International Journal of Biological Macromolecules 189 (2021) 65–71

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International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

Inhibition effect of nicotinamide (vitamin B3) and reduced glutathione

(GSH) peptide on angiotensin-converting enzyme activity purified from
sheep kidney
Zehra Bas *
Van Yüzüncü Yıl University, Faculty of Health Sciences, Department of Nutrition and Dietetics, Van, Turkey

A R T I C L E I N F O A B S T R A C T

Keywords: Angiotensin-converting enzyme (ACE, EC 3.4.15.1) plays a significant role in blood pressure regulation and
Angiotensin-converting enzyme (ACE) inhibition of this enzyme is one of the significant drug targets for the treatment of hypertension. In this work,
Nicotinamide (vitamin B3) ACE was purified from sheep kidneys with the affinity chromatography method in one step. The purity and
Reduced glutathione (GSH)
molecular weight of ACE were designated using the SDS-PAGE method and observed two bands at around 60 kDa
Antioxidant
Purification
and 70 kDa on the gel. The effects of nicotinamide (vitamin B3) and reduced glutathione (GSH) peptide on
Inhibition purified ACE were researched. Nicotinamide and GSH peptide on purified ACE showed an inhibition effect. IC50
values for nicotinamide and GSH were calculated as 14.3 μM and 7.3 μM, respectively. Type of inhibition and Ki
values for nicotinamide and GSH from the Lineweaver-Burk graph were determined. The type of inhibition for
nicotinamide and GSH was determined as non-competitive inhibition. Ki value was calculated as 15.4 μM for
nicotinamide and 6.7 μM for GSH. Also, GSH peptide showed higher inhibitory activity on ACE activity than
nicotinamide. In this study, it was concluded that nicotinamide and GSH peptide compounds, which show an
inhibition effect on ACE activity, may have both protective and therapeutic effects against hypertension.

1. Introduction chamomilla L. plants indicated an inhibition effect on ACE purified from

Hypertension, known as high blood pressure, is one of the most
significant health problems in the world. Also, hypertension can be a risk
factor for stroke, heart attack, atherosclerosis, and myocardial infarc­
tion. Angiotensin-converting enzyme (ACE, dipeptidyl carboxypepti­
dase, E.C 3.4.15.1) in the renin-angiotensin system plays a substantial
role in the regulation of blood pressure by transmuting inactive decap­
eptide angiotensin I to octapeptide angiotensin II. Also, ACE is regulated
electrolyte homeostasis via the equilibrium of salt and water in the body
[1,2]. The most commonly used hypertension drugs are ACE inhibitors
like fosinopril, captopril, and lisinopril. However, these synthetic in­
hibitors have side effects like hyperkalemia, cough, and angioedema [3].
Therefore, many studies investigated the inhibitory effect of antioxidant
compounds such as vitamins, glutathione, peptides, plants, which are
known to have fewer side effects, on ACE activity. For instance, a
dipeptide Ser-Tyr (SY) isolated from the gonad of jellyfish, high protein-
containing, demonstrated an inhibition effect on the ACE activity with
an IC50 value of 1164.179 μM [4]. In our previous studies, the butanol
and water phases of the Juniperus excelsa Bieb. and Matricaria
human plasma, and IC50 values were calculated to be 2.858 mg/mL,
5.790 mg/mL, 0.353 mg/mL, and 1.292 mg/mL, respectively [5,6]. In a
work by Xue et al., a new ACE inhibitor peptide YQKFPQYLQY (YQK)
was isolated from bovine casein and the IC50 value was determined as
11.1 μM. Besides, it was observed that systolic blood pressure decreased
substantially by oral administration of YQK peptide to spontaneously
hypertensive rats [7].
Antioxidant compounds protect the human body against oxidative
stress caused by reactive oxygen species (ROS) like superoxide anion
radicals (O2.-), singlet oxygen (1O2), hydrogen peroxide (H2O2), and
hydroxyl radicals (OH•-). However, it has been observed that oxidative
stress is effective in the progression of hypertension [8,9]. For instance,
in middle-aged male hypertensive patients, levels of red blood cell
reduced glutathione (GSH) decreased, and oxidized glutathione (GSSG)
levels increased. At the same time, a higher GSSG/GSH ratio (P < 0.001)
and higher plasma homocysteine levels (P < 0.004) were found in hy­
pertensive patients compared to controls [10]. In a study performed by
Túri et al., decreased plasma nitrate levels and increased lipid peroxi­
dation levels were observed in hypertensive patients, and plasma lipid

