Industrial Crops and Products 42 (2013) 587–594

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Industrial Crops and Products

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Optimization of extraction and enrichment of phenolics from pomegranate

(Punica granatum L.) leaves
Caiyun Wang, Lingling Shi, Litong Fan, Youfang Ding, Shan Zhao, Yujun Liu ∗ , Chao Ma ∗
National Engineering Laboratory for Tree Breeding, College of Biological Sciences and Biotechnology, Beijing Forestry University, Beijing 100083, China

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

Article history: Pomegranate (Punica granatum L.) leaves, as byproducts, are rich sources of phenolic compounds and
Received 1 May 2012 possess strong antioxidant activities. The aim of the present study is to optimize the extraction and
Received in revised form 21 June 2012 enrichment conditions of phenolics from pomegranate leaves. Firstly, four solvents (water, 50% methanol,
Accepted 21 June 2012
50% ethanol and 50% acetone) were used to assess the effect of solvent polarity on the extraction yield
and total phenolics content in extracts. Then, the response surface methodology based on three-level,
Keywords:
three-variable Box–Behnken design was used to optimize and evaluate three independent variables:
Phenolics
solvent concentration (25–50–75%), temperature (40–60–80 ◦ C) and extraction time (20–40–60 min) on
Antioxidant
Pomegranate leaf
the extraction yield and total phenolics content. Thereafter, the macroporous resin chromatography was
Macroporous resin used to enrich the phenolics in the crude extract and the resin types, pH values of samples and elution
Response surface methodology solvent, and the concentration of elution solvent were optimized based on the absorption and desorp-
Ellagic acid tion capacity of phenolics. Finally, the optimum conditions of extraction and enrichment were selected:
the highest temperature (80 ◦ C), the longest extraction time (60 min) and 61% ethanol solvent concen-
tration for extraction; HPD-100 macroporous resin for chromatography, pH 2.0 for absorption sample
solution, and 50% ethanol with pH 2.0 as desorption solvent. The extraction and enrichment process
established here can be easily and effectively achieved, and the content of phenolics in the resulting
extracts can reach 669.35 ± 19.50 mg GAE/g. Moreover, DPPH and ABTS radical scavenging abilities were
significantly increased in the resulting extracts. This is the first time to study the enrichment of phenolics
with macroporous resin chromatography from pomegranate leaves.
© 2012 Elsevier B.V. All rights reserved.

1. Introduction predominantly contributed to their health benefits (Lan et al.,

Pomegranate (Punica granatum L.) is a shrub or a small
tree widely distributed in both tropical and subtropical areas.
Pomegranate, including its roots, barks, fruit juice, and dried peels,
contain abundant anthocyanins and/or hydrolysable tannins, and
possess strong antioxidant (Chidambara Murthy et al., 2002), anti-
tumor (Hora et al., 2003; Afaq et al., 2005), and hypolipidemia
activities (Huang et al., 2005). Recently, pomegranate leaf, used to
be byproducts after harvest of fruits, has attracted much atten-
tion due to its wide range of bioactivities. It was reported that
pomegranate leaf extracts could inhibit the development of obe-
sity and hyperlipidemia in high fat diet-induced obese mice and
the effects appear to be partly mediated by inhibiting the pancre-
atic lipase activity and suppressing energy intake (Lei et al., 2007).
Ponytail antiparasitic activity of pomegranate leaves against Giar-
dia lamblia was also reported (El-Shennawy et al., 2010). Usually it
was regarded that the phenolics contained in pomegranate leaves
∗ Corresponding authors. Tel.: +86 10 6233 6012; fax: +86 10 6233 6012.
E-mail addresses: yjliubio@163.com (Y. Liu), machao@bjfu.edu.cn (C. Ma).
2009). So far, no studies have been reported on the extraction and
enrichment of phenolic compounds from pomegranate leaves, thus
there is an urgent need to optimize the extracting and enriching
conditions.
As a collection of statistical techniques, response surface
methodology (RSM) has been widely used to analyze or to opti-
mize the independent factors which influenced the extraction yield
or extract profiles of bioactive components from herbs or natu-
ral materials. For example, it was used to optimize the extraction
parameters and to evaluate their effects on antioxidant capacity
and total phenolic contents from sunflower shells (Szydłowska-
Czerniak et al., 2011) and from chestnut burs (Vázquez et al., 2012).
In this study, the RSM was also used to optimize the extraction
conditions and to evaluate the effect of solvent polarity, extraction
time and temperature on the extraction yields of phenolics and
phenolics content in pomegranate leaves and crude extracts.
As for separation and enrichment of phenolics from crude
extracts of herbs, the conventional methods were usually per-
formed by solid–liquid extraction or liquid–liquid extraction using
different solvents, then followed by polyamide chromatography,
gel chromatography, silica gel column, ODS (octadecylsilyl), and

