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
See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
Downloaded via UNIV AUTONOMA DE MADRID on June 8, 2020 at 14:23:05 (UTC).

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

■ 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, posttran-
scriptional, translational, or posttranslational mechanisms as
well as by diverse protein−protein interactions.5−8 Endothe-
lium-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
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-poly-
phenol-content apples had an influence on brachial flow-
mediated 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 nitro-
sylated 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
Received: October 19, 2015
Revised: December 18, 2015
Accepted: December 18, 2015
Published: December 18, 2015

© 2015 American Chemical Society 185 DOI: 10.1021/acs.jafc.5b05061

J. Agric. Food Chem. 2016, 64, 185−194
Journal of Agricultural and Food Chemistry
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 plant-
derived extracts.18 To evaluate whether apple waste is a source
for eNOS-activating compounds, we performed a bioassay-
guided 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.
■
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-rutino-
side (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 Fractio-
nation. 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
Article
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 stand-
ardization 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
DOI: 10.1021/acs.jafc.5b05061
J. Agric. Food Chem. 2016, 64, 185−194
Journal of Agricultural and Food Chemistry Article

pomace EtOAc extract). Structure-elucidation was performed by MS immortalized endothelium-derived cell line EA.hy926. For that,
and spectroscopic methods in reconciliation of published literature extracts with solvents of different polarities were prepared from
(see the Supporting Information, Structure elucidation).22−25 apple pomace by ASE. As shown in Figure 1a, none of the
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-epoxy-
2,3,19-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)-3,19-
dihydroxy-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 HEX-
dominated 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 positive- Figure 1. Biological activities of apple pomace extracts (MeW, EtOAc,
ion mode using direct infusion. The sum formulas were determined DCM, HEX) (a) and apple pomace apolar fractions (APFs) (b). NC,
using Bruker Compass DataAnalysis 4.2 based on the mass accuracy negative control (solvent vehicle-treated cells); PC, positive control
(Δm/z ≤ 2 ppm) and isotopic pattern matching (SmartFormula (100 μM ascorbic acid); statistical analysis, one-way ANOVA
algorithm). (Dunnett’s post-test compared to NC: ∗, p < 0.05; ∗∗, p < 0.01;
Nuclear Magnetic Resonance (NMR). Spectra were recorded on ∗∗∗, p < 0.001). eNOS activity in EA.hy926 cells was evaluated after
a Bruker Avance 500 NMR spectrometer (UltraShield) using a 5 mm 24 h of stimulation with 50 μg/mL extract or APF, assessed by 14C-L-
switchable probe (PA BBO 500SB BBF-H-D-05-Z, 1H, BB = 19F and arginine to 14C-L-citrulline conversion assay (ACCA). Results indicate
31
P − 15N) with z-axis gradients and automatic tuning and matching 14
C production normalized to PC (mean ± SD, n = 3, black columns).
accessory (Bruker BioSpin). The resonance frequency for 1H NMR Metabolic activity of EA.hy926 cells was evaluated after 24 h of
was 500.13 MHz and that for 13C NMR, 125.75 MHz. All stimulation with 50 μg/mL of extract or APF, assessed by resazurin
measurements were performed for a solution in fully deuterated assay. Results show resorufin production normalized to NC (mean ±
methanol at 298 K. Standard 1D and gradient-enhanced (ge) 2D SD, n = 3, gray columns).
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 obtained extracts was able to enhance eNOS activity in
solvent signal for methanol 1H (δ 3.31) and to the carbon signal of EA.hy926 cells significantly. As the extracts are complex
methanol 13C (δ 49.00). mixtures of various phytochemicals, the activity of potentially
Circular Dichroism. Spectra were measured on a J-810 CD bioactive compounds may be masked due to the presence of
spectrometer from Jasco (Tokyo, Japan), equipped with a 0.1 cm cell.
The instrument was operated with a scanning speed of 100 nm/min
inactive bulk compounds. Via fractionation of pomace extracts,
and a response of 4 nm at 20 °C using MeOH as solvent. bioactive compounds may be enriched. These fractions might
Statistical Analysis. Raw data from the ACCA were normalized to be used for nutraceutical purposes or for the isolation of pure
the positive control (PC, ascorbic acid, 100 μM), whereas raw data bioactive compounds. For the evaluation of this hypothesis, the
from the resazurin assay were normalized to the negative control (NC, apple pomace methanol/water (70:30) (MeW) extract was
solvent vehicle treated cells). Normalized data were incorporated in chosen for fractionation for the following reasons: (a) cell
Graph Pad Prism version 4.0 software, and significant differences to metabolic activity was not affected by this extract (Figure 1a);
the NC were assessed using one-way ANOVA/Dunnett’s post-test (∗, (b) methanol has dissolving properties comparable to those of
P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001). Shown results are the mean ± ethanol, a solvent that obliges minor restrictions in the food
SD of three independent experiments.

