The effect of hydroxytyrosol and its nitroderivatives on catechol-O-methyl transferase activity in rat striatal tissue

Article abstract of DOI:10.1039/c4ra09872b

Hydroxytyrosol is a well-known phenolic compound with antioxidant properties that is found in virgin olive oil. Studies have shown that virgin olive oil has neuroprotective effects in rats; thus the purpose of the present study was to investigate the neuroprotective effect of hydroxytyrosol in rats. Additionally, this study aimed to investigate the neuroprotective potential of a homologous series of compounds with better lipophilic profiles in order to increase the assortment of compounds with a putative effect against Parkinson's disease (PD). In this context, the inhibition of catechol-O-methyl transferase (COMT) activity by hydroxytyrosol, nitrohydroxytyrosol, nitrohydroxytyrosol acetate and ethyl nitrohydroxytyrosol ether was investigated by measuring intracellular dopamine and its metabolite levels in the corpus striatum by high performance liquid chromatography (HPLC) with electrochemical detection. The animals received an acute (single dose; 20 mg kg^-1; i.p.) or chronic (one daily dose for 5 days; 20 mg kg^-1; i.p.) treatment of hydroxytyrosol and its nitroderivatives. For comparison, a commercial COMT inhibitor, Ro 41-0960, was also included. Our data show that acute and chronic systemic administration of these compounds produced a clear and statistically significant increase in the intracellular levels of dopamine and its metabolite, 3,4-dihydroxyphenylacetic acid. The increase in dopamine levels was very similar to the increase seen with Ro 41-0960 treatment. The effect of chronic treatment was stronger than that of acute treatment. With respect to the intracellular level of homovanillic acid, Ro 41-0960 produced a statistically significant decrease which it was not observed when hydroxytyrosol and its nitroderivatives were systemically administered. However, the chronic homovanillic acid treatment effect was stronger than the acute treatment. The results suggest that these compounds could inhibit COMT activity.

Full text of DOI:10.1039/c4ra09872b

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The effect of hydroxytyrosol and its nitroderivatives
on catechol-O-methyl transferase activity in rat
striatal tissue
Cite this: RSC Adv., 2014, 4, 61086
Elena Gallardo,ab Andres Madrona,a Rocıo Palma-Valdes,a Mariana Trujillo,a
´
´
´
a
b
*
Jose Luis Espartero and Marti Santiago
´
Hydroxytyrosol is a well-known phenolic compound with antioxidant properties that is found in virgin olive
oil. Studies have shown that virgin olive oil has neuroprotective effects in rats; thus the purpose of the
present study was to investigate the neuroprotective effect of hydroxytyrosol in rats. Additionally, this
study aimed to investigate the neuroprotective potential of a homologous series of compounds with
better lipophilic profiles in order to increase the assortment of compounds with a putative effect against
Parkinson's disease (PD). In this context, the inhibition of catechol-O-methyl transferase (COMT) activity
by hydroxytyrosol, nitrohydroxytyrosol, nitrohydroxytyrosol acetate and ethyl nitrohydroxytyrosol ether
was investigated by measuring intracellular dopamine and its metabolite levels in the corpus striatum by
high performance liquid chromatography (HPLC) with electrochemical detection. The animals received
an acute (single dose; 20 mg kgꢀ1; i.p.) or chronic (one daily dose for 5 days; 20 mg kgꢀ1; i.p.) treatment
of hydroxytyrosol and its nitroderivatives. For comparison, a commercial COMT inhibitor, Ro 41-0960,
was also included. Our data show that acute and chronic systemic administration of these compounds
produced a clear and statistically significant increase in the intracellular levels of dopamine and its
metabolite, 3,4-dihydroxyphenylacetic acid. The increase in dopamine levels was very similar to the
increase seen with Ro 41-0960 treatment. The effect of chronic treatment was stronger than that of
acute treatment. With respect to the intracellular level of homovanillic acid, Ro 41-0960 produced a
statistically significant decrease which it was not observed when hydroxytyrosol and its nitroderivatives
were systemically administered. However, the chronic homovanillic acid treatment effect was stronger
than the acute treatment. The results suggest that these compounds could inhibit COMT activity.
