Molecular Analysis of the Melanogenesis Inhibitory Effect of Saponins-Rich Fraction of Argania spinosa Leaves Extract

Plant saponins are abundant and diverse natural products with a great potential for use in drug-discovery research. Here, we evaluated extracts of saponins-rich fractions of argan leaves and argan oil extraction byproducts (shell, pulp, press cake) for their effect on melanogenesis. Results show that from among the samples tested, only the saponins-rich fraction from leaves (ALS) inhibited melanin production in B16 murine melanoma (B16) cells. The mechanism of the melanogenesis inhibition was elucidated by determining the protein and mRNA expression of melanogenesis-associated enzymes tyrosinase (TYR), tyrosinase-related protein 1 (TRP1), and dopachrome tautomerase (DCT), and microphthalmia-associated transcription factor (MITF), and performing DNA microarray analysis. Results showed that 10 µg/mL ALS significantly inhibited melanogenesis in B16 cells and human epidermal melanocytes by 59% and 48%, respectively, without cytotoxicity. The effect of ALS on melanogenesis can be attributed to the decrease in TYR, TRP1, and MITF expression at the protein and mRNA levels. MITF inhibition naturally led to the downregulation of the expression of Tyr and Trp1 genes. Results of the DNA microarray analysis revealed the effect on melanogenesis-associated cAMP and Wnt signaling pathways’ genes. The results of this study suggest that ALS may be used in cosmeceuticals preparations for hyperpigmentation treatment.


Introduction
Skin pigmentation is imparted by melanin produced by melanocytes found at the skin's epidermal layer. Aside from giving the skin its characteristic color, melanin also functions as a free radical scavenger, protecting melanocytes from damage caused by ultraviolet radiation (UVR), the main causative factor in the induction of melanoma [1]. Melanin, in human and animal skin, eyes, and hair, is synthesized via the catalytic action of melanogenesis enzymes tyrosinase (TYR), tyrosinase-related protein 1 (TRP-1), and dopachrome tautomerase (DCT), also called tyrosinase-related protein 2 (TRP-2) [2]. The rate-limiting enzyme TYR is a copper-containing enzyme that converts L-tyrosine to 3,4dihydroxyphenylalanine (DOPA) and catalyzes the oxidation of DOPA into DOPA quinone. DCT functions as DOPA-chrome tautomerase and catalyzes the rearrangement of DOPAchrome into 5,6-dihydroxyindole-2-carboxylic acid (DHICA), whereas TRP-1 oxidizes DHICA into a carboxylated indole-quinone [3]. Melanocytes produce the pigment melanin in membrane-bound organelles called melanosomes. The active export of melanosomes through dendritic processes to surrounding cells, the keratinocytes, is a crucial step in the effect of saponins-rich fractions of argan leaves, shell, pulp, and press cakes on skin pigmentation. This study was conducted to evaluate the melanogenesis regulatory effect of saponins-rich fractions of extracts of argan leaves, shell, press cakes, and pulp, and to elucidate the mechanism underlying the effect of the saponin fractions with melanogenesis inhibitory effect.

Qualitative Evaluation of Saponins in Argan Polar Fractions
Saponins-rich fractions of argan were subjected to frothing test and thin layer chromatography (TLC) to test for the presence of saponins. The formation of a foam in the frothing test, which was stable for a while, is an indication of the surface-active properties of the molecules of these fractions, properties that are characteristics of saponins [27,28]. TLC analysis results confirmed the presence of the saponins. After spraying with vanillinperchloric acid reagent, blue and purple spots develop in the TLC, which is characteristic of saponins (Supplementary Materials: Figure S1) [28].

Saponins-Rich Fraction of Argan Leaves, Fruit Pulp, Shell, and Press Cakes Modulated Melanogenesis without Cytotoxicity
B16 cells treated for 48 h with the saponins-rich fraction of argan samples were subjected to MTT and melanin assays. The results showed that the saponin fractions of argan leaves, shell, pulp, and press-cake extract were not cytotoxic to B16 cells at up to 20 µg/mL. Furthermore, while a slight decrease in proliferation was observed in cells treated with 5 µg/mL argan shell and 10 µg/mL argan press cake I, the difference (vs. control) was not significant ( Figure 1A). Melanogenesis assay results showed that the different saponins-rich fractions at 5 µg/mL had either melanogenesis inhibition or promotion effects ( Figure 1B). Crude saponins fractions from argan shell, pulp, and press cake promoted melanogenesis by 50 to 80%, whereas ALS inhibited melanogenesis.
of saponins-rich fractions of extracts of argan leaves, shell, press cak elucidate the mechanism underlying the effect of the sapon melanogenesis inhibitory effect.

