Prenylated Flavonoids from Roots of Glycyrrhiza uralensis Induce Differentiation of B16-F10 Melanoma Cells

Roots of Glycyrrhiza uralensis have been used as herbal medicine and natural sweetener. By activity-guided phytochemical investigation of the extracts from G. uralensis root, ten flavonoids, namely GF-1–GF-10, of which five were prenylated flavonoids, were found to show antiproliferative effects in melanoma B16-F10 cells. Three of the prenylated flavonoids, namely GF-1, GF-4 and GF-9, significantly induced the differentiation of B16-F10 cells; the inductions included increase of tyrosinase activity, tyrosinase protein, and melanin content. In GF-1 and GF-9 induced melanoma differentiation, the phosphorylation of p38 MAPK (mitogen activated potein kinase) was identified; while GF-4 could trigger the phosphorylation of PI3K/AKT (phosphatidylinositol 3-kinase/Protein Kinase B) signaling. However, application of GF-6 to the melanoma cells did not induce differentiation; but which promoted cell apoptotic signaling, i.e., increase levels of cleaved-PRAP, cleaved-caspase 3, and cleaved-caspase 9. These results suggested that different types of prenylated flavonoids from G. uralensis might have potential anticancer effects against melanoma cells by acting through different signaling pathways.


Introduction
The roots of Glycyrrhiza uralensis Fisch (GlycyrrhizaeUralensis Radix; GUR), named as licorice or gancao, have been extensively used as a herbal drug in both Eastern and Western countries [1]. Clinical studies have shown GUR is highly effective in the treatment of respiratory, gastrointestinal, cardiovascular and genitourinary conditions [2]. According to traditional Chinese medicine (TCM) theory, GUR is described as a beneficial herb to enhance therapeutic effects; meanwhile, this herb is being employed commonly to detoxify potential adverse effects in many herbal mixtures during clinical application. Phytochemical studies revealed that triterpene saponins and flavonoid glycosides were two major active substances in GUR [3]. In the extractive of GUR, the nonpolar fraction of GUR is rich in various types of prenylated flavonoids, e.g., flavones, isoflavones, flavanones, chalcones, and coumestans [4]. These flavonoids are being considered to be active ingredients of GUR, and indeed which have been commonly used in food industries. In recent pharmacological studies, GUR flavonoids have been proposed to have anti-oxidation [5], anti-inflammatory [6], antiproliferative, and cytotoxic pharmacological studies, GUR flavonoids have been proposed to have anti-oxidation [5], antiinflammatory [6], antiproliferative, and cytotoxic effects in various cells [7]. Despite the aforementioned proposed actions, the roles of GUR prenylated flavonoids in cancer cells have not been extensively investigated.
Malignant melanoma is a highly aggressive and invasive skin cancer with high metastatic potential and extraordinary resistance to cytotoxic agents [8]. The occurrence of melanoma is related to different factors, e.g., sun exposure, fair pigmentation, and genetic mutation [9]. In recent years, the incidence of melanoma is rapidly increasing throughout the world, especially in America and Europe [10]. Although drug therapy for this cancer has been developed rapidly, e.g., immunotherapies with PD-1 inhibition drugs (ipilimumab and nivolumab) and T-cell checkpoint blockade therapies [11,12], the usage of herbal products is still one of the alternative approaches for melanoma cancer treatment.
Considering the pathogenesis and clinical treatment of melanoma, the recent targets in cancer therapy is focusing on discovery of natural products that are able to suppress cancer cell proliferation and promote cell differentiation [13,14]. Here, the CH2Cl2 extract of GUR (GURCH2Cl2) was shown to exhibited potent effect in antiproliferation and inducing differentiation of cultured melanoma B16-F10 cells: these effects were significant higher than that deriving from water extract (GURwater) and ethanol extract of GUR (GUREtOH). To explore the possible underlying mechanism for anticancer effect of GUR, ten flavonoids (GF1-GF10) of which five were prenylated flavonoids, were isolated. The roles of these flavonoids in inducing the differentiation of cultures B16-F10 cells were illustrated, and subsequently the signaling cascades, triggered by various flavonoids, were revealed and compared.

