Nada Khazal Kadhim Hindi et al. / Vol 4 / Issue 1 / 2014/ 43-48.

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International Journal of

Pharmacological Screening Methods

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ANTIBACTERIAL EVALUATION OF AQUEOUS EXTRACTS OF

FOUR CITRUS SPECIES IN HILLA, IRAQ
Nada Khazal Kadhim Hindi1*, Zainab Adil Ghani Chabuck2, Shatha Khazal Kadhim Hindi3
1

Basic and Medical science Department, College of Nursing, Babylon University, Babylon Province, Iraq.
2
Department of Microbiology, College of Medicine, Babylon University, Babylon Province, Iraq.
3
Department of Microbiology, College of Medicine, Babylon Province, Iraq.

ABSTRACT
The use of medicinal plants as natural antimicrobial agents is gaining popularity. Natural products provide unlimited
opportunities for new drug leads because of the unmatched availability of chemical diversity. The aim of this study is to
evaluate the antimicrobial activity of different types and part of citrus species against different microbial isolates. The
antmicrobial effects of aqueous extracts of peel, juice and leaves from fresh Citrus aurantium (sour orange), Citrus sinensis
(sweet orange), Citrus reticulata (tangerine, mandarin) and Citrus grandis (pummelo) against 3 Gram-positive and 6 Gramnegative bacterial, including S. aureus, S. pyogenes, E. feacalis, P. aeruginosa, K. pneumoniae, E. coli, S. typhi, Proteus spp.,
M. catarrhalis, all of them were studied. Citrus juices showed the highest antibacterial activity against most of the study
bacterial isolates. Moderate activity produced by the citrus peels and the lowest effect produced by the extract of the citrus
leaves. Different aqueous extracts from various citrus species can be used as effective antibacterial reagents even against
multidrug resistant bacteria, and home-available, safe, cheap and with no side effect like the synthetic drugs.
key words: Aqueous extracts, Citrus, Antimicrobial activity.
INTRODUCTION
Plants are rich source of antibacterial agents
because they produce wide array of bioactive molecules,
most of which probably evolved as chemical defense
against predation or infection. A major part of the total
population in developing countries still uses traditional
folk medicine obtained from plant resources [1].
Many medicinal plants have been found effective
in the cure of bacterial diseases. Due to increasing
antibiotic resistance in microorganisms and side effects of
synthetic antibiotics, medicinal plants are now gaining
popularity in the treatment of bacterial infections. The use
of traditional medicines and medicinal plants in most
developing countries as therapeutic agents for the
maintenance of good health has been widely observed.

Furthermore, an increasing reliance on the use of medicinal

plants in the industrialized societies has been traced to the
extraction
and
development
of
drugs
and
chemotherapeutics from these plants as well as from
traditionally used herbal remedies. Medicinal plants are
considered as clinically effective and safer alternatives to
the synthetic antibiotics [2].
According to World Health Organization (WHO),
medicinal plants would be the best source to obtain a
variety of drugs. Extensive research in the area of isolation
and characterization of the active principles of these plants
are required so that better, safer and cost effective drugs
for treating bacterial infections can be developed [3].Over
80% of the populations in some Asian and African
countries depend on traditional medicine for primary

Corresponding Author:- Nada Khazal Kadhim Hindi Email:- nadakhazal@yahoo.com

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health care. The WHO estimates that in many developed

