Chemical Constituents of Mangifera indica and Their Antiausterity

Article pubs.acs.org/jnp

Chemical Constituents of Mangifera indica and Their Antiausterity Activity against the PANC‑1 Human Pancreatic Cancer Cell Line Hai Xuan Nguyen,† Truong Nhat Van Do,† Tho Huu Le,† Mai Thanh Thi Nguyen,*,† Nhan Trung Nguyen,† Hiroyasu Esumi,‡ and Suresh Awale*,§ †

Faculty of Chemistry, University of Science, Vietnam National University-Hochiminh City, 227 Nguyen Van Cu Street, District 5, Vietnam ‡ Research Institute for Biomedical Sciences, Tokyo University of Science, Chiba 278-8510, Japan § Division of Natural Drug Discovery, Department of Translational Research, Institute of Natural Medicine, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan S Supporting Information *

ABSTRACT: Human pancreatic cancer cell lines such as PANC-1 have an altered metabolism, enabiling them to tolerate and survive under extreme conditions of nutrient starvation. The search for candidates that inhibit their viability during nutrition starvation represents a novel antiausterity strategy in anticancer drug discovery. A methanol extract of the bark of Mangifera indica was found to inhibit the survival of PANC-1 human pancreatic cancer cells preferentially under nutrient-deprived conditions with a PC50 value of 15.5 μg/mL, without apparent toxicity, in normal nutrient-rich conditions. Chemical investigation on this bioactive extract led to the isolation of 19 compounds (1−19), including two new cycloartane-type triterpenes, mangiferolate A (1) and mangiferolate B (2). The structures of 1 and 2 were determined by NMR spectroscopic analysis. Among the isolated compounds, mangiferolate B (2) and isoambolic acid (12) exhibited potent preferential cytotoxicity against PANC-1 human pancreatic cancer cells under the nutrition-deprived condition with PC50 values of 11.0 and 4.8 μM, respectively.

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hostile tumor microenvironment, pancreatic tumor cells adopt countermeasures for their survival, such as a “metabolic switch” to tolerate the starvation conditions.10,11 Our earlier studies have shown that human pancreatic PANC-1 cancer cells survive for over 3 days even in the complete absence of nutrients such as glucose, amino acids, and serum. This phenomenon of survival of pancreatic cancer cells under extreme nutrientdeficient conditions is referred to as “austerity”.10 Discovery of anticancer agents that inhibit the tolerance of cancer cells to nutrient starvation has emerged as an attractive “antiausterity” strategy in anticancer drug discovery.10,11 In this strategy, the plant extract and purified compounds are tested for their cytotoxic activity against a human pancreatic cancer cell line such as PANC-1 in both a normal nutrient-rich medium (Dulbecco’s modified Eagle medium, DMEM) and a nutrientdeprived medium (NDM). Compounds possessing preferential cytotoxicity in the NDM without toxicity in DMEM are

ancreatic cancer is one of the most aggressive types of cancer and has the lowest five-year survival of 11, 6, 7, 8). However, the presence of a β-acetyl group at C-3 was found to be favorable (2 > 11) over a hydroxy group (Table 2). Further, an ethidium bromide and acridine orange (EB/AO) staining assay was performed to observe the morphological change of PANC-1 cells induced by isoambolic acid (12). AO is a cell-permeable dye and emits bright green fluorescence in live cells. EB is permeable in dead cells and gives a red fluorescence.35 In dead cells, although both EB and AO

2 δC 32.1 27.0 80.5 39.6 47.3 21.1 26.0 47.9 20.2 26.1 26.6 33.0 45.5 49.0 35.6 28.3 52.3 18.2 29.9 36.1 18.3 34.9 25.9 146.0 126.7 172.7 12.1 25.6 14.3 19.4

173.9 35.0 25.3 29.0−30.0 27.3 130.0 130.1 27.4 29.0−30.0 22.8 15.4

δH 1.60 1.25 1.76 1.61 4.56

m m m m dd (10.5, 5.0)

1.39 1.57 0.79 1.32 1.09 1.51

dd (12.5, 4.2) m m m m dd (12.2, 4.7)

1.99 m 1.13 m 1.61 m, 2H

1.30 1.90 1.29 1.59 0.96 0.57 0.34 1.41 0.88 1.58 1.15 2.21 1.97

m m m m s d (3.9) d (3.9) m brs m m m m

3.16 q (7.0) 1.31 0.84 0.90 0.88 4.96 4.92

d (7.0) s brs brs brs brs

2.06 s

δC 31.8 27.0 80.9 39.6 47.3 21.1 26.0 48.0 20.3 26.2 26.7 33.0 45.5 49.0 35.7 28.2 52.4 18.4 29.9 36.2 18.1 34.7 31.7 148.8 45.7 179.6 16.5 25.6 15.3 19.5 111.3 171.2 21.4

orientations. These data also suggested that the orientation of an unsaturated fatty ester group at C-3 is β-equatorial. The NOESY correlations also indicated that rings A and C adopt a chair conformation, while rings B and D adopt a boat conformation. This was supported further by the large transC

DOI: 10.1021/acs.jnatprod.6b00381 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Figure 1. Connectivities (bold lines) deduced by the COSY and HSQC spectra and significant HMBC correlations (solid arrows) of 1 and 2.

