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Abstract

Obective:The present study deals with in vitro antioxidant activity of lemon grass essential oil of family Poaceae Methods:by using DPPH assay,Nitrogen Oxide assay , reducing power assay and β-carotene bleaching assay. Results:IC50 values observed for DPPH and NO scavenging method was 0.5 mg/ml and 2mg/ml respectively. The reducing activity gave positive results of increase in absorbance with increase in the concentration of oil,in β-carotene bleaching method also there is 84.1% bleaching in first one hour and it went to 46.8% by the completion of second hour. In all the methods BHT and Gallic acid were kept as standards. Conclusion:The results clearly indicate lemon grass essential oil is effective in scavanging free radical and has the potential to be powerful antioxidant.
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Antioxidant activity of lemon grass ESSENTIAL OIL (Cympopogon
citratus) grown in North Indian plains
Reena Lawrence*, Kapil Lawrence**,Rashmi Srivastava*** and
Deepti Gupta****
*Assistant Professor,Chemistry Department, Sam Higginbottom Institute of Agriculture, Technology
and Sciences (Formerly AAI-DU),Allahabad,India, email - reena_lawrence74@yahoo.co.in (author with
whom all correspondance to be made) **Associate Professor, Biochemistry Department, Sam
Higginbottom Institute of Agriculture, Technology and Sciences, (Formerly AAI-DU),
Allahabad,India.email-kapillawrence@yahoo.com*** Scientist, Biotech Research Institute****Assistant
Professor, Venkateshwar Instt. Of Technology,Meerut, India
ABSTRACT:
Obective:The present study deals with in vitro antioxidant activity of lemon grass essential oil of family Poaceae
Methods:by using DPPH assay,Nitrogen Oxide assay , red ucing power assay and β-carotene bleaching assay.
Results:IC50 values observed for DPPH and NO scavenging method was 0.5 mg/ml and 2mg/ml respectively. The reducing
activity gave positive results of increase in absorbance with increase in the concentration of oil,in β-carotene bleaching
method also there is 84.1% bleaching in first one hour and it went to 46.8% by the completion of second hour. In all the
methods BHT and Gallic acid were kept as standards.
Conclusion:The results clearly indicate lemon grass essential oil is effective in scavanging free radical and has the potential
to be powerful antioxidant.
Key Words: Lemon grass essential oil,Poaceae,DPPH, BHT,Gallic Acid and antioxidant activity.
INTRODUCTION
Lemon grass (Cympopogon citratus) belonging to
family Poaceae is a plant containing 1-2%
essential oil on a dry basis with wide variation of
chemical composition as a function of genetic
diversity, habitat and agronomic traetment of
culture. Lemon grass has been used in traditional
Indian medicine for a long time to treat fever,
rheumatism, headaches,cold and stomach pain.
Lemongrass oil has been reported to have various
activties like antimicrobial [1,2],
antiinflammatory, astringent [3],carminative[4],
insecticidal[5], antioxidant and antifungal [6].
It is a well known fact that free radicals and other
reactive species formed in living cells play an
important role in origin of life and biological
evolution [7]. Free radicals can also cause lipid
peroxidation in foods and lead to their
deterioration. Although there ae some synthetic
antioxidants also like BHT, BHA, but all these are
associated with some side effects [8]. A majoriy of
plant species has been investigated time to time for
their use as antioxidant and antimicrobial
agents.Since the biological activities of essential
oils usually varies depending on the place where
they are grownso our studyaims at the study of
antimicrobial and antioxidant activities of
essential oil of leaves lemongrass from North
Indian plain. For this purpose antioxidant activity
has been checked by the following methods:
DPPH assay, Reducing activity assay and Nitrogen
oxide method.
METERIALS AND METHODS
ESSENTIAL OIL-The essential oil has been
obtained from CIMAP (Central Institute of
medicinal and Aromatic Plants, CSIR,
Lucknow,U.P, India.)The specimen voucher has
been submited to the Institue’s herbarium.
