A novel insulin mimetic vanadium-flavonol complex: Synthesis, characterization and in vivo evaluation in STZ-induced rats

Article abstract of DOI:10.1016/j.ejmech.2013.02.002

Since 1985, when Heyliger et al., first demonstrated a serendipitous discovery that oral administration of 0.8 mg/ml of sodium orthovanadate in drinking water to streptozotocin-induced diabetic rats resulted in normoglycemia, numerous extensive studies have been pursued on the anti-diabetic and insulinomimetic actions of vanadium. The acceptance of vanadium compounds as promising therapeutic antidiabetic agents has been slowed due to the concern for chronic toxicity associated with vanadium accumulation. In order to circumvent the toxic effects of vanadium, we have taken up a combinational approach wherein a novel vanadium-flavonol complex was synthesized, characterized and its toxic as well as insulin mimetic potential was evaluated in STZ-induced experimental diabetes in rats. The results indicate that the complex is non-toxic and possess anti-diabetic activity.

Full text of DOI:10.1016/j.ejmech.2013.02.002

European Journal of Medicinal Chemistry 63 (2013) 109e117
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
A novel insulin mimetic vanadiumeflavonol complex: Synthesis, characterization
and in vivo evaluation in STZ-induced rats
,
Subramanian Iyyam Pillai a, Sorimuthu Pillai Subramanian b, Muthusamy Kandaswamy a
*
a Department of Inorganic Chemistry, University of Madras, Guindy Campus, Chennai 600 025, Tamil Nadu, India
b Department of Biochemistry, University of Madras, Guindy Campus, Chennai 600 025, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Since 1985, when Heyliger et al., first demonstrated a serendipitous discovery that oral administration of
0.8 mg/ml of sodium orthovanadate in drinking water to streptozotocin-induced diabetic rats resulted in
normoglycemia, numerous extensive studies have been pursued on the anti-diabetic and insulinomi-
metic actions of vanadium. The acceptance of vanadium compounds as promising therapeutic antidia-
betic agents has been slowed due to the concern for chronic toxicity associated with vanadium
accumulation. In order to circumvent the toxic effects of vanadium, we have taken up a combinational
approach wherein a novel vanadiumeflavonol complex was synthesized, characterized and its toxic as
well as insulin mimetic potential was evaluated in STZ-induced experimental diabetes in rats. The results
indicate that the complex is non-toxic and possess anti-diabetic activity.
Received 7 April 2012
Received in revised form
31 January 2013
Accepted 2 February 2013
Available online 16 February 2013
Keywords:
Vanadium
Flavonol
Insulin mimetic
Diabetes
Ó 2013 Elsevier Masson SAS. All rights reserved.
Spectroscopic analysis
1. Introduction
increasingly important over the last couple of decades result in a
variety of exciting and valuable metallo pharmaceutical drugs such
The prevalence of diabetes mellitus is rapidly emerging at an
alarming rate and is considered to be one of the biggest health
catastrophes in the world causing significant health and economic
burdens on patients and communities [1]. The number of diabetic
patients is estimated to rise to over 439 million by 2030, making
diabetes the second most frequent cause of death after cancer [2].
Diabetes mellitus is characterized by chronic hyperglycemia
resulting in the disturbances of carbohydrate, lipid and protein
metabolism with a high risk of morbidity and mortality from pri-
mary as well as secondary complications [3]. Most of the oral hy-
poglycemic drugs currently used for the treatment of diabetes such
as cis-platin and auranofin as anticancer and antiarthritic drugs
respectively [5]. Studies on various structureepharmacological
activity relationships and therapeutic aspects of metals binding to
cellular targets have paved immense scope for designing novel
coordination compounds that are suitable to bind and elicit bene-
ficial pharmacological activities.
Vanadium, an element from the transition metal group of the
periodic table is proposed to be an essential trace element in ani-
mals and humans. It is present in plant and animal cells at con-
centrations ranging from 10 to 20 nmol [6]. Vanadium derivatives
have been shown to possess insulin-mimetic and antidiabetic ac-
tivities in animal models of type 1 and type 2 diabetes mellitus as
well as in a small number of diabetic human subjects [7e10]. The
coordination chemistry of vanadium has great versatility for
adjustment of pharmacological characteristics [11]. However, most
of the vanadium complexes so far investigated for their possible
antidiabetic activity were poorly absorbed in their inorganic forms
and required high doses which have been associated with unde-
sirable side effects. In order to circumvent the toxicity, various
organo vanadium complexes have been formulated and tested for
their antidiabetic activity [12].
as sulphonylureas, biguanides,
a-glycosidase inhibitors and thia-
zolidenes are often associated with undesirable side effects or
diminution in response after prolonged use [4]. Hence, the search
continues for novel drugs with effective antidiabetic activity at low
concentration without side effects.
Mineral elements serve as structural components of tissues as
well as constituents of the body fluids and vital enzymes in major
metabolic pathways. The use of metals in therapeutic drugs become
Flavonoids have a basic structure of 2-phenyl-benzo-g-pyrones,
mostly polyphenolic in nature [13]. A multitude of substitution
patterns in the two benzene rings of the basic structure exists in
* Corresponding author. Tel./fax: þ91 44 22300488.
E-mail
addresses:
mkands@yahoo.com,
gsriramprasath@gmail.com
(M. Kandaswamy).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.02.002

110
S.I. Pillai et al. / European Journal of Medicinal Chemistry 63 (2013) 109e117
nature. Variations in their heterocyclic rings give rise to flavonols,
flavones, catechins, flavonones, anthocyanidins and isoflavones.
