Kinetic mechanism and product characterization of the enzymatic peroxidation of pterostilbene as model of the detoxification process of stilbene-type phytoalexins

Article abstract of DOI:10.1016/j.phytochem.2010.10.009

The enzymatic peroxidation of pterostilbene, a strong antifungal belonging to the stilbene family, by peroxidase (POX), is reported for the first time as a model of phytoalexin detoxification carried out by the enzymatic pool of pathogens. Kinetic charact

Full text of DOI:10.1016/j.phytochem.2010.10.009

Phytochemistry 72 (2011) 100–108
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Kinetic mechanism and product characterization of the enzymatic peroxidation of
pterostilbene as model of the detoxification process of stilbene-type phytoalexins
⇑
Pilar Rodríguez-Bonilla, Lorena Méndez-Cazorla, José Manuel López-Nicolás , Francisco García-Carmona
Department of Biochemistry and Molecular Biology-A, Faculty of Biology, University of Murcia, Campus de Espinardo, 30071 Murcia, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 July 2010
Received in revised form 14 October 2010
The enzymatic peroxidation of pterostilbene, a strong antifungal belonging to the stilbene family, by per-
oxidase (POX), is reported for the first time as a model of phytoalexin detoxification carried out by the
enzymatic pool of pathogens. Kinetic characterization of the pterostilbene oxidation reaction pointed
to an optimum pH of 7.0, at which value the thermal stability of POX was studied. Moreover, the data
showed that pterostilbene inhibits POX activity at high concentrations of substrate. Several kinetic
parameters, including Vmax, Km and KI, were calculated and values of 0.16
31.41 M were reported. To understand the possible physiological role of this reaction in the phytoalexin
D lM, and
Abs minꢀ1, 14.61
Keywords:
Pterostilbene
Peroxidase
l
detoxification process, the products of pterostilbene oxidation were identified using HPLC-MS and a rad-
ical–radical coupling reaction mechanism was proposed. Three main products with a high molecular
weight and pronounced hydrophobicity were identified: pterostilbene cis dehydromer, pterostilbene
trans dehydromer and pterostilbene open dimer. The dimeric structures of these molecules indicate that
the pterostilbene oxidation reaction took place at the 40-OH position of the hydroxystilbenic moieties and
the three above mentioned dimeric products were found, due to the ability of electron-delocalized rad-
icals to couple at various sites. Finally, the capacity of cyclodextrins (CDs) as starch model molecules in
plants to complex both the substrate and the products of the oxidation reaction was evaluated. The inhi-
bition process of POX activity was modified at high pterostilbene concentrations due to sequestering of
the substrate reaction and to the different affinity of the reaction products for CDs.
Phytoalexin
Cyclodextrin
Detoxification
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
Pont, 1988). It also appears to be a constituent of the bark of
Guibourtia tessmanii, a tree found in central Africa and is commonly
In response to infection by agents such as fungus, some plants
can synthesize different antifungal compounds belonging to the
phytoalexins family like resveratrol, piceid or some viniferins
(Wilkens et al., 2010). Although a great number of authors have
focused on the study of these three compounds belonging to the
stilbenes family, the most important phytoalexins, in recent years
the importance of pterostilbene (trans-3,5-dimethoxy-40-hydroxy-
stilbene), another stilbene-type phytoalexin, has been reported in
different papers as a potent antifungal compound (e.g., Breuil
et al., 1999). Pterostilbene, a naturally occurring phytoalexin
belonging to a group of phenolic compounds known as stilbenes,
has been identified in several plant species such as the heartwood
of sandalwood (Sehadri, 1972), leaves of Vitis vinifera (Langcake
et al., 1979), peanuts (Medina-Bolivar et al., 2007), infected grape
berries of var. Chardonnay and Gamay (Adrian et al., 2000), healthy
and immature berries of var. Pinot Noir and Gamay (Pezet and
Pont, 1988) and berries of some Vacciunium species (Pezet and
used in folk medicine (Fuendjiep et al., 2002). In addition to its
antifungal role (Pezet and Pont, 1990; Mazullo et al., 2000), ptero-
stilbene presents other biological activities, including antihyper-
glycemic (Manickam et al., 1997), antioxidative (Rimando et al.,
2002; Kathryn et al., 2006; Remsberg et al., 2008), anticancer
(Rimando et al., 2005; Kathryn et al., 2006; Remsberg et al.,
2008; Pan et al., 2007; Nanjoo et al., 2007; Ferrer et al., 2005), anti-
inflammatory (Remsberg et al., 2008), anticholesterol (Mizuno
et al., 2008; Mazullo et al., 2000), hypolipidemic (Mazullo et al.,
2000) and analgesic (Mazullo et al., 2000) activities.
