treated with corn silk extract and arbutin for 3 days and incubated for another 24 h.
3.4. Cell Viability
After the elimination of the culture medium, cells were washed with PBS and then 200 μL of a crystal violet solution (0.1% crystal violet, 10% EtOH, the rest is PBS, w/w) was added to each well. Cells were incubated for 5 min at room temperature, and then washed with distilled water twice, and 1 mL of EtOH was added. After shaking for 10 min at room temperature, absorbance was measured at 590 nm.
3.5. Melanin Production
After the elimination of the culture medium, cells were washed with PBS, and 1 mL of 1 N NaOH was added to each well to dissolve the melanin. Absorbance was then measured at 400 nm.
3.6. Tyrosinase Inhibitory Effect
L-Dopa (120 μL, 8.0 mM) dissolved in 67 mM phosphate buffer (pH 6.8) and sample (40 μL) dissolved in methanol were placed in a 96-well microplate, and mushroom tyrosinase (40 μL, 125 U/mL) was then added. After incubation at 37 °C for 20 min, the amount of dopachrome produced was measured at 492 nm.
3.7. Protein Expression Level in the Cell
Melan-A cells (5 × 105) were inoculated onto a culture dish and incubated for 24 h, and then treated with the samples for 3 days. Cells were incubated for another 24 h and subsequently collected, and lysis buffer (50 mM Tris-HCl, pH 8.0, 0.1% SDS, 150 mM NaCl, 1% NP-40, 0.02% sodium azide, 10 μg/mL PMSF, 1 μg/mL aprotinin) was added and the mixture sonicated to extract the proteins inside the cells. After electrophoresis of 50 μg of extracted protein using 8% SDS-polyacrylamide gel, it was transferred onto the membrane and blocked with 5% skim milk. The membrane was made to react with tyrosinase and dopachrome tautomerase (TRP-2) primary antibody followed by anti-goat secondary antibody and was detected using enhanced chemiluminescence detection (Bio-Rad Laboratories, Hercules, CA, USA).
3.8. Improvement of Hyperpigmentation in Human Skin
The subjects were 42 females aged 36–50 years (average: 42.9 ± 3.9) with hyperpigmentation on the face. The subjects were randomly divided into two groups and treated with either corn silk extract 0.75% aqueous solution or 1.5% aqueous solution (w/w) for 8 weeks twice a day (morning and evening) by applying all over the face after washing. Skin color was evaluated before use and at 4 weeks and 8 weeks after use by skin color measurement, and area and density of hyperpigmentation in subjects who washed their face and waited for 20 min at constant temperature and humidity (22 ± 2 °C, 50 ± 5%). At each time point, skin color on the side of the face was measured using a spectrophotometer CM-2500d (Minolta, Tokyo, Japan) for the L* value and ITA value. The formula of ITA value is [Arc tangent ((L* − 50)/b*)] × 180/3.14159. Likewise, the area and density of hyperpigmentation were measured using a facial image capturing system, VISIA ver.5 (Canfield, Fairfield, NJ, USA), and an image analysis program called Image-pro plus (MediaCybernetics, Bethesda, MD, USA).
3.9. Safety Evaluation on Human Skin
For safety evaluation, the corn silk extract aqueous solution was applied to the skin at 4 weeks and 8 weeks after use; subjective irritation and objective irritation by the subject's observation were evaluated.
3.10. Statistical Analysis
The statistical significance of the data was analyzed using the SPSS Package Program (IBM, Armonk, NY, USA). For human experiments, the Shapiro-Wilks test was applied for the normality test, the comparison by time point used repeated measures ANOVA for the parametric test and Wilcoxon’s signed-ranks test followed by post hoc evaluation for the non-parametric test.