G. uralensis Extracts in Proliferation and Differentiation of Melanoma Cells
Different extractives of GUR, i.e., GUwater, GUEtOH, and GUCH2Cl2, were subjected to HPLC analyses. As shown in HPLC chromatograms ( Figure 1A), saponins and flavonoid glycosides were the major constituents in both GUwater (water extract) and GUEtOH (ethanol extract). Besides, a small amount of free flavonoids could be detected in GUEtOH. In comparison with GUwater and GUEtOH, GUCH2Cl2 (dichloromethane extract) exhibited a comparable enrichment of free flavonoids. To a certain extent, difference in chemicals might cause possible difference in their biological capacities.  To investigate the effect of G. uralensis extracts, cultured B16-F10 melanoma cells were treated with different extracts at indicated concentrations for 48 h. As shown in Figure 1B, GUCH2Cl2 inhibited the proliferation of B16-F10 cells with an IC50 value of 48.7 ± 2.5 μg/mL,whereas GUwater and GUEtOH exhibited little effects (IC50 > 100 μg/mL). In addition to the differentiation induction, as shown in Figure 1C, the GUCH2Cl2-treated (50 μg/mL) cells showed typical dendrite-like cellular protrusions, as compared with control cells. Although GUEtOH had a certain degree of differentiation-inducing activity, as compared with the control group, its effect was far lower than that of GUCH2Cl2. Over 40% cells were induced to differentiate under the treatment of GUCH2Cl2 ( Figure 1C). Similar to antiproliferative activity, GUwater showed little morphological changes towards cultured B16-F10 cells. These results suggested that free flavonoid content in the extract could be responsible for differentiation and antiproliferation of B16-F10 cells.

Structure Identification of Isolated Compounds
To explore active compounds in GUR extracts corresponding for activitieson cell differentiation, phytochemical investigation of GUCH2Cl2 was performed, and there after 10 compounds were isolated. Their structures and types were elucidated on the basic of ESI-Q-TOF/MS and 1 H and 13 C NMR and by comparison with the literatures. The 13 C NMR data were presented in Table 1, and the MS and 1 H NMR data of each compound were available in the supplementary information.  To investigate the effect of G. uralensis extracts, cultured B16-F10 melanoma cells were treated with different extracts at indicated concentrations for 48 h. As shown in Figure 1B, GU CH2Cl2 inhibited the proliferation of B16-F10 cells with an IC 50 value of 48.7 ± 2.5 µg/mL, whereas GU water and GU EtOH exhibited little effects (IC 50 > 100 µg/mL). In addition to the differentiation induction, as shown in Figure 1C, the GU CH2Cl2 -treated (50 µg/mL) cells showed typical dendrite-like cellular protrusions, as compared with control cells. Although GU EtOH had a certain degree of differentiation-inducing activity, as compared with the control group, its effect was far lower than that of GU CH2Cl2 . Over 40% cells were induced to differentiate under the treatment of GU CH2Cl2 ( Figure 1C). Similar to antiproliferative activity, GU water showed little morphological changes towards cultured B16-F10 cells. These results suggested that free flavonoid content in the extract could be responsible for differentiation and antiproliferation of B16-F10 cells.

Structure Identification of Isolated Compounds
To explore active compounds in GUR extracts corresponding for activitieson cell differentiation, phytochemical investigation of GU CH2Cl2 was performed, and there after 10 compounds were isolated. Their structures and types were elucidated on the basic of ESI-Q-TOF/MS and 1 H and 13 C NMR and by comparison with the literatures. The 13 C NMR data were presented in Table 1, and the MS and 1 H NMR data of each compound were available in the supplementary information.
As shown in Figure 2, all isolated compounds were identified as free flavonoids, including three isoflavones, three flavones, two chalcones, one coumarin, and one flavanone, namely GF-1-GF-10. Compounds GF-1, 2, and 5 were isoflavones, identified as licoisoflavone B, glabrone, and formononetin, respectively. Compounds GF-3, 9, and 10 were classified as flavones and elucidated successively as 7, 4 -dihydroxyflavone, licoflavone, and kumatakenin A, respectively. With the data of NMR and MS, two chalcones, GF-6 and GF-8 were established as licochalcone A and isoliquiritigenin, respectively. GF-4 was identified as a coumarin namely as neoglycyrol, which could be regarded as an isoflavone derivative with methoxyl, hydroxyl, and prenyl groups. GF-7 was identified as a common flavanone, namely as liquiritigenin. The HPLC of isolated flavonoids and dichloromethane extract of G. uralensis were available in the supplementary information. The purities of these ten flavonoids were tested to be higher than 95% by HPLC-UV area normalization method. Table 1. Table of 13 C NMR Spectroscopic data of 10 flavonoid compounds in DMSO-d6.