Figure 2. HRESIMS/MS of compound 1.

Figure 3. Key NOESY correlations observed for compounds 1 and 2.

mobility, leading to a marked change in PANC-1 cell morphology within 6 h of treatment. A typical plasma membrane blebbing was observed from 9 h of treatment, leading to >50% cell death within 24 h (Figure 6). The supporting video S1 (Supporting Information) shows the detailed events of cell death induced by isoambolic acid (12, 5 μM) treatment for 48 h. As human pancreatic cancer cells are resistant to conventional anticancer drugs in clinical use such as paclitaxel, gemcitabine, and 5-fluorouracil, with PC50 values of >200 μM,17 analogues of isoambolic acid (12) should be candidates of further interest for antiausterity drug development.

Table 2. Preferential Cytotoxicity of Compounds 1−19 against the PANC-1 Human Pancreatic Cancer Cell Line in Nutrient-Deprived Medium (NDM) compound

PC50, μMa

compound

PC50, μMa

compound

PC50, μMa

1 2 3 4 5 6 7

69.6 11.0 >100 >100 32.3 >100 52.1

8 9 10 11 12 13 14

>100 44.2 22.8 >100 4.8 >100 >100

15 16 17 18 19 arctigeninb paclitaxelc

>100 >100 >100 >100 >100 0.8 >100

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a

Concentration at which 50% of cells were killed preferentially in NDM. bPositive control. cNegative control.

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were recorded on a JASCO DIP-140 digital polarimeter. IR spectra were measured with a Shimadzu IR-408 spectrophotometer in CHCl3 solution. NMR spectra were taken on a Bruker Advance III 500 spectrometer with tetramethylsilane as an internal standard, and chemical shifts are expressed in δ values. HRESIMS measurements were carried out on a Bruker microTOF-QII spectrometer. Silica gel 60, 40−63 μm (230−400 mesh ASTM), for column chromatography was purchased from Scharlau. Analytical and preparative TLC were carried out on precoated Kiesegel 60F254 or RP-18F254 plates (0.25 or 0.5 mm thickness) from Merck. Plant Material. The bark of Mangifera indica was collected in Giong Trom District of Ben Tre Province, Vietnam, in March 2013, and was identified by Ms. Hoang Viet, Faculty of Biology, University of Science, Vietnam National University Hochiminh City (VNUHCM). A voucher specimen (MCE0035) has been deposited at the

penetrate, the bright red fluorescence due to EB predominates over AO. As shown in Figure 5, PANC-1 cells in NDM (control) after 24 h showed intact morphology and stained only with AO to give bright green fluorescence, suggesting all the cells were alive. Treatment with isoambolic acid (12, 10 μM) for 24 h showed dramatic morphological alterations with exclusive red fluorescence indicative of dead cells. Isoambolic acid (12) was studied further for its effects against PANC-1 cells in NDM in real time using a live-cell imaging system. PANC-1 cells were treated with 5 μM isoambolic acid (12, PC50 value) in NDM and incubated in a stage-top incubator at 37 °C and 5% CO2; images were captured every 5 min under phase-contrast mode on an EVOSFL cell imaging system for 48 h. Isoambolic acid (12) was found to inhibit cell D



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Figure 4. Preferential cytotoxic activity of (A) mangiferolate B (2) and (B) isoambolic acid (12) against the PANC-1 human pancreatic cancer cell line in nutrient-deprived medium (NDM) and Dulbecco’s modified Eagle medium (DMEM).

