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Effect of gamma irradiation on the anti-oxidant and anti-melanogenic activity of black ginseng extract in B16F10 melanoma cells
Radiation Physics and Chemistry 149 (2018) 33–40
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Effect of gamma irradiation on the anti-oxidant and anti-melanogenic activity of black ginseng extract in B16F10 melanoma cells
T
Ha-Yeon Songa,b, Hye-Min Kima, Woo Sik Kima, Eui-Hong Byunb, Beom-Su Janga, ⁎ Dae Seong Choia, Eui-Baek Byuna, a b
Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup 580-185, Republic of Korea Department of Food Science and Technology, Kongju National University, Yesan 340-800, Republic of Korea
A B S T R A C T
Black ginseng extract (BGE) was irradiated at various doses (10, 30, 50, 70, and 100 kGy) to improve its biological activity. The content of reducing sugar was significantly increased by gamma irradiation up to 100 kGy. The gamma-irradiated BGE showed its highest anti-oxidant activity (DPPH radical scavenging activity-IC50 = 4.33 µg, ABTS radical scavenging activity-IC50 = 2.39 µg, FRAP AEAC = 6.46), and mushroom tyrosinase inhibition activity (IC50 = 12.46 mg) when irradiated at the dose of 100 kGy. The gamma-irradiated BGE showed a greater inhibitory effect on IBMX-induced melanin synthesis (121.43%), intracellular tyrosinase activity (91.80%), melanogenic enzyme expression (tyrosinase, TRP-1, and TRP-2), as well as H2O2-induced reactive oxygen species production, than was observed with the non-irradiated BGE-treated group (melanin synthesis146.21% and intracellular tyrosinase activity-117.11%) in B16F10 melanoma cells without cytotoxicity. In addition, we also found that the anti-melanogenic effect of gamma-irradiated BGE is mediated by the cyclic AMP (cAMP) signaling pathway (MITF and CREB), ERK, and p38 MAPK signaling pathway. Our findings suggest that gamma-irradiated BGE could be a potential anti-melanogenic agent for use in skin-whitening cosmetics. Consequently, gamma irradiation could become an effective tool for developing functional foods and cosmetics.
1. Introduction Korean ginseng (Panax ginseng Meyer) has been used as a valuable herbal medicine for more than 2000 years in East Asia (Yun, 2001). Black ginseng is manufactured from fresh ginseng by a process involving nine periods of steaming and drying (Jo et al., 2009). During this intensive and long manufacturing process, ginseng undergoes a nonenzymatic browning reaction, otherwise known as a Maillard reaction, and the bright yellow color of ginseng changes to a dark black (Suzuki et al., 2004). It has been reported that black ginseng has greater pharmacological effect, including anti-cancer, neuroprotective, and anti-diabetic activity, compared with white and red ginseng (Kim and Kim, 2015; Lee et al., 2011; Kim and Kang, 2009). A recent report by Jin et al. (2015) also revealed that black ginseng has substantial amounts of biological components, such as Maillard reaction products, non-polar ginsenosides, acidic polysaccharides, and phenolic compounds.
Melanin, a major determinant of skin color, is synthesized by melanocytes in the basal layer of the epidermis (Agar and Young, 2005). Melanin is necessary to protect our skin against exposure to ultraviolet radiation (UV), however, abnormal melanin synthesis and accumulation cause a hyperpigmentation disorder (Briganti et al., 2003). The melanogenesis process is regulated through melanogenic enzymes, including tyrosinase, tyrosinase related protein (TRP)-1, and TRP-2 (Slominski et al., 2012). Among these enzymes, tyrosinase is a key enzyme in melanin synthesis because it functions as a catalyst for the rate-limiting reaction of melanogenic process (Chan et al., 2011). Tyrosinase converts tyrosine into dopaquinone, which is oxidized to form 5,6-dihydroxyindole-2-carboxylic acid (DHICA) by TRP-2. Subsequently, the TRP-1 catalyzes the oxidation of DHICA and forms eumelanin (Chan et al., 2014; Costin et al., 2005; Chao et al., 2013). The microphthalamia-associated transcription factor (MITF) is a major transcription factor of tyrosinase and TRPs (Widlund and Fisher, 2003). Recent reports revealed that anti-melanogenic ingredients inhibit
Abbreviations: DPPH, 2,2-diphenyl-1-picrylhydrazyl; ABTS, 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid; FRAP, ferric reducing antioxidant power; AEAC, ascorbic acid equivalent antioxidant capacity; TRP-1, tyrosinase related protein-1; TRP-2, tyrosinase related protein-2; IBMX, 3-isobutyl-1-methylxanthine; MITF, microphthalamia-associated transcription factor; cAMP, cyclic adenosine monophosphate; CREB, cAMP response element binding protein; ERK, extracellular regulated kinase; P38 MAPK, p38 mitogen-activated protein kinases ⁎ Corresponding author. E-mail address: [email protected] (E.-B. Byun). https://doi.org/10.1016/j.radphyschem.2018.03.008 Received 26 July 2017; Received in revised form 2 March 2018; Accepted 18 March 2018 Available online 21 March 2018 0969-806X/ © 2018 Elsevier Ltd. All rights reserved.
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hot water extraction method modified from Park (Park and Yoon, 2017). Then, 40 g of dried black ginseng powder was extracted using 800 mL of distilled water at 80 °C for 2 h. The extract was passed through filter paper No. 4 (GE Healthcare, Uppsala, Sweden) and then freeze-dried. The yield of black ginseng extract (BGE) was 43.13%. The BGE was dissolved in distilled water to a concentration of 10 mg/mL (w/v); then irradiated at doses of 10, 30, 50, 70, and 100 kGy (absorbed doses). This was done using a cobalt-60 irradiator (point source AECL, IR-221, MDS Nordion International Co. Ltd., Ottawa, ON, Canada) with an 11.1 peta Becquerel (PBq) source strength. This was performed at a dose rate of 10 kGy/h in the Advanced Radiation Technology Institute (ARTI), a branch of the Korea Atomic Energy Research Institute (KAERI, Jeoung-Eup, Republic of Korea). The cobalt-60 irradiator was operated with an activity of 3.6 × 105 Ci. Dosimetry was calibrated using 5 mmdiameter alanine dosimeters (Bruker Instruments, Rheinstetten, Germany).
activation of MITF through several signaling pathways, such as mitogen-activated protein kinases (MAPKs) and phosphatidylinositol (PI3K)-3-kinase/Akt signaling pathway (Jang et al., 2011; Oka et al., 2000). It has been reported that oxidative stress induces overproduction of reactive oxygen species (ROS), which are linked to melanogenesis (Kang et al., 2007). Accumulation of ROS leads to oxidative stress-induced damage of melanocytes, resulting in imbalance between ROS and the antioxidant system (Meyskens et al., 1997; Fuchs and Kern, 1998). Various anti-melanogenic agents, such as ascorbic acid, kojic acid, and hydroquinone (Huang et al., 2013; Gonçalez et al., 2013; Briganti et al., 2003) are used in cosmetic and medical applications. However, they have a variety of side effects, for example, ascorbic acid can easily degrade (Spínola et al., 2013); and the long-term use of kojic acid and hydroquinone can trigger allergic reactions (Serra-Baldrich et al., 1998; Tatebayashi et al., 2014). For these reasons, many studies have recently focused on developing antioxidants and whitening agents from natural biomaterials. In the past few decades, radiation technology has been used to enhance the shelf-life and safety of food materials through sterilization of pathogenic microorganisms (Hugo, 1995). In recent years, many scientific approaches have been applied to improvement of the functional properties of natural ingredients using gamma irradiation (Byun et al., 2016, 2015). Various chemical changes occur during the irradiation process, and these reactions are mediated through the radiolysis of water (Cho and Song, 2000). Some previous studies, as illustrated by Oh et al. (2005), have determined that a non-enzymatic browning reaction is induced in a gamma-irradiated aqueous sugar solution. In addition, antioxidant compounds were produced after gamma irradiation of sugar-amino acid combinations (Chawla et al., 2007). Likewise, in our previous study, we observed that gamma irradiation increased the content of arginyl-fructose (AF) in black ginseng extract (BGE); as a result, the anti-allergic effect of BGE was enhanced by gamma irradiation (Kang et al., 2018). The AF produced during the Maillard reaction, has specific physiological activities, including anti-oxidant, antidiabetic, and anti-hypertensive effects (Gao et al., 2012; Lee et al., 2014, 2016). Based on these previous reports, we put forward the hypothesis that gamma irradiation contributes to the increase of antioxidant and anti-melanogenic potential in BGE by increasing AF. Therefore, the aim of this study was to examine the anti-oxidant and anti-melanogenic effect of gamma-irradiated BGE, and explore the potential of using gamma-irradiated BGE as a cosmeceutical for skin whitening.
