Ginseng-Lactobacillus Ferment Exerts Synergistic Anti-Melanogenic Effects with Low Doses of Niacinamide In Vitro

Ferment Synergistic Anti-Melanogenic Effects with Low Doses of Niacinamide In Abstract The fermentation of ginseng increases the efficacy and bioavailability of ginseng. However, the ginsenoside Rb 2-specific molecular mechanism in the ginseng lactobacillus ferment (GLF) with niacinamide has not been elucidated in murine B16F10 melanoma cells. In this study, we investigated the possible synergistic anti-melanogenic effects of the GLF or ginsenoside Rb 2 and low dose of niacinamide (100 μM) on B16F10 cells and explored the potential synergistic role of anti-melanogenesis. Cell proliferation analysis was evaluated by MTT assay. The effects on melanogenesis were evaluated spectrophotometrically. The expression levels of melanogenesis-related proteins were analyzed by Western blot. Combining of GLF (0.75 mg/mL) or ginsenoside Rb 2 (10 μM) with of niacinamide (100 μM) significantly decreased cellular melanin content. In the western blotting assay, the combination of GLF or Rb 2 and niacinamide decreased the protein expression levels of tyrosinase, tyrosinase-related protein 1 (TRP-1) and PMEL17 compared with control alone. The results demonstrated that ginsenoside Rb 2 in GLF may play a role in the synergistic anti-melanogenic effects of GLF with niacinamide on murine melanoma cells. The findings suggested GLF or ginsenoside Rb2 can be added into skin whitening skin care product with niacinamide to enhance its anti-melanogenesis effects. the of a


Introduction
In the field of research on skin health, much effort has focused on developing functional skin-whitening products for people with unwanted pigments. Hyperpigmentation of the skin is a result of abnormal accumulation of melanin, a skin pigment that is essential for photoprotection of the human body against ultraviolet (UV) hurts. Previous studies have reported that the first steps of melanogenesis pathway in humans are the hydroxylation of L-tyrosine to 3-4-dihydroxy phenylalanine (L-DOPA) and the oxidation of L-DOPA to o-dopaquinone [1]. Tyrosinase is known as the rate-limiting enzyme and catalyzes both of the reactions in melanogenesis. Antioxidants, such as arbutin or kojic acid [2], may inhibit these oxidation steps and have been used in the treatment of hyperpigmentation [3]. Niacinamide (also known as nicotinamide, 3-pyridinecarboxamide) is the amide of niacin (vitamin B3) that is a physiologically active component in various cosmetics and medicines. Niacinamide is well tolerated by the skin, in contrast to other common forms of this vitamin family (e.g. nicotinic acid and its esters), which often induce uncomfortable skin flushing reactions [4]. It has been reported that oral niacinamide (or niacin) could prevent the development of insulin-dependent diabetes mellitus [5].
Moreover, niacinamide has been reported to promote the repair of DNA damage induced by UV in keratinocytes [11,12]. Previous study has been demonstrated that niacinamide affect pigmentation that works by inhibiting melanosome transfer from melanocytes to keratinocytes [13]. Also, several benefits in terms of improved appearance of photo-aged skin including redness and wrinkles, reduced sebum production and improved barrier function have been described by topical usage of niacinamide [14,15]. As a typical medicine for treating hyperpigmentation disorders, niacinamide blocks the melanosome migration between melanocytes and keratinocytes and suppresses skin pigmentation [16]. Panax ginseng C.A. Meyer has been used in traditional folk medicine in Asian countries.
The cells were exposed to GLF (0.75 mg/mL), ginsenoside Rb 2 (10 μM) or niacinamide (100 μM) for 24 h, then the MTT solution was added to the wells. The insoluble derivative of MTT that was produced by intracellular dehydrogenase was solubilized with DMSO-ethanol (1:1 mixture solution). The absorbance of the wells at 570 nm was read using a microplate reader. The results are expressed as percent viability relative to control. Each sample was measured in triplicate, and each experiment was repeated at least three times. The mushroom tyrosinase activity assays were conducted as previously described [33]. In brief, the aqueous solution of mushroom tyrosinase (200 units/10 μL) was added to a 96-well microplate to result in a mixture with a total volume of 200 μL that contained L-DOPA (5 mM), which was dissolved in phosphate-buffered saline (PBS, 50 mM, pH 6.8), and GLF (0.75 mg/ mL), ginsenoside Rb 2 (10 μM) or niacinamide (100 μM), arbutin (2 mM) or kojic acid (200 μM) were then added into the mixture. The assay mixture was incubated at 37°C for 30 min, and the absorbance of the dopachrome that was produced was measured at 490 nm.
The intracellular melanin content in the B16F10 melanoma cells was measured as described by Tsuboi, et al. [34].
The cells were treated with α-MSH (100 nM) for 24 h, and the melanin content was determined after treatment with either GLF (0.75 mg/mL), ginsenoside Rb 2 (10 μM) or niacinamide (100 μM) or arbutin (2 mM) for an additional 24 h. After the treatment, the cell pellets containing a known number of cells were solubilized in 1 N NaOH at 60°C for 60 min. The melanin content was assayed at 405 nm. The intracellular tyrosinase activity of the B16F10 cells was determined as described previously [35]. The cells were treated Differences were considered as statistically significant at p < 0.05.

Results
The UPLC chromatograms of the GLF are shown in ( Figure   1) and the analysis of the ginsenosides in the preparations are represented in (Table 1). The concentration s of ginsenoside Rb 1, Rb 2 and Rg 3 in GLF were 554.72 ± 24.09, 231.68 ± 19.29, and 549.03 ± 20.03 (μg/mL), respectively (Table 1)     on the proliferation of B16F10 cells. Cell viability was measured by the MTT assay method after 24 h of incubation. Data are expressed as a percentage of the number of viable cells observed in the control group, and each column presents the mean values ± SD from three independent experiments performed in triplicate.

