Journal of Ethnopharmacology 151 (2014) 784790

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jep

Research paper

Antidiabetic and antioxidant effects of Annona muricata (Annonaceae),

aqueous extract on streptozotocin-induced diabetic rats
Ngueguim Tsofack Florence a, Massa Zibi Benoit a, Kouamouo Jonas b,
Tchuidjang Alexandra b, Dzeuet Djomeni Paul Dsir a, Kamtchouing Pierre a,
Dimo Thophile a,b,n
a
b

Department of Animal Biology and Physiology, Laboratory of Animal Physiology, University of Yaounde I, PO Box 812, Cameroon
Department of Pharmacy, Universit des Montagnes (UdM), PO Box 208, Bangangt, Cameroon

art ic l e i nf o

a b s t r a c t

Article history:
Received 15 May 2013
Received in revised form
16 September 2013
Accepted 17 September 2013
Available online 25 September 2013

Ethnopharmacological relevance: The leaves of Annona muricata are used in Cameroon to manage diabetes
and its complications. The aim of this study was to evaluate the antidiabetic, antioxidant activities and the
potential toxicity of aqueous extract of Annona muricata in streptozotocin-induced diabetic rats.
Material and methods: Oral administration of Annona muricata aqueous extract (100 mg/kg or 200 mg/kg) was
studied in normal and streptozotocin-induced diabetic rats. In long term treatment, 2 weeks after
streptozotocin-induced diabetic rats, animals received plant extract during 28 consecutive days. For a protective
effect, extract was administered 3 days prior to streptozotocin exposure and animals were observed 2 weeks
without treatment.
Results: The plant extract was not effective in normal rats. In diabetic rats, single administration of the extract
signicantly reduced blood glucose levels by 75% and 58.22% respectively at the dose of 100 mg/kg and
200 mg/kg as compared to the initial value. Treatment of normal rats 3 days prior to diabetes induction showed
that, Annona muricata extract has no effect within 72 h following STZ injection. However, after 14 days posttreatment, the extract at the dose of 100 mg/kg signicantly reduced blood glucose levels as compared with
initial value and diabetic control rats. Immunohistochemical staining of pancreatic -cells of diabetic rats
treated with the dose of 100 mg/kg expressed strong staining for -cell compared to diabetic control. In a longterm study daily administration of Annona muricata aqueous extract for 28 days to diabetic rats, reduced blood
glucose levels, serum creatinine, MDA, AST, ALT activity, and nitrite levels LDL-cholesterol. Total cholesterol,
triglycerides, SOD, and CAT activity contents were restored.
Conclusion: These different results show that the antidiabetic activity of Annona muricata aqueous extract can
be explained by its hypolipidaemic effect, its antioxidant and protective action on pancreatic -cells, which in
turn improve glucose metabolism.
& 2013 Published by Elsevier Ireland Ltd.

Keywords:
Streptozotocin
Annona muricata
Antidiabetic
Hypolipidemic
Antioxidant

1. Introduction
The increasing worldwide incidence of diabetes mellitus constitutes a global public health problem. It is predicted that by 2025
the world will have an increase of 72% of diabetic patients
(Lefbvre, 2005). Despite the development of new drug and their
validation by scientic criteria, the research still continues in
scientic community around the world to evaluate antidiabetic
activities of raw material or isolated natural product without
adverse affects.
n
Corresponding author at: Department of Animal Biology and Physiology,
Laboratory of Animal Physiology, University of Yaounde I, PO Box 812, Cameroon.
Tel.: 237 765 7442.
E-mail address: tdimo@uycdc.uninet.cm (D. Thophile).

0378-8741/$ - see front matter & 2013 Published by Elsevier Ireland Ltd.
http://dx.doi.org/10.1016/j.jep.2013.09.021

Annona muricata belongs to the annonaceae family and is

commonly known as Sour-sop (Adjanohoun et al., 1996). It is used
for hypertension, diabetes, stomach pain, fever, and against worms
and vomiting (Adjanohoun et al., 1996). With regard to the literature,
several pharmacological studies have shown that, Annona muricata
possess antihypertensive, vasodilatator, antispasmodic, cardiodepressive (Feng, 1962), antiplasmodial, anti-mutagen, anti-convulsivant
(N'gouemo, 1997), antiviral (Padma et al., 1998), anti-bactrienne
(Takahaski et al., 2006), antidiabetic, hypolipidmique and antioxydante properties (Adewole and Caxton-Martins, 2006; Adewole and
Ojewole, 2009; Adeyemi et al., 2009). Moreover, phytochemical
investigations revealed the presence of alkaloids, tannins, coumarins,
avonoids, terpenoids, stearic acid, myristique acid, ellagic acid, (Watt
and Breyer-Brandwijk, 1962; TDRG, 2002). Despite these multitude
studies in Annona muricata plant, its toxicological studies have not