2.3. Analysis of total carbohydrates, acidic polysaccharides, and reducing sugar The total carbohydrates were measured using a phenol-sulfuric acid method (DuBois et al., 1956). Acidic polysaccharides were determined using a method modified from Bitter and Muir (1962). The reaction mixture: 0.5 mL of gamma-irradiated BGE (1 mg/mL), 0.25 mL of a solution of 0.125% carbazole (in 0.1% ethanol) solution, and 3 mL of sulfuric acid, was incubated for 30 min at room temperature. The absorbance was measured at 525 nm using galacturonic acid as a standard. The reducing sugars were measured using the dinitrosalicyclic acid (DNS) reagent method (Miller, 1959). The reaction mixture, containing 1 mL of gamma-irradiated BGE solution and 3 mL of DNS reagent, was incubated at 98 °C for 5 min; then cooled for 15 min. The absorbance was measured at 550 nm using glucose as a standard. 2.4. Measurement of anti-oxidant activity The free radical scavenging activities of gamma-irradiated BGE were measured using the DPPH and ABTS methods (Blois, 1958; Re et al., 1999). To determine the DPPH radical scavenging activity, 0.1 mM solution of DPPH in methanol was prepared, and 0.1 mL of BGE solution was added. After incubating the mixture at room temperature in the dark for 15 min, the absorbance was measured at 517 nm. To determine the ABTS radical scavenging activity, a mixture of 2.45 mM potassium persulfate and 7 mM ABTS solution was incubated at room temperature in the dark for 16 h to create a stable, dark blue-green radical solution. Then, 50 µL of gamma-irradiated BGE was added to 1 mL of the diluted ABTS solution. After incubating the solution at room temperature in the dark for 30 min, the absorbance was measured at 760 nm. The antioxidant capacity of the gamma-irradiated BGE was determined using a ferric reducing antioxidant power (FRAP) assay as described by Benzie and Strain (Benzie and Strain, 1996) with some modification. Next, 10 mM of 2,4,6-tripyridyl-s-triazine (TPTZ) was dissolved in 40 mM HCl. The working solution was prepared by mixing a 0.3 M acetate buffer (pH 3.6), TPTZ solution, and 20 mM ferric chloride. Gamma-irradiated BGE (50 µL) was added to the FRAP working solution (750 µL) and incubated at room temperature in the dark for 30 min. The absorbance was measured at 593 nm. The FRAP values were expressed as AEAC (ascorbic acid equivalent antioxidant capacity: mg ascorbic acid per 100 g of sample) using an ascorbic standard curve (10–100 µg/mL).
2. Materials and methods 2.1. Materials Arbutin, carbazole; 2,2-diphenyl-1-picrylhydrazyl (DPPH); L-3,4dihydroxy phenylalanine (L-DOPA); 2′, 7′-dichlorofluorescein diacetate (DCFH-DA) phenylmethylsulfonyl fluoride (PMSF); ascorbic acid; thiazolyl blue tetrazolium bromide (MTT); 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS); sulfuric acid; potassium persulfate; and mushroom tyrosinase were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies were obtained from Santa Cruz biotechnology (Santa Cruz, CA, USA) including anti-tyrosinase, anti-TRP1, and anti-TRP-2. Antibodies, including anti-phospho-ERK 1/2, antiERK 1/2, anti-phospho-p38, anti-phospho-CREB, and anti-MITF, were obtained from Cell Signaling Technology (Beverly, MA, USA). Horseradish peroxidase (HRP)-conjugated anti-mouse immunoglobulin G (IgG) and HRP-conjugated anti-rabbit IgG antibodies were obtained from Calbiochem (San Diego, CA, USA).
2.5. Measurement of mushroom tyrosinase inhibition activity The inhibition activity of mushroom tyrosinase was measured using a previous method after slight modification (Alam et al., 2016). Briefly, the reaction mixture, containing a 0.1 M potassium phosphate buffer (pH 6.8) (100 µL), 10 mM of L-DOPA (200 µL), mushroom tyrosinase
2.2. Sample preparation Black ginseng powder was obtained from Sam Kwang Bio Co., Ltd. (Geumsan, Republic of Korea). The black ginseng was extracted using a 34
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membranes were washed, they were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. The proteins were visualized using an ECL advanced kit (GE Healthcare).
(100 unit/mL) (100 µL), and gamma-irradiated BGE (100 µL); was incubated at 37 °C for 30 min. The absorbance was determined at 490 nm. 2.6. Cell culture and cell viability assay
2.11. Statistical analysis B16F10 melanoma cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were maintained in Dulbecco's modified Eagle's medium (Gibco BRL, Carlsbad, CA, USA) supplemented with 10% Fetal bovine serum (Gibco BRL) and 100 units/mL of penicillin/streptomycin (Gibco BRL) at 37 °C in a humidified atmosphere containing 5% CO2. The reagent 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was used to conduct the cell viability assay. Briefly, the cells were seeded into a 96-well plate (1 × 104 cells/well) with non-irradiated- or gamma-irradiated BGE solution and incubated in a 5% CO2 humidified chamber at 37 °C. After incubation for 72 h, the cells were washed, and then treated with 50 µL of MTT solution (0.5 mg/mL in DMEM) for 2 h. The insoluble formazan crystals were dissolved using DMSO. Absorbance was measured at 570 nm using a SpectraMax M3 multi-detect microplate reader.
All the experiments were repeated at least three times with consistent results. The results were presented as mean ± SEM. All statistical analyses were calculated using GraphPad Prism version 5 (Graphpad Prism Software, San Diego, CA, USA). Experimental values were evaluated using a Student's two-tailed t-test. The statistical significance of the differences was defined as *p < 0.05, **p < 0.01, and *** p < 0.001. 3. Results and discussion 3.1. Effect of gamma irradiation on content of total carbohydrates, acidic polysaccharides, and reducing sugar in BGE Gamma irradiation induced a significant increase of reducing sugar content in BGE, whereas acidic polysaccharide content was relatively unaffected, as shown in Table 1. The total carbohydrate content also increased at the dose of 10 kGy, but total carbohydrates did not increase at doses ranging from 30 to 100 kGy. The content of reducing sugar gradually increased with rise in the irradiation dose, and the highest content of reducing sugars in BGE was observed at doses of 70 and 100 kGy. It has been reported that increase of the reducing sugar can induce accelerated production of Maillard reaction products (MRPs) (Ghafoor et al., 2012). The MRPs are generated by a non-enzymatic glycation reaction between the amino group of free amino acids and the carbonyl group of reducing sugars, which contribute to aroma, flavor, taste, and color, as well as to functionality (Sun et al., 2009). It is already known that steamed ginseng has various MRPs, such as arginylfructose, arginyl-fructosyl-glucose, and maltol, which have shown a variety of biological activities (Gao et al., 2012; Han et al., 1981). Yaylayan (1997) revealed that generation of Amadori rearrangement products or Heyns products (belonging to the MRPs) is associated with the richness of reducing sugar. It has been reported that gamma irradiation induces the breakage of glycosidic linkages in disaccharides and starch, which contributes to generation of the Maillard reaction products (Oh et al., 2005). In our previous study, we observed that gammairradiation increased a specific Maillard reaction product (AF) in the BGE (Kang et al., 2018). In this regard, we can put forward that reducing sugars increased by gamma irradiation promote formation of AF through an amino-carbonyl reaction (Ryu et al., 2001).
