Innovare International Journal of Pharmacy and Pharmaceutical Sciences

Academic Sciences
ISSN- 0975-1491 Vol 6, Issue 11, 2014

Original Article

ACACIA ATAXACANTHA (BARK): CHEMICAL COMPOSITION AND ANTIBACTERIAL ACTIVITY

OF THE EXTRACTS

A. M. O. AMOUSSA, L. LAGNIKA*, A. SANNI

Unité de Biochimie et de Biologie Moléculaire, Team of Biochemistry and Bioactives Natural Substances, Faculté des Sciences et
Techniques, University of Abomey-calavi, Benin.
Email: [email protected]
Received: 09 Sep 2014 Revised and Accepted: 10 Oct 2014
ABSTRACT
Objective: The present work aimed to investigate the phytochemical constituents and antibacterial activity of several extracts of Acacia
ataxacantha bark on pathogenic microbes.
Methods: The phytochemical study was performed using tube test and Thin layer chromatography methods. The growth inhibitory effect and the
Minimum Inhibitory Concentration of hexane, dichloromethane, ethyl acetate, methanol and hydroalcolic extracts were determined using the
microplate dilution method.
Results: Phytochemical screening revealed the presence of alkaloid, tannins, lignan, triterpenoids, anthracene derivatives, flavonoids, saponins and
coumarins at different level. The extracts exhibited different effect against the tested bacteria. The minimum inhibitory concentrations of extracts
ranged from 325 µg/ml to 5 mg/ml. Ethyl acetate extract was more potent than other extracts with the MIC values of 325 µg/ml against S. aureus
and 625 µg/ml against all other tested bacteria. Escherichia coli was resistant to all extracts.
Conlusion: These findings suggest that the rich phytochemical content of Acacia ataxacantha and its good antibacterial activity may be responsible
for its popular and wide traditional use.
Keywords: Acacia ataxacantha bark, Phytochemical constituent, Antibacterial activity.

INTRODUCTION In the best of our knowledge, there is no scientific information

concerning the in vitro antibacterial activity of this medicinal plant.
Infectious diseases are becoming a crisis as a major cause of
mortality and human and animal morbidity [1]. They cause massive In our search for natural products with antibacterial activity, we
mortality in developing countries due to the extreme poverty of the investigated a traditional plant used in Benin (West Africa) to treat
population compared to developed countries. This situation is infectious diseases. Polar and apolar extracts were prepared from
aggravated by the lack of appropriate vaccine, inaccessibility and/or selected plant and then evaluated for in vitro antibacterial activity
the absence of antibiotics and the emergence of antibiotic resistant against four Gram positive and two Gram negative bacteria. First, by
strains. Many efforts have been made by researchers to discover assessing the ability of each extract to inhibit the growth of bacteria
new antibacterial compound. One important source of discovery of at a concentration of 10 mg/ml, secondly, by determined the
the antibacterial drugs is medicinal plants through traditional Minimum Inhibitory Concentration and total activity of extracts. The
medicines. Traditional treatments have been investigated and a chemical constituent of the plant was also determined.
significant success was achieved. In addition, it is known that, unlike
synthetic drugs, antimicrobials of plant origin are not associated MATERIALS AND METHODS
with many side effects and have an enormous therapeutic potential Plant material
to cure many infectious diseases [2, 3].
The barks of Acacia ataxacantha were collected in September 2012
The use of herbal medicine in Benin is a long history of human from Ouidah, Department of Atlantic, South Bénin. Botanical
interaction with the environment. The plants used in traditional determination was performed by taxonomists from the Herbier
medicine contain a wide range of substances that can be used to National of Abomey-Calavi University in Benin and voucher
treat chronic and infectious diseases. Acacia species have had a long specimens were deposited at the same herbarium (AA 6509/ HNB).
history of medicinal use in the treatment of diarrhea, urinary The collected material was dried for two weeks in the laboratory
infections, throat inflammation, gastritis, tuberculosis and (22°C) and ground to a fine powder using an electric grinder (Excella
headaches [4]. mixer grinder).
Acacia Ataxacantha (Fabaceae) is widespread in much of sub- Preparation of the extracts
Saharan Africa. This species is a very thorny shrub scandent 5 to 8 m
in height. The leaves are alternate, pinnate with spine that carries 5 Two hundred and fifty grams (250 g) of dry powder of the barks of
to 12 pairs of pinnae. On twigs, spines are short, clearly pointing Acacia ataxacantha were successively extracted by maceration with
down. The white flowers with a long transition axillary 4 to 5 cm hexane, dichloromethane, ethyl acetate and methanol for 72 h
long and arranged on stem 10 to 15 mm are sometimes isolated in stirring. Fifty grams (50 g) of dry powder was also extracted with a
pairs. The fruit pods are flattened, brownish red in the dry state. The mixture of ethanol-water (20:80). Each extraction is repeated three
dough sheet is used topically in the treatment of abscesses. The leaf times. The macerates were filtered and concentrated using a rotary
decoction is used orally in febrile convulsions. Its bark is used evaporator (RE 300, stuart) and the extracts were stored at 4° C
against tooth decay, by inhalation in case of bronchitis and cough until biological assay.
[5]. In Nigeria, pods and seeds of Acacia ataxacantha were also used Phytochemical analysis
against dysentery [6]. The roots are used in Kenya in the treatment
of joint and back pain [7]. Acacia ataxacantha is also known for the Phytochemical screening of the plant was carried out according to
treatment of pneumonia [8]. the methods described by Wagner and Blat [9] and Bruneton [10]
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Int J Pharm Pharm Sci, Vol 6, Issue 11, 138-141

