Triterpenoic Acids from Apple Pomace Enhance the Activity of the

Article pubs.acs.org/JAFC

Triterpenoic Acids from Apple Pomace Enhance the Activity of the Endothelial Nitric Oxide Synthase (eNOS) Katharina Waldbauer,† Günter Seiringer,† Dieu Linh Nguyen,† Johannes Winkler,‡ Michael Blaschke,† Ruxandra McKinnon,† Ernst Urban,‡ Angela Ladurner,† Verena M. Dirsch,† Martin Zehl,*,†,‡ and Brigitte Kopp† †

Department of Pharmacognosy and ‡Department of Pharmaceutical Chemistry, University of Vienna, Althanstrasse 14, 1090 Vienna, Austria S Supporting Information *

ABSTRACT: Pomace is an easy-accessible raw material for the isolation of fruit-derived compounds. Fruit consumption is associated with health-promoting effects, such as the prevention of cardiovascular disease. Increased vascular nitric oxide (NO) bioavailability, for example, due to an enhanced endothelial nitric oxide synthase (eNOS) activity, could be one molecular mechanism mediating this effect. To identify compounds from apple (Malus domestica Borkh.) pomace that have the potential to amplify NO bioavailability via eNOS activation, a bioassay-guided fractionation of the methanol/water (70:30) extract has been performed using the 14C-L-arginine to 14C-L-citrulline conversion assay (ACCA) in the human endothelium-derived cell line EA.hy926. Phytochemical characterization of the active fractions was performed using the spectrophotometric assessment of the total phenolic content, as well as TLC, HPLC-DAD-ELSD, and HPLC-MS analyses. Eleven triterpenoic acids, of which one is a newly discovered compound, were identified as the main constituents in the most active fraction, accompanied by only minor contents of phenolic compounds. When tested individually, none of the tested compounds exhibited significant eNOS activation. Nevertheless, cell stimulation with the reconstituted compound mixture restored eNOS activation, validating the potential of apple pomace as a source of bioactive components. KEYWORDS: apple pomace, Malus domestica Borkh., endothelial nitric oxide synthase (eNOS), triterpenoic acids, total phenolic content

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juices.10,11 The pomace, however, still represents a rich source of potentially bioactive apple ingredients.12 The acute influence of apple consumption on endothelial function was investigated in two intervention studies but remains controversial. In hypercholesterolemic adults, neither the consumption of polyphenol-rich apples nor the consumption of low-polyphenol-content apples had an influence on brachial flowmediated dilatation (brFMD).13 In contrast, a significant increase of the brFMD as well as the concentration of plasma S-nitrosothiols, nitrite, total nitrogen oxides and other nitrosylated species, which all are indicators for enhanced in vivo NO bioavailability, was observed in healthy subjects consuming whole apples (considered as flavonoid-rich).14 Furthermore, a Finnish cohort study, in which apples were a primary flavonoid source, associated the increased flavonoid consumption with a risk reduction in coronary mortality.15 There also exists growing evidence that single fruit compounds, which can be isolated from apple waste, beneficially influence the vascular NO status. For instance, the consumption of quercetin or (−)-epicatechin increased plasma S-nitrosothiols, plasma nitrite, and urinary nitrite in

INTRODUCTION Epidemiological studies correlate the consumption of fruits and vegetables with the prevention of cardiovascular diseases. Fruit components seem to mediate cardiovascular protective effects by diverse molecular mechanisms, of which maintenance of nitric oxide (NO) homeostasis may be one.1,2 This is achieved either via the reduction of NO degradation, for instance, by decreasing the level of reactive oxygen species (ROS, e.g., O2•−), or via the increase of its production by the endothelial nitric oxide synthase (eNOS).3 eNOS activation in endothelial cells is dependent on an increase of the intracellular Ca2+ concentration, which leads to the formation of the Ca2+/calmodulin complex. This triggers the dimerization of two inactive eNOS monomers, which again facilitates the e−-transfer within the enzyme, resulting in the conversion of L-arginine to L-citrulline in two consecutive mono-oxygenation reactions under the production of NO.4 eNOS activity is dependent on substrate and cofactor availability and can be enhanced by transcriptional, posttranscriptional, translational, or posttranslational mechanisms as well as by diverse protein−protein interactions.5−8 Endothelium-released NO mediates vascular relaxation via the induction of soluble guanylyl cyclase (sGC). Furthermore, NO decreases platelet adhesion and aggregation as well as vascular smooth muscle cell proliferation and vascular inflammation.9 During apple juice production, predominantly the polar, water-soluble fruit components pass to the respective © 2015 American Chemical Society

Received: Revised: Accepted: Published: 185

October 19, 2015 December 18, 2015 December 18, 2015 December 18, 2015 DOI: 10.1021/acs.jafc.5b05061 J. Agric. Food Chem. 2016, 64, 185−194

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Journal of Agricultural and Food Chemistry

