Skin Lightening Effect of the Dietary Intake of Citrus Peel Extract Against UV-Induced Pigmentation

Abstract

Recent studies revealed that citrus peel has beneficial effects in various disorders associated with nitric oxide and/or oxidative stress. In this study, we investigated the effects of Jeju citrus (Citrus unshiu) peel using various in vitro and in vivo methods. First, the inhibitory effect of citrus peel extract (CPE) on enzymatic activity of tyrosinase was evaluated. Tyrosinase activity was dose-dependently decreased by CPE. Second, the effect of CPE on melanogenesis was determined by measuring the melanin content in melan-a cells. The inhibitory effect of CPE on melanin synthesis was greater than that of vitamin C. Finally, the effect of long-term supplementation with CPE on ultraviolet B-induced skin pigmentation was examined in guinea pigs. Administration of CPE improved ΔL-value compared with the nontreated ultraviolet control group. As a strong inhibitor of melanogenesis, CPE could be used as a depigmentation agent and a supplement for skin lightening.

Melanogenesis is the procedure of synthesis and distribution of melanin inside skin and hair follicles. As a powerful absorbent of light, melanin is responsible for defensing skin from the dangerous effects of ultraviolet (UV) irradiation. ¹ Mainly, dysregulation of the biosynthesis and distribution of melanin makes many troubles consisting of age spot, melasma, and freckles within the epidermis. Considering the fact that tyrosinase is the key enzyme concerned in melanogenesis, materials that exert an inhibitory effect toward tyrosinase are anticipated to inhibit melanogenesis. Several tyrosinase inhibitors which include arbutin, kojic acid (KA), and ellagic acid were recognized from herbal merchandise used as skin lightening agents, cosmetics, and practical ingredients. ^{2 -4}

Oral supplementation for protection against skin damage has attracted attention. ⁵ For systemic photoprotection, several compounds have been assessed for their ability to provide photoprotective activity after oral administration to develop new functional foods for skin. Since reactive oxygen species (ROS) formed in response to solar radiation play a central role in initiating and driving detrimental signaling events, antioxidant supplementation is considered to exert a photoprotective effect against UV irradiation. ⁶

Citrus fruits are representative dietary sources of abundant antioxidants and have been used for traditional medicines in Eastern Asian Countries, including Korea, Japan, and China. Many compounds present in citrus fruits have been identified and reported to possess diverse biological activities. ^{7 -9} In particular, the peels, the waste parts of citrus fruits, have been shown to be more efficient in eliminating free radical species than the corresponding juice-containing portions. Various components of citrus peel extract (CPE) are also suggested to exert inhibitory effects in skin aging through diverse mechanisms including inhibition of collagenase activity, induction of procollagen synthesis, and anti-inflammatory activity. ^{10 -12} However, little information is available about the inhibition of melanogenesis by citrus peel. The purpose of this study was to investigate the inhibitory effect of CPE on tyrosinase enzyme and melanin synthesis in murine melanocytes and to research whether oral intake of CPE exerts a useful effect on skin brightening in vivo model.

Fresh citrus peel (Citrus unshiu) harvested on Jeju Island in 2012 was extracted with 70% ethanol solvent, pulverized by a freeze-drying method, and flavonoid components in the powder were quantified using High Performance Liquid Chromatography (HPLC) (Table 1).

Table 1

Flavonoids Composition of Citrus Peel Extract (Citrus unshiu).

Flavonoid ingredients	Content (mg/g extract)
Hesperidin	220 ± 5.0
3,3′,4′,5,6,7,8-Heptamethoxyflavone	106 ± 10.1
β-Cryptoxanthin	3.4 ± 0.4

Data are expressed in milligrams of flavonoids per gram of powdered extract.

The content of hesperidin, heptamethoxyflavone, and β-cryptoxanthin in the powdered extract was approximately 220, 106, and 3.4 mg/g powder, respectively. Hesperidin, a flavanone glycoside, has a disaccharide rutinoside moiety that is found abundantly in citrus and an aglycone form called hesperetin. Hesperidin induces apoptosis and suppresses proliferation in human cancer cells, and its many beneficial effects can be attributed to its antioxidant activity. ^9,10 3,3′,4′,5,6,7,8-Heptamethoxyflavone (HMF) that makes up polymethoxy-flavonoids with tangeretin and nobiletin is a major ingredient of citrus peel and has anti-inflammatory activity as well as antioxidant activity. ^7,11 β-Cryptoxanthin is one of the 6 major carotenoids found in human blood, in addition to a-carotene, b-carotene, lutein, zeaxanthin, and lycopene. It was revealed that β-cryptoxanthin has preventive effects against osteoporosis, diabetes, arteriosclerosis, and liver malfunction and also has antioxidant activity, anticancer activity, and stimulatory effects on the immune system. ^2,12 Our CPE contained a high level of hesperdin, HMF, and β-cryptoxanthin.