N.T. Florence et al. / Journal of Ethnopharmacology 151 (2014) 784790

been shown. In the present study we examined the toxicity of Annona

muricata aqueous extract, the antidiabetic and antioxidant effects of its
chronic administration in streptozotocin-induced diabetic rat with
complications.

2. Material and methods

2.1. Animals
Three months old male albinos Wistar rats, weighting between
150 and 250 g were used. Animals were raised in the Animal
House of the Faculty of Science, University of Yaounde I. They were
maintained in a temperature room (22 72 1C) on a 12 h light-dark
natural cycle. Rats were fed with standard diet and water ad
libitum. These studies were conducted with the approval of the
Cameroon National Ethical Committee (Ref no. FW-IRB00001954).
2.2. Plant material
Annona muricata leaves were collected at Mvog-Mbetsi quarter in
Yaounde (Cameroon). The plant was authenticated at the National
Herbarium, Yaounde, where voucher specimen no. 32879 was deposited. The sample was dried in the shade and crushed to ne powder.
The aqueous extract was prepared by soaking 1 kg of powder in 3 L of
distilled water for 48 h following the traditional method. The ltrate
was evaporated in an oven at 40 1C, giving 50 g of a dried powder
(yield 5%).
2.3. Acute oral toxicity study
The acute oral toxicity study was conducted using test guidelines on acute oral toxicity test 423 according to OCDE (2001).
Eighteen Wistar rats fasted overnight, but allowed free access to
water ad libitum were randomly assigned into the following three
groups of six rats of either sex (three males and three females).
Control received distilled water and two groups received the
extract at the doses of 2000 mg/kg and 5000 mg/kg. The rats were
not fed for 3 h following administration. The signs of toxic
effects and/or mortality were observed 3 h after administration
then, for next 48 h. The body weight was recorded for consecutive
14 days.
2.4. Sub-chronic toxicity study
Subacute toxicity was evaluated using the OECD (2008) test
guidelines 407. The animals were randomly divided into four groups
containing 10 rats each (ve females and ve males). Annona muricata
aqueous extract was administered to groups of rats at the doses of
200, 400 and 800 mg /kg daily by gavage for 4 weeks. The control
group received distilled water (10 ml/kg). The weight of each rat was
recorded at weekly intervals throughout the course of the study. At the
end of 4 weeks experiment, animals were sacriced under anaesthesia. Blood was collected into two tubes: tube 1 containing EDTA was
processed immediately for haematological parameters and tube
2 without additive was centrifuged at 3000g at 4 1C for 10 min to
obtain serum (stored at  20 1C until analysis).
2.5. Protective effect of aqueous extract from Annona muricata
against streptozotocin
Normoglycemic rats were randomly divided into four groups of
eight rats each: two groups received vehicle (distilled water) as
control; two other groups received the plant extract at 100 mg/kg/
day and 200 mg/kg/day during 3 days. Beforehand, animals were
treated with plant extract treated during 3 days then;

