1,1-diphenyl-2-picryl hydrazyl (DPPH), mushroom tyrosinase, α-MSH, L-DOPA, and betulinic acid were purchased from Sigma (St. Louis, MO, USA). Dulbecco's modified Eagle medium (DMEM), fetal bovine serum (FBS), and L-glutamine were obtained from Invitrogen Corporation (Carlsbad, CA, USA). Antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) for tyrosinase, actin, and secondary antibodies. Nitrocellulose membrane was purchased from GE Healthcare (Little Chalfont, Buckinghamshire, UK). All other chemicals were of the highest commercial grade available.

V. amurensis Ruprecht root, which was purchased from Daehan Natural Drug (Busan, Republic of Korea), originated from Yeong cheon in Korea. Dried powder of V. amurensis Ruprecht root (5 kg) was extracted four times with five volumes of absolute (99.8%) methanol at 65℃. The extract was evaporated, freeze dried, and stored at 4℃ until use. Methanol extract of V. amurensis Ruprecht root (VARM, 527.6 g) was suspended in water and serially fractionated into dichloromethane (CH₂Cl₂, C fraction, 21.3 g), ethyl acetate (EtOAc, E fraction, 81.1 g), n-butanol (n-BuOH, B fraction, 71.3 g), and water (H₂O, W fraction, 246.1 g). The C fraction, which exhibited potent anti-melanogenic activity without cytotoxicity, was applied onto a silica gel (70-230 mesh, Merk, Darmstadt, Germany) column. The column was eluted using mixtures of n-hexane:EtOAc under gradient conditions [10:1, 5:1, 3:1, 2:1, 1:1 (v/v)] to yield the four fractions (fraction 1 to 4). Among four fractions, fraction 3 was further purified by a silica gel once more to yield six fractions (fraction 1 to 6). Among them, fraction 3 was identified as a betulinic acid through ¹H nuclear magnetic resonance (NMR) and ¹³C-NMR analyses. For identification of betulinic acid, TLC was performed on a precoated Merck Kieselgel 60 F₂₅₄ plate (0.25 mm) and the spots were compared with standard betulinic acid purchased from Sigma.

Antioxidative activity of VARM and its solvent fractions were determined using a DPPH radical scavenging assay as described previously [33]. Various concentrations of reagents and standard compound (Ascorbic acid) were added to DPPH solution. Absorbance was measured at 520 nm after 30 min incubation protected from light.

The effect of VARM on mushroom tyrosinase activity was determined as previously described [34]. In brief, 50 µL of mushroom tyrosinase (700 unit/mL), 50 µL of various concentrations of reagents, and 100 µL of 0.1 M phosphate buffer (pH 6.8) were added to a 96-well plate and mixed, 100 µL of 2.5 mM_L-DOPA was added, followed by incubation for 20 min at room temperature. The amount of dopachrome formed in the reaction mixture was measured at 475 nm. Kojic acid was used as a positive control.

B16F10 murine melanoma cells (CRL-6475) were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). Cells were maintained in DMEM supplemented with 10% FBS and 100 units/mL of penicillin/streptomycin at 37℃ in a humidified atmosphere containing 5% CO₂. Cells were treated with either α-MSH alone or reagents together. Cells not treated with α-MSH served as the negative control, while cells treated with α-MSH alone served as the positive control.

Cell viability was determined using a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay purchased from Daeil Lab Service (Seoul, Korea) according to the manufacturer's instructions [35]. After treatment with various concentrations of VARM and its solvent fractions, cells were incubated with MTS for 1 h and the viability was quantified by measurement at OD₄₅₀ using a multi-plate reader (Paradigm, Beckman, CA, USA).

Effect of VARM and its solvent fractions on α-MSH induced melanogenesis on B16F10 cells was investigated according to the previous research, which was slightly modified [36]. Briefly, cells were stimulated with α-MSH (200 nM) and treated with VARM and its solvent fractions for 72 hr. After treatment, the cells were dissolved in 1 N NaOH/10% DMSO for 1 hr at 80℃, and solubilized melanin was measured at 475 nm.

Effect of VARM and its solvent fractions on α-MSH induced tyrosinase activity on B16F10 cells was investigated according to the previous research, which was slightly modified [36]. Briefly, cells were stimulated with α-MSH (200 nM) and treated with VARM and its solvent fractions for 72 hr. After treatment, the cells were washed with 1 × phosphate buffered saline (PBS), and then lysed into protein lysis buffer (Cell Signaling Technology, Boston, MA, USA). Each extract was placed in each well of a 96-well plate and the enzymatic assay was commenced by addition of L-DOPA. After incubation, dopachrome formation was assayed by measuring absorbance at 475 nm.

