J Food Sci Technol (August 2017) 54(9):2890–2901

DOI 10.1007/s13197-017-2727-0

ORIGINAL ARTICLE

Antioxidant properties and phenolic profile characterization

by LC–MS/MS of selected Tunisian pomegranate peels
Mouna Abid1 • Héla Yaich1 • Salma Cheikhrouhou1 • Ibtihel Khemakhem1 •

Mohamed Bouaziz2 • Hamadi Attia1 • M. A. Ayadi1

Revised: 24 May 2017 / Accepted: 30 May 2017 / Published online: 19 June 2017
Ó Association of Food Scientists & Technologists (India) 2017

Abstract Antioxidant contents and activities of different Introduction

extracts from four Tunisian pomegranate peels, locally
called ‘‘Acide’’, ‘‘Gabsi’’, ‘‘Nebli’’ and ‘‘Tounsi’’, were Over the last five decades, there has been a trend to find
studied. Peels samples were extracted with three solvents new sources of natural antioxidants, such as agronomic by-
(water, ethanol and acetone). For each extract, the total products that have traditionally been undervalued.
phenol contents and antioxidant activity were evaluated. The toxicological effects of synthetic antioxidants
The highest values of polyphenol, tannins, flavonoids and together with the consumer preference for natural products
anthocyanins were recorded in the acetone extract of Acide have resulted in increased interest in the search for and use
ecotype with 304.6 mg gallic acid equivalent/g; 292.23 mg of natural antioxidants present in fruits and vegeta-
gallic acid equivalent/g; 15.46 mg Quercetin/g and bles (Püssa et al. 2009). Some examples of fruit by-prod-
54.51 mg cy-3-glu/100 g, respectively. The acetone extract ucts that can show the profitability of bioactive compounds
of Acide ecotype also showed the highest free radical- extraction are citrus fruits (oranges, lemons and man-
scavenging and reducing power activity compared to other darins). Rich in carotenoids, the citrus peel can be used as a
extracts. Besides, the phytochemical analysis by LC–MS/ natural colorant in the food industry (Wang et al. 2008).
MS revealed a high content of ellagitannins with puni- Other successful examples of exotic fruit by-products that
calagin and punicalagin derivatives as the major com- may be considered as sources of bioactive compounds are
pounds that might be responsible for promising antioxidant coffee and mango (Miljkovic and Bignami 2002). Indeed
activity of pomegranate peel extracts. Two compounds coffee by-product showed an antioxidant capacity and
(Castalagin derivative and Galloyl-bis-HHDP-hex deriva- could be considered as a new potential functional ingre-
tive) were detected only in ‘‘Acide’’ ecotype in important dient (Borrelli et al. 2004). Mango peels were also reported
contents. to be a good source of antioxidants such as polyphenols
and carotenoids and can be utilized for the preparation of
Keywords Antioxidant propreties  Punica granatum L.  macaroni with improved antioxidant activity (Ajila et al.
LC–MS/MS analysis  Punicalagin 2010).
Tropical exotic fruit by-products are known as sources
of a great variety of antioxidants, and their particular
properties may be useful in maintaining food quality and
stability against oxydative phenomenon. Pomegrenate
(Punica granatum) is one of these exotic fruits widely
& M. A. Ayadi cultivated and consumed in the Mediterranean countries. In
ayadimedali@yahoo.fr; ayadimedali@gmail.com
Tunisia, pomegranate has been cultivated since ancient
1
Food Analysis Laboratory, National Engineering School of times under diverse agroclimatic conditions. The produc-
Sfax (ENIS), University of Tunisia, BP 3038, Sfax, Tunisia tion of pomegranate, consumed exclusively as fresh fruit
2
Laboratory of Electrochemistry and Environnement, ENIS, and juice in Tunisia, is in a constant evolution. However,
Université de Sfax, BP ‘1175’, 3038, Sfax, Tunisia this progress of production and industrial transformation is

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accompanied with an increase of the low-value peel samples at 105 ± 3 °C to constant weights (AOAC 1997).
quantities. Pomegranate peel, which constitutes approxi- The total ash was determined by calcination in muffle
mately 40% of the whole fruit, possess higher antioxidant furnace at 550 °C until constant weight was obtained
activity than the edible portion (Venkataramanamma et al. (AOAC 1997). The total nitrogen concentration was
2016). obtained using Kjeldahl method (AOAC 1997), and the
Pomegranates are reported to be a rich source of protein concentration was estimated using a nitrogen con-
polyphenolic compounds that include flavonoids (antho- version factor of 6.25. Fat content was determined by
cyanins, catechins and other complex flavonoids) and Soxhlet extraction with hexane at boiling point of the
hydrolyzable tannins (punicalin, pedunculagin, puni- solvent (AOAC 1997). Total, soluble and insoluble fibre
calagin, gallagic acid and ellagic acid esters of glucose) contents were determined according to the AOAC enzy-
which account for 92% of their antioxidant activities (Afaq matic–gravimetric method of Prosky et al. (1988). The total
et al. 2005). The pomegranate fruit was widely studied for sugar content was determined by the phenol–sulfuric acid
its phenolic content (Gil et al. 2000; Nuncio-Jáuregui et al. method of Dubois et al. (1956).
2015); however, there are only very few studies evaluating Colour measurements were carried out with a col-
the phenolic content of pomegranate peel, particularly, orimeter (Minolta CR-300, Minolta, Osaka, Japan). Mea-
Tunisian cultivars. surements were taken using the Commission International
Thus, the purpose of this study was to investigate the de l’Eclairage (CIE) L*a*b* system.
effects of different extracting solvents on the total
polyphenols and antioxidant activities of four Tunisian Extracts preparation
pomegranate peels ecotypes. An attempt was made to
determine individual phenolic compounds, especially tan- Five grams of finely-powdered peels were shaken for 1 h
nins, in pomegranate extracts using the high-performance (25 °C- 180 rpm) with 25 ml of solvents (acetone or
liquid chromatography (HPLC)-linear ion trap mass spec- ethanol or distilled water). For each solvent, the mixture
trometry with negative electrospray ionization was centrifuged at 3000g for 15 min and the supernatant
methodology. was recovered. The residue was re-extracted two times
with the same procedure described above. Then the
supernatants were combined and evaporated (concentrated
Materials and methods to dryness) at the adequate temperature under vacuum
(rotary evaporator, Heidolph, Schwabach, Germany), then
Plant material stored at -20 °C.

