High-Resolution Screening Combined with HPLC-HRMS-SPE-NMR

May 16, 2014 - Crude extracts of 33 plant species were assessed for fungal plasma membrane (PM) H+-ATPase inhibition. This led to identification of 18...
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High-Resolution Screening Combined with HPLC-HRMS-SPE-NMR for Identification of Fungal Plasma Membrane H+‑ATPase Inhibitors from Plants Kenneth T. Kongstad,†,§ Sileshi G. Wubshet,†,§ Ane Johannesen,† Lasse Kjellerup,‡ Anne-Marie Lund Winther,‡ Anna Katharina Jag̈ er,† and Dan Staerk*,† †

Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark ‡ PCOVERY, Ole Maaløes Vej 3, DK-2200 Copenhagen, Denmark S Supporting Information *

ABSTRACT: Crude extracts of 33 plant species were assessed for fungal plasma membrane (PM) H+-ATPase inhibition. This led to identification of 18 extracts showing more than 95% inhibition at a concentration of 7.5 mg/mL and/or a concentrationdependent activity profile. These extracts were selected for semi-high-resolution fungal PM H+-ATPase inhibition screening, and, on the basis of these results, Haplocoelum foliolosum (Hiern) Bullock and Sauvagesia erecta L. were selected for investigation by high-resolution fungal PM H+-ATPase inhibition screening. Structural analysis performed by high-performance liquid chromatography-high-resolution mass spectrometry-solid-phase extraction-nuclear magnetic resonance spectroscopy (HPLCHRMS-SPE-NMR) led to identification of chebulagic acid (1) and tellimagrandin II (2) from H. foliolosum. Preparative-scale isolation of the two metabolites allowed determination of IC50 values for PM H+-ATPase, and growth inhibition of Saccharomyces cerevisiae and Candida albicans. Chebulagic acid and tellimagrandin II are both potent inhibitors of the PM H+-ATPase with inhibitory effect on the growth of S. cerevisiae. KEYWORDS: fungal plasma membrane H+-ATPase, high-resolution screening, HPLC-HRMS-SPE-NMR, chebulagic acid, tellimagrandin II, antifungal, Haplocoelum foliolosum

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INTRODUCTION Fungi are capable of colonizing a large variety of food and animal feed, and it has been estimated that 5−10% of the world’s food production is lost due to fungal spoilage.1 Fungal infections lead to substantial damage manifested as general spoilage such as discoloration and off-flavors as well as nutritional losses. The spoilage is often caused by excretion of exoenzymes during growth, and once inside the food the enzymes may continue their deteriorating activities despite the removal of the fungal mycelium. However, the most important aspect of fungal spoilage is the production of mycotoxins, which are often toxic to vertebrates and may lead to immune suppression and different types of cancers.2 Thus, in addition to the financial consequences, fungal spoilage poses a serious threat to the health of immuno-compromised as well as healthy individuals. Fungi are also known to cause systemic infections in immunocompromised patients and are considered as a major healthcare problem. There are an increased number of infections that cannot be treated with the medicines available today, in part caused by infections with fungi that have become resistant to existing drugs,3,4 but also due to infections with newly emerging fungal pathogens, which are not susceptible to the existing drugs.5 A large number of fungal proteins have been proposed as potential targets for novel antifungal agents.3,6 However, current available antifungal agents are primarily targeting the intracellular membrane and cell wall biosynthesis,3 and thus need to enter the fungus to act. In our search for novel and © 2014 American Chemical Society

more efficient antifungal compounds, we are focusing on the plasma membrane (PM) H+-ATPase enzyme as target. The PM H+-ATPase enzyme consists of 10 trans-membrane helices and plays a pivotal role in eukaryotic cell physiology in maintaining the trans-membrane electrochemical proton gradient necessary for nutrient uptake.7,8 Characterization of the PM H+-ATPase from various fungi has shown it to comprise a single 100 kDa subunit, containing both a membrane-spanning transport domain as well as a catalytic ATP hydrolyzing domain located in the cytoplasm. The PMA1 gene encoding for the enzyme has been shown to be highly conserved among fungi, but having only 32% sequence identity to its counterparts in plants9,10 (sequence alignment of plant H+-ATPase AHA2 (accession number P19456) and Saccharomyces cerevisiae H+-ATPase PMA1 (accession number P05030) made with ClustalW2). Gene-disruption experiments with S. cerevisiae have confirmed the essential nature of the gene product of PMA1.11 Thus, the low sequence identity with plant PM H+-ATPases as well as its accessibility from the cell exterior, that is, circumventing potential problems with multidrug resistance pumps,12,13 make the fungal PM H+-ATPase a good target for new antifungal drugs. Received: Revised: Accepted: Published: 5595

April 3, 2014 May 14, 2014 May 16, 2014 May 16, 2014 dx.doi.org/10.1021/jf501605z | J. Agric. Food Chem. 2014, 62, 5595−5602

Journal of Agricultural and Food Chemistry

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Table 1. Percent Inhibition of Plasma Membrane H+-ATPase at Crude Extract Concentrations of 7.5, 15, and 30 mg/mLa percent inhibition no.

species

family

plant part

7.5 mg/mL

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 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

