Antimicrobial Activity of Dichloromethane-Methanol (1:1 v/v) Extract from the Stem Bark of Coula edulis Bail. (Olacaceae)
D.J. Mpetga Simo,
P.H. Amvam Zollo
In order to confirm the traditional uses of Coula
edulis, the CH2Cl2-MeOH (1:1 v/v) extract of
stem bark of this plant and its column fractions were screened for antimicrobial
activity. The plant was dried and extracted by maceration in CH2-Cl2-MeOH
(1:1 v/v). The dry extract was fractionated by silica gel column chromatography.
Phytochemical screening was performed using common chemical standard methods.
Antimicrobial activity was assayed by disc diffusion method and broth
macro dilution method. From the results, it appeared that the crude extract
of Coula edulis stem bark displayed antibacterial activities against
four clinical isolates of bacteria and antifungal activities against six
strains of Candida species. The Minimum Inhibitory Concentration
(MIC) values ranged from 12.5 to 25 mg mL-1 for bacteria and
1.56 to 6.25 mg mL-1 for yeasts. The fractionation of crude
extract gave eight fractions. Fractions F3 and F4 showed higher antibacterial
activities while fractions F5 and F6 displayed higher antifungal activity
compared to the crude extract. Their MICs ranged from 0.19 to 12.5 mg
mL-1. Phytochemical screening indicated that the crude extract
contains tannins, flavonoids, anthraquinones, anthocyanins, sterols and
phenols. Coula edulis crude extract has the ability to inhibit
bacterial and yeast growth. Fractionation enhanced the antimicrobial activity
in some fractions. These results justify the traditional use of this plant
for the treatment of infectious diseases.
to cite this article:
J.D. Tamokou, J.R. Kuiate, G.S.S. Njateng, D.J. Mpetga Simo, A.J. Njouendou, P. Tane and P.H. Amvam Zollo, 2008. Antimicrobial Activity of Dichloromethane-Methanol (1:1 v/v) Extract from the Stem Bark of Coula edulis Bail. (Olacaceae). Research Journal of Microbiology, 3: 414-422.
The search for plants with antimicrobial activity has gained increasing
importance in recent years due to the development of antimicrobial drug
resistance and often the occurrence of undesirable side effects of some
antibiotics (Soberon et al., 2007). For example, over the last
three decades, methicillin resistant Staphylococcus aureus has
caused major problems in hospitals throughout the world (Waldyogel, 1995).
With the advent of ever-increasing resistant bacterial and yeast strains,
there has been a corresponding rise in the universal demand for natural
antimicrobial therapeutics (Kishore et al., 1996; Soberon et
al., 2007). Indeed, even though pharmacological industries and researchers
have produced a good number of antibiotics in the last three decades,
resistance to these drugs by micro organisms is increasingly high. Herbal
medicine has been widely used and formed an integral part of primary health
care in many countries (Akinyemi et al., 2005) and may constitute
a reservoir of new antimicrobial substances to be discovered. According
to WHO, medicinal plants would be the best source of a variety of drugs
(Nascimento et al., 2000). On the other hand, about 80% of developing
countries, citizens used traditional medicine based on plant products.
This explains why numerous studies have been conducted on various medicinal
plant extracts, screening their antimicrobial activities to better understand
their properties and efficacy and also with the hope to discover new antimicrobial
Coula edulis Bail. (Family, Olacaceae), commonly known as African
walnut, is a medicinal plant that originated from Tropical Western Africa.
It is an evergreen tree growing to a height of 25-38 m and native to Tropical
Western Africa. It can be found in the top canopy of forest as well as
the lower story and has no special soil requirements. Ethnobotanical studies
indicate that the stem or fruits of C. edulis are commonly used
in West Africa for the treatment of stomach ache, skin diseases and tonic
effect (Iwu, 1993). The bark is used to produce rinses or enemas for loin
pains or kidney problems (Davidson, 1999). There is no report regarding
the antimicrobial activities of this plant. Therefore, the aim of the
present work is to evaluate the antimicrobial activities of the crude
dichloromethane-methanol (1:1 v/v) extract of the stem bark of C. edulis
and its column fractions on several bacteria and yeasts that can cause
stomach ache, skin diseases and urinary problems in man.
MATERIALS AND METHODS
The stem bark of Coula edulis was collected in Buea (South
West Province of Cameroon) in January 2005. Plant material was identified
at the Cameroon National Herbarium in Yaoundé where a voucher specimen
was kept under the accession number 19357 HNC.