Received 5th September 2014
Accepted 6th November 2014
DOI: 10.1039/c4ra09872b
www.rsc.org/advances
neuroinammation,6,7 another factor in PD progression and this
effect is related to its aforementioned antioxidant properties.
Introduction
Due to these remarkable biological properties, many HTy
derivatives have been prepared in the past few years, and their
structure/antioxidant activity relationships (SAR) have been
studied. These studies have identied a key role of the free
catechol in their function.8 Furthermore, acylation or alkylation
of HTy with variable side chain lengths gives rise to lipophilic
HTy esters9 and ethers,10 respectively. In the literature, most of
the studies on lipophilic antioxidants reported an increase in the
antioxidant activity for these derivatives as compared with free
precursors.8–10 Moreover, some recent contributions revealed a
nonlinear trend in this increase in emulsied, liposomal and
cellular systems, with a maximum antioxidant activity for
medium chain length, which lead to the cut-off effect theory.11–13
Some reviews have been recently published concerning
different biological activities of nitrocatecholic compounds,14,15
as enzymatic inhibitors or antioxidants.16 Among these nitro-
catechols are tolcapone17 and entacapone,18 which are highly
effective as inhibitors of catechol-O-methyl transferase (COMT),
which are part of the second-generation of COMT inhibitors
Natural polyphenols present in the Mediterranean diet exert a
variety of biochemical and pharmacological roles in several
diseases; this has been especially documented in neurodegen-
erative disorders. Among virgin olive oil (VOO) phenols,
hydroxytyrosol (HTy) stands out as a powerful antioxidant in
both in vitro and in vivo studies,1 and a wide range of associated
biological activities have been described for it.2 For example,
HTy is a potent inhibitor of the low density lipoprotein (LDL)
oxidation.2,3 HTy has the capacity to scavenge free radical
species4 as well as prompt the activity of antioxidant enzymes.5
These activities are central to cellular processes which protect
against oxidative stress. Oxidative stress has been identied as
one of the underlying factors of neurodegenerative diseases
such as Parkinson's (PD). Furthermore, HTy modulates
aDepartment of Organic and Pharmaceutical Chemistry, Faculty of Pharmacy,
University of Seville, 41012-Seville, Spain
bDepartment of Biochemistry and Molecular Biology, Faculty of Pharmacy, University
of Seville, 41012-Seville, Spain. E-mail: msantiago@us.es
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Fig.
1 Chemical structure of hydroxytyrosol (HTy), nitro-
hydroxytyrosol (NO2HTy), nitrohydroxytyrosyl acetate (NO2HTy-A)
and ethyl nitrohydroxytyrosyl ether (NO2HTy-E).
Fig. 2 Effect of the acute and chronic systemic treatments on the
striatal content of DA. Each column represents mean ꢁ SEM (vertical
lines), expressed as ng gꢀ1 wet tissue (N ¼ 4–8). Statistical significance:
(a) compared with saline treatment, (b) compared with Ro 41-0960
treatment, and (c) compared with the same treatment under acute
conditions. See text for statistical details.
used in the treatment of PD. Although they were approved and
reached the marketplace as adjuncts to levodopa therapy, tol-
capone was subsequently withdrawn due to hepatotoxicity
concerns.18–20 Entacapone unfortunately has a very short in vivo
half-life and, thus, its clinical efficacy has been questioned.21
Thus, given the lack of pharmacological availability of COMT
inhibitors, there has been a great effort in recent years in drug
discovery and the development of new selective and potent
COMT inhibitors with safer and long-acting proles.22–25
For these reasons, nitroderivatives of HTy, nitrohydroxytyrosol
(NO2HTy), nitrohydroxytyrosyl acetate (NO2HTy-A) and ethyl
nitrohydroxytyrosyl ether (NO2HTy-E) (Fig. 1), which contain a
catechol pharmacophore with a highly electronegative nitro
group at ortho-position relative to the side-chain substituent,
have been synthesized as a new class of potential COMT inhibi-
tors. These inhibitors could play a role in the prevention of PD.
