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Three new cardiac glycosides obtained from the roots of Streblus asper Lour. and their cytotoxic and melanogenesis-inhibitory activities

Three new cardiac glycosides strophanthidin-3-O-α-l-rhamnopyranosyl-(1→4)-6-deoxy-β-d-allopyranoside (1), 5βH-16β-acetylkamaloside (2), and mansonin-19-carboxylic acid (3) along with seven known steroids including five cardiac glycosides were isolated from the methanol extracts of Streblus asper Lour. roots. The structures of these compounds were established by spectroscopic analyses. The cytotoxicities of crude extracts and all the isolated compounds were evaluated against four human cancer cell lines (HL60, A549, AZ521, and SKBR3). Furthermore, the selective index (SI) of each compound was measured by the ratio of cytotoxic effect on a normal cell line (WI38) to the cytotoxic effect on cancer cell line (A549). The results suggested that cardiac glycosides (2, 4, and 6–8) exhibited significant cytotoxicities with IC50 values from 0.01 to 3.77 μM as well as high selective index for WI38/A549 (SI 1.50–24.26), and they displayed superior selectivities when compared with the reference cisplatin (SI 1.09). Preliminary structure–activity relationships (SARs) were also discussed regarding the type of C-10 group in the cardiac glycosides being a crucial factor in determining the cytotoxic activities and regarding the sugar moieties having much less of an active role than the type of C-10 group. In addition, the melanogenesis-inhibitory abilities of these compounds were also evaluated. Cardiac glycosides (3 and 6–8) displayed moderate inhibition effects on melanogenesis with melanin content (MC) of 26.22–74.90% at a concentration of 100 μM, thus showing high cell viability (CV: 77.94–111.70%) compared with that of the reference arbutin (MC: 82.50% and CV: 107.60%). Furthermore, western blot analysis of melanogenesis-related proteins suggested that 3 could inhibit melanogenesis by suppressing the protein expressions of TRP-2 and tyrosinase.


Introduction
The small trees of Streblus asper Lour. (Moraceae) are mainly distributed in tropical regions including India, Sri Lanka, Malaysia, Thailand, Vietnam, and South-west China. 1,2 S. asper is traditionally used in Ayurveda and in the medicine of Chinese minorities for the treatment of different ailments. 1,2 In Ayurveda, the roots of S. asper are used to treat unhealthy ulcers, sinuses, epilepsy, and obesity, and they are applied locally as an antidote to snake bite. 1 In current pharmacologic research, S. asper has exhibited antilarial, 3,4 antimicrobial, 5 antimalarial, 6 anti-HBV, 7,8 antidiabetic, 9 cytotoxic, and antineoplastic activities. [10][11][12] The chemical constituents of S. asper including avonoids, 7,13 volatile oils, 14 phenolic acids, 4 fatty acids, 15 phenylpropanoids, 7,8,16,17 and cardiac glycosides [18][19][20][21] have been extensively studied. Numerous cardiac glycosides have also been discovered from the roots of this plant. 18 In this study, three new cardiac glycosides (1-3) were discovered through phytochemical research on the roots of S. asper. The structures of the isolated compounds were characterized by spectroscopic analyses and compared with the results of the literature data. The cytotoxicities of the crude extracts and isolated compounds were evaluated against HL60, A549, AZ521, and SKBR3 human cancer cell lines as well as WI38 human normal cell line for obtaining the selective index (SI). Combining the structural characteristics, the structure-activity relationships (SARs) of potential bioactive cardiac glycosides were also evaluated. Bufalin is a cardiac aglycone, which belongs to bufanolide, and it can stimulate melanin synthesis in B16F10. 22 Thus, cardiac glycosides (3)(4)(5)(6)(7)(8) having aglycones belonging to cardenolides were selected for the evaluation of their ability to stimulate melanin synthesis in a-melanocyte (a-MSH)-stimulated B16F10 cells. It was interesting that 3-8 showed melanogenesis-inhibitory abilities. Hence, we chose four key melanogenesis-related proteins to further explore the mechanisms of melanogenesis-inhibitory activities using western blot.

