Review Article

Isolation and Identification of Candida from the Oral Cavity

Smitha Byadarahally Raju1 and Shashanka Rajappa2
1

Department of Oral Pathology & Microbiology, Sri Hasanamba Dental College &
Hospital, Karnataka, Hassan 573201, India
2
Department of General Surgery, Hassan Institute of Medical Sciences, Karnataka,
Hassan 573201, India
Received 30 June 2011; Accepted 8 August 2011
Academic Editors: E. T. Giampaolo, A. Jger, and H.-S. Kho
Copyright 2011 Smitha Byadarahally Raju and Shashanka Rajappa. This is an open
access article distributed under the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium, provided the
original work is properly cited.

Abstract
Various techniques are available for the isolation of Candida within the oral cavity. Such
methods play an important role in the diagnosis and management of oral candidosis. The
growing importance of Candida is in part related to the emergence of HIV infection and
the more widespread use of immunosuppressive chemotherapy. Along with the Candida
albicans there has been a greater recognition of the importance of the
nonalbicans Candida species in oral candidosis. Identification of infecting strains
of Candida is important because isolates of Candida species differ widely, both in their
ability to cause infection and also in their susceptibility to antifungal agents. Thus this
review provides an overview of the reliable methods of candidal isolation and
identification of isolates from the oral cavity.

1. Introduction
The term Candida originates from the Latin word candid, meaning white. The spores
of Candida are a commensal, harmless form of a dimorphic fungus that becomes invasive
and pathogenic pseudohyphae when there is a disturbance in the balance of flora or in
debilitation of the host [1].
The translation of this endogenous commensal to the disease-causing parasite may be
associated with factors other than the pathogenic attributes of the organism itself, which
is rather unique compared with most of the other infectious diseases, where the virulence
of the organisms considered being the key factor in the pathogenesis.
Hence Candida species are strictly opportunistic. It could be stated with that neither the
superficial nor the systemic forms of Candida infections could be initiated in the absence
of underlying pathology [2].

There are many species of Candida (Table 1), [3] but the most prevalent one which is
recovered from the oral cavity, in both commensal state and in cases of oral candidosis,
is C. albicans. It is estimated that this species accounts for over 80% of all oral yeast
isolates.

Table 1: Species of Candida.

In recent years there has been an increased interest in infections caused by the
opportunistic pathogen Candida. The growing importance of Candida is in part related to
the emergence of HIV infection and the more widespread use of immunosuppressive
chemotherapy [4, 5]. Identification of infecting strains of Candida is important because
isolates of Candida species differ widely, both in their ability to cause infection [6] and
also in their susceptibility to antifungal agents [7].
Along with the C. albicans there has been a greater recognition of the importance of the
non-albicans Candidaspecies in human disease. C. glabrata and C. krusei are species that
have received attention due to their enhanced resistance to certain antifungal agents. C.
dubliniensis is a recently identified pathogenic species, first described in 1995 when it
was coisolated with C. albicans from cases of oral candidosis in HIV infected individuals
[8].
This review provides an overview of the reliable methods of candidal isolation and
identification of isolates from the oral cavity.

2. Pathogenic Attributes of Candida

The transition of Candida from a harmless commensal to a pathogenic organism is
complex and is related to subtle environmental changes that lead to expression of a range
of virulence factors (Table 2). It is the combined effect of both host and candidal factors
that ultimately contribute to the development of oral candidosis [8].

Table 2: Virulence factors associated with Candida Albicans.

Regardless of the type of candidosis, the ability of Candida species to persist on mucosal
surfaces of healthy individuals is an important factor contributing to its virulence. This is
particularly important in the oral cavity, where the organism has to resist the mechanical
washing action of a relatively constant flow of saliva toward the esophagus [9].

No single predominant virulence factor for Candida is recognized although there are a
number of factors that have been implicated in promoting the infection process. These
include attributes involved in the adhesion ofCandida to oral surfaces (e.g., relative cell
surface hydrophobicity and the presence of specific adhesin molecules), the ability to
resist host immune defence mechanisms (e.g., high frequency phenotypic switching and
morphological transition), and the release of hydrolytic enzymes (e.g., secreted aspartyl
proteinases and phospholipases) that can induce damage to host cells [8].

3. Oral Candidosis
Samaranayake [10] proposed a classification where the oral candidosis lesions were
subdivided into two main groups: Group I, or primary oral candidoses confined to lesions
localized to the oral cavity with no involvement of skin or other mucosae; Group II or
secondary oral candidoses, where the lesions are present in the oral as well as extraoral
sites such as skin (Table 3). Group I lesions consist of the classic triad
pseudomembranous, erythematous, and hyperplastic variantsand some have suggested
further subdivision of the latter into plaque-like and nodular types [11].

Table 3: Classification of oral candidosis.

4. Diagnosis of Oral Candidosis

Diagnosis of oral candidosis can often be made on the nature of the clinical presenting
features although microbiological specimens should be taken if possible in order to both
identify and quantify any Candida that may be present and provide isolates for antifungal
sensitivity testing.

5. Methods of Isolation
Techniques available for the isolation of Candida within the oral cavity include the use of
a smear, a plain swab [8], an imprint culture [12], collection of whole saliva [13], the
concentrated oral rinse [14], and mucosal biopsy. Each method has particular advantages
and disadvantages and the choice of sampling technique is primarily governed by the
nature of the lesion to be investigated (Table 4). Where an accessible and defined lesion
is evident, a direct sampling approach such as the use of a swab or an imprint is often
preferred as this will provide information of the organisms present at the lesion itself. In
cases where there are no obvious lesions or in instances where the lesion is difficult to
access, an indirect sample based on culturing saliva specimens or an oral rinse is more
acceptable.

Table 4: Methods of recovering Candida from the oral cavity.

Quantitative estimation of fungal load can be done using imprints, concentrated oral
rinse, and culturing of oral rinse, as a means of differentiating between commensal
carriage and pathogenic existence of oral Candida, with higher loads considered likely in
the latter [8].

6. Direct Microscopy
Morphological features of Candida species [15] (Table 5) need to be examined for
identification. A smear is of value in differentiating between yeast and hyphal forms but
is less sensitive than cultural methods [16]. Potassium hydroxide (KOH) preparation of
the specimen reveals nonpigmented septate hyphae with characteristic dichotomous
branching (at an angle of approximately 45) [17]. In KOH-Calcofluor fluorescent-stain
method fungal characteristics like hyphae, yeast cells, and other fungal elements will
fluoresce [18].

Table 5: Morphological features of Candida species.

A smear taken from the lesional site is fixed on to microscope slides and then stained
either by the gram stain or by the periodic acid Schiff (PAS) technique. Using these
methods, candidal hyphae and yeasts appear either dark blue (Gram-stain) or red/purple
(PAS) [19].
In case of chronic hyperplastic candidosis, a biopsy of the lesion is necessary for
subsequent detection of invading Candida by histological staining using either the PAS or
Gomori's methenamine silver stains. Demonstration of fungal elements within tissues is
done as they are dyed deeply by these stains. The presence of blastospores and hyphae or
pseudohyphae may enable the histopathologist to identify the fungus as a species
ofCandida and, given the presence of other histopathological features, make a diagnosis
of chronic hyperplastic candidosis [20].

7. Laboratory Culture
7.1. Swab
A swab of a lesional site is a relatively simple method of detecting growth and
semiquantitative estimation ofCandida can be obtained. The sampling approach involves
gently rubbing a sterile cotton swab over the lesional tissue and then subsequently
inoculating a primary isolation medium such as Sabourauds dextrose agar (SDA) [21].
7.2. Concentrated Oral Rinse

The oral rinse technique involves the patient holding 10mL of sterile phosphate-buffered
saline (0.01M, pH7.2) in the mouth for 1 minute. The solution is then concentrated (10fold) by centrifugation and a known volume, usually 50L, inoculated on an agar
medium using a spiral plating system. After 2448hrs incubation at 37C, growth is
assessed by enumeration of colonies and expressed as candidal colony forming units per
mL (cfumL1) of rinse [16].
7.3. Imprint Culture
The imprint method utilises a sterile foam pad of known size (typically 2.5cm2),
previously dipped in an appropriate liquid medium, such as Sabourauds broth,
immediately before use. The pad is then placed on the target site (mucosa or intraoral
prosthesis) for 30seconds and then transferred to an agar for culture [16].

8. Culture Media
The most frequently used primary isolation medium for Candida is SDA [22] which,
although permitting growth of Candida, suppresses the growth of many species of oral
bacteria due to its low pH. Incorporation of antibiotics into SDA will further increase its
selectivity [8]. Typically SDA is incubated aerobically at 37C for 24
48hrs. Candida develops as cream, smooth, pasty convex colonies on SDA and
differentiation between species is rarely possible [17]. It is estimated that more than
one Candida species occurs in approximately 10% of oral samples and in recent years the
ability to detect nonalbicans species has become increasingly important [16].
In recent years, other differential media have been developed that allow identification of
certain Candidaspecies based on colony appearance and colour following primary
culture. The advantage of such media is that the presence of multiple Candida species in a
single infection can be determined which can be important in selecting subsequent
treatment options [8]. Examples of these include Pagano-Levin agar or commercially
available chromogenic agars, namely, CHROMagar Candida, Albicans ID, Fluroplate, or
Candichrom albicans [16].
Pagano-Levin agar distinguishes between Candida species based on reduction of
triphenyltetrazolium chloride. The medium produces pale-coloured colonies of C.
albicans, whilst colonies of other Candida species exhibit varying degrees of pink
coloration. Pagano-Levin agar has a similar sensitivity to SDA but is superior for the
detection of more than one species in the sample [23], CHROMagar Candida identifies C.
albicans, C. tropicalis, and C. krusei based on colony colour and appearance [24], whilst
Albicans ID and Fluroplate have proven beneficial for the presumptive identification
of C. albicans [25]. The specificity of identification is reported to be 95% for
CHROMagar Candida [26] and 98.6% for Albicans ID and Fluroplate agars [25]. The use
of CHROMagar Candida as a primary isolation agar has been cited as an approach that
permits discrimination of the newly described C. dubliniensis [27] from C. albicans. On
CHROMagar Candida, C. dubliniensis reportedly develops as darker green colonies
compared with those of C. albicans [28]. However, discrimination between these two

species using CHROMagar appears to decline upon subculture and storage of isolates.
Failure of C. dubliniensis to grow on agar media at the elevated incubation temperature
of 45C has recently been suggested as an alternative test to discriminate between these
two species [29].

