The Antimicrobial efficacy of Elaeis guineensis: characterization, in vitro and in vivo studies.

Abstract

The urgent need to treat multi-drug resistant pathogenic microorganisms in chronically infected patients has given rise to the development of new antimicrobials from natural resources. We have tested Elaeis guineensis Jacq (Arecaceae) methanol extract against a variety of bacterial, fungal and yeast strains associated with infections. Our studies have demonstrated that E. guineensis exhibits excellent antimicrobial activity in vitro and in vivo against the bacterial and fungal strains tested. A marked inhibitory effect of the E. guineensis extracts was observed against C. albicans whereby E. guineensis extract at ½, 1, or 2 times the MIC significantly inhibited C. albicans growth with a noticeable drop in optical density (OD) of the bacterial culture. This finding confirmed the anticandidal activity of the extract on C. albicans. Imaging using scanning (SEM) and transmission (TEM) electron microscopy was done to determine the major alterations in the microstructure of the extract-treated C. albicans. The main abnormalities noted via SEM and TEM studies were the alteration in morphology of the yeast cells. In vivo antimicrobial activity was studies in mice that had been inoculated with C. albicans and exhibited good anticandidal activity. The authors conclude that the extract may be used as a candidate for the development of anticandidal agent.

Full text

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Molecules 2012, 17, 4860-4877; doi:10.3390/molecules17054860

molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
The Antimicrobial Efficacy of Elaeis guineensis:
Characterization, in Vitro and in Vivo Studies
Soundararajan Vijayarathna 1, Zuraini Zakaria 2, Yeng Chen 3, Lachimanan Yoga Latha 1,
Jagat R. Kanwar 4 and Sreenivasan Sasidharan 1,*
1 Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia,
11800 Minden, Penang, Malaysia; E-Mails: vijaya_r_1984@yahoo.com (S.V.);
latha_usm@yahoo.com (L.Y.L.)
2 Biology Program, School of Distance Education, Universiti Sains Malaysia, 11800 Minden,
Penang, Malaysia; E-Mail: zuraini@usm.my
3 Dental Research & Training Unit, and Oral Cancer Research and Coordinating Centre (OCRCC),
Faculty of Dentistry, University of Malaya, 50603 Kuala Lumpur, Malaysia;
E-Mail: chenyeng@um.edu.my
4 Nanomedicine-Laboratory of Immunology and Molecular Biomedical Research (LIMBR),
Centre for Biotechnology and Interdisciplinary Biosciences (BioDeakin),
Institute for Frontier Materials (IFM), Deakin University, Waurn Ponds,
Victoria 3217, Australia; E-Mail: jagat.kanwar@deakin.edu.au
* Author to whom correspondence should be addressed; E-Mail: srisasidharan@yahoo.com;
Tel.: +604-653-4820; Fax: +604-653-4803.
Received: 6 March 2012; in revised form: 14 April 2012 / Accepted: 16 April 2012 /
Published: 26 April 2012

Abstract: The urgent need to treat multi-drug resistant pathogenic microorganisms in
chronically infected patients has given rise to the development of new antimicrobials from
natural resources. We have tested Elaeis guineensis Jacq (Arecaceae) methanol extract
against a variety of bacterial, fungal and yeast strains associated with infections. Our
studies have demonstrated that E. guineensis exhibits excellent antimicrobial activity
in vitro and in vivo against the bacterial and fungal strains tested. A marked inhibitory
effect of the E. guineensis extracts was observed against C. albicans whereby E. guineensis
extract at ½, 1, or 2 times the MIC significantly inhibited C. albicans growth with a
noticeable drop in optical density (OD) of the bacterial culture. This finding confirmed the
anticandidal activity of the extract on C. albicans. Imaging using scanning (SEM) and
transmission (TEM) electron microscopy was done to determine the major alterations in
OPEN ACCESS

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Molecules 2012, 17

the microstructure of the extract-treated C. albicans. The main abnormalities noted via
SEM and TEM studies were the alteration in morphology of the yeast cells. In vivo
antimicrobial activity was studied in mice that had been inoculated with C. albicans and
exhibited good anticandidal activity. The authors conclude that the extract may be used as
a candidate for the development of anticandidal agent.
4861

Keywords: antimicrobial activity; Candida albicans; Elaeis guineensis; scanning electron
microscopy; transmission electron microscopy

