Inhibition of catechol-O-methyltransferase (COMT) by some plant-derived alkaloids and phenolics

Article abstract of DOI:10.1016/j.molcatb.2009.04.014

In this study, as an alternative to the medicines, natural compounds extracted from plant species (Peganum harmala, Cistus parviflorus and Vitex agnus-cactus) were investigated in order to inhibit the catechol-O-metyhltranferase (COMT) activity. In fluoro

Full text of DOI:10.1016/j.molcatb.2009.04.014

Journal of Molecular Catalysis B: Enzymatic 64 (2010) 162–166
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Inhibition of catechol-O-methyltransferase (COMT) by some plant-derived
alkaloids and phenolics
∗
Dilek Yalcin, Oguz Bayraktar
Department of Chemical Engineering, Bioreaction Engineering Laboratory, Izmir Institute of Technology, Gulbahce Koyu, 35437 Urla-Izmir, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 3 May 2009
In this study, as an alternative to the medicines, natural compounds extracted from plant species (Peganum
harmala, Cistus parviflorus and Vitex agnus-cactus) were investigated in order to inhibit the catechol-
O-metyhltranferase (COMT) activity. In fluorometric enzyme assay, S-adenosylmethionine (SAM) and
aesculetin (ES) were used as methyl donor and acceptor substrates, respectively. Their Km values were
determined as 3.5 ± 0.3 ␮M and 6.4 ± 0.4 ␮M in absence of inhibitor. Inhibition performances of the plant-
derived polyphenolics and alkaloids were determined. Inhibitory effect of alkaloids extracted from P.
harmala seeds was found the highest among the plant extracts; however, it was lower than that of 3,5-
DNC. In case of inhibition mechanism, mixed type inhibition was observed for alkaloid extract whereas
uncompetitive inhibition was observed for 3,5-DNC. In case of polyphenolic extracts obtained from C.
parviflorus and V. agnus-cactus leaves, mechanism were also explained as mixed type inhibition and their
Keywords:
COMT
Inhibition
Alkaloids
Phenolics
Parkinson’s disease
˛
Ki values were calculated as 1.99 ± 0.35 ␮g/ml and 9.48 ± 0.58 ␮g/ml, respectively.
©
2009 Elsevier B.V. All rights reserved.
1
. Introduction
All of them revealed that flavonoids in C. sinensis have great
inhibitory potency against COMT. In this study, the natural com-
pounds extracted from the leaves of Cistus parviflorus and Vitex
agnus-cactus and from the seeds of Peganum harmala were used as
natural inhibitors of COMT in order to investigate the inactivation
performance of methylation of aesculetin to scopoletin.
Since ancient times, plants have been used as medicine against
certain human diseases. In the last century, the use of natu-
ral sources as medicine has expanded and there is a growing
interest in obtaining biologically active compounds from natu-
ral sources. These compounds have antioxidant, antimicrobial,
antiinflammatory and neuroprotective properties and classified
as phytochemicals include most of catecholic structures [1,2]. In
administration of catechols, it is known that they are methylated
by an enzyme known as catechol-O-metyhltranferase (COMT; EC
2. Experimental
2.1. Materials
2
.1.1.6) that plays an important role in Parkinsonism [3]. The pri-
HCl used as alkaloid extraction solvent was purchased from
Merck (Germany). Petroleum ether used during the liquid-liquid
extraction of alkaloids was purchased from Riedel-de Haen
mary function of COMT is to deactivate biologically active catechols
like catecholamine neurotransmitters (dopamine, epinephrine),
levodopa (l-DOPA), carbidopa and flavonoids [4,5].
(
Germany). All other extraction solvents were purchased from
In current therapies for Parkinson’s disease, selective COMT
inhibitors, mainly synthetic nitrocatechol compounds (entacapone,
tolcapone), are used in combination with l-DOPA and dopa decar-
boxylase inhibitors, although there are several well-known adverse
side effects of these drugs [4,5]. Therefore, researchers have focused
on the potency of natural products having ability of inhibiting
COMT. Among several natural sources, tea catechins have been
mostly investigated compounds by several research groups [6,4,7].