While the skin is exposed to UV, the quantity of ROS is robustly increased. Due to the fact that ROS can cause directly DNA damage that leads to consequences in induction of melanocyte proliferation and melanin production, ¹³ the activity of antioxidants is liable for maintaining our skin bright. Therefore, we measured the antioxidant capacity of CPE by 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. As shown in Figure 1(a), CPE exhibited antioxidant activity in a dose-dependent manner. Interestingly, the antioxidant effect of CPE was greater than that of vitamin C at the same dose (50 µg/mL). According to these data, CPE would appear to be a more potent antioxidant than vitamin C. Melanin is synthesized from tyrosine in a metabolic pathway called melanogenesis that involves several enzymes such as tyrosinase, 5,6-dihydroxyindole-2-carboxylic acid oxidase (TRP-1), and dopachrome tautomerase (TRP-2) (2). Among these, tyrosinase is regarded as the rate-limiting enzyme. We investigated the inhibitory activity of CPE on tyrosinase activity using mushroom tyrosinase and showed that CPE had an inhibitory effect (Figure 1b). The inhibitory activity of CPE (50 µg/mL) on tyrosinase activity was similar to that of KA, a representative inhibitor of tyrosinase (10 µM, equivalent to 1.4 µg/mL).

Figure 1

Antioxidant capacity and inhibitory effect of citrus peel extract against tyrosinase activity. (a) Scavenging activity of 2,2-diphenyl-1-picrylhydrazyl radicals for different concentrations of citrus peel extract. (b) Mushroom tyrosinase activity after treatment with different concentrations of citrus peel extract. Each value represents the mean ± SE of 3 independent experiments. Values not followed by a common letter are significantly different (P < 0.05). KA, kojic acid; CPE, citrus peel extract.

Comparison of the dose and effect of CPE and KA suggests that CPE would be a mild inhibitor of tyrosinase enzyme. Interestingly, the inhibitory effect of CPE on the enzymatic activity of tyrosinase appears to be through direct interaction as we also tested a cell-free experimental system containing only enzyme, buffer, and CPE. In conclusion, our data indicate that CPE contains one or more components that directly inhibit the activity of tyrosinase enzyme.

We found that CPE reduces oxidative stress and directly inhibits the enzymatic activity of tyrosinase, a key enzyme for melanogenesis. To elucidate whether CPE reduces melanin synthesis, melan-a cells were treated with the indicated concentrations of CPE and total melanin content was measured 3 days later. Quantitative analysis showed that CPE treatment significantly decreased melanin content in melan-a cells in a dose-dependent manner (Figure 2) without inducing cytotoxicity (1-50 µg /mL) (data not shown).

Figure 2

Dose-response effects of citrus peel extract on the melanin content of melan-a melanocytes. Melan-a cells were treated with citrus peel extract or vitamin C at the concentrations indicated in the figure and incubated for 72 hours. Vitamin C (50 µg/mL) was used as the positive control. Values not followed by a common letter are significantly different (P < 0.05). CTL, negative control.

To examine the effect of CPE on ultraviolet B (UVB)-induced skin pigmentation, brown guinea pigs were orally administered CPE. A reduction of pigmentation was observed in the skin area of the CPE-administered group compared with that of the nontreated group. Although a low dose of CPE (50 mg/kg) treatment did not meaningfully improve skin luminosity, the ΔL value of the CPE group (250 mg/kg) was significantly higher than that of the nontreated group on all days of measurement (P < 0.05) (Figure 3). Therefore, CPE intake is considered to have an inhibitory effect on skin pigmentation.

Figure 3

Citrus peel extract intake and the change in lightness value. Guinea pigs were given test materials by oral administration every day during the experiment. The L-value for the ultraviolet B-irradiated area on the back of brownish guinea Pigs was measured by chromameter (Minolta). ΔL-values were calculated from the L-values as follows: ΔL-values = L-value (at each day measured) – L -value (at Day 1). Each value is expressed as the mean ± SE (n = 6). *Significantly different from the nontreated group (P < 0.05).

