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Journal of Pharmacy Research Vol.1.Issue 1. July-September 2008
In vitro Antioxidant Activity from Leaves of Oroxylum
indicum (L.) Vent. -A North Indian Highly Threatened
and Vulnerable Medicinal Plant
Raghbir C. Gupta1 *, Vivek Sharma1, Nisha Sharma1, Neeraj Kumar2, Bikram Singh2
1Botany Department, Punjabi University, Patiala-147002 (Punjab) India.
2N.P.P. Division, Institute of Himalayan Bioresource Technology, CSIR, Palampur-176061
(Himachal Pradesh) India.
For correspondence: Raghbir C. Gupta Professor & Head,Department of Botany, Punjabi University,Patiala-
147002 (Punjab) India.
Email: rcgupta53@gmail.com
Received on: 08-06-2008; Accepted on :16-07-2008
ABSTRACT
Free radical scavenging potential of the different extracts of leaves of Oroxylum indicum (L.) Vent.
(Bignoniaceae), one of the widely used medicinal plant, was evaluated in vitro by using diphenyl-picryl-hydrazyl
(DPPH) assay. In this method the antioxidants present in the plant extracts reacted with DPPH, which is a stable
free radical and converted it to 1,1-diphenyl-1,2-picryl, hydrazine. The scavenging effect of plant extracts and standard
(L-ascorbic acid) on the DPPH radical decreases in the following order: L-ascorbic acid > Ethyl acetate (I) >Methanol
(II) > Water (III) and it was found to be 97.4%, 61.4%, 40.8% and 29.2% at concentration of 100 µg/mL, respectively.
The results were expressed as IC50. Ascorbic acid which was used as a standard showed an IC50 of 24.0 µg/mL,
whereas, the crude ethyl acetate (I), methanolic (II) and water (III) extracts of leaves of O. indicum showed
antioxidant activity with IC50 values of 49.0, 55.0 and 42.5 respectively at 100 µg/mL concentration. None of the
extracts were found to be more active than the standard i.e. ascorbic acid. But still, free radical scavenging activity
of water (III) and ethyl acetate (I) extracts of leaves of O. indicum (L.) was confirmed in the present investigation.
In the present study, different extracts of O. indicum leaves showed concentration dependent free radical scavenging
activity. Significant results were recorded first time in the given estimated parameters from different extracts of
leaves of O. indicum, a North Indian highly threatened and vulnerable medicinal plant.
Key words: Oroxylum indicum, antioxidant activity, extracts, DPPH assay.
Research Article
INTRODUCTION
Oroxylum indicum (L.) Vent. (Bignoniaceae)
is a medicinally important, medium sized, deciduous tree
distributed in India, Srilanka, China, Thailand, Philippines
and Indonesia. In India it is distributed in Eastern and
Western Ghats and North-East regions. The existence
of O. indicum in natural population is highly threatened
and it has been categorized as vulnerable medicinal plant
by the government of India [1]. Many flavones and
traces of alkaloids are present in pods, seeds, stem and
root bark [2]. Various parts of this plant are utilized to
cure many diseases. In India roots are used in Ayurvedic
preparation called “Dasamoola” i.e. used as an astringent,
anti-inflammatory, anti-helminthic, anti-bronchitic, anti-
leucodermatic, anti-rheumatic, anti-anorexic and for
treatment of leprosy and tuberculosis [3,4]. Decoction
of root bark is also effective to cure nasopharyngeal
cancer[5]. Leaves are emollient, that contain
anthraquinone and aloe-emodin [6]. According to
scientific observations, fruits are used in treating
bronchitis, leucoderma and helminthosis [7]. Seed
extract exhibits anti-microbial, analgesic, anti-tussive and
anti-inflammatory properties [8].
Oxidation is one of the most important
processes, which produce free radicals in food,
chemicals and even in living systems. Free radicals have
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Journal of Pharmacy Research Vol.1.Issue 1. July-September 2008
an important role in the processes of food spoilage,
chemical materials degradation and also contribute to
more than hundred disorders in human beings [9]. Active
oxygen and in particular, free radicals are considered to
induce oxidative damage in biomolecules and to play an
important role in aging, cardiovascular diseases, cancer,
and inflammatory diseases [10]. On the other hand,
antioxidants are now known to be protective or
therapeutic agents. The applications of antioxidants are
industrially widespread in order to prevent polymers
oxidative degradation, auto-oxidation of fats, synthetic
and natural pigments discoloration, etc. There is an
increased interest of using antioxidants for medical
purposes in the recent years, particularly in those
intended to prevent the presumed deleterious effects of
free radicals in the human body, and to prevent the
deterioration of fats and other constituents of foodstuffs
[11]. In the past few years, there is an increased
preference for antioxidants from natural sources rather
than from synthetic sources because of the health risks
and toxicity of synthetic antioxidants [12]. Plants are
being utilized frequently as sources of natural
antioxidants, and some of the compounds present in
plants have significant antioxidative properties and health
benefits [13]. The potential of the antioxidant constituents
of plant materials for the maintenance of health and
protection from coronary heart disease and cancer is
also raising interest among scientists and food
manufacturers as consumers move toward functional
foods with specific health effects [14]. At present, most
of the natural antioxidants such as traditional nutrients,
polyphenols, and flavonoids are obtained from plants.