countries, 70% to 80% of the population has used some
form of alternative or complementary medicine [4, 5].
The practice of herbal medicine in Iraq is
becoming so intense due to the high cost of medicines in
pharmacies and appearance of different and untreatable
diseases especially after wars. Studies still reveal that the
plant kingdom has not been exhausted based on the species
of medicinal plants which are yet to be discovered [6].
Citrus aurantium (sour orange), Citrus sinensis
(sweet orange), Citrus tangerine (tangerine, mandarin) and
Citrus grandis (pummelo) belong to a family Rutaceae. In
Iraq they called Naringe, Purtuqal, Lalingi and Sindi
respectively. In Latin American areas, these fruits are called
naranja de China, widely distributed in the tropics. The
fruit originated in Southern China, North Eastern India and
Southern Asia. They are perennial trees. The entire parts of
the trees are useful medically [6].
Citrus fruits make up the largest sector of the
worlds fruit production, with more than 100 million tons
produced each season. About 34% of citrus fruits are made
into juices; therefore, large amounts of residues are formed
every year (7). Citrus peels, which comprise the dominant
residue, exhibit potent antioxidant, antimicrobial and antiinflammatory activities [8], and are considered potential
sources of functional components. Except for ascorbic acid,
citrus peels contain more bioactive compounds, such as
phenolic acids, flavonoids, limonoids, and fibre than do
juices [9].
Sweet orange constitutes about 60% of the total
citrus world production. Peels represent between 50 to 65%
of total weight of the fruits and remain as the primary
byproduct. If not processed further, it becomes waste
produce odor, soil pollution, harborage for insects and can
give rise to serious environmental pollution [10].
Orange peel oil has lethal effect on fleas, fireants,
houseflies due to its 90-95% limonene. Oranges are eaten to
allay fever. The roasted pulp is prepared as poultice for skin
diseases. The fresh peel is rubbed on acne. A decoction of
the dried leaves and flower is given in Italy as an
antispasmodic, cardiac sedative and antiemetic. Orange
peel is medically used against fungi [6].
Flavanones, flavones and flavonols are three types
of flavonoids that occur in Citrus fruit. The main flavonoids
found in citrus species are hesperidine, narirutin, naringin
and eriocitrin [11]. Phytochemicals that contribute to health
(e.g., flavonoids, carotenoids and pectin) are abundant in
citrus peels [12]. The highest amount of flavonoids (a major
group of citrus secondary metabolites) occurs in the peel.
Flavonoids have a wide range of biological effects, such as
inhibition of key enzymes in mitochondrial respiration,
protection against coronary heart disease and antiinflammatory, antitumour, and antimicrobial activities [13].
Due to their high flavonoid content, citrus peels
could be exploited by both pharmaceutical and food
industries. In spite of this, the compounds present in citrus

peel are usually processed as by-products or wasted,

resulting in environmental pollution. One of the main
reasons for this is the absence of effective extraction
procedures to obtain the flavonoids from the citrus peels
[14].
One of the important products of citrus fruits is the
essential oil, which is obtained from citrus peels [15]. A
cosmetic lotion from citrus limetta essential oil for the
treatment of corns, warts and other skin lesions [16].
The aim of this study was to appraise the
antibacterial activity of different parts (peel, juice and leaf)
of various citrus species.
MATERIALS AND METHODS
Microbial isolates
Different nine clinical microbial isolates (Gram
positive and Gram negative) were collected and identified
by using conventional biochemical tests [17]. and cultivated
in pure culture, at microbiological laboratory/college of
Medicine / Babylon University. These include
Staphylococcus
aureus,
Streptococcus
pyogenes,
Enterococcus
feacalis,
Pseudomonas
aeruginosa,
Klebsiella pneumoniae, E. coli, Salmonella typhi, Proteus
spp., Moraxella catarrhalis.
Plants collection
Fruit extracts were prepared according to [18].
Fresh fruits [Citrus aurantium (sour orange), Citrus
sinensis (sweet orange), Citrus reticulata (tangerine) and
Citrus grandis (pummelo] were obtained from the local
market at Hilla City, Iraq, 2013. The fresh fruits were
washed in running tap water in laboratory, surface sterilized
with 70% alcohol, rinsed with sterile distilled water and cut
open with a sterile knife and the juice pressed out into a
sterile universal container separately and then filtered
(using Millipore 0.45 filter paper) into another sterile
container to remove the seeds and other tissues and used
freshly as crude without refrigeration.
Extracts of peels and leaves were prepared as 50%
extract (by adding 50 gm solid materials for each 100ml
water), hot water was primed, peel and leaves were added
each one in separated container and left to cool, the
containers were covered in order to keep all active
elements. The mixtures were vigorously swirled by the
blender. The mixtures were filtered using filter paper into a
clean beaker and stored at 4 C when not in use.
Antimicrobial activities
The screening of antimicrobial activities of each
aqueous citrus extract on the tested bacteria used in this
investigation was determined on nutrient agar media, by the
agar diffusion techniques using agar well diffusion method.
Wells of 6 mm diameter and 5 mm depth were made on the
solid agar using a sterile glass borer [19, 20].
Approximately 20l of each extract was inoculated onto
wells were made in the spread plate culture of each

