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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