Swertiajaponin Inhibits Skin Pigmentation By Dual Mechanisms To Suppress Tyrosinase Part 1

ABSTRACT

Many skin-whitening compounds target tyrosinase because it catalyzes two rate-limiting steps in melanin synthesis. Although many tyrosinase inhibitors are currently available for skin–whitening purposes, undesirable adverse effects are also reported. Thus, numerous efforts have been made to develop safer tyrosinase inhibitors from natural products. In line with this, we tested fifty flavonoids, a group of naturally occurring antioxidants and metal chelators, and screened swertiajaponin as the strongest tyrosinase inhibitor in cell-free experiments. Swertiajaponin did not show cytotoxicity in B16F10, HaCat, and Hs27 cells and exhibited strong antioxidative activity in experiments using the cell-free system and B16F10 cells. It markedly inhibited αMSH- or UVB-induced melanin accumulation in B16F10 cells and suppressed skin pigmentation in a human skin model. As underlying mechanisms, in silico and Lineweaver-Burk plot analyses exhibited that swertiajaponin may directly bind to and inhibit tyrosinase activity by forming multiple hydrogen bonds and hydrophobic interactions with the binding pocket of tyrosinase. In addition, western blotting results indicated that swertiajaponin inhibited oxidative stress-mediated MAPK/MITF signaling, leading to a decrease in tyrosinase protein level. Together, swertiajaponin suppresses melanin accumulation by inhibiting both activity and protein expression levels of tyrosinase. Thus, it would be a novel additive for whitening cosmetics.

Keywords

swertiajaponin; melanogenesis; pigmentation; human skin; tyrosinase

INTRODUCTION

Skin is a major site where melanin pigments are accumulated. Melanin synthesis and accumulation occur as a protective mechanism against ultraviolet (UV) irradiation. Thus, people with black skin are at lower risk for skin cancers than those with pale skin because they are protected from the mutagenic effects of UV [1]. However, uncontrolled melanogenesis is closely associated with pigmentation disorders including melisma, freckles, and senile lentigines [2]. In addition, abnormal alterations in skin color result in unfavorable cosmetic problems and eventually degrade the quality of life. Thus, the prevention of abnormal melanogenesis has attracted substantial attention for the development of skin-whitening cosmetics [3–5].

The melanocyte is the main cell type that synthesizes melanin in the epidermis of the skin. After synthesis, melanocytes transfer melanin to keratinocytes through the dendrites, leading to skin pigmentation. Melanin can be synthesized from L-tyrosine through hydroxylation and oxidation processes, in which a multifunctional copper-containing tyrosinase plays a pivotal role in melanin synthesis. Tyrosinase catalyzes two rate-limiting steps in melanin synthesis; the mycophenolate hydroxylation of L-tyrosine to 3,4-dihydroxy-L-phenylalanine (L-DOPA), and the oxidation of L-DOPA to DOPA quinine [6, 7]. Accordingly, tyrosinase inhibition is a good strategy to brighten the skin, but cytotoxicity and lack of stability have limited the application of various tyrosinase inhibitors in the field of medical products and cosmetics. Thus, unceasing efforts have been made to find safer tyrosinase inhibitors with stronger stability and selectivity from natural sources.

To find compounds for better and safer whitening effects for the skin, we tested fifty flavonoids available naturally for their inhibitory effects on tyrosinase and screened swertiajaponin as the strongest tyrosinase inhibitor. We further investigated its effect on skin pigmentation using cell and human skin models with various biological assays and in silico analysis.

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.

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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.

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.

RESULTS AND DISCUSSION

Swertiajaponin is the strongest tyrosinase inhibitor of fifty flavonoids

Of various natural compounds, flavonoids, a group of naturally occurring antioxidants and metal chelators, have been known to suppress tyrosinase activity because of their ability to form copper-flavonoid complexes [8, 9]. We used fifty flavonoids that were commercially available to test whether they have inhibitory activity against mushroom tyrosinase. Kojic acid, a well-known tyrosinase inhibitor, was used as a positive control to screen better tyrosinase inhibitors (Figure 1A-1B). As a result, sample number 40 (swertiajaponin) (Figure 1C) exhibited the strongest inhibitory activity against tyrosinase than that of other flavonoids (Figure 1A-1B and Supplementary Figure 1). When the inhibitory activity was further examined by a concentration-dependent experiment, the IC50 value of kojic acid was 41.26 μM and that of swertiajaponin was 43.47 μM (Figure 1D), indicating that tyrosinase activity inhibition of swertiajaponin is comparable to that of kojic acid based on test tube experiments.