* Van Yüzüncü Yıl University, Faculty of Health Sciences, Department of Nutrition and Dietetics, Van 65080, Turkey.
E-mail address: zehrabas@yyu.edu.tr.

https://doi.org/10.1016/j.ijbiomac.2021.08.109
Received 21 April 2021; Received in revised form 13 August 2021; Accepted 14 August 2021
Available online 19 August 2021
0141-8130/© 2021 Elsevier B.V. All rights reserved.
Z. Bas
peroxidation / nitric oxide ratio increased (P < 0.01) and glutathione
level decreased compared to controls with the same body mass index (P
< 0.001) [11]. Also, in various clinical studies, it has been observed that
hypertension is treated with antioxidant compounds such as tocopherol
(Vitamin E), alpha ascorbic acid (Vitamin C), coenzyme Q10, alpha-
lipoic acid, glutathione, and acetyl-L-carnitine [12].
wReduced glutathione (GSH) peptide is a water-soluble, low molecular
weight tripeptide composed of cysteine, glutamic acid, and glycine and
found in comparatively high concentrations in many tissues in the body
[13]. GSH peptide is a significant endogenous intracellular antioxidant that
plays a role in the detoxification of harmful compounds such as lipid
peroxides, heavy metals, drugs, and xenobiotic compounds. Maintaining
GSH levels in tissues is very important in terms of protecting the immune
system and protecting the body against diseases. Low GSH levels in the
body are linked to increased risks of various diseases such as hypertension,
cardiovascular diseases, cancer, and diabetes [14,15]. Also, in many clin­
ical studies, it has been observed that glutathione has a therapeutic effect
against many diseases with oral and intravenous administration [13,16].
The nicotinamide compound is the water-soluble active form of
vitamin B3. Nicotinamide is the precursor to the nicotinamide adenine
dinucleotide (NAD+) compound for ATP production, which is essential
for cellular energy. At the same time, the nicotinamide compound is the
essential coenzyme of NADH and NADPH [17,18]. Nicotinamide in­
adequacy causes pellagra disease, which is characterized by dermatitis,
dementia, and diarrhea, and this deficiency especially affects organs
with high cellular energy needs [17,19]. Many studies have shown that
nicotinamide is protective against diseases such as nonmelanoma skin
cancer, hypertension, Huntington's, Alzheimer's, and Parkinson's dis­
eases [20].
In the present work, the in vitro inhibition effect of nicotinamide
(vitamin B3) and GSH peptide on ACE purified from sheep kidneys was
studied. These compounds indicated an important inhibition effect on
purified ACE. The effect of nicotinamide and GSH peptide on ACE pu­
rified from sheep kidneys has not been investigated to date.
2. Materials and methods
2.1. Materials
Nicotinamide (vitamin B3), reduced glutathione (GSH) peptide,
Coomassie Brillant Blue R-250, N-[3-(2-Furyl)acryloyl]-L-phenylalanyl-
glycyl-glycine (FAPGG), sodium tetraborate (Na2B4O7.10H2O),
HepesNa, Coomassie Brillant Blue G-250, and lisinopril were bought
from Sigma-Aldrich. NHS-activated Sepharose 4 Fast Flow was bought
from GE Healthcare Life Sciences. SM0671 fermentas (electrophoresis
marker) from Life Science Thermo Fisher Scientific was received.
2.2. Obtain of the sheep kidney
Healthy sheep kidneys, which were slaughtered in the slaughter­
house, were brought to the laboratory. Approximately 20 g of tissue was
cut from different areas of the kidneys. This section was cut into small
cubic pieces with a scalpel. The disrupted kidney was added to 50 mM
Tris (pH 7.4) buffer. This mix was subjected to disintegration for 3 min
with the help of a mixer. Meanwhile, ice was placed around the mixer to
prevent it from heating. The homogenate obtained was applied for 3 min
in an ultrasonic homogenizer for further disintegration. Then, the
mixture in the beaker was placed in the centrifuge tubes and centrifuged
in a cooled centrifuge device at 8500 ×g at +4 ◦ C for 1 h. This was done
