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Journal of Environmental Biology, Vol. 36, 1329-1336, November 2015© Triveni Enterprises, Lucknow (India)
Introduction
From antiquity, nature has been a rich source of remedies
for relief from various ailments affecting mankind. Use of plants
for treating diseases is as old as human species. Plants produce a
wide variety of secondary metabolites such as vitamins,
terpenoids, tannins, flavonoids, alkaloids and other metabolites,
which are rich in antimicrobial and antioxidant activities (Wong et
al., 2006; Baker et al., 2010). Popular observations on the use
and efficacy of medicinal plants significantly contribute to
disclosure of their therapeutic properties, so that they are
frequently prescribed, even if their chemical constituents are not
always completely known. Essential oils are volatile aromatic oils
obtained by steam or hydro distillation of aromatic plant. Most
essential oils are primarily composed of terpenes and their
oxygenated derivatives (Ramya et al., 2013). Essential oils have
been shown to possess antibacterial, antifungal, antiviral,
insecticidal and antioxidant proprieties (Burt, 2004; Kordali et al.,
2005). Due to these properties, essential oils and essential oil
blends have become an essential addition to health and wellness,
and their versatile nature, accessibility and affordability makes
them a safe, non-toxic addition to a person's lifestyle.
Enteric or diarrhoeal infections are major public health
problems in developing countries. Enteric bacteria comprise of
Salmonella sp., Shigella sp., Proteus sp., Klebsiella sp., E. coli,
Pseudomonas sp., Vibrio cholerae and Staphylococcus aureus
which are major the etiologic agents of sporadic and epidemic
diarrhea both in children and adults (Tambekar and Dahikar,
2011). There are many pathogenic organisms known to spoil
refrigerated and ready to eat products, often leading to food
poisoning. Bacillus subtilis is a foodborne pathogen. They are
In vitro antibacterial, antioxidant activity and total phenolic content
of some essential oils
pooja.ddu@gmail.com
Upma Srivastava, Swati Ojha, N.N. Tripathi and Pooja Singh*
Department of Botany, DDU Gorakhpur University, Gorakhpur-273 009, India
*Corresponding Author E-mail:
Abstract
Key words
In vitro antibacterial activity of 16 essential oils was investigated by disc diffusion method against two Gram
positive bacteria Bacillus subtilis and Staphylococcus aureus and two Gram negative bacteria, Shigella
flexneri and Escherichia coli. Oils of Cymbopogon citratus and Ocimum basilicum showed highest
antibacterial activity. Gram positive bacteria were found to be more sensitive than Gram negative.
Antioxidant activities were tested by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay and
ABTS radical cation decolourization assay while Folin-Ciocalteu method was used to determine the total
phenolic content. In DPPH assay, highest antioxidant activity was observed in O. basilicum oil followed by
Azeratum conyzoides, A. marmelos and C. citratus, with percent inhibition and IC value ranging from
50
-1
66.11-71.93% and 14.10-17.92 µl ml respectively. In ABTS assay, similar results were obtained but with
-1
higher percent inhibition which ranged from 67.48-76.23% and lower IC value (12.12-17.21 µl ml ).
50
Moreover, radical scavenging activity of essential oils was lower than that observed for the synthetic
-1
antioxidant BHA and BHT. The total phenolic content of the essential oils as GAE in mg 100µl of EO was
found to be highest in O. basilicum (0.406) oil followed by A. conyzoides (0.322), A. marmelos (0.238) and
C. citratus (0.231). The results provide evidence that the oils of C. citratus and O. basilicum can be further
recommended for treatment of infections caused by these bacterial pathogens and are potential source of
natural antioxidants having appreciable amount of total phenolic content.
