Protective Effects of an Oxovanadium(IV) Complex with N2O2 Chelating Thiosemicarbazone on Small Intestine Injury of STZ-Diabetic Rats

Article abstract of DOI:10.1007/s12011-020-02269-7

Vanadium compounds are being investigated as potential therapeutic agents in the treatment of many health problems, primarily diabetes. We aimed to provide the effect of N(1)-4-hydroxysalicylidene-N(4)-salicylidene-S-methyl-isothiosemicarbazidato-oxovanadium(IV) (VOL) on small intestinal injury in experimental male diabetic rats. Four groups were created of 3.0–3.5-month-old rats. The rats were made diabetic by a single dose of streptozotocin (STZ) at 65 mg/kg and grouped as follows: control animals, VOL-given control animals, STZ-induced diabetic animals and STZ-induced diabetic animals given VOL. A daily dose of 0.2 mM/kg vanadium complex was administered orally for 12 days after the inducement of diabetes. On the 12th day, small intestine tissue samples were taken. According to the data obtained from the biochemical analysis, reduced glutathione (GSH) level, catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase (GST), superoxide dismutase (SOD), Na⁺/K⁺-ATPase and paraoxanase (PON) activities were increased, whereas sialic acid (SA), xanthine oxidase (XO) and disaccharidases (maltase and saccharidase) activities were decreased in the small intestine tissue of VOL-treated diabetic rats. Microscopic examinations revealed a remarkable decrease in the mucosal necrotic areas, discontinuity in the brush border, deterioration of the villi integrity and oedema inside the villi, but with a mild decrease in the inflammatory cells, deterioration and loss of integrity of the gland in the small intestine of VOL-treated diabetic rats. Moreover, VOL treatment markedly decreased the proliferation of villus cells and especially inflammatory cells in the small intestine of diabetic rats. According to the obtained data, the administration of VOL is a potentially convenient strategy to reducing small intestine injury in diabetic rats.

Full text of DOI:10.1007/s12011-020-02269-7

Biological Trace Element Research
https://doi.org/10.1007/s12011-020-02269-7
Protective Effects of an Oxovanadium(IV) Complex with N2O2
Chelating Thiosemicarbazone on Small Intestine Injury
of STZ-Diabetic Rats
Sevim Tunali1 & Selda Gezginci-Oktayoglu2 & Sehnaz Bolkent2 & Ediz Coskun2 & Tulay Bal-Demirci3
Bahri Ulkuseven3 & Refiye Yanardag1
&
Received: 18 May 2020 /Accepted: 23 June 2020
#
Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
Vanadium compounds are being investigated as potential therapeutic agents in the treatment of many health problems, primarily
diabetes. We aimed to provide the effect of N(1)-4-hydroxysalicylidene-N(4)-salicylidene-S-methyl-isothiosemicarbazidato-
oxovanadium(IV) (VOL) on small intestinal injury in experimental male diabetic rats. Four groups were created of 3.0–3.5-
month-old rats. The rats were made diabetic by a single dose of streptozotocin (STZ) at 65 mg/kg and grouped as follows: control
animals, VOL-given control animals, STZ-induced diabetic animals and STZ-induced diabetic animals given VOL. A daily dose
of 0.2 mM/kg vanadium complex was administered orally for 12 days after the inducement of diabetes. On the 12th day, small
intestine tissue samples were taken. According to the data obtained from the biochemical analysis, reduced glutathione (GSH)
level, catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase (GST), superoxide dismutase (SOD), Na+/K+-
ATPase and paraoxanase (PON) activities were increased, whereas sialic acid (SA), xanthine oxidase (XO) and disaccharidases
(maltase and saccharidase) activities were decreased in the small intestine tissue of VOL-treated diabetic rats. Microscopic
examinations revealed a remarkable decrease in the mucosal necrotic areas, discontinuity in the brush border, deterioration of
the villi integrity and oedema inside the villi, but with a mild decrease in the inflammatory cells, deterioration and loss of integrity
of the gland in the small intestine of VOL-treated diabetic rats. Moreover, VOL treatment markedly decreased the proliferation of
villus cells and especially inflammatory cells in the small intestine of diabetic rats. According to the obtained data, the admin-
istration of VOL is a potentially convenient strategy to reducing small intestine injury in diabetic rats.
.
.
.
Keywords Diabetes mellitus Vanadium complex Small intestine Oxidative stress
Introduction
incidence of adult diabetic patients in the world will increase
from 177 to 370 million by 2030 [3]. The nature of metabolic
disturbances in DM has a significant adverse effect on the or-
ganism resulting in cardiovascular, kidney and neurological dis-
eases [4–6], all contributing to a decreased quality of life [7].
Dysfunction caused by the destruction of β cells in the pancreas
is common in both type 1 diabetes (T1DM) and type 2 diabetes
(T2DM). The extremely increase in diabetes worldwide reveals
the need for insulin and various problems associated with insulin
injection. Oral insulin mimic compounds would be ideal to
overcome the problems caused by diabetes [8].
Diabetes mellitus (DM) is a type of metabolic disorder, charac-
terized by impairments in protein, lipid and carbohydrate me-
tabolism as a result of insulin resistance, chronic
hyperglycaemia and insufficient insulin secretion [1–3].
According to the World Health Organization (WHO), the
* Sevim Tunali
stunali@istanbul.edu.tr
The small intestine tissue plays an essential role in the reg-
ulation of glucose metabolism and the pathogenesis of DM.
The gastrointestinal symptoms in diabetes are associated with
manifestations such as diarrhoea, faecal incontinence, constipa-
tion, dyspepsia, nausea and vomiting [9]. Therefore, improve-
ment of hypoglycaemic condition with glycaemic control is a
target of diabetic small intestine therapy. The permanent
1
Department of Chemistry, Biochemistry Division, Istanbul
University-Cerrahpasa, Avcilar, 34320 Istanbul, Turkey
2
Faculty of Science, Department of Biology, Istanbul University,
Vezneciler, 34134 Istanbul, Turkey
3
Department of Chemistry, Inorganic Chemistry Division, Istanbul
University-Cerrahpasa, Avcilar, 34320 Istanbul, Turkey

Tunali et al.
