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Research Article
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Antioxidant Activity of Aqueous Methanol and Ethyl Acetate Extract of Leaves of Annona senegalensis Pers from Togo Versus the One Originates from Burkina Faso
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Y. Potchoo,
I.P. Guissou,
M. Lompo,
E. Sakie
and
B. Yaro
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ABSTRACT
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The aim of the present study is to evaluate and compare
the antioxidant potential of the leaves extracts of Annona senegalensis
(Annonaceae) of Togo versus the one of Burkina Faso. To this end, aqueous
methanol and ethyl acetate extracts by splitting and by steeping were
achieved and the determination of total polyphenols of which flavonoids
was carried out. A survey of the antioxidant activity using the DPPH methods
was performed. The content in total polyphenol (3.47 ± 0.03%) and
flavonoid (2.33 ± 0.17%) of 70% (v/v) aqueous methanol extract
of the specimen from Togo was significantly higher than the one from Burkina
(2.66 ± 0.08 and 1.64 ± 0.04%, respectively) (p<0.00001
for total polyphenol; p<0.05 for total flavonoid), whereas, the amount
of total flavonoid in the ethyl acetate extract of the species from Burkina
(40.38%) was triplicated. For the two types of extracts, the species of
Burkina Faso showed an improved antioxidant activity than the one of Togo
(IC50 = 8.51 and 21.08 μg mL-1 versus 12.46
and 29.22 μg mL-1, respectively) (p<0.05). These free
radicals inhibition activity of the extracts may be due at least to polyphenolic
flavonoids identified by means of HPLC assay performed in the preliminary
study. These flavonoids were rutin and isoquercetrin as flavanols (specimen
from Togo) of which are added epicatechin and catechin derivatives (flavanols)
in the specimen from Burkina. The traditional use of plant leaves may
imply in part this activity against the free radicals.
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How
to cite this article:
Y. Potchoo, I.P. Guissou, M. Lompo, E. Sakie and B. Yaro, 2008. Antioxidant Activity of Aqueous Methanol and Ethyl Acetate Extract of Leaves of Annona senegalensis Pers from Togo Versus the One Originates from Burkina Faso. International Journal of Pharmacology, 4: 120-124. DOI: 10.3923/ijp.2008.120.124 URL: https://scialert.net/abstract/?doi=ijp.2008.120.124
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INTRODUCTION
The free radicals are chemical species (O2•G,
OH•, RO•, ROO•,
H2O2, O2*) (Coudert et al., 1994)
produced in the organism during the chemical reactions that contribute
to the development and the maintenance of the cellular life. These are
very reactive species towards endogenous molecules (DNA, proteins, lipids)
having a beneficial role (antimicrobial activity) but implied especially
in the pathological physiology of numerous affections: atherosclerosis,
heart failure, ageing, neurodegenerative disorders, cancer, diabetes mellitus
and a plethora of other diseases (Coudert et al., 1994; Abdi and
Ali, 1999). So, one constantly attends nowadays the increasing development
of substances having the suitability to stop the initiation and/or the
propagation of reactions (oxidization) leading up to the production of
these free radicals: These are the free radical scavenging or antioxidant
agents which are synthesis substances but more and more the phytochemical
principles of medicinal plants.
In the preliminary phytochemical study, we have also reported the circumstances
of choice of Annona senegalensis as subject of study; we have described
its physical characteristics, its ecosystem of growing and its numerous
traditional uses not only in Togo but also in many other countries of
Africa.
A review of the literature showed that the free radical scavenging activity
has not yet been evaluated nor in the leaves nor in any other part of
the plant. In order to value the antioxidant potential of this plant,
our work intends to do this compared to the species originates from Burkina
Faso using extracts from the leaves.
MATERIALS AND METHODS
This study has been carried out during the year 2006 at the Institute
of Research in Science of Health (IRSS/CNRST), Department of Medicine
and Traditional Pharmacopeia, Ouagadougou, Burkina Faso.
Collection of the plant material, extraction and phytochemical screening
of the extracts: To this end, a preliminary study has previously been
performed.