0926-6690/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.indcrop.2012.06.031
588 C. Wang et al. / Industrial Crops and Products 42 (2013) 587–594
so forth. However, all these methods have similar disadvantages,
including long time taking, large amount of organic solvent
consuming and low recoveries (Zhang et al., 2009). Recently,
macroporous resins, with durable non-polar, middle polar, or polar
macroporous polymers (Zhang et al., 2009), have been successfully
applied to the separation and enrichment of bioactive components
from many natural products, as an efficient method with a mod-
erate purification effect, high absorption capacity, low operating
costs, low solvent consumption, and easy regeneration (Zhao et al.,
2007; Silva et al., 2007; Sun et al., 2007). In this study, to avoid the
toxic organic solvents used in conventional separation techniques
such as gel and high-speed counter-current chromatographic sep-
aration process and to increase the safety of the process (Zhao et al.,
2011), the absorption and desorption on macroporous resins were
utilized for the enrichment of phenolics from pomegranate leaves.
The present study focused on the optimization of extraction
and enrichment of phenolics with high antioxidant capacities from
pomegranate leaves, which has not yet been reported so far. In
extraction process, RSM was used to optimize three independent
variables including temperature, solvent composition and polar-
ity, and extracting time. For enrichment of phenolics, macroporous
resin chromatography was used to separate the crude extracts into
four fractions with different phenolic contents and different antiox-
idant capacities.
2. Materials and methods
2.1. Raw materials and reagents
Pomegranate (P. granatum L.) leaves were collected after har-
vest of fruits in Beijing of China in October, 2010. The leaves
were washed with distilled water and air-dried till equilibrium
humidity, then ground and transferred to darkness for further
use. Macroporous resins (ADS-17, AB-8, D-101, HPD-826 and HPD-
100) were bought from Cangzhou Bonchem Co., Ltd., China. Other
chemicals and reagents, unless specialized, were purchased from
Sigma–Aldrich, Inc.
2.2. Solvent extraction
Extractions with four different solvent systems [water, 50:50
(v/v) methanol/water, 50:50 (v/v) ethanol/water and 50:50 (v/v)
acetone/water] were carried out using a soxhlet apparatus with
constant-temperature water bath at 70 ◦ C for 40 min, and the
solid/liquid ratio was 1/10 (w/v). The resulting slurries were cen-
trifuged to obtain supernatant and the residues were subject to
extraction twice under the same condition. All the supernatants
collected were combined and then evaporated under vacuum at
50 ◦ C. After concentration, the extracts were lyophilized at −50 ◦ C
and 0.1 MPa, and finally stored at −20 ◦ C in vacuum-sealed light-
proof pouches for further analysis.
2.3. Response surface modeling
A 33 factorial experimental design was used to evaluate
the effects of three independent variables: temperature (T,
40–60–80 ◦ C), solvent concentration (C, 25–50–75%) and time (t,
20–40–60 min) on the extraction efficiency of phenolics, which
were reflected by three dependent variables including extraction
yield (Y1 , g extract/100 g leaves), phenolics content based on dry
weight of the extract (Y2 , g gallic acid equivalents (GAE)/100 g) and
phenolics content based on dry weight of leaves from which the
extract was obtained (Y3 , g GAE/100 g). All experiments were car-
ried out according to the experimental design listed in Table 1.The
statistical software package ‘Design Expert 8.0’ was used for regres-
sion analysis of the data and estimation of the regression equation
coefficients. A multiple regression analysis was applied to obtain
an empirical model that relates the response measured to the
independent variables. The second-order response function for the
experiments was predicted by the following equation:
Yn = ˛0 + ˛1 · C + ˛2 · T + ˛3 · t + ˛11 · C 2 + ˛22 · T 2 + ˛33 · t 2
+˛12 · C · T + ˛13 · C · t + ˛23 · T · t
where Yn is the measured responses, ˛0 is the intercept term, ˛1 ,
˛2 and ˛3 are linear coefficients, ˛11 , ˛22 and ˛33 are quadratic
coefficients, ˛12 , ˛13 and ˛23 are interaction coefficients, T, C, and t
are the three independent variables coded at three levels −1 (lower
limit), 0 (central point) and +1 (upper limit) as shown in Table 1.
2.4. Enrichment
2.4.1. Static absorption and desorption tests
In order to get the most appropriate type of macroporous resin
for enrichment of phenolics from pomegranate leaves, five different
types of resins were tested, including ADS-17, AB-8, D-101, HPD-
826 and HPD-100, whose physical properties were summarized
in Table 2. After pretreated and activated according to the man-
ufacturer’s recommendation, 1.0 g (dry weight) resins were put
into a flask with a lid, then 40 mL crude pomegranate leaf extracts
(4.17 mg/mL) were added for absorption with shaking (200 rpm) for
12 h at 25 ◦ C. After reaching absorption equilibrium, all the resins
were washed with 100 mL distilled water, and then transferred to
another flask with 40 mL ethanol–water (60:40, v/v) solutions for
desorption with shaking (200 rpm) for 12 h at 25 ◦ C.
To study the effects of the initial sample solution pH on absorp-
tion capacity and the elution solvent pH on desorption capacity, a
series of solutions with pH values ranged from 2 to 9 were tested.
2.4.2. Capacities and ratios of absorption and desorption
The absorption and desorption properties of different resins for
phenolics are quantified with the following equations.
C0 Ce C0 Ce
Absorption : Qe = ·E = × 100%
W C0
where Qe is the absorption capacity for phenolics at absorption
equilibrium (mg GAE/g resin); C0 and Ce are the initial and equi-
librium concentrations of phenolics in the solutions (mg GAE/mL),
respectively; Vi is the volume of the initial sample solution (mL);
W is the weight of the tested dry resins (g) and E is the absorption
ratio (%).
Cd Vd Cd Vd
Desorption : Qd = ·D= × 100%
W (C0 − Ce )Vi
where Qd is the desorption capacity for phenolics after absorption
equilibrium (mg GAE/g dry resin); Cd is the concentration of the
phenolics in the desorption solutions (mg GAE/mL); Vd is the vol-
ume of the desorption solution; D is the desorption ratio (%); C0 , Ce ,
W, and Vi are the same as those defined for absorption.
2.4.3. Column chromatography for enrichment
Enrichment of phenolics from crude pomegranate leaves
extracts was carried out by macroporous resin chromatography.
40 g pomegranate leaves was extracted by employing the optimum
conditions obtained in Section 2.3, and supernatants were com-
bined after centrifugation and evaporated till 20% volume left. Then,
the extracts were replenished with distilled water to the initial vol-
ume and centrifuged at 5000 × g for 10 min to obtain supernatant
again. Subsequently, the combined supernatant was subject to a
column chromatography (600 mm × 60 mm i.d.) packed with HPD-
100 macroporous resins and equilibrated with distilled water. Fr.
 C. Wang et al. / Industrial Crops and Products 42 (2013) 587–594 589

