European Journal of Medicinal Plants

4(9): 1036-1045, 2014

SCIENCEDOMAIN international
www.sciencedomain.org

Antimicrobial Studies of Aqueous and

Ethanolic Extracts of Enantia chlorantha
Leaves and Stem Bark and Their Combined
Effect on Selected Bacteria and Fungi
C. P. Atukpawu1* and P. T. E. Ozoh1
1
Department of Biotechnology, Federal University of Technology, Owerri, Imo State, Nigeria.

Authors’ contributions

This work was carried out in collaboration between all authors.

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Received 27 March 2014
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Original Research Article Accepted 27 May 2014
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Published 9 June 2014

ABSTRACT

Aim: To revalidate the antimicrobial effects of aqueous and ethanolic extracts of Enantia
chlorantha leaves and stem bark against Enterococcus faecalis, Escherichia coli,
Salmonella Typhi, Shigella sonnei, Staphylococcus aureus, Proteus vulgaris and Candida
albicans; and to check the effects on the isolates when the stem bark and leave extracts
are combined.
Place and Duration of Study: Department of Biotechnology, School of Science, Federal
University of Technology Owerri, Nigeria, between January 2013 and May 2013.
Methodology: Agar well diffusion method was used for the susceptibility studies while the
Minimum Inhibitory Concentrations were determined using the broth dilution method. The
Minimum bactericidal/fungicidal concentration was determined by plating on nutrient agar.
Results: The ethanolic extract of E. chlorantha stem bark showed antimicrobial activity on
all 7 isolates tested with zones of inhibition in the range of 5mm to 33mm, while its
aqueous extracts showed activity on only 3 of the 7 isolates with diameter zones of
inhibition ranging between 5mm to 20mm. The aqueous leaf extracts showed activity
against 3 of the 7 isolates while the ethanolic extracts had activity on 6. The minimum
inhibitory concentration (MIC) of the ethanolic extract of both stem bark and leaves was
between 1.56 and 12.5mg/ml, while that of aqueous extracts ranged from 6.25 to
12.5mg/ml. There was no obvious difference in the activity of the extracts when
combined.
____________________________________________________________________________________________

*Corresponding author: Email: phoebechidimma@yahoo.com;

 European Journal of Medicinal Plants, 4(9): 1036-1045, 2014

Conclusion: This study validates potent antimicrobial activity of ethanol and aqueous
extracts of Enantia chlorantha leaves and stem bark in line with similar studies. Further
work is however needed to determine the toxicity of the plant extracts and also identify
active components of the plant.

Keywords: Enantia chlorantha; minimum inhibitory concentrations; agar-well diffusion;

toxicity; antimicrobial.

1. INTRODUCTION

Resistance to antimicrobial agents has become a global problem. Strategies to improve the
current situation include finding new antimicrobial agents some of which are plant-based.
Although antibiotics have been used in the therapy and prevention of animal and human
infectious diseases since their discovery, their judicious use is necessary to minimize the
development of resistant organisms. A large proportion of the population in developing
countries utilize medicinal plants for the treatment of infectious diseases largely because
most current and effective antibiotics are expensive, inaccessible and unaffordable to many
Africans especially those residing in rural areas. According to the World Health
Organization’s estimation, about 75% of the population in Africa use herbal medicines.
Hence the need to subject the various plants/herbs to pharmacological and biological tests.
Besides, a substantial number of the new and current antibiotics are obtained from natural or
semi-synthetic sources.

Much attention has been recently directed towards plant extracts and biologically active
compounds isolated from plants. The use of medicinal plants play a vital role in combating
diseases in developing parts of the world where poverty and drug resistance limit access to,
and effectiveness of synthetic drugs for chemotherapy [1]. This study is necessary; coming
on the heels of an explosion of trado-medicine in Nigeria, as plant-based drugs are seen
marketed and sold on the streets, offices and even on radio and television, with strong
claims being made by trado-medical practitioners about these plants treating a wide array of
diseases. Worthy of note is the large number of people who patronize the practioners
without concern to the potency and safety of the said plant products.

The advantages of herbal medicines over orthodox drugs include; minimal or no side effects,
consistent potency and the fact that they are well absorbed and distributed in the area of
infection [2,3]. “The success story of chemotherapy lies in the continuous search for new
drugs to counter the challenges posed by resistant strains of microorganisms” [4]. Natural
products of higher plants may provide a new source of antimicrobial agents with potentially
novel mechanisms of action [5,6]. Secondary metabolites from higher plants could serve as
defense agents against invading microorganisms [7].