■
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
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
DOI: 10.1021/acs.jafc.5b05061
J. Agric. Food Chem. 2016, 64, 185−194
Journal of Agricultural and Food Chemistry Article

EtOH 96% (v/v)/H2O (10:90), did not exhibit any influence Table 2. Quantification of Quercetin Derivatives and
on eNOS or metabolic activity in EA.hy926 cells (data not Phloridzin in APFs Using External Standardization by
shown). APF3 and APF4, eluted with EtOH 96% (v/v), HPLC-DAD at 280 nm
showed a significant increase of eNOS activity in EA.hy926
mg/g apple pomace apolar fractiona
cells, whereas their metabolic activity was not affected
significantly (Figure 1b). APF2 APF3
Phytochemical Characterization of Apple Pomace quercetin-3-rutinoside 2.8 ± 0.8 1.6 ± 0.1
Apolar Fractions. In previous studies, the cardiovascular quercetin-3-galactoside 16.0 ± 5.1 6.7 ± 0.1
protective effects of apples were mainly associated with quercetin-3-glucoside 4.8 ± 1.5 2.4 ± 0.2
polyphenolic compounds.15,27,28 Thus, it was evaluated whether quercetin-pentoside 1 6.1 ± 1.8 3.5 ± 0.3
there exists a correlation between the total phenolic content quercetin-pentoside 2 3.5 ± 0.7 1.6 ± 0.1
(TPC) of the apple pomace fractions and their eNOS- quercetin-pentoside 3 1.2 ± 0.2 1.2 ± 0.1
activating potential in EA.hy926 cells. APF2 exhibited the quercetin-pentoside 4 13.4 ± 4.1 4.5 ± 0.2
highest content of total phenolics (77.2 mg/g, as gallic acid quercetin-desoxyhexoside 13.4 ± 0.4 7.0 ± 0.1
equivalents, GAE) assessed by Folin−Ciocalteu method (Table quercetin ndb 9.9 ± 0.5
1), but did not show any influence on eNOS activity (Figure Σ quercetin derivatives 61.2 38.4
phloridzin 28.7 ± 9.1 0.6 ± 0.1
Table 1. Total Phenolic Content of APFs Measured by the a
Results are reported as the mean ± SD (n = 3). nd, not detected.
b

Folin−Ciocalteu Method
gallic acid equivalents (GAE)
(mg/g)
fraction mean SDa % RSDb
APF1 16.6 0.3 1.5
APF2 77.2 1.4 1.8
APF3 62.8 0.5 0.7
APF4 13.3 0.5 3.4
APF5 18.2 0.3 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 APF-
induced eNOS activation in EA.hy926 cells. Eight quercetin-
glycosides, quercetin, and phloridzin were detected by HPLC-
MS 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 eNOS-
activating 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
188
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-L-
citrulline 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
DOI: 10.1021/acs.jafc.5b05061
J. Agric. Food Chem. 2016, 64, 185−194
Journal of Agricultural and Food Chemistry Article

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) identified compd APF3 (mg/g) APF4 (mg/g) identified byd
e
1 6.6 502.3 ni
2 7.1 502.3 cuneataol 25.5 ± 2.3 nd #
3 7.9 502.2 pomaceic acid 232.2 ± 5.2 28.7 ± 2.1 *
4 8.8 488.3 euscaphic acid 139.4 ± 4.3 16.6 ± 1.1 #
tormentic acid
5 14.3 486.3 annurcoic acid 65.6 ± 2.0 180.4 ± 3.1 #
6 14.6 486.3 pirolonic acid 5.3 ± 0.8 31.7 ± 0.3 #
7 17.7 472.3 pomolic acid 35.0 ± 3.2 15.1 ± 0.6 #
8 19.5 472.2 maslinic acid 17.4 ± 0.8 14.6 ± 0.3 #
9 20.5 472.2 corosolic acid 14.5 ± 1.1 19.2 ± 0.6 #
10 35.5 456.3 oleanolic acid 9.6 ± 0.3 20.4 ± 0.6 #
11 35.9 456.3 ursolic acid 7.9 ± 0.3 17.1 ± 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.