Table 1
Three level Box–Behnken experimental design for pomegranate leaf extraction.

Exp. Coded level Independent variables Dependent variables

T C t T (◦ C) C (%, v/v) t (min) Y1 Y2 Y3

1 0 1 −1 60 75 20 23.80 35.22 8.38

2 0 0 0 60 50 40 37.00 38.95 14.41
3 0 0 0 60 50 40 34.00 39.45 13.41
4 −1 0 1 40 50 60 28.60 37.89 10.84
5 0 0 0 60 50 40 36.00 38.27 13.78
6 0 0 0 60 50 40 37.80 39.67 15.00
7 0 1 1 60 75 60 37.80 39.03 14.75
8 0 −1 1 60 25 60 33.40 39.18 13.09
9 −1 0 −1 40 50 20 16.20 34.28 5.55
10 −1 1 0 40 75 40 19.40 34.66 6.72
11 1 0 −1 80 50 20 40.40 41.15 16.62
12 1 1 0 80 75 40 44.80 40.90 18.32
13 −1 −1 0 40 25 40 22.00 34.16 7.52
14 0 0 0 60 50 40 38.00 39.07 14.85
15 1 0 1 80 50 60 44.60 42.59 19.27
16 1 −1 0 80 25 40 39.00 39.16 15.00
17 0 −1 −1 60 25 20 26.40 37.38 9.87

R2 0.9883 0.9607 0.9887

Adj. R2 0.9732 0.9101 0.9741
SD 1.42 0.74 0.65
Sig. 0.000 0.000 0.000

Y1 , extraction yield (%); Y2 , total phenolic content in extracts (g GAE/100 g); Y3 , phenolic content in pomegranate leaves (g GAE/100 g); SD, standard deviation; Sig., model
significance.