Enantia chlorantha is an ornamental tree belonging to the Annonaceae family. It can grow up
to 30m high, with dense foliage and spreading crown. It has a fluted and aromatic stem, thick
and brown bark with a dark yellow color beneath the bark. It is commonly found in the forest
and coastal areas of West Africa, and the Democratic Republic of Congo [8,9]. E.
chlorantha, locally known as “Erumeru” in Igbo land; “Awogba”, “Oso pupa” or “Dokita Igbo”
in Yoruba land helps in the treatment of yellow fever infections [10]. The plant is used in
trado-medicine for the treatment of conditions such as rickettsia fever, typhoid fever and
infective hepatitis [11].

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Research on E. chlorantha stem bark shows that “the aqueous stem bark maintained the
cytological integrity of testes and viability of sperm motility and sperm count in rats” [12]. It is
reported to also possess antiviral activity [13] and antipyretic activity [14]. It is used for
treating leprosy spots, as hemostatic agents and as uterus stimulants. It is also used in
Cameroun to treat jaundice and urinary tract infections [15]. Leaves of E. chlorantha have
substantial antisickling activity [16], while the potentials of the plant extract in relieving
pyrogen-induced fever in albino rats was reported by Agbaje & Onabanjo [17].

2. MATERIALS AND METHODS

2.1 Collection of Plant Materials

The leaves and stem bark of Enantia chlorantha were obtained in a garden at Egbelu,
Nguru, Ngor-okpala, Imo state. All the collections were done in the month of January. The
plants were identified and authenticated by the taxonomist at Green Finger gardens, Okigwe
road, Owerri, Imo state.

2.2 Preparation of Extracts

The plant samples were thoroughly washed with tap-water to remove debris, and then rinsed
with distilled water. A voucher specimen was kept at the Biotechnology laboratory, Federal
University of Technology, Owerri. The samples were shade-dried for about two weeks.
Exposure to sunlight was avoided to prevent the possible loss of active compounds. The
shade-dried plants samples were milled to fine powder using a Kenitone blender (model:
MS-223).

2.3 Extraction

The powdered plant samples (100g) were soaked in 400ml distilled water and absolute
ethanol (to make aqueous and ethanolic extracts) respectively in a 500ml sterile conical flask
for 72 hours at ambient temperature with vigorous shaking at intervals. The extracts were
then filtered first using a sterile muslin cloth and then a Whatman No. 1 filter paper. The
procedure was used for both the aqueous and ethanolic extracts. The extracts produced
were kept under a ceiling fan to evaporate the solvents to dryness (this took from 48 to 96
hours). The dry extracts were weighed and tested for purity by plating them on nutrient agar
for 24 hours at 37ºC and then used to prepare stock solutions for the susceptibility test.

2.4 Preparation of Stock Solution for Susceptibility Studies

The extracts were dissolved in distilled water and absolute ethanol to get stock extracts of
concentration 200mg/ml. The stock of the extract mixtures were prepared in a ratio of 1:1;
that is, one portion of the bark extract (ECB) to an equal portion of the leaves extract (ECL).

2.5 Test Microrganisms

Six bacterial isolates (Enterococcus faecalis, Escherichia coli, Salmonella typhi, Shigella
sonnei, Vancomycin-resistant Staphylococcus aureus and Proteus vulgaris) and the fungal
isolate (Candida albicans) used in this study were obtained from the Biotechnology
laboratory, of the Federal University of Technology, Owerri. They were identified and
authenticated by the Microbiologist/Senior Laboratory technologist there.

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2.6 Standardization of Innoculum

This was done using the method described by the Manual of Antimicrobial Susceptibility
Testing of the American Society of Microbiology (2005) [18]. Briefly, five colonies of each
organism used were picked into 5ml Nutrient Broth (Oxoid, Hampshire, England) and
incubated at 37ºC for 18-24 hours for bacteria, and C. albicans at 25ºC for 3 days. Turbidity
produced was adjusted to 0.5 McFarland’s standard.