HO
O
hyperglycaemic state in DM causes the formation of oxidative
stress and reactive oxygen species (ROS) [10, 11]. In order to
combat excess of ROS production, the activity of the body’s
natural protective mechanisms is increased. Eventually, antiox-
idant depletion occurs against natural defences in small intesti-
nal enterocytes, resulting in uncontrolled ROS formation [12,
13]. In addition, in a typical hyperglycaemic state, disruption of
antioxidant balance can lead to a decrease in both circulating
repair factors and levels of inhibition factors involved in muco-
sal healing. This can cause damage to healing mechanisms as in
diabetic enteropathy [13]. Increased oxidative stress excessive-
ly increases cytokine production through numerous mecha-
nisms, with various derivatives of oxygen molecules acting as
secondary messengers that cause transcription of immune-
related cytokines in the small intestine tissue [14].
O
O
V
1
4
N
N
N
S
CH
3
Fig. 1 The oxovanadium(IV) complex with dibasic N2O2-chelating
thiosemicarbazidato ligand (symbolized by VOL)
VOL The complex was obtained from 4-Hydroxysalicylidene-S-
methyl-isothiosemicarbazone and salicylaldehyde in the pres-
ence of VOSO4.5H2O in 1:1:1 molar ratio by the previously
described method [30]. The physical properties of amorphous
brown powder were controlled: yield, 72%; m.p, > 380 °C.
Vanadium is an abundant element on earth which distribu-
tion takes a large place in all organisms [15–17]. Some previ-
ous studies indicated that some synthesized vanadium com-
plexes have exhaustive antidiabetic activities and a wide vari-
ety of other pharmacological properties in vitro and in vivo
[18–21]. Many researches are widespread on the development
and creation of new metalopharmaceuticals, and these types of
pharmaceuticals are extremely desirable for the treatment of
DM. Based on our previous works, we noticed that vanadium
might be very useful in the treatment of DM [22, 23].
Thiosemicarbazones are an important class of ligands for
metal complexes because of biological and therapeutic proper-
ties such as antioxidant [24, 25], cytotoxic [24, 26–29], antidi-
abetic [30], antimalarial [31, 32], antiviral [33], antibacterial
[34], anticancer [35, 36] and antiprotozoal [29, 37]. In the pres-
ent study, we aimed to demonstrate biochemical and patholog-
ical effects of N(1)-4-hydroxysalicylidene-N(4)-salicylidene-S-
methyl-isothiosemicarbazidato-oxovanadium(IV) (VOL),
which is a vanadium compound bearing a thiosemicarbazone,
on small intestinal injury in male streptozotocin (STZ)-induced
diabetic rats.
Animals Approval
Twenty one clinically healthy Swiss Albino male rats (of be-
tween 3.0 and 3.5 months old) were used. Experiments were
reviewed and approved according to the Animal Care and Use
Institute’s Committee of Istanbul University.
Induction of Diabetes
STZ was freshly prepared by dissolving in cold 0.01-M sodi-
um buffer of citrate–hydrochloric acid (pH = 4.5) [38] and
used for induction of diabetes by a single intraperitoneal in-
jection (i.p) of 65 mg/kg body weight.
Experimental Design
The animals were randomly allocated according to the order,
nondiabetic group (n = 5), VOL-treated nondiabetic group (n
= 5), STZ-diabetic group (n = 6) and STZ-diabetic animals
given with VOL group (n = 5). VOL was given to the rats by
gavage technique (upon dissolution in gum Arabic solution) at
a dose of 0.2 mM/kg/day for 12 days after rats became diabet-
ic. Body weights of all animals were taken on 0, 1 and 12
experimental days and tail vein blood samples were measured
for the same days.
Materials and Methods
Preparation of VOL
VOL complex was obtained through 4-hydroxysalicylidene-
S-methylisothiosemicarbazone and salicylaldehyde in the
presence of vanadyl sulphate by template reaction (Fig. 1).
Biochemical Assays
On day 12 of the experiment, animals were fasted overnight
and were subsequently sacrificed under ether anaesthesia. The
small intestine of all animals was removed and processed for
study. The ortho-toluidine method was used for the determi-
nation of 18-h fasting blood glucose for all animals [39]. The
data of body weights and fasting blood glucose levels have
been published in a previous article describing the effects of
VOL on the pancreas tissue [30].
Starting Material 4-Hydroxysalicylidene-S-methyl-
isothiosemicarbazide was prepared by refluxing from the reac-
tion of 4-hydroxysalicylaldehyde (1 g, 1 mmol) with S-methyl-
isothiosemicarbazide (0.776 g, 1 mmol) in ethanol (25 ml). After
4 h, the precipitated powder with pinkish cream colour was fil-
tered and washed with ethanol [30]. The yield was 91% (1.48 g)
and m.p is 180–181 °C.

Protective Effects of an Oxovanadium(IV) Complex with N2O2 Chelating Thiosemicarbazone on Small...
Oxidative Stress Markers and Antioxidant Enzyme
Activities
Statistical Evaluation of Data
The obtained all biochemical data were evaluated using an
unpaired t test and analysis of variance (ANOVA) using the
statistical computer package NCSS. The results were
expressed as mean SD. GraphPad Prism Software, version
4.00 (San Diego, CA), was used for immunohistochemical
analysis. Data were stated as mean S.E.M. One-way
ANOVA and Tukey’s test were used for statistical analysis.
p < 0.05 was accepted as significant.
The small intestinal tissue homogenates were made 10%
(w/v) by using a glass equipment. After centrifugation,
the clear supernatants were collected and used for the
estimation of reduced glutathione (GSH), sialic acid
(SA), protein levels, antioxidant levels and disaccharidase
enzyme activities. The small intestine GSH, SA and pro-
tein levels were determined by the methods of Beutler
[40], Warren [41] and Lowry et al. [42], respectively.
The activities of enzymes were measured with following
techniques: of catalase (CAT) according to Aebi [43],
glutathione peroxidase (GPx) according to Paglia and
Valentine [44] as modified by Wendel [45], glutathione-
S-transferase (GST) according to the Habig and Jacoby
[46], superoxide dismutase (SOD) according to Mylorie
et al. [47], Na+/K+-ATPase according to Ridderstap and
Bonting [48], paraoxanase (PON) according to Furlong
et al. [49], xanthine oxidase (XO) according to Corte
and Stirpe [50] and disaccharidase (maltase and
saccharidase) according to the Dahlqvist [51] by diluting
in appropriate rates.