It has already been reported that the pathogenicity of fungi like
Botrytis cinerea or Trabetes pubescens is strongly associated with
their ability to degrade stilbene phytoalexins such as pterostilbene
or resveratrol (Ponzoni et al., 2007). These observations suggest
that the enzymes of these pathogens attack the phytoalexins which
represent the first line of the defence of the plant. Indeed, this type
of fungus has an enzymatic pool able, for example, to lyse cell walls
or to oxidize polyphenols (Gil-ad et al., 2000). Subsequently, direct
evidence was obtained that resveratrol and pterostilbene can un-
dergo degradation by enzymes such as the laccase produced by
B. cinerea (Sbaghi et al., 1996).
⇑
Corresponding author. Tel.: +34 868884777; fax: +34 868364765.
E-mail address: josemln@um.es (J.M. López-Nicolás).
0031-9422/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2010.10.009

P. Rodríguez-Bonilla et al. / Phytochemistry 72 (2011) 100–108
101
2.5
Although most works published concerning the detoxification
of phytoalexins by the enzymes present in B. cinerea only focus
on the role of laccase-like stilbene oxidase produced by this fungus
(Breuil et al., 1999; Ponzoni et al., 2007; Gil-ad et al., 2000), Gil-ad
et al. (2000) showed that B. cinerea has a peroxidase (POX) (E.C.
1.11.1.7) enzymatic activity not reported previously. However, no
work has reported, as is done for first time in this paper, on
whether the enzymatic peroxidation of pterostilbene is involved
in the detoxification of phytoalexins. Thus, although POX catalyzes
the oxidation of a wide variety of substrates, the potential oxida-
tion of pterostilbene by this oxidative enzyme as a model of phyto-
alexin degradation by B. cinerea has not been reported.
As occurs with the detoxification of other phytoalexins carried
out by laccase (Ponzoni et al., 2007; Sbaghi et al., 1996; Pezet,
1998; Breuil et al., 1998, 1999), a knowledge of both the mecha-
nism by which POX acts on pterostilbene and the nature of the oxi-
dation reaction products, is essential for understanding the
possible physiological role of this reaction in the phytoalexin
detoxification process. Indeed, metabolism of phytoalexins by B.
cinerea or T. pubescens may manifest itself as an insolubilization
of the products formed when different substrates are used. The
fungus could, therefore, avoid the action of phytoalexins such as
pterostilbene by transforming these compounds into other prod-
ucts of increasing molecular weight and hydrophobicity. Moreover,
in the case of another phytoalexin-type stilbenes such as resvera-
trol, Dercks and Creasy (1989) stated that e-viniferin, a dimeric
form of resveratrol analogous to the dimer produced by B. cinerea
(Breuil et al., 1998), is less stable (soluble) in water than resveratrol
itself, its biological activity against B. cinerea being lower. Similar
results were found when pterostilbene was incubated in the pres-
ence of laccase from B. cinerea. These observations demonstrate the
interest of indentifying the reaction products and underline the
involvement of the enzyme-mediated oxidation of stilbenes in
the pathogen-plant interaction.
Bearing the above in mind, and in order to study the possible
role of POX in the detoxification of antifungal compounds and, con-
sequently, in the pathogenesis of the organisms which contain POX
activity, the four main objectives of this work were to: (i) to deter-
mine the enzymatic kinetic parameters of the oxidation of ptero-
stilbene by POX, (ii) to identify the oxidation reaction products,
(iii) to propose a reaction mechanism to justify the results ob-
tained, (iv) to evaluate the use of cyclodextrins (CDs) as modula-
tors of both the enzymatic activity and the oxidation products.
