Food Research International 44 (2011) 1161–1167

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Food Research International

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f o o d r e s

Identification of phenolic compounds from pomegranate (Punica granatum L.) seed

residues and investigation into their antioxidant capacities by HPLC–ABTS+ assay
Li He, Honggao Xu, Xuan Liu, Wenhao He, Fang Yuan, Zhanqun Hou, Yanxiang Gao ⁎
College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 10083, PR China

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

Article history: In this study, phenolic compounds were extracted and isolated from pomegranate seed residue (PSR).
Received 1 April 2010 Phytochemicals and antioxidant capacity of extracts from PSR were firstly investigated. Total phenolic (TP)
Accepted 31 May 2010 and proanthocyanidin (PC) contents of the extracts were determined as 2427.90 and 505.63 mg catechin
equivalent of 100 g dry weight respectively. To evaluate the antioxidant capacity of individual compounds,
Keywords:
on line assay of coupling high performance liquid chromatographic separation with ABTS free radical
Pomegranate seed residue
Antioxidant activity
reaction system (HPLC–ABTS+) was carried out. 17 compounds in PSR extracts were detected with
Phenolic compounds antioxidant capacity, and HPLC associated with electrospray ionization mass spectrometry (HPLC–ESI–MS)
HPLC–ESI–MS was used to identify them. The main phenolics in PSR identified were flavol-3-ols, phenolic acids, flavonoid
HPLC ABTS+ assay glycosides, and hydrolysable tannin. The results showed that PSR contained some amount of antioxidant
compounds, and the HPLC–ABTS+ on line method was fast, sensitive and effective.
Crown Copyright © 2010 Published by Elsevier Ltd. All rights reserved.

1. Introduction however, the antioxidant and chemical constitutes of pomegranate

Recently, chemical constituents and their bioactivities in all parts
of pomegranate (Punica granatum L.), including leaf, seed, juice, husk
and peel, have been investigated (Lansky & Newman, 2007; Singh,
Chidambara Murthy, & Jayaprakasha, 2002; Gil, Tomas-Barberran,
Hess-Pierce, Holcroft, & Kader, 2000). In China, pomegranate has been
recognized as economic potential fruit with pharmaceutical value,
resulting in a steadily growing cultivation area of pomegranate
recently. A range of phenolics had been found in pomegranate.
Galloylglucose, punicalagin, punicalin, ellagic acid and gallic acid were
main active compounds in pomegranate juice and husk (Gil et al.,
2000; Seeram, Lee, Hardy, & Heber, 2005). Pomegranate juice contains
plenty of anthocyanins such as delphinidin 3-glucoside, cyaniding 3-
glucoside, pelargonidin 3, 5-diglucoside, et al. (Hernandez, Melgarejo,
Tomas-Barberran, & Artes, 1999). Pomegranate seed, the by-product
of pomegranate juice processing, contains a range of nutraceutical
components such as sterols, γ-tocopherol, punicic acid and hydro-
xybenzoic acids (Liu, Xu, Hao, & Gao, 2009). Phenyl aliphatic
glycosides as phenethyl rutinoside were also found in pomegranate
seed (Wang et al., 2004). The extracts of pomegranate seed were
reported with antidiarrhoeal and antioxidant bioactivities (Das et al.,
1999; Schubert, Lansky, & Neeman, 1999; Singh et al., 2002). Most of
these functional components were rich in pomegranate seed oil,
seed residue (PSR, defatted pomegranate seed) have not been
reported yet. The seed residues of pomegranate have become
agricultural wastes and need to be converted into value-added
products.
The technique coupling high performance liquid chromatographic
(HPLC) separation and post-column detection of radical scavenging
individual compound (s) based on a model oxidation system has been
reported in many articles (Niederländer Harm, Van Beek, Bartasiutec,
& Koleva, 2008). It has been proved sensitive, selective, and relatively
simple for antioxidant capacity analysis, and has been applied for
quick screening of complex matrix of various plant extract, foods and
beverages for radical trapping compounds (Pellegrini, Rio, Colombi,
Bianchi, & Brighrnti, 2003; Nuengchamnong et al., 2005; Bandonien &
Murkovic, 2002).
The purpose of the current study was to analyze the total phenolic
(TP), proanthocyanidin (PC) contents of the pomegranate seed
residue (PSR) extract and fractions, and evaluate their antioxidant
capacities by DPPH, ABTS+ assay and on line HPLC–ABTS+ method,
additionally, antioxidant compounds were identified by HPLC and
electrospray ionization mass spectrometry (HPLC–ESI–MS).
2. Materials and methods
2.1. Chemicals