I was eluted with distilled water, Fr. II was obtained after elut-
ing with 20% ethanol, then Fr. III and Fr. IV were yield with 50%
and 80% ethanol as mobile phase, respectively. All the fractions
obtained were evaporated, lyophilized and finally stored at −20 ◦ C
in darkness for further analysis.
2.5. Determination of total phenolics
and left to stand for 20 min at room temperature in the dark, and
then the absorbance was read at 517 nm. The RSA was determined
as %RSA = 100 (Ablank − Asample )/Ablank , where Asample and Ablank is
the absorbance of the extract solution and control solution, respec-
tively. The Trolox equivalent of the extracts (TRE) was calculated
and expressed as mmol Trolox equivalent (TRE) per g extract on
dried basis.

The content of the total phenolics in pomegranate leaf extracts
was estimated using a modified Folin–Ciocalteu method as
described by Wolfe et al. (2008). Briefly, 0.5 mL of deionized water
and 0.125 mL of diluted pomegranate leaf extracts (0.5 mg/mL)
were added into a brown volumetric flask. Folin Ciocalteu reagent
(0.125 mL) was subsequently added to the flask and allowed to
react for 6 min. Then, 1.25 mL of 7% sodium carbonate solution was
transferred to the flask, and then the mixture was diluted to 3 mL
with deionized water. The absorbance was recorded after 90 min
at 760 nm. The measurement was compared to a standard curve
of gallic acid concentrations and expressed as milligrams of gal-
lic acid equivalents (GAE) per 100 g extract (on a dried basis) ± SD
(standard deviation) for triplicate extracts.
2.6. Antioxidant activity
2.6.1. DPPH radical scavenging activity
The DPPH radical scavenging activities (RSA) of the crude
extracts and four fractions were assessed using a method described
by Alasalvar et al. (2009). Briefly, aqueous solutions (0.1 mL) of the
crude extracts or fractions with concentration 40–320 ␮g/mL were
mixed with 0.25 mL of freshly prepared DPPH solution (1 mM in
methanol) and 2 mL ethanol. The mixture was shaken vigorously
2.6.2. ABTS radical scavenging activity
The free radical scavenging activity using ABTS radical was also
carried out according to the method of Alasalvar et al. (2009).
Briefly, an ABTS solution (7 mM) was mixed with potassium per-
sulfate (140 mM) with a ratio of 62.5:1 for 16 h in the darkness at
room temperature to produce ABTS radical cation (ABTS+ ) stock
solutions. The ABTS+ stock solution was diluted with ethanol
to an absorbance of 0.70 at 734 nm as a working solution. An
aliquot (0.15 mL) of aqueous solution containing 10–80 ␮g/mL of
pomegranate leaf extract or fraction was mixed thoroughly with
the ABTS+ solution (2.85 mL) and after 6–10 min in the darkness
at room temperature; the absorbance was read at 734 nm. The
radical-scavenging activity (RSA) for DPPH and ABTS radicals was
calculated as %RSA = 100 (A0 − As )/A0 , where As is the absorbance of
the extract solution and A0 is the absorbance of a control solution
prepared without extracts. The Trolox equivalent of the extracts
(TRE) was calculated and expressed as mmol Trolox equivalent
(TRE) per g extract on dried basis.
2.7. HPLC analysis
A Shimadzu HPLC system (Shimadzu, Kyoto, Japan) with a SPD-
M20A ultraviolet detector and a SIL-20AC TH autosampler coupled

Table 2
Physical properties of the 5 macroporous resins used.