To perform the study, UV–VIS method which makes use of
changes in the spectrophotometric parameters of pterostilbene in
the presence of POX and an HPLC-MS tandem to identify the oxida-
tion products in the absence or presence of CDs were used. More-
over, horseradish POX, the most studied POX type, was used as an
enzyme model for oxidizing pterostilbene.
2.0
1.5
1.0
0.5
0.0
250
275
300
325
350
375
400
Wavelength (nm)
Fig. 1. Evolution of the spectrum of the peroxidation of pterostilbene by POX in the
presence of H2O2. The reaction medium at 25 °C contained 20 M of pterostilbene
in the presence of 114 M H2O2 at pH 7.0. The scans were carried out every 0.5 min.
l
l
reaction medium when pterostilbene was incubated with POX and
H2O2 pointed to maximal spectral changes in the 250–400 nm re-
gion, with a decrease in pterostilbene absorbance at 310 nm and
an increase at 360 nm. Since the spectral changes observed were
not detectable in the absence of H2O2, or in the absence of enzyme
(data not shown), they were considered to be the result of POX
activity. In addition, the dependence of these spectral changes on
enzyme concentration confirms that the changes in the UV spec-
trum are a reliable measure of the enzymatic oxidation rate.
Finally, the nature of the spectral changes of pterostilbene with
time at 250–400 nm (Fig. 1) indicated that the formation of ptero-
stilbene oxidation products was proportional to time during the
first 11 min of the reaction. Moreover, the presence of two isos-
bestic points at 268 and 342 nm in the consecutive spectra of the
reaction medium suggests two things: (i) the presence of one oxi-
dation product formed during the course of pterostilbene oxidation
by POX or (ii) the presence of several oxidation products with a
constant stoichiometry during the time of the reaction. As will be
demonstrated in following sections, the oxidation of pterostilbene
by POX provides a mixture of products with a constant stoichiom-
etry during the reaction.
2.2. Determination of the optimum physico-chemical conditions for
studying the oxidation of pterostilbene by peroxidase
To ascertain the best physico-chemical conditions for kineti-
cally characterizing the oxidation of pterostilbene by POX, the next
step of this investigation was to study both the optimum pH and
the thermal stability of the reaction. Reports in the literature de-
scribe how the optimum pH of any POX depends on different fac-
tors, such as the H-donor in the activity assay (Halpin et al., 1989),
the substrate used (Chisari et al., 2007, 2008) or the POX source
(Duarte-Vázquez et al., 2000). In the present study, the influence
of the incubation medium pH on the rate of pterostilbene oxidation
by POX is demonstrated (Fig. 2A). It was concluded that the rate of
oxidation is strongly pH-dependent. As can be seen, the enzymatic
activity showed an optimum at pH 7.0, above which it fell sharply.
The pH profile depicted in Fig. 2A, with an optimum at pH 7.0 and a
strong decrease in the region of basic pH, agrees with the observa-
tions of several authors (O’Brien, 2000). For these reasons, the opti-
mum pH value selected for studying the oxidation of pterostilbene
by POX was 7.0.
2. Results and discussion
2.1. Scanning spectrophotometric studies of the oxidation of
pterostilbene by POX
Although many studies have focused on POX and its capacity to
metabolize new substrates (O’Brien, 2000), no work has reported
on the potential oxidation of the potent antifungal pterostilbene
by this enzyme. For this reason, the first step in this investigation
was to monitor the possible oxidation of pterostilbene by POX,
observing the changes that occur in the UV spectrum of this phyto-
alexin with time.
The pterostilbene spectrum shows a single absorption band at
around 300 nm with a bandwidth of 20 nm and two small maxima
centred at 304 and 316 nm (Fig. 1). The changes in absorbance of a
To continue the identification of the optimum physico-chemical
parameters for studying the oxidation of pterostilbene by POX, the

102
P. Rodríguez-Bonilla et al. / Phytochemistry 72 (2011) 100–108
0.06
0.05
0.04
0.03
0.02
0.01
0.00
(A)
0.20
(A)
0.15
0.10
0.05
0.00
0
2000
4000
6000
2O2] (μM)
8000
10000
3
4
5
6
7
pH
8
9
10
11
[H
0.08
0.06
0.04
0.02
0.00
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
(B)
(B)
0
50
100
[Pterostilbene
150
200
20
40
60
80
100
] (μM)
Temperature (ºC)
Fig. 3. (A) Effect of H2O2 concentration on the peroxidation of perostilbene by POX.