1. Introduction
Antimicrobial agents, particularly antibiotics, have been the standard therapy for managing
microbial infections, but in recent years, genetic variation has given to pathogenic microbes a great
advantage by creating antibiotic resistance so the search for new antimicrobial substances or drugs
continues to be necessary. Major clinical issues arise when pathogenic microbes develop multi-drug
resistance intertwined with other problems such as level of toxicity of antimicrobial drugs on host
tissues. Further, reports from the scientific community have raised concerns that antibacterial drug
development will not be adequately addressing the problems posed by antibiotic resistance among
important bacterial pathogens [1–4]. For example, in the First European Communicable Disease
Epidemiological Report, the European Centre for Disease Prevention and Control (ECDC) had rated
antimicrobial resistance as the main factor that contributes to infectious disease in Europe due to the
increase in infections owing to multidrug resistant bacteria [5]. Hospitals globally are facing the recent
emergence of bacteria that are totally or almost totally resistant to currently available antibiotics is
even more threatening since treatment options for infected patients are extremely limited [6,7].
The various strategies which have been identified to defeat drug resistance, the investigation of new
and effective natural products exhibiting antimicrobial activity against pathogenic microorganisms is
likely to play a significant role to overcome drug resistance. Malaysia, being one of the 12 mega-diversity
centers of the World, is rich in all three levels of biodiversity, namely species diversity, genetic
diversity and habitat diversity with many plants used for medicinal and nutritional purposes [8]. One of
such plant known to have healing potential with various pharmacological activities is Elaeis guineensis
Jacq (Arecaceae). E. guineensis has many therapeutic uses in traditional medicine practice. Every part
of the plant can be used medicinally. The leaves of E. guineensis are squeezed and the juice that is
obtained is placed on wounds to enhance healing [9]. The sap of this plant is also used as a laxative
and the partially fermented palm wine is administered to nursing mothers to improve lactation.
Fruit-husk ash is used for the preparation of a soap used for skin infections. A root decoction is used in
Nigeria for headaches. The pulverized roots are added to drinks for gonorrhea, menorrhagia and as a
cure for bronchitis [9]. The fruit mesocarp oil and palm kernel oil are administered as poison antidote
and used externally with several other herbs as lotions for skin diseases. Palm kernel oil is applied to
convulsant children to regulate their body temperature. Folk remedies of oil palm include treatment for
cancer, headache and rheumatism and as an aphrodisiac, diuretic and liniment [9]. Recent studies also
reported various pharmacological activity of this plant extract namely standardization of the extract,

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Molecules 2012, 17

antimicrobial activity, infected wound healing activity and antioxidant activity [10–13]. Syahmi et al. [14]
also tested the toxicity of E. guineensis leaf methanol extract against brine shrimp (A. salina) and mice.
The results of both tests confirmed that E. guineensis is nontoxic and they recommended as safe
natural product for commercial utilization. Hence, this work was attempted to study the potential
in vitro and in vivo antimicrobial activity of E. guineensis.
4862

2. Results
2.1. Antimicrobial Activity
Antimicrobial activity of leaf extract of E. guineensis expressed as zone of inhibition (mm) is
shown in Table 1.
Table 1. Antimicrobial activity of E. guineensis.
Microorganisms
Zone of Inhibition (mm) a
Leaf extract


14
12

13
11
14
12
13

15
-

-
-
13
-
-
-
-
-
-
-
Minimum Inhibitory
Concentration (mg/mL)
C


21
22

21
24
24
22
23

ND
ND

ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
M


ND
ND

ND
ND
ND
ND
ND

24
24

23
22
24
23
24
22
23
22
24
24
BACTERIA
Gram-positive
Staphylococcus aureus
Bacillus subtilis
Gram-negative
Escherichia coli
Klebsiella pneumoniae
Pseudomonas aeruginosa
Salmonella typhi
Proteus mirabilis
YEAST
Candida albicans
Saccharomyces cerevisiae
FUNGI
Fusarium sp.
Fusarium oxysporium
Aspergillus niger
Aspergillus flavus
Microsporum canis
Mucor sp.
Penicillium sp.
Rhizophus sp.
Trichoderma viride
Trichophyton mentagrophytes
a The values ( average of triplicate) are diameter of zone of inhibition at 100 mg/mL crude
extract, 30 µg/mL chloramphenicol and 30 µg/mL of miconazole nitrate. (ND: Not determined;
C: chloramphenicol; M: miconazole nitrate).


