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Food Research International 44 (2011) 957963

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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

Dog rose and pomegranate extracts as agents to control enzymatic browning

Federico Zocca, Giovanna Lomolino, Anna Lante
Dipartimento di Biotecnologie Agrarie, Universit di Padova, Viale dell'Universit 16, Agripolis, 35020 Legnaro, Padova, Italy

a r t i c l e

i n f o

Article history:
Received 12 October 2010
Accepted 12 February 2011
Dog rose
Polyphenol oxidase
Enzymatic browning

a b s t r a c t
To demonstrate that two natural products obtained with minimal processing can be used as antibrowning
agents, extracts of dog rose hips and pomegranate arils were assayed for inhibition of tyrosinase and
polyphenol oxidase activity. The efciency of antibrowning activity was evaluated in terms of absorbance and
polyacrylamide gel zymograms. In addition to the in vitro studies, melanosis in foods such as artichokes,
mushrooms and pear juice was evaluated. The results revealed that dog rose hip extract was more effective
than pomegranate aril extract. Moreover, high performance liquid chromatography analysis showed that
extracts from both sources had many potential inhibitors of polyphenol oxidase and tyrosinase activity.
2011 Elsevier Ltd. All rights reserved.

1. Introduction
Polyphenol oxidase (PPO; EC catalyzes the oxidation of
phenolic compounds to their corresponding quinones and is responsible for enzymatic browning, which is one of the food industry's
major problems. Enzymatic browning of plant-derived food and
beverages takes place in the presence of oxygen when PPO and its
polyphenolic substrates are mixed after brushing, peeling and
crushing operations, which leads to the rupture of cell structure
(Hurrel & Finot, 1984). The normal approach to inhibiting enzymatic
oxidative browning in foods has been the application of sultes. Due
to consumers' demand for natural food additives, studies have been
devoted to controlling this phenomenon, using other methods, and
several chemicals of plant origin have been tested (Kim & Uyama,
2005; Parvez, Kang, Chung, & Bae, 2007). Fruit of the dog rose
(Rosaceae family) is highly valued for its high vitamin C content
(Halasova, 1988) and is also rich in organic acids and phenolics
(Olsson, Andersson, Werlemark, Uggla, & Gustavsson, 2005). This fruit
is used in the production of jam and for medicinal purposes. In
addition, it is employed as an additive for fruit and vegetable juices
which have low levels of ascorbic acid (Halasova & Jicinska, 1988).
Pomegranate fruit (Punica granatum L.) has recently attracted much
attention for its health benets due to numerous studies showing that
pomegranate juice contains high levels of antioxidants in comparison
to other fruit beverages (Seeram et al., 2008). For instance, a glass of
pomegranate juice contains about 40% of the Recommended Daily
Allowance (RDA) of Vitamin C (Food and Nutrition Board of the

Corresponding author. Tel.: +39 049 8272920; fax: +39 049 8272929.
E-mail address: (A. Lante).
0963-9969/$ see front matter 2011 Elsevier Ltd. All rights reserved.

Institute of Medicine of the National Academies). Moreover, the

antioxidant activity of the pomegranate juice can be correlated to its
phenolic composition (Gil, Toms-Barbern, Hess-Pierce, Holcroft, &
Kader, 2000).
In this study, we have investigated extracts of dog rose hips and
pomegranate arils as natural inhibitors of polyphenol oxidase or
tyrosinase derived from mushrooms and vegetables to demonstrate
that these two natural products, obtained with minimal processing,
can be used as antibrowning agents.
To verify the inhibition of PPOs by these two natural products,
enzymatic browning was investigated in vitro and in vivo. The rst
approach was carried out by spectrophotometric and zymographic
analyses, while the second was performed treating mushroom slices,
artichoke slices, and pear extract with the inhibitors. The composition
of each extract was evaluated by high performance liquid chromatography analysis.
2. Materials and methods
2.1. Enzyme sources
Commercial mushroom tyrosinase was obtained from Sigma (St.
Louis, MO, USA). Artichokes (Cynara cardunculus subsp. Scolymus L.),
pears (Pyrus communis L. cv. Abate), and mushrooms (Agaricus bisporus
L.) were purchased at commercial maturity from a local store. Potato
tubers (Solanum tuberosum L. cv. Agata) were purchased from Ghisetti
1870 Enterprise (Rovigo, Italy). Plant material was washed under
running water to eliminate any surface contamination and wiped with
blotting paper. Potatoes and pears were hand-peeled. PPOs were
extracted as reported by Zocca, Lomolino, and Lante (2010).