The reaction medium at 25 °C contained 0.1 M phosphate buffer pH 7.0, 8
pterostilbene and increasing concentrations of H2O2 from 0 to 10 mM. (B) Effect of
pterostilbene concentration on POX activity in the presence of 114 lM H2O2 at pH
7.0.
Fig. 2. (A) Effect of pH on the peroxidation of pterostilbene by POX. The reaction
medium consisted of 8 M pterostilbene, 114 M H2O2 at different pH values at
25 °C. (B) Thermal stability of POX on pterostilbene at different temperatures for
l
M
l
l
40 min. The reaction medium consisted of 8
lM pterostilbene, 114 lM H2O2 at pH
7.0.
enzyme catalyzed-reaction, evaluating the changes in the oxidation
rate with changes in pterostilbene concentration.
As can see in Fig. 3B, the dependence of the pterostilbene oxida-
tion rate on a variation in the pterostilbene concentration was
complex. Thus, although this parameter showed a Michaelis–
Menten type kinetic at pterostilbene concentrations of less than
next step was to evaluate the thermal stability of POX at increasing
temperatures from 15 to 90 °C. For this, the enzyme solutions were
incubated at ten temperatures for 40 min and, after heating, the
samples were assayed at 25 °C. As can be seen in Fig. 2B, a strong
dependence of the oxidation rate of pterostilbene by POX on the
incubation temperature was observed, being stable between 15
and 50 °C and strongly decreasing above 50 °C. As a consequence
of these results, 25 °C was selected as the optimum temperature
for the next sections.
Finally, as shown in Fig. 3A, pterostilbene oxidation was also
seen to be dependent on the concentration of H2O2, being inhib-
ited at high concentrations. These results are in good agreement
with those described for asparagus and turnip peroxidases
(Duarte-Vázquez et al., 2000; Rodrigo et al., 1996), and so a low
27 lM, it showed strong inhibition at higher concentrations. For
this reason, Eq. (1), which calculates the main kinetic parameters
of an enzymatic reaction when an inhibition of the oxidation rate
at high concentrations of substrate is observed, was used.
mmax½Sꢁ
v
¼
ð1Þ
ðKm þ ½Sꢁ ꢂ ð1 þ ½Sꢁ=KIÞÞ
where Vmax is the maximum enzyme velocity, Km is the Michaelis–
Menten constant and KI is the constant for substrate inhibition.
Fitting the experimental data to Eq. (1), the kinetic constants
H2O2 concentration of 114
the investigation.
lM was selected for the next steps of
Vmax, Km and KI were calculated and values of 0.16
14.61 M, and 31.41 M were obtained.
D ,
Abs minꢀ1
l
l
2.3. Study of the inhibition of POX activity at high pterostilbene
concentrations
2.4. Characterization of the reaction products of the oxidation of
pterostilbene by peroxidase
The results obtained in the previous sections show that the
optimum reaction conditions for characterizing the oxidation of
pterostilbene by POX are as follows: (i) pH: 7.0, (ii) temperature:
Characterization of the pterostilbene metabolites allowed us to
understand more about phytoalexin metabolism by different pathogens
with POX activity and the relation with fungal pathogenicity. As
25 °C, and (iii) H2O2 concentration of 114
lM. These conditions
were selected to calculate the main kinetic parameters of the

P. Rodríguez-Bonilla et al. / Phytochemistry 72 (2011) 100–108
103
mentioned above, the production of phytoalexins is considered
HO
to form part of a general defence mechanism of plants. In this
species, such a response includes the formation, mediated by
laccase enzyme, of a range of biosynthetically related di- and
oligomers of different stilbenes. For this reason, the next step of
our investigation was to identify the products of pterostilbene
oxidation by POX.
The products formed upon incubation of pterostilbene with POX
at pH 7.0 were analyzed by HPLC-MS. As shown in Fig. 4, the HPLC
chromatogram resulted in the presence of three reaction products
(peaks A–C).
