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Akoué et al.
1 Int. J. Biomol. Biomed.
RESEARCH PAPER OPEN ACCESS
Phytochemical composition and antiradical activity of Sakersia africana
Hook. f. medicinal plant from Gabon
Gontran Nsi Akoué1,2
, Louis-Clément Obame2*
, Joseph Privat Ondo2
, Ibrahim Brama1
,
Elvis Simplice Otogo N’Nang2
, Skitt Yvan Tapoyo2
, Alain Souza1
1
Laboratoire de Physiologie Animale: Electrophysiologie-Pharmacologie-URAB, Université des Sciences
et Techniques de Masuku, BP 913, Franceville Gabon
2
Laboratoire de Substances Naturelles et de Synthèses Organométalliques, URCHI-Université des
Sciences et Techniques de Masuku, BP 943, Franceville Gabon
Article Published: 17 May 2013
Key words: Sakersia africana Hook.f., phytochemical screening, total phenols,
proanthocyanidins, antiradical activity.
Abstract
The valorization of the medicinal plants of our country and determination of their impact on health due to their
abundance of substances with various pharmacological effects are our principal objective. This study was evaluated
the phytochemical screening and radical 2, 2-Diphenyl-1-picrylhydrazyl (DPPH) scavenging activity of different
extracts of Sakersia africana Hook. f.. The results revealed that Sakersia africana Hook. f. is rich in phenols
compounds, sterols, triterpenes, alkaloids and reducing compound. The values in total phenols and
proanthocyanidines are ranging respectively from 391.58 ± 0.04 to 777 ± 0.03 mg/100 g of drugs and 113.5 ± 3.17 to
653.5 ± 36.83 mg/100 g of drugs. Results also show that different extracts tested present antiradical activity with
values of IC50 ranging from 164.21± 0.014 to 195.54± 0.012 % and abundance in bioactive compounds. This study
could justify the use of Sakersia africana of some chronic diseases.
*Corresponding Author: Louis-Clément Obame  obamengonga@gmail.com, yacth58@hotmail.com
International Journal of Biomolecules and Biomedicine (IJBB)
ISSN: 2221-1063 (Print) 2222-503X (Online)
Vol. 3, No. 3, p. 1-8, 2013
http://www.innspub.net
Akoué et al.
2 Int. J. Biomol. Biomed.
Introduction
The plants were used for prehistory by Human for
nutritional and therapeutic requirement. Medicinal
plants are the major source of drugs through their rich
secondary metabolites (Fouché et al., 2000; Nostro et
al., 2000). Thus, medicinal plants are the main means
to fight against diseases by including populations of
underdeveloped countries. This practice was
abandoned in favor of modern medicine. We are
witnessing a return to use of medicinal plants over the
past decade. World Health Organization (WHO), in
2007 estimated that about 80 % of the population of
developing countries can be cured from plants
(Obame, 2009). In 2001, the studies have revealed that
approximately 25 % of prescriptions in the world for
drugs herbal and 60 to 70 % of antibacterial and
anticancer drugs are substances of natural origin (Rate,
2001). Hence the useful achieve the phytochemical
studies of medicinal plants for discovery of news
bioactive compounds. As such the WHO is currently
supporting clinical validation of some traditional
medicines. It is a shrub of the Melastomataceae family
that grows on moist soils and in secondary forest
(Walker and Sillans 1961). The leaves and twigs in a
hairy very steep, and the protruding ribs of a green-
red, turn yellow or red before falling. Fruit globular
form berries are very small and contain many tiny
seeds. The decoction of the leaves has healing
properties crushed. It is used as healing by gabonese
populations in southern and the treatment of several
diseases as dysentery, diarrhea, stomach ulcers and
stomach aches (Walker and Sillans, 1961). The
objective of this study was to evaluate the
phytochemical characteristics and radical scavenging
activity of different extracts of Sakersia africana Hook.
f.
Material and methods
Plant material
The branches of S. africana were collected in
september 2012 in Mbomo village, at 28 km of Oyem
city (Northern Province of Gabon). The plant was
toxonomically authenticated at the Laboratory of Plant
Biology and Ecology of University of Science and
Technology of Masuku (Franceville, Gabon), where a
voucher specimen was deposited.
Preparation of plant extract
The branches of Sakersia africana was air-dried at
room temperature for a total period six weeks and
pulverized to powder using a clean electric blender
(Model Phillips 190). A 25 g sample of the pulverized
branches of Sakersia africana was soaked in 500 mL
of solvent (ethanol, ethanol-water (50/50 v/v) and
water) and allowed to stand for 72 h with intermittent
stirring. This was filtered through a whatman No. 1
filter paper and the filtrate obtained was evaporated to
a dry mass using a rotary rotavaporator at 40 °C. The
residues recovered were dried in an oven at a
temperature of 65 °C. The extract obtained is stored in
vials protected from light until the completion of
various tests. The yields of the extracts (%) were
calculated (Boulenouar et al., 2009; Salem, 2009).