Determination of total polyphenolic compounds: The concentration
of total polyphenol compounds in the 70% (v/v) aqueous methanol extract
(Ehm) were determined spectrophotometrically on the basis of a standard
curve (R2 = 0.9988) plotted using tannic acid (serial dilutions
to give a range of 3.33-23.33 μg mL-1). Briefly, 50 μL
of extract and the standard previously dissolved in 60% methanol were
diluted (3-fold replicated). The absorbance of the studied extract and
the serial concentrations of the standard were measured with an UV-Visible
Agilent spectrophotometer 8453 E at 280 nm against a blank.
The content in total polyphenolic compounds was calculated using the
equation of the standard curve and the results expressed as mg of Tannic
Acid Equivalent (TAE) per 100 mg of lyophilised extract.
Determination of total flavonoid compounds: The total flavonoids
(a class of polyphenols) content of 70% aqueous methanol extract and ethyl
acetate extract (Eea) was determined with the help of Dowd method adapted
by Arvouet-Grand et al. (1994) using 2% aluminium trichloride in
methanol as reagent. The absorbance was measured using an Agilent UV-Visible
spectrophotometer 8453 E at 415 nm against a blank. The standard curve
(R2 = 0.9986) was plotted using the serial concentrations of
quercetin.
The content in total flavonoid constituents was calculated using the
equation of the standard curve and the results expressed as mg of Quercetin
Equivalent (QE) per 100 mg of lyophilised extract.
Evaluation of the antioxidant activity
Qualitative determination of the antioxidant activity: It is a
rapid test in order to evaluate the antioxidant activity on the precipitated
fraction of ethyl acetate extracts of both samples. Based on the method
previously described (Burits and Bucar, 2002), the solutions of each extract
and quercetin as standard were spotted on the starting point on silica
gel F254 of a thin layer chromatography sheet and developed in an appropriate
migration solvent system. After this, the silica gel sheet is allowed
to dry and sprayed with 4% 2, 2-diphenyl-1-picryl-hydrazil (DPPH, a purple
coloured free radical generator) in methanol. Any bleaching of the purple
color background of DPPH reagent to the site of the spots within 30 min
was taken as positive result.
Quantitative determination of the antioxidant activity: The free
radical scavenging potency of 70% aqueous methanol and ethyl acetate extracts
was determined using the method of Mensor et al. (2001) with some
modifications. Briefly, each extract and quercetin (21 mg mL-1),
as positive control, were diluted to give concentrations ranging 1050-0.5
μg mL-1 with dimethyl sulfoxide (DMSO). The solution of
4% DPPH in methanol was prepared. Two hundred microliter of this solution
was added to 10 μL of each extract or quercetin solutions in various
concentrations and allowed to react in the steam room at 37 °C for
30 mn. The blank and the negative control were prepared with appropriate
solvents. Each assay was 4 fold replicated. The decrease in absorbance
(deep violet to light yellow) of the mixtures of tested extracts or quercetin
was measured at 490 mn on a spectrophotometer Biorad Model 680 Microplate
Reader. These absorbance values were converted to the percentage of inhibition
of free radical scavenging activity (I%) using the formula:
where, As was the absorbance of the sample, Ao
was the absorbance of the control. The IC50 (concentration
causing 50% inhibition of a maximum effect estimate in 100%) value of
each extract was calculated using the equation of linear regression of
plots (excel software) of concentrations of tested samples (μg mL-1).
The inhibition of DPPH activity of quercetin was also measured under the
same condition to serve as standard antioxidant agent.
RESULTS
Phytochemical contents of the extracts: The phytochemical principles
identified in the preliminary study were mainly polyphenols (flavonoids,
catechol tannins, anthocyanosides), saponosides and carotenoids for the
70% aqueous methanol extract and anthocyanosides, flavonoids and carotenoids
for ethyl acetate extract. The High Performance Liquid Chromatography
(HPLC) analysis of the extracts has confirmed the presence of flavonoids,
especially rutin and isoquercetrin (flavonols) in the specimen from Togo
and the preceding compounds plus epicatechin and catechin derivatives
(flavanols) in the specimen from Burkina.