O
H
a
H CO
3
OCH
3
Hb
OCH
OCH
3
3
(A)
HO
The mass analysis of a sample of this less polar product (Fig. 4A
inset) showed a quasi-molecular ion of 511 Da and a molecular for-
mula C32H30O6. Identical results were reported for the oxidation of
pterostilbene by B. cinerea laccase (Breuil et al., 1999) and for the
incubation of pterostilbene with the T. pubescens laccase (Ponzoni
et al., 2007). For this reason, peak A shown in Fig. 4 is consistent
with the structure of a pterostilbene trans dehydromer presented
in Fig. 5A. Moreover, mass analysis of peak B (Fig. 4) showed an
identical quasi-molecular ion of 511 Da and a molecular formula
O
H CO
3
H
H
b
a
H CO
3
OCH
3
C
32H30O6. According to the results reported by Breuil et al.
OCH
(1999) for the oxidation of pterostilbene by laccase, this fact would
be due to the existence of a photochemical isomerization of the
product presented in Fig. 5B, which is a typical reaction for trans-
stilbenes. For this reason, although pterostilbene degradation to a
major compound that was in the trans ethylenic form (Fig. 5A),
the HPLC chromatogram shown in Fig. 4 also presented (as second-
ary product) a pterostilbene cis dehydromer (Fig. 5B) with an iden-
tical molecular ion and molecular formula.
3
(B)
OH
O
H CO
3
OH
To confirm that the structures of peaks A and B correspond to
pterostilbene trans dehydromer and pterostilbene cis dehydromer,
respectively, the UV spectrum from 240 to 360 nm of these mole-
cules are showed (Fig. 6). As can be seen in Fig. 6A, the UV spec-
H CO
3
OCH
3
OCH
3
(C)
trum of pterostilbene trans dehydromer presents
absorption band around 300 nm with a bandwidth of 20 nm and
two small maxima at 309 and 325 nm. This type of spectrum is
a single
Fig. 5. Reation’s products of the pterostilbene oxidation reaction products by POX.
(A) pterostilbene trans dehydromer, (B) pterostilbene cis dehydromer and (C)
pterostilbene open dimer.
Fig. 4. Reversed-phase HPLC analysis of pterostilbene oxidation products by POX. (A) trans dehydromer pterostilbene; (B) cis dehydromer pterostilbene and (C) pterostilbene
open dimer. The eluent was methanol/water (80:20 v/v). Inset: mass spectrum of A and C pterostilbene oxidation products.

104
P. Rodríguez-Bonilla et al. / Phytochemistry 72 (2011) 100–108
which may frequently produce a complex mixture of polyphenolic
oligomers.
As regards our investigation, the dimeric structures indicate
(A)
20
15
10
5
that the pterostilbene oxidation reaction took place at the 40-OH
(4-OH) position of the hydroxystilbenic moieties. Due to the capa-
bility of electron-delocalized radicals to couple at various sites, dif-
ferent dimeric products were found, depending on the structural
features of the starting substrates. In turn, the phenoxy radicals
formed could delocalize as reported in Fig. 7.
Successively, the coupling of one radical ‘‘B’’ and one radical ‘‘C’’,
followed by tautomeric rearrangement and intramolecular nucleo-
philic attack on the intermediate quinone, produced both cis and
trans dihydrofuran pterostilbene dehydromers identified as the
two least polar isomeric products in the HPLC chromatogram
shown in Fig. 4 (peaks A and B).
In turn, the formation of the ‘‘open’’ hydroxylated (Fig. 4 peak
C), the most polar of the reaction products, can be easily explained
by the coupling of one radical ‘‘A’’ with one radical ‘‘C’’, followed by
addition of a water molecule to the intermediate quinones.
The results of our experiments clearly supports our hypothesis
that the reaction proceeded through radical–radical coupling and
not through radical addition to the double bond of a non-radical
substrate. These results are in accordance with most of the litera-
ture reports (Ponzoni et al., 2007) and not with the hypothesis sug-
gested by Szewczuk et al. (2005), who proposed a mechanism in
which the another stilbene-type molecules dimerization occurs
via radical attack on a second molecule of stilbene.
