317 - In-vitro Antioxidant studies on ethanolic extracts of Boswellia ovalifoliolata and saccharum spontaneum by DPPH, Nitric Oxide and Lipid perooxidation methods

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* Corresponding author: Geetha kodali
E-mail address: geetha_pharma1@yahoo.co.in
Available online at www.ijrpp.com
Print ISSN: 2278 – 2648
Online ISSN: 2278 - 2656 IJRPP | Volume 2 | Issue 1 | 2013 Research article
In-vitro Antioxidant studies on ethanolic extracts of Boswellia ovalifoliolata
and Saccharum spontaneum by DPPH, Nitric oxide and
Lipid peroxidation methods.
*
Geetha Kodali, Ganapaty Seru.
Department of Pharmacognosy and Phytochemistry, University College of Pharmaceutical
Sciences, Andhra University, Visakhapatnam, Andhra Pradesh, India.
ABSTRACT
Oxidative stress is currently suggested a mechanism underlying various degenerative disorders such as
atherosclerosis, cancer, aging, diabetes and Alzheimer’s. Chemical principles from nature such as flavonoids
and other phenolic compounds were reported to act as scavengers of free radicals that are continuously
generated in the human body. The aim of the present study was to investigate the antioxidant properties of
ethanolic extracts of Boswellia ovalifoliolata and Saccharum spontaneum. The antioxidant properties of the
extracts were determined by scavenging 1, 1-diphenyl-2-picrylhydrazyl radical (DPPH), Nitric oxide (NO) and
lipid peroxidation (LPO). The results of in-vitro studies showed higher percentage of inhibition of free radicals
by ethanolic extract of Boswellia ovalifoliolata and saccharum spontaneum in a concentration dependent
manner. The antioxidant effect may be attributed to the presence of phenolic compounds in the extracts which
were quantified by Folin-ciocalteau method. From the results obtained, it may be concluded that Boswellia
ovalifoliolata and Saccharum spontaneum possess significant antioxidant activities.
Key words: Boswellia ovalifoliolata, Saccharum spontaneum, total phenolics, antioxidant, DPPH, Nitric
oxide, Lipid peroxidation.
INTRODUCTION
Free radicals of different forms such as superoxide,
hydroxyl, nitric oxide and hydrogen peroxide are
constantly generated for specific metabolic
requirement and quenched by an efficient
antioxidant network in the body. When the
generation of these species exceeds the levels of
antioxidant mechanism, it leads to oxidative
damage of tissues and biomolecules, eventually
leading to disease conditions, especially
degenerative diseases[1]
. Numerous synthetic
antioxidants like BHA (Butylated Hydroxy
Anisole) and BHT (Butylated Hydroxy Toluene)
are available commercially but their use is
restricted especially in foods as they were reported
to posess toxic and mutagenic effects. This finding
has prompted the search for antioxidant principles
from natural sources.
Several types of plant materials such as vegetables,
fruits, leaves, bark, roots, spices and herbs are
potential sources of antioxidant compounds. Most
of the isolated compounds with antioxidant activity
were found to be phenolic compounds[2]
and studies
have shown that a number of plant products
containing polyphenols, flavonoids, terpenes and
various plant extracts exerted an antioxidant
action[3]
. There is currently immense interest in
natural antioxidants and their role in human health
and nutrition [4]
.
International Journal of Research in
Pharmacology & Pharmacotherapeutics

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The genus Boswellia belonging to the family
Burseraceae is well known for the fragrant resin
they produce and was also claimed to have several
health benefits including anti-inflammatory, cardio
protective, anti-hyperlipidemic etc. Boswellia
ovalifoliolata bal.henry is an endemic medicinal
tree found on the Eastern Ghats. It is commonly
known as Kondasambrani and adaviguggilam. The
leaves, bark and gum of the plant are highly
medicated and are traditionally used in the
treatment of arthritis, dysentery, diabetes and
obesity[5]
. Leaves are used in the treatment of
mouth and throat ulcers [6]
, stem bark is used as
anti-inflammatory [7]
, antibacterial [8]
, anti-
rheumatic and gum is used in amoebic dysentery[9]
.
Phyto chemical studies showed that leaves and gum
contain higher amounts of flavonoids, tannins,
alkaloids, saponins etc. [10].
