Plant Science 162 (2002) 115– 119

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Arbutin oxidation by pear (Pyrus communis L.) peroxidases

Demetrios Petkou a, Grigorios Diamantidis b,*, Miltiadis Vasilakakis a
a
Laboratory of Pomology, School of Geotechnical Sciences, Department of Agriculture, Aristotle Uni6ersity of Thessaloniki,
540 06 Thessaloniki, Greece
b
Laboratory of Agricultural Chemistry, School of Geotechnical Sciences, Department of Agriculture, Aristotle Uni6ersity of Thessaloniki,
540 06 Thessaloniki, Greece
Received 23 July 2001; received in revised form 25 September 2001; accepted 25 September 2001

Abstract

The oxidation of arbutin (hydroquinone-b-D-glucopyranoside) by soluble and ionically bound to cell wall peroxidases (EC.
1.11.1.7) from pear (Pyrus communis L.) shoots is described for the first time. Following the initiation of the reaction with
hydrogen peroxide, arbutin was transformed to a product that was detected by UV spectrophotometry. The stoichiometry of the
oxidized product formation versus hydrogen peroxide was nearly 2:1 suggesting that arbutin oxidation is a one-electron process.
Higher oxidation rates were observed in a narrow pH zone and ionically bound peroxidases showed lower apparent Km for
arbutin than soluble ones. Arbutin is considered to be an antioxidant and peroxidases from pear may influence the pro- or
antioxidant properties of this compound. © 2002 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Arbutin; Pear (Pyrus communis L.); Peroxidases; Oxidation

1. Introduction inhibit phospholipase A2 (PLA2) activity in mostly de-

Arbutin (hydroquinone-b-D-glucopyranoside) is a
natural phenolic glucoside found in various plant spe-
cies of diverse families such as Ericaceae (Vaccinium
spp., Arctostaphylos spp.), Asteraceae (Achillea mille-
folium), Betulaceae (Betula alba) and Rosaceae (Pyrus
communis L.). Arbutin is commonly used in urinary
therapeutics [1], and as a human skin-whitening agent
[2]. This latter action was attributed mainly to its
inhibitory effect on melanosomal tyrosinase activity,
rather than suppression of the expression and synthesis
of tyrosinase [3]. Arbutin is found in extremely high
concentrations in certain resurrection plants [4,5], and
as the species that accumulate it survive extreme envi-
ronmental stresses, such as frost and drought, arbutin
may contribute to their stress hardiness. The physiolog-
ical role of arbutin in resurrection plants is unknown,
but it is believed that arbutin contributes to the protec-
tion of membrane components in the dry state [6], as it
has been shown to be an antioxidant [7] and also to
* Corresponding author. Tel./fax: + 30-31-998-607.
E-mail address: diagreg@agro.auth.gr (G. Diamantidis).
hydrated systems [8].
In the past, the presence of arbutin in pear (Pyrus
communis L.) has been correlated with the biochemical
processes that operate as defense mechanisms against
bacterial invasion [9,10]. It has been suggested that the
oxidation pathway of arbutin degradation may be in-
volved in fire blight resistance of some pear varieties via
the formation of toxic substances [11]. In addition, the
degradation of arbutin has been considered as a possi-
ble cause of graft incompatibility between pear and
quince (Cydonia oblonga Mill.) [12].
Despite the number of studies concerning the possi-
ble involvement of arbutin in a number of physiological
phenomena, little is known about its metabolism and
particularly about its oxidation. Arbutin is a well-
known substrate of the enzyme b-glucosidase [8] until
present, it has been reported that arbutin is oxidized
directly by commercial polyphenoloxidases [11] and by
isolated chloroplasts of arbutin-containing plants [13].
Preliminary experiments in our laboratory showed
that among pear varieties the concentration of arbutin
in their leaves was different. In order to gain some
more information about the physiological role of ar-