0
3
(B)
2
1
0
2.6. Effect of the presence of cyclodextrins on the rate of pterostilbene
oxidation by peroxidase
240
260
280
300
320
340
360
Wavelength (nm)
Different studies have demonstrated that cyclodextrins (CDs)
can modify the enzymatic activity of an oxidorreductase enzyme,
such as LOX, when a stilbene is used as substrate (López-Nicolás
et al., 2009a). As regards the physiological role in plants, several
works have reported that the oxidation of some antioxidant com-
pounds forming inclusion complexes with CD by oxidative en-
zymes can be regarded as a model system of their oxidation in
storage tissues complexed with starch (López-Nicolás et al.,
1997). For these reasons, and in order to evaluate the effect of
CDs on the structure and availability of this potent antifungal com-
pound in the enzymatic reaction, the next step was to evaluate the
response of POX to the presence of CDs in the reaction medium.
CDs are torus-shaped oligosaccharides made up of 6–8 gluco-
pyranose units and originated by the enzymatic degradation of
starch through the action of CD-glucano-transferase (Szente and
Szejtli, 2004). Poorly water-soluble compounds and hydrophobic
moieties of amphiphilic molecules interact non-covalently with
the CD cavity to form so-called inclusion complexes, which are also
highly water-soluble. Several publications have reported the
aggregation behaviour of different enzymatic substrates such as
fatty acids, phenols, and stilbenes, in the presence of CD, and evi-
dence has been presented concerning the formation of guest/CD
inclusion complexes with several stoichiometries (Bru et al.,
1995; López-Nicolás et al., 1997, 2009a,b,c). Indeed, our group re-
cently reported the formation of pterostilbene/CD complexes using
fluorescence techniques (López-Nicolás et al., 2009b).
Fig. 6. (A) UV spectrum of pterostilbene trans dehydromer; (B) UV spectrum of
pterostilbene cis dehydromer.
characteristic of the molecules belonging to the stilbene family
which present a trans configuration as it is cited in different papers
(Abert Vian et al., 2005; López-Nicolás et al., 2009a). However, the
UV spectrum of molecules belonging to the stilbene family which
present a cis configuration shows a single absorption band around
285 nm but with a bandwidth of few nm (Abert Vian et al., 2005)
identical to the spectrum presented in Fig. 6B. These data confirm
again the structures of pterostilbene trans dehydromer and ptero-
stilbene cis dehydromer.
Finally, the HPLC study of the oxidation of pterostilbene by POX
showed a third peak (C). The mass spectrum of the most polar
product had a quasi-molecular ion of 529 Da (Fig. 4C inset), that
is 18 amu more than the value obtained with the other products,
and C32H31O7 the formula molecular. This product was identified
as an pterostilbene open dimer with two 3,5-dimethoxybenzoyl
moieties (Fig. 5C) by Ponzoni et al. (2007) during the oxidation of
pterostilbene by T. pubescens laccase.
2.5. Reaction mechanism of the oxidation of pterostilbene by
peroxidase
On the basis of the results obtained, a mechanism for the syn-
thesis of the different dimeric derivatives can be proposed. As sta-
ted above, phenols are among the most suitable POX substrates
(O’Brien, 2000). Oxidations proceed via the formation of radical
cations and the subsequent deprotonation of the phenolic hydroxy
groups to give phenoxy radicals, which can undergo a broad vari-
ety of coupling reactions (Ponzoni et al., 2007). The main drawback
of these biotransformations is the extensive polymerization that
may occurs due to the radical mechanism of the oxidative process,
Fig. 8A shows the Hydroxy-propyl-b-CD dependence of POX at
pH 7.0 when three pterostilbene concentrations were used. As
can be seen, the addition of increasing concentrations of HP-b-CD
produced a change in the POX enzymatic activity that depended
on the pterostilbene concentration used. In the presence of the
24 lM pterostilbene in the reaction medium, the POX activity de-
creased when HP-b-CD concentration was increased (Fig. 8A filled
circle). This typical behaviour is due to the ability of both natural
and modified CDs to sequester part of the pterostilbene to form

P. Rodríguez-Bonilla et al. / Phytochemistry 72 (2011) 100–108
105
OH
O
H3CO
OH
Pterostilbene open dimer
H3CO
OCH3
O
OCH3
O
H
O
H
CO
H
3
H
CO
OCH
3
3
OCH
3
O
OH
O
O
H
CO
H
CO
3
H3CO
3
H
CO
3
POX
OCH
OCH3
OCH
3
OCH
3
3
pterostilbene
radical A
radical B
radical C
O
H
CO
O
3
H
H
CO
3
OCH
3
H
CO
3
O
H
H
CO
O
3
H
CO
3
OCH
3
H CO
3
HO
CO
HO
O
O
H
3
H
CO
3
H
H
b
a
H
CO
3
Isomerisation
OCH
3
OCH
OCH
3
3
OCH
3
H
CO
3
Pterostilbene cis dehydromer
Pterostilbene trans dehydromer
Fig. 7. Radical–radical coupling reaction mechanism proposed for the synthesis of pterostilbene trans dehydromer, pterostilbene cis dehydromer and pterostilbene open
dimmer.
