Anti-Melanoma Activities and Phytochemical Compositions of Sorbus commixta Fruit Extracts

Sorbus commixta Hedl. (Rosaceae family) has a long history as a medicinal plant in East Asian countries. In this study, we evaluated the effect of S. commixta fruit extracts prepared with different ethanol concentrations on anti-melanoma activity, and the extraction yield of phenolic compounds and flavonoids. Using the partitioned fractions from the EtOH extract, we found that the butanol fraction (BF) possessed strong cytotoxic activity against SK-MEL-2 cells (human melanoma cells) but not against HDFa cells (human dermal fibroblast adult cells). Additionally, BF-induced cell death was mediated by the inhibition of the mitogen-activated protein kinase/extracellular regulated kinase (MEK/ERK) signaling pathway, coupled with the upregulation of caspase-3 activity in SK-MEL-2 cells. Furthermore, HPLC analysis of polyphenolic compounds suggested that S. commixta fruits contained several active compounds including chlorogenic acid, rutin, protocatechuic acid, and hydroxybenzoic acid, all of which are known to possess anti-cancer activities. Although this study has been carried out by cell-based approach, these results suggest that S. commixta fruits contain promising anti-melanoma compounds.


Introduction
Malignant melanoma, an aggressive and fatal form of skin cancer, is a malignant melanocyte tumor that accounts for approximately 75% of skin cancer-related deaths worldwide [1][2][3]. In recent years, melanoma incidence has continued to increase worldwide, and the onset of malignant melanomas is reportedly affected by various environmental and genetic factors, such as ultraviolet exposure and carcinogenic BRAF mutations [4][5][6]. Despite the wide variety of therapies available to treat melanoma, including surgery, radiation therapy, immunotherapy, and chemotherapy [7][8][9], phytochemicals have been recognized as better anti-cancer therapies to prevent or inhibit carcinogenesis [10]. Camptothecin, vincristine, vinblastine, taxol, topotecan, podophyllotoxin, and irinotecan are good examples of plant-based anticancer molecules [11]. Growing evidence supports that plant-derived natural products are important sources of novel candidates, with pharmacological applicability.

Effects of S. commixta Fruit Extracts and Solvent Fractions on Human Melanoma SK-MEL-2 Cell
Extraction efficiencies are affected by the solvent type and concentration [23]. Particularly, EtOH is considered an excellent solvent and is therefore often used to recover polyphenols from plant matrices, as it is also safe for human consumption [24]. In addition, EtOH is completely miscible with water. Therefore, our study conducted further experiments to define the effect of S. commixta fruit extracts prepared with different EtOH concentrations on anti-melanoma activity. As shown in Figure 1A, incubation with 100 µg/mL of EtOH extracts significantly inhibited the proliferation of SK-MEL-2 cells (28.85 ± 3.38%), although ethanol concentration did not have a substantial impact on SK-MEL-2 cell cytotoxicity. Among the bioactive compounds of S. commixta fruit [22], phenolic and flavonoid compounds have been proposed. Binary-solvent systems have been found to be more effective for the extraction of these compounds from plants compared to mono-solvent systems [25,26]. However, in the case of S. commixta fruit, EtOH extract contained higher TPC (total phenolic content) levels than water extracts, but solvent concentrations did not significantly affect the extraction of phenolic compound yields. TFC (total flavonoid content) in the extracts increased with increasing ethanol concentrations, and the mono-solvent system examined herein (EtOH) was more effective in extracting phenolic and flavonoid compounds than EtOH/water solvents (Table 1). Additionally, higher concentrations of chlorogenic acid, rutin, protocatechuic acid, and hydroxybenzoic acid in EtOH extracts were observed compared to other extracts, suggesting that the differences in the cytotoxic activity of S. commixta fruit extracts were due to their concentration and composition of polyphenolic compounds.
Plants 2020, 9, x FOR PEER REVIEW 2 of 9 diabetic, diuretic, and vasorelaxant properties [12,19]. Phytochemical analysis of S. commixta fruits has revealed the presence of active ingredients often used in cosmetics, including rutin, isoquercitrin, caffeoylquinic acid, dicaffeoylquinic acid, neosakuranin, chlorogenic acid, neochlorogenic acid, carotenoids, and ascorbic acid [20][21][22], suggesting the potential applicability of S. commixta fruits for the production of herbal cosmetics with anti-melanoma activity. Nonetheless, the anti-melanoma properties of S. commixta fruits are yet to be comprehensively characterized. Therefore, our study analyzed the effect of S. commixta fruit extracts prepared with different ethanol (EtOH) concentrations on anti-melanoma activity, as well as the role of the mitogen-activated protein kinase/extracellular regulated kinase (MEK/ERK) pathways in the activation of cytotoxicity. Additionally, seven polyphenolic compounds were identified in S. commixta fruit extracts via HPLC. Therefore, we expect our study to motivate further interest in the use of S. commixta fruits as a natural anti-melanoma source in the cosmetic and pharmaceutical industries.