Thus, recent work has examined the syntheses of some nitroester
derivatives of HTy with variable acyl side chain lengths from 2 to
16 carbon atoms and has also evaluated their antioxidant activity
by FRAP, ABTS, and ORAC assays.26 These nitrocatechols,
obtained from HTy recovered from olive oil wastewaters, have
demonstrated a remarkable antioxidant capacity as reducing
agents and free radical scavengers.26 Moreover, they did not show
any cytotoxic effect in human HepG2 cells, thus they have the
value of protecting against oxidative stress without any toxicity
concerns.27
with respect to saline, but statistically signicant when they
were compared to Ro 41-0960 (Fig. 2).
Chronic systemic administration produced an increase in
the striatal content of DA, which was higher and statistically
signicant than that produced by the acute systemic adminis-
tration in HTy (137.0%) and NO2HTy-E (123.6%) (Fig. 2).
However, NO2HTy (113.7%) and NO2HTy-A (114.3%) chronic
treatment did not show any statistical difference when
compared with acute treatment (Fig. 1). Ro 41-0960 chronic
systemic administration produced similar increase (122.5%)
when compared with the acute systemic administration (Fig. 2).
3,4-Dihydroxyphenylacetic acid (DOPAC)
Both acute and chronic systemic administrations produced a
strong and signicant increase in the striatal content of DOPAC
(Fig. 3). The biggest increase in the striatal content of DOPAC
was found when Ro 41-0960 was systemically administered
(acute, 234.6% and chronic, 225.5%; Fig. 3). HTy systemic
administration produced very similar increases in the striatal
content of DOPAC for both treatments (acute, 152.7% and
chronic, 155.1%). NO2HTY-A and NO2HTy-E systemic admin-
Considering the above evidence, nitroderivatives of HTy have
been proposed as potential and suitable options for the
prevention of PD. Therefore, the aim of the present study was to
evaluate the effect of HTy and its nitroderivatives on COMT
activity in the rat's brain, by measuring the striatal content of
intracellular dopamine (DA) and its metabolites, 3,4-dihydrox-
yphenylacetic acid (DOPAC) and homovanillic acid (HVA), using
HPLC with electrochemical detection. A known commercial
inhibitor, Ro 41-0960 was also included in our study, in order to
compare data with a typical COMT inhibitor.
istrations produced
a bigger and statistically signicant
increase in the striatal content of DOPAC when chronic treat-
ment was compared with acute treatment: NO2HTy-A (acute,
146.7% and chronic, 181.8%) and NO2HTy-E (acute, 122.7%
Results and discussion
Dopamine (DA)
The acute treatment with a single systemic administration
(20 mg kgꢀ1, i.p.) showed a clear and signicant increase in the
striatal content of DA when Ro 41-0960 (123.6%), HTy (118.9%)
and NO2HTy (119.6%) were compared to saline (Fig. 2).
However, NO2HTy-A (105.8%) and NO2HTy-E (104.8%) showed
Fig. 3 Effect of the acute and chronic systemic treatments on the
striatal content of DOPAC. Each column represents mean ꢁ SEM
(vertical lines), expressed as ng gꢀ1 wet tissue (N ¼ 4–8). Statistical
significance: (a) compared with saline treatment, (b) compared with Ro
41-0960 treatment, and (c) compared with the same treatment under
only small increases, which were not statistically signicant acute conditions. See text for statistical details.
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and chronic, 195.8%) (Fig. 3). However, NO2HTy systemic decreased DA metabolism and, simultaneously increase DOPAC
administration produced a high increase in the striatal content levels due to monoamine oxidase activity (Fig. 5). We have
of DOPAC, which was not statistically signicant (acute, 131.3% demonstrated that in HTy and NO–HTy treated animals, there
and chronic, 167.6%) (Fig. 3).
was extensive COMT inhibition in the striatal tissue based on
HTy, NO2HTy, NO2HTy-A and NO2HTy-E acute and chronic DA and DOPAC levels. In the second generation inhibitors,
systemic administration were statistically signicant when potency was enhanced with the incorporation of the nitro
compared with Ro 41-0960, with the exception of the NO2HTy-E group.23 Although a trend is observed our data does not nd a
chronic systemic administration (Fig. 3).