Alafia barteri Alchornea cordifolia Avicennia marina Avicennia marina Baissea leonensis Baissea leonensis Baissea leunensis Caloncoba echinata Caloncoba echinata Caloncoba gilgiana Croton longiracemosus Croton longiracemosus Croton membranaceus Dovyalis macrocalyx Euadenia eminens Euadenia eminens Gymnema sylvestre Haplocoelum foliolosum Hubertia ambavilla Hubertia tomentosum Hypericum scabrum Lophira alata Momordica charantia Mussaenda tristigmatica Nepeta glomerulosa Oncinotis glabrata Oncinotis glabrata Oncinotis glabrata Oncinotis gracilis Oncinotis pontyi Oncinotis pontyi Orthopichonia barteri Orthopichonia barteri Pierreodendron kerstingii Pierreodendron kerstingii Pierreodendron kerstingii Pleiocarpa mutica Protomegabaria stapfiana Pycnocycla spinosa Pyrenacantha acuminatab Pyrenacantha acuminata Pyrenacantha acuminatab Salacia pyriformis Secamone afzelii Sphenocentrum jollyanum Sauvagesia erecta

Apocynaceae Euphorbiaceae Acanthaceae Acanthaceae Apocynaceae Apocynaceae Apocynaceae Achariaceae Achariaceae Achariaceae Euphorbiaceae Euphorbiaceae Euphorbiaceae Salicaceae Capparaceae Capparaceae Apocynaceae Sapindaceae Asteraceae Asteraceae Hypericaceae Achnaceae Curcurbitaceae Rubiaceae Lamiaceae Apocynaceae Apocynaceae Apocynaceae Apocynaceae Apocynaceae Apocynaceae Apocynaceae Apocynaceae Simaroubaceae Simaroubaceae Simaroubaceae Apocynaceae Euphorbiaceae Apiaceae Icacinaceae Icacinaceae Icacinaceae Celastraceae Apocynaceae Menispermaceae Ochnaceae

leaves + twigs leaves + fruits root stembark leaves + twigs fruit root leaves branches leaves branches root bark leaves + twigs stem aerial parts root aerial parts aerial parts leaves + twigs leaves whole plant leaves aerial parts leaves + twigs whole plant leaves branches stem leaves + twigs leaves branches stem leaves stem bark leaves branches leaves fruit root branches leaves branches leaves + twigs stem leaves + twigs whole plant

27 99 −19 24 99 98 −3 50 10 99 −8 20 40 −4 33 2 34 99 27 36 59 41 31 99 9 0 −12 17 −1 17 8 22 21 3 99 35 44 −4 -1 −16 48 6 99 −2 39 98

11 98 −12 13 99 98 −6 39 −28 99 −13 12 18 −13 15 −47 19 99 29 19 51 38 27 98 8 −10 −8 22 −20 7 −5 30 22 9 98 21 40 −10 -6 −20 38 −4 98 −19 24 98

15 mg/mL 33 98 −1 31 99 98 −3 54 14 98 3 27 40 21 43 4 38 100 56 50 96 60 33 99 7 16 4 29 3 22 16 45 25 18 99 38 43 14 34 −16 52 7 99 11 55 99

19 98 −7 17 99 98 6 46 −18 97 9 19 28 1 25 −36 26 99 58 31 75 56 35 98 14 12 2 32 −14 12 11 51 25 29 99 25 50 4 29 −13 39 −20 99 −4 36 98

30 mg/mL 33 97 8 40 99 97 9 57 29 99 19 34 53 35 49 13 59 100 84 75 97 96 42 99 23 30 16 42 20 29 29 97 35 55 99 47 70 48 87 16 51 24 98 25 76 97

27 97 −6 26 100 97 15 46 −10 97 7 18 41 18 32 −7 46 100 86 71 96 90 39 98 24 28 13 40 6 29 18 95 37 49 98 36 65 34 76 0 37 −2 97 10 68 98

a Extracts showing inhibition higher than 95% for all concentrations or extracts showing a concentration-dependent activity profile are marked with bold and were selected for semi-high-resolution screening. bDifferent collections.

developed a bioanalytical platform that combines highresolution microplate bioassays18,19 with a hyphenated system consisting of high-performance liquid chromatography, solidphase extraction, and nuclear magnetic resonance, that is, HPLC-SPE-NMR.20,21 HPLC-SPE-NMR has proven successful for fast and efficient chemical analysis of crude plant extracts;22−24 including the combination with circular dichroism to establish the absolute configuration of the separated analytes.25 However, HPLC-SPE-NMR alone does not give any information about the pharmacological activity of the analytes,

Plants are exposed to a wide array of phytopathogenic fungi in their natural habitat, and have been forced to develop antifungal metabolites to survive.14,15 Hence, as was previously suggested by Monk and co-workers,16 it is reasonable to assume that the PM H+-ATPase enzyme is a target for antifungal plant metabolites. However, plant extracts are very complex mixtures, and the traditional bioassay-guided fractionation used for identification of individual bioactive components is very timeconsuming and suffers from inherently low resolution during the fractionation process.17 To circumvent this, we have 5596

