Triterpenoid Components from Oak Heartwood ... - ACS Publications

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Triterpenoid Components from Oak Heartwood (Quercus robur) and Their Potential Health Benefits Andy J. Pérez,*,†,‡ Łukasz Pecio,† Mariusz Kowalczyk,† Renata Kontek,§ Gabriela Gajek,§ Lidija Stopinsek,⊥ Ivan Mirt,⊥ Wiesław Oleszek,† and Anna Stochmal† †

Department of Biochemistry and Crop Quality, Institute of Soil Science and Plant Cultivation, State Research Institute, ul. Czartoryskich 8, 24-100 Puławy, Poland ‡ ́ Area Productos Químicos, Unidad de Desarrollo Tecnológico (UDT) − Universidad de Concepción, Av. Cordillera No. 2634, Coronel 4191996, Concepción, Chile § Laboratory of Cytogenetics, Faculty of Biology and Environmental Protection, University of Lodz, Banacha 12/16, 90 237 Lodz, Poland ⊥ Tanin Sevnica d.d., Hermanova 1, 8290 Sevnica, Slovenia S Supporting Information *

ABSTRACT: For centuries oak wood (Quercus robur) has been used in aging of wines and spirits, which is based on pleasant flavors given to beverages by phenolics transferred to the liquid during the maturation process. Other metabolites, such as triterpenoids, can also be released. Searching for extractable triterpenoids in oak heartwood, 12 new, 1−12, and five known, 13− 17, oleanane types were isolated and characterized. Their cytotoxicities were tested against cancer cells (PC3 and MCF-7) and lymphocytes. Breast cancer cells (MCF-7) were the most affected by triterpenoids, with roburgenic acid, 4, being the most active compound (IC50 = 19.7 μM). Selectivity was observed for compounds 1−3, 8, 9, and 16, exhibiting an IC50 > 200 μM against lymphocytes, while active against cancer cells. A galloyl unit attached to the triterpenoid moiety was established as the key feature for such effect. These results highlight the occurrence of triterpenoids in oak heartwood and their relevance for chemoprevention of breast cancer. KEYWORDS: Quercus robur, heartwood, triterpenoids, breast cancer, cytotoxicity, structure−activity relationships

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INTRODUCTION Quercus robur L. (Fagaceae) is a large deciduous tree native to most of Europe, commonly known as pedunculate oak. Together with sessile oak (Q. petraea), it occurs widely across Europe, reaching northwards to southern Norway and Sweden, and southwards to the northern part of the Iberian Peninsula, South Italy, the Balkan Peninsula, and Turkey.1 The quality of oak wood is well recognized in the furniture industry due to its hardness and durability, and it has also been widely used in the manufacture of barrels used for the aging of wines and spirits. During the maturation process of these beverages, the transfer of volatile and nonvolatile compounds takes place from the wood matrix to the liquid. Mainly aromatic aldehydes, originated from lignin decomposition after heat treatment, and hydrolyzable tannins are released, playing an important role in the sensorial characteristics of the final product.2−4 Q. robur wood is known for having high ellagitannin content, which together with its low aromatic potential compared to American white oak (Q. alba), makes it more suited for aging spirits.2 As a consequence, secondary metabolites other than polyphenols, such as triterpenoids and their derivatives, can also be extracted due to the higher alcohol percentage of spirits relative to wines.5 Bitter and astringent tastes have been correlated to triterpenoids when present in alcoholic beverages.5 However, recent investigations searching for the molecular determinants of sweetness of oak heartwood, and dry wines aged in oak © 2017 American Chemical Society

barrels, have concluded that some of the examined triterpenoids are in fact natural sweeteners.6,7 Despite the significant flavor altering properties of triterpenoids, they could also be considered as potential cancer preventative constituents,8 among other multiple health benefits.9,10 The anticancer properties of triterpenoids and their glycosides (saponins) have been broadly reviewed.11,12 Promoting apoptosis is not the only mechanism through which triterpenoids target the suppression of tumors.13,14 Indeed, this class of metabolites has also been described as multifunctional groups of compounds, capable of targeting tumor cells at various levels, therefore being recognized as promising anticancer drugs,14 or used in combination with other drugs for chemotherapy as a strategy to overcome multidrug resistance (MDR) in cancer treatments.15 Given the impact that triterpenoids could have for the industry of viticulture as potential bioactive natural supplements or substances able to modify censorial characteristics, the literature still lacks investigations focused on them. This study was aimed, therefore, in performing a complete characterization of triterpenoids and their derivatives in heartwood of Q. robur, and evaluation of their cytotoxic activity against three types of Received: Revised: Accepted: Published: 4611

March 27, 2017 May 20, 2017 May 24, 2017 May 24, 2017 DOI: 10.1021/acs.jafc.7b01396 J. Agric. Food Chem. 2017, 65, 4611−4623

Article

Journal of Agricultural and Food Chemistry

Figure 1. Total ion chromatograms in negative ion mode for the heartwood 70% aqueous methanol extract of Q. robur, and the organic and aqueous layers after liquid−liquid extraction with ethyl acetate. Peaks corresponding to isolated triterpenoids are shown with their assigned numbers. Darmstadt, Germany), developed with acetonitrile/water/formic acid (4:6:0.5, v/v), observed under 254 and 366 nm UV light, and then visualized after spraying with Liebermann−Burchard reagent and heating at 130 °C. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), penicillin-streptomycin solution stabilized, Histopaque 1077 and buffered saline (PBS) were purchased from Sigma Chemical Co. (Steinheim, Germany), Fetal bovine serum (FBS), phytohemagglutinin (PHA), RPMI 1640 medium with glutamine, F12K medium and Dulbecco’s modified Eagle’s medium (DMEM), and trypsin-EDTA were supplied by CytoGen (Łódź, Poland). Plant Material. Oak heartwood used in this study came from a 78years old Q. robur tree, grown in forest near to Velika Gorica, Croatia. Wood was reduced to chips, air-dried and ground to obtain a powder of 200 μm particle size. A voucher specimen was deposited in the own herbarium of the Institute of Soil Science and Plant Cultivation (IUNG-PIB), Poland (No. 11/19082014). Extraction and Isolation. Air-dried and ground oak heartwood (1.0 kg) was extracted by soaking in 70% aq. MeOH (6 L × 4, 2 days each) at room temperature in the dark. Combined filtered solutions were concentrated under vacuum, suspended in distilled water and exhaustively partitioned with ethyl acetate (yield: 2.1% of dry weight). Chemical composition of samples after each fractionation and purification step was monitored by LC-MS (Figure 1), using an LCQ Thermo Advantage Max ion trap mass spectrometer (Thermo Finnigan, Waltham, MA) coupled with a Surveyor HPLC system or a TQ detector coupled to an ACQUITY UPLC system (Waters Corp.). The ethyl acetate soluble extract (5 g × 4) was subjected to open column chromatography on a 20.5 cm × 7.5 cm glass column packed with 140 μm Cosmosil C18−PREP (120 g) (Nacalai Tesque, Inc., Kyoto, Japan), and eluted with MeOH/H2O step gradient (5:95, 20:80, 80:20, 100:0; 1 L each, v/v). Four fractions were collected according to their characteristics checked by TLC: Qr-1 (0.96 g), Qr-2 (1.56 g), Qr-3 (15.34 g), and Qr-4 (2.34 g). LC-MS analyses of these fractions (Figure S1) confirmed that Qr-3 contained the crude triterpenoids, therefore it was further fractionated until the pure compounds were obtained. Fraction Qr-3 (750 mg ×20) was applied onto a 44.5 cm × 3.2 cm i.d. glass column (Millipore Corp., Bedford, MA), packed with Sephadex LH-20 (Sigma-Aldrich, Steinheim, Germany) and eluted with methanol at 1 mL/min. Seven further fractions were obtained

cell lines: human prostate cancer (PC3), estrogen dependent breast adenocarcinoma (MCF-7), and human normal lymphocytes. At the same time, we attempt to provide experimental data for the unequivocal identification of these metabolites in other matrices, such as beverages aged in oak barrels.