several times. After centrifugation, the liquid from the top of the tube
was taken and stored in the freezer for use in the purification process.
2.3. Purification by affinity chromatography method
NHS-activated Sepharose 4 Fast Flow (25 mL) kept in 100% iso­
propanol was taken and arranged according to the procedure specified
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International Journal of Biological Macromolecules 189 (2021) 65–71
by the manufacturer. Primarily, the affinity gel (column packing mate­
rial) was washed using cold 1 mM HCl. Following, assembling buffer (5
mM Lisinopril, 0.2 M NaHCO3, and 0.5 M NaCl) on the column packing
material was supplemented. The assembling reaction for one night at
4 ◦ C occurred. The affinity gel was supplemented into a beaker inclusive
0.1 M Tris-HCl (pH 8.5) solution and the mixture was carefully stirred
for several hours to inhibit any non-reacted groups on the affinity gel.
First, the affinity gel was laved using Tris-HCl tampon (0.1 M, pH 8.5) 3
times. After, the affinity gel was laved using an acetate tampon (0.1 M,
pH 4.5) 3 times.
The affinity gel was loaded onto the column (1.5 cm × 30 cm) with a
washing and equilibration tampon (20 mM Tris and 0.3 M NaCl, pH 8.0).
The flow rates for washing and equalization were set to 40 mL/h using a
peristaltic pump. The sheep kidney was filled onto an affinity column
and the column was laved and balanced by the equalization tampon. The
washing of the gel was maintained till the final absorbance was 0.1 at
280 nm. Later, Na2B4O7.10H2O (50 mM sodium tetraborate, pH 9.0), an
eluting tampon, was conducted from the column by a peristaltic pump.
The elution with Na2B4O7.10H2O was received as 1.5 mL fractions. The
ACE activity in the fractions was measured at 345 nm. The ACE activity-
inclusive tubes were assembled and repeat the ACE activity was gauged.
The purified ACE was split into portions and stocked in the freezer.
Entire the processes were done at 4 ◦ C [21,22].
2.4. ACE activity determination
The ACE activity was defined as the reduction in absorbance at 345
nm. The ACE activity was found with a Spectrophotometer at 35 ◦ C
using the Holmquist method [23]. The assay cuvette contained the ACE,
50 mM HepesNa tampon (10 μM ZnCl2 and 0.3 M NaCl, pH 7.5), and 1
mM FAPGG. One unit of the activity was described to be the amount of
ACE that produces a ΔA345 /min of 1.0 [23,24].
2.5. Protein determination
The protein concentrations of the purified fractions and the sheep
kidney homogenate were designated using Coomassie Brillant Blue G-
250 dye solution at 595 nm with the Bradford method and bovine serum
albumin was prepared to be the standard protein solution [25].
2.6. Molecular weight determination by SDS-PAGE
The purity of ACE was defined with SDS-PAGE (sodium dodecyl
sulfate-polyacrylamide gel electrophoresis). Also, the molecular weight
and purity of ACE were found with the SDS-PAGE method. Herein, 4%
and 10% acrylamide, respectively, for stacking and running comprising
1% SDS were performed using the Laemmli procedure [26]. The gel was
dyed for 2 h in 0.025% Coomassie Brillant Blue R-250 comprising 40%
methanol, 7% acetic acid, and distilled water. The gel was laved with a
first wash solution (10% acetic acid, 50% methanol, and 40% distilled
water). Subsequently, the gel was laved with a second wash solution (7%
acetic acid, 5% methanol, and 88% distilled water). The clear protein
band was occurred [26].
2.7. Preparation of nicotinamide (vitamin B3) and reduced glutathione
(GSH) peptide solution
1 mg of nicotinamide (vitamin B3) was solved in some distilled water
and it was completed to 20 mL using distilled water.
0.1 mg of GSH peptide was solved in some distilled water and it was
completed to 1 mL with distilled water.
2.8. In vitro inhibition effects of nicotinamide (vitamin B3) and reduced
glutathione (GSH) peptide on purified ACE
The effects of nicotinamide (vitamin B3) and GSH peptide on ACE
Z. Bas