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Protocol for Growth Assay. Minimum inhibitory concentrations (MICs) were determined on S. cerevisiae (FuSc041 − De Danske Gærfabrikker, Grenå, Denmark) and Candida albicans (SC5314 − LGC Standards, Borås, Sweden) as the lowest concentration that inhibited fungal growth with 50%. Fungal freeze stocks were transferred to YPD-agar plates (10 g/L yeast extract, 20 g/L bactopeptone, 20 g/L glucose, 20 g/L agar) and incubated at 30 °C overnight (O/N). Cells were suspended in sterile H2O, and the start OD600 was adjusted to C. albicans OD600 at 0.025, S. cerevisiae OD600 at 0.05. Three microliters of sample or DMSO, 100 μL of cell suspension, and 97 μL of 2 × RPMI-medium (20.8 g/L RPMI-1640 medium, 0.33 M MOPS, 36 g/L glucose) were pipetted into a microtiter plate and incubated for 24 h at 30 °C, followed by OD measurement at 490 nm. Plant Material and Extractions. Ground plant material (0.3 g of dried material) of 46 plant samples from an in-house collection was extracted with 3.6 mL of ethanol (96%) by sonication for 2 h. Extracts were filtered (Q-Max RR 13 mm 0.45 μm Nylon, Frisenette, Denmark) and concentrated in vacuo before defatting by partitioning between 2 mL of 90% aqueous methanol and 1.5 mL of petroleum ether (bp 40−65 °C). Defatted extracts were dissolved in DMSO to a concentration of 30 mg/mL. Samples selected for HPLC-HRMS-SPENMR/bioassay analysis were extracted with 35 mL of ethanol (ca. 1.2 g of ground plant material) by sonication for 2 h. Extracts were concentrated in vacuo and defatted by partitioning between 20 mL of 90% aqueous methanol and 15 mL of petroleum ether (bp 40−65 °C). Defatted extracts were dissolved in methanol to a concentration of 10 mg/mL for semi-high-resolution bioassay analysis. Crude Extract Screening. The defatted extracts of 46 plant samples (Table 1) were tested for PM H+-ATPase inhibition at concentrations of 30, 15, and 7.5 mg/mL. The assay was performed in duplicate according to the protocol described above. Semi-High-Resolution PM H+-ATPase Inhibition Assay. Chromatographic separation of crude extracts was performed with an Agilent 1200 series instrument (Santa Clara, CA) consisting of a quaternary pump, a degasser, a thermostated column compartment, a photodiode-array detector, a high-performance auto sampler, and a fraction collector, all controlled by Agilent ChemStation ver. B.03.02 software and equipped with a reversed phase Luna C18 (2) (Phenomenex, 150 × 4.6 mm, 5 μm, 100 Å) maintained at 40 °C. The aqueous eluent (A) consisted of water/acetonitrile (95:5, v/v), and the organic eluent (B) consisted of water/acetonitrile (5:95, v/v), both acidified with 0.1% formic acid. The eluent flow rate was maintained at 0.5 mL/min with the following elution profile: 0 min, 0% B; 5 min, 0% B; 35 min, 100% B; 43 min, 100% B; 45 min, 0% B, and 10 min equilibration with five successive injections of 10 μL. The column eluent was directed to an automated fraction collector, and eluent from 10 to 40 min was collected in 183 μL aliquots in 80 wells of a 96-well V-shaped microplate (Brand, Wertheim, Germany) (excluding outer columns), concentrated in vacuo, and redissolved in 25 μL of DMSO. Two microliters was transferred to a 96-well microplate, and the PM H+-ATPase assay was conducted as described above. High-Resolution PM H+-ATPase Inhibition Assay. Chromatographic separation of the two selected plant extracts (Haplocoelum foliolosum and Sauvagesia erecta L.) was performed on the Agilent 1200 series instrument described above with similar conditions (column, solvent composition, temperature, and flow rate). The elution profiles were: 0 min, 5% B; 50 min, 40% B; 52 min, 100% B; 57 min, 100% B; 58 min, 5% B; and 10 min equilibration for H. foliolosum; and 0 min, 5% B; 50 min, 30% B; 52 min, 100% B; 57 min, 100% B; 58 min, 5% B; and 10 min equilibration for S. erecta. Single chromatographic separations after injection of 7 μL (from 170 mg/mL solution) and 30 μL (from 50 mg/mL solution) were used for microfractionation of H. foliolosum and S. erecta, respectively. The eluate from 10 to 40 min was collected in 94 μL aliquots in 160 wells of 2 × 96-well V-shaped microplates (excluding the outer columns). The chromatographic solvent was evaporated to dryness in vacuo, and the contents of each well were redissolved in 40 μL of DMSO. From each well, 3 × 2 μL and 3 × 3 μL were used for the PM H+-ATPase and growth assays, respectively.

and therefore the extension with high-resolution assays, that is, HR-BIOASSAY/HPLC-SPE-NMR,26−31 is probably the most important improvement to the basic HPLC-SPE-NMR setup in recent years. Here, we report crude extract screening of 46 plant extracts (from 33 different plant species) for fungal PM H+-ATPase inhibitors, followed by high-resolution bioassay and HPLCHRMS-SPE-NMR analysis for identification of individual active constituents.