Figure 5. Morphology of PANC-1 cells under the control and following treatement with isoambolic acid (12, 10 μM) at 24 h and stained by ethidim bromide (EB)/acridine orange (AO). Images were acquired under the fluorescent, phase-contrast, and overlay modes. Live cells were stained with AO and emitted a bright green fluorescence, while dead cells were stained with EB and emitted a red fluorescence. Treatment with isoambolic acid (12) at 10 μM led to dramatic alteration of PANC-1 cell morphology and total death of PANC-1 cells within 24 h. Division of Medicinal Chemistry, Faculty of Chemistry, University of Science, VNU-HCM. Extraction and Isolation. Dried powdered bark of M. indica (1.5 kg) was extracted with MeOH (15 L, reflux, 3 h × 3) to yield a methanol extract (100 g). The MeOH extract was suspended in H2O (1 L) and then partitioned successively with n-hexane (3 × 1 L) and EtOAc (3 × 1 L) to give n-hexane (21 g), EtOAc (35 g), and H2O (44 g) fractions, respectively. The n-hexane fraction (16 g) was subjected to silica gel column (6.5 × 120 cm) chromatography, eluted with acetone−hexane gradient mixtures (0−50%) to yield 15 fractions (H1, 1.1 g; H-2, 0.3 g; H-3, 3.1 g; H-4, 0.7 g; H-5, 0.4 g; H-6, 1.0 g; H-7, 0.4 g; H-8, 3.0 g; H-9, 1.4 g; H-10, 0.8 g; H-11, 1.2 g; H-12, 0.3 g; H13, 0.4 g; H-14, 0.3 g; H-15, 1.6 g). Fraction H-2 (300 mg) was applied to silica gel column chromatography using an EtOAc−hexane gradient system to give three subfractions (H-2-1, 130 mg; H-2-2, 70 mg; H-2-3, 100 mg). Subfraction H-2-3 was further chromatographed using acetone−hexane mixtures (0−30%) to afford 10 (95 mg). Fraction H-5 (400 mg) was rechromatographed on silica gel with an acetone−hexane gradient system to yield four subfractions (H-5-1, 60 mg; H-5-2, 167 mg; H-5-3, 76 mg; H-5-4, 100 mg). Subfraction H-5-1 was chromatographed with EtOAc−hexane mixtures (0−50%) and then purified by normal-phase preparative TLC with CHCl3 (100%) to give 1 (5.0 mg). Subfraction H-5-2 was subjected elution with MeOH−CHCl3 mixtures (0−30%) to afford 13 (26.5 mg). Fraction H-5-3 was applied to silica gel chromatography with EtOAc−hexane mixtures (0−50%) to afford 2 (6.5 mg). Fraction H-9 (1.4 g) was

applied to silica gel column chromatography, eluted with an acetone− hexane gradient system, to yield five subfractions (H-9-1, 207 mg; H9-2, 110 mg; H-9-3, 515 mg; H-9-4, 185 mg; H-9-5, 15 mg). Subfraction H-9-2 was further purified by chromatography on silica gel with acetone−hexane mixtures (0−50%), followed by normal-phase preparative TLC with acetone−hexane mixtures (15:85), to give 3 (14.8 mg) and 9 (14.5 mg). Repeated purification of subfraction H-9-3 on silica gel column chromatography followed by normal-phase preparative TLC with acetone−hexane mixtures (30:70) gave 14 (12.3 mg). Fraction H-10 (800 mg) was chromatographed on silica gel column chromatography, eluted with an acetone−hexane gradient system, to give four subfractions (H-10-1, 25 mg; H-10-2, 301; H-10-3, 291 mg; H-10-4, 69 mg). Subfraction H-10-3 was subjected to silica gel chromatography with CHCl3 (100%), followed by purification with normal-phase preparative TLC with EtOAc−CHCl 3 −hexane (85:5:10), to furnish 6 (15.0 mg) and 12 (5.0 mg). Subfraction H10-4 was applied to silica gel chromatography using CHCl3 (100%) for elution, followed by normal-phase preparative TLC with EtOAc− hexane (5:95), to obtain 4 (5.0 mg), 11 (6.7 mg), and 5 (13.1 mg). Fraction H-15 (1.6 g) was separated by silica gel chromatography with a MeOH−CHCl3 gradient system to yield six subfractions (H-15-1, 100 mg; H-15-2, 300 mg; H-15-3, 350 mg; H-15-4, 250 mg; H-15-5, 330 mg; H-15-6, 270 mg). Subfraction H-15-1 was chromatographed with MeOH−CHCl3 mixtures (0−50%), followed by purification with normal-phase preparative TLC with MeOH−CHCl3 (10:90), to give 7 (15.0 mg) and 8 (8.0 mg). The EtOAc fraction (35 g) was E