Phytochemical screening
Phytochemical tests the dry extracts (ethanolic,
ethanolic-water and aqueous) bioactive compounds
was realized using standard procedures. Two
extractions methods (maceration and decoction) were
used (Bruneton, 1999; Bidié et al., 2008). The extracts
were tested qualitatively for the presence of chemical
constituents such as sterols, terpenoids, tannins,
polyphenols, flavonoids, quinones and saponins (Paris
and Moyses, 1969; Bouquet and Debray 1971). The
alkaloids were tested by the method of Dragendorff
and Meyer (Bruneton, 1999).
Phenols and proanthocyanidins content extracts
The Folin-Ciocalteu method was used to measure total
amount of total phenols content (Singleton et al.,
1999). Aliquots of 0.25 mL of leaf extracts (1 mg/mL)
were mixed with 1.25 mL Folin–Ciocalteu reagent (0.2
N diluted in Methanol). A reagent blank using
methanol instead of sample was prepared. After 5 min
Akoué et al.
3 Int. J. Biomol. Biomed.
incubation at room temperature, 1 mL sodium
carbonate solution (7.5 %) was added. Samples were
incubated at room temperature for 1 h and the
absorbance was measured at 765 nm versus the
prepared blank. All tests were carried out in triplicate
and total phenols content was expressed as mg of gallic
acid equivalents (GAE) per 100 g of drug.
Table 1. Composition phytochemical extracts of Sakersia africana Hook.f.
Chemical compounds Aqueous Ethanolic-water Ethanolic
Saponins +++ + -
Gallic tannins ++ +++ +++
Catechic tannins +++ +++ +++
Anthraquinones - - -
Total phenols +++ +++ +++
Total flavonoids - +++ +++
Flavanonols - +++ -
Flavones - - +
Flavanones - - -
Coumarins +++ +++ ++
Digitoxin +++ +++ ++
Digitoxigenin - - -
Gitoxin - - -
Gitoxigenin - - -
Alkaloids +++ +++ +++
Sterols and triterpenes +++ +++ +++
Reducters compounds +++ +++ +++
+++ = High, ++ = Moderate; + = Low; -: negative test.
Table 2. Comparison of total phenolic compound, proanthocyanidins and antiradical activity of extracts de Sakersia
africana Hook.f.
Extracts
Yields (%) Total phenols
(mg GAE/100 g
of drug)
PAs (mg
APE/100 g of
drug)
Quota of PAs in
Total phenols (%)
DPPH:
IC50 (µg/ml)
Aqueous 4.04 507.42 ± 0.17 113.5 ± 3.17 22.37± 3.17 195.54± 0.012
Ethanolic-water 4.36 391.58 ± 0.04 341.83 ± 36.5 87.30 ± 36.5 164.21± 0.014
Ethanolic 5.56 777 ± 0.03 653.5 ± 36.83 84.11 ± 36.83 177.02± 0.013
Proanthocyanidins (PAs) were quantified with the
hydrolysis test of proanthocyanidins in a hot acid-
alcohol medium into anthocyanidins. This method
allows taking into account all the units of flavans-3-ols
constituting the polymers (Prigent, 2005). The heating
step destroys the anthocyanidins pigments generated
by flavan-4-ols and eliminates part of the chlorophyll
pigments. The routine assay is performed by mixing
0.16 mL (1 mg/mL) of the extract with 2.33 mL of 30 %
HCl-butanol solution (v/v). The mixture was put in
tightly closed tube and vortexed for 1 min.
Subsequently, the tube was heated at 100°C for 2 h and
after cooling, the absorbance was read at 550 nm.
Apple procyanidins (DP ≈ 7.4) treated as
Akoué et al.
4 Int. J. Biomol. Biomed.
aforementioned were used as a standard. Results were
expressed as apple procyanidins equivalent (APE).
Antiradical activity
DPPH spectrophotometric (quantitative) assay was
performed with some modifications (Brand-Williams
et al., 1995). Method was widely used to test the ability
of antioxidant bioactive compounds to activity as free
radical scavengers or hydrogen donors. This test is
based on the capacity of stable free radical 2, 2-
diphenyl-1-picrylhydrazyl to react with hydrogen (H)
donors, including phenols. It is used for the
quantification of antioxidants in the complex of
biological systems (Miliauskas et al., 2004). Each
sample of extract was prepared at different
concentrations (100, 200, 300 and 400 μg/mL). The
reaction mixture contained 1 mL of DPPH prepared at
a concentration 20 mg/L in methanol, and 1 ml of test
samples. After a 30 min reaction, the absorbance was
read at 517 nm and converted into percentage of
antiradical activity (AA %), using the following the
formula (Abdoul-Latif et al., 201O).