Saccharum spontaneum
Linn. Commonly known as kasa and wild
sugarcane belonging to family poaceae is
considered as a valuable medicinal herb in
traditional systems of medicine. This occurs
throughout India along the sides of the river and is
widely distributed in Andhra Pradesh and
Tamilnadu. Although many benefits were claimed,
very few scientific reports were available on the
phytochemistry and pharmacology of Saccharum
spontaneum. It is used traditionally in the treatment
of several disorders including mental disturbances,
dyspnoea, anaemia, diuretic, obesity, lithotriptic,
purgative, tonic, aphrodisiac, dyspepsia,
obesity,[11,12]
gynecological troubles, respiratory
troubles etc[13]
. The whole plant including the
stems, leaves and roots are used medicinally.
Vhuiyan et al,[14]
reported the Anti-diarrhoel and
CNS depressant activity of the whole plant and
Anti urolithiatic activity was reported by Satya et
al.[15]
in root. The Phytochemical studies revealed
presence of steroids, terpenoids, quinones and other
poly phenolic compounds. [16]
Taking in view the important phyto constituents
such as phenols reported from the plants and the
lack of systematic studies on the free radical
scavenging activities of the selected plants, the
present study was undertaken to study the
antioxidant potential of the ethanolic extracts by
different methods.
MATERIALS AND METHODS
Chemicals and Instruments
All the chemicals and solvents used in the study
were of analytical grade and are obtained from
Ranbaxy fine chemicals, India. Folin-Ciocalteau’s
Phenol reagent, gallic acid, catechin, 1, 1-diphenyl-
2-picryl hydrazyl (DPPH), Griess reagent, dimethyl
sulphoxide, nitrobluetetrazolium(NBT), vitamin E,
ascorbic acid, deoxy ribose and sodium
nitroprusside were obtained from Sigma Aldrich, St
Louis, USA. UV Spectrophotometer (Shimadzu),
Tissue Homogeniser, Research centrifuge (Remi
Instruments Ltd) and pH meter (Elico Ltd., India)
were used for the study.
Plant material
Fresh whole plant material of Saccharum
spontaneum and gum resin of Boswellia
ovalifoliolata were collected from the hills of
Tirumala region, Chittoor (dist).A.P, India and
authenticated by Dr. Madhavachetty, Assistant
Professor, Department of Botany, S.V. University,
Tirupathi.
Extraction procedure
Freshly collected plant material was dried under
shade and the dried material was milled to obtain a
coarse powder. The coarsely powdered material (1
Kg) was extracted with petroleum ether to remove
the fatty material and further plant material was
packed in Soxhlet apparatus and subjected to
continuous extraction with ethanol (95%). The
liquid extract was collected and concentrated under
reduced pressure until a waxy mass was obtained.
The mass obtained was weighed in each case [17]
.
Preliminary Phytochemical Screening
The ethanolic extracts of Boswellia ovalifoliolata
gum and Saccharum spontaneum whole plant were
tested for the presence or absence of
phytoconstituents like steroids, alkaloids, saponins,
tannins, cardohydrates, proteins, tannins,
flavonoids and other phenolic compounds
according to the standard procedures available in
the literature. [18]
Determination of total phenolic content
The total phenolic content of the extracts was
determined by the Folin-ciocalteau assay. The
extract was diluted with ethanol to a suitable
concentration for analysis. To 1 mL of the extract
or a standard solution of gallic acid (20, 40, 60, 80
and 100 mg/mL), 9 ml of water was added in a 25
ml volumetric flask. Reagent blank was prepared
with water. One milliliter of the Folin-Ciocalteau’s
phenol reagent was added to the mixture and
shaken. After 5 min, 10 mL of 7% sodium

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carbonate solution was added to the mixture. The
solution was diluted to 25 mL with water and
mixed. It was incubated for 90 min at room
temperature and the absorbance against the
prepared reagent blank was determined at 750 nm
with an UV-VIS Spectrophotometer. The data for
the total phenolic content of the extracts were
expressed as milligrams of gallic acid equivalents
(GAE) per 100 grams dry mass (mg GAE/100 g
dw). All the samples were analysed in duplicates
[19]
.
