International Journal of Pharmacy Research and Technology

2012, Volume 2, Issue 4, 23-27

ISSN 2250 – 0944 (Online)
ISSN 2250 – 1150 (Print)
ResearchArticle

Comparison of Antioxidant and Free Radical Scavenging Potential of Polyherbal Formulations of SIDITI

Dipak S. Patel*, P. B. Shah, N. B. Managoli

Department of Pharmaceutical sciences, Jodhpur national university, Jodhpur, Rajasthan
Sahajanand Life Sciences Pvt. Ltd., Surat - 395004, Gujarat, India
Shri B. M. Shah Diploma Pharmacy college, College campus, Dhansura road, Modasa
*Corresponding author Email:gajeradipak@gmail.com
Received: 29/08/2012, Revised: 05/10/2012 Accepted: 17/11/2012
ABSTRACT
The present investigation was undertaken to compare the in vitro potential of SIDITI formulations (Existed (EF), New
(NF)). In-vitro comparison of antioxidant potential of SIDITI formulations was done by Hydroxyl radical scavenging
activity, Hydrogen peroxide‐scavenging assay, Super oxide scavenging activity, Reducing power, Chelating capacity, Total
antioxidant activity. Siditi NF exhibits powerful super oxide radical scavenging activity, reducing power, total antioxidant
activity as compare to Siditi EF. It was also observed that Siditi NF possesses better hydroxyl radical scavenging activity and
hydrogen peroxide‐scavenging potential compare to Siditi EF. While chelating power of both the formulation was almost
similar. The overall antioxidant activity of both the formulations might be attributed to its polyphenolic content as well as
other phytochemicals constituents. The findings of the present study suggest that Siditi NF could be a potential source of
natural antioxidant that could have great importance as therapeutic agents in preventing or slowing the progress of gastric
ulceration and lesion compare to Siditi EF.

Keywords: Hydroxyl radical scavenging activity, hydrogen peroxide‐scavenging activity, super oxide radical scavenging
activity, chelating activity, total antioxidant activity.

INTRODUCTION scavenging activity, reducing power, chelating activity,

Reactive oxygen species (ROS) such as singlet oxygen
(.O2), superoxide anion (O2-) and hydroxyl radical (.OH)
and hydrogen peroxide (H2O2) are often generated as by-
products of biological reactions or from exogenous factors
[1-3]
.These reactive species exert oxidative damaging effects
by reacting with nearly every molecules found in living
cells[4] including DNA, if excess ROS are not eliminated by
antioxidant system. Many synthetic antioxidant compounds
have shown toxic and/or mutagenic effects, which have
stimulated the interest of many investigators to search
natural antioxidant. In view of this the search for natural
antioxidants as alternatives is therefore of great interest
among researchers [5, 6]. It is believed that the herbal
products have less side effects and toxicities.
Siditi is polyherbal Ayurvedic medicine used as
antacid, for peptic ulcer and Gastro Oesophageal Reflux
Disease (GERD). New formulation was prepared by using
some of the extracts of existing formulation at different
concentration. Existed Formulation (EF) contains
standardized herbal extracts of Glycyrrhiza glabra
(Increase gastric mucous, Gastric motility, Regulate gastric
acidity and bile secretion), Cyperus rotundus (Regulate bile
secretion, Antiemetic, Regulate gastric acidity), Aloe vera
(Regulate gastric mucous level, Improves gastric motility),
Zingiber officinale (Antiemetic, Digestive tonic), Withania
somnifera (Antioxidant, Antistress), Triphala (Gastric tonic,
Improves gastric motility), while New Formulation (NF)
contains standardized herbal extracts of Glycyrrhiza glabra,
Triphala, Cyperus rotundus, Zingiber officinale, Withania
somnifera.
In the present study, two different formulations of
Siditi was evaluated for its antioxidant activities by in vitro
methods, including hydroxyl radical scavenging activity,
hydrogen peroxide‐scavenging activity, super oxide radical
total antioxidant activity. Combination of herbs with
specific concentration will help to get synergistic
antioxidant potential of polyherbal formulation.
MATERIALS AND METHODS
Preparation of Siditi (EF and NF)
All standardized extracts for formulation, chemicals,
and solvents were of A. R. grade and provided by
Sahajanad Life sciences Pvt. Ltd., Surat. All the
standardized herbal extracts passed through 80 # sieve and
then mixed together in specified given proportions to get
uniformly blended Siditi-EF and Siditi-NF.
Preparation of sample
Aqueous extract – 10gm mixture of both the
formulation individually extracted with distilled water by
cold extraction process for 24 h. After completion of the
extraction, the solution was recovered by filtration. Fresh
sample was prepared and used for each test. Both the
formulations (Siditi-EF and Siditi-NF) were tested at the
concentration of 25, 50, and 100 µg/ml.
Methods
Hydroxyl radical scavenging activity[6]
The scavenging activity for hydroxyl radicals was
measured with fenton reaction. Reaction mixture contained
60 µL of 1.0 mM FeCl2, 90 µl of 1mM 1,10-phenanthroline,
2.4 mL of 0.2 M phosphate buffer (pH 7.8), 150 µL of 0.17
M H2O2, and 1.0 mL of extract at various concentrations.
Adding H2O2 started the reaction. After incubation at room
temperature for 5 min, the absorbance of the mixture at 560
nm was measured with UV visible spectrometer Shimadzu,
UV-1800, Japan. The percentage inhibition of hydroxyl
scavenging activity was calculated using the following
formula,

