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EVALUATION OF IN-VITRO ANTIOXIDANT ACTIVITY OF PANCHAGAVYA: A TRADITIONAL AYURVEDIC PREPARATION

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Panchagavya, a classical Ayurvedic preparation, was evaluated for its antioxidant potential by HPTLC-DPPH bioautography method as well as assays for Ferric reducing antioxidant power (FRAP), DPPH – free radical scavenging activity (DPPH) and Superoxide radical scavenging activity. In addition total phenolic content was also estimated which was in fairly good amount. HPTLC-DPPH bioautography study revealed the presence of several antioxidant compounds in Panchagavya. In all the assays performed, it showed considerable antioxidant activity. On comparison of the data of three different batches of the samples studied, it showed 98.3 -99.8% correlation between total phenolic content, FRAP and DPPH assays. INTRODUCTION: Ayurveda, the ancient Indian system of medicine, with its holistic approach takes into account the aetiological factors, disease condition, patient's psycho-somatic condition, food and even activities altogether while deciding the line of treatment. It mentions use of various plant, animal and mineral products as such and in unique combinations for treatment and positive health maintenance.
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Athavale et al., IJPSR, 2012; Vol. 3(8): 2543-2549 ISSN: 0975-8232
Available online on www.ijpsr.com 2543
IJPSR (2012), Vol. 3, Issue 08 (Research Article)
Received on 21 April, 2012; received in revised form 18 May, 2012; accepted 21 July, 2012
EVALUATION OF IN-VITRO ANTIOXIDANT ACTIVITY OF PANCHAGAVYA: A TRADITIONAL AYURVEDIC
PREPARATION
Arun Athavale, Nikhil Jirankalgikar, Pankaj Nariya and Subrata De*
RMD Research and Development Center, Waghaldhara, Dist-Valsad- 396 375, Gujarat, India
ABSTRACT
Panchagavya, a classical Ayurvedic preparation, was evaluated for its
antioxidant potential by HPTLC-DPPH bioautography method as well as
assays for Ferric reducing antioxidant power (FRAP), DPPH free radical
scavenging activity (DPPH) and Superoxide radical scavenging activity. In
addition total phenolic content was also estimated which was in fairly good
amount. HPTLC-DPPH bioautography study revealed the presence of several
antioxidant compounds in Panchagavya. In all the assays performed, it
showed considerable antioxidant activity. On comparison of the data of three
different batches of the samples studied, it showed 98.3 - 99.8% correlation
between total phenolic content, FRAP and DPPH assays.
INTRODUCTION: Ayurveda, the ancient Indian system
of medicine, with its holistic approach takes into
account the aetiological factors, disease condition,
patient’s psycho-somatic condition, food and even
activities altogether while deciding the line of
treatment. It mentions use of various plant, animal and
mineral products as such and in unique combinations
for treatment and positive health maintenance.
Many materials, which can hardly be termed as drug
by prevalent norms, are also utilized in the treatment.
Panchagavya- a peculiar combination of five cow
products namely dung, urine, milk, curd and ghee 1, is
one such drug which has been advocated in Ayurvedic
classics as such in Vishamajvara 2 (malaria/typhoid), for
detoxification of body/toxicity management 1 while in
processed form (viz. Panchagavya ghrita) for
psychogenic and neurogenic disorders 3.
In recent days, its use in management of cancerous
conditions is also a practice in vogue.
Free radicals play an important role in degenerative
diseases like cancer, cataract, immune system
weakness and brain disorders, collectively called
oxidative stress 4. Amount/availability of free radicals
are controlled by systems called antioxidants which
can reduce oxidation rate considerably and are
synthesized in the body as well as supplied by dietary
sources and nutraceuticals. Applicability of
antioxidants in management of oxidative stress related
disorders has been suggested 5.
Since oxidative stress plays major role in aetio-
pathogenesis of cancer, present study of in vitro
antioxidant activity of Panchagavya was undertaken to
evaluate its role in cancer therapy. This is probably the
first report of antioxidant activity of Panchagavya.
