EVALUATION OF THE DPPH FREE RADICAL SCAVENGING ACTIVITY OF WRIGHTIA TINCTORIA R Br LEAF, BARK AND SEED EXTRACTS

www.wjpr.net Vol 3, Issue 3, 2014. 5041

EVALUATION OF THE DPPH FREE RADICAL SCAVENGING

ACTIVITY OF WRIGHTIA TINCTORIA R. Br. LEAF, BARK AND

SEED EXTRACTS

Beena Jose1*, Jesy E.J.1, Ritty J. Nedumpara2

1

Department of Chemistry, Vimala College, Thrissur, Kerala, 680009, India. 2

Department of Physics, Vimala College, Thrissur, Kerala, 680009, India.

ABSTRACT

The DPPH free radical scavenging activity of the leaf, bark and seed

extracts of Wrightia tinctoria (Roxb.) R. Br. (Apocynaceae) was

evaluated. Among these, DPPH radical could be scavenged most

effectively by Wrightia tinctoria bark ethyl acetate extract with IC50 value 67.30±2.4µg/ml and was quite comparable with the standard

antioxidant L-ascorbic acid (IC50 value 62.5±2.5µg/ml). The antioxidant activity of W. tinctoria leaf methanol extract

(IC50:72.9±3.5µg/ml) was not significantly different from that of L- ascorbic acid (IC50: 62.5 ±2.5µg/ml). The remarkable 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 bark ethyl

acetate and leaf methanol extracts of Wrightia tinctoria can be considered as good sources of

natural antioxidants and can be incorporated into the drug formulations.

Key words: Wrightia tinctoria, Apocynaceae, DPPH radical scavenging activity, natural antioxidants, drug formulations.

INTRODUCTION

In the recent years, the antioxidant actions have received much attention. It is well known

that reactive oxygen species (ROS) formed in vivo, such as superoxide anion, hydroxyl

radical and hydrogen peroxide, are highly reactive and potentially damaging transient

chemical species [1]. ROS, causing damage to DNA, proteins and lipids, have been associated with carcinogenesis, coronary heart disease, and many other health problems [2]. Free radicals are known to play a definite role in a wide variety of pathological manifestations of pain,

Volume 3, Issue 3, 5041-5048. Research Article ISSN 2277 – 7105

Article Received on 12 March 2014,

Revised on 05 April 2014, Accepted on 28 April 2014

*Correspondence for Author

Dr. Beena Jose

www.wjpr.net Vol 3, Issue 3, 2014. 5042 inflammation, cancer, diabetes, alzheimer; hepatic damage etc. antioxidants fight free radicals

and protect us from various diseases. They exert their action either by scavenging the reactive

oxygen species or protecting the antioxidant defense mechanisms [3]. Minimizing oxidative damage may well be one of the most important approaches to the primary prevention of these

oxidative stress-related diseases and health problems, since antioxidants terminate direct ROS

attacks and radical-mediated oxidative reactions [4]. Plants are the primary sources of naturally occurring antioxidants for humans [5].

W. tinctoria R.Br. Belongs to family Apocynaceae [6], is a small deciduous tree, generally up to 1.8 m tall and often under 60 cm girth, sometimes up to 7.5 m high, distributed all over

India. It is commonly known as “indrajav”, has been important in the traditional healing and

widely recognized medicinal plant [7]. The wrightial a new terpene and other phytoconstituents such as cycloartenone, cycloeucalenol were isolated identified by

fractionation of methanol extract of the immature seed pods [8]. The ursolic acid and isoricinolic acid has been also isolated from the seed pods [9]. The characterization of lingo-cellulosic seed fiber from W. tinctoria has been carried out [10].Almost every part of plant is useful - leaves pungent chewed for relief from tooth ache, bark and seeds are antidysenteric,

antidiarrhoel and antihaemorrhagic [11]. Oil emulsion of leaves and pods is used to treat psoriasis [12, 13]. The plant is also reported for its antimicrobial, wound healing and hepatoprotective activity [14, 15, 16, 17].

Earlier chemical investigations on Wrightia tinctoria reported alkaloids [18], triterpenoids [19], steroids, flavonol [20] etc. as major chemical constituents in the leaves of this plant. However, the antioxidant activity of the seed, bark and leaf extracts and leaf coconut oil extract of this

plant were not revealed completely. DPPH radical scavenging activity of the leaf methanol

extract was reported [21]. In the present study, the antioxidant activity of the leaf, bark and seed extracts of Wrightia tinctoria were evaluated by DPPH radical scavenging assay. The

results showed that the methanolic and ethyl acetate extracts of the leaf and bark of Wrightia

tinctoria is a good source of active compounds and antioxidants.