The micro-organisms used in this study consisted of two gram positive
(Enterococcus faecalis, Staphylococcus aureus) and six gram
negative (Pseudomonas aeruginosa, Proteus mirabilis,
Shigella flexneri, Escherichia coli, Klebsiella
pneumoniae, Salmonella typhi) bacteria (clinical isolates)
collected from Centre Pasteur (Yaoundé-Cameroon). Also, two strains
of Candida albicans (ATCC 9002 and ATCC 1663.86) and six
clinical isolates (Candida parapsilosis, C. lusitaniae,
C. tropicalis, C. glabbrata, C. krusei,
C. albicans) of yeasts originally obtained from Centre Pasteur
(Paris-France) were tested. The bacterial and yeast isolates were grown
at 35°C and maintained on nutrient agar (NA, Conda, Madrid, Spain)
and Sabouraud Dextrose Agar (SDA, Conda) slants, respectively.
Extraction and Fractionation
The stem bark of C. edulis Bail was dried at room temperature
(25±2°C) for 3 weeks and crushed. Five kilograms of obtained
powder was macerated into 12 L dichloromethane-methanol (Merck) (1:1 v/v)
mixture for two days and this process was repeated twice. After filtration,
the filtrate was successively evaporated to dryness at 40°C (for the
dichloromethane) and at 65°C (for the methanol) under reduced pressure
using rotary vacuum evaporator. The dried crude extract was stored at
+4°C. The crude extract (104 g) was then subjected to column chromatography
(30x8 cm column) using 500 g of silica gel 40 (particle size 0.2-0.5 mm).
The column was successively eluted with hexane (1000 mL), Hexane-ethyl
acetate [19:1 v/v (2500 mL), 9:1 v/v (3750 mL), 4:1 v/v (1500 mL), 7:3
v/v (1750 mL), 3:2 v/v (1750 mL), 1:1 v/v (1750 mL), 2:3 v/v (2750 mL)
and 1:4 v/v (5000 mL)] mixtures, ethyl acetate (5000 mL), ethyl acetate-methanol
[9:1 v/v (2750 mL), 4 : 1(2250 mL), 13:7 (2500 mL), 1:1 v/v (2500 mL),
3:7 v/v (5250 mL)] mixtures and methanol (750 mL). One hundred and seventy
one fractions of 250 mL each were collected and combined on the basis
of their Thin Layer Chromatography (TLC) profiles to afford eight main
fractions. Fractions from 1-59, 60-122, 123-124, 125-126, 127-138, 139-149,
150-156 and 157-171 were referred as F1, F2, F3, F4, F5, F6, F7 and F8,
respectively. TLC analyses were carried out on silica Gel 60 GF254
precoated plates (20x20 cm) and visualised under a UV light (254 and 366
nm), UV lamp Model 52-58 mineralight and sprayed with 50% v/v H2SO4
followed by heating at 100°C.
Phytochemical Screening of Extracts
The Phytochemical screening of the crude extract and its column fractions
were carried out using standard methods (Silva et al., 1998; Bruneton,
1999). The plant material was screened for the presence of different classes
of compounds including alkaloids, flavonoids, sterols, triterpenes, coumarins,
anthraquinones, tannins, anthocyanins, saponins and phenols.
The susceptibility tests were performed by disc diffusion method as
recommended by National Committee for Clinical Laboratory Standards (1993)
with slight modifications. Stock solutions of the extracts (crude extract
and fractions) were prepared in 5% v/v aqueous dimethyl sulphoxide (DMSO,
Fisher chemicals) at concentration of 125 mg mL-1. The inocula
of micro-organisms were prepared from 24 h old broth cultures. The absorbance
was read at 530 nm and adjusted with sterile distilled water to match
that of a 0.5 McFarland standard solution. From the prepared microbial
solutions, other dilutions with sterile distilled water were prepared
to give a final concentration of 106 Colony-Forming Units (CFU)
per millilitre for bacteria and 2x105 spores per millilitre
for yeasts. Bottles containing 19.8 mL of sterile Sabouraud Dextrose Agar
(Conda, Madrid, Spain) or Mueller Hinton Agar (MHA) (Conda, Madrid, Spain)
were maintained in a steam bath set at 40°C to prevent solidification
of the medium and then inoculated aseptically with 0.2 mL of bacteria
or yeast suspension followed by thorough mixing. Sterile Petri dishes
(Diameter, 90 mm) were filled to 20 mL final volume of each bottle to
give a solid plate. Discs of 6 mm in diameter previously impregnated with
10 μL of stock solution of extracts were placed aseptically on the
solid plates and allowed for 2 h at +4°C for the extract to diffuse.
