Rauwolfia serpentina improves altered glucose and lipid homeostasis in fructose-induced type 2 diabetic mice

Abstract

Rauwolfia serpentina is well-reported in traditional medicines for the treatment of hypertensive and neurological disorders. However, its antidiabetic potential has been currently described in both alloxan-treated and normoglycemic mice. Present effort was carried out to investigate the effect of methanol root extract (MREt) of R.serpentina in fructose-induced type 2 diabetic mice. Experimental mice were grouped into normal control (distilled water 1ml/kg) and fructose-induced type 2 diabetic groups (10% fructose 1 ml/kg).The second group subdivided into negative (0.05% DMSO 1ml/kg) control, positive (pioglitazone 15mg/kg) control and three test groups (MREt 10, 30 & 60 mg/kg). Each treatment was given orally for 14 days consecutively then mice were sacrificed in order to collect serum and liver samples to analyze physical, biochemical as well as hematological markers. MREt significantly improved percent body weight and glycemic change along with serum insulin, total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL-c), very low-density lipoprotein (VLDL-c), high-density lipoprotein-cholesterols (HDL-c), total hemoglobin, glycosylated hemoglobin, hepatic glycogen, coronary risk and fasting insulin resistance indices while suppressed down the activity of 3-hydroxy-3-methylglutaryl Coenzyme A reductase enzyme in test groups when compared with diabetic controls. The present findings conclude that MREt of R. serpentina can effectively betters the carbohydrate and lipid homeostasis by either inhibiting fructose absorption in intestine or decreasing insulin resistance in fructose-induced type 2 diabetic mice.

Full text

Pak. J. Pharm. Sci., Vol.29, No.5, September 2016, pp.1619-1624 1619
Rauwolfia serpentina improves altered glucose and lipid homeostasis
in fructose-induced type 2 diabetic mice
Muhammad Bilal Azmi1, 2 and Shamim A Qureshi1
1Department of Biochemistry, University of Karachi, Karachi, Pakistan
2 Department of Biochemistry, Dow Medical College, Dow University of Health Sciences, Karachi, Pakistan
Abstract: Rauwolfia serpentina is well-reported in traditional medicines for the treatment of hypertensive and
neurological disorders. However, its antidiabetic potential has been currently described in both alloxan-treated and
normoglycemic mice. Present effort was carried out to investigate the effect of methanol root extract (MREt) of
R.serpentina in fructose-induced type 2 diabetic mice. Experimental mice were grouped into normal control (distilled
water 1ml/kg) and fructose-induced type 2 diabetic groups (10% fructose 1 ml/kg).The second group sub-divided into
negative (0.05% DMSO 1ml/kg) control, positive (pioglitazone 15mg/kg) control and three test groups (MREt 10, 30 &
60 mg/kg). Each treatment was given orally for 14 days consecutively then mice were sacrificed in order to collect serum
and liver samples to analyze physical, biochemical as well as hematological markers. MREt significantly improved
percent body weight and glycemic change along with serum insulin, total cholesterol (TC), triglycerides (TG), low-
density lipoprotein (LDL-c), very low-density lipoprotein (VLDL-c), high-density lipoprotein-cholesterols (HDL-c),
total hemoglobin, glycosylated hemoglobin, hepatic glycogen, coronary risk and fasting insulin resistance indices while
suppressed down the activity of 3-hydroxy-3-methylglutaryl Coenzyme A reductase enzyme in test groups when
compared with diabetic controls. The present findings conclude that MREt of R. serpentina can effectively betters the
carbohydrate and lipid homeostasis by either inhibiting fructose absorption in intestine or decreasing insulin resistance in
fructose-induced type 2 diabetic mice.
Keywords: Fructose, insulin resistance, 3-hydroxy-3-methylglutarylcoenzyme A, Rauwolfia serpentina.