Determination of total polyphenolic and flavonoid content: Table
1 shows the content in total polyphenols and flavonoids (a group of
polyphenol) of Ehm and Eea from
Table 1: |
Total polyphenolic and flavonoid content and the antioxidant
activity (IC50) of 70% aqueous methanol and ethyl acetate
extract of plant material from Togo versus the one originates from
Burkina Faso |
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Comparison of total polyphenolic and flavonoid content
(Student`s t-test): a vs b (p<0.00001), c vs d (p<0.05), e vs
f (p<0.0001), Comparison of IC50 values (Student`s t-test):
g vs s (p<0.001), h vs s (p<0.001), i vs s (p<0.0001), j
vs s (p<10-8), g vs h (p<0.05), i vs j (p<0.05)
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both plants. The quantitative composition of Ehm was 3.47 ± 0.03%
of total polyphenols and 2.33 ± 0.17% of total flavonoids for Annona
senegalensis of Togo (AsTg) versus, respectively 2.66 ± 0.08%
and 1.64 ± 0.04% for Annona senegalensis from Burkina Faso
(AsBf).
For Eae, the content in total polyphenols of which mainly flavonoids
was 13.27 ± 0.20% in Togolese sample versus 40.38 ± 1.08%
in the plant originates from Burkina Faso.
Comparative antioxidant activity of the extracts Qualitative
determination of the antioxidant activity: This chromatogram photography
(Fig. 1 ) shows the positive test of DPPH bleaching
to the site of spots issued from ethyl acetate extract of sample originates
from Togo (Eea Tg) versus the one from Burkina Faso (Eea Bf) and Quercetin
(Q) as standard.
Quantitative determination of the antioxidant activity: Table
1 shows the antioxidant potential of Ehm and Eea expressed as the
IC50 value in comparison to the positive control (quercetin).
For both plants, 70% aqueous methanol extract exhibited a highest antioxidant
activity (IC50 = 8.51 ± 0.66 μg mL-1
for AsBf versus 12.46 ± 1.05 μg mL-1 for As Tg)
than ethyl acetate extract (IC50 = 21.08 ± .31 μg
mL-1 for AsBf versus 29.22 ± 2.03 μg mL-1
for AsTg).
The two extracts from Annona senegalensis of Burkina Faso showed
a potent antioxidant activity (IC50 = 8.51 ± 0.66 μg
mL-1 for Ehm versus 21.08 ± 0.31 μg mL-1
for Eea) compared to those from Togo (IC50 = 12.46 ±
1.05 μg mL-1 for Ehm versus 29.22 ± 2.03 μg
mL-1 for Eea).
Statistical analysis: The results of total polyphenolic and flavonoid
content, the IC50 values of the antioxidant activity are expressed
as mean ± SEM of three or five determinations. The determination
of total polyphenolic and flavonoid content were issued from the
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Fig. 1: |
Chromatogram of ethyl acetate extracts of Eae Tg versus
Eae Bf and quercetin (Q) as standard. The arrow indicates the migration
steering of the solvent |
concentration-absorbance curves. The IC50 values were obtained
from the dose-response regression plots. In the two cases, good coefficients
of correlation R2 were observed: 0.9986 and 0.9643 to 0.9842,
respectively. A low IC50 value is an indication of a strong
antioxidant activity. The two samples were compared in regard to the quantitative
determination and the antioxidant activity using the Student`s t-test.
A probability P of 0.05 or less was considered significant.
DISCUSSION
The determination of total polyphenols and its related group flavonoids
showed that the content of these constituents is significantly higher
in Ehm Tg (3.47 ± 0.03 and 2.33 ± 0.17%, respectively) than
in Ehm Bf (2.66 ± 0.08 and 1.64 ± 0.04%, respectively) (p<0.00001
for total polyphenol; p<0.05 for total flavonoid). Concerning Eea,
as reported by Ibewuike et al. (1997) and Aderogba et al.
(2003, 2004, 2005), the main polyphenolic group that have been isolated
in ethyl acetate extract of leaves were flavonoids. In this study, the
highest amount (40.38 ± 1.08%) of flavonoids were found in ethyl
acetate extract of the specimen collected at Burkina Faso (p<0.0001)
(Table 1). These flavonoids are represented at least
by rutin, isoquercetrin, epicatechin and catechin derivatives as has detected
HPLC assay.
These quantitative and qualitative differences noted in the extract content
of phytochemical principles of both species may be explained, as we have
discussed in a previous phytochemical screening study, by the climatic
conditions (Pieters and Vlietinck, 2005; Reynolds, 2002; Metting and Pyne,
2007), the geological environment (Gomes and Silva, 2007) of the sites
of harvest, the period of the collect, the enzymatic content responsible
for the biosynthesis pathways (Pieters and Vlietinck, 2005) and the regulation
of gene expression by environmental factors (Boudet, 2007). Indeed, if
one considers that the substances elaborated by the plant allow it to
adapt to its environment (role of signal, defense against the predators
and the parasites, resistance against the harmful bugs and the diseases)
(Pieters and Vlietinck, 2005), the one of Burkina being more arid (Sub-Saharan
climate), this difference can be explained.
