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A The study was designed to examine the in vitro
antioxidant activities of ethanolic extract of whole plant of Lactuca
runcinata. The antioxidant activity was evaluated by DPPH radical
scavenging , Nitric Oxide Scavenging and Iron chelating activity
with reference standard Rutin, Ascorbic acid and EDTA respectively
and total phenol content was estimated. An IC50 value was found that
ethanolic extract of Lactuca runcinata is more effective in DPPH
scavenging activity and nitric oxide radical scavenging activity when
compared with standard . In addition, the ethanolic extract was found
to contain a noticeable amount of total phenols, which play a major
role in controlling antioxidants. So, the in-vitro studies clearly
showed that the ethanolic extract of Lactuca runcinata has a
significant antioxidant activity. These in-vitro assays indicate that
this plant extracts is a better source of natural antioxidant, which
might be helpful in preventing the progress of various oxidative
stresses.
Keywords---DPPH assay, In vitro antioxidant, Iron chelating
activity, Lactuca runcinata, Nitric oxide radical scavenging.
I. INTRODUCTION
HE in vitro antioxidant activities study was designed to
examine the of ethanolic extract of whole plant of Lactuca
runcinata. The antioxidant activity was evaluated by DPPH
( -diphenyl-β-picrylhydrazyl) radical scavenging activity,
Nitric Oxide Scavenging Activity and Iron chelating activity
with reference standard Rutin, Ascorbic acid and EDTA
respectively and total phenol content was estimated. The
whole plant of ethanolic extract of Lactuca runcinata, which
contains large amounts of phenolic compounds, exhibits high
antioxidant and free radical scavenging activities. These in
vitro assays indicate that this plant extracts is a better source
of natural antioxidant, which might be helpful in preventing
the progress of various oxidative stresses. Lactuca runcinata
DC. Synonym L. heyneana DC. Family Compositae;
Asteraceae. Habitat Many parts of India, as a common weed.
Folk Undir-chaa-kaan (Maharashtra).
J. Amutha Iswarya Devi, M.Pharm., Annamalai University, Mobile . No:
91-9790311920, E-mail: shreeenkay@yahoo.com
A. Kottai Muthu, M.Pharm, Ph.D, Assistant Professor of Pharmacy,
Annamalai University. Mobile .No: 91-9443171712, E-mail:
arthik03@yahoo.com
Action Diuretic, slightly aperient. It is used as a diuretic in
calculous affections, also for chronic obstruction of liver and
bowels. A smaller var., found in western Uttar Pradesh,
Rajasthan, Saurashtra and the Deccan Penninsula, is equated
with L. remotiflora DC.
However, no data are available in the literature on the
antioxidant activity of whole plant of Lactuca runcinata.
Therefore we undertook the present investigation to examine
the antioxidant activities of ethanolic extract of whole plant of
Lactuca runcinata through various in vitro models.
II. MATERIAL AND METHODS
A. Collection and Identification of Plant materials
The whole plant of Lactuca runcinata (DC), were collected
form Thoothukkudi District of Tamil Nadu, India. Taxonomic
identification was made from Botanical Survey of Medical
Plants Unit Siddha, Government of India, Palayamkottai. The
whole plant of Lactuca runcinata (DC), were dried under
shade, segregated, pulverized by a mechanical grinder and
passed through a 40 mesh sieve.
B. Preparation of Extracts
The above powered materials were extracted with Ethanol
(70-800C) by hot continuous percolation method in Soxhlet
apparatus [1] 24 hrs. The extract was concentrated by using a
rotary evaporator and subjected to freeze drying in a
lyophilizer till dry powder was obtained.
III. EVALUATION OF ANTIOXIDANT ACTIVITY BY IN
VITRO TECHNIQUES
A. DPPH photometric assay
The effect of extract on DPPH radical was assayed using
the method [2]. A methanolic solution of 0.5ml of DPPH
(0.4mM) was added to 1 ml of the different concentrations of
plant extract and allowed to react at room temperature for 30
minutes. Methanol served as the blank and DPPH in methanol
without the extracts served as the positive control. After 30
min, the absorbance was measured at 518 nm and converted
into percentage radical scavenging activity as follows.
100Control A
Sample A -Control A(%)
518
518518 activityScavenging
In Vitro Anti Oxidant Activity And Total
Phenolic Content of Ethanolic Extract of
whole Plant of Lactuca runcinata (DC)
J. Amutha Iswarya Devi, and A. Kottai Muthu
J.Amutha Ishwarya devi
T
3rd International Conference on Medical, Biological and Pharmaceutical Sciences (ICMBPS'2014) March 19-20, 2014 Abu Dhabi (UAE)
112
Where A518 control is the absorbance of DPPH radical+
methanol; A518 sample is the absorbance of DPPH radical+
sample extract/ standard.
