Acetylcholinesterase and carbonic anhydrase isoenzymes i and II inhibition profiles of taxifolin

Article abstract of DOI:10.3109/14756366.2015.1036051

Taxifolin, also known as dihydroquercetin, is a flavonoid commonly found in plants. Carbonic anhydrase (CA, EC 4.2.1.1) plays an important role in many critical physiological events including carbon dioxide (CO₂)/bicarbonate () respiration and pH regulation. There are 16 known CA isoforms in humans, of which human hCA isoenzymes I and II (hCA I and II) are ubiquitous cytosolic isoforms. In this study, the inhibition properties of taxifolin against the slow cytosolic isoenzyme hCA I, and the ubiquitous and dominant rapid cytosolic isoenzyme hCA II were studied. Taxifolin, as a naturally bioactive flavonoid, has a K_i of 29.2 nM against hCA I, and 24.2 nM against hCA II. For acetylcholinesterase enzyme (AChE) inhibition, K_i parameter of taxifolin was determined to be 16.7 nM. These results clearly show that taxifolin inhibited both CA isoenzymes and AChE at the nM levels.

Full text of DOI:10.3109/14756366.2015.1036051

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2015 Informa UK Ltd. DOI: 10.3109/14756366.2015.1036051
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RESEARCH ARTICLE
Acetylcholinesterase and carbonic anhydrase isoenzymes I and II
inhibition profiles of taxifolin
1
2
2
3
3
3,4
4
Hulya Gocer , Fevzi Topal , Meryem Topal , Murat K u¨ c¸ u¨ k , Dilek Teke , Ilhami G u¨ l c¸ in , Saleh H. Alwasel , and
_
5,6
Claudiu T. Supuran
1
2
Central Researching Laboratory, Agri Ibrahim Cecen University, Agri, Turkey, Department of Medical Services and Techniques, Vocational School
3
of Health Services, Gumushane University, Gumushane, Turkey, Department of Chemistry, Faculty of Sciences, Atat u¨ rk University, Erzurum, Turkey,
4
Zoology Department, College of Science, Fetal Programming of Diseases Research Chair, King Saud University, Riyadh, Saudi Arabia,
6
5
Dipartimento di Chimica Ugo Schiff, Universita degli Studi di Firenze, Sesto Fiorentino, Firenze, Italy, and Neurofarba Department, Section of
Pharmaceutical and Nutriceutical Sciences, Universita degli Studi di Firenze, Sesto Fiorentino, Florence, Italy
Abstract
Keywords
Taxifolin, also known as dihydroquercetin, is a flavonoid commonly found in plants. Carbonic Acetylcholinesterase, carbonic anhydrase,
anhydrase (CA, EC 4.2.1.1) plays an important role in many critical physiological events
ꢀ
enzyme inhibition, enzyme purification,
taxifolin
including carbon dioxide (CO
2
)/bicarbonate (HCO ) respiration and pH regulation. There are 16
3
known CA isoforms in humans, of which human hCA isoenzymes I and II (hCA I and II) are
ubiquitous cytosolic isoforms. In this study, the inhibition properties of taxifolin against the
slow cytosolic isoenzyme hCA I, and the ubiquitous and dominant rapid cytosolic isoenzyme
History
Received 10 March 2015
Revised 24 March 2015
Accepted 27 March 2015
Published online 20 April 2015
hCA II were studied. Taxifolin, as a naturally bioactive flavonoid, has a K
I, and 24.2 nM against hCA II. For acetylcholinesterase enzyme (AChE) inhibition, K
i
of 29.2 nM against hCA
parameter of
i
taxifolin was determined to be 16.7 nM. These results clearly show that taxifolin inhibited both
CA isoenzymes and AChE at the nM levels.
Introduction
More important, taxifolin produces effective antioxidant effects,
which contribute to its cardiovascular protective effects. Due to
the reducing properties of its hydroxyl groups, taxifolin exhibits
In recent years, natural flavonoids have attracted significant
interests in the scientific arena because of their versatility of uses
and health-promoting effects. Flavonoids are important com-
pounds that can be found in many plants, including edible fruits
and vegetables. Flavonoids act as efficient antioxidants because of
their ability to chelate transition metal ions, to scavenge free
radicals and to interact with enzymes . It was reported that
in vivo and in vitro studies of flavonoids showed that they have a
positive impact on many important diseases, such as those
affecting cardiovascular system
Phenolic compounds contain at last a hydroxyl group (–OH)
bonded to aromatic ring and are mildly acidic
compounds are classified as simple phenols or polyphenols based
on the number of phenol units in the molecule. This chemical class
in terms of biological and pharmaceutical is very reactive
Taxifolin (dihydroquercetin) is a representative flavonoid
3
antioxidant activity and reacts with free radicals .
Researchers have demonstrated that taxifolin decreased the
production of lipid radicals in a concentration-dependent manner
and reduced the peroxidase activity of the complex of cytochrome c
combined with dioleoyl cardiolipin, which is critical for the onset
of apoptosis. Taxifolin also ameliorated cerebral ischemia-
1
–3
reperfusion injury by inhibiting oxidative enzymes and reducing
27,28
1
,3
.
the overproduction of ROS
. Taxifolin inhibited recombinant
human aldose reductase and the accumulation of sorbitol in human
erythrocyte in diabetes. Taxifolin also retained the clarity of rat
lenses incubated with glucose, suggesting that taxifolin might be
4
–11
. Phenolic
29
effective in preventing osmotic stress in hyperglycemia .
1
2–24
However, there has been little work on possible beneficial effects
of taxifolin for diabetic cardiomyopathy. Caspase enzymes are
important factors for modulating the apoptotic cascade. These
results have demonstrated that caspase-3 and caspase-9 activities
.
compound. It is present in the plants from the Pinus genus and
2
5
it is also found in citrus fruits and milk thistle seeds . Taxifolin
has many a large area of biological effects, including anti-
30
were significantly inhibited by taxifolin . Additionally, it was
shown that taxifolin decreases the angiotensin-converting enzyme
2
inflammatory, hepatoprotective effects and antitumor effects .
6
31
activity in the aorta of aging rats . Studies have shown the
modulatory effects of flavonoids upon multiple enzymes
involved in xenobiotic metabolism such as on various cytochrome
P450 monooxygenase isoforms and phase II conjugation
enzymes and on membrane transport systems including in drug
_
Address for correspondence: Prof. Dr. Ilhami G u¨ l c¸ in, Department of
Chemistry, Faculty of Sciences, Atat u¨ rk University, Erzurum 25240,
Turkey. Tel: +90 442 2314375. Fax: +90 442 2314109. E-mail:
igulcin@atauni.edu.tr, igulcin@yahoo.com
3
2–35
excretion
.