Effects of S. commixta Fruit Extracts and Solvent Fractions on Human Melanoma SK-MEL-2 Cell
Extraction efficiencies are affected by the solvent type and concentration [23]. Particularly, EtOH is considered an excellent solvent and is therefore often used to recover polyphenols from plant matrices, as it is also safe for human consumption [24]. In addition, EtOH is completely miscible with water. Therefore, our study conducted further experiments to define the effect of S. commixta fruit extracts prepared with different EtOH concentrations on anti-melanoma activity. As shown in Figure  1A, incubation with 100 μg/mL of EtOH extracts significantly inhibited the proliferation of SK-MEL-2 cells (28.85 ± 3.38%), although ethanol concentration did not have a substantial impact on SK-MEL-2 cell cytotoxicity. Among the bioactive compounds of S. commixta fruit [22], phenolic and flavonoid compounds have been proposed. Binary-solvent systems have been found to be more effective for the extraction of these compounds from plants compared to mono-solvent systems [25,26]. However, in the case of S. commixta fruit, EtOH extract contained higher TPC (total phenolic content) levels than water extracts, but solvent concentrations did not significantly affect the extraction of phenolic compound yields. TFC (total flavonoid content) in the extracts increased with increasing ethanol concentrations, and the mono-solvent system examined herein (EtOH) was more effective in extracting phenolic and flavonoid compounds than EtOH/water solvents (Table 1). Additionally, higher concentrations of chlorogenic acid, rutin, protocatechuic acid, and hydroxybenzoic acid in EtOH extracts were observed compared to other extracts, suggesting that the differences in the cytotoxic activity of S. commixta fruit extracts were due to their concentration and composition of polyphenolic compounds.   Organic solvents such as ethyl acetate (relative polarity 0.228), butanol (relative polarity 0.586), and hexane (relative polarity 0.009) were used to partition the crude extract via the liquid-liquid extraction technique, which is a method to separate compounds, based on their relative solubilities in two different immiscible liquids with different polarities. Therefore, this method is commonly used for the separation of a substance from mixture [27]. It is well known that ethanol (relative polarity 0.654) can dissolve polar compounds, such as sugar, amino acid, glycoside compounds, phenolic compounds with medium polarity, aglycon flavonoids, anthocyanins, terpenoids, flavones, and polyphenols [28], indicating that EtOH extract is a complex mixture of organic compounds. Based on the liquid-liquid extraction technique, highly polar substances, such as organic acids, polysaccharides, free sugars, and proteins stay in the aqueous phase, whereas other relatively less polar compounds, such as terpenoids and polyphenols, are more soluble in organic solvents [29,30]. To enrich the active compounds in S. commixta fruit EtOH extract, the extracts were partitioned with different solvents, after which the cytotoxicity of each solvent fraction against SK-MEL-2 cells was evaluated. As shown in Figure 1B, the anti-melanoma activities of solvent fractionated EtOH extracts exhibited the following order: BF > EF > HF > AF (83.47%, 33.57%, 9.43%, and 5.56%, respectively). Although several drugs have been used for the treatment of cancer, their cytotoxicity towards normal cells remains a major drawback, which results in secondary malignancy risk [31]. To investigate the effect of BF on the viability of normal cells, HDFa and SK-MEL-2 cells were treated with different concentrations (25 µg/mL, 50 µg/mL, and 100 µg/mL) of BF ( Figure 1C). BF exhibited very little cytotoxicity against HDFa cells (5.98%) up to 50 µg/mL, whereas BF showed cytotoxic effects on SK-MEL-2 cells at concentrations of 50 µg/mL, indicating that BF can be a potent source of anticancer agents due to its selective cytotoxicity against melanoma cells. Further experiments were performed at exposure concentrations below (≤50 µg/mL).
Plant-derived polyphenolic compounds (i.e., including flavonoids) have been reported to possess a wide range of pharmacological properties and many polyphenolic compounds have been shown to induce cell cycle arrest and apoptosis in various types of cancer cells [32,33]. Moreover, the anticancer activity of plant extracts has been widely linked to their TPC and TFC. Therefore, our study analyzed TPC and TFC in solvent fractions of S. commixta fruit ethanol extracts. As shown in Table 2, BF contained the highest amount of phenolic compounds (87.15 ± 4.66 µg GAE/mg of extract), whereas the lowest level of TPC was observed in HF (19.89 ± 2.65 µg GAE/mg of extract). Additionally, total flavonoid levels were measured in the following order: BF (22.25 ± 0.77 µg QE/mg of extract) > EF (13.54 ± 0.43 µg QE/mg of extract) > HF (6.25 ± 1.25 µg QE/mg of extract) > AF (5.96 ± 1.11 µg QE/mg of extract). These results suggested that the inhibitory effect of S. commixta fruit solvent fractions on SK-MEL-2 cell proliferation was significantly related to their TFC and TPC. To further identify the active substance that caused SK-MEL-2 cell cytotoxicity, the polyphenolic compounds of the solvent fractions of S. commixta fruit were analyzed and quantified via HPLC. We identified and quantified seven polyphenolic compounds, including chlorogenic acid, ferulic acid, gallic acid, hydroxybenzoic acid, protocatechuic acid, rutin, and sinapinic acid ( Table 2). BF contained the highest amount of chlorogenic acid (912.72 ± 68.04 µg/g of extract), which is a known antioxidant, anti-inflammatory, and anti-cancer polyphenol compound [34,35].