After 2 h of incubation with resazurin, fluorescence intensity at 580 nm (excitation wavelength, 535 nm) was measured on a Tecan Genios Pro (Männedorf, Switzerland). Total Phenolic Content of Apolar Fractions (APF). This value was assessed on the basis of the Folin−Ciocalteu method as described previously.21 The quantification was performed by external standardization with gallic acid (Phytolab), dissolved in either MeOH or water, in two concentration ranges each [water, r2 (2.5−50 mg/L) = 0.9931; r2 (50−500 mg/L) = 0.9932); or methanol, r2 (2.5−50 mg/L) = 0.9970; r2 (50−250 mg/L) = 0.9932)]. Analyses were performed in triplicate. Identification and Quantification of the Main Compounds by HPLC. The separation was performed on an Agilent Hypersil BDS C18, 4.0 × 250 mm, 5 μm column, using water (pH 2.8 with formic acid) and acetonitrile (modified with the same amount of formic acid) as mobile phases A and B, respectively. HPLC method I, optimized for the analysis of the main phenolic compounds, was performed at 20 °C at a flow rate of 1 mL/min as follows: in 8 min from 15 to 19% B, 10 min at 19% B, and in 30 min from 19 to 90% B. HPLC method II, applied to analyze the triterpenoic acids, started at 45% B followed by a linear increase to 80% B in 40 min, using a column oven temperature of 25 °C. After each run, the column was washed with 90 or 95% B for 10 min, followed by a 10 min re-equilibration step prior to the next injection. LC-MS was carried out on an UltiMate 3000 RSLC-series system (Dionex/Thermo Fisher Scientific, Germering, Germany) coupled to an HCT 3D quadrupole ion trap mass spectrometer equipped with an ESI source (Bruker Daltonics, Bremen, Germany). Before MS analysis, the eluate flow was split 1:4. The ESI ion source was operated as follows: capillary voltage, +3.5/−3.7 kV; nebulizer, 26 psi (N2); dry gas flow, 9 L/min (N2); and dry temperature, 340 °C. Positive and negative ion mode multistage mass spectra up to MS4 were obtained in automated data-dependent acquisition mode. Identification of the respective peaks was performed using an in-house MS spectra database and standard addition experiments (Figures S33−S37). Quantitative analysis of the main phenolic compounds was carried out on a PerkinElmer HPLC series 200 equipped with a UV/vis detector and TotalChrom software (Waltham, MA, USA). Assessed parameters of external calibration at the wavelength 280 nm were, for quercetin, y = 9,617,718x − 143,093 (r2 = 0.9949); LOD (S/N ∼ 3) = 1.3 μg/mL; LOQ (S/N ∼ 10) = 5.0 μg/mL; and for phloridzin, y = 12,321,275x − 63,185; LOD (S/N ∼ 3) = 1.6 μg/mL; LOQ (S/N ∼ 10) = 2.1 μg/mL. Correction factors for the quantification of quercetin derivatives were calculated to compensate for the differences in their molecular weights. Standard addition and quantification of triterpenoic acids were performed on a Shimadzu HPLC series 20 system (Kyoto, KY, Japan) equipped with a diode array detector and coupled to a Shimadzu ELSD-LT detector (nebulizer temperature, 40 °C; nebulizer pressure, 350 kPa; gain, 8). Assessed parameters for external calibration at the wavelength 210 nm for ursolic acid were y = 2,262,153x + 12,187 (r2 = 0.9993); LOD (S/N ∼ 3) = 7.5 μg/mL; LOQ (S/N ∼ 10) = 25.0 μg/ mL. Correction factors for the quantification of other triterpenoic acids were determined experimentally by comparison of the peak areas from equally concentrated solutions of ursolic acid and the respective triterpenoic acid at the 210 nm wavelength (annurcoic acid, 1.3; corosolic acid, 1.4; cuneataol, 1.9; euscaphic acid, 1.2; maslinic acid, 1.0; oleanolic acid, 1.0; pirolonic acid, 1.4; pomaceic acid, 2.3; pomolic acid, 1.0). All quantitative analyses were performed in triplicates. Isolation of Triterpenoic Acids from Apple Pomace. As not all of the compounds needed for the identification of the peaks in bioactive fractions were commercially available, apple triterpenoic acids were isolated. An apple pomace sample from Obstgut Stift Klosterneuburg (September 2011) was extracted with EtOAc under conditions similar to those described above for the MeW extract. The triterpenoic acids were isolated from the EtOAc extract by column chromatography (CC) on silica gel and subsequent reversed-phase semipreparative HPLC with chloroform/methanol/H2O and water/ acetonitrile mixtures as mobile phases, respectively (see the Supporting Information, Isolation of triterpenoic acids from apple

healthy men, indicating an enhanced in vivo bioavailability of NO.16,17 The 14C-L-arginine to 14C-L-citrulline conversion assay (ACCA) is a robust tool for the detection of eNOS activity in human endothelial cells that are stimulated with plantderived extracts.18 To evaluate whether apple waste is a source for eNOS-activating compounds, we performed a bioassayguided fractionation of apple pomace extracts, followed by a detailed phytochemical analysis of the active fractions as well as the assessment of the eNOS-activating potential of their main compounds.

