Melanogenesis inhibitory effect of aerial part of Pueraria thunbergiana in vitro and in vivo
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Melanin is major factor that determines skin color as well as one of the defense systems that prevent the UV-induced damage. In case of abnormal concentration of melanin, skin diseases or problems occur such as albinism, leukoplakia, melasma, freckles, moles, and lentigo. With the lifespan of humans has been extended, importance of ‘life quality’ has been increased. White and clean skin is very important part of the satisfaction of appearance, especially for Asia women. The aim of this study was to find an anti-melanogenesis activity for which the aerial part of Pueraria thunbergiana can be utilized based on the increase in demands for cosmetics, particularly natural products. We demonstrated anti-pigmentation effects of aerial part of P. thunbergiana by measuring melanin content and through staining in the B16F10 melanoma cell line. The aerial part of P. thunbergiana decreased tyrosinase activity significantly in B16F10 cell cultures, while there is no direct effect on enzyme in cell-free conditions. To define the mechanisms, real-time PCR, western blot, glucosidase activity and antioxidant activity assay were implemented. As results, we demonstrated that aerial part of P. thunbergiana has anti-melanogenesis activity via two mechanisms. One is downgrading microphthalmia-associated transcription factor by activating Akt/GSK-3β. Consequently, transcription of tyrosinase and tyrosinase-related protein 1 is decreased. Another is interrupting maturation of tyrosinase through inhibiting α-glucosidase. Furthermore, aerial part of P. thunbergiana showed great efficacy on pigmentation in vivo. These results suggest that aerial part of P. thunbergiana can be used as an anti-melanogenic agent.
KeywordsPueraria thunbergiana Anti-melanogenesis GSK-3β Glucosidase In vivo
Pueraria thunbergiana, also known as the kudzu, is a species of climbing plant belonging to the Leguminosae family. The root and flower of P. thunbergiana, used in traditional medicine, have various medicinal properties [15, 33, 35, 36, 38]. However, the vine of P. thunbergiana is commonly discarded as a waste product and presents an environmental problem. The vine grows by climbing adjoining structures and trees, and destroys forests and landscape because of its weight and fast rate of growth. In some countries, P. thunbergiana is considered among the invasive species and seen as a threat to the ecosystem, with its management exacting a high cost, both financially and in terms of manpower [9, 16].
Melanin is a major factor that determines skin color, as well as one of the defense systems that prevent UV-induced skin damage. Abnormal concentrations of melanin manifest as skin diseases or problems, such as albinism, leukoplakia, melasma, freckles, moles, and lentigo. Skin-whitening agents are commonly applied for treating pigmentation and pigmentary diseases. Because of an increasing interest in herbs, many studies focused on discovering novel natural skin-whitening agents that are currently underway . We investigated, therefore, whether the aerial part of P. thunbergiana, which is currently considered a waste product, has potentially useful skin-whitening effects.
Melanin is synthesized in melanocytes by enzyme-related mechanism, such as tyrosinase, tyrosinase-related protein 1 (TRP1) and tyrosinase-related protein 2 (TRP2). The first step of melanogenesis is a transformation of l-tyrosine into 3,4-dihydroxyphenylalanine (l-DOPA), and this l-DOPA is converted into DOPA quinine . Then from this product, melanin is synthesized by subsequent transformation. First two ensuing stages, regulated by tyrosinase, are rate-limiting steps because the rest of the melanin synthesis process can go along naturally at a physiological pH . Since tyrosinase is the key enzyme of melanogenesis, many whitening agents are targeting the tyrosinase by various mechanisms such as interference with tyrosinase catalytic activity directly, inhibition of mRNA expression, interruption of tyrosinase maturation, and acceleration of tyrosinase degradation .
Transcription levels of melanogenic genes are regulated by microphthalmia-associated transcription factor (MITF) . MITF is expressed by that kind of stimulating factors. On the other hand, mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinases (ERK) and Akt/glycogen synthase kinase-3β (GSK-3β) signals pathways are involved in the down-regulation of melanogenesis by phosphorylating MITF [18, 34]. The phosphorylated MITF decreases its activity, binding to tyrosinase promoter site, and eventually degrades. Thereby, activation of MEK/ERK or Akt/GSK-3 inhibits melanin synthesis [14, 18].
