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



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.
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
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
1:20 1:40 1:60 1:80 1:100
O.D. at 530 nm
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.
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
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
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,
Br. J. Pharmacol. Toxicol., 2(3): 119-122, 2011
O.D. at 500 nm
1:20 1:40 1:60 1:80 1:100
01:20 1:40 1:60 1:80 1:100
O.D. at 530 nm
20 40 60 80 100 120 140 160 180 200 220 240 260 300280 320 340 360 380 400 420 440
41 53 65 77 91107
121 136
10 20 30 4
60 7
80 9
130 140
59 72
87 98 115 129
Fig. 2: Minimum inhibitory concentrations for Salmonella
Fig. 3: Minimum inhibitory concentrations for Micrococcus
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
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
Ahmad, M.M., Z. Salim-ur-Rehman, F.M. Iqbal-Anjum
and J.I. Sultan, 2006. Genetic variability to essential
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Al-Shuneigat, J., S.D. Cox and J.L. Markham, 2005.
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formulation on biofilm-forming coagulase-negative
staphylococci. Lett. Appl. Microbiol., 41: 52-55.
Aronson, J.K., 2001. Nature Publishing Group. Retrieved
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Dorman, H.J. and S.G. Deans, 2000. Antimicrobial agents
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The tangerine fruit peels of Citrus reticulata contain significant amounts of three flavanones: hesperidin, naringin, and narirutin. Citrus is a rue group species of blooming trees and shrubs. In contrast to flavonol and quercetinRutaceae, citrus peel complex compounds quantities of flavonol and quercetinRutaceae. DM is one of most chronic diseases in the world. Citrus reticulata is a kind of citrus fruit with a various degrees of Hesperidin, a glycosylated flavanone of hesperetin, suppresses gluconeogenic pathways and diminishes intestinal glucose absorption in diabetes patients, leading in anti-hyperglycemic properties. Recent research looks into the efficacy of naringin to improve cholesterol levels by reducing HMGCoA reductase. Citrus sinensis, Citrus paradisi, and their combination have antihyperlipidemic and antiatherosclerosis properties. Hyperlipidemia is a collection of disorders characterized by elevated blood levels of lipids and lipoproteins, such as cholesterol, low-density lipoprotein (LDL), and very low-density lipoprotein (VLDL), which can lead to atherosclerosis. Atherosclerosis and coronary heart disease (CHD) are the leading causes of death and sickness around the world.
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A novel alternative to synthetic preservatives is the use of natural products such as essential oil (EO) as a natural food-grade preservative. EOs are Generally Recognized as Safe (GRAS), so they could be considered an alternative way to increase the shelf-life of highly perishable food products by impeding the proliferation of food-borne pathogens. The mounting interest within the food industry and consumer preference for “natural” and “safe” products means that scientific evidence on plant-derived essential oils (EOs) needs to be examined in-depth, including the underlying mechanisms of action. Understanding the mechanism of action that individual components of EO exert on the cell is imperative to design strategies to eradicate food-borne pathogens. Results from published works showed that most EOs are more active against Gram-positive bacteria than Gram-negative bacteria due to the difference in the cell wall structure. In addition, the application of EOs at a commercial scale has been minimal, as their flavour and odour could be imparted to food. This review provides a comprehensive summary of the research carried out on EOs, emphasizing the antibacterial activity of fruit peel EOs, and the antibacterial mechanism of action of the individual components of EOs. A brief outline of recent contributions of EOs in the food matrix is highlighted. The findings from the literature have been encouraging, and further research is recommended to develop strategies for the application of EO at an industrial scale.
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Essential oils from the peels of Malta (C. sinensis), Mousami (C. sinensis), Grapefruit (C. paradisi) and Eureka lemon (C. limon) were extracted through cold pressing method. Highest oil yield (1.21%) was obtained from Malta peel followed by Eureka lemon (1.12%), Mousami (0.98%) and Grapefruit (0.73%). The extracted oils so obtained were investigated for composition by GC/FID on Carbowax 20 M packed glass column. Main constituents separated in Malta peel oil were limonene (61.08%), α-thujene (0.11%), α-pinene (0.84%), camphene (0.32%), citronellol (4.18%), citral (7.74%), capraldehyde (5.62%), caprinaldehyde (2.10%), borneol (7.63%), α-terpinolene (2.06%), linalool (1.28%) and citranelyl acetate (0.22%). In Mousami, the principal compounds were limonene (76.28%), α-pinene (1.26%), β-pinene (5.45%), α-terpinolene (1.56%), citral (1.74%), capraldehyde (0.35%), 2-hexene 1-ol (1.26%), decanol (0.35%) and linalool (2.32%). In Grapefruit peel oil, limonene (86.27%), α-thujene (0.15%), myrcene (6.28%), α-terpinene (2.11%), α-pinene (1.26%), citronellol (0.50%) and caprinaldehyde (0.31%) were among the principal components. Major constituents present in Eureka lemon oil were limonene (53.61%), α-thujene (0.45%), γ-terpinene (18.57%), camphene (0.13%), β-pinene (11.80%), sabinene (0.63%), α-terpinolene (0.25%), myrcene (11.16%), α-pinene (2.63%), citral (0.27%), citronellol (0.15%), caprinaldehyde (0.26%), borneol (0.16%), ∇ 3 -carene (0.45%) and p-cymene (0.12%). Chemical composition of essential oils of these species varied significantly, which may be due to the difference in their genetic make up.