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MATERIALS AND METHODS

Chemicals. Methanol (MeOH) and hexane (HEX), both AnalaR Normapur, ethyl acetate (EtOAc), dichloromethane (DCM), and chloroform (CHCl3), all GPR Rectapur, as well as acetonitrile (ACN) HiPerSolv Chromanorm were obtained from VWR (Vienna, Austria). Ethanol 96% (v/v) (EtOH) was from Brenntag CEE (Vienna, Austria), and formic acid (purity > 98.0%) and concentrated ammonia (24%) were from Gatt-Koller GmbH (Absam, Austria). Water, for extraction and phytochemical analysis, was deionized and distilled. Quercetin-3-O-glucoside (purity = 95%), phloridzin (purity = 93%), maslinic acid (purity = 89%), corosolic acid (purity = 90%), oleanolic acid (purity = 94%), and ursolic acid (purity = 94%) were provided from Phytolab (Vestenbergsgreuth, Germany). Quercetin-3-O-rutinoside (purity = 99%, HPLC) and quercetin-3-O-galactoside (purity = 98%, HPLC) were from Extrasynthèse (Lyon, France), and quercetin (purity > 95%, HPLC) was from Sigma-Aldrich (St. Louis, MO, USA). Pomolic acid (purity = 98%, HPLC) and tormentic acid (purity = 97%, HPLC) were obtained from Quality Phytochemicals LLC (East Brunswick, NJ, USA). Euscaphic acid was isolated (see below) and additionally obtained from BioBioPha (purity = 97%, NMR, Kunming, China). Extraction of Apple Pomace and Bioassay-Guided Fractionation. Dry apple pomace was obtained from HAAS Wildfutter (St. Leonhard/Forst, Austria) in 2011. Extraction was performed on a Dionex ASE 200 (Dionex/Thermo Scientific Austria, Vienna, Austria). The instrument was equipped with 11 mL stainless steel extraction cells and 60 mL glass collection bottles. Extraction parameters for the extraction with solvents of different polarities [MeOH/H2O (70:30) (MeW), EtOAc, DCM, and HEX] were as follows: 40 °C, 1500 psi, three extraction cycles, 5 min heat-up time, 5 min static extraction time, 0.6% flush volume, and 60 s nitrogen purge. For bioassay-guided fractionation, a further 207 g of apple pomace was extracted with MeW using 22 mL extraction cells. The obtained extract was fractionated on an 85 × 6 cm styrene−divinylbenzene matrix column (DIAION-HP 20 from Sigma-Aldrich). The elution with 18 L eluent 1 [EtOH 96% (v/v)/H2O (10:90)] led to the formation of five cumulative polar fractions (PF1−PF5) and the subsequent elution with 5 L of eluent 2 [EtOH 96% (v/v)] to five cumulative apolar fractions (APF1−APF5). Assessment of Endothelial Nitric Oxide Synthase and Metabolic Activity of EA.hy926 Cells. The immortalized human endothelial cell line EA.hy926 was kindly provided by Dr. C.-J. S. Edgell (University of North Carolina, Chapel Hill, NC, USA). Cell culture and experiments were performed as described previously.18 Cells were stimulated for 24 h with the respective fraction, compound, or compound mixture using the indicated concentrations. Before stimulation, 100 U of bovine liver catalase (Sigma-Aldrich) per milliliter of cell culture medium was added to avoid false-positive results due to H2O2 production in the cell culture media due to phenolic compounds present in the test samples.19 The ACCA indirectly measures the production of NO by human endothelium-derived cell lines and was performed as described previously.18 Cellular metabolic activity was assessed by the resazurin conversion assay.20 Viable, metabolically active cells reduce the nonfluorescent dye resazurin (Sigma-Aldrich) to the highly fluorescent resorufin. The fluorescence intensity linearly correlates to the number of viable cells. 186