Antibacterial, Antibiotic susceptibility, Antioxidant, Phenolic content
Publication Info
Paper received:
21 May 2014
Revised received:
17 December 2014
Re-revised received:
23 January 2015
Accepted:
20 March 2015
JEB Journal Website : www.jeb.co.in
E-mail : editor@jeb.co.in
Journal of Environmental Biology
ISSN: 0254-8704 (Print)
ISSN: 2394-0379 (Online)
CODEN: JEBIDP
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Journal of Environmental Biology, November 2015
common soil inhabitants but may frequently contaminate food
and are widely distributed in hospital environments (Yassin and
Ahamd, 2012). Survival and spread of resistant bacteria is the
result of consistent and broad use of antibiotics from so many
years. Although the bulk of traditional antibiotics can still manage
drug-resistant bacteria, many commonly used antibiotics are no
longer effective (Khan and Malik, 2001). Bacteria have the
genetic ability to transmit and acquire resistance to drugs, which
are utilized as therapeutic agents. A 'threshold' hypothesis
proposed that resistance could be curtailed if total antibiotic use in
a particular environment stayed below a critical quantitative level
(Barbosa and Levy, 2000). Limited treatment options for
infections caused by such multiresistant microorganisms
prompted the search for novel plant antimicrobial compounds
with a broad spectrum of activity and new therapeutic strategies.
Besides being spoiled by pathogenic organisms, all
refrigerated and packed food also undergo autooxidation during
storage leading to formation of reactive oxygen species (Gupta
and Gupta, 2011). Oxygen is one of the most essential
components for living, it is also a double edged sword. Oxygen is
a highly reactive atom that is capable of becoming part of
potentially damaging molecules called “free radicals''. Cell
damage caused by free radicals appears to be a major contributor
to aging and diseases like cancer, heart disease, decline in brain
function, decline in immune system etc. Humans have evolved
highly complex antioxidant systems (enzymic and nonenzymic),
which work synergistically and in combination with each other to
protect the cells and organ systems of the body against free
radical damage. Commonly used synthetic antioxidants like
butylated hydroxyanisole (BHA) and butylated hydroxy toluene
(BHT) are restricted by legislative rules because they are
suspected to have some toxic effects and as possible
carcinogens (Feng et al., 2006). Due to the negative and toxic
effects of synthetic antioxidants, natural phenolic antioxidants are
being promoted as food preservatives and diet supplements
(Gharib and Teixeira da Silva, 2012). Several reports have
revealed that majority of the antioxidant activity are achieved from
biochemicals such as flavonoids, isoflavones, flavones,
anthocyanins, catechins and other phenolics (Alothman et al.,
2009; Isabelle et al., 2010).
In view of the above, the present study was carried out to
investigate the antibacterial potential and antioxidant properties
of some essential oils.
Materials and Methods
Plant materials : Aerial parts (leaves) of 16 angiospermic
aromatic plants were collected from different regions of
Gorakhpur district. Leaves were plucked and packed in polythene
bags. Plants were initially identified by morphological features
and then confirmed from the herbarium database present in the
herbarium of DDU Gorakhpur University, Gorakhpur. The
scientific names and family of the plants are detailed in Table 1 .
Microbial strains and preparation of inoculums : Two Gram
positive bacteria Bacillus subtilis (MTCC No. 3053) and
Staphylococcus aureus (MTCC No. 9542) and one two Gram
negative bacteria Shigella flexneri (MTCC No. 9543) and
Escherichia coli (MTCC No. 1698) were used for evaluation of
antibacterial assay. Stock cultures were maintained on nutrient
agar (NA) slant at 4°C and sub-cultured monthly. Working
cultures were prepared by inoculating a loopful of each test
microorganism in 10 ml of nutrient broth (NB) from NA slants.
Broths were incubated at 37°C for 18-20 hours. The suspension
6
was diluted with sterile distilled water to obtain approximately 10
-1
CFU ml using Mc Farland standard.
Extraction of essential oils : Air-dried aerial parts of plants
(200gm) were subjected to hydrodistillation for 3 hr with distilled
water (1000ml) using a Clevenger-type apparatus. Crude oil
obtained was collected and dried over anhydrous sodium sulfate
and stored in sealed glass vials at 4 ºC prior to analysis.
Determination of antibacterial activity : Antibacterial activity of
essential oils was evaluated by disc diffusion method (Andrews,
2001a) with slight modifications. 10 ml of sterilized nutrient agar
medium was poured in Petri dishes and was allowed to solidify.
The plates were seeded by spreading 0.1 ml of overnight cultures
(adjusted to 0.5 McFarland turbidity standards according to
Andrews, 2001b) and allowed to set for 20-25 min. For screening,
sterile 6mm diameter filter paper discs were impregnated with 5µl
of essential oil and placed on the surface of inoculated media agar
plates using sterile forceps and then gently pressed down onto
the agar surface. Control sets contained only sterile disc without
essential oil. Antibiotics were used as positive reference
standards to determine the sensitivity of bacteria tested (Table 3).