Mature pomegranate fruits, ecotypes ‘‘Acide’’ (Ac), Antioxidant compound quantification

‘‘Gabsi’’ (Ga), ‘‘Nebli’’ (Ne) and ‘‘Tounsi’’ (To), having no
visible external cuts or spoilage, were collected from the Total phenolic content (TPC) of the concentrated extracts
same oasis at Gabes region (southeast of Tunisia). Fruits was determined spectrophotometrically (UV mini 1240,
were manually peeled then the collected peels were cut into UV/VIS spectrophotometer, SHIMDZU, Kyoto, Japan) at
small pieces and maintained at -12 °C for further 760 nm according to the Folin–Ciocalteu method (Slinkard
physicochemical characterization. For antioxidant extrac- and Singleton 1977). TPC was expressed as mg gallic acid
tion, samples were lyophilized (Thermo Electro Corpora- equivalent (GAE) per gram of extract.
tion, USA), ground and maintained at room temperature. Total tannin content was determined by Folin–Ciocalteu
procedure, after the removal of tannins by their adsorption
Physicochemical characterization of pomegranate on insoluble matrix (polyvinylpolypyrrolidone, PVPP)
peel (Kollidon CL, BASF, Germany) and non-absorbed phe-
nolics were determined as described. The calculated values
Fruits of each ecotype were individually analyzed for were subtracted from the total polyphenol contents and
physical characteristics. Pomegranates were weighed, total tannin contents expressed as mg gallic acid equivalent
then the peels were manually separated from the fruits, (GAE) per gram of extract.
and the percentage of pulps and peels per fruit were Total flavonoid content of extracts was determined by
measured. the method described by Zhishen et al. (1999). The
Proximate composition (dry matter, lipid, ash and pro- absorbance of the mixture was then measured spec-
tein content) was determined according to the method trophotometrically at 510 nm. The total flavonoid content
described by the Association of Official Analytical Che- was expressed as mg Quercetin Equivalent (QE) per gram
mists (AOAC 1997). Dry matter was determined by drying of extract.

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The amount of carotenoids was determined according to LC–MS/MS analysis

the method described by kuti (2004) and was carried out on
an aliquot of hexane extract by measuring absorbance LC–MS/MS (liquid chromatography–tandem mass spec-
spectrophotometrically at 450 nm. The total carotenoids trometry) analyses were performed as described by Ben
were calculated using an extinction Coefficient of b-car- Mansour et al. (2015) on a Thermo Scientific System
otene, E1% = 2505. consisting of an Accela U-HPLC unit with a photodiode
The total anthocyanins of pomegranate peel extract were array detector and an LTQ Orbitrap XL mass spectrometer
determined by a pH differential method using 2 buffer fitted with an electrospray source. Chromatography was
systems: potassium chloride buffer (pH 1.0, 0.025 M) and performed on 5 lL sample injections onto a 150 9 3 mm
sodium acetate buffer (pH 4.5, 0.4 M). Peel extracts were i.d., 3 um, Luna C18(2) column (Phenomenex) using a 400
mixed with the corresponding buffers and read against lL/min linear mobile phase gradient of methanol/water/
water as a blank at 510 and 700 nm (Cam et al. 2009). acetonitrile ?1% formic acid changing from 0:90:10 to
Absorbance (A) was calculated using: 90:0:10 over 20 min followed by an isocratic phase for
5 min and then a column wash phase and equilibrium of
A ¼ ðA510  A700ÞpH1:0  ðA510  A700ÞpH4:5
column for 3 min before the next injection. The electro-
with a molar extinction coefficient e of 29,600. The results spray ionization (ESI) source of the mass spectrometer was
were expressed as mg of cyanidin-3-glucoside equivalents operated in negative mode since the phenolic compounds
(CGE) per 100 g of extract. in question ionize better in this mode. The orbitrap mass
analyzer was set to scan in range m/z 200–2000 at 30,000
Antioxidant activity resolutions in negative polarity, while the linear ion-trap
analyzer performed MSn analyses on the most abundant
DPPH free radical scavenging activity ions in both polarities using an ion isolation window of
±2 m/z and relative collision energy of 35%. Phenolic
The DPPH radical-scavenging activity of the extracts compounds were identified on the basis of their Rt values,
(50 lg/ml) was determined spectrophotometrically at UV spectra and mass spectra, as well as by comparison of
517 nm as described by Bersuder et al. (1998). the spectra with those of the available authentic standards.
The control was conducted in the same manner, except The structure assignment of compounds for which no
that distilled water was used instead of extract. Butylat- standards were available was based on a systematic search
edhydroxyanisole (BHA) was used as a standard. for molecular ions using extracted ion mass chro-
matograms and comparing those with data in the literature.
Reducing power assay
Statistical analysis
The ability of the extracts (20 lg/ml) to reduce iron (III)
was determined spectrophotometrically at 700 nm accord- All experiments were conducted in triplicate and the dif-
ing to the method of Kumaran and Joel Karunakaran ferences between treatment means were determined by
(2007). The more the reducing power increased, the more Duncan’s procedure at p \ 0.05 using the SPSS statistics
the absorbance of the reaction mixture increased. Butylat- 19. The expressed values are mean ± standard deviation of
edhydroxyanisole (BHA) was used as a standard. triplicate measurements.