DOI: 10.1021/acs.jafc.5b05061 J. Agric. Food Chem. 2016, 64, 185−194

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Journal of Agricultural and Food Chemistry pomace EtOAc extract). Structure-elucidation was performed by MS and spectroscopic methods in reconciliation of published literature (see the Supporting Information, Structure elucidation).22−25 Euscaphic acid [(2α,3α)-2,3,19-trihydroxy-urs-12-en-28-oic acid; purity = 95.1% (ELSD); 1.7 mg], cuneataol [(2β,3β)-2,25-epoxy2,3,19-trihydroxy-urs-12-en-28-oic acid; purity = 96.5% (ELSD), 2.3 mg], annurcoic acid [(1α)-1,19-dihydroxy-3-oxo-urs-12-en-28-oic acid; purity = 93.2% (ELSD), 1.8 mg], and pirolonic acid [(3β)-3,19dihydroxy-2-oxo-urs-12-en-28-oic acid; purity = 88.4% (ELSD); 1.8 mg] were further used in this study. Isolation of Compound 3 from APF3. One hundred and seven milligrams of APF3 was separated on a 6 cm3 Varian Bond Elut SPE C18 cartridge (Varian/Agilent Technologies, Santa Clara, CA, USA). Fraction A and fraction B were eluted with two reservoir volumes (RV) of water/acetonitrile (60:40) and water/acetonitrile (40:60), respectively, and fraction C was eluted with 4 RV of acetonitrile. An enrichment of compound 3 was detected by HPLC method II in fraction B. The fraction (38 mg of yellowish-white powder) was further separated by liquid−liquid partition in a four-solvent biphasic system of HEX/EtOAc/MeOH/H2O (1:1:1:1). Eighteen milligrams of a white powder was recovered from the upper phase after solvent evaporation. As compound 3 showed about 1:1 partition between the upper and lower phases of the HEX/EtOAc/MeOH/H2O (2:3:2:3) biphasic system, this solvent mixture was used for high-performance counter current chromatography (HPCCC) on a Dynamic Extractions Ltd. instrument (Slough, UK) equipped with a 40 mL analytical column and a 3 mL sample loop. The instrument was operated with a spinning rate of 1600 rpm and a flow rate of 1 mL/min. The HEXdominated upper phase was used as the mobile phase. With a retention volume of 27−32 mL, 5.7 mg of compound 3 (purity = 86.7%, ELSD) was eluted. High-Resolution Mass Spectrometry (HRMS). HRESIMS spectra were obtained on a maXis HD ESI-Qq-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany) in the positiveion mode using direct infusion. The sum formulas were determined using Bruker Compass DataAnalysis 4.2 based on the mass accuracy (Δm/z ≤ 2 ppm) and isotopic pattern matching (SmartFormula algorithm). Nuclear Magnetic Resonance (NMR). Spectra were recorded on a Bruker Avance 500 NMR spectrometer (UltraShield) using a 5 mm switchable probe (PA BBO 500SB BBF-H-D-05-Z, 1H, BB = 19F and 31 P − 15N) with z-axis gradients and automatic tuning and matching accessory (Bruker BioSpin). The resonance frequency for 1H NMR was 500.13 MHz and that for 13C NMR, 125.75 MHz. All measurements were performed for a solution in fully deuterated methanol at 298 K. Standard 1D and gradient-enhanced (ge) 2D experiments, such as double quantum filtered (DQF) COSY, NOESY, HSQC, and HMBC, were used as supplied by the manufacturer. Chemical shifts are referenced internally to the residual, nondeuterated solvent signal for methanol 1H (δ 3.31) and to the carbon signal of methanol 13C (δ 49.00). Circular Dichroism. Spectra were measured on a J-810 CD spectrometer from Jasco (Tokyo, Japan), equipped with a 0.1 cm cell. The instrument was operated with a scanning speed of 100 nm/min and a response of 4 nm at 20 °C using MeOH as solvent. Statistical Analysis. Raw data from the ACCA were normalized to the positive control (PC, ascorbic acid, 100 μM), whereas raw data from the resazurin assay were normalized to the negative control (NC, solvent vehicle treated cells). Normalized data were incorporated in Graph Pad Prism version 4.0 software, and significant differences to the NC were assessed using one-way ANOVA/Dunnett’s post-test (∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001). Shown results are the mean ± SD of three independent experiments.

immortalized endothelium-derived cell line EA.hy926. For that, extracts with solvents of different polarities were prepared from apple pomace by ASE. As shown in Figure 1a, none of the

Figure 1. Biological activities of apple pomace extracts (MeW, EtOAc, DCM, HEX) (a) and apple pomace apolar fractions (APFs) (b). NC, negative control (solvent vehicle-treated cells); PC, positive control (100 μM ascorbic acid); statistical analysis, one-way ANOVA (Dunnett’s post-test compared to NC: ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001). eNOS activity in EA.hy926 cells was evaluated after 24 h of stimulation with 50 μg/mL extract or APF, assessed by 14C-Larginine to 14C-L-citrulline conversion assay (ACCA). Results indicate 14 C production normalized to PC (mean ± SD, n = 3, black columns). Metabolic activity of EA.hy926 cells was evaluated after 24 h of stimulation with 50 μg/mL of extract or APF, assessed by resazurin assay. Results show resorufin production normalized to NC (mean ± SD, n = 3, gray columns).

obtained extracts was able to enhance eNOS activity in EA.hy926 cells significantly. As the extracts are complex mixtures of various phytochemicals, the activity of potentially bioactive compounds may be masked due to the presence of inactive bulk compounds. Via fractionation of pomace extracts, bioactive compounds may be enriched. These fractions might be used for nutraceutical purposes or for the isolation of pure bioactive compounds. For the evaluation of this hypothesis, the apple pomace methanol/water (70:30) (MeW) extract was chosen for fractionation for the following reasons: (a) cell metabolic activity was not affected by this extract (Figure 1a); (b) methanol has dissolving properties comparable to those of ethanol, a solvent that obliges minor restrictions in the food and pharmaceutical industries. Activities of Polar and Apolar Fractions from Apple Pomace Methanol/Water Extract on eNOS. Thirty-eight milligrams of the MeW extract were fractionated on styrene− divinylbenzene (DIAION-HP 20) resulting in five polar (PF1− PF5) and five apolar (APF1−APF5) cumulative fractions that underwent bioassay testing. PF1−PF5, obtained by elution with

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RESULTS AND DISCUSSION Biological Activities of Apple Pomace Extracts. Apple pomace could be a valuable, easily accessible source for bioactive compounds.26 We tested this hypothesis by investigating its ability to enhance eNOS activation in the 187


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Journal of Agricultural and Food Chemistry EtOH 96% (v/v)/H2O (10:90), did not exhibit any influence on eNOS or metabolic activity in EA.hy926 cells (data not shown). APF3 and APF4, eluted with EtOH 96% (v/v), showed a significant increase of eNOS activity in EA.hy926 cells, whereas their metabolic activity was not affected significantly (Figure 1b). Phytochemical Characterization of Apple Pomace Apolar Fractions. In previous studies, the cardiovascular protective effects of apples were mainly associated with polyphenolic compounds.15,27,28 Thus, it was evaluated whether there exists a correlation between the total phenolic content (TPC) of the apple pomace fractions and their eNOSactivating potential in EA.hy926 cells. APF2 exhibited the highest content of total phenolics (77.2 mg/g, as gallic acid equivalents, GAE) assessed by Folin−Ciocalteu method (Table 1), but did not show any influence on eNOS activity (Figure