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Use of essential oils for controlling Candida albicans growth has gained significance due to the resistance acquired by pathogens towards a number of widely-used drugs. The aim of this study was to test the antifungal activity of selected essential oils against Candida albicans in liquid and vapour phase and to determine the chemical composition and mechanism of action of most potent essential oil. Minimum Inhibitory concentration (MIC) of different essential oils in liquid phase, assayed through agar plate dilution, broth dilution & 96-well micro plate dilution method and vapour phase activity evaluated through disc volatilization method. Reduction of C. albicans cells with vapour exposure was estimated by kill time assay. Morphological alteration in treated/untreated C. albicans cells was observed by the Scanning electron microscopy (SEM)/Atomic force microscopy (AFM) and chemical analysis of the strongest antifungal agent/essential oil has been done by GC, GC-MS. Lemon grass (Cymbopogon citratus) essential oil exhibited the strongest antifungal effect followed by mentha (Mentha piperita) and eucalyptus (Eucalyptus globulus) essential oil. The MIC of lemon grass essential oil in liquid phase (288 mg/l) was significantly higher than that in the vapour phase (32.7 mg/l) and a 4 h exposure was sufficient to cause 100% loss in viability of C. albicans cells. SEM/AFM of C. albicans cells treated with lemon grass essential oil at MIC level in liquid and vapour phase showed prominent shrinkage and partial degradation, respectively, confirming higher efficacy of vapour phase. GC-MS analysis revealed that lemon grass essential oil was dominated by oxygenated monoterpenes (78.2%); α-citral or geranial (36.2%) and β-citral or neral (26.5%), monoterpene hydrocarbons (7.9%) and sesquiterpene hydrocarbons (3.8%). Lemon grass essential oil is highly effective in vapour phase against C. albicans, leading to deleterious morphological changes in cellular structures and cell surface alterations.
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The antimicrobial activity of plant extracts and phytochemicals was evaluated with antibiotic susceptible and resistant microorganisms. In addition, the possible synergistic effects when associated with antibiotics were studied. Extracts from the following plants were utilized: Achillea millifolium (yarrow), Caryophyllus aromaticus (clove), Melissa offficinalis (lemon-balm), Ocimun basilucum (basil), Psidium guajava (guava), Punica granatum (pomegranate), Rosmarinus officinalis (rosemary), Salvia officinalis (sage), Syzygyum joabolanum (jambolan) and Thymus vulgaris (thyme). The phytochemicals benzoic acid, cinnamic acid, eugenol and farnesol were also utilized. The highest antimicrobial potentials were observed for the extracts of Caryophyllus aromaticus and Syzygyum joabolanum, which inhibited 64.2 and 57.1% of the tested microorganisms, respectively, with higher activity against antibiotic-resistant bacteria (83.3%). Sage and yarrow extracts did not present any antimicrobial activity. Association of antibiotics and plant extracts showed synergistic antibacterial activity against antibiotic-resistant bacteria. The results obtained with Pseudomonas aeruginosa was particularly interesting, since it was inhibited by clove, jambolan, pomegranate and thyme extracts. This inhibition was observed with the individual extracts and when they were used in lower concentrations with ineffective antibiotics.
Citrus peel is rich in flavanone glycosides and polymethoxyflavones. In view of their importance for industrial application as well as for their pharmacological properties, their content was analyzed in the mature fruits of several Citrus paradisi (grapefruit) and Citrus sinensis (orange) varieties, with a view to select the most interesting for isolation. The results shows that the Star Ruby grapefruit and the Sanguinelli orange stand out for their high contents of naringin and hesperidin, respectively. The presence of the polymethoxyflavones nobiletin, heptamethoxyflavone and tangeretin, could be ascertained in all the grapefruit varieties analysed. Higher polymethoxyflavone levels were recorded in orange, with Valencia Late showing the greatest nobiletin, sinensetin and tangeretin contents and Navelate the highest heptamethoxyflavone levels. An in vitro study revealed that these compounds acted as antifungal agents against Penicillium digitatum, the polymethoxyflavones being more active than the flavanones in this respect. The possible participation of these phenolic compounds in the defence mechanism of Citrus against P. digitatum is discussed.