INTRODUCTION
Type 2 or non-insulin dependent diabetes mellitus (T2D)
becomes health problem globally which affects both
genders in every country especially developing countries
like Pakistan (Ijaz and Ajmal, 2011). It is a hormone-
linked metabolic disorder associated with relative insulin
deficiency or insulin resistance (Kadowaki, 2000). Insulin
resistance reflects the incompatibility of insulin with its
receptors on target tissues including liver, muscle and
adipose tissues (Patel et al., 2013) which results in
persistent hyperglycemia by impairing carbohydrate, lipid
and protein metabolism (Hsu, 2013).Genetic and acquired
factors are increasing the risk of insulin resistance
worldwide (Singh, 2011). Among the acquired factors,
diet with high-sugar (sucrose/fructose) content plays a
significant role in the growth of insulin resistance by
producing hypercholesterolemia and hypertriglyceridemia
(Salas-Salvadó et al., 2011; Adeneye, 2012; Khitan and
Kim, 2013) which in turn increases the number of patients
with vascular complications (Ahmed et al., 2010).
Fructose is an obesity inducer ketohexose that produced
dihydroxy acetone phosphate (DHAP) and
glyceraldehydes-3-phosphate (GA3P) without passing
through the step catalyzed by phosphofructokinase, one of
the rate-regulatory enzymes of glycolysis, thereby
accelerating the synthesis of glucose via gluconeogenesis
and triglycerides (TG) that lead to hyperglycemia and
hypertriglyceridemia (Feinman and Fine, 2013; Bray,
2007). This increased amount of TG not only mask the
insulin receptors on target tissues and lead to
hyperinsulinemia but also act as an alternate source of
energy for the body results in increased production of
acetyl coenzyme A that cannot be easily handled by
tricarboxylic acid cycle (TCA) and induced to cholesterol
biosynthesis that leads to hypercholesterolemia (Feinman
and Fine, 2013; Bray, 2007; Johnson et al., 2009). In spite
of these harmful effects, high fructose corn syrup (HFCS)
is one of the widely used sweeteners in commercial food
stuffs where it acts as a slow poison for increasing the risk
of metabolic disorders especially T2D globally (Bray et
al., 2004; Gorana et al., 2012). Many hypoglycemic
agents like biguanides and thiozolidenediones have been
used for the management of this health hazard but possess
few side effects (Masuda and Terauchi, 2010). However,
literature also witnesses the importance of herbal
remedies with no or negligible side effect in the treatment
of T2D (Chang et al., 2013).
The well-known antihypertensive medicinal plant
Rauwolfia serpentina Benth (family Apocynaceae) is also
famous for its variety of ethno-medicinal effectiveness
like in the treatment of snake bite, gastrointestinal tract
disorders, breast cancer, skin problems, etc (Azmi and
Qureshi, 2012a). Currently its short- and long-term
antidiabetic activities have been reported in alloxan-
*Corresponding author: e-mail: qureshi29@live.com

Rauwolfia serpentina improves altered glucose and lipid homeostasis in fructose-induced type 2 diabetic mice
Pak. J. Pharm. Sci., Vol.29, No.5, September 2016, pp.1619-1624
1620
induced diabetic mice where it was found to improve the
atherogenic, arteriosclerosis and cardioprotective indices
(Qureshi et al., 2009; Azmi and Qureshi, 2012b; Azmi et
al., 2015). Another investigation has explored the
antioxidant and heamatinic properties of methanolic root
extract of R.serpentina in alloxan-induced (type 1
diabetic) mice (Azmi and Qureshi, 2013). However,
antidiabetic activity of same plant has not been reported
in insulin resistance diabetes so far. Therefore, for the first
time, present effort was designed to evaluate the
antidiabetic and lipid lowering efficiencies of methanol
root extract of R. serpentina in fructose-induced type 2
diabetic mice.
