Industrial Crops and Products 49 (2013) 794–804

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Industrial Crops and Products

journal homepage: www.elsevier.com/locate/indcrop

Evaluation of apple pomace extracts as a source of bioactive

compounds
Cristina G. Grigoras a,b , Emilie Destandau a,∗ , Laëtitia Fougère a , Claire Elfakir a
a
Institut de Chimie Organique et Analytique, Université d’Orléans – CNRS UMR 7311, rue de Chartres, BP 67059, 45067 Orléans Cedex 2, France
b
“Vasile Alecsandri” University of Bacau, Faculty of Engineering, 157 Calea Marasesti, 600115 Bacau, Romania

a r t i c l e i n f o a b s t r a c t

Article history: Pomaces of four different apple cultivars (Gala v. Royal Gala Tenroy, Golden v. Golden, Granny Smith,
Received 30 January 2013 and Pink Lady v. Cripps Pink) were submitted to microwave assisted extraction with various solvents
Received in revised form 14 May 2013 (MeOH/H2 O mixture, ethanol and ethyl acetate) in order to recover their bioactive compounds. The
Accepted 13 June 2013
analyses of the obtained extracts by HPLC coupled to different detection modes such as UV, ELSD or
MS revealed the presence of various phenolic and terpenic compounds. Major phenols were benzoic
Keywords:
acids (gallic acid), hydroxycinnamic acids (chlorogenic acid), flavanols (catechin), flavonols (rutin) and
Apple pomace
chalcones (phloridzin). Among the terpenes, triterpenic acids as ursolic acid were the most abundant
Microwave assisted extraction
Phenolic compounds
but triterpenic acid derivatives with coumaryl group were also detected. The apple pomace extracts
Triterpenes and fractions obtained by successive liquid–liquid extraction were able to inhibit the DPPH free radical.
HPLC–UV–ELSD According to the FRAP assay results, these extracts presented also good reducing properties. The total
Mass spectrometry phenolic and total flavonoid contents were correlated to the antioxidant activity thus confirming that
Antioxidant activity apple pomaces are a valuable source of bioactive molecules.
© 2013 Elsevier B.V. All rights reserved.

1. Introduction
Apple pomace is the main by-product obtained by crushing
and pressing apples during the clear juice process recovery. It
represents approximately 30% of the original fruits and is highly
susceptible to biodegradation. Therefore apple pomace is a real
problem for manufacturers who have to manage extremely large
volumes of waste generated daily. This wet by-product is often used
as animal fodder (Mirzaei-Aghsaghali and Maheri-Sis, 2008) or as
fertilizer (Laufenberg et al., 2003). This last process can damage the
environment due to the high acidity and anti-germinant activity
and to the important content of organic compounds in such by-
products. Other more intensive practices include the production of
agro fuels (Balat et al., 2008) or coal (Walter and Sherman, 1975).
Apple pomace can be evaluated according to the compounds ini-
tially present in the fruit and which remain in the pomace after the
pressing step. Among the components contained in the apple fruit,
the polyphenolic ones are widely studied in recent years and are
well known for their beneficial effects on human health and their
ability to limit damage from oxidative stress due to radical species
(Alonso-Salces et al., 2005; Cetkovic et al., 2008; Diñeiro García
et al., 2009; Sánchez-Rabaneda et al., 2004; Schieber et al., 2001;
∗ Corresponding author. Tel.: +33 238417074; fax: +33 238417281.
E-mail address: emilie.destandau@univ-orleans.fr (E. Destandau).
Suárez et al., 2010). However, apple fruit also contains terpenoids
(Ma et al., 2005; Frighetto et al., 2008; Cefarelli et al., 2006), less
studied in apple than polyphenols. These compounds have many
interesting properties such as anti-inflammatory, antimicrobial,
antimycotic, antioxidant, liver protective, antiviral, immunomod-
ulatory, hemolytic or cytostatic effects (Muffler et al., 2011).
As consequence, the aim of this work was to evaluate if apple
pomace can constitute an interesting source of bioactive molecules
that could be added in various food, cosmetic or pharmaceutical for-
mulations for improving the composition and the activity of these
finished products. Hence an overall process, from the raw mate-
rial apple pomace to the bioactive molecules was developed. This
process needs to assess different steps and was applied on four dif-
ferent apple cultivars (Gala v. Royal Gala Tenroy, Golden v. Golden,
Granny Smith, and Pink Lady v. Cripps Pink). Extraction was first
studied to obtain the most representative extract of the pomace
content while limiting solvent, energy and time consumption. Then
extracted compounds were separated by HPLC and most of them
were identified by mass spectrometry. Finally, crude extracts were
fractionated by liquid–liquid extraction to separate molecular fam-
ilies and each crude extract and each fraction were then tested
against 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,4,6-tripyridyl-
S-triazine (TPTZ) for their antioxidant activity evaluation. The total
phenolic and total flavonoid contents were also determinate by col-
orimetric methods. This process affords a better knowledge of the
whole molecular content of apple pomace.

