Antibacterial Properties of Limau Kasturi (C.

microcarpa) Peels Extract

1Rubiatul, A.S., 2Nor Helya I. K., 3Zarina Z., 4Dachyar A., 5NurulAin H.A.
1,4School of Bioprocess Engineering, Universiti Malaysia Perlis, Jejawi 3 Engineering Complex,
02600 Arau, Perlis, Malaysia.
2,3,5Faculty of Engineering Technology, Universiti Malaysia Perlis, P.O Box 77, D/A Pejabat
Pos Besar Kangar, 01000 Kangar, Perlis,
Malaysia.
Citrus microcarpa or Citrus mitis, also known as limaukasturi is an edible plant
and have potential as antibacterial agent. The presence of bioactive compound in
limaukasturi peels extract has possessed antibacterial property by killing or inhibits the
growth of pathogenic bacteria. In this research, limaukasturi peels were dried and
extracted using soxhlet extraction method. The solvents used for the extraction were
methanol, ethanol, acetone, hexane and water. The antibacterial effect of limaukasturi
peels extract was evaluated on Gram-positive bacteria like Bacillus subtilis and
Streptococcus spp., and Gram-negative bacteria such as Escherichia coli and Salmonella spp.
The extracted products were subjected to antibacterial susceptibility assay by agar welldiffusion method with measurement diameter of inhibition zone around the extracts.
From the result, methanol extracts showed the highest inhibition zone compared to
other solvent extracts. The zone of inhibition of methanol extracts on
E. coli, Salmonella spp., B. subtilis, and Streptococcus spp. were19 mm, 17 mm, 13 mm and
12 mm, respectively. Overall, the bacterial are susceptible and moderately susceptible to
methanol extracts whereas the other extracts of ethanol, acetone, hexane and water
showed low inhibition and no inhibition effects towards four bacterial tested. Thus, the
results obtained in the present study suggest that methanol extracts of limaukasturi
peels can be used for further study in treating diseases caused by the test organisms.
METHODS AND MATERIALS
Sample Preparation:
Limaukasturi were purchased from local market in Kangar, Perlis. The fruits were
washed with tap water and peeled manually. The peels were heated at 45 for 24 h in a
convection oven and the dried peels were grinded to fine powder using a grinder. The
ground powders then were packed in a tight plastic sealer and stored at room
temperature until use.
Soxhlet Extraction:
About 20 g of sample were packed in a nylon fabric before be placed in an extraction
chamber, which is suspended above a flask that containing of solvent and below a
condenser. After that, the flask containing samples is heated using heating mantle until

the solvent is evaporates and move up into the condenser until it is converted into a
liquid form that trickles into the extraction chamber containing the sample. The time
taken for the solvent to evaporate will be regulated. At the end of the process, the round
bottom flask is taken out to undergo separation process using rotary evaporator. This
process aims to separate the solvent from the extract.
Antibacterial Activities Test:
Bacteria used:
Antibacterial activities were carried out according to method that suggested by Archana
with some modification, where bacteria were obtained from School of Bioprocess,
Universiti Malaysia Perlis (UniMAP).
Bacterial cultures used are such Escherichia coli, Bacillus subtilis, Salmonella spp., and
Streptococcus spp.
Culture media and inoculum:
The culture media and inoculum were prepared according to the method as suggested
by Archana with slight modification. Before use, bacteria cultures were revived in
nutrient broth (NB). For maintenance, bacteria cultures were maintained in nutrient
agar (NA) at 4. The inoculum of bacteria cultures were incubated at 37. The
inoculated broth was incubated overnight for 24 h. Then, a freshly grown microbial
cultures were appropriately diluted in sterile both media to obtain cell suspension of
106cfu/ml.
Antibacterial assay:
For antibacterial assay, agar well-diffusion method has been modified slightly from Sen
and Batra.
Nutrient agar (NA) was swabbed (sterile cotton swabs) with 8 hour old-broth culture of
respective bacteria. Then, wells (7mm diameter and 20mm a part) were made in each
plates using tips 1 ml. About 100mg/ml of peels extracts concentration were used for
each solvent. 100l of peels extracts will be pipetted into the wells. An antibiotic, named
ciprofloxacin was used as positive control while distilled water as negative control.
Next, the plates were incubated at 37for 18-24 h. The diameter of the inhibition zone
(mm) was measured. The tests were performed in triplicate.
RESULTS AND DISCUSSION
Results of antibacterial activity of limaukasturi peel extracts against Gram-positive and
Gram-negative bacteria by the agar-well diffusion method. From overall result, the
inhibition zone of extracts against Gram-negative bacteria which are E. coli and
Salmonella spp. is higher than Gram-positive bacteria (B. subtilis and Streptococcus spp.).