MATERIALS AND METHODS
Plant material and preparation of methanol extract
The roots of R.serpentina were procured, authenticated
and kept (KU/BCH/SAQ/02) at Biochemistry
Department, University of Karachi (UoK), Karachi-
75270, Pakistan. The methanol roots extract (MREt) was
prepared as described by Azmi and Qureshi in 2012 and
stored in refrigerator below 10°C until used (Azmi and
Qureshi, 2012a).
Induction of fructose-induced type 2 diabetes
It was done by giving 10% fructose solution (1ml/kg)
orally once a day for 14 days connectively in overnight
fasted mice (Neeharika et al., 2012).
Antidiabetic medicine and vehicle for MREt
Commercially available pioglitazone (Zolid, 15mg/kg) of
Getz Pharma, Pakistan Ltd. and 0.05% dimethyl
sulphoxide (DMSO) of Fisher Chemicals (United
Kingdom) were used as positive control and vehicle for
MREt in present study.
Experimental mice and their grouping
Wister male albino mice (n= 42) from 25 to 35 grams in
weight were procured from the commercial breeding
center of Dow University of Health Sciences (DUHS),
Karachi, Pakistan and kept in animal house of same
university according to the international guidelines of
animal care and handling. These mice were provided
standard laboratory diet with easy access to water ad
libitum and divided in different groups on the basis of
treatments (fig. 1).The present research protocol was
approved by Institutional Ethical Review Board (IERB –
Authority Reference Number: IRB-186/DUHS-10 ) and
Board of Advance Studies and Research (BASR) of
DUHS and UoK respectively. All treatments were given
to their respective groups orally once per day for
consecutive 14 days. At the end of 14th day, mice were
sacrificed; whole blood, serum and liver samples were
collected and used to analyze biochemical markers.
Determination of physical parameter
Percent change in body weights of all mice of each group
was calculated (Azmi and Qureshi, 2012b) after
measuring their weights on initial and final day of trial
with the help of weighing balance.
Determination of biochemical parameters
Percent glycemic change of all mice was calculated after
measuring fasting blood glucose (FBG) at initial and final
day of trial by using glucomemter (Abbott Laboratories,
Pakistan) from tail vein (Azmi and Qureshi, 2012b).
Insulin level in serum was determined by the help of
cobas e411 analyzer, Hitachi (Roche Diagnostics GmbH,
Mannheim, Germany) whereas fasting insulin resistance
index (FIRI) was calculated by using following formula
(Duncan et al., 1995).
Serum total cholesterol (TC), triglycerides (TG) and high-
density lipoprotein-cholesterol (HDL-c) were determined
by enzymatic kits (Randox, United Kingdom). However,
low density lipoprotein-cholesterol (LDL-c), very low
density lipoprotein-cholesterol (VLDL-c) and coronary
risk index (CRI) were calculated through following
formulae (Azmi and Qureshi, 2012b).
Determination of hematological parameters
Total hemoglobin (Hb) and glycosylated hemoglobin
(HbA1c) levels were estimated by Automated Analyzer,
Sysmex (XS-1000i) and Nycocard Kit of USA,
respectively.
Determination of hepatic parameters
Estimation of glycogen content in liver homogenate was
performed by colorimetric method (Dubois et al.,
1956).Whereas 3-hydroxy-3-methylglutaryl-coenzyme A
(HMG-CoA) reductase activity was determined in terms
of HMG-CoA/Mevalonate ratio (Rao and Ramakrishnan,
1975).
STATISTICAL ANALYSIS
Data of present research was analyzed by using one-way
ANOVA, followed with least significant difference (LSD)
test, through SPSS version 18. Results are mentioned as
mean ± SEM (Standard Error Mean). All values are
considered significant at p<0.0001, p<0.01 & p<0.05.

Muhammad Bilal Azmi and Shamim A Qureshi
Pak. J. Pharm. Sci., Vol.29, No.5, September 2016, pp.1619-1624 1621
RESULTS
Effect of MREt on physical parameter
Pioglitazone and two doses (10 and 30 mg/kg) of MREt
induced 1.6, 4.9 and 2.09% increase in body weights of
mice in positive control and test (IV, V& VI) groups when
compared with diabetic control groups (II and III) which
showed prominent gain (6-9%) in their body weights.
