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Asian Journal of Biochemical and Pharmaceutical Research Issue 2 (Vol.

2) 2012
ISSN: 2231-2560
CODEN (USA): AJBPAD
Research Article

Asian Journal of Biochemical and Pharmaceutical Research


Evaluation of Antibacterial & Antioxidant Activities of The Leaf Essential Oil & Leaf
Extracts of Citrus Aurantifolia
L. Joji Reddy1, Reshma Devi Jalli1, Beena Jose2* & Spandana Gopu1
1. Department of Biotechnology, Loyola Academy Degree & P.G. College, Alwal, Secunderabad, Andhra Pradesh, India
2*. Department of Chemistry, Vimala College, Thrissur, Kerala, 680009, India

Received: 24 April 2012; Revised: 04 May 2012; Accepted: 28 May. 2012

Abstract: The antibacterial and DPPH radical scavenging activities of the leaf extracts and leaf essential oil of
Citrus aurantifolia were investigated. The antibacterial potential of the leaf essential oil and petroleum ether,
chloroform, ethyl acetate and methanol extracts of the leaves of Citrus aurantifolia were studied against human
pathogenic bacteria viz. Bacillus cereus, Enterobacter faecalis, Salmonella paratyphi, Staphylococcus aureus,
Escherichia coli, Proteus vulgaris, Klebsiella pneumoniae, Pseudomonas aeruginosa and Serratia marcescens
by agar well diffusion method. Leaf essential oil as well as ethyl acetate, chloroform and methanol extracts of
Citrus aurantifolia leaves exhibited pronounced activity against Gram-positive and Gram-negative bacteria and
their activity is quite comparable with the standard antibiotics such as tobramycin, gentamicin sulphate,
ofloxacin and ciprofloxacin screened under similar conditions. Among the leaf extracts and leaf essential oil of
C. aurantifolia studied, leaf methanol and ethyl acetate extracts showed potent scavenging activity on 1, 1diphenyl-2-picrylhydrazyl (DPPH) radical. The remarkable antibacterial and antioxidant activity exhibited by
the plant extracts can be attributed to the synergic effect of the active compounds present in it. The results
obtained showed that the leaf methanol and ethyl acetate extracts of C. aurantifolia can be considered as good
sources of natural antioxidants and antimicrobial compounds and can be incorporated into the drug
formulations.
Key words: Citrus aurantifolia, antibacterial activity, agar well diffusion method, antioxidant activity, DPPH
radical scavenging activity, drug formulations

INTRODUCTION:
Citrus aurantifolia (family: Rutaceae) fruit juice contains lots of water and vitamin C. Leaves,
fruit, and flower oil contain limonene and linalool. In addition to being a refreshing drink, a fruit has
been consumed for thousands of years ago to cure various diseases. Roots, leaves and flowers of lime
are often used as a drug. The leaf essential oil of lemon can inhibit the growth of Staphylococcus
aureus. Lime is also used to overcome dysentery, constipation, diphtheria, acne, dizziness, cough,
body odor, increase appetite, prevent hair loss, dandruff, flu, fever, too fat, tonsils, and inflammation
of the nose. The activity of compounds from Mexican Lime (Citrus aurantifolia) juice to induce
apoptosis in human pancreatic cells was studied [1].
Based on review of literature no reports are available regarding antibacterial and DPPH radical
scavenging activities of Citrus aurantifolia leaf essential oil and leaf extracts. In this work, the
antibacterial property of the Citrus aurantifolia leaf oil and leaf extracts were checked against nine
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pathogenic bacteria namely Bacillus cereus, Enterobacter faecalis, Salmonella paratyphi,