Zone of inhibition was recorded in mm. All the plates were
incubated at 35-37 ºC for 24-48hr. Clear inhibition zones around
the discs indicated the presence of antibacterial activity. Diameter
of inhibition zones were measured in millimeters. An inhibition
zone of 10mm or more was considered as high antibacterial
activity.
Determination of Minimum inhibitory concentration values:
Minimum inhibitory concentration value for bacterial pathogen
was determined by agar dilution technique of Andrews (2001b)
with slight modifications. A series of doubling dilution of oil
-1
concentrations (0.5, 1, 2, 4, 8, 16, 32 µl ml ) was prepared in Petri
dishes. 10 ml of sterilized and molten nutrient agar medium was
poured in each dish already containing various dilutions of oil.
0.5% (v/v) tween-80 was incorporated into agar medium to
enhance solubility of oil. Agar plates were allowed to set for 30 min
at room temperature. With the help of a sterilized inoculating
needle, a loopful of overnight bacterial culture (adjusted to 0.5
MacFarland standard) was delivered on to the agar plate. Agar
plates without oil were used as control sets. The inoculum spots
U. Srivastava et al.
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Journal of Environmental Biology, November 2015
were allowed to dry at room temperature and the plates were
incubated at 35-37 ºC for 24hr. MICs were determined as the
lowest concentration of oil inhibiting visible growth of
microorganisms on agar plate, disregarding the presence of 1 or 2
colonies.
Determination of Minimum bactericidal concentration (MBC)
values : The MBC of the oil was determined as described by
Mishra et al. (2008). The plates of MIC, which showed no visible
growth that were cultured on fresh nutrient agar plates. The
lowest concentration of antimicrobial agent from which bacteria
do not recover on fresh medium was treated as MBC.
Antioxidant activity
DPPH free radical scavenging activity: Effect of oils on DPPH
radical was estimated using method of Güllüce et al. (2003) with
slight modifications. 0.004% of DPPH (Hi Media) was prepared in
methanol and 2ml of this solution was mixed with different
-1
concentrations of oil (10, 20, 30, 40, 50 µl ml ) dissolved in
methanol. Reaction mixture was vortexed thoroughly and left for
30 min. After 30min absorbance of the mixture was measured at
517 nm by UV spectrophotometer (Hitachi) against a blank (pure
methanol). Control sample was also prepared as above without
any oil. Butylated hydroxyanisole (BHA) and Butylated
hydroxytoluene (BHT) was taken as reference standards.
Experiments were performed in triplicate and averaged. IC
50
value was determined from percent inhibition versus
concentration graph.
ABTS radical scavenging assay : For ABTS assay, the method
of Adedapo et al. (2008) was adopted. Stock solution contained 7
mM ABTS solution and 2.4 mM potassium persulfate solution.
The working solution was then prepared by mixing two stock
solutions in equal quantities and allowing them to react for 12 hr at
room temperature in dark. The solution was then diluted by mixing
1 ml ABTS solution with 60 ml methanol to obtain an absorbance
of 0.706 ± 0.001 units at 734 nm by spectrophotometer. Fresh
ABTS solution was prepared for each assay. 1 ml of different
-1
concentrations of oil (10, 20, 30, 40, 50 and 60 µg ml ) dissolved
in methanol was allowed to react with 1 ml of ABTS solution and
the absorbance was taken at 734 nm after 7 min using the
spectrophotometer. ABTS scavenging capacity of oil was
compared with that of BHT and BHA and percentage inhibition
was calculated as ABTS radical scavenging activity.
Determination of total phenolic contents : Total phenolic
content of four essential oils was determined according to the
method of Taga et al. (1984) with slight modifications. 100 µl of
each pure essential oil was dissolved in 10 ml of methanol. Now, 2
ml of this solution was made up with 0.3% HCl to 5 ml. A 100 µl
aliquot of this resulting solution was added to 2 ml of 7.5% Na CO
23
and after 2 min 100 µl of Folin Ciocalteau (Hi Media) reagent
(diluted tenfold with distilled water) was added and mixed well.