2.7. Measurement of ROS production The cells were cultured in a 24-well plate and treated with nonirradiated- or 100 kGy gamma-irradiated BGE for 24 h. The cells were stimulated by 24 mM H2O2 at 37 °C for 30 min. The cells were stained with 10 μM DCFH-DA for 20 min, and then washed with a potassium phosphate buffer (PBS, Gibco BRL). The stained cells were immediately observed using a multispectral imaging system: Nuance FX camera and Nuance 2.10 software (Cri, Woburn, MA, USA). 2.8. Measurement of melanin content and intracellular tyrosinase activity B16F10 melanoma cells were seeded in a 6-well plate (5 × 104 cells/mL). After 4 h, the cells were stimulated with IBMX (50 μM); then treated with non-irradiated- or 100 kGy gamma-irradiated BGE for 72 h. After media removal, the cells were lysed with 1 N NaOH in 10% DMSO at 80 °C for 1 h. Absorbance was measured at 405 nm. To determine the intracellular tyrosinase activity, the cells were lysed with PBS containing 1% triton X-100 and 1 mM PMSF. The lysates were centrifuged at 13,000 rpm for 15 min. Then 90 μg of lysate was placed in each well of a 96-well plate, and the enzymatic assay was initiated by adding 10 µL of 1 mM L-DOPA. After incubation, the generated dopachrome was measured at an absorbance of 475 nm. 2.9. Measurement of intracellular cAMP level
3.2. Effects of gamma irradiation on anti-oxidant activity and mushroom tyrosinase inhibition activity in BGE
Measurement of the cAMP level was carried out according to the manufacturer protocol in the ELISA kit (Cell Biolabs, San Diego, CA, USA). The cells were treated with non-irradiated- or 100 kGy gammairradiated BGE and IBMX (50 μM) for 24 h. The cells were lysed in 0.1 M HCl to inhibit phosphodiesterase activity, and the reaction was carried out as described in the manufacturer protocol in the ELISA kit.
Arginyl-fructose (AF) is produced by reaction between glucose and arginine during the Maillard reaction, and has strong anti-oxidant Table 1 Total sugar, acidic polysaccharides, and reducing sugar contents of gammairradiated BGE.
2.10. Western blot analysis For the western blot analysis, the cells were lysed using an NP-40 lysis buffer (Invitrogen, Carlsbad, CA, USA) containing 1 mM PMSF. The cell lysates were incubated on ice for 30 min, and then centrifuged at 13,000 rpm for 20 min at 4 °C. The protein concentration of lysates was determined by the bicinichonic acid assay (Thermo scientific, Rockford, IL, USA). The lysates were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to a polyvinylidene difluoride membrane (PVDF, Invitrogen). The PVDF membrane was blocked with 5% skim milk, and incubated overnight at 4 °C with several primary antibodies. After the
Irradiation dose (kGy)
Total sugar
0 10 30 50 70 100
5.85 6.52 6.51 6.29 6.76 6.24
± ± ± ± ± ±
0.337b 0.460a 0.538a 0.644a 0.381a 0.264a
Acidic polysaccharides 33.70 31.42 33.02 34.60 31.94 31.57
± ± ± ± ± ±
1.348a 0.321a 0.275a 0.772a 0.835a 0.639a
Reducing sugar
22.39 28.06 32.16 36.54 38.18 42.89
± ± ± ± ± ±
0.161e 0.773d 0.097c 0.580b 1.063a 1.095a
Values represent means of three replicates ± SEM. Value with different letters (a–e) within the same column differ significantly (P < 0.05). 35
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(protons) or electrons (Alam et al., 2016). The DPPH radical scavenging activity was determined for non-irradiated- or gamma-irradiated BGE at doses ranging from 6.25 to 50 µg/mL. As shown in Table 2, 100 kGy gamma-irradiated BGE shows greater DPPH radical scavenging activity (IC50 = 4.33 µg) than non-irradiated BGE (IC50 = 2.39 µg) does. The ABTS radical scavenging activity of BGE (dose ranging from 0.125 to 1 mg/mL) also increased from IC50 = 3.82 mg to IC50 = 2.53 mg. Similarly, the 100 kGy gamma-irradiated BGE showed stronger FRAP value (6.46 g AA/100 g) than did non-irradiated BGE (4.30 g AA/100 g). To investigate further whether increasing anti-oxidant activity is associated with anti-melanogenic potential, we measured the mushroom tyrosinase inhibition activity of gamma-irradiated BGE. Interestingly, the mushroom tyrosinase inhibition activity of gamma-irradiated BGE increased with increasing irradiation dose (to 100 kGy), while non-irradiated BGE did not show any inhibitory effect on mushroom tyrosinase (Fig. 1C). These findings are consistent with previous studies showing that increasing the anti-oxidant capacity led to anti-melanogenic effects (Huang et al., 2011; Peng et al., 2014). This is thought to be caused primarily by the increase of AF through the gamma irradiation process (Kang et al., 2018). We further investigated
Table 2 Anti-oxidant and tyrosinase inhibition activity of gamma-irradiated BGE. Irradiation dose (kGy)
DPPH radical scavenging activity (IC50)
ABTS radical scavenging activity (IC50)
FRAP AEAC (g AA/ 100 g)
Tyrosinase inhibition activity (IC50)
0 10 30 50 70 100
39.43 µg 39.61 µg 38.72 µg 33.21 µg 10.01 µg 4.33 µg
3.82 mg 3.31 mg 3.09 mg 2.95 mg 2.53 mg 2.39 mg
4.30 4.72 4.69 4.97 5.64 6.46
– – 48.66 mg 43.51 mg 14.78 mg 12.46 mg
AEAC = Ascorbic acid equivalent antioxidant capacity: mg ascorbic acid per 100 g of sample.
capacity (Lee et al., 2009). Therefore, as a first step to confirming the effect of gamma irradiation on the increase of anti-oxidant capacity in BGE, we measured DPPH and ABTS radical scavenging activity. Antioxidant activity can be regarded as the scavenging activity of free radicals, reactive oxygen, and nitrogen species by donation of hydrogen
Fig. 1. Effect of 100 kGy gamma-irradiated BGE on cell viability and H2O2-induced ROS production in B16F10 melanoma cells. The cell viability was measured by MTT assay (A). The data represent the mean ± SEM (n = 3). Statistical analysis was performed by unpaired Student's two-tailed t-test within * P < 0.05 compared to control group. Inhibitory effect of gamma-irradiated BGE on H2O2-induced ROS production (B). The data represent the mean ± SEM (n = 3). Statistical analysis was performed by unpaired Student's two-tailed t-test within *P < 0.05, **P < 0.01, and ***P < 0.001 compared to only H2O2-treated group. 36
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Fig. 2. Anti-melanogenic effects of gamma-irradiated BGE in IBMX-stimulated B16F10 melanoma cells. Inhibitory effect of gamma-irradiated BGE on melanin production (A) and intracellular tyrosinase activity (B) and melanogenic enzymes expression (C) in IBMX-stimulated B16F10 melanoma cells. B16F10 melanoma cells treated for 72 h with 0 kGy- or 100 kGy gamma-irradiated BGE at specific concentrations (125 and 250 μg/mL). Arbutin (500 μg/mL) was used as a positive control. The data represent the mean ± SEM (n = 3). Statistical analysis was performed by Student's two-tailed t-test within ##P < 0.01, and ###P < 0.001 compared to control group, and **P < 0.01, and ***P < 0.001 compared to the only IBMX-treated group.