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116 D. Petkou et al. / Plant Science 162 (2002) 115–119
butin we investigated the possibility arbutin to be oxi-
dized by peroxidases from the two pear cultivars
‘Tsakoniki’ and ‘Williams’. The former is the main pear
cultivar grown in Greece and also in Spain under the
name ‘Blanquilla’. It is highly compatible when grafted
onto quince rootstocks and is well adapted under the
Greek xerothermic conditions. ‘Williams’ is incompat-
ible with quince and is adapted in less xerothermic
conditions than ‘Tsakoniki’. In this study, we report for
the first time the capacity of soluble and ionically
bound cell wall peroxidases obtained from young pear
shoots to oxidize arbutin. Kinetic properties and zymo-
graphic patterns of soluble and ionically bound pear
peroxidases are also presented.
2. Materials and methods
2.1. Enzymatic extracts preparation
Actively growing shoots, approximately 2 cm long, of
two pear cultivars (Pyrus communis L., cvs. ‘Tsakoniki’
and ‘Williams’) were harvested in early spring from
trees grown at the Farm of Aristotle University of
Thessaloniki, Greece. The shoots were homogenized in
a cold pestle with a mortar in a three-fold volume of 10
mM sodium phosphate buffer (pH 6.5) containing 10%
(w/v) polyvinylpolypyrrolidone (PVPP) and 1 mM
phenylmethylsulfonyl fluoride (PMSF). The superna-
tant obtained after centrifugation (27 000g, 20 min,
4 °C) was considered as the ‘soluble fraction’ contain-
ing the so-called ‘soluble’ peroxidases.
The remaining cell material was washed twice in a
three-fold volume of Triton (0.1%) in order to remove
all the membrane debris and incubated for 12 h in 100
mM sodium phosphate buffer (pH 6.5) containing 1 M
NaCl. After centrifugation (27 000g, 20 min, 4 °C) the
obtained supernatant was concentrated and desalted in
an Amicon PM 10 ultafiltration cell (Amicon, Lexing-
ton, USA) and was considered as the ‘ionically bound
cell wall’ fraction containing the so-called ‘ionically
bound’ peroxidases.
2.2. Kinetic assays
All spectra and absorbance measurements were ob-
tained with a Shimadzu UV-1601 spectrophotometer
(Shimadzu, Kyoto, Japan) at room temperature (259
2 °C). The oxidation of arbutin, unless otherwise
noted, was determined in 1 ml reaction mixtures con-
taining 50 mM sodium phosphate (pH 6.5), 0.75 mM
arbutin, 1.3 mM H2O2 and suitable amounts of enzyme
preparation. The increase in A 234 nm against a blank
containing all the reagents except enzyme preparation
was measured over a period of 3 min, the A increase
always being linear with respect to time. The oxidation
rate was expressed as DA234 nm/min.
Peroxidase activity was determined in 3 ml reaction
mixtures containing 50 mM sodium phosphate (pH
6.5), 7.2 mM guaiacol, 13 mM H2O2 and variable
amounts of enzyme preparations. Oxidation of guaiacol
was followed by the increase of absorbance at 470 nm.
Enzyme specific activity was expressed as DA470 nm/
(min mg) protein.
2.3. Electrophoretic assays
Native PAGE was performed with a mini protean II
cell (Bio-Rad, Hercules, CA, USA) according to
Laemmli [14]. Peroxidase isozymes were visualized in
staining solutions containing 100 mM sodium phos-
phate (pH 6.5), 5.55 mM H2O2 and 0.1% o-dianisidine.
Protein concentrations were determined by the
method of Brandford [15] using bovine serum albumin
(BSA) as the standard.
3. Results
In the presence of H2O2 and an enzymatic prepara-
tion containing ionically bound cell wall proteins from
pear shoots, the difference spectrum of arbutin exhib-
ited two peaks in the UV region, at 234 and 299 nm,
respectively (Fig. 1A). The same difference spectrum of
arbutin appeared when catalytic amounts of soluble
fraction from pear shoots (Fig. 1B) or horseradish
peroxidase (not shown), instead of the ionically bound
wall fraction, were added to the reaction mixture. No
oxidation was observed in the absence of H2O2, or
enzymatic preparation, in a time scale up to 30 min.
The increase of A at 234 nm was linear to the reaction
time up to 3 min and the initial rate of the A change at
234 nm was linear to increasing volumes of enzymatic
preparation (Fig. 2).
These observations suggest that the oxidation of
arbutin must be attributed to peroxidase activities and
that the oxidation rate can be followed spectrophoto-
metrically from the increases in A at 234 nm.
Performing titration tests, it was found that the
stoichiometric ratio of the formation of the arbutin’s
oxidized product at 234 nm versus hydrogen peroxide
was approximately 2:1 (Fig. 3).
In order to characterize the peroxidative oxidation of
arbutin by pear peroxidases, the oxidation rate was
determined at different pHs and arbutin concentrations.
Soluble peroxidase activity showed a maximum oxida-
tion rate at pH 6.5, which was reduced to 90% at pH 4
and to 85% at pH 8. Ionically bound peroxidase also
showed a maximum activity at pH 6.5, which was
reduced to 83% at pH 4 but only to 60% at pH 8 (Fig.
4). Although valid Kms values cannot be defined for
oxidations catalyzed by peroxidases [16], apparent Kms
for arbutin, estimated by double reciprocal plots (Fig.
 D. Petkou et al. / Plant Science 162 (2002) 115–119 117