F. Zocca et al. / Food Research International 44 (2011) 957963

Fig. 1. SDS-PAGE 12% zymograms of TYR and PPO activities of extracts in the absence (a) and presence (b) of inhibitors. Panel A: TYR 16 U per lane with DR extract as inhibitor. Panel B: TYR
with P extract as inhibitor. Panel C: pear enzyme ( 8 g of protein loaded per lane) with DR extract as inhibitor. Panel D: pear enzyme with P extract as inhibitor. Panel E: potato enzyme
( 16 g of protein loaded per lane) with DR extract as inhibitor. Panel F: potato enzyme with P extracts inhibitor. Panel G: artichoke enzyme ( 5 g of protein loaded per lane) with DR
extract as inhibitor. Panel H: artichoke enzyme with P extract as inhibitor.

2.2. Extraction of dog rose hips and pomegranate arils

Frozen fully mature fruits of dog rose (Rosa canina L.,) plants grown
in the Asiago plateau were cut into pieces. After seeds were removed,
the pieces were homogenized and extracted under constant agitation in
distilled water at room temperature for 10 min. The ratio of vegetableto-water was 1:2. Frozen arils from pomegranate (P. granatum L.) were
crushed with a mortar and pestle and the juice collected. The extracts
were centrifuged at 14,000 rpm for 2 min at 4 C, ltered using Millipore
0.45 m lter membranes (MA, USA) and stored at 20 C in dark
2.3. Determination of total protein
Protein content was determined from buffer dilutions of lyophilized enzymes using Bradford's (1976) dye-binding method, with BSA
(BioRad, Milano, Italy) as a standard.
2.4. Spectrophotometric PPO assay
The lyophilized enzymes were dissolved in 0.1 M sodium citrate
buffer at pH 6.0 for commercial tyrosinase; potato, artichoke, and pear
PPOs were dissolved in buffers with pH values of 5.7, 5.9, and 5.0,
respectively. PPO activity was assayed spectrophotometrically at 400 nm
with 10 mM catechol (Prolabo, Paris, France) in a sodium citrate buffer.

The reaction mixture included 1.0 ml of 10 mM catechol and 5 l of

commercial tyrosinase (45 U) or 100 l of PPOs in sodium citrate
buffer (pH values as reported above). The inhibitors used were:
pomegranate extract (P) 100 l; dog rose extract (DR) 100 l;
ascorbic acid (AA) 100 l of 0.5 g/l ascorbic acid; a combination of
agents P and AA (PAA); and a combination of agents DR and AA
(DRAA). Absorbance at 400 nm was monitored continuously at 25 C
for 30 min using a JASCO 7800 UVVis spectrophotometer (Tokyo,
Japan). Only the linear part of the curve ( absorbance vs. time) was
taken into account to calculate enzyme activity. One unit of enzyme
activity is herein dened as the amount of enzyme that would cause
an increase of 0.001 in absorbance per minute, at 400 nm and 25 C.
The percent inhibition of PPO activity was calculated as follows
(Baurin, Arnoult, Scior, Do, & Bernard, 2002):
%PPO inhibition =


Acontrol Asample = Acontrol 100%

where Acontrol = absorbance at 400 nm without test sample and

Asample = absorbance at 400 nm with test sample.
2.5. Electrophoresis (SDS-PAGE) and PPO zymography
The SDS-PAGE procedure was carried out in a Mini Protean II (BioRad, Milano, Italy) at room temperature. Non-reducing SDS-PAGE was

F. Zocca et al. / Food Research International 44 (2011) 957963


Fig. 2. Antibrowning effect of DR extract on mushroom slices using cathecol and L-DOPA as substrates. The slices were observed after incubating at 25 C for 15 min. C is control; DR is
dog rose extract.