9. Identification of Candida Species

Identification of yeasts based on primary culture media can be confirmed through a
variety of supplemental tests traditionally based on morphological (Table 5) and
physiological characteristics of the isolates.
9.1. Morphological Criteria
The germ-tube test is the standard laboratory method for identifying C. albicans. The test
involves the induction of hyphal outgrowths (germ tubes) when subcultured in horse
serum at 37C for 24hours. Approximately 95% of C. albicans isolates produce germ
tubes, a property also shared by C. stellatoidea and C. dubliniensis [16].
C. albicans and C. dubliniensis can also be identified from other species based on their
ability to produce morphological features known as chlamydospores. Chlamydospores
are refractile, spherical structures generated at the termini of hyphae following culture of
isolates on a nutritionally poor medium such as cornmeal agar. Isolates are inoculated in
a cross hatch pattern on the agar and overlaid with a sterile coverslip. Agars are incubated
for 2448 hours at 37C and then examined microscopically for chlamydospore presence
[8].
9.2. Physiological Criteria/Biochemical Identification
Biochemical identification of Candida species is largely based on carbohydrate
utilization. Traditional testing would have involved culture of test isolates on a basal agar
lacking a carbon source. Carbohydrate solutions would then be placed within wells of the
seeded agar or upon filter paper discs located on the agar surface. Growth in the vicinity
of the carbon source would indicate utilization. Commercial systems are based on the
same principle but test carbohydrates are housed in plastic wells located on a test strip.
Growth in each well is read by changes in turbidity or colour changes in certain kit
systems. Numerical codes obtained from the test results are used to identify the test
organism based on database comparison [30].
9.3. Serology
Serological tests are frequently used to ascertain the clinical significance
of Candida species isolates. Rising titers of lgG antibodies to C. albicans may reflect
invasive candidiasis in immunocompetent individuals. The detection of IgA and IgM
antibodies is important to identify an acute infection. Immunosuppressed individuals
often show variability in antibody production and in such a case the use of an antigen
detection test is recommended. Tests like enzyme linked immunosorbent assay (ELISA)
and radio immuno assay (RIA) for detection of candidial antigen, either cell-wall mannan
or cytoplasmic constituents are now available in developed countries [31].

Serological diagnosis is often delayed and the tests still lack sensitivity and specificity.
Furthermore, antibody production in immunocompromised patients is variable, making
diagnosis complicated [32]. This is due to the fact that fungal antigens and metabolites
are often cleared rapidly from the circulation and the presence of antibodies does not
always imply a Candida infection, especially in patients with serious underlying disease
or who are taking immunosuppressive drugs [33].
Serologic tests are normally not a diagnostic tool for oral candidosis. However, such tests
may be a prognostic instrument in patients with severe oral candidosis who respond
poorly to antimycotic therapy [34].
9.4. Molecular-Based Identification Methods
Identification by analysis of genetic variability is a more stable approach than using
methods based on phenotypic criteria. For the identification of Candida based on genetic
variation are analyses of electrophoretic karyotype differences and restriction fragment
length polymorphisms (RFLPs) using gel electrophoresis or DNA-DNA hybridization
[16].
Species-specific PCR approaches have also been used for Candida species identification.
Several target genes have been reported for Candida species discrimination, although
those most frequently amplified are the sequences of the ribosomal RNA operon.
Identification can be obtained based on PCR product sizes obtained following gel
electrophoresis resolution, or PCR product sequence variation determined either by direct
sequencing or through the use of restriction fragment analysis following cutting of PCR
sequences with restriction endonucleases [8].
Fluorescence in situ hybridization with peptide nucleic acid method (PNA Fish) is a new
detection technique which targets highly conserved species-specific sequences in the
abundant rRNA of living C. albicans. Individual cells can be detected directly without the
need for amplification [35]. This technique achieves a sensitivity of 98.7100%, with a
specificity of 100%, allowing for the discrimination of C. albicans from the
phenotypically similar C. dubliniensis [36].
Molecular-based technology can also be used to identify strains of Candida species
although the use of techniques such as Pulsed Field Gel Electrophoresis (PFGE), Random
Amplified Polymorphic DNA (RAPD) analysis, and repeat sequence amplification PCR
(REP) are largely reserved for epidemiological investigations in research of oral
candidosis [8].

10. Conclusion
In recent years a greater emphasis has been given for reliable identification
of Candida species from human clinical samples. A schematic representation for candidal
isolation and identification is presented in Figure 1. Since Candida is the resident
microflora, appropriate isolation methods are required to ascertain the presence in the
mouth along with their number. It is also important to identify the infecting strains
of Candida because isolates of Candida species differ widely, both in their ability to

cause infection and also in their susceptibility to antifungal agents. Various phenotypic
techniques are available for identifying isolated Candida including using morphological
culture tests, differential agar media, and biochemical assimilation tests. These methods
are supplemented with recent molecular techniques largely reserved for epidemiological
investigations.

Figure 1: Schematic representation of isolation and identification of Candida species

from the oral cavity.

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Antibacterial activities of locally used

toothpastes against dental pathogens

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Journal of Institute of Medi
cine, August,

2008;
30:2
www.jiom.com.np

Antibacteri
al activities
of locally
used
toothpastes

againstden
tal
pathogens
K. B. Tiwari, U. T.
Shrestha, A.
Acharya, B. Subedi,

B. Paudyal, M.
Jnawali, P. Shakya,
U. K.C., V. P.
Agrawal
Central Department
of Microbiology,
Tribhuvan
University, Kirtipur,
Kathmandu, Nepal,

Research Laboratory
forAgricultural
Biotechnology and
Biochemistry
(RLABB), Maitidevi,
Kathmandu, Nepal
Correspondence to
: Kiran Babu Tiwari,
Research Laboratory

for Biotechnology and

Biochemistry
(RLABB),e-mail:
kiranbabu.babukiran
@gmail.com
Background:
Toothpastes need to
contain various
antimicrobial agents

in order to reduce,
controland prevent
different kinds of
dental diseases.
Different brands have
their own composition
andconcentration of
ingredients for their
efficacy. The

consumers should
aware about the facts
associatedwith their
health.
Methods:
The bacterial
pathogens were
isolated and
identified from

various dental
samples.Antibacterial
activities of
11 different
toothpastes available
locally in markets
were assessed
againstthe isolates by

standard agar well

diffusion method.
Result:
Monomicrobial
infections were
observed in all cases.
The bacterial
pathogens were
foundto be

Streptococcus mutans,
S. salivarius, S.
sanguis, S. sobrinus
and S. mitis

. Of the
assayedtoothpastesColgate Total,
Colgate, Anchor
White and Pepsodont

were found to be
highly
effectiveagainst the
pathogens.
Conclusion:
The result showed
that the toothpastes
containing Triclosan
as a major

chemicalingredient
posses significant
antibacterial
activities.
Keywords:
Streptococcus,
Triclosan, Zone-ofinhibition

Introduction
Toothpaste has a
history that stretches
back nearly
4000years. Different
abrasives, green lead,
incense were used
toclean stain from
teeth until mid

nineteenth century. In
middleages, fine sand
and pumice were the
primary ingredients
inthe tooth
cleaning formulas
used by Arabs. In
1950 AD,
Dr.Washington

Wentworth Sheffield,
a dental surgeon
andchemist, invented
the first toothpaste.
1

Then, the market

of the toothpaste has
never been slowed
down.