IJPRT | October – December | 23

 Patel et al / International Journal of Pharmacy Research & Technology 2012 2(4)
% inhibition of Hydroxyl scavenging activity
Absorbance !"#
1 ) * 100
Absorbance $%&'(#
Where, Abs (Blank): Absorbance of the control (Without
extract) andAbs (Test): Absorbance of the test (With
extract)
Figure 1: % inhibition of hydroxyl radical scavenging
activity (Values are mean ± SD of triplicate determinations)
Hydrogen peroxide‐‐scavenging activity [7, 8]
A solution of hydrogen peroxide (2 mmol/l) was
prepared in phosphate buffer (pH 7.4). Extract at various
concentrations were added to hydrogen peroxide solution
(0.6 ml). For each concentration, a separate blank sample
was used for background subtraction. Absorbance of
hydrogen peroxide at 230 nm was determined after 10 min
against a blank solution containing phosphate buffer
without hydrogen peroxide by UV visible spectrometer
Shimadzu, UV-1800, Japan.. The percentage inhibition of
H2O2 scavenging activity was calculated using the
following formula,
% inhibition of H O scavenging activity
Absorbance !"#
1 ) * 100
Absorbance $%&'(#
% inhibition of H2O2 scavenging activity = [1 – (Abs (Test)
/ Abs (Blank)] × 100
Where, Abs (Blank): Absorbance ce of the control (Without
extract) and Abs (Test): Absorbance of the test (With
extract)
Figure 2: % inhibition of hydrogengen peroxide scavenging
activity (Values are mean ± SD of triplicate determinations)
Super Oxide radical scavenging activity[9, 10]
100 µll Riboflavin solution (20 µg),
µ 200 µl EDTA
solution (12 mM), 200 µll methanol and 100 µl NBT (Nitro-
blue tetrazolium) solution (0.1 mg) were mixed in test tube
and reaction mixture was diluted up to 3 ml with phosphate
24 | IJPRT | October – December
2( 23-27
buffer (50 mM). The absorbance of solution was measured
at 590 nm using phosphate buffer as blank after 5min. This
is taken as control (Blank). Different concentrations of
extracts were diluted up to 100 µl with methanol, to each of
this, 100 µl Riboflavin, 200 µl EDTA, 200 µl methanol and
100 µll NBT were mixed in test tubes, further diluted up to 3
ml with phosphate buffer. Absorbance was measured after 5
min. at 590 nm was measured with UV visible spectrometer
Shimadzu, UV-1800, Japan.. The percentage scavenging
activity was calculated using the following formula,
Absorbance !"#
% scavenging activity 1 ) * 100
Absorbance $%&'(#
Where, Abs (Blank): Absorbance of the control (Without
extract) and Abs (Test): Absorbance of the test (With
extract)
Reducing power[11]
In this assay the ability of the extract to reduce Fe3+ to
2+
Fe was estimated. Reducing power of the extracts was
determined by using potassium ferricyanide-ferric
ferricyanide chloride
system. Briefly, 1 ml of extract at different concentration
was added with 2.5ml of 0.2 M phosphate buffer (pH 6.6),
mixed with 2.5ml of potassium ferricyanide (0.1%) and the
mixture was incubated at 50°C for 20 min. 2.5ml of
trichloroacetic acid (10%) was added to the reaction
mixture and centrifuged at 8000g for 10 min and 2.5ml of
supernatant was mixedd with equal volume of distilled water
& 0.5ml of 0.1% ferric chloride was added and the
absorbance was measured at 700 nm using UV visible
spectrometer Shimadzu, UV-1800, 1800, Japan. Increased
absorbance of the reaction mixture indicated the increased
reducing power.
Chelating activity[12]
To 1 ml of different concentration of extract was
treated with an equivalent amount of reaction mixture
which contains 1 ml, 0.05% ortho – phenanthroline in
methanol, 2ml ferric chloride (200 mM).
mM The treated
compound was incubated at ambient temperature for 10
min and the absorbance of same was measured at 510 nm.
Absorbance !"#
% Chelating activity 1 ) * 100
Absorbance $%&'(#
Where, Abs (Blank): Absorbance of the control (Without
extract) and Abss (Test): Absorbance of the test (With
extract)
Total antioxidant activity[13]
The assay is based on the reduction of Mo (VI) - Mo
(V) by the extract and subsequent formation of a green