12.5
6.25

12.5
>50.0
12.5
6.25
>50.0

6.25
-

-
-
12.5
-
-
-
-
-
-
-
The extract had great in vitro potential of antimicrobial activities against tested Gram-positive and
Gram-negative and fungi with inhibition zone diameters ranging from 11 to 15 mm. Maximum activity

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was observed against C. albicans (15 mm) while Klebsiella pneumoniae (11 mm) exhibited a weak
inhibition zone. The antimicrobial activity of leaf extract was also observed on the growth of
filamentous fungi A. niger (13 mm). In contrast, the inhibition zone of solvent control methanol
(negative control) was zero so that it was not active against any of the tested microorganisms.
However, the two antibiotics (positive control), 30 µg/mL of chloramphenicols for bacteria and
miconazole nitrate for fungi were found more effective than the leaf extract of E. guineensis with the
inhibition zone diameters ranging between 21 and 24 mm. Based on the initial antimicrobial screening
assay the strains which showed positive results against leaf extract of E. guineensis were selected for
further studies to determine the MIC values as shown in Table 1. The MIC values against all the tested
Gram-positive and Gram-negative bacteria and fungi ranged from 6.25 to 50.00 mg/mL. The MIC
values indicated that the seed extract was more effective against Gram-positive bacteria at lower
concentration than Gram-negative bacteria (Table 1). The lowest concentration was recorded as
6.25 mg/mL for Bacillus subtilis, Salmonella typhi and particularly Candida albicans.
4863

2.2. Time Kill Study
The growth profile curves for C. albicans in Mueller-Hinton Broth which being exposed to 1, ½ and
2 MIC of E. guineensis leaf extract over a period of 48 h are shown in Figure 1. The various MICs of
seed extract shifted the normal growth profile for C. albicans. The control curve of the C. albicans
demonstrated a typical microbial population growth cycle. The growth profile could be divided into
three phases, from 0 to 8 h the control curve exhibited the “lag phase”, from 8 to 20 h the “exponential
phase” and finally 20 to 48 h is the “stationary phase”. At ½ MIC value (3.125 mg/mL), the
E. guineensis leaf extract demonstrated the microbial growth increases until maximum value of 0.361
OD at 16 h. At the MIC value (6.25 mg/mL), E. guineensis leaf extract caused a large drop in OD after
8 h, then the biomass decreased gradually and finally lead to a stationary phase where there were no
visible growth changes. At 2 times the MIC value (12.5 mg/mL), E. guineensis leaf extract exhibited
complete eradication within 2 h with a reading of 0.2 OD.
Figure 1. Growth profile for Candida albicans in Mueller-Hinton broth with 0 (Control)
½, 1 and 2 times MIC of Elaies guineensis leaf extract.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
0481216 20 24
Time (h)
2832364044 4852
Growth OD ( 540 nm)
Control
1/2 MIC
1MIC
2 MIC

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Molecules 2012, 17

2.3. In Vivo Antifungal Activity
4864

The antifungal study was carried out with using infected mice to contemplate the effectiveness of
the extract in combating infection in vivo (Figure 2).
Figure 2. Mortality rate of mice in different group of treatment for seven days. Group 1
(control); Group 2 (treatment with ketoconazole); Group 3 (treatment with E. guineensis
extract). p < 0.05.
0
10
20
30
40
50
60
70
123
Experimental groups
% Mortality
Day 1Day 2
Day 3 Day 4
Day 5Day 6
Day 7

Sixty percent of Group 1 animals died within 7 days of receiving phosphate buffer solution (PBS).
The survival of mice was higher after ketoconazole treatment compared to other experimental groups
with 70% of animals surviving to 7 days. In contrast, survival of mice with E. guineensis leaf extract
was relatively high, with a survival rate of 60%. However, 70% of Group 2 mice receiving
ketoconazole survived up to 7th day.
The extract had a good effect on the reduction of the mortality when the treatment was given after
infection by the C. albicans, decreasing the mortality from 50% (Group 1) to 20 % (Groups 2 and 3).
In addition, the effect of the extract was comparable to the commercial antibiotic ketoconazole. Figure 3
shows the mean of CFU/g of the kidney from the three experimental groups. Group 3 mice received a
2.5 g/kg body weight dose of the plant extract followed by inoculation of C. albicans, a significant
reduction (p < 0.05) in CFU was observed in the studied group compared with control group.
2.4. SEM Observation
Scanning electron microscopy study was used to view any surface alterations and or general
morphological changes of C. albicans cells after exposure to E. guineensis leaves extract. Comparisons
were made between the control C. albicans cells and the treated C. albicans cells.