785

streptozotocin at the dose of 55 mg/kg was injected by intravenous route. Blood glucose levels were determined 72 h later using
glucose oxidase method (Dimo et al., 2006; Ngueguim et al., 2007)
then, animals were observed without treatment for 2 weeks. The
weight of each rat and the mortality were recorded throughout the
course of the study. At the end of experiment, rats were sacriced
by decapitation under anaesthesia. Pancreas samples were harvested and xed in formalin (10%) for immunohistochemistry
analysis using the avidinbiotin peroxidase complex method.
2.6. Immunochemistry
Sections of 4 m were mounted on charged slides (Superfrost
Plus) for immunochemistry. Parafn was removed from samples.
Endogen peroxidases were blocked by exposure to 0.5% hydrogen
peroxide. Then, the sections were washed with PBS and incubated
overnight with primary insulin-antibody. Biotinylated secondary
antibody was applied for 10 min. The sections were washed with
PBS again and incubated with Avidine/Biotine/Peroxydase complexe kit (ABC Kitt Vector) for 10 min. The sections were incubated
for revelation with diaminobenzidine (DAB). A counter staining
with haematoxylin-eosin was performed.
2.7. Induction of experimental diabetes in rats
Diabetes mellitus was induced by intravenous (penile vein)
injection of 55 mg/kg of streptozotocin in 0.9% sterile sodium
chloride solution to non-fasted rats anaesthetised with ether.
Control group received normal saline through the same route.
Three days later, blood glucose levels of streptozotocin treated
fasted rats greater than 200 mg/dL were considered as diabetic
and used in this study.
2.8. Single oral administration of Annona muricata extract
Normoglycemic rats were assigned to four different groups of
ve rats each. Group I made up of control rats treated with 1 mL/
kg of distilled water. Group II containing rats which received
glibenclamide (5 mg/kg). Group III and Group IV were given the
plant extract at the dose of 100 and 200 mg/kg respectively. Blood
samples were collected from the tail vein at intervals 0, 1, 2, 3 and
5 h. Glucose content in each sample was assessed using the
glucose oxidase method (Accuchek glucometer, Boehringer Mannheim, Germany) (Dimo et al., 2006; Ngueguim et al., 2007).
The hypoglycaemic effect of Annona muricata extract was also
evaluated in diabetic rats. One group of ve normal rats was
treated with distilled water (used in previous experiment). Two
groups of diabetic rats received either distilled water or glibenclamide (5 mg/kg) while two other groups were administered a
single dose of extract at 100 and 200 mg/kg. Blood sample
collection and analysis were done as previously mentioned.
2.9. Sub-chronic effects of aqueous extract of Annona muricata
In this study we examine the effects of long term administration of the plant extract on streptozotocin-induced diabetic rats.
Rats were kept for 14 days before the beginning of the treatment
to stabilise the diabetic conditions and to allow a permanent and
chronic hyperglycaemia (Jyoti et al., 2002). The animals were
randomly divided into four groups containing ve rats each:
normal control and diabetic control rats treated with distilled
water, one group of diabetic rats treated with insulin at the dose of
10 UI/kg, two groups of diabetic rats treated with aqueous extract
from Annona muricata at the doses of 100 mg/kg and 200 mg/kg.
The animal received treatment for 28 consecutive days by gastric
intubation. Glycaemia was determined weekly. At the end of the

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N.T. Florence et al. / Journal of Ethnopharmacology 151 (2014) 784790

experiment, rats were sacriced by decapitation under anaesthesia. Blood was collected and centrifuged at 3000g at 4 1C for
10 min to obtain serum (stored at  20 1C until analysis) for
biochemical analysis (creatinine, ALT, AST, triglycerides, total
chlolesterol, HDL chlolesterol, LDL chlolesterol and atherogen
index determined by the ratio of total cholesterol/HDL cholesterol)
(Youmbissi et al., 2001). Lipids, creatinine, hepatic enzymes levels
were quantied spectrophotometrically according to the commercial instructions for the kits.

was observed respectively at the doses of 100 mg/kg and 200 mg/kg
compared to the diabetic control. Glibenclamide at the dose of 5 mg/
kg induced a reduction of 21.77% 5 h post-dosing as compared to the
initial value.

5. Protective effect of aqueous extract of Annona muricata

against streptozotocin
5.1. Blood glucose levels

2.10. Statistical analysis

Data are expressed as the mean 7standard error mean. Statistical signicance was determined by one way analysis of variance
(ANOVA) followed by the Turkey test as the post-test. Po0.05
indicates signicant difference between group means.

3. Results
3.1. Acute toxicity
A single dose of 2000 mg/kg or 5000 mg/kg did not indicate
modication of behaviour. No mortality was recorded during the
study. After sacrice on the 14th day, macroscopic pathology observations, revealed no visible lesions in any animals. The oral LD50 value of
Annona muricata aqueous extract must be greater than 5000 mg/kg.