Moderntoothpaste
was invented to aid in
the removal of
foreignparticle and
food substance in
addition to cleaning
of tooth.During 194060 AD, fluoride was
added which aided

inprotection from
tooth decay. Many of
the innovations
weremade in
toothpaste after the
fluoride break through
whichinvolved the
addition of
ingredients with

special abilitiesto
toothpaste and
toothpaste packaging.
2,3

Dental problem is the

most common health
problem in thehuman
communities.
4

Dental infections are

mainly of threetypes,
viz
.: formation of dental
plaques, dental caries
andperiodontal
diseases.
3

Dental plaque is
material adheringto
teeth, which consists
of bacterial cell (6070% of thevolume of
plaque) salivary
polymers and
bacterialextracellula
r products. Plaque is

a naturally
constructedbiofilm of
bacteria, which may
reach thickness of
300-500cells on the
teeth. The very
normal flora of the
oral cavity,
S. mutants

and
S. sanguis
, are the most
dominant bacterial

Original article
15-18

16

Journal of Institute of Medi

cine, August,

2008;
30:2
www.jiom.com.np

Fig-1: Selection of diluent

for toothpastesagainst
S. mutans

121189202114160510152025
Pepsodent-d/w Colgate totald/wPepsodentTw Colgate total-Tw
Diluents (d/w, distilled
water; Tw, Tween-20)
Z o n e o f i n h i b i t
i o n ( m m )

1:101:50

species in dental
plaque. After initial

weak attachment
of streptococcal
cells to salivary
glycoprotein,
strongerattachment
takes place by
polymer of glucose
(glucan)synthesized
by bacteria. Dental

caries is the
destruction of enamel,
dentin or cement of
teeth due to bacterial
activities.Caries are
initiated by
demineralization of
the enamel of teeth

due to Lactic-acid
bacteria.
Actinomyces
spp. andvarious
proteolytic bacteria
are commonly found
in humancaries as
secondary invaders,
contributing to the

progressionof the
lesion. Periodontal
diseases are bacterial
infectionsthat affect
the supporting
structure of the teeth
(gingival,cementum,
periodontal
membrane and

alveolar bone).
Theendotoxins,
hydrolytic enzymes
and toxic
bacterialmetabolites
are involved in this
disease. Gingivitis,
aninflammatory
condition of gum, is

the most common

formof periodontal
disease. Serious forms
of periodontal
diseasethat affect the
periodontal
membrane and
alveolar bone
mayresults in tooth

loss. Streptococci,
actinomycetes,spiro
chetes and
bacteroids are the
possible
bacteriaresponsible
for the disease.

Materials and
Methods

Collection of
Sample:
Thirty-four different
samples
werecollected from
34 patients in Samaj
Dental Clinic
andPeoples Dental

Hospital, Kathmandu,
Nepal.
5,6

Collectedsamples
were transferred in
nutrient broth and
immediatelytranspor
ted to RLABB
(Research

Laboratory
forBiotechnology and
Biochemistry,
Kathmandu) where
thestudy was carried
out.
Isolation of
organisms:

The samples were

enriched innutrient
broth at 37
o

C for 4 hours and

streaked on
nutrientagar plate.
Corresponding pure
culture was obtained

bystreak plate
method.
5,6

Identification:
The organism were
identified by
standardmicrobiolo
gical techniques
including

colonialcharacteris
tics,
morphological
characteristics
andbiochemical
characteristics.
7

Assessment
of Toothpastes

(Antibacterial
Activity):
1,2,5,6-10

The toothpaste
solutions were made
by mixing
thecalculated amount
of the toothpaste in
measured volume

of the solvent
followed by
continuous stirring for
half an hour.In order
to investigate
antimicrobial activity
of
differenttoothpastes,
toothpastes were

diluted in two
differentdiluents
viz
: distilled water and
Tween-80. Five
differentdilutions of
1:5, 1:10, 1:20, 1:50
and 1:100 were made
ineach of the diluents.

Muller-Hinton Agar
(MHA) agar
platewere prepared
to assess
antimicrobial
activity of
thetoothpastes against
the pathogens.

Results

Isolation and
Identification:
Of the total 34 dental
subjects,14 (41.2%)
had plaques, 12
(35.2%) had Dental
caries and8 (23.6%)
had Gingivitis.
Altogether 15

isolates
wererecovered and
identified as given
in Table- 1.
Higherpercentage of
isolates was found
from plaque samples
(7/ 14, 50.0%).
Table 1:

Dental pathogens
isolated from different
dentalsamples
T y p e o f N o .
o f N o . o f O r
g a n i s m s * sa
mplessample
sisolates

Plaques (Pla)14 (4
1.2%)7 (50.0%)
S. sobrinus

(2),
S. sanguis

(1),
S. mutans

(2),
S. mitis

(2)Dental caries (Dc)

12 (35.2%)5 (41.7%)
S. sobrinus

(1),
S. sanguis

(1),
S. mitis

(3)G i n g i v i t i s ( G g ) 8
(23.6%)3 (37.5%)

S. sanguis

(1),
S. salivarius

(2)

Note: *= No. of
corresponding isolates
in parentheses

Assessment of
antimicrobial
activity
The distilled water
extracts of the
toothpaste were found
tohave marked
antimicrobial
properties compared

to that of 2% Tween20 (Fig-2). The

optimum dilution of
thetoothpaste for
antimicrobial activity
assessment against
S.mutans

,
S. sobrinus

and
S. mitis
was determined to be
1:50(Fig-2). The
zones of inhibition
(ZOI) against the
pathogensoffered by
the toothpastes are
shown in Table-2.

Statisticalanalysis
showed that the
zones-of-inhibition of
toothpastesagainst the
test organism were
not differed
significantly
onrepeated attempts
(P > 0.05).

K. B. Tiwari, U. T.
Shrestha, A. Acharya,
et. al.
15-18

17
Journal of Institute of Medi
cine, August,

2008;
30:2
www.jiom.com.np

Fig-2: Selection of
optimum dilution
oftoothpastes against
S. mutans

122024131518211605101520253
01 : 1 0 1 : 2 0 1 : 5
0 1 : 1 0 0

Dilutions
Z o n e o f i n h i b i t i
o n ( m m )

PepsodentColgate total

Table- 2:
Mean Zone of
inhibition shown by
differenttoothpastes
(mm) against the
dental pathogens

ToothpastesDental
pathogens
S . S . S
. S . S .
s
alivariusmutanssobrinu
smitissanguis
C
o
2
o

o l g a t e
t
t a l 1 0 2 3
1 1 6 1 8 C
l g a t e 1

3
6
o
2
2
o
1
C
3

2
1
r
2
P
n
7

2 2 0 1
8 A n c h
w h i t e 1
0 1 8 1 6 2
e p s o d
t 1 1 2 4
1 7 1 8
l o s e
U p 9 1
8 5 6

K
i
d
o
o
s
0
1
0
0
0
0
D a b u r
p o w d e r
0 9 0 0 0

Note: Absolutely no
ZOI was observed for
Dabur paste,Neem,
Babool and Brighter

against the tested

bacteria.

Discussion
The viridian
streptococciS. mutans, S. sanguis,
S. sobrinus

and

S. mitis

are the major

pathogens while
S. salivarius

isan initiator of the

dental infection.
These oral
streptococciposes
the significant

health risks if they

enter
intobloodstream via.
Wounds, oral
infection,
dental proceduresand
can cause
endocarditis.
Followed by primary

invaders,oral cavities
are vulnerable for
secondary invaders
like
Candida albicans
and species of
Actinomyces
,

Bacteroids

,
Spirochetes

and
Lactobacillus
etc. inviting
severeconditions.
5-7

Dental problems are

the most frequent
cases in the
generalpopulation
associated mainly
with dental
hygienepractices.
11

Further, the
efficacies of the
toothpastesregarding
their chemical
composition is not
less
importantespecially in
developing countries
like Nepal where

lowgrade products
can be found in local
markets and
consumersare forced
unknowingly to
choose the
products.Plaque
formation is the
primary process of

dental infectionsand
hence the numbers of
the cases are likely to
be highamong the
common populations.
Further, dental caries
andgingivitis in
Nepalese
communities were

found to befrequent
cases too. The results
clearly indicate that
thepeople should
aware about their
dental hygiene.The
aqueous diluent
(water) was better
than Tween-20 andthe

assessment was done

in aqueous fractions,
which,excludes nonpolar components that
might be present inthe
toothpastes/powder. It
is self evident that the
tooth-brushing is
aqueous based

procedure. Figure- 1
shows,however, nonpolar components
extracted in Tween-20
maybe important and
second major
fractions. The
optimumdilution of
1:50 (Figures 1& 2)

of the pastes was

selectedas the more
concentrated
dilutions showed
weakerantimicrobial
activities, possibly,
because of
diffusionkinetics of
the active ingredients

in higher
concentrationsthan
optimum one that is
achieved during
tooth-brushing.Only
few of the locally
available toothpastes
were found toposses
efficient antimicrobial

properties, especially,
thosethat have
triclosan as a major
ingredient (Colgate
total,Colgate,
Anchor white and
Pepsodont).
Triclosan,
achlorophenol

derivative, kills germs

by interfering with
theenzymes required
for fatty acid
synthesis. Next to
triclosan,fluorinated
products, e.g., Closeup and Kedoos, were
foundto posses

marked antibacterial
activities. These
activecompounds,
besides reducing
cariogenic
microorganisms,along
with other compounds
in the paste/powder
formula(Perooxides,

silica, pyrophosphates
and polymers,
bakingsoda, chlorides
and nitrates,
detergents and
surfactants aswell as
various plant extracts)
helps to strengthen
the teethby reducing

demineralization
and
increasingreminerali
zation of the
teeth.Most commonly
used and recommend
by the WHO,
ADA,FDI is the
fluoride and triclosan.

But the excess use of

thefluoride can cause
the dental fluorisis so
the
recommendedamount
of the fluoride should
be used as the
ingredients inthe
toothpaste. And the

regular evaluation of
the efficacyof the
fluoridated toothpaste
by the private
laboratory havebeen
recommended by the
WHO.
11

References

1.Lee SS, Zhang W,

Li Y. The
antimicrobial
potential of 14
natural herbal
dentifrices results of
an in vitrodiffusion
method study.

Journal of
American Dental Asso
ciation

2004;
135
: 1135-41.2.Hawkins
R, Locker D, Noble
J, Kay EJ. Professio
nallyapplied topical

fluorides for caries

prevention.
British Dental Journal
2003;
6
:313-7.3.Clarke JK.
On the bacterial
factor in the

etiology of dental
caries.
British Journal of Exp
ert Pathology
1942;
5
: 141-7.

