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Original Article
Comparative evaluation of antioxidant potential of extracts of Vitex negundo,
Vitex trifolia, Terminalia bellerica, Terminalia chebula, Embelica officinalis and
Asparagus racemosus

Sonal Shah, Tushar Dhanani, Satyanshu Kumar*

Directorate of Medicinal and Aromatic Plants Research (Indian Council of Agricultural Research) Boriavi,
Anand-387310, Gujarat, India

Abstract

The antioxidant potential of six important Indian medicinal plants V. negundo, V. trifolia, T.
bellerica, T. chebula, E. officinalis and A. racemosus were evaluated. Extracts prepared using
solvents of varying polarity were screened for their free radical scavenging and reducing
capacity by chemical assays. Total phenolic content was determined in order to assess its effect
on the antioxidant activity of the extract. The properties of extracting solvents affected the
measured total phenolic content and antioxidant activity. Extracts of all the six medicinal plants
exhibited antioxidant potential but T. bellerica , T. chebula, E. officinalis proved more active.
The presence of antioxidant activity in the extracts showed that these plants have the potential
to be an alternate source of natural antioxidants. In vivo study is needed for successful
commercialization and to benefit the food and pharmaceutical industries.
Keywords: Free radicals, reactive oxygen species, oxidative stress, antioxidant, total phenols, scavenger

* Corresponding author: Satyanshu Kumar, Directorate of Medicinal and Aromatic Plants Research
(Indian Council of Agricultural Research), Boriavi, Anand-387310, Gujarat, India. E- mail:
satyanshu66@gmail.com

1. Introduction

Plants and herbs have been used an many diseases such as cancer, arthrosclerosis, aging,
important contributor to the quality of human life for diabetes etc [3, 4]. The potential targets for ROS in
thousands of years. They provide abundant natural cells are membrane lipids, DNA and proteins.
antioxidants which are virtually important for human Antioxidants are very important for human health.
health. Oxidants and antioxidants in humans are Natural antioxidants or other compounds such as
maintained in balance in a normal physiological state. vitamins, minerals or proteins can neutralize free
However, the overproduction of oxidants in certain radicals. Antioxidant supplementation is
conditions may cause oxidative stress leading to recommended to provide cellular protection from the
oxidative damage to biomolecules and cells. [1, 2]. deleterious effects of excessive reactive oxygen
Reactive oxygen species (ROS) are formed during species concentration [5]. Antioxidants are our first
normal cellular metabolism but when present in high line of defense against free radical damage and are
concentration they become toxic. Oxidative stress or critical for maintaining optimum health and
excessive production of ROS is being implicated in wellbeing. The need for antioxidants becomes even

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 Satyanshu K. IPP, Vol 1 (1), 44-53, 2013

