Evaluation of antioxidant and antiradical properties of Pomegranate (Punica granatum L.) seed and defatted seed extracts This is Journal Club activity Presentation with the reference of various research papers. This Presentation Contain following... #Info about Paper #Abstract #Materials #Methods #Results #Discussion #Conclusion #References *Important Methods used #Moisture content #Fat content #Acid value #Peroxide value #Oxidative stability index #Total phenols content #Preparation of Pomegranate seed extracts and calculate extract yield #Evaluation of antioxidant properties of Pomegranate seed extracts using -DPPH radicals scavenging activity -FRAP assay #Antioxidant efficiency of seed extract (Oxidative stability extract) #Statistical analysis Journal Club Presentation at Bharati Vidyapeeth College of Pharmacy, Kolhapur. Thanks for Help and Guidance of Dr. P. B. Choudhari (Assistant Professor, Pharmaceutical Chemistry) and Dr. A. J. Shinde (Assistant Professor, Department of Pharmaceutics)

1. Evaluation of antioxidant and antiradical properties
of Pomegranate (Punica granatum L.) seed and defatted
seed extracts
Presented by:-
Mr. Pritam P. Kolge
M. Pharm 2nd Year Sem III
Department of Pharmaceutical Quality
Assurance
1
Journal Club Faculty:-
Dr. A. J. Shinde
Assistant Professor,
Department of Pharmaceutics
Guide:-
Dr. P. B. Choudhari
Assistant Professor,
Pharmaceutical Chemistry
Bharati Vidyapeeth College of Pharmacy, Kolhapur

2. Information about Paper
• Authors- Shadi Basiri
• Journal- Journal of Food Science and Technology
• Revised Date- 9 August 2012
• Accepted Date- 1 July 2013
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3. • This study was aimed to evaluate the effect of solvents on extraction from
Pomegranate seed and Pomegranate defatted seed and to measure the yield extract and
phenolic content and antioxidant properties.
• The seeds and defatted seeds were directly isolated from fruits and seeds by cold
pressing respectively, then were crushed and extracted with different solvents,
including water, Methanol, Acetone, Ethyl acetate and Hexane and finally the extracts
of them were evaluated.
• Phenolic compounds, ferric reducing-antioxidant power and radicals scavenging
property of extracts were measured.
• The highest phenolic content was obtained from Methanol seed extract.
Abstract
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4. • Ripened Pomegranates were collected from trees growing in a Pomegranate garden
at Agricultural Research Station of Sabzevar, Khorasan province in Iran.
• The mean weight of each fruit was 250 g.
• The Pomegranate seeds (PS), Pomegranate Defatted Seeds (PDS)
• Refined and antioxidant free Soybean oil was purchased from Shadgol edible oil
factory (Neyshabor- Iran).
• All chemicals and solvents were analytical reagent grade purchased from Merck
(Darmstadt, Germany) and Sigma Aldrich (St. Louis, MO).
Materials
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5. • Moisture content
• Fat content
• Acid value (AV)
• Peroxide value (PV)
• Oxidative stability index (OSI)
• Total phenols (TP) content
• Preparation of Pomegranate seed extracts and calculate extract yield
• Evaluation of antioxidant properties of Pomegranate seed extracts using
-DPPH radicals scavenging activity
-FRAP assay
• Antioxidant efficiency of seed extract (Oxidative stability extract)
• Statistical analysis
Methods
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6. Moisture content
The moisture content of PS and PDS were calculated using AOAC (Association of Official Agricultural
Chemistry) method No. 984.25 (AOAC 2005).
Acid value (AV)
The Soxhlet method as described by AOAC method No.963.15 was used to extract the oil with petroleum
ether (40–60 °C) for 8 h (AOAC 2005).
Peroxide value (PV)
The AV was determined according to AOCS (American Oil Chemists’ Society official method cd 3d-63
(AOCS 1993).
Oxidative stability index (OSI)
The OSI was determined by a Rancimat instrument (Model 743; Metrohm Ltd., Herisau, Switzerland).
Oxidation was carried out with 3 g oil sample at temperature of 110 °C at an air flowrate of 15 liter.
Total phenols (TP) content
The determination of TP content was done spectrophotometrically using Folin-Ciocalteau’s reagent. A
calibration curve of Gallic acid in Methanol was performed in a concentration range of 0.04–0.40 mg/ml.
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7. Preparation of Pomegranate seed extracts to
calculate Extract Yield
PS & PDS were crushed to a particle size of less than
0.5mm+ mixed with solvent at a ratio of 1:10 (w/v).
Residues were re-extracted by the same solvent.
The solvents were Water, Methanol, Acetone, Ethyl
acetate and Hexane + 24 h of shaking at room
temperature
liquid was separated from the solid using vacuum
filtration through a Whatman No.1 filter paper
All extracts were concentrated under vacuum at 60
°C+ dried, desiccated and stored at −18 °C
Calculate Yield extract
DPPH radicals scavenging activity
0.1 ml Pomegranate seed extract at various
concentrations
0.49 ml of Methanol and 0.39 ml of DPPH
Methanolic solution (4 mg/100 ml).
mixtures were vortexed vigorously and allowed to
stand in the dark for 60 min.
Check Absorbance at 512 nm
The sample concentration providing 50 % of radical
scavenging activity (EC50) was obtained through
interpolation of linear regression analysis.