cutaneous tissue causes lipid peroxidation and inactivation of antioxidant enzymes, which leads to skin aging (Kohen, 1999). In the melanogenic process, H2O2 is overproduced, and this triggers a cascade of events leading to overproduction of ROS, lipid peroxidation, and inactivation of antioxidant enzymes (Pandel et al., 2013). Tyrosinase is a major melanogenic enzyme that converts L-tyrosine to L-DOPA and dopaquinone (Chan et al., 2014). After the formation of dopaquinone, dopachrome is oxidized into 5,6-dihydroxyindole-2-carboxylic acid (DHICA) by TRP-2. Subsequently, indole-2-carboxilic acid5,6-quinone is formed from DHICA by TRP-1 (Costin et al., 2005; Park et al., 2011). In view of these findings, we can infer that gamma-irradiated BGE inhibits melanogenesis by regulating ROS overproduction and suppressing melanogenic enzymes, including tyrosinase, TRP-1, and TRP-2.
the anti-melanogenic activity of gamma-irradiated BGE in B16F10 melanoma cells. 3.3. Effect of gamma-irradiated BGE on melanogenesis in B16F10 melanoma cells To determine the anti-melanogenic effect of gamma-irradiated BGE, the 100 kGy dose of BGE and non-irradiated and were chosen because the highest anti-oxidant activity and tyrosinase inhibition activity were observed at 100 kGy. Prior to investigating the anti-melanogenic effect of gamma-irradiated BGE, the cell viability was tested using a MTT assay. We observed that both non-irradiated- and 100 kGy gamma-irradiated BGE did not induce cytotoxicity in B16F10 melanoma cells within the concentration range 31.25–250 μg/mL (Fig. 2A). At noncytotoxic concentrations, 100 kGy gamma-irradiated BGE showed a much stronger inhibitory effect on ROS production (125 μg/mL and 250 μg/mL, 46.61 ± 4.57 and 22.50 ± 3.93%) than did non-irradiated BGE (125 and 250 μg/mL; 81.96 ± 8.08 and 61.49 ± 9.25%) in H2O2-stimulated B16F10 melanoma cells (Fig. 2B). A significantly lower percentage of melanin contents in the 100 kGy BGE-treated group (125 and 250 μg/mL; 132.98 ± 6.94 and 121.43 ± 8.58%) compared with the group treated with IBMX only (161.44 ± 8.20%, Fig. 2A). Similarly, the 100 kGy gamma-irradiated BGE treatment group showed efficiently inhibited intracellular tyrosinase activity (125 and 250 μg/ mL; 105.84 ± 1.94 and 91.80 ± 9.22%; see Fig. 2B) than did the group treated with IBMX only (127.97 ± 7.69%). The expression of melanogenic enzymes, including tyrosinase, TRP, and TRP-2, was effectively reduced by treatment with 100 kGy gamma-irradiated BGE of IBMX-stimulated B16F10 melanoma cells (Fig. 3). Melanogenesis is closely related to oxidative stress, in particular, excess ROS levels in
3.4. Effects of gamma-irradiated BGE on the cAMP/ERK/p38 signaling pathway Several 3′,5′-cyclic adenosine monophosphate (cAMP) elevating agents, such as IBMX, forskolin, and α-melanocyte stimulating hormone, are common inducers of melanogenesis for evaluating the value of a product as a skin whitening product (Englaro et al., 1995). Among them, IBMX is a strong stimulus for inducing melanogenesis, and is widely used as an inducer of melanogenesis in cell culture (Brown et al., 1998). Thus, we investigated whether gamma-irradiated BGE has antimelanogenic activity through regulation of the cAMP signaling pathway. As shown in Fig. 3A, 100 kGy gamma-irradiated BGE significantly reduced the intracellular cAMP level (125 and 250 μg/mL; 67.64 ± 6.04 and 42.24 ± 11.78%; Fig. 3B) compared with the IBMX-only treatment group (146.37 ± 40.24%) in IBMX-stimulated 37
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Fig. 3. Effect of gamma-irradiated BGE on melanogenesis-related signaling pathways. B16F10 cells were treated with 0 kGy- or 100 kGy gamma-irradiated BGE (125 and 250 μg/mL) with IBMX (50 μM) for 1 h (A) Statistical analysis was performed by Student's two-tailed t-test within #P < 0.05 compared to control group, and *P < 0.05 compared to the only IBMX-treated group. The expression levels of p-CREB and MITF proteins were determined by Western blotting at 2 h after treatment (B). Time-dependent effect of gamma-irradiated BGE on the expression of p-ERK and p-p38 (C). Effect of ERK (PD98059) and p38 (SB203580) inhibitors on gamma-irradiated BGE-treated B16F10 cells (D). Statistical analysis was performed by Student's two-tailed t-test within ***P < 0.001 compared to the IBMX and 100 kGy gamma-irradiated BGE-treated group.
4. Conclusions
B16F10 cells. Likewise, the protein expression of phospho-CREB and MITF was significantly decreased by treatment with 100 kGy gammairradiated BGE (Fig. 3B). The elevated cAMP level led to phosphorylation of the cAMP response element binding protein (CREB), which binds to and activates the promoter of microphthalamia-associated transcription factor (MITF) (Park et al., 2006). MITF promotes the melanogenic pathway as a major transcription factor of the tyrosinase gene, and as a result, activated MITF promotes the melanogenesis pathway (Levy et al., 2006). On the other hand, previous reports demonstrated that ERK and p38 signaling pathways are associated with the inhibition of melanogenesis because activated ERK and p-38 block the binding of the tyrosinase promoter site to the MITF-M box (Jang et al., 2011; Ko et al., 2014). To investigate whether gamma-irradiated BGE has an effect on activating ERK and p38, we performed a western blot analysis. Treatment of 100 kGy gamma-irradiated BGE induced phosphorylation of ERK and p38 in a time-dependent manner (Fig. 3C). To rule out that the possibility of ERK and p38 signaling on the anti-melanogenic effect of gamma-irradiated BGE, we used an ERK (PD98059) and p38 (SB203580) inhibitors. As shown in Fig. 3D, the anti-melanogenic effect of gamma-irradiated BGE was hindered through the treatment of PD98059 and SB203580. These results indicate that the ERK and p38 signaling pathway was importantly involved in anti-melanogenic activity of gamma-irradiated BGE, and these findings are consistent with previous studies showing that various natural biomaterials inhibit melanogenesis by activating ERK and the p38 signaling pathway (Baek and Lee, 2015; Alam et al., 2016).
In this study, black ginseng extract was exposed to gamma irradiation to improve it for utilization in the cosmetic industry. The reducing sugar content was increased with increase in the irradiation dose to 100 kGy, and it may be speculated that the production of Maillard reaction products was accelerated through gamma irradiation. The anti-oxidant properties and anti-melanogenic activity of black ginseng extract were improved through gamma irradiation. The antimelanogenic action of gamma-irradiated black ginseng extract is mediated by the cAMP/MITF signaling pathway. This action is attributed to activation of the ERK and p38 signaling pathway. Consequently, gamma irradiation could be a useful tool for improving the utilization of black ginseng extract in the cosmetic industry. However, further study will be necessary to characterize the Maillard reaction products of gamma-irradiated black ginseng and to confirm the anti-melanogenic activity of refined Maillard reaction products in an artificial skin model. Acknowledgements This work was supported by the National Research Foundation of Korea grant funded by Korea government (Grant no. 2017-02-EE-004) and Nuclear R&D Program of the Ministry of Science and ICT (MSIT), Republic of Korea. References Agar, N., Young, A.R., 2005. Melanogenesis: a photoprotective response to DNA damage? Mutat. Res. 571, 121–132.