Antibacterial
activities of locally
used toothpastes
15-18

18
Journal of Institute of Medi
cine, August,

2008;
30:2
www.jiom.com.np

4.Loesche WJ.
Microbiology of

dental
decay andperiodon
tal disease. In:
Barons
MedicalMicrobiolog
y. 4
th

Ed. University of
Texas

MedicalBranch.
1996.5.Cheesbroug
h M.
Medical Laborator
y Manual
forTropical
Countries.
Butterworth and Co.

(Publishers)Ltd. 1984;
Vol.
2
. pp. 227-32.6.Collee
JG, Fraser AG,
Marmion BP,
Simmons (Eds).A
Mackie and
McCartney

Practical
MedicalMicrobiolo
gy. 14
th

edition. Singapore:
LongmanSingapore
Publishers (Pre)
Limited.
1996.7.Holt JG (Ed

). Bergeys manua
l of systematicbact
eriology, vol. 4
th

ed. S.T. Williams

and M.E.Sharpe,
Baltimore, Md:
Williams and
Williams.8.Fine DH,

Furgang D, Bonta Y
et al. Efficacy of
atriclosan/NaF
dentifrice in the
control of plaque
andgingivitis and
concurrent oral
microflora
monitoring.American

Journal of Dentistry
1998;
11
:259-70.9.Loveren
CV, Buijs JF, Cate
JM. The effect of
triclosantoothpaste
on enamel
demineralization in a

bacterialdemineraliza
tion model.
Journal of Antimicrob
ialChemotherapy
2000;
45
:153-8.10.Gomes
BPFA et al. In vitro
antimicrobial

potential of calcium
hydroxide pastes and
their vehicles
againstselected
microorganisms.
Brazilian Dental
Journal2002;
13:

155-61.11.Peterson
PE. World Oral
Health Report
2003. OralHealth
Programme Noncommunicable
DiseasePrevention
and Health
Promotion, world

HealthOrganization,
Geneva, Switerland.
2003.
K. B. Tiwari, U. T.
Shrestha, A. Acharya,
et. al.
15-18

Antibacterial Activity of Leaf Extracts of Baeckea frutescensagainst

Methicillin-Resistant Staphylococcus aureus
Somayeh Razmavar, Mahmood Ameen Abdulla, Salmah Binti Ismail, and Pouya
Hassandarvish
Department of Molecular Medicine, Faculty of Medicine, University of Malaya,
Malaysia
Received 3 February 2014; Accepted 16 April 2014; Published 16 June 2014
Academic Editor: Fabio Ferreira Perazzo
Copyright 2014 Somayeh Razmavar et al. This is an open access article distributed
under the Creative Commons Attribution License, which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly
cited.

Abstract
This study was based on screening antibacterial activity of the ethanol extract of Baeckea
frutescens L. against MRSA clinical isolates, analyzes the potential antibacterial
compound, and assesses the cytotoxicity effect of the extract in tissue culture. Leaves
of Baeckea frutescens L. were shade dried, powdered, and extracted using solvent
ethanol. Preliminary phytochemical screening of the crude extracts revealed the presence
of alkaloids, flavonoids, steroids, terpenoids, phenols, and carbohydrates. The presence of
these bioactive constituents is related to the antibacterial activity of the plant. Disc
diffusion method revealed a high degree of activity against microorganisms. The results
confirm that Baeckea frutescens L. can be used as a source of drugs to fight infections
caused by susceptible bacteria.

1. Introduction
In recent years, there has been an increasing awareness about the importance of medicinal
plants. Drugs from these plants are easily available, inexpensive, safe, efficient, and
rarely accompanied by side effects. Plants which have been selected for medical use over
thousands of years constitute the most obvious starting point for new therapeutically
effective drugs such as anticancer drugs [1] and antimicrobial drugs [2]. Recently,
medicinal plants usage has increased in spite of the advances made in the field of
chemotherapy. The reasons proposed [3] are the use of medicinal plants as materials for
the extraction of active pharmacological agents or as precursors for
chemicopharmaceutical hemisynthesis. There is also the increased use of medicinal
plants in industrialized countries for galenic preparations and herbal medicines.
Baeckea frutescens L. of the family Myrtaceae and subfamily Myrtoideae is a medicinal
plant that has an essential oil which has been used as a traditional drug in South East

Asia. Baeckea frutescens L. is a small tree found in mountainous areas of South China,
Hong Kong, South East Asia, and Australia. The local Malay name of this plant is Cucur
Atap. The needle-like leaves are small and narrow in only about 615mm long. When
crushed, the leaves give off a resinous aromatic fragrance. The tiny fruits split, releasing
minute angular seeds. Tea made from these leaves is used to treat fever in China [4]. It is
one of the traditional folk medicine in Indonesia [4]. Packets of leaves are burned under
the bed of colic sufferers.
The essential oil has been used for aromatherapy and is inhaled for mental clarity and to
ease mental distress [5]. The oil is also used when massaging aching muscles and to treat
pain on the surface of the body in addition to its use as a bath or tonic [5].
This paper presents a preliminary phytochemical investigation of Baeckea frutescens L.,
which is responsible for the antibacterial activity of the extracts of the leaves on
methicillin-resistant Staphylococcus aureus (MRSA) bacterial species.

2. Materials and Methods

2.1. Preparation of Plant Extracts
Test plant was first collected from the Rimba Ilmu, University of Malaya. All parts of the
plant except the roots were oven-dried at 56C for several days until fully dried and then
ground to fine powder with a blender machine. The extraction was done at room
temperature. The powder was soaked in absolute ethanol at a 1:20 ratio for 7 days and
then filtered by Whatman filter paper number 1. The filtrate was collected and evaporated
under vacuum using the BUCHI Switzerland Rotary Evaporator to obtain concentrated,
powdered extracts. All extracts were stored at 4C for further use. The ranged yield of
extracts is 520% (w/w).
2.2. Bacterial Culture
A bacterial culture is a method of bacteria organisms by allowing them to reproduce in
predetermined culture media under controlled laboratory conditions. For any bacterial
culture, it is necessary to provide the suitable environmental and nutritional conditions
that exist in its natural habitat.
The methicillin-resistant Staphylococcus aureus (MRSA) pure isolates used in this study
were kindly provided by Professor Dr. Yassim of the Microbiology Laboratory of
University Malaya Medical Centre. The streak plate method is the most common way of
separating bacterial cells on the agar surface.
Confirmation of the identity of working strains was done by colony morphology and
gram staining as described in the Textbook of Diagnostic Microbiology [6]. The bacterial
isolate was maintained in Brain Heart Infusion (BHI) agar (Pronadisa, Spain) slants at
4C.
2.3. Disc Diffusion Method

Disc diffusion method was used for antibacterial activity. A stock solution of extract was
prepared by dissolving 0.1g of extract with 100mL of their respective solvents (distilled
water and absolute ethanol) to produce a final concentration of 100mg/mL. The stock
solution was then diluted to concentrations of 2.5, 5, 10, 20, 50, and 100mg/mL of
extract. 20L of each dilution was impregnated into sterile, blank discs 6mm in
diameter. 5L of extract was spotted alternately on both sides of the discs and allowed to
dry before the next 5L was spotted to ensure precise impregnation. Distilled water and
ethanol-loaded discs were used as negative controls for aqueous and ethanol extracts,
respectively. All discs were fully dried before the application on bacterial lawn. The
positive controls used were vancomycin antibiotic discs (Becton-Dickinson, USA) for
all S. aureus strains. Antibacterial activity was evaluated by measuring the diameter of
the inhibition zone (IZ) around the discs. The assay was repeated trice. Antibacterial
activity was expressed as the mean zone of inhibition diameters (mm) produced by the
leaf extract.
2.4. Column Chromatography (CC) Spectral Analysis
The sample is dissolved in a solvent and applied to the front of the column (wet packing)
or alternatively adsorbed on a coarse silica gel (dry packing). Using a ratio of 100g of
silica gel/g of crude sample allows for relatively easy separation. The solvent elutes the
sample through the column, allowing the components to separate. The ethanol soluble
phase was subjected to silica gel column chromatography using AcOH-MeOH (90:10)
solvent system.
2.5. HPLC Analysis
A HPLC test was performed using an Agilent Zorbax column (Xdb-C18 Type MG 5m,
4.6 250mm). The detection wavelengths were 200, 230, 254, and 320nm. Elution was
carried out with CH3CN-H2O at the flow rate of 1.2mL/min. The injection volume was
100m. Samples were mixed and vacuum dried to 29.6mg. Then the samples were
dissolved in 1.0mL of distilled water. A stock solution (12,00ppm) was prepared by
adding 405.4L of sample solution (29.6mg/mL) to 594.6L of distilled water, which
was kept refrigerated at 4C. The samples were filtered using a SRP-4 membrane
0.45 M before they were injected into HPLC. The fractions were collected and subjected
to profiling.
2.6. Liquid Chromatography-Mass Spectrometry
Liquid chromatography-mass spectrometry (LC-MS, or alternatively HPLC-MS) is an
analytical chemistry technique that combines the physical separation capabilities of liquid
chromatography (or HPLC) with the mass analysis capabilities of mass spectrometry.
LC-MS is a powerful technique used for many applications which has very high
sensitivity and selectivity.

2mg of sample was prepared by dissolving in 2mL methanol in volumetric flask.