2.7 Determination of Antimicrobial Susceptibility of the Plant Extracts (Agar

Well Diffusion Method)
This was done using the standard method employed by the American Society of
Microbiology (2005) [18]. Briefly, One ml of 18-24 hours test culture, adjusted to 0.5
McFarland was spread (using a sterile swab) on a sterile plate containing 25ml of solidified
Mueller Hinton agar ( Oxoid, Hampshire, England) for the bacterial strains and Sabouraud
Dextrose Agar ( Oxoid, Hampshire, England) for C. albicans. Using a sterile cork borer of
6mm, four ditches were made on the surface of the agar medium in each plate; into each
ditch was dispensed 0.1ml of the stock extract of E. chlorantha leaves, stem bark, the
mixture (ECL/ECB) and ethanol or water respectively. Water was used as negative control
for plates containing water extracts. Chloramphenicol discs of 30µg concentrations were
used as positive control for the bacteria while Nystatin discs (Oxoid) of 100 units were used
for C. albicans. The inoculated plates were left on the laboratory table for about an hour to
allow the extracts diffuse into the agar. Each analysis was done in triplicates. The bacterial
culture plates were then incubated at 37ºC for twenty-four hours while the C. albicans plates
were incubated for seventy-two hours. Antimicrobial activity was determined by measuring
the diameter of the zone of inhibition in millimeters.

2.8 Determination of Minimum Inhibitory Concentration (Mic)

The MIC of the extracts against the test organisms was determined using the broth dilution
method as described by the American Society for Microbiology (2005) [18]. Nutrient broth
(Oxoid, Hampshire, England) was used. Briefly, One ml of the stock extract at
concentrations of 200mg/ml was added to one ml of nutrient broth and serially diluted to
obtain extract concentrations of 100, 50, 25, 12.5, 6.25, 3.125 and 1.56mg/ml in 7 different
test tubes. One ml of an 18 hour culture adjusted to 0.5 McFarland turbidity standard
8
(1.0x10 cfu/ml) was inoculated in each test tube. Control tubes were also set up containing
the extract alone and 18 hour cultures only for each organism. The tubes were then
incubated at 37ºC for 24h. After incubation, the tube with lowest dilution with no detectable
growth when checked visually for turbidity was considered the MIC.

2.9 Determination of Minimum Bactericidal/Fungicidal Concentration

(Mbc/Mfc)
This was done using the National Committee for Clinical Laboratory Standard (1990) method
[19]. Briefly, One ml sample from tubes used in MIC determination which didn’t show any
visible growth after the period of incubation was streaked out on Nutrient Agar for 24 hours
for bacteria and Sabouraud Dextrose Agar for 72 hours C. albicans, to determine the
minimum concentration of the extract required to kill the organisms. The lowest
concentration of the extract that indicated a bactericidal effect after incubation was regarded
as the Minimum Bactericidal Concentration (MBC), while the lowest concentration that
prevented the growth of C. albicans was taken as the Minimum Fungicidal Concentration
(MFC).

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3. RESULTS AND DISCUSSION

Aqueous extracts showed minimal antimicrobial activity against the isolates at 200mg/ml
concentrations. The ethanolic plant extracts gave wider zones of inhibition than the aqueous
extracts (as shown in Tables 1 and 2). The diameter of each zone of inhibition denotes the
rate of extract diffusion and was used in this study to estimate an organism’s sensitivity to a
particular extract. E. faecalis, S. aureus and S. sonnei were resistant to all the aqueous
extracts but sensitive to the ethanolic extracts of E. chlorantha, they were also sensitive to
the combined ethanolic extracts of E. chlorantha bark and leaves (ECL/B) (see Table 1 and
2). It is note-worthy that all the organisms were more sensitive to the ethanolic extracts of E.
chlorantha bark in its single and combined form, than to the antibiotics used as standard
(Chloramphenicol and Nystatin). The ethanolic extract of E. chlorantha stem bark showed
broad spectrum activity as all the isolates were sensitive to it, with zones of inhibition
comparable to the antibiotics used as standard.

It was observed that the Minimum Inhibitory Concentration (MIC) at which the isolates were
sensitive to the various extracts varied in most cases (Tables 3 and 4). The MIC values
obtained for the entire test organisms ranged from 1.56mg/ml to 25mg/ml. The MBC/MFC
(Tables 5 and 6) in most cases varied from the MIC, indicating a different concentration
needed to inhibit the growth of the microorganisms and an entirely different one to kill them.

From the results of this study, it is clear that E. chlorantha leaves and stem bark possess
promising antimicrobial activities against the test organisms.