The melanogenesis is regulated by nutritional and hormonal factors. l-tyrosine and l-DOPA are not only substrates but also bioregulator for melanogenesis. Regulation for melanocyte function of l-tyrosinase and l-DOPA can be mediated through specific receptors or without receptors . In addition, there are complex multiple hormonal stimulators such as MSH, adrenocorticotropic hormone (ACTH), endorphin and stem cell factor (SCF) and inhibitors such as serotonin, melatonin, dopamine, acetylcholine and melanin-concentrating hormone (MCH) .
Since in vivo study is also important regarding the transmembrane or transdermal permeation, it was undertaken using cream formulations. There are differences between epidermal and follicular melanogenesis although basic features such as existence of melanocyte and melanosome are common. The follicular melanogenesis depends on anagen stage of hair cycle, whereas epidermal melanogenesis is continuous and follicular melanocyte is more influenced by aging . Consequently, melanin-generating hairless mice irradiated UVB were used for in vivo study . When skin exposes to UV, α-melanocyte-stimulating hormone (α-MSH), one of the important hormones which modulate pigmentation, is produced as a response .
Materials and methods
Antibiotic–antimycotic, bovine serum albumin (BSA), Dulbecco’s modified Eagle’s medium (DMEM), and fetal bovine serum were purchased from Life Technologies (Carlsbad, CA, USA). Acarbose, dimethyl sulfoxide (DMSO), 3,4-dihydroxy-l-phenylalanine (l-DOPA), 2,2-diphenyl-1-picrylhydrazyl (DPPH), ethylenediaminetetraacetic acid (EDTA), α-glucosidase (EC 126.96.36.199, from baker’s yeast, 77 U/mg), LY294002, α-melanocyte-stimulating hormone (α-MSH), mushroom tyrosinase and ρ-Nitrophenyl-α-d-glucopyranoside were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fontana–Masson staining kit was obtained from IHCWORLD (Woodstock, MD, USA). Phosphate-buffered saline (PBS) was obtained from Bioworld Technology, Inc. (BioWorld, USA). easy-Blue™ Total RNA extraction kit was purchased from Intron Biotechnology, Inc. (iNtRON Biotechnology, Seongnam, Korea). TaqMan® RNA-to-Ct™ 1-Step Kits were purchased from Applied Biosystems (USA). RIPA buffer was obtained from Biosesang Inc. (Seongnam, Korea). Protease inhibitor cocktail tablets were purchased from Roche Diagnostics (Switzerland). Antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA) and Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). ECL reagent was purchased from GE Healthcare.
Extraction of plant material
Aerial parts of P. thunbergiana were collected from Jinan, Jeonbuk, Korea, in November 2010, and extracted by the Hanpoong Pharm and Foods Company (Hanpoong Pharm. CO., Ltd.). Briefly, dried and pulverized materials (2 kg) were boiled with 2 L of distilled water and a range of ethanol concentrations (0, 30, 70, and 95 %) for 3 h. The solvent was then removed under reduced pressure in a rotary evaporator (N-1000S, EYELA, Japan) to yield a water extract (439.5 g), 30 % ethanolic extract (409.2 g), 70 % ethanolic extract (436.7 g), and a 95 % ethanolic extract (284.8 g).
High-performance liquid chromatography analysis
Composition of isoflavones from the aerial part of P. thunbergiana
Aerial part of P. thunbergiana
Cell culture and treatment with extracts
Murine B16F10 melanoma cells purchased from ATCC (Manassas, VA, USA) were cultured in DMEM with 10 % (v/v) fetal bovine serum and 1 % (v/v) antibiotic–antimycotic. The cells were incubated in a humidified incubator under 5 % CO2 at 37 °C. Aqueous fractions are dissolved in DDW and EtOAc fractions are dissolved in DMSO. Controls of B16F10 cells were added DDW or DMSO at the same concentration as in the treated cells.
