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Article history:
Received 12 October 2010
Accepted 12 February 2011
Keywords:
Dog rose
Pomegranate
Polyphenol oxidase
Tyrosinase
Enzymatic browning
Inhibitors
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 1.10.3.1) 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: anna.lante@unipd.it (A. Lante).
0963-9969/$ see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodres.2011.02.010
958
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.
h
i
Acontrol Asample = Acontrol 100%
959
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.
960
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.
961
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.
Table 1
HPLC mobile phase gradient.
Time (min)
Water pH 2.5
MeOH
0
15
75
85
100
100%
100%
20%
20%
100%
0%
0%
80%
80%
0%
0.35
0.35
0.45
0.45
0.35
962
Table 2
The inhibitory effect of antibrowning agents on commercial TYR and plant PPOs.
PPO source
Inhibitors
None
AA
PAA
DR
DRAA
Commercial tyrosinase
Units
Inhibition percentage (%)
Lag phase (s)
304.3 15.98a
0
No
147.75 2.19b
51.44
42.5 10.61
220.47 10.18c
27.55
No
227.62 3.58c
25.2
23.33 2.89
4.55 0.21d
98.45
905 7.07
0e
100
1800 0
Artichoke
Units
Inhibition percentage (%)
Lag phase (s)
141.1 6.92a
0
No
100.25 4.74b
28.95
No
72.6 11.75c
48.55
No
70.73 2.55c
49.87
No
19.01 5.09d
86.53
No
7.12 2.43e
94.96
No
Potato
Units
Inhibition percentage (%)
Lag phase (s)
125.05 2.9a
0
No
19.5 0.42b
84.4
215 0
147.50 6.51c
0
No
nd
nd
nd
61.27 1.38d
51.01
No
52 4.49e
58.42
No
Pear
Units
Inhibition percentage (%)
Lag phase (s)
172.05 0.21a
0
No
93.67 7.08b
45.6
95 7.07
314.05 14.35c
0
No
nd
nd
nd
28.90 7.98d
83.2
No
31 0.42d
81.98
No
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).
a,b,c,d,e
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
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.
Acknowledgments
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.
Extracts
Oxalic acid
Malic acid
Ascorbic acid
Lactic acid
Acetic acid
Citric acid
Fumaric acid
DR
P
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
0
0
6054.53 64.72
13,721.01 53.78
9.39 0.21
8.66 0.26
963
Table 4
HPLC quantication of phenols and polyphenols (mg/l) in dog rose and pomegranate extracts.
Phenols and polyphenols
DR
Pyrogallol
Gallic acid
Epigallocatechin
Catechin
-hydroxybenzoic acid
Chlorogenic acid
Epigallocatechin gallate
Caffeic acid
Epicatechin
Syringic acid
Epicatechin gallate
-coumaric acid
Ferulic acid
Benzoic acid
Ellagic acid
0
0
2433.51 80.45
124.76 1.24
485.37 12.85
574.45 12.21
0
0
37.69 0.82
17.46 0.38
230.59 3.81
12.64 0.42
46.09 0.64
0
176.91 2.28
0
0
IC50 (mg/l)
76.96 1.53
10.45 0.3
14.03 0.25
71.99 1.25
335.15 8.16
0
13.09 0.3
4.23 0.14
158.25 2.5
12.10 0.42
11.87 0.78
0
0
10.71
179.55
Reference
15.58
7.52
182.2
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