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microbial isolates. The plate was then allowed to incubate

at 37C for overnight. After 12-24 hrs of incubation, each
extract was noted for zone of inhibition for all isolates. The
diameters of the zone of inhibitions were measured by
measuring scale in millimeter (mm).
RESULTS
Results of antibacterial activity of extracts from
different parts from Citrus sinensis, Citrus aurantium,
Citrus reticulata and Citrus grandis against Gram positive
and negative isolates by the agar well diffusion method
were shown on Figure (1, 2 and 3) respectively. As these
figures, showing the inhibitory effect (inhibition zones) of
these extracts against bacterial isolates measured in
millimeter (mm).
Results in figure (1) showed that the aqueous
extract of peels of C. sinensis produced inhibition activity
against five isolates including S. pyogenes, P. aeruginosa,
K. pneumoniae, S. typhi and Proteus spp with inhibition
zone ranging from 10-25mm and highest inhibition activity
was against S. pyogenes with the 25mm zone. On the other
hand, leaves and juice of C. sinensis produced less
inhibition as they affect only three and two isolates
respectively.
Additionally, from figure (1) we can see that E.

feacalis, M. catarrhalis and E. coli were resistant to all

extracts of C. sinensis. S. aureus, S. pyogenes and Proteus
spp. each one were susceptible to only one extract.
Surprisingly, P. aeruginosa showed susceptibility with
inhibition zone 10mm with all these three extracts.
Results in figure (2) showed that the juice of C.
aurantium produced inhibition activity (inhibition zones
10-30mm) against all the study isolates except one (P.
aeruginosa). In contrast, aqueous extract of leaves of C.
aurantium produced inhibition activity against only one
isolate (K. pneumoniae) with 20mm zone. Aqueous extract
of peels of C. aurantium showed activity against four
isolates S. aureus, E. feacalis, K. pneumonia and M.
catarrhalis. P. aeruginosa showed resistant to all extracts
of bitter orange.
Finally, on Figure (3) results showed that the juice
of C. reticulata produced inhibition activity (inhibition
zones 10-25mm) against all the study isolates except one
(P. aeruginosa). While the peels of C. reticulate and C.
grandis affect four and five isolates respectively.
In comparison between these results, citrus juices
showed the highest antibacterial activity against most of the
study bacterial isolates. Moderate activity produced by the
citrus peels and the lowest effect produced by the extract of
the citrus leaves.

Figure 1. Effect of Aqueous extracts of different parts of Citrus sinensis (sweet orange) [peel, leaf and juice] against
different bacterial isolates

Figure 2. Effect of Aqueous extracts of different parts of Citrus aurantium (bitter orange) [peel, leaf and juice] against
different bacterial isolates

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Figure 3. Effect of Aqueous extracts of different parts of Citrus reticulata (tangerine) [peel and juice] and peel of Citrus
grandis (pummelo) against different bacterial isolates