BF Blocks the MEK/ERK Signaling Pathway
The mitogen-activated protein kinase (MAPK) cascade is an important signaling pathway involved in cellular processes, such as proliferation, and apoptosis. Importantly, dysregulation of MAPK cascades has been linked to several cancers and other diseases [36]. For instance, the MEK/ERK signaling pathway has been shown to play an important role in tumorigenesis and cancer progression [37], suggesting that MEK and ERK are key protein kinases to target for the discovery of anticancer drugs. Therefore, to investigate the involvement of the MEK/ERK signaling pathway in cell death induced by BF, we analyzed the activation level of the MEK/ERK pathway after treatment with BF. As shown in Figure 2A, activation of MEK1/2 and ERK1/2 was inhibited by BF treatment in a dose-dependent manner, indicating that BF reduces the survival of SK-MEL-2 cells by inhibiting the activation of MEK and ERK.

BF Blocks the MEK/ERK Signaling Pathway
The mitogen-activated protein kinase (MAPK) cascade is an important signaling pathway involved in cellular processes, such as proliferation, and apoptosis. Importantly, dysregulation of MAPK cascades has been linked to several cancers and other diseases [36]. For instance, the MEK/ERK signaling pathway has been shown to play an important role in tumorigenesis and cancer progression [37], suggesting that MEK and ERK are key protein kinases to target for the discovery of anticancer drugs. Therefore, to investigate the involvement of the MEK/ERK signaling pathway in cell death induced by BF, we analyzed the activation level of the MEK/ERK pathway after treatment with BF. As shown in Figure 2A, activation of MEK1/2 and ERK1/2 was inhibited by BF treatment in a dose-dependent manner, indicating that BF reduces the survival of SK-MEL-2 cells by inhibiting the activation of MEK and ERK. Caspases (aspartate-specific cysteine proteases) are a family of protease enzymes with fatedetermining roles involved in many cellular processes, including programmed cell death, differentiation, neuronal remodeling, and inflammation [38]. During apoptosis, caspase-3 (i.e., a Caspases (aspartate-specific cysteine proteases) are a family of protease enzymes with fate-determining roles involved in many cellular processes, including programmed cell death, differentiation, neuronal remodeling, and inflammation [38]. During apoptosis, caspase-3 (i.e., a major executioner caspase) is cleaved at an aspartate residue to yield a p12 and a p17 subunit to form the active caspase-3 enzyme [39], resulting in the cleavage of key structural proteins, cell cycle proteins, and DNase proteins, such as poly (ADP-ribose) polymerase, gelsolin, ICAD/DFF, and DNA-dependent kinase [40]. When SK-MEL-2 cells were treated with 50 µg/mL BF, caspase-3 activity was strongly increased (Figure 2B), indicating that BF induced cell death via caspase-3 activation. ERK has been found to directly phosphorylate pro-caspase-9 to inhibit caspase-9 processing and caspase-3 activation, resulting in the inactivation of the caspase cascade during apoptosis [41], suggesting that BF increased caspase-3 activity by inhibiting ERK activation in SK-MEL-2 cells. Additionally, chlorogenic acid has been shown to inactivate ERK in hepatocellular carcinoma cells [42]. Furthermore, rutin, which exhibits several promising pharmacological properties including antitumor activity [43], was approximately 22 times more abundant in BF (2.59 ± 0.17 µg/g of extract) than in EF (0.12 ± 0.03 µg/g of extract) and AF (0.12 ± 0.02 µg/g of extract) ( Table 2). Taken together, these relationships suggest that rutin and chlorogenic acid might be potential anti-melanoma active compounds in S. commixta fruit.