Solution was then filtered by using SRP-4 membrane 0.45mm. Stock solution 1mg/mL
was kept in fridge at 4C.
LCMS was performed with an Acquity BEH C18, 2.1 50mm, 1.7m UPLC columns.
Elution was carried out with %H20 + 0.1% F.A at the flow rate 0.5mL/min. The
injection volume was 3L.

3. Results and Discussion

Phytochemical screening of the crude extracts of Baeckea frutescens L. revealed the
presence of flavonoids and phenolic compounds (Table 1).

Table 1: Result of the phytochemical screening of ethanol extracts of leaves of Baeckea

frutescens L.
The presence of alkaloid is interesting as significant quantities are used as antimalarial,
analgesics, and stimulants [7]. Flavonoids, which are known to prevent tumor growth and
also used to protect against gastrointestinal infections, are of pharmacognostic
importance thus lending credence to the use of the plant in ethnomedicine [8]. Some of
these bioactive compounds that are synthesized as secondary metabolites as the plant
grows are also used to protect the plant against microbial attacks and predation by
animals [8].
According to the results of disc diffusion assay, this plant has active compounds that are
effective for the prevention of infections caused by MRSA.
There are a number of factors that could influence the results of the disc diffusion assay.
Firstly, the diameter of the zones is affected by the rate of diffusion of the antimicrobial
compound [9, 10] and thus may not exactly represent the potency of the extracts
antimicrobial activity. Where studies of plant extracts are concerned, the disc preparation
technique could present with another problem wherein the extract was not properly and
evenly impregnated into the paper discs. Another important factor is the standardization
of the inoculum size to 0.5 McFarland turbidity. This inoculum size is important to
ensure confluent or almost confluent lawn growth as a smaller inoculum size (such that
single colonies are seen) may produce falsely large inhibition zones while a bigger
inoculum size (thick bacterial lawn) may produce falsely smaller zones instead [11].
The ethyl acetate, methanol, and acid acetic solvents were more effective than other
solvents to show inhibition zone against MRSA (Table 2).

Table 2: Antibacterial activity of ethanol extract of Baeckea frutescens in different

solvents against MRSA.
The zone produced by the plant extract against the MRSA was from acid acetic and
methanol solvents were the largest zone. The lowest zone of growth inhibition was ethyl
acetate and hexane.
To determine the chemical constituents of the biological activity in ethyl acetate:normalhexane and acid acetic:methanol soluble phases, HPLC analysis was performed
(Figure 1).

Figure 1: HPLC profiling of samples by UV 254nm. Indicating the compounds shown in

Figure 2.
One principal peak and several lesser peaks were observed in the ethyl acetate (EtOAc):
normal-hexane soluble phases. Compound 1 was isolated from the EtOAc soluble phase
by repeated column chromatography on silica gel.
The molecular formula of the main peak was determined to be C 5H11NO2, C5H5N5 or
C8H9N by liquid chromatography-mass spectrometry (Table 3 and Figure 2).

Table 3: Liquid chromatography-mass spectrometry.

Figure 2: Chemical structures of compounds.

Results obtained for antibacterial activity against MRSA for 3 main peaks were in
Table 4.
Table 4: Antibacterial activity of liquid chromatography-mass spectrometry results
against MRSA.
From the results of the disc diffusion screening, B. frutescens is shown to clearly possess
antibacterial properties against MRSA. As B. frutescens seems to give appreciable
antibacterial activity against all gram-positive staphylococcal strains, this may indicate
that the plant extract acts specifically against the gram-positive cell wall, particularly the
staphylococcal cell wall [12] because they have a much thicker peptidoglycan layer than

gram-negative bacteria. This outer membrane is composed of lipopolysaccharides that

give gram-negative bacteria extra resistance against antibiotics that cannot penetrate it,
for example, glycopeptides like vancomycin [13].
Antibacterial activity of ethanol extract of B. frutescens leaf has been assessed by
measuring the diameters of zones of growth inhibition on some strain of bacteria and the
results are presented as shown in Table 5.

Table 5: Zone of inhibition against some bacteria strains by ethanol extract of Baeckea
frutescens L.
Inhibition growth of the highest zone has been shown by ethanol extract against grampositive
bacteria
like
MRSA
(14.5mm), Staphylococcus
aureus (13mm),
and Bacillus (9.5mm). The growth inhibition was moderately active against gramnegative bacteria Escherichia coli (8.5mm) and Klebsiella (0mm).

4. Conclusion
The result of this study showed that Baeckea frutescens L. extract contains
phytochemical components. Potentially, these compounds have the most important
applications against human pathogens, including those that cause enteric infections. The
results of various screening tests indicate that the leaves have some measurable inhibitory
action against gram-positive bacteria such as Staphylococcus aureus (MRSA).

Conflict of Interests
All authors have nothing to disclose and have no commercial or financial interests in the
products described in this paper.

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Jorgensen, M. L. Landry, and M. A. Pfaller, Eds., pp. 11521172, ASM press,
Washington, DC, USA, 9th edition, 2007.
12. A. N. Sudjana, C. D'Orazio, V. Ryan et al., Antimicrobial activity of commercial Olea
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Saunders Elsevier, Beijing, China, 3rd edition, 2007.

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Iran J Med Sci. Jan 2014; 39(1): 3643.

PMCID: PMC3895893

In Vitro Antibacterial Activity of Several Plant

Extracts and Oils against Some GramNegative Bacteria
Ayman Al-Mariri, PhD1 and Mazen Safi, PhD1
Author information Article notes Copyright and License information

Abstract
Go to:
Introduction
Medicinal and aromatic plants are used on a large scale in
medicine against drug-resistant bacteria, which are
considered one of the most important reasons for the lack of
success of treatment in infectious diseases. Medicinal plants
are the major sources of new medicines and may constitute
an alternative to the usual drugs.1
Aromatic oils are used in many industries, including food
preservation,2 pharmacy, and medicine.3,4 They are expected
to form new sources of antimicrobial drugs, especially
against bacteria.5 The antibacterial effectiveness of aromatic
oils has been divided into a good, medium, or bad.6,7 These
oils can also produce some defense products against several
natural enemies.8 In addition, and in order to continue their
natural growth and development, aromatic oils may produce
some secondary metabolites in response to some external
stress.9

The extracts and oils of 28 plants used in this work have

been traditionally employed by people for various purposes
in different parts of the world. Cinnamomum
zeylanicumessential oil has antibacterial and antifungal
activities10 as well as anti-diabetic properties;11Citrus
limon and Rosmarinus officinalis L. essential oils possess
antioxidant properties;12,13Citrus aurantium has
immunological effects in humans;14 Eucalyptus globulus oil
has good antimicrobial activities;15,16 Thymus
pannonicus essential oil has an excellent effect againstE.
coli O157:H7;17 light thyme essential oil inhibits the growth
of E. coli O157:H7 in foods;18Brillantaisia lamium extract
exhibits antibacterial and antifungal effects
againstStaphylococcus aureus, Enterococcus faecalis,
Candida tropicalis, and Cryptococcus neoformans;19 and
finally Crinum purpurascens herb extract has antimicrobial
activities against Salmonella paratyphi A and
B.20 Traditionally, many plant extracts and oils are used as
medicinal plants in Syria for many purposes, particularly for
respiratory and gastrointestinal disorders.
The aim of this study was to screen the in vitro antibacterial
activity of 28 plant extracts and oils against some Gramnegative bacteria, including: E.
coli O157:H7, Yersinia enterocoliticaO9, Proteus spp.,
and Klebsiella pneumoniae.
Go to:
Materials and Methods

Microorganisms and Growth Conditions

Fifteen local isolates of E. coli O157:H7, Y.
enterocolitica O9, Proteus spp., and K. pneumoniae were
grown for 24-48 h in 2YT agar (peptone, 16 g/liter; yeast
extract, 10 g/liter; NaCl, 5 g/liter; agar, 13 g/liter [Difco, BD,
Spars, MD]). The bacteria were suspended in a sterile
phosphate-buffered saline (PBS). Bacteria abundance in the
PBS was monitored by recording the optical density (OD) at
590 nm.21 The exact doses were assessed retrospectively by
viable counts on 2YT agar plates.
Plant Samples Collection
Rosmarinus officinalis L., Origanum syriacum L., Thymus
syriacus Boiss., Salvia palaestinaBenth., Mentha piperita L.,
and Lavandula stoechas L. (Lamiaceae); Citrus
aurantium L. and Citrus medica L. (Rutaceae); Syzygium
aromaticum L., Myrtus communis L., andEucalyptus
camaldulensis Dehnh. (Myrtaceae); Cinnamomum
zeylanicum L. and Laurusnobilis L. (Lauraceae); Juniperus
foetidissima Wild (Cupressaceae); Pelargonium roseumL.
(Geraniaceae); Scilla maritima Squill and Allium sativum L.
(Liliaceae); Pinus halepensis Miller. (Pinaceae); Artemisia
herba-alba Asso. (Compositae); Anabasis
haussknechtii Boiss. (Chenopodiaceae); Crataegus aronia L.
(Rosaceae); Mercurialis annua L.
(Euphorbiaceae); Matthiola crassifolia
Boiss. (Brassicaceae); Myristica
fragransHoutt. (Myristicaceae); Brassica nigra Koch.
(Cruciferae); Coriandrum sativum L. (Apiaceae); Zingiber

officinale Rosc. (Zingiberaceae);

and Achillea fragrantissima Forssk. (Asteraceae) samples
were collected during the flowering season from different
regions in Syria between March and July 2010, or purchased
from local markets (table 1). The samples were cleaned from
any strange plants, dust, or any other contaminants.