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Effect of gamma irradiation on the anti-oxidant and anti-melanogenic activity of black ginseng extract in B16F10 melanoma cells
T
Ha-Yeon Songa,b, Hye-Min Kima, Woo Sik Kima, Eui-Hong Byunb, Beom-Su Janga, ⁎ Dae Seong Choia, Eui-Baek Byuna, a b
Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup 580-185, Republic of Korea Department of Food Science and Technology, Kongju National University, Yesan 340-800, Republic of Korea
A B S T R A C T
Black ginseng extract (BGE) was irradiated at various doses (10, 30, 50, 70, and 100 kGy) to improve its biological activity. The content of reducing sugar was significantly increased by gamma irradiation up to 100 kGy. The gamma-irradiated BGE showed its highest anti-oxidant activity (DPPH radical scavenging activity-IC50 = 4.33 µg, ABTS radical scavenging activity-IC50 = 2.39 µg, FRAP AEAC = 6.46), and mushroom tyrosinase inhibition activity (IC50 = 12.46 mg) when irradiated at the dose of 100 kGy. The gamma-irradiated BGE showed a greater inhibitory effect on IBMX-induced melanin synthesis (121.43%), intracellular tyrosinase activity (91.80%), melanogenic enzyme expression (tyrosinase, TRP-1, and TRP-2), as well as H2O2-induced reactive oxygen species production, than was observed with the non-irradiated BGE-treated group (melanin synthesis146.21% and intracellular tyrosinase activity-117.11%) in B16F10 melanoma cells without cytotoxicity. In addition, we also found that the anti-melanogenic effect of gamma-irradiated BGE is mediated by the cyclic AMP (cAMP) signaling pathway (MITF and CREB), ERK, and p38 MAPK signaling pathway. Our findings suggest that gamma-irradiated BGE could be a potential anti-melanogenic agent for use in skin-whitening cosmetics. Consequently, gamma irradiation could become an effective tool for developing functional foods and cosmetics.
1. Introduction Korean ginseng (Panax ginseng Meyer) has been used as a valuable herbal medicine for more than 2000 years in East Asia (Yun, 2001). Black ginseng is manufactured from fresh ginseng by a process involving nine periods of steaming and drying (Jo et al., 2009). During this intensive and long manufacturing process, ginseng undergoes a nonenzymatic browning reaction, otherwise known as a Maillard reaction, and the bright yellow color of ginseng changes to a dark black (Suzuki et al., 2004). It has been reported that black ginseng has greater pharmacological effect, including anti-cancer, neuroprotective, and anti-diabetic activity, compared with white and red ginseng (Kim and Kim, 2015; Lee et al., 2011; Kim and Kang, 2009). A recent report by Jin et al. (2015) also revealed that black ginseng has substantial amounts of biological components, such as Maillard reaction products, non-polar ginsenosides, acidic polysaccharides, and phenolic compounds.
Melanin, a major determinant of skin color, is synthesized by melanocytes in the basal layer of the epidermis (Agar and Young, 2005). Melanin is necessary to protect our skin against exposure to ultraviolet radiation (UV), however, abnormal melanin synthesis and accumulation cause a hyperpigmentation disorder (Briganti et al., 2003). The melanogenesis process is regulated through melanogenic enzymes, including tyrosinase, tyrosinase related protein (TRP)-1, and TRP-2 (Slominski et al., 2012). Among these enzymes, tyrosinase is a key enzyme in melanin synthesis because it functions as a catalyst for the rate-limiting reaction of melanogenic process (Chan et al., 2011). Tyrosinase converts tyrosine into dopaquinone, which is oxidized to form 5,6-dihydroxyindole-2-carboxylic acid (DHICA) by TRP-2. Subsequently, the TRP-1 catalyzes the oxidation of DHICA and forms eumelanin (Chan et al., 2014; Costin et al., 2005; Chao et al., 2013). The microphthalamia-associated transcription factor (MITF) is a major transcription factor of tyrosinase and TRPs (Widlund and Fisher, 2003). Recent reports revealed that anti-melanogenic ingredients inhibit
Abbreviations: DPPH, 2,2-diphenyl-1-picrylhydrazyl; ABTS, 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid; FRAP, ferric reducing antioxidant power; AEAC, ascorbic acid equivalent antioxidant capacity; TRP-1, tyrosinase related protein-1; TRP-2, tyrosinase related protein-2; IBMX, 3-isobutyl-1-methylxanthine; MITF, microphthalamia-associated transcription factor; cAMP, cyclic adenosine monophosphate; CREB, cAMP response element binding protein; ERK, extracellular regulated kinase; P38 MAPK, p38 mitogen-activated protein kinases ⁎ Corresponding author. E-mail address: [email protected] (E.-B. Byun). https://doi.org/10.1016/j.radphyschem.2018.03.008 Received 26 July 2017; Received in revised form 2 March 2018; Accepted 18 March 2018 Available online 21 March 2018 0969-806X/ © 2018 Elsevier Ltd. All rights reserved.
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hot water extraction method modified from Park (Park and Yoon, 2017). Then, 40 g of dried black ginseng powder was extracted using 800 mL of distilled water at 80 °C for 2 h. The extract was passed through filter paper No. 4 (GE Healthcare, Uppsala, Sweden) and then freeze-dried. The yield of black ginseng extract (BGE) was 43.13%. The BGE was dissolved in distilled water to a concentration of 10 mg/mL (w/v); then irradiated at doses of 10, 30, 50, 70, and 100 kGy (absorbed doses). This was done using a cobalt-60 irradiator (point source AECL, IR-221, MDS Nordion International Co. Ltd., Ottawa, ON, Canada) with an 11.1 peta Becquerel (PBq) source strength. This was performed at a dose rate of 10 kGy/h in the Advanced Radiation Technology Institute (ARTI), a branch of the Korea Atomic Energy Research Institute (KAERI, Jeoung-Eup, Republic of Korea). The cobalt-60 irradiator was operated with an activity of 3.6 × 105 Ci. Dosimetry was calibrated using 5 mmdiameter alanine dosimeters (Bruker Instruments, Rheinstetten, Germany).
activation of MITF through several signaling pathways, such as mitogen-activated protein kinases (MAPKs) and phosphatidylinositol (PI3K)-3-kinase/Akt signaling pathway (Jang et al., 2011; Oka et al., 2000). It has been reported that oxidative stress induces overproduction of reactive oxygen species (ROS), which are linked to melanogenesis (Kang et al., 2007). Accumulation of ROS leads to oxidative stress-induced damage of melanocytes, resulting in imbalance between ROS and the antioxidant system (Meyskens et al., 1997; Fuchs and Kern, 1998). Various anti-melanogenic agents, such as ascorbic acid, kojic acid, and hydroquinone (Huang et al., 2013; Gonçalez et al., 2013; Briganti et al., 2003) are used in cosmetic and medical applications. However, they have a variety of side effects, for example, ascorbic acid can easily degrade (Spínola et al., 2013); and the long-term use of kojic acid and hydroquinone can trigger allergic reactions (Serra-Baldrich et al., 1998; Tatebayashi et al., 2014). For these reasons, many studies have recently focused on developing antioxidants and whitening agents from natural biomaterials. In the past few decades, radiation technology has been used to enhance the shelf-life and safety of food materials through sterilization of pathogenic microorganisms (Hugo, 1995). In recent years, many scientific approaches have been applied to improvement of the functional properties of natural ingredients using gamma irradiation (Byun et al., 2016, 2015). Various chemical changes occur during the irradiation process, and these reactions are mediated through the radiolysis of water (Cho and Song, 2000). Some previous studies, as illustrated by Oh et al. (2005), have determined that a non-enzymatic browning reaction is induced in a gamma-irradiated aqueous sugar solution. In addition, antioxidant compounds were produced after gamma irradiation of sugar-amino acid combinations (Chawla et al., 2007). Likewise, in our previous study, we observed that gamma irradiation increased the content of arginyl-fructose (AF) in black ginseng extract (BGE); as a result, the anti-allergic effect of BGE was enhanced by gamma irradiation (Kang et al., 2018). The AF produced during the Maillard reaction, has specific physiological activities, including anti-oxidant, antidiabetic, and anti-hypertensive effects (Gao et al., 2012; Lee et al., 2014, 2016). Based on these previous reports, we put forward the hypothesis that gamma irradiation contributes to the increase of antioxidant and anti-melanogenic potential in BGE by increasing AF. Therefore, the aim of this study was to examine the anti-oxidant and anti-melanogenic effect of gamma-irradiated BGE, and explore the potential of using gamma-irradiated BGE as a cosmeceutical for skin whitening.