After treatment, the cells were washed with ice-cold 1 × PBS and lysed with cell lysis buffer (pH 7.4). The cell lysates were incubated on ice for 30 min and the homogenates were centrifuged at 13,000 × g for 10 min at 4℃. The supernatants were collected and protein concentration was determined using the Bradford protein assay. Protein samples (50 µg) were mixed with 2 × loading buffer, and boiled at 100℃ for 5 min. The samples were separated on a 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel and electrotransferred to nitrocellulose membranes (WhatmanTM, GE Healthcare, Little Chalfont, UK). The membranes were blocked with 5% nonfat dry milk in 1 × PBST buffer (0.1% Tween 20 in PBS) for 1 hr at room temperature and incubated overnight with primary antibodies in blocking buffer at 4℃. The membranes were washed, followed by incubation with anti-rabbit-IgG or anti-goat-IgG with horseradish peroxidase for 1 hr at room temperature. After washing the membrane, final detection and quantification were performed using a chemiluminescence detection system (FluoChem® FC2, Alpha Innotech, CA, USA).

The data were expressed as mean ± standard deviation (SD). The values were compared to the control using analysis of variance (ANOVA) followed by unpaired Student's t-tests. A P value < 0.05 was considered significant.

Thus far, a potent DPPH scavenger among natural resources has been regarded as a potential antioxidant candidate. In this study, VARM was evaluated for its anti-oxidative activity by DPPH, a stable free radical, scavenging capacity. As shown in Fig. 1A, VARM scavenged free radicals in a dose dependent manner, with an inhibitory rate of 15.6, 50.2, and 95.8% at 2.56, 12.8, and 64 µg/mL, respectively. Dose for IC₅₀ was calculated as 12.8 µg/mL. As shown in Fig. 1B, VARM suppressed the enzyme activity of mushroom tyrosinase, with an inhibitory rate of 6.7, 46.0, and 87.9% at 20, 100, and 500 µg/mL, respectively. The IC₅₀ value was calculated as 148.1 µg/mL. IC₅₀ values of DPPH radical scavenging and tyrosinase inhibitory activity issued from VARM-treated B16F10 cells are shown in Table 1.

The cytotoxicity of VARM for 48 hr was determined using a colorimetric MTS assay in B16F10 cells. Fig. 2A shows the percent cell viability of VARM-treated cells compared with that of vehicle treated cells. No remarkable cytotoxicity was observed in cells treated with VARM under the concentration of 200 µg/mL, which was applied for the following cascade of experiments. As shown in Fig. 2B, melanin content of α-MSH stimulated cells was 2.5 times higher than that of vehicle treated cells, indicating that melanogenesis was successfully induced by α-MSH treatment. On the other hand, α-MSH-stimulated melanin production was significantly (P < 0.5) inhibited by treatment with VARM in a dose-dependent manner (Fig. 2B).

VARM was serially fractionated with CH₂Cl₂, EtOAc, n-BuOH, and H₂O for identification of active compounds possessing anti-melanogenic activity (Fig. 3). The effect of each fraction on B16F10 cell viability was evaluated in advance of the downstream experiment. As shown in Fig. 4A, VARM, C, and B fractions did not show any cytotoxicity below 250 µg/mL, while the E fraction exhibited a cytotoxic effect over 50 µg/mL. Followed by the result for cell viability, anti-melanogenic effect of each fraction was evaluated on non-cytotoxic doses. As shown in Fig. 4B, the C fraction induced the most potent inhibition of α-MSH induced melanogenesis among all fractions that exhibited an anti-melanogenic effect. In the W fraction, neither cytotoxic nor anti-melanogenic activities were detected.

To confirm the anti-melanogenic effect of VARM and its solvent fractions, cellular tyrosinase enzymatic activity and protein expression level were analyzed. As shown in Fig. 5A, elevated tyrosinase enzyme activity induced by α-MSH was down-regulated by VARM and its solvent fractions, including C, E, and B. In addition, tyrosinase protein expression was also inhibited by VARM and its solvent fractions, which was in accordance with melanin contents and tyrosinase activity (Fig. 5B). According to the results, anti-melanogenic effects of VARM and its solvent fractions originated from the inhibited tyrosinase activity. Among all fractions of VARM, the C fraction showed the most potent anti-melanogenic activity without cytotoxicity (Fig. 6A).

Identification and purification of the functional compounds contained in the C fraction, which showed the most potent anti-melanogenic activity, was attempted. As a result, various compounds were identified as methyl palmitate, palmitate, methyl stearate, oleanolic acid, and betulinic acid. Among them, only betulinic acid significantly attenuated α-MSH-induced melanogenesis in a dose dependent manner, which was stronger than that of arbutin, a well-known depigmenting agent (Fig. 6B).