(see the Supporting Information, Structure elucidation).
Chromatographic comparison with APF3 and addition experi-
ments 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 reversed-
phase 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
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
DOI: 10.1021/acs.jafc.5b05061
J. Agric. Food Chem. 2016, 64, 185−194
Journal of Agricultural and Food Chemistry Article

giving characteristic shift values for an urs-12-en-28-oic acid acids were individually tested for their potential to enhance
skeleton. Comparison with cuneataol (2) (Table 4) revealed a eNOS activity in EA.hy926 cells. In addition, their impact on
the metabolic activity (as a measure of cell viability) of
Table 4. Comparison of 1H and 13C NMR Data of Pomaceic EA.hy926 cells was evaluated by the resazurin assay. The most
Acid and Cuneataol in CD3OD abundant compounds in APF3, pomaceic acid (3), euscaphic
acid (4a), and annurcoic acid (5), did not affect cell metabolic
13
C, δ pomaceic 13
C, δ
1
H, δ pomaceic acid acid cuneataol activity in the tested concentration range from 1 to 80 μM
(Table 5). The minor compounds tormentic acid (4b) and
1 2.60 (1H, dd, J = 13.9 and 10.4 Hz) 46.49 41.56
pomolic acid (7) revealed IC50 values of 55.2 and 16.5 μM,
1.11 (1H, m)
respectively, whereas cuneataol (2) and pirolonic acid (6) did
2 3.84 (1H, dd, J = 10.4 and 3.8 Hz) 73.84 106.57
not affect metabolic activity up to 80 μM. Maslinic acid (8),
3 98.80 81.55
corosolic acid (9), oleanolic acid (10), and ursolic acid (11)
4 40.68 39.27
5 1.36 (3H, m) 51.60 51.95
inhibited cell metabolic activity at significantly lower test
6 1.51 (2H, m) 20.71 21.39
concentrations (Table 5). It can be hypothesized that 3β-OH-
7 1.40 (1H, m) 32.72 33.37
ursan-type triterpenoic acids favor inhibition of cell metabolism
1.51 (1H, m)
compared to 3β-OH-olean-type triterpenoic acids. Hydroxy
8 40.08 40.42
groups on positions 2 and 19 seem to lower inhibiting effects
9 1.87 (1H, dd, J = 12.0 and 5.4 Hz) 42.16 41.03
on cell metabolism as does the α-conformation of the hydroxy
10 37.17 49.45
group in position 3 (Figure 4; Table 5).
11 2.11 (1H, ddd, 25.08 24.54
Surprisingly, none of the triterpenoic acids identified in APF3
J = 18.6, 5.4, and 3.2 Hz) was able to increase 14C-L-citrulline production when tested as a
1.77 (1H, m) single compound (see the Supporting Information, Biological
12 5.31 (1H, s, br) 129.42 128.79 activities). Thus, even for ursolic acid (11) no positive
13 140.12 140.03 modulation of eNOS activity was observed in EA.hy926 cells
14 42.85 42.22 in this study, which is in contrast to in vitro data from the
15 1.76 (1H, m) 29.73 29.95 literature.36,37 On the other hand, an induction of eNOS
1.09 (1H, m) activity by ursolic acid was observed at concentrations >7.5 μM
16 2.55 (1H, dd, J = 13.8 and 4.7 Hz) 26.77 26.72 (data not shown). However, these concentrations already
1.52 (1H, m) caused a significant decrease of cell metabolic activity.
17 49.30 49.39 Therapeutic relevance of ursolic acid (11) stays ambiguous
18 2.51 (1H, s) 55.66 55.42 with regard to in vivo data. For diabetic mice and Dahl-salt-
19 73.37 73.55 sensitive/insulin-resistant rats, a decrease of atherosclerotic
20 1.34 (1H, m) 43.09 43.10 plaque formation and systolic blood pressure was determined
21 1.24 (1H, m), 1.72 (1H, m) 27.27 27.32 under ursolic acid (11) treatment,38,39 whereas increased
22 1.61 (1H, m), 1.74 (1H, m) 38.74 38.89 atherosclerotic plaque formation was observed in ApoE−/−
23 1.21 (3H, s) 27.44 28.48 mice under ursolic acid (11) treatment.40
24 0.97 (3H, s) 20.85 25.38 Oleanolic acid (10) was the only tested triterpenoic acid that
25 4.21 (1H, dd, J = 8.2 and 0.3 Hz) 67.64 67.36 exhibited at least a nonsignificant trend of eNOS activation in
3.73 (1H, d, J = 8.2 Hz) EA.hy926 cells (Figure S59), which could be mediated by
26 0.76 (3H, s) 17.93 17.06 enhanced eNOS expression and Ser1177 phosphorylation of
27 1.37 (3H, s) 24.16 24.03 eNOS, according to the literature.41 In vivo relevance for eNOS
28 182.21 182.63 activating properties of oleanolic acid (10) seem to be given. A
29 1.18 (3H, s) 26.99 27.04 high-fat diet containing 15% of pomace olive oil enriched in
30 0.93 (3H, d, J = 6.90 Hz) 16.55 16.60 oleanolic acid (10) was able to improve endothelial function of
Wistar Kyoto and spontaneous hypertensive rats compared to
high similarity of signals in rings B, C, D, and E,35 but the control groups under a corn oil diet or pomace olive oil diet
remarkable differences of signals in ring A. Both compounds without the supplementation of oleanolic acid.42
contain a secondary carbinol, a hemiacetal carbon, and an For the other triterpenoic acids present in APF3, to the best
epoxy bridge. Structural differences between cuneataol (2) and of our knowledge, in vivo data are lacking. Nevertheless,
compound 3 were elucidated by heteronuclear multiple-bond pomolic acid (7), isolated from Licania pittieri (Prance), was
correlation (HMBC) experiments. The position of the identified to elicit endothelium-dependent relaxation in in vitro
hemiacetal carbon (C-2) in cuneataol (2) could be proved, experiments performed with rat aortic rings.43 Equivalent
because the methyl group C-24 and the carbinol C-25 gave a results are reported for maslinic acid (8), isolated from olive
correlation to the hemiacetal carbon, whereas in compound 3 a pomace.44 Furthermore, a methanolic extract from the Mexican
correlation signal to the C-3 was detectable. Compound 3 hawthorn Crataegus gracilior (Phipps), which was enriched in
(Figure 4) has not been reported previously and was named ursolic acid (11) and corosolic acid (9) derivatives, mediated an
pomaceic acid. endothelium-dependent aortic relaxant activity.45
Biological Activities Mediated by the Triterpenoic Reconstitution of APF3. None of the individual
Acids from APF3. Eight of the main compounds present in compounds identified in the apple pomace-derived APF3 was
APF3 were identified either by standard addition using two able to induce a significant eNOS activation in EA.hy926 cells
independent chromatographic methods (reversed-phase HPLC at relevant concentrations. Thus, we hypothesized that the
and normal-phase TLC) or by isolation and structure eNOS-activating potential of the fraction must be elicited by
elucidation (Table 3; Figure 4). The respective triterpenoic the additive effects of the compound mixture present in APF3.
190 DOI: 10.1021/acs.jafc.5b05061
J. Agric. Food Chem. 2016, 64, 185−194
Journal of Agricultural and Food Chemistry Article