more critical with increased exposure to free radicals. convulsant activities [13]. V. negundo is used in
Pollution, cigarette smoking, drugs, illness, stress, and several commercial formulations and in the Ayurvedic
even exercise can increase free radical exposure [6]. System of Medicine, leaves of V. negundo is used as a
Free radical scavenger may prevent the oxidative drug of choice for pain, inflammation and related
stress by peroxidation, inhibiting free radicals and diseases [14, 15]. Vitex trifolia is a shrub or small tree,
also by other mechanism they prevent diseases. In its leaves contains flavonoids, sterols and terpenoids.
addition to endogenous antioxidant defense systems, The leaves are considered useful as an external
consumption of dietary and plant-derived application to all rheumatic pain, sprains etc. The
antioxidants appears to be a suitable alternative as petroleum and ethanol extracts of leaves of V. trifolia
the synthetic antioxidants are reported to be harmful exhibited moderate inhibiting activity against most of
for human health. Further, synthetic antioxidants the tested Gram positive and Gram negative bacteria
such as butylated hydroxyl anisole (BHA) and [13, 16]. Significant hepatoprotective activity of
butylated hydroxyl toluene (BHT) have restricted use aqueous and ethanol extracts of V. trifolia in carbon
in food because of their carcinogenic properties [7]. tetrachloride induced liver damage of male wistar rats
Also, the use of the most potent synthetic food was also reported [17].
antioxidant tertiary butylated hydroquinone (TBHQ) is
not permitted in Japan and Europe [8]. Therefore, the Terminalia chebula belongs to the family
search for effective, nontoxic natural compounds with Combretaceae and is found throughout India chiefly
antioxidative activity has been intensified in recent in deciduous forests. It occurs abundantly in North
years [9]. Medicinal plants contain substantial India and range extends southwards at 300 to 900 m
amounts phytochemicals with capacity to scavenge attitude [18]. Its yellowish brown fruits are included
the free radical and can serve as a potential source in the Indian pharmacopoeia under the category
of natural antioxidants [10].The majority of astringent. It possesses laxative, diuretic, cardio tonic
antioxidant belongs to flavonoids, isoflavones, and hypoglycemic properties. The dried fruit of T.
anthocyanins, coumarins, lignans, catechins and chebula is used extensively in the indigenous system
isocatechins class of phytochemicals. of medicine (Ayurvedic) for its homoeostatic,
antitussive, laxative, diuretic and cardio tonic
The genus Vitex belongs to the family activities [19]. Gallic acid, ellagic acid, tannic acid,
Verbenacea. It includes 80 genera and about 800 ethyl gallate, chebulic acid, chebulagic acid, corilagin,
species. Vitex is a genus of trees and shrubs widely mannitol, ascorbic acid and other compounds had
distributed in the tropic and warm region. Vitex been reported from T. chebula [20]. Terminalia
negundo Linn is a shrub quite prolific in India, bellirica plant is also common throughout India in the
occurring up to an altitude of about 1500 m. It is one plains and lower hills, chiefly in the deciduous forests
of the common plants used in traditional medicine at 900 m elevation where the climate is not very dry
and reported to have a number of biological activities [21]. Emblica officinalis is a medium sized tree found
[11]. Flavonoids, iridoids, terpenes and steroids are cultivated or growing wild in mixed deciduous forests
the major classes of compounds isolated from V. of India ascending up to 1300 m on hills [22].
negundo [12]. A large number of compounds with Triphala, a combination drug is a composite mixture
varying structural classifications including of T. chebula, T. bellerica and E. officinalis and used in
glucononitol, camphene, -pinene, citral, - popular traditional medicine for the treatment of
caryophyllene, casicin, orientin, isorientin, many chronic diseases such as ageing, heart ailments,
corymbosin, flavonoid glycosides, diterpenoid, hepatic diseases etc. [23, 24]. Fruit of E. officinalis is a
triterpenoids, long chain unsaturated fatty acids, rich source of phenolic compounds [25]. A.
iridoid glycosides and lignin have been reported from racemosus is distributed in tropical and subtropical
the leaves, twigs, seeds and roots of V. negundo parts of India including the Andamans and ascending
[13]. Although all parts of V. negundo are used as in the Himalayas up to an altitude of 1500 m. The root
medicine in the indigenous system of medicine, the of A. racemosus is the source of the drug shatavar
leaves are most potent for medicinal use. Decoction and is highly mucilaginous, antidiarrhoeal, refrigerant,
of the leaves of V. negundo is used as a bath in the diuretic, antidysenteric, nutritive, tonic, and
puerperal state of women in India. The leaf extract of demulcent, galactagogue, aphrodisiac and
V. negundo has been reported to exhibit a wide range antispasmodic. The root contained proteins, saponins,
of pharmacological activities. The methanolic extract carbohydrates, crude fibre, inorganic matter and
of V. negundo had been found to exhibit very potent mucilage .The sapogenin present in the root was
antifeedent, anti-inflammatory, analgesic and anti identified as sarsapogenin [26, 27].