Article

Figure 6. Morphological change of PANC-1 cells following treatement with isoambolic acid (12, 5 μM) at different intervals of time in phasecontrast mode for 24 h. chromatographed on a silica gel column (9 × 120 cm), eluted with a MeOH−CHCl3 gradient system, to yield 12 fractions (E-1, 3.2 g; E-2, 2.4 g; E-3, 0.6 g; E-4, 0.2 g; E-5, 1.2 g; E-6, 1.1 g; E-7, 2.8 g; E-8, 1.2 g; E-9, 3.0 g; E-10, 2.6 g; E-11, 3.2 g; E-12, 13.4 g). Fraction E-6 (1.1 g) was applied to silica gel chromatography with a EtOAc−hexane gradient system, to give four subfractions, E-6-1 to E-6-4. Subfraction E-6-2 (100 mg) was dissolved in MeOH−CHCl3 and stored overnight to produce a precipitate of 16 (20.0 mg). Fraction E-7 (2.8 g) was subjected to silica gel chromatography with an acetone−hexane gradient system followed by purification with reversed-phase preparative TLC with MeOH−CH3CN−H2O (10:70:20), to afford 17 (5.0 mg), 18 (5.0 mg), and 19 (6.6 mg). Fraction E-10 (1.0 g) on was dissolved in MeOH−CHCl3 to afford a precipitate of 15 (130 mg). Mangiferolate A (1): white, amorphous powder; [α]25 D +32 (c 0.01, CHCl3); IR νmax (CHCl3) 3400, 3030, 2950, 2870, 1720, 1680, 1630, 1450 cm−1; 1H and 13C NMR (CDCl3 500 MHz, see Table 1); HRESIMS m/z 743.5930 [M + Na]+ (calcd for C48H80O4Na, 743.5954). Mangiferolate B (2): white, amorphous powder; [α]25 D +40 (c 0.01, CHCl3); IR νmax (CHCl3) 3500, 3035, 2960, 2860, 1730, 1690, 1630, 1460 cm−1; 1H and 13C NMR (CDCl3 500 MHz, see Table 1); HRESIMS m/z 535.3744 [M + Na]+ (calcd for C33H52O4Na, 535.3763). Preferential Cytotoxicity Assay against PANC-1 Cells. The PANC-1 (RBRC-RCB2095) human pancreatic cancer cell line was purchased from the Riken BRC cell bank and maintained in standard Dulbecco’s modified Eagle medium with 10% fetal bovine serum supplement at 37 °C under a humidified atmosphere of 5% CO2 and 95% air. Briefly, human pancreatic cancer cells were seeded in 96-well plates (1.5 × 104/well) and incubated in fresh DMEM at 37 °C under 5% CO2 and 95% air for 24 h. After the cells were washed twice with phosphate-buffered saline (PBS), the medium was changed to serially diluted test samples in both nutrient-rich medium (DMEM) and

nutrient-deprived medium (NDM)11 with a control and blank in each test plates. The composition of the nutrient-deprived medium was as follows: 265 mg/L CaCl2(2H2O), 0.1 mg/L Fe(NO3)(9H2O), 400 mg/L KCl, 200 mg/L MgSO4(7H2O), 6400 mg/L NaCl, 700 mg/L NaHCO3, 125 mg/L NaH2PO4, 15 mg/L phenol red, 25 mM/L HEPES buffer (pH 7.4), and MEM vitamin solution (Life Technologies, Inc., Rockville, MD, USA); the final pH was adjusted to 7.4 with 10% NaHCO3. Arctigenin, a positive control in this study, was isolated from the seed of Arctium lappa.11 After 24 h incubation with test compounds in DMEM and NDM, the cells were washed twice with PBS and replaced with 100 μL of DMEM containing a 10% WST-8 cell counting kit solution. After a 3 h incubation, the absorbance at 450 nm was measured (PerkinElmer EnSpire multilabel reader). Cell viability was calculated from the mean values of data from three wells by using the following equation:

Cell viability (%) = [Abs(test sample) − Abs(blank)/Abs(control) − Abs(blank)]× 100% Morphological Assessment of Cancer Cells. PANC-1 cells were seeded in 96-well plates (1.5× 104/well) and incubated in fresh DMEM or RPMI-1640 at 37 °C under 5% CO2 and 95% air for 24 h. After the cells were washed twice with PBS, the medium was changed to NDM (control) or serially diluted test samples in NDM (treated). After a 24 h incubation, 2 μL of EB/AO reagent was added to each test well and incubated for 5 min, and morphology was captured using the EVOS FL cell imaging system (20 × objective) under fluorescent and phase contrast mode. Live-Cell Imaging. PANC-1 cells (3.0× 106) were plated in 35 mm cell culture dishes and allowed to attach overnight in a complete medium. The cells were then washed twice with PBS, treated with 5 μM isoambolic acid (12) in NDM, and immediately placed inside a stage-top incubator (BLAST CP-089 AR) that was maintained at 37 F



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°C and 5% CO2. Live images were captured under the phase contrast mode every 5 min on an EVOS FL cell imaging system for 48 h.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00381. Copies of spectroscopic data for 1 and 2 (PDF) Live imaging movie of the effect of isoambolic acid (12) against PANC-1 cells (AVI)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail (M. T. T. Nguyen): [email protected]. Tel: +84907-426-331. Fax: +84-838-353-659. *E-mail (S. Awale): [email protected]. Tel: +81-76434-7640. Fax: +81-76-434-7640. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by a grant from the Vietnam National University Hochiminh City (No. C2015-18-11) to H.X.N. and by a Grant in Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS), Japan, to S.A.

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Chemical Constituents of Mangifera indica and Their Antiausterity

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