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MATERIALS AND METHODS

General Experimental Procedure. Optical rotations were measured on a P-2000 automatic digital polarimeter (JASCO, Tokyo, Japan). Melting point was obtained by differential scanning calorimetry using a DSC 204F1 Phoenix (NETZSCH GmbH, Selb, Germany). 1D and 2D NMR spectra (1H, 13C decoupled UDEFT,16 DEPT-135, HSQC, F2-coupled perfect CLIP-HSQC, HMBC, H2BC, 1 H−1H COSY DQF, TOCSY, HSQC-TOCSY, TROESY) were recorded on an Avance III HD Ascend-500 spectrometer (Bruker BioSpin, Rheinstetten, Germany), equipped with 5 mm 1 H {109Ag−31P} broad-band inverse (BBI) probe, in methanol-d4 at 30 °C. Exact masses, MS/MS fragmentation patterns, and molecular formulas were determined on an Impact II HD high-resolution quadrupole time-of-flight mass spectrometer (HR/Q-TOF/MS) (Bruker Daltonik GmbH, Bremen, Germany). The mass spectrometer was operated in the negative electrospray ionization mode using the following ion source parameters: capillary voltage set at 3.0 kV; nebulizer 1.8 bar; dry gas 8.0 L/min; drying temperature of 220 °C; and mass scan range set as m/z 50−2000. MS/MS spectra were acquired using variable collision energy in the range 35−45 eV. Data were collected and processed by the DataAnalysis 4.3 software (Bruker Daltonik GmbH, Germany). Semipreparative HPLC was performed on a chromatographic system equipped with a model 321 pump, a GX271 liquid handler with a 2 mL sample loop, and a Prep ELS II detector (Gilson, Middleton, WI), and either a semipreparative reversed phase column #1, 250 mm × 10 mm i.d., 5 μm, Atlantis Prep T3 (Waters, Milford, MA), or a column #2, 250 mm × 10 mm i.d., 5 μm, Cosmosil Cholester (Nacalai Tesque INC., Kyoto, Japan). Acetonitrile hypergrade for LC-MS and HPLC grade, and methanol HPLC grade were purchased from Merck (Darmstadt, Germany). Water was purified in-house with a Milli-Q water purification system (Millipore Co., Billerica, MA). Formic acid MS-grade, eluent additive for LC-MS, was obtained from Sigma-Aldrich (Steinheim, Germany). TLC were performed on silica gel RP-18 F254S plates (Merck, 4612

DOI: 10.1021/acs.jafc.7b01396 J. Agric. Food Chem. 2017, 65, 4611−4623

1.83b (2H)

1.53 (td, 12.6, 12.6, 5.0) 1.37 (dt, 13.7, 3.6, 3.6)

1.83b (2H)

1.50 (td, 12.3, 12.3, 5.3) 1.38 (dt, 12.8, 3.3, 3.3)

4613

24 25 26 27 28

17 18 19 20 21 22ax 22eq 23

12 13 14 15β 15α 16β 16α

10 11

8 9

7eq

6ax 6eq 7ax

1.05 (s) 0.78 (s) 1.31 (s)

1.02b; 1.76b 1.79b 1.67b 1.47 (s)

1.04 (s) 0.80 (s) 1.32 (s)

1.01b; 1.75b 1.78b 1.62b 1.47 (s)

3.06 (brs) 3.24 (d, 3.7)

1.64b 1.03b 1.62b 2.29 (td, 13.5, 12.8, 3.8)

1.68b 1.03b 1.73b 2.33 (td, 12.7, 12.1, 3.5)

3.06 (brs) 3.26 (d, 3.3)

1.98 (2H) (dd, 9.3, 3.5) 5.31 (t, 3.5, 3.5)

1.98 (2H) (dd, 9.7, 3.3) 5.33 (t, 3.3, 3.3)

1.81b

1.16b

1.15b

3 4 5

1.79b

2.12 (dd, 12.4, 5.0) 5.68 (ddd, 11.4, 10.1, 5.0) 3.25 (d, 10.3)

2.12 (dd, 12.4, 4.9) 5.68 (td, 10.8, 10.8, 4.9) 3.25 (d, 10.8)

1eq

2

1.12b

δH (multi, J in Hz)

1.12b

No.

1ax

2

1


1.02 (s) 0.79 (s) 1.34 (s)

1.01b; 1.77b 1.78b 1.63b 1.25 (s)

3.07 (brs) 3.27 (d, 3.8)

1.64b 1.03b 1.61b 2.31 (td, 13.2, 12.3, 3.6)

2.03 (2H) (dd, 9.5, 3.7) 5.34 (t, 3.7, 3.7)

1.84b

1.70b 1.86b 1.55 (ddd, 13.2, 13.1, 4.2) 1.36b

1.24b

1.08 (dd, 12.7, 11.2) 2.08 (dd, 12.7, 5.0) 4.59 (ddd, 11.2, 10.0, 5.0) 4.74 (d, 10.0)


3

0.92 (s) 0.78 (s) 1.31 (s)

1.01b; 1.75b 1.78b 1.62b 3.81 (d, 10.2); 4.02 (d, 10.2)

3.06 (brs) 3.26 (d, 3.8)

1.62b 1.01b 1.61b 2.29 (td, 13.2, 12.3, 3.7)

1.96 (ddd, 18.4, 10.4, 3.7); 2.03 (ddd, 18.4, 7.5, 3.7) 5.33 (t, 3.7)

0.93 (s) 0.76 (s) 1.30 (s)

1.01b; 1.76b 1.78b 1.66b 3.81 (d, 10.1); 4.01 (d, 10.1)

3.06 (brs) 3.27 (d, 3.8)

1.67b 1.01b 1.72b 2.32 (td, 13.4, 13.2, 3.8)

5.34 (t, 3.7)

2.00b (2H)

1.84 (dd, 10.4, 7.6)

1.31b

1.30b

1.85 (dd, 10.4, 7.5)

1.80b 1.54b 1.46 (brt, 12.6)

1.53b

4.27 (brs)

1.81b 1.57b 1.49 (td,12.9,12.6, 3.5)

1.55b

4.28 (d, 2.9)

4.23 (brd, 12.4)

1.62 (dd, 12.1, 4.0)

1.62b 4.22 (ddd, 11.9, 4.8, 2.9)

1.25 (t, 12.1)


5

1.26 (t, 11.9)


4

Table 1. 1H NMR Spectroscopic Data of Compounds 1−9 (in CD3OD).a 6

1.01 (s) 0.77 (s) 1.19 (s)

1.00b; 1.74b 1.77b 1.60b 4.45 (d, 11.1); 4.82b

3.04 (brs) 3.24 (d, 3.8)

1.58b 0.96b 1.57b 2.25 (td, 14.3,14.3, 4.1

5.31 (t, 3.6)

2.01b (2H)

1.79b

1.26b

1.38b

1.51 (dd, 10.4, 3.6) 1.74b (2H)

4.31 (ddd, 11.3, 9.8, 4.1) 3.45 (d, 9.8)

2.04b

0.97b


1.01b; 1.76b 1.77b 1.61b 3.79 (d, 11.1); 3.85 (d, 11.1) 9.93 (s) 0.88 (s) 0.74 (s) 1.31 (s)

3.06 (brs) 3.25 (d, 3.9)

1.62b 1.01b 1.60b 2.29 (td, 13.2, 12.4, 3.8)

5.32 (t, 3.4)

1.98b; 2.05b

1.86 (dd, 10.5, 7.3)

1.28b

1.34b 1.71b 1.57b

1.51 (dd, 12.3, 2.0)

4.09 (ddd, 11.5, 9.7, 4.7) 3.56 (d, 9.7)

2.00b

0.95b


7

1.00b; 1.74b 1.75b 1.60b 4.32 (d, 11.5); 4.77 (d, 11.8) 9.97 (s) 0.94 (s) 0.73 (s) 1.20 (s)

3.04 (brs) 3.24 (d, 3.7)

1.56b 0.95b 1.57b 2.25 (td, 14.3, 14.1, 3.7)

1.98 (ddd, 18.4, 10.9, 3.8); 2.06b 5.32 (t, 3.7)

1.87 (dd, 10.5, 7.3)

1.31b 1.67b 1.39 (td, 12.1, 12.1, 3.2) 1.25 (dt, 12.1, 2.8, 2.8)

1.57b

4.18 (ddd, 11.5, 9.7, 4.7) 3.62 (d, 9.7)

2.07 (dd, 11.5, 4.7)

1.03 (t, 11.5)


8

10.09 (s) 0.93 (s) 0.76 (s) 1.34 (s)

1.01b; 1.76b 1.77b 1.62b 1.11 (s)

3.07 (brs) 3.26 (d, 3.9)

1.64b 1.04b 1.62b 2.31 (td, 13.1, 12.1, 2.9)

5.34 (t, 3.7)

1.99b; 2.07b

1.90 (dd, 10.3, 7.4)

1.44b 1.81b 1.56 (td, 12.9, 12.9, 3.4) 1.35b

1.34b

2.10 (dd, 12.1, 4.8) 4.33 (ddd, 12.1, 10.1, 4.8) 4.90 (d, 10.1)

1.15 (t, 12.1)


9

Journal of Agricultural and Food Chemistry Article


a

No.