Although many plant species have been investigated in
the search for novel antioxidants [15-16] but there is
still a demand to find more information concerning the
antioxidant potential of medicinal plant species.
However, there is not even a single report of
antioxidant activity for the leaves of this highly threatened
and vulnerable medicinal plant from North Indian
Himalayan region. Therefore, the chief objective of this
present study is to assess the antioxidant activity for first
time from different extracts of leaves of O. indicum by
the use of stable free radical diphenyl picryl hydrazyl
(DPPH) assay and to further check the “IC50” Inhibition
Concentration values.
MATERIAL AND METHODS
Plant Material
Leaves of O. indicum were collected from
Dhameta, (Himachal Pradesh) in the month of November,
2006, which come under the foot hills of North-Indian
Himalayan region. The specimens were authenticated
by Department of Botany, Punjabi University, Patiala
(Punjab) India and were deposited in Departmental
Herbarium.
Preparation of Crude Plant Extracts
The air dried 500g leaves of O. indicum were
ground into powder and extracted for three days with
1000mL of 80% (v/v) methanol; extract was then filtered
with Whatman’s filter paper No.1. The filtrate was
collected and solvent was evaporated under reduced
pressure with the help of vacuum evaporator. After
removal of the solvent, 120g of dry crude extract was
obtained. Again, dry crude extract was re-extracted three
times with 500mL of each ethyl acetate, methanol and
water. Further, these three extracts were filtered and
evaporated with the help of vacuum evaporator. After
re-extraction, quantity of ethyl acetate (I), methanol (II)
and water (III) extracts were 35g, 28g and 32g
respectively and stored in air tight tubes under low
temperature i.e. 4oC till further analysis (Flow chart 1).
Finally, 2mg of each extract i.e. ethyl acetate (I), methanol
(II) and water (III) were dissolved in 10mL of methanol
and different concentrations of each extract were
prepared for the free radical scavenging activity.
Preparation of Chemical Solutions
For antioxidant activity, chemical solution of L-
ascorbic acid was used as a control and prepared by
dissolving 2mg of L-ascorbic acid in 10ml of methanol.
For Diphenyl-picryl-hydrazyl assay, 0.1mM stock
solution of DPPH in methanol was prepared. All the
chemicals, including solvents, were of analytical grade
from E. Merck, India and Sigma (New Delhi, India).
Reaction Time
In the original method a reaction time of 30min
was recommended [17]. There were so many different
views for reaction time, given by workers all over the
world. Shorter time had also been used for the reaction,
Raghbir et al. In vitro Antioxidant Activity of Oroxylum indicum (L.) vent. -A North Indian Highly Threatened and Vulnerable Medicinal Plant
66
Journal of Pharmacy Research Vol.1.Issue 1. July-September 2008
such as 10min [18]. However, in view of the fact that the
rate of reaction varies widely among substrates, the best
practice seems to be to follow the reaction until it had
gone to completion. In present attempt of research work,
30min reaction time was followed, to check the antioxidant
activity.
Absorbance Measurements - Wavelength and
Instrument Used
During evaluation of antioxidant activity of the
three extracts (I, II, III), we followed the methodology
given by Blois in 1958 [19]. Absorbance was measured
at 517nm by using XP 2001 Explorer
spectrophotometer. The working wavelength of
maximum absorbance, to be used for the absorbance
measurements, were given variously as 515nm by Brand-
Williams, 516nm by Schwarz, 517nm by Blois and
518nm by Leitao [20].