⁎ Corresponding author. Tel.: + 86 10 62737034; fax: + 86 10 62737986. 1, 1-Diphenyl-2-picrylhydrazyl (DPPH), 2, 2′-Azinobis (3-
E-mail address: gyxcau@126.com (Y. Gao). ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS),

0963-9969/$ – see front matter. Crown Copyright © 2010 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodres.2010.05.023
1162 L. He et al. / Food Research International 44 (2011) 1161–1167
6-hydroxy-2, 5, 7, 8-tetramethylchroman-2-carboxilic acid (Tro-
lox), Folin–Ciocalteu's reagent, vanillin and (+)-catechin hydrate
were purchased from Sigma-Aldrich (Shanghai, China). All the reagents
(HPLC grade) were obtained from Merck (Shanghai, China). Other
chemicals and solvents (analytical grade) used were purchased from
Beijing Chemical Co. (Beijing, China).
2.2. Plant material
Pomegranate seeds were obtained from Huiyuan Fruit Juice
Company (Xinjiang, China); and were ground to 0.3–0.9 mm by a
high-speed mill (Model HY-200, Beijing Huanya Scientific Ltd., China).
Moisture content was determined by drying samples (10 g) at 100 ±
0.5 °C until constant weight and the moisture was 4.05%. The seed
powder was defatted using supercritical CO2 by a Hua'an supercritical
fluid apparatus (Model HA220-50-06, Nantong, China) at a pressure of
37.9 MPa and temperature of 47 °C with a flow rate of 21.3 l/h (Liu
et al., 2009), the residues were stored in the dark at an ambient
temperature until use.
2.3. Preparation of the crude extracts and fractions
PSR was extracted for 200 min (2 × 100 min) with acetone–water
(7:3, v/v) in a shaking bath (Model DSHZ-300, Suzhou, China). The
extracts were filtered though Whatman No. 1 paper. The filtrate was
centrifuged at 2000 ×g for 10 min and supernatants were collected,
vaporized to dry under vacuum at ≤40 °C. The extracts were then
dissolved in methanol and kept at − 18 °C for analysis.
The crude extract of PSR was successfully fractionized into four
fractions (Fa, Fb, Fc, and Fd) with ethyl ether, ethyl acetate and n-
butanol, respectively (Wu, Huang, Tung, & Chang, 2008). The fractions
were evaporated to dry under vacuum at ≤50 °C. All the fractions
were dissolved in methanol and kept at − 18 °C for analysis.
2.4. Determination of total phenolic (TP) content
Total phenolic (TP) content of the extract was estimated according
to the method described by Amarowicza, Pegg, Rahimi-Moghaddam,
Barl, & Weil, 2004) and Singleton et al. with minor modification
(Singleton, Orthofer, & Lamuela-Raventos, 1999; Singleton & Rossi,
1965). Four milliliters of extract dilute was mixed with 0.5 ml of
Folin–Ciocalteu's reagent (1 mol/l, 1 N). After 10 min, 1.5 ml of
sodium carbonate solution (20%, w/v) was added. The mixture was
vortexed and stood for 60 min at room temperature. The absorbance
was measured at 750 nm spectrophtometrically (Model UV-1800,
Beijing, China), the final result was expressed as milligram of catechin
equivalents (CE) per 100 g of dry weight (DW).
2.5. Determination of proanthocyanidin (PC) content
Total proanthocyanidin (PC) content in PSR extract was performed
according to Sun, Ricardo-da-Silva, and Spranger (1998) and Li et al.
(2006) with slight modification. Half milliliter of extract solutions was
mixed with 3 ml, 4% (w/v) vanillin–methanol solution and 1.5 ml of
hydrochloric acid in 10 ml tube packed with foil. The mixture was
allowed to stand in the dark at room temperature for 15 min. The
absorbance at 500 nm was measured and the final result was
expressed as milligram of catechin equivalents (CE) per 100 g of dry
weight (DW).
2.6. Evaluation of radical scavenging capacity (RSC)
2.6.1. ABTS radical scavenging capacity
The ABTS+ assay was based on the procedure described by Re et al.
(1999) and Siddhuraju (Siddhuraju, 2006). Briefly, 10 mg ABTS was
diluted in 2.6 ml of potassium persulfate solution (2.45 mM) and final
