Antioxidant potential of aqueous leaf extract of Ageratum conyzoides Linn. in diabetic rats

Abstract

The antioxidant activity of aqueous extract of leaves of Ageratum conyzoides (AC) in the serum of male diabetic rats was evaluated using Ferric Reducing Antioxidant Power (FRAP) assays, by determination of malonadehydes, lipid hydroperoxydes and protein thiol groups. The plant extract was tested at doses of 100, 200 and 300 mg/kg on diabetic rats during three weeks of treatment; glibenclamide (10 mg/kg) was used as positive control. Glycaemia of diabetic rats was also determined, at the beginning and at the end of the experimentation. The results showed that A. conyzoides did not have an incidence in serum protein thiols and serum malonaldehydes level. Nevertheless, the aqueous extract induced lowering of lipid hydroperoxides in the groups treated with 100 mg/kg (p � 0.01) and 200 mg/kg (p � 0.01) when compared to the negative control group. Power (FRAP) was also higher in the 100 mg/kg group. In addition, glycaemia was decreased at the ferric reducing antioxidant the third week in the group receiving 200 mg/kg (p � 0.01) and 300 mg/kg (p � 0.01). A. conyzoides had a positive effect on the oxidation-reduction system on streptozotocin induced diabetic rats and improved glycaemia of diabetic rats.

Full text

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Journal of Pharmacognosy and Phytotherapy Vol. 1(4) pp. 041-046, October, 2009
Available online at http://www.academicjournals.org/jpp
© 2009 Academic Journals



Full Length Research Paper

Antioxidant potential of aqueous leaf extract of
Ageratum conyzoides Linn. in diabetic rats

Nyunaï Nyemb1, 2*, Manguelle-Dicoum Biyong Adèle1, Njifutié Njikam2 and Abdennebi El
Hassane3

1Centre de Recherches Médicales, Institut de Recherches Médicales et d’Etudes des Plantes Médicinales, B. P. 3805
Yaoundé, Cameroun.
2Faculté des sciences, Université de Yaoundé 1, B. P. 812 Yaoundé, Cameroun.
3Département des Sciences Biologiques et Pharmaceutiques Vétérinaires, Institut Agronomique et Vétérinaire Hassan
II, B. P. 6202, Rabat-Instituts, Maroc.

Accepted 7 September, 2009

The antioxidant activity of aqueous extract of leaves of Ageratum conyzoides (AC) in the serum of male
diabetic rats was evaluated using Ferric Reducing Antioxidant Power (FRAP) assays, by determination
of malonadehydes, lipid hydroperoxydes and protein thiol groups. The plant extract was tested at
doses of 100, 200 and 300 mg/kg on diabetic rats during three weeks of treatment; glibenclamide
(10 mg/kg) was used as positive control. Glycaemia of diabetic rats was also determined, at the
beginning and at the end of the experimentation. The results showed that A. conyzoides did not have an
incidence in serum protein thiols and serum malonaldehydes level. Nevertheless, the aqueous extract
induced lowering of lipid hydroperoxides in the groups treated with 100 mg/kg (p ? 0.01) and 200 mg/kg
(p ? 0.01) when compared to the negative control group. Power (FRAP) was also higher in the 100 mg/kg
group. In addition, glycaemia was decreased at the ferric reducing antioxidant the third week in the
group receiving 200 mg/kg (p ? 0.01) and 300 mg/kg (p ? 0.01). A. conyzoides had a positive effect on
the oxidation-reduction system on streptozotocin induced diabetic rats and improved glycaemia of
diabetic rats.

Keys words: Ageratum conyzoides, antioxidant, antihyperglycemic, streptozotocin, diabetic rats.


INTRODUCTION

Ageratum conyzoides is widely utilized in traditional
medicine systems wherever
applications vary by region. Traditional communities in
India use this plant as a bacteriocide, antidysenteric, and
antilithic (Okunade, 2002), and in Asia, South America,
and Africa, aqueous extract of this plant is used as a
bacteriocide (Okunade, 2002). In Central Africa it is used
to treat pneumonia, but the most common use is to cure
wounds and burns (Okunade, 2002). A. conyzoides is
also utilized to treat fever, rheumatism, headache and
colic (Okunade, 2002., 1993). In Cameroon aqueous
extracts of leaves or the whole plant are used as an anti-
diabetic (Tsabang et al., 2001).
In diabetes mellitus, hyperglycemia may depress the



