Khan et al
Tropical Journal of Pharmaceutical Research July 2017; 16 (7): 1573-1578
ISSN: 1596-5996 (print); 1596-9827 (electronic)
© Pharmacotherapy Group, Faculty of Pharmacy, University of Benin, Benin City, 300001 Nigeria.
All rights reserved.
Available online at http://www.tjpr.org
http://dx.doi.org/10.4314/tjpr.v16i7.15
Original Research Article
Anti-diabetic potential of aerial parts of Galium tricornutum
(Dandy) Rubiaceae
Shah Tamas Khan, Mushtaq Ahmed, Rahmat Ali Khan*, Nadia Mushtaq and
Nisar Khan
Department of Biotechnology, University of Science and Technology Bannu 28100, Pakistan
*For correspondence: Email: rahmatul_81@yahoo.com; Tel: +92 928633425
Sent for review: 8 April 2016
Revised accepted: 8 June 2017
Abstract
Purpose: To evaluate the anti-diabetic potential of methanol extract of the aerial parts of Galium
tricornutum (Dandy) in diabetic rats.
Methods: The methanol extract of the aerial parts of Galium tricornutum was first subjected to acute
toxicity studies. Thereafter, the effect of the extract on oral glucose tolerance was determined. In
addition, the effect of the extract on fasting blood glucose, as well as serum lipid profile, urea,
creatinine, alanine transaminase (ALT), aspartate transaminase (AST), bilirubin, alkaline phosphatase
(ALP) and protein were investigated in alloxan-induced diabetic rats.
Results: No acute toxicity were observed in the rats after administration of the plant extract up to a
dose of 2000 mg/kg. The effect of the extract on glucose tolerance test was significant from 30 to 180
min after treatment. In the diabetic rats, the extract showed significant (p < 0.05) anti-hyperglycemic
activity at 400 mg/kg. It also led to significant increases in body weight and HDL-cholesterol, and
significant reductions in serum LDL, triglycerides and transaminases (p < 0.05).
Conclusion: These results indicate that the aerial parts of G. tricornutum possess significant antidiabetic potential.
Keywords: Diabetes, Galium tricornutum, Glibenclamide, Glucose tolerance tes, Lipid profile
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INTRODUCTION
Diabetes mellitus is one of the most common
metabolic disorders. It is caused by inability of
pancreatic cells to produce insulin, or defects in
insulin utilization. The disease is associated with
significant morbidity and mortality, and is
considered one of the five leading causes of
death in the world [1]. There are no drugs that
can effectively treat diabetes mellitus. Moreover,
the use hypoglycemic drugs and insulin results in
unwanted side effects. Thus there is increasing
interest in the management of diabetes through
the use of natural products with hypoglycemic
potential.
Many medicinal plants have been reported to
possess hypoglycemic properties. These include
Ocimum santum (Tulsi), Momordica charantia
(bitter round), Trigonella foenum (Fenugreek),
Allium sativum (garlic), Vinca rosea (nayantara),
and Azadirachta indica (neem). However, in
severe diabetes, many of these plants are not
very effective in lowering blood glucose levels.
Galium tricornutum is an annual herb that
belongs to the family Rubiaceae. It has trailing or
climbing stems and is found almost all over the
world. The plant is used as an anti-scorbutic
agent, and as a refrigerant, diuretic and aperient
Trop J Pharm Res, July 2017; 16(7): 1573
Khan et al
[2]. It is also used as anti-malarial, antipyretic
and emetic [3]. In the northern parts of Pakistan,
the plant is used for the treatment of skin
infections [4]. However, despite its medicinal
importance, no studies have been carried out on
the anti-diabetic potential of Galium tricornutum
to date.
The present study was carried out to investigate
the anti-diabetic effects of methanol extract of
aerial parts of Galium tricornutum in alloxaninduced diabetic rats. Antidiabetic potential and
biochemical parameters such as LDL, HDL,
cholesterol, urea, creatinine, ALP, bilirubin,
triglycerides, protein, ALT and SPT were studied
in male Wistar albino rats exposed to two doses
of the extract for 21 days.
EXPERIMENTAL
Plant collection and extraction
Galium tricornutum was collected from the
outskirts of Bannu district, Khyber Pakhtunkhwa,
Pakistan. The plant was identified and
authenticated by Professor Abdur Rahman,
Department
of
Botany,
Government
Postgraduate College, Bannu.
mg/kg did not result in mortality or any signs of
toxicity or changes in general behavior.