Staphylococcus aureus, Escherichia coli, Proteus vulgaris, Klebsiella pneumoniae, Pseudomonas
aeruginosa and Serratia marcescens. The antioxidant activity of the leaf essential oil and extracts were
studied by DPPH radical scavenging assay. The results showed that the leaf essential oil and
methanolic and ethyl acetate extracts of the leaves of Citrus aurantifolia is a good source of active
compounds and antioxidants.
MATERIALS AND METHODS:
Plant Material: The leaves of Citrus aurantifolia were collected from Thrissur district of Kerala,
South India and authenticated by Dr. Kochuthressia M.V., HOD, Department of Botany, Vimala
College, Thrissur. Voucher specimen is deposited in the specially maintained herbarium, Department
of Botany, Vimala College, Thrissur.
Essential Oil Extraction: The fresh leaves (250g) of Citrus aurantifolia were cut into small pieces
and ground to a paste using an electric mixer grinder and subjected to steam distillation for three
hours. About 2 liters of the distillate were collected and extracted with diethyl ether (3X100 mL) and
dried using anhydrous sodium sulphate. The dry ether extract on evaporation yielded 0.50g (0.20% of
fresh weight of the sample) of pale yellow oil.
Preparation of Plant Extracts: Fifty grams of the powered plant material were extracted successively
with 150mL of petroleum ether, chloroform, ethyl acetate and methanol as solvents for 24hours by
Soxhlet equipment [2].
Test Microorganisms: The microorganisms used for antibacterial activity evaluation were obtained
from Microbial Type Culture Collection and gene bank (IMTECH, Chandigarh, India). They were
Gram-positive bacteria such as Bacillus cereus (MTCC-1305), Staphylococcus aureus (MTCC-96)
and Enterobacter faecalis (MTCC-5112) and Gram-negative bacteria such as Salmonella paratyphi
(MTCC-735), Escherichia coli (MTCC-729), Klebsiella pneumoniae (MTCC-109), Pseudomonas
aeruginosa (MTCC-647), Proteus vulgaris (MTCC-426) and Serratia marcescens (MTCC-86).
Culture medium and inoculums
The stock cultures of microorganisms used in this study were maintained on Plate Count Agar slants at
40C. Inoculum was prepared by suspending a loop full of bacterial cultures into 10mL of nutrient broth
and was incubated at 370C for 24hours. On the next day Muller-Hinton agar (MHA) (Merck) sterilized
in a flask and cooled to 45-500C was distributed by pipette (20mL) into each sterile Petri dish and
swirled to distribute the medium homogeneously. About 0.1mL of bacterial suspension was taken and
poured into Petri plates containing 20mL nutrient agar medium. Using the L-shaped sterile glass
spreader bacterial suspensions were spread to get a uniform lawn culture.
Antibacterial Activity Assay: The agar diffusion method is used for the antimicrobial evaluations.
Wells of 8mm (0.8cm) diameter were dug on the inoculated nutrient agar medium with sterile cork
borer and 50L of Citrus aurantifolia leaf essential oil (at various concentrations) were added in each
well. The essential oil of required concentrations 20%, 10%, 5% and 1% were prepared by dissolving
the oil into appropriate quantities of DMSO, which did not influence the growth of bacteria was used
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as a negative control. Antibacterial activities of the leaf extracts in various solvents were studied and
wells introduced with 50L pure petroleum ether, chloroform, ethyl acetate and methanol served as
negative controls. The plates were incubated at 370C over night and examined for the zone of
inhibition. The diameter of the inhibition zone was measured in mm. The standard antibiotic drugs
such as tobramycin, gentamicin sulphate, ofloxacin and ciprofloxacin were also screened under similar
conditions for comparison. An extract was classified as active when the diameter of the inhibition was
equal to or larger than 8mm [3]. All the assays were performed in triplicate and expressed as average
values.
Preliminary Phytochemical Analysis : The sample extracts were analysed for the presence of various
phytoconstituents like of flavonoids, alkaloids, glycosides, steroids, phenols, saponins and tannins
according to standard methods [4].
DPPH free radical scavenging assay : The DPPH free radical is a stable free radical, which has been
widely accepted as a tool for estimating free radical-scavenging activities of antioxidants [5].
Hydrogen or electron donation abilities of the compounds were measured from the bleaching of the
purple-colored ethanol solution of 1, 1-diphenyl-2-picrylhydrazyl (DPPH). This spectrophotometer
assay uses the stable radical DPPH as a reagent. The sample solution of material (50 L) at four
concentrations (1.0, 0.5, 0.25 and 0.125 mg/mL) was mixed with freshly prepared methanolic solution
of DPPH (634 M) and allowed to stand for 30 min at room temperature. The absorbance was then
measured at 515nm using a spectrophotometer and the inhibition of free radical DPPH in percent (%)
was calculated using the formula below:
The percent of inhibition of DPPH reduction (decolourization)
% of inhibition =