MATERIALS AND METHODS:
Chemicals and Instrument: 2, 2 Diphenyl-1-picryl
hydrazil (DPPH; Sigma-Aldrich, Germany), 2, 4, 6-Tri-(2-
pyridyl)-s-triazine (TPTZ; SRL chemicals, Mumbai),
Key words:
Panchagavya,
Antioxidant,
HPTLC-Bioautography,
FRAP,
DPPH,
Superoxide
Correspondence to Author:
Dr. Subrata De
Director, RMD Research and Development
Center, NH No. 8, Waghaldhara, Dist-
Valsad- 396 375, Gujarat, India
E-mail: subratde@gmail.com
Athavale et al., IJPSR, 2012; Vol. 3(8): 2543-2549 ISSN: 0975-8232
Available online on www.ijpsr.com 2544
Nitroblue tetrazolium chloride (NBT), Riboflavin,
Methionine, Gallic acid (Lobachemie, Mumbai), all
other chemicals of Merck India pure or GR grade.
Absorbance was noted using a Perkin-Elmer Lambda
25 UV-Visible spectrometer.
The experiments were carried out at RMD Research
and Development Center during October 2011 to
January 2012.
Preparation of Panchagavya: 25 ml of freshly collected
cow urine was added to 12.5 g freshly collected cow
dung and uniformly mixed by stirring. The mixture was
passed through a cotton cloth by squeezing and then
strained repeatedly (7 times) through a cloth. To the
filtrate 5 ml cow milk (boiled and cooled) and 5 ml curd
(prepared by using cow milk) were added, mixed
thoroughly, to it 4 ml molten cow ghee was added and
again mixed well to prepare a homogeneous mixture.
This freshly prepared Panchagavya was used for the
study.
For the present study three batches of Panchagavya
were prepared in three different days.
Preparation of sample: To 1 g Panchagavya 10 ml
methanol was added, stirred well and sonicated for 60
minutes. It was filtered; the volume of the filtrate was
made to 10 ml with methanol (PG) and was used for
further study.
A. Determination of Total Phenolic Content: The
total phenolic content (TPC) of the samples was
determined with Folin-Ciocalteu reagent according
to method described by Andrew6 with slight
modifications. It is based on the reduction of the
reagent (a mixture of tungsten and molybdenum
oxides) and measuring the absorbance of the
product (blue colour) at 765 nm.
Reagents/Solutions: Working Folin-Ciocalteu
reagent - 50% v/v aqueous solution of Folin-
Ciocalteu reagent, Sodium carbonate solution -
20% w/v, Standard Gallic acid solution - 0.1 mg/ml
(freshly prepared).
To 0.1 ml of the sample (PG), 3.9 ml distilled water
and 0.25 ml of working Folin-Ciocalteu reagent
were added. After 5 minutes (but before 8
minutes), 0.75 ml of sodium carbonate solution
was added, mixed and incubated at room
temperature for 60 minutes. The absorbance was
measured at 765 nm. Different volumes of Gallic
acid solution were used in same manner for
calibration of standard curve and quantification
was done in terms of mg equivalent of Gallic acid.
The blank was prepared by using distilled water in
place of sample/standard.
B. Evaluation for Antioxidant Activity:
i. Rapid screening for in vitro Antioxidant Activity by
HPTLC-Bioautography method: For preliminary
screening of the sample, the antioxidant activity
was evaluated by using HPTLC-DPPH
bioautography method 7, 8. HPTLC was performed
on 10 cm x 10 cm aluminum backed plates coated
with 0.2 mm layers of silica gel 60F254 (Merck,
Germany). Defatted methanol extract of
Panchagavya was applied in band with a Linomat V
applicator (CAMAG, Switzerland), equipped with a
100-μl syringe. Plates were developed vertically, in
a CAMAG twin trough chamber previously
saturated with mobile phase vapor for 20 min at
room temperature. Toluene - Ethyl acetate -
Glacial acetic acid (6:4:0.2) was used as mobile
phase. After development the plates were dried at
room temperature, sprayed with 0.2% methanolic
DPPH solution and observed after keeping the
plate in dark for 30 minutes. Presence of
antioxidant compounds were detected by yellowish
spots against a purple background.
ii. In vitro Antioxidant Assay:
a. Assay for in vitro Ferric Reducing Antioxidant
Power (FRAP assay): The FRAP assay was carried
out by the method described by Benzie and
Strain9 with slight modifications. It is based on
the principle of reduction of Fe3+-TPTZ to Fe2+-
TPTZ complex at low pH which gives blue color
and can be measured at 593 nm.