7.08 (s)

7.08 (s)

2-O-galloyl

0.93 (s) 0.95 (s)


0.93 (s) 0.94 (s) 28-O-Glc 5.39 (d, 8.1) 3.32 (dd, 8.1, 9.2) 3.41 (dd, 8.1, 8.9) 3.35b 3.35b 3.68 (dd, 12.1, 4.5) 3.82 (dd, 12.1, 1.6) 2-O-galloyl 7.14 (s)

3-O-galloyl

0.94 (s) 0.97 (s)


3

0.94 (s) 0.96 (s)


4

3.82 (brd, 11.4)

0.94 (s) 0.95 (s) 28-O-Glc 5.38 (d, 8.1) 3.32 (dd, 8.1, 8.9) 3.40b 3.35b 3.35b 3.68 (dd, 12.0, 4.2)


5

6

7.09 (s)

23-O-galloyl

0.93 (s) 0.95 (s)

δH (multi, J in Hz) 0.94 (s) 0.96 (s)


7

7.09 (s)

2-O-galloyl

0.93 (s) 0.95 (s)


8

7.07 (s)

2-O-galloyl

0.94 (s) 0.96 (s)


9

Assignments were confirmed by COSY DQF, 2D and 1D TOCSY, 2D and 1D TROESY, HSQC, HSQC-TOCSY, H2BC, HMBC, and DEPT-135 experiments. bOverlapped with other signals.

1″ 2″/6″ 3″/5″ 4″ 7″

6b′

1′ 2′ 3′ 4′ 5′ 6a′

29 30

2

1


Table 1. continued


4614


Article

Journal of Agricultural and Food Chemistry Table 2. 1H NMR Spectroscopic Data of Compounds 10−12 (in CD3OD)a

No. 1ax 1eq 2 3 4 5 6ax 6eq 7ax 7eq 8 9 10 11 12 13 14 15β 15α 16β 16α 17 18 19 20 21 22ax 22eq 23 24 25 26 27 28 29 30 1′ 2′ 3′ 4′ 5′ 6a′ 6b′

10

11

12




Unit A

Unit B

0.91b 1.98b 4.24 (td, 10.7, 10.5, 4.7 2.98 (d, 9.7)

1.41b 1.78b 4.42 (ddd, 12.3, 4.9, 2.5)

1.16 (dd, 10.5, 3.8) 1.81b (2H)

Unit A

Unit B

Unit A

Unit B

1.40b 1.78b 4.42 (ddd, 12.3, 4.9, 2.6)

1.28 (t, 12.4) 1.58 (dd, 12.4, 4.1) 4.13b

5.52 (d, 2.5)

0.90b 1.96b 4.27 (td, 10.6, 10.6. 4.7) 3.46 (d, 9.8)

5.55 (d, 2.6)

4.33 (d, 2.7)

1.37 (t, 12.7) 1.73b 4.45 (ddd, 12.7, 3.2, 2.5) 5.72 (d, 2.5)

1.40b

1.52b

1.42b

1.51b

1.54b

1.75b

1.48b 1.38b

1.84b 1.56b 1.44b 1.35b

1.52b 1.34b

1.84b 1.56b 1.44b 1.35b

2.03b 1.62b 1.40b 1.36b

1.84b 1.53b 1.51b 1.33b

1.77 (d, 9.0)

1.88 (dd, 10.7, 7.0)

1.80b

1.88 (dd, 10.6, 7.0)

1.83b

1.90 (dd, 9.7, 8.5)

1.99 (dd, 9.0, 3.7) 5.33 (t, 3.7)

1.98 (ddd, 18.2, 10.7, 3.8); 2.07 (ddd, 18.4, 7.0, 3.2) 5.35 (t, 3.7)

1.99b (2H)

1.99b (2H)

1.99b (2H)

5.32 (t, 3.6)

1.98 (ddd, 18.4, 10.6, 3.7); 2.07 (ddd, 18.4, 7.0, 3.3) 5.34 (t, 3.5)

5.31b

5.32b

1.69b 1.02b 1.74b 2.34b

1.68b 1.03b 1.69b 2.35b

1.70b 0.99b 1.68b 2.34b

1.68b 1.03b 1.74b 2.36b

1.73b 0.96b 1.63b 2.34b

1.68b 1.02b 1.74b 2.36b

3.08 (brs) 3.26 (d, 3.9)

3.06 (brs) 3.32 (d, 3.8)

3.08 (brs) 3.26 (d, 3.9)

3.06 (brs) 3.33 (d, 3.7)

3.10 (brs) 3.25 (d, 4.1)

3.06 (brs) 3.36 (d, 3.7)

1.02b; 1.77b 1.87b 1.59b 1.51 (s)

1.01b; 1.77b 1.78b 1.67b 3.36b; 4.06 (d, 11.0)

1.01b; 1.73b 1.89b 1.57b 3.92 (d, 11.0); 4.02 (d, 11.0)

1.01b; 1.76b 1.80b 1.68b 3.44b; 4.06 (d, 11.1)

1.01b; 1.74b 1.97b 1.49b 3.91 (d, 10.5); 4.09 (d, 10.5)

1.01b; 1.76b 1.78b 1.68b 3.63 (d, 11.3); 4.12 (d, 11.3)

1.04 (s) 0.75 (s) 1.30 (s)

0.97 (s) 0.79 (s) 1.33 (s)

1.07 (s) 0.75 (s) 1.30 (s)

0.97 (s) 0.79 (s) 1.34 (s)

0.97 (s) 0.75 (s) 1.29 (s)

0.96 (s) 0.78 (s) 1.34 (s)

0.94 (s) 0.97 (s) 28-O-Glc 5.40 (d, 8.0) 3.32b 3.40b 3.41b 3.32b 3.71 (dd, 11.9, 4.6) 3.79 (dd, 11.9, 2.6)

0.94 (s) 0.96 (s) 28-O-Glc 5.38 (d, 8.0) 3.32 (dd, 8.0, 9.1) 3.40b 3.35b 3.34b 3.68 (dd, 12.0, 3.8)

0.94 (s) 0.98 (s) 28-O-Glc 5.40 (d, 8.1) 3.32 (dd, 8.1, 9.3) 3.40b 3.43b 3.41b 3.72 (dd, 11.9, 4.3)

0.94 (s) 0.96 (s) 28-O-Glc 5.38 (d, 8.1) 3.32 (d, 8.1, 9.2) 3.41b 3.39b 3.35b 3.68 (dd, 12.1, 4.5)

0.94 (s) 0.99 (s) 28-O-Glc 5.42 (d, 8.0) 3.33 (dd, 8.0, 9.6) 3.39 (t, 9.0) 3.48 (t, 9.2) 3.29 (dt, 9.1, 3.0) 3.75 (d, 3.3) (2H)

0.94 (s) 0.97 (s) 28-O-Glc 5.38 (d, 8.1) 3.32 (dd, 8.1, 9.5) 3.41b 3.39b 3.35b 3.68 (dd, 11.9, 4.4)

3.82 (brd, 11.9)

3.79 (dd, 11.9, 2.5)

3.82 (dd, 12.1, 1.6)

3.82 (brd, 11.9)

a Assignments were confirmed by COSY DQF, 2D and 1D TOCSY, 2D and 1D TROESY, HSQC, HSQC-TOCSY, H2BC, HMBC, and DEPT-135 experiments. bOverlapped with other signals.

subfractions (Qr-3-1-A − Qr-3-1-C). Separation of Qr-3-1-B by HPLC in isocratic CH3CN/H2O/FA (36:64:0.2, v/v/v), 3 mL/min, using column #2 at 30 °C, gave pure compound 11 (6.8 mg). Separation of Qr-3-1-C under the same conditions as for Qr-3-1-B, but eluting with CH3CN/H2O/FA (39:61:0.2, v/v/v), gave compounds 10 (11.3 mg) and 12 (2.3 mg). Fractionation of Qr-3-2 (200 mg ×9) by LPLC under the same conditions as described for Qr-3-1, but eluting with

