Journal of Enzyme Inhibition and Medicinal Chemistry

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Inhibition properties of propolis extracts to some

clinically important enzymes

Nimet Baltas, Oktay Yildiz & Sevgi Kolayli

To cite this article: Nimet Baltas, Oktay Yildiz & Sevgi Kolayli (2016) Inhibition properties of
propolis extracts to some clinically important enzymes, Journal of Enzyme Inhibition and Medicinal
Chemistry, 31:sup1, 52-55, DOI: 10.3109/14756366.2016.1167049

To link to this article: http://dx.doi.org/10.3109/14756366.2016.1167049

Published online: 07 Apr 2016.

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ISSN: 1475-6366 (print), 1475-6374 (electronic)

J Enzyme Inhib Med Chem, 2016; 31(S1): 5255

! 2016 Informa UK Limited, trading as Taylor & Francis Group. DOI: 10.3109/14756366.2016.1167049

RESEARCH ARTICLE

Inhibition properties of propolis extracts to some clinically important

enzymes
Nimet Baltas1, Oktay Yildiz2, and Sevgi Kolayli3
1
Department of Chemistry, Faculty of Arts & Science, Recep Tayyip Erdogan University, Rize, Turkey, 2Macka Vocational School, Karadeniz Thecnical
University, Trabzon, Turkey, and 3Department of Chemistry, Faculty of Science, Karadeniz Thecnical University, Trabzon, Turkey

Abstract
Downloaded by [114.124.138.238] at 23:04 13 September 2017

Keywords
The present study was conducted to envisage inhibition effects of propolis on the crucial Acetylcholinesterase, enzyme inhibition,
enzymes, urease, xanthine oxidase (XO) and acetylcholinesterase (AChE). Some of the propolis, urease, xanthine oxidase
antioxidant properties of the propolis samples were determined using the total phenolic
content (TPE) and total flavonoids in the eight different ethanolic propolis extracts (EPE) History
samples. Inhibition values of the enzymes were expressed as inhibition concentration (IC50; mg/
mL or mg/mL) causing 50% inhibition of the enzymes with donepezil, acetohydroxamic acid and Received 5 February 2016
allopurinol as reference inhibitors. All the propolis extracts exhibited variable inhibition effects Revised 7 March 2016
on these enzymes, but the higher the phenolic contents the lower the inhibitions values (IC50 Accepted 9 March 2016
0.074 to 1.560 mg/mL). IC50 values of the P5 propolis sample having the highest TPE, obtained Published online 5 April 2016
from Zonguldak, for AChE, urease and XO were 0.081 0.009, 0.080 0.006 and
0.074 0.011 mg/mL, respectively. The EPE proved to be a good source of inhibitor agents
that can be used as natural inhibitors to serve human health.