purified from sheep kidneys were explored. Accordingly, several con­
centrations of nicotinamide and GSH peptide were annexed to the
evaluation tube (50 mM HepesNa, 10 μM ZnCl2, 0.3 M NaCl, 1 mM
FAPGG, 100 μL ACE solution) for the definition of the concentration
range and ACE activities. Activity% vs. inhibitor graph using these
concentrations was drawn. The IC50 value of nicotinamide (vitamin B3)
and GSH peptide was calculated from the equation of the inhibition
graph. Lineweaver-Burk graph was plotted with five distinct FAPGG
concentrations and three different concentrations of nicotinamide and
GSH peptide. The inhibition type and Ki constants of nicotinamide and
GSH peptide from this graph were determined [27].
A (ACE Activity) = (∆ODOD/0.517) × (Vc/Ve) × f
where ∆OD is the decrease in the absorbance at 345 nm per minute, Vc is
total reaction volume, Ve is the volume of enzyme solution (sheep kid­
ney homogenate, pure enzyme), f is the dilution coefficient, and 0.517 is
the millimolar absorption coefficient of FAPGG.
The ACE inhibitory activity (inhibition %) was calculated from the
calibration curve and found by the following equation:
%ACE Inhibition = Uninhibited activity − Inhibited activity/Uninhibited activity × 100
3. Results and discussion
Herein, angiotensin-converting enzyme (ACE, EC 3.4.15.1) in the
sheep kidney was purified in a single step using the affinity chroma­
tography method. NHS-activated Sepharose 4 Fast Flow column filler to
be matrix and lisinopril, specifically an ACE inhibitor, was used to be a
ligand. At the same time, the in vitro inhibition effect of nicotinamide
(vitamin B3) and reduced glutathione (GSH) peptide on the purified ACE
was investigated.
NHS (N-hydroxysuccinimide) is a compound that forms a chemically
stable amide bond with ligands containing primary amino groups.
Sepharose 4 Fast Flow is activated with NHS to form a spacer arm and
bind small enzyme ligands. With the high flow and stability properties of
the NHS-activated Sepharose 4 Fast Flow column filler, purification can
be performed successfully in a single step and with very high purity
[28]. Therefore, NHS-activated Sepharose 4 Fast Flow column filler
provides a great advantage in terms of efficiency, time, and cost
compared to other column fillers.
The ACE enzyme is a high molecular weight integral membrane
protein situated on the lumen surface of the cell membrane [29]. There
are two forms of the ACE enzyme, the somatic form that is abundant on
the endothelial surface of the lung vessels and a smaller germinal form
found only in the testis. The somatic isoform has a molecular weight that
varies from 130 to 180 kDa and was described in the endothelial,
mesangial, epithelial, and neuronal cells, and in the subsequent tissues
as intestine, kidney, lung, heart, placenta, pancreas, and liver
[30,31,32]. Germinal ACE has a molecular mass of 90-100 kDa and is
similar to the C-terminal part of the endothelial ACE. Germinal ACE is
found only in the testis in germinal cells during the maturation of
spermatogenesis [33]. Since the ACE enzyme has two isoenzymes,
different molecular weights were determined in different purified tis­
sues. Also, in our previous studies, the molecular weight of the ACE
enzyme purified from human plasma was defined as 60 and 70 kDa with
SDS-PAGE [34,35]. In a study by Hooper and Turner, two forms (170
kDa and 180 kDa) from the pig striatum of the ACE and merely one form
(180 kDa) from the pig kidney of the ACE were found with SDS-PAGE
[36]. The soluble forms of the ACE of the bovine lung by size exclu­
sion and affinity chromatography were purified. Then, the molecular
weight of the membrane-bound enzyme was identified as 170 kDa and
the ACE form dissolved with trypsin was determined as 160 kDa using
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International Journal of Biological Macromolecules 189 (2021) 65–71
the SDS-PAGE method [37]. The molecular weight found with SDS-