concentration of ABTS+ was 7 mM. The mixture was kept in the dark
at room temperature for 12–16 h before use. The ABTS+ was diluted
to an absorbance of 0.70 ± 0.02 and stocked for off line and on
line assays. One milliliter of diluted PSR extract was added with 3 ml
ABTS+ solution and kept in the dark at room temperature for 60 min.
The absorbance was measured at 734 nm. The scavenging capacity
was calculated as (1 − Ab/A0) × 100% (where Ab is the absorbance at
734 nm; A0 refers to the absorbance of ABTS+ without sample added
at 734 nm).
2.6.2. DPPH radical scavenging capacity
DPPH was dissolved in methanol to a concentration of 10− 4 mol/l.
DPPH radical scavenging capacity was estimated according to Blois
(1958) and Shyu and Hwang (2002) with slight modification. Two
milliliters of diluted extract was mixed with 2 ml of DPPH solution,
the mixture was kept in the dark for 60 min and the absorbance
at 517 nm was measured. The scavenging capacity was calculated as
(1 − Ab/A0) × 100% (where Ab is the absorbance at 517 nm; A0 refers
to the absorbance of DPPH without sample added at 517 nm).
2.7. On line HPLC–ABTS+ radical scavenging activity analysis
The antioxidant activity of the PSR extracts was further monitored
by the HPLC–ABTS+ method, based on the capacity of antioxidant
compounds to scavenge radicals in ABTS+ solution (Koleva, Nieder-
länder Harm, & Van Beek, 2001). The scheme of the instrumental set
up was displayed in Fig. 1. The diode-array detector (DAD) was
connected to a reaction coil (Peek, 15 m × 0.25 mm) loaded into a
temperature controller. 10 μL of sample was firstly separated on an
Agilent 1100 HPLC system equipped with a DAD. The separation of
phenolics was carried on an Agilent Zorbax SB C-18 column
(250 mm × 4.6 mm i.d., 5 μm) which was protected by a Zorbax SB
C-18 guard column (12.5 mm × 4.6 mm i.d., 5 μm). The HPLC condi-
tion was expressed as follows: mobile phase was 0.1% (v/v) formic
acid (solvent A) and methanol (solvent B); flow rate was kept at
0.7 ml/min. The elution gradient was carried out as follows: solvent A
was 100%–40% from 0 to 45 min, 40%–0% in 5 min, and 0%–100% in
5 min. The temperature of the column was 35 °C. The DAD
wavelengths were set at 280 nm and 350 nm.
While the sample was separating, an ABTS+ stock solution was
delivered via a pulse pump (Waters Corporation, USA) with a flow
rate of 0.8 ml/min. If the ABTS+ stock solution mixed with the fraction
from column and reacted in the peak coil. The mixture was then
transported into the DAD detector monitoring absorbance at 734 nm
and signals of the positive–negative peaks were recorded.
Fig. 1. Schematics of on line HPLC–ABTS+ for rapid screening and identification of
antioxidants in PSR extract.
 L. He et al. / Food Research International 44 (2011) 1161–1167
2.8. Identification of phenolics
In order to know the active phenolic compounds presented in PSR
extracts, a HPLC–MS fitted with an electrospray ionization (ESI)
source was used. The HPLC separation program of PSR extract was the
same as described previously, MS was operated in negative ion mode.
The electrospray capillary voltage was set to 3500 V. Nitrogen was
used as nebulizing gas at a pressure of 30 psi with a flow rate of 9.0 l/
min and drying gas temperature of 300 °C. Scan range of the mass
spectrometry was m/z 50–1500.
3. Results and discussion
3.1. Radical scavenging capacity (RSC) of crude extract and fraction
Two in vitro assays based on DPPH and ABTS+ radical scavenging
capacity, respectively, were used to evaluate antioxidant capacity of
crude extract and various fractions. The relatively stable free radical
accepts an electron or hydrogen radical to be a stable diamagnetic
molecule. As shown in Figs. 2 and 3, radical scavenging capacities of