Metal chelating activity

Results and discussion
The chelating activity of the extracts (12 mg/ml) was
measured spectrophotometrically at 562 nm according to Physicochemical characterization
the methods described by Dinis et al. (1994). The chelating
anti-oxidant activity for Fe2? was calculated according to The physical characteristics of analyzed pomegranate cul-
the following formula: tivars are described in Table 1. The average fruit weight of
pomegranate cultivars ranged between 223.0 g (Ac) and
ðAc  AsÞ 546.3 g (Ga). Similarly, the lowest (33.5%) and the highest
Chelating rate (% ) ¼  100
Ac (51.4%) peel’s percentages were observed in (Ac) and
where, Ac is the absorbance of the control reaction and (Ga), respectively (Table 1). In general, the obtained
As is the absorbance of the sample extract. Ethylene- results are close to those found by a previous study of
diaminetetraacetic acid (EDTA) was used as a pomegranate fruits grown in Iran, which are between 164.9
standard. and 375.8 g (Sarkhosh et al. 2009).

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Table 1 Physicochemical
Parameters Ac Ga Ne To
characteristics of four selected
pomegranate peels Fruit weight (g) 222.95 ± 65.12a 546.28 ± 154.33b 530.08 ± 87.29b 456.43 ± 47.34b
b a b
Pulp percentage (%) 64.33 ± 10.12 47.41 ± 1.85 60.97 ± 4.71 54.06 ± 6.09ab
a b a
Peel percentage (%) 33.5 ± 8.04 51.37 ± 1.64 36.98 ± 5.22 42.99 ± 6.51ab
a b b
Moisture (%) 67.26 ± 0.23 73.23 ± 0.15 72.58 ± 0.67 72.68 ± 0.79b
a c b
Proteins (%, DW) 3.96 ± 0.49 7.13 ± 0.53 5.42 ± 0.01 5.84 ± 0.38b
Fat (%, DW) nd nd nd nd
Total sugar (%, DW) 30.65 ± 0.70a 33.58 ± 0.21ab 33.00 ± 2.99ab 34.83 ± 0.79b
a b ab
Solubles 27.33 ± 0.70 30.60 ± 0.79 30.14 ± 2.99 32.33 ± 0.21b
ab ab ab
Insolubles 3.31 ± 0.07 2.98 ± 0.37 2.86 ± 0.04 2.49 ± 0.44a
a a b
Total fibre (%, DW) 28.27 ± 0.90 28.10 ± 1.20 33.81 ± 0.42 33.93 ± 0.66b
Insolubles 27.11 ± 0.65a 27.04 ± 0.77a 32.51 ± 0.36b 32.13 ± 0.46b
ab a b
Solubles 1.16 ± 0.11 1.06 ± 0.04 1.35 ± 0.21 1.80 ± 0.09c
b ab a
Ash (%, DW) 4.97 ± 0.22 4.44 ± 0.31 3.71 ± 0.37 4.52 ± 0.75ab
d c b
Carbohydrates (%, DW) 32.14 ± 0.45 26.73 ± 0.48 24.04 ± 0.89 20.87 ± 0.50a
± a b d
L* 35.10 0.18 37.74 ± 0.26 47.83 ± 0.04 38.32 ± 0.22c
a* 14.47 ± 0.09c 15.22 ± 0.06d 8.52 ± 0.02a 12.57 ± 0.17b
b* 10.43 ± 0.10a 11.96 ± 0.19b 22.61 ± 0.02d 15.90 ± 0.28c
Ac Acide, Ga Gabsi, Ne Nebli, To Tounsi, DW dry weight basis, L*a*b*, lightness and chromaticity
coordinates in the L*a*b* color space (CIELAB)
Each value in the table is represented as mean ± SE (n = 3). Significant differences between values in the
same row are indicated by different letters a–d (P \ 0.05)