B. Nitric oxide radical scavenging activity
Nitric oxide generated from sodium nitroprusside in
aqueous solution at physiological pH interacts with oxygen to
produce nitrite ions, which were measured by the method [3].
The reaction mixture (3ml) containing 2 ml of sodium
nitroprusside (10mM), 0.5 ml of phosphate buffer saline (1M)
were incubated at 250C for 150 mins. After incubation, 0.5 ml
of the reaction mixture containing nitrite was pipetted and
mixed with 1 ml of sulphanilic acid reagent (0.33%) and
allowed to stand for 5 min for completing diazotization. Then
1 ml of naphthylethylene diamine dihydrochloride (1%
NEDA) was added, mixed and allowed to stand for 30 mins.
Sodium nitroprusside in aqueous solution at physiological pH
spontaneously generates nitric oxide, which interacts with
oxygen to produce nitrite ions which can be estimated by the
use of Griess Illosvery reaction at 540 nm.
C. Iron chelating activity
The method [4] was adopted for the assay. The principle is
based on the formation of O-Phenanthroline-Fe2+
complex and
its disruption in the presence of chelating agents. The reaction
mixture containing 1 ml of 0.05% O-Phenanthroline in
methanol, 2 ml ferric chloride (200µM) and 2 ml of various
concentrations ranging from 10 to 1000µg was incubated at
room temperature for 10 min and the absorbance of the same
was measured at 510 nm. EDTA was used as a classical metal
chelator. The experiment was performed in triplicates.
D. Total phenol
The measurement of total phenol is based on [5]. To
0.25g of sample, added 2.5 ml of ethanol and centrifuged at
2oC for 10 mins. The supernatant was preserved. Then, the
sample was re-extracted with 2.5 ml of 80% ethanol and
centrifuged. The pooled supernatant was evaporated to
dryness. Then, added 3 ml of water to the dried supernatant.
To which added 0.5 ml of Folins phenol reagent and 2 ml of
sodium carbonate (20%). The reaction mixture was kept in
boiling water bath for 1 min. the absorbance was measured at
650 nm in a spectrophotometer.
VI. RESULTS AND DISCUSSION
Free radical is a molecule with an unpaired electron and is
involved in bacterial and parasitic infections, lung damage,
inflammation, reperfusion injury, cardiovascular disorders,
atherosclerosis, aging and neoplastic diseases [6].They are
also involved in autoimmune disorders like rheumatoid
arthritis etc [7].
Antioxidant compounds may function as free radical
scavengers, initiator of the complexes of pro-oxidant metals,
reducing agents and quenchers of singlet oxygen formation
[8]. Phenolic compounds and flavonoids are major
constituents of most of the plants reported to possess
antioxidant and free radical scavenging activity [9]. Therefore,
the importance of search for natural antioxidants has increased
in the recent years so many researchers focused the same
[10].
A. DPPH scavenging activity
DPPH is a stable free radical at room temperature often
used to evaluate the antioxidant activity of several natural
compounds. The reduction capacity of DPPH radicals was
determined by the decrease in its absorbance at 518 nm, which
is induced by antioxidants.
The percentage of DPPH radical scavenging activity of
ethanolic extract of Lactuca runcinata presented in Table I.
The ethanolic extract of Lactuca runcinata exhibited a
maximum DPPH scavenging activity of 63.46% at 1000 µg/ml
whereas for Rutin(standard) was found to be 69.83% at 1000
µg/ml. The IC50 of the ethanolic extract of Lactuca runcinata
and Rutin were found to be 510µg/ml and 480µg/ml
respectively. TABLE I
EFFECT OF ETHANOLIC EXTRACT OF LACTUCA RUNCINATA (DC)
ON DPPH ASSAY
S.No
Concentration
(µg/ml)
% of activity(±SEM)*
Sample
(Ethanolic
extract)
Standard
(Rutin)
1 125 21.64±0.049 18.85 ± 0.076
2 250 30.36±0.087 22.08 ± 0.054
3 500 48.54±0.069 52.21 ± 0.022
4 1000 63.46±0.018 69.83 ± 0.014
IC50 = 510
µg/ml
IC50 = 480
µg/ml
*All values are expressed as mean ± SEM for three determinations
B. Nitric oxide radical scavenging activity
Free radical scavenging activity of the ethanolic extract of
Lactuca runcinata was determined by Nitric oxide radical
scavenging activity.