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CHEMICALS: All the chemicals used in the work
were purchased from HI-MEDIA Pvt. Ltd,
Bombay. The chemicals used were of analytical
grade.
DETERMINATION OF ANTIOXIDANT
ACTIVITY
The antioxidant activity was evaluated by three
methods which are as follows;
FREE RADICAL SCAVENGING
ACTIVITY (DPPH METHOD)
The hydrogen atom or electron donating
ability of essential oil and standards -
Gallic Acid and BHT was determined
from bleaching of purple colored
methanol solution of DPPH.This
spectrophotometric assay uses the stable
radical DPPH as a reagent.The diluted
working solution of essential oil were
prepared in methanol ( 2.0,1.0,0.5,0.25
and 0.125 mg/ml).DPPH was prepared at
a concentration of 0.002%. Different
concentrations of essential oils was taken
in each test tube and volumes was made
upto 2 ml . Then 2ml of DPPH solution
was added in each test tube and these
solutions were kept in dark for thirty
minutes. The same procedure was
followed for BHT and Gallic Acid as
well.All the samples were tested in
triplicate. Later optical density was
recorded at 517nm using UV-Visible
spectrophotometer. Methanol with DPPH
was used as control.The method was
same as used by Khalaf et al [9] with
slight modification . The formula used for
calculation is
% Inhibition of DPPH activity = (A-B/A ) X 100
Where A- Optical Density of control
B-Optical density of sample
REDUCING POWER
The reducing antioxidant activity of the essential
oil has been analyzed by the method given by
Huda Fajan et al [10] with slight alterations. In this
method different concentrations of essential oil
(2.0,1.0,0.5,0.25 and 0.125 mg/ml) were taken in
different tubes and volume of all the working
solutions is made upto 1ml by adding distilled
water, in these added 2.5 ml Phosphate buffer
(0.2M,pH-6.6)and 2.5 ml of potassium
ferricyanide (1%).The mixture was incubated for
20 min. at 500C. Then 2.5 ml TCA (
Trichloroacetic acid,10%) was added to each
mixture and these were centrifuged for 10 min. at
3000 rpm. Then 2.5ml of the upper layer was
mixed with distilled water (2.5 ml) and 0.5ml
FeCl3 (0.1%).Then absorbance was measured at
700nm against a blank using UV-Visible
spectrophotometer. The same procedure was
repeated with gallic acid used as standard and
sample without the oil was used as control.
Increased absorbance of reaction mixture indicates
increase in reducing power.
NITRIC OXIDE SCAVANGING ACTIVITY
METHOD
Nitric oxide was generated from sodium
nitroprusside (SNP) and was measured by Griess
reagent. SNP in aqueous solution at physiological
pH spontaneously generates NO, which interacts
with oxygen to produce nitrite ion that can be
estimated by use of Griess Reagent, sodium
nitroprussisde (5mM) in phosphate buffer saline
(PBS) was mixed with different concentrations (
100µg/ml, 50 µg/ml,25 µg/ml and 12.5 µg/ml)
respectively and volume was made upto 3.0ml.
The solution was kept at 250C for 180 min. Then
the sample from the above were reacted with
Griess reagent ( a solution of 1% sulphanilic acid
in 2% phosphoric acid and 0.1% napthylamine in
distilled water).The absorbance of the
chromophore produced by diazotization of nitrite
ion with sulphanilic acid and subsequent coupling
with napthylamine was read a 546nm. BHT and
gallic acid were used as standards. The method
used has been taken from Ghosh et al [11]
The formula used for calculation is
Nitric Oxide scavenged (%) = (A-B/A ) X 100
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Where A- Optical Density of control.
B- Optical density of sample of
essential oil.
β-CAROTENE BLEACHING METHOD
The method followed is same as used by Geckil et
al [12] with some moderations. 0.02 mg of
crystalline β-carotene was dissolved in 10 ml of
chloroform and then added in this 20 mg linoleic
acid and 200 mg of Tween-80 reagent (Merck).