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Medicinal plants have been extremely 2. Material and Methods

studied for their antioxidant assay. Selection of the
plants in the present study was based on their use in Plant material and chemicals:
traditional Indian Systems of Medicine (ISM). Leaves of V. negundo , V. trifolia, barks of T.
Extraction solvent significantly alter the antioxidant bellerica, T. chebula, fruits of E. officinalis and roots of
estimation, therefore, selection of extracting solvent A. racemosus were collected from the herbal garden
of matching polarity is one of the most important of Directorate of Medicinal and Aromatic Plants
factors in the extraction of antioxidants. Water, Research (DMAPR), Boriavi, Anand ,Gujarat , India
methanol and ethanol have been widely used for during the year 2011. Analytical grade solvents
extraction of antioxidants. hexane, chloroform, ethyl acetate, methanol and n-
butanol were purchased from Sisco Research
Polar solvents and alcoholic solutions Laboratories (SRL), Mumbai, India. Reagent grade
frequently provide satisfactory extraction [28, 29]. trichloroacetic acid (TCA), ammonium persulphate,
However, more than one extragent or sequential gallic acid, Folin-Ciocalteau reagent, sodium
multi solvent extraction are preferred in very few carbonate, potassium ferricyanide , ferric chloride,
cases. Herbs are traditionally ingested as hot water ascorbic acid were also obtained from Sisco Research
infusion. Recovery of antioxidant phytochemicalas Laboratories (SRL), Mumbai, India. 2, 2-diphenyl-1-
from plant materials is controlled by their chemical picrylhydrazyl (DPPH), 2, 2-azinobis(3-
properties and distribution in plant matrix. Because of ethylbenzothiazoline-6-sulphonic acid, ABTS),
the presence of different groups of antioxidants with butylated hydroxyl toluene (BHT), tertiary
differing chemical behaviour in plant matrix, extract butylhydroquinone (TBHQ), 6-hydroxy-2,5,7,8-
yield and resulting antioxidant activities are strongly tetramethylchroman-2-carboxylic acid (trolox) were
dependent on the nature of extracting solvent as purchased from Sigma-Aldrich, Bangalore, India.
polarity of the solvent used for extraction purposes Preparation of extract:
may or may not be matching. Plants extracts made Plant materials were divided into small
with water are nutritionally more relevant and also pieces and dried at room temperature under shade.
required for clinical usages. Further, the powder of suitable mesh size of dried
In the present report, antioxidant potential plant materials was prepared using an electric grinder
of six important medicinal plants V. negundo, V. and powder samples were used for preparation of the
trifolia, T. bellerica , T. chebula, E. officinalis and A. extracts. The powder samples of plant materials were
racemousus was determined with non-polar (hexane, extracted with methanol at room temperature for 24
chloroform), semipolar (ethyl acetate) and polar hrs. This procedure was repeated for five times and
(methanol, n-butanol and water) solvents. Although, the solvent was removed from the combined extract
there have been numerous researches on crude under vaccum. A part of the methanol extract
extraction and bioactive compounds of potential obtained was dissolved in water and sequentially
plants for natural and possibly economic and effective extracted with hexane, chloroform, ethyl acetate and
antioxidants to replace the synthetic ones. These n-butanol to obtain hexane, chloroform, ethyl
sources particularly leaves, bark and roots have not acetate, n-butanol and aqueous extracts respectively.
been received much attention as antioxidant sources Measurement of total phenolics:
as compared to the other plants/fruits. The aim of the Total phenolics in the extracts were quantified using
present research work was to determine the best Folin- Ciocalteau reagent and expressed as gallic acid
solvent for extraction of antioxidants from six equivalent [30, 31]. Extract (0.5ml) was mixed with
important Indian medicinal plants and to make a Folin-Ciocalteau reagent (0.5ml) and distilled water
comparative evaluation of the antioxidant potential (7.5 ml). The resulting mixture was incubated at room
of the extracts. Plant materials of six medicinal plants temperature for 10 minutes. Thereafter, sodium
were extracted with five solvents hexane, chloroform, carbonate solution (1.5ml, 20%) was added to the
ethyl acetate, n-butanol and water using conventional mixture and further incubated in a water bath for 20
extraction method. Total phenols in the extracts were min at 40C. The absorbance of the solution was
determined and antioxidant potential of the extracts measured using a UV-visible spectrophotometer at
was assayed using chemical assays. The result would 755 nm. A calibration curve was prepared by plotting
be useful for the optimization of the antioxidant concentration vs absorbance of gallic acid as
yielding fractions for development of up scaling standard. Total phenolics in the extract samples were
process from these medicinal plants. quantified from the calibration curve and were