Results and discussion
2.1 Cytotoxic and melanogenesis-inhibitory activities of the extracts of S. asper roots Dried S. asper roots were extracted with n-hexane rst and then, the defatted residue was extracted with MeOH. The methanol extract was successively fractionated into EtOAc, n-BuOH and H 2 O fractions. Both the extracts and fractions were evaluated for cytotoxic activities against four human cancer cell lines (HL60, A549, AZ521, and SKBR3) and inhibitory potentials on the production of melanin in a-MSH-activated B16F10 cells. As shown in Table 1, the EtOAc and n-BuOH fractions exhibited strong activities and IC 50 values ranging from 0.05 to 1.79 mg mL À1 , which were higher than those of the reference cisplatin (IC 50 1.14-5.60 mg mL À1 ). As shown in Fig. 1, the n-hexane extract and EtOAc fraction inhibited melanin production signicantly by 36.6% and 8.3% (MC), respectively, at a high concentration of 100 mg mL À1 ; moreover, the n-hexane extract and EtOAc fraction displayed values of 86.7% and 86.6% (MC) at a low concentration of 10 mg mL À1 along with weak toxicities (CV: 86.8% and 91.4%, respectively), which were similar to those of the reference arbutin (MC: 87.1%, CV: 96.5%). Herein, the EtOAc and n-BuOH fractions, which exhibited reasonable cytotoxicities and melanogenesis inhibition, were chosen for further investigation of their constituents in this study.
Compound 2 was obtained as white powder. It was assigned the molecular formula C 33 29,30 which is scarce in Streblus species. Furthermore, the resonance characteristics at d H 4.27 (1H, d, J ¼ 7.6 Hz, H-1 0 ) and d C 101.1 (C-1 0 ) suggested the presence of a b-conguration of glycosyl in the structure of 2, which was similar to that of glycosyl of 5. Analyses of 1 H-1 H COSY, HSQC, and HMBC data revealed that the sugar moiety was 6-deoxy-2,3-dimethoxyl-b-D-galactopyranoside. 16 HMBC correlation between H-1 0 and C-3 was used to establish the position of the sugar unit. The stereo structure was further conrmed by 1D-NMR and 2D-NMR. The combined results showed that compound 2 was 5bH-16bacetylkamaloside. Compound 3 was obtained as light yellow powder. It was assigned the molecular formula C 31 H 46 O 11 with nine degrees of unsaturation, which was deduced from HR-ESI-MS (m/z 617.2944 [M + Na] + , calcd for 617.2938). The aglycone was similar to that of 1 except that in 3, a carboxyl group (d C 176.5) was replaced with an aldehyde group at C-10, and it was determined to have the strophanthidinic acid skeleton by 1D-NMR and 2D-NMR data ( Table 2). 31 The resonance Fig. 1 Melanin content and cell viability were determined for melanogenesis-inhibitory activities of S. asper in B16F10 cell lines, which with and without a-MSH treatment were used as negative controls, whereas arbutin was used as the positive control. *P < 0.05, **P < 0.01, ***P < 0.001 compared with a-MSH model group in three separate experiments.  Hz, H-1 0 ) and d C 100.0 (C-1 0 ) suggested the presence of a b-conguration of sugar, which was similar to that of the glycosyl of 4. Analyses of 1 H-1 H COSY, HSQC, and HMBC data revealed that the sugar moiety was 6-deoxy-2,3-dimethoxyl-b-D-gluopyranoside. 10 HMBC correlation between H-1 0 and C-3 was used to establish the position of the sugar unit. The combined results showed that compound 3 was mansonin-19-carboxylic acid.