crude extract and fractions (Fa, Fb, Fc, and Fd) against DPPH and ABTS+
radicals were investigated. Both of the methods indicated that the n-
butanol phase fraction (Fc) possessed the strongest radical scavenging
capacity, with the inhibition of 92.75% against DPPH· (the concen-
tration of Fc was 0.4 mg/ml) and 99.80% against ABTS+ (the
concentration of Fc was 0.2 mg/ml). The ethyl ether phase fraction
(Fa) exhibited lower radical scavenging capacity than crude extract
and ethyl acetate fraction (Fb) with both of the methods. The Fd
(residue after the fractionation with different solvents) showed the
weakest activity, with the inhibition of 30.48% against DPPH (the
concentration of Fd was 0.4 mg/ml) and 11.27% against ABTS+ (the
concentration of Fd was 0.2 mg/ml). The results based on the inhibi-
tion of PSR phenolics to DPPH free radical were generally consistent
with Singh et al. (2002).
The Fc exhibited the highest radical scavenging ability since more
amounts of phenolic compounds dissolved in n-butanol. The results
showed a similar agreement with the A. confusa flower extracts
reported by Wu et al. (2008) and indicated that n-butanol could be a
good solvent for phenolic compounds in PSR. The HPLC profiles in
Fig. 4 revealed that the n-butanol fraction (Fc) contained more
compounds possessing high antioxidant activity.
Fig. 2. Radical scavenging activity of crude extract and fractions against DPPH· solution
(Results were presented as mean ± SD (n = 3); RSC, Radical Scavenging Capacity).
1163
Fig. 3. Radical scavenging activity of crude extract and fractions against ABTS+·solution
(Results were presented as mean ± SD (n = 3); RSC, Radical Scavenging Capacity).
3.2. The correlation between total antioxidant activity and total phenolic
(TP), proanthocyanidin (PC) contents of PSR extracts
TP content of the extract (with 70% of acetone) was 2427.90 (mg
CE/100 g DW) and PC content was 505.63 (mg CE/100 g DW). TP
content was slightly lower than the result (2.6%) from Singh et al.
(2002) on defatted pomegranate seed extraction with methanol, and
could be equivalent with that of the pomegranate pulp. Li et al.
extracted antioxidants from pomegranate pulp and peel with a
mixture of methanol, acetone, ethanol and water, found that TP
content of peel extract (294.4 ± 17.2 mg/g) was 10-folds than that of
the pulp extract (24.4 ± 2.7 mg/g) (Li et al., 2006). TP and PC contents
in the PSR extracts were larger than that of soybean seed extracts
reported by Malencic, Maksimovic, Popovic, and Miladinovic (2008).
However, TP and PC contents of PSR extract were less than those of
grape seed residues (Xu, Zhang, Cao, & Lu, 2010) and seabuckthorn
seed (Arimboor, Kumar, & Arumughan, 2008).
Presence of phenolic compounds in plant extracts contributes
significantly to their antioxidant activity potential. The correlation
between phenolic compounds and antioxidant activity has been
described in many studies (Anesini, Ferraro, & Filip, 2008; Pasko et al.,
2009). The correlation coefficients (R2) between TP, PC contents and
Trolox equivalent of antioxidant capacity (TEAC) values of ABTS and
DPPH assays were calculated. As shown in Table 1, the ABTS and DPPH
assays showed a different relationship between TP, PC and TEAC
values. Generally, the ABTS assay showed higher linear relationship
with TP and PC contents due to that ABTS+ solution was more stable
and suitable for determination of water-soluble phenolics in PSR
which had been defatted. The results were in agreement with the
report from Pellegrini, Rio, Colombi, Bianchi, and Brighrnti (2003).
3.3. HPLC–ABTS+ assay of crude extract and fractions in PSR
ABTS+ solution was stable during the reaction with PSR extract
and fractions. HPLC–ABTS+ assay is more sensitive than the HPLC–
DPPH for an estimation of the water-soluble antioxidants (Pellegrini