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

Table 5. Influence of Triterpenoic Acids on Cell Metabolic To evaluate this hypothesis, the content of the respective
Activity of EA.hy926 Cells triterpenoic acids in APF3 was determined. Quantification was
performed using HPLC-DAD at 210 nm and external
compd metabolic activity, IC50 (μM)
standardization with ursolic acid (11), whereby experimentally
cuneataol >80.0 evaluated correction factors were elucidated for the other
pomaceic acid >80.0
triterpenoic acids present in the fraction. Because euscaphic
euscaphic acid >80.0
(4a) and tormentic acid (4b) were coeluting (Figure 3), both
tormentic acid 55.2
were quantified as the main compound euscaphic acid (Table
annurcoic acid >80.0
3). Then, EA.hy926 cells were stimulated with the reconstituted
pirolonic acid >80.0
pomolic acid 16.5
mixture of triterpenoic acids (TTA) present in 50 μg of APF3
maslinic acid 26.8
per milliliter of cell culture medium (Table 3). Furthermore,
corosolic acid 22.1 TTA under the addition of the amount of quercetin present in
oleanolic acid 50.4 APF3 (Table 2) was tested (TTA+Q). Metabolic activity was
ursolic acid 12.6 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
191 DOI: 10.1021/acs.jafc.5b05061
J. Agric. Food Chem. 2016, 64, 185−194
Journal of Agricultural and Food Chemistry Article

■
*
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)

