The biodiversity of the nature, particularly the one of Togo remains
largely unexplored. Since only 5-15% of the higher plants have been systematically
investigated for the presence of bioactive compounds (Pieters and Vlietinck,
2005). One of the first steps leading to drug discovery and development
process is the phytochemical screening of the plants used in traditional
medicine. In the setting of the revalorization of the traditional medicine
of Togo, we identified among the plants entering in the composition of
the recipes used by the healer Sakiè E. a medicinal plant named
Annona senegalensis. The healer uses the leaves macerate or decoction
as medication in the localized or generalized oedema, the fever, the snakebite,
the fractures and sprains (poultice for applying).
Annona senegalensis Persoon (Annonaceae) is a shrub or small tree
with large ovate or oblong alternate leaves, widespread in the Saharan
savannahs to Guinean (Neuwinger, 1996). Traditionally (in Togo and in
many other countries of Africa), the leaves are used as medication in
various diseases (diarrhoea, diseases of the joints, respiratory diseases,
conjunctivitis, wounds, snakebite, jaundice, haemorrhoids, dracunculosis,
feminine barrenness, convulsions, fever, asthenia) (Neuwinger, 1996).
In order to value the pharmacological activities of this plant, we were
interested in this preliminary study in the extraction and the characterization
of the active principles (phytochemical screening) of the leaves powder
of Annona senegalensis originates from Togo. This screening has
been undertaken simultaneously on the species collected at Burkina Faso
where the study has been carried out as comparison.
MATERIALS AND METHODS
This study has been achieved 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: The leafed twigs of the two
plants were harvested in localities situated to 633 km in the south of
Ouagadougou (Burkina Faso) on the Togolese territory at Pya-Kadjika (Kozah
Prefecture) either to a few 437 km in the North of Lomé (Togo)
for Annona senegalensis originates from Togo. The species from
Burkina Faso has been obtained to about 40 km in the west of Ouagadougou
in Kokologho on the road of Bobo-Dioulasso. The period of the collect
was between the months of August and October 2005. The plant was then
identified by the botanists of the Faculties of the Sciences of the Universities
of Lomé and Ouagadougou.
The leafed twigs were dried safe from the sun, to the plain temperature
(about 27 to 29 °C) for 2 weeks. After removal of the twigs, the green
dried leaves were ground into fine powder using a dry grinder provided
with a sifter of 1.5 mm stitches. The ground samples were kept in a tight
plastic bag (1 kg) for further extraction.
Determination of the relative humidity rate (RHR) of the leaves powder:
Before submitting the plant powder to different operations of active
principles extraction, one of the first parameters to determine was the
relative content in water of this powder. To make it, an amount of 1 g
(P) has been placed in the oven (105 to 110 °C) during 1 h in order
to get a dry weight (P') constant. Four tests allowed us to calculate
the average RHR according to the formula:
The RHR of both leaves powder were compared using statistical t-test
Extraction of the active principles Solvent partitioning extraction
of vegetable material (percolation): The extraction was a partitioning
method which separates the various chemical groups according to their
physico-chemical properties using organic solvents of different polarities.
One hundred grams of plant powder have been subjected to the successive
extraction, in an adapted percolator, with the following solvents (5 to
8 times the volume of vegetable material amount): ether of petroleum,
dichloromethane, ethyl acetate, distilled water. After each percolation,
the residue was dried in the oven at 40 °C in order to remove the
residual solvent and then macerated for 5 h in the following solvent.
After percolation, ether of petroleum (Eep), ethyl acetate (Eea) and water
(Ea) extracts were collected and then freeze-dried and kept in desiccator
for qualitative chemical analysis.
In this study, the dichloromethane solvent is used mainly to remove chlorophyll
pigment from leaves powder.
Steeped extracts: Twenty five grams of leaves powder have been
macerated during 5 h either with distilled water (10-12 volumes) for aqueous
macerate (Maq) either with 70% (v/v) aqueous methanol (5-8 volumes) for
aqueous methanol extract (Ehm). The extracts were separated from residue
by filtration and centrifugation (2000 rpm for 15 min). The extract obtained
was freeze-dried and kept in desiccator.
Hydrolysed extracts: This method aims to hydrolyse and characterize
the heterosidic contents of the different extracts (Eae, Ehm, Maq). The
10% hydrochloric acid solution is added to the extract (5:3, v/v) in refluxing
system and heated up for 30 min (Constantinescu et al., 1964).