0926-6690/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.indcrop.2013.06.026
 C.G. Grigoras et al. / Industrial Crops and Products 49 (2013) 794–804
2. Materials and methods
2.1. Reagents
Methanol (MeOH), ethanol (EtOH), acetonitrile (MeCN), ethyl
acetate (EtOAc), heptane (hept) and isopropanol (iPrOH) used for
extraction and analyses were of analytical grade and were provided
by SDS Carlo Erba (Val-de-Reuil, France).
Water was purified (resistance < 18 M) from distilled water
using an Elgastat UHQ II system (Elga, Antony, France).
Formic acid, glucose (GLU), gallic acid (GA), chlorogenic
acid (CA), p-coumaric acid (pCA), betulinic acid (BA), oleanolic
acid (OA), erythrodiol (ER), palmitic acid (PA), 2,2␣-diphenyl-1-
picrylhydrazil (DPPH) free radical, Folin–Ciocâlteu reagent (FC),
trolox (TX), sodium acetate trihydrate, hydrochloric acid and alu-
minum chloride were purchased from Sigma Aldrich (Saint Quentin
Fallvier, France). Quercetin (QUE), catechin (CAT), rutin (RUT), phlo-
ridzin (PH), ursolic acid (UA), and uvaol (Uv) were obtained from
Extrasynthese (Genay, France). Ferric chloride hexahydrate was
obtained from Acros organics (Geel, Belgium). The 2,4,6-tripyridyl-
S-triazine (TPTZ) was obtained from JT Baker Chemicals (Deventer,
Holland) and sodium carbonate from Merck (Val de Fontenay,
France).
2.2. Plant material
1 kg of four different apple cultivars (Gala v. Royal Gala Ten-
roy, Golden v. Golden, Granny Smith, and Pink Lady v. Cripps Pink)
purchased from local market was cut in quarters and immedi-
ately pressed. The obtained pomaces were frozen at −80 ◦ C and
lyophilized. After drying, the plant material was ground to a pow-
der with a basic electric grinder and stocked at room temperature
in hermetically closed glass recipients.
2.3. Extraction procedure
2.3.1. Maceration (M)
1 g of dried apple pomace was introduced in 20 mL of ethanol
and stirred for 1 h at room temperature.
2.3.2. Pressurized fluid extraction (PLE)
Extraction was carried out by Pressurized Liquid Extraction
(PLE), using an Accelerated Solvent Extraction (ASE 100) system
from Dionex (Voisins le Bretonneux, France), with a 34 mL stain-
less steel vessel. 3 g of dried apple pomace were mixed with 3 g
of Na2 SO4 and extracted with ethanol using three static cycles for
5 min each, a flush volume of 65% and a purge with nitrogen gas
of 100 s at the end of each extraction cycle. Extractions were car-
ried out at 40 ◦ C and under a pressure of 100 bar. Around 60 mL of
ethanolic extract was obtained.
2.3.3. Ultrasound assisted extraction (UAE)
Extraction was carried out in a Touzart et Matignon ultrasonic
bath (Les ulis, France). 3 g of dried apple pomace were introduced
in 60 mL of ethanol and submitted to ultrasounds during 30 min at
room temperature.
2.3.4. Microwave assisted extraction (MAE)
Microwave assisted extraction was achieved in a Milestone
MicroSYNTH (Sorisole, Italy) system. The microwave power and
the irradiation time were controlled by the “easyCONTROL” soft-
ware. 1 g of dried apple pomace and 20 mL of solvent (ethanol,
ethyl acetate or water/methanol mixture) were introduced in a
50 mL closed reactor and submitted to 3 extraction cycles of 30 s at
1000 W. Because temperature increased during extraction process,
795
the reactor was cool down at room temperature on ice between
each extraction cycle.
All the extracts were centrifuged at 5000 rpm for 5 min at 15 ◦ C.
The supernatant was evaporated under vacuum at 40 ◦ C until dry-
ness. The mass of dried extract was compared to plant material
mass used for extraction in order to calculate the extraction yield
(expressed in percentages). Dried extracts were diluted in 5 mL
MeOH, centrifuged in the above mentioned conditions and fil-
tered on syringe-filters (0.45 ␮m). Filtrates were stocked at 4 ◦ C
and diluted at adequate concentration just before analysis.
2.4. HPLC analysis
The obtained extracts were analyzed by using a LaChrom Elite
HPLC system controlled by EZChrome Elite software (VWR Fonte-
nay sous bois, France). It was equipped with a quaternary pump,
a degasser, an automatic injector, a diode array detector (DAD) set
at four wavelengths (210, 254, 280 and 360 nm) and an evapora-
tive light scattering detector (ELSD) type Sedex 85 from SEDERE
(Alfortville, France) used in the following conditions: nebuliza-
tion temperature, 52 ◦ C; nebulization gas pressure, 3.2 bar; gain,
6. Analyses were carried out at 1 mL min−1 at room temper-
ature and aliquots of 20 ␮L samples were injected. Separation
of compounds from apple pomace extracts was performed on
a Pursuit XRsC18 (150 mm × 4.6 mm, 5 ␮m) column from Varian
(Courtaboeuf, France). The mobile phase employed for the separa-
tion was composed of water (solvent A) and methanol (solvent B)
both acidified with 0.1% formic acid and it was used according to
the following elution gradient program: 0 min, 80% A; 30 min, 10%
A; 45 min, 10% A.
A porous Graphitic Carbon (PGC) column (30 mm × 4.6 mm;
5 ␮m) from ThermoHypersil (Courtaboeuf, France) was used for
separation of triterpenic compounds. In this case, the mobile phase
was composed of MeOH (solvent A); MeCN (solvent B) and iPrOH
(solvent C) all acidified with 0.1% formic acid and the employed
elution gradient was established as follows: 0 min, 50% A, 50% B;
10 min, 100% B; 15 min, 50% B, 50% C; 20 min, 50% B, 50% C.
2.5. HPLC–MS analysis
An Agilent 1100 (Palto Alto, CA, USA) system equipped with a
binary pump, an oven and a DAD detector was coupled to a mass
spectrometer (API 3000 triple quadrupole AB Sciex, Forster City, CA,
USA). A TurboIonSpray source (electrospray ESI) was employed in
positive and negative modes; nitrogen was used as nebulization gas
and curtain gas. Optimization of mass parameters was performed
using the standard mixture containing phenolic and terpenic com-
pounds.
For ESI source in negative mode the following conditions were
used: nebulizer gas flow rate, NEB = 8 (1.2 L min−1 ); curtain gas
flow rate, CUR = 8 (1.2 L min−1 ); ion spray voltage, IS = −4000 V;
vaporization temperature, Tem = 200 ◦ C; declustering potential,
DP = −100 V; focusing potential, FP = −400 V; entrance potential,
EP = −10 V, For ESI source in positive mode the established
parameters were: nebulizer gas flow rate, NEB = 8 (1.2 L min−1 );
curtain gas flow rate, CUR = 8 (1.2 L min−1 ); ion spray voltage,
IS = 4200 V; vaporization temperature, Tem = 200 ◦ C; declustering
potential, DP = 70 V; focusing potential, FP = 300 V; entrance poten-
tial, EP = 10 V, The analyses were performed in full scan mode. Total
ionic courant (TIC) was measured between 100 and 1000 u with a
step size of 0.5 u. All results were recorded with the Analyst soft-
ware version 1.4.2 (Sciex Applied Biosystems).
The chromatographic columns were used at 20 ◦ C. The mobile
phases and elution gradient programs were the same as those used
for HPLC–DAD–ELSD analyses; the effluent flow was reduced to
0.3 mL min−1 before entrance into the ESI source.
796 C.G. Grigoras et al. / Industrial Crops and Products 49 (2013) 794–804
For a better molecular mass precision, a high resolution