Among the flavonoids, flavonol is known to chelate metal ions with
obtained at a maximum of pH around 2.5. Above the pH 3.5, the
dimeric species, (VO)-hydroxyflavone formed is stable. By
employing the stability constants and hydrolysis constants in the
origin 8.5 version, species distribution diagram was generated as
depicted is Fig. 1.
great affinity owing to the presence of an
a hydroxy-carbonyl group
[14]. It was observed that some flavonoids possess antidiabetic
properties [15,16]. The interaction of flavonoids with metal ions may
change the antioxidant properties and some biological effects of the
flavonoids [17]. Recently we have reported that the oral adminis-
tration of zinc-3-hydroxyflavone at a concentration of 5 mg/kg b.w
to STZ induced experimental diabetic rats significantly improved
altered biochemical parameters and its non-toxic nature [18].
Having in view these aspects, an attempt has been made to
synthesize a novel vanadium complex using 3-hydroxyflavone
(flavonol) as an organic ligand. The metallo complex was charac-
terized by spectral studies. Chronic toxicity studies as well as
antidiabetic activity of the complex were evaluated in
streptozotocin-induced experimental diabetes in rats.
H
þ þ HFꢀ#HHF log Ka ¼ 8:38
(1)
(2)
(3)
(4)
VO2þ þ HFꢀ#VOðHFÞþ log K1 ¼ 8:65
VOðHFÞþ þ HFꢀ#VOðHFÞ2 log K2 ¼ 7:39
VO2þ þ 2ðHFÞꢀ#VOðHFÞ2 log
b
¼ 16:14
2.3. FTIR e spectral data
The IR spectra of 3-hydroxyflavone and vanadiumeflavonol
complex are depicted in Fig. 2. In the IR spectra, the lowering of
the carbonyl carbon stretching coupled with the absence of (eOH)
clearly indicates that the vanadyl anion is attached to both 4-keto
and 3-hydroxy groups. The shift may be due to the VO (IV) coor-
dination that occurs through the oxygen of the carbonyl carbon
atom and the 3-OH groups of the ligand after deprotonation. The
band around 1530 cmꢀ1 can be considered as associated with the
antisymmetric stretching mode, of the g(CeO) groups at the
chelating site. The band related to the g(CeOeC) mode at 1294 cmꢀ1
is slightly shifted by complex formation (1287 cmꢀ1), indicating a
weak alteration of the ring structure and the lack of interaction of
the O ring atom with the metal. The band at 978 cmꢀ1 is assigned to
2. Result and discussion
2.1. Synthesis of vanadiumeflavonol complex
The possible reaction of vanadium with 3-hydroxyflavone for
the formation of vanadiumeflavonol complex was depicted in
Scheme 1. Bordbar et al. (2009) have reported that the higher po-
tency of oxovanadium complexes with maltol or ethylmaltol as
antidiabetic agents has been attributed to a number of possible
factors. Besides improved absorption, protection of vanadium ion-
induced gastrointestinal irritation by the ligands is a possibility
[19]. Since, the binding of flavonol is similar to the maltol or eth-
ylmaltol, it can be concluded that the vanadyl ions in vanadiume
flavonol complex readily oxidized to vanadate ions to facilitate
pharmacological studies.
g
]O), this band was observed as a new peak for the vanadyle
(V
flavonol complex and not existed in the spectrum of free ligand.
The band at 1026 cmꢀ1 may be due to the presence of SOꢀ4 ion. Thus,
it can be induced that 3-hydroxyflavone anion forms a chelate with
vanadium. This observation is in accordance with those reported by
others workers [23,24].
2.2. Potentiometric pH titration
Potentiometric titrations were carried out for determination of
the formation constant of the successive complex formed by the
vanadyl ion and hydroxyflavone molecule. The stability constants
of the vanadium-hydroxyflavone complex determined are shown
in Eqns. (1)e(4). Since the flavonol moiety is much similar to maltol
and their derivatives, the potentiometric titrations of vanadium-
hydroxyflavone complex were in analogous pattern of equilibria
described earlier with Bis(maltolato) oxovanadium(IV) [20], Bis(e-
thylmaltolato) oxovanadium(IV) [21], Bis(isomaltolato) oxovana-
dium(IV) [21], and bis(kojato) oxovanadium(IV) [22] complexes.
The monomeric species exists in between the pH range of 2e3 and
2.4. UVeVisible spectral data
The UV spectra of 3-hydroxyflavone and vanadiumeflavonol
complex dissolved in methanol (10ꢀ3) have been measured and
the data obtained were depicted in Fig. 3. In the UV region of
spectrum, two major absorption maxima are typically observed for
the flavonol structure. The first absorption maximum, observed at
241 nm can be considered as originating from
p
ꢀ
p* transitions in
the A ring, a benzene system, and the second absorption maximum
observed around 341 nm may be assigned to transition in the B
ring, a cinnamoyl system. The absorptions at around 908 nm and
623 nm are assigned to the spin allowed 2B2
/
2E and 2B2 / 2B1.
Scheme 1. Synthesis of vanadiumeflavonol complex.
Fig. 1. Speciation diagram for the VO2þ/3HF system.

S.I. Pillai et al. / European Journal of Medicinal Chemistry 63 (2013) 109e117
111
Fig. 2. IR spectra of the free ligand and its complex.
Moreover a shoulder observed around 446 nm can be assigned to
the 2B2 2A1. This observation is in accordance with a previous
investigation shows that 3-hydroxyflavone itself exhibits a strong
fluorescence, but the fluorescence intensity of the complex is
stronger than that of the ligand (fig 3-OH, V-3-OH) under the same
conditions (lex. ¼ 290 nm, lem. ¼ 330 nm). The enhancement in the
fluorescence intensity of the complex may be related to the coor-
dination of the ligand to the oxovanadium (IV) ion. The coordina-
tion increases the rigidity of the ligand structure and enhances the
fluorescence quantum yield by reducing the probability of non-
radioactive dissipation process.
/
report [25]. To validate the stoichiometric composition of the
chelate, Job’s method (continual variation method) was applied.