However, dose of 60 mg/kg of same extract effectively
(p<0.01 & p<0.05) prevent gain in body weights of mice
of group VII (fig. 2).
Effect of MREt on FBG and other biochemical
parameters
Three of the doses of MREt showed 25.38, 18.64 and
17.40% gain (p<0.01, p<0.0001) in FBG respectively in
group V, VI and VII which was compared to diabetic
control groups that depicted 76.8-80% increase in same
parameter (fig. 2). A gradual decrease (p<0.01
&p<0.0001) in serum insulin levels (6.01 to 3 pmol/l) and
FIRI (4.35-1.93) was observed in test groups when
compared with diabetic controls which showed increased
insulin level and insulin resistance while pioglitazone
showed more significant effect on both of these
parameters (fig. 3). Similarly, MREt (10, 30 & 60 mg/kg)
showed significant decrease in serum levels of TC, TG,
LDL-c, VLDL-c and increase in HDL-c level in their
respective test groups whereas completely opposite
picture of lipid profile was observed in diabetic group II
& III. On contrary, pioglitazone was only found effective
(p<0001 & p<0.05) in decreasing the levels of TG and
VLDL-c (table 1). Prominent decrease (p<0.05 &
p<0.01) in CRI from 3.5 to 1.48 in MREt-treated test
groups while diabetic control groups showed much
increase (i.e., up to 4.09) in the same ratio. Beside this,
positive control group showed high value of CRI (fig. 4).
Effect of MREt on hematological parameters
High HbA1c (7.13%) and low total Hb (7.55%) levels
were observed in diabetic control groups. However, three
of the doses of MREt gradually improved (p<0.01 &
p<0.0001) the magnitude of total Hb and HbA1c in their
respective test groups. Similarly, positive control also
improved HbA1c but not total Hb level in group IV (fig.
5).
Fig. 1: Animal Grouping
Table1: Effect of MREt on biochemical parameters
Groups TC (mg/dl) TG (mg/dl) HDL-c (mg/dl) LDL-c (mg/dl) VLDL-c (mg/dl) Glycogen
(g/g of
tissue)
I 139.74±14.32 140.21±7.32 54.52±7.59 65.22±9.71 28.04±1.46 1.39±0.27
II 199.63±21.35 261.51±13.61 58.16±14.09 94.64±29.49 52.30±2.72 0.49±0.06
III 237.80±21.16 219.54±31.43 61.25±5.28 132.64±18.30 43.91±6.29 0.56±0.13
IV 193.50±17.21 167.74±7.72***a*b 43.37±7.40 115.47±19.39 33.55±1.54***a*b 0.79±0.06
V 176.73±8.35**b 178.14±10.58**a 50.82±1.54 90.30±4.75 35.63±2.12**a 0.52±0.11
VI 163.50±12.75**b 144.46±12.28***a**b 77±5.55 57.61±5.73**b 28.89±2.45***a**b 0.74±0.10
VII 165.75±3.88**b 146.66±6.24***a**b 113.17±3.49***a**b 23.25±2.81**a***b 29.33±1.25***a**b 0.86±0.11
Values are expressed as mean ± SEM (n= ). *
p
<0.05, **
p
<0.01 and ***
p
<0.0001, when compared with respective group II (a) and III (b).

Rauwolfia serpentina improves altered glucose and lipid homeostasis in fructose-induced type 2 diabetic mice
Pak. J. Pharm. Sci., Vol.29, No.5, September 2016, pp.1619-1624
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Effect of MREt on hepatic parameters
MREt (10-60mg/kg) and pioglitazone improved liver
glycogen content from 0.5-0.86g/g of hepatic tissue in test
and positive control groups while diabetic control groups
showed decreased glycogen content in liver tissues (table
1). HMG-Co A reductase activity was found inhibited by
observing improved (p<0.0001 & p<0.01) HMG-
CoA/Mevalonate ratio in MREt-treated test groups
especially group VI & VII as compared diabetic control
groups (fig. 4).