soluble inclusion complexes (López-Nicolás et al., 2009b), thereby
reducing the concentration of the free pterostilbene. Thus, CDs act
as substrate reservoir in a dose-dependent manner. Indeed, free
pterostilbene is the only effective substrate and the oxidation of
the complexed substrate requires the prior dissociation of the
complex.
Moreover, when a pterostilbene concentration of 47 lM was
used, the POX activity practically remained constant when HP-b-CD
concentrations below of 0.5 mM were tested. However, when
the HP-b-CD concentration was above 0.5 mM, the POX activity
decreased, as occurred at the pterostilbene concentration of
24 lM (Fig. 8A filled squares).

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P. Rodríguez-Bonilla et al. / Phytochemistry 72 (2011) 100–108
2.7. Effect of the presence of cyclodextrins on the stoichiometry of the
pterostilbene oxidation products by POX
As shown in the previous sections, the oxidation of pterostil-
bene by POX produced three main products, which were identified
as pterostilbene cis dehydromer, pterostilbene trans dehydromer
and pterostilbene open dimer. Although CDs have usually been
used for complexing the substrates of several enzymatic reactions,
due to the importance of the pterostilbene oxidation products in
the detoxification of phytoalexins, we studied the potential effect
of the addition of CDs on the three main products of the oxidation
of pterostilbene by POX.
Although in both the absence and presence of HP-b-CD, the sum
of the three reaction products concentration remained constant
(Fig. 9 filled diamonds), the presence of increasing concentrations
of HP-b-CD had different effects on the concentration of the three
reaction products determined by HPLC. For example, the area of
the pterostilbene trans dehydromer decreased when increasing
concentrations of HP-b-CD were added to the reaction medium
(Fig. 9 filled circles), pointing to a significant interaction between
this main reaction product and HP-b-CD. Moreover, the area of
pterostilbene cis dehydromer increased (Fig. 9 filled squares) with
increasing HP-b-CD concentrations due to the capacity of HP-b-CD
to modify the isomerization equilibrium shown in Fig. 10. Thus,
pterostilbene cis dehydromer presents higher affinity for HP-b-CD
than the observed for pterostilbene trans dehydromer, and so a
higher complexation constant (Kcis > Ktrans). Finally, no significant
variation in the area of pterostilbene open dimer was found when
HP-b-CD was added to the reaction medium (Fig. 9 filled triangles),
showing that this third pterostilbene oxidation product by POX
cannot been complexed by HP-b-CD.
Although further investigations are necessary to determine the
biological activity of the three pterostilbene oxidation products
identified in this work, the fact that HP-b-CDs, complexant agents
defined as starch model molecules in plants (López-Nicolás et al.,
1997), can form inclusion complex with both pterostilbene cis
dehydromer and pterostilbene trans dehydromer pterostilbene
may be used in the detoxification process of stilbene-type phytoal-
exins. As mentioned above, an increase in the solubility of the
formed products is related with the detoxification process. Thus,
metabolism of phytoalexins by the enzymes present in pathogens
may manifest itself as an insolubization of the products formed.
Fig. 8. (A) Effect of HP-b-CD concentrations on the reaction rate of POX-catalyzed
pterostilbene oxidation at different substrate concentrations: (d) 24
47 M, (N) 140 M. The reaction medium at 25 °C contained increasing HP-b-CD
concentrations, 114 M H2O2 in 0.1 M sodium phosphate buffer pH 7.0. (B) Effect of
lM, (j)
l
l
l
the addition of different HP-b-CD concentrations on peroxidation of pterostilbene
by POX: (d) no agent, (j) HP-b-CD 0.5 mM, (N) HP-b-CD 1.0 mM. The reaction
medium at 25 °C contained increasing pterostilbene concentrations, 114 lM H2O2
in 0.1 M sodium phosphate buffer pH 7.0.
