. Evaluation of antibacterial & antioxidant activities of the leaf essential oil & leaf extracts of citrus aurantifolia

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1

L. Joji Reddy , Reshma Devi Jalli , Beena Jose

2*

& Spandana Gopu

1

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. 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 aurantifolia  were studied against human E nterobacter faecalis, Salmonella paratyphi, Staphylococcus aureus, pathogenic bacteria viz.  Bacillus cereus, Enterobacter  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 0 4 C. Inoculum was prepared by suspending a loop full of bacterial cultures into 10mL of nutrient broth 0 and was incubated at 37 C for 24hours. On the next day Muller-Hinton agar (MHA) (Merck) sterilized 0 in a flask and cooled to 45-50 C 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 50µL 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 50µL pure petroleum ether, chloroform, ethyl acetate and methanol served as 0 negative controls. The plates were incubated at 37 C 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 µL) at four concentrations (1.0, 0.5, 0.25 and 0.125 mg/mL) was mixed with freshly prepared methanolic solution of DPPH (634  µ 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 (IC 50) 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/50µL 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 (10µg each) screened under similar conditions. The activities of 10% (21-33mm/50µL inhibition zone), 5% (19-30mm/50µL inhibition zone) and 1% (16-27mm/50µL 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/50µL inhibition zone), ethyl acetate (18-24mm/50µL inhibition zone), chloroform (1522mm/50µL inhibition zone) and petroleum ether (12-19mm/50µL 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/50µL inhibition zone). Leaf chloroform extract was also found to be active against  Escherichia coli, Pseudomonas aeruginosa  and  Enterobacter faecalis  (19-22mm/50µL inhibition zone). Leaf petroleum ether extract inhibited the growth of  Escherichia coli, Enterobacter faecalis and Pseudomonas aeruginosa (17-19mm/50µL inhibition zone) where as it had little effect on Salmonella  paratyphi (12mm/50µL 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/50µL inhibition zone) and Pseudomonas aeruginosa  (26mm/50µL inhibition zone) their activities were quite comparable with that of standard antibiotics such as tobramycin and ciprofloxacin (10µg 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 (10µg). 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 IC 50. Lower IC50  value indicates higher antioxidant activity. IC 50  values of C. aurantifolia  leaf essential oil, leaf extracts and the authentic antioxidant L-ascorbic acid are given in table 5. By comparing the IC 50 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 µg/mL) was quite comparable with that of L-ascorbic acid (IC50: 57.0 µg/mL). IC50 values of Citrus aurantifolia leaf ethyl aetate extract (IC 50: 86.5µg/mL) and leaf chloroform extract (IC 50: 76.9µg/ml) were not significantly different from that of L-ascorbic acid (IC 50: 57.0 µg/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/50µL) C.aurantifolia leaf oil

C.aurantifolia leaf extracts

Microorganisms

20%

10% 5% 1%

A

B

C

D

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: 50 µL of 20%, 10%, 5% and 1% essential oil samples in DMSO & 50µL 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/50µL)

Microorganisms

Tob

Gen

Oflo

Cip

Control

10µg

10µg

10µg

10µg

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 s ulphate, 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 ���

+++Appreciable amount

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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; CHCl 3: 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 (µg/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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