Biomed 4(3) July-Sept 2009.
Pp 303-308
Comparative Antioxidant activity of Propolis from Indian Sub-continent using 2,2-
diphenyl-1-picrylhydrazyl (DPPH)
Nilesh Kumar*, Mueen Ahmad KK.1, Raman Dang2, Santanu Mallik3, Bimlesh Kumar4
*,3,4 Dept. of Pharmacy, Lovely Professional university, Jalandhar.
2,3
Dept of Pharmacognosy, Al-Ameen College of Pharmacy, Bangalore.
Corresponding address - mpharmnilesh @[Link]
Abstract
Antioxidant compounds in food play an important role as a health-protecting factor.
Scientific evidence suggests that antioxidants reduce risk for chronic diseases including
cancer and heart disease. Primary sources of naturally occurring antioxidants are whole
grains, fruits and vegetables. Plant sourced food antioxidants like vitamin C, vitamin E,
carotenes, phenolic acids, phytate and phytoestrogens have been recognized as having the
potential to reduce disease risk. The free radical scavenging activity of the EEP of the Uttar
Pradesh, Manipur, Gujarat and Tamil Nadu was 57.34%, 55.21%, 48.36% and 51.30%
respectively at a concentration of 10µg while it was 65.19%, 63.11%, 56.99% and 57.62%
respectively at a concentration of 50 µg. At the 100 µg it was found to have maximum free
radical scavenging activity of 83.20% EEP of Uttar Pradesh zone and the least of 70.96% of
the Tamil Nadu zone. Similarly free radical scavenging activity of the MEP of the Uttar
Pradesh, Manipur, Gujarat and Tamil Nadu was47.60%, 54.35%, 43.78% and 50.66%
respectively at a concentration of 10µg while it was 63.95%, 56.10%, 49.99% and 53.14%
respectively at a concentration of 50 µg. The maximum free radical scavenging activity was
found 74.43% at 100µg for Uttar Pradesh zone and the least for the Tamilnadu extracts. In
the case of 40% MEP the maximum free radical scavenging activity was in Manipur zone
with 73.60% and the least for Tamil Nadu zone 59.26% at 100µg respectively. The results of
the free radical scavenging effect of vitamin C were 94.7% at a concentration of 100µg but
the activity at a concentration of 50µg was 93.4% respectively.
Key words - EEP (Ethanolic extract of propolis), MEP (Methanolic extract of propolis)
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Biomed 4(3) July-Sept 2009. Pp 303-308
Introduction region of India i.e. Ahmedabad (Gujarat),
Basti (Utter Pradesh), Javadi hills (Tamil
The term Propolis derives from the Greek
Nadu) and Leikai (Manipur) in the month of
pro (for “in front of”) and polis
June and was used for the extraction.
(“community”) and means a substance in
Propolis was stored in the dark place at 4˚C.
defense of the hive. Propolis was used
effectively on wounds by doctors during the Preparation of extracts- Successive solvent
Anglo- Boer war and during the World War extraction method was used for the
II. Hippocrates prescribed propolis to help extraction of active constituents present in
heal sores as well as ulcers, both external the drug using various solvents ranging from
and internal. The ancient Egyptians looked nonpolar to polar. For phytochemical and
upon the bees and their propolis as the microbiolocical screening, the propolis were
source of external health and life. In the 17th subjected to successive solvent extraction
century, propolis was a major ingredient of viz. petroleum ether, chloroform, ethanol
healing ointments in the European (95%), methanol and methanol (40%). Raw
Pharmacopoeia. In Folk Georgian medicine, propolis samples (50 gm) were chopped in a
they used ointments with propolis to cure very small piece and extracted with the
some diseases. There was the custom of different solvents (250 ml). It was kept for 5
placing a propolis cake on the belly button days with occasional shaking. Then, it was
of the newborn baby and also they rubbed filtered with # Whatman 1 paper and
children’s toys with propolis. Also in folk concentrated at 50°C to obtain the crude
medicine, the use of propolis is widely extract in paste form and kept in a dry and
known especially for the treatment of corns. dark place i.e. amber colored bottle.6
People inhale propolis in case of infections DDPH preparations- 2.366 mg of 1, 1-
of respiratory tracts and of the lungs. It is diphenyl-2-picrylhydrazyl (DPPH) was
also efficient for burns and angina. dissolved in 100ml of absolute ethanol to
Material method: obtain 60 μM DPPH and measure it at 520
nm and this reading was used control
Collection and drying of Drug- The dried reading.2,5
propolis was collected from four different
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Biomed 4(3) July-Sept 2009. Pp 303-308
Standard preparations- 25 mg of ascorbic ml) was mixed with the same volume of
acid was dissolved to 25 ml of absolute DPPH solution and allowed to stand for 30
ethanol and then it was further diluted to min at room temperature. The absorbance
obtain 10 μg, 15 μg, 20 μg, 25 μg and 30 μg was then measured at 520 nm. The sample
and it was measured in UV and DPPH were dissolved in ethanol. The
spectrophotometer at 520 nm.1,2 percentage scavenging effect was
determined by comparing the absorbance of
Sample preparations- 25 mg of the extract
solution containing the test sample to that of
was dissolved in 25 ml of absolute ethanol
control solution without the test sample
and then it was further diluted to obtain 10
taking the corresponding blanks. Then this
μg to 140 μg.
was again measured after 1 hour. The result
Procedure is tabulated as mean of the 3 readings for
The scavenging effect of propolis sample as each sample. The vitamin C was used as
well as vitamin C corresponding to the positive control samples.