Gallic acid equivalents (GAE) = Absorbance (at
765 nm) / 0.0508
DPPH radical scavenging activity
The assay was performed as described by Bondet et
al [20]
.4.3 mg of DPPH (1, 1-Diphenyl –2-
picrylhydrazyl) was dissolved in 3.3 mL ethanol; it
was protected from light by covering the test tubes
with aluminum foil. A 150 µL of DPPH solution
was added to 3 mL of ethanol and absorbance was
taken immediately at 517nm for control reading. 50
µL of various concentrations of test extracts (15.6
to 1000 µg/mL) as well as standard compound
(ascorbic acid) were taken and the volume was
made uniformly to 150 µL using ethanol. Each of
the samples was then further diluted with ethanol
up to 3 mL and to each 150 µL DPPH was added.
Absorbance was taken after 15 min at 517nm using
ethanol as blank on UV-visible spectrometer
Shimadzu, UV-1601, Japan. The IC50 values for
each extract as well as standard preparation were
determined as the concentration of the text mixture
that gave 50% reduction in absorbance from that of
the control blank. The DPPH free radical
scavenging activity was calculated using the
following formula:
% scavenging = [Absorbance of control - Absorbance
of test sample/Absorbance of control] X 100
Nitric oxide free radical scavenging activity
The principle involved in the assay is that sodium
nitro-prusside in aqueous solution at physiological
pH spontaneously generates nitric oxide, which
interacts with oxygen to produce nitrite ions that
can be estimated using Griess reagent[21]
. 1 mL of
10 mM sodium nitroprusside was mixed with 1 mL
of test solution of different concentrations (15.6 to
1000 μg/ml) as well as the standard compound (α-
Tocopherol)) in phosphate buffer (pH 7.4) and the
mixture was incubated at 25°C for 150 min. From
the incubated mixture, 1 mL was taken out and 1
mL of Griess’ reagent (1% sulphanilamide, 2% o-
phosphoric acid, 0.1% naphthyl ethylene diamine
dihydro chloride) was added to it. Absorbance of
the chromophore formed by the diazotization of
nitrite with sulfanilamide and subsequent coupling
with naphthyl ethylene diamine dihydrochloride
was read at 546 nm and percentage inhibition was
calculated by comparing the results of the test with
those of the control using equation.
% scavenging/Reduction = [Absorbance of (control - test
sample)/Absorbance of control] X 100
Inhibition of lipid peroxidation
Antioxidant activity in linoleic acid
emulsion system
Alkoxy radicals are formed by the redox reaction
with ferrous ions (reducing agents) in a linoleic
acid emulsion at physiological pH. The degree of
oxidation was measured by the thiocyanate method
[22]
. The reaction mixture containing 0.5 mL of the
extract, 2 mL of 0.2 M sodium phosphate buffer,
pH 7.0, and 2.5 mL linoleic emulsion were
incubated at 37°C. 1 mL aliquots were taken at
different intervals during incubation which was
further diluted with 5 mL of 75% ethanol , 200 mM
FeCl2 (0.1 ml) in 3.5% HCl and 30% KSCN were
added to it. The resulting red chromogen of the
ferric (III)-thiocyanate complex could be measured
at 500 nm. The inhibition of lipid peroxidation
(LPO) in percent was calculated by the following
equation:
LPI (%) = [1 - (A1-A2) /A0] x 100
Where A0 was the absorbance of control (the
reaction mixture without the extract), A1 was the
absorbance in the presence of the extract, and A2
was the absorbance without potassium thiocyanate
solution. α-Tocopherol was used as a standard.
Statistical analysis
The results obtained in the studies were expressed
as mean of three parallel measurements. The IC50
values were obtained by linear regression analysis
of dose-response curve plotting between %
inhibition and concentrations [23]
. The results of
antioxidant studies of the selected extracts were
presented in Table 2.
RESULTS AND DISCUSSION
The preliminary phytochemical investigations of
the ethanolic extracts showed the presence of phyto
constituents like steroids, terpenoids, saponins,
flavonoids, alkaloids, glycosides, tannins, and
phenols. The results were presented in Table 1. The
total phenolic content of the extracts was

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determined spectrophotometrically by Folin-
ciocalteu method and calculated as Gallic acid
equivalents (GAE). The amount of phenols in
Boswellia ovalifoliolata and Saccharum
spontaneum were found to be 7.45 and 9.82 (mg/g),
respectively. It was widely accepted that plant
phenolics are effective free radical scavengers.
