Anti-Hyperglycemic And Antidyslipidemic Potential Of Azadirachta indica Leaf Extract In STZ- Induced Diabetes Mellitus

Abstract

Azadirachta indica, an Indian medicinal plant, has been studied for its role in diabetes and its effect on lipid profile. This study was conducted to elucidate whether treatment of Azadirachta indica leaf extract after streptozotocin (STZ) - induced diabetes has anti-hyperglycemic and anti-dyslipidaemic action or not. The experiment involved four groups of rat; one group was control group, second diabetic control, third diabetic group received alcoholic extract of Azadirachta indica and fourth diabetic group received Glibenclamide as a reference standard. Oral glucose tolerance test was performed before induction of diabetes. Blood was collected by retro-orbital puncture for glucose estimation, and to evaluate serum triglyceride, total cholesterol, HDL-cholesterol, LDL-cholesterol levels. Blood Glucose level as well as serum lipid profile parameters such as total-cholesterol, triglyceride, low-density lipoprotein and very low-density lipoprotein cholesterol were also elevated, whereas, the level of high-density lipoprotein-cholesterol was reduced significantly (P<0.05) in diabetic rats. Ethanolic extract of A.indica after induction of diabetes, normalized glucose level and lipid profile. It can be concluded that STZ-induced hyperglycaemia can be ameliorated by treatment with ethanolic extract of A indica. A.indica ethanolic leaves extract after diabetic induction, reverses dyslipidaemia.

Full text

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Anti-Hyperglycemic And Antidyslipidemic Potential Of Azadirachta indica
Leaf Extract In STZ- Induced Diabetes Mellitus
Shradha Bisht1, S.S.Sisodia2
B.N.College of Pharmacy, Udaipur, Rajasthan
Abstract:
Azadirachta indica , an Indian medicinal plant, has been studied for its role in diabetes and its effect on lipid profile.
This study was conducted to elucidate whether treatment of Azadirachta indica leaf extract after streptozotocin
(STZ) - induced diabetes has anti-hyperglycemic and anti-dyslipidaemic action or not. The experiment involved four
groups of rat; one group was control group, second diabetic control, third diabetic group received alcoholic extract
of Azadirachta indica and fourth diabetic group received Glibenclamide as a reference standard. Oral glucose
tolerance test was performed before induction of diabetes. Blood was collected by retro-orbital puncture for glucose
estimation, and to evaluate serum triglyceride, total cholesterol, HDL-cholesterol, LDL-cholesterol levels. Blood
Glucose level as well as serum lipid profile parameters such as total-cholesterol, triglyceride, low-density
lipoprotein and very low-density lipoprotein cholesterol were also elevated, whereas, the level of high-density
lipoprotein-cholesterol was reduced significantly (P<0.05) in diabetic rats . Ethanolic extract of A.indica after
induction of diabetes, normalized glucose level and lipid profile. It can be concluded that STZ-induced
hyperglycaemia can be ameliorated by treatment with ethanolic extract of A indica. A.indica ethanolic leaves extract
after diabetic induction, reverses dyslipidaemia.
Key words: Diabetes mellitus, serum lipid, Streptozotocin, Azadirachta indica.
Introduction:
India has one of the oldest, richest and
diverse cultural traditions associated with
the use of the plants and herbs for human,
liverstock and plant health. Many of the
ingredients of Indian cooking which have
been handed down from ages contain
medicinal properties. A vast ethnobotanical
knowledge exists in India from ancient
times. However, very few plants used by
locals for medicines are subjected to
scientific investigation. The need for
conservation of medicinal plants and
traditional knowledge, particularly in
developing countries like India, taking into
account the socio cultural and economic
conditions is urgent.[1]
Diabetes mellitus (DM) is a serious
metabolic disease which has several
complications including diabetic
nephropathy, diabetic neuropathy, coronary
heart disease and hypertension.[2] It has
been estimated that by the year 2010, the
prevalence of DM worldwide will reach
approximately 240 million.[3] Patients with
DM are more likely to develop and die from
microvascular and macrovascular
complications than the nondiabetic
population.[4] There is usually an
association between coronary heart disease
or atherosclerosis and dyslipidaemia.[5, 6]
Dyslipidaemia is a frequent complication of
DM and is characterized by low levels of
HDL-cholesterol and high levels of LDL-
cholesterol and triglyceride. Several groups
of hypoglycaemic drugs are currently
available to treat DM. However, their toxic
side effects and sometimes diminution in
response after prolonged use are
problematic. Management of DM to avoid
these problems is still a major challenge. In
the indigenous Indian system of medicine,
good number of plants was mentioned for
the cure of diabetes and some of them have
been experimentally evaluated and the
active principles isolated.[7] However
search for new antidiabetic drugs were
continues.