This is because of the cell wall of Gram-negative bacteria is thinner than Gram-positive
bacteria, so that the extract can penetrate the wall of Gram-negative bacteria more and
inhibit the growth of bacteria. In this test, ciprofloxacin was used as positive control. It
is an antibiotic in a group of drugs called fluoroquinolones that have been commercially
used to treat bacterial infections on humans. From observations, ciprofloxacin is very
effective antibiotic against bacteria. The bacterial susceptibility of E.coli was observed
where the highest activity of methanol extracts with zone of diameters of 19 mm. For
Salmonella spp. methanol extracts has inhibited about 17 mm of bacterial growth while
for B. subtilis and Streptococcus spp., its inhibited about 13 mm and 12 mm, respectively.
From this result, it can emphasized that Gram negative bacteria are susceptible to
methanolic extracts of limaukasturi peels while Gram-positive bacteria are moderately
susceptible to the extracts. From previous study, Gopa infer that C. aurantium leaves
extracts was significantly effective against both Gram-positive and Gram-negative
bacteria with inhibition zone 12-14 mm.
Next, inhibition zone of ethanol extracts of limaukasturi peel. A very little zone was
inhibited by ethanol extracts on E. coli and Salmonella spp. with zone of diameter 8 mm,
respectively. While for Streptococcus spp. the extracts only covered 7 mm and no
inhibition on B. subtilis. Thus, from the observation, it can be concluded that ethanol
extracts has a low inhibition towards bacterial pathogens, except B. subtilis which is
resistant to the extracts. Unnisaet al. has reported that lowest antibacterial effect of
ethanolic extracts from sweet lime was observed on E. coli with diameter of zone is 8
mm.From the previous study, Kumar et al. considered that the aqueous extract of C.
sinensis peel has a moderate percentage with inhibition zone 9 mm against E. coli. For
acetone extracts, only E. coli is resistant to acetone extracts. While the other three
bacteria shows low inhibition on the plates. Salmonella spp. has recorded about 7 mm of
diameter zone, while B. subtilis and Streptococcus spp. are inhibited only 6 mm,
respectively. Jwannyet al. concluded that aqueous extract of orange peels has
antimicrobial activity against microbes with zone of inhibition 7 12 mm. For hexane
extracts, no inhibitions are detected in each bacterial plate, therefore, all the bacteria are
resistant towards both hexane and water extracts. Its possibly that there are no or less
phenolic compound are presence in the extracts which responsible for antibacterial
activity.
Conclusion:
As the conclusion, limaukasturi peel extracts has possessed antibacterial activity
towards all tested bacterial pathogens. However, only certain extracts shows a good
inhibition effects on agar plates. The most goods appearance of inhibition zone was
found using methanolic extracts where its inhibit 19 mm on E. coli, 17 mm on

Salmonella spp., 13 mm on B. subtilis and and 12 mm on Streptococcus spp. For overall

result, Gram-positive bacteria are susceptible to methanol extracts and Gram-negative
bacteria are moderately susceptible to the extracts. While forethanol extracts, it gave low
inhibition on all bacteria except B. subtilis. For acetone extracts, its covered low
inhibition on all bacteria except E. coli, while hexane and water extracts showed no
inhibition for all bacteria. Thus, the results obtained in the present study suggest that
methanol extracts of limaukasturi peels can be used for further study in treating
diseases caused by the test organisms.
https://www.researchgate.net/publication/298643732_Antibacterial_Properties_of_Li
mau_Kasturi_C_microcarpa_Peels_Extract