*Corresponding author: nyunain@yahoo.fr. Tel: 00(237)77816
636. Fax: 00(237)22239239.


it grows, although
natural antioxidant system (Pavana et al., 2009). Several
reports indicate that modified oxidative stress is due to
chronic hyperglycemia (Bhor et al., 2004). Enhanced
oxidative stress has been well documented in both
experimental and human diabetes mellitus (Baynes,
1991).
Several pharmacological investigations have been
conducted to determine the efficacy of this plant
(Okunade, 2002). The chemical composition shows that
A. conyzoides contain many bioactive compounds
including flavonoids, alkaloids, cumarins, essential oils,
chromenes, benzofurans,
(Okunade, 2002).
However, we have not found any studies on the
antihyperglycemic and antioxidant
conyzoides in experimental diabetes mellitus. Thus, in
the present study, we evaluated the antihyperglycemic
and antioxidant effects of A. conyzoides in streptozotocin
induced diabetic rats.
terpenoids and tannins
effects of A.

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042 J. Pharmacognosy Phytother.



MATERIALS AND METHODS

Collection and preparation of plant material

Mature A. conyzoides was collected during the month of February,
2006 in Yaoundé, Centre province, Cameroon. Botanical iden-
tification was performed at the National Herbarium of Yaoundé, in
comparison with the voucher specimen N° 19050/SFR/Cam. The
leaves were shade-dried and ground into powder.


Preparation of the extracts

138 g of the powdered leaves was boiled in distilled water (2.25 L)
for 30 min. The decoction was taken and allowed to cool for 30 min
at room temperature (24 ± 5° C). This decoction was filtered twice
and the filtrate was dried in an oven (55° C) for 3 days. The yield
after extraction was about 29% (w/w).


Drugs and chemicals

Glibenclamide (Glib) was purchased from Strides Arcolat Ltd.
Bangalore, India and Streptozotocin from Sigma-Aldrich Co Ltd,
United Kingdom.


Animals

Male albino Wistar rats (180 - 220 g) were maintained under
standard laboratory diet and tap water ad libitum in the Animal
House of the Institute of Medical Research and Medicinal Plants
Studies, Cameroon.
Prior to the experiment, the rats were divided into 5 experimental
groups of 6 animals each. The animals were subjected to fasting for
16 h (before the determination of glycaemia and at the end of the
experiment when collecting blood sample) but they had free access
to water. The study was carried out with the approval by the
Institutional Animal Ethics Committee.


Induction of diabetes

Animals were rendered diabetic by an intravenous injection of a
freshly prepared streptozotocin (STZ) solution at a dose of
55 mg/kg body weight in acidified saline solution (0.9%; pH 4.5), as
described by Szkudelski (2001). In this case, the control animals
received only the acidified saline solution (pH 4.5). After 72 h, when
the condition of diabetes was stabilized, the animals with blood
glucose levels above 200 mg/dL were selected for the study and
divided into 5 groups.


Experimental design

Groups I, II and III were given the aqueous extract of leaves of A.
conyzoides (suspended in distilled water 10 ml/kg) orally daily at
doses of 100, 200 and 300 mg/kg respectively during three weeks.
Animals of group IV received glibenclamide at a dose of 10 mg/kg
as positive control while those of group V served as a negative
control and received appropriate volumes of vehicle (distilled water)
orally.


Determination of antihyperglycemic activity

Blood samples for glucose determination were obtained from the tip
of the tails of the rats before administration of drugs at the initial




week, and at the third week thereafter. Blood glucose level was
determined using a glucometer, Glucotrend®2 (An Accu-Chek
system of the Roche Group Germany, Roche diagnostics GmbH D-
68298 Mannheim, Germany) in all animals.


Determination of antioxidant activity

At the end of the experimental period, all animals were sacrificed by
cervical dislocation and biochemical studies were conducted on
serum of control and experimental animals in each group.
FRAP (Ferric Reducing Antioxydant Potential) were estimated by
the method of Benzie and Strain (1996), while protein thiol level
was assayed by the method of Ellmann (1959). Serum malo-
naldehydes (Yagi, 1976) were assayed and lipid hydroperoxides
(Wolf, 1994) was estimated.


Statistical analysis

All values were expressed as mean ± S.D. The data were
statistically analysed by the classical student’s paired t-test. Data of
treated groups were compared to those of negative control.