Table 2 shows the protective effect of Annona muricata extract

against streptozotocin. Intravenous injection of streptozotocin to
normal animal induced a signicant increase in blood glucose
levels of 72.76% as compared to normal rats. Throughout the
experimental period, the hyperglycaemia increase considerably.
Animals pre-treated with Annona muricata aqueous extract before
the injection of streptozotocin have shown increased blood glucose levels of 70.90% and 70.80% respectively at the doses of 100
and 200 mg/kg. However, 14 days after streptozotocin injection,
the plant extract at the dose of 100 mg/kg induced a reduction of
31.77% and 45.77% respectively compared to the initial value and
to the diabetic control. At the dose of 200 mg/kg the plant extract
did not show a signicant reduction on blood glucose levels. At the
end of treatment, 0% and 25% of animal died respectively at the
doses of 100 and 200 mg/kg vs. 50% in diabetic control group.
5.2. Immunohistochemistry

3.2. Subacute toxicity

No toxicity sign or death was recorded after daily administration of aqueous extract of Annona muricata during 4 weeks. In
addition there was no signicant difference between the initial
and nal body weight of treated animals vs. control groups at the
end of treatment.

4. Single oral dose of aqueous extract of Annona muricata

on blood glucose levels in normal and diabetic rats.
A single oral administration of Annona muricata aqueous
extract at the doses of 100 mg/kg and 200 mg/kg did not affect
signicantly blood glucose levels in normal rat (data not shown).
Blood glucose levels of diabetic control rat signicantly increased
throughout the experimental period compared to the normal control
rat (Table 1). Single administrations of the plant extract at all doses
exhibit a signicant reduction on blood glucose level, 2 h post-dosing.
At the end of experiment, a signicant decrease of 75.00% and 58.22%

Immunohistostaining of different pancreas of each group was

examined (Fig. 1). The staining for -cell for diabetic control group
Table 2
Effect of aqueous extract of Annona muricata against streptozotocin-induced
diabetic rats. Plant extract was given 3 days prior the injection of streptozotocin.
Treatment

Blood glucose (mg/dL)

Day 4

Mortality
percentage (%)

Day 15

Normal control
93.007 3.86
93.40 7 1.40
0
Diabetic control
261.40 7 17.48### 308.25 7 16.55###
50
Extract 100 mg/kg 245.007 12.03### 167.177 10.36##nn$$
0
Extract 200 mg /kg 244.007 15.94### 270.127 18.31###
25
Values are expressed as mean 7 S.E.M, 4Z n r 8.
###

P o0.001 compared to normal control.

Po 0.01 compared to normal control.
nn
P o 0.01 compared to the diabetic control.
$$
Po 0.01 compared to the initial value.
##

Table 1
Effect of a single oral administration of Annona muricata on blood glucose levels of diabetic rats.
Treatment

Normal control
Diabetic control
Glibenclamide (5 mg/kg)
Extract (100 mg/kg)
Extract (200 mg/kg)

Blood glucose levels (mg/dL)

0h

1h

2h

3h

5h

93.007 3.86
220.40 7 7.21
218.337 10.60
217.80 7 7.55
217.007 5.77

95.40 7 1.63
226.20 7 9.66
193.40 7 2.42
187.40 7 11.99
213.40 7 5.11

100.60 7 2.48
228.60 7 5.19###
190.80 7 5.38nn###
149.207 7.37nnn###
195.40 7 5.30nn###

102.20 7 0.66
306.607 6.21###
175.20 7 7.07nnn###
134.20 7 0.73nnn##
185.50 7 5.95nnn###

93.40 7 1.40
313.007 3.42###
170.807 2.87nnn###
88.40 7 8.23nnn
130.75 7 3.57nnn###

Data are expressed as mean 7 S.E.M, n 5.

nn

Po 0.01, as compared with the diabetic control group.

Po 0.001 as compared with the diabetic control group.
##
P o0.01, as compared with normal control group.
###
Po 0.001 as compared with normal control group.
nnn

N.T. Florence et al. / Journal of Ethnopharmacology 151 (2014) 784790

Normal control

Diabetic control

Extract 100 mg/kg

787

Extract 200 mg/kg

Fig. 1. Immunohistochemistry staining of -cells of diabetic rat treated with plant extract after injection of streptozotocin. Animals were administered plant extract 3 days
prior to streptozotocin injection. Rats were then sacriced and the pancreatic tissues were isolated for histology and immunohistochemistry.