2
H. Gocer et al.
J Enzyme Inhib Med Chem, Early Online: 1–7
6
6
Carbonic anhydrase enzymes (CAs) are ubiquitous in all the catalytic activity . Carbamate pesticides show their toxicity by
living organisms. They have crucial physiological and patho- irreversibly modifying the catalytic serine residue of AChE, and
6
7
logical roles such as in fluid balance, calcification, pH regulation, the inhibiting the AChE . Recently, several studies supported
carboxylation reactions, tumorigenicity, bone resorption, the that many active compounds from plant origins, including
ꢀ
68,69
synthesis of HCO and in many other pathophysiological anthocyanins and terpenoids have AChE inhibition activity
3
.
. The CA catalyzes the reversible hydration of Additionally, some studies demonstrated that grape and blueberry
3
6–39
processes
carbon dioxide (CO ) and water (H O) to bicarbonate (HCO ) anthocyanin
ꢀ
70,71
72
, grape skin anthocyanin lycodine-type alkal-
oids from Lycopodiastrum casuarinoides have neuroprotective
roles.
2
40–46
2
3
+
and a proton (H )
73
.
−
+
In this study, we identify the potential inhibition profile and
associated mechanisms of taxifolin for human CA isoenzymes
I and II (hCA I and II) and for AChE which are widely used
enzyme in pharmacological industry.
CO + H O ⇔ H CO ⇔ HCO + H
2
2
2
3
3
An enzyme inhibitor is a molecule that engages to an enzyme
and decreases its activity. An inhibitor can prevent a substrate
from binding the active site of the enzyme, thus hindering
catalysis. It is well known that CA inhibitors (CAIs) bind to a
Experimental
2
+
catalytic zinc ion (Zn ) in the active site of CA isoenzymes and
7–52
4
Purification of CA isoenzymes
block their activity
established as antiglaucoma, and as anticonvulsant agents ,
. The clinical use of CAIs had been
4
8
Both hCA isoenzymes were purified by sepharose-4B-L-tyrosine-
4–77
4
diuretics and antiobesity drugs
2
53–55
7
in the management of
mountain sickness, gastric and duodenal ulcers, neurological
4
disorders or osteoporosis . Additionally, CAIs have recently been
used as hypoxic tumors management agent
clinically used heterocyclic and aromatic sulfonamides were
clinically used derivatives of acetazolamide (AZA), a known
sulphanilamide affinity chromatography
. For this purpose,
the lysate was adjusted with Tris buffer to pH 8.7 and applied to
the affinity column. Then, protein content in the eluates was
recorded spectrophotometrically at wavelength of 280 nm.
SDS–PAGE (sodium dodecyl sulphate-polyacrylamide gel elec-
trophoresis) was performed after purification of the enzymes. The
isoenzymes purities were controlled by SDS–PAGE, and a single
7
5
6,57
. The first
58,59
CAI
idiopathic intracranial hypertension, epilepsy and altitude sick-
. AZA is an inhibitor of CA and used for glaucoma,
7
8
band was found for each CA isoenzyme . SDS–PAGE method
7
+
ness. To regenerate the basic form of CA isoenzyme, a H is
9
has been detailed described previously and was performed using
acrylamide in the running (10%) and the stacking gel (3%), with
+
transferred from the active site to the solvent. This H transpor-
80,81
tation may be supported by active site residues or by present
buffers in the reaction medium. The fourth position is occupied by
H O at an acidic pH and is catalytically inactive. At higher pH, a
0
.1% SDS
.
2
2
+
Determination of CA isoenzymes activities
H O molecule binds to Zn within the CA active site. Then, this
2
+
proton transfer reaction transfers a H to the solvent, leaving an Both CA isoenzyme activities were performed according to the
4
7,49,50,52,57
82
procedure of Verpoorte et al. and as described previously by our
–
OH group
.
8
1,83
Alzheimer’s disease (AD) is a neurological disorder in which group
. The absorbance change was spectrophotometrically
ꢁ
the patient suffers from memory loss and impaired cognitive reordered at 348 nm during 3 min at room temperature (25 C).
abilities. AD is a chronic neurological disorder in which the One unit of enzyme activity is expressed as 1 mol/L of released
patient suffers from loss impaired cognitive abilities, deficits in p-nitrophenol per minute at 25 C . The protein quantity was
ꢁ
84
3
7,60
activities of daily living and behavioral disturbances
.
spectrophotometrically determined at 595 nm during purification
8
5
According to the cholinergic hypothesis, memory impairment in steps by the Bradford method . Bovine serum albumin was used
86,87
patients suffering from AD results from decreased levels of as the standard protein
.
3
9
the neurotransmitter acetylcholine (ACh) in the cortex .
Acetylcholinesterase (AChE, EC: 3.1.1.7) is a hydrolase that
plays a key role in cholinergic transmission through catalyzing the
CA isoenzymes inhibition assay
6
1
rapid hydrolysis of the neurotransmitter ACh . AChE is a The inhibition property of taxifolin against both CA isoenzymes
special carboxylic ester hydrolase that hydrolyses the esters of was determined by hydrolysis of p-nitrophenylacetate to
39,60
62
to produce acetic acid and choline .
choline
p-nitrophenol. The later molecule can be monitored spectro-
O
CH3
CH3
N
O
AChE
CH3
CH3
CH3
CH3
N
−
H C
O
HO
H C
O
3
3
Acetylcholine
Choline
Acetate
4
AChE is found in high concentrations in the brain and in photometrically . The CO hydration reaction catalyzed by CA
9
2
6
3
erythrocytes . AChE is a necessary enzyme for the nervous was first observed in the absence of taxifolin; the resulting rates
system. AChE inhibitors (AChEI) are used in the treatment of were measured and used as a control for the CA isoenzymes.
several neuromuscular diseases, and were studied for treatment of Also, the same reaction was measured in the presence of taxifolin.