Results
VOL complex was obtained via template synthesis method as
pr eviously report ed [ 30]. Firstly, S-methyl-
isothiosemicarbazide was prepared through the reaction of a
methyl halogenide and thiosemicarbazide. Thereafter, a 4-
hydroxysalicylidene-S-methyl-isothiosemicarbazide as
starting material was obtained from the reaction of 4-
hydroxysalicylidene with the prepared S-methyl-
isothiosemicarbazide. Then, VOL was synthesized by
reacting to the starting material with salicylaldehyde in the
presence of VOSO4.5H2O in 1:1:1 molar ratio. All processes
were monitored via thin-layer chromatography. The reaction
was tracked by IR spectroscopy. In the IR spectrum of the
VOL complex, the disappearance of stretching and bending
vibration bands of NH2 group of the starting material at 3445,
3337 and 1624 cm−1 and appearance of the characteristic
(V=O) and (V-O) bands at 987 and 478-434 cm−1 were proofs
that VOL complex was formed [30].
Histopathology
The small intestine tissue pieces were kept in Bouin’s fixative.
The water in the tissue pieces was removed by passing
through the rising alcohol series, and after xylene application,
they were embedded in the paraffin blocks. After applying
periodic acid-Schiff (PAS) dye to 5-μm-thick sections, they
were analysed with an Olympus CX-45 microscope with
DP71 digital camera attachment.
Impact of VOL on Small Intestine GSH and SA Levels
The data showing the reduced GSH and SA levels in nondia-
betic and treated animals are presented in Table 1. A insignif-
icant decrease of GSH level was shown in the diabetic group
Immunohistochemistry
Proliferative cell nuclear antigen (PCNA) was labelled
using the Streptavidin-Biotin-Peroxidase method. Three
percent hydrogen peroxide was applied to suppress the
activity of endogenous peroxidases. Following the block
solution which is a component of the Histostain Plus
Broad Spectrum Kit (Zymed 85-9043), the mouse poly-
clonal antibody against PCNA (NeoMarkers MS-106-P)
was incubated at 1:50 dilution for 1 h at room tempera-
ture. After applying biotinylated secondary antibody and
streptavidin peroxidase, which are the other components
of the same kit, the reaction was developed with 3-amino-
9-ethylcarbazole. Mayer’s Haematoxylin was applied for
counter staining. Sections were analysed with an Olympus
CX-45 microscope with DP71 digital camera attachment.
Ten fields (0.0506 mm2) were randomly counted per sec-
tion from 5 animals of each group.
Table 1 The levels of reduced glutathione and SA in the control and
experimental animals’ small intestine tissues
Groups
GSH (nmol GSH/
mg protein)*
SA (mmol/mg
protein)*
Control
9.11 0.43
5.66 0.08a
7.79 0.87
8.56 0.33
0.001
344.42 32.35
302.48 72.37
469.34 10.53a
209.40 64.71b
0.014
Control + VOL
Diabetic
Diabetic + VOL
PANOVA
*Mean SD
a P < 0.05 compared with control group
b P < 0.05 compared with diabetic group

Tunali et al.
when compared with control rats. VOL administration result-
ed in a nonsignificant increase of GSH level in the diabetic
group. Interestingly, there was a significant decrease in the
GSH level in VOL-treated rats in comparison with non-
treated control rats (p < 0.05). A profound increase of SA
levels in diabetic animals in comparison with the control
group (p < 0.05) was observed. Orally treated VOL signifi-
cantly decreased the level of SA in diabetic group (p < 0.05).
control rats (p < 0.005) was noticed. The administration of
VOL significantly raised PON activity in the diabetic group
(p < 0.0001). XO activity was significantly increased in the
diabetic rats compared with the control (p < 0.05). However
VOL treatment significantly decreased XO activity in diabetic
group (p < 0.05). On the other hand, the small intestinal XO
activity was increased significantly in the VOL-given control
rats in comparison with non-treated control animals (p <
0.0001). In comparison with the control, the activities of in-
testinal maltase and saccharase in diabetic rats were signifi-
cantly elevated (p < 0.005, p < 0.0001). The administration of
VOL reduced the activity of these enzymes significantly (p <
0.05, p < 0.0001).
Impact of VOL on Small Intestine Antioxidant
Enzymes and Na+/K+-ATPase Activities
The small intestinal CAT, GPx, GST, SOD and Na+/ K+-ATPase
activities are given in Table 2. Small intestinal CAT, GPx, GST
and SOD activities were significantly decreased in diabetic group
compared with the control group (p < 0.005 for CAT, p < 0.0001
for GPx and GST, p < 0.05 for SOD). The vanadium complex
significantly increased the activities of these enzymes to near
normal in VOL-treated diabetic group (p < 0.0001 for CAT,
GPx and GST; p < 0.05 for SOD). Another remarkable output
is that the control animals given with VOL showed a significant
decrease in GPx and GST activities compared with control rats (p
< 0.005, p < 0.0001). A mean decrease in Na+/K+-ATPase ac-
tivity was noticed in diabetic rats as compared with the controls
(p < 0.05). On the other hand, diabetic rats that received VOL
had a significantly increased Na+/K+-ATPase activity in compar-
ison with non-treated diabetic rats (p < 0.005).
Impact of VOL on Small Intestine Histopathology
Microscopic examination reveals that VOL treatment re-
stored intestinal morphological alterations observed in dia-
betic rats. A remarkable decrease in mucosal necrotic areas,
discontinuity in the brush border, deterioration of the villi
integrity and oedema inside the villi were observed.
Moreover, a mild decrease in the inflammatory cells, as well
as the deterioration and loss of integrity of glands in the
small intestine by VOL treatment to diabetic rats (Fig. 2),
was detected.
Impact of VOL on Small Intestinal Cell Proliferation
Impact of VOL on Small Intestine PON, XO and
Disaccharidase Activities
PCNA immunostaining was evaluated for three different
types of cells that included crypt, villus and inflammatory
cells in duodenum sections. There were no statistically sig-
nificant changes in PCNA+ crypt cells between the groups.