Sigma–Aldrich Chemie GmbH (Germany). COMT from porcine liver
was purchased from Sigma (Germany). Other reaction mixture
components including SAM, aesculetin (ES), MgCl2 anhydrous and
l-cysteine non-animal source were purchased from Sigma (Ger-
many). Potassium phosphate buffer solutions were purchased from
Fluka and Merck (Germany). Dimethylsulfoxide (DMSO) used in
reaction mixture was purchased from Carlo-Erba Reagents (Spain).
2.2. Preparation of raw plant materials and extraction procedure
The leaves of C. parviflorus and V. agnus-cactus were washed,
∗ Corresponding author. Tel.: +90 232 750 6681; fax: +90 232 750 6645.
E-mail addresses: dilekyalcin@iyte.edu.tr (D. Yalcin), oguzbayraktar@iyte.edu.tr
O. Bayraktar).
dried and ground. The leaves of these two plants were extracted
◦
(
with70%aqueousethanolsolutionat30 Cfor2 h. Afterevaporation
1381-1177/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2009.04.014

D. Yalcin, O. Bayraktar / Journal of Molecular Catalysis B: Enzymatic 64 (2010) 162–166
163
of alcohol by using a rotary evaporator, the aqueous crude extracts
were dried by using lyophilizator. The unshelled seeds of P. har-
mala were ground. The ground seeds were extracted with methanol
inhibition. If it is around 1 (˛ < 1 or ˛ > 1) at a certain value, mecha-
nism can be described as mixed type inhibition which explains the
deviation from noncompetitive and competitive nature [10]. If it is
equal to 1, noncompetitive inhibition takes place.
◦
by using a Soxhlet apparatus at 70 C for 8 h. Then, methanol was
evaporated to dryness. Dried particles were dissolved with 5% HCl
solution and it was extracted two times with 30 ml petroleum ether,
3. Results and discussion
the acidic part was collected and basified to pH 9.0 with NH OH
4
which was further extracted with 50 ml chloroform for four times.
Finally, chloroform layer was collected and evaporated to dryness.
3.1. Kinetic analysis to determine enzyme–substrates relationship
Before performing inhibition study, a previous study was per-
formed by changing substrates and enzyme concentrations in
order to obtain the linear working concentration ranges of enzyme
and also substrates. Enzyme concentrations were kept constant at
2
.3. Determination of enzyme activity and inhibition study
Enzymatic assays were performed according to the method
16.5–11.0–8.5 ␮g/ml for this experiment while seven different SAM
reported by J. Veser’s and M. Kurkela et al.’s studies with minor dif-
ferences by using Microfluor white, 96 well plate [8,9]. The followed
reactionwhichwascatalyzedbyCOMTwasconversionofaesculetin
to scopoletin at 37 C. Aesculetin was dissolved in dimethyl sulfox-
ide (DMSO) and diluted with aqueous buffer solution containing
concentrations varied from 10 ␮M to 600 ␮M were used against
six different ES concentrations prepared at the range of 2–10 ␮M.
As a result, it was found that the concentration of SAM should
be kept lower than 200 ␮M and the lowest enzyme concentration
was determined as 11.0 ␮g protein/ml. It was also revealed that the
used concentration range of ES did not deviate from linearity. From
this experiment, Km values both for SAM and ES was obtained as
◦
100 mM phosphate, 5 mM MgCl , 20 mM l-cysteine (pH 7.4), for a
2
final DMSO concentration of 2% in the 200 ␮l of reaction mixture.
All other reagents were dissolved in the same buffer solution. Flu-
orometric measurements were performed at 355 nm emission and
3
.5 ± 0.3 ␮M and 6.4 ± 0.4 ␮M which was reported as 6.2 ␮M in
Veser’s study [8].
4
60 nm excitation wavelengths for 2 h by using Thermo Varioskan
Flash microplate reader. Enzyme concentration was kept constant
at 11.0 ␮g/ml while five different SAM and five different aesculetin
concentrations varied from 10 ␮M to 100 ␮M and from 2 ␮M to
3
.2. Kinetic analysis for inhibition study
In inhibition study, ES concentration was kept constant at 4 ␮M
6
␮M, respectively. 3,5-DNC and the crude extracts obtained from
in 250 ␮l total reaction volume while SAM concentrations were
changed from 10 ␮M to 100 ␮M to determine the inhibition per-
formance with respect to SAM. Besides, inhibition with respect
to ES was investigated by varying its concentrations from 2 ␮M
to 6 ␮M at constant SAM concentration of 100 ␮M. Enzyme con-
centration was kept constant at 11.0 ␮g/ml for the whole assay.