Regulation of pigmentation in human skin has been a longstanding goal for cosmetic pharmaceutical applications. Important factors that regulate the pigmentation of human skin are melanocytes in the epidermis that synthesize the melanin and neighboring keratinocytes that transport melanin to the upper layers of the skin. ¹⁴ Hesperidin, a major component of citrus flavonoid shown to be active against various oxidative stress–mediated diseases, did not inhibit melanin production in B16F10 murine melanoma cells stimulated with a-melanocyte stimulating hormone or affect the catalytic activity of tyrosinase. Instead, hesperidin inhibited melanosome transport in melanocytes and exerted a skin lightening effect in a pigmented reconstructed epidermis model. Given that skin is the outermost barrier of the body, it is very vulnerable to outside disturbances and directly interacts with dangerous environments such as UV irradiation, pollutants, and mechanical stress. Oxidative stress that is induced by UV radiation is thought to play an important role in initiating and driving the signaling events that cause skin issues such as aging and pigmentation. ^15,16

Especially, UVB radiation is 1000 times more genotoxic and strongly absorbed by DNA, causing sunburn and skin cancer. ^5,6,17 Lately, it has been shown that the regular intake of Epigallocatechin gallate strengthens the tolerance of skin by increasing the minimal erythema dose and thus prevents UV-induced perturbation of epidermal barrier function and skin damage. ^5,18 Oxidative stress, which causes various undesirable effects in the living body, is considered a major casual factor for aging and aging-related disease, and antioxidant substances such as vitamin C or a vitamin E are known to reduce such risks. ^19,20 As the antioxidant ability of carotenoids is well known and believed to be beneficial for a healthy life, it is likely that CPE possesses the potential to improve the quality of life through its activity as an antioxidant agent.

According to the previous reports, various components of CPE are suggested to exert various inhibitory effects in melanogenesis: (i) decreased melanin content through the inhibition of melanogenesis (by β-cryptoxanthin), (ii) suppression of melanosome transport in melanocyte (by hesperidin), and (iii) antioxidant activity against intracellular ROS stimulated by UVB (by HMF). As a mixture of these functional ingredients, CPE is expected to possess a strong skin lightening ability against harmful stress, and our study confirmed that CPE effectively ameliorated oxidative stress and inhibited melanogenesis to alleviate skin pigmentation induced by UV radiation.

Taken together, in this study, we analyzed the useful effects of CPE and its flavonoids on skin pigmentation in vivo and in vitro. Our findings suggest that consumption of CPE suppresses melanin production induced by UVB irradiation in the living body. But, additional researches are needed to decide whether the findings defined in this research for the guinea pig model are relevant to human skin. In addition, the precise mechanism by which CPE flavonoids are metabolized to active forms remains to be elucidated.

Experimental

Samples, Analysis, and Melan-a Cell Culture

Melan-a cells had been maintained in RPMI-1640 supplemented with 10% (v/v) fetal bovine serum, 1% penicillin-streptomycin (10 000 units and 10 000 µg/mL respectively), and 200 nM phorbol 12-myristate 13-acetate in 10% CO₂ at 37°C. All reagents and solvents for interest tests had been bought from commercial enterprise carriers (Sigma-Aldrich, St Louis, MO, United States) and used without further purification. All other chemical compounds of analytical grade have been purchased from Merck (Kenilworth, NJ, United States). All reagents for cell culture and analysis were purchased from Lonza (Basel, Switzerland), Gibco (Waltham, MA, United States), Wako (Osaka, Japan), and Sigma-Aldrich. Citrus peel harvested on Jeju Island in South Korea was purchased from Bioland Co. Ltd. (Cheonan, Korea), extracted with 70% ethanol at room temperature for 48 hours, evaporated the solvent in vacuum, and pulverized by a freeze-drying method. The extraction yield is 40.6% and its flavonoids in the powder were quantified using HPLC. The HPLC analysis was carried out using an Alliance Waters 2695 (Waters Co., Milford, MA, United States) system including degasser, quaternary pump, automatic sampler, column oven, and diode array detector. The compounds were eluted with a Prevail C18-column (250 × 4.6 mm i.d.; Alltech Associates, Inc., IL, United States) and detected at 285 nm with a flow rate of 0.8 mL/min. Analysis of β-cryptoxanthin was carried out using a YMC carotenoid column (3 µm, 150 × 4.6 mm i.d.; YMC Co., Kyoto, Japan) by the same system.