ANTIBACTERIAL ACTIVITY OF FRUITS AGAINST Escherichia coli

Misbah Manzoor, Sangeen Naseer, Dr.Rukhsana Jabeen and Mirfa Manzoor
Sardar Bahadur Khan Womens University Quetta, Balochistan, Pakistan.
ABSTRACT
Numerous fruits are unquestionably utilized to prevent food borne illness diseases.
Fruits were analyzed for their antibacterial activity. The antibacterial activity was
determined by disc diffusion method. Nearly eight fruits with their various
concentrations (25%, 50%, 75%, 100%,) were prepared in order to check their
antibacterial activity opposing E. coli. Mango (Mangifera indica L), Apricot (Prunus
armeniaca), Grapes (Vitisvinifera), Apple (Malus domestica), Peach (Prunus Persica), Lemon
(Citrus limonum), Melon (Cucumismelo) and watermelon (Cirtrullus lanatus) were the
selected fruits. The highest inhibition zone was observed in the juice extract of Apricot
with concentration of 100%. The mean value of inhibition zone was (8.2 1.1121). The
minimum inhibition was surely noticed in the juice extract of mango with also
concentration of 100%. The mean value of inhibition zone was (5 0.9574). Other fruits
showed different inhibition zones along with different concentrations and observed that
the effect of fruits against E. coli was concentration dependent. Response against in
increase and decrease in concentration was varied among all the fruits.
THE KIRBY BAUER ANTIBIOTIC SENSITIVITY
TEST PROCEDURE
Purpose
The main motive of this test procedure is undoubtedly to become familiar with the
Kirby- Bauer producer for the assessment of the antimicrobial activity of
chemotherapeutic agents.

Principle
The available chemotherapeutic agent probably differs in their scope or potential of
antimicrobial activity. Some chemotherapeutic agent has a limited spectrum of
microbial activity, being effective agents only one group of microorganism. Other
exhibits or has broad and wide spectrum activity agents a range of microorganism. The
drug susceptibilities many disease causing microorganisms are known, but it sometimes
becomes surely necessary to test a multitude of agents in order to determine the drug of
choice. The producer of standardized filter paper disc agar diffusion is known as Kirby
-Bauer. Most importantly, this method undoubtedly permits the rapid and quick
determination of the efficiency of a drug by measuring diameter of the zones of
inhibition that result from diffusion of the agent into soaked in concentration of various
substances that can prevent the growth of bacteria, antibiotic, and then kept on the
surface of the agar plates that have been seeded with organism to be tested. MullerHutton agar is the medium of choice with the pH of 7.2 - 7.4 is poured up to 5mm to
plates and refrigerator in solidification. Before utilizing them, the plates are transferred
to a box like machine incubator at
37c for approximately 10-20 minutes to dry of that developed mixture on the agar
surface. The plates with the help of a cotton swab are then inoculated with inoculums in
order to ensure the full growth and development of the organism. As well-spaced
intervals, the discs are transferred to the surface of the agar plates, after incubation with
the assistance of the incubator, the plates are carefully examined with the growth
inhibition, which is indicated or represented by the clear and a vivid zone around each
disc. The size of the zone determines the susceptibility of an organism to the drug.
CONCLUSIONS
Several fruits were undoubtedly analyzed for its antibacterial activity opposing E. coli,
the fruits were collected from a well- known city Quetta. The fruits were Apricot,
Mango, Lemon, Melon, Watermelon, Peach, and Apple and Grapes. Various juice
extracts of the fruits were unquestionably put to action for the determination of
antibacterial activity with undoubtedly various concentrations (25%, 50%, 75%, and
100%). The maximum inhibition was certainly found in the juice extracts of Apricot
essentially with concentration of 100% and the value of mean inhibition zone was
(8.41.1121). The minimum inhibition was surely noticed in the juice extract of mango
with also concentration 100% of inhibition zone of 5 0.9574. Apricot undoubtedly plays
a tremendous role in manufacturing the various products essentially jam and nectar.

http://www.arpnjournals.com/jabs/research_papers/rp_2013/jabs_0313_547.pdf

Study Antimicrobial Activity of Lemon (Citrus lemon L.) Peel Extract

Maruti J. Dhanavade, Chidamber B. Jalkute, Jai S. Ghosh and Kailash D. Sonawane
Department of Microbiology, Shivaji University, Kolhapur-416004, Maharashtra, India
Abstract:
The main objective of the study is extraction, identification of antimicrobial compounds
and demonstration of antimicrobial activity of lemon (Citrus lemon L.) peel against
bacteria. As microorganism are becoming resistant to present day antibiotics, our study
focuses on antimicrobial activity and future prophylactic potential of the lemon peel.
Biologically active compounds present in the medicinal plants have always been of great
interest to scientists. The peel of citrus fruits is a rich source of flavanones and many
polymethoxylated flavones, which are very rare in other plants. These compounds, not
only play an important physiological and ecological role, but are also of commercial
interest because of their multitude of applications in the food and pharmaceutical
industries. The citrus peel oils show strong antimicrobial activity. The antimicrobial
activity has been checked in terms of MIC by using different solvents against
microorganisms like Pseudomonas aeruginosa NCIM 2036 for which MIC was 1:20 in
presence of methanol, for Salmonella typhimurium NCIM 5021 the observed MIC was
1:20 in presence of acetone. In case of Micrococcus aureus NCIM 5021 the observed MIC
was 1:20 when ethanol was used as solvent. The compounds like coumarin and
tetrazene were identified by GC/MS of lemon peel extract.
MATERIALS AND METHODS
The study was conducted between June 2010 and January 2011. The study was carried
out at the Department of Microbiology, Shivaji University, Kolhapur, India.
Preparation of extract:
The peel of lemon was homogenized in different solvents individually and mixed well.
The solvents used were ethanol, acetone, and methanol. The extracts were collected
separately for further study.
Cultures used for antimicrobial activity:
The microorganisms used were as follows, Pseudomonas aeruginosa NCIM 2036,
Salmonella typhimurium NCIM 5021, and Micrococcus aureus NCIM 5021.