2.3. Analysis of total carbohydrates, acidic polysaccharides, and reducing sugar The total carbohydrates were measured using a phenol-sulfuric acid method (DuBois et al., 1956). Acidic polysaccharides were determined using a method modified from Bitter and Muir (1962). The reaction mixture: 0.5 mL of gamma-irradiated BGE (1 mg/mL), 0.25 mL of a solution of 0.125% carbazole (in 0.1% ethanol) solution, and 3 mL of sulfuric acid, was incubated for 30 min at room temperature. The absorbance was measured at 525 nm using galacturonic acid as a standard. The reducing sugars were measured using the dinitrosalicyclic acid (DNS) reagent method (Miller, 1959). The reaction mixture, containing 1 mL of gamma-irradiated BGE solution and 3 mL of DNS reagent, was incubated at 98 °C for 5 min; then cooled for 15 min. The absorbance was measured at 550 nm using glucose as a standard. 2.4. Measurement of anti-oxidant activity The free radical scavenging activities of gamma-irradiated BGE were measured using the DPPH and ABTS methods (Blois, 1958; Re et al., 1999). To determine the DPPH radical scavenging activity, 0.1 mM solution of DPPH in methanol was prepared, and 0.1 mL of BGE solution was added. After incubating the mixture at room temperature in the dark for 15 min, the absorbance was measured at 517 nm. To determine the ABTS radical scavenging activity, a mixture of 2.45 mM potassium persulfate and 7 mM ABTS solution was incubated at room temperature in the dark for 16 h to create a stable, dark blue-green radical solution. Then, 50 µL of gamma-irradiated BGE was added to 1 mL of the diluted ABTS solution. After incubating the solution at room temperature in the dark for 30 min, the absorbance was measured at 760 nm. The antioxidant capacity of the gamma-irradiated BGE was determined using a ferric reducing antioxidant power (FRAP) assay as described by Benzie and Strain (Benzie and Strain, 1996) with some modification. Next, 10 mM of 2,4,6-tripyridyl-s-triazine (TPTZ) was dissolved in 40 mM HCl. The working solution was prepared by mixing a 0.3 M acetate buffer (pH 3.6), TPTZ solution, and 20 mM ferric chloride. Gamma-irradiated BGE (50 µL) was added to the FRAP working solution (750 µL) and incubated at room temperature in the dark for 30 min. The absorbance was measured at 593 nm. The FRAP values were expressed as AEAC (ascorbic acid equivalent antioxidant capacity: mg ascorbic acid per 100 g of sample) using an ascorbic standard curve (10–100 µg/mL).
2. Materials and methods 2.1. Materials Arbutin, carbazole; 2,2-diphenyl-1-picrylhydrazyl (DPPH); L-3,4dihydroxy phenylalanine (L-DOPA); 2′, 7′-dichlorofluorescein diacetate (DCFH-DA) phenylmethylsulfonyl fluoride (PMSF); ascorbic acid; thiazolyl blue tetrazolium bromide (MTT); 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS); sulfuric acid; potassium persulfate; and mushroom tyrosinase were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies were obtained from Santa Cruz biotechnology (Santa Cruz, CA, USA) including anti-tyrosinase, anti-TRP1, and anti-TRP-2. Antibodies, including anti-phospho-ERK 1/2, antiERK 1/2, anti-phospho-p38, anti-phospho-CREB, and anti-MITF, were obtained from Cell Signaling Technology (Beverly, MA, USA). Horseradish peroxidase (HRP)-conjugated anti-mouse immunoglobulin G (IgG) and HRP-conjugated anti-rabbit IgG antibodies were obtained from Calbiochem (San Diego, CA, USA).
2.5. Measurement of mushroom tyrosinase inhibition activity The inhibition activity of mushroom tyrosinase was measured using a previous method after slight modification (Alam et al., 2016). Briefly, the reaction mixture, containing a 0.1 M potassium phosphate buffer (pH 6.8) (100 µL), 10 mM of L-DOPA (200 µL), mushroom tyrosinase
2.2. Sample preparation Black ginseng powder was obtained from Sam Kwang Bio Co., Ltd. (Geumsan, Republic of Korea). The black ginseng was extracted using a 34
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membranes were washed, they were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. The proteins were visualized using an ECL advanced kit (GE Healthcare).
(100 unit/mL) (100 µL), and gamma-irradiated BGE (100 µL); was incubated at 37 °C for 30 min. The absorbance was determined at 490 nm. 2.6. Cell culture and cell viability assay
2.11. Statistical analysis B16F10 melanoma cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were maintained in Dulbecco's modified Eagle's medium (Gibco BRL, Carlsbad, CA, USA) supplemented with 10% Fetal bovine serum (Gibco BRL) and 100 units/mL of penicillin/streptomycin (Gibco BRL) at 37 °C in a humidified atmosphere containing 5% CO2. The reagent 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was used to conduct the cell viability assay. Briefly, the cells were seeded into a 96-well plate (1 × 104 cells/well) with non-irradiated- or gamma-irradiated BGE solution and incubated in a 5% CO2 humidified chamber at 37 °C. After incubation for 72 h, the cells were washed, and then treated with 50 µL of MTT solution (0.5 mg/mL in DMEM) for 2 h. The insoluble formazan crystals were dissolved using DMSO. Absorbance was measured at 570 nm using a SpectraMax M3 multi-detect microplate reader.
All the experiments were repeated at least three times with consistent results. The results were presented as mean ± SEM. All statistical analyses were calculated using GraphPad Prism version 5 (Graphpad Prism Software, San Diego, CA, USA). Experimental values were evaluated using a Student's two-tailed t-test. The statistical significance of the differences was defined as *p < 0.05, **p < 0.01, and *** p < 0.001. 3. Results and discussion 3.1. Effect of gamma irradiation on content of total carbohydrates, acidic polysaccharides, and reducing sugar in BGE Gamma irradiation induced a significant increase of reducing sugar content in BGE, whereas acidic polysaccharide content was relatively unaffected, as shown in Table 1. The total carbohydrate content also increased at the dose of 10 kGy, but total carbohydrates did not increase at doses ranging from 30 to 100 kGy. The content of reducing sugar gradually increased with rise in the irradiation dose, and the highest content of reducing sugars in BGE was observed at doses of 70 and 100 kGy. It has been reported that increase of the reducing sugar can induce accelerated production of Maillard reaction products (MRPs) (Ghafoor et al., 2012). The MRPs are generated by a non-enzymatic glycation reaction between the amino group of free amino acids and the carbonyl group of reducing sugars, which contribute to aroma, flavor, taste, and color, as well as to functionality (Sun et al., 2009). It is already known that steamed ginseng has various MRPs, such as arginylfructose, arginyl-fructosyl-glucose, and maltol, which have shown a variety of biological activities (Gao et al., 2012; Han et al., 1981). Yaylayan (1997) revealed that generation of Amadori rearrangement products or Heyns products (belonging to the MRPs) is associated with the richness of reducing sugar. It has been reported that gamma irradiation induces the breakage of glycosidic linkages in disaccharides and starch, which contributes to generation of the Maillard reaction products (Oh et al., 2005). In our previous study, we observed that gammairradiation increased a specific Maillard reaction product (AF) in the BGE (Kang et al., 2018). In this regard, we can put forward that reducing sugars increased by gamma irradiation promote formation of AF through an amino-carbonyl reaction (Ryu et al., 2001).