Cell viability assay
Cell proliferation was evaluated by performing the MTT assay. The cells (3 × 103 cells/well) were seeded into a 96-well plate and incubated for 24 h; the cells were subsequently exposed to the P. thunbergiana extracts at a range of concentrations (0, 5, 10, 50, 100, 500, and 1,000 μg/mL) for 2 days. Thereafter, serum-free MTT medium was added to each well to the final concentration of 1 mg/mL, and incubation performed for 2–4 h at 37 °C. The MTT medium was removed from the wells, and DMSO was added; thereafter, the plate was placed on a shaker for 5 min. Absorbance was read at 540 nm using a microplate spectrophotometer (SpectraMax 190, USA).
The B16F10 cells (3 × 104 cells/well) were seeded in a 6-well plate and exposed to α-MSH (100 nM) for 1 day. The treatment was performed with a combination of α-MSH (100 nM) and P. thunbergiana extracts for 2 days. The cells were harvested by trypsinization and washed twice with PBS and alcohol. 2 × 105 cells were dissolved in 200 μL of 1 N NaOH with 10 % DMSO at 90 °C for 1 h. The resulting melanin concentration was quantified by measuring the absorbance at 475 nm.
Mushroom tyrosinase activity
Cellular tyrosinase assay
Another method of measuring tyrosinase activity was repeated since there are complex regulations of tyrosinase activation . The B16F10 cells were exposed to α-MSH (100 nM) for 1 day and treated with a combination of extracts and α-MSH for 2 days. The pellet was obtained by trypsinization and washed with PBS. The cells were lysed with 0.1 M sodium phosphate buffer (pH 6.8) containing 5 mM EDTA, 1 % Triton X-100, and 0.1 % phenylmethylsulfonyl fluoride (PMSF) in ice for 30 min. After centrifugation of the lysate at 15,000 rpm for 30 min at 4 °C, cellular tyrosinase activity was measured in the resulting supernatant. Enzyme activity was normalized to protein concentration, as determined by Bradford assay. The cellular tyrosinase and 0.1 % l-DOPA reaction were performed in 0.1 M sodium phosphate buffer at 37 °C for 1 h. Tyrosinase activity was quantified by measuring the absorbance at 475 nm.
Quantification of selected genes transcript by real-time PCR was performed using a TaqMan® RNA-to-Ct™ 1-Step Kit according to the manufacturer’s instructions. The mRNA was extracted with easy-Blue™ Total RNA extraction kit and quantified by Nano Drop. Relative ratio of a target gene expression was calculated with the ΔC t method.
Western blot analysis
After treatment with a range of concentrations (0, 10, 50, and 100 μg/mL) of extracts, the B16F10 cells were washed with PBS, and lysed by RIPA buffer in ice to get the protein. Next, 20–50 μg of proteins was subjected to electrophoresis on 10–15 % sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE). Following electrophoresis, the proteins were transferred to nitrocellulose membranes, and the membranes were blocked with 5 % skimmed milk or 5 % BSA in PBS with 0.1 % Tween 20 (PBST). Then, the proteins were probed using antibodies. The immunoblots were developed and visualized using an enhanced chemiluminescence (ECL) detection system (Amersham Biosciences, Piscataway, NJ, USA). The western blot was imaged using the Chemi Documentation Imaging System and quantified using densitometric program (Image J). GAPDH and β-actin were used as an internal control.
Glucosidase activity assay
Inhibition activities of α-glucosidase were evaluated according to the chromogenic method described by McCue et al. . Yeast α-glucosidase (0.5 unit/mL) 20 µL, 0.1 M phosphate buffer (pH 6.9) 120 and 10 µL of test sample dissolved in DMSO at various concentrations were mixed in a microplate well. After incubating for 15 min at 37 °C, 20 µL of ρ-Nitrophenyl-α-d-glucopyranoside (5 mM) was added to substrates and incubated for an additional 15 min. The reaction was terminated by 80 µL of 0.2 M sodium carbonate (Na2CO3) solution. The absorbance was measured at 405 nm. Acarbose was used as positive control. Each experiment was conducted in triplicate, and the IC50 values of samples were calculated.