performed according to Laemmli (1970) with a total polyacrylamide

concentration of 12% at 100 V. Freeze-dried powders were solubilized
with 700 l of Milli-Q water and 300 l of 1.33 M Tris (pH 7.4),
glycerol (40% v/v), 8% SDS w/v buffer. Samples were centrifuged at
19,000 rpm for 2 min before loading gel. After electrophoresis, the
gels were exhaustively washed for 15 min in 50 ml of appropriate
buffer (see 2.4). For each enzyme tested, two samples were run: 1) a
control treated with 4 ml of distilled water and 2) a sample treated
with 3.5 ml of distilled water and 0.5 ml of the inhibitor being tested.
After 15 min, enzyme activity was detected by incubating each gel
lane in 4 ml of sodium acetate buffer containing 5 mM L-DOPA and
3 mM MBTH (Sigma, St. Louis, MO, USA) at 25 C for 45 min (NezDelicado, Serrano-Megas, Prez-Lpez, & Lpez-Nicols, 2005). After
deep pink bands appeared on a lighter background, the gel images
were acquired by a digital camera (Fig. 1).

(C) was obtained by adding 1 ml of 10 mM catechol or L-DOPA to

200 l of buffer and 100 l of pear extract. In the inhibitor sample,
100 l of buffer was substituted by 100 l of DR extract. The browning
was visually observed within 15 min of incubation at 25 C, and the
images were acquired with a digital camera (Fig. 4).

2.8. Total phenolic content of extracts

Extracts of dog rose hips (DR) and pomegranate arils (P) were
examined for their total phenolic content as determined by the Folin
Ciocalteau method and expressed as gallic acid equivalent (GAE) per
ml of extract (Ercisli, 2007).

2.9. HPLC analysis of extracts

2.6. Antibrowning effect on artichokes and mushrooms
The antibrowning effect on artichoke and mushrooms (Figs. 2 and
3) was determined using a method modied from Lee et al. (2002).
Artichoke stems and mushrooms were cut into 1 cm slices, and each
slice was placed in a Petri dish. For the control (C), the whole surface
of cross-sections of artichoke stems and mushroom slices were
sprayed with 1 ml of distilled water using a syringe. Experimental
samples were sprayed with total volume of 1 ml (500 l of P or DR and
500 l of distilled water). After 15 min at 25 C, the surfaces were
wiped, and each sample was mixed with a substrate solution (2 ml of
10 mM catechol or L-DOPA). Browning was visually observed after
incubation at 25 C for 25 min, and the images were acquired with a
digital camera.
2.7. Antibrowning effect on pear extract
130 mg of lyophilized pear extract was dissolved in 1 ml of pH 5.0,
0.1 M sodium citrate buffer (Zocca et al., 2010). The control condition

Polyphenols, organic acids, and phenolic acids were quantied by

HPLC analysis performed using a Thermo Finnigan SpectraSystem
UV6000LP HPLC system (Thermo Finnigan, San Jose, CA, USA) with a
diode array detector and a Supelcosil LC-18 column (SigmaAldrich). The mobile phase, a mixture of water acidied with sulfuric
acid (pH 2.5) and 100% methanol, was programmed as follows
(Table 1).
The column temperature was 40 C, and the detector was set to a
wavelength range of 200600 nm. Analysis time course was 100 min.
All experiments were done in triplicate.

2.10. Statistical analysis

Statistical analysis was performed by subjecting the data to an
analysis of variance (ANOVA). Signicant difference was determined
by Tukey's multiple range test (p b 0.05) using the CoHort software
package (CoHort Software, Monterey, CA, USA).


F. Zocca et al. / Food Research International 44 (2011) 957963

Fig. 3. Antibrowning effect of DR and P extracts on cross-sections of artichoke stems using cathecol (cat) and L-DOPA as substrates. The slices were observed after incubating at 25 C
for 5 (t0) and 15 (t1) min. C is control; DR or P is dog rose or pomegranate extracts, respectively.