5A and B) were 1.6 mM for ionically bound peroxidase

and 7.1 mM for soluble peroxidase.
Table 1 shows the guaiacol peroxidase activities and
arbutin peroxidase activities in the soluble and ionically
bound fractions from shoots of two pear cultivars.
Considering as total arbutin peroxidase activity the sum
of activities measured in both fractions, cv. ‘Tsakoniki’
showed 16.4% of activity in the soluble fraction and
83.6% in the ionically bound fraction. Cv. ‘Williams’
showed 24.8% of arbutin peroxidase activity in the
soluble fraction and 75.2% of activity in the ionically
bound fraction. Peroxidase activity, estimated as guaia-
col oxidation, was found to be 11.5% of total activity in
the soluble fraction and 88.5% in the ionically bound
fraction for cv. ‘Tsakoniki’ and 30.7% in the soluble
and 69.3% in the ionically bound fraction for cv. Fig. 2. Effect of enzymatic preparation (ionically bound fraction cv.
‘Williams’. ‘Tsakoniki’) on arbutin oxidation. The reaction mixtures contained
50 mM sodium phosphate (pH 6.5), 0.75 mM arbutin and 1.3 mM
Fig. 6 shows the band patterns of anionic peroxidases
H2O2. The inset shows time course of A increase at 234 nm in
in the soluble and ionically bound fractions of cv. reaction mixtures containing 50 mM sodium phosphate (pH 6.5), 0.75
‘Williams’. Two anionic isozymes (A1, A2) were visual- mM arbutin, 3 mg protein of ionically bound fraction (cv.
ized in soluble fraction and one (A2) in the ionically ‘Tsakoniki’) and 1.3 mM H2O2.

bound fraction. The same zymograms obtained for

different quantity of loaded protein up to 345 mg and
for extended incubation period of the gel in the sub-
strate solution up to 90 min (data not shown).

4. Discussion

Spectrophotometric analysis in the UV spectrum

showed that arbutin was oxidized by pear’s young
shoot peroxidases present in soluble and ionically
bound cell wall fractions. Soluble and ionically bound
peroxidases showed almost identical pH profiles for

