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Effect of Ethanolic Leaf Extract of Psidium guajava Linn. (Guava) in Alloxan-Induced Diabetic Rats

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British Journal of Pharmaceutical Research
15(4): 1-6, 2017; Article no.BJPR.31987
ISSN: 2231-2919, NLM ID: 101631759
SCIENCEDOMAIN international
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Effect of Ethanolic Leaf Extract of Psidium guajava
Linn. (Guava) in Alloxan-Induced Diabetic Rats
P. C. Ofoha
1*
and R. I. Nimenibo-Uadia
1
1
Department of Biochemistry, Faculty of Life Sciences, University of Benin, Benin City, Nigeria.
Authors’ contributions
This work was carried out in collaboration between both authors. Author RINU designed the study,
wrote the first draft of the manuscript and wrote the protocol. Author PCO managed the literature
searches, managed the analyses of the study and performed the statistical analysis. Both authors
read and approved the final manuscript.
Article Information
DOI: 10.9734/BJPR/2017/31987
Editor(s):
(1) Syed A. A. Rizvi, Department of Pharmaceutical Sciences, College of Pharmacy, Nova Southeastern University, USA.
Reviewers:
(1) Lorna T. Enerva, Polytechnic University of the Philippines, Philippines.
(2)
Armando Cuéllar Cuéllar, Havana University, Cuba.
Complete Peer review History:
http://www.sciencedomain.org/review-history/18575
Received 1
st
February 2017
Accepted 19
th
March 2017
Published 10
th
April 2017
ABSTRACT
Effect of water soluble components of ethanolic leaf extract of Psidium guajava Linn. on blood
glucose and cholesterol levels in alloxan-induced diabetic male albino wistar rats was evaluated. A
total of 30 male albino wistar rats were randomized into 4 groups. Ten (10) animals each were
randomly assigned to groups 1 and 2 and 5 each for groups 3 and 4. Group 1 animals were induced
with 150 mg of alloxan/kg body weight of animal, after which they were administered 400 mg of
extracts/kg body weight of animal. Group 2 animals serving as diabetic control were administered
only 150 mg of alloxan/kg body weight of animal, no extract was given. Group 3 animals received
only 400 mg of extract/kg body weight of animal while Group 4 animals served as normal control
and were given distilled water in place of the extract. Extracts were administered twice daily and
treatments lasted for 10 consecutive days. Blood samples were collected for analysis every two
days from the tail tips of the rats. Results obtained showed that there were significant (p < 0.05)
reductions in blood glucose and cholesterol levels in the diabetic treated rats (Group 1) when
compared with the diabetic control rats (Group 2). However, administration of the extract did not
show any hypoglycaemic effect on the normal rats (p ˃ 0.05) compared with the control. The results
suggest that leaf extract of P. guajava Linn. is antihyperglycaemic and not hypoglycaemic. In view
of the fact that it also showed significant antihypercholesterolaemia, P. guajava Linn. may be a
potential antidiabetic agent.
Original Research Article
Ofoha and Nimenibo-Uadia; BJPR, 15(4): 1-6, 2017; Article no.BJPR.31987
2
Keywords: Psidium guajava; diabetes; glucose; cholesterol.
1. INTRODUCTION
Diabetes mellitus is a metabolic disorder that is
characterized by hyperglycemia and alterations
in carbohydrate, fat and protein metabolisms
associated with absolute or relative deficiencies
in insulin secretion and/ or insulin action [1]. The
causes are not completely understood, but a
more sedentary lifestyle, the consumption of an
energy rich diet, obesity, a longer life span [2],
long-term diabetes, poor glycemic control,
hypertension and dyslipidemia are linked to
diabetes-related vascular complications [3].