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Molecules 2012, 17

Figure 3. Effect of methanol extract of E.guineensis on Candida albicans recovered from
kidney of mice. Group 1 (control); Group 2 (treatment with ketoconazole); Group 3
(treatment with E. guineensis extract). p < 0.05.
4865

0.00E+00
2.00E+04
4.00E+04
6.00E+04
8.00E+04
1.00E+05
1.20E+05
1.40E+05
1.60E+05
1.80E+05
2.00E+05
12
Group 2
3
Experimental group
CFU / g kidneys
Group 1
Group 1 Group 3

All the control C. albicans cells were generally smooth-walled bodies, spherical in shape and were
mostly present in the yeast form. All the cells were lying apart showing normal budding stage (Figure 4A).
After 12 h of exposure to the leaf extract of E. guineensis, several small invaginations and
convolutions appeared on the C. albicans cell surfaces (Figure 4B). Other remaining cells showed a
smooth surface as observed in control cells. More invaginations and convolutions appeared in the 24 h
treated cells (Figure 4C). Cracks in the cell wall were detected in the last group, which was treated
with the leaf extract for the duration of 36 h (Figure 4D). Thus, it was assumed at this stage that the
cells had completely lost their metabolic functions.
2.5. TEM Observation
The scanning electron microscopy studies results were compared with those seen on the yeast cells
by means of transmission electron microscope. It was found that the untreated (control)
C. albicans cells as examined by TEM showed typical C. albicans morphology with a uniform central
density (Figure 5A). Control cells typically had a structured nucleus and a cytoplasm with several
elements of endomembrane system enveloped by a regular, intact cell wall plasma membrane lying
closely to the cell wall. After being exposed to E. guineensis extract for 12 h (Figure 5B), the
C. albicans cells appeared to possess large vesicles while membranous bodies were found disposed
within the cell. On the other hand, the cell walls of the 24 h treated cells (Figure 5C) appeared
vertically more oblong accompanied by the shrinkage of protoplast and disruption of cytoplasmatic
membrane. In addition, the cytoplasmic volume is contemplated decreasing following the cell
membrane invagination causing notable structural disorganization within the cell cytoplasm. It can be
deduce that the leaf extract had triggered cell membrane to become dysfunctional. The significant
a
b
b

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Molecules 2012, 17

effect of the extract can be viewed after 36 h of treatment (Figure 5D). All the inner organelles are
completely discomposed while cell membranes appear undulant. Yeast cells were found collapsed.
4866

Figure 4. SEM micrographs of the untreated (A), 12 h (B), 24 h (C) and 36 h (D) extract
treated cells of Candida albicans.

Figure 5. TEM micrographs of the untreated (A), 12 h (B), 24 h (C) and 36 h (D) extract
treated cells of Candida albicans.

AB
CD
A
B

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Molecules 2012, 17

4867

Figure 5. Cont.

2.6. Identification of Antifungal Bioactive Compound (s)
2.6.1. FTIR Analysis
FTIR spectroscopic studies were carried out to investigate the possible antimicrobial and antifungal
agents that might present in the extract of E. guinensis (Figure 6). The spectra of extracts were
recorded in the form of an interferrogram to which results of various functional groups were exhibited
between the wavelength of 100–1,700 cm−1 and 2,900–3,500 cm−1. Eight functional groups were
found. The spectrum denoted a broad at 3,406.05–3,436.91 cm−1 which is assigned to (OH) stretching
vibrations from phenols present in the extract. Strong to medium intensities broad bands are observed
at 2,983.67–2,870.84 cm−1 which suggest energetically favored carboxylic acid groups. Other bands
are observed to be appearing at 1,514.00–1,450.00 cm−1 and 1,038.45 cm−1 are attributed to the
existence of aromatic, alkene C=C, primary alcohol and phenol groups, respectively.
2.6.2. Gas Chromatography-Mass Spectrometry (GC-MS) Analysis
GC-MS analysis was done to identify the compounds responsible for observed antimicrobial
activity. The gas chromatogram of the methanolic leaf extract of E. guineensis showed eleven
compounds (Figure 7). The retention times (Rt) were reported in minutes. The eleven major
compounds identified in the methanolic extract were dimethyl sulfoxide, which was eluted twice with
different retention times (Rt 3.14; 3.17); tetrahydro-trans-3,4-Furandiol (Rt 5.35); 1-Amino-2,6-
dimethylpiperidine (Rt 6.53); 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-Pyran-4-one (Rt 8.43);
4-methyl-1-(1-methylethyl)-3-Cyclohexen-1-ol (Rt 9.07); 5-(hydroxymethyl)-2-Furancarboxaldehyde
(Rt 10.05); D-mannose (Rt 13.83); 1,6-anhydro-α-D-glucopyranose (levoglucosan) (Rt 14.66);
3-tert-butyl-4-hydroxyanisole (Rt 15.42); 3,4-dihydro-2(1H)-isoquinolinecarboximidamide (Rt 15.65);
and 5-isopropenyl-2-methylcyclopent-1-ene-carboxaldehyde (Rt 16.11).