Qualitative Evaluation of Saponins in Argan Polar Fractions
Saponins-rich fractions of argan were subjected to frothing chromatography (TLC) to test for the presence of saponins. The forma frothing test, which was stable for a while, is an indication of the surfa of the molecules of these fractions, properties that are characteristics TLC analysis results confirmed the presence of the saponins. After spr perchloric acid reagent, blue and purple spots develop in the TLC, w of saponins (Supplementary Materials: Figure S1) [28].

Saponins-Rich Fraction of Argan Leaves, Fruit Pulp, Shell, and Press C Melanogenesis without Cytotoxicity
B16 cells treated for 48 h with the saponins-rich fraction of a subjected to MTT and melanin assays. The results showed that the argan leaves, shell, pulp, and press-cake extract were not cytotoxic to µg/mL. Furthermore, while a slight decrease in proliferation was obse with 5 µg/mL argan shell and 10 µg/mL argan press cake I, the differe not significant ( Figure 1A). Melanogenesis assay results showed saponins-rich fractions at 5 µg/mL had either melanogenesis inhib effects ( Figure 1B). Crude saponins fractions from argan shell, pu promoted melanogenesis by 50 to 80%, whereas ALS inhibited melan  Cell proliferation of B16 cells determined using MTT assay. B16 cells were seeded at a density of 3 × 10 3 cells per well of 96-well plate. After overnight incubation, cells were treated for 48 h with 5 µg/mL, 10 µg/mL, and 20 µg/mL of saponins-rich fraction of argan leaves (ALS), argan shell (SS), argan fruit pulp (SP), press cake from roasted argan (SPCI), and press cake from nonroasted argan (SPCII). (B) Melanin content was quantified using melanin assay and expressed as melanin content/cell (% of control). B16 cells were seeded at a density of 5 × 10 5 cells per 100 mm Petri dish. After overnight incubation, cells were incubated for 48 h with 5 µg/mL of saponins-rich fraction of ALS, SS, SP, SPCI, and SPCII. (C) Melanin extracted from B16 cells, as described in (B), dissolved in 99.5% ethanol. *indicates significance at p < 0.05, ** at p < 0.01.

Saponins-Rich Fraction of Argan Leaves (ALS) Extract Inhibits Melanogenesis in B16 Cells
Based on the preliminary test results, we decided to focus the succeeding experiments on the effect of the saponins-rich argan leaves (ALS), which inhibited melanogenesis. Dose-dependent melanogenesis assay results showed that B16 cells, treated with ALS at 5 µg/mL and 10 µg/mL, had 20% and 40% lower melanin content (vs. control), respectively ( Figure 2A). The decrease in the melanin content was also evident in the precipitated melanin dissolved in NaOH, as shown in Figure 2B. Cell proliferation of B16 cells determined using MTT assay. B16 cells were seeded at a density of 3 × 10 3 cells per well of 96-well plate. After overnight incubation, cells were treated for 48 h with 5 µg/mL, 10 µg/mL, and 20 µg/mL of saponins-rich fraction of argan leaves (ALS), argan shell (SS), argan fruit pulp (SP), press cake from roasted argan (SPCI), and press cake from nonroasted argan (SPCII). (B) Melanin content was quantified using melanin assay and expressed as melanin content/cell (% of control). B16 cells were seeded at a density of 5 × 10 5 cells per 100 mm Petri dish. After overnight incubation, cells were incubated for 48 h with 5 µg/mL of saponins-rich fraction of ALS, SS, SP, SPCI, and SPCII. (C) Melanin extracted from B16 cells, as described in (B), dissolved in 99.5% ethanol. * indicates significance at p < 0.05, ** at p < 0.01.

Saponins-Rich Fraction of Argan Leaves (ALS) Extract Inhibits Melanogenesis in B16 Cells
Based on the preliminary test results, we decided to focus the succeeding experiments on the effect of the saponins-rich argan leaves (ALS), which inhibited melanogenesis. Dose-dependent melanogenesis assay results showed that B16 cells, treated with ALS at 5 µg/mL and 10 µg/mL, had 20% and 40% lower melanin content (vs. control), respectively ( Figure 2A). The decrease in the melanin content was also evident in the precipitated melanin dissolved in NaOH, as shown in Figure 2B.