(Figure S2): Qr-3-1 (0.810 g), Qr-3-2 (1.95 g), Qr-3-3 (2.11 g), Qr-34 (2.68 g), Qr-3-5 (1.04 g), Qr-3-6 (0.65 g), and Qr-3-7 (2.47 g). Fraction Qr-3-1 (200 mg ×4) was fractionated by low pressure liquid chromatography (LPLC), on a 29.0 cm × 1.6 cm i.d. glass column (Millipore Corp., Bedford, MA) packed with 25-40 μm LiChroprep RP-18 (30 g) (Merck, Darmstadt, Germany), and eluted with 70% aq. MeOH containing 0.2% formic acid (FA) at 1 mL/min, to afford three 4615


Article

Journal of Agricultural and Food Chemistry 60% aq. MeOH (0.2% FA), gave four subfractions (Qr-3-2-A − Qr-32-D). Separation of Qr-3-2-B by HPLC in isocratic CH3CN/H2O/FA (25:75:0.2, v/v/v), at 3 mL/min, using column #1 at 30 °C, yielded compound 5 (21.0 mg). Isolation of Qr-3-2-C in HPLC under the same conditions as for Qr-3-2-B, but eluting with CH3CN/H2O/FA (30:70:0.2, v/v/v), gave compound 15 (20.5 mg). Fractionation of Qr3-3 (250 mg ×4) by LPLC under the same conditions as described for Qr-3-1, gave two subfractions (Qr-3-3-A and Qr-3-3-B). Separation of Qr-3-3-B by HPLC in isocratic CH3CN/H2O/FA (45:55:0.2, v/v/v), 3 mL/min, using column #1 at 30 °C, gave compound 14 (29.9 mg). Fractionation of Qr-3-4 (300 mg ×8) by LPLC under identical conditions as previously described for Qr-3-2, gave five subfractions (Qr-3-4-A − Qr-3-4-E). Subsequent HPLC separation of Qr-3-4-B, using identical conditions as in Qr-3-2-C, afforded compounds 16 (31.4 mg) and 1 (15.2 mg). Separation of Qr-3-4-C by HPLC, eluting with CH3CN/H2O/FA (33:67:0.2, v/v/v) and the rest of condition identical to those used for Qr-3-1-B, gave compounds 17 (4.3 mg), 13 (22.7 mg), 6 (3.1 mg), and 7 (6.2 mg). The HPLC isolation of Qr-3-4D, using MeOH/H2O/FA (60:30:0.2, v/v/v) and the rest of conditions the same as described for Qr-3-4-C, yielded compound 4 (49.4 mg). Fractionation of Qr-3-5 (200 mg ×5) by LPLC using 65% aq. MeOH (0.2% FA), yielded five subfractions (Qr-3-5-A − Qr-3-5E). Pure compound 3 (144.7 mg) was the only one component of fraction Qr-3-5-D. Compounds 2 (29.6 mg) and 9 (5.7 mg) were obtained after purification of Qr-3-5-C by HPLC, using CH3CN/ H2O/FA (45:65:0.2, v/v/v) and the rest of conditions identical to those used for purification of Qr-3-1-B. Finally, fractionation of Qr-3-6 (200 mg ×3) by LPLC, eluting with a gradient from 60% to 70% aq. MeOH (0.2% FA), afforded five subfractions (Qr-3-6-A − Qr-3-6-E). Compound 8 (24.5 mg) was then obtained after purification of Qr-36-C by HPLC, eluting with CH3CN/H2O/FA (40:60:0.2, v/v/v), and the rest of conditions equal to those used for purification of Qr-3-1-B. Characteristic Data of Compounds. 2-O-Galloyl bartogenic acid 28-O-β-D-glucopyranosyl ester (1) (15.2 mg). White amorphous powder; [α]21 D + 21.2 (c 0.11, MeOH); HR-ESI-MS (neg.) m/z 831.3796 [M − H]− (calcd for C43H59O16, 831.3809); HR-MS/MS (% of base peak) give diagnostic fragment ions at m/z 831.3785 (4) [M − H]−, 679.3684 (11) [M − H − 152]− (C36H55O12), 517.3164 (6) [M − H − 152 − 162]− (C30H45O7), 169.0142 (100) [M − H − 662]− (C7H5O5), 125.0244 (18) [M − H − 662 − 44]− (C6H5O3); 1H and 13 C NMR spectroscopic data (Tables 1 and 3). 2-O-Galloyl bartogenic acid (2) (29.6 mg). White amorphous powder; [α]21 D + 17.3 (c 0.11, MeOH); HR-ESI-MS (neg.) m/z 669.3259 [M − H]− (calcd for C37H49O11, 669.3280); HR-MS/MS (% of base peak) give diagnostic fragment ions at m/z 669.3248 (3) [M − H]−, 517.3149 (22) [M − H − 152]− (C30H45O7), 169.0133 (100) [M − H − 500]− (C7H5O5), 125.0237 (21) [M − H − -500 − 44]− (C6H5O3); 1H and 13C NMR spectroscopic data (Tables 1 and 3). 3-O-Galloyl bartogenic acid (3) (144.7 mg). Pale yellow amorphous powder; [α]21 D + 33.8 (c 0.10, MeOH); mp 251.6 °C (dec); HR-ESIMS (neg.) m/z 669.3270 [M − H]− (calcd for C37H49O11, 669.3280); HR-MS/MS (% of base peak) give a diagnostic fragment ion at m/z 517.3162 (100) [M − H − 152]− (C30H44O7); 1H and 13C NMR spectroscopic data (Tables 1 and 3). 2α,3α,19α,23-Tetrahydroxyolean-12-en-24,28-dioic acid (roburgenic acid, 4) (49.4 mg). White amorphous powder; [α]21 D + 58.6 (c 0.10, MeOH); HR-ESI-MS (neg.) m/z 533.3119 [M − H]− (calcd for C30H45O8, 533.3120); HR-MS/MS (% of base peak) give diagnostic fragment ions at m/z 533.3112 (0.2) [M − H]−, 485.2906 (100) [M − H − 48]− (C29H41O6), 471.3112 (27) [M − H − 62]− (C29H43O5), 453.3009 (30) [M − H − 62-18]− (C29H41O4); 1H and 13C NMR spectroscopic data (Tables 1 and 3). Roburgenic acid 28-O-β-D-glucopyranosyl ester (5) (21.0 mg). White amorphous powder; [α]21 D + 44.5 (c 0.10, MeOH); HR-ESI-MS (neg.) m/z 695.3648 [M − H]− (calcd for C36H55O13, 695.3648); HRMS/MS (% of base peak) give diagnostic fragment ions at m/z 647.3432 (9) [M − H − 48]− (C35H51O11), 527.3013 (16) [M − H − 168]− (C31H43O7), 485.2907 (100) [M − H − 48 − 162]− (C 29 H 41 O 6 ), 471.3114 (40) [M − H − 48 − 162 − 14] − (C29H43O5); 1H and 13C NMR spectroscopic data (Tables 1 and 3).