MATERIALS AND METHODS Plant Material

The leaf, bark and seeds of Wrightia tinctoria were collected from Thrissur district of Kerala,

South India and authenticated by Dr. Kochuthressia M.V., HOD, Department of Botany,

www.wjpr.net Vol 3, Issue 3, 2014. 5043 herbarium, Department of Botany, Vimala College, Thrissur.

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. Leaf coconut oil extracts were prepared by soaking the fresh leaves of Wrightia

tinctoria (500g) in 1Kg coconut oil and kept for 1, 4, 7 and 15 days in the sunlight.

Preliminary Phytochemical analysis

The sample extracts were analysed for the presence of various phytoconstituents like

flavonoids, alkaloids, glycosides, steroids, phenols, saponins and tannins according to

standard methods [22].

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 [23]. Hydrogen or electron donation abilities of the compounds were measured from the bleaching of the purple-colored

methanol 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) were mixed with freshly prepared

methanolic solution of DPPH (634 µM) 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)

Asample

A₀

A₀ X 100

% of inhibition =

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 (IC50) 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

www.wjpr.net Vol 3, Issue 3, 2014. 5044 RESULTS

Antioxidant activity

The antioxidant activity of Wrightia tinctoria leaf, bark and seed extracts in solvents of

varying polarity were measured in terms of hydrogen donating or radical scavenging ability,

using the stable radical, DPPH. The method is based on the reduction of alcoholic DPPH·

solutions in the presence of a hydrogen donating antioxidant. DPPH· solutions show a strong

absorption band at 515 nm appearing as a deep violet color. The absorption vanishes and the

resulting decolourization is stoichiometric with respect to degree of reduction. The remaining

DPPH·, measured after a certain time, corresponds inversely to the radical scavenging

activity of the antioxidant. The results of the free radical scavenging activity of the leaf

extracts of W.tinctoria assessed by DPPH assay were summarized in table 1. The DPPH

radical scavenging activity of the bark and seed extracts is given in table 2.

Table 1: The DPPH radical scavenging activity of the leaf extracts of Wrightia tinctoria

Wrightia tinctoria leaf extracts and positive

control

Concentration (mg/ml)

1.0 0.5 0.25 0.125

Radical scavenging effect (%) W. tinctoria leaf methanol extract

W. tinctoria leaf ethyl acetate extract W. tinctoria leaf chloroform extract W. tinctoria leaf petroleum ether extract W. tinctoria leaf coconut oil extract (15d) W. tinctoria leaf coconut oil extract (7d) W. tinctoria leaf coconut oil extract (4d) W. tinctoria leaf coconut oil extract (1d) L-ascorbic acid 95.16 93.55 90.32 85.48 19.55 26.32 31.58 33.08 98.39 93.55 82.26 70.96 64.52 18.79 22.56 27.82 28.57 96.77 88.71 77.42 58.06 41.94 14.29 17.29 26.32 26.32 95.16 83.87 70.97 25.80 16.12 12.78 13.53 14.29 17.29 93.55

Table 2: The DPPH radical scavenging activity of the seed and bark extracts of Wrightia

tinctoria

Wrightia tinctoria

bark & seed extracts, positive control

Seed extracts Bark extracts

Concentration (mg/ml) Concentration (mg/ml) 1.0 0.5 0.25 0.125 1.0 0.5 0.25 0.125

Radical scavenging effect (%) Methanol extract

EA extract

www.wjpr.net Vol 3, Issue 3, 2014. 5045 The amount of the extract needed for 50% inhibition of free radical activity is expressed by

IC50. Lower IC50 value indicates higher antioxidant activity. IC50 value of Wrightia tinctoria leaf methanol, leaf ethyl acetate, seed methanol, bark ethyl acetate extracts and the authentic

antioxidant L-ascorbic acid are given in table 3.