mass spectrometer UHR-QTof maXis (Bruker, Bremen, Germany)
equipped with an ESI source was coupled to a UHPLC Ultimate 3000
RSLC (Dionex, Germering, Germany) chromatographic system. The
same HPLC Pursuit XRsC18 (150 mm × 4.6 mm, 5 ␮m) column was
used under the same chromatographic conditions for separation
of compounds before mass acquisition and the LC effluent was
reduced to 0.3 mL min−1 before entrance into the ESI source. Data
acquisition and treatment have been possible thanks to Data Anal-
ysis v.4.0 (Bruker Daltonik) software capable to propose different
brute formulas for one molecular mass thanks to SmartFormulaTM
Editor specific tool.
2.6. Antioxidant activity
2.6.1. Crude extracts fractionation procedure
150 mg of dried crude extracts of apple pomace obtained by
microwave assisted extraction were submitted to a fractionation by
conventional liquid–liquid extraction. Crude extract was dissolved
in water/heptane (1/1, v/v) mixture and extracted successively with
equal volumes (3× 50 mL) of heptane and then ethyl acetate and
led to three different fractions: two organics (heptane and ethyl
acetate) and one aqueous (residue obtained at the end of the proce-
dure). All fractions were evaporated until dryness under vacuum at
40 ◦ C. Solutions of apple pomace crude extracts (5 mg L−1 in MeOH),
three extract fractions (5 mg L−1 in MeOH) and standard solutions
(1 mg L−1 in MeOH) were used for determination of antioxidant
activity.
2.6.2. Determination of total phenolic content
A miniaturized method based on that proposed by Singleton
and Rossi (1965) was employed. 10 ␮L of standard solutions, crude
extracts, fractions or water (blank) and 10 ␮L of Folin-Ciocâlteu
(FC) reagent were placed in 96-well microplates. After 1–8 min,
30 ␮L of Na2 CO3 aqueous solution (20%) were added. Ultra pure
water was used to reach a final volume of 200 ␮L. The blue mixture
was incubated at room temperature for 2 h and its absorbance was
read at 760 nm with a ␮Quant Universal Microplate spectropho-
tometer (Montigny le Bretonneux, France). Results were expressed
in mg L−1 of gallic acid equivalents (GAE mg L−1 ) using a linear
equation based on the calibration curve of gallic acid from 5 to
1000 mg L−1 . All samples were analyzed at least four times.
2.6.3. Determination of flavonoid content
Total flavonoid content was determined by the aluminum col-
orimetric method presented by Ismail et al. (2010) with some
modifications. 100 ␮L of standard solutions, crude extracts, frac-
tions, or water (blank) were mixed with 100 ␮L of AlCl3 aqueous
solution (2%) in 96-well microplates. After 10 min of incubation at
room temperature, the absorbance of the mixture was measured
at 435 nm. At least four replicates were performed for each sam-
ple. The total flavonoid content was expressed as mg L−1 of rutin
equivalents (RE mg L−1 ) based on the linear equation expression of
a calibration curve of rutin (from 5 to 150 mg L−1 ).
2.6.4. Determination of DPPH radical-scavenging activity
The DPPH radical scavenging activity was determined according
to the method introduced by Blois (Blois, 1958). Aliquots of 10 ␮L
of each crude extract, fraction or standard solution were mixed
with a 190 ␮L DPPH (75 ␮M in MeOH) in 96-well microplates. The
discoloration of purple free radical DPPH solution by the added
samples was measured at 517 nm after 30 min of incubation at
room temperature in the dark. Results were expressed as inhibition
percentages using the following relation:
AbsDPPH t=30 min − AbsS t=30 min
I% = × 100
AbsDPPH t=30 min
AbsDPPH t = 30 min : absorbance of DPPH blank sample after 30 min of
incubation; AbsS t = 30 min : absorbance of tested samples after 30 min
of incubation.
2.6.5. Determination of ferric-reducing antioxidant power (FRAP)
The FRAP assay was carried out according to the method devel-
oped by Benzie and Strain (1996) with some modifications. The
FRAP reagent was prepared daily by mixing acetate buffer (300 mM
at pH 3.6), FeCl3 ·6H2 O (20 mM) and TPTZ (10 mM in HCl 40 mM)
in a 10:1:1 (v/v/v) ratio. The mixture was heated up to 37 ◦ C and
immediately used for measurements.
10 ␮L of standard solutions, crude extracts, fractions or ultra-
pure water (blank) were introduced in 96-well microplates and
first mixed with 20 ␮L of ultrapure water. 170 ␮L of the prepared
FRAP reagent were added in order to obtain a 200 ␮L final volume.
The change in absorbance from the red to the blue was followed at
593 nm after 10 min of incubation. A trolox calibration curve was
done between 50 and 1000 mg L−1 and the obtained results were
expressed in mg L−1 of trolox equivalents (TE mg L−1 ).
3. Results and discussion
3.1. Extraction procedure
In order to evaluate the influence of the extraction process on
the extraction yield (calculated as the ratio of dried extract mass on
the initial raw material dried mass) and on the extract composition,
different extraction techniques were tested: maceration (M), pres-
surized liquid extraction (PLE), extraction assisted by ultrasound
(UAE) or by microwave (MAE). For the four apple pomace culti-
vars (Gala, Golden, Granny Smith, and Pink Lady), MAE leads to
the higher amount of extracted molecules with the best extraction
yield (54 ± 1%). Furthermore, whatever the extraction method used
similar HPLC chromatographic profiles were observed for the dif-
ferent crude extracts. Hence, MAE was defined as the most suitable
extraction method for our studies seeing that the extraction yield
was the best one and the extraction time the lowest one (less than
30 min for 3 extraction cycles of 30 s each including the cooling step
between each cycles).
If the extraction technique did not have a large influence on the
extract composition, the extraction solvent should have one. Thus,
three extraction solvents (H2 O:MeOH (90:10) mixture, EtOH and
EtOAc) were tested with the MAE conditions described above (3
cycles of 30 s of microwave irradiation at 1000 W). Fig. 1 depicts
the HPLC–UV and HPLC–ELSD chromatographic profiles of each
extract. It appears that the H2 O:MeOH (90:10) solvent mixture
extracts preferentially the most polar solutes (0 < tr < 12 min). The
intensity of this first group of peaks is obviously higher in the
H2 O:MeOH extract than in the EtOH one, and is not present in the
EtOAc extract (Fig. 1A). The less polar compounds (30 < tr < 40 min)
were extracted only with both EtOH and EtOAc solvents with sim-
ilar chromatographic peak intensities (Fig. 1B). EtOH seems to be
a good compromise solubilizing compounds with a larger polarity
range, from polar compounds eluted in void time to quite non polar
compounds eluted with 90% of MeOH in mobile phase. Hence EtOH
was chosen as extraction solvent to perform the most complete
extract, representative of the whole apple pomace content, for the
following experiments. Thanks to the ELSD detection which is able
to detect all non volatile compounds (even without chromophore
group) with quite similar response coefficients, it is obvious that
the less polar compounds are the most concentrated with higher
 C.G. Grigoras et al. / Industrial Crops and Products 49 (2013) 794–804 797