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 Satyanshu K. IPP, Vol 1 (1), 44-53, 2013

expressed as gallic acid equivalent (GAE) per gm of Reducing power assay:

dry extract. The total reducing power of standard antioxidants
Antioxidant Activity Assay and extracts were determined as described by Oyaizu
DPPH (2, 2-diphenyl-l-picryl hydrazyl) radical [34]. Different concentrations of extracts were mixed
scavenging assay: with distilled water (2.5 ml), phosphate buffer (2.5ml,
The antioxidant activity of the plant extracts was 0.2 M, pH 6.6) and potassium ferricyanide (2.5ml,
determined using the DPPH free radical scavenging 1%). The resulting mixture was incubated at 50C for
method with some modifications [32]. DPPH solution 20 min in a water bath. After cooling, trichloroacetic
(0.1mM litre-1) was prepared in methanol. Stock acid (2.5ml, 10 %,) was added to the mixture. The
solution (1mg ml-1) of extracts and the standard upper layer of solution (2.5ml) was taken and mixed
antioxidants (ascorbic acid, BHT, BHQ and Trolox) with distilled water (2.5 ml) and ferric chloride (0.5ml,
were also prepared in methanol. Working 0.1%). The absorbance was measured using a UV-
concentration of the standard antioxidants and visible spectrophotometer at 700 nm. The increasing
extracts were prepared using appropriate dilution. absorbance value was interpreted as increased
Freshly prepared DPPH solution (1 ml) was added to reducing activity [35]. The extract concentration
standard antioxidants and extracts solution (3ml). The providing absorbance value of 0.5 (EC50) was
mixture was incubated in the dark for 30 min and calculated from the graph.
thereafter, absorbance was recorded at 517 nm
against the blank. For control, DPPH solution (1 ml) 3. Results and Discussion
was mixed with methanol (3 ml) and used for
measuring the absorbance value. The decrease in Solvent polarity plays a key role in increasing
absorbance of DPPH solution on addition of test phenolic solubility [36]. Phenolic compounds such as
samples in relation to the control was used to BHT, propyl gallate etc. are known to be effective
calculate the antioxidant activity in terms of antioxidants. Phenolic compounds have been known
percentage inhibition of DPPH radical. The capability to act as antioxidants not only because of their ability
of scavenging DPPH radical (%RSA) was calculated to donate electrons also because of their stable
using the following equation as described by Lee et al radical intermediates which can effectively prevent
[33] the oxidation at cellular and physiological level [37].
(Acontrol Atest) In the present study five solvents with different
Radical scavenging percentage = X 100 polarity were used and they can be arranged as
(Acontrol) following (starting from more non- polar solvent):
The IC50 values of the standard antioxidants as well hexane< chloroform< ethyl acetate< n- butanol<
as the plant extracts corresponded to concentration methanol< water. Even though it is unclear whether
inhibiting fifty percent of initial concentration of active compounds are active against free radicals
DPPH and values were obtained from the graph of the after being absorbed and metabolized by cells in the
percentage inhibition versus the concentration. body, radical scavenging assays have gained
Obtained IC50 values of the standards and extracts acceptance for their capacity to rapidly screen
were verified experimentally. materials of interest [38]. Antioxidant activity of
plants is well correlated with the content of their
ABTS radical assay: phenolic compounds [39]. The most commonly used
ABTS radical cation decolorization assay was antioxidant methods DPPH. And ABTS.+ were used to
performed as described by Re et al [33]. ABTS cation assess the free radical scavenging activity of the
was generated by the reaction between ABTS (7 mM) different extracts.
and potassium persulfate (2.45 mM) at room Extract yield of plant material:
temperature in the dark for 1216 h before use. ABTS The extraction yield varied depending on the type of
solution was diluted with methanol to get a final extraction solvent and extract yield of plant materials
absorbance of 0.7 0.02 at 734 nm. For measuring of V. negundo , V. trifolia, T. bellerica , T. chebula, A.
the decrease of absorbance of blue- green colour of racemousus and E. officinalis has been described in
cation generated, ABTS solution (990 l) was mixed Figure 1. For V. negundo and V. tripholia leaves,
with test solution (10 l). The reduction in absorbance extract yield was maximum for aqueous extract and
was recorded after 6 min and compared with the minimum for chloroform extract and it varied in the
control. following order: aqueous>methanol>hexane>ethyl
acetate>chloroform. In case of T. chebula and T.