ALS Decreased the Protein Expression of Melanogenesis Enzymes
To determine the mechanism underlying the observed effect of ALS on melanogenesis, the effect of ALS on the protein expression of the melanogenesis enzymes tyrosinase (TYR), tyrosinase-related protein 1 (TRP1), and dopachrome tautomerase (DCT), and of these enzymes' transcription factor, microphthalmia-associated transcription factor (MITF), was investigated. Western blotting results showed that compared with the control, the TYR and TRP1 expression level decreased when the cells were treated with 10 µg/mL of ALS ( Figure 3A). No significant change was observed on the expression of DCT. MITF expression was also decreased by ALS treatment ( Figure 3B). Quantification of the Western blot signals detected revealed that ALS reduced the TYR and TRP1 expression level by 40% (24 h) and 20-25% (at 24 h, 48 h), respectively (Supplementary Figure S2). The expression of MITF was also decreased by ALS by 50%, 48 h after treatment ( Figure 2B).

ALS Decreased the Protein Expression of Melanogenesis Enzymes
To determine the mechanism underlying the observed effect of ALS on melanogenesis, the effect of ALS on the protein expression of the melanogenesis enzymes tyrosinase (TYR), tyrosinase-related protein 1 (TRP1), and dopachrome tautomerase (DCT), and of these enzymes' transcription factor, microphthalmia-associated transcription factor (MITF), was investigated. Western blotting results showed that compared with the control, the TYR and TRP1 expression level decreased when the cells were treated with 10 µg/mL of ALS ( Figure 3A). No significant change was observed on the expression of DCT. MITF expression was also decreased by ALS treatment ( Figure 3B). Quantification of the Western blot signals detected revealed that ALS reduced the TYR and TRP1 expression level by 40% (24 h) and 20-25% (at 24 h, 48 h), respectively (Supplementary Figure S2). The expression of MITF was also decreased by ALS by 50%, 48 h after treatment ( Figure 2B).

ALS Downregulated the Expression of Tyr and Trp1 Genes
TaqMan real-time PCR (qPCR) was then used to determine the effect of ALS on the expression of melanogenesis enzyme genes Tyr, Trp1, and Dct in B16 cells. ALS significantly reduced the expression levels of Tyr ( Figure 3C) and Trp1 ( Figure 3D), whereas no difference was observed in the expression of Dct ( Figure 3E), compared with the untreated cells.

ALS Inhibited Melanogenesis in Human Epidermal Melanocytes (HEMs)
To validate if the observed effect of ALS on murine B16 cells is true for human

ALS Downregulated the Expression of Tyr and Trp1 Genes
TaqMan real-time PCR (qPCR) was then used to determine the effect of ALS on the expression of melanogenesis enzyme genes Tyr, Trp1, and Dct in B16 cells. ALS significantly reduced the expression levels of Tyr ( Figure 3C) and Trp1 ( Figure 3D), whereas no difference was observed in the expression of Dct ( Figure 3E), compared with the untreated cells.

ALS Inhibited Melanogenesis in Human Epidermal Melanocytes (HEMs)
To validate if the observed effect of ALS on murine B16 cells is true for human epidermal melanocytes, cell proliferation and MTT assays were performed. MTT assay results showed that ALS was not cytotoxic to HEMs at the concentrations tested (0, 2.5, 5, and 10 µg/mL) ( Figure 4A). The result of the MTT assay was the basis for the ALS concentrations used in the melanogenesis assay. For this experiment, HEMs were treated with ALS, arbutin, and phorbol 12-myristate 13-acetate (PMA). The results show that treatment with ALS reduced the melanin produced in HEMs by 51% compared with control (untreated), and this effect of ALS at 10 µg/mL was comparable with the effect of arbutin (Arb, 100 µM), a known melanogenesis inhibitor ( Figure 4B). ALS at 5 µg/mL and 10 µg/mL concentrations decreased the melanin content of HEMs by 20% and 50%, respectively. The cell pellets after the cells were harvested for melanin assay show the decreased melanin content of HEMs following treatment with ALS and arbutin (Arb) ( Figure 4C). results showed that ALS was not cytotoxic to HEMs at the concentrations tested (0, 2.5, 5, and 10 µg/mL) ( Figure 4A). The result of the MTT assay was the basis for the ALS concentrations used in the melanogenesis assay. For this experiment, HEMs were treated with ALS, arbutin, and phorbol 12-myristate 13-acetate (PMA). The results show that treatment with ALS reduced the melanin produced in HEMs by 51% compared with control (untreated), and this effect of ALS at 10 µg/mL was comparable with the effect of arbutin (Arb, 100 µM), a known melanogenesis inhibitor ( Figure 4B). ALS at 5 µg/mL and 10 µg/mL concentrations decreased the melanin content of HEMs by 20% and 50%, respectively. The cell pellets after the cells were harvested for melanin assay show the decreased melanin content of HEMs following treatment with ALS and arbutin (Arb) ( Figure 4C). Data are expressed as mean ± SD (n = 5). **indicates significance at p < 0.01.