signicant difference between the natural precursor phenolic
compound (HTy) and the nitroderivative (NO2HTy). HTy may be
playing a role in PD as a free radical scavenger, and may not be
directly affecting COMT activity. Accordingly, some COMT
inhibitors have demonstrated protection against free radical
mechanisms that might be involved in PD. In a cell culture
model of cerebral ischemia nitecapone and tolcapone are
reported to scavenge peroxyl and hydroxyl radicals and inhibit
lipid peroxidation32 accounting for cytoprotection following
hypoxia and hypoglycaemia.33 Although NO2HTy has already
been identied as having antioxidant activity in vitro,26 the
present paper elucidates the biological effect attributed to
nitrocatechol structure on COMT activity of these novel
compounds.
Additionally this study shows higher levels of DOPAC from
lipophilic nitroderivatives, NO2HTy-A and NO2HTy-E, in
comparison with their precursor, NO2HTy. The chemical nature
of the side chain substituent is emerging as an important factor
in the biological activity of these compounds. Structure–activity
relationship studies (SAR) have demonstrated that, although
the nitrocatechol structure was mainly responsible for the
“anchoring” of the inhibitor to the enzyme active site, variations
in the side chain substituent exert a profound inuence on both
the peripheral selectivity and duration of COMT inhibition.23
These results are in accordance with previous studies in which
the side chain substituent also exerts an inuence on the anti-
oxidant capacity. Regarding nitroester derivatives of HTy, we
have demonstrated that antioxidant activity varies depending
Homovanillic acid (HVA)
Lastly, striatal content of HVA differed signicantly when
comparing Ro 41-0960 with phenolic compounds. In fact, Ro 41-
0960 systemic administration showed a strong reduction in the
striatal content of HVA, while no decrease was found with the
systemic administration of the other compounds (Fig. 4).
However, in all compounds studied, a clear reduction in the
striatal content of HVA was found when comparing chronic and
acute systemic administration. In these experiments, the
biggest difference was found with systemic Ro 41-0960 treat-
ment: acute, 58.0% and chronic, 13.1% (Fig. 4). Similar results,
but to a lesser extent, were found with HTy (acute, 138.3% and
chronic, 102.5%); NO2HTy (acute 114.9% and chronic, 95.7%);
NO2HTy-A (acute, 107.7% and chronic, 83.9%); and NO2HTy-E
(acute, 117.6% and chronic, 86.5%) (Fig. 4).
The naturally occurring biophenol, HTy, along with some of
its nitroderivatives, have been studied in order to demonstrate
their potential neuroprotection against PD. This work is valu-
able in the light of emerging concerns about availability and
relatively short half-lives of the current clinical COMT inhibi-
tors28,29 This work looked to identify compounds that could be
used as potent and long-acting COMT inhibitors as an adjunct
to L-DOPA/AADC inhibitor therapy for PD.30,31 Therefore, we
have designed novel lipophilic hydroxytyrosyl derivatives as
COMT inhibitors and we have evaluated their capability to cross
the blood brain barrier and, their effects on the neurochemical
markers of PD in rat striatal tissue. Considering the controver-
sial nature of the proposed selectivity of COMT inhibitors;30 we
studied the potency of novel inhibitors of COMT activity in
brain tissue aer intraperitoneal administration.
In order to be considered a COMT inhibitor a compound
must increase the amount of DA present in the brain due to
Fig. 4 Effect of the acute and chronic systemic treatments on the Fig. 5 The metabolism of dopamine. Dopamine can be catabolised by
striatal content of HVA. Each column represents mean ꢁ SEM (vertical the joint action of the enzymes catechol-O-methyl transferase
lines), expressed as ng gꢀ1 wet tissue (N ¼ 4–8). Statistical significance: (COMT), aldehyde dehydrogenase (AH) and monoamine oxidase
(a) compared with saline treatment, (b) compared with Ro 41-0960 (MAO). Other abbreviations: dopamine (DA), 3,4-dihydroxyphenyl-
treatment, and (c) compared with the same treatment under acute acetic acid (DOPAC), 3-methoxytyramine (3-MT) and homovanillic
conditions. See text for statistical details.
acid (HVA).