Different concentrations of ethanolic extracts (15.6
to 1000 µg/ml) were tested for the antioxidant
activity using different in-vitro methods. The
ethanolic extracts of Boswellia ovalifoliolata and
Saccharum spontaneum exhibited dose dependent
scavenging of free radicals.
Table1: Preliminary phytochemical screening of ethanolic extracts of
Boswellia ovalifoliolata and Saccharum spontaneum
S.No Name of the test EBOG ESSW
1. Sterols + +
2. Glycosides + +
3. Saponins + +
4. Tannins + +
5. Alkaloids + +
6. Triterpenoids + +
7. Flavonoids + +
8. Phenols + +
EBOG :Ethanolic extract of Boswellia ovalifoliolata gum,
ESSW: Ethanolic extract of Saccharum spontaneum whole plant
Table 2: Concentration dependent percentage inhibition of ethanolic extracts and
standard in in-vitro studies.
EBOG ESSW Ascorbic acid
DPPH 15.6 24.73 ± 2.08 12.41 ± 1.00 34.11 ± 2.61
31.2 37.79 ± 1.61 37.05 ± 4.36 51.01 ± 1.89
62.5 62.81 ± 1.07 48.65 ± 3.06 65.18 ± 3.48
125 70.63 ± 3.59 60.42 ± 4.05 72.33 ± 2.64
250 77.75 ± 2.09 64.58 ± 3.26 78.34 ± 3.01
500 81.96 ± 1.76 73.99 ± 2.55 83.24 ± 2.39
1000 85.56 ± 0.76 83.7 ± 2.30 85.72 ± 0.62
IC50 (µg/mL) 49.16 88.71 32.05
EBOG ESSW α- Tocopherol
NO 15.6 11.63 ± 1.22 19.96 ± 1.46 21.75 ± 1.35
31.2 18.58 ± 0.97 31.71 ± 3.20 32.01 ± 0.87
62.5 38.95 ± 3.06 43.57 ± 2.33 41.18 ± 0.34
125 54.68 ± 3.9 50.99 ± 1.82 54.82 ± 1.74
250 63.01 ± 1.82 66.22 ± 4.35 65.72 ± 0.57
500 68.21 ± 2.35 74.02 ± 4.28 72.03 ± 1.84
1000 69.95 ± 1.12 85.45 ± 1.50 81.75 ± 1.65
IC50 (µg/mL) 151.13 122.33 101.63
EBOG ESSW α- Tocopherol
LPO 15.6 12.95 ± 1.04 13.47 ± 2.51 28.31 ± 2.51
31.2 28.61 ± 3.09 19.21 ± 2.02 45.03 ± 1.17
62.5 39.58 ± 2.00 37.04 ± 1.66 62.63 ± 3.22
125 63.05 ± 2.50 54.73 ± 1.77 74.81 ± 4.48
250 72.20 ± 1.53 70.84 ± 1.28 82.03 ± 1.87
500 81.62 ± 0.31 77.19 ± 2.17 85.41 ± 0.90
1000 80.56 ± 0.90 87.72 ± 1.58 88.56 ± 1.09
IC50 (µg/mL) 91.67 112.27 39.64
Values are mean ± S.D (n=3)
Method
Conc.
(µg/mL)
% Inhibition
Method
Conc.
(µg/mL)
% Inhibition
Method
Conc.
(µg/mL)
% Inhibition
EBOG: Ethanolic extract of Boswellia ovalifoliolata gum,
ESSW: Ethanolic extract of Saccharum spontaneum whole plant.

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Figure 1: In-vitro concentration dependent percentage inhibition of DPPH
radical by ethanolic extracts and ascorbic acid.
8 16 32 64 128 256 512 1024
0
25
50
75
100
EBOG ESSW Ascorbic acid
Concentration (µg/mL)
%Inhibition
EBOG: Ethanolic extract of Boswellia ovalifoliolata gum.
ESSW: Ethanolic extract of Saccharum spontaneum whole plant.