3
9,61,64
AD
of ACh is therefore considered to be a promising approach for the A ) ꢂ 100], where A is the absorbance of the sample containing
C S
. The use of AChEI to block the cholinergic degradation The percent inhibition was determined with (%) ¼ [100 ꢀ (A /
S
treatment of AD. Natural products might slow the progression of taxifolin and AC is the absorbance of the control sample. The
AD by simultaneously protecting neurons from oxidative stress activity (%)–[taxifolin] graphs were drawn and the half maximal
5
and by acting as an AChEI . Certain organophosphorus and inhibitory concentration (IC ) values of taxifolin demonstrated
6
5
0
carbamate derivatives are known to be the best inhibitors of AChE 450% inhibition of CA isoenzymes were calculated after

DOI: 10.3109/14756366.2015.1036051
Acetylcholinesterase and carbonic anhydrase isoenzymes I and II
3
2
suitable dilutions. K values for taxifolin were determined for both to the metal coordination sphere when the Zn has trigonal
+
i
1
isoenzymes. For this purpose to determine the K values, taxifolin bipyramidal geometry . (ii) Inhibition by anchoring of the
i
2
was tested at three different concentrations. K is the binding inhibitor to the Zn -bound solvent molecule, i.e. an H O/–OH.
+
i
2
affinity constant of the taxifolin to CA isoenzymes. NPA was used Phenolic compounds and polyamine molecules can bind to CA in
as the substrate at five different concentrations, and Lineweaver– this way, as shown schematically for phenol or (iii) inhibition by
8
8
Burk curves were drawn in detail as described previously
89–92
.
inhibitor occlusion of the active site or activator-binding site of
1
00–102
CA
. Thus, by binding in a non-classical way to CAs,
phenols and their derivatives provide interesting leads for
identifying novel types of CAIs. Taxifolin has two classical
Acetylcholinesterase inhibition assay
Acetylcholinesterase enzyme inhibition assay was determined on structural characteristics in one molecule: phenols and ketone.
commercially available purified AChE (Product no: C3389-
It has been reported that phenols act as an inhibitors of the
Sigma–Aldrich, St. Louis, MO) from electric gel (Electrophorus Zn -containing CA isoenzymes . Phenol binds to CA in a
2
+
103
9
3
electricus) based on the method of Ellman procedure . Acetyl diverse manner when compared to the classic sulfonamide
2
+
thiocholine iodide (ATCI) was used as the substrate. Also, 5,5- inhibitors. Sulfonamides coordinate to the Zn ion in the CA
dithiobis(2-nitrobenzoic) acid (DTNB) was used for the monitor- active site by replacing the fourth non-protein ligand, which is
ing of AChE activity. Briefly, 150 ml of sodium phosphate buffer typically an H
0.1 M, pH 8.0), 10 ml test compound solution and 20 ml of enzyme classical way to CAs, phenols and their derivatives constitute
O molecule or a –OH ion. By binding in a non-
(
solution (0.09 units/ml) were mixed and incubated for 15 min at interesting leads for identifying novel types of CAIs
2
94,100,102
. In
room temperature. Then, 10 ml of DTNB (10 mM) was transferred the present study, we report the inhibition profiles of taxifolin
and the reaction was initiated by the addition of substrate solution against the slower cytosolic isoform, hCA I and the more rapid
(
10 ml of ATCI, 14 mM solution). The hydrolysis of the ATCI was isoenzyme hCA II. Taxifolin showed effective inhibition of both
measured by the formation of the product, 5-thio-2-nitrobenzoate, isoforms (Table 1).
which is released by AChE hydrolysis. Absorbance of final
solution was spectrophotometrically measured at 412 nm value; however, a more suitable measure is the K
To describe inhibitory effects, researchers often list an IC50
constant. K
i
i
(
Beckhman Coulter DU 730) after 10-min incubation. Tacrine, values were calculated from Lineweaver–Burk graphs (Figures 1,
a standard AChE inhibitor, was used as a positive control. 2 and 3), and both the K
and IC50 parameters of the taxifolin were
i
The percent of AChE inhibition was calculated as follows:
determined in this study. The Lineweaver–Burk plot is a graphical
representation of the Lineweaver–Burk equation of enzyme
Inhibition ð%Þ ¼ 100 ꢀ ½AS=ACꢃ ꢂ 100
where A is the absorbance of the sample containing taxifolin and
AC is the absorbance of the control sample.
104
kinetics . The plot provides a useful graphical method for
analysis of the Michaelis–Menten equation:
S
½Sꢃ
V ¼ Vmax þ
Km þ ½Sꢃ
Results and discussion
Taking the reciprocal gives
Carbonic anhydrases catalysis the crucial pathophysiological
ꢀ
processes that are connected with CO /HCO3 transport and
2
1
K þ ½Sꢃ
m
Km
1
1
¼
¼
ꢂ
þ
homeostasis, biosynthetic reactions including gluconeogenesis,
ureagenesis and lipogenesis, respiration, calcification, tumorigen-
icity, electrolyte secretion in a variety of tissues and organs, and where V is the reaction velocity, K
V
Vmax½Sꢃ
Vmax ½Sꢃ Vmax
is the Michaelis–Menten
m
9
4
bone resorption . Phenolic compounds are a class of chemicals constant, Vmax is the maximum reaction velocity and [S] is the
containing of a –OH bonded directly to an aromatic hydrocarbon substrate concentration.
group and are categorized either as simple phenols or as
polyphenols depending on the number of phenol units in the found for taxifolin against both CA isoenzymes. For the cytosolic
As shown in Table 1 and Figure 1, the IC50 and K values were
i
2
4
molecule . Recently, a lot of phenolic acid, phenols and phenolic isoenzyme hCA I, taxifolin had an IC50 value of 27.72 nM and a
2
+
derivatives were investigated in detail as inhibitors of the Zn
containing CA isoenzymes by our group
-
K
i
value of 29.20 ± 2.85 nM (Table 1). On the other hand, AZA is
. It was reported all a hCA I that is used for the medical treatment of glaucoma,
CA isoforms are inhibited by three different mechanisms: (i) altitude sickness, epileptic seizure, idiopathic intracranial hyper-
9
5–99
2
+
inhibition by coordination of the inhibitor to the Zn located tension, central sleep apnoea, cystinuria, periodic paralysis and
in the active site of CA isoenzymes, thereby replacing the dural ectasia. AZA was extensively used as positive control for
2
Zn -bound H O/–OH, which leads to a tetrahedral geometry for both CA isoenzymes. In the present study, AZA demonstrated
+
2
2
+
Zn . This geometry can also arise by the addition of an inhibitor IC50 values of 315.52 and 123.53 nM against hCA I and II,
Table 1. The inhibition profiles of taxifolin on purified hCA I and II from human erythrocytes by sepharose-4B-L-tyrosine-
sulfanilamide affinity chromatography and inhibition of AChE purified from electric gel (Electrophorus electricus).