A representative percentage values of COMT inhibition obtained
for the assay performed at 100 ␮M SAM concentration were given
in Fig. 1. Inhibition performances of crude extract of alkaloids and
their standards were found as comparable to that of obtained for
positive control, 3,5-DNC. In Fig. 1, it was also revealed that among
polyphenolic extracts, C. parviflorus leaf extracts showed higher
inhibition than that of V. agnus-cactus. As a result, alkaloids were
found to be more potent COMT inhibitors than the polyphenolics
and it was thought that nitro groups content of them plays impor-
tant role in COMT inhibition.
plant species were dissolved in DMSO and diluted with the buffer
solution for a final DMSO concentration of 2%. Final concentrations
of inhibitors in 250 ␮l total reaction volume were given on resulting
figures.
2.4. Curve fitting and data analysis
In data analysis, reciprocals of velocities and substrate concen-
trations gave the linear relationship by which Km and Vmax values
could be calculated. The kinetic mechanism and inhibition con-
stants were obtained by fitting the initial steady state velocity as
a function of substrate concentration to the following equations
defined in R.A. Copeland’s book using nonlinear regression analy-
sis program in GraphPad Prism 5.0 software [10]. The goodness of
curve fit was evaluated statistically by one-way of ANOVA following
with Tukey’s multiple comparison test.
Michaelis kinetic calculations supported these findings. A sharp
decrease of formation rate of scopoletin, V (nmol/min mg protein)
was observed in presence of the positive control. The nonlinear
fitting of data given in Fig. 2A obtained by using GraphPad soft-
ware which was supported also by the reciprocal plot (Fig. 2B)
demonstrated the uncompetitive nature of the positive control,
as expected. Because, almost all nitrocatechols are known that
they behave uncompetitively with respect to SAM and tight bind-
ing inhibitors of COMT [5]. From these kinetic data, the Km
Vmax[S]
Km + [S](1 + ([I]/˛K ))
v =
v =
v =
v =
uncompetitive inhibition
i
Vmax[S]
competitive inhibition
Km(1 + ([I]/K )) + [S]
i
Vmax[S]
noncompetitive inhibition
mixed type inhibition
Km(1 + ([I]/K )) + [S](1 + ([I]/K ))
value was calculated as 6.83 ± 0.77 ␮M and V
max
was found as
i
i
1
3
9
.07 ± 0.02 nmol/min. mg protein. Dissociation constant (˛K ) for
i
,5-DNC which was an uncompetitive inhibitor was obtained as
.17 ± 0.46 ng/ml (i.e., 45.60 ± 2.29 nM). Also, IC50 was calculated
Vmax[S]
Km(1 + ([I]/K )) + [S](1 + ([I]/˛K ))
i
i
as 44.18 ± 0.78 nM from dose response curve of 3,5-DNC which was
very similar to that value reported in Kurkela et al.’s study as 35 nM
[9].
where Vmax is the maximum enzyme velocity without inhibitor. Km
is the Michaelis–Menten constant without inhibitor. K is the inhi-
i
bition constant indicating the dissociation of EI complex. AlphaKi
Moreover, the nonlinear fit of data obtained for alkaloid extract
was given in Fig. 3A and reciprocals of velocities were given in
(
˛K ) is also inhibition constant indicating the dissociation of ESI
i
complex. In this term, Alpha (˛) determines the mechanism by
revealing the binding degree of the inhibitor which explains the
affinity of enzyme for substrate [10]. At very high ˛ values that
is much greater than 1, mechanism is explained by competitive
inhibition and very low ˛ values (˛ ꢀ 1) indicate uncompetitive
Fig. 3B. Dissociation constant, ˛K was found as 2.24 ± 0.75 ␮g/ml
i
where ˛ was equal to 18.72 ± 2.28. Based on the criteria given
in Section 2.4, the inhibition mechanism for alkaloid extract was
determined as mixed type inhibition with respect to SAM unlike
the uncompetitive mechanism found for positive control, 3,5-

1
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D. Yalcin, O. Bayraktar / Journal of Molecular Catalysis B: Enzymatic 64 (2010) 162–166
Fig. 1. Inhibition percentages of crude extracts and alkaloid standards with respect to 3,5-DNC. (SAM concentration was 100␮M.)