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EXPERIMENTAL SECTION

Chemicals. Amphotericin B, RPMI 1640 medium, dimethyl sulfoxide, analytical grade HPLC solvents, and methanol-d4 (99.8 atom % of deuterium) were purchased from Sigma-Aldrich (St. Louis, MO). Water used for HPLC was purified by deionization and 0.22 μm membrane filtration (Millipore, Billerica, MA). Purification of the Yeast Plasma Membrane H+-ATPase by Recombinant Expression. Heat competent S. cerevisiae RS72 yeast cells were prepared and transformed according to Gietz and Woods32 with a yeast multicopy vector,33 containing the full-length cDNA of the S. cerevisiae plasma membrane H+-ATPase isoform PMA1 under control of the PMA1 promoter.34,35 Transformed yeast cells were precultured in 100 mL of sterile SGAH medium (7.04 g/L yeast nitrogen base, 19.8 g/L galactose, 64 mg/L adenine, and 64 mg/L histidine) for 3 days at 25 °C and 150 rpm. The preculture was transferred to 500 mL of sterile SGAH medium and further incubated for 3−4 days. 100 mL from the cell culture was transferred to 1 L of YPAD medium (10 g/L yeast extract, 20 g/L bacto-peptone, 20 g/L glucose, 20 mg/L adenine) and incubated at 25 °C for 18−20 h. Recombinant yeast was harvested by 2−3 min of centrifugation at 3000g and 4 °C, followed by two times washing in milli-Q water. Harvested cells were incubated in 10% glucose for 10 min on a shaking table, and centrifuged at 3000g and 4 °C. Cells were resuspended in homogenization buffer (50 g/L glucose, 28.3% glycerol, 0.1 M TrisHCl pH 7.25, 10 mM EDTA pH 8.0, 50 mM KCl, 1 mM DTT, 200 μM PMSF, 2 μg/mL Pepstatin A), and disrupted with 165 g of glassbeads (500 μm) by runs in a BeadBeater (Biospec Products, Bartlesville, OK). The disrupted cells were centrifuged at 4 °C for 5 and 15 min at 1400g and 12 000g, respectively. The supernatant was collected and centrifuged at 251 000g for 1 h with 112 μM phenylmethylsulfonyl fluoride (PMSF) and 1.1 μg/mL Pepstatin A. The resulting pellet was resuspended in GTEK20 buffer (20% glycerol, 10 mM Tris-HCl pH 7.25, 25 mM KCl, 0.5 mM EDTA pH 8.0, 1 mM DTT, 0.2 mM PMSF, 2 μg/mL Pepstatin A) and centrifuged for 45 min at 251 000g and 4 °C. Pellet was then resuspended in STKED20 buffer (200 g/L sucrose, 40 g/L glucose, 50 mM Tris-HCl pH 7.25, 50 mM KCl, 1 mM EDTA pH 8.0, 1 mM DTT, 0.2 mM PMSF, 2 μg/mL Pepstatin A), homogenized, and diluted with STKED20 buffer. The plasma membranes were recovered at the interface of a 43%/53% (w/ w) step sucrose gradient containing sucrose in 50 mM Tris-HCl pH 7.25, 50 mM KCl, 1 mM EDTA, and 1 mM DTT. Centrifugation was done for 16 h at 154 000g and 4 °C. The plasma membrane fraction was collected and diluted with GTEK20 buffer and centrifuged for 1 h at 274 000g. Pellet was collected and homogenized in GTEK20 buffer and stored at −80 °C. Determination of Protein Activity. The protein activity was determined by measuring the amount of liberated phosphate (Pi) from the ATP hydrolysis reaction. Reactions, with or without 2 μL sample, were performed in an assay buffer consisting of 20 mM MOPS-NaOH pH 6.5, 8 mM MgSO4, 50 mM KNO3, 25 mM NaN3, 250 μM Na2MoO4, and initiated by the addition of Na-ATP to a final concentration of 2.5 mM, followed by incubation for 30 min at 30 °C. The amount of Pi was measured after addition of STOP-solution (mixture of A, 170.3 μM C6H8O6 in 0.5 M HCl; and B, 28.3 mM (NH4)6Mo7O24·4H2O in H2O) with an incubation of 10 min at room temperature and thereafter addition of arsenite solution (154 mM NaAsO2, 68 mM Na3C6H5O7·2H2O, 0.35 M CH3COOH). OD was measured at 860 nm after an additional incubation for 30 min at room temperature. 5597

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Phenomenex Luna C18 (5 μm) column operated at room temperature. Repeated injection (3 × 800 μL) of the above solution, followed by a gradient elution (similar to the one used for HR screening), afforded 9.5 mg of fraction A (collected in a retention time range 22− 24 min). Fraction A was reconstituted in 800 μL of 50% MeOH and subjected to further preparation purification using an isocratic elution (12% B) for 30 min. Single injection of 400 μL was performed, and peaks 1 and 2 were manually collected. After concentration under reduced pressure and lyophilization, 0.82 mg of 1 and 0.49 mg of 2 were obtained. The two compounds were reconstituted in DMSO to a final concentration of 5 mM, and subsequently IC50 values in PM H+ATPase and fungal growth assays with S. cerevisiae and C. albicans were assessed.