3. Results and discussion

3.1. Spectrophotometric PPO assay
Since the extracts had no PPO activity when using catechol and
substrates, all possibility of interference from the use of the
spectrophotometric assay was ruled out. Table 2 shows, under the
given experimental conditions, the capacity of DR and P extracts to
inhibit both commercial tyrosinase and PPOs. In particular, DR extract
alone and in association with AA showed strong inhibition of all
enzymes tested. Tyrosinase activity was reduced by 98.45% when
using 100 l of DR extract, a reduction about two times that obtained
with the same volume of AA. Moreover, a signicant difference was
found in the lag phase length; the length of lag phase seen with the DR
extract exceeded the lag phase length when using only AA by about
twenty times. There is an apparent synergistic effect between DR
extract and AA on the inhibition of PPO/TYR activities, with the
exception of pear PPO. Pomegranate extract, lauded by the cosmeL-DOPA as

ceutical industry for its antioxidant and anti-TYR effect (Yoshimura,

Watanabe, Kasai, Yamakoshi, & Koga, 2005; Rout & Banerjee, 2007),
actually increased PPO activity in the case of pear and potato. Only
when using artichoke as a sample did P extract reach the IC50 (100 l
of P extract). P inhibited TYR by 27.55%, showing a result similar to
that obtained when using the same volume of Brassicacaea cooking
water under the same experimental conditions (Zocca et al., 2010). It
is interesting to note that DR and P extracts are more efcient than AA
as artichoke PPO inhibitors, with inhibitory values of 19.6% and
57.58%, respectively. Moreover, unlike the DR extract, there is no
apparent synergistic effect between AA and P.
3.2. Electrophoresis (SDS-PAGE) and PPO zymography
Martnez-Alvarez, Gmez-Guilln, and Montero (2008) reported
that zymography techniques are effective tools for visualizing the
specic inhibition of PPO activity, therefore as a further demonstration of the capacity of the extracts to specically inhibit TYR and PPO

F. Zocca et al. / Food Research International 44 (2011) 957963


Fig. 4. Antibrowning effect of DR and P extracts on reconstituted pear juice using cathecol (cat) and L-DOPA as substrates. The slices were observed after incubating at 25 C for 0 (t0),
5 (t1) and 15 (t2) min. C is control; DR or P is dog rose or pomegranate extract, respectively.

activities, zymographic analyses were performed using L-DOPA as a

substrate. In Fig. 1, electrophoretic patterns showed that the same
concentrations of DR and P extracts inhibited all enzymes analyzed.
The only difference between the two inhibitors is observed when
using PPO from pear (Fig. 1, panels C and D), where the enzyme
isoforms were affected differently by each extract. For potato and
pear, there is no correlation between spectrophotometric and
zymographic results when P extract was used as inhibitor. This
behavior is probably due to different substrate specicity. It is possible
that the inhibition capacity of P extract could depend on the presence
of either a noncyclizable (catechol) or cyclizable (L-DOPA) diphenol as
a substrate of PPO/TYR (Snchez-Ferrer, Rodrguez-Lpez, GarcaCnovas, & Garca-Carmona, 1995).
3.3. Antibrowning effect on artichoke and mushrooms
The inhibitory effect of the two extracts on the browning of freshcut artichoke stems and mushrooms was also investigated in vivo by
using mushroom slices and artichoke as models (Figs. 2 and 3). Based
on the spectrophotometric results, DR extract was tested on
mushroom and artichoke, and P extract was only tested on artichoke.
A strong difference in color and browning development, likely due to
the different enzyme afnity for catechol and L-DOPA, was visible in
mushroom, but not in artichoke. As reported by Zhang, van Leeuwen,

Table 1
HPLC mobile phase gradient.
Time (min)

Water pH 2.5


Flow rate (ml/min)