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on the acyl side-chain length; short chains maintained or even may arise from differences in pharmacokinetic behavior.
enhanced the antioxidant activity compared to nitro- Nitrocatechols have been kinetically characterized as reversible
hydroxytyrosol. Activity decreased with longer side chains in tight-binding inhibitors of COMT.37–39 They bind to the enzyme
accordance with the lipophilic nature of these compounds.26 with such a high affinity that the amount of free inhibitor
This nonlinear association between biological activity and the molecules is signicantly depleted by the formation of the
lipophilic nature of molecules describes the emerging applica- enzyme–inhibitor complex.40
tion of lipophilic phenolic compounds in lipid systems.11–13
Laatikainen et al.41 have studied the gender differences in the
With this in mind, lipophilic nitroderivatives with short chains effects of COMT inhibition on DA metabolism by tolcapone (30
(NO2HTy-A and NO2HTy-E) seem to be a suitable option to mg kgꢀ1, i.p.). In the striatum, they found that tolcapone did
address biological studies. Moreover, the nitroderivatives' lip- not affect DA content, in fact it decreased in female rats.
ohilic nature might also related to the bioavailability and their Moreover, they demonstrated a clear increase of DOPAC striatal
potential clinical efficacy. At present, entacapone remains the content in female rats, but not in male rats. In contrast, our data
only COMT inhibitor currently used in clinical practice without show a clear increase of DA striatal content using male rats (see
special restrictions. Nevertheless, it has low oral bioavailability Fig. 2) and also, a strong DOPAC increase in all treatments (see
(approximately 35%)23 which explaining the short half-life of Fig. 3). In conclusion, with a lower systemically administered
this compound. These data seem to be attributed to extensive dose, a stronger effect was observed with the proposed COMT
and variable rst-pass metabolism of the current clinical COMT inhibitors.
inhibitors. However, phenolic derivatives from olive oil have
In terms of HVA, there was an increase in its intracellular
been previously described as being slowly metabolized by levels, especially in the acute HTy treatment (see Fig. 4). In
intestinal cells in vitro. Evidence suggests that over 80% of the accordance with this, some authors have described that HTy is
precursor HTy that reaches the bloodstream remains a non- converted into homovanillic alcohol (HVAL) by COMT activity42
metabolized compound.34 Moreover, it has been demonstrated and this metabolite is subsequently converted into HVA.43,44 Hu
that NO2HTy and its lipophilic derivatives are efficiently et al.2 offers another explanation that assumes that HTy is part
absorbed in accordance with their lipophilic nature and of the DA metabolic pathway, and this is mediated by COMT
partially metabolized by intestinal epithelial cell monolayers activity yielding HVAL and HVA as end products.2,44 The nitro-
(unpublished results).
derivatives of HTy, due to the nitrocatechol structure, do not
Our results conrmed a signicant increase in the striatal participate in this metabolism pathway thus there is differential
content of DA and DOPAC observed for HTy and its derivatives, yield in metabolites.
especially for the nitroderivatives aer a chronic treatment in
comparison to acute administration. Our results suggest that
novel catechols administrated in peripheral tissues can reach
the brain aer chronic treatment producing longer lasting
Experimental procedures
effects compared with those from acute treatment. Parada and Male albino Wistar rats weighing 270–320 g were used for all the
Soares-Da-Silva35 demonstrated that the levels of DOPAC and experiments. The rats were kept, three or four rats per cage, at
HVA observed in the striatum were not signicantly affected by constant room temperature (22 ꢁ 2 ꢂC), relative humidity (60%)
a single dose of nebicapone, but, with higher doses of the with a 12 h light–dark cycle and food and water ad libitum.
inhibitor, an increase in DOPAC and a decrease in HVA levels Experiments were carried out in accordance with the Guidelines
were observed, indicating that some level of COMT inhibition in of the European Union Council (2010/63/EU), following the
the brain may occur at higher doses of the inhibitor; this could Spanish regulations (BOE 34/11370-421, 2013) for the use of
explain our results from chronic administration.