Elevated levels of both fasting and post-prandial
blood sugar is the specific characteristic of
diabetes. Auto-immune and non-auto-immune
responses cause destruction of pancreatic β-
cells resulting in type 1 diabetes [4]. There are
circulating immune markers against pancreatic
islets known as anti-islet cell antibodies or β-cell
antigens [5]. That is the reason for patient’s
dependency on external supply of insulin but
those suffering from Type II diabetes known as
non-insulin dependent, cannot properly respond
to insulin. Type II diabetics can be treated by
changes in diet intake, exercise and by use of
medicines. Type II diabetes is the more prevalent
form among the two and constitutes about 90%
of the whole. Symptoms can be same for both
types such as: (i) raised levels of blood glucose;
(ii) increased need to drink water; (iii) repeated
urine output; (iv) increased food intake and
weight loss; (v) blurred vision; (vi) nausea and
vomiting; (vii) fatigue and weakness; (viii)
restlessness and changes of mood etc.
Diabetes is characterized by a high incidence of
cardiovascular disease [6]. The primary goal of
diabetes treatment is the prevention of
macrovascular complications (myocardial
infarction, heart failure, ischemic stroke), as well
as the microvascular complications (retinopathy,
neuropathy, and nephropathy); for that reason,
most patients require not only a good glycemic
control but also treatment for dyslipidemia [7].
Alloxan is administered parenterally:
Intravenously, intraperitoneally or
subcutaneously, while the dose necessary to
induce diabetes is a function of the animal
species, nutritional status and route of
administration. This diabetogenic agent causes
an insulin-dependent diabetes mellitus with
characteristics similar to type I diabetes in
humans [8,9]. It has been employed in
experimental models for the study of diabetes.
Plants are sources of potential therapeutic
agents against various diseases due to their
biodiversity and the presence of a wide array
of bioactive phytochemicals and secondary
metabolites [10]. Several investigations into the
chemical and biological activities of plants have
yielded compounds with properties useful for the
development of modern synthetic drugs for
management of several diseases including
diabetes [11]. In fact, there has been increasing
demand for the use of plant products with
antidiabetic activity due to low cost, easy
accessibility and lesser side effects [12].
Psidium guajava Linn. (Family: Myrtaceae) is an
economically important plant of high medicinal
value [13,14,15]. Psidium guajava is commonly
known as guava. It is used in many parts of the
world for the treatment of a number of
diseases, e.g. as an anti-inflammatory, anti-
ulcer, analgesic, anticough, antimicrobial,
antiplasmodial, antioxidant, anti-diabetes, anti-
hypertensive, and for reducing fever [16]. In this
study, the effect of the water soluble
components of ethanolic leaf extract of
P. guajava on blood glucose and cholesterol
levels in alloxan-induced diabetes in male albino
wistar rats was evaluated.
2. MATERIALS AND METHODS
2.1 Plant Material
Fresh leaves of Psidium guajava Linn. were
harvested from the premises of University of
Benin, Benin City, Nigeria, during the month of
June. The leaves were authenticated at the
Department of Plant Biology and Biotechnology
of the University of Benin, Benin City, Nigeria.
2.2 Extract Preparation
Ethanolic extract of the plant was prepared by
adding 5 litres of absolute ethanol (BDH,
England) to 500 grams of plant powder with
repeated stirring for 3 days. The extract was then
filtered and the recovered filtrate was
concentrated at 60°C using a rotary evaporator
(Buchi Labortechnik, Flawil, Switzerland). The
semi-solid extract was further dried to a constant
weight by evaporation in a water bath at 60°C
and the yield determined. 5 grams of the extract
was dissolved in 100 ml of distilled water to form
the extract stock solution from which estimated
Ofoha and Nimenibo-Uadia; BJPR, 15(4): 1-6, 2017; Article no.BJPR.31987
3
doses were administered. The doses of the
extract administered was estimated using the
method described by Tedong et al., [17].
2.3 Animals
Male albino wistar rats weighing 100–150 g were
used. They were housed and maintained at room
temperature with day/night cycles of 12 h in the
animal house at the Department of Biochemistry,
University of Benin. They were fed with standard
rodent diet and water ad libitum.