Introduction diseases3, and the inhibition of urease prevents gastric disorders

caused by Helicobacter pylori3,13,14.
Bee products like honey, pollen and propolis have been used in
Additionally to the literature, this work covers the investigations
traditional and complementary medicine from ancient times.
of urease, AChE and XO enzymes inhibition properties of the
Scientific studies reveal that the natural products are effective in
different propolis extracts and the relation between their phenolic
treating and protecting human health. The treatment with bee
contents and inhibition values. The aim of these observations was
products is called apitherapy14. Propolis is a resinous mixture
to evaluate inhibitory potentials of the bee product and that it can
used to protect hives from many diseases and threats. The
be used for clinical purposes as pharmaceutical agents.
composition of propolis depends mainly on floral sources, it
contains nearly 4050% resins, 30% wax, 510% essential oils and
Materials and methods
310% phenolic substances, such as phenolic acids, flavonoids,
tannins, etc.5,6 Propolis is a good pharmaceutical mixture that Chemicals
includes many biological active compounds and has many
Folin-Ciocalteus phenol reagent, Trolox (6-hydroxy-2,5,7,8-
biological active features such as antioxidants, antimicrobial,
tetramethylchroman-2-carboxylic acid), gallic acid and quercetin
anti-inflammatory, anti-tumoral, hepatoprotective and anti-neu-
were purchased from Sigma Chemical Co. (St Louis, MO).
rodegenerative activities611.
Enyzmes of AChE (from Electric Eel), urease (Jack Bean Urease)
As planned in this study, inhibitory effects of propolis samples
and XO (bovine milk xanthine oxidase), standard inhibitors and
were investigated on acetylcholinesterase (AChE), urease and
substrates such as acetohydroxamic acid, allopurinol, donepezil,
xanthine oxidase (XO) enzymes which are crucial for human
xanthine, acetylthiocholine chloride (ATC) and 5,5-dithio-bis(2-
health. The inhibition of the catalytic functions of these enzymes
nitrobenzoic) acid (DTNB) were obtained from Sigma-Aldrich,
is important for the treatment of many diseases. For example,
St. Louis, MO.
AChE inhibition is related to many neurodegenerative diseases
such as Alzheimers disease and Parkinsons12, XO inhibition
Samples
obstructs accumulation of uric acid and stops the growth of gout
Raw propolis samples were obtained from experienced bee-
keepers in 2015 in different areas of Turkey. Only one raw
Address for correspondence: Prof. Dr. Sevgi Kolayli, Department propolis sample was purchased from bee product markets in
of Chemistry, Faculty of Science, Karadeniz Technical University, Brazil. Raw propolis samples were frozen at 20  C than grinded
61080 Trabzon, Turkey. Tel: +90-0462-3773000. E-mail:
skolayli@yahoo.com
to powder. For extraction, 5 g of the powdered propolis was
Asst. Prof. Dr. Nimet Baltas, Department of Chemistry, Faculty of Arts placed with 50 mL 70% ethanol in a glass flask and stirred on a
and Sciences, Recep Tayyip Erdogan University, 53100 Rize, Turkey. Tel: shaker (Heidolph Promax 2020, Schwabach, Germany) at room
+90-464-2236126. E-mail: nimet.baltas@erdogan.edu.tr temperature for 24 h. The suspension was centrifuged at 10 000 g
 DOI: 10.3109/14756366.2016.1167049
for 15 min, then supernatants were evaporated. The residue was
resolved in minimal volumes of 70% ethanol.
Total phenolic contents
The total phenolic compounds of the ethanolic extracts of propolis
samples were determined using the Folin-Ciocalteu method15.
Briefly, 680 mL distilled water, 20 mL propolis extract, 400 mL of
0.2 N Folin reagent and 400 mL Na2CO3 (7.5%) were added in
a test tube. After 2 h of incubation at room temperature, the
absorbance was measured at 740 nm. The result was expressed as
mg of gallic acid equivalents per g samples.
Total flavanoid contents
The total flavonoid concentration was measured using a spectro-
metric assay. Briefly, 0.5 mL samples, 0.1 mL of 10% Al(NO3)3
and 0.1 mL of 1 M NH4(CH3COO) were added to a test tube and
incubated at room temperature for 40 min. The absorbance was
measured against a blank at 415 nm. Quercetin was used for the
standard calibration curve. The total flavonoid concentration was
expressed as mg of quercetin equivalents per 100 g sample16.
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Urease inhibition assay
Urease catalyzes the hydrolysis of urea into carbon dioxide and
ammonia. The production of the ammonia was measured using the
indophenol method17. Reaction mixtures including 200 mL of Jack
Bean Urease, 500 mL of buffer (100 mM urea, 0.01 M K2HPO4,
1 mM EDTA and 0.01 M LiCl, pH 8.2) and 100 mL of the propolis
extract were incubated at room temperature for 20 min. The phenol
reagent (550 mL, 1% w/v phenol and 0.005% w/v sodium
nitroprusside) and alkali reagent (650 mL, 0.5% w/v sodium
hydroxide and 0.1% v/v NaOCl) were added to each tube and an
increasing absorbance at 625 nm was measured after 50 min, using
a UV/vis spectrophotometer (1601UV-Shimadzu, Australia).
Acetohydroxamic acid (AA) was used as standard inhibitor. In
order to calculate IC50 values, different concentrations of each
extracts and standards were assayed at the same reaction conditions.