phosphate / Mo (V) complex at acidic pH. Different
concentration of (0.3 ml) extracts was combined with 3 ml
of reagent solution (0.6 M sulfuric acid, 28 mM sodium
phosphate and 4 mM ammonium molybdate). The tubes
containing the reaction solution were incubated at 95°C for
90 min. Then the absorbance
bance of the solution was measured
at 695 nm using spectrophotometer against blank after
cooling to room temperature. Ascorbic acid equivalents
were calculated using standard graph of ascorbic acid. The
experiment was conducted in triplicates and values are a
expressed as equivalents of ascorbic acid in µg per mg of
extract.
 Patel et al / International Journal of Pharmacy Research & Technology 2012 2(4)
Statistical analysis
All assays were carried out in triplicates and results are
expressed as mean ± SD.
Figure 3: % Superoxide radical scavenging activity
(Values are mean ± SDof triplicate determinations)
RESULTS AND DISCUSSION
The antioxidant activity has been assigned to various
mechanisms such as prevention of chain initiation, binding
of transition-metal
metal ion catalysts, decomposition of
peroxides, and prevention of continued hydrogen
abstraction, reductive capacity and radical scavenging. In
the present study, we investigated the antioxidant activity of
Siditi EF and Siditi NF by Hydroxylydroxyl radical scavenging
activity, hydrogen peroxide‐scavenging
scavenging activity,
activity super
oxide radical scavenging activity,, Reducing power,
chelating activity and total antioxidant activity.
activity
A single hydroxyl radical can result in formation of
many molecules of lipid hydroperoxides in the cell
membrane, which may severely disrupt its function and
lead to cell death. Hydroxyl radicals are most reactive
species, initiating the peroxidation of the cell membrane
[20]
. The lipid radical, thus generate would initiate chain
reaction in the presence of oxygen, giving rise to lipid
peroxide, which break down to aldehydes such as
malondialdehyde, which are known to be mutagenic and
carcinogenic. Hydroxyl radical is highly reactive oxygen
centered radical formed from the reaction of various
hydroperoxides with transition metal ions. It attacks
proteins, DNA, polyunsaturated fatty acid in membranes,
and most biologicalical molecule it contacts [7, 8] and is known
to be capable of abstracting hydrogen atoms from
membrane lipids and brings about peroxidic reaction of
lipids. Activity of the extract on hydroxyl radical has been
shown in Figure 1 (At high concentration (100 µg / ml)
siditi NF shown better hydroxyl radical scavenging
activity). Hydrogen peroxide can cross cell membranes
rapidly, once inside the cell, H2O2 can probably react with
Fe2+, and possibly Cu2+ ions to form hydroxyl radical and
this may be the origin of many of its toxic effects [14, 15]. As
shown in Figure 2,, both the formulations demonstrated
hydrogen peroxide decomposition activity in a
concentration dependent manner. The decomposition of
H2O2 by Siditi-EF and Siditi-NF may ay at least partly result
from its antioxidant and free radical scavenging activity.
Superoxide has been observed to directly initiate lipid
peroxidation [14, 15]. Superoxides are produced from
molecular oxygen due to oxidative enzymesof body as well
as via ia non enzymatic reactions such as auto oxidation by
catecholamines.Superoxides
Superoxides are produced from molecular
oxygen due to oxidative enzymes [21] of body as well as via
2( 23-27
non enzymatic reactions such as auto oxidation by
catecholamines. Figure 3 shown superoxide scavenging
activity of both the formulation was increased markedly
with the increase in concentrations. Both the formulations
found to scavenge the superoxides generated by photo
reduction of riboflavin. Siditi - NF shown excellent
percentage
tage scavenging inhibitory effects in dose dependant
manner.
Figure 4: Comparisons in Reduction power of both of the
formulations(Values
Values are mean ± SD of triplicate
determinations)
Ferrous iron can stimulate lipid peroxidation by the
Fenton reaction and also accelerates peroxidation by