Neem (Azadirachta indica A. Juss) is
perhaps the most useful traditional
medicinal plant in India. Each part of the
neem tree has some medicinal property and
is thus commercially exploitable. During the
last five decades, apart from the chemistry
of the neem compounds, considerable
progress has been achieved regarding the
biological activity and medicinal
applications of neem. It is now considered as
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a valuable source of unique natural products
for development of medicines against
various diseases and also for the
development of industrial products.[8]
Every part of the tree has been used as
traditional medicine for household remedy
against various human ailments, from
antiquity[9-14]. Neem has been extensively
used in ayurveda, unani and homoeopathic
medicine and has become a cynosure of
modern medicine. The importance of the
neem tree has been recognized by the US
National Academy of Sciences, which
published a report in 1992 entitled ‘Neem –
a tree for solving global problems’. The
advancement of neem research has earlier
been documented [15, 16]. The neem tree
has been described as A. indica as early as
1830 by De Jussieu [17] and its taxonomic
position is as follows:
Order Rutales
Suborder Rutinae
Family Meliaceae (mahogany family)
Subfamily Melioideae
Tribe Melieae
Genus Azadirachta
Species indica
Neem oil, bark and leaf extracts have been
therapeutically used as folk medicine to
control diseases like leprosy, intestinal
helminthiasis, respiratory disorders,
constipation, and skin infections.[18]
However, apart from these uses, there are
several reports on the biological activities
and pharmacological actions based on
modern scientific investigations, such as
antiviral [19], antibacterial [20], antifungal
[21], anti-inflammatory and antipyretic [22],
antiseptic, antiparalitic [23], antioxidant [24,
25]etc.
In this study, we investigated the effects of
A. indica leaf extract on blood glucose level,
serum lipid profile changes in normal and
stz induced diabetic rats with a view to
finding out its possible effect on
cardiovascular disease induced by
hyperglycemia.
Material and Methods:
Animal preparation:
stz- induced model :
Male Wistar albino rats weighing between
180-220g were used in the study with the
approval of the animal ethical committee of
Bhopal Nobels College of Pharmacy,
Udaipur, Rajasthan. Rats were housed in a
12-hr light-dark cycle at 25 ± 2 °C. The
animals were provided standard rat pellet
feed and tap water ad libitum. All animals
were cared for in accordance with the
principles and guidelines of the Institutional
Animal Ethics Committee of B.N.College of
pharmacy, Udaipur. Diabetes was induced in
rats by tail vein injection of streptozotocin
(50 mg/kg, i.v.) (Sigma chemicals) dissolved
in normal saline. (One group of identical rats
was kept without streptozotocin
administration as normal control, group I).
Forty eight hours after streptozotocin
administration blood samples were drawn by
retroorbital puncture and glucose levels
determined to confirm diabetes. The diabetic
rats exhibiting blood glucose levels in the
range of 275 to 300 mg/100 ml were
selected for the studies. Glibenclamide (500
µg/kg) was used as reference standard. The
dose of Glibenclamide was selected based
on previous reports.[26]
Following four groups of rats, were taken.
Group I : normal control (NC)
Group II: diabetic control (DC) -given
(untreated rats) 0.5 ml of 5% Tween 80.
Group III: diabetic rats given (200 mg/kg)
ethanolic extract of A.Indica (ET) in 0.5 ml
5% Tween 80
Group IV: diabetic rats treated with
glibenclamide (500 μg/kg) (GT) in 0.5ml
5% tween 80 (GT)
Preparation of Azadirrachta indica leaf
ethanol extract:
Fresh leaves of A.indica obtained from the
local market of Jaipur, were washed in tap
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water and then left to dry at room
temperature for 2-3 days. The dried leaves
were then ground to fine powder in a mixer.