PAGE is the molecular weight of the subunits of the ACE enzyme.
Since oligomeric enzymes are denatured by the SDS-PAGE electropho­
resis method, they are divided into subunits. In this work, molecular
weight and purity of the ACE enzyme purified from sheep kidneys were
identified with SDS-PAGE, and two bands, 60 kDa and 70 kDa were
observed on the gel (Fig. 1). Herein, it has been determined that ACE
purified from sheep kidneys is a dimer enzyme with two subunits.
Angiotensin-converting enzyme (ACE) inhibitors have often been
utilized as first-line treatment drugs in the treatment of kidney and
cardiovascular diseases. These inhibitors have proven to be influential in
the treatment of hypertension and decrease mortality in left ventricular
dysfunction and congestive heart failure after myocardial infarction
[38,39]. However, synthetic ACE inhibitors such as captopril, lisinopril,
and fosinopril cause many side effects such as hypotension, hyper­
kalemia, angioedema, and cough [39,40,41]. For this reason, the effects
of natural compounds such as herbs, antioxidant compounds, and pep­
tides, which are found to have fewer side effects, on ACE activity have
been examined in recent studies. For example; peptides IPPAYTK,
TFQGPPHGIQVER, and LVLPGELAK were purified from broccoli protein
hydrolysates. These peptides indicated an important inhibition effect on
ACE activity with IC50 constants of 23.5, 3.4 μM, and 184.0, respectively
[42]. In another study, two new peptides from the natural herb Bromelia
antiacantha Bertol. with strong ACE inhibitor activity; GYDTQAIVQ and
TTFHTSGY were isolated, and IC50 values were determined as 1.0 mM
and 142 μM, respectively [43]. In our previous study, the effect of
reduced glutathione (GSH) and oxidized glutathione (GSSG) on ACE
purified from human plasma was investigated. GSSG demonstrated an
activation effect on ACE, while GSH indicated an inhibition effect. IC50
constant for GSH was designated as 16.2 μM [35]. The in vivo and in
vitro effects of Leu-Ser-Gly-Tyr-Gly-Pro (LSGYGP) obtained from tilapia
fish skin gelatin hydrolyzate on ACE activity were studied. It has been
determined that LSGYGP peptide, which has an IC50 constant of 2.577
μmol L-1, has an antihypertensive effect in spontaneously hypertensive
rats [44] (Table 1).
In this work, the inhibition effect of nicotinamide (vitamin B3) and
GSH peptide compounds on pure ACE activity was studied. Nicotin­
amide and GSH peptide showed a substantial inhibition effect with IC50
values of 14.3 μM and 7.3 μM, respectively (Figs. 2, 3). Inhibition type
and Ki values were found from Lineveawer-Burk graphs. The type of
inhibition for nicotinamide and GSH peptide was determined as non-
competitive inhibition (Table 1). Ki values were calculated as 15.4 μM
for nicotinamide and 6.7 μM for GSH (Figs. 4, 5). These results show that
nicotinamide (vitamin B3) and GSH peptide compounds that have an
inhibitory effect on ACE activity can be used as antihypertensive agents
in hypertension treatment.
It has been observed that oxidative stress caused by free radicals is
effective in the development of hypertension. Moreover, the production
of hydrogen peroxide and lipid hydroperoxide is higher in hypertensive
patients. In many experimental and clinical studies, it was determined
that antioxidant compounds such as glutathione, beta-carotene, vita­
mins A, C, and E treat hypertension while preventing oxidative stress
[9,45]. In a study, a randomized double-blind, placebo-controlled clin­
ical trial was performed on 110 men with essential hypertension. Pa­
tients were randomly administered either vitamins E + C [vitamin C (1
g/day) plus vitamin E (400 international units/day)] or placebo for 8
weeks. Measurements of 24-h ambulatory blood pressure and parame­
ters related to oxidative stress in erythrocytes (GSH / GSSG ratio,
malondialdehyde, and antioxidant enzymes) and plasma were taken.
Z. Bas International Journal of Biological Macromolecules 189 (2021) 65–71