The UV absorbance of ligand at 340 nm decreases and a new
characteristic band of complex was observed at 445 nm by the
addition of VOSO4.5H2O. Aliquots of the solutions were varied
alternatively from 0.1 to 0.9 ml for free ligand and VOSO4.5H2O
solution to hold the total volume 1.0 ml in the cuvette by using a
1.0 ml Pipette. The plot of absorbance at 445 nm against the molar
fraction of hydroxyflavone (X) exhibit maximum absorbance at
XL ¼ 0.67, close to the typical ligand mole fraction (0.66) for a 2:1
ligand-to-metal complex.
2.6. Mass spectral analysis
The mass spectra of the vanadiumeflavonol complex (Fig. 5)
indicated the molecular weight corresponding to the structure
consisting of VO2þ: ligand 1:2, as supported by the elemental an-
alyzes. In the mass spectrum, a peak at 541 corresponds to Mþ ion
and the peak at 270 corresponds to M2þ ion. The other two peaks at
170 and 360 correspond to various fragmentations as presented in
2.5. Fluorescence emission spectral data
Fig. 4 represents the fluorescence spectra of 3-hydroxyflavone
and vanadiumeflavonol complex. The fluorescence emission
Fig. 3. UVeVisible spectrum of free ligand and its complex. Inset shows spectrophotometric of the stoichiometric of the vanadiumeflavonol complex.

112
S.I. Pillai et al. / European Journal of Medicinal Chemistry 63 (2013) 109e117
stages of pre-diabetes more accurately than glycosylated hemo-
globin (HbA1c) and the ability to investigate postprandial glucose
levels in a physiological way [31]. It is a more sensitive measure of
early abnormalities in glucose regulation than fasting plasma
glucose. Impaired glucose tolerance due to pancreatic dysfunction
results in the defective utilization of glucose by the tissues and
increased hepatic gluconeogenesis. Streptozotocin-induced dia-
betic rats showed a marked raise (P < 0.001) in glucose levels which
were unsubsidized even at the end of 2 h after glucose load due to
impaired glucose homeostasis; whereas vanadiumeflavonol com-
plex treated diabetic rats showed significant (P < 0.001) decrease in
the blood glucose levels at fasting, 30, 60, 90 and 120 min indicating
that vanadiumeflavonol complex may exhibit insulin mimetic ef-
fect. Diabetic rats treated with 3-HF showed significant (P < 0.001)
reduction in blood glucose at fasting and 30 min; a significant
(P < 0.05) reduction in blood glucose at 90 and 120 min. However,
there was no significant difference in blood glucose concentration
at 60 min. There was no significant difference in blood glucose
concentration in normal rats treated with vanadiumeflavonol
complex as well as 3-HF when compared with normal control rats.
Table 1 shows the levels of blood glucose, glycosylated hemo-
globin, and urine sugar in all the groups of rats. The increase in the
levels of glucose and glycosylated hemoglobin in diabetic rats was
significantly (P < 0.001) reduced in vanadiumeflavonol complex
treated diabetic rats. Diabetic rats treated with 3-HF showed sig-
nificant (P < 0.05) decrease in blood glucose as well as glycosylated
hemoglobin. Urine sugar was present in diabetic rats as well as
diabetic rats treated with 3-HF whereas urine sugar was absent in
vanadiumeflavonol complex treated diabetic rats. However,
normal rats treated with the complex and ligand did not show any
significant alterations in the levels of fasting blood glucose, glyco-
sylated hemoglobin and urine sugar.
The management of diabetes is mainly focused at glycemic
control and international guidelines recommend reducing HbA1c
to 6.5e7% [32]. Persistent hyperglycemia results in glycation of
hemoglobin that leads to the formation of glycosylated hemoglo-
bin. HbA1c was found to increase in uncontrolled diabetes mellitus
and the increase is directly proportional to the blood glucose level
for about 3 months. Every percentage point decrease in HbA1c
reduces the risk of microvascular complications by 35% and every
10% reduction in HbA1c is associated with 21% reduction in car-
diovascular diseases [33]. Diabetic rats showed higher levels of
HbA1c indicating their poor glycemic control. Oral administration
of vanadiumeflavonol complex to diabetic rats significantly
declined the level of HbA1c indicating the improved glycemic
control.
Fig. 4. Fluorescence emission spectrum of free ligand and its complex.
the figure. These observations are in accordance with those re-
ported by other workers [26,27].
2.7. EPR spectroscopy
The X-band ESR spectra of an oxovanadium (IV) complex was
measured in DMSO at room temperature as presented in Fig. 6. The
EPR spectrum gives an eight line pattern (I ¼ 7/2), confirming the d1
VO (IV) oxidation state. This confirms the presence of single oxo-
vandium (IV) cation as the metallic center in the complex. The
isotropic EPR parameters giso ¼ 1.961 and Aiso ¼ 95.5 can be
calculated from the position spacing of the resonance lines from
room temperature solution spectrum of the complex. From the
anisotropic spectrum, the anisotropic parameters were calculated.
The observed order (Ak ¼ 166.5 > At ¼ 60; gj ¼ 1.972 > gk ¼ 1.938)
indicates that the unpaired electron is present in the dxy orbital
with square-pyramidal geometry around the VO(IV) chelates [28].
2.8. Antidiabetic activity of vanadiumeflavonol complex
STZ-induced diabetic rat model is widely used to study the
in vivo effects of vanadium compounds. STZ-induced diabetes is
considered a type 1 diabetic model since it arises from destruction
of the insulin producing pancreatic
b cells [29]. Streptozotocin is a
nitrosourea analog, preferentially uptaken by pancreatic beta cells
via the glucose transporter GLUT2 and causes DNA alkylation fol-
lowed by the activation of poly ADP ribosylation leading to deple-
tion of cytosolic concentration of NADþ and ATP. Enhanced ATP
dephosporylation after STZ administration, supplies the substrate
for xanthine oxidase resulting in the formation of superoxide rad-
Table 2 depicts the plasma protein, blood urea and serum
creatinine levels in control and experimental groups of rats. The
increased levels of urea and creatinine and reduced levels of plasma
protein in diabetic rats were significantly (P < 0.001) improved in
vanadiumeflavonol complex treated diabetic rats. Whereas, the
diabetic rats treated with 3-HF showed significant improvement in
urea (P < 0.05) and creatinine (P < 0.01) levels but not in protein
levels. Normal rats treated with vanadiumeflavonol complex and
3-HF showed no significant difference in these parameters.