%DPPH radical scavenging = ((Abscontrol - Abssample))/
Abscontrol) X 100
Where,
Abscontrol is the absorbance of the blank;
Abssample is the absorbance of the sample.
Control contained 1 mL of DPPH solution and 3 mL of
methanol. The measurements of DPPH radical
scavenging activity were carried out for three sample
replications, and values are an average of three
replicates. IC50 is defined as the concentration of the
test sample leading to a 50 % inhibition of the DPPH
free radicals. IC50 value was calculated from the
separate linear regression of plots of the mean
percentage of the antioxidant activity against
concentration of the test compounds (mg /mL)
obtained from three replicate assays.
Results and discussion
Phytochemical screening
The results show that all extracts are rich in
sterols,triterpenes, tannins (catechic and gallic),
polyphenols, alkaloids, and reducing compounds.
Coumarins and cardiac glycosides (digitoxins) are
abundant in aqueous and ethanolic-water extracts.
Only the aqueous extract contains saponins and no
anthraquinons free flavanones, digitoxigenin, and
gitoxin gitoxigenine. Also, some extracts (ethanolic and
ethanolic-water) are rich in flavonoids total (Table 1).
Phytochemical analysis is very useful in the evaluation
of some active biological components of Sakersia
africana. The qualitative and quantitative analyses
were carried out in both dry extract show that Sakersia
africana is rich in phenolic compounds (polyphenols,
tannins, flavonoids) in terpenic and nitrogen
compounds. This wealth of pharmacological
compounds conferred to said plant several
pharmacological properties. The abundance of tannins
explains the use of Sakersia africana ethnotherapy for
the treatment of pathology identified during this
investigation. These are diseases such as hypertension,
dysentery, diarrhea, stomach ulcers and stomach
aches. Indeed, it has been shown that the higher the
plant is rich in tannic compounds will be more effective
for the treatment of diseases of hypertension,
dysentery, diarrhea, hemorrhoids, stomach ulcer,
stomach ache and some various venereal diseases
(Kerharo and Adjanohoun 1989; Pousset, 1989). In
addition, the healing properties of plants are correlated
with the abundance of tannins. This is the case for
example Lannea acida, rich in tannins, is used in
traditional medicine for wound healing (Sereme et al.,
2008). This could also justify the healing properties
attributed to Sakersia africana (Walker and Sillans
1961).
Total polyphenols, proanthocyanidins contents
The yields of different extracts of Sakersia africana
were respectively 4.04 % (aqueous), 4.36 % (ethanolic-
water) and 5.56 % (ethanolic) (table 2). The
concentrations of total phenols in the different extracts
of the plant of study are more significant in
Akoué et al.
5 Int. J. Biomol. Biomed.
comparison with those of the cereals (0,481 à 0,896
mg/g of matter) appreciably equal with those of other
plants such as Broccolis (11.7 mg /g of matter), the
gallic ones (9.9 mg/g of matter) and the fruits (23.1
mg/g of matter for the blackberry) (Vinson et al., 1995;
Wang and Lin, 2000).
Fig. 1. Antiradical activity extracts of Sakersia
africana Hook.f.
Fig. 2. Correlation between antioxidant activity and
total phenols, (R2 = 0.8605).
Levels of phenolic content were expressed in terms of
gallic acid equivalent (GAE). The equation of the right
and side of the proportioning of total phenolic content
by the method of Folin-Ciocalteu gave Y = 0.0012 X –
0.0004 with R2 = 0.9902 (Abdoul-Latif et al., 2012).
The HCl/butanol assay used here for the determination
of proanthocyanidins is more specific than many other
tests such as the vanillin assay (Makkar, 2000; Santos-
Buelga and Scalbert, 2000). The interferences, which
might result from flavan-4-ols conversion into
proanthocyanidins or from chlorophylls, may have
been minimized during the heating step (Prigent,
2005; Santos-Buelga and Scalbert, 2000).
Levels of proanthocyanidins were expressed in terms
of apple proanthocyanidins equivalent (APE). The
equation of the right-hand side of the proportioning of
the proanthocyanidins by the HCl- Butanol method
gave Y = 0.0006 X + 0.0024 with R2 = 0.9869
(Abdoul-Latif et al., 2012) Among extracts,
proanthocyanidins contents ranged between 113.5 and
653.5 mg APE/100 g of drug (Table 2).