Fig. 2: Effect of MREt on percent weight and glycemic
change. Each bar represents the mean ± SEM (n=6).
*p<0.05, **p<0.01 and ***p<0.0001, when compared
with group II (a) and III (b).
Fig. 3: Effect of MREt on serum insulin and FIRI in
insulin resistance mice. Each bar represents the mean ±
SEM (n=6). *p<0.05, **p<0.01 and ***p<0.0001, when
compared with respective group II (a) and III (b).
DISCUSSION
Insulin resistance is the classical feature of T2D and well-
accepted as the initiator of disability and death worldwide
(Patel et al., 2013; Hsu, 2013). Excessive intake of HFCS
is also reported as one of the leading causes of insulin
resistance which slowly and gradually induce obesity by
producing hypertriglyceridemia that eventually decrease
the sensitivity of receptors for its agonist insulin, this
situation leads to hyperinsulinemia, hyperglycemia,
hyperlipidemia and many other important signs of T2D
(Khitan and Kim, 2013; Ahmed et al., 2010; Johnson et
al., 2009; Bray et al., 2004; Gorana et al., 2012).
Therefore, the preliminary and most reported effect of
T2D is the weight gain (Bray, 2007). The same feature
was observed in present study that all mice of diabetic
control groups showed percent gain from 6-9% in their
body weights after consuming 10% fructose solution
(1ml/kg) consecutively for 14 days. Whereas gradual
improvement in body weights of mice in three test groups
was observed in dose-dependent manner. In this respect,
the highest dose (60 mg/kg) of MREt completely prevents
gain in body weights of mice in its respective group.
Similarly, all three doses of MREt found effective in
controlling fructose-induced hyperglycemia in
experimental groups when compared to diabetic control
groups which showed extreme increase in FBG level.
Pioglitazone, a well-known medicine used in T2D also
found effective in preventing fructose-induced percent
glycemic and weight gain in positive control group. The
percentage of HbA1c is about 4-6% in non-diabetic
condition whereas in T2D persistent hyperglycemia
induced non-enzymatic glycation of total Hb thus elevates
the amount of HbA1c from 6% to onwards and creates an
unrecognized mild anemia which gradually increases with
the age of diabetes (Minshawy and El-Bassuoni, 2010;
Cederberg et al., 2010). Similar situation was observed in
present study where high HbA1c and low total Hb levels
were found in diabetic control groups. On contrary, three
of the doses of MREt gradually improved the magnitude
of total Hb and HbA1c in their respective test groups by
showing good control of HbA1c from 4.4-4.8%.
Interestingly, pioglitazone was only found effective in
improving HbA1c but did not show any effect on
decreased total Hb concentration in positive group.
Fig. 4: Effect of MREt on CRI and HMG Co-A
/Mevalonate ratio. Each bar represents the mean ± SEM
(n=6). *p<0.05, **p<0.01 and ***p<0.0001, when
compared with respective group II (a) and III (b).
The hypoglycemic effect of MREt observed in present
study may be due to its extra-pancreatic action via
inhibiting fructose absorption in intestine or reducing
insulin resistance in fructose-induced T2D mice. The last
possibility was clarified by observing a significant
gradual decrease in serum insulin levels in MREt-treated
groups which also lowers FIRI in same experimental
groups when compared to the both diabetic controls

Muhammad Bilal Azmi and Shamim A Qureshi
Pak. J. Pharm. Sci., Vol.29, No.5, September 2016, pp.1619-1624 1623
which showed hyperinsulinemia and increased insulin
resistance (FIRI). Therefore, MREt may be effective in
improving insulin resistance which improves glucose
uptake in target tissues and stimulate anabolic processes
of insulin like glycogenesis, lipogenesis, etc, thereby
decrease fructose-induced hyperglycemia and HbA1c level
in test groups. It was also confirmed by noticing the dose-
dependent increased in hepatic glycogen in three MREt-
treated test groups. However, the possibility of inhibiting
the fructose absorption in intestine by MREt cannot be
overlooked as the same extract was found to improve
glucose tolerance in glucose-fed mice earlier (Azmi and
Qureshi, 2012a).