In microbiology, large diameters of zones of inhibition correlate with small minimum inhibitory
concentrations (MIC). The MIC is usually described as the lowest concentration of an
antimicrobial agent that results in inhibition of visible growth (that is colonies on an agar plate
or turbidity in broth culture) under standard conditions, while the MBC is the lowest
concentration of the antibiotic that kills 99.9% of the original inoculum in a given time [14].
The basic quantitative measures of the In vitro activity of antibiotics and plant extracts with
antibacterial potentials are the minimum inhibitory concentration (MIC) and the minimum
bactericidal concentration (MBC) [14]. The MIC values obtained for the entire test organisms
were very high, ranging from 1.56mg/ml to 25mg/ml, when compared to the values of 0.01-
10µg/ml usually recorded for typical antibiotics. This difference may be due to the fact that
the extracts were in impure forms and would definitely contain substances which do not have
antibacterial activities [20].

The result of the antimicrobial analyses on Enantia chlorantha stem bark was consistent with
that obtained by Adesokan et al. [14] and Razaq et al. [21] respectively. The aqueous
extracts had no effect on Gram-positive organisms believed to be largely as a result of cell
wall thickening of Gram-positive organisms and the diffusion problem of water.

The microorganisms were not as sensitive to the aqueous extracts as they were to the
ethanolic extracts. In cases where they were sensitive in aqueous extracts, the diameter of
their zones of inhibition were smaller, and their MICs higher. The observed difference
between the aqueous and ethanolic plant extracts may be due to insolubility of active
compounds in water or the presence of inhibitors to the antimicrobial components in water
[22]. Amadioha & Obi [23], Okigbo & Ajale [24] or possible diffusion problem with water;
Okigbo et al. [25] also reported that inactivity of plant extracts may be due to the age of
plant, extracting solvent, method of extraction and time of harvesting of plant materials which
should not apply in this case as all plant samples used in this study was obtained from the
same E. chlorantha plant.

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Table 1. Antibacterial and antifungal activity of aqueous extracts of bark and leaf of E. chlorantha alone and combined
# + - - - - +
C. albicans E. faecalis E. coli P. vulgaris S. typhi S. sonnei S. aureus
ECB 5.66±1.81 0 9.33±2.66 20.00±2.00 0 0 0
ECL 0 0 12.00±0.00 16.00±4.00 25.00±2.0 0 0
ECL/B 3.33±2.77 0 5.00±1.00 0 5.00±0.66 0 0
WATER 0 0 0 0 0 0 0
KEY: ECB= Enantia chlorantha bark; ECL= Enantia chlorantha leaf; ECL/B= Enantia chlorantha leaves+Enantia chlorantha bark; #=Mycotic isolate;
- = Gram-negative isolate; += Gram-positive isolate; Mean values in millimeters, and standard deviations of the zones of inhibition are expressed

Table 2. Antibacterial and antifungal activity of ethanolic extracts of bark and leaf of E. chlorantha alone and combined
# + - - - - +
C. albicans E. faecalis E. coli P. vulgaris S. Typhi S. sonnei S. aureus
ECB 30.00±1.00 22.33±2.52 30.33±2.74 33.00±3.00 18.33±2.89 25.00±2.65 30.00±1.00
ECL 24.00±4.00 19.33±3.06 16.66±1.77 22.33±1.40 0 5.00±2.00 14.33±2.04
ECL/B 35.00±3.00 28.00±1.00 29.33±1.02 33.00±1.73 24.33±2.03 28.00±4.00 25.66±1.13
Ethanol 13.33±4.16 15.00±0.00 8.33±1.53 10.00±0.00 9.33±1.15 3.00±1.00 8.00±2.00
Antbiotics 22.33±2.52 18.33±2.89 26.33±4.16 16.00±3.46 23.00±2.00 22.00±1.00 21.33±3.51
KEY: ECB= Enantia chlorantha bark; ECL= Enantia chlorantha leaf;ECL/B= Enantia chlorantha leaves+Enantia chlorantha bark; #=Mycotic isolate; -
= Gram-negative isolate; += Gram-positive isolate; Mean values in millimeters, and standard deviations of the zones of inhibition are expressed

Table 3. The minimum inhibitory concentration (MIC) of aqueous extracts of bark and leaf of E. chlorantha alone and
combined
# - - +
Microorganism/extract C. albicans E. coli P. vulgaris S. typhi
ECB 3.125 6.25 6.25
ECL 12.5 12.5 6.25
ECL/B 6.25 12.5 25
KEY: ECB= Enantia chlorantha bark; ECL= Enantia chlorantha leaf; ECL/B= Enantia chlorantha leaves+Enantia chlorantha bark; #=Mycotic isolate;
- = Gram-negative isolate; += Gram-positive isolate; Results in mg/ml