Culture medium:
Nutrient agar medium and a mineral based medium were used in all further studies.

Antimicrobial effect: Sterile molten nutrient agar at around 40C was taken and seeded
with different microbial cultures and plates were prepared. After solidification 4 mm
wells were prepared. In these wells solvent extracts of the peel were added. The plate
was incubated overnight at 37C. After incubation the zones of inhibition were
measured and recorded. Respective solvent controls were also run simultaneously. The
above procedure was repeated using mineral based medium with added yeast extract at
0.02%.
Determination of minimum inhibitory concentration of crude extracts:
Different concentration of crude extract as 1:20, 1:40, 1:60, 1:80 and 1:100 were added
respectively into mineral based medium containing glucose (1%), yeast extract (0.1%).
The organisms were inoculated respectively and incubated at 37C, overnight on shaker.
Detection of phytochemicals by GCMS:
Peel supernatant obtained in different solvents was analyzed by GCMS.
DISCUSSION
The study shows that the peel of lemon is not only an astringent but also is a good
antimicrobial agent. This is an important finding as certain skin flora like Pseudomonas
and Micrococcus can grow in presence of sebum, especially when it is secreted in excess
(in certain person), and cause purulent skin infections. Some time it can serve as a
predisposing factor for other types of skin infections like acne. Simple use of lemon juice
can prevent such types of infections and could help in keeping a good and healthy skin.
Of course it is needless to point out that good personal hygiene, exercise and a good diet
is equally essential too.
http://www.jonnsaromatherapy.com/pdf/Dhanavade_Antimicrobial_Activity_of_Lem
on_2011.pdf

ANTIMICROBIAL PROPERTIES OF PEELS OF CITRUS FRUITS

Akhilesh Khushwaha*1, Raghvendra Pratap Singh2, Vikas Gupta2, Madhulika Singh1
1: Institute of Transgene Life Sciences, Lucknow, U.P., India
2: School of Biotechnology, Shobhit University, Meerut, U.P., India
ABSTRACT

Current study targeted the extraction and assay of antimicrobial metabolites from the
peels. Peels were dried and antimicrobial metabolites were extracted from them by
soxhlet extraction procedure. Extraction was done by solvents like cold water, hot water,
methanol, ethanol, ethyl acetate and acetone. Extracts were subjected to antibacterial
and antifungal susceptibility assay by agar well diffusion method. All the extracts of
Lemon (Citrus lemon) were found to be effective against the used bacterial pathogens
except hexane extracts. Methanol and acetone extract showed maximum zone of
inhibition of 18 mm. Only methanol extract was effective against fungal pathogens
showing a zone of inhibition of 18 mm. In case of orange (Citrus sinensis) hexane extract
was found to be most effective against bacterial pathogens giving a zone of 13 mm. Only
the cold water extract of orange was effective against fungal pathogens used in the
study. Acetone extract of Mosambi (Citrus limetta) was most effective giving a zone of
20 mm against bacterial pathogens. Only cold water andethyl acetate extracts of
mosambi were effective against fungal pathogens giving a zone of inhibition of 17mm
and 15 mm respectively.
MATERIALS AND METHODS
Plant Material
Fresh and healthy fruits of Lemon (Citrus lemon L.), Orange (Citrus sinensis) and
Mosambi (Citrus limetta) were collected from Chinhat, Lucknow, India. They were
identified according to their taxonomical classification by the botanists at ITLS,
Lucknow. Fruits were washed with the help of tap water followed by sterilized distilled
water, air dried and peeled off further the peels were dried in sun, packed in envelops
for drying in hot air oven at 50C for 5 days and used as raw material for the extraction
of antimicrobial compounds.
Bacterial/ Fungal Strains and Culture Preparation
The bacterial strains used in this study were Pseudomonas aeruginosa, Salmonella
typhimurium and Micrococcus aureus. The fungal strains used in this study were
Trichophyton mentagrophytes, Microsporum canis and Candida albicans. Bacterial strains
were maintained on Nutrient Agar plates. They were sub-cultured weekly and
subsequently stored at 4C. The strains was inoculated in the nutrient broth (pH 7.0)
and incubated at 37oC for 24 hours. Fungal strains were maintained on Potato dextrose
agar plates. They were sub-cultured weekly and subsequently stored at 4C. The strains
were inoculated in the potato dextrose broth (pH 5.2) and incubated at 28oC for 48
hours.
Preparation of antimicrobial extracts by solvent extraction via soxhlet apparatus