The total contents of phenols ranged between 391.58
and 777 mg GAE/100 g of drug (table 2). However,
87.30 and 84.11 % of total phenols in ethanolic-water
extracts and ethanolic respectively are
proanthocyanidins. Only the aqueous extract has a low
percentage of proanthocyanidins (22.37 %).
Correlation between antioxidant activity and total
phenols
Several comprehensive works have been done on the
effects of phenolic compounds on total antioxidants (Li
et al., 2008; Kim et al., 2009; Kouamé et al., 2009 and
Abdoul-Latif et al., 2010), and correlations between
phenolic compounds and total antioxidants
(Chanwitheesuk et al., 2005; Li et al., 2008). This
same trend was also obtained in our study. There was a
good linear correlation (R2 = 0.8605, p < 0.05)
between the total phenolic content and the scavenging
of DPPH radical in each extract (Figure 1). These
results are similar to those obtained by Njintang and
collaborators in their study on antiradical activity and
polyphenol content of ethanolic extracts of Propolis
deed. These authors showed that the antiradical
activity was correlated with the polyphenol content in
different extracts (Njintang et al. 2012).
Antiradical activity
Antioxidant activity using DPPH radical-scavenging
assay expressed as IC50 value is showed in table 2 lower
IC50 indicating the higher antioxidant activity of
extract. The results of DPPH antiradical activity were
differed significantly between different extracts. This
difference is explained by the fact that an antiradical
activity of phenolic compounds depends on their
molecular structure, on the availability of phenolic
Akoué et al.
6 Int. J. Biomol. Biomed.
hydrogens and on the possibility for stabilization of the
resulting phenoxyl radicals formed by hydrogen
donation (Catherine et al., 1996; Ramarathnam et al.,
1997; Sundaram et al., 2011). These results of
antiradical activity of Sakersia africana show that
extracts exhibit antiradical activity from a
concentration of 50 μg/mL (aqueous and ethanolic-
water) (figure 1 and table 2). The radical scavenging
activity of ethanolic extract starts at concentrations
that are higher than 50 μg/mL. The lowest
concentration IC50 that inhibits half of the DPPH
radical was obtained with ethanolic-water extract
(164.21 mg/mL) followed by ethanol extract (177.02
mg/mL). The aqueous extract show the highest
inhibitory concentration (IC50 = 195.54 mg/mL) (table
2). These results also show that Sakersia africana have
a significant antiradical activity. However, ethanolic-
water and ethanol extract were the higher antioxidant
activity with an IC50 respectively 164.21μg/mL and
177.02 mg/mL. Aqueous extract show lower activity
(IC50 = 195.54 mg/mL). In terms of concentrations of
phenolic compounds in different extracts, it appears
that ethanolic-water extract is less rich in total
polyphenols (391.58 mg EGA/g of drug) and more
active (IC50 = 164.21 mg/mL). It is observed probably
result from the fact that the determination by the
Folin-Ciocalteu reagent is not specific to polyphenols.
Other compounds may interact with said reagent and
provide a high level of polyphenols (Catherine et
al.,1996; Ramarathnam et al, 1997). Thus ethanolic
and aqueous extracts are apparently rich while the
actual content of total phenols is low.
Conclusion
The present study was aimed to evaluate the
phytochemical and antiradical allow us to infer that the
use of Sakersia africana traditherapy by people
against various diseases would depend on its relative
wealth in saponins, phenolic compounds (tannins,
Coumarins and flavonoids) and nitrogen (alkaloids)
endowed of pharmacological properties. This
abundance of active plant gives remarkable properties,
which could justify its multiple therapeutic indications
for which it is used traditherapy. These preliminary
results could provide a scientific basis for the research
of new therapeutic molecules. Further pharmacological
investigations allow us to determine precisely the
different biological activities and to evaluate the acute
and subacute Sakersia africana.
Acknowledgements
The authors thank anyone who contributed to the
completion of this work. Especially we thought Mrs
Daniel Ebozogho Metou, Benoit Ndong Mozogho and
Jean-Calixte Nguema Akoué for her contribution
related to the informations on traditional use of the
plant like this Dr Alain Ondo Azi and Pr Crépin Ella
Missang for the complete support throughout the work
with timely and valuable discussions.