Table 1
Plants and their families, collection sites, and parts used
Essential Oil Extraction
Essential oils from fresh, clean, weighed aerial parts,
flowers, leaf fruits, barks, seeds, rhizomes, and bulbs (table
1) extracted by hydro-steam distillation using the Clevenger
apparatus were collected and stored in sterile vials.22 Briefly,
100 to 150 g of each plant was introduced in the distillation
flask (1 L), which was connected to a steam generator via a
glass tube and to a condenser to retrieve the oil. This was
recovered in a funnel tube. Aromatic molecules of the
essential oils were released from the plant material and
evaporated into hot steam. The hot steam forced the plant
material to release the essential oil without burning the plant
material itself. Then, steam containing the essential oil was
passed through a cooling system in order to condense the

steam. The steam was applied for 3 h. After settling the

recovered mixture, essential oil was withdrawn. The
supernatant essential oil was filtered through anhydrous
Na2SO4 to dry the yielded essential oil. Afterward, the
essential oil was collected in tightened vials and stored in a
refrigerator. For the antimicrobial activity test, several
dilutions of the oils were done using dimethyl sulfoxide
(DMSO).
Preparation of Ethanolic Extracts
Successive solvent extraction was performed for some plants
(table 1). Leaves and bulbs were washed, air dried for 7-8
days, and ground into powder before they were placed into
the flask of the Soxhlet apparatus for extraction using
ethanol with increasing order of polarity to extract the
phytoconstituents separately at 20C for 3-4 h. (The ethanol
used was HPLC grade obtained from Sigma-Aldrich,
Germany.) Whatman No.1 filter papers were then applied to
filter the extracts. After that, reduced pressure was applied to
evaporate and dry the filtrates, which were stored at -20C in
labeled, sterile, screw-capped bottles.
Antibacterial Susceptibility Assay
Muller-Hinton Broth (MHB, Merck) medium was used to
grow the test isolates for 22 h at 37C. Final bacterial
numbers were standardized to 110 6 CFU/ml. A total of 0.1
ml of bacterial suspension was poured on each plate,
containing Muller-Hinton Agar (MHA, Merck). The lawn
culture was prepared by sterile cotton swab and allowed to
remain in contact for 1 min. Thereafter, a 5% concentration

of each plant extracts was prepared. The sterile filter paper

discs (6-mm diameter) were placed on the lawn cultures, and
24 h after incubation at 37C, the inhibition zone was
measured in mm.
Antibiotics Minimum Inhibitory Concentration
Determination
In order to estimate the antibiotics susceptibility, the well
broth microdilution method was used with 96-well plates
(TPP, Switzerland). The antibiotics were diluted twofold in
LB broth (Acumedia, Michigan, USA), and the wells were
inoculated with 110 6 CFU of bacteria (in a 0.2 ml final
volume). The incubation period was 24 h at 37C. The
lowest concentration that inhibited 50% of visual growth was
recorded and interpreted as the MIC50. The MIC testing was
performed according to the recommendations of the Clinical
and Laboratory Standards Institute (CLSI).23 The range of the
concentrations assayed for each antibiotic was 0.064 to 128
g/ml. The absorbance was determined at 590 nm (ThermoLab Systems Reader, Finland). All the tests were performed
in triplicate and then averaged. The investigated antibiotics
were Ciprofloxacin, Levofloxacin, Ofloxacin, Sparfloxacin,
Ceftazidime, Ceftriaxone, and Cefotaxime. Positive control
was done without adding any antibiotics.
Plants Extracts and Oils Minimum Inhibitory Concentration
Determination
The microdilution broth susceptibility assay was
used.24 Three replicates of the serial dilutions of each
essential oil were prepared in LB broth medium in 96-well

microtiter plates, using a range of concentrations for each

essential oil from 0.75 to 50 l/ml. Next, 100 l of freshly
grown bacteria, standardized until a bacterial number of
110 6 CFU/ml in LB broth was achieved, was added to each
well. Positive and negative controls were also done. The
plate was incubated with shaking for 24 h at 37C. The
lowest concentration that inhibited 50% of visual growth was
recorded and interpreted as the MIC50.
Statistical Analysis
Optimal concentrations for the most effective essential oils
and plant extracts were estimated by Probit Analysis (SPSS
Inc. 2010; Finney, 1971). Minimum concentrations to
achieve 50% inhibition of the various bacteria (MIC50) were
considered significantly different if their 95% confidential
limits did not overlap.
Go to:
Results
Table 2 demonstrates that O. syriacum. L., T. syriacus, S.
aromaticum, C. zeylanicum, L. nobilis L., J. foetidissima, A.
sativum L., and M. fragrans Houtt. had good antibacterial
activities against the Gram-negative bacteria, whereas the
rest of the studied extracts were ineffective.

Table 2
Number of Gram-negative isolates susceptible to each plant
extract
The MIC50 values for these plant extracts and oils were 12.5,
12.5, 25, 12.5, 12.5, 25, 12.5, and 6.25 l/ml, respectively,
against E. coli O157:H7; and 1.5, 6.25, 6.25, 6.25, 6.25, 25,
6.25, and 12.5 l/ml, respectively,
against Y. enterocolitica O9; and 1.5, 3.125, 1.5, 1.5, 3.125,
12.5, 3.125, and 12.5 l/ml, respectively,
against Proteus spp.; and 6.25, 3.125, 1.5, 3.125, 6.25, 12.5,
6.25, and 6.25 l/ml, respectively,
against K. pneumoniae (table 3).

Table 3
Minimum inhibitory concentrations (MICs) for the selected
essential oils and extracts against some Gram-negative
bacteria
In contrast, when studying the optimal concentrations that
could inhibit 50% of the bacterial isolates, the X 2 values
were not significant (P>0.05) for all the studied

concentrations, indicating adequate fit of the Probit

regression models (table 4).

Table 4
Optimal inhibitory concentrations of the selected essential
oils and extracts against some Gram-negative bacteria
Table 5 also shows that Ceftazidime, Cefotaxime, and
Ciprofloxacin were the most effective antibiotics against E.
coli O157:H7 (MIC50= 0.25, 0.5, and 2 g/ml, respectively).
Moreover, Ceftazidime and Ciprofloxacin were the most
effective antibiotics against Y. enterocoliticaO9 (MIC50=
0.25 and 0.5 g/ml, respectively) and against Proteus spp.
(MIC50= 4 and 2 g/ml, respectively) and Ceftriaxone,
Cefotaxime, and Ciprofloxacin were the most effective
antibiotics against K. pneumoniae (MIC50= 0.25, 0.25, and
0.5 g/ml, respectively).

Table 5

Minimum inhibitory concentrations (MICs) of some

antibiotics against Gram-negative bacteria
Go to:
Discussion
Because of their safety and low cost as well as their impact
on a large number of microbes,25medicinal plants may have
the ability to treat bacterial resistance to many types of
antibiotics. The antimicrobial effects of aromatic oils
extracted from a large number of plants have been evaluated
and reviewed,26,27 and the mechanisms that enable the natural
ingredients of herbs and spices to resist microbes have been
discussed.28 The results show that these mechanisms vary
greatly depending on the components of the essential oil.29,30
In the present study, the efficacy of some plant extracts and
oils was determined, quantitatively, by measuring the
diameter of the inhibition zones around the discs (table 2).
Only O. syriacum. L., T. syriacus Boiss., S.
aromaticum L., C. zeylanicum L., L. nobilis L., J.
foetidissima Wild, A. sativum L., and M. fragrans Houtt.
extracts inhibited the growth of the tested bacteria. In
addition, O. syriacum. L., T. syriacus Boiss., S.
aromaticum L., and C. zeylanicum L. essential oils were the
most effective, and their MIC50 values varied from 1.5 l/ml
to 25 l/ml against various kinds of bacteria. Because the
values of minimum bactericidal concentration (MBC) and
MIC are usually very similar,31 it can be logically assumed
that the above-mentioned plant extracts and oils have a

bactericidal effect on Gram-negative bacteria, especially

against Proteus spp. and K. pneumoniae.
The Probit Analysis (table 4) revealed that the minimum
concentrations of the essential oils that could inhibit 50% of
the various bacteria were T. syriacus Boiss. for E.
coli O157H7 (7.85 l/ml), O. syriacum. L. for Proteus spp.
and Y. enterocolitica (1.12 and 1.59 l/ml, respectively),
and S. aromaticum for K. pneumoniae (1.33 l/ml).
Ooi et al.32 reported that Cinnamomum verum shows
excellent activities against E. coli andProteus vulgaris.
Preuss et al.33 found that origanum essential oil proves cidal
to E. coli andK. pneumoniae. In addition, Barbosa et
al.34 found that the MIC90 of Origanum vulgareessential oil is
0.46% (v/v) against E. coli. Lpez et al.35 found that 8-10%
(v/v) concentrations of Origanum vulgare essential oil can
completely inhibit the growth of E. coliand other Gramnegative bacteria. Elsewhere, Mkaddem et al.36 reported
that Menthaessential oils are very active against K.
pneumoniae bacteria, whereas they are less effective
against E. coli. Furthermore, Mentha longifolia oil is thought
to exhibit an antimicrobial activity against some Grampositive bacteria such as Streptococcus
mutans andStaphylococcus aureus, but without
affecting Pseudomonas aeruginosa.37
Since the antibacterial effectiveness of medicinal plants
varies dramatically depending on the phytochemical
characteristics of plant families and subfamilies, it is not
surprising to note the difference in this efficacy even when

using samples taken from the same plant, but from two
different regions.38 Our results reveal that the cephalosporins
were the most effective antibiotics against almost all the
studied bacteria, and only Ciprofloxacin, one of the
fluoroquinolones group, was effective against these bacteria.
Go to:
Conclusion
O. syriacum. L., T. syriacus Boiss., S. aromaticum L., C.
zeylanicum L., J. foetidissima Wild,A. sativum L., and M.
fragrans Houtt. oils and L. nobilis L. extract were the most
effective plant extracts against the Gram-negative bacteria
studied in this work. These plant extracts could be a potential
source of new antibacterial agents.
Further and more specific studies, in vivo, are recommended
to determine the efficacy of these essential oils in the
treatment of gram-negative bacterial infections.
Go to:
Acknowledgment
The authors would like to thank the Director General of the
Atomic Energy Commission of Syria (AECS) and the head
of the Department of Molecular Biology and Biotechnology
for their support.
Conflict of Interest: None declared.
Go to:

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Antibacterial activity and phytochemical screening of some medicinal

plants commonly used in Saudi Arabia against selected pathogenic
microorganisms

Sooad Al-Daihan,

Manar Al-Faham,

Nora Al-shawi,

Rawan Almayman,

Amal Brnawi,

Seema zargar,

Ramesa shafi Bhat,

Show more

doi:10.1016/j.jksus.2012.11.003
Get rights and content
Open Access funded by King Saud University
Under a Creative Commons license

Abstract
In the present study aqueous and methanol extracts of Zingiber officinale, Curcuma longa, Commiphora
molmol and Pimpinella anisum were investigated for antimicrobial activity. The microorganisms employed
were Streptococcus pyogenes, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa.
The susceptibility of bacteria strains against the two extracts was determined using the disk diffusion
method. The most susceptible micro organisms were S. pyogenes, S. aureus, while the least susceptible
was E. coli. Highest antibacterial activity was observed with methanol extract of C. longa and C.
molmol against S. pyogenes and S. aureus (19 mm) respectively while minimum activity was observed
with aqueous extract ofP. anisum against E. coli and P. aeruginosa (7 mm). Methanolic extracts of almost
all samples dominated aqueous extracts in inhibiting the growth of the pathogenic bacteria under study,
but were less potent when compared to those of kanamycin used as positive controls. Phytochemical
analyses revealed the presence of carbohydrates and saponins in all samples. Alkaloids were found in Z.
officinale and C. myrrha whereas flavonoids in C. longa, and P. anisum. Steroids and tannins were found
only in Z. officinale and C. longa, respectively.

Keywords

Z. officinale;

C. longa;

C. myrrha;

P. anisum;

Antibacterial activity;

Microorganism

1. Introduction
For ages nature has gifted us plenty of herbs and plants which form the main source of traditional
medicines used to help in relief from illness and are still widely used all over the world. Herbal treatment is
still used for many health problems. Herbs are safe, less toxic, economical and a reliable key natural
resource of drugs all over the world. Use of traditional medicine among the tribal local people and
medicinal healers (Hakim) is a significant part of Saudi Arabias tradition and it is widely practiced till date
( Al-Essa et al., 1998).
Zingiber officinale, Curcuma longa, Commiphora myrrha and Pimpinella anisum have been used as
herbal drugs by local inhabitants from ancient time and even today ( Amal, 2010). Z. officinale and C.
longabelonging to the family Zingiberaceae, are most commonly used rhizomes for their medical values.
In Ayurveda, Z. officinale has

been used in the treatment of

inflammation. Gingerols

and

diarylhepatanoids present in Z. officinale are powerful inhibitors against prostaglandin biosynthesizing

enzyme and arachidonate 5-lipoxygenase which results in inhibition of prostaglandin and leukotriene
biosynthesis ( Kiuchi et al., 1992). Z. officinale is known for pain relief ( Black et al., 2010), soothing and
antibacterial properties and is very helpful in constipations ( Hara et al., 1998). Z. officinale is very helpful
in pregnancy to cure nausea and morning sickness, also it is useful for treating nausea caused by
chemotherapy ( Ernst and Pittle, 2000), inflammation, rheumatism, cold, heat cramps, and diabetes
( Amin-Al, 2006 and Afshari, 2007). C. longacommonly known as turmeric possesses many therapeutic
properties and is used as a remedy for various problems especially in lowering cholesterol and
triglyceride levels, inhibiting platelet aggregation, as antiseptic and also possesses anti-inflammatory and
antioxidant properties ( Luer et al., 2012). It is full of essential oils, yellow pigment, starch and oleoresin
( Leung and Foster, 2003). As a strong anti oxidant it employs various protective effects on the
gastrointestinal tract, and is very good for the cardiovascular system ( Luthra et al., 2001).
C. myrrha commonly known as myrrh is a tree belonging to Burseraceae family. It has been used as a
traditional remedy in Arab countries for long time. Originally it was found in Northern Africa, Arabia and
Northern Somalia ( Hanus et al., 2005). Early Muslim scholars reported many medicinal uses
of Commiphora molmol. It has been found helpful in treating intestinal disorder, diarrhea, wound healing
( Al-Said, 2010), respiratory conjunction ( Ghazanfar, 1994). Also found effective for treating fascioliasis
( Massoud et al., 2001). It is also used to treat gum diseases ( Serfaty and Itid, 1988) and inflammations
( Kimura et al., 2001).
P. anisum is a flowering plant belonging to Apiaceae family, originated in India and Southwest Asia. P.
anisum has been used as a traditional aromatic herb in stimulating digestion and is known for its
antiparasitic, antifungal ( Soliman and Badea, 2002) and antipyretic ( Afifi et al., 1994) antibacterial
( Parasa et al.,2012) properties. Its essential oil has been reported for treatment of some disease like
seizures and epilepsy ( Avicenna, 1988 and Abdul-Ghani et al., 1987). Commonly P. anisum is used for
the treatment of constipation ( Chicouri and Chicouri, 2000) and as a muscle relaxant ( Albuquerque et
al., 1995). Also, its oil is used as an antibiotic substitute in rations for broilers ( Mehmet et al., 2005).
The current investigation was carried out to screen the antibacterial activity of four medicinal plants used
for herbal treatment by local communities against some pathogenic bacterial strains.

2. Material and methods

2.1. Collection of plant materials
Samples of dry Z. officinale, C. longa, C. molmol and P. anisum were collected from a local market in
Riyadh. All the samples were washed and rinsed with distilled water. Samples were dried and crushed
using mortar pestle and finally reduced to fine particles using Waring laboratory blender (MX-7011G) for
5 min at high speed and then stored in airtight closed bottles for two days before being used for analysis.

2.2. Microorganisms
Reference bacterial strains were obtained from the Botany Department of King Saud University, which
included Staphylococcus
Aureus, Streptococcus
Pyogenes, Escherichia
coli and Pseudomonas
Aeruginosa. (Clinical isolate). The strains were kept at 4 C on agar slant and sub cultured at 37 C for
24 h on nutrient agar (SigmaAldrich, Germany) before any susceptibility test.

2.3. Extraction of material

2.3.1. Aqueous extraction
10 g of powdered sample was dissolved in 100 ml of distilled water and boiled for 2 h on slow heat. The
residue was removed by filtering through 8 layers of muslin cloth; the filtrate was then centrifuged at
5000g for 10 min. The supernatant was collected and further boiled till the volume was reduced to onefourth of the original volume of the solvent used [that was 100 ml] giving the concentration of 400 mg/ml.
( Harborne, 1973). It was then autoclaved at 121 C and at 15 lbs pressure and stored at 4 C ( Jigna and
Sumitra, 2007).

2.3.2. Methanol extraction

Ten grams of powdered sample was dissolved in 100 ml of methanol in a conical flask, plugged with
cotton wool and then kept on a rotary shaker at 190220 rpm for 24 h. The supernatant was collected
slowly and evaporated in wide mouthed evaporating bowls at room temperature for 23 days till the final
volume was reduced to one fourth of the original volume of the solvent used [that was 100 ml] giving the
concentration of 400 mg/ml. (Harborne,1973) and stored at 4 C in airtight bottles.

2.4. Phytochemical analysis

Phytochemical analysis of all the samples was determined as follows:

2.4.1. Molischs test for Carbohydrates

Five hundred milligram of powdered sample was taken and dissolved in 5 ml of distilled water and then
filtered. Filtrate was added with few drops of Molischs reagent, followed by addition of 1 ml of conc.
H2SO4 by the side of the test tube. After two minutes, 5 ml of distilled water was added. Red or dull violet
color formation at the interphase of the two layers was taken as positive test (Sofowora, 1993).

2.4.2. Test for alkaloids

100 mg of powdered sample was dissolved in 5 ml of methanol and then filtered. Then 2 ml of filtrate was
mixed with 5 ml of 1% aqueous HCl. One milliliter of mixture was taken separately in two test tubes. Few
drops of Dragendorffs reagent were added in one tube and occurrence of orange-red precipitate was
taken as positive. To the second tube Mayers reagent was added and appearance of buff-colored
precipitate was taken as positive test for the presence of alkaloids (Sofowora, 1993).

2.4.3. LiebermannBurchard test for steroids

200 mg of powder sample was dissolved in 2 ml of acetic acid separately; solutions were cooled followed
by the addition of few drops of conc. H 2SO4. Color development from violet to blue or bluish-green was
taken as positive test steroidal ring (Sofowora, 1993).