■
Figure 5. Biological activities of the reconstituted APF3 using either a
mixture of triterpenoic acids (TTA) or the same mixture of
AUTHOR INFORMATION
triterpenoic acids with the addition of quercetin (TTA+Q) in
comparison to APF3 (50 μg/mL). Data were assessed by the 14C-L- Corresponding Author
arginine to 14C-L-citrulline conversion assay (black columns) and *(M.Z.) Mail: Department of Pharmacognosy, Althanstrasse
resazurin assay (gray columns) in EA.hy926 cells. Cells were 14, 1090 Vienna, Austria. Phone: 0043 4277 55219. Fax: 0043
stimulated for 24 h with the solvent vehicle (NC, negative control), 4277 9552. E-mail: martin.zehl@univie.ac.at.
100 μM ascorbic acid (PC, positive control), or the respective test
fraction. Results indicate 14C-L-citrulline production normalized to PC Funding
and resorufin production normalized to NC (mean ± SD, n = 3). This work was funded by the Initiative Group Biopromotion of
Statistical analysis: one-way ANOVA (Dunnett’s post-test compared to the University of Vienna.
NC; ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001). Notes
The authors declare no competing financial interest.

quercetin (TTA+Q), no further significant increase of eNOS

activity was observed (Figure 5). Therefore, synergistic effects
■
ACN
ABBREVIATIONS USED
acetonitrile
of triterpenoic acids and the flavonoid quercetin can be AMPK AMP-activated protein kinase
excluded, at least for the given concentrations. In conclusion, APF apolar fraction
additive or even synergistic effects of the triterpenoic acids ApoE−/− mice apolipoprotein E knockout mice
present in APF3 were shown to fully explain the eNOS- BAEC bovine aortic endothelial cells
activating principle of the fraction. brFMD brachial flow mediated dilatation
The majority of the performed epidemiological and CAD charged aerosol detection
intervention studies correlated the pharmacological effects of cGMP guanosine 3′,5′-cyclic monophosphate
apples to their content of polyphenols.14,46 In this study, the CHCl3 chloroform
eNOS-activating effect of an apple pomace-derived fraction was ELSD evaporative light-scattering detection
adjudged to a mixture of triterpenoic acids. It has been shown eNOS endothelial nitric oxide synthase
that polyphenols are mainly located in apple peels,47 which is EtOAc ethyl acetate
also the case for triterpenoic acids. The latter comprise a major EtOH ethanol
part of the apple skin as they are part of the apple’s cuticular HEX hexane
waxes.48 Flavonoids and other polyphenols can be detected HPCCC high-performance counter current chromatog-
easily by spectrophotometric methods or HPLC-UV/DAD raphy
detection, whereas triterpenoic acids absorb only weakly MeOH methanol
around the wavelength of 200 nm. Therefore, their content NO nitric oxide
as well as their impact on pharmacological activities might have nd not detected
been underestimated in the past. Prospective research in the ns not significant
form of in vivo intervention studies should apply a more ROS reactive oxygen species
holistic approach regarding the analytical profile of the sGC soluble guanylyl cyclase
(ingested) plant material potentially influencing vascular VSMC vascular smooth muscle cells
function.
This study showed that apple pomace is a valuable resource
for the isolation of apple compounds. One, so far unknown,
■ REFERENCES
(1) Miura, K.; Greenland, P.; Stamler, J.; Liu, K.; Daviglus, M. L.;
triterpenoic acid was isolated, together with several known Nakagawa, H. Relation of vegetable, fruit, and meat intake to 7-year
substances that might be used for industrial or pharmaceutical blood pressure change in middle-aged men: the Chicago Western
purposes. Via the fractionation of a methanol/water apple Electric Study. Am. J. Epidemiol. 2004, 159, 572−580.
pomace extract, a pharmacologically active fraction was (2) Ascherio, A.; Rimm, E. B.; Giovannucci, E. L.; Colditz, G. A.;
Rosner, B.; Willett, W. C.; Sacks, F.; Stampfer, M. J. A prospective
obtained, which exhibited eNOS-activating properties in vitro.
study of nutritional factors and hypertension among United-States
Furthermore, it was demonstrated that this apple pomace- men. Circulation 1992, 86, 1475−1484.
derived compound mixture exhibits advanced pharmacological (3) Michel, T.; Vanhoutte, P. Cellular signaling and NO production.
efficacy compared to the isolated single compounds of the Pfluegers Arch. 2010, 459, 807−816.
fraction but that the same activity can be achieved by the (4) Daff, S. NO synthase: structures and mechanisms. Nitric Oxide
reconstituted mixture of triterpenoic acids. 2010, 23, 1−11.