Swertiajaponin shows no cytotoxicity in vitro

Swertiajaponin belongs to the family of flavonoid C-glycosides. It contains a carbohydrate moiety that is C-glycosidically linked to one of the flavonoid backbones (Figure 1C). It has been reported as a compound found in the whole herb of Swertia japonica [10]. Although some studies reported that Swertia japonica is used clinically as a remedy for gastrointestinal symptoms in Japan [11], few studies are available about the biological functions of swertiajaponin. Thus, we further examined its effects of it on melanogenesis. Because cytotoxicity is a major issue of synthetic tyrosinase inhibitors, we tested whether swertiajaponin showed cellular toxicity using multiple cell lines including HaCat (human keratinocyte), B16F10 (mouse melanoma), and Hs27 (human fibroblast).  Swertiajaponin showed no cytotoxicity in these cell lines (Figure 2A-2C).

Swertiajaponin inhibits melanogenesis in B16F10 cells

We studied its effect on melanogenesis using B16F10 cells pre-treated with kojic acid or swertiajaponin followed by UVB or αMSH exposure, the strongest melanogenic inducers. As expected, UVB or αMSH exposure notably increased melanin levels in B16F10 cells (Figure 2D-2F), and swertiajaponin treatment reduced cellular melanin content in a dose-dependent manner post-UVB or αMSH exposure (Figure 2D-2F). In cell studies, the anti-melanogenic effect of swertiajaponin appears to be stronger than that of kojic acid (Figure 2E- 2F). Similarly, the color of the cell pellet pre-treated with swertiajaponin before αMSH treatment was brightened compared to the αMSH-treated cells (Figure 2D), indicating that swertiajaponin inhibits UVB or αMSHinduced melanogenesis.

Swertiajaponin inhibits melanogenesis in a human skin model

To confirm that swertiajaponin suppresses skin pigmentation in humans, we cultured a viable, three-dimensional, reconstituted human epidermis consisting of human melanocytes and keratinocytes that have been known to undergo spontaneous melanogenesis with time [12]. We pretreated the human skin model with swertiajaponin for 1 h and cultured it in the maintenance medium for 5 d. Compared to day 1, the human skin model was darkened on day 5 and swertiajaponin treatment prevented it in a concentration-dependent manner (Figure 3A-3B). Similarly, Fontana-Masson staining exhibited that swertiajaponin markedly reduced melanin levels in the epidermis (Figure 2C), suggesting that swertiajaponin decreases pigmentation in the human skin model.

Swertiajaponin may bind to and inactivate tyrosinase

We next investigated mechanisms underlying the swertiajaponin-mediated anti-melanogenic effects using B16F10 cells. Because swertiajaponin suppressed tyrosinase activity in the cell-free experiments using mushroom tyrosinase (Figure 1B), we hypothesized that it directly binds to and deactivate tyrosinase. To test this, protein-ligand docking simulation was performed to predict binding affinity using AutoDock Vina. The predicted binding affinity between tyrosinase and kojic acid was -5.4 kcal/mol and the affinity between tyrosinase and swertiajaponin was -6.3 kcal/mol (Figure 4A and 4C), suggesting that swertiajaponin may bind to tyrosinase with stronger affinity than kojic acid does. To further investigate the binding mode between compounds and tyrosinase, pharmacophore analysis was performed using LigandScout 3.1 software. While kojic acid may bind to tyrosinase mainly via hydrogen bonds with the ASN260 or GLU256 residue of tyrosinase (Figure 4B), swertiajaponin forms multiple hydrogen bonds with HIS85, ASN81, ASN260, HIS244, and ARG268 residues of tyrosinase and hydrophobic interactions with MET257, VAL248, and PHE264 residue of tyrosinase (Figure 4D), which likely explains the higher binding affinity of swertiajaponin to tyrosinase.

Because a predefined binding site of tyrosine was applied as a docking pocket in the protein-ligand docking simulation [3], it is likely that swertiajaponin may competitively bind to tyrosinase with tyrosine. To examine this, a Lineweaver-Burk analysis was performed. As the concentration of swertiajaponin elevated, Km values also increased while Vmax values were consistent (Figure 4E-4F), indicating that swertiajaponin is a competitive inhibitor of tyrosinase. 

Swertiajaponin is an antioxidant that suppresses UVB-induced MITF/tyrosinase signaling

Although the IC50 value of swertiajaponin is similar to that of kojic acid in the cell-free experiment, actual inhibition of melanogenesis is greater in the swertiajaponin-treated cells than the kojic acid-treated cells, indicating that the direct binding to tyrosinase may not be the only mechanism underlying swertiajponin mediated inhibition of melanogenesis. It is well-documented that the biological functions of flavonoids are associated with their anti-oxidative effects [13]. Therefore, we studied whether swertiajaponin regulates oxidative stress by cell-free reactive oxygen (ROS) and peroxynitrite (ONOO- ) scavenging assays. Swertiajaponin exhibited strong ROS and ONOO-scavenging activities comparable to Trolox and penicillamine, well-known ROS and ONOO inhibitors, respectively (Figure 5A-5B). We further tested whether the anti-oxidative activity of swertiajaponin was also shown in B16F10 cells post-UVB exposure. UVB exposure significantly increased ROS and ONOO- production, whereas swertiajaponin decreased them in a concentration-dependent manner, suggesting that swertiajaponin is an antioxidant (Figure 5C-5D).

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