Name Particle diameter (mm) Surface area (m2 /g) Average pore diameter (nm) Polarity

ADS-17 0.3–1.25 90–120 25.0–30.0 Middle-polar

HPD-826 0.3–1.20 500–600 90.0–100.0 Middle-polar
AB-8 0.3–1.25 480–520 13.0–14.0 Weak-polar
HPD-100 0.3–1.25 650–700 85.0–90.0 Non-polar
D-101 0.2–0.60 400–600 10.0–12.0 Non-polar
590 C. Wang et al. / Industrial Crops and Products 42 (2013) 587–594
with an analytical software (LC Solution-Release 1.23SP1) were
applied to figure out the chromatographic profiles difference of
the pomegranate extracts and fractions, which could reflect the
chemical components profiles of them. A 250 mm × 4.6 mm i.d.,
5 ␮m, Eclipse XDB-C18 (Agilent) column was used, the column
thermostat was set at 30 ◦ C, and the mobile phase was 0.2% (v:v)
phosphoric acid in water (A) and acetonitrile (B) at a total flow rate
of 1 mL/min. Elution gradient was 15–25% B (0–25 min), 25–42% B
(25–35 min), 42–50% B (35–50 min), and the eluate was detected
at 254 nm. Also, ellagic acid was used as a standard and the con-
tent of it in different extracts were determined. Each extract was
centrifuged, filtered through a 0.45 ␮m nylon filter and analyzed
three times directly by HPLC running. After each run, the column
was equilibrated for 10 min under initial conditions.
2.8. Statistical analysis
Statistical analysis was performed using ANOVA (two-tailed
Student’s t-tests) of Microsoft Excel Statistical Software (Microsoft
Office Excel 2007), and statically significant differences were con-
sidered when p < 0.05. Correlations were determined using linear
regression on log-transformed data.
3. Results and discussion
3.1. Effects of solvents on extraction of phenolics
The extraction yield and total phenolics in pomegranate
leaves using different solvents, including water, 50:50 (v/v)
methanol/water, 50:50 (v/v) ethanol/water and 50:50 (v/v) ace-
tone/water, were shown in Table 3. It is clear that acetone/water
and ethanol/water showed the highest extraction yield and
Y1 = −36.82500 + 0.98525 · T + 0.22180 · C + 0.87350 · t + 4.20000E − 003 · T · C − 5.12500E − 003 · T · t + 3.50000E − 003 · C · t − 3.95000E − 003 · T 2 − 5.88800E − 003 · C 2 − 6.32500E − 003 · t 2
Y2 = +20.33625 + 0.25386 · T + 0.17321 · C + 0.059950 · t + 6.20000E − 004 · T · C − 1.35625E − 003 · T · t + 1.00500E − 003 · C · t − 7.33750E − 004 · T 2 − 2.50960E − 003 · C 2 + 4.72500E − 004 · t 2
Y3 = −14.76875 + 0.33200 · T + 0.14265 · C + 0.28856 · t + 2.06000E − 003 · T · C − 1.65000E − 003 · T · t + 1.57500E − 003 · C · t − 1.06563E − 003 · T 2 − 3.15800E − 003 · C 2 − 1.98438E − 003 · t 2
phenolics content based on both dry weight leaves and
extracts. Water exhibited almost the same extraction yields with
methanol/water, but with significantly lower phenolics contents.
The results obtained appears to differ from those reported by
Kaneria et al. (2011), who measured the extraction yield from
pomegranate leaves and stems using five different solvents includ-
ing petroleum ether, toluene, ethyl acetate, acetone, and water,
and found that the yield was maximum in water, followed by
acetone, while the total phenolics content in extracts of acetone
(40 g GAE/100 g extract) and water (22 g GAE/100 g) were similar
to the present study.
Free radical scavenging ability (RSA) of extracts obtained using
various solvents were evaluated with the change of absorbance
caused by the reduction of DPPH and ABTS+ radicals. The more
rapidly the absorbance changes, the more potent the antioxi-
dant activity of the extract presents in terms of its hydrogen
atom-donating capacity (Alasalvar et al., 2009). The percentage
scavenging activity of each extract against DPPH and ABTS+ were
shown in Fig. 1. It was obvious that all four different solvent extracts
of pomegranate leaf extracts exhibited great DPPH and ABTS+ rad-
ical scavenging activities, and both the DPPH and ABTS+ radical
scavenging activity of water extract was lower than the others.
In Fig. 1A, the extracts of 50% acetone and 50% ethanol had the