GF-1 GF-2 GF-3 GF-4 GF-5 GF-6 GF-7 GF-8 GF-9
GF-10 As shown in Figure 2, all isolated compounds were identified as free flavonoids, including three isoflavones, three flavones, two chalcones, one coumarin, and one flavanone, namely GF-1-GF-10. Compounds GF-1, 2, and 5 were isoflavones, identified as licoisoflavone B, glabrone, and formononetin, respectively. Compounds GF-3, 9, and 10 were classified as flavones and elucidated successively as 7, 4'-dihydroxyflavone, licoflavone, and kumatakenin A, respectively. With the data of NMR and MS, two chalcones, GF-6 and GF-8 were established as licochalcone A and isoliquiritigenin, respectively. GF-4 was identified as a coumarin namely as neoglycyrol, which could be regarded as an isoflavone derivative with methoxyl, hydroxyl, and prenyl groups. GF-7 was identified as a common flavanone, namely as liquiritigenin. The HPLC of isolated flavonoids and dichloromethane extract of G. uralensis were available in the supplementary information. The purities of these ten flavonoids were tested to be higher than 95% by HPLC-UV area normalization method. In recent years, flavonoids, especially the prenylated ones, have attracted extensive attention because of its resulting radicals can enhance their biological activities [15]. In this study, several flavonoids, including GF-1, 2, 4, 6, and 9, were found to contain a substituent of prenyl moiety or dimethylpyran (formation from prenyl) in their structures.

Isolated Flavonoidsin Proliferation and Differentiation of Melanoma Cells
The isolated flavonoids were tested for their antiproliferative capacity using MTT bioassay on cultured B16-F10 cells ( Figure 3A). In cell inhibition, the most active component was GF-4 (neoglycyrol), with an IC50 of 17.5 μM, while compounds GF-1, 2, 6, and 9 showed good activities in the range of 26.0-35.8 μM. The other nonprenyl flavonoids, GF-3, 5, 7, 8, and 10, possessed low activities of cell inhibition. Thus, the prenyl could be an important group for inhibition activity of licorice flavonoids in cancer cell growth. In recent years, flavonoids, especially the prenylated ones, have attracted extensive attention because of its resulting radicals can enhance their biological activities [15]. In this study, several flavonoids, including GF-1, 2, 4, 6, and 9, were found to contain a substituent of prenyl moiety or dimethylpyran (formation from prenyl) in their structures.

Isolated Flavonoidsin Proliferation and Differentiation of Melanoma Cells
The isolated flavonoids were tested for their antiproliferative capacity using MTT bioassay on cultured B16-F10 cells ( Figure 3A). In cell inhibition, the most active component was GF-4 (neoglycyrol), with an IC 50 of 17.5 µM, while compounds GF-1, 2, 6, and 9 showed good activities in the range of 26.0-35.8 µM. The other nonprenyl flavonoids, GF-3, 5, 7, 8, and 10, possessed low activities of cell inhibition. Thus, the prenyl could be an important group for inhibition activity of licorice flavonoids in cancer cell growth. In recent years, flavonoids, especially the prenylated ones, have attracted extensive attention because of its resulting radicals can enhance their biological activities [15]. In this study, several flavonoids, including GF-1, 2, 4, 6, and 9, were found to contain a substituent of prenyl moiety or dimethylpyran (formation from prenyl) in their structures.