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Phytochemical composition and antiradical activity of Sakersia africana Hook. f. medicinal plant from Gabon

  • 1. Akoué et al. 1 Int. J. Biomol. Biomed. RESEARCH PAPER OPEN ACCESS Phytochemical composition and antiradical activity of Sakersia africana Hook. f. medicinal plant from Gabon Gontran Nsi Akoué1,2 , Louis-Clément Obame2* , Joseph Privat Ondo2 , Ibrahim Brama1 , Elvis Simplice Otogo N’Nang2 , Skitt Yvan Tapoyo2 , Alain Souza1 1 Laboratoire de Physiologie Animale: Electrophysiologie-Pharmacologie-URAB, Université des Sciences et Techniques de Masuku, BP 913, Franceville Gabon 2 Laboratoire de Substances Naturelles et de Synthèses Organométalliques, URCHI-Université des Sciences et Techniques de Masuku, BP 943, Franceville Gabon Article Published: 17 May 2013 Key words: Sakersia africana Hook.f., phytochemical screening, total phenols, proanthocyanidins, antiradical activity. Abstract The valorization of the medicinal plants of our country and determination of their impact on health due to their abundance of substances with various pharmacological effects are our principal objective. This study was evaluated the phytochemical screening and radical 2, 2-Diphenyl-1-picrylhydrazyl (DPPH) scavenging activity of different extracts of Sakersia africana Hook. f.. The results revealed that Sakersia africana Hook. f. is rich in phenols compounds, sterols, triterpenes, alkaloids and reducing compound. The values in total phenols and proanthocyanidines are ranging respectively from 391.58 ± 0.04 to 777 ± 0.03 mg/100 g of drugs and 113.5 ± 3.17 to 653.5 ± 36.83 mg/100 g of drugs. Results also show that different extracts tested present antiradical activity with values of IC50 ranging from 164.21± 0.014 to 195.54± 0.012 % and abundance in bioactive compounds. This study could justify the use of Sakersia africana of some chronic diseases. *Corresponding Author: Louis-Clément Obame  obamengonga@gmail.com, yacth58@hotmail.com International Journal of Biomolecules and Biomedicine (IJBB) ISSN: 2221-1063 (Print) 2222-503X (Online) Vol. 3, No. 3, p. 1-8, 2013 http://www.innspub.net
  • 2. Akoué et al. 2 Int. J. Biomol. Biomed. Introduction The plants were used for prehistory by Human for nutritional and therapeutic requirement. Medicinal plants are the major source of drugs through their rich secondary metabolites (Fouché et al., 2000; Nostro et al., 2000). Thus, medicinal plants are the main means to fight against diseases by including populations of underdeveloped countries. This practice was abandoned in favor of modern medicine. We are witnessing a return to use of medicinal plants over the past decade. World Health Organization (WHO), in 2007 estimated that about 80 % of the population of developing countries can be cured from plants (Obame, 2009). In 2001, the studies have revealed that approximately 25 % of prescriptions in the world for drugs herbal and 60 to 70 % of antibacterial and anticancer drugs are substances of natural origin (Rate, 2001). Hence the useful achieve the phytochemical studies of medicinal plants for discovery of news bioactive compounds. As such the WHO is currently supporting clinical validation of some traditional medicines. It is a shrub of the Melastomataceae family that grows on moist soils and in secondary forest (Walker and Sillans 1961). The leaves and twigs in a hairy very steep, and the protruding ribs of a green- red, turn yellow or red before falling. Fruit globular form berries are very small and contain many tiny seeds. The decoction of the leaves has healing properties crushed. It is used as healing by gabonese populations in southern and the treatment of several diseases as dysentery, diarrhea, stomach ulcers and stomach aches (Walker and Sillans, 1961). The objective of this study was to evaluate the phytochemical characteristics and radical scavenging activity of different extracts of Sakersia africana Hook. f. Material and methods Plant material The branches of S. africana were collected in september 2012 in Mbomo village, at 28 km of Oyem city (Northern Province of Gabon). The plant was toxonomically authenticated at the Laboratory of Plant Biology and Ecology of University of Science and Technology of Masuku (Franceville, Gabon), where a voucher specimen was deposited. Preparation of plant extract The branches of Sakersia africana was air-dried at room temperature for a total period six weeks and pulverized to powder using a clean electric blender (Model Phillips 190). A 25 g sample of the pulverized branches of Sakersia africana was soaked in 500 mL of solvent (ethanol, ethanol-water (50/50 v/v) and water) and allowed to stand for 72 h with intermittent stirring. This was filtered through a