CD

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Molecules 2012, 17

Figure 6. Fourier transform infrared (FTIR) spectroscopy analysis obtained for the leaf
extract of Elaeis guineensis.
4868


Figure 7. Gas chromatogram of E. guineensis methanolic extract.

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Molecules 2012, 17

3. Discussion
4869

In this study, we investigated the in vitro and in vivo activity of leaf extract of E. guineensis. The
in vitro activity was tested against a wide panel of clinical isolates including bacterial strains, as well
as fungal and the yeast C. albicans. The results indicate a broad spectrum activity of the leaf extract of
E. guineensis, which was more effective against Gram-positive bacteria than Gram-negative bacteria
(Table 1). This occurrence could be explained by that the structures of cell envelope differ
significantly between Gram-positive and Gram-negative bacteria. Gram-negative bacteria possess an
outer membrane surrounding the cell wall, which restricts diffusion of hydrophobic compounds
through its lipopolysaccharide covering. The cell wall of Gram-positive bacteria without this outer
membrane can be permeated more easily and the constituents of E. guineensis extract can disturb the
various molecular targets including the cytoplasmic membrane, proton motive force (PMF), electron
flow, active transport and coagulation of cell contents [15]. This finding was further verified by our
SEM and TEM study which indicated the outer physical changes that took place on the fungal cells
structures while the TEM describes the inner cell morphology and cytology changes that further
substantiate the aptitude of E. guineensis as a potential anticandidal compound (Figures 4 and 5).
Time killing profile was applied to further corroborate the in vitro antimicrobial results observed.
The time killing study exhibited a prolonged anti-candididal activity when Candida albicans cells were
exposed to the methanolic extract of E. guineensis at ½ MIC, MIC and 2 MIC for 48 h. In vitro
antimicrobial methods are controversial because the results do not always correlate with the in vivo
conditions or clinical outcomes. Such a fact clearly shows that traditional MIC determinations are
inadequate for determining an antimicrobial agent’s clinical effectiveness against pathogens. Time-kill
curves of pathogens exposed to several extract concentrations measure antimicrobial activity over
time, and may reveal differences in activity between agents at different MIC concentrations. Our
results indicate that E. guineensis extract possesses a good anti-yeast activity; concentration dependent
killing was observed against the strains tested.
The results obtained with in vitro assays encouraged us to exploit the in vivo potential of
E. guineensis extract against lethal candidal challenge in animals. The use of an in vivo model in mice
was suggested by the previous results of in vivo oral toxicity tests that indicated a much better LD50 for
oral administration of these E. guineensis extracts. The in vivo results using the E. guineensis extract is
encouraging and parallel those obtained in vitro. E. guineensis extract was highly protective against
C. albicans infection, with 80% protection at respectively 2.5 g/kg. Notably, protection is achieved at
E. guineensis doses used in this study, suggesting a satisfactory therapeutic index for this extract.
In keeping with the in vitro results, E. guineensis also defends mice from lethal i.v. dose of
C. albicans, suggesting that E. guineensis extract are effective in vitro than in vivo. The in vivo results
so far obtained indicate that, the effect of E. guneensis extract was significantly comparable to the
commercial antibiotic ketoconazole.
Our results indicated that E. guneensis extract had good antimicrobial activity towards the tested
pathogenic microbes. Then, the extract was subjected to FTIR and GC-MS analysis, which
revealed the presence of antimicrobial compound and with various functional groups. Both the
3,406.05–3,436.91 cm−1 and 1,038.45 cm−1 absorption bands were attributed to (OH) stretching
vibrations from phenols, a class of chemical compounds containing hydroxyl functional groups (–OH)