A0

A sample
A0

X 100

where (A0) is the absorbance of the control (blank) and (A sample) is the absorbance of the test
compound. The compound concentration demonstrating 50% inhibition (IC50) was calculated from the
plot of inhibition percentage against sample concentration. Tests were carried out in triplicate.
Samples and DPPH were dissolved in methanol. L-ascorbic acid was used as positive control.
RESULTS AND DISCUSSIONS:
Antibacterial screening of essential oil: The antibacterial spectra of the leaf extracts and leaf
essential oil of C. aurantifolia, showing the zone of inhibition in millimeters, for Gram positive and
Gram negative bacteria are summarized in table 1. In addition, the inhibition zones formed by standard
antibiotics and those of negative controls are listed in table 2. The leaf essential oil and leaf extracts of
C. aurantifolia showed pronounced antibacterial activity against all the microorganisms tested. Leaf
essential oil showed more activity than leaf extracts and the activity was found to be concentration
dependent.
C. aurantifolia leaf oil at various concentrations was evaluated for antimicrobial activity and
the leaf oil (20%) showed pronounced activity against Bacillus cereus, Enterobacter faecalis,
Salmonella paratyphi, Escherichia coli, Proteus vulgaris, Pseudomonas aeruginosa, Serratia
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marcescens, Staphylococcus aureus and Klebsiella pneumoniae (23-36mm/50L inhibition zone). The
inhibitory effect of 20% leaf oil of C. aurantifolia on Pseudomonas aeruginosa was comparatively
less than that of standard antibiotics whereas the activity of the leaf oil (20%) against all other tested
bacteria was found to be comparable with the standard antibiotics such as tobramycin, gentamicin
sulphate, ofloxacin and ciprofloxacin (10g each) screened under similar conditions. The activities of
10% (21-33mm/50L inhibition zone), 5% (19-30mm/50L inhibition zone) and 1% (16-27mm/50L
inhibition zone) of the leaf oil samples were also studied against various pathogenic bacteria and were
found to be active on all microorganisms tested and its activity is higher than that of the leaf extracts.
The MIC (minimum inhibitory concentration) of the leaf essential oil was found to be 0.25%.
As the leaf oil exhibited pronounced antibacterial activity comparable with standard antibiotics,
it can be used as an external antiseptic in prevention and treatment of bacterial infections. The
remarkable antibacterial activity exhibited by the C.aurantifolia leaf oil can be attributed to the
synergic effect of the antimicrobial agents present in the oil [1].
Antibacterial screening of leaf extracts: Among the leaf extracts, methanol extract exhibited higher
activity than the other extracts and petroleum ether extract showed least activity. Methanol (2026mm/50L inhibition zone), ethyl acetate (18-24mm/50L inhibition zone), chloroform (1522mm/50L inhibition zone) and petroleum ether (12-19mm/50L inhibition zone) extracts of the leaf
exhibited marked activity against all the tested organisms. The leaf methanol and ethyl acetate extracts
exhibited significant activity against Escherichia coli, Pseudomonas aeruginosa and Enterobacter
faecalis (22-26mm/50L inhibition zone). Leaf chloroform extract was also found to be active against
Escherichia coli, Pseudomonas aeruginosa and Enterobacter faecalis (19-22mm/50L inhibition
zone). Leaf petroleum ether extract inhibited the growth of Escherichia coli, Enterobacter faecalis and
Pseudomonas aeruginosa (17-19mm/50L inhibition zone) where as it had little effect on Salmonella
paratyphi (12mm/50L inhibition zone)
The results obtained were compared with standard antibiotics and it was observed that C.
aurantifolia leaf methanol extract at a concentration of 1mg/mL showed marked activity against
Enterobacter faecalis (25mm/50L inhibition zone) and Pseudomonas aeruginosa (26mm/50L
inhibition zone) their activities were quite comparable with that of standard antibiotics such as
tobramycin and ciprofloxacin (10g each). Leaf ethyl acetate extract at a concentration of 1mg/mL
exhibited remarkable activity against Pseudomonas aeruginosa and was comparable with that of
gentamicin suphate (10g). The Minimum Inhibitory Concentration (MIC) of methanol and ethyl
acetate extracts of leaf was found to be 0.5mg/mL.
The antimicrobial potency of the C. aurantifolia leaf extracts can be attributed to the presence
antimicrobial agents. Plant extracts are potential sources of novel antimicrobial compounds especially
against bacterial pathogens. Plant extracts inhibited bacterial growth but their effectiveness varied [6].
Phytochemical Analysis: Phytochemical evaluation was performed with methanol, ethyl acetate,
chloroform and petroleum ether extracts of C. aurantifolia leaves. The leaf methanol extract of Citrus