2.4 Diabetes Induction
Diabetes was induced by intraperitoneal
administration of freshly prepared alloxan
(Aldrich, Germany) in a single dose of 150 mg/kg
of body weight. Control rats were given distilled
water only. Two days after alloxan
administration, blood glucose levels were
determined using a glucometer (Accu-Check,
Mannheim, Germany) after an overnight fast
(14 h) to confirm diabetes. Rats exhibiting blood
glucose levels 200 mg/dl were considered for
the study.
2.5 Experimental Protocol
Thirty (30) male albino wistar rats were used in
this study. The rats were randomly divided into 4
groups of n animals each (n = number of
animals).
Group 1 (n = 10): Diabetic rats treated with
Psidium guajava extract (400mg/kg body weight)
twice daily by gavage.
Group 2 (n = 10): Diabetic control rats
Group 3 (n = 5): Normal rats treated with
Psidium guajava extract (400 mg/kg body weight)
twice daily by gavage.
Group 4 (n = 5): Normal control rats.
The rats were acclimatized for a period of two (2)
weeks, after which, they were fasted overnight
(14 h) and blood collected from the tail tips of
each animal for the determination of baseline
values for glucose and cholesterol (pre-alloxan),
followed by the induction of diabetes into some of
the animals. After the 48 h adaptation period
following alloxan administration, animals were
fasted overnight (14 h) and blood samples
collected (post-alloxan) and thereafter the extract
was given for 10 consecutive days by oral
administration using a gavage. Blood samples
were collected (post-treatment) at intervals of 2
days.
For cholesterol determination, whole blood was
collected into clean plain bottles and immediately
placed on ice. The sample was allowed to stand
for about 15 min and centrifuged (B. Bran
Scientific, 80 – 2, England) at 10,000 r.p.m. for 5
min. Serum was separated from the clot with
Pasteur pipette into sterile sample tubes for
biochemical analysis.
2.6 Biochemical Analysis
The blood glucose concentration was estimated
using One Touch Glucometer (Accu–Check,
Mannheim, Germany) while cholesterol was
determined spectrophotometrically after enzymic
hydrolysis and oxidation as described by Randox
reagent kit (Randox Laboratory Ltd, UK).
2.7 Statistical Analysis
The results were expressed as the mean ± SEM.
All data were analyzed by the analysis of
variance (ANOVA) followed by Duncan’s multiple
test using SPSS Advanced Statistical Version 21
package with statistical significance level at p
0.05.
3. RESULTS
Table 1 shows the mean values of blood glucose
of controls, normal and diabetic treated rats. The
results revealed a significant (p ˂ 0.05) increase
in fasting blood glucose in the diabetic control
group when compared with the normal control
rats. A significant (p ˂ 0.05) decrease was
observed in the diabetic rats treated with the
ethanolic leaf extract of P. guajava, however no
such decreases were shown in the normal
treated rats (p ˃ 0.05).
Table 2 revealed the mean values of serum
cholesterol of the control, treated and diabetic
rats. The mean values of cholesterol were
significantly (p < 0.05) higher in diabetic control
rats when compared with the normal control rats.
The cholesterol levels in the diabetic rats treated
with the ethanolic leaf extract of P. guajava
decreased significantly (p < 0.05) when
compared with the control. There was no
significant difference in serum cholesterol levels
between normal treated rats with P. guajava
extract and normal control rats (p > 0.05).