The Petri dishes were then incubated at 35°C for 24 h (for bacteria)
and for 48 h (for yeasts). The final disc charges were 1.25 mg of extract
per disc. The susceptibility was recorded by measuring the clear zone
of growth inhibition on agar surface around the discs.
All the experiments were carried out in triplicates. Gentamicin (Sigma-Aldrich,
Steinheim, Germany) and Nystatin (Merck, Darmstadt, Germany) at 10 μg
per disc (for bacteria and yeasts respectively) were used as positive
controls and 5% v/v aqueous DMSO as a negative control.
Determination of the Minimum Inhibitory Concentration (MIC)
MIC was determined by broth macro dilution method with slight modifications
from the one described by Gulluce et al. (2003). The two-fold serial
dilutions in concentration of the extracts (25-0.19 mg mL-1)
were prepared in Mueller Hinton Broth (MHB) (Conda, Madrid, Spain) for
bacteria and Sabouraud Dextrose Broth (SDB) (Conda, Madrid, Spain) for
yeasts. For every experiment, a sterility check (5% v/v aqueous DMSO and
medium), negative control (5% v/v aqueous DMSO, medium and inoculum) and
positive control (5% v/v aqueous DMSO, medium, inoculum and water-soluble
antibiotics) were included. In general, the 24 macro well plates (Nunclon,
Roskilde, Danmark) were prepared by dispensing into each well 880 μL
of an appropriate medium, 100 μL of test extracts (crude extract
or fractions) and 20 μL of the inoculum (106 cfu per mL
for bacteria and 5x105 spores per mL for yeasts). The content
of each well was mixed thoroughly with a multi-channel pipette and the
macro well plates were covered with the sterile sealer and incubated at
35°C for 24 h (for bacteria) and 48 h (for yeasts) under shaking by
using a plate shaker (Flow Laboratory, Germany) at 300 rpm. Microbial
growth in each well was determined by observing and comparing the test
wells with the positive and negative controls. The absence of microbial
growth was interpreted as the antibacterial or antifungal activities.
The MIC was the lowest concentration of the test substances that prevented
visible growth of micro-organisms. Minimum Bactericidal Concentrations
(MBCs) or Minimum Fungicidal Concentrations (MFCs) were determined by
plating 10 μL from each negative well and from the positive growth
control on Mueller Hinton Agar (for bacteria) and Sabouraud Dextrose Agar
(for yeasts). MBCs or MFCs were defined as the lowest concentration yielding
negative subcultures or only one colony. All the experiments were performed
in triplicate. Gentamicin and Nystatin at the concentration ranging between
400 and 0.79 μg mL-1 served as positive controls for antibacterial
and antifungal activities respectively.
The inhibition diameters of crude extract and its column fractions
were expressed as the Mean±Standard Deviation and compared using
Student-Waller Duncan test at p≤0.05.
The crude extract of Coula edulis and eight fractions obtained
presented variable physical aspects (Table 1). The phytochemical
screening of crude extract showed the presence of phenols, flavonoids,
sterols, tannins, anthocyanins and anthraquinones but alkaloids, saponins,
coumarins and triterpenes were absent. These main classes of compounds
varied within the fractions.