Table 2. Quantification of Quercetin Derivatives and Phloridzin in APFs Using External Standardization by HPLC-DAD at 280 nm

Table 1. Total Phenolic Content of APFs Measured by the Folin−Ciocalteu Method

a

mg/g apple pomace apolar fractiona APF2 quercetin-3-rutinoside quercetin-3-galactoside quercetin-3-glucoside quercetin-pentoside 1 quercetin-pentoside 2 quercetin-pentoside 3 quercetin-pentoside 4 quercetin-desoxyhexoside quercetin Σ quercetin derivatives phloridzin

2.8 16.0 4.8 6.1 3.5 1.2 13.4 13.4 ndb 61.2 28.7

± ± ± ± ± ± ± ±

APF3 0.8 5.1 1.5 1.8 0.7 0.2 4.1 0.4

1.6 6.7 2.4 3.5 1.6 1.2 4.5 7.0 9.9 38.4 0.6

± 9.1

± ± ± ± ± ± ± ± ±

0.1 0.1 0.2 0.3 0.1 0.1 0.2 0.1 0.5

± 0.1

Results are reported as the mean ± SD (n = 3). nd, not detected. b

gallic acid equivalents (GAE) (mg/g) fraction

mean

SDa

% RSDb

APF1 APF2 APF3 APF4 APF5

16.6 77.2 62.8 13.3 18.2

0.3 1.4 0.5 0.5 0.3

1.5 1.8 0.7 3.4 1.6

a

Standard deviation. bRelative standard deviation as percentage of mean. n = 3.

1b). APF3, however, with a marginally lower TPC of 62.8 mg/g GAE, showed a significant eNOS activation, as did APF4 with a TPC of only 13.3 mg/g GAE (Figure 1b; Table 1). Thus, no correlation between the eNOS-activating effects and the polyphenol content of the APFs was found in this study. These results did not rule out the influence of specific phenolic compounds on eNOS activity. Quercetin-glycosides and phloridzin have been identified as the main monomeric phenolic compounds present in apple pomace.29 Quercetin is able to enhance eNOS activity in bovine aortic endothelial cells (BAEC) via increased eNOS Ser1179 phosphorylation.30,31 Thus, we evaluated whether quercetin or its glycosylated derivatives identified in the APFs are responsible for the APFinduced eNOS activation in EA.hy926 cells. Eight quercetinglycosides, quercetin, and phloridzin were detected by HPLCMS in the APFs (Table S12). Their content was assessed by HPLC-UV detection using external standardization (HPLC method I). The total concentrations of quercetin derivatives and phloridzin in APF2 and APF3 were 89.9 and 39.0 mg/g, respectively (Table 2), which confirmed the trend determined by the Folin−Ciocalteu method (Table 1). APF2, which did not increase eNOS activity in EA.hy926 cells, solely contained quercetin-glycosides and phloridzin. In the significantly eNOSactivating fraction APF3, 9.9 mg/g of quercetin-aglycon was present (Table 2). This corresponds to a final quercetin concentration of 1.6 μM if the cells are stimulated with a concentration of 50 μg/mL of APF3. However, a significant eNOS activation in EA.hy926 cells needed a quercetin concentration of at least 25 μM, when determined by the ACCA (Figure 2). It can be concluded that the quercetin concentration in the APF3 solution (50 μg/mL) was too low to account for the significant eNOS activation mediated by this

Figure 2. eNOS activation by quercetin assessed by the ACCA. EA.hy926 cells were stimulated for 24 h with the solvent vehicle (NC, negative control), 100 μM ascorbic acid (PC, positive control), or the indicated quercetin concentrations (μM). Bars represent 14C-Lcitrulline production normalized to PC (mean ± SD, n = 3). Statistical analysis: one-way ANOVA (Dunnett’s post-test compared to NC: ns, not significant; ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001).

fraction. Thus, the evaluation of the eNOS-activating properties of the main compounds in APF3 was continued. Using evaporative light scattering detection (ELSD), further prominent compounds were detected (Figure 3). HPLC-MS analysis revealed the presence of numerous, partly isomeric, triterpenoic acids (Table 3). Three compounds had a molecular weight of 472.2 Da. Pomolic acid (7) and maslinic acid (8), exhibiting this molecular weight, were previously isolated from apple,32,33 and their presence could be confirmed in APF3 (Figures S45 and S46; Tables S16 and S17). The third dihydroxytriterpenoic acid was identified as corosolic acid (9) (Figure S47; Table S17). The molecular weight and MS fragmentation pattern of compound 5 corresponded to annurcoic acid, which was isolated for the first time from Malus domestica (Borkh.) cv. Annurca in 200624 and was later also found in apple peels from other cultivars.34 Annurcoic acid was not available as a commercial authentic standard. Therefore, annurcoic acid (5) and its low-abundant isomer pirolonic acid (6) were isolated from the apple pomace EtOAc extract (see the Supporting Information, Isolation of triterpenoic acids from apple pomace EtOAc extract). Their structure was confirmed by NMR, MS, and CD experiments 188


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Figure 3. ELSD chromatogram of APF3 using HPLC method II for the separation of triterpenoic acids. Peak numbers correspond to compounds listed in Table 3, which were identified by LC-MS and chromatographic techniques. Column, Hypersil BDS C18 (250.0 × 4.0 mm; 5 μm); mobile phase A, H2O, pH 2.8 (HCOOH); mobile phase B, ACN (HCOOH).