DPPH was dissolved in methanol to a 0.1 mM concentration, and mixed with the diluted P. thunbergiana extracts (0, 10, 50, and 100 μg/mL) in a 96-well plate in a 1:1 ratio. Reaction was allowed to proceed in the dark for 30 min, and the absorbance was measured at 520 nm. Ascorbic acid was used as a standard. The data are expressed as mean ± SD.
Six-week-old male hairless mice, Hos: HRM2, were purchased from Hoshino Laboratory Animals Inc. (Yashio, Saitama, Japan). The animals were housed in a SPF animal facility room at 23 ± 1 °C and 50 ± 10 % relative humidity with 12-h light/dark cycle with free access to standard commercial diet and water. Creams containing 1 or 3 % of No.6 extracts were used for assessing in vivo anti-melanogenesis efficacy. There are five groups of mice in the animal model; control group (applied cream base, CON), UVB-exposed control group (UVB + applied cream base, UVB), positive control group (UVB + applied 1 % kojic acid cream, kojic acid), experiment group 1 [UVB + applied 1 % No.6 cream, No.6 (1 %)] and experiment group 2 [UVB + applied 3 % No.6 cream, No.6 (3 %)].
In vivo skin pigmentation determinations
The B16F10 melanoma cells were cultured and treated in a chamber slide. After removal of the medium, the cells were fixed with 4 % formalin for 20 min and washed twice by PBS containing 0.1 % Triton X-100. The cells were stained with the Fontana–Masson staining kit according to manufacturer’s instructions.
Each specimen was soaked in formaldehyde to fix. Then, paraffin processing and embedding were carried out. The tissue blocks were sectioned at 4 μm. The skins were stained with Fontana–Masson staining kit.
Briefly, the B16F10 cells or specimens were incubated in pre-warmed FM silver nitrate solution, toned in gold chloride solution, and soaked in 5 % sodium thiosulfate solution. Finally, counterstaining was performed with the Nuclear Fast Red Solution.
All results obtained from at least three independent experiments were combined and analyzed using the Student’s t test. All values were expressed as the mean ± SD. The acceptable level of significance was established at p values <0.05
The aerial part of P. thunbergiana hinders melanogenesis in B16F10 cells
The aerial part of P. thunbergiana indirectly inhibits tyrosinase activity
Effects on expression of mRNA and protein of tyrosinase
Inhibitory effects on α-glucosidase
Effects on expression of mRNA and protein of TRP1
Aerial part of P. thunbergiana inhibits MITF expression through the activation of Akt/GSK-3β signaling pathway
Evaluation of the in vivo depigmenting activity of aerial part of P. thunbergiana
Microscopic observation of the epidermis
Effects of aerial part of P. thunbergiana on pigmentation in UVB-irradiated mice were confirmed by Fontana–Masson staining (Fig. 15c). In the UVB-exposed-control epidermis, an induction of melanogenesis was observed compared to controls. In mice applied with No. 6 (1 and 3 %), melanogenesis was reduced.
Currently, numerous compounds are used for skin whitening, such as arbutin, hydroquinone, and kojic acid. However, there is growing interest in finding alternative agents since the existing ones possess mutagenic properties and can induce skin disorders or low depigmenting activity [8, 20].We investigated, therefore, the effects of the aerial part of P. thunbergiana, an overlooked resource, on melanin synthesis. Both aqueous and EtOAc fractions of the extracts obtained using a range of EtOH densities (0, 30, 70, and 95 %) were studied.
We found that the EtOAc fraction significantly reduced melanin synthesis in the B16F10 melanin-processing cell lines (Fig. 5) without causing cytotoxicity (Fig. 4). The P. thunbergiana extracts exhibited dose-dependent inhibition, with extract Nos. 6 and 7 (30 and 70 % ethanol, respectively) proving to be excellent melanogenesis inhibitors (Fig. 6). In addition, anti-pigmentation effects of the aerial part of P. thunbergiana were clearly demonstrated by the intracellular melanin accumulation observed using staining techniques (Fig. 7).