Table 3
Body weight, water and food intake in STZ-diabetic rats after oral treatment with Annona muricata once a day for 4 weeks.
Treatment

Normal control
Diabetic control
Insulin (10 UI/kg)
Ext (100 mg/kg)
Ext (200 mg/kg)

Body weight (g)

Food intake (g/rat/day)

Water intake (mL/rat/day)

Initial

Final

Initial

Final

Initial

Final

225.80 7 14.01
195.7077.06
190.50 713.09
198.40 75.45
186.20 78.32

226.337 3.48
175.60 7 6.16###$$$
231.007 4.77nnn
240.25 7 4.85nnn
220.75 7 4.62nnn

25.007 1.58
23.22 7 3.55
28.007 5.07
26.54 7 2.25
24.427 7.70

25.20 7 1.05
50.40 7 2.67###$$$
30.20 7 0.71nnn
27.50 7 1.41nnn
34.60 7 2.28nnn

19.167 0.60
21.55 7 2.82
22.23 7 4.35
20.717 9.21
23.127 1.07

20.00 71.41
40.80 74.30###$$$
24.2070.96nnn
18.50 71.12nnn
25.60 71.03nnn

Data are expressed as mean7 S.E.M., n 5.

nnn

###
$$$

Po 0.001 compared to diabetic control.

Po 0.001 compared to normal control.
Po 0.001 compared to initial value.

Table 4
Effect of repeated administration of aqueous extract of Annona muricata in diabetic rats with a chronic hyperglycaemia.
Treatment

Normal control
Diabetic control
Insulin (10 UI/kg)
Extract 100 mg/kg
Extract 200 mg/kg

Blood glucose (mg/dL)

Day0

Day7

Day14

Day21

Day28

91.20 7 4.21
344.20 7 8.22
308.25 7 3.57
344.60 7 9.78
338.807 12.47

94.80 7 5.03
316.20 7 10.28###
220.75 7 8.72###
79.80 7 4.34nnn
106.757 5.12nnn

92.007 4.02
318.007 11.02###
211.007 11.79###
118.20 7 11.70nnn
112.25 7 7.21nnn

93.007 2.08
329.60 7 11.84###
187.337 8.37###
90.20 7 5.37nnn
131.007 5.96nnn

93.337 3.76
367.40 7 12.28###
81.007 9.00
80.75 7 5.68nnn
141.25 7 10.25##nnn

Data are expressed as mean7 S.E.M., n 5.

nnn

Po 0.001 compared to diabetic control.

Po 0.001 compared to normal control.
P o0.01 compared to normal control.

###
##

was less intense than that of control normal rats. However, animals
treated with plant extract at the doses of 100 and 200 mg/kg have
shown intense staining compared to diabetic control rat. Interestingly,
animal treated with the dose of 100 mg/kg expressed strong staining
for -cell compared to diabetic control.

6. Effect of repeated dose of aqueous extract of Annona

muricata

diabetic control. Parallel, food intake was increased signicantly in

diabetic rats by 50% compared to normal control rats. Treatment with
plant extract at the doses of 100 and 200 mg/kg signicantly reduced
food intake respectively by 45.43% and 31.34% compared to diabetic
control. On the other hand, uid intake elevated in diabetic control
rats (50.98%) was signicantly reduced by 54% and 37% respectively at
the doses of 100 and 200 mg/kg compared to diabetic control rats.
Administration of insulin 10 UI/kg induced a signicant decrease of
40% and 40.68% respectively in food and water intake compared to
diabetic control rats.

6.1. Body weight, food and uid intake

6.2. Effect on blood glucose levels
Table 3 shows the effect of repeated administration of Annona
muricata aqueous extract on body weight, food and uid intakes of
diabetic and treated rats. At the end of treatment, diabetic control rats
showed a considerably reduction of body weight by 21.73% and 22.41%
compared to the initial value and the normal control. When administered Annona muricata aqueous extract during 4 weeks at the dose of
100 mg/kg, there was a signicant weight gain as compared to

As shown in Table 4, intravenous injection of streptozotocin

induced a signicant increase in blood glucose levels throughout
the experimental period. Daily administration of Annona muricata
aqueous extract at all doses results in a signicant (Po0.001)
decrease of blood glucose levels near normal values on day 7. The
reduction was 76.56% and 58.31% respectively at the doses of 100 and

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N.T. Florence et al. / Journal of Ethnopharmacology 151 (2014) 784790