Taxifolin
Kinetic parameters
IC50 (nM)
hCA I
27.7
hCA II
43.3
AChE
30.1
OH
OH
2
R
(nM)
Inhibition type
0.9858
29.2 ± 2.9
Non-competitive
0.9682
24.2 ± 6.5
Non-competitive
0.9723
16.7 ± 3.9
Non-competitive
HO
K
i
OH
OH
O

4
H. Gocer et al.
J Enzyme Inhib Med Chem, Early Online: 1–7
(
B) 50
40
Figure 1. Determination of the half maximal
inhibitory concentration (IC50) (A) and
inhibition constant (K ) values (B) of taxifolin
i
for human erythrocyte carbonic anhydrase I
isoenzyme (hCA I) by using a Lineweaver–
Burk graph.
(A) 100
[Control]
[8.20 nM]
[16.40 nM]
[32.90 nM]
7
5
2
5
0
5
0
3
2
1
0
0
0
0
0
20
40
−1
1
3
5
7
[
Taxifolin] (nM)
−1
1
/ [S] (mM)
(A) 100
(B) 60
Figure 2. Determination of the half maximal
inhibitory concentration (IC50) (A) and
[ Control]
[ 32.87 nM]
5
4
3
2
1
0
0
0
0
0
0
[
[
49.30 nM]
65.74 nM]
i
inhibition constant (K ) values (B) of taxifolin
7
5
2
5
0
5
0
for human erythrocyte carbonic anhydrase II
isoenzyme (hCA II) by using a Lineweaver–
Burk graph.
0
15
30
45
60
75
−
1
1
3
5
7
[
Taxifolin] (nM)
−
1
1
/ [S] (mM)
(A) 100
Figure 3. Determination of the half maximal
inhibitory concentration (IC50) value (A) and
(B) 250
[
Control]
i
inhibition constant (K ) value (B) of taxifolin
for acetylcholinesterase enzyme (AChE) by
using a Lineweaver–Burk graph.
[ 8.217 nM]
2
1
1
00
50
00
7
5
2
5
0
5
0
[
[
32.868 nM]
49.302 nM]
5
0
0
0
20
40
60
80
−
8
12
32
[
Taxifolin] (nM)
−1
/ [S] (mM)
1
2
respectively. On the other hand, its K values for both isoenzymes In addition to the Zn binding ligands (His 94, His 96 and His
+
i
were found as 184.34 and 61.12 nM, respectively. These results 119) discussed in the introduction, the His 64 residue of CA I play
clearly show that taxifolin had more CA isoenzyme inhibitor an important role in catalysis. Another difference between the two
effects than that of AZA. isozymes is that CA II contains a histidine cluster consisting of
As seen in Figure 2, for the physiologically predominant CA II, the following residues: His 64, His 4, His 3, His 10, His 15 and
taxifolin had an IC50 value of 43.31 nM and a K value of His 17 which are absent in CA I. Hence, these two isozymes
i
2
CA II is brought about by an inhibitor’s ability to bind to the a higher affinity for the inhibitor than CA I .
4.15 ± 6.45 nM. Many studies have shown that the inhibition of exhibit different affinities for the inhibitors. In general, CA II has
4
7
2
catalytic Zn in the CA active site and mimic the tetrahedral
+
hCA I is highly abundant in red blood cells and is found in
many tissues but its precise physiological function is unknown.
4
7,49,50
transition state
.
There are important differences in inhibition between the two CA I is associated with cerebral and retinal edema; thus, the
isoenzymes. The main difference is found in the active site inhibition of CA I may be a valuable tool for fighting these
architectures of the two hCA isoenzymes and is due to the conditions. The physiologically predominant cytosolic
4
7
presence of more histidine residues in the CA I isoform . isoform hCA II is ubiquitous, and it is associated with several

DOI: 10.3109/14756366.2015.1036051
Acetylcholinesterase and carbonic anhydrase isoenzymes I and II
_
¨ _
¨
l c¸ in I, B u¨ y u¨ kokuro g˘ lu ME, Oktay M, K u¨ frevio g˘ lu OI. On the
5
diseases including epilepsy, edema, glaucoma and altitude
6. Gu
in vitro antioxidant properties of melatonin. J Pineal Res 2002;33:
67–71.
5
6,57,105
sickness
AZA is a well-known example of a clinically established
CAI
.
1
_
¨ _
7. G u¨ l c¸ in I, B u¨ y u¨ kokurog
hydrogen peroxide scavenging effects of melatonin. J Pineal Res
003;34:278–81.
˘lu ME, K u¨ frevio g˘ lu OI. Metal chelating and
1
06,107
and in recent years we have reported its strong
inhibition of both human cytosolic CA I and II. CAI effects are
also exhibited by a wide spectrum of phenolic compounds
2
8
.
Gul c¸ in I, Beydemir S, Buyukokuroglu ME. In vitro and in vivo
effects of dantrolene on carbonic anhydrase enzyme activities. Biol
Pharm Bull 2004;27:613–16.
¨
4
including melatonin , morphine , vitamin E , CAPE , anti-
0
41
74
84
1
00
98
oxidant phenols , phenolic acids , natural product polyphenols
9
and phenolic acids , natural phenolic compounds
6
51,92,95
_
9
.
G u¨ l c¸ in I, Beydemir S¸ , Alici HA, et al. In vitro antioxidant properties
of morphine. Pharmacol Res 2004;49:59–66.
, anti-
9
2,95
oxidant polyphenol products
trihydroxyphenyl)methanone and its derivatives , natural and
(3,4-dihydroxyphenyl)(2,3,4-
5
_
2
10. G u¨ l c¸ in I, Berashvili D, Gepdiremen A. Antiradical and antioxidant
activity of total anthocyanins from Perilla pankinensis decne.
J Ethnopharmacol 2005;101:287–93.
5
7,91,108
synthetic bromophenols
aminoindanes and anilines , novel phenolic sulfamides , novel
, novel sulfonamide derivatives of
108
4
4
_
1
1
1
1. G u¨ l c¸ in I, Elias R, Gepdiremen A, Boyer L. Antioxidant activity of
lignans from fringe tree (Chionanthus virginicus L.). Eur Food Res
Technol 2006;223:759–67.
4
phenolic benzylamine derivatives , sulfonamide derivatives
5
60
3
8
and novel sulfamide analogues of dopamine-related compounds ,
5
new benzotropone derivatives , guaiacol and catechol deriva-
0
_
2. G o¨ c¸ er H, G u¨ l c¸ in I. Caffeic acid phenethyl ester (CAPE): correlation
5
tives , capsaicin , hydroquinone
5
109
110
of structure and antioxidant properties. Int J Food Sci Nutr 2011;62:
821–5.