192 DOI: 10.1021/acs.jafc.5b05061

J. Agric. Food Chem. 2016, 64, 185−194
Journal of Agricultural and Food Chemistry
(5) Fleming, I.; Busse, R. Molecular mechanisms involved in the
regulation of the endothelial nitric oxide synthase. Am. J. Physiol. 2003,
284, R1−R12.
(6) Fleming, I. Molecular mechanisms underlying the activation of
eNOS. Pfluegers Arch. 2010, 459, 793−806.
(7) Heiss, E. H.; Dirsch, V. M. Regulation of eNOS enzyme activity
by posttranslational modification. Curr. Pharm. Des. 2014, 20, 3503−
3513.
(8) Eisenreich, A. Regulation of vascular function on posttranscrip-
tional level. Thrombosis 2013, 2013, 948765.
(9) Gkaliagkousi, E.; Ferro, A. Nitric oxide signalling in the regulation
of cardiovascular and platelet function. Front. Biosci., Landmark Ed.
2011, 16, 1873−1897.
(10) Lanzerstorfer, P.; Wruss, J.; Huemer, S.; Steininger, A.; Müller,
U.; Himmelsbach, M.; Borgmann, D.; Winkler, S.; Höglinger, O.;
Weghuber, J. Bioanalytical characterization of apple juice from 88
grafted and nongrafted apple varieties grown in Upper Austria. J. Agric.
Food Chem. 2014, 62, 1047−1056.
(11) Markowski, J.; Baron, A.; Le Quere, J.-M.; Plocharski, W.
Composition of clear and cloudy juices from French and Polish apples
in relation to processing technology. LWT−Food Sci. Technol. 2015,
62, 813−820.
(12) Grigoras, C. G.; Destandau, E.; Fougere, L.; Elfakir, C.
Evaluation of apple pomace extracts as a source of bioactive
compounds. Ind. Crops Prod. 2013, 49, 794−804.
(13) Auclair, S.; Chironi, G.; Milenkovic, D.; Hollman, P. C. H.;
Renard, C.; Megnien, J. L.; Gariepy, J.; Paul, J. L.; Simon, A.; Scalbert,
A. The regular consumption of a polyphenol-rich apple does not
influence endothelial function: a randomised double-blind trial in
hypercholesterolemic adults. Eur. J. Clin. Nutr. 2010, 64, 1158−1165.
(14) Bondonno, C. P.; Yang, X. B.; Croft, K. D.; Considine, M. J.;
Ward, N. C.; Rich, L.; Puddey, I. B.; Swinny, E.; Mubarak, A.;
Hodgson, J. M. Flavonoid-rich apples and nitrate-rich spinach augment
nitric oxide status and improve endothelial function in healthy men
and women: a randomized controlled trial. Free Radical Biol. Med.
2012, 52, 95−102.
(15) Knekt, P.; Jarvinen, R.; Reunanen, A.; Maatela, J. Flavonoid
intake and coronary mortality in Finland: a cohort study. Br. Med. J.
1996, 312, 478−481.
(16) Loke, W. M.; Hodgson, J. M.; Proudfoot, J. M.; McKinley, A. J.;
Puddey, I. B.; Croft, K. D. Pure dietary flavonoids quercetin and
(−)-epicatechin augment nitric oxide products and reduce endothelin-
1 acutely in healthy men. Am. J. Clin. Nutr. 2008, 88, 1018−1025.
(17) Shen, Y.; Ward, N. C.; Hodgson, J. M.; Puddey, I. B.; Wang, Y.;
Zhang, D.; Maghzal, G. J.; Stocker, R.; Croft, K. D. Dietary quercetin
attenuates oxidant-induced endothelial dysfunction and atherosclerosis
in apolipoprotein E knockout mice fed a high-fat diet: a critical role for
heme oxygenase-1. Free Radical Biol. Med. 2013, 65, 908−915.
(18) Schmitt, C. A.; Handler, N.; Heiss, E. H.; Erker, T.; Dirsch, V.
M. No evidence for modulation of endothelial nitric oxide synthase by
the olive oil polyphenol hydroxytyrosol in human endothelial cells.
Atherosclerosis 2007, 195, e58−e64.
(19) Halliwell, B. Oxidative stress in cell culture: an under-
appreciated problem? FEBS Lett. 2003, 540, 3−6.
(20) O’Brien, J.; Wilson, I.; Orton, T.; Pognan, F. Investigation of the
Alamar Blue (resazurin) fluorescent dye for the assessment of
mammalian cell cytotoxicity. Eur. J. Biochem. 2000, 267, 5421−5426.
(21) Gridling, M.; Popescu, R.; Kopp, B.; Wagner, K. H.; Krenn, L.;
Krupitza, G. Anti-leukaemic effects of two extract types of Lactuca
sativa correlate with the activation of Chk2, induction of p21,
downregulation of cyclin D1 and acetylation of α-tubulin. Oncol. Rep.
2010, 23, 1145−1151.
(22) Cheng, J. J.; Zhang, L. J.; Cheng, H. L.; Chiou, C. T.; Lee, I. J.;
Kuo, Y. H. Cytotoxic hexacyclic triterpene acids from Euscaphis
japonica. J. Nat. Prod. 2010, 73, 1655−1658.
(23) Reher, G.; Buděsí̌ nský, M. Triterpenoids from plants of the
sanguisorbeae. Phytochemistry 1992, 31, 3909−3914.
193
Article