Preparation of FRAP working reagent: Acetate
buffer of 300 mM concentration and pH 3.6 was
prepared by using appropriate volumes of
sodium acetate anhydrous, glacial acetic acid and
distilled water. TPTZ solution of 10 mM
concentration was prepared in 40 mM
hydrochloric acid. Aqueous ferric chloride
Athavale et al., IJPSR, 2012; Vol. 3(8): 2543-2549 ISSN: 0975-8232
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solution of 24.998 mM concentration was
prepared using ferric chloride anhydrous. Acetate
buffer, TPTZ solution and freshly prepared ferric
chloride solution were mixed in 10: 1: 1
proportions to prepare the FRAP working
reagent.
0.1 ml of PG was added to 3.0 ml of FRAP
working reagent, mixed well and absorbance was
measured after 10 minutes. Freshly prepared
aqueous ascorbic acid solution (0.1 mg/ml) was
used as standard. Different volumes of ascorbic
acid solution (equivalent to 10 80 µg) were
used in same manner for calibration of standard
curve and quantification was done in terms of mg
equivalents of ascorbic acid. The blank was
prepared by using distilled water in place of
sample/standard.
b. Assay for in vitro DPPH- Free Radical Scavenging
Activity (DPPH Assay): DPPH radical gives strong
absorbance at 517 nm (deep violet color) due to
its unpaired electron. When this radical pairs off
in presence of a free radical scavenger, the
absorption vanishes and the resulting
discoloration is stoichiometric with respect to the
number of electrons taken up.
DPPH free radical scavenging activity assay of
Panchagavya was carried out using reported
method 10-12 with suitable modifications.
Reagent/solutions: DPPH solution - 0.3 mM in
methanol (freshly prepared), Standard Ascorbic
acid solution 1 mg/ml in methanol.
Sample preparation: 1 ml of PG was dried on mild
heat in a water bath; the residue was taken with
methanol to make 1mg/ml (PGE1) and used for
the test.
Different volumes (equivalent to 5 - 300 µg) of
PGE1/standard were taken in a set of test tubes
and methanol was added to make the volume to
3 ml. To this, 1 ml of DPPH reagent was added
mixed thoroughly and absorbance was recorded
at 517 nm after 30 minutes incubation in dark at
room temperature. 1 ml of DPPH reagent diluted
to 4 ml with methanol was taken as reagent
blank. Percent scavenging activity was calculated
as
% Scavenging =
A0-AS
X 100
A0
Where, A0 = Absorbance of reagent blank, AS =
Absorbance of sample/standard
c. Assay for Superoxide radical scavenging: The
method is based on scavenging of superoxide
anion generated in light induced reaction mixture
having absorbance at 560 nm due to blue
complex formed by NBT; antioxidants inhibit blue
complex formation. The decrease of absorbance
at 560 nm in presence of sample thus indicates
the consumption of superoxide anion in the
reaction mixture 13, 14. The method was adapted
with minor modifications.
Reagent/solutions: Stock solutions of 130 µM
riboflavin, 1 mg/ml NBT and 40 µM ethylene di-
amine tetra acetic acid (EDTA) di-sodium salt (all
in distilled water) were prepared. Phosphate
buffer 50 µM, pH 7.8 was prepared by using
appropriate quantities of di-sodium hydrogen
phosphate (Na2HPO4) and potassium di-hydrogen
phosphate (KH2PO4).
1.539 ml of Riboflavin, 6.132 ml of NBT, 2 ml of
EDTA stock solutions were mixed in this
sequence. To it 65-70 ml of phosphate buffer was
added and mixed thoroughly. To this mixture,
193.96 mg of methionine was dissolved
completely and the resultant solution was diluted
to 120 ml with phosphate buffer and was used as
reaction mixture.
Freshly prepared 1 mg/ml aqueous solution of
ascorbic acid was used as standard.
Sample preparation: 1 ml of PG was dried on mild
heat in a water bath, the residue was taken with
water to make 1mg/ml, sonicated for 15 minutes
to prepare a homogeneous suspension (PGE2)
and used for the test in different concentration
range. Different volumes of PGE2/standard were
taken in test tubes, 3 ml of the reaction mixture
were added, mixed and phosphate buffer was
added to make the volume to 4.5 ml.