Wichers, and Flurkey (1999) TYR of mushroom is able to use mono-,

di-, and trihydroxyphenols as substrates but has greater afnity for
dihydroxyphenols. Furthermore, it was also reported that among the
monohydroxyphenols (p-cresol and tyrosine), dihydroxyphenols
(catechol, L-DOPA, D-DOPA, catechin, and chlorogenic acid), and
trihydroxyphenols (pyrogallol), catechol showed maximum activity,
indicating that the enzyme is most active with catechol as substrate.
Our results indicated that degree of browning was lower in artichoke
and mushroom treated with extracts than control.
3.4. Antibrowning effect on pear extract
Reconstituted pear juice was also used to evaluate the effectiveness
of DR extract as an antibrowning agent (Fig. 4). A lyophilized powder
was rehydrated with sodium citrate, pH 5.0, and the development of
browning was monitored at different times after the addition of
catechol or L-DOPA. Samples treated with DR extract showed a slower
browning rate, particularly in the presence of L-DOPA. The development of browning in the control (C) is mainly due to the high afnity of
PPO/TYR enzymes for catechol, which is easily oxidizable although this
compound does not normally occur in pear (Rivas & Whitaker, 1973).
3.5. Total phenolic content and HPLC analysis of extracts
Many studies of dog rose hips and pomegranate arils report their
strong antioxidant activity to be attributable to the high levels of organic
acids and phenols that they contain (Wenzig et al., 2008; Lansky &
Newman, 2007). Tables 3 and 4 show the chromatographic proles of
these extracts. Citric acid was the predominant organic acid, with an
average content of 13,721 mg/l in P and 6054 mg/l in DR extracts,
respectively, followed by a malic acid concentration of 3008 mg/l in DR
extract and 2410 mg/l in P extract. The concentration of citric acid
normally employed for the prevention of enzymatic browning in fruits
and vegetables is between 0.5 and 2% (w/v). As reported by Marshall,


F. Zocca et al. / Food Research International 44 (2011) 957963

Table 2
The inhibitory effect of antibrowning agents on commercial TYR and plant PPOs.
PPO source






Commercial tyrosinase
Inhibition percentage (%)
Lag phase (s)

304.3 15.98a

147.75 2.19b
42.5 10.61

220.47 10.18c

227.62 3.58c
23.33 2.89

4.55 0.21d
905 7.07

1800 0

Inhibition percentage (%)
Lag phase (s)

141.1 6.92a

100.25 4.74b

72.6 11.75c

70.73 2.55c

19.01 5.09d

7.12 2.43e

Inhibition percentage (%)
Lag phase (s)

125.05 2.9a

19.5 0.42b
215 0

147.50 6.51c


61.27 1.38d

52 4.49e

Inhibition percentage (%)
Lag phase (s)

172.05 0.21a

93.67 7.08b
95 7.07

314.05 14.35c


28.90 7.98d

31 0.42d

nd: Values not determined because P extract was shown to have no inhibitory effect on potato and pear PPOs. AA: 100 l of 0.5 g/l ascorbic acid. P: 100 l of pomegranate extract.
PAA: the combination of agents P and AA. DR: 100 l of dog rose hip extract. DRAA: the combination of agents DR and AA. Lag phase: is the time necessary for activation of the enzyme.
The inhibitory effect was calculated using Eq. (1).
All values were based on three different samples. All assays were done in triplicate, and data is presented as mean SD. In each samples row, values followed by the same letter
are not signicantly different (p b 0.05), as measured by the Tukeys multiple range test.