The two extracts of both samples belonging to two different ecosystems
exhibited a substantial but a least antioxidant activity (IC50
= 8.51-29.22 μg mL-1) in relation to the quercetin (IC50
= 2.99 μg mL-1) (p<0.001). The DPPH inhibition activity
of aqueous methanol extracts (IC50 = 8.51 μg mL-1
for AsBf versus 12.46 μg mL-1 for AsTg) was more potent
compared to the one of the ethyl acetate extracts (IC50 = 21.08
μg mL-1 for AsBf versus 29.22 μg mL-1
for AsTg) (p<0.0001). Indeed, the antioxidant activity of the plant
extracts depends on: the type and the polarity of the extracting solvent,
the extracting technique, the purity of the active principle, the antioxidant
test, the substrate used (Tsuda et al., 1994) and the structural
requirements (a number of phenolic hydroxyl groups on ring structures)
(Harborne, 1986; Saskia et al., 1996; de Beck et al., 2003).
Although the content in total polyphenols and their related flavonoids
was higher in Ehm Tg (p<0.00001 for total polyphenol; p<0.05 for
total flavonoid), the Ehm Bf has showed the best scavenging activity with
IC50 value of 8.51 μg mL-1 versus IC50
value of 12.46 μg mL-1 for Ehm Tg (p<0.05). Relating
to the ethyl acetate extracts, the total flavonoids content of Eea Bf
was 3-fold higher than Eae Tg (40.38 ± 1.08% versus 13.27 ±
0.20%) (p<0.0001). However, this Eea Bf exhibited a weak scavenging
potential compared to the one of Ehm Bf (21.08 versus 8.51 μg mL-1)
but more potent than the one of Eae Tg (IC50 = 21.08 μg
mL-1 versus 29.22 μg mL-1) (p<0.05).
The effectiveness of the antioxidant activity of the extracts of Annona
senegalensis from Burkina may be explained by the environmental factors
such as the climatic conditions, the stage of plant maturation, the temperature
(Gazzani et al., 1998). McCune and Johns (2005) reported that this
antioxidant activity was increased under growing conditions of decreased
water and fertility, what is the case of the Sub-saharan climate like
the growing ecosystem of Annona senegalensis originates from Burkina.
Besides, a big diversity exists in the efficiency of the antioxidant activity
of different phenolic compounds (Montalleb et al., 2005). The
antioxidant activity reported in this study is related in part to the
previous flavonoids (rutin, isoquercetrin, epicatechin and catechin derivatives)
which involve at least a catechol group necessary to radical scavenging
in any flavonoid that possesses this activity (Saskia et al.,
1996; de Beck et al., 2003).
Despite the highest amount of total flavonoids of Eea Bf (40.38 ±
1.08%), this one didn`t exhibited a highest scavenging activity than Ehm
Bf which contained only 1.64 ± 0.04%. This finding confirms the
data of the literature according to which no any relationship exist between
the antioxidant activity and the content of total flavonoids (Galvez et
al., 2005; Sawadogo et al., 2006), even though their free radical
scavenging activity is not anymore to demonstrate.
The free radical scavenging activity of the 70% aqueous methanol and
the ethyl acetate extracts of Annona senegalensis leaves is due
in part to their phytochemical constituents of polyphenols of which the
flavonoids as reported numerous authors (Pieters and Vlietinck, 2005;
Galvez et al., 2005; Aderogba et al., 2004, 2005); other
polyphenols like the tannins (Pieters and Vlietinck, 2005), the anthocyanins
(Wang et al., 1999) and the saponins (Alaoui et al., 1998)
as terpenoid identified in the extracts could also participate to this
antioxidant activity. Indeed, these chemical principles have been reported
to be involved in the antioxidant activity of various medicinal plants.
The present study showed that the leaves extracts of Annona senegalensis
from the two ecosystems, especially the one of Burkina Faso constitutes
a potent natural source of antioxidant agents. This antioxidant activity
could justify in part, the use of this medicinal plant in traditional
practice.
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