Fig. 2. (A) Plot of formation rate of scopoletin; (B) double reciprocal plot of formation rate against various SAM concentrations in presence of 3 different 3,5-DNC concentrations
P < 0.01, means of inhibited ones are significantly different from control).
(
DNC. Besides, the inhibitory effect of this natural alkaloid extract
was found significantly lower than that of 3,5-DNC. From the
kinetic data, the Km and Vmax values were also determined as
mance was found higher than that of harmalol which inhibited
COMT by uncompetitive manner like positive control, 3,5-DNC.
From the data given in Figs. 4 and 5, it was found that the
obtained dissociation constant for harmaline were closer to that
of crude alkaloid extract compared to those determined for har-
6
.05 ± 0.69 ␮M and 1.06 ± 0.02 nmol/min. mg protein, respectively.
IC of alkaloid extracts was calculated as 1.09 ± 0.33 ␮g/ml.
50
It was known that P. harmala contains several ␤-carbolines
malol. The ˛K value for harmaline were found as 1.36 ± 0.11 ␮g/ml
i
like harmaline, harmine, harmalol, harmane, vasicinone, vasicine,
etc. [11,12]. It was also reported that among these alkaloids har-
maline and harmine are known as monoamineoxidase (MAO-B)
inhibitors and they have several types of activities such as antioxi-
dant, antiviral and anticancer [12,13]. Our HPLC analysis has shown
that the major alkaloid in this P. harmala seed extract was har-
maline. Therefore, in this study harmaline and harmalol standards
were used to determine which type of alkaloids could be responsi-
ble for the inhibitory effect. In Figs. 4 and 5, the results obtained for
these two alkaloid standards were given. As expected, harmaline
strongly inhibited COMT by mixed type manner and its perfor-
having ˛ value of 2.49 ± 0.78. The lower ˛ value obtained for harma-
line compared to that of found for P. harmala extract (18.72 ± 2.28)
can be explained by shift in the dominant inhibition mechanism
from competitive to noncompetitive. The total net inhibitory effect
was reflected and explained by mixed type inhibition. The ˛K of
i
harmalol was obtained as 3.21 ± 0.16 ␮g/ml. Besides, IC50 values
of harmaline and harmalol were calculated as 0.98 ± 0.12 ␮g/ml
(4.57 ± 0.14 ␮M) and 3.59 ± 0.37 ␮g/ml (13.19 ± 0.31 ␮M), respec-
tively.
It was reported by several researchers that polyphenolics mainly
tea catechins, gallic acid and hydroxybenzoic acid derivatives have
Fig. 3. (A) Plot of formation rate of scopoletin; (B) double reciprocal plot of formation rate against various SAM concentrations in presence of 3 different concentrations of
crude alkaloid extracts obtained from P. harmala seed (P < 0.01, means of inhibited ones are significantly different from control).

D. Yalcin, O. Bayraktar / Journal of Molecular Catalysis B: Enzymatic 64 (2010) 162–166
165
Fig. 4. (A) Plot of formation rate of scopoletin; (B) double reciprocal plot of formation rate against various SAM concentrations in presence of 3 different concentrations of
harmaline (P < 0.01, means of inhibited ones are significantly different from control).
Fig. 5. (A) Plot of formation rate of scopoletin; (B) double reciprocal plot of formation rate against various SAM concentrations in presence of 3 different concentrations of
harmalol (P < 0.01, means of inhibited ones are significantly different from control).
inhibitory effect on COMT [4,7,16]. Therefore, extracts of V. agnus-
cactus and C. parviflorus leaves having high polyphenol content
were used in inhibition study. As seen in Fig. 1, the crude alka-
loid extract and its standards showed higher inhibition potency
than polyphenolic extracts obtained from V. agnus-cactus and C.
parviflorus leaves.