aurantifolia was rich in alkaloids, phenolics, saponins, tannins, steroids and flavonoids. Antibacterial
and antioxidant potential of leaf extracts can be attributed to the presence of these phytochemicals
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(Table 3). Saponins and flavonoids are present in appreciable amount in ethyl acetate extract. Ethyl
acetate, chloroform and petroleum ether extracts gave positive test for steroids and alkaloids.
Evaluation of DPPH Radical Scavenging Activity: The antioxidant activity of essential from leaves
of C. aurantifolia and leaf extracts in solvents of varying polarity were measured in terms of hydrogen
donating or radical scavenging ability, using the stable radical, DPPH. In the present study, the results
of the free radical scavenging activity of leaf essential oil and leaf extracts of C. aurantifolia assessed
by DPPH assay was summarized in (Table 4). The DPPH free radical scavenging activity of the leaf
essential oil and leaf extracts of C. aurantifolia are sorted in descending order: leaf methanol extract >
leaf ethyl acetate extract >leaf chloroform extract > leaf oil > leaf petroleum ether extract.
Out of the five samples tested, C. aurantifolia leaf methanol extract showed the highest
scavenging activity (% inhibition 93.55, 90.04, 82.33 and 79.52 at 1.0, 0.5, 0.25 and 0.125mg/mL
respectively), followed by C. aurantifolia leaf ethyl acetate extract. Leaf petroleum ether extract
exhibited least DPPH radical scavenging ability with % inhibition 53.29, 36.19, 27.91 and 20.19 at
1.0, 0.5, 0.25 and 0.125mg/mL respectively.
C. aurantifolia leaf methanol extract possess potent free radical-scavenging activity. The
amount of the sample needed for 50% inhibition of free radical activity is expressed by IC50. Lower
IC50 value indicates higher antioxidant activity. IC50 values of C. aurantifolia leaf essential oil, leaf
extracts and the authentic antioxidant L-ascorbic acid are given in table 5. By comparing the IC50
value of the leaf extracts and leaf essential oil of C. aurantifolia with that of the authentic antioxidant
L-ascorbic acid, it was found that the antioxidant activity of C. aurantifolia leaf methanol extract
(IC50:76.9 g/mL) was quite comparable with that of L-ascorbic acid (IC50: 57.0 g/mL). IC50 values
of Citrus aurantifolia leaf ethyl aetate extract (IC50: 86.5g/mL) and leaf chloroform extract (IC50:
76.9g/ml) were not significantly different from that of L-ascorbic acid (IC50: 57.0 g/mL).
CONCLUSIONS:
The essential oil from the leaf of Citrus aurantifolia showed varying degrees of antibacterial
activity on the microorganisms tested. The variation of the susceptibility of microorganisms towards
the essential oils could be attributed to their intrinsic properties that are related to the permeability of
their cell surface to the essential oils. Due to the emergence of the antibiotic resistant pathogens, plants
are being looked upon as an excellent alternate to combat the spread of multi drug resistant
microorganisms.
From the above experiment it can be inferred that Citrus aurantifolia leaf essential oil as well
as leaf methanol extract showed significant activity against Gram-positive and Gram-negative bacteria.
The activity of leaf oil and leaf methanol extract was found to be quite comparable with the standard
antibiotics screened under similar conditions. So they can be used as an external antiseptic in the
prevention and treatment of bacterial infections caused by various pathogenic bacteria such as Bacillus
cereus, Enterobacter faecalis, Salmonella paratyphi, Staphylococcus aureus, Escherichia coli, Proteus
vulgaris, Klebsiella pneumoniae, Pseudomonas aeruginosa and Serratia marcescens, which have
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developed resistance to antibiotics. The incorporation of these samples into the drug formulations is
also recommended.
Phytochemical studies revealed the presence of various secondary metabolites in the leaf
extracts of C. aurantifolia. The various phytochemical compounds detected are known to have
beneficial importance in medicinal sciences [7, 8]. Among the leaf essential oil and leaf extracts of C.
aurantifolia studied, the leaf methanol extract and leaf ethyl acetate extract showed potent scavenging
activity on DPPH free radical. Antioxidant activity of the leaf methanol extract can be attributed to the
presence of phytochemicals such as alkaloids, phenolics, saponins, tannins, steroids and flavonoids.
Phenolic compounds and saponins are reported to have antioxidant activities [9, 10]. The results
obtained showed that the leaf methanol extract and leaf ethyl acetate extract of Citrus aurantifolia can
be considered as good sources of natural antioxidants.