2.4.4. Test for saponins

One gram of powdered sample was boiled in 10 ml of distilled water and then filtered. 3 ml of distilled
water was added to filtrate and shaken vigorously for about 5 min. Formation of foam after shaking was
taken as a confirmation for the presence of saponins (Sofowora, 1993).

2.4.5. Shinodas test for flavonoids

Five hundred milligram of sample was dissolved in 5 ml of ethanol, slightly warmed and then filtered. Few
pieces of magnesium chips were added to the filtrate followed by addition of few drops of conc. HCl. A
pink, orange, or red to purple coloration was taken as a confirmation for the presence of flavonoids
(Trease and Evans, 2002).

2.4.6. Test for tannins

500 mg of powdered sample was mixed with 10 ml of distilled water and then filtered followed by the
addition of few drops of 1% ferric chloride solution. Occurrence of a blue-black, green or blue-green
precipitate indicates the presence of tannins (Trease and Evans, 2002).

2.5. Media preparation

Twenty three grams of nutrient agar (SigmaAldrich, Germany) was dissolved in 1000 ml of distilled water
and bring to boil. Agar was then autoclaved for 15 min at 121 C and left to cool at room temperature.
Once the LB medium was cooled (about 45 C), it was poured into Petri dishes. Each Petri dish was left
on the flat surface for 3040 min until completely set.

2.6. Antimicrobial assay

Antibacterial activity was assayed by disc diffusion method. For all bacteria strains, overnight culture
grown in broth was adjusted to an inoculums density of 100 l: 0.1A600 culture containing
3.2 108 colony forming unit. Further, 20 l was spread onto 20 ml of sterile agar plates by using a sterile
cotton swab. The surface of the medium was allowed to dry for about 3 min. Sterile filter paper discs

(5 mm in diameter) impregnated with 100 l of different test extracts (40 mg/disc) were then sited on the
surface of these agar plates. Kanamycin (30 g/disc) was used as positive control. The plates were then
incubated at 37 C for 24 h for bacteria after which microbial growth was determined by measuring the
diameter of the inhibition zone (mm) using a transparent scale. Each extract was analyzed in triplicate,
the mean values are presented. Kanamycin disc (30 g/disc) was used for comparing the bioassay.

3. Results
Antibacterial activities of the extracts obtained from Z. officinale, C. longa, C. molmol and P. anisum,
against the tested organisms are shown in Table 1. All the plant extracts tested showed antibacterial
activity; however, the plants differ in their activities against the micro-organisms tested. Extracts of Z.
officinale, C. longa and C. molmol showed antimicrobial activity against S. pyogenes, S. aureus and P.
aeruginosa than against E. coli. Highest antibacterial activity was observed with methanol extract of C.
longa and C. molmolagainst S. pyogenes and S. aureus (19 mm) respectively while minimum activity was
observed with aqueous extract of P. anisum against E. coli and P. aeruginosa (7 mm) ( Table 1). Results
obtained in the current investigation revealed that studied herbal extracts possess potential antibacterial
activity against entire tested organisms, albeit methanol extract was found to have shown the strongest
and broadest spectrum. Phytochemical analysis of selected plant samples is shown in Table 2.
Carbohydrates and saponins were found in all samples. Alkaloids were found in Z. officinale and C.
myrrha whereas flavonoids in C. longa, andP. anisum. Steroids and tannins were found only in Z.
officinale and C. longa, respectively ( Table 2).
Table 1.
Antimicrobial activity of methanol and aqueous extracts of selected medicinal plants against different
microorganisms.
Zone of inhibition

Plant extract

Micro organism

AQ

ME

KA

Z. officinal

S. pyogenes

10 0.20

12 0.58

28 0.57

S. aureus

10 0.33

12 0.70

26.5 0.33

E. Coli

9 0.88

10 0.23

20 0.33

P. aeruginosa

14 0.50

12 0.00

25 0.10

S. pyogenes

11 0.80

19 0.20

S. aureus

11 0.80

15 0.10

E. Coli

11 0.55

12 0.65

P. aeruginosa

14 0.55

12 0.30

C. longa

Zone of inhibition

Plant extract

Micro organism

AQ

ME

C. molmol

S. pyogenes

12 0.88

8.5 0.36

S. aureus

14 0.66

19 0.41

E. Coli

9 0.33

9 0.55

P. aeruginosa

12 0.90

13 1.10

S. pyogenes

10 0.80

8 0.70

S. aureus

12 0.10

E. coli

7 0.88

8 0.55

P. aeruginosa

7 0.44

14 0.20

P. anisum

KA

Values are mean inhibition zone (mm) S.D of three replicates.

[AQ-aqueous, ME-methanol and KA-Kanamycin(30 g/discolor)] [ = no inhibition].
Table options
Table 2.
Phytochemical analysis of selected plant samples.
Constituents
Z. officinal C. longa C. molmol P. anisum
Carbohydrates

Alkaloids

Steroids

Saponins

Flavonoids

Tannins

Key: + = present; = absent.

Table options

4. Discussion

Antibacterial activity obtained in this study varied with solvents used for extraction. Z. officinale extracts
showed moderate inhibition activity with the zone range of 914 mm. Maximum inhibition was observed
against P. aeruginosa (14 mm) and minimum inhibition against E. coli (9 mm). Malu et al. (2009) reported
anti bacterial activity of various extracts of Z. officinale against C. bacillus, S. epidermidis and S.
viridians. Bele et al. (2009) also confirmed that the methanol extract of Z. officinale showed a significant
zone of inhibition against E. coli, S. aureus and Z. officinale is known to contain resins and volatile oils
such as borneol, camphene, citral, eucalyptol, linalool, phenllandrene, zingiberine and zingiberol phenols
( Ahmad et al., 2008 and Hirasa and Takemasa, 1998) which may be responsible for its potent
antimicrobial activities.
The crude extracts of C. longa were active against all bacterial strains showing maximum zone of
inhibition (19 mm) against S. pyogenes from methanol extract. This may be due to the presence of
tannins ( Table 2). Tannins are known for their astringent property and antimicrobial activity ( Cowan,
1999). The antibacterial activity of extracts of C. longa may be attributed to the presence of active
ingredients p-tolymethyl-carbinol, curcumin ( Lutomoski et al., 1974, Ramprasad and Sirsi,
1956 and Huhtanen, 1980) and essential oils (Banerjee and Nigam, 1978). Methanol extract of Z.
officinale and C. longa displayed effective antimicrobial activity against selected pathogens with the
inhibition zone in the range of 719 mm. These results are in parallel to the finding of previously reported
study by Anbu Jeba et al. (2009).
Oleo-resin of C. molmol is likely of same potential as of ciprofloxacin and tetracycline against various
strains

of S.

aureus and

has

also

shown

antibacterial

activities

against S.

enterica and K.

pneumonia ( Rahman et al., 2008). Phytochemical analysis by Emad et al. (2009) revealed the presence
of the carbohydrates in C. molmol extracts which is in agreement with our results ( Table 2). Alkaloids and
saponins detected in C. molmol may be responsible for the antibacterial activity of the plant species
( Table 2).
Among all the extracts, P. anisum was found to have least antibacterial activity which has also been
reported in previous study by Akhtar et al. (2008). Now a days its oil is being used as antibiotic substitute
in rations for broilers ( Mehmet et al., 2005).
Medicinal and healing properties of herbs are closely related to their chemical components which are
classified into some major groups like alkaloids, acids, essential oils, steroids, saponins, tannins etc. and
getting these chemicals out into the herbal remedy depends upon the solubility of these compounds in
various solvents. Against all the tested bacterial strain, we observe methanol extract of all the samples
showing much better antibacterial activities in contrast to aqueous extract, which may be because of
organic nature of methanol and also for the reason of its high capacity to dissolve more organic and
active antimicrobial compounds (Cowan, 1999). The antimicrobial action of the aqueous extracts could be
ascribed to the anionic components such as thiocyanate, nitrate, chlorides and sulfates besides other
water soluble components which are naturally occurring in the plant material (Darout et al.,2000). These
results confirmed the substantiation of previous studies which have reported that methanol is a better
solvent for more consistent extraction of antimicrobial substances from medical plants compared to other
solvents, such as water (Ahmad et al., 1998, Eloff, 1998, Lin et al., 1999, Karaman et al., 2003, Emad et
al., 2009, Parekh et al., 2005 and Mothana and Lindequist, 2005).
The use of plant extracts with known antimicrobial properties can be of great significance in therapeutic
treatments but several studies have also reported various types of contamination of herbal medicines
which include microorganisms and toxins produced by microorganisms, pesticides and toxic heavy metals

(Talaly and Talaly, 2001). As a result, sterilization is needed especially for aqueous extracts before use to
get rid of these contaminations. In present study aqueous extracts were autoclave-sterilized before use
as autoclaving is reported to cause less damage to the antibacterial activities of the aqueous extract
(Hashemi et al., 2008).

5. Conclusion
Our results suggest that Z. officinale and C. longa can serve as potential source of bioactive healthy
compounds in the diet and their consumption could be useful in the prevention of diseases. Further
research is needed toward isolation and identification of active principles present in the extracts which
could possibly be exploited for pharmaceutical use.

Acknowledgment
This research project was supported by a grant from the Research Center of the Center for Female
Scientific and Medical Colleges, Deanship of Scientific Research,King Saud university.

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