Oral glucose tolerance test
Four groups of rats (6 rats/group) were used.
The rats were fasted for 16 hr before the test.
Group I served as normal control and received
normal saline p.o. only. Group II received
glucose (3 g/kg) only. Groups III and IV received
methanol extract at 200 mg/kg and 400 mg/kg,
respectively. Thirty minutes after extract
administration, rats in groups III and IV were
given glucose (3 mg/kg, p.o.). Blood samples
were collected from the retro-orbital plexus just
prior to extract administration, and 30, 90 and
150 min after glucose load, for measurement of
serum
glucose levels
using ACCUCHEK® Active meter (Indianapolis, USA).
Effect of extract on alloxan-induced diabetes
in rats
The rats were divided into five groups, each with
six rats, and were treated as summarized below:
Group I: Normal control (saline).
Group II: Alloxan-treated control (150 mg/kg.ip).
The aerial parts of the plant were shade-dried,
and made into coarse powder by pulverization.
The coarse powder (200 g) put extracted by
shaking with 80 % commercial grade methanol in
a beaker for 3 days, and the extract was filtered
through qualitative Whatman no. 1 filter paper.
Animals
Wistar albino rats of both sexes (8 - 10 weeks
old) weighing 170 – 200 g were obtained from
the Animal House of Quaid-i-Azam University,
Islamabad. The rats were fed with standard rat
feed before and during the experiment, and were
randomly assigned to various groups. Prior to
commencement of the study, the rats were
acclimatized to laboratory conditions for 7 days
at room temperature, 12 hour-dark/light cycle
and relative humidity.
Acute oral toxicity studies
The experiment was conducted according to the
guide lines for experimental animal model [5]
approved by the ethical committee of University
of Science and Technology Bannu. Two groups
of rats (3 rats/group) were treated orally with the
Galium tricornutum extract at a dose of 5000
mg/kg. A third group received an equivalent
volume of normal saline. The dose of 5000
Group III: Alloxan (150 mg/kg. i.p.) + Galium
tricornutum extract (200 mg/kg, p.o.).
Group IV: Alloxan (150 mg/kg. i.p.) + Galium
tricornutum extract (400mg/kg, p.o.).
Group V: Alloxan (150 mg/kg.ip) + Standard
drug, glibenclamide (5mg/kg, p.o.).
All treatments lasted for 21 consecutive days.
The extract, standard drug glibenclamide (5
mg/kg) and saline were administered via a
feeding cannula.
Induction of diabetes in experimental animals
Diabetes was inducted in the experimental
animals by a single intraperitoneal (i.p.) dose of
alloxan monohydrate (150 mg/kg). Alloxan was
dissolved in normal saline prior to administration.
Since alloxan causes fatal hypoglycemia
because of massive release of insulin, the rats
were given 20 % glucose solution i.p. after 6
hours. In addition, 5 % glucose solution bottles
were kept handy for 24 hr to prevent
hypoglycemia [5]. After 48 hours of alloxan
injection, rats with blood glucose levels less than
200 mg/dL were included in the study. Treatment
with extract and glibenclamide was started 48 h
after alloxan injection.
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Khan et al
Collection of blood samples and blood
glucose determination
Blood samples were taken from tail tip of the
rats. Estimations of fasting blood glucose and
body weight measurement were done onset, and
on the 1st, 7th, 14th and 21st days of the study.
Blood glucose levels were checked by one-touch
electronic glucometer using glucose test strips.
On day 21, fasting blood glucose was estimated
in overnight-fasted rats, with blood taken from
the retro-orbital plexus under mild ether
anesthesia [6]. Serum was separated and
analyzed for LDL [7] and HDL [8] cholesterol, as
well as triglycerides [9], urea [10], creatinine [11],
ALP [12], bilirubin [13], protein [14] and ALT/AST
[15].
Statistical analysis
Data were expressed as mean ± SEM.
Differences between groups were assessed by
one-way analysis of variance (ANOVA) using the
Statistical Package for Social Sciences (SPSS)
software package for Windows (version 13.0).
Post hoc testing was performed for inter-group
comparisons using the least significant difference
(LSD) test. P values less than 0.05 were deemed
statistically significant.