et al., 2003). Furthermore, the reaction between DPPH·and an
antioxidant exhibited slow kinetics in methanolic medium (Barta-
siute, Westerink, Verpoorte, & Niederländer Harm, 2007). Accord-
ingly, on line HPLC–ABTS+ rapid screening of individual antioxidants
in PSR extract and fractions was established. ABTS+ solution has a
deep green color with maximum absorbance at 734 nm, and any
quenching of the radical results in a loss of color was indicated by a
negative peak on the HPLC profile.
1164 L. He et al. / Food Research International 44 (2011) 1161–1167
Fig. 4. HPLC profiles (280 nm) of four fractions from PSR extract (A—ethyl ether phase; B—ethyl acetate phase; C—n-butanol phase; D—aqueous phase).
The four fractions showed the similar chromatographic finger-
prints according to Fig. 4, and all the HPLC peaks involved could
generally be found in the profiles of Fa and Fc. On line HPLC–ABTS+
profiles of Fa and Fc were shown in Fig. 5. All the main antioxidant
compounds in PSR extracts could be clearly seen in profiles of the two
fractions. Compounds 3, 5, 6, 7, 11, 14 and 15 showed relatively high
radical scavenging capacity, and peaks 4, 13, 16 and 17 exhibited little
or no radical scavenging capacity even though they had relative high
response value in HPLC profiles.
3.4. Identification of the main phenolic compounds in PSR extracts
The main antioxidants of Fa and Fc were monitored by diode-array
and mass spectrometry. A typical chromatogram was shown in Fig. 5.
Table 2 described detailed information of the MS (MS/MS) and
ultraviolet (UV) characteristics for the peaks of major phenolics, and
17 main phenolic compounds were detected from the four fractions
based on their retention times, absorbance spectrum, and MS (MS/
MS) data. These phenolics could be classified into 4 categories:
Table 1
Correlation coefficient (R2) between total phenolic (TP), proanthocyanidin (PC)
contents and antioxidant activity.
ABTS assay-TEAC DPPH assay-TEAC
(mmol/g DW) (mmol/g DW)
TP(mg CE/100 g DW) 0.9837 0.7513
PC(mg CE/100 g DW) 0.8832 0.8769
phenolic acid derivatives, flavan-3-ols, flavonoid glycosides and
hydrolysable tannins.
3.4.1. Phenolic acid derivatives
Compounds 1 and 13 presented in the aqueous and n-butanol
fractions with relatively low ABTS radical scavenging capacity.
Compound 1 had λmax of 325 nm with a shoulder at 270 nm. Mass
spectrum of m/z 683 and fragments of m/z 341, 179 and 161 indicated
that it's dimmers of caffeic acid derivative (Bravo, Goya, & Lecum-
berry, 2007; Regos, Urbanella, & Treutter, 2009). The MS spectrum
showed an ion at m/z 845, it probably be dicaffeic acid dim-glycoside
with elimination of a glucose moiety of [M-H]− at m/z 683 and a
caffeic acid glycoside unit of [M-H]− at m/z 503. Therefore, compound
1 was the mixture of dicaffeic acid glycoside and dicaffeic acid dim-
glycoside. Compound 13 had [M-H]− ion at m/z 517 and fragments of
m/z 193,175,337. It was identified as a ferulic acid derivative
(Bystrom, Lewis, Brown, Rodriguez, & Obendorf, 2008; Kammerer,
Carle, & Schieber, 2004). No free phenolic acid was found in the
fractions.
3.4.2. Flavan-3-ols
Compounds 6, 7, 8 and 9 exhibited very strong antioxidant activity
with the same λmax of 280 nm. Compound 6 showed [M-H]− ion of m/
z 865 and fragments of m/z 849, 739, 713, 577, 407 and 425, and it was
identified as proanthocyanidin trimer. The [M-H]− ion fragment at m/
z 849 was obtained after losing a water unit from m/z 865, m/z 739
after elimination of a phloroglucinol unit from extension Epicatechin
(E) or Catechin(C) subunit and m/z 713 was the product of Retro Diels
Alder (RDA) reaction fragmentation, m/z of 577 was obtained due to a
loss of extension (E)C subunit. Hokkanen and his coworkers linked
 L. He et al. / Food Research International 44 (2011) 1161–1167 1165