Cytotoxic activities of compounds
The cytotoxic activities of ten compounds, 1-10, and the reference chemotherapeutic drug, cisplatin, against human cell lines (HL60, A549, AZ521, SKBR3, and WI38) were evaluated using MTT assays (  C-10 substituent in these cardiac glycosides was a crucial factor in determining the cytotoxic activity. 32,33 However, the results showed that the methyl group linked to C-10 seemed to have superior selectivity than the aldehyde group, and the aldehyde group linked to C-10 seemed to have better selectivity than the carboxyl group, which was evidenced from the comparisons between 5 (SI 1.00) and 7 (SI 15.00), 3 (SI 0.66) and 4 (SI 1.50), and 6 (SI 8.00) and 8 (SI 24.26). In addition, OH-5 and OCOCH 3 -16 were the potential cytotoxic groups compared with these compounds, and the results were supported by the results given in the literature. 34,35 Furthermore, observation of two pairs of related compounds 5/6, and 7/8 indicated that cardiac glycosides displayed weaker cytotoxicities with an increase in the number of sugar units. The sugar moiety is another factor while determining cytotoxic activity, 34 but the sugar moieties were less active than C-10 groups.

Melanogenesis-inhibitory activities of compounds
To identify the active constituents, compounds (3-8) from the EtOAc fraction were investigated for anti-melanogenic effects, with arbutin as positive control. The cytotoxic activities of these compounds against B16F10 cells were also determined by MTT assays. To assess the risk/benet ratio of each compound, relative activities vs. toxicities were calculated by dividing the melanin content (MC, %), by the cell viability (CV, %), and this was expressed as an activity-to-cytotoxicity ratio (A/C ratio) for each compound. 36 As shown in Table 4, the compounds were treated with three concentrations (100 mM, 30 mM, and 10 mM).

Western blot analysis of melanogenesis-related proteins
Depigmentation can be achieved by regulating the transcriptions and activities of tyrosinase, tyrosinase-related protein-1 (TRP-1) and tyrosinase-related protein-2 (TRP-2). 37 Tyrosinase, a rate-limiting enzyme, catalyzes the hydroxylation of L-tyrosine  to l-(3,4-dihydroxyphenyl)-alanine (L-DOPA) and the oxidation of L-DOPA to L-DOPA quinone. 38 TRP-1 catalyzes the oxidation of 5,6-dihydroxyindole-2-carboxylic acid (DHICA), and TRP-2 functions as DOPAchrome tautomerase. 39 Transcription for the expression of these enzymes is regulated by the microphthalmia transcription factor (MITF). 38 To clarify the mechanism involved in melanogenesis inhibition by 3, which was the most potent melanogenesis inhibitor at 100 mM (MC: 26.22%, CV: 77.94%, A/C 0.34) in this study, the protein levels of tyrosinase, TRP-1, TRP-2, and MITF were evaluated in B16F10 cells treated with 3 (30 mM and 100 mM) by western blot analysis. As shown in Fig. 4, the protein levels of TRP-2 and tyrosinase decreased in a dose-dependent manner, whereas those of TRP-1 indicated a slight change. However, the protein levels of MITF treated with 3 at 30 mM were higher compared with those of the control group (the second line), but they were much lower at 100 mM. These results indicated that 3 possessed almost no melanogenesis-inhibitory activity at 30 mM (MC: 89.29%).
Although the protein levels of MITF and TRP-1 were higher than those of the control group, tyrosinase, which is a rate-limiting enzyme, showed lower level compared with the control group, and this was in accordance with the changes in the melanin contents at 30 mM and 10 mM. It was inferred that 3 exhibited melanogenesis inhibitory activity on a-MSH-stimulated B16F10 cells by decreasing the expressions of TRP-2 and tyrosinase.