greatest DPPH RSA, followed by 50% methanol extract and water
extract. In Fig. 1B, the extracts of 50% acetone, 50% ethanol and
50% methanol had the similar ABTS RSA, and water extract pre-
sented lowest antioxidant activity. These results indicated that
both the DPPH RSA and ABTS RSA decreased with the increase of
solvent polarity, which was similar to the trends of total pheno-
lic content. These results agree with those reported by Turkmen
et al. (2006), who studied effect of the use of water and different
organic solvents such as acetone, N,N-dimethylformamide (DMF),
ethanol or methanol on the total polyphenol content and antiox-
idant activity of the black tea and mate tea, and found that 50%
acetone showed the highest polyphenol content for mate tea, 50%
ethanol extract from mate tea and 50% acetone from black tea had
the greatest antioxidant activity. It was obvious that solvent with
different polarity had significant effect on polyphenol content and
antioxidant activity, and it showed that there is a high correlation
between phenolic content and antioxidant activity of pomegranate
leaf extract.
Since acetone and ethanol aqueous solvents showed the highest
extraction yield, phenolics content and antioxidant activities, and
no significant difference was observed between them, both of them
would be the most appropriate solvent for phenolics extraction.
However, ethanol was generally recognized as safe, therefore, it
was considered to be the best in the four solvents assessed in this
study.
3.2. Optimization of extraction condition
To assess the effects of three independent variables including
extraction time (t), temperature (T), and ethanol concentration (C)
on three dependent variables, which reflect the extraction effi-
ciencies of phenolics from pomegranate leaves, RSM was applied,
and the coefficients R2 , adjusted R2 , standard deviation (SD) and
model significance (Sig.) were shown in Table 1. Significant models
were found for all the three dependent variables. Multiple regres-
sion coefficients were determined by the least-squares technique
in order to predict quadratic polynomial models for the tested
response variables and the regression equations were obtained as
following:
Fig. 2 shows the response surfaces for extraction yield
(Y1 , 16.2–44.8%), phenolics content based on both dry weight
of the extract (Y2 , 34.16–42.59 g GAE/100 g) and leaves (Y3 ,
5.55–19.27 g GAE/100 g) in function of temperature, ethanol con-
centration and extraction time, respectively. Y1 increased with
increasing of the temperature (below 80 ◦ C) and the extraction
time (less than 60 min). Long time heating might make pheno-
lics dissolve and diffuse quickly and thoroughly. The extraction
yield increased with increasing of ethanol concentration in the
range from 25% to 60%, while decreased when above 60%. Simi-
lar findings were reported by Vázquez et al. (2012), who observed
that the highest extraction yield of chestnut burs occurred when
ethanol concentration was approximately 65%, then the yield
decreased with increasing of ethanol concentration. The maximum
yield (46.683%) predicted by the model was achieved at 80 ◦ C,
61% ethanol and 60 min. Similar to Y1 , Y2 and Y3 increased with
increasing of the temperature (below 80 ◦ C), time (less than 60 min)
and ethanol concentration (below 60%), whereas at concentration
above 60%, both Y2 and Y3 decreased with increasing of the ethanol
concentration. Predicted by the model, the maximum Y2 (42.668%)
and Y3 (19.991%) were both located at 80 ◦ C, 61% ethanol, 60 min.
Since the best conditions for Y1 , Y2 and Y3 were the same, the opti-
mum conditions for extraction was selected as 80 ◦ C, 61% ethanol
and 60 min.
3.3. Macroporous resins selection
Macroporous resin chromatography was used to enrich and sep-
arate the phenolics of pomegranate leaf extracts. To obtain the
most appropriate resins with the best absorption and desorption
 C. Wang et al. / Industrial Crops and Products 42 (2013) 587–594 591