The results for chemical characteristics of pomegranate 13.9%; respectively (Gorinstein et al. 2001). Singh et al.
peel from different cultivars are displayed in Table 1. It (2016) reported that total dietary fibre content of different
can be noticed that moisture content in peel ranged from fruits (pomegranate, kinnow, mango, banana, jambolan,
67.26% in (Ac) ecotype to 73.23% in (Ga) ecotype. (Ga) grapes and sapodilla) was positively related with the
ecotype has the highest protein content (7.13 g/100 g dry insoluble dietary fibre content. It was also observed that
weight (dw)). These contents are higher than those found fruit peels had higher total dietary fibre content than the
in other Turkish cultivars, ‘Lefon’, ‘Seedless’, ‘Kadi’, respective pulps.
‘Siyah’ and ‘Koycegiz’, whose contents are 3.19; 3.11; The four cultivars under study varied in colour from
3.06; 2.67 and 2.58% dw, respectively (Hepaksoy et al. yellow to orange to red. The results show that all samples
2000). exhibited red coloration, with positive a* values (Table 1).
Total sugars account for 30.65–34.83%. This content, (Ga) and (Ac) ecotypes showed the highest a* values
consisting mainly of soluble fraction (27.33–32.33% dw), (15.22 and 14.47, respectively) followed by (To) (12.57)
was comparable to that reported for a Pakistan ecotype and (Ne) (8.52). Compared with other ecotypes, (Ga), (Ac)
(Ullah et al. 2012) (31.38% dw). These carbohydrates and (To) had significantly higher red coloration (a* value)
would be held in the fibrous structure of the peel. than ‘Jabal 20 grown in the Sultanate of Oman
Pomegranate peel is considered a low-fat by-product, (10.16 ± 1.41) (Al-Said et al. 2009). On the other hand,
compared with that of other fruits such as bananas and the (Ac) ecotype had significantly less whiteness, while (Ne)
prickly pear (0.7 and 3.68, respectively) (Espiard 2002). had the most whiteness (L* value) and yellowness
Indeed, as shown in Table 1, the peels of all ecotypes are (b* value).
devoid of fat. Skin colour is an important quality attribute in pome-
The peel of pomegranate could be considered as a rich granate marketing and the fruit with deep red colouration
source of dietary fibre, especially of insoluble fibres. It or blush tends to have greater consumer appeal in the local
contains considerable contents of fibres ranging from 28.10 market.
to 33.93% dw, respectively, in cultivars (Ga) and (To). As anthocyanins are a group of phenolics compounds
These contents were higher than that of a Pakistan ecotype which contributes to the red, blue, or purple colour of many
(Ullah et al. 2012) (21% dw). These quantities are rela- fruits, especially of pomegranate juice, these characterizing
tively very important compared with those found in the values of colour in pomegranate peels may be due to the
peels of lemons, oranges and grapefruit; 14; 13.9 and presence of anthocyanins.

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Evaluation of antioxidant properties highest content was achieved with acetone in (Ac) ecotype
(304 mg GAE/g = 541.89/100 g fresh weight). It should
Yield of extraction and antioxidant compounds content be noted that, because of polarity differences between
solvents, the solubility of the solute into the solvent is
Table 2 shows the percentage yield and antioxidant expected to be different. Water and ethanol are polar protic
compounds content in different extracts. The pome- solvents of dielectric constants of 80 and 24 respectively,
granate peel extracted with water gave the highest total while acetone is polar aprotic solvent of dielectric constant
extract yield (43.03–73.12%), followed by ethanol of 21 (Wang et al. 2011).
(16.88–32.16%) and acetone (1.68–9.72%). Thus, the The comparison between the four ecotypes shows that
majority of compounds are hydrosoluble. The highest the content of polyphenol in (Ac) ecotype is significantly
yields were in (Ac) and (Ga) ecotypes, which indicates higher than the other ecotypes using the ethanol and ace-
that these ecotypes are richer in hydrosoluble com- tone as extracting solvent.
pounds than (Ne) and (To). Acetone was found to be the Since all four pomegranate cultivars used in this
most efficient for extracting total phenolics from (Ne) research were grown in the same location using similar
ecotype, thus it can be considered as the richest in apolar agronomic practices, the differences in phenolic com-
compounds. This difference in extraction yield by dif- pounds have shown that the genetic variability leads to the
ferent solvents can be explained by the fact that the yield variation in the biosynthesis of phenolic secondary
of extraction and the extracts’ antioxidant activity metabolites in these cultivars.
extremely depend on the solvent polarity, which deter- The best contents in tannins were obtained in (Ac)
mines both quantitatively and qualitatively the extracted ecotype by using the ethanol and the acetone with the
antioxidant compounds. contents of 129 and 290 mg GAE/g, respectively. Thus we
Quantitatively, our results are in agreement with the come to the conclusion that the (Ac) ecotype is a potential
study of Wang et al. (2011) on pomegrante peel of source of tannins.
‘Wonderful’ variety from Madera, CA. They reported that The amount of total tannins ranged from 90 to 95% of
the extract yield was 43.19% with water, 17.71% with the total rate of polyphenols. We can deduce that
ethanol and 3.81% with acetone. These results are nearly polyphenols consist essentially of tannins.
the same in (To), (Ne) and (Ga) ecotypes extracted with The total flavonoids varied from 9.98 to 15.25; 5 to 7.49
water, ethanol and acetone, respectively. It is clear that the and 10.27 to 15.46 mg Quercetin/g in water, ethanol and
yield of extraction in (Ac) ecotype is higher than that of acetone extracts, respectively. Our results demonstrated
«Wonderful» variety for all solvents. that the amount of total flavonoids ranged from 4 to 6%.
As shown in Table 2, acetone was the best extracting Therefore, the flavonoids can be said to constitute a small
solvent of phenolic compounds. As a matter of fact, the part of the total phenolics.