Isolated Flavonoidsin Proliferation and Differentiation of Melanoma Cells
The isolated flavonoids were tested for their antiproliferative capacity using MTT bioassay on cultured B16-F10 cells ( Figure 3A). In cell inhibition, the most active component was GF-4 (neoglycyrol), with an IC50 of 17.5 μM, while compounds GF-1, 2, 6, and 9 showed good activities in the range of 26.0-35.8 μM. The other nonprenyl flavonoids, GF-3, 5, 7, 8, and 10, possessed low activities of cell inhibition. Thus, the prenyl could be an important group for inhibition activity of licorice flavonoids in cancer cell growth. In order to evaluate the differentiating effect of isolated flavonoids, cultured B16-F10 cells were treated with α-MSH (positive drug) or these compounds at two concentrations for 48 h. Accordingly, typical dendrite-like cellular protrusions of cells, induced by GF-1, 2, 4, and 9, were similar with those induced by 20 nM α-melanocyte-stimulating hormone(α-MSH) ( Figure 3B) [16]. Surprisingly, GF-6 had no such phenotype induction regardless of its role in cell inhibition ( Figure 3B). After comparison of structures of above compounds, the opening of central C-ring in GF-6 could be a possible domain in inducing differentiation in cultured B16-F10 cells. In order to evaluate the differentiating effect of isolated flavonoids, cultured B16-F10 cells were treated with α-MSH (positive drug) or these compounds at two concentrations for 48 h. Accordingly, typical dendrite-like cellular protrusions of cells, induced by GF-1, 2, 4, and 9, were similar with those induced by 20 nM α-melanocyte-stimulating hormone(α-MSH) ( Figure 3B) [16]. Surprisingly, GF-6 had no such phenotype induction regardless of its role in cell inhibition ( Figure 3B). After comparison of structures of above compounds, the opening of central C-ring in GF-6 could be a possible domain in inducing differentiation in cultured B16-F10 cells.

Isolated Flavonoids in Melanogenesis
Melanoma cell differentiation is generally accepted to be related to inhibition of cell proliferation, dendritic-like morphology, increased tyrosinase activity, and melanin production [17]. To validate the implications of morphological observations, the amounts of intracellular melanin content, tyrosinase activity and protein were evaluated in B16-F10 cells with the treatment of α-MSH or selected prenylated flavonoids, i.e., GF-1, 4, 6, and 9. The application of α-MSH (positive drug) at 20nM, GF-1, 4, and 9 at 40 µM significantly increased the melanin amounts by about 2-fold, as compared to the control ( Figure 4A). However, GF-6 showed no effect. Except for GF-6, the prenylated flavoniods could induce tyrosinase activity in a dose-dependent manner in cultured B16-F10 cells ( Figure 4A). The level of tyrosinase protein in the melanoma cells was measured by Western blot analysis. The expression of tyrosinase protein was enhanced to about 2-fold in GF-1, 4, and 9 treated cells ( Figure 4B), demonstrating that the melanogenesis promotion was partly mediated by increased tyrosinase in melanoma cells.