After 30 min incubation, absorbance of mixtures was recorded
spectrophotometrically at 750 nm. Total phenolic content was
calculated as gallic acid equivalent (GAE) from a calibration curve
of gallic acid standard solutions and expressed as mg of gallic
acid per 100 µl of essential oil sample.
Statistical analysis : Experimental results were expressed as
mean ± SD of three parallel measurements. Analysis of
antioxidant activity and total phenolic content were carried out
using Microsoft office excel programme. IC value of antioxidant
50
activity was calculated by Sigma plot. Antibacterial activity, MIC
1331
Antibacterial activity of essential oils
Table 1 : Antibacterial activity of different essential oils against the bacterial strains tested based on disc diffusion assay
Essential oils from aromatic plants Local name Family Zone of inhibition (mm)
B. subtilis S. aureus S. flexneri E. coli
Aegle marmelos (L.) Correa Bel Rutaceae 24±0.81 19.66±0.47 21±0.81 17.33±0.47
Azeratum conyzoides L. Ajagandha Asteraceae 9.66±1.28 - - -
Callistemon lanceolatus (Sm.) DC. Bottlebrush Myrtaceae 13.33±0.46 14±1.41 11.33±1.24 -
Chenopodium ambrosioides L. Ban bhathuwa Amranthaceae 23.66±0.94 14.33±1.69 13.33±0.47 12.66±0.47
Citrus aurantifolia (Christm.) Swingle Kaghzi nimbu Rutaceae 23.66±0.45 - 11.66±1.24 11.33±0.46
Citrus limone (L.) Burm. f. Bara nimbu Rutaceae 18.66±0.45 10±1.61 - -
Citrus sinensis (L.) Osbeck Mausami Rutaceae 14.66±0.45 10±0.81 - -
Curcuma domestica Valeton Haldi Zingiberaceae 16.67±0.94 16.66±0.45 15±0.81 14±0.81
Cymbopogon citratus (DC.) Stapf Lemmongrass Poaceae # 40.33±1.24 32.33±0.46 35.67±0.47
Eucalytus citridora Hook Eucalyptus Myrtaceae 20.33±0.46 21.66±0.45 18±0.81 21±0.81
Hyptis suaveolens (Linn.) Poit Wilayati tulsi Lamiacece 18±1.61 - - -
Murraya koenigii Spreng. Kurry pattha Rutaceae 17.33±1.24 10±0.81 10.67±0.47 12.66±0.47
Ocimum basilicum Linn. Kali tulsi Lamiaceae 30±1.61 29.33±1.38 27±0.81 31±0.81
Ocimum canum Linn. Bantulsi Lamiaceae 16.33±0.45 10±0.81 - 10.33±0.47
Ocimum gratissimum Linn. Ramtulsi Lamiaceae - 11.33±1.24 13.67±0.47 -
Ocimum sanctum Linn. Krishna tulsi Lamiaceae 20.33±0.45 - 10±0.81 11.33±0.47
# complete zone of inhibition
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Journal of Environmental Biology, November 2015
Table 2 : MIC and MBC data of effective oils, C.citratus oil and O.basilicum oil against Gram positive and Gram negative bacteria
Bacterial Strains C. citratus oil O. basilicum oil
MIC MBC MIC MBC
B. subtilis 2µl 4µl 8 µl 8 µl
S. aureus 8 µl £16 µl 16 µl £32µl
S. flexneri 8 µl £16 µl 16 µl 32 µl
E. coli 16 32 16 16
Table 3 : Effect of antibiotics against tested Gram positive and Gram negative pathogenic bacteria
Gram positive Antibiotics B. subtilis S. aureus Gram negative antibiotics E. coli S. flexneri
Azithromycin (15µg) 29±0.81 25.66±0.47 Amphicillin (30µg) - -
Chloremphenicol (30µg) 25.66±0.81 10±1.25 Cefotaxime (30µg) - 27.66±0.81
Ciproflaxacin (30µg) 26±0.81 16.66±0.47 Cefpodoxime (10 µg) - -
Clindamycin (2µg) - - Ceftriaxone (30 µg) 21±0.81 28.66±0.47
Erythromycin (15µg) 23±0.812 26.33±0.45 Ceftizoxime (30 µg) 29±0.81 -
Gatifloxin (5µg) 29±0.91 20.66±0.88 Ciprofloxacin (5µg)28±0.81 29.33±0.45
Lincomycin (15µg) 14.33±0.47 - Gatifloxacin (5µg) 23.66±0.47 15.66±0.47
Lomefloxacin (10µg) 28.66±0.47 17.33±0.61 Gentamycin (10µg) - 10±1.25
Moxifloxacin (5µg) 25.33±0.45 21.66±0.88 Levofloxacin (5µg) 22±0.61 17.33±0.61
Penicillin (10µg) - - Nalidixic acid (30 µg) 19.33±0.45 30.33±0.45
Roxithromycin (30µg) 31±0.81 28±0.61 Nitrofurantoin (30 µg) 15±1.20 14.33±0.47
Telcoplanin (30µg) 24.66±0.48 - Norfloxacin (10µg) 20.33±1.38 22.66±0.88
Tetracycline (30µg) 24.66. ±0.47 13.66±0.48 Ofloxacin (5µg) 26.66±0.45 24.66±0.47
Vancomycin (30µg) 31.33±0.88 14.66±0.81 Sparfloxacin (5µg) 19.33±0.45 23.33±0.91
- No sensitivity
1332
and MBC were obtained by calculating average of three
experiments.