200 mg/kg compared to the initial value. After 28 days of treatment

with plant extract, the decrease in blood glucose levels was 73.72%
and 77.95%. At the end of treatment, animals treated with insulin
(10 UI/kg) have shown a decrease of 73.72% and 77.95% respectively
compared to their initial value and to the diabetic control.
6.3. Effect on serum lipid prole, renal and hepatic parameters
Table 5 shows serum lipid prole in diabetic rats with chronic
hyperglycaemia, as compared to the normal control rats, diabetic

control rats have shown a signicant increase in triglycerides

(Po0.001; 56.03%), total cholesterol (Po0.001; 31.95%), LDL-cholesterol (Po0.001; 70.71%), and atherogen index (Po0.001, 59.25%)
with a signicant decrease in HDL-cholesterol (Po0.001; 40.11%).
Daily administration of Annona muricata aqueous extract at the
dose of 100 mg/kg signicantly decreased (Po0.001) triglycerides
(52.95%), total cholesterol (Po0.001; 31.51%), LDL-cholesterol
(81.22%) and atherogen index (65.15%) compared to the diabetic
control. The plant extract at the dose of 200 mg/kg, decreased (Po
0.001) triglycerides (43.97%), total cholesterol (Po0.001; 21.35%),

Table 5
Effect of Annona muricata aqueous extract on lipid prole, renal and hepatic functions of streptozotocin-induced diabetic rats.
Treatment
Parameters
Total cholesterol (mg/dL)
Triglycerides (mg/dL)
LDL-cholesterol (mg/dL)
HDL-cholesterol (mg/dL)
Atherogen index
Creatinine (mg/mL)
AST (U/L)
ALT (U/L)

Normal control
65.337 1.70
37.20 7 1.41
11.127 2.53
42.917 4.77
1.52 7 0.02
2.96 7 0.28
13.007 1.64
27.067 0.70

Diabetic control
###

96.007 2.00
84.60 7 1.41###
37.977 5.54##
25.707 1.70##
3.737 0.03###
4.007 0.13##
28.127 3.07##
53.55 7 6.20##

Insulin (10 UI/kg)

nnn###

79.677 1.41
60.80 7 1.70nnn###
17.917 1.65n
40.007 2.25nn
1.99 7 0.06nnn#
3.067 0.06n
18.677 1.33
37.40 7 3.60

Extract (100 mg/kg)

nnn

65.75 7 1.70
39.80 7 1.41nnn
7.137 1.8nnn
50.517 2.01nnn
1.30 7 0.05nnn
2.28 7 0.12nnn
10.50 7 1.06nn
22.86 7 0.87nn

Extract (200 mg/kg)

75.50 7 1.14nnn##
47.40 7 1.14nnn###
13.40 7 2.73nn
47.767 4.49nn
1.58 7 0.04nnn
2.65 7 0.18nnn
14.217 2.12n
34.50 7 2.10n

Data are expressed as mean7 S.E.M., n 5.

n

Po 0.05, compared to diabetic control.

Po 0.01, compared to diabetic control.
nnn
Po 0.001 compared to diabetic control.
#
Po 0.05, compared to normal control.
##
P o 0.01, compared to normal control.
###
Po 0.001, compared to normal control.
nn

Fig. 2. Effect of aqueous extract of Annona muricata on tissue superoxide dismutase activity (A), tissue catalase (B), tissue malondialdehyde levels (C) and tissue nitrites (D) in
diabetic rat with chronic hyperglycaemia. nPo 0.05, nnPo 0.01, and nnnP o 0.001: compared to diabetic control. #Po 0.05, ##P o 0.01, and ###Po 0.001: compared to normal
control. Animal were diagnose diabetic after injection of STZ. The treatment with plant extract or insulin started two weeks later (Once daily during 28 days). Data
represented in each panel were done in triplicate.