_
3. G u¨ l c¸ in I, Beydemir S¸ , C¸ oban TA, Ekinci D. The inhibitory effect of
and brominated diphenyl-
methanone and its derivatives , novel sulfamides and
4
6
3
9
sulfonamides incorporating a tetralin scaffold . These extensive
studies indicate the importance of CA I and II isoenzyme
inhibitors.
AChE was very strongly inhibited by taxifolin (Table 1).
Taxifolin had an IC50 value of 30.1 nM and a K value of
dantrolene sodium and propofol on 6-phosphogluconate dehydro-
genase from rat erythrocyte. Fresen Environ Bull 2008;17:1283–7.
14. G u¨ l c¸ in I_ , Elias R, Gepdiremen A, et al. Antioxidant secoiridoids
from fringe tree (Chionanthus virginicus L.). Wood Sci Technol
2
009;43:195–212.
i
_
¨
1
5. S¸ i s¸ ecio g˘ lu M, C¸ ankaya M, G u¨ l c¸ in I, Ozdemir M. The Inhibitory
effect of propofol on lactoperoxidase. Protein Peptide Lett 2009;16:
1
6.7 ± 3.9 nM (Figure 3). On the other hand, donepezil hydro-
chloride, which is used to the treatment of mild-to-moderate AD
and various other memory impairments, had been shown to lower
4
6–9.
6. G u¨ l c¸ in I. Antioxidant activity of caffeic acid (3,4-dihydroxycin-
_
1
1
AChE inhibition activity (IC : 55 nM) . Donepezil hydrochlor-
06
5
0
namic acid). Toxicology 2006;217:213–20.
ide contains N-benzylpiperidine and an indanone moiety that 17. G u¨ l c¸ in I_ . Antioxidant and antiradical activities of L-carnitine. Life
shows longer and more selective action.
Sci 2006;78:803–11.
_
¨
1
8. S¸ i s¸ ecio g˘ lu M, C¸ ankaya M, G u¨ l c¸ in I, Ozdemir M. Interactions of
melatonin and serotonin to lactoperoxidase enzyme. J Enzyme Inhib
Med Chem 2010;25:779–83.
Conclusions
_
1
2
9. G u¨ l c¸ in I. Comparison of in vitro antioxidant and antiradical
activities of L-tyrosine and L-Dopa. Amino Acids 2007;32:431–8.
Taxifolin demonstrated unique inhibition profiles against both CA
isoforms I and II. These results demonstrated that in the light of
the high homology between these two CAs, they exhibit similar
activity. Taxifolin was first identified as a potent CAI because
_
0. G u¨ l c¸ in I. Measurement of antioxidant ability of melatonin and
serotonin by the DMPD and CUPRAC methods as trolox equivalent.
J Enzyme Inhib Med Chem 2008;23:871–6.
phenolic compounds with aromatic rings have previously been 21. S¸ i s¸ ecio g˘ lu M, G u¨ l c¸ in I_ , C¸ ankaya M, O¨ zdemir H. The inhibitory
identified as inhibitors of CA. In this study, nanomolar levels of
K and IC values were observed for taxifolin. We show that
effects of L-adrenaline on lactoperoxidase enzyme (LPO) purified
from buffalo milk. Int J Food Propert 2012;15:1182–9.
_
2. G u¨ l c¸ in I. Antioxidant activity of L-adrenaline: an activity-structure
insight. Chem Biol Interact 2009;179:71–80.
_
3. G u¨ l c¸ in I. Antioxidant properties of resveratrol: a structure-activity
insight. Innov Food Sci Emerg 2010;11:210–18.
_
4. G u¨ l c¸ in I. Antioxidant activity of eugenol – a structure and activity
relationship study. J Med Food 2011;14:975–85.
i
50
2
2
2
taxifolin is a selective inhibitor for both cytosolic CA isoenzymes.
These results clearly indicate the potential for use of bioactive
taxifolin to identifying more CAIs and for eventually targeting
additional isoforms. Additionally, taxifolin had effective AChE
inhibition properties and it can be a good candidate for the
treatment of mild-to-moderate AD and various other memory 25. Kim NC, Graf TN, Sparacino CM, et al. Complete isolation and
characterization of silybins and isosilybins from milk thistle
impairments.
(
Silybum marianum). Org Biomol Chem 2003;1:1684–9.
2
2
6. Weidmann AE. Dihydroquercetin: more than just an impurity? Eur J
Pharmacol 2012;684:19–26.
7. Vladimirov YA, Proskurnina EV, Demin EM, et al.
Dihydroquercetin (taxifolin) and other flavonoids as inhibitors of
free radical formation at key stages of apoptosis. Biochemistry
Declaration of interest
The authors declare no conflict of interest. I.G. and S.H.A. would like to
extend his sincere appreciation to the Deanship of Scientific Research at
King Saud University for its funding of this research through the Research
Group Project No. RGP-VPP-254.
(
Mosc) 2009;74:301–7.
2
2
3
8. Wang YH, Wang WY, Chang CC, et al. Taxifolin ameliorates
cerebral ischemia-reperfusion injury in rats through its anti-oxida-
tive effect and modulation of NF-kappa B activation. J Biomed Sci
References
2
006;13:127–41.
1
.
Rice-Evans CA, Miller NJ, Paganga G. Structure antioxitant activity
relationships of flavanoids and phenotic acids. Free Radical Biol
Med 1996;20;933–56.
9. Haraguchi H, Ohmi I, Fukuda A, et al. Inhibition of aldose reductase
and sorbitol accumulation by astilbin and taxifolin dihydroflavonols
in Engelhardtia chrysolepis. Biosci Biotechnol Biochem 1997;61:
_
2
.
.
G u¨ l c¸ in I. Antioxidant activity of food constituents – an overview.
Arch Toxicol 2012;86:345–91.
6
51–4.
0. Sun X, Chen RC, Yang ZH, et al. Taxifolin prevents diabetic
cardiomyopathy in vivo and in vitro by inhibition of oxidative stress
and cell apoptosis. Food Chem Toxicol 2014;63:221–32.
3
Makarova K, Lastawska K, Wagner D, Wawer I. ESR study of spin
trapping in Fenton media in the presence of taxifolin. J Mol Struct
2
014;1067:27–36.
_
31. Arutyunyan TV, Korystova AF, Kublik LN, et al. Effects of taxifolin
on the activity of angiotensin-converting enzyme and reactive
oxygen and nitrogen species in the aorta of aging rats and rats treated
with the nitric oxide synthase inhibitor and dexamethasone. Age
2013;35:2089–97.