The extraction of genins has been performed using apolar solvent such
as dichloromethane (organic phase), the anthocyanins (pigments) remained
in the acidic aqueous phase.
Characterization of the chemical groups of the extracts: The qualitative
chemical determination of the extracted constituents has been performed
with the help of colourful specific reactions from Constantinescu et
al. (1964) using suitable chemical reagents.
Ether of petroleum extract: The ether of petroleum is a suitable
apolar solvent for the fat-soluble chemical principles extraction from
vegetable material such as volatile and fatty compounds, sterols and triterpenes,
emodols, carotenoids, basic alkaloids, coumarins, chlorophyll and aglycones
issued from hydrolysed glycosides (Constantinescu et al., 1964).
Thus, the following specific reactions were tested in order to identify
the phytochemical active principles of interest for present study:
Reaction of Liebermann-Burchard's: The dried ether of petroleum
extract was dissolved in acetic anhydride and then in chloroform. By means
of a pipette, concentrated sulphuric acid was added at the bottom of the
tube containing the extract solution. At the contact zone of the two liquids,
a brownish-red or violet ring denotes the presence of sterols and triterpenes.
Reaction of Bornträger's: A red coloration appears when 25%
ammonia solution or 10% sodium hydroxide is added to the extract (3:1,
v/v) in the presence of emodols (anthracenoside aglycones).
Reaction of Carr Price's: The carotenoids are identified by a
blue coloration that turns to the red in presence of a saturated solution
of antimony chloride in chloroform.
Reaction of Shibata or cyanidine test: This test is used to identify
flavonic aglycones in 50% methanolic middle in presence of metallic magnesium
and some drops of concentrated hydrochloric acid. A red or an orange coloration
is a sign of the presence of flavonols or flavanones, respectively.
Ethyl acetate extract: The ethyl acetate essentially extracts
some glycosides compounds such as the flavonosides and anthocyanosides.
The lipophilic aglycones compounds such as flavonic genins were identified
in the organic phase of hydrolysed ethyl acetate extract by means of Shibata`s
reaction as previously described. If the aqueous acidic phase of hydrolysed
extract is red and it turns either to violet at a neutral pH, either to
green or blue in alkaline medium (NaOH or KOH), the anthocyanin pigments
During the concentration of the ethyl acetate extract under reduced pressure
in a Rotavapor - Büchi 461 Water Bath, a precipitate appears at the
end of the operation. This precipitate which becomes insoluble in the
solvent of extraction but soluble in the ethanol 95 ° has been washed
3 to 4-fold with the ethyl acetate and recovered as powder. The Shibata`s
reaction tested on this precipitated fraction has induced a strong red
coloration. The carotenoids were identified with the help of the Carr
Price's reaction previously described under ether of petroleum extract.
Aqueous methanol extract: Aqueous methanol (70%, v/v) extract
may contain many active principles of natural constituents as for example:
polyphenols (tannins), reducing compounds, alkaloid salts, polyphenolic
glycosides (anthracenosides, flavonosides), sterol glycosides (cardiotoniques,
saponosides), triterpene glycosides, anthocyanosides (Constantinescu et
The chemically active constituents extracted were identified by means
of some specific colourful reactions within 70% aqueous methanol or its
In the aqueous methanol extract, we were particularly interested in the
identification of following groups.
The tannins were characterised using a 2% solution of ferric chloride.
The occurrence of a blackish blue colour shows the presence of gallic
tannins and a green blackish colour indicates catechol tannins.
The saponins (saponosides) were identified by shaking 2 mL of diluted
solution (1:1) in a test-tube of 1.6 cm diameter for 15 min. The occurrence
of a foam column of at least 1 cm in height, persisting minimum 15 min,
indicates the presence of saponins.
The flavonosides and anthocyanosides were detected as previously described
under ether of petroleum and ethyl acetate extracts, respectively.
Aqueous steeped extract: Water can extract from vegetable products
hydrosoluble (polar) constituents such as saponosides, polyphenols (tannins,
flavonosides, anthocyanosides), glucides, alkaloid salts (Constantinescu
et al., 1964). The previously chemical active principles of interest
(flavonosides, tannins, saponosides) were identified using the tests described
under aqueous methanol extract. We tested also the organic phase of the
hydrolysed aqueous extract for the detection of lipophilic constituents
sterols/triterpenes, flavonic aglycones as previously described. The anthocyanins
(pigments) were detected in the acidic aqueous phase of hydrolysed aqueous
extract (see under ethyl acetate extract).