Secondary metabolites which have been found to be responsible for the antimicrobial
properties of the plant extracts are soluble in various solvents ethanol, methanol,
acetone, ethyl acetate, hexane. Dried peels were ground into fine powders by the help of
grinders. 15g of powdered peels of plant material was soxhleted in 50 ml of solvents for
48 hours. The sample the pot after extraction was transferred in a beaker. All extracts
were concentrated over a rotary vacuum evaporator until a solid extract sample was
obtained. The resulting crude extract was freeze-dried. Dried crude extract was used in
a concentration of 1 mg/ml for antimicrobial susceptibility testing. (Extract was
dissolved in sterile distilled water). For cold and hot water extraction 15gm ground
plant material was soaked in cold and hot water respectively for 72 hours. After that the
solution was filtered through Whatmans filter paper No. 1. Filtrate was concentrated
over a rotary vacuum evaporator until a solid extract sample was obtained. The
resulting crude extract was freeze-dried. Dried crude extract was used in a
concentration of 1 mg/ml for antimicrobial susceptibility testing.
Antimicrobial Test
The screening of extract plates for antimicrobial activity was performed by using
Antimicrobial susceptibility assay by agar well diffusion method to compare their
effectiveness against antimicrobial activity.
A) Antibacterial Properties:
In order to determine the antibacterial spectrum of the extract, antibacterial
susceptibility assay was performed by the agar well diffusion assay, also called cup plate
method (Kirby Bauer method). Sterile MH media was prepared and was poured into
sterile petri plates and allowed to solidify. 25 l of bacterial pathogens were spread on
respective plates labelled earlier as
Pseudomonas aeruginosa, Salmonella typhimurium and Micrococcus aureus. Three wells of 8
mm diameter were bored using a sterile cup-borer. 25 l of Ampicillin (1 mg/ml), crude
antimicrobial extract and autoclaved distilled water were poured in the respective wells
and the plates were incubated at 37C overnight. The antibacterial activity of each
extract was expressed in terms of the mean of diameter of zone of inhibition (in mm)
produced by each extract at the end of incubation period.
B) Antifungal Properties:
In order to determine the antifungal spectrum of the extract, antifungal susceptibility
assay was performed by the agar well diffusion assay, also called cup plate method
(Kirby Bauer method).
Sterile PDA media was prepared and was poured into sterile petriplates and allowed to
solidify.

25 l of fungal pathogens were spread on respective plates labelled earlier Trichophyton

