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

Abstract

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.

Full text

British Journal of Pharmacology and Toxicology 2(3): 119-122, 2011
ISSN: 2044-2467
© Maxwell Scientific Organization, 2011
Received: March 16, 2011 Received: April 30, 2011 Published: August 05, 2011
Corresponding Author: Jai S. Ghosh, Department of Microbiology, Shivaji University, Kolhapur 416004, India.
Tel: +91 9850515620 119
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.
Key words: Antimicrobial, coumarin, lemon, prophylaxis, tetrazene
INTRODUCTION
Even though pharmacological industries have
produced a number of new antibiotics in the last three
decades, resistance to these drugs by microorganisms has
increased. In general, bacteria have the genetic ability to
transmit and acquire resistance to drugs, which are
utilized as therapeutic agents (Gislene et al., 2000). For a
long period of time, plants have been a valuable source of
natural products for maintaining human health. The use of
plant extracts and phytochemicals, both with known
antimicrobial properties, can be of great significance in
therapeutic treatments (Seenivasan et al., 2006). Many
plants have been used because of their antimicrobial traits,
which are due to compounds synthesized in the secondary
metabolism of the plant. These products are known by
their active sub stances e.g. the phenolic compounds which
are part of the essential oils, as well as tannin (Tyagi and
Malik, 2010). Essential oils are more effective in
controlling biofilm cultures due to their better diffusibility
and mode of contact (Al-Shuneigat et al., 2005). Hence
the essential oils and other extracts of plants have evoked
interest as sources of natural products. They have been
screened for their potential uses as alternative remedies
for the treatment of many infectious diseases
(Tepe et al., 2004; Dorman and Deans, 2000).
Lemon is an important medicinal plant of the family
Rutaceae. It is cultivated mainly for its alkaloids, which
are having anticancer activities and the antibacterial
potential in crude extracts of different parts (viz., leaves,
stem, root and flower) of Lemon against clinically
significant bacterial strains has been reported
(Kawaii et al., 2000). Citrus flavonoids have a large
spectrum of biological activity including antibacterial,
antifungal, antidiabetic, anticancer and antiviral activities
(Burt, 2004; Ortuno et al., 2006). Flavonoids can function
as direct antioxidants and free radical scavengers, and
have the capacity to modulate enzymatic activities and
inhibit cell proliferation (Duthie and Crozier, 2000). In
plants, they appear to play a defensive role against
invading pathogens, including bacteria, fungi and viruses
(Sohn et al., 2004). Flavonoids are generally present in
glycosylated forms in plants, and the sugar moiety is an
important factor determining their bioavailability.
Preparation from peel, flowers and leaves of bitter orange
(Citrus aurantium L.) are popularly used in order to
minimize central nervous system disorders
(Pultrini et al., 2006).The peel of Citrus fruits is a rich
source of flavonoid glycosides, coumarins, $ and
(- sitosterol, glycosides and volatile oils
(Shahnah et al., 2007). Many polymethoxylated flavones
have several important bioactivities, which are very

Br. J. Pharmacol. Toxicol., 2(3): 119-122, 2011
120
0
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
1:20 1:40 1:60 1:80 1:100
O.D. at 530 nm
Dilution
Methanol
Ethanol
Acetone
rare in other plants (Ahmad et al., 2006). In addition
the fiber of citrus fruit also contains bioactive
compounds, such as polyphenols, the most important
being vitamin C (or ascorbic acid), and they certainly
prevent and cure vitamin C deficiency-the cause of scurvy
(Aronson, 2001). Antimicrobial ac tivity of the peel extract
is directly concerned with the components that they
contain. The studies showed that essential oils, protopine
and corydaline alkaloids, lactons, polyacetylene, acyclic
sesquiterpenes, hypericin and pseudohypericin
compounds are effective toward various bacteria.
Nevertheless, other active terpenes, as well as alcohols,
aldehydes, and esters, can contribute to the
overall antimicrobial effects of the essential oils
(Keles et al., 2001). The lemon peel extracts in different
solvents such as ethanol, methanol and acetone were
subjected to antibacterial assay. The extract in solvent
ethanol shows higher antimicrobial activity against tested
microorganisms in comparison with the extracts of lemon
peel in other solvents like methanol and acetone. The aim
of this study was to evaluate the potential of plant extracts
and phytochemicals on standard microorgan ism strains by
using routine antibacterial assay techniques.
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. The
compositions are as shown in Table 1 and 2, respectively.
Antimicrobial effect: Sterile molten nutrient agar at
around 40ºC was taken and seeded with different
microbial cultures and plates were prepared. After
Table 1: Composition of nutrient broth
Components (%)
Peptone 1.0
Yeast extract 1.0
Sodium chloride 0.5
Agar 2.5
Table 2: Composition of mineral based medium
Components (%)
Sodium nitrate 0.20
Dipotassium hydrogen phosphate 0.10
Potassium chloride 0.05
Glucose 1.00
Yeast extract 0.02
Fig. 1: Minimum inhibitory concentrations for Pseudomonas
aeruginosa
solidification 4 mm wells were prepared. In these wells
solvent extracts of the peel were added. The plate was
incubated overnight at 37ºC. 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 37ºC, overnight
on shaker.
Detection of phytochemicals by GCMS: Peel
supernatant obtained in different solvents was analyzed by
GCMS.
RESULTS
The minimum inhibitory concentration assay
conducted in nutrient broth using solvent extract are
reported in Fig. 1, 2 and 3 for Pseudomonas aeruginosa,
Salmonella typhimurium and Micrococcus aureus,
respectively.

Br. J. Pharmacol. Toxicol., 2(3): 119-122, 2011
121
Methanol
Ethanol
Acetone
0
0.05
0.10
0.15
0.20
0.25
0.30
0.35
O.D. at 500 nm
1:20 1:40 1:60 1:80 1:100
Dilution
0.30
0.25
0.20
0.15
0.10
0.05
01:20 1:40 1:60 1:80 1:100
O.D. at 530 nm
Dilution
Methanol
Ethanol
Acetone
20 40 60 80 100 120 140 160 180 200 220 240 260 300280 320 340 360 380 400 420 440
100
41 53 65 77 91107
121 136
149
164
178
206
NH
H
NN
H N
2
10 20 30 4
0
5
0
60 7
0
80 9
0
10
0
11
0
12
0
130 140
29
30
44
59 72
87 98 115 129
Tetrazene
Fig. 2: Minimum inhibitory concentrations for Salmonella
typhimurium
Fig. 3: Minimum inhibitory concentrations for Micrococcus
aureus
GCMS analysis of the extracts of the lemon peels: The
lemon peel extracts prepared in ethanol, methanol and
acetone when analyzed using GCMS shows the presence
of following compounds as shown in Fig. 4, 5 and 6
respectively. Figure 4, shows the presence of coumarin
and Fig. 5 shows the presence of Tetrazene. Both the
substances are good antimicrobials with broad spectrum
activity.
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.
Fig. 4: GCMS analysis of lemon peel extracts shows presence of coumarin
Fig. 5: GCMS analysis of lemon peel extracts shows presence of Tetrazene

Br. J. Pharmacol. Toxicol., 2(3): 119-122, 2011
122
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