laboratory animals and approved by the Scientic Committee of
For comparison, we carried out experiments with the the University of Seville.
commercial COMT inhibitor Ro 41-0960. This compound
The following drugs were used: 20-uoro-3,4-dihydroxy-5-
decreased the formation of the striatal HVA with a concomitant nitrobenzophenone (RO 41-0960, Sigma Chemical Co., MO,
increase in striatal DA content as well as DOPAC. This was USA). HTy was extracted and puried from olive oil wastewaters
expected due its proposed activity as an inhibitor of brain as previously described Fernandez-Bolanos et al.45 NO2HTy and
COMT activity. This strong inhibition on DA catabolism NO2HTy-A were obtained in our lab accordingly to the synthetic
observed aer Ro 41-0960 systemic administration suggests procedures described by Trujillo et al.26 NO2HTy-E was obtained
that this compound is a selective and brain penetrant COMT in a similar way from ethyl hydroxytyrosyl ether.10
´
˜
inhibitor. A similar compound, Ro 40-7592 (40-methyl-3,4-
Animals were divided into two main groups: (a) acute treat-
dihydroxy-5-nitro-benzophenone) also showed potent and cen- ment: animals received one single dose (20 mg kgꢀ1; i.p.)
trally active COMT inhibition.36 When comparing acute and between 10:00 and 11:00 a.m. and two hours later they were
chronic treatments, it is important to point out that Ro 41-0960 sacriced and (b) chronic treatment: animals received a daily
has similar effects on the striatal content of DA and DOPAC, but dose (20 mg kgꢀ1; i.p.) for ve days between 10:00 and 11:00
HVA striatal content is more affected in the chronic treatment. a.m. and, the last day, two hours later they were sacriced. Each
DOPAC data showed the higher effect on striatal tissue of Ro main group was divided into six subgroups which were
41-0960 inhibitor in comparison to novel compounds. This systemically administered saline, Ro 41-0960, HTy, NO2HTy,
result suggests that the differences in the potency of the effect NO2HTy-A and NO2HTy-E.
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Animals were decapitated between 12:00 and 13:00 h and the
whole brain was quickly removed. The caudoputamen was
dissected out, frozen in liquid nitrogen and stored at ꢀ80 C
NO2HTy-E
DA
DOPAC
HVA
COMT
i.p
Ethyl nitrohydroxytyrosyl ether
Dopamine
3,4-Dihydroxyphenylacetic acid
Homovanillic acid
Catechol-O-methyl transferase
Intraperitoneal
ꢂ
until analysis (total time for brain area isolation was <3 min).
Postmortem changes of amines were not a problem in this study
in light of the relatively short time lapse between decapitation
and perchloric acid extraction. The brain tissue was weighed to
the nearest milligram and homogenized in 0.1 M perchloric
acid (1 mg fresh tissue by 30 ml perchloric acid) by ultrasonic
disintegration over ice using an Hielscher UP100H Ultrasonic
Processor (Teltow, Germany). Samples were centrifuged at
30 000g for 15 min at +4 ꢂC and the supernatant was then
ltered through 0.22 mm lter nylon (Spin-X centrifuge tube
lter, Corning Incorporated, NY, USA). Material was injected by
high-pressure injection valve (Rheodyne, CA, USA), with a 10 ml
sample loop.
Acknowledgements
This work was supported by the Grant P09-AGR-5098 from Junta
´
´
de Andalucıa. E.G. thanks Junta de Andalucıa for a pre-doctoral
fellowship. We thank Ms Rianne Stowell for revising the
manuscript.
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Conclusion
The new nitroderivative compounds synthesized from the
natural olive oil phenol, hydroxytyrosol, show a remarkable
activity in the DA metabolism, suggesting a putative effect
against PD as novel and lipophilic COMT inhibitors.
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Abbreviations
HTy
Hydroxytyrosol
NO2HTy
NO2HTy-A
Nitro-hydroxytyrosol
Nitrohydroxytyrosyl acetate
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