Biofunctional Constituents from Michelia compressa var. lanyuensis with Anti-Melanogenic Properties

Seven compounds were extracted and purified from the roots of Michelia compressa var. lanyuensis. These compounds are liriodenine, (−)-N-acetylanonaine, pressalanine A, p-dihydroxybenzaldehyde, 3,4-dihydroxybenzoic acid, (−)-bornesitol and β-sitostenone. These compounds were screened for anti-proliferation and anti-tyrosinase activities in B16F10 cells. Liriodenine, pressalanine A, (−)-bornesitol and β-sitostenone displayed cytotoxicity at high concentration (100 μM), but liriodenine (5 μM), (−)-N-acetylanonaine (10 μM), and β-sitostenone (5 μM) inhibit tyrosinase activity and reduce the melanin content in B16F10 cells without cytotoxicity, suggesting that liriodenine and β-sitostenone could be safe and potentially used in cosmetic skin whitening.

Tyrosinase is well-known enzyme in the synthesis of the human pigment melanin responsible for coloring skin, eyes and hair. In animals, the melanin pigments are produced by melanocytes [9,10]. The abnormal accumulation of melanin pigments is responsible for hyperpigmentations, including freckles and nevus. Skin hyperpigmentation is the most common complaint of patients. Depigmenting agents are very important in the cosmetic and medicinal industries. They should provide safety and efficacy without side effects. In the clinic, whitening agents are used for treating dermatological disorders related to melanin hyperaccumulation. UV exposure, sunburn and family history can cause skin cancers [11], which can be divided into non-melanoma and melanoma skin cancers. Non-melanoma skin cancer includes squamous cell carcinoma [12]. Non-melanoma skin cancer is common in Western countries, especially in the United States. Melanoma has a poor prognosis and low patient survival rates.

Anti-Proliferative Properties of Compounds 1-7 from M. compressa var. lanyuensis on B16F10 Cells
The XTT assay is a colorimetric assay used to measure cell viability. The XTT assay was used to investigate the anti-proliferation activity of tested compounds in B16F10 cells after 24 h treatment. The cells were treated with compounds at different concentrations (1, 10, and 100 μM) as demonstrated in

Inhibitory Effects of the Test Compounds on Tyrosinase Activity and Melanin Content
Melanin is a vitally important factor in determining the skin color of humans. The melanogenesis pathway consists of the enzymatic hydroxylation of L-tyrosine and the oxidation of L-dopa to dopaquinone [13]. The whitening effects of compounds and kojic acid (positive control) were evaluated by measuring melanin content in B16F10 cells. The cellular tyrosinase-inhibition abilities of the compounds from M. compressa var. lanyuensis were also examined in B16F10 cells (Table 1). In order to confirm the inhibition of enzyme activity, the effect of test compounds were evaluated by measuring tyrosinase activity with L-tyrosine as substrate. Liriodenine (1) and β-sitostenone (7) showed minor tyrosinase activity inhibition, with 14.52% ± 3.58% and 25.48% ± 6.30% inhibition on B16F10 cells (5 μM). In contrast, 3,4-dihydroxybenzoic acid (5) significantly increased the tyrosinase activity in B16F10 cells at the concentration of 5 μM. Further, we also observed that liriodenine (1), (−)-N-acetylanonaine (2) and β-sitostenone (7) showed a small decrease in the melanin content in B16F10 cells. Kojic acid (10 μM) inhibited the tyrosinase activity by 25.38% ± 1.40% and decreased melanin content (20.00% ± 2.12%) in B16F10 cells. We discovered that the decrease in melanin contents matched the tyrosinase activity inhibition with the same tendencies. Based on the XTT, tyrosinase activity and melanin content results, liriodenine (1) and β-sitostenone (7) might have a potential use in skin lightening without cytotoxicity and safety in skin whitening.

Plant Material
The roots of Michelia compressa var. lanyuensis were collected from Chiayi County, Taiwan

Cell Viability Assay-XTT Assay
The XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) assay was used to determine cell viability and proliferation. The cell lines were seeded in 96-well culture plates (1 × 104 cells/well). XTT were obtained from Sigma-Aldrich GmbH (Stenheim, Germany). After seeding cells for 24 h, various different concentrations of compounds were added. After the treatment, the medium was replaced with fresh medium without drugs. XTT reagent was added to each well and cultured for 3 h. The optical density (OD) values of the supernatant were measured at 492 nm and 690 nm (reference wavelength). All experiments were repeated at least three times.

Determination of Melanin Content
Briefly, we followed the previous method with minor modifications [14,15]. B16F10 cells (10 5 /well) were seeded into a 24 well culture dish with or without treatment in 300 μL of culture medium and incubated for 48 h. Cell pellets were dissolved in 2.0 N NaOH containing 10% dimethyl sulfoxide (DMSO) and heated at 80 °C for 1 h, and suspensions were clarified by centrifugation for 10 min at 10,000× g. Amounts of melanin in the NaOH solution were spectrophotometrically measured at 405 nm. A melanin standard curve was prepared by dissolving synthetic melanin in 0.01 M sodium carbonate (pH 7.8) and treated as above.

Tyrosinase Activity
The tyrosinase activity was estimated by measuring the rate of dopachrome formation, based on the method described previously with minor modifications [14]. Briefly, B16F10 cells (10 5 /well) were seeded into a 24 well culture dish with or without treatment in 300 μL of culture medium and incubated for 48 h. Cells were then solubilized in phosphate buffer (0.1 M; pH 6.8) containing 0.1% Triton X100. The enzyme extract of cellular lysate was added to 10 μL of 10 mm L-tyrosine and 10 mm L-dopa as substrates mixed in 0.1 m phosphate buffer (pH 6.8). This reaction was then incubated at 37 °C for 3 h in a dark environment, and the absorbance at 490 nm was measured on a spectrophotometer. Tyrosinase inhibitory activity was determined at 490 nm by the following equation: Tyrosinase inhibitory activity was calculated using the following formula: Tyrosinase inhibition (%) = [1 − (OD490 nm of sample/OD490 nm of control)] × 100 (1)

Statistical Analysis
All experiments were carried out at three times and at least triplicate. The results were expressed as the average of the mean values ± standard deviation (SD). Statistical differences were estimated by one-way analysis of variance (ANOVA) followed by Dunnett's test. Analysis of the data was done with SigmaPlot software (Version 8.0, SPSS Scientific, Chicago, IL, USA) and SigmaStat (Version 2.03, SPSS Scientific) run on an IBM-compatible computer. Statistical significance is expressed as * p < 0.05.