Fig. 5. HPLC–ABTS+ on line analysis of antioxidants from PSR extract of n-butanol and ethyl ether fractions.

this fragmentation pattern to C-type proanthocyanidin trimer
(Hokkanen, Mattila, Jaakola, Pirttila, & Tolonen, 2009). Compound 7
was identified as procyanidin dimer, the molecular ion was m/z 577
with fragments of m/z at 451, 425, 407, 289 and 245. Fragment of m/z
425 was RDA fission product which gave rise to m/z 407 after the loss
of water. [M-H]− ion of m/z 289 was monomer catechin resulted from
a cleavage of inter-flavanoid C–C linkages with a elimination of 288
unit, m/z 245 was obtained by losing a –CH–CHOH– group from
Epicatechin(Catechin). The literature reported that this fragmentation
pattern was a character of procyanidin type B (Verardo, Bonoli,
Marconi, & Caboni, 2008; Tomas-Barberan et al., 2001). Compound
8 was identified as another type of proanthocyanidin dimmer
(Beekwilder et al., 2005) with UV spectrum of 280 nm. The
main fragments of m/z 289 and m/z 273 were catechin and afzelechin

Table 2
Retention time, maximum absorbance and mass spectrum of phenolic compounds in fractions of PSR extract.

No. tR (min) λ (nm) [M-H]−(m/z) MS/MS of [M-H]−(m/z) Identification

a
1 5.7 325,270sh 683 341,179,161 Caffeic acid glycoside dimmer
845 503,341,179,161 Caffeic acid dimglu dimer
2 11.2 262 481 301,275,421 2,3-O-hexahydroxydiphenoyl-glu
3 13.3 258 783 UDb Unknown
4 14.0 255,274sh 565 282,150,133 Unknown
5 14.8 252 783 481,301 Pedunculagin
6 17.8 280 865 739,713,695,577,407,849 Procyanidin timer type C
7 20.4 280 577 451,425,407,289,245 Procyanidin drimer type B
8 22.2 280 561 289,273,425,543,435,407 Procyanidin dimer
9 23.9 280 289 245,205,125 (E) catechin
10 28.1 265,286sh 635 465,483 Trigalloylglucopyranose
11 29.2 278,353 934 897,915,783,633,301 Unknown
487 325,307,265,163,145,235 Coumaric acid derivative
12 30.4 278 787 465,617,635 Tetragalloylglucopyranose
13 31.1 326,298sh 517 193,175,337,295,235,265 Feuric acid derivate
14 31.8 268 939 787,769,617 Pentagalloylglucopyranose
15 39.5 254,368 447 301 Quercitrin 3-O-rhamnoside
16 44.2 266,348 447 285,255,227 Kaempferol 3-O-glucoside
17 49.5 268,350,316sh 593 447,285 Kaempferol 3-O-rutinoside

a, sh: shoulder peak; b, UD: undetected MS/MS.

