Swertiajaponin Inhibits Skin Pigmentation By Dual Mechanisms To Suppress Tyrosinase Part 2
Mar 21, 2023
Cistanche is a common herb that is known as "the miracle herb that prolongs life". Its main component is cistanoside, which has various effects such as antioxidant, anti-inflammatory, and immune function promotion. The mechanism between cistanche and skin whitening lies in the antioxidant effect of cistanche glycosides.
Melanin in human skin is produced by the oxidation of tyrosine catalyzed by tyrosinase, and the oxidation reaction requires the participation of oxygen, so the oxygen-free radicals in the body become an important factor affecting melanin production. Cistanche contains cistanoside, which is an antioxidant and can reduce the generation of free radicals in the body, thus inhibiting melanin production.
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In addition, cistanche also has the function of promoting collagen production, which can increase the elasticity and luster of the skin and help repair damaged skin cells.
Cistanche Phenylethanol Glycosides have a significant down-regulating effect on tyrosinase activity, and the effect on tyrosinase is shown to be competitive and reversible inhibition, which can provide a scientific basis for developing and utilizing the whitening ingredients in Cistanche.
Therefore, cistanche has a key role in skin whitening. It can inhibit melanin production to reduce discoloration and dullness; and promote collagen production to improve skin elasticity and radiance. Due to the widespread recognition of these effects of cistanche, many skin whitening products have begun to infuse herbal ingredients such as Cistanche to meet consumer demand, thus increasing the commercial value of Cistanche in skin whitening products.
In summary, the role of cistanche in skin whitening is crucial. Its antioxidant effect and collagen-producing effect can reduce discoloration and dullness, improve skin elasticity and luster, and thus achieve a whitening effect. Also, the wide application of Cistanche in skin whitening products demonstrates that its role in commercial value cannot be underestimated.
Swertiajaponin inhibits UVB-induced MAPK activation
Oxidative stress has been shown to stimulate melanogenesis by upregulation of microphthalmia-associated transcription factor (MITF), a transcription factor to induce tyrosinase gene expression [4, 14]. We studied whether the antioxidative effect of swertiajaponin can regulate MITF activity. Western blotting data showed that UVB exposure increased phosphorylated MITF, an active form of MITF, whereas swertiajaponin treatment at 10 μM decreased it (Figure 6A). Consistently, a UVB-mediated increase in tyrosinase protein level was reduced by swertiajaponin treatment (Figure 6A). Because oxidative stress has been shown to activate MITF through mitogen-activated protein kinase (MAPK) [15, 16], it was investigated whether swertiajaponin can control MAPK signaling. UVB exposure dramatically increased the phosphorylation of ERK, JNK, and p38 (Figure 6B), which is associated with the UVB-induced ROS and ONOO- production in B16F10 cells (Figure 5C- 5D). Swertiajaponin treatment only at 10 μM decreased the protein levels of MAPKs (Figure 6B). This result is consistent with the antioxidative activity of swertiajaponin because the decrease in UVB-induced cellular oxidative stress was clear only at 10 μM of swertiajaponin (Figure 5C-5D).
Although swertiajaponin is the main compound of the whole herb of Swertia japonica that has been used as a Japanese medicine, our study did not show conclusive evidence about the safety of swertiajaponin for its application to human skin. However, swertiajaponin did not exhibit cytotoxicity in cell lines of Hs27 (human fibroblast), HaCat (human keratinocyte), and B16F10 (mouse melanoma) in our experimental setting. Moreover, there were no visible signs of cytotoxicity including cell debris formation or cell detachment based on microscopic observation when swertiajaponin was treated in the human skin model with concentrations that did not show cytotoxicity in the cell lines. Considering safety issues are the main concerns of skin whitening compounds that are currently available, further in vivo studies are needed to examine their safety in physiology.
Together, swertiajaponin inhibited melanin accumulation up to a satisfactory limit both in the cell and human skin models by dual mechanisms to suppress tyrosinase through direct binding to and competitively inhibiting tyrosinase and suppressing oxidative stress-mediated MAPK/MITF signaling (Figure 7). Considering the adverse effects and lack of long-term effectiveness of known skin whitening agents such as kojic acid and arbutin [17], swertiajaponin may be more safely applied to suppress skin pigmentation and would be a novel additive for whitening cosmetics.
MATERIALS AND METHODS
Tyrosinase activity assay using mushroom tyrosinase
Swertiajaponin and kojic acid (50 μM) were loaded into a 96-well microplate (Nunc, Denmark) in tyrosinase buffer (200 μL) containing mushroom tyrosinase (1000 U), 1 mM L-tyrosine solution, and 50 mM phosphate buffer (pH 6.5) [5]. The plate was incubated at 37 °C for 15 min and dopaquinone was evaluated by spectrophotometry (450 nm). Based on the measurement, the IC50 was calculated using log-linear curves and their equations.
Docking simulation of swertiajaponin and tyrosinase
AutoDock Vina was used for the in silico protein–ligand docking simulation. The three-dimensional structure of tyrosinase was used in the crystal structure of Agaricus bisporus (PDB ID: 2Y9X). The predefined binding site of tyrosine was applied as a docking pocket. After docking simulations between tyrosinase and swertiajaponin or kojic acid were performed, the LigandScout 3.0 software was used to predict binding residues between different compounds and tyrosinase.