2.7. Measurement of ROS production The cells were cultured in a 24-well plate and treated with nonirradiated- or 100 kGy gamma-irradiated BGE for 24 h. The cells were stimulated by 24 mM H2O2 at 37 °C for 30 min. The cells were stained with 10 μM DCFH-DA for 20 min, and then washed with a potassium phosphate buffer (PBS, Gibco BRL). The stained cells were immediately observed using a multispectral imaging system: Nuance FX camera and Nuance 2.10 software (Cri, Woburn, MA, USA). 2.8. Measurement of melanin content and intracellular tyrosinase activity B16F10 melanoma cells were seeded in a 6-well plate (5 × 104 cells/mL). After 4 h, the cells were stimulated with IBMX (50 μM); then treated with non-irradiated- or 100 kGy gamma-irradiated BGE for 72 h. After media removal, the cells were lysed with 1 N NaOH in 10% DMSO at 80 °C for 1 h. Absorbance was measured at 405 nm. To determine the intracellular tyrosinase activity, the cells were lysed with PBS containing 1% triton X-100 and 1 mM PMSF. The lysates were centrifuged at 13,000 rpm for 15 min. Then 90 μg of lysate was placed in each well of a 96-well plate, and the enzymatic assay was initiated by adding 10 µL of 1 mM L-DOPA. After incubation, the generated dopachrome was measured at an absorbance of 475 nm. 2.9. Measurement of intracellular cAMP level
3.2. Effects of gamma irradiation on anti-oxidant activity and mushroom tyrosinase inhibition activity in BGE
Measurement of the cAMP level was carried out according to the manufacturer protocol in the ELISA kit (Cell Biolabs, San Diego, CA, USA). The cells were treated with non-irradiated- or 100 kGy gammairradiated BGE and IBMX (50 μM) for 24 h. The cells were lysed in 0.1 M HCl to inhibit phosphodiesterase activity, and the reaction was carried out as described in the manufacturer protocol in the ELISA kit.
Arginyl-fructose (AF) is produced by reaction between glucose and arginine during the Maillard reaction, and has strong anti-oxidant Table 1 Total sugar, acidic polysaccharides, and reducing sugar contents of gammairradiated BGE.
2.10. Western blot analysis For the western blot analysis, the cells were lysed using an NP-40 lysis buffer (Invitrogen, Carlsbad, CA, USA) containing 1 mM PMSF. The cell lysates were incubated on ice for 30 min, and then centrifuged at 13,000 rpm for 20 min at 4 °C. The protein concentration of lysates was determined by the bicinichonic acid assay (Thermo scientific, Rockford, IL, USA). The lysates were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to a polyvinylidene difluoride membrane (PVDF, Invitrogen). The PVDF membrane was blocked with 5% skim milk, and incubated overnight at 4 °C with several primary antibodies. After the
Irradiation dose (kGy)
Total sugar
0 10 30 50 70 100
5.85 6.52 6.51 6.29 6.76 6.24
± ± ± ± ± ±
0.337b 0.460a 0.538a 0.644a 0.381a 0.264a
Acidic polysaccharides 33.70 31.42 33.02 34.60 31.94 31.57
± ± ± ± ± ±
1.348a 0.321a 0.275a 0.772a 0.835a 0.639a
Reducing sugar
22.39 28.06 32.16 36.54 38.18 42.89
± ± ± ± ± ±
0.161e 0.773d 0.097c 0.580b 1.063a 1.095a
Values represent means of three replicates ± SEM. Value with different letters (a–e) within the same column differ significantly (P < 0.05). 35
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(protons) or electrons (Alam et al., 2016). The DPPH radical scavenging activity was determined for non-irradiated- or gamma-irradiated BGE at doses ranging from 6.25 to 50 µg/mL. As shown in Table 2, 100 kGy gamma-irradiated BGE shows greater DPPH radical scavenging activity (IC50 = 4.33 µg) than non-irradiated BGE (IC50 = 2.39 µg) does. The ABTS radical scavenging activity of BGE (dose ranging from 0.125 to 1 mg/mL) also increased from IC50 = 3.82 mg to IC50 = 2.53 mg. Similarly, the 100 kGy gamma-irradiated BGE showed stronger FRAP value (6.46 g AA/100 g) than did non-irradiated BGE (4.30 g AA/100 g). To investigate further whether increasing anti-oxidant activity is associated with anti-melanogenic potential, we measured the mushroom tyrosinase inhibition activity of gamma-irradiated BGE. Interestingly, the mushroom tyrosinase inhibition activity of gamma-irradiated BGE increased with increasing irradiation dose (to 100 kGy), while non-irradiated BGE did not show any inhibitory effect on mushroom tyrosinase (Fig. 1C). These findings are consistent with previous studies showing that increasing the anti-oxidant capacity led to anti-melanogenic effects (Huang et al., 2011; Peng et al., 2014). This is thought to be caused primarily by the increase of AF through the gamma irradiation process (Kang et al., 2018). We further investigated
Table 2 Anti-oxidant and tyrosinase inhibition activity of gamma-irradiated BGE. Irradiation dose (kGy)
DPPH radical scavenging activity (IC50)
ABTS radical scavenging activity (IC50)
FRAP AEAC (g AA/ 100 g)
Tyrosinase inhibition activity (IC50)
0 10 30 50 70 100
39.43 µg 39.61 µg 38.72 µg 33.21 µg 10.01 µg 4.33 µg
3.82 mg 3.31 mg 3.09 mg 2.95 mg 2.53 mg 2.39 mg
4.30 4.72 4.69 4.97 5.64 6.46
– – 48.66 mg 43.51 mg 14.78 mg 12.46 mg
AEAC = Ascorbic acid equivalent antioxidant capacity: mg ascorbic acid per 100 g of sample.
capacity (Lee et al., 2009). Therefore, as a first step to confirming the effect of gamma irradiation on the increase of anti-oxidant capacity in BGE, we measured DPPH and ABTS radical scavenging activity. Antioxidant activity can be regarded as the scavenging activity of free radicals, reactive oxygen, and nitrogen species by donation of hydrogen
Fig. 1. Effect of 100 kGy gamma-irradiated BGE on cell viability and H2O2-induced ROS production in B16F10 melanoma cells. The cell viability was measured by MTT assay (A). The data represent the mean ± SEM (n = 3). Statistical analysis was performed by unpaired Student's two-tailed t-test within * P < 0.05 compared to control group. Inhibitory effect of gamma-irradiated BGE on H2O2-induced ROS production (B). The data represent the mean ± SEM (n = 3). Statistical analysis was performed by unpaired Student's two-tailed t-test within *P < 0.05, **P < 0.01, and ***P < 0.001 compared to only H2O2-treated group. 36
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Fig. 2. Anti-melanogenic effects of gamma-irradiated BGE in IBMX-stimulated B16F10 melanoma cells. Inhibitory effect of gamma-irradiated BGE on melanin production (A) and intracellular tyrosinase activity (B) and melanogenic enzymes expression (C) in IBMX-stimulated B16F10 melanoma cells. B16F10 melanoma cells treated for 72 h with 0 kGy- or 100 kGy gamma-irradiated BGE at specific concentrations (125 and 250 μg/mL). Arbutin (500 μg/mL) was used as a positive control. The data represent the mean ± SEM (n = 3). Statistical analysis was performed by Student's two-tailed t-test within ##P < 0.01, and ###P < 0.001 compared to control group, and **P < 0.01, and ***P < 0.001 compared to the only IBMX-treated group.