Results and Discussion
Results of antibacterial disc diffusion assay are
summarized in Table 1. Zones of inhibition ranged from 10-40
mm. Oils of Cymbopogon citratus, Ocimum basilicum, Aegle
marmelos and Chenopodium ambrosioides showed potential
antibacterial activity against specific pathogens. C. citratus oil
formed the highest zone of inhibition against all the four bacterial
pathogens followed by O. basilicum oil. No other oil was found
effective against all the pathogens collectively irrespective of
being effective against single bacterial pathogen (Table 1).
C. citratus oil showed complete inhibition of B. subtilis
while 40.33, 32.33 and 35.67 mm inhibition zone was recorded
against S. aureus, S. flexneri and E. coli. C. citratus oil exhibited
strong antibacterial activity against B. subtilis, S. aureus, S.
flexneri and E. coli with MIC values of 2, 8, 8 and 16 µl
respectively. O. basilicum oil inhibited the growth of B. subtilis, S.
aureus, S. flexneri and E. coli forming zones of 30, 29.33, 27 and
31 mm respectively. MIC value for B. subtilis was found to be 8 µl
and 16 µl for rest of the three pathogens. MIC and MBC values
are given in Table 2.
Out of 16 oils tested in the present study, 2 oils exhibited
strong antibacterial action against B. subtilis, S. aureus, S.
flexneri and E. coli, while 3 oils showed moderate activity. C.
citratus oil showed highest inhibitory activity against all the four
bacterial pathogens. The present study is in confirmation with the
reports of Naik et al. (2010); Arputha et al. (2012) and Singh et al.
(2011). In the present study, Gram positive strains were found to
be more susceptible to C. citratus oil than the Gram negative
strains. Similar results were reported by Barbosa et al. (2009). O.
basilicum oil proved good in inhibiting S. aureus after C. citratus
oil as reported by Stefan et al. (2013).
The MIC results of C. citratus and O. basilicum oil against
S. aureus and E.coli are in fair correlation with the studies of Singh
et al. (2011) and Mohaddam et al. (2011) respectively. An
important characteristic of essential oils and their components is
their hydrophobicity which enables them to partition lipids of the
bacterial cell membrane, disturbing the cell structures and
rendering them more permeable. Extensive leakage from
bacterial cells or exit of critical molecules and ions will lead to
death (Prabuseenivasan et al., 2006). The mode by which
microorganisms are inhibited by essential oils and their chemical
compounds, seem to involve different mechanisms. It has been
hypothesized that inhibition involves phenolic compounds
because these compounds sensitize phospholipid bilayer of the
U. Srivastava et al.
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Journal of Environmental Biology, November 2015
Fig. 1 : Free radical-scavenging activity of 16 essential oil evaluated
by DPPH assay and comparison with that of reference BHA and BHT
BHT
BHA
Aegle marmelos
Azeratum conyzoides
Callistemon lanceolatus
Chenopodium ambrosioides
Citrus aurantifolia
Citrus limone
Citrus sinensis
Curcuma domestica
Cymbopogon citratus
Eucalytus
Hyptis suaveolens
Murraya koenigii
Ocimum basilicum
Ocimum canum
Ocimum gratissimum
Ocimum sanctum
97.99
97.61
68.81
70.47
65.07
44.07
46.98
56.52
51.55
64.39
66.11
55.05
49.68
54.88
71.93
48.44
44.9
64.65
0 50 100
% Inhibition
Fig. 2 : Free radical-scavenging activity of 16 essential oils evaluated by
the ABTS assay and comparison with that of reference BHA and BHT.