decomposing lipid hydroperoxides into peroxyl and alkoxyl
radicals that can themselves abstract hydrogen and
perpetuate the chain reaction of lipid peroxidation [16–18].
For the measurements of the reducing ability, the Fe3+–Fe2+
transformation was investigated in both of the formulation.
The reducing capacity of a compound may serve as a
significant indicator of its potential antioxidant activity.
Figure 4 shown Siditi-EF EF and Siditi-NF
Siditi have marked
capacity for iron binding, suggesting the presence of
polyphenols that has potent iron chelating capacity. The
reducing power of a compound is related to its electron
transfer ability and may serve as a significant indicator of
its potential antioxidant activity [14, 15].
Figure 5: % Chelating power of both of the formulations
(Values are mean ± SD of triplicate determinations)
O-phenanthroline
phenanthroline quantitatively forms complexes with
Fe+2 which get disrupted in the presence of chelating agents
[22]
. Both of the formulations interfered with the formation
of a ferrous-o-phenanthroline
phenanthroline complex, thereby
the suggesting
that the formulations have metal chelating activity. Ortho –
substituted phenolic compounds may exert pro-oxidant
pro
effects by interacting with iron. ron. O – phenanthroline
quantitatively forms complexes with ferric ion which get
IJPRT | October – December | 25
 Patel et al / International Journal of Pharmacy Research & Technology 2012 2(4)
disrupted in the presence of chelating agents. The extracts
interfered with the formation of ferrous – O –
phenanthroline complex, thereby suggesting that the extract
has metal chelating activity [22]. As per the assay, Figure5
presents the reductive capabilities of both the formulation.
The reducing power of both the formulation extract might
be due to their hydrogen‐donating
donating ability. Possibly,
formulations could react with radicals
dicals to stabilize and
terminate radical chain reactions. The data suggest that
reducing power of Siditi NF was better than Siditi EF.
The total antioxidant activity of the extracts was
calculated based on the formation of phosphomolybdenum
complex which was measured spectrophotometrically [24].
Total antioxidant capacity by Phosphomolybdenum method
assay is based on the reduction ofMo(VI) toMo(V) by the
sample analyte and the subsequent formation of green
phosphate/Mo (V) complex at acidic pH. The
phosphomolybdenum
omolybdenum method is quantitative since the total
antioxidant activity is expressed as the number of
equivalents of ascorbic acid [25]. Aqueous test extracts of
ten different vegetables showed very potent total
antioxidant capacity. The total antioxidant capacity [18, 19]
was concluded from Figure 6,, Both the formulation has
shown the dose dependant efficacy among which the
maximum total antioxidant potential was found with Siditi
NF.
Figure 6: Total antioxidant activity (Values
Values are mean ± SD
S 14. Nabavi SM, Ebrahimzadeh MA, Nabavi SF and
of triplicate determinations)
CONCLUSION
In present study, antioxidant activities of Siditi NF and
Siditi EF were investigated. The results obtained in the
present study indicate that Siditi NF exhibits powerful
super
uper oxide radical scavenging activity,
activity reducing power,
total antioxidant activity as compare to Siditi EF.EF It was
also observed that Siditi NF possesses better hydroxylh
radical scavenging activity and h
hydrogen
peroxide‐scavenging
scavenging potential compare to Siditi EF. While
chelating power of both the formulation was almost similar.
The overall antioxidant activity of both the formulation
might be attributed to its polyphenolic content and other
phytochemicals constituents. The findings of the present
study suggest that Siditi NF could be a potential source of
natural antioxidant that could have great importance as
therapeutic agents in preventing or slowing the progress of
gastric ulceration and lesion compare to Siditi EF.
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