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Molecules 2012, 17

attached to an aromatic hydrocarbon group. Numerous recent studies have reported that phenolic
compounds from natural resources display antifungal activity. Duke reported that the common herbs
tarragon and thyme both possessed a phenylpropane-derived compound (phenol) which is very active
against fungi. The location site(s) and the amount of hydroxyl groups found on the phenols are related
to their relative toxicity towards microorganisms with evidence that increased hydroxylation results in
increased toxicity [16]. Likewise, it was also reported that more highly oxidized phenols show more
inhibitory activity [17,18] which includes enzyme inhibition by the oxidized compounds, possibly
through reaction with sulfhydryl groups or through more nonspecific interactions with the proteins [19].
Literature searches also reveal that phenolics also serve as an antimicrobial agents [20–22] with
activity that includes adsorption and disruption of microbial membranes, interactions with enzymes
and substrates and metal ion deprivation [23,24].
Strong to medium intensity bands were observed at 2,983.67–2,870.84 cm−1 confirming the
presence of carboxylic acid functional groups. Carboxylic acids are essential groups found in other
important plant secondary metabolites such as olean-27-carboxylic acid, a type of triterpene reported
to shows antibacterial activity [25]. Likewise carboxylic acids were found to be linked with many
antimicrobial and antifungal activities which are found to exist in various plant metabolite molecular
structures such as ursolic acid which had been reported as a strong antibacterial agent [26]. In other
cases, Jabeen et al. [27] have mentioned that the elimination of fungal pathogens by the seed extract of
Moringa oleifera can be attributed to the presence of carboxylic acids. This fact is further substantiated
by Shittu et al. [28] who had earlier reported on carboxylic acids being responsible for the
antimicrobial activity in Sesame radiatum. Other bands being detected in the FTIR analysis appeared
at 1,514.00–1,450.00 cm−1 and 1,038.45 cm−1 that correspond respectively to the presence of C=C
aromatic, alkenes, primary alcohol, and again phenol groups. Plants produce vast and diverse numbers
of secondary metabolites that include these active groups. Other chemical components of the extract
also certainly could contribute although lack of chemical profiling has never been reported on this.
In addition, GC-MS analysis also revealed the presence of several potentially antimicrobial
components in the E. guineensis extract. The antimicrobial properties of E. guineensis extract are
suspected to be associated with the dimethyl sulfoxide, 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-
pyran-4-one, 5-(hydroxymethyl)-2-furancarboxaldehyde, and D-mannose, which was detected by the
GC-MS analysis in this study. All these components have been tested previously and reported to have
a significant antimicrobial activity [29–32]. It is possible that these compounds are mainly responsible
for the antimicrobial effects observed in this study.
4870

4. Experimental
4.1. Plant Collection
Fresh leaves of E. guineensis were collected Semeling, Kedah in January 2010 and authenticated at
the Herbarium of the School of Biological Sciences, Universiti Sains Malaysia, Pulau Pinang,
Malaysia where a sample (voucher number 11037) has been deposited. The leaves were separated and
cut into small pieces, which were first washed with tap water and then with distilled water. The leaves

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were then dried in an oven at 60 °C for 7 days, after which the dried leaves were ground into a fine
powder using a grinder and stored in clean, labeled airtight bottles.
4871

4.2. Preparation of Plant Extract
Dried sample (approximately 100 g) was added to methanol (300 mL) and soaked for 4 days at
room temperature (30 ± 2 °C). The suspension was stirred from time to time to allow the leaf powder to
fully dissolve in the methanol. Removal of the sample from the solvents was done by filtration through
cheesecloth followed by filter paper (Whatman No. 1); the filtrate was concentrated under vacuum
(vacuum pressure: 500 N/m2) at 40 °C [33] to one-fifth its volume using a rotary evaporator and then
sterilized by filtration using a 0.22-mm membrane for antimicrobial assay. The thick paste obtained
was further dried in an oven at 40 °C. The resultant extract was kept at 4 °C for further analysis.
4.3. Determination of the Antimicrobial Activity
4.3.1. Antimicrobial Disc Diffusion Assay
Antimicrobial and antifungal activities of E. guinensis extract were investigated using the disc
diffusion method [34,35]. The test microbes were removed aseptically with an inoculating loop and
transferred to a test tube containing 5 mL of sterile distilled water. Sufficient inoculums were added
until the turbidity became equaled to 0.5 McFarland (106 colony-forming units (CFU)/mL of bacteria
or 2 × 105 (CFU)/mL fungi cells/spore) standards (bioMerieux, Marcy Petoile, France). The test tube
suspension (1 mL) was added to 15 to 20 mL of Mueller-Hinton Agar and Sabouraud Dextrose Agar
before setting aside the seeded agar plate (9 cm in diameter) 15 min to solidify. Three Whatman’s filter
paper No.1 discs of 6 mm diameter were used to screen the antimicrobial activity. Each sterile
disc was impregnated with 20 μL of the extract (corresponding to 100 mg/mL of crude extract),
chloramphenicol or miconazole nitrate (30 µg/mL, as positive control for bacteria and yeast/fungus
respectively) and 80% methanol (v/v) (as negative control) after the discs were placed on the surface
of the seeded plates. The plates were incubated in the incubator (Memmert, Schwabach, Germany) at
37 °C and at 28 °C for yeast/fungi. The zones of inhibition around the discs were measured after 18 to
24 h of incubation for bacteria, and 48 to 96 h for fungi. Sensitivity of the microorganism species to
the plant extract was determined by measuring the sizes of inhibitory zones (including the diameter of
disc) on the agar surface around the discs, and values less than 8 mm were considered as not active
against microorganisms [36]. All the microbial strains used in this study were local clinical isolates.
All of the experiments were performed in triplicate and results were reported as the average of
three experiments.
4.3.2. Minimum Inhibitory Concentration (MIC) Determination
Due to the display of significant activity by the E. guinensis leaf extract against the tested
microorganisms, further determination of the minimum inhibitory concentration was investigated.
Two-fold broth dilution method was implied as described by [37] with slight modification. The plant
extract (500.00 mg) was dissolved in distilled water (10 mL) to reconstitute an extract solution of
50.00 mg/mL as stock. Subsequently, a serial dilution technique was carried out with 2.5 mL of stock