HPLC-HRMS-SPE-NMR. The HPLC-HRMS-SPE-NMR system consisted of an Agilent 1100 chromatograph comprising quaternary pump, degasser, thermostated column compartment, auto sampler, and photodiode array detector (Santa Clara, CA), a Bruker micrOTOF-Q II mass spectrometer (Bruker Daltonik, Bremen, Germany) equipped with an electrospray ionization source and operated via a 1:99 flow splitter, a Knauer Smartline 100 pump for postcolumn dilution (Knauer, Berlin, Germany), a Spark Holland Prospekt 2 SPE unit (Spark Holland, Emmen, The Netherlands), a Gilson 215 liquid handler equipped with a 1 mm needle for automated filling of 1.7 mm NMR tubes, and a Bruker Avance III 600 MHz NMR spectrometer (1H operating frequency 600.13 MHz) equipped with a Bruker SampleJet sample changer and a cryogenically cooled gradient inverse triple-resonance 1.7 mm TCI probe-head (Bruker Biospin, Rheinstetten, Germany). Mass spectra were acquired in both positive and negative ion modes, using drying temperature of 200 °C, capillary voltages of −4100 and +4000 V for positive and negative ion modes, respectively, nebulizer pressure of 2.0 bar, and drying gas flow of 7 L/ min. The negative ion mode HPLC-HRMS analysis was performed in a different experiment using identical chromatographic condition. A solution of sodium formate clusters was injected in the beginning of each run to enable internal mass calibration. Cumulative SPE trapping of H. foliolosum (peaks 3−6) was performed after eight consecutive separations using the chromatographic conditions (column, solvent composition, temperature, flow rate, and elution profile) described above. The HPLC eluate was diluted with Milli-Q water at a flow rate of 1.5 mL/min prior to trapping on 10 × 2 mm i.d. resin GP (general purpose, 5−15 μm, spherical shape, polydivinyl-benzene phase) SPE cartridges from Spark Holland (Emmen, The Netherlands), and analytes were trapped using absorption thresholds (254 nm). Overlapped peaks (peaks 1 and 2 of H. foliolosum) were manually collected from repeated chromatographic separations, evaporated to dryness in vacuo, and reconstituted in methanol to afford 9 mg/mL. This solution was subjected to HPLC-HRMS-SPE-NMR analysis using the following isocratic elution profile: 0 min, 13% B; 24 min, 13% B; 25 min, 100% B; 30 min, 100% B; 31 min, 13% B; and 4 min equilibration. A total of eight cumulative trappings was performed using absorption thresholds (254 nm). SPE cartridges were conditioned with 1000 μL of methanol at 6 mL/min and equilibrated with 500 μL of Milli-Q water at 1 mL/min prior to trapping. Loaded cartridges were dried with pressurized nitrogen gas for 45 min each. Separations were controlled by Bruker Hystar version 3.2 software, automated filling of NMR tubes was controlled by PrepGilsonST version 1.2 software, and automated NMR acquisition was controlled by Bruker IconNMR version 4.2 software. NMR data processing was performed using Bruker Topspin version 3.2 software. NMR Experiments. All NMR spectra were recorded in methanold4 at 300 K. 1H chemical shifts were referenced to the residual solvent signal (δ 3.31). One-dimensional 1H NMR spectra were acquired in automation (temperature equilibration to 300 K, optimization of lock parameters, gradient shimming, and setting of receiver gain) with 30° pulses, 3.66 s interpulse intervals, 64k data points, and multiplied with an exponential function corresponding to line-broadening of 0.3 Hz prior to Fourier transform. Phase-sensitive DQF-COSY spectra were recorded using a gradient-based pulse sequence with a 20 ppm spectral width and 2k × 256 data points (processed with forward linear prediction to 1k data points). Multiplicity-edited HSQC spectra were acquired with the following parameters: spectral width 20 ppm for 1H and 165 ppm for 13C, 2k × 256 data points (processed with forward linear prediction to 1k data points), and 2.0 s relaxation delay. HMBC spectra (without a low-pass filter) were optimized for nJC,H = 10 Hz and acquired using the following parameters: spectral width 20 ppm for 1H and 222 ppm for 13C, 2k × 128 data points (processed with forward linear prediction to 1k data points), and 1.5 s relaxation delay. Targeted Isolation and Determination of IC50 Values of 1 and 2. An injection solution of 0.17 g/mL (H. foliolosum) was subjected to a preparative chromatography using the Agilent 1100 system equipped with two preparative solvent delivery units, a multiple wavelength detector, an autosampler, and an optional fraction collector. Separation was performed using a 250 mm × 21.2 mm i.d.

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RESULTS AND DISCUSSION Crude Extract Screening. From our in-house plant collection, 33 different plant species were selected for this study. For some of the species, different parts were available, yielding a total of 46 samples of ground plant material (see Table 1). The material was extracted with ethanol, and the dried extracts were defatted by partitioning between 90% aqueous methanol and petroleum ether. The defatted extracts were tested for their ability to inhibit the PM H+-ATPase enzyme at concentrations of 30, 15, and 7.5 mg/mL, and extracts showing inhibition higher than 95% for all concentrations or extracts showing a concentration-dependent activity profile (indicated with bold in Table 1) were selected for semihigh-resolution screening. Thus, Alchornea cordifolia, Baissea leonensis (fruits as well as leaves + twigs), Caloncoba gilgiana, Haplocoelum foliolosum, Mussaenda tristigmatica, Pierreodendron kerstingii (leaves), Salacia pyriformis, and Sauvagesia erecta were selected on the basis of the former criteria, whereas Hubertia ambavilla, Hubertia tomentosum, Hypericum scabrum, Lophira alata, Orthopichonia barteri, Pierreodendron kerstingii (stem bark), Pleiocarpa mutica, Pycnocycla spinosa, and Sphenocentrum jollyanum were selected on the basis of the latter criteria. Semi-High-Resolution PM H+-ATPase and Growth Inhibition Profiles. The 18 samples selected for semi-highresolution screening were separated using a HPLC method starting at 5% acetonitrile (plus 0.1% formic acid) for 5 min, and using a linear gradient from 5% to 95% acetonitrile (plus 0.1% formic acid) from 5 to 35 min. The eluate from 10 to 40 min was collected in 80 wells of a 96-well microplate (omitting the first and last columns), yielding fractions of 183 μL. The eluate was evaporated, and the dried material in each well was redissolved in DMSO. These solutions were assayed for their ability to inhibit the PM H+-ATPase as well as their ability to inhibit growth of C. albicans and S. cerevisiae. The results were plotted under the HPLC traces at 254 nm, and all semi-highresolution biochromatograms are given in Supporting Information, Figures S1−S18. The results show that the tested extracts can be placed in three different groups as illustrated in Figure 1. The first group consists of Hubertia ambavilla, Hubertia tomentosum, Hypericum scabrum, Lophira alata, Orthopichonia barteri, Pierreodendron kerstingii (stem bark), Pleiocarpa mutica, Pycnocycla spinosa, Salacia pyriformis, and Sphenocentrum jollyanum, which show very low PM H+-ATPase inhibition and/or no distinct peaks with PM H+-ATPase inhibition. The second group consists of Alchornea cordifolia, Baissea leonensis (fruits as well as leaves + twigs), Caloncoba gilgiana, Mussaenda tristigmatica, and Pierreodendron kerstingii (leaves), where the observed PM H+-ATPase inhibition is correlated with a broad hump at approximately 10−30 min. This hump is attributed to tannin-rich fractions, and the PM 5598