Kim, and Wei (2000), citric acid reduces PPO activity by lowering the pH
and chelating a Cu2+ ion in the active site. Ascorbic acid is a moderately
strong reducing compound and also acts as an oxygen scavenger for the
removal of molecular oxygen in PPO reactions. Walker (1977) suggested
that PPO inhibition by ascorbic acid can be attributed to the reduction of
enzymatically formed o-quinones to their precursor diphenols. Ascorbic
acid is, however, irreversibly oxidized to dehydroascorbic acid during
the reduction process, thus allowing browning to occur upon its
depletion. It is usually applied in conjunction with citric acid in order to
maintain a more acidic pH. In addition, it is also believed to have a
chelating effect on the Cu prosthetic group of PPO. DR extract had the
greatest concentration of ascorbic acid (925 mg/l), 4.6 times higher than
P extracts.
Chang (2009) consider polyphenols to be the primary category of
inhibitors of TYR. Using the FolinCiocalteau method, we determined
that the total phenolic content of the P and DR extracts was 1.45 and
9.16 mg GAE/ml, respectively. These values are very close to concentrations obtained by Gil et al. (2000) and Ghazghazi et al. (2010).
Among the avanols, epigallocatechin, as reported by Kim and Uyama
(2005), is a competitive inhibitor of TYR that requires a concentration
of 0.035 mM to reach the IC50 of the enzyme activity. The epigallocatechin concentration of DR extract was 8 mM (Table 4). DR can thus be
considered a good source for the recovery of this inhibitory compound.
1 g of DR fruit contained 5.3% of the epigallocatechin present in 1 g of
green tea leaves (Hollman & Arts, 2000; Bronner & Beecher, 1998).
Another avanol that possesses similar properties to epigallocatechin
is epigallocatechin gallate. P extract contained an average of 335 mg/
l (0.7 mM) of epigallocatechin gallate (Table 4); this value is higher
than the IC50 value (0.034 mM) reported by Kim and Uyama (2005).
The same authors also reported an IC50 value of 0.017 mM for
epicatechin gallate when inhibiting TYR; in the DR extract, epicatechin

gallate was found to have a concentration of 230 mg/l (0.52 mM;

Table 4). Another phenolic compound found in DR extract that
possesses anti-PPO/TYR activity is p-hydroxybenzoic acid, (485 mg/l,
3.5 mM; Table 4). According to Chen et al. (2005), a concentration of
1.3 mM is sufcient to reach the IC50. The hydroxybenzoic acids also
include ellagic acid, which has anti-TYR activity (Yoshimura et al.,
2005). Ellagic acid is normally present in the juice, leaves and seeds of
pomegranate (Lansky & Newman, 2007). In this study, ellagic acid was
detected only in the DR extract (177 mg/l; Table 4). According to
Yoshimura et al. (2005), an ellagic acid concentration of 182 mg/l is the
IC50 for TYR activity.

4. Conclusions
Our results suggest that an extract of dog rose hips and, to a lesser
extent, pomegranate arils juice could potentially be used as natural
inhibitors of PPO and TYR to preserve the quality of fresh-cut
vegetables and fruit. Products enriched with bioactive compounds
such as those present in dog rose hips and pomegranate may prove to
be an effective tool to both develop functional foods and to increase
the overall intake of plant products. Further studies will focus on
evaluating the effect of these natural additives on the sensory quality
attributes of minimally processed products.

The authors thank Dr. Federica Tinello, Dr. Luca Greggio and
Stefania Zannoni for their technical help. This research project was
supported by a grant from the University of Padova.

Table 3
HPLC quantication of organic acids (mg/l) in dog rose and pomegranate extracts.

Oxalic acid

Malic acid

Ascorbic acid

Lactic acid

Acetic acid

Citric acid

Fumaric acid


274.27 7.51
231.19 0.27

3008.49 77.65
2410 6.7

925.11 6.81
199.71 0.74

618.27 16.13
302.6 4.42


6054.53 64.72
13,721.01 53.78

9.39 0.21
8.66 0.26

DR: dog rose. P: pomegranate.

F. Zocca et al. / Food Research International 44 (2011) 957963


Table 4
HPLC quantication of phenols and polyphenols (mg/l) in dog rose and pomegranate extracts.
Phenols and polyphenols


Gallic acid
-hydroxybenzoic acid
Chlorogenic acid
Epigallocatechin gallate
Caffeic acid
Syringic acid
Epicatechin gallate
-coumaric acid
Ferulic acid
Benzoic acid
Ellagic acid

2433.51 80.45
124.76 1.24
485.37 12.85
574.45 12.21
37.69 0.82
17.46 0.38
230.59 3.81
12.64 0.42
46.09 0.64
176.91 2.28


IC50 (mg/l)

76.96 1.53
10.45 0.3
14.03 0.25
71.99 1.25
335.15 8.16
13.09 0.3
4.23 0.14
158.25 2.5
12.10 0.42
11.87 0.78



Kim and Uyama (2005)

Chen et al. (2005)


Kim and Uyama (2005)


Kim and Uyama (2005)


Yoshimura et al. (2005)

DR: dog rose. P: pomegranate.

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