A
Water : MeOH (90 : 10) EtOH EtOAc UV 280 UV
nm
110000
99000
88000
77000

Intensity (uA)
66000
55000
44000 aca
33000
b
22000
11000 c
a

0
0 5 10 15 20 25 30 35 40 45 50
Time (minutes)

B
Water : MeOH (90 : 10) EtOH EtOAc ELSD
900000

800000

700000

600000
Intensity (uA)

500000

400000

300000
ca
200000
b
100000 c
0
0 5 10 15 20 25 30 35 40 45 50
Time (minutes)

Fig. 1. Chromatographic profiles of Granny Smith pomace MAE extracts. Influence of the extraction solvent (a: H2 O:MeOH (90:10) mixture; b: EtOH; c: EtOAc). Column:
Pursuit XRs C18 (Lx = 150 mm × 4.6 mm, 5 ␮m); mobile phase: A. H2 O; B. MeOH both acidified with 0.1% HCOOH; flow rate: 1 mL min−1 ; injected volume: 20 ␮L; elution
gradient: 0-30 min, 20-90% B; 30-45 min, 90% B; detections A: UV at 280 nm and B: ELSD: drift tube temperature: 52 ◦ C; nebulizer gas pressure: 3.2 bar; gain: 6.

peak intensities while the more polar, well detected by UV, appear the apple pomace extract, the first group of peaks (5 < tr < 15 min)
in fact no detected by ELSD and consequently less concentrated in can correspond to benzoic, hydroxycinnamic and flavanol deriva-
the crude ethanolic extract. tives, hypothesis confirmed by the absorption spectra which show
maxima at 280-330 nm. The second group can be attributed to
flavonoids molecules (some absorbance spectra show a maximum
3.2. Identification of the main groups of bioactive compounds
at 366 nm corresponding to flavonols and the other ones show
extracted from apple pomaces
a maximum at 280 nm corresponding to chalcones). Some com-
pounds (with a maximum absorbance at 310 nm) were eluted
3.2.1. Development of a generic HPLC–DAD–ELSD method from
between 27 and 35 min, whereas none of the standard molecules
standards
were eluted in this range. The last group of peaks with reten-
The first identification of the main groups of apple pomace
tion time around 38-40 min well detected by ELSD but not by
extracted compounds was based on the comparison of their reten-
UV (absorbance below 220 nm) can be composed of triterpenes.
tion times and absorption spectra with those observed for different
In these chromatographic conditions triterpenic standards com-
standards described as present in apple and analyzed in the same
pounds (oleanolic and ursolic acids, uvaol and erythrodiol) were
conditions. In order to cover different molecular families with a
coeluted (Fig. 3B), so the intense peak observed at the same reten-
wide polarity range, the chosen standards were six phenolic com-
tion time for the apple extract also could be the result of coelution
pounds (gallic acid, chlorogenic acid, catechin, rutin, quercetin and
of several triterpenic compounds.
phloridzin), five triterpenic compounds (betulinic, oleanolic, urso-
Separation of these compounds was improved starting from
lic acids, uvaol and erythrodiol) and one fatty acid (palmitic acid),
the conditions described by Rhourri-Frih (Rhourri-Frih et al.,
whose structures are presented in Fig. 2.
2012) using Porous Graphitic Carbon (PGC) column. Betulinic acid,
Fig. 3 reports the HPLC–DAD–ELSD separation obtained, on the
oleanolic acid, ursolic acid, erythrodiol and uvaol were separated in
Pursuit XRsC18 (150 mm × 4.6 mm, 5 ␮m) column in the conditions
less than 20 min (Fig. 4) using MeOH–MeCN–iPrOH gradient mobile
described in Section 2.4, for the twelve standards mixture solution.
phase.
Benzoic acids (gallic acid), hydroxycinnamic acids (chlorogenic
acid) and flavanols (catechin) are eluted between 3 and 10 min
(Fig. 3A). Then it can be observed the elution of flavonols (rutin 3.2.2. Comparison of HPLC–DAD–ELSD analysis of four apple
and quercetin) and chalcones (phloridzin) between 15 and 20 min pomace varieties
and the elution of less polar triterpenic compounds and fatty acid Crude extracts of the four apple cultivars were analyzed in the
between 35 and 45 min. Retention time of these standard molecules same HPLC–UV–ELSD conditions on both C18 (Fig. 5A) and PGC
can be compared to those of extracted compounds. Thereby in (Fig. 5B) columns. The comparison of the four varieties shows the
798 C.G. Grigoras et al. / Industrial Crops and Products 49 (2013) 794–804

COOH
O OH HO OH
O
HO O
HO O OH
OH
HO OH OH OH
OH OH OH

GA – Gallic acid CA – Chlorogenic acid CAT – Catechin

OH

OH OH HO O
OH
OH OH
HO OH HO
O O OH
HO O
OH OH O O

O HO O
OH HO O O
HO HO
OH O OH OH

QUE – Quercetin PH – Phloridzin RUT – Rutin

COOH COOH COOH

HO HO HO

OA – Oleanolic acid UA – Ursolic acid BA –Betulinic acid

O
OH

OH OH

HO HO

ER – Erythrodiol Uv – Uvaol PA – Palmitic acid

Fig. 2. Standard molecule structures.

same chromatographic profiles for both phenolic (C18 column) and
triterpenic (PGC column) composition. Same compounds seem to
be present in the four varieties, only some differences in the rela-
tive proportion of molecules were noticed. For all cultivars, peak
groups containing phenolic acids (5 < tr < 10 min) and flavonoid
compounds (12 < tr < 20 min) could be observed with UV detec-
tion. Compounds eluted between 30 and 40 min were also present
in all extracts. For triterpenic compounds same profiles were also
observed. The last peak which has the same retention time as urso-
lic acid seems to correspond to the main component. Pink lady
extract shows the highest peak intensity for all compounds fam-
ilies and appears to be more concentrated in bioactive molecules.
Whereas, Granny smith extract shows lower amount of triterpenic
compounds. Gala and Golden extracts present intermediate con-
centrations for both polyphenolic and triterpenic compounds.
3.2.3. HPLC–MS analysis of apple pomace extracts
From the four apple pomace cultivar, Gala extract appears to
have an average composition representative of the four cultivars,
with relative similar proportions for all detected compounds then
it was appropriated to develop further HPLC–MS identification. In
order to detect the maximum of compounds without any mass
selection, experiments were achieved in full scan analysis in the
mass range from 100 to 1000 Da.
3.2.3.1. Phenolic compounds HPLC–MS analysis. Phenolic com-
pounds of Gala ethanolic pomace extract were analyzed by
HPLC–ESI-MS on the Pursuit XRs C18 column with the same elution
program used for HPLC–DAD–ELSD. In ESI positive ionization mode
phenolic compounds were detected as sodium adduct [M+Na]+
(rutin: m/z 633.5, phloridzin: m/z 459.5, quercitrin: m/z 471.5).
In negative mode, a higher signal intensity was obtained and the
deprotonated molecule ion [M−H]− could be observed.
As the negative mode gave a better sensitivity and allowed to
obtain easily nominal molecular mass it was chosen to performed
identification of phenolic molecules from apple pomace extract.
Fig. 6 shows the superposition of extracted ions detected with ESI
ionization in negative mode. Hence, 12 phenolic compounds can
be detected. As on HPLC-DAD chromatogram (Figs. 1A and 5A) two
groups of molecules can be distinguished; the first one between
6 and 12 min containing peaks from 1 to 6 and the second one
between 14 and 18 min containing peaks from 7 to 12 that present
higher intensities.
Moreover, in ESI negative mode, loss of neutral molecules (H2 O
(18 Da), CO (28 Da)) may be noticed and the glycoside phenolic
compounds loss the sugar moiety directly in the source allowing
an attempt of molecule identification even in full scan analysis.
Similar type of flavonoid fragmentation was reported by other
studies (Cuyckens and Claeys, 2002; March et al., 2006; Shu et al.,
 C.G. Grigoras et al. / Industrial Crops and Products 49 (2013) 794–804 799