4
5
.
.
G u¨ l c¸ in I, Da s¸ tan A. Synthesis of dimeric phenol derivatives and
determination of in vitro antioxidant and radical scavenging
activities. J Enzyme Inhib Med Chem 2007;22:685–95.
_
Ak T, G u¨ l c¸ in I. Antioxidant and radical scavenging properties of
curcumin. Chem Biol Interact 2008;174:27–37.

6
H. Gocer et al.
J Enzyme Inhib Med Chem, Early Online: 1–7
3
2. Middleton Jr E, Kandaswami C, Theoharides TC. The effects of 56. Sethi KK, Verma SM, Tanc M, et al. Carbonic anhydrase inhibitors:
plant flavonoids on mammalian cells: implications for inflamma-
tion, heart disease, and cancer. Pharmacol Rev 2000;52:673–51.
3. Cermak R, Wolffram S. The potential of flavonoids to influence
synthesis and inhibition of the human carbonic anhydrase isoforms I,
II, IX and XII with benzene sulfonamides incorporating 4- and 3-
nitrophthalimide moieties. Bioorg Med Chem 2014;22:1586–95.
3
drug metabolism and pharmacokinetics by local gastrointestinal 57. Bozta s¸ M, C¸ etinkaya Y, Topal M, et al. Synthesis and carbonic
mechanisms. Curr Drug Metab 2006;7:729–44.
4. Egert S, Rimbach G. Which sources of flavonoids: complex diets or
dietary supplements? Adv Nutr 2011;2:8–14.
anhydrase isoenzymes I, II, IX, and XII inhibitory effects of
dimethoxy-bromophenol derivatives incorporating cyclopropane
moieties. J Med Chem 2015;58:640–50.
3
3
5. Bou sˇ ov a´ I, H a´ jek J, Dr sˇ ata J, Sk a´ lov a´ L. Naturally occurring 58. Franchi M, Vullo D, Gallori E, et al. Carbonic anhydrase inhibitors:
flavonoids as inhibitors of purified cytosolic glutathione S-transfer-
ase. Xenobiotica 2012;42:872–9.
inhibition of human and murine mitochondrial isozymes V with
anions. Bioorg Med Chem Lett 2003;13:2857–61.
_
3
3
6. Supuran CT, Scozzafava A. Carbonic anhydrases as targets for 59. Taslimi P, Gulcin I, Ozgeris B, et al. The human carbonic anhydrase
medicinal chemistry. Bioorg Med Chem 2007;15:4336–50.
7. Zolnowska B, Slawinskia J, Pogorzelska A, et al. Carbonic
anhydrase inhibitors. Synthesis, and molecular structure of novel
isoenzymes I and II (hCA I and II) inhibition effects of
trimethoxyindane derivatives. J Enzyme Inhib Med Chem 2015;
DOI:10.3109/14756366.2015.1014476.
60. G o¨ c¸ er H, Akıncıo g˘ lu A, O¨ zta s¸ kın N, et al. Synthesis, antioxidant
0
series N-substituted N -(2-arylmethylthio-4-chloro-5-methyl benze-
and antiacetylcholinesterase activities of sulfonamide derivatives of
dopamine related compounds. Arch Pharm 2013;346:783–92.
1. Gocer H, Akıncıo g˘ lu A, Goksu S, et al. Carbonic anhydrase
and acetylcholine esterase inhibitory effects of carbamates
and sulfamoylcarbamates. J Enzyme Inhib Med Chem 2015;30:
nesulfonyl) guanidines and their inhibition of human cytosolic
isozymes I and II and the transmembrane tumor-associated isozymes
IX and XII. Eur J Med Chem 2014;71:135–47.
6
6
6
6
6
3
3
4
4
8. Aksu K, Nar M, Tanc M, et al. The synthesis of sulfamide analogues
of dopamine related compounds and their carbonic anhydrase
inhibitory properties. Bioorg Med Chem 2013;21:2925–31.
9. Akincioglu A, Topal M, G u¨ l c¸ in I, Goksu S. Novel sulfamides and
sulfonamides incorporating tetralin scaffold as carbonic anhydrase
and acetylcholine esterase inhibitors. Arch Pharm 2014;347:68–76.
0. Beydemir S, G u¨ l c¸ in I. Effect of melatonin on carbonic anhydrase
from human erythrocyte in vitro and from rat erythrocyte in vivo.
J Enzyme Inhib Med Chem 2004;19:193–7.
3
16–20.
2. Fulton MH, Key PB. Acetylcholinesterase inhibition in estuar-
ine fish and invertebrates as an indicator of organophosphorus
insecticide exposure and effects. Environ Toxicol Chem 2001;20:
3
7–45.
3. Allam AR, Sridhar GR, Das UN. Elevated butrylcholinesterase and
acetylcholinesterase may predict the development of type
diabetes mellitus and Alzheimer’s disease. Med Hypotheses 2007;
9:1272–6.
2
1. Coban TA, Beydemir S, G u¨ l c¸ in I, Ekinci D. Morphine inhibits
erythrocyte carbonic anhydrase in vitro and in vivo. Biol Pharm Bull
6
4. Greenblatt HM, Dvir H, Silman I, Sussman JL.
Acetylcholinesterase: a multifaceted target for structure-based drug
design of anticholinesterase agents for the treatment of Alzheimer’s
disease. J Mol Neurosci 2003;20:369–83.
2
007;30:2257–61.
4
4
2. Supuran CT. Carbonic anhydrases: novel therapeutic applications for
inhibitors and activators. Nat Rev Drug Discov 2008;7:168–81.
3. Akbaba Y, Akincioglu A, Gocer H, et al. Carbonic anhydrase
inhibitory properties of novel sulfonamide derivatives of aminoin-
danes and aminotetralins. J Enzyme Inhib Med Chem 2014;29:
5. Costa P, Gonccalves S, Valentao P, et al. Accumulation of phenolic
compounds in in vitro cultures and wild plants of Lavandula viridis
0
L H e´ r and their antioxidant and anti-cholinesterase potential. Food
Chem Toxicol 2013;57:69–74.
3
5–42.
4
4
4
4. Akbaba Y, Bastem E, Topal F, et al. Synthesis and carbonic
anhydrase inhibitory effects of novel sulfamides derived from 1-
aminoindanes and anilines. Arch Pharm 2014;347:950–7.
5. Cetinkaya Y, Gocer H, Goksu S, G u¨ l c¸ in I. Synthesis and carbonic
anhydrase isoenzymes inhibitory effects of novel benzylamine
derivatives. J Enzyme Inhib Med Chem 2014;29:168–74.