PCNA+- crypt cell numbers were as follows: C: 12.68
3.84, VOL: 24.95 0.82, D: 23.03 4.06 and D + VOL:
The small intestinal PON, XO and disaccharidases (maltase
and saccharidase) activities are given in Table 3. A significant
decrease in PON activity of diabetic rats compared with
Table 2 Small intestinal antioxidant enzymes and Na+/ K+-ATPase activities in control and experimental groups
Groups
CAT
GPx
GST
SOD
Na+/ K++/ K+-ATPase
(U/g protein)*
(U/g protein)*
(U/g protein)*
(U/g protein)*
(μmol Pi/g protein/h)*
Control
24.43 2.64
23.76 3.22
13.06 2.07a
30.88 1.94b
0.0001
25.91 1.15
21.36 0.93a
19.23 1.04c
29.25 0.56b
0.0001
73.31 2.97
59.01 2.39c
50.36 5.31c
74.26 3.70b
0.0001
6.31 0.60
6.43 0.69
4.98 0.31d
6.65 0.43e
0.016
129.48 4.11
141.02 1.86
113.69 0.08d
134.78 1.70f
0.001
Control + VOL
Diabetic
Diabetic + VOL
PANOVA
*Mean SD
a P < 0.005 compared with control group
b P < 0.0001compared with diabetic group
c P < 0.0001 compared with control group
d P < 0.05 compared with control group
e P < 0.05 compared with diabetic group
f P < 0.005 compared with diabetic group

Protective Effects of an Oxovanadium(IV) Complex with N2O2 Chelating Thiosemicarbazone on Small...
Table 3 Small intestine PON,
XO and disaccharidase activities
Groups
PON
XO
Maltase
Saccharase
in all groups
(U/mg protein)*
(U/g protein)*
(U/g protein)*
(U/g protein)*
Control
8.51 1.07
8.76 0.68
2.27 0.70a
4.88 0.62b
0.0001
1.51 0.09
3.59 0.78c
2.79 0.65d
0.46 0.15e
0.002
5.09 2.10
6.79 3.74
17.79 0.59a
11.07 0.63e
0.003
4.67 2.45
7.43 3.31
24.68 3.74c
10.54 3.55b
0.002
Control + VOL
Diabetic
Diabetic + VOL
PANOVA
*Mean SD
a P < 0.005 compared with control group
b P < 0.0001compared with diabetic group
c P < 0.0001 compared with control group
d P < 0.05 compared with control group
e P < 0.05 compared with diabetic group
23.93 5.84. Although the number of PCNA+- villus cell
index were very low in the control group, it was observed
that PCNA+- villus cell number increased in diabetic group
(p < 0.01), while VOL administration to these rats resulted
in a reduction in PCNA+ - villus cell number (p < 0.01).
PCNA+ - villus cell numbers were as follows: C: 0.03
0.03, VOL: 0.0 0.0, D: 0.64 0.07 and D + VOL: 0.19
0.06. The most prominent findings were in PCNA+ in-
flammatory cells. PCNA+ inflammatory cell number in-
creased in diabetic group (p < 0.01), while VOL treatment
to these rats resulted in a decrease in PCNA+ inflammatory
cell number (p < 0.05). PCNA+ inflammatory cell numbers
were as follows: C: 2.7 0.29, VOL: 6.26 0.26, D: 18.59
2.13 and D + VOL: 10.11 1.24 (Fig. 3).
Fig. 2 Histological appearance of
duodenum from each group.
Normal histological appearance
of duodenal mucosa of control or
VOL-treated control rats can be
seen in the photos. We observed a
remarkable decrease in the mu-
cosal necrotic areas (NA), dis-
continuity in the brush border
(arrow), deterioration of integrity
(*) of the villi (V), while mild
decrease in the mononuclear in-
flammatory cells (MCI) and dete-
rioration of crypt (cry) integrity in
the small intestine by VOL treat-
ment to diabetic rats. (B)
Brunner’s gland, (C) control rats,
VOL: oxovanadium-treated rats,
(D) diabetic rats, D + VOL:
oxovanadium-treated diabetic
rats. PAS, X135

Tunali et al.
Fig. 3 PCNA+ crypt, villus and inflammatory cells in the duodenum.
PCNA+ villus and inflammatory cell number increased in diabetic rats.
VOL administration caused a significant decrease in the PCNA+ villus
and inflammatory cell number in diabetic rats. PCNA+ inflammatory cells
(thin arrow), PCNA+ crypt cell (thick arrow).**p < 0.01 vs control and #p
< 0.05, ##p < 0.01 vs diabetic rats. Values are mean S.E.M. n = 5
animals for each group. C, control rats; VOL, oxovanadium-treated rats;
D, diabetic rats; D + VOL, oxovanadium-treated diabetic rats. X540
Discussion
compared with non-treated diabetic animals. Regulation of
glycoprotein component level under DM conditions further
emphasizes the therapeutic effect of VOL in relieving DM-
related complications.
Due to the increasing prevalence of DM in the world, studies
are underway to investigate new antidiabetic agents [52].
Researches involving biochemistry, immunohistochemistry,
histopathology and bioinorganic chemistry indicate that vana-
dium ligands and vanadium complexes are useful in the treat-
ment of experimental diabetes [30, 53, 54]. In the present
study, a vanadium complex was synthesized and tested for
its hypoglycaemic effect in STZ-induced diabetic rats [30].
Increased ROS production or low ROS sweeping capacity
causes oxidative stress to the pathogenesis of diabetic compli-
cations [55]. GSH is a cysteine-containing tripeptide whose
sulfhydryl group (–SH) is involved in the reduction and con-
jugation reactions [56]. In our study, it was seen that GSH
level was decreased in diabetic small intestinal tissue. The
administration of VOL to diabetic rats improved GSH level,
and this indicates the positive function of the VOL complex
on improving the antioxidant status [30, 52].