0 50 100
% Inhibition
150
99.25
98.96
70.85
72.19
66.14
45.06
48.2
57.39
52.91
65.02
67.48
59.19
51.79
54.93
76.23
49.1
45.29
65.24
microbial cytoplasmic membrane causing increased
permeability, unavailability of vital intracellular constituents and
impairment of bacterial enzymes system (El-Mougy et al., 2012).
In the present study, lemon grass and basil oils were found to be
effective against both Gram positive and Gram negative bacterial
strains but Gram positive strains were found more susceptible.
Various antibiotics showed varying degree of
susceptibility pattern in the present study (Table 3). B. subtilis was
sensitive to all antibiotics except penicillin, and with clindamycin
and lincomycin intermediate resistance was recorded. S. aureus
was found resistant to clindamycin, lincomycin, penicillin and
telcoplanin and sensitive to azithromycin, moxifloxacin,
roxithromycin, whereas with other antibiotics intermediate
resistance was recorded (Table.3). Antibiotic susceptibility
pattern of test organisms in the present study was in correlation
with the studies of Jeyakumar et al. (2011) where a difference in
the sensitivity pattern of B. subtilis was reported. B. subtilis
showed resistance against penicillin which is in correlation with
the study of Hashemi et al. (2008). However, the findings of
Adewumi et al. (2009) demonstrated that B. subtilis exhibited
resistance to erythromycin which is in contrast with the findings of
present study. S. aureus was found sensitive to chloremphenicol
and tetracycline which is also comparable to the study of
Onwubiko and Sadiq (2011). S. aureus showed resistance to
clindamycin while for same antibiotic S. aureus was found to be
sensitive in the study of INSAR group, India (INSAR, 2013).
It was observed that indiscriminate use of antibiotics
without prescriptions in the developing countries where there are
no regulatory policies in this respect, has rendered the
commonly used antibiotics completely ineffective in treatment of
S. aureus infections (Onwubiko and Sadiq, 2011). Ciprofloxacin,
nitrofurantoin, ceftizoxime exhibited highest sensitivity against
1333
Antibacterial activity of essential oils
Fig. 3 : DPPH free radical scavenging activity of essential oils at different
concentration
80
78
76
74
72
70
68
66
64
62
60
58
% Inhibition
-1
Concentration (ml ml )
10 20 30 40 50
Aegle marmelos
Ageratum conyzoides
Cymbopogon citratus
Ocimum basilicum Fig. 4 : ABTS radical cation decolourization assay of essential oils at
different concentrations
82
80
78
76
74
72
70
68
66
64
62
60
% Inhibition
-1
Concentration (ml ml )
10 20 30 40 50
Aegle marmelos
Ageratum conyzoides
Cymbopogon citratus
Ocimum basilicum
BHT
BHA
Aegle marmelos
Azeratum conyzoides
Callistemon lanceolatus
Chenopodium ambrosioides
Citrus aurantifolia
Citrus limone
Citrus sinensis
Curcuma domestica
Cymbopogon citratus
Eucalytus
Hyptis suaveolens
Murraya koenigii
Ocimum basilicum
Ocimum canum
Ocimum gratissimum
Ocimum sanctum
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Journal of Environmental Biology, November 2015
E. coli. Similar results were demonstrated by Khoshbakht et al.
(2013). E. coli showed poor degree of sensitivity towards
gentamycin which is contrary with the findings of Khalili et al.
(2012) and in correlation with the study of Khoshbakht et al.
(2013). Gram negative S. flexneri was found to be highly
sensitive to all antibiotics with highest degree of sensitivity
towards nalidixic acid and ciprofloxacin and these results are in
confirmation with the study of Mandomando et al. (2009). S.
flexneri showed resistance to amphicillin, likewise Reda et al.
(2011). Susceptibility of gentamycin was low in the present study
while other studies mentioned above showed effectiveness of
this drug, which can be attributed to its uncontrolled use.