RESULTS
Acute oral toxicity
The OECD guidelines AOT-425 was used for
determination of acute toxicity of the extract in
rats. A single oral dose of Galium tricornutum
(5000 mg/kg) did not cause any signs of toxicity
or mortality in the rats within 4 hrs. The animals
were observed continuously and were found to
be safe at a dose of up to 5000 mg/kg.
Oral glucose tolerance test
The effects of methanol extracts of Galium
tricornutum (200 and 400 mg/kg) on oral glucose
tolerance test are shown in Table 1. The extract
improved glucose tolerance in fasted normal rats,
with significant decreases in serum glucose
levels at 90 minutes and at 150 minutes postglucose load.
Effect of G. tricornutum on fasting blood
glucose levels of alloxan-diabetic rats
The anti-diabetic effect of Galium tricornutum
on the fasting blood glucose levels of diabetic
rats is shown in Table 2. Administration of
alloxan (150 mg/kg, i.p.) led to increases in
fasting blood glucose levels, which were
maintained over a period of three weeks.
However, daily administration of G. tricornutum
extract for 3 weeks led to dose-dependent
decreases in blood glucose levels. The
decreases in blood glucose peaked on day 21 of
treatment. Rats in normal control showed slight
increases in body weight but the body weights of
the diabetic rats were significantly decreased
during the 21–day study period.
Effect of G. tricornutum extract on body
weight and on some serum, biochemical
indices in alloxan-diabetic rats
Alloxan caused decreases in body weight, which
were reversed by the extract (400 mg/kg and 200
mg/kg) after 21 days of treatment (Table 3). It
also bought about significant increases in serum
cholesterol, LDL, creatinine, urea and ALP; and
significant decreases in HDL levels (p ˂ 0.05;
Table 4).
Table 1: Changes in serum glucose in the glucose tolerance test (mg/dL)
Group
Normal
Glucose (3g/kg)
Glucose (3mg/kg) + 200mg/kg G.
tricornutum
Glucose (3g/kg) + 400mg/kg G.
tricornutum
0 min
69.5±2.2
69.3±5.4
70.6±4.6
30 min
68.5±5.4
110.1±4.7
100±4.9
90 min
71±5.1
121.3±4.3
93.8±3.7
150 min
69.3±4.1
129.6±4.5
89.1±5.1
70.3±5.4
95.3±4.7
89.3±2.9
84.6±2.7
Table 2: Effect of extract on fasting blood glucose levels of alloxan-diabetic rats (mg/dL)
Group
Normal
Diabetic control
Diabetic + 200m/k G. tricornutum
Diabetic + 400m/kg G. tricornutum
Diabetic + glibenclamide
Initial
70.66±2.6
222.6±3.1
261.3±2.8
257.6±3.0
249.8±7.1
st
1 day
70.16±2.8
222.5±7.1
248.6±6.1
246.5±5.6
189.16±3.8
th
7 day
70±2.4
240±2.4
206.16±1.8
190.3±3.8
140.3±4.0
th
14 day
70±3.5
246.16±3.1
148.5±4.0
144.83±2.5
88.5±3.1
st
21 day
72.83±4.1
260.16±3.7
130.5±3.1
123.16±3.2
69.66±2.6
Trop J Pharm Res, July 2017; 16(7): 1575
Khan et al
Table 3: Effect of extract on body weight of rats (grams)
Group
Normal
Diabetic control
Diabetic+200mg/kg extract
Diabetic+400mg/kg extract
Diabetic + glibenclamide
Day 1
170±2.5
181.8±3.8
170±2.4
170±3.1
175.8±5.0
Day 7
174.6±5.0
177.6±4.7
165.3±5.2
168±6.0
174.5±2.4
Day 14
176±2.7
169.8±3.1
160.3±4.7
163±3.2
175.8±2.9
Day 21
178.6±3.8
161±4.3
157±5.1
160±3.1
173.3±2.8
Table 4: Effect of extract on serum biochemical profile of alloxan-diabetic rats after 21 days
Group
Normal
Diabetic
control
Diabetic +
200mg/kg
extract
Diabetic +
400mg/kg
extract
Diabetic +
glibenclamide
LDL
(mg/dl)
93±4.6
171.8±1.2
HDL
(mg/dl)
37±1.8
28.8±1.9
Cholesterol
(mg/dl)
155±5.1
261.3±4.5
Urea
(mg/dl )
25±1.0
60.3±3.2
Creatinine
(mg/dl )
0.71±0.01
2.18±1.0
ALP
(mg/dl )
128±4.1
265.8±5.1
Bilirubin
(mg/dl )
0.73±0.1
2.11±0.5
TG
(mg/dl )
95±5.4
171.5±4.7
Protein
(g/dl )
6.7±0.4
4.24±0.3
ALT
(U/l)
49±1.4
90.6±1.5
129±3.2
32±3.1
209.8±6.1
39.8±2.1
0.97±0.4
146.5±2.6
0.62±0.2
159.6±4.1
4.86±0.5
78.3±2.1
99.8±5.0
33.6±2.0
170±3.1
33.6±2.3
0.60±0.3
129.6±4.5
0.62±0.1
144.8±3.8
5.23±0.1
67.8±3.4
91.8±3.7
35.5±1.7
147±2.4
30±1.2
0.57±0.2