High performance liquid chromatography (HPLC) analysis of the extracts:
The HPLC profiling of 70% aqueous methanol and aqueous steeped extract
was carried out in the Laboratory of Toxico pharmacology of CHU of Clermont
Ferrand (France) using a HPLC/UV System (Waters 2690 Separations Module)
equipped with an autosampler, a Waters® 2696 pomp, a Symmetry C8 Waters®
column and a Diode Array Detector. Data were processed and analyzed using
Millenium32 Software (Waters Product). The samples were separated
at 30 °C on a 5 μm C18 column (4.6 mm x 25 cm). The solvents
of elution were (A) 0.6% monosodium orthophosphate buffer and (B) acetonitrile
for HPLC. The internal standard was Proadifen (SKF525A). Quercetin (Extrasynthese
S. A., France) and rutin (Sarsynthese, France) were used as standard flavonoids.
The HLPC was monitored at 200-350 nm and the two main criteria used for
identification of molecules were the time of retention and the UV spectrum
which were compared to those of Waters Toxicol data base.
RESULTS AND DISCUSSION
Determination of the relative humidity rate (RHR) of the leaves powder:
The content in relative humidity of the leaves powder of Annona
senegalensis of Togo and the one harvested in Burkina Faso was respectively
9.7 ± 0.9% and 7.0 ± 1.1%.
Characterization of the chemical groups of the extracts: In Table
1, sterols/triterpenes, carotenoids, flavonoids, anthocyanosides,
saponosides and catechol tannins were
||Comparative phytochemical screening of the leaves powder
partitioning (percolation) extracts of Annona senegalensis
of Togo and the one originates from Burkina Faso
|nd: No detected; ± : Doubtful reaction; +: Weakly
positive reaction; ++: Moderately positive reaction; +++: Strongly
positive reaction, Tg*: Vegetable material from Togo; Bf*: Vegetable
material from Burkina Faso
||Comparative phytochemical screening of the steeped leaves
powder and hydrolysed aqueous extracts (Maq) of Annona senegalensis
of Togo and the one originates from Burkina Faso
|nd: No detected; +: Weakly positive reaction; ++: Moderately
positive reaction; +++: Strongly positive reaction, Tg*: Vegetable
material from Togo; Bf*: Vegetable material from Burkina Faso
detected in different extracts obtained by partitioning extraction. Concerning
carotenoids and flavonoids in Eea, the reaction of identification was
doubtful or moderately positive respectively for Togolese sample.
The flavonic aglycones (Eep) were identified in Togolese sample but not
in Burkinabe corresponding sample. Emodols were not detected in both samples.
In Table 2, the main phytochemical constituents detected
in steeped extracts (Ehm, Maq) were: polyphenols (catechol tannins, flavonosides,
anthocyanosides.), saponosides. Sterols/triterpenes were identified in
organic phase of hydrolysed aqueous steeped extract (Maq) in both samples;
whereas, flavonic aglycones were found only in the apolar phase of the
sample originates from Burkina Faso.
The aqueous phase of hydrolysed aqueous extract (Maq) was constituted
of mainly anthocyanins in both species.
HPLC analysis of the extracts: The HPLC chromatogram of Ehm (Fig.
1) and Maq (Fig. 2) confirmed the presence of flavonoids
including rutin and isoquercetrin (flavonols) respectively in Togolese
sample; whereas rutin, isoquercetrin, epicatechin and catechin derivatives
(flavanols) were detected in extracts of the specimen from Burkina.
The determination of the relative humidity rate was justified by the
fact that this parameter has been considered in weighing the plant powder
for the different tests. Indeed, the weighted samples were carried out
with 5% of residual humidity (European pharmacopoeia) in the plant powder
(dry weight). The difference in the RHR detected in both plant powders
may be explained by the difference of climatic conditions between the
two sites of origin of the plant: Pya-Kadjika in Togo (savannah climate)
and Kokologho in Burkina (Sub Saharan climate). The site of origin of
Togolese species is the most watered compared to the one of Burkina Faso;
this may explained the significantly highest RHR in the plant powder originates
from Togo (9.7 ± 0.9%)(p<0.05).