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bellerica bark extract, aqueous extract had highest content in the following order: n-butanol (25.1 mg g-1
yield. The extract yield for T. chebula and T. bellerica GAE)> methanol (10.0 mg g-1 GAE)> aqueous (4.8 mg
varied in the following order: aqueous> methanol> n- g-1 GAE).
butanol> ethyl acetate> hexane> chloroform. For Free radical scavenging activity by stable DPPH
fruits of E. officinalis, yield of methanol extract was radical:
highest followed by aqueous and n-butanol extract. For DPPH assay, four synthetic antioxidants such as
The extract yield varied in the following order: ascorbic acid, BHT, BHQ and trolox were used as
methanol> aqueous> n-butanol> ethyl acetate> reference and their IC50 values were found to be 4.2,
-1
hexane> chloroform. For A. racemosus, methanol was 4.3, 1.7 and 2.1 g mL respectively, these values are
used for preparation of the extract. comparable to their reported IC50 values [42, 43].
Results of DPPH assay of different extracts of V.
Total phenol content (TPC): negundo , V. trifolia, T. bellerica , T. chebula, A.
Total phenol content expressed as gallic acid racemousus and E. officinalis has been summarized in
equivalent (mg /g GAE) in different extracts of V. Table 1. For V.negundo and V. trifolia, ethyl acetate
negundo, V. trifolia, T. bellerica , T. chebula, A. extract had the lowest IC50 value followed by
racemousus and E. officinalis has been summarized in methanol, aqueous and chloroform extract. IC50
Table 1. For V. negundo and V. trifolia leaves extract, value of ethyl acetate extract of V. negundo (4.2 g
total phenol content was maximum in ethyl acetate mL-1) was found to be comparable to the IC50 value
extract (172.1, 150.3 mg g-1 GAE) followed by of ascorbic acid and BHT and comparatively lower
methanol (90.1, 74.5 mg g-1 GAE) , chloroform (56.3, than reported value for ethanol extract of V. negundo
74.1 mg g-1 GAE) , aqueous (53.6, 62.2 mg g-1 GAE) leaves extract [44]. All the four extracts of T. chebula
and hexane extract (35.5, 33.8 mg g-1 GAE). Kumar et and T. bellerica exhibited very low IC50 value. For T.
al [12] reported total phenol content (27.7 + 0.3 mg g- chebula, the IC50 varied in the following order:
1 GAE) in aqueous - methanol extract of V. neguno aqueous (1.6 g mL-1) < methanol (2.0 g mL-1) < n-
leaves. However, in case of T. chebula, aqueous butanol (2.4 g mL-1) < ethyl acetate (2.7 g mL-1).
extract had highest total phenol content (595.4 mg g- These values were comparable to the IC50 of
1 GAE) followed methanol (531.5 mg g-1 GAE), ethyl standard antioxidants such as trolox and BHQ.
acetate (410.8 mg g-1 GAE) and n-butanol (380.5 mg However, in case of T. bellerica extracts, IC50 values
g-1 GAE). Similar trend was reported for fruit extracts varied in the following order: n-butanol (2.7 g mL-1)
of T. chebula by Chang and Lin [40]. For T. bellerica, n- < ethyl acetate (2.8 g mL-1) < methanol (3.1 g mL-
butanol extract had highest total phenol content 1) < aqueous (3.5 mL-1). IC50 value of ethyl acetate
(500.7 mg g-1 GAE) followed by aqueous (376.4 mg g- extract of T. chebula bark was observed to be lower
1 GAE), ethyl acetate (367.4 mg g-1 GAE) and than the IC50 values reported for the methanol
methanol (362.5 mg g-1 GAE). Here, T. chebula and T. extract of the fruits of T.chebula and T. bellerica. Also
bellerica bark extracts had higher total phenol for E. officinalis, IC50 values comparable to standard
content than the total polyphenolic compounds in the antioxidants were recorded for n-butanol (1.5 g mL-
methanol extracts of fruits from T.horrida, T. chebula 1), methanol (1.6 g mL-1) and aqueous extracts (4.1
and T. bellerica [41] .Ethyl acetate extract of E. g mL-1). For A. racemosus extracts very high IC50
officinalis had highest total phenol content (404.0 mg values (methanol extract: 614 g mL-1, n-butanol
g-1 GAE) followed by n-butanol (362. 3 mg g-1 GAE), extract: 348.6 g mL-1) were in agreement to the
methanol (221.6 mg g-1 GAE). Aqueous extract values reported by Dohare et al [45].
(153.8 mg g-1 GAE) and chloroform extract (153.1 mg
g-1 GAE) had similar total phenolic content. A.
racemosus extracts had very low total phenolic