whatman No. 1 filter paper and the filtrate obtained was evaporated to a dry mass using a rotary rotavaporator at 40 °C. The residues recovered were dried in an oven at a temperature of 65 °C. The extract obtained is stored in vials protected from light until the completion of various tests. The yields of the extracts (%) were calculated (Boulenouar et al., 2009; Salem, 2009). Phytochemical screening Phytochemical tests the dry extracts (ethanolic, ethanolic-water and aqueous) bioactive compounds was realized using standard procedures. Two extractions methods (maceration and decoction) were used (Bruneton, 1999; Bidié et al., 2008). The extracts were tested qualitatively for the presence of chemical constituents such as sterols, terpenoids, tannins, polyphenols, flavonoids, quinones and saponins (Paris and Moyses, 1969; Bouquet and Debray 1971). The alkaloids were tested by the method of Dragendorff and Meyer (Bruneton, 1999). Phenols and proanthocyanidins content extracts The Folin-Ciocalteu method was used to measure total amount of total phenols content (Singleton et al., 1999). Aliquots of 0.25 mL of leaf extracts (1 mg/mL) were mixed with 1.25 mL Folin–Ciocalteu reagent (0.2 N diluted in Methanol). A reagent blank using methanol instead of sample was prepared. After 5 min
  • 3. Akoué et al. 3 Int. J. Biomol. Biomed. incubation at room temperature, 1 mL sodium carbonate solution (7.5 %) was added. Samples were incubated at room temperature for 1 h and the absorbance was measured at 765 nm versus the prepared blank. All tests were carried out in triplicate and total phenols content was expressed as mg of gallic acid equivalents (GAE) per 100 g of drug. Table 1. Composition phytochemical extracts of Sakersia africana Hook.f. Chemical compounds Aqueous Ethanolic-water Ethanolic Saponins +++ + - Gallic tannins ++ +++ +++ Catechic tannins +++ +++ +++ Anthraquinones - - - Total phenols +++ +++ +++ Total flavonoids - +++ +++ Flavanonols - +++ - Flavones - - + Flavanones - - - Coumarins +++ +++ ++ Digitoxin +++ +++ ++ Digitoxigenin - - - Gitoxin - - - Gitoxigenin - - - Alkaloids +++ +++ +++ Sterols and triterpenes +++ +++ +++ Reducters compounds +++ +++ +++ +++ = High, ++ = Moderate; + = Low; -: negative test. Table 2. Comparison of total phenolic compound, proanthocyanidins and antiradical activity of extracts de Sakersia africana Hook.f. Extracts Yields (%) Total phenols (mg GAE/100 g of drug) PAs (mg APE/100 g of drug) Quota of PAs in Total phenols (%) DPPH: IC50 (µg/ml) Aqueous 4.04 507.42 ± 0.17 113.5 ± 3.17 22.37± 3.17 195.54± 0.012 Ethanolic-water 4.36 391.58 ± 0.04 341.83 ± 36.5 87.30 ± 36.5 164.21± 0.014 Ethanolic 5.56 777 ± 0.03 653.5 ± 36.83 84.11 ± 36.83 177.02± 0.013 Proanthocyanidins (PAs) were quantified with the hydrolysis test of proanthocyanidins in a hot acid- alcohol medium into anthocyanidins. This method allows taking into account all the units of flavans-3-ols constituting the polymers (Prigent, 2005). The heating step destroys the anthocyanidins pigments generated by flavan-4-ols and eliminates part of the chlorophyll pigments. The routine assay is performed by mixing 0.16 mL (1 mg/mL) of the extract with 2.33 mL of 30 % HCl-butanol solution (v/v). The mixture was put in tightly closed tube and vortexed for 1 min. Subsequently, the tube was heated at 100°C for 2 h and after cooling, the absorbance was read at 550 nm. Apple procyanidins (DP ≈ 7.4) treated as
  • 4. Akoué et al. 4 Int. J. Biomol. Biomed. aforementioned were used as a standard. Results were expressed as apple procyanidins equivalent (APE). Antiradical activity DPPH spectrophotometric (quantitative) assay was performed with some modifications (Brand-Williams et al., 1995). Method was widely used to test the ability of antioxidant bioactive compounds to activity as free radical scavengers or hydrogen donors. This test is based on the capacity of stable free radical 2, 2- diphenyl-1-picrylhydrazyl to react with hydrogen (H) donors, including phenols. It is used for the quantification of antioxidants in the complex of biological systems (Miliauskas et al., 2004). Each sample of extract was prepared at different concentrations (100, 200, 300 and 400 μg/mL). The reaction mixture contained 1 mL of DPPH prepared at a concentration 20 mg/L in methanol, and 1 ml of test samples. After a 30 min reaction, the absorbance was read at 517 nm and converted into percentage of antiradical activity (AA %), using the following the formula (Abdoul-Latif et al., 201O). %DPPH radical scavenging = ((Abscontrol - Abssample))/ Abscontrol) X 100 Where, Abscontrol is the absorbance of the blank; Abssample is the absorbance of the sample. Control contained 1 mL of DPPH solution and 3 mL of methanol. The measurements of DPPH radical