Isolated Flavonoids in Melanogenesis
Melanoma cell differentiation is generally accepted to be related to inhibition of cell proliferation, dendritic-like morphology, increased tyrosinase activity, and melanin production [17]. To validate the implications of morphological observations, the amounts of intracellular melanin content, tyrosinase activity and protein were evaluated in B16-F10 cells with the treatment of α-MSH or selected prenylated flavonoids, i.e., GF-1, 4, 6, and 9. The application of α-MSH (positive drug) at 20nM, GF-1, 4, and 9 at 40 μM significantly increased the melanin amounts by about 2-fold, as compared to the control ( Figure 4A). However, GF-6 showed no effect. Except for GF-6, the prenylated flavoniods could induce tyrosinase activity in a dose-dependent manner in cultured B16-F10 cells ( Figure 4A). The level of tyrosinase protein in the melanoma cells was measured by Western blot analysis. The expression of tyrosinase protein was enhanced to about 2-fold in GF-1, 4, and 9 treated cells ( Figure 4B), demonstrating that the melanogenesis promotion was partly mediated by increased tyrosinase in melanoma cells. To explore potential mechanism of the four prenylated flavonoids (GF-1, 4, 6, and 9) on antiproliferation and differentiation-inducing activities in B16 cells,the activations of MAPKs (ERK 1/2, JNK, and p38), AKT (Protein Kinase B), and PARP (poly ADP-ribose polymerase) signaling pathwayswere probed in the treated B16-F10 cells. These signaling pathways have been identified as important regulators of cell proliferation, differentiation, and tumor development of the malignant phenotype of tumor [18]. The levels of phospho-ERK1/2 (p-ERK1/2) and phospho-JNK (p-JNK) remained unchanged after treatment with the four prenylated flavonoids ( Figure 5). However, the level of phospho-p38 (p-p38) was significantly induced by treatment of GF-1 and GF-9, with ~16-and ~13fold of increase, in respect to the control. In the phosphorylation of p-AKT, only GF-4 showed a reduction ( Figure 5). In the cultures, GF-6 showed cell inhibition, but not in cell differentiation, and did To explore potential mechanism of the four prenylated flavonoids (GF-1, 4, 6, and 9) on antiproliferation and differentiation-inducing activities in B16 cells, the activations of MAPKs (ERK 1/2, JNK, and p38), AKT (Protein Kinase B), and PARP (poly ADP-ribose polymerase) signaling pathwayswere probed in the treated B16-F10 cells. These signaling pathways have been identified as important regulators of cell proliferation, differentiation, and tumor development of the malignant phenotype of tumor [18]. The levels of phospho-ERK1/2 (p-ERK1/2) and phospho-JNK (p-JNK) remained unchanged after treatment with the four prenylated flavonoids ( Figure 5). However, the level of phospho-p38 (p-p38) was significantly induced by treatment of GF-1 and GF-9, with~16-and 13-fold of increase, in respect to the control. In the phosphorylation of p-AKT, only GF-4 showed a reduction ( Figure 5). In the cultures, GF-6 showed cell inhibition, but not in cell differentiation, and did not cause phosphorylation of p-ERK1/2, p-JNK, p38, and p-AKT. In contrast, GF-6 application in B16-F10 cells induced the phosphorylation of Cl-PRAP by~30-fold ( Figure 5). Thus, the four tested prenylated flavonoids showed distinction in inducing the signaling cascades, i.e., p-ERK1/2 and p-JNK triggered by GF-1 and GF-9, p-AKT triggered by GF-4, and Cl-PARP triggered by GF-9. not cause phosphorylation of p-ERK1/2, p-JNK, p38, and p-AKT. In contrast, GF-6 application in B16-F10 cells induced the phosphorylation of Cl-PRAP by ~30-fold ( Figure 5). Thus, the four tested prenylated flavonoids showed