A
800000 PH UV 280 nm
UV

700000
GA
600000 QUE

(uA)
Intensité(uA)
500000

Intensity 400000 CA RUT

300000

200000 CAT

100000

0
0 5 10 15 20 25 30 35 40 45 50
Time (minutes)
Temps (minutes)
B
600000 ELSD
OA ER
UA Uv
500000

400000
(uA)
Intensité(uA)
Intensity

300000

BA|
200000

QUE
100000 RUT PA
PH
GA CAT CA
0
0 5 10 15 20 25 30 35 40 45 50
Time (minutes)
Temps (minutes)

Fig. 3. HPLC–DAD–ELSD separation of twelve phenolic, triterpenic and fatty acid compounds in standard mixture on Pursuit XRS C18 column. GA, gallic acid; CAT, catechin;
CA, chlorogenic acid; RUT, rutin; PH, phloridzin; QUE, quercetin; BA, betulinic acid; OA, oleanolic acid; UA, ursolic acid; ER, erythrodiol; Uv, uvaol; PA, palmitic acid; injected
concentrations: 100 mg L−1 ; column: Pursuit XRs C18 (Lx = 150 mm × 4.6 mm, 5 ␮m); detections A: UV at 280 nm and B: ELSD; same conditions as in Fig. 1.

2010; Wu et al., 2004; Xing et al., 2007). Table 1 presents the
[M−H]− nominal mass and the main ion-source fragments used
to propose molecules identification. For example, the in-source
fragmentation of peak 8 mainly gave an ion at m/z 300.5 due to the
loss of 309 from the depotonated molecule ion at m/z 609.5 which
indicated the presence of a rutinose moiety [M−H−309]− . Genine
fragmentation due to Retro Diels Alder (RDA) rearrangements
leads to ions at m/z 179, 151, 121, 107. This fragmentation scheme
and the suitable retention time (compared to standard injection)
allowed the identification of compound 8 as rutin molecule
(Table 1). In the same way, the compound 10 can be identified as
phloridzin (Table 1); the ion at m/z 273.5 was due to the loss of

Table 1
Characteristic ions of phenolic compounds from Gala apple pomace ethanolic extract.

Peaks HPLC–MS HPLC–HRMS Proposed compound

identification

[M−H]− (m/z) Fragment ions (m/z) (−) Fragment ions [M-H]− (m/z) Proposed molecular
(m/z) (+) formula

1 577.5 451; 425; 407; 289; 125 577.135 C30 H26 O12 Procyanidin dimer
2 353.5 191 163 353.08772 C16 H18 O9 Chlorogenic acida
3 289 245; 205; 187; 109; 203; 151; 125 289.07204 C15 H14 O6 Epicatechin
4 517.5 385; 293; 233; 223; 205; 191; 153; 149 517.22863 C24 H38 O12 Unknown
5 351 101; 113 351.0932 C14 H24 O10 3-Digitalose-quinic acid
6 337.5 173 337.09297 C16 H18 O8 Coumaroylquinic acid
7 568 273 437; 275 567.17191 C26 H32 O14 Phloretin xyloglucoside
8 609.5 463 465; 303 609.14592 C27 H30 O16 Rutina
9 433.5 301 303 433.07782 C20 H18 O11 Quercetin pentoside
10 435.5 273; 167 275 435.12955 C21 H24 O10 Phloridzina
11 433.5 301 303 433.07764 C20 H18 O11 Quercetin pentoside
12 447.5 301 447.09307 C21 H20 O11 Quercitrina
a
Identification confirmed by injection of standard molecules.
800 C.G. Grigoras et al. / Industrial Crops and Products 49 (2013) 794–804

1150000 BA
5.9e6
5.9e6 8 ESI -

950000
5.0e6
5.0e6 10
11
Intensity (uA)

750000 7

Intensity, cps
4.0e6
4.0e6
550000 12
3.0e6
3.0e6
Uv
350000 OA
2.0e6
2.0e6 3 9
UA
150000 ER 4
2
1.0e6
1.0e6
-50000 1 5 6
0 2 4 6 8 10 12 14 16 18 20 0.0
0.0
Time (minutes) 4 6 8 10 12 14 16 18 20
Time, min
Fig. 4. HPLC–ELSD analysis of five triterpenic acids in standard mixture separa-
tion on Porous Graphitic Carbone column. BA, betulinic acid; OA, oleanolic acid; UA, Fig. 6. Superposition of extracted ions for phenolic compounds from Gala pomace
ursolic acid; ER, erythrodiol; Uv, uvaol; column: Hypercarb (Lx = 50 mm × 4.6 mm, ethanolic extract. Column: Pursuit XRs C18 (Lx = 150 mm × 4.6 mm, 5 ␮m) same
5 ␮m); ELSD detection: drift tube temperature: 52 ◦ C; nebulizer gas pressure: conditions as in Fig. 1, ESI in negative mode.
3.2 bar; gain: 6; mobile phase: A. MeOH; B. MeCN; C. iPrOH all acidified with 0.1%
HCOOH; flow rate: 1 mL min−1 ; elution gradient: 0 min, 50% A, 50% B; 10 min, 100%
B; 15 min, 50% B, 50% C; 20 min, 50% B, 50% C; injected volume: 20 ␮L. (5), coumaroylquinic acid (6), phloretin-2 -xyloside (7) as reported
in Table 1. The first identification proposed by HPLC–DAD analysis
and standard injection was confirmed by mass spectrometry; the
162 from the ion at m/z 435.5 which indicated the presence of a first molecular group (with retention times between 6 and 12 min)
glucose moiety [M−H−162]− . corresponds to phenolic acids and catechin derivatives while the
The identification of other compounds in absence of corre- second group of compounds (14 < tr < 18 min) is composed mainly
sponding standards cannot be performed under these conditions. of flavonoids derivatives.
Consequently, in order to obtain exact mass and molecular for-
mula of unknown compounds high resolution mass spectrometry 3.2.3.2. Triterpenic compounds HPLC–MS analysis. Triterpenic com-
(HRMS) was coupled to HPLC. Table 1 also gathered the [M−H]− pounds contained in the Gala pomace ethanolic extract were
exact mass recorded by HRMS associated to the molecular formula studied with the same procedure as the phenolic compounds:
proposed by the HRMS software. The comparison of our results with first analyses were carried out coupling HPLC to mass spectrom-
those reported in literature (Alonso-Salces et al., 2004; Rodríguez- etry (HPLC–MS). ESI ionization was tested in both positive and
Medina et al., 2009; Tsao et al., 2003) leads to the identification negative mode. Negative mode was more sensitive and allowed
of other polyphenolic molecules such as 3-digitalose-quinic acid deprotonated molecule ion [M−H]− detection. Concerning in
source fragmentation both ionization modes gave complemen-
tary information . As the Pursuit XRs C18 and the Hypercarb PGC
A columns provided different selectivities for separation for triter-
Gala apple Golden apple Granny Smith apple Pink Lady apple