Table 2 reports the inhibition zones of crude extract
and its column fractions determined for eight clinical isolates of bacteria
and eight Candida species. The results showed that Escherichia
coli and Klebsiella pneumoniae were resistant since no inhibition
zone was observed. Other micro-organisms
||Physical characteristics and phytochemical analysis
of crude extract of Coula edulis stem bark and its column fractions
|a: The crude extract and fractions are reported
as a percentage with respect to the plant material and dichloromethane-methanol
extract respectively. b: Thin-layer chromatography plates
were performed with ethyl acetate-methanol solvent system as mobile
phase at different polarity. Spots and bands were visualized by UV
irradiation (254 and 366 nm) and by spraying with 50% (v/v) sulphuric
acid reagent followed by heating at 100°C
||Diameter of the inhibition zones* (mm) of crude extract
of Coula edulis stem bark and its column fractions
|The results are the mean values of triplicate tests
measured in two directions after 24-48 h incubation at 35°C, *:
Zone diameter±SD at 1.25 mg per disc, **: Gentamicin
and Nystatin were used as reference drugs for bacteria and yeasts
respectively. For the same line, values affected by the same superscripts
letter (a-h) are not significantly different (test of student-Waller-Duncan
at p>0.05). 1.25 mg of extract per disc is the minimum charge of
disc after testing 5 and 0.625 mg of extract per disc (results have
||Minimal Inhibitory Concentration (MIC)/Minimum Bactericidal
or Fungicidal concentration (MBC or MFC) of extracts (mg mL-1)
of Coula edulis stem bark
|- : Absence of inhibition at concentrations up to 25
mg mL-1 for crude extract and 12.5 mg mL-1 for
column fractions, a: Gentamicin and Nystatin were used
as reference drugs for bacteria and yeasts respectively
tested showed sensitivity for at least two extracts with inhibition zones
ranging from 7 to 21 mm. Staphylococcus aureus, Proteus
mirabilis and Shigella flexneri were in general,
found to be more sensitive among the bacteria tested while Candida
albicans ATCC 9002 and Candida parapsilosis showed the best
susceptibility among the yeasts tested. The fractionation increased the
antibacterial and antifungal activities of the crude extract in fractions
F2, F3, F4, F5 and F6. However, these activities were low in fractions
F7 and F8. No activity was noticed in fraction F1 for all the micro organisms
The bacterial and fungal growth inhibitions by the crude stem bark extract
and column fractions indicated by the MIC values are summarized in Table
3. In general, the results obtained confirm the considerations retained
in Table 2. Pseudomonas aeruginosa, Proteus
mirabilis, Shigella flexneri, Salmonella typhi, Staphylococcus
aureus, Enterococcus faecalis and Candida species were
all inhibited by crude extract and fractions F5 and F6 with the MIC/MBC
or MFC values ranging from 0.19 to 25/0.39 to >25 mg mL-1.
Klebsiella pneumoniae and Escherichia coli were not inhibited
at concentrations up to 25 mg mL-1 for crude extract and 12.5
mg mL-1 for column fractions.
Differences were noticed between the crude extract and its fractions
as far as the antibacterial and antifungal activities are concerned. This
can be linked to the differences in chemical composition of these substances.
The crude extract was more active on yeast while the fractions were more
active on bacteria, showing that fractionation increased the antibacterial
activity and in some cases decreased the activity against yeast. These
results suggest possible synergetic effects between some of the extract
constituents for antifungal activity. The antifungal activity is more
concentrated in fractions F5 and F6, indicating that the antifungal active
principle may belong to anthocyanins group. On the other hand, fractionation
may have increased the concentration and the activity of antibacterial
principles in the fractions. This activity is found in almost all the
fractions but particularly in fractions F2 to F6. These fractions are
characterised by the presence of phenols, flavonoids and in some cases
tannins. These groups of compounds are known to possess antibacterial
activities (Rojas et al., 1992; Scalbert, 1991) and they may act
by complexing with extracellular and soluble proteins as well as cell
wall of micro-organisms (Cowan, 1999). In addition, tannins also complex
with polysaccharides (Ya et al., 1988). Comparable results were
obtained by Ogunleye et al. (2003) while working on Ximenia
americana. It is also important to mention the presence of anthraquinones
and anthocyanins in some of the fractions since Ali et al. (2000),
Mohamed (2003) and Lenta et al. (2007) reported the antibacterial
and antifungal activities of some individual anthraquinones and anthocyanins.
The results also indicated that crude extract and its column fractions
are less active compared to reference drugs. This may be due to the low
concentration of active compound(s) in these extracts suggesting that
purification of bioactive compound(s) from the more active fractions are
The fact that the fractions F5 and F6 showed relatively good antifungal
activity against the eight clinical isolates of Candida species
is interesting because there are currently only a few antifungal agents
e.g., Amphotericin B, fluconazole, itraconazole that are effective on
candidoses particularly those caused by C. albicans. The treatment
of candidoses often requires a combination of these agents. Resistance
and significant adverse effects including gastrointestinal disturbances,
nephrotoxicity and arachnoiditis have been observed following treatment
with the above antifungal drugs, while the success rate has been rather
limited (Rahalison et al., 1991; Carrillo-Munoõz et al.,
From this study, we can conclude that the crude stem bark extract of
C. edulis possesses antibacterial and antifungal properties. It
is interesting to notice that the fractionation enhanced the antibacterial
and antifungal activities and then can be used at the local level to produce
phytomedecine that can be used at affordable prise by the populations
to cure stomachache, skin diseases and urinary tract problems in man.
Purification of bioactive compound(s) from the more active fractions is
underway and further investigations may improve our understanding of possible
antimicrobial and antifungal activities.
Financial support from the international Program in the chemical Sciences,
IPICS Uppsala, Sweden (Grant No. Cam 02) is gratefully acknowledged.
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