Table 3
Extraction yield and phenolics contents in crude extracts and pomegranate leaves.

Water 50% methanol 50% ethanol 50% acetone

Extraction yield (%) 34.8 ± 0.8A 34 ± 0.35A 39.53 ± 0.58B 40.87 ± 1.50B
PC in extracts (g GAE/100 g) 25.97 ± 0.57A 37.08 ± 0.54B 39.89 ± 0.58C 40.23 ± 1.2C
PC in leaves (g GAE/100 g) 9.04 ± 0.4A 12.61 ± 0.07B 15.77 ± 0.3C 16.44 ± 0.87C
A,B,C,D
In each line, different letters mean significant differences (p < 0.05). PC, phenolics content; GAE, gallic acid equivalent.

Fig. 1. DPPH radical scavenging activity (DPPH RSA) (A) and ABTS radical scavenging activity (ABTS RSA) (B) of pomegranate leaf extracts.

Fig. 2. Response surfaces for extraction yield (Y1 ), phenolics content in extracts (Y2 ) and phenolics content in leaves (Y3 ) in function of ethanol concentration, temperature
and time.
592 C. Wang et al. / Industrial Crops and Products 42 (2013) 587–594
Fig. 3. Effect of different macroporous resins on capacities (A) and ratios (B) of absorption and desorption of phenolics.
capacities, five types of macroporous resins with different physical
properties were assessed and the results were shown in Fig. 3. It
is indicated that the absorption capacities of different resins corre-
lated with their physical properties such as polarity and surface
area. The nonpolar resins (HPD-100 and D-101) and the weak-
polar resin (AB-8) have better absorption and desorption capacities
than the middle-polar resins (HPD-826 and ADS-17), indicating
that there might be more nonpolar phenolics than polar pheno-
lics in pomegranate leaves. Therefore, HPD-100 was selected as
the best one with the highest absorption and desorption capacities,
followed by D101, AB-8, HPD-826 and ADS-17.
3.4. Effects of pH on the absorption and desorption capacities
The pH plays an important role in absorption and desorption
processes, because the ionization of solutes can be influenced by
pH, thus affecting the absorption affinity between solutes and solu-
tions (Fu et al., 2006). The effects of initial sample solution pH on
the absorption capacity and the eluting solution pH on the desorp-
tion capacity were shown in Fig. 4. It shows that both the absorption
and desorption capacities decreased with increasing of pH of initial
sample solutions and elution solutions. Especially, absorption and
desorption capacities decreased sharply when the pH was above 6.
Similar results were reported by Fu et al. (2006), who found that
the absorption capacities of vitexin and isovitexin decreased with
the increase of pH. The reason for these sharp decreases is that
the phenolic hydroxyl groups in phenolics dissociated to form H+
and their corresponding anions with increasing of pH, thus resulted
in the decrease of absorption and desorption capacities. Based on
Fig. 4. Effects of pH on the absorption and desorption capacities of phenolics.
these results, the lowest pH of sample and elution solutions were
selected for absorption and desorption.
3.5. Fractionation of pomegranate leaf extracts
After HPD-100 macroporous resin chromatography, the crude
extracts of pomegranate leaves were fractionated into four parts:
Fr. I eluted with water, Fr. II with 20% ethanol, Fr. III with 50%
ethanol and Fr. IV with 80% ethanol successively. The transfer ratios
of both extracts and phenolics in each fraction were presented in
Table 4. It shows that about 68.34% of the crude extract was trans-
ferred to Fr. III, while over 80% of the phenolics in the crude extracts
were transferred to this fraction. Therefore, the phenolics were
significantly enriched in Fr. III, which was about 1.72-fold higher
than the crude extract. Also, as shown in Fig. 5, Fr. III exhibited
the highest DPPH RSA and ABTS RSA, which was much higher than
the other fractions and crude extracts. Although significant differ-
ence between Fr. II, Fr. IV and crude extracts was not observed
in phenolics contents, it was found in DPPH RSA and ABTS RSA
between them. Fr. II showed the highest DPPH RSA and ABTS RSA,
followed by the crude extracts and Fr. IV, and Fr. I showed both
the lowest DPPH RSA and ABTS RSA, indicating that their contents
of bioactive phenolics with antioxidant activities were different.
Therefore, macroporous resin chromatography effectively enriched
the phenolics, especially the phenolics with antioxidant activities
in the extracts. Similar results were reported by Zhao et al. (2011),
who used macroporous resin chromatography to enrich antioxi-
dant phenolics in chestnut burs effectively.
3.6. HPLC profiles of pomegranate leaf extracts and fractions
The HPLC chromatographic profile of pomegranate leaf extracts
and fractions was shown in Fig. 6. It is clear that the chromato-
graphic profile of different fraction was different, which indicated
that the components of them were significantly different. Also, it
showed that the macroporous resin chromatography effectively
fractionated the crude extracts into different fractions with differ-
ent components. The predominant component in crude extract was
identified as ellagic acid by comparing the retention time with the
ellagic acid standard. As shown in Fig. 7, Fr. IV presented the highest
content of ellagic acid, followed by crude extracts, Fr. III, while Fr. I
and Fr. II showed the lowest content. Obviously, the DPPH RSA and
ABTS RSA were not correlated with the content of ellagic acid con-
tent in different fractions, which indicated that ellagic acid might
not be the dominant component responsible for the DPPH RSA and
ABTS RSA, although its antioxidant activities were found in some
 C. Wang et al. / Industrial Crops and Products 42 (2013) 587–594 593

Table 4
Total phenolics content in crude and fractionated extracts.