121.8±2.5
0.59±0.2
125±3.9
6.34±0.5
58.6±2.7
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Khan et al
Similarly, serum levels of other biochemical
parameters (bilirubin, triglycerides, ALT and
AST) were significantly increased, while total
protein was significantly decreased by alloxan (p
˂ 0.05). These alloxan-induced changes were
reversed by glibenclamide and G. tricornutum
extract.
CONCLUSION
The results obtained in this study indicate that
methanol extract of G. tricornutum possesses
significant anti-diabetic effects. This finding is
considered useful for the development of new
and safer anti-diabetic drugs from natural
sources.
DISCUSSION
DECLARATIONS
The management of diabetes mellitus is faced
with the major challenge of finding antidiabetic
agents without side effects. This has led to
increased interest in natural products with antihyperglycemic potential, which have the
advantage of producing much less side effects
when compared with orthodox hypoglycemic
drugs. Alloxan exerts its effect by destroying
insulin-producing beta cells of the pancreas [16].
In vitro studies have revealed that alloxan is
selectively toxic to beta cells of pancreas and
can induce cell necrosis [17]. The cytotoxic effect
of alloxan is mediated by ROS, which increase
cytosolic calcium concentrations, leading to
destruction of beta cells of pancreas [18]. Many
plant extracts exert anti-diabetic effects by
promoting beta cells regeneration or by
protecting these cells from destruction, through
enhancement of unrestricted endogenous insulin
action. Plant extracts may also induce release of
insulin by beta cells, or activate insulin receptors
[19].
Results from the present study showed that oral
administration of G. tricornutum extract (400
mg/kg) for 21 days produced significant
improvements in alloxan-induced diabetes and
glucose tolerance test in rats. These effects are
evident from results obtained in assays of LDL,
HDL, cholesterol, urea, creatinine, ALP, bilirubin,
triglyceride, protein, ALT and AST. Interestingly,
the anti-diabetic effect of the extract was
comparable to that of glibenclamide, a standard
hypoglycemic agent. In addition, the anti-diabetic
effect of the extract was higher at a dose of 400
mg/kg than at the lower dose of 200 mg/kg. Thus
the observed anti-diabetic potential was dosedependent. These results indicate that G.
tricornutum has very good anti-diabetic
properties. It has been established that the
antioxidant properties of plant extracts are due to
the presence of flavonoids and tannins [20].
Preliminary phytochemical screening of the G.
tricornutum extract also revealed the presence of
flavonoids and tannins. Thus these components
are likely to be responsible for the observed antidiabetic properties of the extract.
Acknowledgement
The authors are thankful to the Department of
Animal Sciences, Quaid-i-Azam University,
Islamabad, for providing animals for this study.
We are also thankful to Dr. Sarwat Jahan for her
guidance and support.
Conflict of Interest
No conflict of interest associated with this work.
Contribution of Authors
The authors declare that this work was done by
the authors named in this article and all liabilities
pertaining to claims relating to the content of this
article will be borne by them.
Open Access
This is an Open Access article that uses a funding model which does not charge readers or their
institutions for access and distributed under the
terms of the Creative Commons Attribution
License (http://creativecommons.org/licenses/by/
4.0) and the Budapest Open Access Initiative
(http://www.budapestopenaccessinitiative.org/rea
d), which permit unrestricted use, distribution,
and reproduction in any medium, provided the
original work is properly credited.
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