800000 UV 280 nm
penic compounds, HPLC–MS analyses were performed on both
columns and the obtained results were gathered in Table 2 to
700000
a propose molecule identifications. Fig. 7 shows extracted ions chro-
600000
matograms recorded in ESI negative mode on the Pursuit XRs C18
Intensity (uA)

500000
b
column for this solute group.
400000 Two groups of molecules can be distinguished. The first one,
300000 presented in Fig. 7A, with molecular mass ranging from 450 to
c
200000 500 u which absorbs only below 220 nm and the second one,
100000 shown in Fig. 7B, with molecular mass around 610-650 u which
d
0
absorbs up to around 310 nm. For each group, several chromato-
0 5 10 15 20 25 30 35 40 45 50 55 60 graphic peaks with different retention times but with identical
Time (minutes) [M−H]− mass could be observed. For example several peaks with
B m/z 469.5 or 455.5 can be detected. In order to distinguish these
185000
Gala apple Golden apple Granny Smith apple Pink Lady apple compounds thanks to their exact mass and their molecular formula
166500
ELSD and to attempt their identification HPLC was coupled with HRMS
148000 (in the same conditions as describe in Section 2.5). Exact mass
129500 and molecular formula proposed by the HRMS software are given
Intensity (uA)

111000 Table 2. Unfortunately no difference can be found between these

a
92500 molecules which correspond to isomeric forms. Indeed the three
74000 peaks observed with m/z 455.5 were also all detected in HRMS with
b
55500
m/z 455.35364 corresponding to molecular formula C30 H48 O3 . In
37000 c
this case, the injection of isomeric standard molecules of betulinic,
18500
d ursolic and oleanolic acids allowed the identification of each peak
0
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 (Table 2). This identification was consistent with literature that
Time (minutes) describes these compounds in apples (McGhie et al., 2012). Nev-
ertheless at least 6 chromatographic peaks were detected with a
Fig. 5. HPLC–DAD–ELSD profiles of four apple cultivar pomace ethanolic MAE m/z 471.34787 and correspond to a molecular formula C30 H48 O4 .
extracts (a: Gala G; b: Golden; c: Granny Smith; d: Pink Lady). (A) Column: Pursuit
Unfortunately, in absence of any standard molecule with this spe-
XRs C18 (Lx = 150 mm × 4.6 mm, 5 ␮m) UV detection at 280 nm; same conditions
as in Fig. 1. (B) Column: Hypercarb (Lx = 50 mm × 4.6 mm, 5 ␮m); ELSD detection, cific m/z ratio it was not possible to attribute these different
same conditions as in Fig. 4. chromatographic peaks to specific compounds. However pomolic
 C.G. Grigoras et al. / Industrial Crops and Products 49 (2013) 794–804 801

Table 2
Characteristic ions of triterpenic compounds from Gala apple pomace ethanolic extract.

Peaks HPLC–MS HPLC–HRMS Proposed compound

identification

[M−H]− (m/z) Fragment ions (m/z) (−) Fragment ions (m/z) (+) [M−H]− (m/z) Proposed molecular
formula

13 503.5 503.33706 C30 H48 O6 Tetrahydroxy-urs-12-

en-28-oic
14, 15 501.5 409; 453; 471; 483 501.32209 C30 H46 O6 Unkown
16 488 469; 426 407; 425; 453; 471 487.34281 C30 H48 O5 Euscaphic acid or
2␣,3␤,13␤-
trihydroxyurs-11-en-
28-oic acid or
isomer
17, 21 486 468; 423; 406 423; 469; 405; 451 485.32724 C30 H46 O5 Annurcoic acid and
isomer
20, 22, 27, 28 469.5 451; 423; 407 453; 425; 407; 219; 201 469.33292 C30 H46 O4 1-Hydroxy-3-oxours-
12-en-28-oic acid and
isomer
18, 19, 23–26 472 454; 410; 407 455; 437; 409; 391; 191; 201 471.34787 C30 H48 O4 Pomolic acid and
isomer
29 455.5 325 439; 411; 203; 191 455.35364 C30 H48 O3 Betulinic acida
30 455.5 407; 325 439; 411; 203; 191 455.35383 C30 H48 O3 Oleanolic acida
31 455.5 407 439; 411 455.35359 C30 H48 O3 Ursolic acida
a
Identification confirmed by injection of standard molecules.