Table 1: Inhibition zones formed by Citrus aurantifolia leaf essential oil and leaf
extracts
Diameter of inhibition zones( mm/50L)
C.aurantifolia leaf oil

C.aurantifolia leaf extracts

Microorganisms

20%

10% 5% 1%

1. Bacillus cereus

27

25

22

19

20

19

16

14

2. Enterobacter faecalis

36

33

30

27

25

22

19

17

3. Salmonella paratyphi

28

26

21

18

21

18

15

12

4. Staphylococcus aureus

29

26

23

19

20

19

17

15

5. Escherichia coli

33

30

27

22

26

24

22

19

6. Proteus vulgaris

30

26

22

19

21

19

17

15

7. Klebsiella pneumoniae

33

29

27

21

21

19

16

14

8. Pseudomonas aeruginosa

23

21

19

16

26

24

20

19

9. Serratia marcescens

30

26

24

20

21

19

17

15

A: methanol; B: ethyl acetate; C: chloroform; D: petroleum ether


Used concentrations: 50L of 20%, 10%, 5% and 1% essential oil samples in
DMSO & 50L of 10mg/mL of plant extracts

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Table 2: Inhibition zones formed by the standard antibiotics and negative controls
Diameter of inhibition zones (mm/50L)

Microorganisms

Tob

Gen

Oflo

Cip

Control

10g

10g

10g

10g

A, B, C, D

1. Bacillus cereus

28

32

34

30

--

2. Enterobacter faecalis

26

32

32

26

--

3. Salmonella paratyphi

25

30

28

30

--

4. Staphylococcus aureus

26

28

24

24

--

5. Escherichia coli

30

36

32

34

--

6. Proteus vulgaris

26

30

24

32

--

7. Klebsiella pneumoniae

26

32

32

36

--

8. Pseudomonas aeruginosa

26

24

32

28

--

9.Serratia marcescens

24

32

30

30

--

Controls- A: methanol; B: ethyl acetate; C: chloroform; D: petroleum ether


Tob: tobramycin, Gen: gentamicin sulphate, Oflo: ofloxacin, Cip:ciprofloxacin

Table 3: Phytochemical screening of Citrus aurantifolia leaf extracts


Phytoconstituents

Methanol
extract

Ethyl
acetate
extract

Chloroform
extract

Petroleum
ether extract

Alkaloids

+++

++

Phenolics

+++

++

Saponins

+++

+++

Tannins

+++

++

Steroids

+++

++

Flavonoids

+++

+++

Glycosides

+ Present

++Moderately present
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Table 4: The DPPH free radical scavenging activity of the leaf essential oil and
leaf extracts of Citrus aurantifolia.
Concentration(mg/mL)
Samples

1.0

0.5

0.25

0.125

Radical scavenging effect (%)


1. Citrus aurantifolia leaf oil

62.55

52.45

45.02

38.43

2. C.aurantifolia leaf MeOH extract 93.55

90.04

82.33

79.52

3. C.aurantifolia leaf EA extract

89.90

85.97

78.40

75.88

4. C.aurantifolia leaf CHCl3 extract 81.91

76.86

73.49

60.73

5. C.urantifolia leaf PE extract

53.29

36.19

27.91

20.19

98.87

94.47

L-ascorbic acid

93.26

90.88

MeOH: methanol; EA: ethyl acetate; CHCl3: chloroform; PE; petroleum ether

Table 5: Antioxidant activities of the Citrus aurantifolia leaf oil, leaf extracts and
positive control using the (DPPH) free radical-scavenging assay
Antioxidant activity
Samples

IC50/DPPH (g/mL)

1. Citrus aurantifolia leaf oil

428

2. C.aurantifolia leaf methanol extract

76.9

3. C.aurantifolia leaf ethyl acetate extract

86.5

4. C.aurantifolia leaf chloroform extract


5. C.aurantifolia leaf pet.ether extract

96.2

942

L- ascorbic acid

57.0

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*Correspondence Author: Beena Jose, Department of Chemistry, Vimala College, Thrissur, Kerala,
680009, INDIA.

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