mentagrophytes and Microsporum canis and Candida albicans. Three wells of 8 mm
diameter were bored using a sterile cup-borer. 25 l of fluconazole (1 mg/ml), crude
antimicrobial extract and autoclaved distilled water were poured in the respective wells
and the plates were incubated at 28C overnight. The antifungal activity of each extract
was expressed in terms of the mean of diameter of zone of inhibition (in mm) produced
by each extract at the end of incubation period.
DISCUSSION
Citrus fruits have been of interest for extraction of antimicrobial metabolites by large
number of researchers (Kumar et al., 2011; Kumar et al., 2010; Amandeep et al., 2009) but
the peels have been less studied. So the current study targeted the extraction and assay
of antimicrobial metabolites from the peels. Peels were dried and antimicrobial
metabolites were extracted from them by soxhlet extraction procedure as used earlier by
Kumar et al., 2011. Extraction was done by solvents like cold water, hot water, methanol,
ethanol, ethyl acetate and acetone. Similar solvents have been used earlier by Kumar et
al., 2011; Kumar et al., 2010; Amandeep et al., 2009. Extracts were subjected to
antibacterial and antifungal susceptibility assay by agar well diffusion method of
Amandeep et al., 2009. All the extracts of Lemon (Citrus lemon) were found to be
effective against the used bacterial pathogens except hexane extracts. Methanol and
acetone extract showed maximum zone of inhibition of 18 mm. Only methanol extract
was effective against fungal pathogens showing a zone of inhibition of 18 mm. In case of
orange (Citrus sinensis) hexane extract was found to be most effective against bacterial
pathogens giving a zone of 13 mm. Only the cold water extract of orange was effective
against fungal pathogens used in the study. Acetone extract of Mosambi (Citrus limetta)
was most effective giving a zone of 20 mm against bacterial pathogens. Only cold water
and ethyl acetate extracts of mosambi were effective against fungal pathogens giving a
zone of inhibition of 17mm and 15 mm respectively.
Kumar et al., 2011; Kumar et al., 2010; Amandeep et al., 2009; Nurmahani et al., 2012 have
also reported similar results for the various extracts from citrus fruits.
CONCLUSION
Based on the above results it can be concluded that the waste portions of the citrus fruits
i.e the peels could be very good source for the extraction of antimicrobial components.
The extracts could be used as a drug after proper pharmacological evaluation and
clinical trials. The future prospects of the current research includes, further purification
of extract and its evaluation for the presence of Phytochemical components and their
identification.

www.ijupls.com

COMPARATIVE EVALUATION OF ANTIMICROBIAL PROPERTIES OF CITRUS

VARIETIES AVAILABLE IN MALAYSIA MARKET
ABSTRACT
Citrus has been cultivated in many tropical and subtropical countries due to its culinary
and medicinal properties. Its antimicrobial properties are well known. There are few
varieties of citrus available in Malaysian market i.e. Citrus aurantifolia, Citrus reticulata,
Citrus microcarpa, Citrus limon and Citrus sinensis. The present study compares the
antibacterial properties of methanol extract of five varieties of citrus peels by disc
diffusion method against Streptococcus pyogenes, Staphylococcus aureus, Escherichia
coli and Pseudomonas aeruginosa. There is no inhibition were observed for all the citrus
peel extracts at 5 and 10 mg/ml. The methanol extract of Citrus microcarpa, Citrus
reticulata and Citrus sinensis at 20 mg/ml showed better inhibition than compare to
Citrus aurantifolia and Citrus limon against Staphylococcus aureus and Escherichia coli.
However, the standards at lower concentrations showed higher inhibition than compare
to all the extracts against entire organism. These findings can form the basis for further
studies to isolate their active constituents to perform its biological activities.
INTRODUCTION
Citrus family is large and varied. There are several types of citrus plants and fruits are
available all over the world. These plants are widely used in both medicinal and cooking
purposes. In Malaysia Citrus aurantifolia, Citrus reticulata, Citrus microcarpa, Citrus
limon and Citrus sinensis were available in the market. They are different from colour,
size, appearance and taste. Citrus fruits reported for enormous number of biological
activities such as anti-cancer, anti-diarrheal, antibacterial, antifungal, antiviral
insecticidal and antioxidant. Some oils have been used in cancer treatment [1].
Medicinal plants represent a rich source of antimicrobial agents. There is also an urgent
need to search for new antimicrobial compounds with novel mechanisms of action
because there has been an alarming increase in the incidence of new infections diseases,
as well as the development of resistance to the antibiotics in current clinical trials [2]. All
the citrus varieties are well known for antimicrobial properties [3]. However, so far there
is no comparative study has been reported in these varieties available in Malaysia for its
antimicrobial properties. Hence, in the presence study, we aimed to carry out a

comparative investigation of its antimicrobial properties of different varieties of citrus