scavenging activity were carried out for three sample replications, and values are an average of three replicates. IC50 is defined as the concentration of the test sample leading to a 50 % inhibition of the DPPH free radicals. IC50 value was calculated from the separate linear regression of plots of the mean percentage of the antioxidant activity against concentration of the test compounds (mg /mL) obtained from three replicate assays. Results and discussion Phytochemical screening The results show that all extracts are rich in sterols,triterpenes, tannins (catechic and gallic), polyphenols, alkaloids, and reducing compounds. Coumarins and cardiac glycosides (digitoxins) are abundant in aqueous and ethanolic-water extracts. Only the aqueous extract contains saponins and no anthraquinons free flavanones, digitoxigenin, and gitoxin gitoxigenine. Also, some extracts (ethanolic and ethanolic-water) are rich in flavonoids total (Table 1). Phytochemical analysis is very useful in the evaluation of some active biological components of Sakersia africana. The qualitative and quantitative analyses were carried out in both dry extract show that Sakersia africana is rich in phenolic compounds (polyphenols, tannins, flavonoids) in terpenic and nitrogen compounds. This wealth of pharmacological compounds conferred to said plant several pharmacological properties. The abundance of tannins explains the use of Sakersia africana ethnotherapy for the treatment of pathology identified during this investigation. These are diseases such as hypertension, dysentery, diarrhea, stomach ulcers and stomach aches. Indeed, it has been shown that the higher the plant is rich in tannic compounds will be more effective for the treatment of diseases of hypertension, dysentery, diarrhea, hemorrhoids, stomach ulcer, stomach ache and some various venereal diseases (Kerharo and Adjanohoun 1989; Pousset, 1989). In addition, the healing properties of plants are correlated with the abundance of tannins. This is the case for example Lannea acida, rich in tannins, is used in traditional medicine for wound healing (Sereme et al., 2008). This could also justify the healing properties attributed to Sakersia africana (Walker and Sillans 1961). Total polyphenols, proanthocyanidins contents The yields of different extracts of Sakersia africana were respectively 4.04 % (aqueous), 4.36 % (ethanolic- water) and 5.56 % (ethanolic) (table 2). The concentrations of total phenols in the different extracts of the plant of study are more significant in
  • 5. Akoué et al. 5 Int. J. Biomol. Biomed. comparison with those of the cereals (0,481 à 0,896 mg/g of matter) appreciably equal with those of other plants such as Broccolis (11.7 mg /g of matter), the gallic ones (9.9 mg/g of matter) and the fruits (23.1 mg/g of matter for the blackberry) (Vinson et al., 1995; Wang and Lin, 2000). Fig. 1. Antiradical activity extracts of Sakersia africana Hook.f. Fig. 2. Correlation between antioxidant activity and total phenols, (R2 = 0.8605). Levels of phenolic content were expressed in terms of gallic acid equivalent (GAE). The equation of the right and side of the proportioning of total phenolic content by the method of Folin-Ciocalteu gave Y = 0.0012 X – 0.0004 with R2 = 0.9902 (Abdoul-Latif et al., 2012). The HCl/butanol assay used here for the determination of proanthocyanidins is more specific than many other tests such as the vanillin assay (Makkar, 2000; Santos- Buelga and Scalbert, 2000). The interferences, which might result from flavan-4-ols conversion into proanthocyanidins or from chlorophylls, may have been minimized during the heating step (Prigent, 2005; Santos-Buelga and Scalbert, 2000). Levels of proanthocyanidins were expressed in terms of apple proanthocyanidins equivalent (APE). The equation of the right-hand side of the proportioning of the proanthocyanidins by the HCl- Butanol method gave Y = 0.0006 X + 0.0024 with R2 = 0.9869 (Abdoul-Latif et al., 2012) Among extracts, proanthocyanidins contents ranged between 113.5 and 653.5 mg APE/100 g of drug (Table 2). The total contents of phenols ranged between 391.58 and 777 mg GAE/100 g of drug (table 2). However, 87.30 and 84.11 % of total phenols in ethanolic-water extracts and ethanolic respectively are proanthocyanidins. Only the aqueous extract has a low percentage of proanthocyanidins (22.37 %). Correlation between antioxidant activity and total phenols Several comprehensive works have been done on the effects of phenolic compounds on total antioxidants (Li et al., 2008; Kim et al., 2009; Kouamé et al., 2009 and Abdoul-Latif et al., 2010), and correlations between phenolic compounds and total antioxidants (Chanwitheesuk et al., 2005; Li et al., 2008). This same trend was also