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solution being transferred to a test tube containing 2.5 mL nutrient broth medium to give a
concentration of 25.00 mg/mL. Next, 2.5 mL of solution from the first test tube was transferred into
another a second test tube containing nutrient broth medium that gave rise to a concentration of
12.50 mg/mL and similarly technique was continued until a final concentration of 0.098 mg/mL
was achieved.
The test microbes were removed aseptically with an inoculating loop and transferred to a test
tube containing 10 mL of sterile distilled water. Sufficient inoculums was added until the turbidity
was equivalent to 0.5 McFarland (106 CFU/mL) standard (bio-Meriuex, Marcy Petoile, France).
An inoculum size of 0.5 mL bacteria/fungal was added to each test tube by maintaining the final
concentration of the extract in each test tube. After 18 h of incubation at 37 °C, the tubes were
examined for bacterial growth. Growth was observed in those tubes where the concentration of the
extract was below the inhibitory level where the broth medium turned into turbid or looks cloudy. The
MIC value of the extract was taken as the lowest concentration that showed no growth or non-turbid in
the test tube [38].
4872

4.4. Time-Kill Study
The time killing study was conducted with ½, 1 and 2 times MIC over time whereby a growth
profile curve was plotted [39]. A 16 h culture was harvested by centrifugation, washed twice with
phosphate saline and re-suspended in phosphate saline. The suspension was adjusted using the
McFarland standard and was then further diluted in phosphate saline to achieve an approximation of
107 colony forming unit (CFU/mL). Leaf extract of E. guineensis was added to aliquots of 25 mL
Mueller-Hilton broth (MHB) in 50 mL Erlenmeyer flask and was placed in a water bath at 37 °C with
amounts corresponding to the concentration of ½ ,1 and 2 times of MIC value (6.25 mg/mL) upon
the addition of the inoculums. Free medium without extract was used as a control. Next, 1 mL of
C. albicans inoculum was added to all Erlenmeyer flasks. After the addition of the inoculums 1 mL
portion was removed from Erlenmeyer flask and the growth of C. albicans was monitored using this
portion by measuring the Optical Density at 540 nm 9 UV-9100, Ruili Co., Beijing, China). The
growth of C. albicans was measured every 4 h throughout 48 h by the above method.
4.5. In Vivo Antifungal Assay
4.5.1. Laboratory Animals
Swiss albino mice (male) weighing between 25 and 35 g were used. The cages with the mice were
placed in a room (temperature 26 ± 2 C) with controlled cycles of 12 h of light and 12 h of darkness;
light went on at 7 am and relative humidity was 45–55%. Water and food were provided to animals
ad libitum. The experimental protocols were approved by the Institutional Animal Ethics Committee
(IAEC) at Universiti Sains Malaysia. Experiments were conducted in accordance with the
internationally accepted principles for laboratory animal use and care (EEC Directive of 1986;
86/609/EEC).