Athavale et al., IJPSR, 2012; Vol. 3(8): 2543-2549 ISSN: 0975-8232
Available online on www.ijpsr.com 2546
After mixing the tubes were exposed to
fluorescent light (30 W) for 40 minutes (taking
care of equal illumination exposure) and the
absorbance was measured at 560 nm. 3 ml of
reaction mixture diluted to 4.5 ml with
phosphate buffer was taken as reagent blank.
Percent scavenging activity was calculated as
% Scavenging =
A0-AS
A0
Where, A0 = Absorbance of reagent blank, AS =
Absorbance of sample/standard
RESULTS AND DISCUSSION:
1. Total Phenolic Content of Panchagavya: The total
phenolic content was determined by Folin-
Ciocalteu reagent. The method used is the one
basically used for determination of total phenol
content in wine industry and capable of
differentiating inter-batch differences 6. The
standard curve of gallic acid has been presented in
Figure 1.
FIGURE 1: STANDARD CURVE OF GALLIC ACID
The total phenolic content of the samples, expressed in
terms of mg gallic acid / g of Panchagavya has been
presented in Table 1.
TABLE 1: TOTAL PHENOLIC CONTENT OF PANCHAGAVYA
Batch
TPC /g*
Mean + SD
Batch I
0.576
0.551 + 0.031
0.561
0.516
Batch II
1.525
1.655 + 0.115
1.742
1.698
Batch III
0.694
0.712 + 0.023
0.703
0.739
*in terms of Gallic acid equivalents (mg)
The total phenolic content in three batches of
Panchagavya is in between 0.55 to 1.65 mg/gm in
terms of gallic acid equivalents, which is significant
as Panchagavya daily dose is about 150 ml i.e.
about 160 g and is comparable with plant materials
of known antioxidant activity 13, 15-20.
The response of Folin-Ciocalteu reagent depends
on chemical structures of phenolics which are of
various types and of different polarities.
Panchagavya being a natural product combination,
variation is expected.
2. Antioxidant activity:
A. HPTLC-DPPH Bioautography method: The
preliminary screening of Panchagavya for
antioxidant activity was carried out by HPTLC-DPPH
bioautography method. Spraying the plate with
DPPH solution revealed several yellowish coloured
spots (Figure 2) indicating the presence of number
of antioxidant compounds in Panchagavya.
FIGURE 2: CHROMATOGRAM AFTER DPPH SPRAY
i. In vitro antioxidant assay:
a. Ferric Reducing Antioxidant Power (FRAP): The
FRAP activity was determined in terms of
ascorbic acid equivalents. The ascorbic acid
standard curve for FRAP is shown in Figure 3.
Athavale et al., IJPSR, 2012; Vol. 3(8): 2543-2549 ISSN: 0975-8232
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FIGURE 3: STANDARD CURVE OF ASCORBIC ACID
The FRAP value varied between 0.22 - 0.89 mg/g of
Panchagavya (Table 2), keeping a correlation with their
phenolic content.
TABLE 2: FRAP VALUE OF PANCHAGAVYA SAMPLES
Batch
FRAP value/g*
Mean + SD
Batch I
0.256
0.222 + 0.029
0.203
0.208
Batch II
0.784
0.891 + 0.093
0.940
0.951
Batch III
0.401
0.434 + 0.044
0.419
0.484
*in terms of Ascorbic acid equivalents (mg)
ii. DPPH Radical Scavenging Activity: For DPPH
scavenging activity, ascorbic acid was used as
standard and the standard curve is presented in
Figure 4.
FIGURE 4: STANDARD CURVE OF ASCORBIC ACID (DPPH)
DPPH scavenging activity of Panchagavya samples
has been presented in Table 3.
TABLE 3: DPPH SCAVENGING ACTIVITY OF PANCHAGAVYA
SAMPLES
Batch
Concentration (µg)
% scavenging
Batch I
100
9.25
150
11.63
200
14.42
Batch II
100
9.32
150
18.08
200
22.91
Batch III
100
9.05
150
17.07
200
21.08
The DPPH scavenging activity of PGE1 revealed a
peculiar pattern. The activity tends to decrease
beyond the concentration 200 µg (Figure 5).