obtained in our study. There was a good linear correlation (R2 = 0.8605, p < 0.05) between the total phenolic content and the scavenging of DPPH radical in each extract (Figure 1). These results are similar to those obtained by Njintang and collaborators in their study on antiradical activity and polyphenol content of ethanolic extracts of Propolis deed. These authors showed that the antiradical activity was correlated with the polyphenol content in different extracts (Njintang et al. 2012). Antiradical activity Antioxidant activity using DPPH radical-scavenging assay expressed as IC50 value is showed in table 2 lower IC50 indicating the higher antioxidant activity of extract. The results of DPPH antiradical activity were differed significantly between different extracts. This difference is explained by the fact that an antiradical activity of phenolic compounds depends on their molecular structure, on the availability of phenolic
  • 6. Akoué et al. 6 Int. J. Biomol. Biomed. hydrogens and on the possibility for stabilization of the resulting phenoxyl radicals formed by hydrogen donation (Catherine et al., 1996; Ramarathnam et al., 1997; Sundaram et al., 2011). These results of antiradical activity of Sakersia africana show that extracts exhibit antiradical activity from a concentration of 50 μg/mL (aqueous and ethanolic- water) (figure 1 and table 2). The radical scavenging activity of ethanolic extract starts at concentrations that are higher than 50 μg/mL. The lowest concentration IC50 that inhibits half of the DPPH radical was obtained with ethanolic-water extract (164.21 mg/mL) followed by ethanol extract (177.02 mg/mL). The aqueous extract show the highest inhibitory concentration (IC50 = 195.54 mg/mL) (table 2). These results also show that Sakersia africana have a significant antiradical activity. However, ethanolic- water and ethanol extract were the higher antioxidant activity with an IC50 respectively 164.21μg/mL and 177.02 mg/mL. Aqueous extract show lower activity (IC50 = 195.54 mg/mL). In terms of concentrations of phenolic compounds in different extracts, it appears that ethanolic-water extract is less rich in total polyphenols (391.58 mg EGA/g of drug) and more active (IC50 = 164.21 mg/mL). It is observed probably result from the fact that the determination by the Folin-Ciocalteu reagent is not specific to polyphenols. Other compounds may interact with said reagent and provide a high level of polyphenols (Catherine et al.,1996; Ramarathnam et al, 1997). Thus ethanolic and aqueous extracts are apparently rich while the actual content of total phenols is low. Conclusion The present study was aimed to evaluate the phytochemical and antiradical allow us to infer that the use of Sakersia africana traditherapy by people against various diseases would depend on its relative wealth in saponins, phenolic compounds (tannins, Coumarins and flavonoids) and nitrogen (alkaloids) endowed of pharmacological properties. This abundance of active plant gives remarkable properties, which could justify its multiple therapeutic indications for which it is used traditherapy. These preliminary results could provide a scientific basis for the research of new therapeutic molecules. Further pharmacological investigations allow us to determine precisely the different biological activities and to evaluate the acute and subacute Sakersia africana. Acknowledgements The authors thank anyone who contributed to the completion of this work. Especially we thought Mrs Daniel Ebozogho Metou, Benoit Ndong Mozogho and Jean-Calixte Nguema Akoué for her contribution related to the informations on traditional use of the plant like this Dr Alain Ondo Azi and Pr Crépin Ella Missang for the complete support throughout the work with timely and valuable discussions. References Abdoul-Latif F, Edou P, Eba F, Ahmed N, Adwa A, Djama S, Obame LC, Bassolé I, Dicko MH. 2010. Antimicrobial and antioxidant activities of essential oil and methanol extract of Jasminum sambac from Djibouti. African Journal of Plant Science 4, 30-31. Abdoul-Latif F, Edou P, Eba F, Ahmed N, Adwa A, Djama S, Obame LC, Bassolé I, Dicko MH. 2011. Antimicrobial and antioxidant activities of essential oil and methanol extract of Matricaria chamomilla L. from Djibouti; Journal of Medicinal Plants Research 5, 1512-1517. Abdoul-latif FM, Romaric G, Bayili, Obame LC, Bassolé HN and. Dicko MH. 2012. Comparison of phenolic comounds and antioxydant capacities of traditional sorghum beers with other alcoholic beverages. African Journal of Biotechnology 11, 14671- 14678. Bidié AP, Koffi E, N’guessan JD, Djaman AJ and Guédé-Guina F. 2008. Influence of Mitragyna ciliata (Myta) on the microsomalactivity of ATPase
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