Table 2 Yield and total phenolics content in different extracts

Solvent Ecotype Yield (%) Phenolic compounds (mg Tannins (mg Flavonoids (mg Anthocyanins (mg cy-3-glu/
GAE/g) GAE/g) Quer/g) 100 g)

Water Ac 73.12 ± 5.31c 209.83 ± 3.83b 197.69 ± 0.12c 11.67 ± 0.55b 29.11 ± 2.10c
b a a a
Ga 63.08 ± 2.76 146.75 ± 1.75 134.50 ± 0.25 9.98 ± 0.08 21.68 ± 1.10b
a c d d
Ne 43.96 ± 2.01 248.93 ± 14.50 236.69 ± 0.34 15.26 ± 0.43 16.8 ± 1.70a
To 43.03 ± 1.94a 202.11 ± 4.95b 185.03 ± 0.10b 13.38 ± 0.17c 20.37 ± 3.40ab
c c d d
Ethanol Ac 32.16 ± 2.31 140.93 ± 0.53 129.20 ± 0.27 7.49 ± 0.20 42.58 ± 2.00c
c a a b
Ga 30.48 ± 1.77 109.21 ± 0.34 99.18 ± 0.10 5.49 ± 0.17 29.55 ± 3.67b
a b c c
Ne 16.88 ± 0.44 121.10 ± 6.20 110.55 ± 0.12 6.74 ± 0.43 14.27 ± 3.39a
b ab b a
To 25.08 ± 2.02 116.11 ± 5.15 105.71 ± 0.12 5.00 ± 0.02 12.95 ± 0.05a
b d d c
Acetone Ac 4.68 ± 0.31 304.60 ± 14.20 292.23 ± 0.25 15.46 ± 0.11 54.51 ± 8.93d
Ga 3.72 ± 0.12ab 157.06 ± 0.00a 144.96 ± 0.55a 10.28 ± 0.26a 30.05 ± 2.00c
Ne 9.72 ± 1.98b 207.8 ± 0.00c 196.26 ± 0.02c 11.43 ± 0.43b 6.84 ± 0.32a
To 1.68 ± 0.32a 190.95 ± 6.91b 179.35 ± 0.10b 11.26 ± 0.60b 13.86 ± 1.40b
Ac Acide, Ga Gabsi, Ne Nebli, To Tounsi
Each value in the table is represented as mean ± SE (n = 3). Significant differences between the values in the same column for each solvent are
indicated by different letters a–d (P \ 0.05)

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The results for the total anthocyanins of the pome- A study on eight species of fruits most commonly
granate peel indicated that there were significant differ- consumed and grown in Thailand (coconut (Cocos nuci-
ences in the total anthocyanins content of the pomegranate fera), mangosteen (Garcinia mangostana), dragon fruit
cultivars. (Ac) ecotype had the highest amount of total (Hylocereus undatus), long-gong (Lansium domesticum),
anthocyanins compared to other cultivars (42.58 and banana (Musa sapientum), rambutan (Nephelium lap-
54.51 mg cy-3-glu/100 g) in ethanol and acetone extract, paceum), passion fruit (Passiflora foetida) and pome-
respectively. The results obtained from the anthocyanins granate (Punica granatum) has shown that the extract of
content measurements were in accordance with L* mea- pomegranate peel has the highest scavenging activity fol-
surements. As a matter of fact, (Ac) peel with the lowest L* lowed by the peel extracts of rambutan and mangosteen
values had the highest amounts of anthocyannins. There- (Okonogi et al. 2007).
fore, the variation in L* values can be attributed to
anthocyanin content. Reducing power assay Table 3 shows the reductive
Hence, the Ac’s acetone extract showed the highest capability of water, ethanol and acetone extracts compared
values of polyphenol, tannins, flavonoids and anthocyanins to BHA. The reducing activity is generally associated with
with 304.6 mg GAE/g; 292.23 mg GAE/g; 15.465 mg the presence of reductones, which has been proven to exert
Quercetin/g and 54.51 mg cy-3-glu/100 g, respectively. antioxidant action by breaking the free radical chain by
donating a hydrogen atom (Gordon 1990). It was also
Antioxidant activity reported that raductones react with certain precursors of
peroxide, thus preventing the peroxide formation.
DPPH free radical scavenging activity As shown in Acetone extracts have higher reducing power than that
Table 3, the radical-scavenging ranges from 71.11 to of ethanol extracts among all ecotypes. Like phenolic
84.16%, from 71.73 to 96.73% and from 77.32 to 97.82% content, the greatest reducing power were detected in (Ac)
in water, ethanol and acetone extracts, respectively. The extract acetone. Hence, this ecotype might contain the
highest activity was observed with acetone for all ecotypes. highest amount of reductones which could react with free
The results clearly indicate that the acetone extract of (Ac) radicals to stabilise and terminate the radical chain
ecotype exhibits the highest scavenging activity. reactions.
According to the results displayed in Table 2, (Ac) and The study of correlation between the phenolic content
(Ga) acetone extracts have the highest and lowest levels of and antioxidant activity of extracts have shown that total
total phenolics and antioxidant activity, respectively. Thus phenols, tannins and flavonoids in peel are highly corre-
it can be concluded that there is a close relationship lated with the reducing power assay with correlations
between the total phenolics and antioxidant activity. coefficients 0.877, 0.874 and 0.844, respectively. This