The dried leaf powder was then extracted
with 95% ethanol using a soxhlet apparatus
for 15 hr. after filtration through cotton
wool; the filtrate was concentrated at 650C
by a rotavapor. The concentrate was then
freeze dried to yield dried powder and were
designated as A.indica leaf ethanol extract.
[27]
Experimental design:
oral glucose tolerance test [28]:
The oral glucose tolerance test was
performed in overnight fasted (18-h) normal
animals. Rats divided into three groups were
administered 2% gum acacia solution,
ethanolic extract of A.Indica (200 mg/kg),
and Glibenclamide (0.25 mg/kg),
respectively. Glucose (2 g/kg) was fed 30
min after the administration of samples.
Blood was withdrawn from the retro-orbital
sinus at 0, 30, 60, 90 and 120 min of
samples administration.[29] Fasting blood
glucose levels were estimated by glucose
oxidase-peroxidase reactive strips (Accu-
check, Roche Diagnostics, USA).
biochemical estimation in stz- induced
model:
The diabetic rats exhibiting blood glucose
levels in the range of 275 to 300 mg/100 ml
were selected for the studies. The treatments
were continued daily for 40 days. Blood was
collected by retro-orbital puncture for
glucose estimation just before drug
administration on the 1st day and 1 h after
drug administration on days 1, 10, 20, 30,
40. Blood glucose (FBG) concentration of
all the four experimental groups was
determined by glucometer during different
phases of the experiment by withdrawing
blood from the retero orbital vein. For
estimating serum lipid profile, serum was
isolated from the blood collected on 40th day
of A. indica leaf ethanol extract treatment
and serum total cholesterol (TC),
triglyceride (TG) and HDL-cholesterol were
estimated by using diagnostic kits (Erba
Mannheim Cholesterol kit, Transasia Bio-
Medicals Ltd., Daman). VLDL and LDL
cholesterol were calculated as per
Friedevald’s equation [30]:
VLDL-cholesterol = Serum triglyceride-
Cholestrol
LDL-cholesterol = Serum total-cholesterol –
VLDL-cholesterol – HDL-cholesterol.
Results were expressed in mg/dl
Data and statistical analysis:
Results are expressed as mean ±Standard
Error of Mean (SEM). Statistical analysis
was performed using one-way Analysis of
Variance (ANOVA) using SPSS (version
10.0) and student’s ‘t’-test using Sigma Plot
(version 8.0). The values of P<0.05 were
considered as statistically significant.
Results:
effects of a.indica leaf ethanol extract on
blood glucose level in ogtt:
Table 1 shows the changes in fasting blood
sugar level during oral glucose tolerance
test. Fasting Blood sugar level was
determined by collecting the blood from
retro orbital sinus in 0, 30, 60, 90, 120
minute. There was significant increase in
blood sugar level in animal of control group
having 2% gum acacia solution from
82.33±2.58 to 90±.1.78mg/dl. Group
receiving Glibenclamide (0.25 mg/kg)
showed a significant (P<0.05) decrease in
blood glucose level in every 30 minutes
interval from 84.66±2.16 to
78.33±1.36mg/dl, group receiving ethanolic
extract of A.indica leaves showed a
significant and continuous decrease from
81.5±2.73 to 768.33±1.21 in blood sugar
level till the 90 min. When it was observed
after 120 min it was reached near about
normal level i.e 81.33±2.25 mg/dl.
effect of a.indica leaf ethanol extract on
blood glucose level in diabetic rats:
As shown in Table 2, the extract (group III)
produced gradual and moderate
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Table 1: Blood glucose concentration (mg/dl) in OGTT
Group Treatment
Initial 30 min 60 min 90 min 120 min
I Control
82.33±2.58 85.83±2.48 89.5±1.87 91.83±1.47 90±1.78
II Extract treated 81.5±2.73 79.5±2.34 77.5±2.42 78.33±1.21 81.33±2.25
III Glibenclamide
treated
84.66±2.16a76.83±1.47a71.66±1.63a68.5±1.64a 78.33±1.36a
Student’s ‘t’-test is significant at P<0.05. a-significant (P<0.05) difference compared to C; b-significant (P<0.05)
difference compared to GT.