Finally, Fig. 8A shows that when a high substrate concentration
of 140 M was used, increasing HP-b-CD concentrations up to
l
1 mM produced a sharp increase in POX activity, which decreased
again when the concentration of the complexation agent exceeded
1 mM (Fig. 8A filled triangles).
Since of this different pterostilbene concentration behaviour of
POX activity in the presence of HP-b-CD has not been previously
reported, we studied the possible effect of CDs on the inhibition
of POX activity by the high pterostilbene concentrations evaluated
in previous sections.
In Fig. 8B it can be seen that when increasing HP-b-CD concen-
trations (0, 0.5 and 1 mM) were added to the reaction medium, the
previously observed inhibition by substrate excess occurred at a
higher pterostilbene concentration than observed in the absence
of any agent. Thus, the concentration of pterostilbene at which
the POX activity was inhibited in the absence of HP-b-CD increased
in its presence, reflecting the formation of inclusion complexes and
extending the range in which the pterostilbene concentration does
not produce inhibition of the enzymatic activity. These results ex-
plain the different behaviour of POX activity dependent of ptero-
stilbene concentration shown in Fig. 8A.
Fig. 9. Effect of HP-b-CD concentration on the area of the three main pterostilbene
oxidation products by POX determined by RP-HPLC. (d) pterostilbene trans dimer,
(j) pterostilbene cis dimer and (N) pterostilbene open dimer, (ꢀ) sum of the
concentrations of the three products. The eluent system is methanol/water (80:20
v/v). Mau: Miliabsorbance units.

P. Rodríguez-Bonilla et al. / Phytochemistry 72 (2011) 100–108
107
HP-β-CD/Cis-Product
HP-β-CD + Cis-Product
HP-β-CD + Trans-Product
HP-β-CD/Trans-Product
Kcis
Keq
Ktranss
Fig. 10. Isomerization equilibrium between pterostilbene trans dehydromer and pterostilbene cis dehydromer in the presence of HP-b-CD.
Bearing in mind that one of the most important properties of CDs is
it ability for increasing the solubility of the guest molecule com-
plexed (Szente and Szejtli, 2004), the inclusion complexes formed
by the interaction between HP-b-CD and both the pterostilbene
trans dehydromer pterostilbene and cis dehydromer products ob-
tained by the oxidation of pterostilbene by POX may show higher
solubility than these molecules in the absence of HP-b-CD and slow
down the detoxification process thus increasing the defence mech-
anisms of the plant against fungal attack.
eluted isocratically with methanol/water (80:20 v/v) at a flow rate
of 0.7 ml/min. The ratio between the concentrations of the three
reaction products was calculated from the peak areas. Parameters
for mass spectrometric analysis: mass range mode: Std/normal;
ion polarity: positive; ion source type: ESI; dry temperature:
350 °C; nebulizer: 60.00 psi; dry gas: 9.00 l/min. HR-ESI-MS tech-
nique was used for calculating the molecular formula of pterostil-
bene oxidation products.
Acknowledgments
3. Experimental
This work was supported by AGL2007-65907 (MEC, FEDER,
Spain) and by Programa de ayudas a Grupos de Excelencia de Reg-
ión de Murcia, de la Fundación Séneca, Agencia de Ciencia y Tec-
nología de la Región de Murcia (Plan Regional de Ciencia y
Tecnología 2007/2010).
3.1. Materials
Biochemicals were purchased from Fluka (Madrid, Spain).
Pterostilbene was from Sequoia Research Products Limited
(Pangbourne, United Kingdom) and was used without further
purification. HP-b-CD was purchased from Sigma (Madrid, Spain).
Hydrogen peroxide and POX type II from horsedish were obtained
from Sigma (Madrid, Spain). Pterostilbene is sensitive to light, and
intense irradiation of solutions of the analyte induces the forma-
tion of a highly fluorescent compound if the irradiation is intense.
Because of this, the samples were stored in darkness. The hydrogen
peroxide, POX and pterostilbene were freshly prepared every day.
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