Table 3 Antioxidant activity in

Solvent Ecotype DPPH (%) Reducing power Metal chelating activity (%)
different extracts
c b
Water Ac 83.85 ± 0.62 0.25 ± 0.00 89.99 ± 0.54a
Ga 71.11 ± 1.24b 0.18 ± 0.01a 89.20 ± 2.54a
c d
Ne 84.16 ± 0.00 0.32 ± 0.02 97.37 ± 1.62b
c c
To 84.16 ± 1.24 0.29 ± 0.02 94.90 ± 3.03b
BHA 64.03 ± 0.84a 0.64 ± 0.00e –
Ethanol Ac 96.73 ± 0.15e 0.24 ± 0.01c 34.93 ± 0.01c
Ga 71.73 ± 1.24b 0.10 ± 0.02a 26.26 ± 0.31b
c c
Ne 84.47 ± 0.00 0.26 ± 0.02 21.00 ± 0.50a
d b
To 90.83 ± 0.16 0.14 ± 0.00 19.72 ± 2.38a
a d
BHA 64.03 ± 0.84 0.64 ± 0.00 –
Acetone Ac 97.82 ± 0.00e 0.39 ± 0.01c 75.25 ± 2.25c
Ga 77.32 ± 0.62b 0.24 ± 0.01a 61.87 ± 1.25b
c b
Ne 86.48 ± 0.46 0.28 ± 0.03 81.43 ± 0.06d
d ab
To 95.65 ± 0.62 0.26 ± 0.02 54.56 ± 0.94a
a d
BHA 64.03 ± 0.84 0.64 ± 0.00 –
Ac Acide, Ga Gabsi, Ne Nebli, To Tounsi, BHA butylatedhydroxyanisole
Each value in the table is represented as mean ± SE (n = 3). Significant differences between the values of
the same column for each solvent are indicated by different letters a-e (P \ 0.05)

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result suggests that the majority of the antioxidant capacity ionization source (ESI) (Table 4). HPLC analysis of the
of pomegranate extracts results from the contribution of fractions showed the presence of peaks with ellagitannins-
phenolic compounds, tannins and flavonoids. type UV spectra (two bands, kmax of band 1 between 255
Consequently, our peel extracts were electron donors and 290 nm and kmax of band 2 between 326 and
and can react with free radicals to convert them into more 378 nm).
stable products and terminate the radical chain reaction.
Therefore, the total phenolics yield might be one of the Ellagitannins
most important indicators of effective extraction processes
for producing high quality products (Wang et al. 2011). Compound eluted at 1.93 min produced a molecular ion at
m/z 481 [M_H]- and a fragment at m/z 301, indicating the
Secondary antioxidant activity: Metal chelating activ- presence of an ellagic acid moiety. This compound C1 was
ity The chelating activity of pomegranate peel may be tentatively identified as HHDP-hex. Compound C9 eluted at
considered low since such activity was recorded only at 5.36 min showed a maxima in UV 359-255, which is
high concentration in spite of its important content in total characteristic of ellagitannins, and exhibited an [M_H]- ion
phenolic compounds. Similar findings have been reported at m/z 783. The loss of water and ellagic acid in the MS2
for bananas. In fact, these were reported to be a powerful experiment produced fragments at 765 and 481m/z, respec-
secondary antioxidant, though had low phenolic content tively. Based on this fragmentation pathway and the
due to the presence of other active compounds that might occurrence of further typical fragments, compound C9 was
bind to metal ions strongly (Lim et al. 2006). identified as bis-HHDP-hexoside (pedunculagin I). Com-
As a result, all extracts under investigation may be pound C10 produced an [M_H]- ion at m/z 799 and frag-
regarded as unable to strongly obstruct the generation of ments at m/z 781 and at m/z 479, which came from the loss