for the detection of plant secondary metabolites. Tannins, alkaloids, well and microplate was incubated at 37°C. Finally, after 30 min, the
flavonoids, steroids, coumarins, saponins, naphthoquinones, color change (extract color to red) of mix in each well was examined to
triterpenes, lignans, pigments, anthracene derivatives have been select actives extracts. Active extract do not change color.
investigated using tube test. Each extract (10 mg/ml) were
deposited on TLC plate to confirm the results. Minimum Inhibitory Concentration (MIC)

Antibacterial activity The Minimum Inhibitory Concentrations (MICs) of antibacterial

(sensitive bacterial) activity were determined by the method of
Bacterial strains broth microdilution using p-iodonitrotetrazolium (INT) as an
indicator of bacterial viability [12]. To determine the MIC of extracts,
Several extracts of acacia ataxacantha bark were individually tested
100 μl of Mueller Hinton broth (DIFCO) were added to each wells of
against a panel of bacteria including four Gram-positive: a 96-wells microplate and then 100 μl of plant extract (20 mg/ml)
Staphylococcus aureus (ATCC 6538), S. epidermidis (CIP8039), were added to the first well (A) of the plate. A two-fold Successive
Enterococcus faecalis (ATCC 29212), Staphylococcus aureus Methicillin dilution was carried from well (A) to the last wells (H) of the plate.
Resistant (SARM) and two Gram-negative: Escherichia coli (CIP 53126) Then, 100μl of bacterial broth at 106CFU/ml were finally added into
and Pseudomonas aeruginosa (CIP82118) obtained from Laboratoire all the wells. The plate was covered and incubated at 37°C. After 18 h
de Biophotonique et Pharmacologie, University of Strasbourg, France. of incubation, 40 μl of p-iodonitrotétrazolium (0.2 %) was added in
Growth inhibition effect of extracts at 10 mg/ml all wells and the plates were incubated again at 37°C. After 1 h of
incubation, wells were examined and the MIC values were recorded.
Sensivity of different bacterial strains to various extracts was
determined using 96-well microplate. The aim of this method was to Total activity
eliminate the extracts, which at 10 mg/ml do not inhibit the growth To select the extracts that can be used for further testing, the
of bacteria [11]. The extracts were reconstituted to a concentration determination of the total activity is important because since the
of 20 mg/ml in acetone/Muller Hinton broth culture. A volume of MIC value is inversely proportional to the amount of antimicrobial
100 μL of each extract (20 mg/ml) was introduced in triplicate extracts. The total activity of each extract was calculated by dividing
microplate already seeded with 100 μL of the Muller Hinton broth the MICs with the amount of extract obtained from 1 g of plant
culture inoculums (106CFU/ml) of the tested bacteria. The microplate material [13]. This value indicates the volume in which the active
was incubated at 37°C. After 18 h of incubation, 40 μL of 0.2 mg/ml principle (extract) present in 1 g of dry plant material can be diluted
solution of p-iodonitrotetrazolium (Sigma Aldrich) were added to each to always have inhibitory activity against organisms [14].