Chloroform was removed in rotary evaporator
under vacuum at 400C for 5 min and then 50 ml of
dist. Water was added with vigorous stirring to
form an emulsion. Five ml of this emulsion was
taken and added in 0.1ml of essential oil extract
(1µg/ml). Gallic acid was used as standard. The
test tube containing sample and standard were kept
in water bath in incubator at 500C and absorbance
was recorded at an interval of 20min till 2 hrs.
RESULTS AND DISCUSSION
DPPH method
DPPH is a stable free radical at room temperature
and accepts an electron or hydroxyl radical to
become a stable diamagnetic molecule. The
reduction capability of DPPH radical was
determined by the decrease in its absorbance at
517nm . In fig-1 it can be seen that there is
decrease in concentration of DPPH radical due to
scavenging ability in the essential oil and also
because of BHT and Gallic acid which were used
as standard. The IC50 values were found to be 2.0
mg/ml for essential oil .
Reducing Ability
The reducing capacity of any compound serves as
a significant indicator of its potential as
antioxidant. Reducing power of the essential oil
was found to be significant (p<0.01) and as good
as the standard. The reducing power of the garlic
oil increased with increasing concentration of
sample. All the amounts showed significant
activities when compared with control and these
differences were statistically significant.Fig.2
NO Scavenging activity
Nitric oxide radical generated from Nitroprusside
at physiological pH was found to be inhibited by
the essential oil as shown in Fig-3. Gallic acid and
BHT has been used as references.IC50 values have
been found to be 2.0mg/ml and 1.0mg/ml for the
oil and standard BHT respectively as shown in
Fig.3
β-carotene bleaching Method
The anti-bleaching activity of sample of β-carotene
was studied by monitoring the color intensity of
emulsion at 470nm for every 15 min for 2 hrs. The
concentration was taken to be 1µg/ml. The initial
concentration was considered to be 100%. In the
first 15 minutes the sample showed 93.75% effect
and 86% of the effect shown by standard. In one
hour of incubation, percentage decrease was found
to be 71.8%. The percentage decrease of standard
was 66.5% in one hour time. By the end of second
hour the percentage decrease was 59.3% and
31.0% for oil and standard respectively as shown
in Fig.4
DISCUSSION
Lemon grass oil has variety of uses and it shows
number of biological activities also. It is used as
antimicrobial agent, preservative and pesticide.
Apart from these it is used in cooking and for
preparation of tea as it releases stress and is
supposed to help in removing cough, cold and
nasal congestion Present work throws light on its
activity as antioxidant agent. Four methods have
been used to study this activity. Various other
scientists have also worked on the same activity
like Guimaraes L et al 2011[13] worked on
antioxidant and fungitoxic activity of lemon grass
oil. Its acyivity hes been done by DPPH and beta
carotene bleaching method IC50 were found to be
75.83µg/ml Another study has been done by
Amani et al 2010 [14], in this study, the
antioxidant and antibacterial effect of garlic (G),
thyme (T) and lemon grass (L) oils were
investigated in refrigerated minced beef. The
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antioxidant activity was evaluated by employing
the DPPH free radical reduction method and
oxidation tests of the system β-carotene/linoleic
acid The IC50 was found to be 75.83µg/ et al
2012.Mirghami M E et al 2012[15] studied the
total phenolic content of the essential oil analyzed
by Folin Ciocalteau method and the results
indicated that highest amount of phenolic content
was obtained from essential oil extracted from
lemongrasses stalk, with phenolic concentration of
2100.769 mg/l GAE. Anti oxidant activity was
examined by DPPH scavenging testand the highest
inhibition was obtained by essential oil extracted
from lemongrass stalk (89.5%). β-glucosidase
inhibition assay was carried out using an in-vitro
model for anti diabetic test and lemongrass stalk
essential oil and it showed highest degree of
inhibitory activity (89.63%). Anti gout test was
examined by xanthine oxidase inhibition (XOI)
assay with the maximum percentage of xanthine
oxi xanthine oxidase inhibition of 81.34%
obtained from emon grass stalk essential oil. Yet
another scientist Jumepaeng T et al 2013 [16]
determined the antioxidant capacity of and in vitro
α-amylase inhibitory activity of the essential oils
extracted from citronella grass and lemongrass.