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ments (Figure 2 and Supporting Information Figure S19 for H. foliolosum and S. erecta, respectively). From H. foliolosum two

Figure 2. High-resolution antifungal screening of Haplocoelum foliolosum crude extract. (A) HPLC chromatogram at 254 nm. (B) High-resolution fungal plasma membrane H+-ATPase inhibition profile (an average of triplicate measurements with standard deviations are plotted).

overlapping peaks at 23.6 min were correlated with >80% inhibition of the PM H+-ATPase. S. erecta, however, did not show any peaks in the HPLC chromatogram correlating with a discrete peak in the inhibition profile of the optimized highresolution assay (Supporting Information Figure S19). This is despite the noticeable inhibition in both the crude extract screening (Table 1) and the semi-high-resolution assay (Supporting Information Figure S18). This can be caused by loss of synergistic activities of multiple constituents that are separated in the high-resolution biochromatogram but assessed collectively in the semi-high-resolution biochromatogram as well as in the crude extract screening. Therefore, S. erecta was excluded from further investigation. HPLC-HRMS-SPE-NMR. The two active peaks from H. foliolosum (peaks 1 and 2) and four other major metabolites (peaks 3−6) were subjected to HPLC-HRMS-SPE-NMR analysis. Detailed HRMS and NMR analysis resulted in identification of six compounds 1−6 (Figure 3) corresponding to peaks 1−6, respectively. 1H NMR, UV, and HRMS data of 1−6 obtained in the HPLC-HRMS-SPE-NMR mode are given in Table S1 in the Supporting Information. HRMS data of compound 1 showed a base peak at m/z 785.0836, which was attributed to C34H25O22+. However, inverse-detected heteronuclear experiments revealed 41 carbon atoms. After a detailed analysis of 1D and 2D NMR experiments and comparison of the observed NMR data with literature data, the analyte was identified as chebulagic acid (1)36 (molecular formula C41H30O27). The observed base peak for 1 at m/z 785.0836 was assigned to a fragment ion [M + H − H2O − galloyl]+ (C34H25O22+, ΔM −0.5 ppm), similar to the fragment ion at m/z 769.0863 [M + H − H2O − galloyl group]+ (C34H25O21+, ΔM 2.5 ppm) observed as the base peak for compound 2. Such fragmentation product has previously been reported for 1.37 The molecular ion peaks of compound 1 and 2 could not be detected in positive ion mode, even when studying extracted ions chromatograms from HPLC-HRMS. The molecular formulas are, however, supported by the presence of ammonium adducts in positive ion mode of m/z 972.1295 [M + NH4]+ (C41H34NO27+, ΔM 1.8 ppm) and m/z 956.1331 [M + NH4]+ (C41H34NO26+, ΔM 3.5 ppm) as well as molecular ion peaks in negative ion mode of m/z 953.0875 [M − H]− (C41H29NO27−, ΔM 2.8 ppm) and m/z 937.0924 [M −

Figure 1. Three representative inhibition profiles obtained through semi-high-resolution antifungal screening: Hypericum scabrum (A, group 1), Caloncoba gilgiana (B, group 2), and Haplocoelum foliolosum (C, group 3).

H+-ATPase inhibition is therefore likely to be caused by tannins, which are of little interest due to their inherent ability to precipitate proteins. The third group consists of two samples, Haplocoelum foliolosum and Sauvagesia erecta, showing direct correlation between individual peaks and inhibition of the PM H+-ATPase. Therefore, the two plants from the last group were selected for a high-resolution PM H+-ATPase assay to correlate the observed activities with individual metabolites. High-Resolution PM H+-ATPase Profiles. Extracts of H. foliolosum and S. erecta were reanalyzed using the highresolution PM H+-ATPase inhibition assay with optimized separation condition and a higher number of data points per retention time (5.33 points/min). Furthermore, higher chromatographic loading (1.2 and 1.5 mg for H. foliolosum and S. erecta, respectively) allowed triplicate assaying of the time-sliced fractions to assess statistical variations of the assay. The biochromatograms for the two plants were therefore plotted with standard deviation from the triplicate measure5599

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Figure 3. Structures of antifungal compounds chebulagic acid (1) and tellimagrandin II (2) as well as of compounds 3−6 showing no antifungal activity.

Table 2. IC50 Values for Fungal Growth Inhibition Assays and Fungal Plasma Membrane H+-ATPase Inhibition growth inhibitionb chebulagic acid (1) tellimagrandin II (2) vanadate amphotericin B fluconazole a

plasma membrane H+-ATPase inhibitiona

Saccharomyces cerevisiae FuSc041a

15.8 ± 7.9 10.6 ± 3.1 2.3 ± 1.2

1.8 ± 0.9 1.8 ± 0.9 0.08 ± 0.05 7.6 ± 4.9

Candida albicans SC5314a >75 >75 0.08 ± 0.05 0.9 ± 0.6

n = 3, results reported in micromolar. bDetermined as MIC for 50% inhibition.