Discussion
The precision of the developed UPLC method was determined by intra-and inter-day variations. The ginseng lactobacillus ferment (GLF) sample was analyzed as described in the materials and methods sections. There are different ginsenosides in ginseng extract. It has been reported that Rb1 [36], Rb2 [28], and Rg3 [37] exhibit anti-melanogenesis activity. We aim to elucidate which ginsenoside could exert synergistically inhibitory effect on melanogenesis with niacinamide, so we analyzed the content of the three ginsenosides by UPLC (Figure 1). The applications of herbal medicines through adequate compatibility may exert synergistic effects and reduce side effects or drug resistance [38].
The health benefits including prevention of diseases such as cancer can be achieved with increased consumption of vegetables, fruits and botanical dietary supplements containing rich sources of phytochemicals. The action mechanisms by which phytochemicals, including ginsenosides could prevent diseases such as cancer are not clear, but potentially appear to involve the Nrf2-ARE-mediated anti-oxidative stress pathway. In addition, ginseng is often consumed as a mixture comprising various ginsenosides [39,40]. The MTT assay for cell viability is a colorimetric assay method that is used to measure the activity of NADH/NADPH-dependent cellular oxidoreductase enzymes that reduce MTT to formazan dyes, giving the solution a purple color. The results shown in (Figure 2) indicated that GLF (0.75 mg/mL) or ginsenoside Rb 2 (10 μM) had no cytotoxic effect on B16F10 melanoma cell viability. Mushroom tyrosinase is widely utilized as a target enzyme for screening potential activators or inhibitors of melanogenesis. It was reported that all tyrosinases have a binuclear type three copper center within their active site. However, there was no common tyrosinase protein structure found to occur across all specie [41,42]. Mushroom tyrosinase from Agaricus bisporus was reported to be a soluble cytosolic enzyme. In contrast, human tyrosinase is a monomeric membrane-bound glycoprotein. It has been reported that there is no significant correlation between the inhibition of mushroom tyrosinase with human melanoma tyrosinase and melanogenesis [43]. In flavonoids, it has been suggested that anti-tyrosinase activity against mushroom would only appear in parallel with mammalian tyrosinase activity and melanogenesis inhibition [44].
Hence, the structural diversity of the tyrosinase-inhibitory compounds could provide a good source for studying the correlation between enzymatic assay and cell-based assays. In ( Figure 3A), it was shown that niacinamide (100 μM) or 0.75 mg/mL of GLF alone could not significantly inhibit the activity of mushroom tyrosinase. However, combination of niacinamide (100 μM) and GLF (0.75 mg/mL) exerted significantly inhibitory effects on mushroom tyrosinase in vitro. Similar synergistic effect was also found in combination of niacinamide (100 μM) and ginsenoside Rb 2 (10 μM) ( Figure 3B). The results shown in (Figure 3C & 3E) indicated that niacinamide (100 μM) and GLF (0.75 mg/mL) exerts a synergistic inhibitory effect on melanin production and cellular tyrosinase activity, reapectively. The results in ( Figure 3D) were in accordance with the results described in (Figure 3F), which means that niacinamide (100 μM) and ginsenoside Rb 2 (10 μM) synergistically inhibited the B16F10 cellular tyrosinase activity and then decreased the melanin content. The results indicated that ginsenoside Rb 2 may play a role in the synergistic inhibitory effect of GLF and niacinamide on the α -MSH-induced melanogenesis in B16F10 cells. In the present study, GLF and ginsenoside Rb 2 were observed to inhibit melanogenesis by inhibiting tyrosinase. We hypothesized that the modulation of the melanogenesis-related proteins may be responsible for the inhibitory activity of GLF or ginsenoside Rb 2 on melanogenesis. Hence, the cell lysates were subjected to Western blot analysis to determine the expression levels of melanogenesis-related proteins. In mammals, melanogenesis is regulated by multiple enzymes, such as tyrosinase, TRP-1, TRP-2 and PMEL [45,46]. PMEL17 (also known as gp100 or the product of the Silver locus) is a type I integral membrane glycoprotein that localizes to the lumen of melanosome precursors [47][48][49]. It is highly expressed by melanocytes and serves as a common target for tumor-directed T lymphocytes in patients with melanoma [50].
The results shown in (Figure 4) indicated that GLF or ginsenoside Rb 2 decreased the protein expression levels tyrosinase, TRP-1 and PMEL17. Interestingly, either GLF or ginsenoside Rb 2 exerted antimelanogeic effects synergistically with low dose of niacinamide (100 μM).
Hence, the results suggest that the anti-melanogenic activity of GLF or ginsenoside Rb 2 was probably due to its inhibitory effects on cellular tyrosinase activity and the subsequent decrease in melanin production. The data obtained from this study show that GLF or ginsenoside Rb 2 inhibit melanogenesis in B16F10 melanoma cells without causing cytotoxicity. These findings indicate that combination of GLF or ginsenoside Rb 2 with niacinamide may be useful in treating hyperpigmentation disorders. Furthermore, GLF or ginsenoside Rb 2 and niacinamide could also be used in the formulation of skin whitening products.

Conclusion
This is the first report on the mechanism of action of the synergistic effect of GLF or ginsenoside Rb 2 with niacinamide on melanin biosynthesis. The present study concluded that GLF or ginsenoside Rb 2 inhibit tyrosinase activity and melanin production in B16F10 cells by declining the expression of tyrosinase, TRP-1 and PMEL17. This finding is of great cosmeceutical importance for the design of skin whiting products that have the potential to reduce hyperpigmentation-related skin cancer risk (Graphic Abstract).