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Table 4. The minimum bactericidal/fungicidal concentration (MBC/MFC) of aqueous extracts of bark and leaf of
E. chlorantha alone and combined
# - - +
Microorganism/extract C. albicans E. coli P. vulgaris S. typhi
ECB 6.25 12.5 12.5
ECL 12.5 25 25
ECL/B 6.25 25 25
KEY: ECB= Enantia chlorantha bark; ECL= Enantia chlorantha leaf; ECL/B= Enantia chlorantha leaves+Enantia chlorantha bark; #=Mycotic isolate;
- = Gram-negative isolate; += Gram-positive isolate; Results in mg/ml

Table 5. The minimum inhibitory concentration (MIC) of aqueous extracts of bark and leaf of E. chlorantha alone and
combined
# + - - - - +
Microorganism/extract C. albicans E. faecalis E. coli P. vulgaris S. typhi S. sonnei S. aureus
ECB 3.125 3.125 3.125 1.56 3.125 6.25 6.25
ECL 12.5 6.25 6.25 3.125 3.125 12.5
ECL/B 3.125 3.125 6.25 3.125 6.25 6.25 25
KEY: ECB= Enantia chlorantha bark; ECL= Enantia chlorantha leaf; ECL/B= Enantia chlorantha leaves+Enantia chlorantha bark; #=Mycotic isolate;
- = Gram-negative isolate; += Gram-positive isolate; Results in mg/ml

Table 6. The minimum bactericidal/fungicidal concentration (MBC/MFC) of aqueous extracts of bark and leaf of
E. chlorantha alone and combined
# + - - - - +
Microorganism/extract C. albicans E. faecalis E. coli P. vulgaris S. typhi S. sonnei S. aureus
ECB 3.125 12.5 6.25 3.125 3.125 6.25 12.5
ECL 25 12.5 6.25 6.25 6.25 25
ECL/B 3.125 3.125 12.5 6.25 6.25 6.25 12.5
KEY: ECB= Enantia chlorantha bark; ECL= Enantia chlorantha leaf; ECL/B= Enantia chlorantha leaves+Enantia chlorantha bark;
#=Mycotic isolate; - = Gram-negative isolate; += Gram-positive isolate; Results in mg/ml

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When combined, the extracts seemed to have antimicrobial activities more than, or equal to
that of the most active component except the case of P. vulgaris (Table 1) which was
sensitive to aqueous extracts of both ECB and ECL but resistant to ECL/B; this could be as
a result of the presence of inhibitors to the active antimicrobial substance in ECB and ECL
which exerted a synergistic effect when combined, inhibiting the antimicrobial component.
That this was not replicated in ethanolic extracts should be explained better by a
phytochemical analysis of ECB and ECL. This aspect of the research work is important as
multidrug therapy is currently recommended as one way of avoiding the rapid development
of drug resistance by microorganisms, which is partly responsible for treatment failure [26].
Crude plant extracts are known to contain several active components, as such combinations
of different plant extracts may be considered as combinations of different active components
with potentiating or synergistic effects.

Considering the fact that development of resistance to crude extracts has hardly been
reported [26], the use of plants or plant extracts in chemotherapy should really be
considered bearing in mind issues of their toxicity and safety.

4. CONCLUSION AND RECOMMENDATION

The results obtained in this study provide a support to the use of E. Chlorantha in ethno-
medicine in line with similar works. However extensive toxicity tests are recommended to be
run on the plant to determine its toxic effects (if any) as well as further chemical and
pharmacological investigations to isolate and identify the chemical constituents in the plant
parts and to screen their other potential bioactivities. These would be useful in determining
the principles of the plant’s therapeutic actions and elucidate the mechanisms of its action.

Hence if results of the toxicological screening of E. chlorantha shows minimal or no side

effects, more detailed information on its various parts would then be needed so that their
extracts can be considered for the production of cheap phytomedicines as an alternative to
the expensive antibiotic regimens for diseases of public health importance like those studied
in this investigation. This study would, based on the results of further toxicological
examination of the plant, support and justify its use in herbal preparations especially for
people in rural communities where the practice of herbal medicine is prevalent. I believe that
E. chlorantha could be a potential source of drugs if the active ingredients are identified and
adequately characterized.

Thus, Enantia chlorantha plant parts could become promising natural antimicrobial agents
with potential applications in pharmaceutical industries for controlling pathogenic organisms.

CONSENT

Not applicable.

ETHICAL APPROVAL

Not applicable.

COMPETING INTERESTS

Authors have declared that no competing interests exist.

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_________________________________________________________________________
© 2014 Atukpawu and Ozoh; This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.

Peer-review history:
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