RESULTS

Effect of A. conyzoides on blood glucose level after
multiple administrations of A. conyzoides (AC) in
hyperglycemic rats

After three weeks of treatment, there was a significant
decrease (P ? 0.01) in blood glucose in groups that
received 200 and 300 mg/kg of aqueous crude extract,
while glibenclamide was ineffective in diabetic rats as
represented by Figure 1.


Antioxidants effect of A. conyzoides (AC) in
hyperglycemic rats

The groups receiving aqueous crude extract of AC at
100 mg/kg (P ? 0.05) and 200 mg/kg (P ? 0.01) showed a
significant decrease of lipid hydroperoxides respectively
as demonstrated in Figure 2. Serum malonaldehydes in
different groups of diabetic rats was similar to that of
control as represented in Figure 3. At the end of the
experiments, the protein serum thiol was not significantly
different in the groups treated with AC when compared to
the control group. The group receiving glibenclamide
showed a thiol level slightly higher than the control group
as seen in the Figure 4. Ferric reducing antioxydant
potential increased in the group receiving 100 mg/kg (P ?
0.05) of A. conyzoides and treated with glibenclamide (P
? 0.01) compared to control. The level of FRAP in the two
groups were comparable as represented in Figure 5.


DISCUSSION

The aim of the present study was to evaluate the possible

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Nyemb et al. 043








Figure 1. Glycaemia level after 3 weeks of administration of 100, 200 and 300 mg/kg of
aqueous extract of A. conyzoides and glibenclamide (10 mg/kg). Results (?mol/L of serum)
are presented as mean ± standard deviation of the mean. n = 6, number of rats. **P ? 0.01
group vs. control.



**
*
0
2
4
6
8
10
12
100 mg/kg200 mg/kg300 mg/kgGlib Control
??????????????
???????????????

?????????????


Figure 2. Serum lipid hydroperoxide level after 3 weeks of administration
of 100, 200 and 300 mg/kg of aqueous extract of A. conyzoides and
glibenclamide (10 mg/kg). The results (µmol/L of serum) are presented as
mean ± standard deviation of the mean. n = 6 number of rats, *P ? 0.05.
**P ? 0.01 vs. control group.


protective effects of aqueous extract of leaves of A.
conyzoides on glucose level serum and antioxidant
defense systems of plasma in streptozotocin induced
diabetes rats. The levels of glucose in blood FRAP,
protein thiol, lipid hydroperoxide and malonaldéhyde
were estimated in serum of control and experimental
groups of streptozotocin (STZ) induced diabetic rats.
The present study showed that oral administration of A.
conyzoides extract decreased the blood glucose level in
diabetic rats. The result suggests that the extract
produces an antidiabetic action mediated by an increase
in peripheral glucose uptake in diabetic rats, especially at
a concentration of 200 and 300 mg/kg (Figure 1). In this
study, the antihyperglycemic effect of the extract was
more efficacies in lowering blood glucose level than that
of glibenclamide, a standard antidiabetic drug that act by
stimulating insulin secretion from pancreatic ?-cells (Tian
et al., 1998). Taking into consideration the mechanism of
action of glibenclamide which is the stimulation of insulin
liberation, glibenclamide is only effective in moderate
diabetic condition and has little or no effect in a severe
diabetic condition where the ?-cells of the pancreas are
totally destroyed (Suba et al., 2004). Thus, the
antihyperglycemic effect of the extract might be due to a
increase in peripheral uptake glucose. The percentage
fall in blood glucose levels was effective in treated extract

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044 J. Pharmacognosy Phytother.


?
?
?
?
?
?
100 mg/kg200 mg/kg300 mg/kgGlibcontrol
??????????????
???????????????

Malonaldehydes


Figure 3. Serum malonaldehyde level after 3 weeks of administration of 100,
200 and 300 mg/kg of aqueous extract of A. conyzoides and glibenclamide
(10 mg/kg). The results (µmol/L of serum) are presented as mean ± standard
deviation of the mean. n = 6 number of rats.





0
10
20
30
40
50
60
70
80
100 mg/kg200 mg/kg 300 mg/kgGlibControl
??????????????
??????????????

?????????????