distinction in inducing the signaling cascades, i.e., p-ERK1/2 and p-JNK triggered by GF-1 and GF-9, p-AKT triggered by GF-4, and Cl-PARP triggered by GF-9. The phosphorylation of p38 could lead to activation of cAMP response element binding protein (CREB) and induction of microphthalmia associated transcription factor (MITF) expression [19]. MITF is a known downstream effector that is required for melanoblast survival, as well as a key regulator to induce genes associated with the differentiation, e.g., tyrosinase [20]. Here, GF-1 and GF-9 markedly increased the phosphorylations of p-p38 and p-CREB, as well as promoting the expression of MITF in dose-dependent manners ( Figure 6A). The phosphorylation of p38 could lead to activation of cAMP response element binding protein (CREB) and induction of microphthalmia associated transcription factor (MITF) expression [19]. MITF is a known downstream effector that is required for melanoblast survival, as well as a key regulator to induce genes associated with the differentiation, e.g., tyrosinase [20]. Here, GF-1 and GF-9 markedly increased the phosphorylations of p-p38 and p-CREB, as well as promoting the expression of MITF in dose-dependent manners ( Figure 6A).
The PI3K/AKT signaling pathway is another crucial pathway involved in melanoma cell differentiation. Application of GF-4 down-regulated the phosphorylation of p-AKT and PI3K in cultured B16-F10 cells, and this suppression was in a dose-dependent manner ( Figure 6B). Glycogen synthase kinase 3 β, a direct downstream target of AKT, phosphorylates MITF and regulates expression of tyrosinase [21]. Therefore, the protein expression of MITF was determined under the treatment of GF-4 in cultured B16-F10 cells. As expected, the level of MITF was induced by 50% after the treatment of GF-4 ( Figure 6B). The GF-6-induced Cl-PRAP expression suggested that apoptosis could be involved in the inhibition of B16-F10 cells, triggered by GF-6. In line with an increase of Cl-PRAP, the levels of cleaved caspase-3 and cleaved caspase-9 were markedly induced by treatment of GF-6: the maximal induction over 5-fold and 15-fold could be revealed in cleaved caspase-3 and cleaved caspase-9, respectively ( Figure 6C). The increase of apoptotic biomarkers strongly suggested the possible anticancer function of GF-6. , and Cl-Casp 3(~19 kDa). The relative abundance of each band to their own GAPDH was quantified, and the control levels were set at 1.0 (lower panel). Data are expressed as percentage of control, in means ± SEM (n = 3).* p < 0.05, ** p < 0.01, compared with control.
The PI3K/AKT signaling pathway is another crucial pathway involved in melanoma cell differentiation. Application of GF-4 down-regulated the phosphorylation of p-AKT and PI3K in cultured B16-F10 cells, and this suppression was in a dose-dependent manner ( Figure 6B). Glycogen synthase kinase 3 β, a direct downstream target of AKT, phosphorylates MITF and regulates expression of tyrosinase [21]. Therefore, the protein expression of MITF was determined under the treatment of GF-4 in cultured B16-F10 cells. As expected, the level of MITF was induced by 50% after the treatment of GF-4 ( Figure 6B). The GF-6-induced Cl-PRAP expression suggested that apoptosis could be involved in the inhibition of B16-F10 cells, triggered by GF-6. In line with an increase of Cl-PRAP, the levels of cleaved caspase-3 and cleaved caspase-9 were markedly induced by treatment of GF-6: the maximal induction over 5-fold and 15-fold could be revealed in cleaved caspase-3 and cleaved caspase-9, respectively ( Figure 6C). The increase of apoptotic biomarkers strongly suggested the possible anticancer function of GF-6.