Insulin deprivation in diabetic milieu causes a profound increase
in protein catabolism. Moreover, protein catabolism in diabetes is
due to a net increase in protein breakdown rather than a decline in
protein synthesis [34]. The imbalance between synthesis and
catabolism of protein can have remarkable consequences in the
metabolism of many tissues such as gut, skeletal muscle and heart
[35]. The antidiabetic property of vanadiumeflavonol complex may
account for the observed increase in the levels of plasma proteins as
well as improved body weight in diabetic rats treated with the
complex indicate the improved nitrogen balance.
icals. NO moiety is liberated from STZ leading to the destruction of
cells by necrosis [30].
The abnormalities in body weight in control and experimental
groups of rats are shown in Fig. 7. Diabetic group of rats showed
significant reduction in body weight (P < 0.001) when compared
b
with normal control group of rats. There was
a significant
(P < 0.001) improvement in body weight gain in diabetic groups of
rats after oral administration of vanadiumeflavonol complex for 30
days. However, there was no significant improvement in body
weight gain of diabetic rats treated with ligand (3-HF). Also, No
notable change in body weight gain in normal rats treated with
complex and 3-HF alone compared to normal control rats was
observed.
Fig. 8 shows the blood glucose levels of control and experi-
mental groups of rats after oral glucose load. Oral glucose tolerance
test (OGTT) measures the body’s ability to utilize glucose, the
body’s main source of energy. Current evidence suggests an addi-
tive value of the OGTT, its main advantage being the ability to detect

S.I. Pillai et al. / European Journal of Medicinal Chemistry 63 (2013) 109e117
113
Fig. 5. Mass spectra of the vanadiumeflavonol complex.
Urea is the main end product of protein catabolism in the body.
During the diabetic conditions, the pronounced degradation of both
hepatic as well as plasma proteins leads to the excessive accumu-
lation of urea in the systemic circulation than its excretion. In
diabetes, due to the elevated supraphysiological concentration of
glucose, damages occur in tissues like kidney causing impairment
in renal function resulting in the accumulation of nitrogenous
wastes in circulation. This in turn elevates urea and creatinine
levels in blood which acts as a biochemical diagnostic marker for
assessing renal function [36]. The administration of vanadiume
flavonol complex to diabetic rats normalized the blood urea and
creatinine levels indicating the recovered renal function which is
due to improved glycemic control.
Table 3 depicts the levels of activities of AST, ALT and ALP in
serum of the control and experimental groups of rats. The elevated
activities of AST, ALT and ALP were significantly (P < 0.001) altered
in diabetic rats administered with vanadiumeflavonol complex.
Also, a reduction in the levels of activities of AST (P < 0.01), ALT
(P < 0.001) and ALP (P < 0.01) was noted in the diabetic rats treated
with 3-HF alone. But there was no significant alteration in the levels
of activities of these enzymes in normal rats treated with
vanadiumeflavonol complex and 3-HF when compared to normal
control rats.
Under normal physiological conditions, the activities of en-
zymes AST, ALT and ALP is low in serum and their activities are
Fig. 7. The abnormalities in body weight in control and experimental groups of rats.
Values are given as mean ꢁ S.E.M for groups of six rats in each. One-way ANOVA
followed by post hoc test LSD. Statistical significance was compared within the groups
as follows: acontrol rats; bdiabetic control rats; Values are statistically significant at
*P < 0.001; #P < 0.01; @P < 0.05.
Fig. 6. EPR spectrum of the vanadiumeflavonol complex.

114
S.I. Pillai et al. / European Journal of Medicinal Chemistry 63 (2013) 109e117
Table 2
The levels of plasma protein, blood urea and serum creatinine in control and
experimental groups of rats after 30 days experimental period.
Groups
Protein (g/dl)
Urea (mg/dl)
Creatinine
(mg/dl)
Normal control
Normal þ V3HF
(5 mg/kg)
8.14 ꢁ 0.18
38.33 ꢁ 3.08
0.32 ꢁ 0.025
7.92 ꢁ 0.30 a
35.82 ꢁ 2.23 a
0.38 ꢁ 0.035 a
Normal þ 3HF
8.09 ꢁ 0.22 a
42.53 ꢁ 2.39 a
0.37 ꢁ 0.042 a
(50 mg/kg)
Diabetic control
Diabetic þ V3HF
(5 mg/kg)
5.75 ꢁ 0.17 a*
7.26 ꢁ 0.16 b*
78.63 ꢁ 3.73 a*
46.90 ꢁ 2.89 b*
1.08 ꢁ 0.050 a*
0.50 ꢁ 0.041 b*
Diabetic þ 3HF
5.94 ꢁ 0.18 b
69.38 ꢁ 3.16 b@
0.89 ꢁ 0.044 b#
(50 mg/kg)
Values are given as mean ꢁ S.E.M for groups of six rats in each. One-way ANOVA
followed by post hoc test LSD. Statistical significance was compared within the
groups as follows: a e control rats; b e diabetic control rats; Values are statistically
significant at *P < 0.001; #P < 0.01; @P < 0.05.
The improved fasting blood glucose, glycosylated hemoglobin,
glucose tolerance and tissue glycogen content exemplifies the
possible role of the complex on glycemic control. The altered levels
of plasma protein, urea, creatinine and the activities of AST, ALT and
ALP were found to be normalized in the rats treated with the
vanadiumeflavonol complex indicating the tissue protective nature
of the complex.