Table 1: Phytochemical constituents of bark extracts from A. ataxacantha

Plant extracts
Phytochemical component Hex CH 2 Cl 2 AcOEt MeOH H 2 O/Et
Coumarin + + + + +
Flavonoid - + ++ + +
Naphtoquinone - ++ ++ - -
Alkaloid - + + + +
anthracene derivative - + + + +
saponin - - + + +
lignan + + + ++ +
triterpene - ++ + + +
tannin - + + + +
Pigment + + + + -
Hex: hexane, CH 2 Cl 2 :dichloromethane, AcOEt: ethyl acetate, MeOH: Methanol, H 2 O/Et: hydroalcolic; (+) = present in moderate concentration, (++) =
present in high concentration, (-) = indicates the absence of the compound tested.

Table 2: Antibacterial activity of bark extracts from A. ataxacantha at 10 mg/ml

Growth inhibition effect of extract at 10 mg/ml
Extracts Gram (+) Gram (-)
S. aureus S. a. m. r S. epidermidis E. faecalis E. coli P. aeruginosa
Hexane - - - - - -
Dichloromethane - - - + - +
Ethyl acetate + + + + - +
Methanol - - - + - -
Hydroalcolic - - - + - -
+: sensitive;-: not sensitive; S. a. m. r: Staphylococcus aureus meticillin resitant

Table 3: Minimum Inhibitory Concentration and total activity of various extracts from A. ataxacantha
Minimum Inhibitory Concentration (mg/ml)
Extracts Gram (+) Gram (-)
S. aureus S. a. m. r S. epidermidis E. faecalis P. aeruginosa
Dichloromethane - - - 0.625 0.625
Ethyl acetate 0.325 0.625 0.625 0.625 0.625
Methanol - - - 2.5 -
Hydroalcolic - - - 2.5 5
Total activity (ml)
Dichloromethane - - - 8.02 8.02
Ethyl acetate 43.32 22.53 22.53 22.53 22.53
Methanol - - - 16.99 -
Hydroalcolic - - - 41.36 20.68
-: not sensitive; S. a. m. r: Staphylococcus aureus meticillin resitant

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RESULTS use traditionally in Benin to treat numerous diseases. Several