In cancer biology, reactive oxygen species (ROS) are known as a double-edged sword, because the imbalance or accumulation of ROS lead to both the survival and death of cancer cells, respectively [44,45]. Particularly, it has been shown that ROS enhances the activation of the Raf/MEK/ERK signaling pathways to promote cancer cell survival, cell proliferation, cell migration, and differentiation [46], whereas ROS accumulation via pro-oxidants under severe oxidative stress conditions leads to apoptosis and cell death [47]. BF-treated SK-MEL-2 cells exhibited similar levels of ROS as compared with mock control (Figure 3), suggesting that inactivation of MEK by BF-treatment (Figure 2A) was not mediated by ROS levels.
Plants 2020, 9, x FOR PEER REVIEW 5 of 9 major executioner caspase) is cleaved at an aspartate residue to yield a p12 and a p17 subunit to form the active caspase-3 enzyme [39], resulting in the cleavage of key structural proteins, cell cycle proteins, and DNase proteins, such as poly (ADP-ribose) polymerase, gelsolin, ICAD/DFF, and DNAdependent kinase [40]. When SK-MEL-2 cells were treated with 50 μg/mL BF, caspase-3 activity was strongly increased (Figure 2B), indicating that BF induced cell death via caspase-3 activation. ERK has been found to directly phosphorylate pro-caspase-9 to inhibit caspase-9 processing and caspase-3 activation, resulting in the inactivation of the caspase cascade during apoptosis [41], suggesting that BF increased caspase-3 activity by inhibiting ERK activation in SK-MEL-2 cells. Additionally, chlorogenic acid has been shown to inactivate ERK in hepatocellular carcinoma cells [42]. Furthermore, rutin, which exhibits several promising pharmacological properties including antitumor activity [43], was approximately 22 times more abundant in BF (2.59 ± 0.17 μg/g of extract) than in EF (0.12 ± 0.03 μg/g of extract) and AF (0.12 ± 0.02 μg/g of extract) ( Table 2). Taken together, these relationships suggest that rutin and chlorogenic acid might be potential anti-melanoma active compounds in S. commixta fruit.
In cancer biology, reactive oxygen species (ROS) are known as a double-edged sword, because the imbalance or accumulation of ROS lead to both the survival and death of cancer cells, respectively [44,45]. Particularly, it has been shown that ROS enhances the activation of the Raf/MEK/ERK signaling pathways to promote cancer cell survival, cell proliferation, cell migration, and differentiation [46], whereas ROS accumulation via pro-oxidants under severe oxidative stress conditions leads to apoptosis and cell death [47]. BF-treated SK-MEL-2 cells exhibited similar levels of ROS as compared with mock control (Figure 3), suggesting that inactivation of MEK by BFtreatment (Figure 2A) was not mediated by ROS levels.

Plant Materials and Sample Preparation
Fresh S. commixta fruits were harvested from the research forest at Chungbuk National University, after which the air-dried fruits were ground into a fine powder using a blender. The ground materials were soaked in water, EtOH/water mixtures (25:75 v/v (EtOH 25%), 50:50 v/v (EtOH 50%), and 75:25 v/v (EtOH 75%)), or absolute EtOH for 24 h at room temperature, then sonicated in an ultrasonic bath (1 h × 3 times). The suspension was then filtered and evaporated under reduced pressure, and lyophilized to produce dried powder extract. Additionally, 150 g of the crude EtOH extract was suspended in 1 L of water and sequentially partitioned with hexane (HF, 1 L × 2 times), ethyl acetate (EF, 1 L × 2 times), and n-butanol (BF, 1 L × 2 times). The remaining aqueous extract was used as an aqueous fraction (AF). After filtration, each fraction was evaporated using a vacuum evaporator.