N.T. Florence et al. / Journal of Ethnopharmacology 151 (2014) 784790

LDL-cholesterol (64.70%) and atherogen index (57.64%) compared to

the diabetic control. HDL-cholesterol was signicantly increased by
49.12% and 46.65% respectively at the doses of 100 and 200 mg/kg
compared to diabetic control group.
Creatinine, AST and ALT levels were signicantly increased in
diabetic control group compared to the normal control group. In
4 weeks treatment, using the plant extract at the dose of 100 mg/kg,
there was a signicant reduction of creatinine, AST and ALT levels
by 43%, 62.66% and 57.31% respectively as compared to the diabetic
control. The plant extract at the dose of 200 mg/kg signicantly
reduced creatinine, AST and ALT levels by 33.75%, 49.46 and 35.75%
respectively as compared to the diabetic control. Insulin exhibited a
signicant reduction of about 23.5% of creatinine level but did not
signicantly reduced AST and ALT levels.
6.4. Effect on antioxidant parameters
At the end of treatment, SOD, catalase, malondialdehyde and
nitrites were analysed. In comparison to the normal control,
diabetic control group has shown a signicant (P o0.001) decrease
of SOD by 58.33% in the liver, 48.57% in the kidney and 47.82% in
the aorta (Fig. 2A). Treatment with the plant extract exhibited a
signicant increase in SOD at all doses. Annona muricata aqueous
extract at the dose of 100 mg/kg induced a signicant (Po0.001)
reduction of SOD of 60.78%, 62.50% and 65.22% respectively in the
liver, kidney and aorta. At the dose of 200 mg/kg, the decrease was
54.54% in the liver, 53.84% in the kidney and 57.14% in the aorta.
Diabetic control rats were also characterised by a decrease in
catalase by 61.85%, 41.35% and 51.75% respectively in the liver,
kidney and aorta (Fig. 2B). Administration of the plant extract at
the dose of 100 mg/kg induced a signicant (Po 0.001) increase of
catalase in the liver, kidney and aorta respectively by 66.23%,
49.92% and 58.96% as compared to the diabetic control. The
increase was 58.50% (P o0.05), 36.05% (P o0.001) and 51.11%
(P o0.001) in the aorta when the dose of 200 mg/kg was administered. Lipid peroxidation in diabetic control group signicantly
(P o0.001) increased in the liver and kidney respectively by
68.52% and 58.69% (Fig. 2C). Treatment with plant extract at the
dose of 100 mg/kg exhibited a signicant decrease in the extent of
lipid peroxidation of 79.19% and 76.76% respectively in liver and
kidney. At the dose of 200 mg/kg, the decrease was 43.63% in the
liver and 49.64% in the kidney compared to the diabetic control
group. On the other hand, the nitrites were signicantly elevated
in diabetic control rats by 41.38%, 45.45% and 44.44% respectively
in the liver, kidney and aorta compared to the normal rats
(Fig. 2D). In comparison to the diabetic control rats, the plant
extract administered at the dose of 100 mg/kg signicantly
reduced nitrites by 46.13%, 55.81% and 51.52% respectively in the
liver, kidney and aorta. At the dose of 200 mg/kg, the decrease was
36.20%, 45.45% and 44.44% respectively in the liver, kidney and
aorta as compared to the diabetic control rats.

7. Discussion
This study was conducted to evaluate the acute and subacute
toxicity of the aqueous extract of Annona muricata and its effect on
streptozotocin-induced diabetic rats. In light of the present results,
our studies indicate that LD50 value of Annona muricata aqueous
extract must be greater than 5000 mg/kg and there was no sign of
toxicity after sub-chronic administration at the doses used. Single
administration of the plant extract to the normal rat did not affect
their blood glucose levels 5 h post-dosing. However, in the similar
conditions, Annona muricata aqueous extract induced a signicant
decrease in streptozotocin-induced diabetic rats, which reaches
normal value at the dose of 100 mg/kg. It is known that