using disc-diffusion method.
MATERIALS AND METHODS
Collection and authentication of Citrus fruits (Fig.1)
The raw citrus fruits, Citrus reticulata (limau mandarin), Citrus microcarpa (limau
kasturi), Citrus aurantifolia (limau nipis), Citrus sinensis (limaumadu) and Citrus limon
(lemon) were collected from local market, Ipoh District, Perak, Malaysia and
authenticated by botanist.
Extraction of dried peels of citrus fruits
The peels of the citrus fruits were removed from the fruits and washed thoroughly in
distilled water to remove the contaminants; it was chopped into small pieces and dried
under shade, coarsely powdered and separately subjected to extraction by maceration in
methanol (10 g of plant powder and 50 ml of methanol) at room temperature with
occasional shaking for seven days. The macerate was filtered and the filtrate was dried
at low temperature (40-50 C) under vacuum. The extracts were stored in air-tight
containers in a refrigerator at 4 C until further use.
Antimicrobial screening
Test microorganism
A panel of four common pathogenic microorganisms were used in the study, which
includes two gram-positive bacteria (Streptococcus pyogenes and Staphylococcus
aurous) and two gram-negative bacteria (Escherichia coli and Pseudomonas
aeruginosa).
Disc-diffusion method
A suspension of the tested microorganisms was uniformly swabbed on agar. Sterile
blank discs were individually impregnated with different concentration of extracts (5,10
and 20 mg/ml) and placed onto the inoculated agar plates The plates were inverted and
incubated at 37 C for 24 h. The antimicrobial activity was measured by measuring
diameter of the resulting zone of inhibition against the tested organisms. The test for
positive control and negative control were performed in duplicates [2].
RESULTS
The nature of methanol extract of citrus varieties and percentage yields were
mentioned in Table 1. The results of antibacterial activity of methanol extract of citrus

varieties were presented in Table 2. The antibacterial activity was tested by using three
different concentrations includes 5, 10 and 20 mg/ml. There is no inhibition were
observed for negative control and all the citrus peel extracts at 5 and 10 mg/ml. The
concentration at 20 mg/ml showed lower to moderate inhibition against Staphylococcus
aureus and Escherichia coli, but also failed to show inhibition against Streptococcus
pyogenes and Pseudomonas aeruginosa. The methanol extract of Citrus microcarpa,
Citrus reticulata and Citrus sinensis at 20 mg/ml showed better inhibition than
compare to Citrus aurantifolia and Citrus lemon against Staphylococcus aureus and
Escherichia coli. However, the standards at low concentrations showed higher inhibition
than compare to all the extracts against entire organism.
http://www.ijcpr.org/Issues/Vol5Issue4/725.pdf

Essential Oils from the Malaysian Citrus (Rutaceae) Medicinal Plants

Abstract
This review article appraises the extraction methods, compositions, and bioactivities
of the essential oils from the Citrus species (family: Rutaceae) endemic to Malaysia
including C. aurantifolia, C. grandis, C. hystrix, and C. microcarpa. Generally, the fresh
peels and leaves of the Citrus species were extracted using different methods such as
steam and water distillation, Likens-Nikerson extraction, solvent extraction, and
headspace solid-phase micro-extraction (HS-SPME). Most of the Citrus oils were
found to be rich in monoterpene hydrocarbons with limonene (1) as the major
component identified in the peels of C. aurantifolia (39.3%), C. grandis (81.6%96.9%),
and C. microcarpa(94.0%), while sabinene (19) was the major component in the peels
of C. hystrix (36.4%48.5%). In addition, citronellal (20) (61.7%72.5%), linalool (18)
(56.5%), and hedycaryol (23) (19.0%) were identified as the major components in the
oil of C. hystrix leaves, C. grandis blossom and C. microcarpa leaves, respectively.
The C. hystrix essential oil has been experimentally shown to have antimicrobial and
antifeedant activities, while no bioactivity study has been reported on the essential
oils of other Malaysian Citrus species.
1. Introduction
The Citrus species of the family Rutaceae provide several popular edible fruits in the
world. The species are widely distributed in the Indo-Malaysia region, South-East

Asia, India and China, but cultivated worldwide. The fruits and the leaves of
the Citrus species contain a variety of essential oils with various distinct flavors, and
biologically-active compounds, which are important to human nutrition and diet,
which include vitamin C, folic acid, potassium, flavonoids, coumarins, pectin, and
dietary fibers. In Malaysia, the oils from the fruits and the leaves are commercially
used as flavors and fragrances, as well as in cooking, perfumery and medical
treatments, especially in aromatherapy. Recent studies on the Malaysian Citrus plants
have reported the identification and composition of essential oils of
several Citrus species including C. aurantifolia, C. grandis, C. hystrix, and C. microcarpa.
A monoterpene hydrocarbon, limonene (1), is the major component in the essential
oils from the peels of these Malaysian Citrus species. This review focuses on the
details of extraction methods, identification and composition of essential oils from the
Malaysian Citrus species and also on their biological properties.
2.4. Citrus microcarpa (Bunge) Wijnands
Citrus microcarpa (synonym: C. madurensis) (Figure 5), common name: limau kasturi
in Malaysia, is used in the preparation of beverages. C. microcarpa is 35 m tall with
abundant of long spine on the stem, branches and twigs. The dark green leaves of C.
microcarpa are between 2.56.8 cm long and 23 cm thick. The round or oblong-shaped
green leaves of this plant are 2.53.8 cm in diameter. This plant is used to treat fever,
cough, and pharyngitis. The juice is traditionally used to prevent respiratory diseases,
strengthen the bones and act as growth stimulant for children. The juice is also
commonly used in cooking as flavoring ingredients and additives. The leaves of this
plant can be used in the treatment of skin diseases, relieve headache and also act as a
mouth wash to treat sore throat. Essential oil from C. microcarpa is used commercially in
perfumes, food, cosmetics and detergents. It is one of the ingredients in pharmaceutical,
aromatherapy and antiseptic products.