A number of mechanisms exist for the regulation of melanogenesis . The most common target for depigmenting agents is tyrosinase, the rate-limiting enzyme of melanogenesis. Whitening ingredients such as ascorbic acid, thio-containing compounds, phenolic compounds, and kojic acid inhibit tyrosinase enzyme activity . In this study, we have established that the aerial part of P. thunbergiana reduces tyrosinase activity through its effect on cellular tyrosinase in α-MSH-treated B16F10 cells (Fig. 9). However, the aerial part of P. thunbergiana had no direct inhibitory effects on tyrosinase (Fig. 8). These results indicate that the anti-melanogenesis activity of the aerial part of P. thunbergiana is involved in superior levels regulating tyrosinase enzyme such as transcription, translation, and maturation .
To understand the mechanisms of inhibitory effects on melanogenesis, we examined the effects on mRNA and protein expression of tyrosinase and TRP1, enzymes involved in melanogenesis. Aerial part of P. thunbergiana inhibits transcription and protein levels of tyrosinase and TRP1 (Figs. 10, 12).
Transcription of tyrosinase and TRP1 is promoted by MITF [2, 32]. We found out that MITF was down-regulated by aerial part of P. thunbergiana (Fig. 13). It was reported that the activation of Akt/GSK-3β and MEK/ERK signaling pathway downgrades activity and stability of MITF and leads to degradation . Accordingly, we investigated the phosphorylation of GSK-3β and ERK. Phosphorylation of GSK-3β was increased significantly by aerial part of P. thunbergiana in a dose-dependent manner. p-ERK had no change (Fig. 13). In addition, we found that melanin formations, which were inhibited by aerial part of P. thunbergiana, recovered with the Akt/GSK-3β-specific inhibitor LY294002 (Fig. 14). These results mean that aerial part of P. thunbergiana inhibits MITF through modulation of the Akt/GSK-3β pathway, not MEK/ERK.
Tyrosinase, the key enzyme of melanogenesis, is one of the glycoprotein which completes its maturation by processing N-glycosylation. Initial tyrosinase is glycosylated when translated polypeptide chain translocates into the endoplasmic reticulum (ER). After trimming by α-glucosidase, glycosylated tyrosinase interacts with chaperones and folds. Folded tyrosinase is completely matured via acquisition of two Cu2+ in Golgi and transported to melanosome . Our data revealed that aerial part of P. thunbergiana reduces tyrosinase maturation (Fig. 10) by inhibiting α-glucosidase (Fig. 11).
UV radiation is a strong inducer of oxidative stress, contributing to skin pigmentation. Therefore, antioxidants can reduce melanogenesis [3, 19]. In this study, we detected antioxidant activity in the extracts of the aerial part of P. thunbergiana using DPPH assay, with the EtOAc fractions exhibiting stronger antioxidant properties than the aqueous fractions (data not shown). The EtOAc-soluble fractions showed remarkable scavenging effects compared to those of ascorbic acid, a well-known antioxidant . The antioxidant activity could be regarded as one of the reasons that the aerial part of P. thunbergiana has anti-melanogenesis activity. Further study about antioxidant effect will be needed, because there are many mechanism linking antioxidant activity and melanin synthesis [27, 29].
Furthermore, we demonstrated that topical spread of cream contained extract of aerial part of P. thunbergiana can inhibit pigmentation in vivo study (Fig. 15). Application of aerial part of P. thunbergiana reduced UVB-induced-pigmentation, and the effect was more dramatical than kojic acid. In case of treatment of aerial part of P. thunbergiana 3 % contained cream, the skin lightness was higher than control that was not exposed to UVB. Furthermore, by Fontana–Masson staining study, it was identified that melanin distribution of epidermis induced by UVB was inhibited by aerial part of P. thunbergiana (Fig. 15c). These results of our study suggest that aerial part of P. thunbergiana can be used as a skin-whitening agent.
This paper was supported by Wonkwang University in 2014.
Conflict of interest
The authors declare that they have no conflict of interest.
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