acid was described in apple extract (D’Abrosca et al., 2006, 2005;
He and Liu, 2007) and corresponds to the molecular mass, thus one
of these peak could be pomolic acid and the other peaks its isomers.
According to its low absorption, the comparison with some
standards retention time, molecular mass and mass spectrum, the
first group of molecule (Fig. 7A) seems to be composed of pen-
tacyclic triterpenic acids. Complementary ESI-MS experiments, in
positive ionization mode, depict for this first molecule group in-
source fragment ions with m/z ratio equal to 191, 201, 203, 219,
corresponding to the retro-Diels-Alder cleavage of the C cycle char-
acteristic of this kind of compounds (Huang et al., 2007; Siddiqui
et al., 1995). The elution order of these compounds mainly fol-
lowed the polarity of the molecule and the number of hydroxyl
groups. The first eluted compounds posses six oxygen and the
last one only three oxygens. Hence, molecular mass and molecular
formula corresponding to compounds as annurcoic acid, pomolic
acid or 1-hydroxy-3-oxours-12-en-28-oic acid described in apple
fruits (D’Abrosca et al., 2005, 2006) could be observed and confirm
the presence of these solutes in apple pomace. Moreover isomeric
compounds with methyl and/or hydroxyl substituent located on
different position on the pentacyclic moiety could be also detected.
Hence the presence of 19 triterpenic compounds could be detected
in the Gala pomace ethanolic extract (Fig. 7A).
The second group of molecules (Fig. 7B) was characterized by
higher molecular mass and absorbs up to 310 nm. Unfortunately
none in-source fragmentation could be observed with ESI neither
in positive nor in negative ionization mode to help identification.
Extracted ion chromatogram presented in Fig. 7B shows three main
peak groups corresponding to three molecular masses. Mass and
UV data suggest the pentacyclic moiety substitution by a coumaryl
group (He and Liu, 2007) (Table 3). For each molecular formula,
many isomeric molecules could be found according to the position
of the substituents (methyl, hydroxyl, coumaryl). Consequently,
15 coumaryl triterpenic derivatives could be detected in the Gala
pomace ethanolic extract.

3.3. Evaluation of antioxidant activity of apple pomaces extracts

Two main molecule families have been revealed in apple

Fig. 7. Superposition of extracted ions for (A) triterpenic and (B) coumaryl triter-
penic compounds from Gala pomace ethanolic extract. Column: Pursuit XRs C18
(Lx = 150 × 4.6 mm, 5 ␮m); same conditions as in Fig. 1; ESI in negative mode.
pomaces: phenolic and triterpenic compounds. Many researches
were aimed to determine the antioxidant properties of pheno-
lic compounds extracted from this type of plant material (Awad
et al., 2000; Carbone et al., 2011; Diñeiro García et al., 2009; Lu and
Yeap Foo, 2000; Soares et al., 2008) but only few papers (Cefarelli
et al., 2006; He and Liu, 2008) were directed to the study of the
antioxidant character of triterpenic molecules. As consequence,
the antioxidant activity of apple pomace extracts, containing both
phenolic and triterpenic compounds, was measured. Moreover, in
order to evaluate the activity of each compound family and if a
synergetic effect due to the presence of both types of compounds
in the same extract exists, the crude extracts were fractionated by
802 C.G. Grigoras et al. / Industrial Crops and Products 49 (2013) 794–804

Table 3
Characteristic ions of coumaryl triterpenic compounds from Gala apple pomace ethanolic extract.

Peaks HPLC–MS HPLC–HRMS Proposed compound

identification

[M−H]− (m/z) Fragment ions (m/z) (−) Fragment ions (m/z) (+) [M−H]− (m/z) Proposed molecular
formula

33, 34 650 – – 649.37458 C39 H54 O8 Unkown

35, 36 634 – – 633.37981 C39 H54 O7 3␤-Trans-p-
coumaroyloxy-
2␣,3␤,13␤-trihydroxy-
urs-11-en-28-oic acid
or isomer
32, 39, 41–46 617.5 – – 617.38475 C39 H54 O6 3␤-Trans-p-
coumaroyloxy-2␣-
hydroxyurs-12-en-28-
oic acid or
isomer