The inhibition concentrations of the extracts (IC50) were calculated
from the dose response curve, which reduced absorbance by 50%.
In vitro anti-xanthine oxidase assay
The inhibition of XO was measured with the UV spectroscopy
technique at 295 nm which attributes to released uric acid from
xanthine. The inhibitory activity of each extract was determined
using a slight modification of the reference methods18,19. Briefly,
the reaction mixture consisted of 500 mL of the propolis extract,
770 mL of phosphate buffer (pH 7.8) and 70 mL of bovine milk XO
(Sigma-Aldrich, St. Louis, MO), which was prepared immediately
before usage. After preincubation at 25  C for 15 min the reaction
was initiated by the addition 660 mL of substrate solution into the
mixture. The assay mixture was incubated at 25  C for 15 min.
The reaction was stopped by adding 200 mL of 0.5 N HCl and the
absorbance was measured at 295 nm using UV/VIS spectropho-
tometer (1601UV-Shimadzu, Australia). A well-known XO
inhibitor (XOI), allopurinol (Sigma-Aldrich, St. Louis, MO) was
used as a positive control for the inhibition test. The assay was
done in triplicate for calculating standard deviation. The IC50
values of extracts were determined as the concentration of extract
that give 50% inhibition of maximal absorbance.
Acetylcholinesterase inhibition assay
Propolis extracts were subjected to the method of Ellmans test20
in order to evaluate their potency to inhibit the AChE from
Electric Eel (Sigma-Aldrich, St. Louis, MO). This method is
Inhibition properties of EPE 53
based on the reaction of released thiocholine to give color to a
product with a chromogenic reagent DTNB. Enzyme solution was
prepared in gelatin solution (1%), at a concentration of 2.5 units/
mL. AChE (50 mL) and propolis samples (50 mL) were added to
3.0 mL phosphate buffer (pH 8.0, 0.1 M) and incubated at 25  C
for 5 min. The reaction was started by adding DTNB (100 mL) and
ATC (20 mL) to the enzymeinhibitor mixture. The production of
the yellow anion was recorded after 10 min at 412 nm, using a UV/
VIS spectrophotometer (1601UV-Shimadzu, Australia). As a
control, an identical solution of the enzyme without the inhibitor
was processed the same protocol. Donepezil hydrochloride was
used as a positive control. All processes were assayed in triplicate.
Statistical analysis
The data were calculated in the form of arithmetical mean values
and standard deviations. The SPSS 13.00 for Windows software
package was used for the statistical analysis of the gathered data
(SPSS Inc., Chicago, IL). Significance of the results was based on
the KruskalWallis test and Pearson correlations, significant
differences were p50.05.
Results and discussion
Total phenolic contents and enzyme inhibition studies
The results of the phenolic contents and total flavonoids of the
ethanolic propolis extracts (EPE) were summarized in Table 1. The
total phenolic contents (TPC) of the EPE samples were changed
between 88.675 and 261.055 mg GAE/g (Table 1). Eight different
propolis samples have also shown different amounts of the TPE and
total flavonoids. It was interesting that all propolis samples had
significantly different TPC, and the highest phenolic contents were
found in the P5 and P8 samples which were the Zonguldak propolis
and the Brazil red propolis, respectively.
It was reported that the TPE was found in Chinese propolis
ranged from 43 to 302 mg GAE/g21. The TPE was reported to be
between 115210 mg GAE/g in different origin of Turkish
propolis22. A slightly narrow range of TPE was reported in the
Poland propolis samples between 150 and 190 mg GAE/g7.
Therefore, the Zonguldak and the Brazilian red propolis were
distinguished from the other propolis samples. The total flavon-
oids of the propolis samples were ranged from 37.526 to
150.412 mg Q/100 g (Table 1). The highest total flavonoids was
in the P5 (Zonguldak) propolis, followed by the Brazilian red
propolis. Much smaller differentiations in the amount of total
flavonoids in the Poland propolis samples, between 3562 mgQ/
100 g, were reported7. Comparing the studied propolis to other
studies, the range of the TPE and flavonoids was wider which may
be explained by the different origins of the samples7. A strong
significant correlation between the TPE and total flavonoids in
the samples (R2: 0.950, p50.01, Table 2) was found. Many
studies have reported that propolis samples contained both high
total phenolics and high total flavonoids4,7.
All propolis extracts showed variable inhibition values,
ranging from 0.081 to 1.353 mg/mL for AChE (Table 1). While
the P3 sample showed the highest IC50 value (1.353 mg/mL), the
lowest IC50 value (0.081 mg/mL) was found in the P5 propolis
sample (Zonguldak propolis). Water-soluble propolis extract was
reported to be used as food supplements which improved
memorial functions in mice by inhibited AChE23. A strong
significant negative correlation between the TPE and AChE
activity of the EPE extracts (R2: 0.679, p50.01, Table 2) has
been found. In recent years, some in vivo and in vitro investiga-
tions indicated that polyphenol-rich extracts have substantial
inhibitory activity of AChE and improve memory in experimental
animals23,24.
 54 N. Baltas et al. J Enzyme Inhib Med Chem, 2016; 31(S1): 5255