Global Gene Expression in Human Epidermal Melanocytes (HEMs) Elucidated Using DNA Microarray.
Global gene expression was carried out to determine the signaling pathways regulated in HEMs in response to ALS treatment, specifically those that are directly or indirectly related to the observed melanogenesis inhibitory effect of ALS. The results show that 33 genes were significantly modulated (>2 fold change) by ALS, among which 32 genes were upregulated and 1 gene was downregulated (Table 1). These genes are part of the signaling pathways that were significant for MAPK activity, negative regulation of MTT assay was performed on HEM seeded at a density of 3 × 10 3 cells/well of a 96-well dish and treated with different concentrations of argan leaf saponins-rich fraction (2.5 µg/mL, 5 µg/mL, or 10 µg/mL). (B) Melanin assay was carried out on HEMs seeded at a density of 5 × 10 5 cells per 100 mm Petri dish treated with 5 µg/mL or 10 µg/mL of saponins-rich argan leaf sample (ALS), arbutin (Arb, 100 µM), or phorbol 12-myristate 13-acetate (PMA, 10 ng/mL) and incubated further for 48 h. (C) Photograph of HEM pellets before melanin assay was performed. Data are expressed as mean ± SD (n = 5). ** indicates significance at p < 0.01.

Global Gene Expression in Human Epidermal Melanocytes (HEMs) Elucidated Using DNA Microarray
Global gene expression was carried out to determine the signaling pathways regulated in HEMs in response to ALS treatment, specifically those that are directly or indirectly related to the observed melanogenesis inhibitory effect of ALS. The results show that 33 genes were significantly modulated (>2 fold change) by ALS, among which 32 genes were upregulated and 1 gene was downregulated (Table 1). These genes are part of the signaling pathways that were significant for MAPK activity, negative regulation of cGMPmediated signaling, TGFß receptor signaling pathway, response to calcium ion, positive regulation of protein kinase B signaling cascade, and positive regulation of canonical Wnt receptor signaling pathway, among others. Hierarchical clustering results of these 33 genes grouped 5 µg/mL ALS with the positive control arbutin (ARB), whereas 10 µg/mL ALS generated a unique profile. Activation of MAPK activity, response to hypoxia, negative regulation of endothelial cell proliferation, negative regulation of endothelial cell proliferation, negative regulation of cell-matrix adhesion, negative regulation of cGMP-mediated signaling, positive regulation of transforming growth factor beta receptor signaling pathway, response to magnesium ion, response to progesterone stimulus, negative regulation of interleukin-12 production, positive regulation of transforming growth factor beta1 production, cellular response to heat, positive regulation of tumor necrosis factor biosynthetic process, positive regulation of macrophage activation, negative regulation of apoptotic process, response to calcium ion, positive regulation of protein kinase B signaling cascade, positive regulation of reactive oxygen species metabolic process, negative regulation of extrinsic apoptotic signaling pathway, etc.  In utero embryonic development, trophectodermal cell differentiation, apoptotic process, cellular component disassembly involved in execution phase of apoptosis, cell adhesion, homophilic cell adhesion, synapse assembly, sensory perception of sound, response to toxic substance, response to organic substance, cell-cell adhesion, protein metabolic process, etc.
a Treated with 5 µg/mL ALS; b Fold change was 3.1 when treated with 10 µg/mL ALS.
Based on the heat map, arbutin and 5 µg/mL ALS modulated the same set of genes except for PNN and THBS1, which were decreased in the expression in arbutin and 10 µg/mL ALS. For the sample cluster, 5 µg/mL ALS was clustered with arbutin (50% similarity). Validation by real-time PCR of the genes that were highly upregulated, based on DNA microarray results (Table 1), shows that SCM3 and CYP1B1 genes' expressions were indeed increased by ALS (Figure 5C,D). Based on the heat map, arbutin and 5 µ g/mL ALS modulated the same set of genes except for PNN and THBS1, which were decreased in the expression in arbutin and 10 µ g/mL ALS. For the sample cluster, 5 µ g/mL ALS was clustered with arbutin (50% similarity). Validation by real-time PCR of the genes that were highly upregulated, based on DNA microarray results (Table 1), shows that SCM3 and CYP1B1 genes' expressions were indeed increased by ALS ( Figure 5C,D).