Scopoletin formation rate was also decreased in presence of V.
agnus-cactus extract. Mechanism was determined as mixed type
with respect to SAM from nonlinear fit of data given in Fig. 6A sup-
ported with double-reciprocal plot of formation rate that was given
seed extract. This was an expected result, since the main polyphenol
content of V. agnus-cactus extract was gallic acid, p-hydroxybenzoic
acid, rutin and ferulic acid which were known the examples of
first generation COMT inhibitors. It was also known that the inhibi-
tion performances of them much lower than that of nitrocatechols
[5,16]. Beside that, this plant contains some estrogenic compounds
like linoleic acid and also halimane and labdane type diterpenoids
which were also thought having possibility of inhibitory effect [14].
Similar observations were obtained for the crude extracts of C.
parviflorus leaves. Changes in the formation rate against different
SAM concentrations in absence and presence of inhibitors were
given in Fig. 7. From the reciprocal plot (Fig. 7B) of the nonlinearly
fitted curves (Fig. 7A), it was obtained that C. parviflorus leaf extract
inhibited COMT also in mixed type manner with respect to SAM like
it was found for the other plant extracts used in this study. The ˛Ki
value for C. parviflorus leaf extracts was found as 1.99 ± 0.35 ␮g/ml
and ˛ was calculated as 0.79 ± 0.32. The closest ˛ value to 1.0
supported inhibition was more similar to noncompetitive profile
in Fig. 6B. Dissociation constant, ˛K was found as 9.48 ± 0.58 ␮g/ml
i
where ˛ was 2.14 ± 0.23 that indicates inhibition was obviously
closer to noncompetitive when the criteria given in Section 2.4
was taken into consideration. The apparent Km and Vmax values
were calculated as 5.84 ± 0.90 ␮M and 1.03 ± 0.02 nmol/min. mg
protein. From dose-response curve, IC5 value for V. agnus-cactus
0
was determined as 12.96 ± 0.29 ␮g/ml. Inhibition performance of
V. agnus-cactus extract was relatively lower than that of P. harmala
Fig. 6. (A) Plot of formation rate of scopoletin; (B) double reciprocal plot of formation rate against various SAM concentrations in presence of 3 different concentrations
of crude extracts obtained from V. agnus-cactus leaves (means obtained for different inhibitor concentrations are significantly different from each other and from control
(P < 0.01)).

1
66
D. Yalcin, O. Bayraktar / Journal of Molecular Catalysis B: Enzymatic 64 (2010) 162–166
Fig. 7. (A) Plot of formation rate of scopoletin; (B) double reciprocal plot of formation rate against various SAM concentrations in presence of 3 different concentrations of
crude extracts obtained from C. parviflorus leaves (means obtained for different inhibitor concentrations are significantly different from each other and from control (P < 0.01)).
for C. parviflorus leaf extracts as seen from double-reciprocal plot
given in Fig. 7B. The apparent Km and Vmax values were obtained
As a result of this study, it was observed that natural compounds
obtained from P. harmala and C. parviflorus can be an alternative
source of medicine in treatment of Parkinson’s disease.
6
.02 ± 1.29 ␮M and 1.06 ± 0.04 nmol/min mg protein, respectively.
IC value for this plant extract was found as 2.44 ± 0.09 ␮g/ml
50
which revealed that this plant extract showed 5 times stronger
Acknowledgements
inhibitory activity than that of V. agnus-cactus, however it was about
2
times lower when it was compared to the extracts of P. harmala.
The known high terpenoid and flavonoid content of leaves of
Support from the Turkish Scientific Research Council under
Project MAG-107M307 and from the Izmir Institute of Technology
under Project 2007IYTE04 is gratefully acknowledged.
C. parviflorus were thought to be responsible for high inhibitory
effect which could be caused by the relaxation activity reported
by other researchers [17]. Also, it was known that this plant com-
posed of numerous terpenoid essential oils like carvacrol, thymol,
manoyl oxides and also esters like sabinene hydrate acetate, benzyl
benzoate [15]. However, in order to determine the found inhibitory
effect of this polyphenolic extract on COMT, further investigations
are still needed.
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[
The alkaloid extracts obtained from P. harmala seed were found
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