An increase in glycoprotein components such as SA is
another patho-biochemical modification that arises due to
hyperglycaemia. It is proposed that under DM conditions,
the increasing level of SA would cause insulin resistance
and affect the functionality of the tissues [57]. In the present
study, an elevated small intestine SA level of diabetic rats
compared with the control group was observed. The treatment
with VOL decreased the SA level of the diabetic group when
Antioxidant enzymes such as CAT, GPx, GST and SOD
play an important role in scavenging the toxic intermediate of
incomplete oxidation [52, 58]. The excessive production of
H2O2 and O2 from the auto oxidation of extremely high glu-
cose concentration and non-enzymatic glycation of proteins
may result from the decreased activities of SOD and CAT
[59]. GPx and GST are GSH-related enzymes that have de-
pressed or inactivated enzymatic activity in the small intestine
tissue of DM. Higher protein glycation in DM is associated
with inactivation of these enzymes. Indeed, glycation inacti-
vates GPx due to the modification of arginine residues located
in the glutathione binding site of the enzyme [60]. In this
context, our data show decreased activity of these enzymes
in the small intestine tissue of diabetic rats. Oral administra-
tion of VOL to diabetic rats resulted in improved activities of
CAT, GPx, GST and SOD, which in turn indicates the anti-
oxidant potential of the complex. Similarly, a decreased tissue
Na+/ K+-ATPase activity in experimental diabetes was report-
ed [61]. In the present work, a 12-day treatment of diabetic rats
with VOL significantly increased small intestinal Na+/K+-
ATPase activity. It was suggested that the improvement of
carbohydrate metabolism through vanadium’s
insulinomimetic and insulin enhancing effects in diabetic rats

Protective Effects of an Oxovanadium(IV) Complex with N2O2 Chelating Thiosemicarbazone on Small...
reduced oxidative stress and increased the activity of Na+/ K+-
ATPase in the small intestine [62].
absorption in diabetic rats, and these changes are suppressed
by VOL pre-treatment. The most striking outcome is the pos-
sibility of VOL’s preventive effect on necrosis and inflamma-
tion in the villi area through the inhibition of rapid carbohy-
drate digestion and absorption.
Previous studies show the association between lower
PON1 and increased XO activities in DM [63]. According to
Ibrahim et al., the reduced PON1 and elevated XO enzymatic
activity is the reason for oxidative stress scenario in the cardi-
ac tissue of diabetic rats [64]. Similarly to this study, we also
noticed decreased PON and elevated XO activities in the small
intestinal tissue of diabetic rats. According to the data of the
present finding, VOL treatment restored the activities of both
enzymes in the diabetic group.
Interestingly, the present study observed a decreased small
intestinal GSH level as well as that of GSH-related enzyme
activities (GPx and GST) in VOL-treated control rats. XO
activity was also increased in the same group. The decreased
GSH level in normal rats may be caused by the toxic effect of
VOL in the small intestine. This is possibly a result of depleted
GSH function, a compound that plays a role in the elimination
of peroxides and many xenobiotic compounds. The declined
thiol levels lead to GSH depletion and generation of ROS,
thereby leading to a raised XO activity in the VOL-given
control rats.
Conclusion
According to the obtained results, diabetes induces oxidative
stress and disrupts the antioxidant defence system in the small
intestine of rat tissue. The biochemical, immunohistochemical
and histopathological results of the present study suggest that
VOL is a potentially favourable complex for diminishing
small intestine deterioration in diabetic rats. However, the
same concentration of this vanadium complex has also been
observed to cause irreversible damage in the small intestinal
tissue when administered to the control animals.
Funding Information This work was supported by Scientific Research
Projects Coordination Unit of Istanbul University, Project Number: BEK-
2016-22294.
The inhibition of intestinal α-glycosidases (maltase and
saccharase) delays the digestion of starch and causes reduced
glucose absorption. Such effect would control/stabilize post-
prandial hyperglycaemia which is considered an important
approach in the treatment of the DM [65]. In our research,
the activities of intestinal maltase and saccharase due to
hyperglycaemia were increased in diabetic rats. VOL admin-
istration inhibited the α-glycosidase activities in the diabetic
group as to STZ-induced diabetic animals. It is reported that
the expression of sugar carriers increases with duodenal sac-
charase and lactase activities in diabetic individuals, leading to
hyperglycaemia due to excessive carbohydrate consumption/
uptake [66]. Similar to these, an increase in maltase and sac-
charase activities of diabetic animals of our study suggests that
carbohydrate digestion is accelerated. Our histopathological
findings indicate the accumulation of inflammatory cell and
necrotic areas, coupled with discontinuity in the brush border
and oedema in the villi of the diabetic group. It may be pos-
sible to develop these histopathological changes in the villi
area as a result of continuous rapid digestion and absorption.
We observed a remarkable decrease in the mucosal necrotic
areas, with mild decrease in the inflammatory cells, deteriora-
tion and loss of integrity of the small intestine glands upon
VOL pre-treatment to diabetic rats. The low levels of maltase
and saccharase activities in this group compared with diabetic
animals suggest that carbohydrate digestion is maintained at
normal levels. On the other hand, VOL pre-treatment mark-
edly decreased proliferation of villus cells and especially in-
flammatory cells in the small intestine of diabetic rats. When
all the findings are combined, it can be suggested that villus
cells proliferate to meet the need for an increased glucose
Compliance with Ethical Standards
Conflict of Interest The authors declare that there is no any conflict of
interest.
Ethics Approval Experiments were reviewed and approved according to
the Animal Care and Use Institute’s Committee of Istanbul University.
References
1. Lolak N, Akocak S, Türkeş C, Taslimi P, Işık M, Beydemir Ş,
Gülçin İ, Durgun M (2020) Synthesis, characterization, inhibition
effects, and molecular docking studies as acetylcholinesterase, α-
glycosidase, and carbonic anhydrase inhibitors of novel
benzenesulfonamides incorporating 1,3,5-triazine structural motifs.
Bioorg Chem 100:103897. https://doi.org/10.1016/j.bioorg.2020.
103897
2. Artunc T, Menzek A, Taslimi P, Gulcin I, Kazaz C, Sahin E (2020)
Synthesis and antioxidant activities of phenol derivatives from 1,6-
bis (dimethoxyphenyl) hexane-1,6-dione. Bioorg Chem 100:
103884. https://doi.org/10.1016/j.bioorg.2020.103884
3. Aktas A, Barut Celepci D, Gok Y, Taslimi P, Akincioglu H, Gulcin
İ (2020) A novel Ag-N-heterocyclic carbene complex bearing the
hydroxyethyl ligand: synthesis, characterization, crystal and spec-
tral structures and bioactivity properties. Crystals. 10(3):171.
https://doi.org/10.3390/cryst10030171
4. Strain WD, Paldánius PM (2018) Diabetes, cardiovascular disease
and the microcirculation. Cardiovasc Diabetol 17(1):57. https://doi.
org/10.1186/s12933-018-0703-2
5. Yanardag R, Bolkent S, Karabulut-Bulan O, Tunali S (2003)
Effects of vanadyl sulfate on kidney in experimental diabetes.