Figure 5. The (A) fruit; (B) leaf and (C) flower of C. microcarpa. (Photos kredit: Ronald
Escanlar, Forest Starr and Kim Starr, H. Zell).

The essential oil from C. microcarpa peels was reported to be rich in limonene (1)
(94.0%) similar to C. aurantifolia. -Myrcene (11) (1.8%), linalool (18) (0.4%), and terpineol (5) (0.3%) were detected as the minor components in the oil. Sesquiterpene
hydrocarbons were the most abundant in the leaves of C. microcarpa. These include
hedycaryol (23) (19.0%), -sesquiphellandrene (24) (18.3%), -eudesmol (25) (14.4%)
and -eudesmol (26) (8.6%) (Figure 2). The essential oil was extracted by
hydrodistillation for 8 h similar to that of C. grandis and C. aurantifolia oils.
3. Conclusions
Extraction and identification of the essential oils from the Malaysian Citrus species
showed that limonene (1) (96.9%) and sabinene (19) (48.5%) were the major components
in C. grandis and C. hystrix, respectively. Sample collections from different locations, and
differences in extraction methods resulted in different composition and percentage of
yields. Moreover, extraction of essential oils from different parts of Citrus plants also
gave different major components. The bioactivity studies on the C. hystrix essential oil
revealed strong antimicrobial activity against E. coli and good antifeedant properties
against S. litura. More bioactivity studies on the essential oils of the
Malaysian Citrus plants need to be carried out to acquire better bioactivity profiles of
these oils.
http://www.mdpi.com/2305-6320/3/2/13/htm

The antibacterial activity of slk syrup Tamarindus indica linn. (sambag),

Zingiber officinale rosc (luy-a), Citrus microcarpa bunge (kalamansi)

Abstract
SLK syrup is known to relieve cough and has been used by many people. It originated
in the Bukidnon Province by the Community Health Workers.
The antibacterial activity of SLK syrup (which is composed of tamarind
(sambag), ginger (luy-a) and lemon (kalamansi)) was determined by Kirby-Bauer Disk
Diffusion Method using two microorganisms namely, Escherichia coli (Gram -)
and Staphylococcus aureus (Gram +) with Gentamicin 10-mcg Sensi Disk as positive
control and distilled water as negative control. Statistical analysis was done using

Statistical Package for Social Science. Results were evaluated at the 0.05 level of
significance.
The average zone of inhibition (ZOI) if SLK syrup against S. aureus was 14.1mm,
while for the positive control was 20.6mm. The average zone of inhibition of SLK syrup
against E. coli was 14.6mm, while for the positive control was 19.66mm. There was no
zone of inhibition for the negative control. The average antibacterial activity of SLK
syrup for the E. coli was 72.4%. The average antibacterial activity for S. aureus was
68.57%. Based on the result, SLK syrup has an antibacterial activity against both test
micro organism. E. coli appeared to be the susceptible test micro organism against SLK
syrup compared to S. aureus. There is a significant difference of zone of inhibition
between SLK syrup and Gemtamicin (p<0.05). Gentamicin exhibit higher zone of
inhibition by 5.0667 mm for E. coli while 6.4556 mm for S. aureus. There is significance
difference in the Percent Antibacterial Activity of SLK syrup between E. coli and S.
aureus (p0.05). SLK syrup had a higher Percent Antibacterial Activity against E. coli
compared to S. aureus.
The antibacterial activity of the test syrup maybe determined against test micro
organism other than E. coli and S. aureus to determine if the SLK syrup is effective
against the bacteria that cause cough such as Streptococcus pneumonia, Klebsiella
pneumonia, Bordetella pertusis, and Corynebacterium diphtheria. Other parts of luy-a plant
and sambag plant if they will still possess antibacterial activity using positive control
other than Gentamicin Sensi Disk.

http://www.herdin.ph/index.php/component/herdin/?view=research&cid=55182