Ofoha and Nimenibo-Uadia; BJPR, 15(4): 1-6, 2017; Article no.BJPR.31987
4
Table 1. Effect of water – soluble components of ethanolic leaf extract of P. guajava on blood glucose concentration in diabetic and non-diabetic male rats
Fasting blood glucose concentration (mg/dl)
Group
Pre
-
Post
-
Post
-
treatment
Day 1
Day 3
Day 5
Day 7
Day 9
Day 11
Day 13
Day 15
Day 17
1 n = 10 74.30 ± 7.66
a
460.90 ± 19.82
c
415.59 ± 1.30
b
397.13 ± 7.80
b
377.16 ± 7.30
b
345.15 ± 9.50
b
323.11 ± 1.40
b
297.13 ± 6.10
b
273.13 ± 0.00
b
263.20 ± 3.40
b
169.12 ± 7.60
b
2 n = 10 81.20 ± 9.11
a
401.16 ± 3.60
c
424.73 ± 7.70
c
388.14 ± 6.80
b
409.14 ± 4.50
c
395.12 ± 5.50
c
394.16 ± 3.00
c
424.20 ± 2.20
c
430.36 ± 3.00
c
422.30 ± 4.00
c
417.25 ± 6.60
c
3 n = 5 83.40 ± 5.54
a
96.10 ± 8.18
a
91.40 ± 2.49
a
83.30 ± 7.87
a
67.30 ± 9.72
a
84.30 ± 5.77
a
79.30 ± 5.70
a
75.30 ± 5.77
a
75.20 ± 5.37
a
65.20 ± 4.37
a
75.50 ± 3.60
a
4 n = 5 76.00 ± 11.42
a
82.33 ± 7.42
a
93.40 ± 2.49
a
81.40 ± 0.71
a
75.30 ± 6.69
a
73.20 ± 3.85
a
71.20 ± 3.30
a
86.20 ± 3.00
a
86.30 ± 0.00
a
76.30 ± 7.80
a
66.80 ± 7.90
a
Values are expressed as mean ± SEM; n = number of rats. Values with different superscripts in the same column are significantly different (p 0.05)
Table 2. Effect of water – soluble components of ethanolic leaf extract of P. guajava on serum cholesterol concentration in diabetic and non-diabetic male rats
Serum cholesterol concentration (mg/dl)
Group
Pre
-
Post
-
Post
-
treatment
Day 1
Day 3
Day 5
Day 7
Day 9
Day 11
Day 13
Day 15
Day 17
1 n = 10 132.40 ± 7.66
a
224.70 ± 8.30
b
216.70 ± 3.00
b
211.50 ± 0.80
b
208.10 ± 1.90
b
199.70± 7.17
b
190.40 ± 5.70
b
178.70 ± 8.00
b
164.80 ± 6.21
b
147.50± 5.70
a
133.00 ± 1.54
a
2 n = 10 121.50 ± 9.11
a
260.50 ± 5.01
c
262.80 ± 4.40
c
254.14 ± 4.68
c
250.10 ± 4.45
c
265.10 ± 5.55
c
290.10 ± 6.20
c
224.20 ± 6.20
c
250.30 ± 6.30
c
264.30 ± 0.04
c
262.30 ± 0.12
c
3 n = 5 148.80 ± 5.54
a
140.10 ± 6.14
a
144.10 ± 4.00
a
139.30 ± 7.87
a
151.30 ± 9.72
a
147.30 ± 5.77
a
140.80 ± 5.22
a
156.30 ± 5.77
a
147.00 ± 6.34
a
149.20 ± 2.56
a
146.90 ± 1.97
a
4 n = 5 142.20± 14.50
a
146.80 ± 0.58
a
146.60 ± 7.11
a
147.90 ± 7.30
a
140.30 ± 5.63
a
142.80 ± 5.85
a
131.30 ± 3.55
a
145.30 ± 0.00
a
139.20 ± 3.50
a
140.30± 7.80
a
137.40 ± 1.75
a
Values are expressed as mean ± SEM; n = number of rats. Values with different superscripts in the same column are significantly different (p 0.05)
Ofoha and Nimenibo-Uadia; BJPR, 15(4): 1-6, 2017; Article no.BJPR.31987
5
4. DISCUSSION
Scientific studies have revealed the potentials of
plants as sources of novel therapeutic agents.