Table 3. Triterpenoic Acids Identified in APFs peaka

tRb (min)

mol wtc (Da)

1 2 3 4

6.6 7.1 7.9 8.8

502.3 502.3 502.2 488.3

5 6 7 8 9 10 11

14.3 14.6 17.7 19.5 20.5 35.5 35.9

486.3 486.3 472.3 472.2 472.2 456.3 456.3

identified compd

APF3 (mg/g)

APF4 (mg/g)

identified byd

25.5 ± 2.3 232.2 ± 5.2 139.4 ± 4.3

nd 28.7 ± 2.1 16.6 ± 1.1

# * #

± ± ± ± ± ± ±

# # # # # # #

e

ni cuneataol pomaceic acid euscaphic acid tormentic acid annurcoic acid pirolonic acid pomolic acid maslinic acid corosolic acid oleanolic acid ursolic acid

65.6 5.3 35.0 17.4 14.5 9.6 7.9

± ± ± ± ± ± ±

2.0 0.8 3.2 0.8 1.1 0.3 0.3

180.4 31.7 15.1 14.6 19.2 20.4 17.1

3.1 0.3 0.6 0.3 0.6 0.6 0.6

a Peak numbers correspond to peaks in Figure 3. bRecorded during LC-DAD-ELSD analysis (HPLC II). cDetermined experimentally. d#, chromatographic techniques; *, isolation from APF3. eni, not identified.

Compound 3 could not be identified by literature research and standard addition experiments. Isolation and Structure Elucidation of Compound 3. Compound 3 (5.7 mg) was isolated from APF3 using solid phase extraction (SPE) and HPCCC. Purity assessment performed by HPLC-ELSD analysis resulted in 86.7%. The high-resolution ESI-MS experiment displayed an [M + H]+ ion at m/z 503.3370 corresponding to the molecular formula of C30H46O6 (calcd m/z 503.3367 for the [M + H]+). In the 1H NMR spectrum of compound 3, signals from six methyl groups were detected; five of them were singlets and one was a doublet coupled by 6.9 Hz (H-30). In addition, completely separated signals of four deep-field-shifted protons were registered. Two of them showed typical shifts (δ 3.73 and 4.21) for geminal coupled carbinol protons, another carbinol hydrogen showed two vicinal coupling partners, and the third was identified as an olefinic hydrogen (δ 5.31). The 13C NMR spectrum of compound 3, which was measured in attached proton test (APT) mode, exhibited signals for 30 well-separated carbons

(see the Supporting Information, Structure elucidation). Chromatographic comparison with APF3 and addition experiments with the isolated compounds 5 and 6 (Figures S43 and S44; Table S15) confirmed their presence in APF3. Using the same procedure, cuneataol (2) was identified (Figure S39; Table S13). For the two most enriched compounds in APF3 (Figure 3), the molecular weights of 502.2 (3) and 488.3 Da (4) were measured (Table 3). A compound comprising the molecular weight of 488.35 Da was detected previously in an apple pomace ethanolic extract, and the authors of this study hypothesized that this compound is euscaphic acid.12 Standard addition experiments performed with the two isomers euscaphic (4a) and tormentic (4b) acid, the latter having been found in several members of the family of Rosaceae, showed that both compounds were coeluting in the reversedphase HPLC method II. However, a separation of the two isomers could be achieved by normal-phase TLC (Figures S41 and S42; Table S14). According to TLC analysis, euscaphic acid seemed to be the dominant compound present in APF3. 189