6
6
6
6. Hobbiger F. The inhibition of acetylcholinesterase by organophos-
phorus compounds and its reversal. Proc R Soc Med 1961;54:403–5.
7. Massoulie J, Pezzementi L, Bon S, et al. Molecular and cellular
biology of cholinesterases. Prog Neurobiol 1993;41:31–91.
8. Ryu HW, Curtis-Long MJ, Jung S, et al. Anticholinesterase potential
of flavonols from paper mulberry (Broussonetia papyrifera) and
their kinetic studies. Food Chem 2012;132:1244–50.
_
6. C¸ etinkaya Y, G o¨ c¸ er H, G u¨ l c¸ in I, Menzek A. Synthesis and carbonic
anhydrase isoenzymes inhibitory effects of brominated diphenyl-
methanone and its derivatives. Arch Pharm 2014;347:354–9.
7. Supuran CT, Scozzafava A. Carbonic anhydrase inhibitors and their
therapeutic potential. Expert Opin Ther Pat 2000;10:575–600.
8. Mincione F, Scozzafava A, Supuran CT. In drug design of zinc-
enzyme inhibitors: functional, structural, and disease applications.
Hoboken: Wiley; 2009:139.
6
9. Szwajgier D. Anticholinesterase activities of selected polyphenols –
a short report. Pol J Food Nutr Sci 2014;64:59–64.
0. Krikorian R, Shidler MD, Nash TA, et al. Blueberry supplementa-
tion improves memory in older adults. J Agric Food Chem 2010;58:
4
4
7
3
996–4000.
7
1. Gutierres JM, Carvalho FB, Schetinger MR, et al. Anthocyanins
restore behavioral and biochemical changes caused by streptozoto-
cin-induced sporadic dementia of Alzheimer’s type. Life Sci 2013;
4
5
9. Supuran CT. Carbonic anhydrase inhibitors. Bioorg Med Chem Lett
010;20:3467–74.
2
9
6:7–17.
0. G u¨ ney M, Coskun A, Topal F, et al. Oxidation of cyanobenzocy-
cloheptatrienes: synthesis, photooxygenation reaction and carbonic
anhydrase isoenzymes inhibition properties of some new benzo-
tropone derivatives. Bioorg Med Chem 2014;22:3537–43.
1. G u¨ l c¸ in I, Beydemir S¸ . Phenolic compounds as antioxidants: carbonic
anhydrase isoenzymes inhibitors. Mini Rev Med Chem 2013;13:
7
7
7
2. Pervin M, Hasnat MdA, Lee YM, et al. Antioxidant activity and
acetylcholinesterase inhibition of grape skin anthocyanin (GSA).
Molecules 2014;19:9403–18.
3. Zhang DB, Chen JJ, Song QY, et al. Lycodine-type alkaloids from
Lycopodiastrum casuarinoides and their acetylcholinesterase inhibi-
tory activity. Molecules 2014;19:9999–10010.
4. Aras Hisar S, Hisar O, Beydemir S, et al. Effect of vitamin E on
carbonic anhydrase enzyme activity in rainbow trout (Oncorhynchus
mykiss) erythrocytes in vitro and in vivo. Acta Vet Hung 2004;52:
5
5
4
08–30.
2. Nar
3,4-Dihydroxyphenyl)(2,3,4-trihydroxyphenyl)methanone and its
M,
Cetinkaya
Y,
Gulcin
I,
Menzek
A.
(
derivatives as carbonic anhydrase isoenzymes inhibitors. J Enzyme
Inhib Med Chem 2013;28:402–6.
3. De Simone G, Di Fiore A, Supuran CT. Are carbonic anhydrase
inhibitors suitable for obtaining antiobesity drugs? Curr Pharm Des
4
13–22.
7
7
5. Coban TA, Beydemir S, G u¨ l c¸ in I, Ekinci D. The inhibitory effect of
ethanol on carbonic anhydrase isoenzymes: in vivo and in vitro
studies. J Enzyme Inhib Med Chem 2008;23:266–70.
6. Senturk M, G u¨ l c¸ in I, Beydemir S, et al. In vitro inhibition of human
carbonic anhydrase I and II isozymes with natural phenolic
compounds. Chem Biol Drug Des 2011;77:494–9.
5
5
5
2
008;14:655–60.
4. G o¨ ksu S, Naderi A, Akbaba Y, et al. Carbonic anhydrase inhibitory
properties of novel benzylsulfamides using molecular modeling and
experimental studies. Bioorg Chem 2014;56:75–82.
77. Atasaver A, O¨ zdemir H, G u¨ l c¸ in I_ , K u¨ frevio g˘ lu O¨ I_ . One-step
_
5. Scozzafava A, Passaponti M, Supuran CT, G u¨ l c¸ in I, Carbonic
anhydrase inhibitors: guaiacol and catechol derivatives effectively
purification of lactoperoxidase from bovine milk by affinity
chromatography. Food Chem 2013;136:864–70.
inhibit certain human carbonic anhydrase isoenzymes (hCA I, II, IX, 78. Laemmli DK. Cleavage of structural proteins during the
and XII). J Enzyme Inhib Med Chem 2015; DOI:10.3109/
1
assembly of the head of bacteriophage T4. Nature 1970;
227:680–3.
4756366.2014.956310.

DOI: 10.3109/14756366.2015.1036051
Acetylcholinesterase and carbonic anhydrase isoenzymes I and II
7
7
8
8
9. G u¨ l c¸ in I, Kufrevioglu OI, Oktay M. Purification and characteriza-
tion of polyphenol oxidase from nettle (Urtica dioica L.) and
inhibition effects of some chemicals on the enzyme activity.
J Enzyme Inhib Med Chem 2005;20:297–302.
96. Innocenti A, Ozturk Sarıkaya SB, G u¨ l c¸ in I, Supuran CT. Carbonic
anhydrase inhibitors. Inhibition of mammalian isoforms I-XIV with
a series of natural product polyphenols and phenolic acids. Bioorg
Med Chem 2010;18:2159–64.
97. Innocenti A, Vullo D, Scozzafava A, Supuran CT. Carbonic
anhydrase inhibitors. Interactions of phenols with the 12 catalyt-
ically active mammalian isoforms (CA I-XIV). Bioorg Med Chem
Lett 2008;18:1583–7.
0. Beydemir S, G u¨ l c¸ in I, Hisar O, et al. Effect of melatonin on glucose-
6
-phospate dehydrogenase from rainbow trout (Oncorhynchus
mykiss) erythrocytes in vitro and in vivo. J Appl Anim Res 2005;
8:65–8.