Table 1. IC50 values of each enzymes and antioxidant properties and location of the ethanolic propolis extracts*.

Inhibition of
Total phenolic Total flavonoids acetylcholinesterase Inhibition of xanthine Inhibition of urease
Samples Location contents mg GAE/g mg Q/100g IC50 (mg/mL) oxidase IC50 (mg/mL) IC50 (mg/mL)
P1 Ankara, Turkey 138.812 3.831abc 65.406 7.803bc 1.340 0.020d 0.188 0.010b 0.144 0.009ab
P2 Kars, Turkey 170.461 8.090c 72.215 4.605c 0.340 0.045b 0.195 0.002b 0.170 0.027ab
P3 Nahcvan, Azerbaycan 88.675 4.562a 37.526 7.810a 1.353 0.062d 0.763 0.011d 1.560 0.045f
P4 Erzurum 150.013 10.643abc 65.580 3.250bc 0.315 0.014ab 0.165 0.004b 0.411 0.398d
P5 Zonguldak, Turkey 261.055 2.500e 150.412 12.505e 0.081 0.009a 0.074 0.011a 0.080 0.006a
P6 Duzce, Turkey 138.321 6.043abc 52.243 1.452abc 0.882 0.022c 0.356 0.031b 0.301 0.027cd
P7 Giresun, Turkey 114.207 7.207abc 45.086 3.004abc 1.053 0.016c 0.387 0.017b 0.240 0.017bc
P8 Red, propolis, Brazil 220.205 5.101d 98.303 1.702d 0.221 0.013ab 0.317 0.023b 0.680 0.051e
AA 25.110 0.120
Allopurinol 0.520 0.010
Donepezil 16.800 0.110

*Means standard deviations; different letters in the same lines are significantly different at the 5% level (p50.05). IC50 values of acetohydroxamic
acid (AA), donepezil and allopurinol were given in terms of mg/mL.

Table 2. Correlations between studied parameters.

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Total phenolic Total Inhibition of Inhibition of Inhibition

contents flavonoids acetylcholinesterase xanthine oxidase of urease
Total phenolic contents Pearson Correlation 1 0.950** 0.679** 0.542** 0.371
p, Sig. (two-tailed) 0.000 0.000 0.006 0.074
N 24 24 24 24 24
Total flavonoids Pearson Correlation 0.950** 1 0.640** 0.563** 0.364
p, Sig. (two-tailed) 0.000 0.001 0.004 0.080
N 24 24 24 24 24
Inhibition of acetylcholinesterase Pearson Correlation 0.679** 0.640** 1 0.646** 0.372
p, Sig. (two-tailed) 0.000 0.001 0.001 0.073
N 24 24 24 24 24
Inhibition of xanthine oxidase Pearson Correlation 0.542** 0.563** 0.646** 1 0.882**
p, Sig. (two-tailed) 0.006 0.004 0.001 0.000
N 24 24 24 24 24
Inhibition of urease Pearson Correlation 0.371 0.364 0.372 0.882** 1
p, Sig. (two-tailed) 0.074 0.080 0.073 0.000
N 24 24 24 24 24