Discussion
Melanin protects us from ultraviolet radiation-induced photodamage, but abnormal increases in melanin production or hyperpigmentation may occur and require treatment. Hyperpigmentation, although not life-threatening, has a significant negative impact on an individual's quality of life, making patients feel self-conscious and unattractive [32]. Most of the available cosmetics, to cover up hyperpigmentation, and drugs, to lighten the skin
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Discussion
Melanin protects us from ultraviolet radiation-induced photodamage, but abnormal increases in melanin production or hyperpigmentation may occur and require treatment. Hyperpigmentation, although not life-threatening, has a significant negative impact on an individual's quality of life, making patients feel self-conscious and unattractive [32]. Most of the available cosmetics, to cover up hyperpigmentation, and drugs, to lighten the skin color, have unwanted side effects. There is, therefore, an increasing need to discover safer therapeutics for hyperpigmentation. Hydroquinone, for example, is an effective depigmenting drug but is banned in Europe, Japan, Australia, and several African countries because it was carcinogenic [33] and may cause fatal liver [34]. These reported serious side effects, therefore, create a need to find safer depigmenting agents. Moreover, there is an increasing trend in the use of safe drugs that are botanical in origin or naturally occurring, for use as treatments of pigment disorder or for cosmeceutical use, in general [35]. The medicinal properties of these naturally occurring materials are attributed to their bioactive components, such as saponins, that have specific physiological action. Saponins are bioorganic compounds that are made up of at least one glycosylic linkage at C-3, between a triterpene aglycone and a sugar chain [36]. The combination of polar and nonpolar structural elements in their structure gives the saponins their surface-active properties, which explains their soap-like behavior in aqueous solutions (production of foam) [27,28].
We have previously reported that argan oil and argan press cake have melanin synthesis inhibitory effects [17,20], whereas extracts of other parts of the argan tree such as argan fruit shell and leaves promote melanogenesis [18,19]. These findings show that the different parts of argan plants are rich sources of materials for melanogenesis regulation.
Here, we have shown that the polar fraction of argan leaves extract is a saponins-rich fraction due to its significant surface-active properties [27,28]. More importantly, we have demonstrated that this saponins-rich fraction of argan leaves (or ALS) has melanogenesis inhibitory effect (Figure 2). We have previously reported that argan oil has a melanogenesis inhibitory effect. However, because of argan oil's popularity, it is now in high demand worldwide, increasing its price. ALS, therefore, provides a cheap but equally effective and readily available alternative.
The melanogenesis inhibitory effect of ALS was discovered following a screening experiment, wherein we compared the effect of saponins-rich fractions of argan oil extraction byproducts-shell, pulp, press cake, and argan leaves-on cytotoxicity ( Figure 1A) and melanogenesis ( Figure 1B). Recently, we have also reported that argan leaves promote melanogenesis, and that the effect was attributed to 14 polyphenols including catechins, flavonoids, and phenolic acids in argan leaves [19]. The sample used in this study was a saponins-rich fraction of the leaves, a polar fraction, which also contains traces of these compounds, specifically those that contain a sugar moiety in their structure such as quercetin 3-O-glucuronide, myricetin-3-O-galactoside, myricitrin, and quercetrin.
Following tests to determine the effect on melanocyte differentiation or melanogenesis markers, ALS was found to decrease the expression of TYR and TRP1 proteins ( Figure 3A) as well as the MITF ( Figure 3B). Moreover, at the transcription level, ALS treatment for 24 h inhibited the expression of Tyr and Trp1 genes in B16 cells. There was no change, however, in the expression of Dct ( Figure 3E). All three enzymes are under the transcriptional regulation of the master regulator of melanogenesis, the microphthalmia-associated transcription factor (MITF), because of their identical TCATGTG sequence [42].
However, for TRP-2 (or DCT expression), LEF-1 was reported to work in conjunction with MITF [43]. It is the same for TYR and TRP1 genes in the sense that the transcription of TYR and TRP1 may be enhanced by USF-1 or PAX3, respectively [44]. For TRP2 or DCT transcription, LEF-1 protein, a Wnt/β-catenin pathway effector, physically interacts and cooperates with MITF in the transactivation of the TRP-2 promoter. Moreover, an MITF-LEF-1 interaction and the cis-acting motif in the promoter are required for the TRP-2 promoter stimulation [45]. ALS appears not to promote the MITF-LEF1 interaction since it failed to promote DCT expression. Previous studies have identified a CRE-like element in the TRP-2 promoter that might also contribute to gene expression through direct regulation by CREB protein [46].
ALS has effectively inhibited melanogenesis in B16 cells, and the same was observed on melanogenesis in human epidermal melanocytes (HEMs). ALS inhibited melanogenesis in HEM 48 h after treatment with 10 µg ALS, decreasing melanin content in the same level as 100 µM arbutin ( Figure 4B,C) By performing DNA microarray, we gained some insight into how ALS affects the expression of genes in HEMs. The hierarchical clustering of genes that were significantly regulated by ALS showed that ALS dosage affected the gene expression. Treatment with 5 µg/mL ALS clustered together with arbutin (Arb), a known melanogenesis inhibitor ( Figure 5A). In addition, several genes were highly upregulated including the SCM3 (structural maintenance of chromosomes 3; 3.04-fold change) and CYP1B1 (cytochrome P450, family 1, subfamily B, polypeptide 1; 2.08-fold change), whereas CD1 (cadherin 1, type 1, epithelial E-cadherin) was significantly downregulated by treatment with 5 µg ALS. Treatment with 10 µg/mL ALS upregulated by more than twofold, CYP1B1 (3.1-fold), which was also upregulated by treatment with 5 µg/mL ALS. This suggests that in HEMs, lower treatment concentration is enough to elicit the desired signaling to regulate melanogenesis, and that long-term treatment with 5 µg/mL ALS would eventually decrease the melanin content such as what has been observed in HEMs treated with 5 µg/mL ALS.
There are no reports, however, on the association of SCM3 or CYP1B1 with melanogenesis. In both prokaryotes and eukaryotes, SCM3, a member of the multimeric cohesin complex, mediates sister chromatid cohesion and segregation. CYP1B1 encodes a member of the cytochrome P450 superfamily of mono-oxygenases, which catalyze reactions involved in drug metabolism and synthesis of cholesterol, steroids, and other lipids (www.ncbi.nlm.nih.gov/gene/9126; accessed on 27 May 2022).
The results of the DNA microarray analysis also revealed the effect on genes that may have an effect on melanogenesis, albeit indirectly ( Figure 5B). These genes, THBS1, IQGAP1, and CDH1, are significant for pathways reported to have an effect on melanogenesis, which include cAMP and Wnt/β-catenin pathways. THBS1 codes for the protein thrombospondin l (TSP1), an angiogenesis inhibitor that decreases tumor growth. TSP1 has been reported to directly suppress human melanoma cell growth by inhibiting the activation of matrix metalloproteinase-9 and mobilizing the vascular endothelial factor [29]. It also prevents cAMP and protein kinase A (PKA) signaling through a CD36-dependent mechanism [47].
The IQGAP1 gene was also upregulated by ALS. It may have an influence on melanogenesis, and IQGAP1 protein has been reported to modulate disheveled (DVL) localization in Wnt signaling. IQGAP1 depletion during embryogenesis has been associated with the reduction of Wnt target gene expression [30]. CHD1 was the only gene that was downregulated by more than twofold ( Figure 4B). CDH1 is a gene that is important for retinal pigment epithelium (RPE) function and is a direct target of MITF, the master regulator of melanogenesis [31]. Clearly, ALS modulated genes that are significant for the activation of MAPK activity, negative regulation of cGMP-mediated signaling, TGFβ receptor signaling pathway, response to calcium ion, positive regulation of protein kinase B signaling cascade, and positive regulation of canonical Wnt receptor signaling pathway, among others. These signaling pathways are relevant in melanogenesis regulation.
The results of the validation of the expression of highly upregulated genes SCM3 and CYP1B1 showed increasing mRNA levels of both SCM3 and CYP1B1 following ALS treatment ( Figure 4C,D). The SCM3 gene encodes for a protein that occurs in some cell types and in nuclear form and known as the structural maintenance of chromosomes 3. It is a component of the multimeric cohesin complex that holds together sister chromatids during mitosis, enabling proper chromosome segregation. There has been no report on its role in melanogenesis, but it has been associated with MITF regulation. Goding [48] described that SCM3 has been identified as a cohesin subunit that is one of the several nuclear pore components present during the shuttling in and out of MITF in the nucleus.
Gene ontology (GO) annotations for CYP1B1 indicate that this gene functions in biological processes that include angiogenesis, cellular aromatic compound metabolic process, xenobiotic metabolic process, visual perception, steroid metabolic process, estrogen metabolic process, toxin metabolic process, response to toxic substance, response to organic substance, sterol metabolic process, arachidonic acid metabolic process, and epoxygenase P450 pathway, among others. Beta-catenin is found in the plasma membrane, cytoplasm, and nucleus. When present in the nucleus, it can activate MITF gene expression [49].
In this study, the downregulation of CDH1 may have indirectly inhibited MITF expression since unavailable E-cadherin will prevent β-catenin from translocating to the nucleus, causing decreased MITF gene expression ( Figure 5B). The CDH1 gene is also associated with susceptibility to vitiligo [50]. Recent advances in gene expression profiling by DNA microarray have enabled genome-wide elucidation of the functional genomics of genes involved in various cellular pathways.
This study used a crude saponins-rich fraction of argan leaves extract, the active compound that caused melanogenesis inhibition was, however, not determined. Therefore, in order to gain an understanding of the possible bioactive compounds to which we can attribute the observed effect, published reports on the natural products that are present in the sample are listed in Table 2.