Conclusion
In this study, three new cardiac glycosides strophanthidin-3-O-a-L-rhamnopyranosyl-(1/4)-6-deoxy-b-D-allopyranoside (1), 5bH-16b-acetylkamaloside (2), and mansonin-19-carboxylic acid (3) along with seven known steroids were isolated from the methanol extracts of S. asper roots. The cardiac glycosides 2, 4, and 6-8 indicated potent cytotoxicities and high SI values, and they displayed superior selectivities when compared with the reference cisplatin. SARs were also discussed regarding the type of C-10 groups in the cardiac glycosides and the sugar moieties' effects on cytotoxicity. In addition, the cardiac glycosides 3 and 6-8 displayed moderate inhibition of melanogenesis compared with the reference arbutin. The western blot analysis of melanogenesis-related proteins suggested that 3 could inhibit melanogenesis by suppressing the protein expressions of TRP-2 and tyrosinase. The current study contributes to the understanding of SARs of cytotoxicity and mechanisms of the melanogenesis-inhibitory activities of cardiac glycosides, which will hopefully help develop new potential antitumor and melanogenesis-inhibitory agents. . TLC was carried out on silica gel GF254 (Millipore, Ltd.). Spots were visualized under UV light (254 and 365 nm) and sprayed with Keddle reagent and vanillin/H 2 SO 4 (1 : 100, w/v) (the former reagent showed red spots and the latter reagent showed green spots); this was followed by heating if the compounds were cardiac glycosides. CH 3 OH used in the preparative HPLC procedure was of HPLC grade, and the other solvents were of analytical grade (Kanto Chemical Co., Inc.).

Plant materials
The
4.3.4 Acid hydrolysis of 1. Compound 1 (2.0 mg) was heated at reux with 2.0 mL of 2 N HCl for 2 h at 80-90 C. The reaction mixtures were extracted with EtOAc (3 Â 2.0 mL). The H 2 O layer was concentrated to dryness aer repeatedly adding MeOH to remove acid. The residue was then dissolved in pyridine (1.0 mL) and stirred with L-cysteine methyl ester at 60 C for 1.0 h. Sugar (D/L) standards were also derivatized using L-cysteine methyl ester in the same manner. Then, trimethylsilyl (TMS) was added to the reaction mixtures, and the mixtures were heated for 1 h at 60 C. The reaction mixtures were analyzed using GC (column: Agilent J&W DB-17, 30 m Â 0.32 mm, column temp: 200 C, injector temp: 270 C, detector temp: 270 C, detector: FID, He ow rate: 0.5 mL min À1 , a-L-Rha: t R ¼ 13.240 min, 6-deoxy-b-D-allose: t R ¼ 12.240 min).