species of genus Acacia have been studied and it has been
Phytochemical analysis demonstrated that they have hypoglycemic [19] anti-aggregation
Phytochemical Constituents extracts of A. ataxacantha are shown in platelet effect [20], antigenotoxic and antimicrobial [21],
Table 1. On the whole, coumarins, flavonoids, alkaloids, derivatives antihypertensive, antispasmodic [22, 23] and anti-inflammatory
of anthracene, lignans, tannins and triterpenes were identified in all activity [24].
extracts with the exception of the hexane extract. The ethyl acetate The phytochemical examination of Acacia ataxacantha extracts indicated
extract gave a positive result for all groups secondary metabolites the presence of tannins, flavonoids, triterpenes, coumarins, lignans,
investigate. Phytochemical tests have also shown the absence of pigments, saponins, alkaloids, anthracene derivatives and
saponins and naphthoquinone respectively in dichloromethane and naphtoquinones. Some secondary metabolites have been reported in the
methanol extracts. literature to have interesting pharmacological activities.
Antibacterial activity Our results showed that MIC values of extracts range from 0.325 to 5
Growth inhibition effect of extracts at 10 mg/ml mg/ml. Ethyl acetate extract was the most active with MIC value of
325 µg/ml against S. aureus. The activity of the ethyl acetate extract
The purpose of this test was to eliminate the extracts did not inhibit is higher compared to other extracts. This may be due to the capacity
the growth of bacteria at 10 mg/ml. In this study, both polar as well of ethyl acetate to extract compounds which have antibacterial
as non polar solvents were used for the extraction of active properties. The dichloromethane extract of A. ataxacantha inhibited
components from the bark of Acacia ataxacantha plant. The results the growth of Pseudomonas aeruginosa with a MIC of 625 µg/ml.
(Table 2) showed that the tested bacteria are sensitive to one or This means potent antibacterial activity of the dichloromethane
more extracts. However, the dichloromethane, ethyl acetate, extract considering the multidrug resistance of P. aeruginosa (gram
methanol and hydroalcolic extracts inhibited the growth of E. negative bacteria) which is responsible for many nosocomial
faecalis. It was noticed that the ethyl acetate extract inhibited the infections. The development of multidrug-resistant of P. aeruginosa
growth of all strains tested except E. coli while dichloromethane is currently one of the biggest challenges to the effective
extract inhibited P. aeruginosa. The hexane extract of A. ataxacantha management of infections [25]. The variation in the effectiveness of
showed no inhibition of bacterial growth. The growth inhibitory the extracts against different microorganisms may be attributed to
activity exercised by dichloromethane and ethyl acetate extracts on the phytochemical composition of the extracts and/or membrane
the most studied strains (five germs out of six or 80% of cases for permeability of the microbes for the chemicals and their
ethyl acetate extract and two germs on 6 or 35% of cases for metabolism.
dichloromethane extract) reflects the reality of an inherent
antimicrobial property to A. ataxacantha bark with an antibacterial Phytochemical study of ethyl acetate and dichloromethane extracts
spectrum of action that affects indiscriminately Gram positive and revealed the presence of tannins, flavonoids, triterpenes, coumarins,
Gram negative bacteria lignans, pigments, saponins, alkaloids, anthracene derivatives and
naphtoquinones except saponins in dichloromethane extract. These
Minimum Inhibitory Concentration (MIC) and total activity results could justify the antibacterial activity of these extracts.
Several studies have demonstrated the antibacterial potential of
The bacterium growth inhibition produced by A. ataxacantha
these secondary metabolites. Tannins inhibit the microbial growth
extracts varied in relation to the type of extract and to the bacterium
by causing the bacterial colonies to disintegrate which probably
strain used. The Minimum Inhibitory Concentrations (MICs) and
results from their interference with the bacterial cell wall [26]. They
total activity of extracts are recorded in Table 3. The extracts
precipitate proteins covering the surface of the cell or tissue, which
showed MIC values ranging from 0.325 to 5 mg/ml. Ethyl acetate
acts as a barrier between tissue and irritants, and the underlying
extract showed interesting activity against S. aureus with MIC values
tissue is therefore soothed and protected from further damage, so
325 µg/ml. A MIC of 625 µg/ml was obtained with the same extract
that healing can take place [27]. Flavonoids have been found to
on the following strains: S. aureus meticillin resitant, S. epidermidis,
exhibit antimicrobial activity through various mechanisms like
E. faecalis and P. aeruginosa. The dichloromethane extract of A.
inhibition of nucleic acid synthesis, inhibition of cytoplasmic
ataxacantha inhibits the growth of E. faecalis and P. aeruginosa with
membrane function and energy metabolism [28]. Plants containing
a concentration of 625 µg/ml. Regarding the methanolic and
tannins and flavonoids are used in the treatment of diarrhoea and
hydroalcolic extracts, the minimum inhibitory concentration was
dysentery [29, 30]. The phytochemical analysis of extracts also
respectively 2.5 mg/ml on E. feacalis and 5 mg/ml on P. aeruginosa.
revealed the presence of naphthoquinones in dichloromethane and
The extracts with higher total activity (TA) values are considered the ethyl acetate extracts while the methanol and hydroalcolic extracts
best. The most interesting total activities of A. ataxacantha were did not contain. The antibacterial effect of dichloromethane and
obtained with ethyl acetate (43.32 ml) and hydroalcolic (41.36 ml) ethyl acetate extracts could be due or accentuated by the presence of
extracts against Staphylococus aureus and E. feacalis respectively. naphthoquinones. Some studies confirm the antibacterial effect of
naphthoquinones [31, 32]. Generally, naphthoquinones are activated
DISCUSSION inside the microbial cells and become covalently attached to the
cellular nucleophiles such as proteins and basic parts of DNA, often
Medicinal plants are the basis of therapeutic treatments in
leading to inactivation of proteins and loss of function [33]. Others
developing countries. Recent years have also seen an increase in the
authors have also reported the antibacterial effect of triterpenes [34,
use of herbal medicines in developed countries. The plants are used
35]. Their antimicrobial activity has associated with among other
medicinally in different countries of the world and are a good source
bacterial cell membrane disruption by the lipophilic compounds [36].
of many potent and powerful drugs [15]. The resistance of
pathogens to antibiotics commonly used, the increase in The presence of saponins in the ethyl acetate and hydroethanolic
opportunistic infections and the effect of toxicity due to the extracts and its absence in dichloromethane extract indicates that
continued use of several drugs have led to increased attention paid saponins do not play an essential role in inhibiting bacterial growth.
to the search for new therapeutic agents from various sources, Indeed, the mode of action of antibacterial effects of saponins seems to
including plants, which are good starting materials for the discovery involve membranolytic properties [37]. They possess detergent like
of new antimicrobial agents [16, 17]. Secondary metabolites activity and might increase the permeability of the bacterial cell
produced by plants constitute a source of bioactive substances. All membrane without destroying them. In theory, this activity might
over the world, plants scientist interest has increased due to the facilitate antibiotic influx through the bacterial cell wall membrane [38].
search for new drugs from plant origin. Secondary metabolites of
plant have been reported to serve as defense mechanisms against The presence of active chemical groups such as tannins,
predation by many microorganisms, insects and herbivores [18]. naphthoquinones, triterpenes and flavonoids in the studied extracts
could justify interesting results obtained and the indications of this
In the present study, phytochemical investigation and antibacterial plant in traditional medicine, particular for its antibacterial properties.
activity were carried out on the barks of Acacia ataxacantha, an herb Our results also confirm the antibacterial activity of A. ataxacantha as