Free Radical Scavenging Activity (Diphenyl-picryl-
hydrazyl assay)
The free-radical scavenging capacity of different
extracts (I, II, III) of leaves of O. indicum were
evaluated with the DPPH stable radical, by the
methodology described by Blois in 1958. Briefly, 0.1mM
alcoholic solution of DPPH in methanol was prepared
and 2mL of this solution was added to 0.3mL of different
extract concentrations (1-100 µg/mL) and allowed to
react at room temperature. After 30min, the absorbance
values were measured at 517nm against the blank, which
did not contain the extract. L-ascorbic acid was used
as control. The radical scavenging activity (percent
inhibition) was expressed as percentage of DPPH radical
elimination calculated according to the following
equation:
Percentage Inhibition (%) = (Acontrol-Asample/Acontrol) ×
100
Where Acontrol is the absorbance of the control (L-
ascorbic acid) and Asample is the absorbance of reaction
mixture (in the presence of sample). All tests were run in
triplicates (n = 3), and the average values were
calculated.
IC50 Value
Inhibition Concentration (IC50 ) was introduced
by Brand-Williams and his colleagues [21] for the
interpretation of the results from DPPH method. The
discoloration of sample was plotted against the sample
concentration in order to calculate the IC50 value. It is
defined as the amount of sample necessary to decrease
the absorbance of DPPH by 50%.
RESULTS AND DISCUSSION
Like other medicinal plants, different extracts
from leaves of O. indicum showed antioxidative effect
which is expected mainly due to phenolic components,
such as flavonoids, phenolic acids, and phenolic
diterpenes [22]. These phenolic components possess
many hydroxyl groups including o-dihydroxy group
which have very strong radical scavenging effect and
antioxidant power. In the DPPH assay, the antioxidant
was able to reduce the stable radical DPPH to the yellow
colored 1, 1-diphenyl-1, 2-picryl hydrazine. The
molecule of 1, 1-diphenyl-1, 2-picryl hydrazine is
characterised as a stable free radical by virtue of the
delocalisation of the spare electron over the molecule
as a whole. The delocalisation also gives rise to the deep
violet colour, characterised by an absorption band in
methanol solution centred at 517 nm. The observed
scavenging effect of plant extracts and standard on the
DPPH radical decreases in the following order: L-
ascorbic acid > Ethyl acetate (I) >Methanol (II) > Water
(III), it was 97.4%, 61.4%, 40.8% and 29.2% at
concentration of 100 µg/mL, respectively (Table 1). The
potential of L-ascorbic acid to scavenge DPPH radical
became almost stable after 70 or 80 µg/mL (Figure 1).
From the experimental data which obtained from all three
extracts, it is clear that ethyl acetate extract (I) showed
maximum percentage of inhibition effect i.e. 61.4% at
100 µg/mL of concentration. Among these three extracts
and standard tested for the vitro antioxidant activity, using
the DPPH method, the crude ethyl acetate (I), methanolic
(II) and water (III) extracts of leaves of O. indicum
showed antioxidant activity with IC50 values of 49.0,
55.0 and 42.5 respectively at 100 µg/mL concentration
(Table 2). Eventhough their IC50 values found to be
higher however, none of the extracts were found to be
more active than the standard i.e. (L-ascorbic acid with
IC50 value of 24.0 at 100 µg/mL). Free radical
scavenging activity of water (III) and ethyl acetate (I)
extracts was confirmed in the present investigation
(Figure 2). Thus it is clear that polyphenolic antioxidants
in leaves of O. indicum play an important role as
bioactive principles. However, polyphenolic constituents
present in these extracts, which are responsible for this
activity, need to be investigated in O. indicum.
Raghbir et al. In vitro Antioxidant Activity of Oroxylum indicum (L.) vent. -A North Indian Highly Threatened and Vulnerable Medicinal Plant
67
Journal of Pharmacy Research Vol.1.Issue 1. July-September 2008
Leaves Powder (500g)
Extracted with 1000mL of 80% (v/v) Methanol
Crude Extract (120 g) Residue
Re-extracted with 500mL of Ethyl acetate, Methanol and water
Ethyl acetate Extract (35g) Methanol Extract (28g) Water Extract (32g)
Flow Chart 1: Extraction/Fractionation procedure applied on leaves powder of O. indicum.
I
II
II
Raghbir et al. In vitro Antioxidant Activity of Oroxylum indicum (L.) vent. -A North Indian Highly Threatened and Vulnerable Medicinal Plant
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Journal of Pharmacy Research Vol.1.Issue 1. July-September 2008
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Journal of Pharmacy Research Vol.1.Issue 1. July-September 2008
ACKNOWLEDGEMENTS
The authors are grateful to the SAP-II DRS
(UGC), Head Department of Botany, Punjabi Univer-
sity, Patiala (Punjab) and Dr. P.S. Ahuja, Director, IHBT
(CSIR), Palampur (H.P) India for providing necessary
laboratory facilities and support.
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