(24) D’Abrosca, B.; Fiorentino, A.; Monaco, P.; Oriano, P.; Pacifico,
S. Annurcoic acid: a new antioxidant ursane triterpene from fruits of
cv. Annurca apple. Food Chem. 2006, 98, 285−290.
(25) Ragasa, C. Y.; Alimboyougen, A. B.; Rideout, J. A. A triterpene
from Rosa sp. J. Res. Sci. Comput. Eng. 2007, 4, 1−5.
(26) Bhushan, S.; Kalia, K.; Sharma, M.; Singh, B.; Ahuja, P. S.
Processing of apple pomace for bioactive molecules. Crit. Rev.
Biotechnol. 2008, 28, 285−296.
(27) Serra, A. T.; Rocha, J.; Sepodes, B.; Matias, A. A.; Feliciano, R.
P.; de Carvalho, A.; Bronze, M. R.; Duarte, C. M. M.; Figueira, M. E.
Evaluation of cardiovascular protective effect of different apple
varieties − correlation of response with composition. Food Chem.
2012, 135, 2378−2386.
(28) Zhao, S.; Bomser, J.; Joseph, E. L.; DiSilvestro, R. A. Intakes of
apples or apple polyphenols decease plasma values for oxidized low-
density lipoprotein/β(2)-glycoprotein I complex. J. Funct. Foods 2013,
5, 493−497.
(29) Lu, Y. R.; Foo, L. Y. Identification and quantification of major
polyphenols in apple pomace. Food Chem. 1997, 59, 187−194.
(30) Khoo, N. K. H.; White, C. R.; Pozzo-Miller, L.; Zhou, F.;
Constance, C.; Inoue, T.; Patel, R. P.; Parks, D. A. Dietary flavonoid
quercetin stimulates vasorelaxation in aortic vessels. Free Radical Biol.
Med. 2010, 49, 339−347.
(31) Shen, Y.; Croft, K. D.; Hodgson, J. M.; Kyle, R.; Lee, I. L. E.;
Wang, Y.; Stocker, R.; Ward, N. C. Quercetin and its metabolites
improve vessel function by inducing eNOS activity via phosphor-
ylation of AMPK. Biochem. Pharmacol. (Amsterdam, Neth.) 2012, 84,
1036−1044.
(32) Brieskorn, C. H.; Wunderer, H. Ü ber den chemischen Aufbau
der Apfelschale, IV. Pomol- und Pomonsäure. Chem. Ber. 1967, 100,
1252−1265.
(33) He, X.; Liu, R. H. Triterpenoids isolated from apple peels have
potent antiproliferative activity and may be partially responsible for
apple’s anticancer activity. J. Agric. Food Chem. 2007, 55, 4366−4370.
(34) McGhie, T. K.; Hudault, S.; Lunken, R. C. M.; Christeller, J. T.
Apple peels, from seven cultivars, have lipase-inhibitory activity and
contain numerous ursenoic acids as identified by LC-ESI-QTOF-
HRMS. J. Agric. Food Chem. 2012, 60, 482−491.
(35) Ikeda, T.; Ogawa, Y.; Nohara, T. A new triterpenoid from
Crataegus cuneata. Chem. Pharm. Bull. 1999, 47, 1487−1488.
(36) Lee, A.-W.; Chen, T.-L.; Shih, C.-M.; Huang, C.-Y.; Tsao, N.-
W.; Chang, N.-C.; Chen, Y.-H.; Fong, T.-H.; Lin, F.-Y. Ursolic acid
induces allograft inflammatory factor-1 expression via a nitric oxide-
related mechanism and increases neovascularization. J. Agric. Food
Chem. 2010, 58, 12941−12949.
(37) Steinkamp-Fenske, K.; Bollinger, L.; Völler, N.; Xu, H.; Yao, Y.;
Bauer, R.; Förstermann, U.; Li, H. Ursolic acid from the Chinese herb
danshen (Salvia miltiorrhiza L.) upregulates eNOS and downregulates
Nox4 expression in human endothelial cells. Atherosclerosis 2007, 195,
e104−e111.
(38) Ullevig, S. L.; Zhao, Q.; Zamora, D.; Asmis, R. Ursolic acid
protects diabetic mice against monocyte dysfunction and accelerated
atherosclerosis. Atherosclerosis 2011, 219, 409−416.
(39) Somova, L. I.; Shode, F. O.; Ramnanan, P.; Nadar, A.
Antihypertensive, antiatherosclerotic and antioxidant activity of
triterpenoids isolated from Olea europaea, subspecies africana leaves.
J. Ethnopharmacol. 2003, 84, 299−305.
(40) Messner, B.; Zeller, I.; Ploner, C.; Frotschnig, S.; Ringer, T.;
Steinacher-Nigisch, A.; Ritsch, A.; Laufer, G.; Huck, C.; Bernhard, D.
Ursolic acid causes DNA-damage, P53-mediated, mitochondria- and
caspase-dependent human endothelial cell apoptosis, and accelerates
atherosclerotic plaque formation in vivo. Atherosclerosis 2011, 219,
402−408.
(41) Rodriguez-Rodriguez, R.; Stankevicius, E.; Herrera, M. D.;
Ostergaard, L.; Andersen, M. R.; Ruiz-Gutierrez, V.; Simonsen, U.
Oleanolic acid induces relaxation and calcium-independent release of
endothelium-derived nitric oxide. Br. J. Pharmacol. 2008, 155, 535−
546.
DOI: 10.1021/acs.jafc.5b05061
J. Agric. Food Chem. 2016, 64, 185−194
Journal of Agricultural and Food Chemistry Article