Figure 2: In-vitro concentration dependent percentage inhibition of Nitric oxide
radical by ethanolic plant extracts and α-Tocopherol.
8 16 32 64 128 256 512 1024
0
25
50
75
100 EBOG ESSW  Tocopherol
Concentration (µg/mL)
%Inhibition
EBOG: Ethanolic extract of Boswellia ovalifoliolata gum.
ESSW: Ethanolic extract of Saccharum spontaneum whole plant.
Figure 3: In-vitro concentration dependent percentage inhibition of Lipid
peroxidation by ethanolic plant extracts and α-Tocopherol.
8 16 32 64 128 256 512 1024
0
25
50
75
100
EBOG ESSW  Tocopherol
Concentration (µg/mL)
%Inhibition
EBOG: Ethanolic extract of Boswellia ovalifoliolata gum.
ESSW: Ethanolic extract of Saccharum spontaneum whole plant.

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DPPH free radical scavenging activity
DPPH assay is one of the quick methods to
evaluate antioxidant activity in-vitro. The DPPH
assay method is based on the reduction of DPPH, a
stable free radical and the proton radical
scavenging action is an important method of
antioxidant activity [24, 25]
. In the DPPH Free radical
scavenging activity, ethanolic extracts of the
selected plants were evaluated for their free radical
scavenging activity with ascorbic acid as standard
compound. The IC50 was calculated for both the
extracts as well as ascorbic acid and summarized in
Table 2 and graphically represented in Figure 2.
The best free radical scavenging activity was
obtained with Ethanolic extract of Boswellia
ovalifoliolata (49.16) while Saccharum
spontaneum showed comparable levels of free
radical scavenging activity with an IC50 value of
88.71 which were relatively higher than the IC50
(32.05) of ascorbic acid. But the highest percentage
of inhibition exhibited by the extracts and standard
at 1000 µg/mL was almost the same. The
scavenging effect increased with the increasing
concentrations of test extract sand lower IC50 value
reflects higher antioxidant activity. From results, it
may be postulated that the test extracts were able to
reduce the stable free radical DPPH to the yellow-
colored diphenyl picrylhydrazine.
Nitric oxide scavenging activity
Nitric oxide (NO) is involved in a variety of
biological functions, including neurotransmission,
vascular homeostasis, antimicrobial, and antitumor
activities. Despite the possible beneficial effects of
NO, its contribution to oxidative damage is also
reported[26]
. Large amounts of NO may lead to
tissue damage[27]
. Nitric oxide scavenging activity
was performed with the test extracts and α-
Tocopherol as standard. The reductive potential of
the ethanolic extracts and standard preparations
were dose dependent. The IC50 values were found
to be 151.13, 122.33, 101.63, respectively for
Boswellia ovalifoliolata, Saccharum spontaneum
and α-Tocopherol. From the graph, it was observed
that as concentration increases, the % scavenging is
increasing linearly for both the extracts and
standard preparation. Compared to Boswellia
ovalifoliolata (69.95), Saccharum spontaneum
showed higher percentage of inhibition of NO
(85.45) at a concentration of 1000 µg/mL.
Inhibition of lipid peroxidation
The peroxidation of membrane lipids by free
radicals leads to cell injury. Lipid peroxidation by
ferrous sulphate initiates a series of oxidative
reactions [28, 29]
. The selected plant extracts not only
exhibited excellent ability for various radical
scavenging activities, but also suppressed lipid
peroxidation. Boswellia ovalifoliolata and
Saccharum spontaneum significantly inhibited the
oxidation of linoleic acid which was determined
by a ferric thiocyanate (FTC) assay. The result
demonstrated that extracts inhibited lipid
peroxidation in a concentration dependent manner
with IC50 values of 91.67 and 112.27 respectively
for Boswellia ovalifoliolata and Saccharum
spontaneum, while α-tocopherol showed IC50 value
at 39.64.
From the above results it can be concluded that
the ethanolic extracts of the selected medicinal
plants exhibited significant antioxidant activity and
the activity may be related to the phenolic
compounds present in them. Since reactive oxygen
species are important contributors to several
disorders in the body, the extracts might be useful
for the development of newer and more potent
natural antioxidants that are useful in preventing
or slowing the progress of aging and
oxidative stress related degenerative diseases.
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