Fig. 8. The levels of blood glucose in control and experimental groups of rats after oral
glucose load. Values are given as mean ꢁ S.E.M for groups of six rats in each. One-way
ANOVA followed by post hoc test LSD. Statistical significance was compared within the
groups as follows: acontrol rats; bdiabetic control rats; Values are statistically signifi-
cant at *P < 0.001; #P < 0.01; @P < 0.05.
elevated during tissue damage. A rise in AST and ALT activity in-
dicates the hepatocellular damage followed by cardiac tissue
damage. Further, ALP is a marker of biliary function and cholestasis.
The observed increase in activities of these enzymes in the serum of
diabetic rats may be due to the leakage of these enzymes from the
liver cytosol into blood stream as a consequence of the hepatic
tissue damage [37]. The significant reversal in AST, ALT and ALP
activities in vanadiumeflavonol complex treated diabetic rats
indicate the tissue protective nature of the complex.
The levels of glycogen content in liver and muscle tissues of
normal control and experimental groups of rats were depicted in
Figs. 9 and 10 respectively. The decreased glycogen content of liver
and muscle tissues in diabetic rats were significantly (P < 0.001)
restored upon oral administration of complex. Whereas no signif-
icant difference was observed in diabetic rats treated with 3-HF. No
significant difference was observed in normal rats treated with the
vanadiumeflavonol and 3-HF. Glycogen, the storage form of car-
bohydrates is formed by the conversion of excess glucose into
glycogen under the influence of insulin. Glycogen is stored as an
energy fuel in tissues, predominantly in the liver and skeletal
muscle. In diabetes, the reduced glycogen level in tissues is due to
the insulin deficiency or impaired responsiveness of tissues to in-
sulin [38]. In the present study, the improved glycogen content in
both the liver and muscle tissues in diabetic rats treated with
vanadiumeflavonol complex indicates the effective glucose utili-
zation and storage.
3. Conclusion
In conclusion, the present study reveals that the oral adminis-
tration of vanadiumeflavonol complex evoked marked blood
glucose lowering effect in diabetic rats. The normalization of
altered biochemical parameters upon vanadiumeflavonol complex
treatment indicates the improved glucose homeostasis. Further-
more, the results indicate the non-toxic nature of the complex. The
observed antidiabetic effect of the complex might be due to its
insulin mimetic action. Further in-depth studies on the mechanism
of action of the vanadiumeflavonol complex would render us with
an efficient nontoxic hypoglycemic drug for the treatment of dia-
betes mellitus.
4. Experimental protocols
4.1. Synthesis of vanadiumeflavonol complex
To a warm solution of 3-hydroxy-flavone (2.36 g; 10 mmol) in
methanol (40 ml), vanadium sulfate (1.26 g; 5 mmol) was added
dropwise under constant stirring. The solution was refluxed for 6 h.
Table 3
The activities of AST, ALT and ALP in serum of control and experimental groups of
rats after 30 days experimental period.
Table 1
Groups
AST (U/L)
ALT (U/L)
ALP (U/L)
The levels of fasting blood glucose, glycosylated hemoglobin, and urine sugar in
control and experimental groups of rats after 30 days experimental period.
Normal control
Normal þ V3HF
(5 mg/kg)
106.17 ꢁ 5.87
77.01 ꢁ 3.33
135.58 ꢁ 7.12
124.12 ꢁ 6.37 a
81.11 ꢁ 4.15 a
144.53 ꢁ 5.05 a
Groups
FBG (mg/dl)
HbA1c (% Hb)
Urine
sugar
Normal þ 3HF
114.65 ꢁ 5.00 a
79.14 ꢁ 4.48 a
154.83 ꢁ 6.09 a
Normal control
83.42 ꢁ 5.47
5.35 ꢁ 0.20
Nil
Nil
Nil
(50 mg/kg)
Normal þ V3HF (5 mg/kg)
Normal þ 3HF (50 mg/kg)
Diabetic control
79.92 ꢁ 6.21 a
88.87 ꢁ 3.86 a
322.78 ꢁ 14.95 a*
136.45 ꢁ 5.14 b*
287.78 ꢁ 12.78 b@
5.29 ꢁ 0.27 a
5.89 ꢁ 0.23 a
13.35 ꢁ 0.35 a*
7.76 ꢁ 0.24 b*
12.47 ꢁ 0.41 b@
Diabetic control
221.55 ꢁ 13.16 a*
169.65 ꢁ 8.29 a* 316.56 ꢁ 17.40 a*
124.68 ꢁ 6.24 b* 169.64 ꢁ 6.11 b*
Diabetic þ V3HF 156.31 ꢁ 5.35 b*
þþþ
(5 mg/kg)
Diabetic þ V3HF (5 mg/kg)
Nil
Diabetic þ 3HF
187.55 ꢁ 10.22 b# 132.88 ꢁ 7.99 b* 271.45 ꢁ 16.65 b#
Diabetic þ 3HF (50 mg/kg)
þþ
(50 mg/kg)
Values are given as mean ꢁ S.E.M for groups of six rats in each. One-way ANOVA
followed by post hoc test LSD. Statistical significance was compared within the
groups as follows: a e control rats; b e diabetic control rats; Values are statistically
significant at *P < 0.001; #P < 0.01; @P < 0.05.
Values are given as mean ꢁ S.E.M for groups of six rats in each. One-way ANOVA
followed by post hoc test LSD. Statistical significance was compared within the
groups as follows: a e control rats; b e diabetic control rats; Values are statistically
significant at *P < 0.001; #P < 0.01; @P < 0.05.

S.I. Pillai et al. / European Journal of Medicinal Chemistry 63 (2013) 109e117
115
EPR spectra of the complex in DMSO solution were obtained using a
Varian E112 spectrophotometer.