**Correlation is significant at the 0.01 level (two-tailed).

Previous studies as well as our results have shown that the
relationship between propolis phenolic contents and the results of
AChE inhibition gives us important information that the enzyme
was probably inhibited by phenolic substances. We also compared
our inhibition values to donepezil that is frequently used as AChE
inhibitor in Alzheimers disease25. The IC50 value of donepezil
was 0.0168 mg/mL, smaller than our inhibition results of the
propolis samples. However, propolis is a complex extract, specific
phenolics in this mixture may exhibit higher AChE inhibitions at
smaller concentrations.
In this study, the second enzyme used to examine inhibition
effects of EPE extracts was XO which catalyzes the formation of
uric acid from purine degradation. XOI block the production of
uric acid. The enzyme XO is responsible for oxidative damage
that causes many pathological diseases, such as gout, hyperur-
icemia, atherosclerosis, hepatitis, carcinogenesis, vascular endo-
thelium damage and ageing2629. Allopurinol has been the sole
XOI under the clinical application for the past three decades30.
However, this drug gives inevitably rise to severe adverse effects
such as hepatitis, nephropathy, allergic reactions and 6-mercapto-
purine toxicity31.
All of the EPE inhibited XO enzyme at a wide inhibition
degree, ranging from 0.074 to 0.387 mg/mL (Table 1). The P5
sample from the studied EPE was distinguished from the others,
showing the highest inhibition effect. When comparing the results
to their TPE, there was a strong relation between TPC and XO
inhibition results (R2: 0.542, p50.01, Table 2). Similarly, a
number of natural compounds, rich in polyphenolic agents, such as
caffeic acid32,33, rutin34, and chestnut and oak honeys3, have been
reported to inhibit XO, and foods rich in phenolic compounds are
recommended to reduce blood concentrations of uric acid in gout.
Also, correlation coefficients and significant values (Table 2)
show positive strong correlations between inhibition of XO and
inhibition of urease (R2: 0.882; p: 0.000); inhibition of AChE and
inhibition of inhibition of XO (R2: 0.646; p: 0.001).
H. pylori is an anaerobic microorganism that survives in an
acidic environment with the help of the enzyme urease, a Nichel-
metallo enzyme, causing numerous gastric disorders. The main
survival way of this bacterium is being prevented from adhering
in the gastric system by urease inhibition, urease inhibitors are
particularly important in the treatment of gastric ulcers. Propolis
is accepted as a natural antibiotic, and inhibits many infection
bacteria as well as H. pylori35,36. All EPE inhibited urease with
different extract concentration, changing from 0.080 to 0.301 mg/
mL (Table 1). The P5 sample showed the highest activity (the
lowest IC50 value) in lower concentration compared to others
propolis samples (Table 1).
For the results of this study, the P5 propolis sample is
distinguished from other propolis, having the highest total
phenolics as well as total flavonoids and it showed the highest
inhibition effect (the lowest IC50 value) against AChE, urease and
XO enzymes. The P5 propolis sample was obtained from
Zonguldak, in the east of the Black Sea Region, where the flora
is richer in chestnut trees and flowers37. However, chestnut honey
 DOI: 10.3109/14756366.2016.1167049
is also a valuable product having high phenolic contents and
antioxidant capacity38.
Our study indicated that propolis has many pharmacokinetic
and pharmaceutical properties, contains many inhibitor agents, its
inhibition effect is related to the total phenolic substances, which
is effected by floral sources. Consequently, the propolis extracts
exhibited good potentials towards the inhibition of urease, XO and
AchE. Consequently, the propolis extracts exhibited a good
potential towards the inhibition of urease, XO and AchE. Propolis
is a mixture containing many bioactive compounds and which
compounds inhibited these enzymes are not clear. But, phenolic
compounds (phenolic acids and flavonoids) present in the propolis
extracts to be appear responsible for these inhibitions. For this
reason, further studies are necessary in order to account the
inhibition mechanism. Considering that some commercially
urease, XO and AchE inhibitors such as acetohydroxamic,
allopurinol and donepezil could not be developed into therapeutic
agents because of their toxicity and rapid metabolic degradation.
In conclusion, we think that regular consumption of these natural
products rich in phenolic compounds can contribute to a reduction
in several forms of H. pylori-associated diseases, neurodegenera-
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tive and gout disease.
Declaration of interest
This study was supported by the project of Tubitak (114Z370).
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