120

100 IC50: 14.3 µM

80

Activity %
60
y = -3.4665x + 99.647
40 R² = 0.9845

20

0
0 2 4 6 8 10
[Nicotinamide] µM

Fig. 2. The inhibition effect of nicotinamide (vitamin B3) on ACE from sheep
kidneys. Four different nicotinamide (from 2.5 to 8.18 μM) concentrations on
ACE activity were studied.

120

100 IC50: 7.3 µM

80

Activity % 60
y = -6.5106x + 97.534
40 R² = 0.9829
20

0
0 2 4 6 8
Fig. 1. SDS-polyacrylamide gel electrophoresis of ACE purified by affinity [Reduced Glutathione (GSH)] µM
chromatography. Lane 1: Standard proteins (Fermentas unstained protein lad­
der SM0671). Lanes 2, 3, and 4: purified angiotensin-converting enzyme (ACE) Fig. 3. The inhibition effect of reduced glutathione (GSH) peptide on ACE from
from sheep kidney. sheep kidneys. Four different GSH peptide (from 1.63 to 6.5 μM) concentrations
on ACE activity were studied.
Subsequently, administration of vitamins E + C, patients with hyper­
tension had substantially lower systolic blood pressure, diastolic blood (B9) vitamins, which are associated with the control of oxidative stress,
pressure, and mean arterial blood pressure compared to patients treated on spontaneously hypertensive stroke-prone rats was investigated. A
with placebo [46]. significant decrease was observed in systolic blood pressure, malon­
It has been observed in experimental and clinical studies that group B dialdehyde (MDA), and homocysteine blood levels in rats receiving B
vitamins are also effective in the treatment of hypertension. For group vitamin supplements. The results in these studies show that
example, The effect of riboflavin (B2), pyridoxine (B6), and folic acid vitamin therapy is effective in controlling both high blood pressure and
oxidative stress [47]. Therefore, vitamins can be considered as one of the
alternative treatments to prevent hypertension.
Table 1 Nicotinamide (vitamin B3) is an important compound that maintains
Comparison scheme of IC50, Ki values, and inhibition types obtained from cellular energy and affects various metabolic processes. Nicotinamide
regression analysis graphs for the angiotensin-converting enzyme in the pres­
protects against oxidative stress that triggers diseases such as hyper­
ence of different inhibitors and peptides concentrations.
tension, atherosclerosis, cardiovascular diseases, and cancer [19,48]. It
ACE inhibitor IC50 Ki Inhibition References has also been reported to have therapeutic effects against these diseases.
type
In one study, the effect of nicotinamide doses on blood pressure in mice
Reduced glutathione 7.3 μM 6.7 μM Non- This study and humans has been studied. After the volunteers were given 3 or 6 g of
(GSH) competitive oral nicotinamide, their blood pressure evaluations were made. The
Nicotinamide 14.3 μM 15.4 μM Non- This study
(Vitamin B3) competitive
mean (±1 SE) resting diastolic and systolic pressures in humans were
Reduced glutathione 16.2 μM 11.7 μM Non- [35] measured as 80.6 mmHg (±2.1) and 122.8 mmHg (±2.5), respectively,
(GSH) competitive and blood pressures did not change during the first 3 h after nicotin­
Reduced glutathione 32.4 μM 49.7 μM Non- [54] amide administration. In mice, the resting value (±1 SE) was deter­
(GSH) competitive
mined as 115.1 mmHg (±4.0), and this value decreased significantly
Ser-Tyr (SY) peptide 1164.179 – Non- [4]
μM competitive after intraperitoneal injection of 400-1000 mg/kg nicotinamide [49]. In
YQKFPQYLQY 11.1 μM 13 μM Competitive [7] another work, the protective effect of nicotinamide against focal cere­
peptide bral ischemia was studied in spontaneously hypertensive rats (SHR) and
TTFHTSGY peptide 142 μM 69 ± 5.3 Competitive [43] diabetic Fischer 344 rats. Nicotinamide, administered intravenously, 2 h
μM
LSGYGP peptide 2.577 μmol – Non- [44]
after permanent middle cerebral artery occlusion substantially
L-1 competitive decreased the infarct volume of SHR (750 mg/ kg, 31%, P,0.01), non-

68
Z. Bas International Journal of Biological Macromolecules 189 (2021) 65–71

0.05
Control
4.09 µM Nicotinamide y = 0.0032x + 0.0081
6.14 µM Nicotinamide 0.04 y = 0.0028x + 0.0071
8.18 µM Nicotinamide
y = 0.0023x + 0.0059

1/V (µmol (FAP) min)-1mL

y = 0.0019x + 0.0049
0.03

0.02

0.01

0
-4 -2 0 2 4 6 8 10 12
1/FAPGG [mM]-1

Fig. 4. Lineweaver–Burk graph with five different substrate concentrations (FAPGG) and three different nicotinamide (vitamin B3) concentrations used for the
designation of inhibition type and Ki.