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Plant Extract TPC DPPH ABTS RPA

(GAE gm-1) (IC50, gmL-1) (IC50, gmL-1) (IC50, gmL-1)
V. negundo methanol 90.1 23.4 547.2 161.5
hexane 35.5 107.8 >1000 851.7
chloroform 56.3 51.3 632.9 561.7
ethyl acetate 172.1 4.2 211.5 40.1
aqueous 53.6 36.5 465.2 193
V. trifolia methanol 74.5 16.8 493.1 381.7
hexane 33.8 78.9 >1000 605
chloroform 74.1 60.4 418.8 477
ethyl acetate 150.3 9.8 286.9 215
aqueous 62.2 27.9 506.9 549.5
T. chebula methanol 531.5 2.0 34.5 37.9
ethyl acetate 410.8 2.7 56.5 49.4
n- butanol 380.5 2.4 48.5 44.9
aqueous 595.4 1.6 29.2 27.1
T. bellerica methanol 362.5 3.1 42.9 64.2
ethyl acetate 367.4 2.8 40.8 53.0
n- butanol 500.7 2.7 38.8 43.1
aqueous 376.4 3.5 52.6 66.7
E. officinalis methanol 221.6 1.6 68.8 52.7
hexane 129.2 5.4 135.6 113.6
chloroform 153.1 6.7 119.3 101.3
ethyl acetate 403.9 1.8 29.8 35.5
n- butanol 362.3 1.5 39.6 33.9
aqueous 153.8 4.1 80.5 71.7
A. racemosus methanol 10.0 613.9 >1000 1655.9
n- butanol 25.1 348.6 871.9 384.9
aqueous 4.8 >1000 >1000 >1000

Table 1: Total phenol content (TPC) and IC50 values of extracts of V. negundo, V. trifolia, T. chebula , T. bellerica, E.
officinalis and A. racemosus in DPPH, ABTS and reducing power assay (RPA)

Free radical scavenging activity by stable ABTS five V. negundo leaves extract, the following order of
radical cation: scavenging activity in terms of IC50 value was
In the ABTS assay, standard antioxidants ascorbic reported by Tiwari et al [46] : total
acid, BHT, BHQ and trolox had IC50 corresponding to ethanolic>methanolic>water>ethyl acetate>hexane
42.7, 116.5, 64.4 and 47.4 g mL-1 respectively. Here extract. For T. chebula, aqueous extract had lowest
also, IC50 value of V. negundo, V. trifolia, T. bellerica, IC50 value and it varied in the following order:
T. chebula, A. racemousus and E. officinalis exhibited aqueous (29.2 g mL-1 ) < methanol (34.5 g mL-1)<
similar trend as in DPPH assay (Table 1). Ethyl n-butanol (48.5 g mL-1 )< ethyl acetate (56.5 g
acetate of V. negundo , V. trifolia exhibited lowest mL-1 ). Also for T. bellerica, the IC50 values were
IC50 value and hexane extract exhibited the found in the following order: n-butanol (38.8 g mL-
maximum IC50 value. In ABTS assay for IC50 value 1) < ethyl acetate (40.8 g mL-1 ) <methanol (42.9 g
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 Satyanshu K. IPP, Vol 1 (1), 44-53, 2013