4.3. Potentiometric pH titration
The potentiometric studies were carried out with a ELICO LI 120
pH meter fitted with a glass and calomel electrodes calibrated to
read elog[Hþ] directly. Before initiating each titration the electrode
was calibrated by titrating known amount of aqueous HCl with a
known concentration of NaOH and the temperature of the solution
were maintained at 25 ꢂC ꢁ 1 ꢂC. Solutions were prepared freshly
using deionized water. The stock solution of vanadyl ion was pre-
pared by dilution of its atomic absorption standard using 50%
NaOH. The ionic strength (I) of the titration solutions was kept
constant with 0.16 M Naþ. The concentrations were in the range of
1e10 mM. A total of six titrations for hydroxyflavone alone and
fifteen titrations defined the VO(IV)-hydroxyflavone equilibria. All
the titration were carried out over the range of pH 2 to 8. The
protonation constants for the vanadium e hydroxyflavone were
determined using the Origin 8.5 lab version program [39].
Male albino rats of Wistar strain weighing (160e180 g) were
procured from Tamilnadu Veterinary and Animal Sciences Univer-
sity (TANUVAS), Chennai. The rats were housed in spacious poly-
propylene cages lined with husk. The experimental rats were
maintained in a controlled environment (12:12 ꢁ 1 h light/dark
cycle) and temperature (22 ꢂC ꢁ 3 ꢂC). Animals were acclimatized
to standard husbandry conditions for one week to eliminate the
effect of stress prior to initiation of the experiments. The rats were
fed with commercial pelleted rats chow (Hindustan Lever Ltd.,
Bangalore, India), and had free access to water ad libitum. The ex-
periments were designed and conducted in strict accordance with
the current ethical norms approved by Ministry of Social justices &
Empowerment, Government of India and Institutional Animal
Ethical Committee guidelines [IAEC NO: 01/060/09 ].
Fig. 9. The levels of glycogen content in liver tissues of normal control and experi-
mental groups of rats. Values are given as mean ꢁ S.E.M for groups of six rats in each.
One-way ANOVA followed by post hoc test LSD. Statistical significance was compared
within the groups as follows: acontrol rats; bdiabetic control rats; Values are statisti-
cally significant at *P < 0.001; #P < 0.01; @P < 0.05.
The green colored product formed was filtered off, washed with
cold methanol and dried in desiccators over CaCl2. Yield: w82%.
Elemental analysis calculated for C30H18O7V (541.4); C, 66.61; H,
3.39. Found: C, 66.55; H, 3.35.
4.2. General experimental information
All the chemicals were purchased from SigmaeAldrich, reagent
grade and were used without further purification. Elemental anal-
ysis for Carbon and Hydrogen was carried out on a Carlo Erba model
1106 elemental analyzer. FT-IR spectra were recorded with Perkin
Elmer FTIR spectrometer in the range of 400e4000 cmꢀ1 with
samples prepared as KBr pellets. UVeVisible spectra were recorded
with a Perkin Elmer Lambda 35 spectrophotometer operating in the
range of 200e1000 nm with quartz cells. Fluorescence experiments
were performed in Perkin Elmer Spectrofluorimeter. Mass spectral
analysis was performed in Q-TOF Mass Spectrometer. The X-band
4.4. Induction of experimental diabetes
Rats were fasted overnight and diabetes was experimentally
induced by intraperitoneal injection of streptozotocin with a single
dose of 60 mg/kg b.w/rat. Streptozotocin was dissolved in freshly
prepared 0.1 M cold citrate buffer pH 4.5 [40]. Since streptozotocin
is capable of inducing fatal hypoglycemia as a result of massive
pancreatic insulin release, streptozotocin-treated rats were pro-
vided with 10% glucose solution after 6 h for the next 24 h to
prevent STZ induced hypoglycemia [41]. On 3rd day the develop-
ment and aggravation of diabetes in rats was confirmed and rats
with fasting blood glucose concentration more than 250 mg/dL
were selected for the experiments.
4.5. Experimental design
The rats were divided into six groups as follows (comprising of
not less than six animals in each group):
Group 1 e Normal rats.
Group 2 e Normal rats treated orally with vanadiumeflavonol
complex (5 mg/kg b.w/rat/day) as aqueous suspension for 30
days.
Group 3 e Normal rats treated orally with 3-HF (50 mg/kg b.w/
rat/day) as aqueous suspension for 30 days.
Group 4 e Streptozotocin induced diabetic rats
Group 5 e Diabetic rats treated orally with vanadiumeflavonol
complex (5 mg/kg b.w/rat/day) as aqueous suspension for 30
days.
Fig. 10. The levels of glycogen content in muscle tissues of normal control and
experimental groups of rats. Values are given as mean ꢁ S.E.M for groups of six rats in
each. One-way ANOVA followed by post hoc test LSD. Statistical significance was
compared within the groups as follows: acontrol rats; bdiabetic control rats; Values are
statistically significant at *P < 0.001; #P < 0.01; @P < 0.05.

116
S.I. Pillai et al. / European Journal of Medicinal Chemistry 63 (2013) 109e117
coordination mode; in vitro and in vivo evaluations in rats, J. Inorg. Biochem.
80 (2000) 99e105.
[13] B.H. Havsteen, The biochemistry and medical significance of the flavonoids,
Pharmacol. Ther. 96 (2002) 67e202.
Group 6 e Diabetic rats treated orally with 3-HF (50 mg/kg b.w/
rat/day) as aqueous solution for 30 days.
[14] J.P. Cornard, L. Dangleterre, C. Lapouge, DFT and TD-DFT investigation and
spectroscopic characterization of the molecular and electronic structure
of the Zn(II)e3-hydroxyflavone complex, Chem. Phys. Lett. 419 (2006)
304e308.
[15] G.S. Prasath, S.P. Subramanian, Modulatory effects of fisetin, a bioflavonoid, on
hyperglycemia by attenuating the key enzymes of carbohydrate metabolism
in hepatic and renal tissues in streptozotocin-induced diabetic rats, Eur. J.