Crude extract Fr. I Fr. II Fr. III Fr. III

PC in extracts (mg GAE/g) 389.03 ± 19.87B 105.46 ± 3.77A 419.41 ± 20.72B 669.35 ± 19.50C 394.96 ± 0.42B
Transfer ratio of extracts (%) 9.87 12.68 68.34 9.10
Transfer ratio of phenolics (%) 1.97 9.55 82.13 6.45
A,B,C,D
In each line with different letters means significant differences (p < 0.05). PC, phenolics content.

Fig. 5. DPPH radical scavenging activity (DPPH RSA) (A) and ABTS radical scavenging activity (ABTS RSA) (B) of fractionated extracts of pomegranate leaves.

report (Han et al., 2006). Further study is still needed to identify the
component responsible for antioxidant activities in Fr. III.

3.7. Correlation analyses

Using regression analyses, the relationships between total phe-

Fig. 6. HPLC chromatograms of crude and fractionated extracts (A, crude extracts;
B, Fr. I; C, Fr. II; D, Fr. III; E, Fr. IV; and F, ellagic acid reference standard) obtained
from pomegranate leaves (1: ellagic acid).
nolic content, ellagic acid content, EC50 of DPPH and ABTS radicals
scavenging values for fractions were determined. Total phenolics
were significantly correlated to EC50 of DPPH values (R2 = 0.8060),
and significant linear correlations were also found between total
phenolic content and EC50 of ABTS values (R2 = 0.7820). There was
a positive correlations between EC50 of DPPH values and EC50
of ABTS values (R2 = 0.9550). Similar trend was also found by Gil
et al. (2002), who reported that there was a strong correlation
(0.93–0.96) between total phenolics and antioxidant activity of nec-
tarines, peaches, and plums. Dudonné et al. (2009) compared total
phenolic content and antioxidant properties of 30 plant extracts
of industrial interest, and found that there was a significant rela-
tionship between total phenolic content and antioxidant capacity
(correlation coefficient R2 = 0.939 and R2 = 0.966, respectively, for
DPPH and ABTS radicals), DPPH and ABTS assay was also positively
correlated (R2 = 0.906). However, no linear correlations was found
between ellagic acid content and EC50 of DPPH values, ellagic acid
content and EC50 of ABTS values, total phenolic content and ellagic
acid content. This indicates that total phenolics had significant con-
tribution to the antioxidant activity of fractions. These correlation
analyses directly reconfirmed that ellagic acid might not be the
dominant component responsible for the antioxidant activity of
pomegranate leaf extracts.

4. Conclusions

The present study established an extraction and enrichment

Fig. 7. Ellagic acid content in crude and fractionated extracts (A, crude extracts; B,
Fr. I; C, Fr. II; D, Fr. III; E, Fr. IV; and F, ellagic acid reference standard, for each column,
different letters mean significant differences (p < 0.05)).
procedure and assessed the potential antioxidant activity for phe-
nolics from pomegranate leaves. The final results showed that the
optimum extraction condition was 80 ◦ C, 61% ethanol and 60 min.
The best macroporous resin for isolation phenolics was HPD-100.
The optimum pH of sample solution for the absorption of phenolics
was 2.0, and the most appropriate elution solvent for desorption
594 C. Wang et al. / Industrial Crops and Products 42 (2013) 587–594
was 50% ethanol with pH 2.0. Also, the resulting extracts pos-
sessed significantly increased phenolics enrichment thus improved
antioxidant activities. Although well known for its antioxidant
activities, ellagic acid seemed not play the dominant role in DPPH
RSA. To the best of our knowledge, this is the first report on
the extraction optimization of phenolics using response surface
methodology, also the first time to study the enrichment of phe-
nolics with macroporous resin chromatography from pomegranate
leaves.
Acknowledgments
The study was financially supported by the Fundamental
Research Funds for the Central Universities, China (JD2010-2),
Forestry Commonweal Programs (201004008), and Ph.D. Programs
Foundation of Ministry of Education of China (20100014120017).
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