In the limit of the sensitivity and specificity of the characterization
reactions used, our results showed that the profile of chemical content
of the fractioned extracts obtained from the two plants was almost similar,
the main chemical groups detected being: sterols/triterpenes, carotenoids,
flavonoids, anthocyanosides, saponosides and catechol tannins (Table
1). The precipitated fraction issued from ethyl acetate concentration
has reacted positively to Shibata`s test with an intense red coloration;
that suggests the presence of flavonoids especially flavonols at least
in this extract. The solvent like ethyl acetate is the favourite solvent
for flavonoids extracting from leaves as reported by Ibewuike et al.
(1997) and Aderogba et al. (2003, 2004, 2005).
The chemical composition of the extracts from the leaves powders, obtained
by steeping showed a similar chemical profile for both plants (Table
2). On the other hand, the differences observed were about the flavonosides
and their flavonic aglycones in different extracts; the coloration of
the characterization tests was less intense in Eea, Ehm and Maq for the
Togolese species. Concerning the flavonoids, these results were confirmed
using HPLC assay of 70% aqueous methanol and aqueous extracts. The HPLC
profiles (Fig. 1, 2) showed the presence
of rutin and isoquercetrin (flavonols) in Togolese sample; whereas in
addition to the preceding flavonols, epicatechin and catechin derivatives
(flavanols) were detected in the extracts of specimen from Burkina. The
concomitant presence of flavonols and flavanols in the same plant is normal
since they belong to the same biosynthesis pathway of polyphenolic flavonoids
(Rice-Evans et al., 1996). The present findings corroborates with
those reported by other authors who have notified the presence of flavonoids
(quercetin and quercetrin) and sterols (Mackie and Misra, 1956), tannins
and saponins (Ogbadoyi et al., 2007) in the plant leaf.
These differences in the qualitative phytochemical content of the extracts
could be due to some factors such as the climatic conditions (Reynolds,
2002; Pieters and
||The HPLC profiles of 70% aqueous methanol leave extract
of Annona senegalensis from Togo and the one originates from
Burkina Faso compared with the standard flavonoids Quercetin (14,325)
and Rutin (8,423); Ehm Tg: Rutin (8,385); Ehm Bf: Rutin (8,384); Epicatectin
(6,679); Catechin derivatives (5,432)
Vlietinck, 2005; Metting and Pyne, 2007), the geological environment
(Gomes and Silva, 2007) of the sites of harvest, the period of the harvest
(maturation), the enzymatic content responsible for the biosynthesis pathways
(Pieters and Vlietinck, 2005), the regulation of gene expression by environmental
factors (Boudet, 2007). Indeed, if one considers that the biosynthesized
substances of the plant let it to adapt to its environmental conditions
(role of signal, defense against predators and parasites, resistance against
harmful bugs and diseases) (Pieters and Vlietinck, 2005), the one of Burkina
being more arid, these differences could be explained.
The flavonic aglycones have not been detected in the organic phase of
the Maq acidic hydrolysed (sample
||The HPLC profiles of aqueous leave extract of Annona
senegalensis from Togo and the one originates from Burkina Faso
compared with the standard flavonoids Quercetin (14,325) and Rutin
(8,423); Maq Tg: Isoquercetrin (9,303); Maq Bf: Rutin (8,377); Isoquercetrin
(9,303); Epicatechin (6,678); Catechin derivatives (5,430)
originates from Togo). An apolar solvent extraction in the previously
described conditions of the hydrolysis without heating gave a positive
result to the Shibata`s test. This finding suggests the heat sensitivity
of these compounds.
The qualitative phytochemical composition observed in this study corroborates
with the role of the environmental conditions of growth for each specimen,
especially with the hostile environmental conditions.
This preliminary study, carried out in our conditions of work, shows
that the phytochemical profile of the two Annona involves some
differences (RHR, phytochemical content, extract outputs) which could
be due to the ecosystem and numerous other factors (maturation state,
enzymatic content, regulation gene expression, conditions of work). These
differences in qualitative bioactive constituents of extracts may have
some outcomes on the pharmacological activities of each plant.
We are grateful to Agence Universitaire de la Francophonie (AUF)
for providing a grant which allowed us to start this study.