23-O-Galloyl 2α,3β,19α,23-tetrahydroxyolean-12-en-24,28-dioic acid (6) (3.1 mg). White amorphous powder; [α]21 D + 41.3 (c 0.11, MeOH); HR-ESI-MS (neg.) m/z 685.3220 [M − H]− (calcd for C37H49O12, 685.3230); HR-MS/MS (% of base peak) give diagnostic fragment ions at m/z 533.3112 (29) [M − H − 152]− (C30H45O8), 485.2901 (25) [M − H − 152-48]− (C29H41O6), 169.0140 (100) [M − H − 516]− (C7H5O5), 125.0243 (37) [M − H − 516 − 44]− (C6H5O3); 1H and 13C NMR spectroscopic data (Tables 1 and 3). 2α,3β,19α,23-Tetrahydroxy-24-oxo-olean-12-en-28-oic acid (robural A, 7) (6.2 mg). White amorphous powder; [α]21 D + 42.6 (c 0.10, MeOH); HR-ESI-MS (neg.) m/z 517.3165 [M − H]− (calcd for C30H45O7, 517.3171); HR-MS/MS (% of base peak) give diagnostic fragment ions at m/z 517.3165 (12) [M − H]−, 451.2843 (48) [M − H − 66]− (C29H39O4), 407.2950 (100) [M − H − 66 − 44]− (C28H39O2); 1H and 13C NMR spectroscopic data (Tables 1 and 3). 23-O-Galloyl robural A (8) (24.5 mg). White amorphous powder; [α]21 D + 19.1 (c 0.11, MeOH); HR-ESI-MS (neg.) m/z 669.3278 [M − H]− (calcd for C37H49O11, 669.3280); HR-MS/MS (% of base peak) give diagnostic fragment ions at m/z 669.3274 (58) [M − H]−, 517.3154 (0.4) [M − H − 152]− (C30H45O7), 469.2958 (3) [M − H − 152-48]− (C29H41O5), 169.0144 (100) [M − H − 500]− (C7H5O5), 125.0245 (13) [M − H − 500 − 44]− (C6H5O3); 1H and 13C NMR spectroscopic data (Tables 1 and 3). 3-O-Galloyl 2α,3β,19α-trihydroxy-24-oxo-olean-12-en-28-oic acid (robural B, 9) (5.7 mg). White amorphous powder; [α]21 D + 35.1 (c 0.10, MeOH); HR-ESI-MS (neg.) m/z 653.3335 [M − H]− (calcd for C37H49O10, 653.3331); HR-MS/MS (% of base peak) give diagnostic fragment ions at m/z 653.3327 (100) [M − H]−, 501.3225 (0.8) [M − H − 152]− (C30H45O6), 169.0143 (4) [M − H − 484]− (C7H5O5), 125.0236 (0.8) [M − H − 484 − 44]− (C6H5O3); 1H and 13C NMR spectroscopic data (Tables 1 and 3). Bartogenic acid [24-O-(28-O-β-D-glucopyranosyl roburgenic acid)] 28-O-β-D-glucopyranosyl ester (roburoside A, 10) (11.3 mg). White amorphous powder; [α]21 D + 36.9 (c 0.11, MeOH); HR-ESI-MS (neg.) m/z 1357.7285 [M − H]− (calcd for C72H109O24, 1357.7314); HRMS/MS (% of base peak) give diagnostic fragment ions at m/z 1357.7256 (2) [M − H]− , 1313.7363 (1) [M − H − 44] − (C71H108O22), 1195.6744 (0.2) [M − H − 162]− (C66H99O19), 679.3691 (100) [M − H − 162 − 516]− (C36H55O12), 517.3166 (1) [M − H − 162 − 516 − 162]− (C30H45O7); 1H and 13C NMR spectroscopic data (Tables 2 and 3). 23-Hydroxy bartogenic acid [24-O-(28-O-β-D-glucopyranosyl roburgenic acid)] 28-O-β-D-glucopyranosyl ester (roburoside B, 11) (6.8 mg). White amorphous powder; [α]21 D + 38.1 (c 0.11, MeOH); HRESI-MS (neg.) m/z 1373.7248 [M − H]− (calcd for C72H109O25, 1373.7263); HR-MS/MS (% of base peak) give diagnostic fragment ions at m/z 1373.7220 (0.7) [M − H]−, 1329.7296 (0.4) [M − H − 44]− (C71H108O23), 1211.7621 (0.2) [M − H − 162]− (C66H99O20), 695.3640 (100) [M − H- − 162-516]− (C36H55O13), 533.3107 (0.3) [M − H − 162 − 516 − 162]− (C30H45O8); 1H and 13C NMR spectroscopic data (Tables 2 and 3). Roburgenic acid [24-O-(28-O-β-D-glucopyranosyl roburgenic acid)] 28-O-β-D-glucopyranosyl ester (roburoside C, 12) (2.3 mg). White amorphous powder; [α]21 D + 27.3 (c 0.11, MeOH); HR-ESI-MS (neg.) m/z 1373.7221 [M − H]− (calcd for C72H109O25, 1373.7263); HRMS/MS (% of base peak) give diagnostic fragment ions at m/z 1373.7220 (0.9) [M − H]−, 1329.7296 (0.4) [M − H − 44]− (C71H108O23), 1211.7621 (0.3) [M − H − 162]− (C66H99O20), 695.3640 (100) [M − H − 162-516]− (C36H55O13), 533.3107 (0.1) [M − H − 162 − 516 −162]− (C30H45O8); 1H and 13C NMR spectroscopic data (Tables 2 and 3). Cell Culture and Cytotoxicity. Two adherent human tumor cell lines: PC3 and MCF-7 supplied by ATCC (Rockville, Manassas, VA), were used in the MTT assay. Human lymphocytes were also used as a model for normal cells. The cancer cells were cultured in an appropriate culture media (F-12K, DMEM) with glutamine, 10% FBS, penicillin/streptomycin 1%, 37 °C, 5% CO2, and 95% air. The human lymphocytes were separated from leucocyte buffy-coat taken from blood collection of Blood Bank in Łódź, Poland. Blood comes from healthy, nonsmoking donors (aged 20-25) with no signs of infection 4616


No.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″/ 6″ 3″/ 5″

122.1, C 110.2, CH

146.4, C

45.6, CH2 73.2, CH 81.3, CH 51.2, C 57.3, CH 21.4, CH2 33.8, CH2 40.8, C 48.6, CH 40.0, C 24.9, CH2 124.7, CH 144.4, C 42.7, C 29.4, CH2 28.5, CH2 47.1, C 45.1, CH 82.4, CH 35.9, C 29.5, CH2 33.3, CH2 24.6, CH3 180.1, C 14.9, CH3 17.6, CH3 24.8, CH3 178.5, C 28.5, CH3 25.2, CH3 95.8, CH 74.0, CH 78.3, CH 71.1, CH 78.7, CH 62.4, CH2 122.1, C 110.2, CH

146.4, C

45.6, CH2 73.1, CH 81.3, CH 51.2, C 57.3, CH 21.3, CH2 33.9, CH2 40.7, C 48.6, CH 40.0, C 24.9, CH2 124.7, CH 144.6, C 42.7, C 29.4, CH2 28.6, CH2 46.7, C 45.2, CH 82.4, CH 36.0, C 29.5, CH2 34.0, CH2 24.6, CH3 180.1, C 14.8, CH3 17.5, CH3 24.9, CH3 182.2, C 28.6, CH3 25.1, CH3

2

δC, type

1

δC, type

4617

146.3, C

121.9, C 110.5, CH


δC, type

3


δC, type

4

42.8, CH2 67.1, CH 70.7, CH 54.9, C 45.9, CH 21.0, CH2 33.7, CH2 40.7, C 48.4, CH 39.9, C 25.0, CH2 124.9, CH 144.4, C 42.8, C 29.4, CH2 28.5, CH2 47.1, C 45.1, CH 82.4, CH 35.9, C 29.5, CH2 33.3, CH2 67.5, CH2 178.8, C 14.9, CH3 17.7, CH3 24.9, CH3 178.6, C 28.6, CH3 25.2, CH3 95.8, CH 73.9, CH 78.3, CH 71.1, CH 78.7, CH 62.4, CH2

δC, type

5

146.7, C

121.3, C 110.2, CH


δC, type

6

7 δC, type 47.0, CH2 69.5, CH 76.9, CH 60.2, C 50.6, CH 19.8, CH2 33.3, CH2 40.5, C 48.0, CH 39.2, C 25.4, CH2 124.6, CH 144.8, C 42.8, C 29.4, CH2 28.6, CH2 46.7, C 45.2, CH 82.4, CH 36.0, C 29.5, CH2 34.0, CH2 61.2, CH2 207.6, C 17.8, CH3 17.7, CH3 25.0, CH3 182.2, C 28.6, CH3 25.1, CH3

Table 3. 13C NMR Spectroscopic Data of Compounds 1−12 (in CD3OD)a 8

146.7, C

121.3, C 110.1, CH


δC, type

9

146.5, C

121.5, C 110.3, CH


δC, type 48.0, CH2 69.0, CH 84.7, CH 52.8, C 58.0, CH 21.9, CH2 33.8, CH2 40.9, C 48.6, CH 39.8, C 25.1, CH2 124.9, CH 144.4, C 42.6, C 29.3, CH2 28.4, CH2 47.1, C 45.1, CH 82.3, CH 36.0, C 29.3, CH2 33.4, CH2 26.2, CH3 178.8, C 16.7, CH3 17.8, CH3 25.1, CH3 178.7, C 28.6, CH3 25.1, CH3 95.9, CH 73.9, CH 78.5, CH 71.1, CH 78.7, CH 62.4, CH2

Unit A

10

Unit B 44.3, CH2 66.2, CH 76.6, CH 55.3, C 47.9, CH 21.0, CH2 34.1, CH2 40.8, C 49.0, CH 39.9, C 24.9, CH2 124.8, CH 144.5, C 42.8, C 29.3, CH2 28.4, CH2 47.2, C 45.2, CH 82.6, CH 36.0, C 29.6, CH2 33.2, CH2 66.7, CH2 177.3, C 15.2, CH3 17.6, CH3 25.2, CH3 178.6, C 28.5, CH3 25.2, CH3 95.8, CH 73.9, CH 78.4, CH 71.1, CH 78.7, CH 62.4, CH2