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4.5.2. Antifungal Assay
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Standard intravenous (i.v.) inoculation of C. albicans was used in this study where 1 × 107 viable
cells/mL PBS, of which 0.1 mL was injected into the lateral tail vein of mice [40]. Animals were
divided into three groups of 10 mice each and received treatment as described in Table 2. All mice
were killed by cervical dislocation on day 5 after i.v. C. albicans inoculation. The drug and extract
concentration for in vivo model were determined based on the body weight of the mice used in this
study (drug weigh/ kg body weight of mice). The kidneys of each animal were removed aseptically,
and 0.1 mL of blood was withdrawn from the renal artery and 0.1 mL of heparin (25 U/mL), as an
anticoagulant was added into the blood sample. The kidneys were then, placed in sterile centrifuge
tubes and homogenized in 5 mL of sterile PBS. Aliquots from each homogenate and blood samples
were serially diluted, plated on Sabouraud dextrose agar plates, and incubated at 37 C for 24 h.
All cultures were done in triplicate. The colonies were then enumerated and the colony forming units
(CFU) were calculated per gram of organ and per mL of blood sample, respectively.
Table 2. Animal experimental groups and received treatments data summary.
Group
Group 1
Treatment
(Negative control)
Group 2
(Positive control)
i.v. Candida 24h gap, follow by treatment with PBS (o.a. once daily for 7 days)
i.v. Candida 24h gap, follow by treatment with ketoconazole, 10 mg/kg body
weight (o.a. Once daily for 7 days)
i.v. Candida 24h gap, follow by treatment with E. guineensis extract, 2.5 g/kg
body weight (o.a. once daily for 7 days)
i.v.: intravenous; o.a.: oral administration; PBS: phosphate buffer saline.
Group 3 (curative)
4.6. Scanning Electron Microscope (SEM) Observation
Scanning electron microscope (SEM) observations were carried out on C. albicans cells. One
milliliter of the C. albicans cell suspension with the concentration of 1 × 106 cells per mL was
inoculated on four Sabouraud dextrose agar plate and then incubated at 30 °C for 12 h. Two milliliter
of 6.25 mg/mL, E. guineensis leaf extract was dropped into three inoculated agar and further incubated
for another 12, 24 and 36 h at the same incubation temperature. An 80% methanol (v/v) treated culture
was used as a control. A small block of yeast containing agar was withdrawn from the inoculated plate
0, 12, 24 and 36 h and fixed for scanning electron microscope (Leo Supra 50 VP Field Emission SEM,
Carl Zeiss, Oberkochen, Germany) [41]. The SEM study was done under the following analytical
conditions: L = SE1, WD = 21 mm, and EHT = 10.0 kV to study the effect of extract on C. albicans cells.
4.7. Transmission Electron Microscope (TEM) Observation
Transmission electron microscope (TEM) observations were carried out on C. albicans cells. The
preparation of the cells on the plates was as Section 3.4.4. After incubating the 0, 12, 24 and 36 h
plates, all were fixed for TEM observation [42]. TEM analyses was performed on samples fixed in
Mc-Dowell-Trump fixative prepared in 0.1 M phosphate buffer, rinsed in buffer and postfixed for 2 h
in 1% osmium tetroxide prepared in the phosphate buffer. The sample in agar was prepared and

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Molecules 2012, 17

dehydrated in ethanol and finally embedded in resin. The resin with the embedded C. albicans cells
were cut into ultra-thin sections in the ultramicrotomy process. Finally the ultra thin sections were
stained with 2% uranyl acetate and lead citrate, and observed under TEM (Philip CM12, Eindhoven,
Netherlands).
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4.8. Identification of Antifungal Bioactive Compound(s)
4.8.1. FTIR Analysis
The methanol extracts of E. guineensis was mixed with potassium bromide (KBr) using a mortar
and pestle, and compressed into a thin pellet. Infrared spectra were recorded as KBr pellets on a
Schimadzu FTIR Spectrometer 8000 series (Columbia, WA, USA), between 4,000 and 500 cm−1. All
determinations were performed in triplicate.
4.8.2. Gas Chromatography-Mass Spectrometry (GC-MS) Analysis
The GC-MS analysis was done on a thermo gas chromatography mass spectrometer (model
Shimadzu 2010, Tokyo, Japan) equipped with DB-5 capillary column (30 m long, 0.25 mm i.d., film
thickness 0.25 µm). The column temperature program was 50 °C for 6 min, with 5 °C increases per min
to 250 °C; which was maintained for 30 min. The carrier gas was helium at a flow rate of 1 mL/min.
The detector and injector temperatures were both maintained at 250 °C. The quadrupole mass
spectrometer scanned over the range 28–400 amu at 1 scan/ sec, with an ionizing voltage of 70 eV, an
ionization current of 150 Ma and an ion source temperature of 200 °C. To determine the Kovats index
of the components, a mixture of alkenes (C9-C24) was added to the extract before injecting in the
GC-MS equipment and analyzed under the same conditions as above. The compounds were identified
by computer searched in the commercial libraries on NIST (National Institute of Standard and
Technology) and by their Kovats retention indexes [43].
4.9. Statistical Analysis
One-Way ANOVA was used to compare the means of three experimental groups with Tukey’s
post-hoc test to calculate least significant differences. The difference between means was considered
significant when p was <0.05.
5. Conclusions
In conclusion, this study provides new scientific information about E. guineensis, based on its
antimicrobial potential and chemical profiling that has never been reported. The anticandidal activity
of E. guineensis may be attributed to the various phytochemical constituents present in the extract. The
purified components may have even more potency with respect inhibition of microbes. Further work
on the types of phytoconstituents and purification of individual groups of bioactive components could
reveal the full potential of the E. guineensis extract to inhibit several pathogenic microbes and
encourage in the developing a novel broad spectrum antimicrobial herbal formulation in the future,
especially against C. albicans.