Bacteria showed resistance to amphicillin and were found
susceptible to ciprofloxacin and nitrofurantoin. It may be
endorsed to its rare availability and expensiveness as compared
to other antibiotics.
DPPH radicals are widely used to investigate the
scavenging activity of natural compounds. A. marmelos, A.
conyzoides, C. citratus and O. basilicum essential oils notably
reduced the concentration of DPPH free radical (Fig. 1) with an
efficacy lower than that of reference BHA and BHT. Highest
antioxidant activity was observed in O. basilicum (71.93%),
A.conyzoides (70.47%) A. marmelos (68.81%) oils and C. citratus
(66.11%). IC value of these oils was found to be 14.10, 14.81,
50
-1
15.92 and 17.92 µl ml (Table 4). Higher concentration of oils
were more effective in quenching free radicals (Fig. 3).
In ABTS radical cation decolourization assay, highest
antioxidant activity was observed in O.basilicum (76.23%)
followed by A. conyzoides (72.19%) A. marmelos (70.85%) and
C. citratus (67.48%) oils, with IC value of 12.12, 14.81, 14328
50
-1
and 17.21 µl ml respectively (Table 4). These oils were found to
be good scavengers of ABTS free radicals (Fig. 2). Rise in oil
concentration was found to enhance the radical scavenging
-1
abilty. At 50 µl ml concentration, highest activity of oil was
observed (Fig. 4). Results were in correlation with the data found
in DPPH assay or it can be said that per cent inhibition was better
than those provided by radical scavenging activity. Proton radical
scavenging is an important attribute of antioxidants. ABTS, a
protonated radical, has characteristic absorbance maximum at
734 nm which decreases with scavenging of proton radicals
(Mathew and Abraham, 2006).
Many studies have demonstrated correlation between
phenolic content and antioxidant activity (Yang et al., 2002). On
the other hand, Bajpai et al. (2005) reported no correlation
between total phenolic content and antioxidant potential of
several medicinal plants. Phenolic compounds may contribute
directly to the antioxidative action (Lu et al., 2011). Phenolic
compounds are secondary metabolites of plants and also good
hydrogen donors, which makes them good antioxidants
(Dudonne et al., 2009; Sim et al., 2010) and they can act as
antioxidants by many potential pathways such as freeradical
scavenging, oxygen radical absorbance and chelation of
metal ions (Gupta and Gupta, 2011).
Total phenolic equivalent ranged from 0.231 to 0.406 mg
-1
100µl EO as GAE (Table 4). Highest total phenolic content was
-1
observed in O. basilicum (0.406mg 100µl EO as GAE) and
-1
lowest in C. citratus (0.231 mg 100µl EO as GAE). Total phenolic
equivalents among four essential oils were as follows: O.
basilicum> A. conyzoides, > A. marmelos > C. citratus (Table 4).
To a certain level a correlation can be establish between
total phenolic content (TPC) and antioxidant activity in the present
study conducted. Highest antioxidant activity with lowest IC
50
value was observed in O. basilicum oil followed by A. conyzoides,
A. marmelos and C. citratus oils. Total phenolic content showed
the same order of results with highest TPC in O. basilicum and A.
conyzoides oils except the oils of C. citratus and A. marmelos
showing contradictory values. All the four oils were found to have
good antioxidant activity as well as high total phenolic content
along with promising antibacterial activity, except for A.
conyzoides which showed poor antibacterial activity against both
bacterial strains. DPPH and ABTS assay proved that all the four
oils were found to have natural antioxidant activity, especially
source of O. basilicum and can be used as potential natural
source of antioxidants and antibacterials.
Acknowledgment
The authors are thankful to the Department of Botany,
D.D.U. Gorakhpur University for providing laboratory facilities.
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1334 U. Srivastava et al.
Table 4 : Radical scavenging activity and total phenolic contents of tested essential oils
-1 -1
Essential Oils IC value in µl ml IC value in µl ml Phenolic content
50 50
-1
(DPPH assay) (ABTS assay) (mg 100 µl EO as GAE)
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Azeratum conyzoides 14.81 14.81 0.322
Cymbopogon citratus 17.92 17.21 0.231
Ocimum basilicum 14.10 12.12 0.406
Values expressed are mean of three replicates
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Journal of Environmental Biology, November 2015
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