Discussion
It has been estimated that 30-40% of cancers can be prevented by dietary or lifestyle condition [22]. Some anticancer drugs currently used in clinics, such as paclitaxel and camptothecin, are derived from natural products. Thus, the search for natural products having anticancer activity represents an interesting area, probably due to its diversity and distinct action mechanism. Flavonoids, commonly

Discussion
It has been estimated that 30-40% of cancers can be prevented by dietary or lifestyle condition [22]. Some anticancer drugs currently used in clinics, such as paclitaxel and camptothecin, are derived from natural products. Thus, the search for natural products having anticancer activity represents an interesting area, probably due to its diversity and distinct action mechanism. Flavonoids, commonly found in foods or herbal medicines, are a group of compounds with potential antitumor activities. According to their variations of backbone and substitution, flavonoids can be classified into different subclasses, providing an extremely diverse range of derivatives. Among them, prenylated flavonoids have attracted considerable attention due to their promising and diverse bioactivities [15]. For example, the prenylated substituted chalcones were shown to be cytotoxic in MCF-7, HT-29, and A-2780 cancer cells, whereas the conversion of a chalcone to a favanone resulted in reduced antiproliferative activity [23]. In addition, a prenylated flavonoid, artonol A, exhibited cytotoxic activity against human lung cancer cells [24]. Thus, the substitution in flavonoid structure might possess a profound influence on their antiproliferative effect on cancer cells. As shown here, the proliferation of B16-F10 cells could be significantly inhibited by CH 2 Cl 2 extract of GUR. From this extract, ten flavonoids were isolated, of which five flavonoids with a prenylated group, i.e., GF-1, 2, 4, 6, and 9, displayed effectively cytotoxic activity: these results suggested that the prenyl group could be an indispensable moiety for licorice flavonoids to inhibit B16-F10 cell growth. Considering the structure-activity relationship, the free isoprene flavonoids, e.g., deprenylated compound of GF-9 and kumatakenin B, are showing low antiproliferative effect on B16-F10 cells.
The differentiation process represents a crucial point in progression of many types of cancer. A potential cancer treatment is to promote cancer cell differentiation, and thereafter, to reduce its proliferative capacity: this approach is a novel therapeutic method aiming to modify tumor cells to a slower rate of proliferation, and thus which leads to the loss of its earlier neoplastic attributes [25,26]. In melanoma, the differentiation status of cells could be monitored by melanogenesis, which resulted in the conversion of L-tyrosine to L-dopa by tyrosinase [27]. Indeed, the levels of melanin and tyrosinase are being considered as indicators of differentiated melanoma cells. Our current results supported the contrary action of differentiation against proliferation in melanoma. Here, we have shown the prenylated flavonoids GF-1, 4, and 9 inhibited B16-F10 cell growth, and subsequently which induced cell differentiation. The differentiation, induced by the prenylated flavonoids, was illustrated by morphological change and increase of melanin and tyrosinase. Having a distinction here, GF-6 (licochalcone A) showed inhibitory effect only on cell growth but not differentiation. The GF-6-inhibited cell growth was revealed to be mediated by its apoptotic role. In line to this observation, licochalcone A was shown to activate apoptotic processes in both in vitro and in vivo [28]. Thus, different backbone structure of flavonoids, as revealed here for G.uralensis flavonoids, might have distinct mechanisms to inhibit growth of tumor cells.
Numerous studies have shown that the MAPKs and AKT signaling pathways could play role in survival, proliferation, and differentiation of melanoma [18]. The MAPKs include three well characterized sub-families, i.e., extra cellular signal-regulated kinases -1/2(ERK 1/2), c-jun N-terminal or stress-activated protein kinases (JNK), and p38 mitogen activated protein kinase (p38 MAPK). The activations of ERK (1/2) and JNK were shown to relate with suppression of melanogenesis [20]. Activation of p38 MAPK signaling could upregulate melanin synthesis and induce differentiation by promoting expressions of MITF and tyrosinase [19]. In addition, the activation of AKT could inhibit melanogenesis and differentiation of melanoma [29]. These different lines of evidence support the antiproliferation and differentiation-inducing effects of natural products on melanoma, and the regulation of MAPKs and AKT signaling could be one of key mediators. For example, 5, 7-dimethoxycoumarin, from Citrus limon L., showed potential antigrowth and differentiation-inducing effect on melanoma cell, involving Ras/Raf/MEK/ERK pathway [30]. Lupeol, a lupine-type triterpene from Danelion root, showed significant inhibition on melanoma cell growth both in vitro and in vivo: the effect was trigged by its induction on cell differentiation [31]. Ye [27] reported that three sesquiterpenes, isolated from the largehead atractylode rhizome, induced melanoma cell differentiation and inhibited cell migration through inactivating the signals of Ras/ERK MAPK (for AT-I and AT-II) and PI3K/AKT. Here, we isolated and identified several prenylated flavonoids including isoflavone, flavone, chalcone, and coumarin. Interestingly, prenylatedflavone (GF-1) and prenylatedisoflavone (GF-9) could induce activation of p38 MAPK pathway, while prenylated coumarin (GF-4) showed inhibition on PI3K/AKT signaling. The prenylated chalcone GF-6 did not affect cell differentiation, but which induced melanoma cell apoptosis with activation of proteins in caspase-mediated signaling pathway. This result was in good agreement with recent literature [32]. However, a more detailed mechanism by which the prenylated flavonoids in melanoma cell differentiation and cell proliferation are waiting for further elucidation.

Statistical Analysis
The significant difference among groups was statistically performed by one-way analysis of variance (ANOVA) combined with Tukey'stest by SPSS v. 22.0 program (IBM Corp., Armonk, New York, NY, USA). Probability value less than 0.05 (p < 0.05) was considered to be statistically significant. Data are expressed as the means±standard error of mean (SEM) of at least three independent experiments.

Conclusions
Here, we provided different lines of evidence that the dichloromethane extract of G. uralensis roots has potential antiproliferation and differentiation-inducing activities in cultured B16-F10 melanoma cells. Prenylated flavoniods were isolated and demonstrated to be the active constituents within the extract of G. uralensis roots. These prenylated flavonoids have different backbone structures, and thus each of them has distinct activity in inhibiting cancer cell growth. The present results provided the molecular basis for using nonpolar extract of G. uralensis roots, and the identified bioactive prenylated flavonoids could be further developed for the prevention and/or treatment of melanoma cancer.