δC, type

11


Unit A 44.4, CH2 66.4, CH 76.9, CH 55.2, C 48.1, CH 21.0, CH2 34.1, CH2 40.7, C 49.0, CH 39.9, C 24.9, CH2 124.7, CH 144.6, C 42.8, C 29.3, CH2 28.4, CH2 47.3, C 45.2, CH 82.7, CH 36.1, C 29.7, CH2 33.3, CH2 67.0, CH2 177.3, C 15.1, CH3 17.6, CH3 25.4, CH3 178.6, C 28.5, CH3 25.3, CH3 95.8, CH 74.0, CH 78.4, CH 71.1, CH 78.7, CH 62.4, CH2

Unit B

δC, type

12


Unit A


Unit B

δC, type



Article

Unit A

Unit B

Unit A

Unit B

disease during collection. The use of human blood (only leucocyte buffy-coat) was approved by the Bioethics Committee for Scientific Investigation, University of Łódź (agreement no. KBBN-UŁ/I/2015). Lymphocytes were suspended in the RPMI 1640 culture medium with inactivated FBS (15%), penicillin/streptomycin (1%), and PHA (1%). The anticancer drugs oxaliplatin and cisplatin, were included in the test as positive controls. The cancer cells and human lymphocytes were treated with compounds 1−17 for 24 h on 96-well microplates. At the end of the incubation, fresh MTT solution was added to each microplate well and the cells were incubated for 4 h, at 37 °C, and 5% CO2. Then, MTT solution was discarded and 100 μL DMSO/well was added in order to dissolve formazan crystals formed during the reduction of MTT by mitochondrial dehydrogenases of metabolically active cells. In the case of lymphocytes this was achieved by adding 100 μL mixture of 20% SDS and 50% DMF to each well for 24 h. Absorbance was measured spectrophotometrically using a microplate reader at 570 nm. The cytotoxicity of compounds was expressed as IC50 values (concentration resulting in 50% cells growth inhibition), which were calculated by the GraphPad Prism 6.0 software. The results are presented as the mean ± SEM of the replicates from six independent experiments.

■

Assignments were confirmed by HSQC, HMBC, and DEPT-135 experiments. a

140.1, C 167.9, C 139.7, C 168.9, C 139.7, C 168.6, C 139.7, C 168.6, C No.

4″ 7″

9

139.9, C 168.3, C 140.0, C 168.1, C

δC, type

8 7

RESULTS AND DISCUSSION

Identification of Compounds. After multiple chromatographic procedures of the EtOAc soluble fraction of the methanol extract obtained from the heartwood of Q. robur, 12 new (1−12) and five known (13−17) triterpenoids were isolated (Figures 1 and 2). Based on comparison with the NMR and MS data previously reported in the literature, the known compounds were identified as 23-hydroxybartogenic acid (13),17 bartogenic acid (14),18 bartogenic acid 28-O-β-Dglucopyranosyl ester (15),17 3-O-galloyl bartogenic acid 28-Oβ-D-glucopyranosyl ester (16),19 and bellericagenin B (17).20 Compound 1, an amorphous white powder, exhibited a deprotonated molecular ion at m/z 831.3796 in the HR-ESIMS spectrum, in agreement with a molecular formula of C43H59O16. The negative ESI-MS/MS spectrum of 1 displayed neutral losses of galloyl (152 Da) and hexosyl (162 Da) units, to produce fragment ions at m/z 679.3684 and m/z 517.3164, respectively. The molecular formula for the latter ion was calculated as C30H45O7, which suggested a triterpenoid ion. 1H and 13C NMR data of 1 (Tables 1 and 3) showed features common with those previously described for compound 15;17 though, important differences in ring A were observed. The most relevant resonance signals owing to the triterpenoid moiety in the 1H NMR spectrum of 1 (Table 1) comprised six singlets corresponding to tertiary methyl groups [δ 0.78 (3H26), 0.93 (3H-29), 0.94 (3H-30), 1.05 (3H-25), 1.31 (3H-27), and 1.47 (3H-23)], three oxymethines [δ 3.25 (d, H-3), 3.26 (d, H-19), and 5.68 (td, H-2)], and an olefinic proton at δ 5.33 (t, H-12). This was confirmed based on correlations observed for these protons in an HSQC experiment, as well as on 13C NMR and DEPT-135 spectra. In this way, the carbon resonances for six methyls [δ 14.9 (C-25), 17.6 (C-26), 24.6 (C-23), 24.8 (C-27), 25.2 (C-30), and 28.5 (C-29)], eight methylenes, seven methines [including an olefinic methine at δ 124.7 (C-12) and three oxymethines at δ 73.2 (C-2), 81.3 (C3), and 82.4 (C-19)], and nine quaternary carbons [including an olefinic carbon at δ 144.4 (C-13) and two carboxylic carbons at δ 178.5 (C-28) and 180.1 (C-24)] were observed. These spectroscopic data together with a careful analysis of the correlations in 2D HMBC and 2D TROESY spectra, allowing for determination of position, orientation, and relative configuration of substituents, supported 1 as having a

δC, type δC, type

6 5

δC, type δC, type

4 3

δC, type

2

δC, type

1

δC, type

Table 3. continued

δC, type

Unit A

δC, type

10

Unit B

12

δC, type

11

δC, type


4618


Article


Figure 2. Structures of triterpenoids 1−17 isolated from heartwood of Q. robur.

were acquired by selective excitation of the anomeric proton, which confirmed a spin system typical for a β-glucopyranosyl unit. Based on the HSQC-TOCSY and H2BC spectra of 1, the carbon signals for the glucose unit were assigned. Finally, the linkage site with the triterpenoid moiety was determined to be through an ester bond at C-28, due to the three-bond HMBC correlation observed between the anomeric proton and the carbon at δ 178.5. After acid hydrolysis of 1, and following a previously reported methodology,21 the absolute configuration of the glucose was determined as D. Based on the above evidence, the structure of 1 was elucidated as 2-O-galloyl bartogenic acid 28-O-β-D-glucopyranosyl ester. The occurrence of this compound has recently been inferred from toasted wood of Q. robur;22 however, its final chemical structure and substituent positions were not determined. Compounds 2 and 3 exhibited identical ions for their deprotonated molecules (calcd for C37H49O11) in HR-ESI-MS spectra, corresponding to 162 Da (hexose unit) less than compound 1. Indeed, the ESI-MS/MS spectra of 2 and 3 only

pentacyclic triterpenoid skeleton as 2α,3β,19α-trihydroxyolean12-en-24,28-dioic acid, better known as bartogenic acid.18 According to the ESI-MS/MS spectrum of 1, galloyl and hexosyl units were connected to its triterpenoid moiety. The galloyl unit was confirmed by the singlet signal integrated for two protons, observed downfield in the 1H NMR spectrum of 1 at δ 7.08 (H-2″/6″) and by its HSQC correlation with carbon at δ 110.2 (C-2″/6″). In addition, three signals of aromatic quaternary carbons [δ 122.1 (C-1″), 139.7 (C-4″), and 146.4 (C-3″/5″)], together with an esterified carboxylic carbon at δ 168.6 (C-7″), finally supported the presence of a galloyl unit. The C−H long-range correlation shown by the HMBC spectrum between this carboxylic carbon and H-2 (δ 5.68) of the triterpenoid moiety revealed the galloyl moiety to be linked through an ester bond at the C-2 position. On the other hand, the 1H NMR spectrum of 1 also exhibited an anomeric signal at δ 5.39 (d, H-1′). The nature of such a sugar unit was determined by a combination of 1H−1H COSY DQF, 1D TOCSY, and 1D TROESY spectra. The latter two experiments 4619