Table 2: Blood glucose level (mg/dl) in STZ – induced diabetic rats
Grou
p Treatment 1 Day 10 Day 20 Day 30 Day 40 day
I Control
84.83±
1.47
85.33±
1.21
84.5±
1.04
84.33±
1.21
84.33± 0.51
II Diabetic
control
283.16±
3.31a
370.16±
3.12a
404.5±
1.87a
373.83±
2.87a
372.16±
3.31a
III Extract
Treated
293.5±
1.87a
263.5±
3.27a bc
230.83±
3.06abc
214.83±
3.6abc
198.83±
3.97abc
IV Glibenclemid
e (500 µg/kg)
286.6±
3.01a
213.83±
2.31a b
176.83±
3.06a
176.83±
3.06a b
125.16±
2.63a b
Student’s ‘t’-test is significant at P<0.05. a significant (P<0.05) difference compared toC; b significant (P<0.05)
difference compared to DC; c significant (P<0.05) difference compared to GT.
Table 3: Effect of treatment of A.indica leaf extract on serum lipid profile (mg/dl) in
streptozotocin -induced diabetic rats:
Parameters Normal
Control Diabetic Control Extract treated Glibenclamide
Treated
TC 87.7 ± 2.7
217.5±3.5a 89.6±3.7b 78.1±3.7b
TG 75.6± 2.9
161.1±4.2a 73.7±5.6b 61±5 b
HDL 29.7±3
18.7±0.7a 32±2.47b 49.2±3.6b
LDL 46.3±3.4
170±3.1a 48±3.7bc 19.7±1.7b
VLDL 17.4±0.3
34.5±0.7a 18.01±2b 14.4±1b
Student’s ‘t’-test is significant at P<0.05. a significant (P<0.05) difference compared to NC; b significant (P<0.05)
difference compared to DC; c significant (P<0.05) difference compared to GT.
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antihyperglycemic effect on day 10, 20, 30
and 40. The plasma glucose level on these
days was significantly less than the
pretreatment level. Further the
antihyperglycemic effect produced by the
extract was significant (p<0.05) as
compared to what observed in group I and
II (control and diabetic control) on different
days. However, the antihyperglycemic effect
produced by Glibenclamide (group IV) was
more pronounced than A.Indica extract.
effects of a. indica leaf extract on lipid
profile in diabetic rats:
A highly significant increase in total
cholesterol level was recorded in normal
control and diabetic control group (untreated
animals). However, the cholesterol level of
extract treated animals were significantly
less than the animals of group I and II
indicating antihypercholesterolemic effect of
the extract.
The plasma triglyceride levels of group III
were significantly less than the contol and
diabetic control group. Intergroup
comparison revealed that the Glibenclamide
treated and extract treated exerted
antihypertriglyceridemic effect, as the
plasma triglyceride levels were significantly
less than that in the untreated control and
diabetic group. A.Indica leaf extract and
Glibenclamide reduced the level of serum
TC, TG, LDL and VLDL-cholesterol
reduced significantly (P<0.05) whereas, the
level of serum HDL-cholesterol was
significantly increased. A.indica treated
group were showed the increased level of
HDL as compare to Glibenclamide treated
group after the 40 days.
Discussion and Conclusion:
Evaluation of the ethanolic extract of
A.Indica leaves in normoglycemic and STZ
–hyperglycemic rats indicated that the
extract possesses hypoglycemic and
antihyperglycemic activities.
Hyperlipidemia has been reported to
accompany hyperglycemic states15 and the
most common lipid abnormality observed
was hypertriglyceridemia. A significant
increase in the total cholesterol, TG, LDL
and VLDL levels were in accordance to
earlier studies.16, 17 Repeated administration
of ethanolic extract of A. indica prevented
the elevation of TC,TG, LDL and VLDL-
cholesterol level in diabetic rats indicating
the A. indica had a beneficial effect on the
hyperlipidemia induced by STZ.
Our finding indicates that the ethanolic
extract of A. indica leaves may be useful for
treatment of diabetes associated with
hyperlipidemia. Further chemical and
pharmacological investigations are required
to elucidate the exact mechanism of action
of this extract and to isolate the active
principles responsible for such effects.
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