FIGURE 5: DPPH ACTIVITY TREND
The usual trend to show scavenging activity is by
indicating IC50 (Half-maximal Inhibitory concentration)
which Panchagavya is unable to show. The reasons
may be of interference by other chemical molecules
competing for reduction by DPPH or having higher
internal activity scavenging chain reaction, not
permitting DPPH be donated with an electron. IC50 for
ascorbic acid was 15.78 µg.
iii. Superoxide Radical Scavenging Activity: The
Superoxide radical scavenging activity of the
samples has been presented in Table 4.
Athavale et al., IJPSR, 2012; Vol. 3(8): 2543-2549 ISSN: 0975-8232
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TABLE 4: SUPEROXIDE SCAVENGING ACTIVITY OF
PANCHAGAVYA SAMPLES
Sample ID
Concentration (µg)
% scavenging
Batch I
30
4.55
60
19.89
90
23.65
Batch II
30
5.57
60
23.04
90
20.36
Batch III
30
23.57
45
24.07
60
17.69
By this method Panchagavya showed concentration
dependent antioxidant activity in the range of 20-
100 µg and then activity decreases with increase of
concentration like in the case of DPPH scavenging
activity. Superoxide scavenging activity of
Panchagavya in the range of 20-100 µg varied from
4 to 24 % (Figure 6). Required concentration of
ascorbic acid standard for 24 % scavenging activity
was about 45 µg.
FIGURE 6: SUPEROXIDE SCAVENGING ACTIVITY CONCENTRATION
WISE TREND
Superoxide anion is a reduced form of molecular
oxygen and has a role in oxidation reaction
associated with ageing 21. Superoxide can directly
initiate lipid peroxidation 22.
The batch wise trend of TPC as well as FRAP and
DPPH activity was compared and is shown in Figure
7.
FIGURE 7: COMPARISON OF BATCHWISE TRENDS
Their correlation level was between 98.3 to 99.8 %
(Table 5).
TABLE 5: CORRELATION BETWEEN BATCHWISE TRENDS
Correlation between
Percentage
TPC and FRAP
98.38%
TPC and DPPH
99.86%
FRAP and DPPH
99.19%
Among naturally occurring antioxidants, both water
soluble and lipid soluble antioxidants are present. The
major water soluble antioxidants are glutathione, α-
lipoic acid, ascorbic acid, polyphenols and bio-
flavanoids while lipid soluble antioxidants are vitamin
E, α-lipoic acid, co-enzymeQ10, polyphenols and bio-
flavanoids.
Free radicals are generated in both aqueous and lipid
portions of intracellular and extracellular environments;
so it is crucial for the body to have a combination of
water soluble and lipid soluble antioxidants to acquire
the full range of protection. Some types of antioxidants
are synthesized by the body while others are obtained
from external sources like food, nutraceuticals 5.
Panchagavya is a unique preparation having both
water based (colloidal milk without fat portion, urine,
curd and dung) and fat based (ghee, milk with fat
particles) products. It is likely to provide both polar and
non-polar natural antioxidants.
The data of the antioxidant study revealed uncommon
activity limiting behavior. The probability of its higher
activity in low concentration at cellular level cannot be
ruled out but requires detail study.
Athavale et al., IJPSR, 2012; Vol. 3(8): 2543-2549 ISSN: 0975-8232
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Since both water and lipid soluble antioxidants are
needed by the body for intra and extracellular clearance
of the oxidative stress and Panchagavya contains both
water based and lipid based products, it has advantage
as potential antioxidant.
The study indicates some basis for the practice of using
Panchagavya in cancer.
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How to cite this article:
Athavale A, Jirankalgikar N, Nariya P and De S Evaluation of In-
Vitro Antioxidant Activity of Panchagavya: A Traditional Ayurvedic
Preparation. Int J Pharm Sci Res 2012; Vol. 3(8): 2543-2549.
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... The absorbance was measured at 593 nm (Analytik Jena) spectrophotometer. The FRAP value was expressed as mg Ascorbic acid Equivalent Antioxidant Capacity (AEAC) per g of sample [13]. ...
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