OH radicals from Fenton reaction (Fe2? ? H2O2- of water and ellagic acid, respectively. This compound
Fe3? ? OH ?OH-). Our results have shown that pome- (C10) may be attributed either to granatin A (HHDP-
granate’s peel has low chelating activity despite its DHHDP-hexoside) or lagerstannin A (bis-HHDP-gluconic
important radical scavenging activity. acid) (Sentandreu et al. 2013). Compound C11 and C15
This result is confirmed by the study of Lim et al. exhibited an [M_H]- ion at m/z 785. The release of typical
(2006), who have found that although guava has a potent ellagitannin and gallotannin fragments at m/z 483 (digal-
radical scavenging property, its function as a secondary loylhexoside), 301 (ellagic acid) and 633 (galloyl-
antioxidant, as measured by chelating power, is rather low. HHDPhexoside), clearly suggests that C11 and C15 were
This means that some fruits can possess high primary characterised as digalloyl-HHDP-hexoside (pedunculagin
antioxidant activities, but low secondary antioxidant II). Each of the two different retention times corresponds to
activities. an isomeric structure, also differing in their fragmentation
It can be concluded that the antioxidant activity of our patterns. Compound C13, showing an [M_H]- ion at m/z
extracts is not limited to phenolics. It may also be due to 633 and fragment at 301 in the MS2 experiment, was
the presence of other antioxidant secondary metabolites, identified as galloyl-HHDP-hexoside. Compound C14
which is in agreement with a study on antioxidant activity exhibited an [M_H]- ion at m/z 951 and compound C16 at
of olive extracts (Hajimahmoodi et al. 2008). m/z 965. Both compounds produced fragments at m/z 933
Therefore, it was concluded from the three antioxidant and at m/z 301 (ellagic acid) in the MS2 experiment. This
activities that (Ac) ecotype exhibits the highest free radi- fragment (m/z 933), generating fragments at m/z 915 from
cal-scavenging and reducing power activity. Hence, this the loss of water, is typical for castalagin/vescalagin or
ecotype might contain the highest amount of reductones galloyl-gallagyl-hexoside (galloylpunicalin, pedunculagin
which could react with free radicals to stabilise and ter- III). Nevertheless, the characteristic gallagyl-fragment at m/
minate radical chain reactions. Pomegranate peel extract of z 601 was not detected. Furthermore, castalagin/vescalagin
(Ac) ecotype appeared to have strong antioxidant proper- exhibited molecular masses 18 Da lower than compound
ties and merits further intensive study. C14. Based on these results, compound C14 was identified
as granatin B (galloyl-HHDPDHHDP- hexoside), which
Characterization of phenolic compounds by LC–MS forms a part of type III-tannins (dehydroellagitannins)
(Fischer et al. 2011). Granatin A and B were firstly identified
To elucidate the structures of phenolic compounds in as the major components of pomegranate leaves (Tanaka
pomegranate peel, the acetone extracts were subjected to et al. 1986). Analysis of compound C16 shows the depro-
a combination of liquid chromatography–tandem mass tonated molecule ion at m/z 965, which produced ions at m/
spectrometry (LC–MS/MS) high resolution unit with a z 933 and at m/z 301 in the MS2 experiment. Additionally,
photodiode array detector and fitted with an electrospray fragments were observed at m/z 915 and 897. The fragments

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Table 4 Retention times, UV/Vis spectra and characteristic ions of phenolic compounds of pomegranate’s acetone extracts
Comp Assignment Rt kmax(nm) [M–H]- HPLC–ESI(_)-MSn Ac Ga Ne To
(min) m/z experiment m/z

C1 HHDP-hex 1.93 326, 290 481 301;275 ? ? ?

C2 citric acid 2.1 214 191 173;111 ? ?
C3 HHDP-gallagyl-hex (punicalagin) 2.33 378, 258 1083 601;781;575;721;549 ?
C4 punicalagin derivative 3.31 377, 256 541.31 532;481;301 ?
C5 punicalagin derivative 3.7 378, 258 541.33 532;481;301;275 ? ? ?
C6 punicalagin derivative 4.33 377, 257 541.36 301;275;523;549;249 ? ? ?
C7 punicalagin derivative 4.94 378, 258 541.37 301;781;601;532;275 ? ? ?
C8 HHDP-gallagyl-hex (punicalagin) 4.93 378, 258 1083 1083;541 ?
C9 Bis-HHDP-hex (pedunculagin II) 5.36 359, 255 783 765;301;481;275 ?
C10 Ellagic acid der 5.62 265 799 781;479;301 ?
C11 Digalloyl-HHDP-hex (pedunculagin II) 6.06 274 785 483;301;633 ? ? ?
C12 Pgd-3-pentoside 6.62 433, 271 401 271 ?
C13 Galloyl-HHDP-hex 6.8 365, 266, 330, 633 463;301;275 ? ? ?
260
C14 Galloyl-HHDP-DHHDP-hex (granatin 7.83 365, 274 951 933;915;301 ? ? ?
B)
C15 Digalloyl-HHDP-hex (pedunculagin II) 8.55 272 785 615;301;275 ?
C16 Castalagin der 9.35 275 965 933;445;301 ?
C17 Ellagic acid-pent 10.01 267 433 301;300 ?
C18 Galloyl-bis-HHDP-hex (casuarinin) 10.18 377, 258 979.14 932;445;301 ?
derivative
C19 Ellagic acid-rhamnoside 10.39 275 447 301;300 ? ?
C20 Galloyl-bis-HHDP-hex (casuarinin) 10.4 378, 260 979.11 933;445;301 ?
derivative
C21 Cyd-3-pentoside 10.53 515, 278 417 287 ?
C22 Ellagic acid 10.74 367, 275 301 301;229;185 ? ? ?
C23 Cya-rut 11.45 375, 268 593 285;257;229;547 ?
C24 cyanidine 3 o-gluco 11.66 518, 278 447 327;285;255 ? ? ?
Ac Acide, Ga Gabsi, Ne Nebli, To Tounsi, Rt retention time, comp compound