789

streptozotocin acts by destructing -cells (Szkudelski and

Szkudeska, 2002); thus the plant extract could act in this condition
by enhancing peripheral glucose uptake. Similar ndings were
revealed by Pepato et al. (2002) and Dimo et al. (2006) who
reported that, Pterocarpus santalinus and Trema orientalis had no
effect on blood glucose levels of normoglycaemic rats. Two hours
after administration of glibenclamide (5 mg/kg) there was a
signicant reduction in blood glucose levels in normal rats. In
streptozotocin-induced diabetic rats, the reduction was moderated
but signicant. It was reported that, glibenclamide is ineffective
when -cells are destroyed (Hosseinzadeh et al., 2002). In our
study, glibenclamide induced a signicant reduction of blood
glucose levels in streptozotocin-induced diabetic rats suggesting
a partial destruction of pancreas -cells. In normoglycaemic rats,
sulfonylurea agents have been found to induce hypoglycaemia by
their ability to stimulate -pancreatic cells to liberate insulin
(Pepato et al., 2002). These results suggest that the action
mechanism of Annona muricata aqueous extract may be different
from that of sulfonylurea. Daily administration of plant extract
during 3 days, prior to streptozotocin injection, did not inhibit
streptozotocin-induced hyperglycaemia. However animals without treatment during 14 days have shown a signicant reduction
of blood glucose levels implying that the plant extract may act in
long-term. This hypothesis is strengthened by immunohistochemical studies. Immunohistostaining of diabetic animal treated with
plant extract expressed strong staining for -cell attesting the
inhibitory effect of the plant extract against streptozotocin. In
addition, it is known that, Annona muricata possesses antioxidant
compounds (Adewole and Caxton-Martins, 2006; Adewole and
Ojewole, 2009) which have the ability to prevent deleterious effect
of streptozotocin. Assuming that, the decomposition of streptozotocin releases reactive oxygen species (ROS), which act on cellular
membrane, DNA chain and cause cell death. Annona muricata
aqueous extract could act by tripping ROS.
In chronic treatment our results indicate that, daily administration during 4 weeks of Annona muricata aqueous extract
reduced body weight loss, uid and water intakes in streptozotocin induced diabetic rats. Blood glucose level lowered within
1 week. This provides evidence that the plant extract could
contain hypoglycaemic compound. The phytochemistry of Annona
muricata has shown the presence of tannins, avonoids known for
their hypoglycaemic activities (Ojewole, 2005). The plant can exert
its action by increasing the proliferation or the renewal of islet cells following destruction by streptozotocin. This is clearly reinforced by the strong immunostaining of islet -cells of rats treated
with plant extract. Streptozotocin induced the elevation of triglycerides, total cholesterol, LDL-cholesterol, atherogen index and
decrease HDL-cholesterol. Hypertriglyceridaemia and hypercholesterolaemia are major factors of diabetic state involved in the
development of atherosclerosis and coronary heart disease which
are the secondary complications of diabetes (Ananthan et al.,
2003). Dyslipidaemia is characterised by high plasma levels of
total cholesterol, LDL-cholesterol and triglycerides, with low
plasma levels of HDL cholesterol. Our results indicate that, plant
extract administered during 28 days reduced total cholesterol,
LDL-cholesterol, triglycerides and lowed serum levels of HDL
cholesterol. Thus, Annona muricata aqueous extract could have a
potential to reduce long-term cardiovascular complications in
diabetic conditions. Serum creatinine levels signicantly increased
in diabetic rats certies a renal failure or an extracellular deshydratation. This could be conrmed by the decrease of atherogenic
index. Daily administration of the plant extract causes a signicant
reduction of creatinine levels. These results suggest that the plant
extract could reduce renal failure or extracellular deshydratation.
Transaminases (ALT and AST) are enzymatic markers of hepatic
function. In this study, serum transaminase activities increase

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N.T. Florence et al. / Journal of Ethnopharmacology 151 (2014) 784790

indicating the hepatic cellular damage resulting from ROS due to

the chronic hyperglycaemia. This hypothesis is justied by the
increase of MDA in diabetic control compared to normal rats.
Hyperglycaemia observed in diabetic conditions reduced antioxidant levels and increased free radicals (Aragno et al., 2004).
The later damage the membrane through lipid peroxidation of
unsaturated fatty acids and alter its function (Memisogullari and
Bakan, 2004; Ravi et al., 2004). The plant extract signicantly
suppressed the increase of transaminases and MDA suggesting a
protective role of the plant extract against oxidative stress. It has
been shown that in diabetic conditions, antioxidant defences are
altered due to the presence of chronic hyperglycaemia thus
promotes free radical regeneration (Hammers et al., 1991;
Kamalakannan and Prince, 2006; Ngueguim et al., 2011). The
effect of Annona muricata extract was also investigated in SOD,
catalase and NO. In diabetic control rats, SOD activity and catalase
levels were signicantly reduced in diabetic control rats while the
NO levels increased. As compared to diabetic control group,
treatment with plant extract increased SOD activity, catalase
activity and provoked a signicant decrease in oxide nitrite levels.
These results suggest that Annona muricata possesses antioxidant
properties. Our ndings agree with those of Adewole and CaxtonMartins (2006), who reported the antioxidant activities of Annona
muricata aqueous extract in short-term treatment.
8. Conclusion
Overall, these results show that, streptozotocin injection
induced hyperglycaemia, polydipsia, polyphagia, body loss, dyslipidaemia and oxidative stress. Administration of Annona muricata
aqueous extract to STZ-diabetic rats reduced near normal blood
glucose levels, body weight, food and water intake, lipid prole
and oxidative defence after a month with daily treatment. Thus;
this extract possesses an antidiabetic effect which can be
explained by its antioxidant and protective effect of pancreatic cells.
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