The antioxidant activity has been determined by
DPPH radical scavenging activity IC50 values of
the antioxidant capacity of both citronella grass
and lemongrass oils were 0.46±0.012 and
4.73±0.15 μL/mL, respectively.Antoxidant activity
has also been studied in cymbopogon citrates by
Cheah PB et al 2001 [17] in linoleic acid and
chicken fat system
CONCLUSION
Herbal remedies have been used for centuries for
countless treatments for headaches to wound
healing. However only in recent years the exact
function of these compounds have been studied in
details. Garlic is a popular spice which is used in
Indian kitchen since time immemorial.There are
reports of wide range of activities of garlic like
antibacterial, antioxidant, anti-inflammatory , anti
carcinogenic etc.
The free radical scavenging activity of essential oil
and synthetic antioxidants have been evaluated
through their ability to quench the synthetic DPPH
radical, NO scavenging activity method, reducing
ability method and beta-carotene bleaching
method and it has been found that lemon grass
essential oil is a very good antioxidant agent
Fig. 1 DPPH Free radical scavenging activity of standards B.H.T, gallic acid and essential oil of
Cympopogon citratus (lemon grass)
54.1 58 61.2 68.3 76.1
20.1 23.2 27.3 31.1 36
23.4 28.5 31.6
52.04 58
0
10
20
30
40
50
60
70
80
0.125
0.25
0.5
1
2
Inhibition percentage
Concentration (mg/ml)
BHT
Gallic Acid
Lemon grass oil
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Fig.2 Reducing ability of Standards gallic acid, BHT and Garlic essential oil
Fig.3 Nitric Oxide assay showing activity of standards BHT ,Gallic acid and Lemon Grass oil
Fig.4 Relative changes in absorbance of beta carotene emulsions containing BHT and Lemon grass oil.
36.1 38.15 45.2 50 55
35.1 36
44.4 48 52
32.6 33.7 37.2 42.6
52.1
0
10
20
30
40
50
60
0.125 0.25 0.5 1 2
Inhibition Percentage
Concentration (mg/ml)
BHT
Gallic Acid
Lemon grass oil
1.47
1.87 2.01 2.23 2.5
1.72 1.86 1.92 2.1 2.2
0.147 0.19
11.3 1.6
0
0.5
1
1.5
2
2.5
3
0.125 2.5 5 10 20
Absorbancee
Concentration (mg/ml)
BHT
gallic acid
Lemon Grass oil
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... In β-carotene bleaching method also there is 84.1% bleaching in first one hour and it went to 46.8% by the completion of second hour. In all the methods BHT and Gallic acid were kept as standards [28] . The results clearly indicated that lemon grass essential oil is effective in scavenging free radical and has the potential to be powerful antioxidant. ...
... Investigators opined that this could be due to the presence of an endogenous ligand of central-type benzodiazepine receptors known as endozepine octadecaneuropeptide (ODN), which are inhibitors of food intake in small animals [30] . βglucosidase inhibition assay was carried out using an in-vitro model for anti diabetic test and lemongrass stalk essential oil and it showed highest degree of inhibitory activity (89.63%) [28] . Results of this study showed that ethanol leaf extract was active against all the test organisms at the concentration of 100 mg/ml, 50 mg/ml and 25 mg/ml. ...
... Anti gout test was examined by xanthine oxidase inhibition (XOI) assay with the maximum percentage of xanthine oxi xanthine oxidase inhibition of 81.34% obtained from lemon grass stalk essential oil [28] . ...
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