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acids were individually tested for their potential to enhance eNOS activity in EA.hy926 cells. In addition, their impact on the metabolic activity (as a measure of cell viability) of EA.hy926 cells was evaluated by the resazurin assay. The most abundant compounds in APF3, pomaceic acid (3), euscaphic acid (4a), and annurcoic acid (5), did not affect cell metabolic activity in the tested concentration range from 1 to 80 μM (Table 5). The minor compounds tormentic acid (4b) and pomolic acid (7) revealed IC50 values of 55.2 and 16.5 μM, respectively, whereas cuneataol (2) and pirolonic acid (6) did not affect metabolic activity up to 80 μM. Maslinic acid (8), corosolic acid (9), oleanolic acid (10), and ursolic acid (11) inhibited cell metabolic activity at significantly lower test concentrations (Table 5). It can be hypothesized that 3β-OHursan-type triterpenoic acids favor inhibition of cell metabolism compared to 3β-OH-olean-type triterpenoic acids. Hydroxy groups on positions 2 and 19 seem to lower inhibiting effects on cell metabolism as does the α-conformation of the hydroxy group in position 3 (Figure 4; Table 5). Surprisingly, none of the triterpenoic acids identified in APF3 was able to increase 14C-L-citrulline production when tested as a single compound (see the Supporting Information, Biological activities). Thus, even for ursolic acid (11) no positive modulation of eNOS activity was observed in EA.hy926 cells in this study, which is in contrast to in vitro data from the literature.36,37 On the other hand, an induction of eNOS activity by ursolic acid was observed at concentrations >7.5 μM (data not shown). However, these concentrations already caused a significant decrease of cell metabolic activity. Therapeutic relevance of ursolic acid (11) stays ambiguous with regard to in vivo data. For diabetic mice and Dahl-saltsensitive/insulin-resistant rats, a decrease of atherosclerotic plaque formation and systolic blood pressure was determined under ursolic acid (11) treatment,38,39 whereas increased atherosclerotic plaque formation was observed in ApoE−/− mice under ursolic acid (11) treatment.40 Oleanolic acid (10) was the only tested triterpenoic acid that exhibited at least a nonsignificant trend of eNOS activation in EA.hy926 cells (Figure S59), which could be mediated by enhanced eNOS expression and Ser1177 phosphorylation of eNOS, according to the literature.41 In vivo relevance for eNOS activating properties of oleanolic acid (10) seem to be given. A high-fat diet containing 15% of pomace olive oil enriched in oleanolic acid (10) was able to improve endothelial function of Wistar Kyoto and spontaneous hypertensive rats compared to the control groups under a corn oil diet or pomace olive oil diet without the supplementation of oleanolic acid.42 For the other triterpenoic acids present in APF3, to the best of our knowledge, in vivo data are lacking. Nevertheless, pomolic acid (7), isolated from Licania pittieri (Prance), was identified to elicit endothelium-dependent relaxation in in vitro experiments performed with rat aortic rings.43 Equivalent results are reported for maslinic acid (8), isolated from olive pomace.44 Furthermore, a methanolic extract from the Mexican hawthorn Crataegus gracilior (Phipps), which was enriched in ursolic acid (11) and corosolic acid (9) derivatives, mediated an endothelium-dependent aortic relaxant activity.45 Reconstitution of APF3. None of the individual compounds identified in the apple pomace-derived APF3 was able to induce a significant eNOS activation in EA.hy926 cells at relevant concentrations. Thus, we hypothesized that the eNOS-activating potential of the fraction must be elicited by the additive effects of the compound mixture present in APF3.

giving characteristic shift values for an urs-12-en-28-oic acid skeleton. Comparison with cuneataol (2) (Table 4) revealed a Table 4. Comparison of 1H and 13C NMR Data of Pomaceic Acid and Cuneataol in CD3OD 13

H, δ pomaceic acid

1

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

2.60 (1H, dd, J = 13.9 and 10.4 Hz) 1.11 (1H, m) 3.84 (1H, dd, J = 10.4 and 3.8 Hz)

1.36 1.51 1.40 1.51

(3H, (2H, (1H, (1H,

m) m) m) m)

1.87 (1H, dd, J = 12.0 and 5.4 Hz) 2.11 (1H, ddd, J = 18.6, 5.4, and 3.2 Hz) 1.77 (1H, m) 5.31 (1H, s, br)

1.76 1.09 2.55 1.52

(1H, m) (1H, m) (1H, dd, J = 13.8 and 4.7 Hz) (1H, m)

2.51 (1H, s) 1.34 1.24 1.61 1.21 0.97 4.21 3.73 0.76 1.37

(1H, (1H, (1H, (3H, (3H, (1H, (1H, (3H, (3H,

m) m), 1.72 (1H, m) m), 1.74 (1H, m) s) s) dd, J = 8.2 and 0.3 Hz) d, J = 8.2 Hz) s) s)

1.18 (3H, s) 0.93 (3H, d, J = 6.90 Hz)

C, δ pomaceic acid

13 C, δ cuneataol

46.49

41.56

73.84 98.80 40.68 51.60 20.71 32.72

106.57 81.55 39.27 51.95 21.39 33.37

40.08 42.16 37.17 25.08

40.42 41.03 49.45 24.54

129.42 140.12 42.85 29.73

128.79 140.03 42.22 29.95

26.77

26.72

49.30 55.66 73.37 43.09 27.27 38.74 27.44 20.85 67.64

49.39 55.42 73.55 43.10 27.32 38.89 28.48 25.38 67.36

17.93 24.16 182.21 26.99 16.55

17.06 24.03 182.63 27.04 16.60

high similarity of signals in rings B, C, D, and E,35 but remarkable differences of signals in ring A. Both compounds contain a secondary carbinol, a hemiacetal carbon, and an epoxy bridge. Structural differences between cuneataol (2) and compound 3 were elucidated by heteronuclear multiple-bond correlation (HMBC) experiments. The position of the hemiacetal carbon (C-2) in cuneataol (2) could be proved, because the methyl group C-24 and the carbinol C-25 gave a correlation to the hemiacetal carbon, whereas in compound 3 a correlation signal to the C-3 was detectable. Compound 3 (Figure 4) has not been reported previously and was named pomaceic acid. Biological Activities Mediated by the Triterpenoic Acids from APF3. Eight of the main compounds present in APF3 were identified either by standard addition using two independent chromatographic methods (reversed-phase HPLC and normal-phase TLC) or by isolation and structure elucidation (Table 3; Figure 4). The respective triterpenoic 190


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Figure 4. Chemical structures of the compounds identified in APF3.