mL-1)< aqueous (52.6 g mL-1). Although, IC50 1). For T. chebula, aqueous extract had lowest EC50
concentration of T. bellerica extract had higher value and it varied in the following order aqueous
values than the corresponding values of T. chebula (27.1 g mL-1) < methanol (37.9 g mL-1) < n-
extracts but the trend was found to be similar as butanol (44.9 g mL-1) < ethyl acetate (49.4 g mL-
observed in DPPH assay. Also the IC50 values of T. 1). Also for T. bellerica, the EC50 values were found
chebula and T. bellerica extracts were comparable to in the following order: n-butanol (43.1 g mL-1 ) <
the four standard antioxidants. For E. officinalis, ethyl acetate (53.0 g mL-1) <methanol (64.2 g mL-
ethyl acetate (29.8 g mL-1) and n-butanol (39.6 g 1 )< aqueous (66.7 g mL-1). For E. officinalis, n-
mL-1) extracts had IC50 lower than the butanol (33.9 g mL-1) had the lowest EC50
corresponding values for all the four standard concentration followed by ethyl acetate (35.5 g mL-
antioxidants. As observed in DPPH assay, here also, 1 ), methanol (52.7 g mL-1), aqueous (71.7 g mL-1
IC50 values of A. racemosus extracts were found to ), chloroform (101.3 g mL-1 ) and hexane (113.6 g
be very high. mL-1). For A. racemosus, here also, EC50
Reducing power assay: concentration was found to be very high.
The reducing power of an extract may serve as a Correlation between antioxidant activity and
significant indicator of its potential as an antioxidant phenolic content:
and reducing power activity was calculated in terms Different phenolic compounds may show different
of EC50 concentration. Ascorbic acid, BHT, BHQ and antioxidant activities depending on their structure as
trolox had EC50 concentration 35.5, 59.8, 31.3 and well as synergistic or antagonistic effect of other
65.9 g mL-1 respectively. For V. negundo, EC50 compounds present in the extract [47] . Correlation
varied in the following order: ethyl acetate (40.1 g analysis were performed to determine the
mL-1) < methanol (161.5 g mL-1) < aqueous (193 g relationship between the TPC of the extracts and
mL-1 ) < chloroform (561.7 g mL-1)< hexane (851.7 corresponding 1/IC50 values obtained in DPPH,
g mL-1). Ethyl acetate extract of V. trifolia (215 g ABTS and reducing power assay (Figure 2).
mL-1) had the lowest EC50 concentration followed by Significant positive correlation between TPC and
methanol (381.7 g mL-1), chloroform (477 g mL- 1/IC50 values of DPPH, ABTS and reducing power
1), aqueous (549.5 g mL-1) and hexane (605 g mL- assay was established with estimated coefficient of

Figure 1: Extract yield of different fractions of V. negundo, V. trifolia, T. chebula, T. bellerica and E. officinali

50
51
determination R2 values 0.72, 0.91 and 0.79 8. Suja, K P, Jayalekshmy, A, Arumughan, C, 2005,
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material used. Also TPC and free radical scavenging : comparison of two extraction systems, Acta Biochim
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indicated the presence of compounds in the extracts activity of flavonoids, Int J Antimicrob Agents, 26;
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officinalis ) antioxidant activity. Also the lowest value K, Pandey, R, Singh, D, Chattopadhyay, N, Maurya, R,
IC50 values of extracts of T. chebula, T. bellerica and 2010, Anti-osteoporotic constituents from Indian
E. officinalis were below the maximum permissible medicinal plants, Phytomed, 17; 993-999.
level (200 ppm) for the synthetic antioxidants [8]. 13. Sharma, R L, Prabhakar, A, Dhar, K L., Sachar, A,
2009, A new iridoid glycoside from Vitex negundo
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