The volatile oils of black pepper [Piper nigrum L. (Piperaceae)], clove [Syzygium aromaticum (L.) Merr. & Perry (Myrtaceae)], geranium [Pelargonium graveolens L'Herit (Geraniaceae)], nutmeg [Myristica fragrans Houtt. (Myristicaceae), oregano [Origanum vulgare ssp. hirtum (Link) Letsw. (Lamiaceae)] and thyme [Thymus vulgaris L. (Lamiaceae)] were assessed for antibacterial activity against 25 different genera of bacteria. These included animal and plant pathogens, food poisoning and spoilage bacteria. The volatile oils exhibited considerable inhibitory effects against all the organisms under test while their major components demonstrated various degrees of growth inhibition.
Leaf flavonoids were quantitatively determined in 68 representative or economically important Citrus species, cultivars, and near-Citrus relatives. Contents of 23 flavonoids including 6 polymethoxylated flavones were analyzed by means of reversed phase HPLC analysis. Principal component analysis revealed that the 7 associations according to Tanaka's classification were observed, but some do overlap each other. Group VII species could be divided into two different subgroups, namely, the first-10-species class and the last-19-species class according to Tanaka's classification numbers.
Antioxidant nutrients are important for limiting damaging oxidative reactions in cells, which may predispose to the development of major clinical conditions such as heart disease and cancer. There is great interest in the possibility that the antioxidant potential of plant-derived phenolic compounds, such as flavonoids, may reduce the risk of developing these conditions. Antioxidant effectiveness in vivo depends on the bioavailability of these compounds, which was assumed to be low. However, recent studies with improved methodology indicate that some plant phenolics appear in plasma and body tissues and, thus, may be important nutritional antioxidants. However, this cannot be established with certainty until their effects on biomarkers of oxidative stress are established.
This study was designed to examine the in vitro antimicrobial and antioxidant activities of the essential oil and various extracts obtained from aerial parts of Thymus eigii. The essential oil was particularly found to possess stronger antimicrobial activity, whereas other nonpolar extracts and subfractions showed moderate activity and polar extracts remained almost inactive. GC-MS analysis of the oil resulted in the identification of 39 compounds, representing 93.7% of the oil; thymol (30.6%), carvacrol (26.1%), and p-cymene (13.0%) were the main components. The samples were also subjected to a screening for their possible antioxidant activity by using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and beta-carotene-linoleic acid assays. In the former case, the polar subfraction of the methanol extract was found to be superior to all extracts tested, only 16.8 microg/mL of which provided 50% inhibition, whereas all extracts, particularly the polar ones, seem to inhibit the oxidation of linoleic acid in the latter case. These data were further supported by total phenolics analysis, indicating that the antioxidative potential of the extracts was closely related to their phenolic constituents.
In vitro studies have demonstrated antibacterial activity of essential oils (EOs) against Listeria monocytogenes, Salmonella typhimurium, Escherichia coli O157:H7, Shigella dysenteria, Bacillus cereus and Staphylococcus aureus at levels between 0.2 and 10 microl ml(-1). Gram-negative organisms are slightly less susceptible than gram-positive bacteria. A number of EO components has been identified as effective antibacterials, e.g. carvacrol, thymol, eugenol, perillaldehyde, cinnamaldehyde and cinnamic acid, having minimum inhibitory concentrations (MICs) of 0.05-5 microl ml(-1) in vitro. A higher concentration is needed to achieve the same effect in foods. Studies with fresh meat, meat products, fish, milk, dairy products, vegetables, fruit and cooked rice have shown that the concentration needed to achieve a significant antibacterial effect is around 0.5-20 microl g(-1) in foods and about 0.1-10 microl ml(-1) in solutions for washing fruit and vegetables. EOs comprise a large number of components and it is likely that their mode of action involves several targets in the bacterial cell. The hydrophobicity of EOs enables them to partition in the lipids of the cell membrane and mitochondria, rendering them permeable and leading to leakage of cell contents. Physical conditions that improve the action of EOs are low pH, low temperature and low oxygen levels. Synergism has been observed between carvacrol and its precursor p-cymene and between cinnamaldehyde and eugenol. Synergy between EO components and mild preservation methods has also been observed. Some EO components are legally registered flavourings in the EU and the USA. Undesirable organoleptic effects can be limited by careful selection of EOs according to the type of food.