Biol Trace Elem Res 95(1):73–85. https://doi.org/10.1385/BTER:
95:1:73
6. Tunali S, Yanardag R (2006) Effect of vanadyl sulfate on the status
of lipid parameters and on stomach and spleen tissues of

Tunali et al.
streptozotocin-induced diabetic rats. Pharmacol Res 53(3):271–
277. https://doi.org/10.1016/j.phrs.2005.12.004
hydrolysate of soy protein on carbohydrate and lipid metabolism
disorders in male Wistar rats. Bull Exp Biol Med 168(5):637–640.
https://doi.org/10.1007/s10517-020-04769-x
7. Hex N, Bartlett C, Wright D, Taylor M, Varley D (2012) Estimating
the current and future costs of Type 1 and Type 2 diabetes in the
UK, including direct health costs and indirect societal and produc-
tivity costs. Diabet Med 29(7):855–862. https://doi.org/10.1111/j.
1464-5491.2012.03698.x
22. Tunali S, Yanardag R (2013) Protective effect of vanadyl sulfate on
skin injury in streptozotocin-induced diabetic rats. Hum Exp
Toxicol 32(11):1206–1212. https://doi.org/10.1177/
0960327113478445
8. Mukherjee S, Chattopadhyay M, Bhattacharya S, Dasgupta S,
Hussain S, Bharadwaj SK, Talukdar D, Usmani A, Pradhan BS,
Majumdar SS, Chattopadhyay P, Mukhopadhyay S, Maity TK,
Chaudhuri MK, Bhattacharya S (2017) A small insulinomimetic
molecule also improves insulin sensitivity in diabetic mice. PLoS
One 12(1):e0169809. https://doi.org/10.1371/journal.pone.
0169809
9. Gotfried J, Priest S, Schey R (2017) Diabetes and the small intes-
tine. Curr Treat Options Gastroenterol 15(4):490–507. https://doi.
org/10.1007/s11938-017-0155-x
23. Koyuturk M, Tunali S, Bolkent S, Yanardag R (2005) Effects of
vanadyl sulfate on liver of streptozotocin-induced diabetic rats. Biol
Trace Elem Res 104(3):233–247. https://doi.org/10.1385/BTER:
104:3:233
24. Ramachandran E, Thomas SP, Poornima P, Kalaivani P,
Prabhakaran R, Padma VV, Natarajan K (2012) Evaluation of
DNA binding, antioxidant and cytotoxic activity of mononuclear
Co(III) complexes of 2-oxo-1,2-dihydrobenzo[h]quinoline-3-
carbaldehyde thiosemicarbazones. Eur J Med Chem 50:405–415.
https://doi.org/10.1016/j.ejmech.2012.02.026
10. Gulcin İ (2020) Antioxidants and antioxidant methods: an updated
overview. Arch Toxicol 94(3):651–715. https://doi.org/10.1007/
s00204-020-02689-3
11. Gülçin İ, Görenb AC, Taslimid P, Alwasele SH, Kılıc O, Bursal E
(2020) Anticholinergic, antidiabetic and antioxidant activities of
Anatolian pennyroyal (Mentha pulegium)-analysis of its polyphe-
nol contents by LC-MS/MS. Biocatal Agric Biotechnol 23:101441.
https://doi.org/10.1016/j.bcab.2019.101441
12. Bhor V, Raghuram N, Sivakami S (2004) Oxidative damage and
altered antioxidant enzyme activities in the small intestine of
streptozotocin-induced diabetic rats. Int J Biochem Cell Biol
36(1):89–97. https://doi.org/10.1016/s1357-2725(03)00142-0
13. Itoh H, Naganuma S, Takeda N, Miyata S, Uchinokura S,
Fukushima T, Uchiyama S, Tanaka H, Nagaike K, Shimomura T,
Miyazawa K, Yamada G, Kitamura N, Koono M, Kataoka H
(2004) Regeneration of injured intestinal mucosa is impaired in
hepatocyte growth factor activator-deficient mice.
Gastroenterology 127(5):1423–1435. https://doi.org/10.1053/j.
gastro.2004.08.027
25. Bal-Demirci T, Şahin M, Kondakçı E, Özyürek M, Ülküseven B,
Apak R (2015) Synthesis and antioxidant activities of transition
metal complexes based 3-hydroxysalicylaldehyde-S-
methylthiosemicarbazone. Spectrochim Acta A Mol Biomol
Spectrosc 138:866–872. https://doi.org/10.1016/j.saa.2014.10.088
26. Chaves OA, de Castro IS, Goulart CM, Bellieny MSS, Netto-
Ferreira JC, Echevarria-Lima J, Echevarria A (2019) In vitro and
in vivo cytotoxic activity and human serum albumin interaction for
a methoxy-styryl-thiosemicarbazone. Investig New Drugs 37(5):
994–1005. https://doi.org/10.1007/s10637-018-00722-y
27. Bal T, Atasever B, Solakoğlu Z, Erdem-Kuruca S, Ulküseven B
(2007) Synthesis, characterisation and cytotoxic properties of the
N1,N4-diarylidene-S-methyl-thiosemicarbazone chelates with
Fe(III) and Ni(II). Eur J Med Chem 42(2):161–167. https://doi.
org/10.1016/j.ejmech.2006.09.004
28. Guo H, Lu J, Ruan Z, Zhang Y, Liu Y, Zang L, Jiang J, Huang J
(2012) Synthesis, DNA-binding, cytotoxicity, and cleavage studies
of unsymmetrical oxovanadium complexes. J Coord Chem 65:191–
204. https://doi.org/10.1080/00958972.2011.645204
14. Malik A, Morya RK, Bhadada SK, Rana S (2018) Type 1 diabetes
mellitus: complex interplay of oxidative stress, cytokines, gastroin-
testinal motility and small intestinal bacterial overgrowth. Eur J
Clin Investig 48(11):e13021. https://doi.org/10.1111/eci.13021
15. Ordog T (2008) Interstitial cells of Cajal in diabetic
gastroenteropathy. Neurogastroenterol Motil 20(1):8–18. https://
doi.org/10.1111/j.1365-2982.2007.01056.x
16. Sanna D, Ugone V, Serra M, Garribba E (2017) Speciation of
potential anti-diabetic vanadium complexes in real serum samples.