liquid–liquid extraction with heptane (hept), ethyl acetate (EtOAc)
and water (H2 O).
For this purpose, four different chemical tests were used: the
determination of total phenolic content (Folin-Ciocâlteu assay),
the determination of the total flavonoid content, the radical-
scavenging activity on DPPH and the ferric reducing antioxidant
power (FRAP) assay. For each test, crude extract and the three
fractions (heptane, ethyl acetate and the aqueous one) were ana-
lyzed. The results were compared to those obtained for standard
molecules representative of different compounds families: car-
bohydrate: glucose (GLU); phenolic compounds: gallic acid (GA),
p-coumaric acid (pCA), catechin (CAT), phloridzin (PH), quercetin
(QUE), rutin (RUT); triterpenic compounds: oleanolic acid (OA),
ursolic acid (UA), uvaol (Uv); fatty acid: palmitic acid (PA) ana-
lyzed.in the same conditions.
3.3.1. Fractionation of crude apple pomace extracts
Since the apple pomace extracts were rather complex with
different kind of molecules, a fractionation by conventional
liquid–liquid extraction with solvent of different polarity (heptane,
ethyl acetate and water) able to simplify their chemical compo-
sition was performed. This fractionation step was carried out on
the crude extracts obtained from the pomaces of the four apple
varieties. The proportions of each fraction, calculated as the dried
extracted mass on each fraction divided by the dried crude extract
mass used for fractionation, are given for the four cultivars in
Table 4. Heptane fraction was the richest one and represents around
30-40% of the crude extract, while the ethyl acetate fraction repre-
sents only 8-15% of the crude extract.
The obtained fractions were analyzed by HPLC–UV–ELSD (data
not shown) on the Pursuit XRs C18 column using the chro-
matographic conditions described in Section 2.4. Chromatograms
revealed that the developed fractionation system insures a con-
venient separation of the extracted molecules. Heptane fraction
contained mainly triterpenic compounds, while the ethyl acetate
one contained mainly phenolic compounds and the residue of
triterpenic compounds and the aqueous fraction is composed
Table 4
Liquid–liquid extraction of four cultivar apple pomace extracts: proportion of each
fraction.
Apple cultivar Hept fraction (%) EtOAc fraction (%) H2 O fraction (%)
Gala 42 12 27
Golden 41 13 37
Granny Smith 32 14 42
Pink Lady 42 9 28
especially of carbohydrates. This partitioning process was consis-
tent with the ELSD chromatogram obtained for the crude extract
(Fig. 1B) indicating that triterpenic compounds were major com-
pounds in the crude extract followed by carbohydrates while
phenolic compounds represented a low proportion of the crude
extract.
The evaluation of the antioxidant activity of the crude extracts
on the one hand and of each fraction enriched in one compound
family on the other hand could show if a synergetic effect existed
in the crude extract or if only one family hold the activity.
3.3.2. Determination of total phenolic content
The FC assays have shown positive responses for all the apple
pomace crude extracts and fractions tested. Fig. 8A reports the dif-
ferent values of total phenolic content. The hept and EtOAc fractions
seemed to be the richest for all apple cultivars while the water
fraction presents the lowest content. According to the HPLC anal-
ysis, the water fraction was composed especially of carbohydrates
which did not give a good response confirmed by the low response
of glucose standard molecule on the FC test. The EtOAc fraction
contained mainly the phenolic compounds and one part of triter-
penic compounds, so the high response of the EtOAc fraction was
consistent to its chromatographic profile. Surprising was the high
response of the heptane fraction that contained mainly triterpenic
compounds. Ursolic and oleanolic acids (major triterpenic acids
found in apple pomace extract – Fig. 5B) were tested as individ-
ual standard molecules with the FC assay and both molecules show
a low response to the FC test with low blue coloration. Then it is
not the presence of these molecules in hept fraction that explain
its high response. It can be suggested that the coumaryl part of
the triterpenic derivatives may have a positive effect increasing the
response to the FC test, but in the absence of any standard molecule
this hypothesis, unfortunately, could not be verified.
By comparing the FC responses of different apple varieties
(Fig. 8A), it appears that the Pink Lady apple pomace extract and
its fractions were the richest one. That confirms the conclusions
obtained from the HPLC analysis (Fig. 5A). It is followed by the
extracts and fractions of Golden while Granny Smith and Gala apple
pomace extracts and fractions revealed lower phenolic contents.
The obtained results showed that the Folin-Ciocâlteu test is not
specific to phenolic compounds. The reaction of the FC reagent
being possible also with other types of molecules as observed with
the hept fraction. The same conclusion was highlighted by other
studies. Prior et al. (2005) revealed that compounds such as carbo-
hydrates, aromatic amines, enediols and reductones may interfere.
Naczk and Shahidi (2006) stipulated that this test is able to detect
all the phenols groups.
 C.G. Grigoras et al. / Industrial Crops and Products 49 (2013) 794–804
Fig. 8. Diagrams of responses obtained for Gala, Golden, Granny Smith and Pink
Lady cultivars apple pomace crude extracts, hept, EtOAc, H2 O fractions for: total
phenolic content (A), total flavonoid content (B), DPPH radical-scavenging activity
(C) and FRAP ferric reducing antioxidant power (D).
3.3.3. Determination of total flavonoid content
The total flavonoid content (Fig. 8B) of crude extracts varies from
28.46 mg L−1 RE for Gala apple pomace extracts to 93 mg L−1 RE for
those of Golden apple pomace.
The EtOAc fractions enriched in flavonoids compounds such
as quercetin derivatives (Table 1) show the highest content in
flavonoids. As illustrated by Cornard and Merlin (2002) quercetin
is able to form stable compounds in presence of aluminum chlo-
ride since three different complexation sites: 3-hydroxy-4-keto,
5-hydroxy-4-keto, 3 ,4 -o-diphenolic exist in its structure. Hept
fractions also show positive responses to this assay even if they
did not contained quercetin derivatives. Some terpenic compounds
may present hydroxyl, acidic or coumaryl adjacent groups that
favored aluminum complexation. Water fraction did not contain
flavonoid compounds and these results are in good agreement with
those obtained by HPLC analyses.
3.3.4. Antioxidant activity
To investigate the antioxidant activity of crude extracts and dif-
ferent fraction two in vitro assays were used. The DPPH radical
scavenging and the FRAP assays. Results are resumed in Fig. 8C and
D. From the four apple pomace crude extracts, Gala and Golden
803
cultivars presented the most important ability to inhibit the DPPH
free radical (Fig. 8C). The fractionation step induces a beneficial
effect onto the ability to inhibit DPPH indeed for three cultivars
(Golden, Granny Smith and Pink lady) EtOAc fractions were found
significantly more active than crude extract. On the contrary, for
all cultivars, the H2 O fraction was regarded as the least antiox-
idant which can be explained by the presence of no-scavenger
molecules like carbohydrates. The hept fraction depicted antioxi-
dant activity slightly reinforced in comparison with corresponding
crude extract. For Gala cultivar crude extract and its hept and EtOAc
fractions show similar DPPH inhibition around 20% barely higher
than water fraction. In contrast, Pink Lady EtOAc fraction shows the
highest reinforced antioxidant activity reaching 80% of DPPH inhi-
bition. For this cultivar the EtOAc fraction represents only 9% of the
total crude extract, then the concentration of bioactive molecules
in this fraction appears very efficient and lead to high antioxidant
activity.
These results were correlated with FRAP assays where higher
activities were seen in EtOAc fractions (Fig. 8D). According to HPLC
analyses, the major phenolic compounds of crude extract were
found in EtOAc fraction confirming that phenolic derivatives could
be responsible of the antioxidant activity of apple pomace. The
activity of these molecules is related to their structure and depends
mainly on different structural features such as O H bound disso-
ciation energy, resonance delocalization of the phenol radical and
steric hydrance derived from bulky groups substituting hydrogen in
the aromatic ring (Sánchez-Moreno et al., 1998). Hept fraction con-
tained mainly terpenic compounds present in apple pomace which
appear to be not free radical inhibitors. This could be explained by
the low number of available hydroxyl groups which are known as
responsible for the inhibition process.
Upon the four apple pomace cultivars tested, antioxidant
potential (ferric reducing power and radical inhibition) was deter-
mined to be in the following range Pink Lady > Golden > Granny
Smith > Gala.
According to the four assays, the Pink Lady cultivar seemed to
be the most interesting one with higher content in phenolic and
flavonoids compounds and with the best free radical inhibition and
ferric reducing activities. Elimination of sugar compounds, which
represent 30-40% of the crude extract, by a water fractionation
allowed concentrating compounds of interest in the organic frac-
tions that exhibited higher activity than crude extract. It seems not
absolutely necessary to distinguish EtOAc and hept fractions since
both molecular families could participate to the antioxidant activity
of apple pomace extracts and should be beneficial for health.
4. Conclusions
The present study shows that pomaces obtained after pressing
different apple cultivars contain important bioactive compounds
belonging especially to phenolic and triterpenic groups. These com-
pounds may be recovered by microwave assisted extraction with
ethanol or ethyl acetate solvent that ensures high recoveries and
provides general extracts containing both phenolic and triterpenic
compounds.
The HPLC–UV–ELSD and HPLC–MS analyses of crude extracts
revealed the presence of numerous different compounds. Major
identified phenols were benzoic acids (gallic acid), hydroxycin-
namic acids (chlorogenic acid), flavanols (catechin), flavonols
(rutin) and chalcones (phloridzin). Among the triterpenes, urso-
lic acid and oleanolic acid were the most abundant. Many isomeric
molecules were contained in the extracts and their identification
need further experiment with NMR to resolve the isomerization.
Triterpenes with coumaryl substituent deserve to be more investi-
gated.
804 C.G. Grigoras et al. / Industrial Crops and Products 49 (2013) 794–804
For the four cultivars (Gala, Golden, Granny smith and Pink
Lady) crude extracts and their fractions enriched in compounds of
interest show attractive antioxidant activity confirming that apple
pomace could be a valuable source of bioactive molecules including
not only polyphenolic compounds but also triterpenic ones.
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