Cytotoxicity assays
4.4.1 Cell culture. Cell lines HL60 (human acute promyelocytic leukemia cells), A549 (human lung carcinoma cells), AZ521 (human duodenal cancer cells), SKBR3 (human adenocarcinoma cells), and WI38 (human normal fetal cells) were obtained from Riken Cell Bank (Japan), and B16F10 cells (mouse melanoma cells) were obtained from the cell bank of the Chinese Academy of Sciences. The human cancer cell lines A549, AZ521, SKBR3, and B16F10 were maintained in adhesion on Petri dishes with Dulbecco's Modied Eagle's Medium (DMEM, Sigma-Aldrich, Japan) supplemented with 10% heatinactivated fetal bovine serum (FBS, GIBCO), penicillin-streptomycin (100 U mL À1 , GIBCO). HL60 was suspended in RPMI medium 1640 with 10% FBS, penicillin-streptomycin (100 U mL À1 ), and WI38 was maintained in adhesion on Petri dishes with Minimum Essential Medium (MEM, Sigma-Aldrich, Japan) with 10% FBS, penicillin-streptomycin (100 U mL À1 ). Cells were grown at 37 C in an atmosphere of 5% CO 2, 95% air and >95% humidity, and those that passaged three or more times were used for each test. HL60, a oating cell, was recovered with the medium from the dish, and other adherent cells were washed with phosphate buffer saline (PBS) and recovered by the action of 0.05% trypsin-EDTA (GIBCO). The collected cells were centrifuged at 800 rpm at 4 C for 5 min. Aer separating and removing the supernatant liquid, an appropriate amount of medium was added and pipetted to prepare a cell suspension. The cell suspension (10 mL) was taken, the number of cells in the corning chamber were counted, and an appropriate amount of the medium was added in each experiment to obtain the appropriate cell density. 4.4.2 MTT assays. All kinds of adherent cell lines were seeded at 3 Â 10 3 cells per well in a 96-well plate and cultured for 24 h. The oating cell line was seeded at 8 Â 10 3 cells per well in a 96-well plate. Samples were then added at an appropriate nal concentration and allowed to act for 48 h. Followed by the addition of 0.5% 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide solution (MTT, Sigma-Aldrich, Japan) and further culturing for 3 h, 100 mL per well of solution (0.04 M HCl/2-PrOH, Wako Pure Chemical Industries) was added, and the cell injury rate was calculated from absorbance at 570-630 nm. The calculation formula of inhibitory rate is as follows: I% ¼ [(Abs control À Abs sample )/(Abs control À Abs blank )] Â 100%. The 50% inhibition concentration (IC 50 ) was analyzed by GraphPad Prism 5. The SI of compounds were measured by the ratio of cytotoxic effect on the normal cell line (WI38) to the cytotoxic effect on the cancer cell line (A549). An anticancer agent (cisplatin) was used as the reference compound.
4.4.3 Measurement of melanin content. The melanin content of B16F10 cells was determined according to the method in the previous literature with slight modications. 40 The B16F10 cells were cultured at a density of 5 Â 10 4 cells per well in 24-well plates for 24 h. Then, 0.10 mM a-MSH (Sigma-Aldrich, Japan) and the tested compounds were added at varying concentrations, and the cells were incubated for additional 48 h. The cells were washed three times with PBS and centrifuged at 95 000 g for 5 min. The harvested cells were dissolved in 100 mL of 2 N NaOH (Wako Pure Chemical Industries) and 10% dimethyl sulfoxide (DMSO, Sigma-Aldrich, Japan) solution, maintained at 80 C for 0.5 h, and mixed to solubilize the melanin. The absorbance value of the supernatant was measured at 405 nm using a microplate reader. The calculation formula of the melanin content is as follows: MC% ¼ [(Abs sample À Abs blank )/(Abs control À Abs blank )] Â 100%. The calculation formula of activity-to-cytotoxicity ratio is as follows: A/C ¼ (MC%)/(CV%). The statistical signicance of the differences between control and treatment groups was analyzed using the program GraphPad Prism 5. The melanogenesisinhibitory agent arbutin was used as the reference compound.
4.4.4 Western blot analysis. The B16F10 cells were incubated with various concentrations of 3 (30 mM and 100 mM), and they were subsequently treated with 0.10 mM a-MSH for 48 h. Aer incubation, the cells were harvested and washed twice with ice-cold PBS. The cells were lysed in lysis buffer (2% sodium dodecyl sulfate, 50 mM Tris-HCl, pH 7.2, 10% glycerol, 2% 2sulfanylethanol). The cell lysates were centrifuged at 18 650 g at 4 C for 10 min, and the supernatant concentration was determined using the Enhanced BCA Protein Assay Kit (Beyotime, Nanjing, P. R. China). Aliquots of the lysates (30 mg protein) were separated by electrophoresis on 10% sodium dodecyl sulfatepolyacrylamide gel and transferred to a polyvinylidene uoride membrane in glycine transfer buffer [25 mM Tris-HCl, 192 mM glycine, and 20% MeOH (v/v)]. The nonspecic sites were blocked with 5% non-fat skim milk in Tris-buffered saline (TBS)-Tween 20 solution. The membrane was incubated with primary antibodies against MITF, tyrosinase, TRP-1, TRP-2, and b-actin, and it was then incubated with a secondary antibody (1 : 5000; Biogot