2
1. Hisar O, Beydemir S, G u¨ l c¸ in I, et al. The effect of melatonin
hormone on carbonic anhydrase enzyme activity in rainbow trout
¨
9
8. Ozturk Sarikaya SB, Gul c¸ in I, Supuran CT. Carbonic anhydrase
inhibitors. Inhibition of human erythrocyte isozymes I and II with a
series of phenolic acids. Chem Biol Drug Des 2010;75:515–20.
9. Khoddami A, Wilkes MA, Roberts TH. Techniques for analysis of
plant phenolic compounds. Molecules 2013;18:2328–75.
(Oncorhynchus mykiss) erythrocytes in vitro and in vivo. Turk J Vet
Anim Sci 2005;29:841–5.
9
8
8
2. Verpoorte JA, Mehta S, Edsall JT. Esterase activities of human
carbonic anhydrases B and C. J Biol Chem 1967;242:4221–9.
3. Coban TA, Beydemir S, G u¨ l c¸ in I, et al. Sildenafil is a strong
activator of mammalian carbonic anhydrase isoforms I–XIV. Bioorg
Med Chem 2009;17:5791–5.
1
1
00. Nair SK, Ludwig PA, Christianson DW. Phenol as a carbonic
anhydrase inhibitor. J Am Chem Soc 1994;116:3659–60.
01. Innocenti A, Vullo D, Scozzafava A, Supuran CT. Carbonic
anhydrase inhibitors: inhibition of mammalian isoforms I–XIV
with a series of substituted phenols including paracetamol and
salicylic acid. Bioorg Med Chem 2008;16:7424–8.
8
8
8
8
4. Gocer H, G u¨ l c¸ in I. Caffeic acid phenethyl ester (CAPE): a potent
carbonic anhydrase isoenzymes inhibitor. Int J Acad Res 2013;5:
1
50–5.
1
1
02. Davis RA, Innocenti A, Poulsen SA, Supuran CT. Carbonic
anhydrase inhibitors. Identification of selective inhibitors of the
human mitochondrial isozymes VA and VB over the cytosolic
isozymes I and II from a natural product-based phenolic library.
Bioorg Med Chem 2010;18:14–18.
03. Hisar O, Beydemir S, G u¨ l c¸ in I, et al. Effect of low molecular
weight plasma inhibitors of rainbow trout (Oncorhyncytes mykiss)
on human erythrocytes carbonic anhydrase-II isozyme activity
in vitro and rat erythrocytes in vivo. J Enzyme Inhib Med Chem
5. Bradford MM. A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye
binding. Anal Biochem 1976;72:248–51.
_
6. Koksal E, G u¨ l c¸ in I. Purification and characterization of peroxidase
from cauliflower (Brassica oleracea L.) buds. Protein Peptide Lett
2
008;15:320–6.
7. Senturk M, G u¨ l c¸ in I, Ciftci M, Kufrevioglu OI. Dantrolene inhibits
human erythrocyte glutathione reductase. Biol Pharm Bull 2008;31:
2
036–9.
2
005;20:35–9.
8
8
8. Lineweaver H, Burk D. The determination of enzyme dissociation
constants. J Am Chem Soc 1934;56:658–66.
9. Yıldırım A, Atmaca U, Keskin A, et al. N-Acylsulfonamides
strongly inhibit human carbonic anhydrase isoenzymes I and II.
Bioorg Med Chem 2015; DOI:10.1016/j.bmc.2014.12.054.
1
1
1
1
04. Akbaba Y, T u¨ rke s¸ C, Polat L, et al. Synthesis and paroxonase
activities of novel bromophenols. J Enzyme Inhib Med Chem 2013;
2
8:1073–9.
05. Akincioglu A, Akbaba Y, Gocer H, et al. Novel sulfamides as
potential carbonic anhydrase isoenzymes inhibitors. Bioorg Med
Chem 2013;21:1379–85.
06. Ogilvie JM, Ohlemiller KK, Shah GN, et al. Carbonic anhydrase
XIV deficiency produces a functional defect in the retinal light
response. Proc Natl Acad Sci USA 2007;104:8514–19.
07. Avvaru BS, Busby SA, Chalmers MJ, et al. Apo-human carbonic
anhydrase II revisited: implications of the loss of a metal in protein
structure, stability, and solvent network. Biochemistry 2009;48:
_
9
0. Cetinkaya Y, Gocer H, Menzek A, G u¨ l c¸ in I. Synthesis and
antioxidant properties of (3,4-dihydroxyphenyl)(2,3,4-trihydroxy-
phenyl)methanone and its derivatives. Arch Pharm 2012;345:
3
23–34.
9
9
9
1. Akbaba Y, Balaydın HT, Menzek A, et al. Synthesis and biological
evaluation of novel bromophenol derivatives as carbonic anhydrase
inhibitors. Arch Pharm 2013;346:447–54.
2. Ozturk Sarikaya SB, Topal F, Senturk M, et al. In vitro inhibition of
a-carbonic anhydrase isozymes by some phenolic compounds.
Bioorg Med Chem Lett 2011;21:4259–62.
3. Ellman GL, Courtney KD, Andres V, Featherston RM. A new and
rapid colorimetric determination of acetylcholinesterase activity.
Biochem Pharmacol 1961;7:88–95.
7
365–72.
1
08. Balaydin HT, G u¨ l c¸ in I, Menzek A, et al. Synthesis and antioxidant
properties of diphenylmethane derivative bromophenols including
a natural product. J Enzyme Inhib Med Chem 2010;25:685–95.
109. Arabaci B, Gu
¨
l c¸ in I, Alwasel S. Capsaicin: a potent inhibitor of
_
carbonic anhydrase isoenzymes. Molecules 2014;19:10103–14.
110. Scozzafava A, Kalın P, Supuran CT, et al. The impact of
hydroquinone on acetylcholine esterase and certain human car-
bonic anhydrase isoenzymes (hCA I, II, IX and XII).
9
9
4. Topal M, G u¨ l c¸ in I. Rosmarinic acid: a potent carbonic anhydrase
isoenzymes inhibitor. Turk J Chem 2014;38:894–902.
_
5. Innocenti A, G u¨ l c¸ in I, Scozzafava A, Supuran CT. Carbonic
anhydrase inhibitors. Antioxidant polyphenol natural products
effectively inhibit mammalian isoforms I-XV. Bioorg Med Chem
Lett 2010;20:5050–3.
J Enzyme Inhib Med Chem 2015; DOI: 10.3109/
14756366.2014.999236.