at m/z 933, 915 and 897 are typical of the castalagin Gallagic acid and gallagyl esters
derivative (Fischer et al. 2011).
Two monoglycosylated ellagic acid derivatives were Punicalagin (2,3-HHDP-4,6 gallagylglucoside) is the main
detected, such as an ellagic acid-pentoside (m/z 433; phenolic compound in pomegranate and has already been
C17) and deoxyhexoside (m/z 447; C19). The [M-H]- well characterised (Tanaka et al. 1986). This is a complex
ion of C17 was obtained at m/z 433. In the MS2 ellagitannin characteristic of pomegranate peel, which
experiment, the ion at m/z 301 was generated by the loss contains glucose, ellagic acid, and gallagic acid (Gil et al.
of 132 Da, reasonably assigned as the elimination of 2000). Punicalagin was detected as doubly-charged ion
pentose. The occurrence of the ion at m/z 300 was species, displaying an [M_2H]2- ion at m/z 541, which is
attributed to a homolytic rupture of the glycosidic bond equivalent to a molecular weight of 1084 Da. The fragment
(Ferreres et al. 2005). Ellagitannin C19 has been previ- at m/z 601 in the MS2 experiment indicated the loss of a
ously detected during a large scale purification of gallagic acid moiety (Fischer et al. 2011). Several isomers
pomegranate husk polyphenols (Seeram et al. 2005). have been previously described in pomegranate fruit peel
C22 was identified as free ellagic acid. This compound and also in leaves and bark (Gil et al. 2000), which were
was confirmed by its m/z 301 [M–H]- ion, yielding char- confirmed in the present study as illustrated by different
acteristic ions at m/z 185 and 229 upon dissociation. retention times of compound 1083. Fragments for the loss
Ellagic acid has previously been reported for pomegranate of ellagic acid (781 m/z) and for the gallagic (601 m/z) and
husk and juices (Seeram et al. 2005). ellagic acid (301 m/z) residues were the main fragments

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Fig. 1 Identification of phenolic compounds in acetone extracts. a Acide ecotype, b Gabsi ecotype, c Nebli ecotype, d Tounsi ecotype

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observed in the mass spectrum, supporting the nature of Organic acid

these compounds (Fig. 2).
According to the abovementioned informations, Compound C2 exhibited an [M_H]- ion at m/z 191, which
compounds (C3, C8) and (C4, C5, C6, C7) were iden- is typical for citric acid. The same compound was identi-
tified as punicalagin and punicalagin derivatives, fied in pomegranate juice by Sentandreu et al. (2013).
respectively.
The mass spectra in the negative mode of compounds Comparison between different ecotypes
C18 and C20 exhibited a base peak [M_H]- at m/z 979 with
significant fragments at m/z 933, m/z 445 and m/z 301. This The analysis of the four extracts has shown the presence of
result suggest that they could be two isomers of Galloyl- punicalagins derivatives, galloyl-HHDP-hex, Galloyl-
bis-HHDP-hex (casuarinin) derivative. HHDP-DHHDP-hex (granatin B) and Digalloyl-HHDP-hex
(pedunculagin II) in (Ac), (Ne) and (To) ecotypes (Fig. 1).
Anthocyanins However, the relative abundance of these compounds were
more in (Ac) ecotype as compared to the others. These
The detected anthocyanin among the pomegranate peels compounds were absent in (Ga) extract (Fig. 1b).
were identified as mono-substituted anthocyanidin deriva- In addition to these phenolics, two other compounds
tives. A pelargonidin derivative was observed in pome- were detected only in (Ac) ecotype in important content:
granate peel, such as Pgd-3-pentoside (m/z 401; C12). Castalagin derivative (m/z 965) and Galloyl-bis-HHDP-hex
Cynanidin derivatives were also detected, such as cyanidin- (casuarinin) derivative (m/z 979.11) (Fig. 1a). Therefore,
3-rutinoside (m/z 593; C23), cyanidin-3-glucoside (m/ these two compounds might be responsible for the pow-
z 447; C24) and Cyd-3-pentoside (m/z 417; C21). erful antioxidative capacity of (Ac) ecotype.

Fig. 2 MS–MS spectra of punicalagin (M–H m/z 1083) and the subsequent fragment ions of punicalin (M–H m/z 781), ellagic acid (M–H m/
z 301) and then gallagic acid (M–H m/z 601)

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Interestingly, among the various phenolic compounds relationship with phenolic composition and processing. J Agric
(24 compounds) detected in pomegranate peel, the acetone Food Chem 48:4581–4589
Gordon MH (1990) The mechanism of antioxidant action in vitro. In:
fraction of (Ac) ecotype is rich in ellagitannins, a group of Hudson BJF (ed) Food antioxidants. Springer, London, pp 1–18
phenolics that could be responsible for demonstrated Gorinstein S, Martı́n-Belloso O, Park YS, Haruenkit R, Lojek A, Ĉı́ž
antioxidant properties (Fig. 2). M, Caspi A, Libman I, Trakhtenberg S (2001) Comparison of
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Acknowledgement The authors would like to thank the ‘‘Ministère Chem 74:309–315
de l’Enseignement Supérieur et de la Recherche Scientifique, Tuni- Hajimahmoodi M, Sadeghi N, Jannat B, Oveisi MR, Madani S, Kiayi
sia’’ for the support of this research work. They also wish to express M, Akrami MR, Ranjbar AM (2008) Antioxidant activity,
their gratitude to Mrs. Leila MAHFOUDHI, an English teacher at the reducing power and total phenolic content of Iranian Olive
Sfax Faculty of Science, for having proofread this paper. Cultivar. J Biol Sci 8:779–783
Hepaksoy S, Aksoy U, Can HZ, Ui MA (2000) Determination of
relationship between fruit cracking and some physiological
responses, leaf characteristics and nutritional status of some
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