0.05
Control
1.63 µM GSH y = 0.004x + 0.0041
3.25 µM GSH y = 0.0035x + 0.0035
0.04
4.88 µM GSH y = 0.0027x + 0.0028
y = 0.0022x + 0.0023
1/V (µmol (FAP) min)-1mL

0.03

0.02

0.01

0
-2 0 2 4 6 8 10 12
1/FAPGG [mM]-1

Fig. 5. Lineweaver–Burk graph with five different substrate concentrations (FAPGG) and three different reduced glutathione (GSH) peptide concentrations used for
the designation of inhibition type and Ki.

diabetic (500 mg/ kg, 73%, P,0.01) and diabetic (500 mg/ kg, 56%,
P,0.01) in Fischer 344 rats compared to saline-injected controls [50]. In
another study, it was observed that dietary nicotinamide administration
in Apolipoprotein-E-deficient mice prevented the progression of
atherosclerosis and prevented Apolipoprotein-B containing lipoprotein
oxidation [51]. In another study, nicotinamide (vitamin B3) was
observed to significantly inhibit oxidative damage caused by reactive
oxygen species (ROS) produced with ascorbate-Fe2+ and photosensiti­
zation systems in rat brain mitochondria. It was concluded that nico­
tinamide is an antioxidant compound that protects against both lipid
peroxidation and protein oxidation at low concentrations [52]. In this
study, it was observed that nicotinamide can prevent hypertension by
showing a significant in vitro inhibition effect on ACE. At the same time,
in vivo studies support this study by showing that nicotinamide has a
therapeutic effect against both hypertension and cardiovascular dis­
eases. Here the nicotinamide demonstrated a significant inhibition effect
on the ACE enzyme with an IC50 value of 14.3 μM. That is, nicotinamide
indicated a large inhibition effect with a very low IC50 value.
Glutathione (GSH), which is reduced by NADPH produced in the
pentose phosphate pathway, is an antioxidant tripeptide. The GSH
peptide is found in relatively high concentrations in many tissues in the
body. It protects cells against oxidative stress caused by free radicals.
Also, many studies have determined that oral, intravenous, and sublin­
gual administration of GSH peptide can be a positive strategy to develop
the endogenous antioxidant defense required to protect against many
chronic and acute diseases [13,15,16]. For instance, one study evaluated
the effect of novel sublingual glutathione on liver metabolism, lipid
profile, and the circulating effect of oxidative stress and peripheral

69
Z. Bas
vascular function in patients with cardiovascular risk factors (CVRF)
compared to placebo. A reduction in total and low-density lipoprotein
cholesterol was observed in subjects after L-GSH supplementation
compared to placebo (P = 0.023 and P = 0.04, respectively). As a result,
it has been determined that sublingual glutathione can prevent vascular
damage in patients with endothelial dysfunction and CVRF [53]. In an in
vitro study by Hou et al., GSH peptide showed an inhibition effect on the
ACE enzyme and IC50 and Ki values were determined as 32.4 μM and
49.7 μM, respectively [54]. Also, in this study, GSH peptide significantly
inhibited the ACE purified from sheep kidneys. The IC50 and Ki values of
the GSH peptide were calculated as 7.3 μM and 6.7 μM. In vitro and in
vivo studies on the naturally synthesized antioxidant GSH peptide in the
body have proven that this peptide significantly protects the body
against many diseases. At the same time, in this study, GSH peptide
significantly inhibited the ACE enzyme in vitro, showing that this pep­
tide is both therapeutic and protective for hypertension and cardiovas­
cular diseases.
Angiotensin-converting enzyme (ACE) inhibition is thought to be a
useful treatment method in the treatment of hypertension. Therefore,
the development of drugs that inhibit ACE to control high blood pressure
has gained great importance. However, due to the side effects of syn­
thetic ACE inhibitors, the inhibition effect of natural compounds such as
antioxidant compounds, peptides, vitamins, which are known to have
few side effects, on the ACE enzyme has been investigated. In this study,
the inhibition effect of reduced glutathione, an endogenous antioxidant,
and nicotinamide (vitamin B3), an exogenous vitamin, were investi­
gated. It was observed that these compounds had a significant inhibitory
effect on the ACE enzyme. It has been observed in many clinical studies
that antioxidant compounds and vitamins have both protective and
therapeutic effects against diseases like hypertension, cardiovascular
diseases, atherosclerosis, and cancer. In this study, it was concluded that
nicotinamide and GSH peptide compounds may have both protective
and therapeutic effects against hypertension due to their inhibitory ef­
fect on ACE.
CRediT authorship contribution statement
Zehra Bas: Conceptualization, Methodology, Validation, Investiga­
tion, Data curation, Writing-Original draft, Reviewing and Editing,
Visualization, Supervision administration.
Ethical approval
This work involved the chemical analyses of animal sources. No
approval of animal use protocols was required.
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