quenching intensity of 1,1-diphenyl-2-
picrylhydrazyl (DPPH) as carried out.3 The
sample solution of each tested (500 μl or o.5
edible plants, and they have been reported to
Calculations5
have multiple biological effects, including
AbsorbanceControl – AbsorbanceSample
X 100 antioxidant activity. Propolis contains a
AbsorbanceControl
wide variety of phenolic compounds, mainly
Where:
flavonoids. The results of the free radical
Control – DPPH in ethanol
scavenging effect of propolis showed a
Sample - Extract solution in ethanol
concentration-dependent activity (Table-1).
and DPPH
Since the Pet ether extract and chloroform
Results extract of Propolis was not having any
DPPH assays reflect the ability of the antioxidant activity so it was discarded. The
compounds present to scavenge hydrophilic results of the free radical scavenging effect
free radicals. Phenolic compounds are of propolis of all four zones showed a
commonly found in both edible and non- concentration-dependent activity. The free
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Biomed 4(3) July-Sept 2009. Pp 303-308
radical scavenging activity of the EEP of the respectively. The results of the free radical
Uttar Pradesh, Manipur, Gujarat and Tamil scavenging effect of vitamin C were 94.7%
Nadu was 57.34%, 55.21%, 48.36% and at a concentration of 100μg but the activity
51.30% respectively at a concentration of at a concentration of 50μg was 93.4%
10μg while it was 65.19%, 63.11%, 56.99% respectively.
and 57.62% respectively at a concentration Discussion
of 50 μg. At the 100 μg it was found to have
In the present study, we found that propolis
maximum free radical scavenging activity of
from Uttar Pradesh had strong DPPH free
83.20% EEP of Uttar Pradesh zone and the
radical-scavenging activity, whereas the
least of 70.96% of the Tamil Nadu zone.
activity of the extract from Gujarat was
Similarly free radical scavenging activity of
weak. Sforcin [Link] had isolated several
the MEP of the Uttar Pradesh, Manipur,
compounds with antioxidant or radical-
Gujarat and Tamil Nadu was47.60%,
scavenging activity from Brazilian Propolis.3
54.35%, 43.78% and 50.66% respectively at
Brazilian propolis was classified into 12
a concentration of 10μg while it was
groups based on the physicochemical
63.95%, 56.10%, 49.99% and 53.14%
characteristics, and that the biological
respectively at a concentration of 50 μg. The
activities of classified Brazilian propolis
maximum free radical scavenging activity
were also altered.3,4 Thus it may be
was found 74.43% at 100μg for Uttar
considered that the antioxidant activity also
Pradesh zone and the least for the Tamilnadu
differs with the regions. However, more
extracts. In the case of 40% MEP the
detailed qualitative and quantitative analyses
maximum free radical scavenging activity
of the compounds with antioxidant activity
was in Manipur zone with 73.60% and the
are necessary to elucidate the antioxidant
least for Tamil Nadu zone 59.26% at 100μg
activity of propolis.
References: using 2,2-diphenyl-1-picrylhydrazyl
(DPPH). Food Chem 2007;203:662-
1. Lee JM, Chung H, Chang PS.
9.
Development of a method predicting
the oxidative stability of edible oils
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Biomed 4(3) July-Sept 2009. Pp 303-308
2. Georgetti SR, Casagrande R, Mambo Seasonal effect on Brazilian propolis
VM, Azzolini AECS, Fonesca M. antibacterial activity. J
Evaluation of the antioxidant activity Ethnopharmacol 2000;73:243-9.
of different flavonoids by the 5. Rossi M, Alamprese Cristina, Ratti
chemiluminescence method. AAPS Simona. Tocopherols and
Pharm Sci 2003;5(2):1-5. tocotrienols as free redical-
3. Hegazi AG, Abd EI Hady FK. scavengers in refined vegetable oils
Egyptian propolis: antioxidant, and their stability during deep-fat
antimicrobial activities and chemical frying. Food Chem 2007;102:812-7.
composition of propolis from 6. Choi YM, Noh DO, Cho SY, Suh
reclaimed lands. Naturforscin HJ, Kim KM, Kim JM. Antioxidant
2002;57C:395-402. and antimicrobial activities of
4. Sforcin JM, Fernandes A, Lopes propolis from several regions of
CAM, Bankova V, Funari SRC. Korea. LNT 2006;39: 756-761.
Table 1- The DPPH free radical scavenging activity of ethanolic and methanolic and 40%
methanolic extracts of Propolis.
Name of Concentration
Sl. No. the state (µg) x 10-3M EEP MEP 40% MEP
10 µg/ml 57.96 48.28 44.89
1 Manipur 50 µg/ml 66.85 65.25 53.58
sample
100 µg/ml 84.09 76.67 70.62
10 µg/ml 49.88 44.91 46.21
Uttar
2 Pradesh 50 µg/ml 61.08 50.78 52.38
Sample
100 µg/ml 73.52 65.64 65.27
10 µg/ml 53.80 51.46 52.58
3 Gujarat 50 µg/ml 59.45 54.89 56.76
Sample
100 µg/ml 72.97 60.70 62.58
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Biomed 4(3) July-Sept 2009. Pp 303-308
10 µg/ml 56.27 55.04 54.03
4 Tamilnadu 50 µg/ml 65.48 57.40 60.80
sample
100 µg/ml 81.17 66.51 76.92
Note: The DPPH free radical scavenging effect was measured by the absorbance of DPPH
radical at 520 nm in the reaction containing the test sample and 6 x 10-5M DPPH.
308