Table 3: Antioxidant activities of the W. tinctoria leaf, bark and seed extracts and positive control using the (DPPH) free radical - scavenging assay

Samples

Antioxidant activity IC50 (µg/ml)

W. tinctoria leaf methanol extract W. tinctoria leaf ethyl acetate extract W. tinctoria seed methanol extract W. tinctoria bark ethyl acetate extract L-ascorbic acid

72.90±3.5 87.50±3.2 86.53±2.5 67.30±2.4 62.5±2.5

DISCUSSION

DPPH free radical scavenging activity assay

The DPPH free radical scavenging activity of the leaf extracts of W. tinctoria are sorted in

descending order: leaf methanol extract> Leaf ethyl acetate extract> Leaf chloroform

extract> Leaf petroleum ether extract. Out of the four samples tested, W. tinctoria leaf

methanol extract showed the highest scavenging activity (% inhibition 95.16, 93.55, 88.71

and 83.87 at 1.0, 0.5, 0.25 and 0.125 mg/ml respectively), followed by W. tinctoria leaf ethyl

acetate extract. Leaf petroleum ether extract exhibited least DPPH radical scavenging ability

with % inhibition 85.48, 64.52, 41.94 and 16.12 at 1.0, 0.5, 0.25 and 0.125mg/ml

respectively.

The DPPH free radical scavenging activity of the leaf coconut oil extracts of Wrightia

tinctoria are: leaf extract (1d)> leaf extract (4d)> leaf extract (7d)> leaf extract (15d). Leaf

coconut oil extract with one day exposure to sunlight was found to be more active than the

extract with 15days of exposure to sunlight. Leaf coconut oil extracts are comparatively less

active (maximum % inhibition 33.08) than petroleum ether, chloroform, ethyl acetate and

methanol extracts of the leaf, seed and bark of W. tinctoria.

The ability of the W. tinctoria seed extract to scavenge the DPPH free radical is: seed

methanol extract> seed petroleum ether extract> seed ethyl acetate extract> seed chloroform

extract and bark extract is: bark ethyl acetate extract> bark methanol extract> bark

www.wjpr.net Vol 3, Issue 3, 2014. 5046 Among the W. tinctoria extracts studied, seed methanol extract, bark ethyl acetate extract and

leaf methanol extract showed considerable radical scavenging activity. The radical

scavenging activity of these extracts is compared with the standard L-ascorbic acid. Wrightia

tinctoria bark ethyl acetate showed greater ability to quench DPPH radicals and its activity

was found to be same as that of L-ascorbic acid. W. tinctoria leaf methanol extract exhibited

more activity than seed methanol extract.

The bark ethyl acetate extract of W. tinctoria was found to be the significant one for

scavenging DPPH radicals with IC50 value of 67.30±2.4 µg/ml and was quite comparable with the standard antioxidant L-ascorbic acid 62.5±2.5 µg/ml. IC50 value of Wrightia tinctoria leaf ethyl acetate extract 87.5±3.2 µg/ml and seed methanol extract 86.53±2.5 were

almost same. By comparing the of the IC50 value of W. tinctoria leaf methanol extract with that of the authentic antioxidant L-ascorbic acid, it was found that the antioxidant activity of

W. tinctoria leaf methanol extract (IC50:72.9±3.5 µg/ml) was not significantly different from that of L- ascorbic acid (IC50: 62.5 ±2.5 µg/ml).

CONCLUSIONS

Among the leaf, bark and seed extracts of Wrightia tinctoria studied, bark ethyl acetate

extract showed potent scavenging activity on DPPH free radical and its activity was quite

comparable with the standard antioxidant L-ascorbic acid. Antioxidant activities of the

extracts from medicinal plants are mainly attributed to the active compounds present in them.

This can be due to the high percentage of main constituents, but also to the presents of other

constituents in small quantities or to synergy among them [24, 25].

Phytochemical screening of the bark ethyl acetate extract of W. tinctoria revealed the

presence of phenolics and tannins. The leaf methanol extract of W.tinctoria was rich in

phenolics, saponins, flavonoids, alkaloids, glycosides and steroids [26]. Appreciable amounts of phenolics, saponins, flavonoids and glycosides were present in the seed methanol extract.

Bark methanol extract showed appreciable amount of tannins, saponins, flavonoids and

phenolics. The antioxidant potential of leaf, bark and seed extracts can be attributed to the

presence of these phytochemicals. The results obtained shows that the methanolic extracts of

the seeds and leaf, ethyl acetate extracts of the bark and laves of W.tinctoria can be

considered good source of natural antioxidants. The study supports the use of this plant for

drug formulations and reinforces the ethnobotanical importance of plant as a potential source

www.wjpr.net Vol 3, Issue 3, 2014. 5047 ACKNOWLEDGEMENT

One of the authors (Beena Jose) sincerely acknowledges the University Grants Commission

(UGC), New Delhi for the financial support to carry out the Major Research Project.

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