Plant Materials and Sample Preparation
Argan leaves and fruits were collected from the Sidi Ifni region (Morocco) in June 2016. Authenticated samples were kept in the Regional Herbarium of Marrakech under the reference MARK 10888. To collect the fruit parts, the pulp was manually removed, as well as the fruit shell, which was coarsely chopped by hand. The nuts were subjected to oil press extraction, yielding argan oil and press cake. Argan leaves, fruit pulp, fruit shell, and fruit press cake were air-dried then ground to a powder before extraction (saponins preparation).
Saponins were extracted following the method described by Larhsini et al. [58] with slight modifications (Figure 6). Ten grams of plant materials (argan leaves, shell, pulp, and press cake, both roasted or nonroasted) was extracted with 70% ethanol (100 mL) for two weeks in the dark at room temperature using a flask orbital laboratory shaker. The mixture was passed through a Whatman paper (N • 4), then the hydro-alcohol extract was evaporated to dryness in a rotary evaporator under reduced pressure at 40 • C. The residue was then suspended in hot water and was successively defatted and depigmented with n-hexane and ethyl acetate. The aqueous layer was then exhaustively extracted with n-butanol. The organic layer was evaporated to dryness, and the residue dissolved in a small amount of ethanol. Saponins were precipitated by the addition of diethyl ether, then collected by vacuum filtration on a filter paper, and washed with diethyl ether, yielding a crude saponins-rich fraction of argan leaves, pulp, shell, and press cakes in powder form. The saponin yields of the various argan byproducts were subsequently determined and expressed as a percentage of plant material. Prior to use in bioassays, a stock solution of each sample was prepared by dissolving each in 70% ethanol (70% ethyl alcohol and 30% milli-Q water), followed by passing them through a 0.22 µm filter (Merck Millipore). These filter-sterilized samples were then stored at −20 • C until use. Treatment with each saponins-rich fraction was prepared by mixing the sample stock solution with the culture media for B16 cells and HEMs.