The results of this study clearly indicated that the
administration of ethanolic leaf extract of
P. guajava produced antihyperglycaemic and
antihyperlipidemic effects against alloxan–
induced diabetes in rats. As shown in Table 1,
the administration of Psidium guajava
significantly reduced the blood glucose levels in
diabetic rats when compared with the untreated
diabetic rats. This suggests that the plant has
antihyperglycaemic activities. The blood glucose
was significantly increased in the diabetic control
rats but the intervention with the extract tended
to ameliorate the effect. A number of agents
apart from insulin have been implicated in the
control of hyperglycaemia, such as alkaloids and
tannins [18]. During diabetes, the levels of serum
lipids (cholesterol, free fatty acids and
phospholipids) are usually elevated. The results
in Table 2 indicate significant increases in
cholesterol levels in the diabetic rats.
These accumulate in blood to constitute
hypercholesterolaemia of diabetes as evident in
this study. The study showed that oral treatment
with 400 mg/kg body weight of the ethanolic leaf
extract of P. guajava exerted a progressive
reduction on elevated levels of blood glucose
and cholesterol. The presence of phytochemicals
in the extract with reported antidiabetic activities
may be responsible for the observed reductions
in blood glucose and cholesterol levels in the
diabetic rats treated with P. guajava [10].
5. CONCLUSION
Based on our results, administration of the water
– soluble components of ethanolic leaf extract of
P. guajava lowered the blood sugar and
cholesterol levels in the alloxan-induced diabetic
rats. The ethanolic leaf extract showed significant
(p < 0.05) antihyperglycaemic activity in alloxan–
induced hyperglycaemia without causing
hypoglycaemia, showing that an over dose of the
extract may not cause hypoglycaemia.
CONSENT
It is not applicable.
ETHICAL APPROVAL
All authors hereby declare that all experiments
have been examined and approved by the
appropriate ethics committee and have therefore
been performed in accordance with the ethical
guidelines and approval of the University of
Benin, Benin - City, Nigeria.
ACKNOWLEDGEMENT
The authors are grateful to the University of
Benin, Benin City, Nigeria for providing the
facilities for the study and also to Mr Akpeh P.K
for assisting in the laboratory analysis.
COMPETING INTERESTS
Authors have declared that no competing
interests exist.
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© 2017 Ofoha and Nimenibo-Uadia; This is an Open Access article distributed under the terms of the Creative Commons
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Peer-review history:
The peer review history for this paper can be accessed here:
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... Therefore, treating diabetes with plants high in antioxidants as well as diets supplemented with eatable plants may be a creative way to lower complications brought on by diabetes's characteristically abnormal production of free radicals [22,23]. Although there is paucity of information regarding the combination of the leaf extracts of P. guajava and D. edulis as possible antidiabetic agent, but there are some evidences that the plants P. guajava [24,25] and D. edulis [26,27] can prevent pancreatic beta cell apoptosis and preserve function. P. guajava leaf extracts were subjected to phytochemical screening, which revealed the presence of bioactive components including tannins, saponins, steroids, flavonoids, alkaloids, and anthraquinones [25,28], whereas D. edulis leaf extracts revealed the presence of bioactive components including alkaloids, phenols, flavonoids, triterpenoids, tannins, and saponin [29]. ...
... Hence, the combined plants' ethanolic leaf extract may have antihyperglycemic properties and work synergistically in inhibiting diabetic effects in Wistar rats. This is consistent with the findings of Ofoha and Nimenibo-Uadia [24], Ononamadu et al. [27], Parker et al. [25] and Akoko et al. [31]. Blood glucose levels were significantly higher in diabetic control rats, but group D (treated with 10% extract combination), E (treated with 15% extract combination) and F (treated with 20% extract combination) experimental animals' blood glucose levels were significantly reduced as a result of the intervention with the combined ethanolic leaf extract. ...
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