2,2-Diphenyl-1-Picrylhydrazyl Assay and Tyrosinase Activity Assay

The percent antioxidant activity of each substance was assessed by the DPPH free radical assay. Measurement of DPPH radical scavenging activity became accomplished as follows. The samples have been reacted with the stable DPPH radical in an ethanol solution. The reaction mixture consisted of 0.5 mL of sample, 3 mL of absolute ethanol, and 0.3 mL of DPPH solution (0.5 mM in ethanol). While DPPH reacted with an antioxidant compound that can donate hydrogen, it was reduced. The color change from violet to yellow was detected by measuring absorbance (Abs) at 515 nm at 37°C using a UV-VIS spectrophotometer after 30 minutes. A mixture of ethanol (3.3 mL) and sample (0.5 mL) served as a blank. The control solution was prepared by adding ethanol (3.5 mL) to DPPH solution (0.3 mL). The scavenging activity was determined as 100 − [(A − B)/C × 100], wherein A represents the absorbance of the test sample, B represents the absorbance of the blank, and C represents the absorbance of the control. Enzymatic activity of tyrosinase was assessed using mushroom tyrosinase due to its prepared availability. Each sample became dissolved in dimethylsulfoxide (DMSO) and used for the experiment at 100-fold dilution. The reaction mixture contained 0.1 M potassium phosphate buffer (pH 6.8), 3 mM l-tyrosine solution, without or with a sample chemical, and 2000 units/mL tyrosinase in aqueous solution. The mixture became incubated at 37°C for 10 minutes and the reaction was monitored at 475 nm. A control reaction was performed with DMSO alone. The percent tyrosinase activity was calculated as 100 − [(A − B)/A × 100], wherein A represents the difference in the absorbance of the control and B represents the difference in the absorbance of the test sample. Kojic acid (20 µM), a well-known tyrosinase inhibitor, was used as reference molecule.

Melanin Assay

Melan-a cells were seeded into 24-well plates (4.5 × 10⁵ cells/well) and treated the following day with the indicated concentrations of CPE or vitamin C. In the course of incubation, the medium was changed every day with fresh media containing CPE or vitamin C. After 3 days, the medium was removed and the cells have been washed 2 times with Dulbecco’s phosphate-buffered saline. For cell lysis, 1 mL of 1 N NaOH was applied to each well and the plates have been incubated for 30 minutes at 60°C. Cellular lysates had been transferred to a 96-well plate and the absorbance was measured at 470 nm. The entire protein content in each experimental group was measured by the use of DC protein Assay (BioRad, Hercules, CA, United States). After normalization of the calculated melanin content vs total protein content, melanin content per protein of every experimental group was obtained.

Ultraviolet B Irradiation-Induced Hyperpigmentation in Guinea Pigs

Eight-week-old female brown guinea pigs were provided by Central Laboratory Animal Inc. (Seoul, Korea). The animal experimental protocol was accepted by the ethical committee of Kyung Hee University and done in accordance with the authorized guidelines. Guinea pigs were kept in an air-conditioned room below a temperature of 23°C ± 1°C, 55% relative humidity, and 12-hour light/dark cycle. After acclimation for 1 week, the guinea pigs had been housed in individual cages. Using an immobilizer, each animal was immobilized in an abdominal position under anesthesia and a site measuring 2 × 2 cm was prepared as the irradiation site on the right or left side of the median line. The UV source was supplied by a closely spaced array of 5 sun lamps with peak irradiance at 310 nm (Kanagawa, Japan). The irradiation (0.1 mW/cm²) was measured with an IL1700 Research Radiometer (International Light, Inc., Newburyport, MA, United States) equipped with a UVB sensor. The guinea pigs were exposed to 380 mJ/cm² UVB radiation 3 times per week for 2 weeks. The guinea pigs were randomly assigned to 3 experimental groups: negative control (water only as a vehicle) and 2 CPE groups (50 and 250 mg/kg body weight of CPE). Citrus peel extract was administered to guinea pigs for 28 consecutive days in drinking water. Luminosity (L value) at the irradiation site was measured using a chromameter (CR-300, Minolta Co., Ltd., Tokyo, Japan) before irradiation on the first dosing day and 14, 21, and 28 days after the first dosing day, and ΔL values (L value on the day of observation – L value before irradiation) were calculated. Luminosity was measured at the center and the 4 corners of the irradiation site, and the mean value at these sites was used as the L value for each animal. When luminosity was measured at the wrong site due to movement of the animal, the measurement was performed again.

Statistical Analysis

All data are expressed as mean ± SD. Statistical analyses were performed using the SPSS program (SPSS 12.0). One-way Analysis of variance and Tukey-Kramer test were used to examine the differences between groups. P values <0.05 were considered to be statistically significant.