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Int J Pharm Pharm Sci, Vol 6, Issue 11, 138-141

a broad spectrum antimicrobial agent, since it inhibits the growth of 15. Mahesh B, Satish S. Antimicrobial activity of some important
Gram-positive and Gram-negative bacteria. Thus, these results support medicinal plants against plant and human pathogens. World J
the use of A. ataxacantha in traditional medicines for the treatment of Agric Sci 2008;4:839-43.
infections caused by bacteria tested in our study. 16. Sasidharan S, Prema B, Yoga LL. Antimicrobial drug resistance
of Staphylococcus aureus in dairy products. Asian Pac J Trop
CONCLUSION Biomed 2011;1:130-2.
The acacia species used in this study had never been evaluated or 17. Saad S, Taher M, Susanti D, Qaralleh H, Rahim N. Antimicrobial
only partially studied for antibacterial activity. At least, this plant activity of mangrove plant (Lumnitzera littorea). Asian Pac J
seems to be of particular interest for further investigation, as it is Trop Med 2011;7:523-52.
effective against both gram-negative and gram-positive bacteria. It is 18. Upadhyay RK, Dwivedi P, Ahmad S. Asian J Med Sci
an indication that the plant can be a source of bioactive substances. 2010;2(3):152-8.
In addition, our results support the idea that herbal medicines with 19. Wadood A, Wadood N, Wahid-Shah SA. Effects of Acacia arabica
medicinal value have a promising future in regard to the discovery of and Caralluma edulis on blood glucose levels of normal and
new substances able to participate in the development of alloxan diabetic rabbits. J Pak Med Assoc 1989;39:208-12.
pharmaceutical products based drugs or improved traditional plant 20. Shah BH, Safdar B, Virani SS, Nawaz Z, Saeed SA, Gilani AH. The
medicines (MTA). Bioguided fractionation of the corresponding antiplatelet aggregatory activityof Accacia nilotica is due to
extracts is under process. blockage of calcium influx through membrane calcium
channels. Gen Pharmacol 1997;29:251-5.
ACKNOWLEDGEMENT 21. Boubaker J, Hedi BM, Kamel G, Leila CG. Polar extracts from
(Tunisian) Acacia salicina Lindl. Study of the antimicrobial and
The authors are grateful to the medicinal plants seller and
traditional practitioners from Ouidah regions. The authors also wish antigenotoxic activities. Complement Altern Med 2012;12:37.
to thank the University of Abomey-Calavi for financial support of 22. Gilani AH, Shaheen F, Zaman M, Janbaz KH, Shah BH, Akhtar MS.
their project (PFCR/UAC, 2nd phase). Studies on hypertensive and antispasmodic activities of
methanol extract of Acacia nilotica pods. Phytother Res
CONFLICT OF INTERESTS 1999;14:510-6.
23. Arias ME, Gomez JD, Cudmani NM, Vattuone MA, Isla MI.
Declared None Antibacterial activity of ethanolic and aqueous extracts of
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