J Inorg Biochem 173:52–65. https://doi.org/10.1016/j.jinorgbio.
2017.04.023
29. Benítez J, Becco L, Correia I, Leal SM, Guiset H, Pessoa JC,
Lorenzo J, Tanco S, Escobar P, Moreno V, Garat B, Gambino D
(2011) Vanadium polypyridyl compounds as potential antiparasitic
and antitumoral agents: new achievements. J Inorg Biochem
105(2):303–312. https://doi.org/10.1016/j.jinorgbio.2010.11.001
30. Yanardag R, Demirci TB, Ulküseven B, Bolkent S, Tunali S,
Bolkent S (2009) Synthesis, characterization and antidiabetic prop-
erties of N(1)-2,4-dihydroxybenzylidene-N(4)-2-
hydroxybenzylidene-S-methyl-thiosemicarbazidato-
oxovanadium(IV). Eur J Med Chem 44(2):818–826. https://doi.
org/10.1016/j.ejmech.2008.04.023
17. Treviño S, Diaz A (2020) Vanadium and insulin: Partners in met-
abolic regulation. J Inorg Biochem 208:111094. https://doi.org/10.
1016/j.jinorgbio.2020.111094
18. Niu X, Xiao R, Wang N, Wang Z, Zhang Y, Xia Q, Yang X (2016)
The molecular mechanisms and rational design of anti-diabetic va-
nadium compounds. Curr Top Med Chem 16:811–822. https://doi.
org/10.2174/1568026615666150827094652
19. Xie MJ, Yang XD, Liu WP, Yan SP, Meng ZH (2010) Insulin-
enhancing activity of a dinuclear vanadium complex: 5-chloro-
salicylaldhyde ethylenediamine oxovanadium(V) and its perme-
ability and cytotoxicity. J Inorg Biochem 104(8):851–857. https://
doi.org/10.1016/j.jinorgbio.2010.03.018
20. Treviño S, Díaz A, Sánchez-Lara E, Sanchez-Gaytan BL, Perez-
Aguilar JM, González-Vergara E (2019) Vanadium in biological
action: chemical, pharmacological aspects, and metabolic implica-
tions in diabetes mellitus. Biol Trace Elem Res 188(1):68–98.
https://doi.org/10.1007/s12011-018-1540-6
31. Matsa R, Makam P, Kaushik M, Hoti SL, Kannan T (2019)
Thiosemicarbazone derivatives: design, synthesis and in vitro anti-
malarial activity studies. Eur J Pharm Sci 137:104986. https://doi.
org/10.1016/j.ejps.2019.104986
32. Summers KL (2019). A structural chemistry perspective on the
antimalarial properties of thiosemicarbazone metal complexes.
Mini Rev Med Chem 19(7):569–590. https://doi.org/10.2174/
1389557518666181015152657
33. Padmanabhan P, Khaleefathullah S, Kaveri K, Palani G,
Ramanathan G, Thennarasu S, Tirichurapalli Sivagnanam U
(2017) Antiviral activity of thiosemicarbazones derived from α-
amino acids against Dengue virus. J Med Virol 89(3):546–552.
https://doi.org/10.1002/jmv.24655
34. Trotsko N, Kosikowska U, Paneth A, Plech T, Malm A, Wujec M
(2018) Synthesis and antibacterial activity of new thiazolidine-2,4-
dione-based chlorophenylthiosemicarbazone hybrids. Molecules
23(5):1023. https://doi.org/10.3390/molecules23051023
21. Sidorova YS, Zorin SN, Petrov NA, Shumakova AA, Frolova YV,
Mazo VK (2020) Effect of vanadium complex with enzymatic
35. Mrozek-Wilczkiewicz A, Malarz K, Rejmund M, Polanski J,
Musiol R (2019) Anticancer activity of the thiosemicarbazones that

Protective Effects of an Oxovanadium(IV) Complex with N2O2 Chelating Thiosemicarbazone on Small...
are based on di-2-pyridine ketone and quinoline moiety. Eur J Med
Chem 171:180–194. https://doi.org/10.1016/j.ejmech.2019.03.027
36. Anjum R, Palanimuthu D, Kalinowski DS, Lewis W, Park KC,
Kovacevic Z, Khan IU, Richardson DR (2019) Synthesis, charac-
terization, and in vitro anticancer activity of copper and zinc bis
(thiosemicarbazone) complexes. Inorg Chem 58(20):13709–13723.
https://doi.org/10.1021/acs.inorgchem.9b01281
37. Benítez J, Guggeri L, Tomaz I, Arrambide G, Navarro M, Costa
Pessoa J, Garat B, Gambino D (2009) Design of vanadium mixed-
ligand complexes as potential anti-protozoa agents. J Inorg
Biochem 103(4):609–616. https://doi.org/10.1016/j.jinorgbio.
2008.10.018
38. Jound A, Lambert E, Stauffacher W, Renold AE (1969)
Diabetogenic action of streptozotocin. Relationship of dose to met-
abolic response. J Clin Invest 48:2129–2139. https://doi.org/10.
1172/JCI106180
39. Relander A, Raiha CE (1963) Differences between the enzymatic
and toluidine methods of blood glucose determination. Scand J Clin
Lab Invest 15:221–224
40. Beutler E (1975) Glutathione in red blood cell metabolism. In: A
manual of biochemical methods, 2nd ed. Grune and Stratton, New
York, pp 112–114
41. Warren L (1959) The thiobarbituric acid assay of sialic acids. J Biol
Chem 234:1971–1975
42. Lowry OH, Rosebrough WI, Farr AL, Randal RJ (1951) Protein
measurement with the Folin phenol reagent. J Biol Chem 193:265–
275
43. Aebi H (1984) Catalase in vitro. Methods Enzymol 105:121–126.
https://doi.org/10.1016/s0076-6879(84)05016-3
54. Yanardag R, Tunali S (2006) Vanadyl sulfate administration pro-
tects the streptozotocin-induced oxidative damage to brain tissue in
rats. Mol Cell Biochem 286(1-2):153–159. https://doi.org/10.1007/
s11010-005-9107-1
55. Siddiqui MR, Taha A, Moorthy K, Hussain ME, Basir SF, Baquer
NZ (2005) Amelioration of altered antioxidant status and mem-
brane linked functions by vanadium and Trigonella in alloxan dia-
betic rat brains. J Biosci 30(4):483–490. https://doi.org/10.1007/
BF02703722
56. Forman HJ, Zhang H, Rinna A (2009) Glutathione: overview of its
protective roles, measurement, and biosynthesis. Mol Asp Med
30(1-2):1–12. https://doi.org/10.1016/j.mam.2008.08.006
57. Khalili P, Sundström J, Jendle J, Lundin F, Jungner I, Nilsson PM
(2014) Sialic acid and incidence of hospitalization for diabetes and
its complications during 40-years of follow-up in a large cohort: the
Värmland survey. Prim Care Diabetes 8(4):352–357. https://doi.