Qualitative Determination of Saponins in ALS
The presence of saponins in the sample was determined using standard analytical techniques-the frothing test and thin layer chromatography (TLC) techniques [27,28]. For the frothing test, 2 mL of aqueous ALS and 2 mL of distilled water were shaken for 15

Qualitative Determination of Saponins in ALS
The presence of saponins in the sample was determined using standard analytical techniques-the frothing test and thin layer chromatography (TLC) techniques [27,28]. For the frothing test, 2 mL of aqueous ALS and 2 mL of distilled water were shaken for 15 min in a graduated cylinder. A 1 cm foam layer that persists was a positive response to the presence of saponins. For the TLC, a ready-made TLC plate coated with silica gel 60 F254 was used. ALS spots were manually applied using a capillary tube and, after drying using an air blower, were developed at room temperature in a chromatography-developing tank. The mobile phase solvent system used was dichloromethane-ethyl acetate-methanol-water (60:30:8:1). The developed plate was sprayed with a vanillin-perchloric acid reagent, then heated at 100 • C for 1-2 min. The spots were observed under UV-254 nm and UV-365 nm light. Photos were taken with a smartphone camera (Supplementary Figure S2). The appearance of blue or purple spots confirms the presence of saponins in the analyzed fraction [28]. The retention factor (Rf) values were calculated using the formula: Rf = Distance travelled by solute/Distance travelled by solvent
Briefly, B16 cells (3 × 10 3 cells/well) were seeded onto 96-well plates and cultured as described above for 24 h. The medium was then replaced with a medium containing ALS at various concentrations (0, 5, 10, and 20 µg). After incubation for 24 h at 37 • C in a 95% air and 5% CO 2 atmosphere incubator, MTT (5 mg/mL) reagent was added at 10 µL/well (0.45 mg/mL final concentration). The plates were then incubated at 37 • C for 48 h in the dark (wrapped in aluminum foil). Sodium dodecyl sulfate (SDS; 10%) was then added followed by overnight incubation at 37 • C to completely dissolve the formazan crystals. Absorbances were obtained at 570 nm using a microplate reader (Powerscan HT; Dainippon Pharmaceuticals USA Corporation, Osaka City, Osaka, Japan). Blanks containing only the medium, MTT, and SDS were used to correct the absorbances.