Pharmacol. 668 (2011) 492e496.
4.6. Oral glucose tolerance test
Overnight fasted rats of all the groups were subjected to oral
glucose tolerance test on 28th day of the experimental period. The
blood glucose levels were monitored before and 30, 60, 90 and
120 min after administration of 2 g/kg b.w./rat, glucose as aqueous
solution using OneTouch glucometer (Life scan, Johnson & Johnson
Company).
[16] T. Venkatesan, S. Sorimuthu Pillai, Antidiabetic activity of gossypin, a penta-
hydroxyflavone glucoside, in streptozotocin-induced experimental diabetes
in rats, J. Diabetes 4 (2012) 41e46.
[17] V.A. Kostyuk, A.I. Potapovich, E.N. Vladykovskaya, L.G. Korkina, I.B. Afanas’ev,
Influence of metal ions on flavonoid protection against asbestos-induced cell
injury, Arch. Biochem. Biophys. 385 (2001) 129e137.
4.7. Biochemical analysis
[18] K. Vijayaraghavan, S. Iyyam Pillai, S.P. Subramanian, Design, synthesis and
characterization of zinc-3 hydroxyflavone, a novel zinc metallo complex for
the treatment of experimental diabetes in rats, Eur. J. Pharmacol. 680 (2012)
122e129.
[19] A.K. Bordbar, A.L. Creagh, F. Mohammadi, C.A. Haynes, C. Orvig, Calorimetric
studies of the interaction between the insulin-enhancing drug candidate
bis(maltolato)oxovanadium(IV) (BMOV) and human serum apo-transferrin,
J. Inorg. Biochem. 103 (2009) 643e647.
[20] V.G. Yuen, P. Caravan, L. Gelmini, N. Glover, J.H. McNeill, I.A. Setyawati,
Y. Zhou, C. Orvig, Glucose-lowering properties of vanadium compounds:
comparison of coordination complexes with maltol or kojic acid as ligands,
J. Inorg. Biochem. 68 (1997) 109e116.
[21] K.H. Thompson, B.D. Liboiron, Y. Sun, K.D. Bellman, I.A. Setyawati,
B.O. Patrick, V. Karunaratne, G. Rawji, J. Wheeler, K. Sutton, S. Bhanot,
C. Cassidy, J.H. McNeill, V.G. Yuen, C. Orvig, Preparation and characteriza-
tion of vanadyl complexes with bidentate maltol-type ligands; in vivo
comparisons of anti-diabetic therapeutic potential, J. Biol. Inorg. Chem. 8
(2003) 66e74.
[22] P. Caravan, L. Gelmini, N. Glover, F.G. Herring, H. Li, J.H. McNeill, S.J. Rettig,
I.A. Setyawati, E. Shuter, Y. Sun, A.S. Tracey, V.G. Yuen, C. Orvig, Reaction
chemistry of BMOV, bis(maltolato)oxovanadium(IV), a potent insulin mimetic
agent, J. Am. Chem. Soc. 117 (1995) 12759e12770.
[23] Mihaela Badea, Rodica Olar, Dana Marinescu, Valentina Uivarosi,
Victoria Aldea, Teodor Octavian Nicolescu, Thermal stability of new vanadyl
complexes with flavonoid derivatives as potential insulin-mimetic agents,
J. Therm. Anal. Calorim 99 (2010) 823e827.
At the end of 30 days of treatment, rats were fasted overnight,
anaesthetized, using ketamine (80 mg/kg b.w./rat, i.p.) and sacri-
ficed by cervical decapitation. Blood was collected with and
without anticoagulant for plasma and serum separation respec-
tively. Fasting blood glucose, glycosylated hemoglobin, Plasma
protein, Blood urea and serum creatinine were estimated according
to the manual instructions in the kits. The activities of AST, ALT and
ALP in serum were assayed according to the kit procedure. Urine
sugar was detected using urine strip. Liver and muscle tissues were
excised out, washed with ice cold saline and stored at ꢀ70 ꢂC and
used for glycogen estimation [42].
4.8. Statistical analysis
All the data were grouped and statistically evaluated with SPSS
16 software. Hypothesis testing methods included ‘One-way
analysis of variance’ followed by ‘least significant difference’ test
was used. A value of P < 0.001, P < 0.01 and P < 0.05 were
considered to indicate statistical significance. All results are
expressed as mean ꢁ Standard error mean (S.E.M) for six rats in
each group.
[24] F. Yang, D.A. Rice, Synthesis and characterisation of cobalt(II) complexes of 3-
hydroxyflavone, Pertanika J. Sci. Tech. 3 (1995) 211e219.
[25] Mihaela Badea, Rodica Olar, Valentina Uivarosi, Dana Marinescu,
Victoria Aldea, Stefania Felicia Barbuceanu, George Mihai Nitulescu,
Thermal behavior of some vanadyl complexes with flavone derivatives as
potential insulin-mimetic agents, J. Therm. Anal. Calorim. 105 (2011)
559e564.
References
[26] E.G. Lewars, R.E. March, Fragmentation of 3-hydroxyflavone; a computational
and mass spectrometric study, Rapid Commun. Mass. Spectrom. 21 (2007)
1669e1679.
[1] V. Sabetsky, J. Ekblom, Insulin: a new era for an old hormone, Pharmacol. Res.
61 (2010) 1e4.
[2] J.E. Shaw, R.A. Sicree, P.Z. Zimmet, Global estimates of the prevalence of dia-
betes for 2010 and 2030, Diabetes Res. Clin. Pract. 87 (2010) 4e14.
[3] American Diabetes Association, Diagnosis and classification of diabetes mel-
litus, Diabetes Care 33 (2010) S62eS69.
ꢀ
[27] E. Woznick, M. Kopacz, M. Umbreit, J. K1os, New complexes of La(III), Ce(III),
Pr(III), Nd(III), Sm(III), Eu(III) and Gd(III) ions with morin, J. Inorg. Biochem.