org/10.1016/j.pcd.2014.06.002
58. Miyazaki Y, Kawano H, Yoshida T, Miyamoto S, Hokamaki J,
Nagayoshi Y, Yamabe H, Nakamura H, Yodoi J, Ogawa H
(2007) Pancreatic B-cell function is altered by oxidative stress in-
duced by acute hyperglycaemia. Diabet Med 24(2):154–160.
https://doi.org/10.1111/j.1464-5491.2007.02058.x
59. Aragno M, Brignardello E, Tamagno E, Gatto V, Danni O,
Boccuzzi G (1997) Dehydroepiandrosterone administration pre-
vents the oxidative damage induced by acute hyperglycemia in rats.
J Endocrinol 155(2):233–240. https://doi.org/10.1677/joe.0.
1550233
60. Lapenna D, Ciofani G, Calafiore AM, Cipollone F, Porreca E
(2018) Impaired glutathione-related antioxidant defenses in the ar-
terial tissue of diabetic patients. Free Radic Biol Med 124:525–531.
https://doi.org/10.1016/j.freeradbiomed.2018.06.033
61. Morsy MD, Abdel-Razek HA, Osman OM (2011) Effect of vana-
dium on renal Na+, K+-ATPase activity in diabetic rats: a possible
role of leptin. J Physiol Biochem 67(1):61–69. https://doi.org/10.
1007/s13105-010-0049-z
62. Bełtowski J, Jamroz-Wiśniewska A, Borkowska E, Nazar J,
Marciniak A (2005) Antioxidant treatment normalizes renal Na+,
K+-ATPase activity in leptin-treated rats. Pharmacol Rep 57(2):
219–228
63. Ayan D, Şeneş M, Çaycı AB, Söylemez S, Eren N, Altuntaş Y,
Öztürk FY (2019) Evaluation of paraoxonase, arylesterase, and
homocysteine thiolactonase activities in patients with diabetes and
incipient diabetes nephropathy. J Med Biochem 38(4):481–488.
https://doi.org/10.2478/jomb-2019-0014
44. Paglia DE, Valentine WN (1967) Studies on the quantitative and
quantitative characterization of erythrocyte glutathione peroxidase.
J Lab Clin Med 70:158–168
45. Wendel A (1981) Glutathione peroxidase. Methods Enzymol 77:
325–333. https://doi.org/10.1016/s0076-6879(81)77046-0
46. Habig WH, Jacoby WB (1981) Assays for differentiation of gluta-
thione-S-transferases. Methods Enzymol 77:398–405. https://doi.
org/10.1016/s0076-6879(81)77053-8
47. Mylroie AA, Collins AH, Umbles C, Kyle J (1986) Erythrocyte
superoxide dismutase activity and other parameters of copper status
in rats ingesting lead acetate. Toxicol Appl Pharmacol 82:512–520.
https://doi.org/10.1016/0041-008x(86)90286-3
48. Ridderstap AS, Bonting SL (1969) Na+-K+-activated ATPase and
exocrine pancreatic secretion in vitro. Am J Phys 217:1721–1727.
https://doi.org/10.1152/ajplegacy.1969.217.6.1721
49. Furlong CE, Richter RJ, Seidel SL, Motulsky AG (1988) Role of
genetic polymorphism of human plasma paraoxonase/arylesterase
in hydrolysis of the insecticide metabolites chlorpyrifos oxon and
paraoxon. Am J Hum Genet 43:230–238
50. Corte ED, Stirpe F (1968) Regulation of xanthine oxidase in rat
liver: modifications of the enzyme activity of rat liver supernatant
on the storage at 20 degrees. Biochem J 108:349–351. https://doi.
org/10.1042/bj1080349
64. Ibrahim IA, Khwanes SA, El-Desouky MA, Elhakim HKA (2019)
Propolis relieves the cardiotoxicity of chlorpyrifos in diabetic rats
via alleviations of paraoxonase-1 and xanthine oxidase genes ex-
pression. Pestic Biochem Physiol 159:127–135. https://doi.org/10.
1016/j.pestbp.2019.06.006
65. Penislusshiyan S, Chitra L, Ancy I, Kumaradhas P, Palvannan T
(2020) Novel antioxidant astaxanthin-s-allyl cysteine biconjugate
diminished oxidative stress and mitochondrial dysfunction to tri-
umph diabetes in rat model. Life Sci 245:117367. https://doi.org/
10.1016/j.lfs.2020.117367
51. Dahlqvist A (1968) Assay of intestinal disaccharidases. Anal
Biochem 22(1):99–107. https://doi.org/10.1016/0003-2697(68)
90263-7
52. Pillai SI, Subramanian SP, Kandaswamy M (2013) Evaluation of
antioxidant efficacy of vanadium-3-hydroxyflavone complex in
streptozotocin-diabetic rats. Chem Biol Interact 204(2):67–74.
https://doi.org/10.1016/j.cbi.2013.04.012
66. Dyer J, Wood IS, Palejwala A, Ellis A, Shirazi-Beechey SP (2002)
Expression of monosaccharide transporters in intestine of diabetic
humans. Am J Physiol Gastrointest Liver Physiol 282:G241–G248.
https://doi.org/10.1152/ajpgi.00310.2001
53. Akgün-Dar K, Bolkent S, Yanardag R, Tunali S (2007) Vanadyl
sulfate protects against streptozotocin-induced morphological and
biochemical changes in rat aorta. Cell Biochem Funct 25(6):603–
609. https://doi.org/10.1027/cbf.1354
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