Melanin Content Determination
The melanin content was measured as we have previously reported [56]. Briefly, B16 cells were cultured at a density of 5 × 10 5 cells/mL and cultivated as described above. After overnight incubation, the culture medium was replaced with a crude saponins samplecontaining growth medium, and the cells were incubated further for 48 or 72 h. α-MSH was used as a positive control for melanin biosynthesis. For the determination of the melanin content in human epidermal melanocytes, HEMs were cultured in a PMA-containing HEM culture medium at a density of 5 × 10 5 cells/mL and cultivated as described above. After overnight incubation, the culture medium was replaced with a PMA-free culture medium with a crude saponins sample, and the cells were incubated further for 48 h. As positive control, cells were treated with 100 • µM arbutin (Arb) and 10 ng/mL phorbol 12-myristate 13-acetate (PMA). Untreated control cells were cultured in a PMA-free culture medium. After incubation with the sample at the prescribed time, the growth medium was removed, and the cells washed twice with phosphate-buffered saline (PBS) and harvested by trypsinization (0.25% trypsin/0.02% EDTA in PBS; Sigma Aldrich, St. Louis, MO, USA). The harvested cells were solubilized in 0.1% Triton X-100 with sonication, and the melanin was precipitated by adding 10% trichloroacetic acid. The isolated melanin was then dissolved in 8 M NaOH and incubated for 2 h at 80 • C before its quantification spectrophotometrically at 410 • nm. The total melanin content was estimated using the standard curve for synthetic melanin. The cell viability and total number of cells were determined using the ViaCount Program of Guava PCA (GE Healthcare, UK Ltd., Buckinghamshire, UK) following the manufacturer's instructions.

Quantitative Real-Time PCR (qPCR) Analysis
RNA was extracted from B16 cells and HEMs, treated with 10 µg/mL argan leaf saponin and 100 µM arbutin, using ISOGEN (Nippon Gene), following the manufacturer's instructions. One microgram RNA sample was reverse-transcribed using a SuperScript III Reverse Transcription Kit (Invitrogen, Waltham, MA, USA), and the resulting cDNA was used as template for real-time PCR (rt-PCR). qPCR was performed using 7500 Fast Real-time PCR System with 7500 software version 2.0.5 (Applied Biosystems, Waltham, MA, USA). RNA samples were mixed with a TaqMan Gene Expression Master Mix (Applied Biosystems, Waltham, MA, USA) and specific primers for mouse Mitf, Tyr, Trp1, Dct, and Gapdh (internal control) and human SCM3, CDH1, and GAPDH.

DNA Microarray
DNA microarray was performed using an Affymetrix GeneChip 30 IVT Express Kit (Affymetrix, Santa Clara, CA, USA) following the manufacturer's instructions. Total RNA (200 ng) was reverse-transcribed into double-stranded cDNA, and biotin-labeled aRNA was generated using a 30 IVT Express Labeling Kit (Affymetrix, Santa Clara, CA, USA). This was followed by biotin-labeling the resulting aRNA, which was then hybridized to an Affymetrix human HG-U219 Array strip (Affymetrix, Santa Clara, CA, USA) for 16 h at 45 • C at the Hybridization Station (Affymetrix, Santa Clara, CA, USA). Hybridized arrays were washed and stained using the hybridization, wash, and stain kit (Affymetrix, Santa Clara, CA, USA) performed in the Affymetrix GeneAtlas TM Fluidics Station. The washed arrays were scanned using the Affymetrix GeneAtlas TM Imaging Station. To analyze the data, Affymetrix Expression Console Software was used, and the gene expression in treated cells was compared with that in control cells, based on mathematical algorithms. The results were based on the analysis of significance (control vs. treatment) using 1-way betweensubject ANOVA (paired) (p value ≤ 0.05) and fold change (linear) ≤−2 or ≥2. The generated data (2-fold change) were then analyzed using Transcription Analysis Console Software to generate gene ontology and functional annotation charts. Hierarchical clustering was performed using TIGR Mev Software version 3.0.3 (The institute for Genomic Research, Rockville, MD, USA). The 36 genes subjected to hierarchical clustering were chosen as having a fold change of 2 or higher (5 µg ALS vs. untreated control). Red and green color-code for up-and downregulation, respectively.

Statistical Analysis
The results were expressed as mean ± standard deviation (SD), and comparisons between untreated and treated samples were performed using t-test. Comparison between treatments was carried out using ANOVA. A p value of ≤0.05 was considered to be statistically significant.

Conclusions
Saponins remain to be an unexplored important natural product with a great potential for therapeutic uses. Here, we have shown that a saponins-rich fraction of argan leaves extract (ALS) can inhibit melanin biosynthesis in B16 cells and in human epidermal melanocytes, and may be used for hyperpigmentation treatment. Molecular analysis results revealed that ALS inhibits melanogenesis by regulating signaling pathways that downregulate the expression of MITF, and as a result, the expression of the genes that are under its transcriptional regulation, which includes the tyrosinase (Tyr) and tyrosinase-related protein 1 (Trp1), was decreased. This is the first report on the effect of a saponins-rich fraction of extract of argan leaves, providing the proof that plant saponins are vital and a sustainable resource for the development of hyperpigmentation drug.