H]− (C41H29NO26−, ΔM 2.9 ppm), compounds 1 and 2, respectively. The 1H NMR spectrum of 2 shows five deshielded aromatic proton resonances, 7.05 (s, 2H, H-2′/H-6′), 6.95 (s, 2H, H-2″/ H-6″), 6.92 (s, 2H, H-2‴/H-6‴), 6.62 (s, 1H, H-3⁗), and 6.49 (s, 1H, H-3⁗′). Three of these signals were integrated to two protons each, and thorough analysis of characteristic 2J and 3J HMBC correlations to the corresponding quaternary aromatic carbons and a carboxyl carbon revealed three galloyl groups. Seven relatively deshielded resonances with discrete COSY spin system were assigned to a glucose moiety with esterification of all hydroxyls. The esterification positions were established by 3J HMBC correlations of the sugar proton resonances H-1 (δ 6.11, d, J = 8.2 Hz), H-2 (δ 5.55, dd, J = 9.1,8.4 Hz), and H-3 (δ 5.76, t, J = 9.6 Hz) with the galloyl carboxyl carbon resonances C-7′ (166.2 ppm), C-7″ (166.6 ppm), and C-7‴ (167.4 ppm), respectively. After identifying a hexahydroxydiphenoyl (HHDP) moiety bridging two oxygens of glucose at positions 4 and 6 through similar HMBC data analysis, the structure of peak 2 was confirmed as tellimagrandin II (2).38 In addition to the two active metabolites (1 and 2), four major metabolites (peaks 2−6) of H. foliolosum were investigated. HRMS analysis of peak 3 (HR-ESIMS(+) m/z 449.0698 [M + H]+ [C20H17O12+, ΔM −3.7 ppm]) revealed the molecular formula of compound 3 to be C20H16O12. After analysis of 1D and 2D NMR data and comparison of chemical shifts with literature values, compound 3 was identified as ellagic acid 4-O-α-L-rhamnopyranoside.39 Peak 4 was identified as rutin (HR-ESIMS(+) m/z 611.1606 [C27H31O16+, ΔM −0.1 ppm]) by comparing HRMS and 1H NMR data with an authentic reference compound. On the basis of HRMS analysis,

peak 5 was assigned the molecular formula C21H20O11 (HRESIMS(+) m/z 449.1075 [C21H22O11+, ΔM −0.8 ppm]). AMX and AX spin systems and a characteristic flavone H-3 resonance (6.63 ppm) observed in the downfield region of the 1H NMR spectrum were in agreement with luteoline aglycone. A discrete spin system observed in COSY was attributed to a glucose moiety. After establishing the glycosidic linkage at C-4′ with 3J HMBC correlations, compound 5 was identified as luteolin 4′O-β-glucopyranoside.40 The 1H NMR spectrum of compound 6 (HR-ESIMS(+) m/z 463.0868 [M + H]+ [C21H19O12+, ΔM 0.7 ppm]) displayed signals and multiplicity patterns similar to those of compound 3, but with an additional methoxy resonance at 4.19 ppm. These observations and comparison of chemical shifts with literature revealed compound 6 as 3-Omethylellagic acid 4-O-α-L-rhamnopyranoside.41 After observation of their significant PM H+-ATPase inhibitory effects in high-resolution screening, chebulagic acid (1) and tellimagrandin II (2) were isolated by preparative-scale HPLC, and the materials were used for assessing IC50 values in the PM H+-ATPase inhibition assay and fungal growth inhibition assays (Table 2). Chebulagic acid and tellimagrandin II had an inhibitory effect against PM H+-ATPase with IC50 values of 15.8 and 10.6 μM, respectively. The two compounds also showed antifungal activity against S. cerevisiae, and were equally potent with MIC of 1.8 μM. This is better than Fluconazole that possess a MIC of 7.6 μM, but not as potent as Amphotericin B with a MIC of 0.08 μM for S. cerevisiae. Both compounds were also tested against C. albicans, but did not show any antifungal activity at concentrations lower than 75 μM, and this is despite conserved PM H+-ATPase among the fungal species (83% sequence 5600