The percentage of Nitric oxide radical scavenging activity
of ethanolic extract of Lactuca runcinata presented in Table
II. The free radical scavenging potential shown maximum
activity is 57.85% at 1000µg/ml for as Standard (ascorbate)
was found to be 62% at 1000 µg/ml. The IC50 of the ethanol
extract of Lactuca runcinata and standard (ascorbate) was
found to be 425µg/ml and 410µg/ml better antioxidant is
respectively.
TABLE II
EFFECT OF ETHANOLIC EXTRACT OF LACTUCA RUNCINATA (DC) BY NITRIC
OXIDE FREE RADICAL SCAVENGING METHOD
S.No
Concentration
(µg/ml)
% of activity(±SEM)*
Sample
(Ethanolic
extract)
Standard
(ascorbate)
1 125 36.710.09 27.630.076
2 250 45.520.17 49.53 0.054
3 500 52.670.11 55.120.022
4 1000 57.850.09 62.000.014
IC50 = 425
µg/ml
IC50=410
g/ml
*All values are expressed as mean ± SEM for three determinations
3rd International Conference on Medical, Biological and Pharmaceutical Sciences (ICMBPS'2014) March 19-20, 2014 Abu Dhabi (UAE)
113
C. Iron chelating activity
Iron is essential for life because it is required for oxygen
transport, respiration and activity of many enzymes. However,
iron is an extremely reactive metal and catalyzes oxidative
changes in lipids, proteins and other cellular components [11],
[12]. It causes lipid peroxidation through the Fenton and
Haber-weiss reaction [13] and decomposes the lipid hydroxide
into peroxyl and Alkoxyl radicals that can perpetuate the chain
reactions[14].
Iron binding capacity of the ethanolic extract of Lactuca
runcinata and the metal chelator EDTA at various
concentrations (125, 250, 500, 1000 µg/ml) were examined
and the values were presented in table III. Maximum chelating
of metal ions at 1000µg/ml for plant extract and EDTA was
found to be 88.73% and 97.90% respectively. The IC50 value
of ethanolic extract of Lactuca runcinata and EDTA was
recorded as 425µg/ml and 65µg/ml respectively.
TABLE III
EFFECT OF ETHANOLIC EXTRACT OF LACTUCA RUNCINATA (DC) ON
IRON-CHELATING METHOD
S.No
Concentration
(µg/ml)
% of activity(±SEM)*
Sample
(Ethanolic
extract)
Standard
(EDTA)
1 125 20.69 ± 0.081 58.68 ± 0.007
2 250 37.63 ± 0.021 65.87 ± 0.018
3 500 57.53 ± 0.014 83.83 ± 0.012
4 1000 88.73 ± 0.014 97.90 ± 0.019
IC50 = 425 µg/ml IC50 = 65
µg/ml
*All values are expressed as mean ± SEM for three determinations
The results indicted the plant extract possess iron binding
capacity which might be due to the presence of polyphenols
that averts the cell from free radical damage by reducing of
transition metal ions [15], [16]. Various plant extracts were
proved to be good chelators [17] and correlation exists
between phenols, flavonoids and chelating activity.
D. Total phenol
Phenolic compounds are known as powerful chain breaking
antioxidants[18]. Phenols are very important plant constituents
because of their scavenging ability due to their hydroxyl
groups [19]. The total amount of phenolic content of ethanolic
extract of whole plant of Lactuca runcinata is presented in
Table IV. The ethanolic extract of whole plant of Lactuca
runcinata was found higher content of phenolic components.
TABLE IV
THE TOTAL PHENOLIC CONTENT OF ETHANOLIC EXTRACT OF WHOLE
PLANT OF LACTUCA RUNCINATA (DC)
S. No Extract Total phenol content
(mg/g of Catechol)
(±SEM)* 1 Ethanolic extract of Lactuca
runcinata
8.68 ± 0.023
*All values are expressed as mean ± SEM for three determinations
V. CONCLUSION
From the results obtained in the present study, it is
concluded that a whole plant of ethanolic extract of Lactuca
runcinata, which contains large amounts of phenolic
compounds, exhibits high antioxidant and free radical
scavenging activities. These in vitro assays indicate that this
plant extracts is a significant source of natural antioxidant,
which might be helpful in preventing the progress of various
oxidative stresses. Therefore, further investigations need to be
carried out to isolate and identify the antioxidant compounds
present in the plant extract and in vivo antioxidant activity of
this extract needs to be assessed prior to clinical use.
ACKNOWLEDGMENTS
The authors are grateful to UGC - BSR fellowship from
University Grants Commission (UGC), New Delhi, India, for
providing financial support for this investigation.
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