Kinetic analysis of tyrosinase inhibition by swertiajaponin
L-DOPA was prepared at concentrations of 4, 2, 1, 0.5, 0.25, 0.125, and 0.0625 mM, and swertiajaponin was prepared at 20, 40, and 80 μM. The reaction mixture solution was prepared in a 96-well plate, in which 20 μL of tyrosinase substrate (L-DOPA), 10 μL of an aqueous mushroom tyrosinase solution (200 U), and 50 mM potassium phosphate buffer (pH 6.5) were added. The dopachrome production rate of the reaction mixture was measured at a wavelength of 450 nm using a microplate reader. The tyrosinase inhibition rate of swertiajaponin was then calculated using Lineweaver-Burk plot analysis. The Michaelis constant (Km) and maximal velocity (Vmax) were also calculated by Lineweaver-Burk plots with different concentrations of L-DOPA substrate [3].
Cell culture and viability assay
B16F10 melanoma cells were purchased from the Korea Cell Line Bank. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 5% fetal bovine serum (FBS), and 1% penicillin, streptomycin, L-glutamine, and sodium pyruvate. The cells were maintained at 37 °C in a humidified 95% air/5% CO2 atmosphere. For the cell viability assay, cells were seeded in 96-well plates. B16F10 melanoma cells were treated with swertiajaponin at various concentrations for 48 h. Ez-Cytox (10 μL) was added to each well and incubated for 2 h. The formazan crystals formed were measured by spectrophotometry at 450 nm. Cell viability was calculated using cells without swertiajaponin treatment as the control group.
Melanin content in B16F10 cells
Melanin level was measured using the method previously described with slight modifications [18 ]. B16F10 cells were allowed to grow to 70-80% confluence in 6-well plates. The cells were pre-treated with swertiajaponin or kojic acid for 2 h. Afterward, αMSH or UVB was treated to the swertiajaponin- or kojic acid-containing medium and incubated for a further 48 h. After washing with PBS, the cells were detached using trypsin and dissolved in 90 μL 1 N NaOH solution containing DMSO (5%). After incubation at 60 °C for 1 h, melanin content was determined by measuring absorbance at 405 nm. A commercially available UV chamber (Boteck UVX000, UV-AB, LAB24, Korea) was used for the UVB exposure of B16F10 cells.
Western blotting
Protein samples isolated from B16F10 cells (30 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using 10-12% acrylamide gels and transferred to polyvinylidene fluoride membranes, which were then immediately placed in blocking buffer (5% non-fat milk) in 10 mM Tris (pH 7.5), 100 mM NaCl, and 0.1% Tween 20. The membrane was washed in TBS-Tween buffer for 30 min and then incubated with specific primary antibodies (1: 1000 dilution) indicated in the figure legends at 4°C overnight. After washing with TBS-Tween buffer, the membrane was incubated with a horseradish peroxidase-conjugated anti-mouse antibody (Santa Cruz, 1: 10,000), an anti-rabbit antibody (Santa Cruz, 1: 10,000), or an anti-goat antibody (Santa Cruz, 1: 10,000) at 25°C for 1 h. The immunoblots were visualized using Western Bright Peroxide solution (Advansta, CA, USA) and ChemiDoc Touch imaging system (Bio-Rad, U.S.A) according to the manufacturer’s instructions. Antibodies used in this study are as follows: p-CREB (Santa Cruz-81486), CREB (Santa Cruz-81486), tyrosinase (Cell Signaling-104976), pMITF (Abcam-59201), MITF (Abcam-20663), p-p38 (Cell Signaling-921L), p38 (Cell Signaling-9212S), pERK (Cell Signaling-4370L), ERK (Cell Signaling-9201L), pJNK (Cell Signaling-9251L), JNK (Cell Signaling-9252S), and β-actin (Santa Cruz-47778).
Measurement of ROS and ONOO- levels
The antioxidative activity of swertiajaponin was examined using fluorescent 2,7-dichlorodihydrofluorescein diacetate (DCFDA) for ROS and dihydro rhodamine (DHR) 123 for ONOO-. Briefly, to determine ROS level, DCFDA (25μM) was added to cell homogenates for a final volume of 250μl. To measure ONOO- level, 10μl of cell homogenates was added to a rhodamine solution (50mM sodium phosphate buffer, 90mM sodium chloride, 5mM diethylenetriaminepentaacetic acid [DTPA], and DHR123). For both assays, fluorescence was measured every 5min for 30min on a fluorescence plate reader, with excitation and emission wavelengths set at 485 and 530 nm, respectively.
Melanin accumulation in a human skin model
A viable, reconstituted, three-dimensional human epidermis (Neoderm-ME, Tego Science) was used to examine the anti-melanogenic effect of swertiajaponin in a human skin model. The human skin model was pretreated with DMSO (vehicle) or swertiajaponin for 1 h and cultured in the maintenance media provided by the company for 5 d with DMSO or swertiajaponin treatment. Microscopic analysis was performed from day 1 to day 5 to observe skin pigmentation. The microscopic images were analyzed by Image J software to semi-quantify the darkening of the skin. For Fontana-Masson staining, skin samples were fixed in 4% paraformaldehyde overnight at room temperature and the samples were analyzed by a commercially available company (Garam Meditech, South Korea).
Statistical analysis
All data are presented as mean ± SEM. Different groups were compared using a one-way analysis of variance followed by Dunnett’s post-test. P < 0.05 was considered statistically significant.
CONFLICTS OF INTEREST
There are no conflicts of interest to declare.
FUNDING
This work was supported by Grant K17281 from the Korea Institute of Oriental Medicine, Ministry of Education, Science and Technology (MEST), Republic of Korea.
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