To evaluate this hypothesis, the content of the respective triterpenoic acids in APF3 was determined. Quantification was performed using HPLC-DAD at 210 nm and external standardization with ursolic acid (11), whereby experimentally evaluated correction factors were elucidated for the other triterpenoic acids present in the fraction. Because euscaphic (4a) and tormentic acid (4b) were coeluting (Figure 3), both were quantified as the main compound euscaphic acid (Table 3). Then, EA.hy926 cells were stimulated with the reconstituted mixture of triterpenoic acids (TTA) present in 50 μg of APF3 per milliliter of cell culture medium (Table 3). Furthermore, TTA under the addition of the amount of quercetin present in APF3 (Table 2) was tested (TTA+Q). Metabolic activity was not inhibited by the stimulation with the reconstituted fractions (Figure 5). TTA significantly enhanced eNOS activity in EA.hy926 cells to a similar extent as APF3. For the addition of

Table 5. Influence of Triterpenoic Acids on Cell Metabolic Activity of EA.hy926 Cells compd

metabolic activity, IC50 (μM)

cuneataol pomaceic acid euscaphic acid tormentic acid annurcoic acid pirolonic acid pomolic acid maslinic acid corosolic acid oleanolic acid ursolic acid

>80.0 >80.0 >80.0 55.2 >80.0 >80.0 16.5 26.8 22.1 50.4 12.6

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b05061. Part 1 describes the fractionation of apple pomace methanolic water extract and the isolation of triterpenoic acids from apple pomace EtOAc extract and part 2, the structure elucidation of the isolated compounds; part 3 comprises the standard addition experiments to APF2 and APF3, and part 4 contains detailed results of biological tests (PDF)

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Figure 5. Biological activities of the reconstituted APF3 using either a mixture of triterpenoic acids (TTA) or the same mixture of triterpenoic acids with the addition of quercetin (TTA+Q) in comparison to APF3 (50 μg/mL). Data were assessed by the 14C-Larginine to 14C-L-citrulline conversion assay (black columns) and resazurin assay (gray columns) in EA.hy926 cells. Cells were stimulated for 24 h with the solvent vehicle (NC, negative control), 100 μM ascorbic acid (PC, positive control), or the respective test fraction. Results indicate 14C-L-citrulline production normalized to PC and resorufin production normalized to NC (mean ± SD, n = 3). Statistical analysis: one-way ANOVA (Dunnett’s post-test compared to NC; ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001).

AUTHOR INFORMATION

Corresponding Author

*(M.Z.) Mail: Department of Pharmacognosy, Althanstrasse 14, 1090 Vienna, Austria. Phone: 0043 4277 55219. Fax: 0043 4277 9552. E-mail: [email protected]. Funding

This work was funded by the Initiative Group Biopromotion of the University of Vienna. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED ACN acetonitrile AMPK AMP-activated protein kinase APF apolar fraction ApoE−/− mice apolipoprotein E knockout mice BAEC bovine aortic endothelial cells brFMD brachial flow mediated dilatation CAD charged aerosol detection cGMP guanosine 3′,5′-cyclic monophosphate CHCl3 chloroform ELSD evaporative light-scattering detection eNOS endothelial nitric oxide synthase EtOAc ethyl acetate EtOH ethanol HEX hexane HPCCC high-performance counter current chromatography MeOH methanol NO nitric oxide nd not detected ns not significant ROS reactive oxygen species sGC soluble guanylyl cyclase VSMC vascular smooth muscle cells

quercetin (TTA+Q), no further significant increase of eNOS activity was observed (Figure 5). Therefore, synergistic effects of triterpenoic acids and the flavonoid quercetin can be excluded, at least for the given concentrations. In conclusion, additive or even synergistic effects of the triterpenoic acids present in APF3 were shown to fully explain the eNOSactivating principle of the fraction. The majority of the performed epidemiological and intervention studies correlated the pharmacological effects of apples to their content of polyphenols.14,46 In this study, the eNOS-activating effect of an apple pomace-derived fraction was adjudged to a mixture of triterpenoic acids. It has been shown that polyphenols are mainly located in apple peels,47 which is also the case for triterpenoic acids. The latter comprise a major part of the apple skin as they are part of the apple’s cuticular waxes.48 Flavonoids and other polyphenols can be detected easily by spectrophotometric methods or HPLC-UV/DAD detection, whereas triterpenoic acids absorb only weakly around the wavelength of 200 nm. Therefore, their content as well as their impact on pharmacological activities might have been underestimated in the past. Prospective research in the form of in vivo intervention studies should apply a more holistic approach regarding the analytical profile of the (ingested) plant material potentially influencing vascular function. This study showed that apple pomace is a valuable resource for the isolation of apple compounds. One, so far unknown, triterpenoic acid was isolated, together with several known substances that might be used for industrial or pharmaceutical purposes. Via the fractionation of a methanol/water apple pomace extract, a pharmacologically active fraction was obtained, which exhibited eNOS-activating properties in vitro. Furthermore, it was demonstrated that this apple pomacederived compound mixture exhibits advanced pharmacological efficacy compared to the isolated single compounds of the fraction but that the same activity can be achieved by the reconstituted mixture of triterpenoic acids.

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Triterpenoic Acids from Apple Pomace Enhance the Activity of the

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