cutaneous tissue causes lipid peroxidation and inactivation of antioxidant enzymes, which leads to skin aging (Kohen, 1999). In the melanogenic process, H2O2 is overproduced, and this triggers a cascade of events leading to overproduction of ROS, lipid peroxidation, and inactivation of antioxidant enzymes (Pandel et al., 2013). Tyrosinase is a major melanogenic enzyme that converts L-tyrosine to L-DOPA and dopaquinone (Chan et al., 2014). After the formation of dopaquinone, dopachrome is oxidized into 5,6-dihydroxyindole-2-carboxylic acid (DHICA) by TRP-2. Subsequently, indole-2-carboxilic acid5,6-quinone is formed from DHICA by TRP-1 (Costin et al., 2005; Park et al., 2011). In view of these findings, we can infer that gamma-irradiated BGE inhibits melanogenesis by regulating ROS overproduction and suppressing melanogenic enzymes, including tyrosinase, TRP-1, and TRP-2.
the anti-melanogenic activity of gamma-irradiated BGE in B16F10 melanoma cells. 3.3. Effect of gamma-irradiated BGE on melanogenesis in B16F10 melanoma cells To determine the anti-melanogenic effect of gamma-irradiated BGE, the 100 kGy dose of BGE and non-irradiated and were chosen because the highest anti-oxidant activity and tyrosinase inhibition activity were observed at 100 kGy. Prior to investigating the anti-melanogenic effect of gamma-irradiated BGE, the cell viability was tested using a MTT assay. We observed that both non-irradiated- and 100 kGy gamma-irradiated BGE did not induce cytotoxicity in B16F10 melanoma cells within the concentration range 31.25–250 μg/mL (Fig. 2A). At noncytotoxic concentrations, 100 kGy gamma-irradiated BGE showed a much stronger inhibitory effect on ROS production (125 μg/mL and 250 μg/mL, 46.61 ± 4.57 and 22.50 ± 3.93%) than did non-irradiated BGE (125 and 250 μg/mL; 81.96 ± 8.08 and 61.49 ± 9.25%) in H2O2-stimulated B16F10 melanoma cells (Fig. 2B). A significantly lower percentage of melanin contents in the 100 kGy BGE-treated group (125 and 250 μg/mL; 132.98 ± 6.94 and 121.43 ± 8.58%) compared with the group treated with IBMX only (161.44 ± 8.20%, Fig. 2A). Similarly, the 100 kGy gamma-irradiated BGE treatment group showed efficiently inhibited intracellular tyrosinase activity (125 and 250 μg/ mL; 105.84 ± 1.94 and 91.80 ± 9.22%; see Fig. 2B) than did the group treated with IBMX only (127.97 ± 7.69%). The expression of melanogenic enzymes, including tyrosinase, TRP, and TRP-2, was effectively reduced by treatment with 100 kGy gamma-irradiated BGE of IBMX-stimulated B16F10 melanoma cells (Fig. 3). Melanogenesis is closely related to oxidative stress, in particular, excess ROS levels in
3.4. Effects of gamma-irradiated BGE on the cAMP/ERK/p38 signaling pathway Several 3′,5′-cyclic adenosine monophosphate (cAMP) elevating agents, such as IBMX, forskolin, and α-melanocyte stimulating hormone, are common inducers of melanogenesis for evaluating the value of a product as a skin whitening product (Englaro et al., 1995). Among them, IBMX is a strong stimulus for inducing melanogenesis, and is widely used as an inducer of melanogenesis in cell culture (Brown et al., 1998). Thus, we investigated whether gamma-irradiated BGE has antimelanogenic activity through regulation of the cAMP signaling pathway. As shown in Fig. 3A, 100 kGy gamma-irradiated BGE significantly reduced the intracellular cAMP level (125 and 250 μg/mL; 67.64 ± 6.04 and 42.24 ± 11.78%; Fig. 3B) compared with the IBMX-only treatment group (146.37 ± 40.24%) in IBMX-stimulated 37
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Fig. 3. Effect of gamma-irradiated BGE on melanogenesis-related signaling pathways. B16F10 cells were treated with 0 kGy- or 100 kGy gamma-irradiated BGE (125 and 250 μg/mL) with IBMX (50 μM) for 1 h (A) Statistical analysis was performed by Student's two-tailed t-test within #P < 0.05 compared to control group, and *P < 0.05 compared to the only IBMX-treated group. The expression levels of p-CREB and MITF proteins were determined by Western blotting at 2 h after treatment (B). Time-dependent effect of gamma-irradiated BGE on the expression of p-ERK and p-p38 (C). Effect of ERK (PD98059) and p38 (SB203580) inhibitors on gamma-irradiated BGE-treated B16F10 cells (D). Statistical analysis was performed by Student's two-tailed t-test within ***P < 0.001 compared to the IBMX and 100 kGy gamma-irradiated BGE-treated group.
4. Conclusions
B16F10 cells. Likewise, the protein expression of phospho-CREB and MITF was significantly decreased by treatment with 100 kGy gammairradiated BGE (Fig. 3B). The elevated cAMP level led to phosphorylation of the cAMP response element binding protein (CREB), which binds to and activates the promoter of microphthalamia-associated transcription factor (MITF) (Park et al., 2006). MITF promotes the melanogenic pathway as a major transcription factor of the tyrosinase gene, and as a result, activated MITF promotes the melanogenesis pathway (Levy et al., 2006). On the other hand, previous reports demonstrated that ERK and p38 signaling pathways are associated with the inhibition of melanogenesis because activated ERK and p-38 block the binding of the tyrosinase promoter site to the MITF-M box (Jang et al., 2011; Ko et al., 2014). To investigate whether gamma-irradiated BGE has an effect on activating ERK and p38, we performed a western blot analysis. Treatment of 100 kGy gamma-irradiated BGE induced phosphorylation of ERK and p38 in a time-dependent manner (Fig. 3C). To rule out that the possibility of ERK and p38 signaling on the anti-melanogenic effect of gamma-irradiated BGE, we used an ERK (PD98059) and p38 (SB203580) inhibitors. As shown in Fig. 3D, the anti-melanogenic effect of gamma-irradiated BGE was hindered through the treatment of PD98059 and SB203580. These results indicate that the ERK and p38 signaling pathway was importantly involved in anti-melanogenic activity of gamma-irradiated BGE, and these findings are consistent with previous studies showing that various natural biomaterials inhibit melanogenesis by activating ERK and the p38 signaling pathway (Baek and Lee, 2015; Alam et al., 2016).
In this study, black ginseng extract was exposed to gamma irradiation to improve it for utilization in the cosmetic industry. The reducing sugar content was increased with increase in the irradiation dose to 100 kGy, and it may be speculated that the production of Maillard reaction products was accelerated through gamma irradiation. The anti-oxidant properties and anti-melanogenic activity of black ginseng extract were improved through gamma irradiation. The antimelanogenic action of gamma-irradiated black ginseng extract is mediated by the cAMP/MITF signaling pathway. This action is attributed to activation of the ERK and p38 signaling pathway. Consequently, gamma irradiation could be a useful tool for improving the utilization of black ginseng extract in the cosmetic industry. However, further study will be necessary to characterize the Maillard reaction products of gamma-irradiated black ginseng and to confirm the anti-melanogenic activity of refined Maillard reaction products in an artificial skin model. Acknowledgements This work was supported by the National Research Foundation of Korea grant funded by Korea government (Grant no. 2017-02-EE-004) and Nuclear R&D Program of the Ministry of Science and ICT (MSIT), Republic of Korea. References Agar, N., Young, A.R., 2005. Melanogenesis: a photoprotective response to DNA damage? Mutat. Res. 571, 121–132.
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