Fig. 1. Difference spectra of peroxidase-dependent oxidation of ar-

butin. The reaction mixtures (1 ml) in the two cuvettes contained 50
mM sodium phosphate (pH 6.5), 0.75 mM arbutin and 15 mg protein
of soluble fraction (A) (cv. ‘Tsakoniki’) or 30 mg protein of ionically
bound fraction (B) (cv. ‘Tsakoniki’). At zero time 1.3 mM H2O2 were
added to one cuvette (in the other the same volume of buffer) and
scanning was started after 2 min (1), 4 min (2) and 9 min (3). No A
changes were observed in the absence of H2O2, or enzymatic prepara-
tion in a time scale up to 30 min.
Fig. 3. Stoichiometry for oxidation of arbutin by H2O2. The reaction
mixture (1 ml) contained 0.05 or 0.1 mM arbutin, 2 mg protein of
ionically bound fraction (cv. ‘Tsakoniki’) and 50 mM sodium phos-
phate (pH 6.5). Reactions were allowed to proceed to the endpoint
and the extent of absorbance changes at 234 nm was plotted as a
function of H2O2 concentration. Data points represent mean 9 SE
(n = 3).
118 D. Petkou et al. / Plant Science 162 (2002) 115–119
Fig. 4. Dependence of the arbutin oxidation rate on the pH. The
reaction mixture (1 ml) contained 8 mg protein of soluble fraction or
4 mg of ionically bound fraction (cv. ‘Tsakoniki’), 0.75 mM arbutin
and 1.3 mM H2O2. Buffers used were 100 mM citric acid –sodium
phosphate (range 3.5 – 5.5), sodium phosphate (range 6.5 –7.5) and
Tris–HCl (range 8.0 – 8.5). Oxidation rates are expressed as DA(234
nm)/min. Data points represent mean 9 SE (n= 3).
arbutin oxidation and higher oxidation rates were ob-
served in a narrow pH zone. On the other hand, from
the estimation of Km values it was concluded that
ionically bound peroxidases showed higher substrate
specificity for arbutin than soluble peroxidases. Further
Fig. 5. Double reciprocal plots of arbutin oxidation rate vs. arbutin
concentration. The reaction mixture (1 ml) contained 50 mM sodium
phosphate (pH 6.5), 30 ml of ionically bound (A) or soluble fraction
(B) (cv. ‘Tsakoniki’) and 1.3 mM H2O2.
studies would clarify if the ionically bound peroxidase
that oxidizes arbutin is different from the soluble one.
The observed ratio of arbutin oxidized product at 234
nm versus hydrogen peroxide was nearly 2:1, indicating
that arbutin oxidation is a one-electron process. A ratio
of 2:1 is expected for compounds that donate one
electron to the peroxidase since the enzyme requires
two reducing equivalents to return to the resting state.
This has been observed for the peroxidative oxidations
of eugenol (4-allyl-2-methoxyphenol), phenol, serotonin
and estradiol [17].
Differences in arbutin oxidation specific activity be-
tween soluble and ionically bound peroxidase followed
differences in guaiacol peroxidase activity in both pear
cultivars tested. The results shown in Table 1 and the
banding patterns of soluble and ionically bound perox-
idases (Fig. 6), suggest that the isozyme that oxidizes
arbutin will oxidize guaiacol as well, and that this
isozyme is probably A2. In contrast, subsequent experi-
ments showed that crude barks extracts of cvs.
‘Williams’ and ‘Tsakoniki’, where A2, was present and
visualized after PAGE by the use of guaiacol as the
electron donor, were not able to oxidize arbutin. Fur-
ther work is needed in order to clarify whether the
isozyme that oxidizes arbutin is A2 and whether it can
be detected in polyacrylamide gels by the use of stain-
ing solutions commonly used for the detection of per-
oxidase isozymes.
It was shown previously that arbutin can act as an
antioxidant [7] and that this property could be related
to the accumulation of arbutin to high concentration in
drought and frost resistant plants. Arbutin can inhibit
membrane lysis, both free radical-mediated and enzy-
matic in nature, and it has been suggested that arbutin
might contribute to membrane stabilization in these
plants. Recently, it has been shown that the interaction
between arbutin and lipid membranes, and the resulting
effects on membrane stability, depends, in a complex
manner, on the lipid composition of membrane [6]. Is
arbutin involved in membrane stability of pear cells as
proposed for the resurrection plants, or is it involved
and in other biological processes?
Although there are many assays for determining the
antioxidant activity of a phenolic compound, under our
assay conditions, arbutin was capable of inhibiting the
reduction of nitroblue tetrazolium (NBT) by superoxide
anion (O2− ), and of inhibiting the hemoglobin-catalyzed
peroxidation of linoleic acid (not shown). Antioxidant
properties of phenols are highly dependent on their
structure [18], but the critical determination whether
pro- or antioxidative properties are expressed, depends
largely on local concentrations and metabolic balances,
such as the individual reduction charge in the cell
compartment, or the availability of free transition
metals [19]. It has been shown previously that some
flavonoids metabolized by peroxidases form pro-oxi-
 D. Petkou et al. / Plant Science 162 (2002) 115–119
Table 1
Specific peroxidative activity of soluble and ionically bound fractions from barks of two pear cultivars
Cultivar Soluble fraction
Guaiacol oxidationa Arbutin oxidationb
Tsakoniki 0.979 0.02 1.1790.06
Williams 4.379 0.06 2.76 9 0.35
Values are mean 9 SE (n= 3).
a
Expressed as dA(470nm)/(min mg) protein.
b
Expressed as dA(234nm)/(min mg) protein.
Fig. 6. Band patterns of soluble (S) and ionically bound (I.B.)
peroxidases of cv. ‘Williams’. Peroxidase activity was visualized by
o-dianisidine. At each lane 231 ng of protein was loaded.
dant aryloxy radicals that cause extensive oxygen acti-
vation in the presence of NADH [20]. Therefore, pear
peroxidase isozymes that oxidize arbutin are likely to
determine the expression of pro- or antioxidant proper-
ties of arbutin at the early stages of growth
development.
Arbutin is found in several plant species, but its
physiological role in them still remains unknown. The
complete study of the peroxidative oxidation of arbutin
will elucidate the impacts, if any, of such reactions on
the role of arbutin in a number of physiological phe-
nomena, such as frost and drought resistance, defense
mechanisms against bacterial invasion and graft incom-
patibility. Moreover, arbutin is commonly used in com-
mercial whitening preparations and therefore oxidation
of arbutin by various classes of peroxidases should be
examined in order to characterize the safety of these
products.
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