(42) Rodriguez-Rodriguez, R.; Herrera, M. D.; de Sotomayor, M. A.;

Ruiz-Gutierrez, V. Pomace olive oil improves endothelial function in
spontaneously hypertensive rats by increasing endothelial nitric oxide
synthase expression. Am. J. Hypertens. 2007, 20, 728−734.
(43) Estrada, O.; Gonzalez-Guzman, J. M.; Salazar-Bookaman, M.;
Fernandez, A. Z.; Cardozo, A.; Alvarado-Castillo, C. Pomolic acid of
Licania pittieri elicits endothelium-dependent relaxation in rat aortic
rings. Phytomedicine 2011, 18, 464−469.
(44) Rodriguez-Rodriguez, R.; Perona, J. S.; Herrera, M. D.; Ruiz-
Gutierrez, V. Triterpenic compounds from ‘Orujo’ olive oil elicit
vasorelaxation in aorta from spontaneously hypertensive rats. J. Agric.
Food Chem. 2006, 54, 2096−2102.
(45) Hernandez-Perez, A.; Bah, M.; Ibarra-Alvarado, C.; Rivero-Cruz,
J. F.; Rojas-Molina, A.; Rojas-Molina, J. I.; Cabrera-Luna, J. A. Aortic
relaxant activity of Crataegus gracilior Phipps and identification of some
of its chemical constituents. Molecules 2014, 19, 20962−20974.
(46) Balasuriya, N.; Rupasinghe, H. P. V. Antihypertensive properties
of flavonoid-rich apple peel extract. Food Chem. 2012, 135, 2320−
2325.
(47) Lata, B.; Trampczynska, A.; Paczesna, J. Cultivar variation in
apple peel and whole fruit phenolic composition. Sci. Hortic. 2009,
121, 176−181.
(48) Belding, R. D.; Blankenship, S. M.; Young, E.; Leidy, R. B.
Composition and variability of epicuticular waxes in apple cultivars. J.
Am. Soc. Hortic. Sci. 1998, 123, 348−356.

194 DOI: 10.1021/acs.jafc.5b05061

J. Agric. Food Chem. 2016, 64, 185−194