101 (2007) 774e782.
[28] J.C. Dutton, G.D. Fallon, K.S. Murray, synthesis, structure, ESR spectra and
redox properties of (N, N’-ethylenebis(thiosalicylideneaminato))oxovanadiu-
m(IV) and of related {S, N} chelates of vanadium(IV), Inorg. Chem. 27 (1988)
34e38.
[4] D.M. Nathan, J.B. Buse, M.B. Davidson, R.J. Heine, R.R. Holman, R. Sherwin,
B. Zinman, Management of hyperglycemia in type 2 diabetes: a consensus
algorithm for the initiation and adjustment of therapy: a consensus statement
from the American Diabetes Association and the European Association for the
study of diabetes, Diabetes Care 29 (2006) 1963e1972.
[5] E.R. Jamieson, S.J. Lippard, Structure, recognition and processsing of cisplatin-
DNA adducts, Chem. Rev. 99 (1999) 2467e2498.
[6] S.M. Brichard, J.C. Henquin, The role of vanadium in the management of
diabetes, Trends Pharmacol. Sci. 16 (1995) 265e270.
[29] J.H. McNeill, V.G. Yuen, H.R. Hoveyda, C. Orvig, Bis(maltolato)oxovana-
dium(IV) is
1491.
a potent insulin mimic, J. Med. Chem. 35 (1992) 1489e
[30] T. Szkudelski, The mechanism of alloxan and streptozotocin action in B cells of
the rat pancreas, Physiol. Res. 50 (2001) 537e546.
[31] Y.M. Luijf, J. Hermanides, M.J. Serlie, J.B. Hoekstra, M.R. Soeters, The added
value of oral glucose tolerance testing in pre-diabetes, Curr. Diabetes Rev. 7
(2011) 56e60.
[32] D.M. Nathan, J.B. Buse, M.B. Davidson, E. Ferrannini, R.R. Holman, R. Sherwin,
B. Zinman, American Diabetes Association; European Association for the
Study of Diabetes, Medical management of hyperglycaemia in type 2 dia-
betes mellitus: a consensus algorithm for the initiation and adjustment of
therapy: a consensus statement from the American Diabetes Association and
the European Association for the study of diabetes, Diabetologia 52 (2009)
17e30.
[33] S.K. Garg, H. Ulrich, Achieving goal glycosylated hemoglobin levels in type 2
diabetes mellitus: practical strategies for success with insulin therapy, Insulin
1 (2006) 109e121.
[34] N. Møller, K.S. Nair, Diabetes and protein metabolism, Diabetes 57 (2008) 3e4.
[35] P.H. Sugden, S.J. Fuller, Regulation of protein turnover in skeletal and cardiac
muscle, Biochem. J. 273 (1991) 21e37.
[7] B. Ramachandran, D.S. Sekar, M. Kandaswamy, V. Narayanan, S. Subramanian,
Hypoglycemic effect of macrocyclic binuclear oxovanadium (IV) complex on
streptozotocin-induced diabetic rats, Exp. Diabesity Res. 5 (2004) 137e142.
[8] B. Ramachandran, M. Kandaswamy, V. Narayanan, S. Subramanian, Insulin
mimetic effects of macrocyclic binuclear oxovanadium complexes on
streptozotocin-induced experimental diabetes in rats, Diabetes Obes. Metab. 5
(2003) 455e461.
[9] B. Ramachandran, K. Ravi, V. Narayanan, M. Kandaswamy, S. Subramanian,
Effect of macrocyclic binuclear oxovanadium complex on tissue defense
system in streptozotocin-induced diabetic rats, Clin. Chim. Acta 345 (2004)
141e150.
[10] B. Ramachandran, K. Ravi, V. Narayanan, M. Kandaswamy, S. Subramanian,
Protective effect of macrocyclic binuclear oxovanadium complex on oxidative
stress in pancreas of streptozotocin induced diabetic rats, Chem. Biol. Interact
149 (2004) 9e21.
[11] H.K. Thompson, C. Orvig, Design of vanadium compounds as insulin
enhancing agents, J. Chem. Soc. Dalton Trans. (2000) 2885e2892.
[12] H. Sakurai, H. Sano, T. Takino, H. Yasui, An orally active antidiabetic vanadyl
complex, bis(1-oxy-2-pyridinethiolato)oxovanadium(IV), with VO(S2O2)
[36] H. Bräunlich, F. Marx, C. Fleck, G. Stein, Kidney function in rats after 5/6 ne-
phrectomy (5/6 NX); effort of treatment with vitamin E, Exp. Toxicol. Pathol.
49 (1997) 135e139.

S.I. Pillai et al. / European Journal of Medicinal Chemistry 63 (2013) 109e117
117
[37] M. van den Brand, L.D. Elving, J.P. Drenth, J.H. van Krieken, Glycogenic hep-
atopathy: a rare cause of elevated serum transaminases in diabetes mellitus,
Neth. J. Med. 67 (2009) 394e396.
[40] N. Rakieten, M.L. Rakieten, M.V. Nadkarni, Studies on the diabetogenic
action of streptozotocin (NSC-37917), Cancer Chemother. Rep. 29 (1963)
91e98.
[38] M.G. Bischof, M. Krssak, M. Krebs, E. Bernroider, H. Stingl, W. Waldhäusl,
M. Roden, Effects of short-term improvement of insulin treatment and glycemia
on hepatic glycogen metabolism in type 1 diabetes, Diabetes 50 (2001) 392e398.
[39] Origin 8.5, OriginLab Corporation, Northampton, 2010.
[41] L.J. Fischer, D.E. Rickert, Pancreatic islet-cell toxicity, CRC Crit. Rev. Toxicol. 3
(1975) 231e263.
[42] J.H. Roe, R.E. Dailey, Determination of glycogen with the anthrone reagent,
Anal. Biochem. 15 (1966) 245e250.