Article


and it was named roburgenic acid 28-O-β-D-glucopyranosyl ester. The fragmentation of compound 6 in the ESI-MS/MS gave a diagnostic ion at m/z 533.3112 [M − H − 152]−. This suggested a galloyl substitution, as well as a triterpenoid moiety with an ion formula of C30H45O8, isobaric with compound 13. 1 H and 13C NMR data for the triterpenoid moiety of compound 6 (Tables 1 and 3) were very similar to those of 13, confirming the same triterpenoid skeleton. However, the chemical shifts for protons at H-23 were downfield shifted because of substitution with a galloyl residue. Such a link was established by the HMBC correlations between the carboxylic carbon at C-7″ (δ 167.9) and the two protons at C-23 (δ 4.45; 4.82). Consequently, the structure of 6 was established as 23-Ogalloyl 2α,3β,19α,23-tetrahydroxyolean-12-en-24,28-dioic acid. According to the HR-ESI-MS spectrum of compound 7, the molecular formula of its deprotonated molecule was calculated as C30H45O7, the same as for bartogenic acid, 14. Despite this, 1 H and 13C NMR data for both compounds differed considerably (Tables 1 and 3). The majority of variation appeared in ring A of compound 7, especially a new resonance signal at δ 9.93 in its 1H NMR spectrum, typical for an aldehyde proton. This was confirmed by the HSQC correlation observed between this proton and the carbon at δ 207.6. Long range correlations for the aldehyde proton in the HMBC spectrum of 7 with C-4 (δ 60.2), C-23 (δ 61.2), C-5 (δ 50.6), and C-3 (δ 76.9) suggested the location for the formyl group at C-24 instead of a carboxylic group. This position was confirmed by TROESY correlations of H-24 with the methyl at H-25 (δ 0.88) and H-2 (δ 4.09). The structure of 7 was therefore established as 2α,3β,19α,23-tetrahydroxy-24-oxo-olean-12-en28-oic acid, which we named robural A. The NMR data for compounds 8 and 7 showed similarities, except for the upfield shifting of the two protons at C-23 in 8, and additional signals typical for a galloyl unit (Tables 1 and 3). HMBC correlation between the carboxylic carbon at C-7″ (δ 168.1) of the galloyl unit and C-23 (δ 62.2) of the triterpenoid moiety indicated that 8 is the 23-O-galloyl derivative of 7. The ESI-MS/MS spectrum of 8 confirmed this, since a diagnostic fragment ion was observed at 517.3154 [M − H − 152]− (C30H45O7), corresponding to the neutral loss of a galloyl unit. Thus, the structure of 8 was established as 23-O-galloyl robural A. The HR-ESI-MS spectrum of compound 9 showed a molecular weight different from those of all aforementioned compounds [m/z 653.3335, [M − H]−], consistent with a formula of C37H49O10. Its ESI-MS/MS spectrum showed a diagnostic fragment ion at m/z 501.3225 [M − H − 152]−, suggesting, as demonstrated above, a galloyl substituent. Like compounds 7 and 8, the 1H NMR spectrum of 9 also showed the aldehyde proton signal at δ 10.09 (H-24), while typical signals for the 23-hydoxymethylene group were absent. Instead, an additional singlet corresponding to a methyl group at δ 1.11 (H-23) was observed. Based on the HMBC correlation between H-3 (δ 4.90) and C-7″ (δ 168.3), the position for the galloyl substitution was determined to be at C-3 of the triterpenoid moiety. The rest of NMR signals corresponding to rings C−E were almost superimposable with those of 7 and 8. Thus, the structure of compound 9 was established as 3-Ogalloyl 2α,3β,19α-trihydroxy-24-oxo-olean-12-en-28-oic acid, and it was named robural B. Compound 10 presents the molecular formula C72H109O24, according to its HR-ESI-MS, 13C NMR (Table 2), and DEPT-

showed a neutral loss of a galloyl unit (152 Da). The NMR data of compound 2 was almost superimposable with that of 1. Only slight upfield shifts for protons at H-16 and H-22 of 2 were observed, probably caused by the absence of a glucose unit at C-28. Consistent with the lack of esterification for the carboxylic group at this position, the chemical shift of C-28 appeared 3.7 ppm shifted downfield (δ 182.2) compared to 1. The main differences in NMR data between 2 and 3 were due to different substitution positions of the galloyl unit, confirmed to be at C-3 for compound 3, which was visible as a long-range correlation in the HMBC spectrum between H-3 (δ 4.74) and C-7″ (δ 168.9). The structures of compounds 2 and 3 were therefore established as 2-O-galloyl bartogenic acid and 3-Ogalloyl bartogenic acid, respectively. The deprotonated ion at m/z 533.3119 [M − H]− (calcd for C30H45O8) in the HR-ESI-MS spectrum of compound 4 indicated an isobaric compound of 23-hydroxybartogenic acid (13), previously described in Q. robur and Q. petraea.17 The ESI-MS/MS spectrum of 4 showed the same fragment ions as that of 13, but with the intensities of a few ions changed. The most remarkable difference was observed for the fragment ion at m/z 471.3112 (C29H43O5), corresponding to the neutral losses of H2O and CO2 (62 Da), which were almost half as intense in 4 as in 13. This may occur by the combined cleavages of the carboxylic group at C-24 not only with the hydroxyl group at C-23, but also with that at C-3. When the hydroxyl group at C-3 is located toward the “α” plane of the molecule, the abundance of this fragment ion should be lower than that of its corresponding “β” isomer. From the comparison of 1H and 13C NMR data between compounds 4 (Tables 1 and 3) and 13, the main inconsistences were observed for the chemical shifts and coupling constant of ring A. Specifically, the multiplicity for the resonance signal of H-2 in 4 indicated only one proton−proton trans-diaxial coupling (11.9 Hz) and two axial−equatorial couplings (4.8, 2.9 Hz). Consistent with this, the coupling constant for H-3 (d, 2.8 Hz) also indicated its equatorial disposition. Considering that the direct C−H coupling constant in the cyclohexane ring depends also on the stereochemistry, being slightly smaller for axial C−H than for equatorial (ΔJ = 4.0 Hz, typically), and increasing with the presence of electron withdrawing groups,23 the F2-coupled perfect CLIP-HSQC spectra for both compounds 4 and 13 were acquired. This experiment allowed accurate measurement of the direct coupling constant in C-3, which was 149.8 Hz for compound 4 and 143.6 Hz for 13 (ΔJ = 6.2 Hz). This may become a rapid and efficient method for distinguishing between both configurations within these types of metabolite. Compound 4 was then elucidated as 2α,3α,19α,23-tetrahydroxyolean-12-en-24,28-dioic acid, which we named roburgenic acid. It is worth noting that the “α” orientation for the hydroxyl group at C-3 of the oleanane skeleton is quite rare, and it is reported here for the first time within the Quercus genus. The HR-ESI-MS spectrum of compound 5 exhibited its deprotonated ion at m/z 695.3648, in agreement with a molecular formula of C36H55O13. This indicated an additional hexose unit compared to compound 4. The 1H and 13C NMR spectra of 5 showed resonances for an anomeric proton and carbon at δH 5.38 and δC 95.8 (Tables 1 and 3). The rest of the signals were almost superimposable with those of 4, except for the carboxylic carbon at C-28, which resonated 3.7 ppm upfield, due to acetylation with the glucose unit. Thus, the structure of compound 5 was determined as a 28-O-glucosyl derivative of 4, 4620


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Cytotoxicity Evaluation. All the isolated compounds (1− 17) were tested for their in vitro cytotoxic activity against human prostate cancer (PC3), and human estrogen dependent breast adenocarcinoma (MCF-7) cell lines. Normal lymphocytes derived from human peripheral blood were also included. The obtained IC50 values are shown in Table 4. Despite the