Plant Materials and Sample Preparation
Fresh S. commixta fruits were harvested from the research forest at Chungbuk National University, after which the air-dried fruits were ground into a fine powder using a blender. The ground materials were soaked in water, EtOH/water mixtures (25:75 v/v (EtOH 25%), 50:50 v/v (EtOH 50%), and 75:25 v/v (EtOH 75%)), or absolute EtOH for 24 h at room temperature, then sonicated in an ultrasonic bath (1 h × 3 times). The suspension was then filtered and evaporated under reduced pressure, and lyophilized to produce dried powder extract. Additionally, 150 g of the crude EtOH extract was suspended in 1 L of water and sequentially partitioned with hexane (HF, 1 L × 2 times), ethyl acetate (EF, 1 L × 2 times), and n-butanol (BF, 1 L × 2 times). The remaining aqueous extract was used as an aqueous fraction (AF). After filtration, each fraction was evaporated using a vacuum evaporator.
The cytotoxicity of each sample was determined via the MTT assay. Briefly, cells were seeded into 96-well plates at a 5 × 10 3 cells/well seeding density and incubated at 37 • C in a humidified chamber containing 5% CO 2 for 24 h. The cells were then treated with the above-described extracts or fractions at a 100 µg/mL concentration. After incubation for 48 h, the medium was replaced with 20 µL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (1 mg/mL in PBS) for 4 h. Formazan crystals were dissolved in dimethyl sulfoxide (DMSO), and the absorbance was measured at 520 nm using an iMARK microplate reader (Bio-Rad Laboratories GmbH, Munich, Germany).

Caspase-3 Activity and Intracellular ROS Measurement
Protein extraction and the analysis of caspase-3 activity were performed using a Caspase-3/CPP32 Fluorometric Assay Kit (BioVision, Milpitas, CA, USA), according to the manufacturer's instructions. In addition, Intracellular ROS was detected using the DCFH-DA fluorogenic probe as described by Yoo et al. [48]. Caspase-3 activity (400 nm excitation and 505 nm emission) and DCF fluorescence (485 nm excitation and 525 nm emission) were analyzed using a SpectraMax Gemini EM microplate reader (Molecular Devices, San Jose, CA, USA).

Determination of TPC and TFC
TPC and TFC were measured according to the Folin-Ciocalteu and colorimetric methods, respectively, as described by Jin et al. [49] TPC was expressed in milligrams of gallic acid equivalents (mg GAE/g extract) using an equation obtained from a standard gallic acid graph. TFC was determined as milligrams of quercetin equivalents (QE) per gram of extract (mg QE/g extract).

HPLC Analysis
HPLC analysis was performed using a Shimadzu liquid chromatography system (LC-10ADvp) coupled with an ultraviolet-visible detector (SPD-10A; Shimadzu, Kyoto, Japan). The samples were separated with PerkinElmer Brownlee SPP columns (2.7 µm C18 2.1 × 150 mm) at 40 • C at a 0.3 mL/min of flow rate. The mobile phases consisted of water containing 0.1% formic acid (mobile phase A) and acetonitrile containing 0.1% formic acid (mobile phase B). The following gradient elution was performed: 10% B in 0-1.6 min, 20% B in 1.6-11.3 min, 25% B in 11.3-17.8 min, 30% B in 17.8-22.7 min, 60% B in 22.7-25.9, and holding at 80% B in 25.9-28.2 min, the samples were finally reconditioned to the initial conditions. Concentrations were calculated by comparing the sample peak areas with the standard calibration curve.

Statistical Analyses
All experiments were conducted in independent triplicates and significant differences between groups were determined via Duncan's multiple range test. Values of p < 0.05 were considered significant.

Conclusions
Our study analyzed the effect of different ethanol concentrations on the polyphenolic compound composition and anti-melanoma activity of S. commixta fruit extracts. The overall results of the present study suggest that EtOH extract contained a high level of polyphenolic compounds, such as chlorogenic acid, rutin, protocatechuic acid, and hydroxybenzoic acid, all of which are well-known pharmaceutically active compounds, indicating that EtOH extract of S. commixta fruits could be a useful source of natural anti-melanoma agents. Additionally, we found that BF induced SK-MEL-2 cell death by increasing caspase-3 activity and inhibiting the MEK/ERK pathway. Taken together, our results provide valuable information for the development of novel anticancer drugs based on S. commixta fruit, as well as the optimization of extraction conditions in future studies.

Conflicts of Interest:
The authors confirm that there is no conflict of interest to declare.