1166 L. He et al. / Food Research International 44 (2011) 1161–1167
units, respectively. Beekwilder identified this compound in red
raspberry, but the precise structure of compound 8 needs a further
confirmation. Compound 9 presented at 23.9 min in HPLC profile with
MS [M-H]−of m/z 289 and fragments of m/z 245, 205, 125. Rio and his
coworkers identified this fragmentation pattern as catechin or
ecatechin, which were generally found in black and green tea (Rio
et al., 2004).
3.4.3. Flavonoid glycosides
Compound 15 showed relatively strong ABTS radical scavenging
capacity with a negative peak response of 30 mAU, while compounds
16 and 17 had nearly no radical scavenging activity even though they
exhibited high level in the PSR extract fractions. These compounds
presented two maximum absorbances between 190 and 400 nm and
could easily be associated with flavonoid. Flavonoid has two
maximum absorbances in ultraviolet region: 240–285 nm (band II)
and 300–400 nm (band I) (Robards & Antolovich, 1997). Compounds
15 and 16 had the [M-H]− ion at m/z 447. Fragments of m/z 301 and
285 were characteristic fragmentation pattern of quercetin and
kaempferol derivatives. The fragment at m/z 301 [M-H-146]−and
m/z 285 [M-H-162]−corresponded to the loss of a deoxyhexose and a
glycoside, respectively, and it was in agreement with Regos's report
(Regos et al., 2009), compounds 15 and 16 were identified as
quercetin 3-O-rhamnoside and kaempferol 3-O-glucoside according
to their MS, MS/MS data and UV spectrum. Compound 17 showed [M-
H]− ion at m/z 593 with the fragments of m/z 447 [M-H-146]− and m/
z 285 [M-H-146-162]−. Compound 17 could be kaempferol 3-O-
rutincoside: m/z 447, 285 were products of [M-rhamnoside]− and [M-
rhamnoside-glycoside]− (Rio et al., 2004).
3.4.4. Hydrolysable tannin
Hydrolysable tannin as gallotannins and ellagitannins have been
frequently found in the leaves, roots, barks and fruits of pomegranate
(Lansky & Newman, 2007). Compounds 2, 5, 10, 12, 14 performing
maximum UV absorbance in the range of 255–280 nm, was in
agreement with the identification of hydrolysable tannin from Rosa
chinensis flowers (Cai, Xing, Sun, Zhan, & Corke, 2005). Compound 5
could be pendunculagin (Bis-2, 3; 4, 6-O-hexahydroxydiphenoyl-
glucose) with [M-H]− ion at m/z 783 consisted of two HHDP
(Hexahydroxydiphenoyl) groups (302 unit per group) and one
glucosyl group (180 unit) (Tian, Li, Ji, Zhang, & Luo, 2009; Salminen,
Ossipov, Loponen, Haukioja, & Pihlaja, 1999), corresponding to the
fragments of m/z 481 [M-H-302]− and m/z 301 [M-H-302-180]−
illustrated in Table 2. Therefore, compound 2 (m/z 481) might be 2, 3-
O-hexahydroxydiphenoyl-glucose, made up of fragments of m/z 301
and m/z 180 (Martin, Krueger, Rodriquez, Dreherd, & Reed, 2008).
Nevertheless, fragments of m/z 275 and 421 in the MS/MS
information could not be explained yet. Peaks of 10, 12 and 14
appeared during the retention time of 27–34 min and were identified
as trigalloylglucopyranos (3-galloyglucopyranose), tetragalloylgluco-
pyranose (4-galloyglucopyranose), and pentagalloylglucopyranose
(5-galloyglucopyranose) (Tian et al., 2009; Salminen et al., 1999).
4. Conclusion
In this study, the antioxidant potential of phenolics from pomegranate
seed residues was evaluated using off line and on line assays. The results
implied that on line HPLC–ABTS+ was fast, effective for selectively
analysis of the antioxidants. A series of phenolic compounds were
identified from simple phenolic acids to complex ellagitannins involved,
most of which have been well-recognized as antioxidant activities. The
findings showed that PSR extract could be utilized as nutraceutical
resource.
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