135 data. Such a number of carbons and degrees of unsaturation (rings plus double bonds equal to 18) are consistent with a dimeric triterpenoid saponin.24 In accordance, two groups of 1H and 13C NMR signals were distinguished to be correlated to two different triterpenoid units, denoted as A and B (Tables 2 and 3). Unit A was elucidated as bartogenic acid 28-O-β-D-glucopyranosyl ester, by the comparison of its NMR data with that previously reported for compound 15.17 The main difference was given by the slight upfield shifting of the 24-carboxylic carbon signal, from δ 180.5 in 15 to δ 178.8 in 10, indicating acylation. This was confirmed by a clear HMBC correlation observed between C-24 and the proton at δ 5.52 (H-3, unit B), which was in turn evidence of its connection with unit B. The 1H and 13C NMR data of unit B, on the other hand, exhibited features similar to those of compound 5 (Tables 1, 2, and 3). Naturally, the chemical shifts for ring A were slightly modified, due to the substitution at C-3 in the case of 10. Thus, the structure of compound 10 was established as bartogenic acid [24-O-(28-O-β-D-glucopyranosyl roburgenic acid)] 28-O-β-D-glucopyranosyl ester, which we named roburoside A. This is the first report of a dimeric triterpenoid saponin from Quercus spp. The deprotonated molecule of compound 11 displayed at m/ z 1373.7248 in its HR-ESI-MS spectrum suggested a molecular formula of C72H109O25. This indicated an additional oxygen atom compared to compound 10. The ESI-MS/MS spectrum of 11 showed neutral losses of two hexose units, suggesting a dimeric triterpenoid saponin similar to 10. 1H and 13C NMR data for unit A of 11 (Tables 2 and 3) afforded some analogies with that of 13. Nonetheless, differences in the 13C NMR chemical shifts between 11 and 13 were noticeable, especially for C-25 (Δδ 1.9 ppm), C-24 (Δδ −1.1 ppm), and C-28 (Δδ −3.5 ppm). This is a consequence of an esterification of carboxylic carbons at C-24 and C-28 in 11, which was confirmed by two long-range correlations between C-24 (δ 177.2) and H-3 of unit B (δ 5.55), and between C-28 (δ 178.7) and the anomeric proton at δ 5.40 (H-1′). This indicated that unit A of compound 11 is 23-hydroxy bartogenic acid 28-O-β17 D-glucopyranosyl ester. Unit B of 11 was elucidated to be the same as that of compound 10, due to perfect matching of their 1 H and 13C NMR data (Tables 2 and 3). Thus, the dimeric structure of compound 11 was established as 23-hydroxybartogenic acid [24-O-(28-O-β-D-glucopyranosyl roburgenic acid)] 28-O-β-D-glucopyranosyl ester and was named roburoside B. The calculated molecular formula for compound 12 as well as its fragmentation pattern obtained by HR-ESI-MS and ESIMS/MS spectra were identical to those of compound 11. The 13 C NMR data for its unit A was almost superimposable with that of 5 (Table 3), displaying the main differences in the chemical shifts of C-24 (Δδ −1.7 ppm) and C-25 (Δδ 1.7 ppm). As stated above, this is likely to be caused by the acylation of the carboxylic group at C-24. Such functionality was confirmed by the HMBC correlation, between the C-24 (δ 177.1) and H-3 (δ 5.72) of unit B. The structure of unit A was therefore determined as roburgenic acid 28-O-β-D-glucopyranosyl ester. Likewise, the structure of unit B was confirmed to be the same as that of unit A, after the comparison of its NMR data with that of unit B of compounds 10 and 11. This implies that compound 12 is a dimeric saponin in a symmetrical form. The structure of compound 12 is therefore proposed as roburgenic acid [24-O-(28-O-β-D-glucopyranosyl roburgenic acid)] 28-O-β-D-glucopyranosyl ester, and was named roburoside C.

Table 4. In Vitro Cytotoxicity of Compounds 1−17 against Two Different Cancer Cell Lines and Lymphocytes Compound

PC3 IC50 (μM)a

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Oxaliplatinb Cisplatinb

46.8 ± 3.5 58.8 ± 3.2 64.4 ± 3.4 47.3 ± 3.2 47.3 ± 3.2 57.2 ± 3.1 34.7 ± 2.9 54.3 ± 3.6 57.3 ± 2.6 50.8 ± 2.3 50.8 ± 2.8 57.2 ± 2.4 41.3 ± 3.9 57.3 ± 3.4 51.9 ± 5.5 43.8 ± 2.0 45.7 ± 2.1 7.9 ± 3.2 26.0 ± 2.6

MCF-7 IC50 (μM)a

Lymphocytes IC50 (μM)a

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

>200 >200 >200 40.2 ± 1.7 44.5 ± 1.2 62.7 ± 2.4 59.8 ± 2.7 >200 >200 58.4 ± 2.7 64.8 ± 2.2 68.2 ± 1.8 48.7 ± 4.3 48.8 ± 2.0 48.3 ± 1.3 >200 57.3 ± 2.1 >200 6.8 ± 1.1

29.2 46.8 28.7 19.7 29.7 43.3 27.2 34.7 43.3 49.8 34.7 47.2 33.2 34.8 37.8 37.6 37.8 12.8 16.7

3.2 3.7 2.8 1.7 3.0 3.6 5.0 3.7 3.3 4.6 4.2 4.3 3.9 3.2 2.6 2.6 3.9 2.8 3.5

Means ± SEM from 6 independent experiments (n = 6). bPositive control.

a

bioactivities of tested compounds being rather moderate compared to positive controls, these were better against MCF-7 cells than against PC3 cells, reaching a comparable value to those of positive controls for roburgenic acid (4, IC50 = 19.7 μM). Perhaps this is caused by their selective ability to act as reversal agents (inhibitors) for breast cancer resistance protein (BCRP) or for multidrug resistance-associated protein 2 (MRP2)-mediated transport, as it was previously shown for the triterpenoid glycyrrhetic acid.25 Another specific mode of action may involve triggering apoptosis by modulation of glucocorticoid receptor and activator protein-1 in MCF-7 breast cancer cells, as elucidated before for ursolic acid.13 Evidence previously accumulated for triterpenoids was reviewed by Bishayee et al.,26 who recognized the potential of these metabolites and their derivatives in the chemoprevention or even in the chemotherapy of breast cancer. On the basis of cytotoxicity against MCF-7 cells, some structure−activity relationships may be proposed. First of all, a selectivity for compounds 1−3, 8, 9, and 16, all exhibiting IC50 values above 200 μM against lymphocytes, was observed. These compounds have a galloyl unit attached to the triterpenoid moiety as a common feature. Compound 6, however, did not show such selectivity, although it also has a galloyl unit attached. The IC50 value for compound 4 was 13.5 μM lower than that of 13, and the lowest among tested compounds. Their chemical difference consists only in the stereochemistry at C-3. This could indicate a stereospecific requirement of exerted cytotoxicity against MCF-7 cells, with the S-configuration at C3 of the triterpenoid being preferred. Esterification of the 4621


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Journal of Agricultural and Food Chemistry Notes

carboxylic group at C-28 with a glucose unit, as in compound 5, negatively affects the bioactivity when compared to 4. It seems, however, this is not always a rule, since the opposite effect was observed when compounds 1 and 2 were compared. The position of the galloyl unit also affects the cytotoxicity. When it is attached at C-3 of the triterpenoid moiety, the cytotoxicity is enhanced, as shown from the comparison between compounds 2 and 3, while the opposite effect is observed when the triterpenoid is glycosylated at C-28, as in 1 and 16. On the other hand, substitution of the carboxylic group at C-24 with an aldehyde group negatively affects the cytotoxicity when a galloyl unit is linked at the C-3 position, as shown by the comparison between 3 and 9. Contrarily, when the galloyl unit is attached at C-23, as in 6 and 8, the cytotoxicity increases. Furthermore, when no galloyl unit is linked to the triterpenoid moiety, substitution with an aldehyde group at C-24 enhances the cytotoxicity, as is suggested from the comparison between compounds 7 and 13. In conclusion, the structural diversity of new compounds, 1− 12, emphasized by the occurrence of 3α-OH substitution in 4 and 5, besides being reported here for the first time within Quercus genus, also seems to have biological implications. Moreover, the occurrence of dimeric triterpenoid saponins, 10−12, is also reported for the first time within this genus. Despite the fact that it could be assumed that these compounds are artifacts formed by esterification during isolation procedures, this is unlikely, since they were initially detected in the first obtained extract by LC-MS analysis (Figure 1, Ex MeOH 70%). Substitution with a galloyl unit at the triterpenoid moiety was identified as a key feature to exert a selective cytotoxicity. These results also represent a contribution for the unambiguous identification of oak triterpenoids, since their occurrence in beverages aged in oak barrels, in amounts ranging from 1.0 to 69.2 mg/L, has been previously reported.5 However, the results presented here are mainly theoretical, and it should not be assumed in any case that consumption of alcoholic beverages aged in oak barrel brings benefits to human health. Further investigations on bioavailability, toxicological, and other effects of these compounds are therefore needed.

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Bartłomiej Furman (Institute of Organic Chemistry PAS, Warsaw, Poland) for granting access to the digital polarimeter and Jerzy Ż uchowski for recording the optical rotation spectra.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b01396. Most relevant NMR spectra of new compounds (1−12), characteristic MS data and tables for NMR data of known compounds (13−17), among other information (PDF)

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (A. J. Pérez). Tel.: +56 41 266 1835. ORCID

Andy J. Pérez: 0000-0001-6717-2040 Łukasz Pecio: 0000-0002-7407-6716 Funding

This research was supported by the Basal Project (PFB-27) from Unidad de Desarrollo Tecnológico (UDT), Universidad de Concepción, Chile. 4622


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