DISCUSSION
Throughout the history of mankind, many
infectious diseases have been known to be treated with
herbal remedies .The natural herbal products either as pure
compounds or as standardized plant extracts provided
unlimited opportunities for new drug leads because of the
uncompared availability of diversities of chemical. This
results to a never ending and urgent need to discover new
antimicrobial compounds with different chemical structure
and new mechanisms of action for re-emerging and new
infectious diseases [21].
Citrus fruit products are known to potent
antimicrobial agents against bacteria and fungus. These
citrus fruits are rich source of flavonones and many
polymethoxylated flavones which are very rare in other
plants [22].
Many studies have reported antioxidant and
antibacterial effect of juice and edible parts of oranges of
different origin and from different varieties [23].
Peels of Citrus contain a wide variety of secondary
metabolites, such as tannins, terpenoids, alkaloids and
flavonoids that are found to have effective as antimicrobial
properties. The monoterpenes affect the structural and
functional properties of lipid fraction of the plasma
membranes of bacteria and yeasts, causing leakage of
intercellular material and exit of critical molecules and ions
leading to death of microbes. Terpenoids affect respiratory
enzymes inhibiting microbial oxygen uptake and oxidative
phosphorylation [24, 25].
Nevertheless, in our study extract of citrus leaves
showed lowest effect against most of the study bacterial
isolates. This result was simultaneous to results obtained by
different researchers. Ekwenye and Edeha [6] showed that
the aqueous extract of C. sinensis leaf affect only E. coli
with very little zone (7mm). On the other hand, Bouabdelli
et al. [26] showed that S. aureus, Proteus mirabilis and E.
coli were sensitive to aqueous extract of C. aurantium
leaves while P. aeruginosa was resistant to it. In addition,
Gopal illustrated that extracts of C. aurantium leaves were

significantly effective against both Gram-positive and

Gram-negative organisms with inhibition zone 12-14mm.
Preliminary phytochemical screening of the leaf extracts
showed the presence of Alkaloids, carbohydrates,
flavonoids, terpenoids, sterols and tannins. Thus, further
work can be carried on the isolation procedure for finding
out the exact moiety responsible for the biological activity
[27].
The highest efficacy of citrus juice (especially of
C. aurantium) to inhibit bacterial growth in this study were
concurrent with Mathur et al. [28] who showed that the
aqueous extracts from citrus fruit pulp contained significant
antimicrobial activity. Al-Ani et al. [29] demonstrated that
the best antimicrobial activity was observed in juices of C.
aurantium, they were active against the three types of
bacteria used in their study including S. aureus P.
aeruginosa and P. vulgaris with 16-20mm inhibition zones
and this result was nearer to the values obtained by A
dedejl et al. [30].
The effect of pH is very important in case of
microbicidal acids of the fruits juices. These are membraneactive substances, which damage the inner cell membrane
in their undissociated form. They alter the membrane
permeability of the microbial cell and acidify the cytoplasm
[31].
Citrus peels have also been noted to have
antibacterial, and anti-inflammatory properties and they are
also known to reduce the symptoms of disorders such as
hyperthyroidism, diabetes, osteoarthritis and high blood
pressure [32, 33]. Peels of citrus fruits showed a moderate
inhibition to the bacterial isolates, this agreed with kumar et
al. [34] who concluded that the aqueous extract of the C.
sinensis peel showed a moderate percentage with inhibition
zone 9mm against E. coli and S. aureus and no effect
against S. typhi and K. pneumoniae. Additionally, Jwanny
et al. [35] concluded that aqueous extract of orange peels
can produce an antibacterial activity against different gram
positive, negative bacteria and fungi with zone of inhibition
7-12mm.

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CONCLUSION
Different aqueous extracts from various citrus
species can be used as effective antibacterial reagents even
against multidrug resistant bacteria, and home-available,
safe, cheap and with no side effect like the synthetic drugs.

ACKNOWLEDGEMENTS
We are extremely thankful to the College of
Medicine and Nursing, Babylon University for providing
all the needed facilities, which are essential for successful
completion of the present work.

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