Fig. 5: Effect of MREt on Hb and HbA1c levels (gm/dl).
Each bar represents the mean ± SEM (n=6).**p<0.01 and
***p<0.0001 when compared with group II (a) and III
(b).
High dietary fructose is reported as a lipogenic agent,
which after intestinal absorption enters in liver cells
through insulin-independent glucose transporters (GLUT-
5) and stimulates the synthesis of glycerol-3-phosphate,
acyl coenzyme A (acyl-Co A) and acetyl coenzyme A
(acetyl-Co A). The first two components accelerate the
formation of triglycerides (fats) and its transporting
vehicle VLDL-c which enhance the deposition of newly
synthesized triglycerides on membranes of peripheral
tissues, thereby possibly involve in masking of insulin
receptors and induce insulin resistance (Rutledge and
Adeli, 2007). Likewise, acetyl-Co A speeds up the
synthesis of cholesterol and cholesterol transporting
protein, LDL-c thus encouraging the
hypercholesterolemia and discouraging the role of HDL-c
that leads in cholesterol efflux from peripheral tissues
(Lateef and Qureshi, 2014). Both of these hyperlipidemic
effects of fructose provoke the risk of life-threatening
heart problems in insulin resistance diabetes (Hsu, 2013;
Khitan and Kim, 2013; Ahmed et al., 2010). In such type
of diabetes, normally oral hypoglycemic agent is
prescribed in combination with hypocholesterolemic
agent in order to minimize the risk of heart problems
(Gomez et al., 2005). In the present effort, MREt (10, 30
& 60mg/kg) showed significant decrease in serum levels
of TC, TG, LDL-c, VLDL-c and increment in HDL-c
quantity in their respective experimental groups while
pioglitazone was only found effective in decreasing TG
and VLDL-c levels. The hypotriglyceridemic effect of
MREt may be due to its ability of enhancing insulin
sensitivity for its receptor. The hypocholesterolemic effect
of MREt in test groups may be associated with inhibition
of HMG-CoA reductase activity, the rate-limiting enzyme
involved in the cholesterol biosynthesis (Lateef and
Qureshi, 2014).This possibility was also evidenced by
observing gradually improved HMG-CoA/Mevalonate
ratio in all extract-treated groups as compared to decrease
values of same ratio found in diabetic control groups.
These findings are more confirmed by observing a
significant decrease in CRI of test groups while diabetic
control groups showed marked increase in the same
index. Despite this, positive control (group IV) showed
high value of CRI. CRI reflects the susceptibility towards
cardiovascular problems (Azmi and Qureshi,
2012b).Therefore, MREt of R.serpentina strongly
minimized the risk of cardiovascular problems in
fructose-induced diabetic mice as same as it was
efficiently improved cardio-protective indices in alloxan-
induced (type 1 diabetic) mice (Azmi and Qureshi,
2012b). A significant amount of alkaloids and
polyphenolic compounds have been estimated in MREt
previously (Azmi and Qureshi, 2012b) which could be
involve in improving the glucose and lipid homeostasis in
fructose-induced diabetic mice.
CONCLUSION
Results from present effort concluded that MREt of
R.serpentina improves hyperinsulinemia, hyperglycemia,
hypertriglyceridemia and hypercholesterolemia in
fructose-induced T2D mice either by inhibiting fructose
absorption in intestine or reducing insulin resistance.
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