Figure 4. Proteinic serum thiols after 3 weeks of administration of 100, 200
and 300 mg/kg of aqueous extract of A. conyzoides and glibenclamide
(10 mg/kg). The results (µmol/L of serum) are presented as mean ± standard
deviation of the mean. n = 6, number of rats.


diabetic’s rats and not with glibenclamide (Figure 1); it
implies that the antihyperglycemic effect of AC is not
dependent on the degree of ?-cell destruction. From the
results of the present study, it may be suggested that the
mechanism of action of AC is different to glibenclamide
action.
Hyperglycemia induces the generation of free radicals
which can affect antioxidant defenses thus leading to the
disruption of cellular functions, oxidative damage to
membranes and increased susceptibility to lipid peroxi-
dation (Giugliano et al., 1996). In addition, an increased
of lipid peroxidation has been reported both in clinical and
experimental diabetes (MacRury et al., 1993). In this aim,
we chose to work on a model of severe experimental
diabetes.
Treatment with plant extract at 100, 200 and 300 mg/kg
showed lowering hydroperoxides (p ? 0.05 and p ? 0.01)
and increase in ferric reducing antioxidant potential in
rat’s serum comparable to that of glibenclamide. The
extract had no effect on proteinic serum thiols and on

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Nyemb et al. 045





**
*
0
10
20
30
40
50
60
70
100 mg/kg 200 mg/kg300mg/kgGlibControl
??????????????
??? !?"???"??????????????
Frap


Figure 5. Serum ferric reducing antioxidant power (FRAP) level after 3 weeks of
administration of 100, 200 and 300 mg/kg of aqueous extract of A. conyzoides and
glibenclamide (10 mg/kg). The results (µmol/L of serum) are presented as mean ± standard
deviation of the mean. n = 6 number of rats. *P ? 0.05. **P ? 0.01 vs control group.




serum malonaldehyde level (Figure 3 and 4). The
hydroxyl radical scavenging activity of the aqueous
extract of AC is shown in Figure 2, at 100 and 200 mg/kg,
the aqueous extracts exhibited significant scavenging
activity. The ability of those above doses to quench
hydroxyl radicals seem to be directly related to the
prevention of propa-gation of the process of lipid
peroxidation. It is known that hydroperoxide are primary
products of lipid-peroxidation, the lowering effect of lipid
hydroperoxide level may be due to an antioxidant activity
of A. conyzoides crude extract. Many plants like mulberry
fruit extracts showed their ability effect to scavenge
hydroxy radical (Song-Hwan and Hyung-Joo, 2007). In
this study the level of hydroperoxide in treated extract
groups may be related to scavenging lipid hydroperoxide
radical’s activity of one or more of its components,
suggesting its potent antilipid peroxidative like previously
observed by Pavana et al. (2009).
We reported an increase of frap serum level which is
synonym of potentialization of antioxidant potential
(Figure 5). Many studies showed increase in antioxidant
activity proportionally to the polyphenol content. A linear
relationship between FRAP values and total polyphenol,
tannin, proanthocyanidin and flavonoid contents was well
established (Maksimovic et al., 2005).
Thus, the saponins or/and polyphenols in the extract
may be suspected to possess the activity that may be
attributed to their protective action on lipid peroxidation
and at the same time the enhancing effects on cellular
antioxidant defence contributing to the protection against
oxidative damage in streptozotocine induced diabetes.
Hence from our results, it is suggested that AC has a
significant protective effect against streptozotocin-
induced diabetes in rats, and this can be attributed to the
combined effect of various chemical constituents of this
plant. Saponines, flavonoids and tannins were reported to
be the main constituent of this plant (Okunade, 2002).
They are known to have antioxidant activities in many
diseases. Their role in the treatment of diabetes
(Yoshikawa et al., 2001; Rhemann and Zaman, 1989)
and antioxidant activity (Yoshiki et al., 1998) is well
established. Presence of
flavonoïdes in the aqueous extract was confirmed
through our preliminary phytochemical screening also.
Therefore the improving role of AC aqueous extract in
diabetes may be attributed at least to those components
which can act with other miscellaneous compound to
produce these antioxidant and antihypeglycemic effects.
In conclusion, the present study showed that AC leaves
possess potent antioxidant activity, which may be directly
or indirectly responsible for then hypoglycaemic property.
These potent antioxidant properties may contribute
towards preventing peroxidative damage. Further studies
are in progress to identify the active components in AC
and their role in controlling diabetes.


ACKNOWLEDGEMENTS

This research was funded by “Agence Universitaire de la
Francophonie”. Institut Agronomique et Vétérinaire
Hassan II and the University of Yaoundé 1 were the hosts
of this grant.


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