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(6) De Backer, M. D.; Van Dijck, P.; Luyten, W. H. M. L. Functional genomics approaches for the identification and validation of antifungal drug targets. Am. J. PharmacoGenomics 2002, 2, 113−127. (7) Scarborough, G. A. The plasma membrane proton-translocating ATPase. Cell. Mol. Life Sci. 2000, 57, 871−883. (8) Serrano, R. Structure and function of plasma membrane ATPase. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1989, 40, 61−94. (9) Morsomme, P.; Slayman, C. W.; Goffeau, A. Mutagenic study of structure, function and biogenesis of the yeast plasma membrane H+ATPase. Biochim. Biophys. Acta, Rev. Biomembr. 2000, 1469, 133−157. (10) Palmgren, M. G. Plant plasma membrane H+-ATPase: Powerhouses for nutrient uptake. Annu. Rev. Plant Physiol. Plant Mol. Biol. 2001, 52, 817−845. (11) Serrano, R.; Kielland-Brandt, M. C.; Fink, G. R. Yeast plasma membrane ATPase is essential for growth and has homology with (Na+ + K+), K+- and Ca2+-ATPases. Nature 1986, 319, 689−693. (12) Seto-Young, D.; Monk, B.; Mason, A. B.; Perlin, D. S. Exploring an antifungal target in the plasma membrane H+-ATPase of fungi. Biochim. Biophys. Acta, Biomembr. 1997, 1326, 249−256. (13) Soteropoulos, P.; Vaz, T.; Santangelo, R.; Paderu, P.; Huang, D. Y.; Tamás, M. J.; Perlin, D. S. Molecular characterization of the plasma membrane H+-ATPase, an antifungal target in Cryptococcus neoformans. Antimicrob. Agents Chemother. 2000, 44, 2349−2355. (14) Bisson, J.; Waffo-Téguo, P.; Papastamoulis, Y.; Richard, T.; Corio-Costet, M.; Mérillon, J.; Cluzet, S. Phenolics and their antifungal role in grapevine wood decay: focus on the botryosphaeriaceae family. J. Agric. Food Chem. 2012, 60, 11859−11868. (15) Maor, R.; Shirasu, K. The arms race continues: battle strategies between plants and fungal pathogens. Curr. Opin. Microbiol. 2005, 8, 399−404. (16) Monk, B. C.; Perlin, D. S. Fungal plasma membrane proton pumps as promising new antifungal targets. Crit. Rev. Microbiol. 1994, 20, 209−223. (17) Kool, J.; Giera, M.; Irth, H.; Niessen, W. M. A. Advances in mass spectrometry-based post-column bioaffinity profiling of mixtures. Anal. Bioanal. Chem. 2011, 399, 2655−2668. (18) Shi, S.-Y.; Zhou, H.-H.; Zhang, Y.-P.; Jiang, X.-Y.; Chen, X.-Q.; Huang, K.-L. Coupling HPLC to on-line, post-column (bio)chemical assays for high-resolution screening of bioactive compounds from complex mixtures. TrAC, Trends Anal. Chem. 2009, 28, 865−877. (19) Giera, M.; Heus, F.; Janssen, L.; Kool, J.; Lingeman, H.; Irth, H. Microfractionation revisited: a 1536 well high resolution screening assay. Anal. Chem. 2009, 81, 5460−5466. (20) Kesting, J. R.; Johansen, K. T.; Jaroszewski, J. W. Hyphenated NMR techniques. In Advances in Biomedical Spectroscopy; Dingley, A. J., Pascal, S. M., Eds.; IOS Press: Amterdam, Netherlands, 2011; Vol. 3, pp 413−434. (21) Seger, C.; Sturm, S. HPLC-SPE-NMR: A new hyphenation technique. LC-GC Eur. 2007, 11, 587−597. (22) Sprogøe, K.; Stærk, D.; Jäger, A. K.; Adsersen, A.; Hansen, S. H.; Witt, M.; Landbo, A.-K. R.; Meyer, A. S.; Jaroszewski, J. W. Targeted natural product isolation guided by HPLC-SPE-NMR: Constituents of Hubertia species. J. Nat. Prod. 2007, 70, 1472−1477. (23) Staerk, D.; Kesting, J. R.; Sairafianpour, M.; Witt, M.; Asili, J.; Emami, S. A.; Jaroszewski, J. W. Accelerated dereplication of crude extracts using HPLC-PDA-MS-SPE-NMR: Quinolone alkaloids of Haplophyllum acutifolium. Phytochemistry 2009, 70, 1055−1061. (24) Johansen, K. T.; Wubshet, S. G.; Nyberg, N. T.; Jaroszewski, J. W. From retrospective assessment to prospective decisions in natural product isolation: HPLC-SPE-NMR analysis of Carthamus oxyacantha. J. Nat. Prod. 2011, 74, 2454−2461. (25) Sprogøe, K.; Stærk, D.; Ziegler, H. L.; Jensen, T. H.; HolmMøller, S. B.; Jaroszewski, J. W. Combining HPLC-PDA-MS-SPENMR with circular dichroism for complete natural product characterization in crude extracts: Levorotatory gossypol in Thespesia danis. J. Nat. Prod. 2008, 71, 516−519. (26) Pukalskas, A.; van Beek, T. A.; de Waard, P. Development of a triple hyphenated HPLC-radical scavenging detection-DAD-SPE-

identity; sequence alignment of C. albicans PMA1 (accession number P28877) and S. cerevisiae H+-ATPase PMA1 (accession number P05030) made with ClustalW2). Furthermore, the compounds have a higher IC50 value against the PM H+ATPase than MIC value against S. cerevisiae. Taken together, this could indicate that not only the inhibition of PM H+ATPase contributes to the antifungal effect of chebulagic acid and tellimagrandin II, but other factors may also play a role. Hydrolyzable tannins, including tellimagrandin II, have been shown to have membrane-damaging activity on liposomes prepared from egg-yolk phosphatidylcholin, but not affecting the viability of MKN-28 cells derived from human gastric epithelium.42 It remains to be determined if chebulagic acid and tellimagrandin II, in addition to their PM H+-ATPase inhibitory effect, have membrane-damaging activity on S. cerevisiae, which contributes to the observed antifungal activity. In the literature, chebulagic acid has been identified as an active inhibitor of xanthione oxidase with an IC50 = 48.5 μM43 and with both antibacterial and antifungal effect in doses of 100 μM. In conclusion, chebulagic acid and tellimagrandin II are two potent inhibitors of the plasma membrane H+-ATPase with inhibitory effect on the growth of S. cerevisiae. Further studies on isolated and/or semisynthetic analogues are planned to investigate the structure−activity relationship and specificity.

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ASSOCIATED CONTENT

S Supporting Information *

Semi-high-resolution biochromatograms of selected plants, and UV, HRMS, and NMR data of 1−6. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +45 3533 6177. Fax: +45 35336001. E-mail: ds@sund. ku.dk. Author Contributions §

These authors contributed equally.

Funding

K.T.K. and S.G.W. supported by The Danish Research Council for Strategic Research − Food and Health. HPLC equipment used for high-resolution bioassay profiles was obtained via a grant from The Carlsberg Foundation. The 600 MHz HPLCHRMS-SPE-NMR system used in this work was acquired through a grant from “Apotekerfonden af 1991”, The Carlsberg Foundation, and the Danish Agency for Science, Technology and Innovation via the National Research Infrastructure funds. Notes

The authors declare no competing financial interest.

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