The lower EC50 indicates higher radical scavenging
activity.
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8. FRAP reagent - Acetate buffer (0.3 M, pH
3.6)+ TPTZ+ Ferric solution in ratio {10:1:1}
FRAP assay
warmed to 37 °C.
Aliquots (200 μl) of each extract were mixed
with 1.8 ml of FRAP reagent
Absorbance 593 nm after incubation at 37 °C
for 10 min
Aqueous standard solutions of FeSO4·7H2O
for the calibration curve.
Antioxidant efficiency of seed extract {Oxidative
stability index (OSI)}
(2 % W/W) of the different Pomegranate seed
extracts added to
purified and antioxidant free Soybean oil
exposed to the thermal resistance (oxidative
stability)
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9. The moisture, fat contents, Peroxide Value, Oxidative stability index (OSI) of PS and
Pomegranate seed oil (PSO) were presented in Table.
Measured parameters Value
PS
Moisture Content (%)
Fat content (% dwb)2
3.75±0.200
17.33±1.330
PSO
Peroxide value (meq g O2/Kg oil)3
Oxidative stability index (h)
Free fatty acid (mg NaOH/g oil)
0.79±0.190
3.03±0.150
0.56±0.010
Where,
Mean ± SD (n=3)
dwb- Dry weight base
meq g O2/Kg oil- Mili equivalent gram of O2 in each Kg of oil
Results
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10. Total phenolic content
Solvent type Phenolic content (mg/l seed extract)
Water 22.61±0.391
Methanol 27.93±0.301
Acetone 3.41±0.043
Butanol 0.57±0.002
Ethyl acetate 0.37±0.010
Hexane 0.29±0.016
• The Table shows that Methanol extract of Pomegranate seed was significantly higher
than other solvents.
• They reported that Methanol extract of Pomegranate peel had the highest antioxidant
activity among all of the extracts.
• Efficiencies of the solvents for extraction of the phenolic compounds were in the order:
Methanol > Water > Acetone > Butanol > Ethyl acetate > Hexane
• Methanol extract of PS gave highest phenolics and Hexane extract showed the minimum
of phenolics.
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11. Solvent extract Extract yield (%)
PS PDS
Water 5.0±0.07 4.5±0.06
Methanol 6.1±0.06 6.3±0.05
Acetone 14.9±0.09 10.1±0.11
Butanol 17.0±0.11 9.0±0.08
Ethyl acetate 15.0±0.08 8.6±0.09
Hexane 17.9±0.09 7.9±0.06
Calculation of Extract Yield-
Extraction yield (%)= (weight of dried extract X 100)/ (initial weight of seed powder)
• Hexane extraction of PS and Acetone extraction of PDS gave maximum yield, whereas
water extract gave minimum yield.
• The polarity of Acetone is intermediate, it able to solve both polar (phenolic compounds)
and inpolar (oil) compounds.
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12. Ferric reducing/antioxidant power (FRAP assay)
Solvent Extract Ferric reduction power (μMol Fe2+/l)
PS PDS
Water 217.3±0.21 207.6±0.23
Methanol 557.0±0.37 721.8±0.35
Acetone 40.7±0.29 155.1±0.21
Butanol 59.3±0.27 60.5±0.11
Ethyl acetate 22.2±0.19 35.1±0.18
Hexane 6.4±0.11 9.9±0.17
• The FRAP assay measures the antioxidant effect in the reaction medium as reducing ability.
• Antioxidant potential of the PS and PDS extracts were estimated from their ability to reduce
TPTZ-Fe(III) complex to TPTZ-Fe(II) complex.
• The PS and PDS Methanol extracts showed highest FRAP antioxidant activity in all
extracts of PS and PDS.
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13. Radical-scavenging activity (DPPH assay)
• Antiradical activities (free radical-scavenging) of extracts of PS and PDS were determined using the
DPPH method.
• The highest antiradical potential observed in Methanol extracts of PS and PDS because lower value
of EC50 indicates a higher antioxidant activity.
• Antioxidants can deactivate or scavenge stable free DPPH radicals by two major mechanisms: by
reduction via electron transfer or by hydrogen atom.
EC50 (mg/ml)
PS PDS
Water 0.30±0.015 0.32±0.022
Methanol 0.15±0.003 0.19±0.006
Acetone 0.34±0.002 0.24±0.008
Butanol 1.77±0.010 1.69±0.008
Ethyl acetate 2.01±0.055 1.83±0.015
Hexane 4.23±0.067 3.88±0.081
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14. Oxidative stability extracts
• In this study, the effects of addition of PS extracts on oxidative stability index (OSI) of
antioxidant free Soybean oil, were evaluated.
• Addition Methanol extract to antioxidant free soybean oil (blank sample) had the highest OSI.
• Addition of the extracts with less polarity such as Acetone, Butanol, Ethyl acetate and Hexane to
oil caused to decrease OSI of antioxidant free Soybean oil.
Sample Oxidative stability (h)
Antioxidant free Soybean oil (blank) 4.4±0.01
Blank + Water extract 3.8±0.02
Blank + Methanol extract 6.2±0.02
blank + Buthanol extract 3.7±0.01
blank + Acetone extract 3.4±0.06
blank + Ethyl acetate extract 3.1±0.03
blank + Hexane extract 2.8±0.03
blank + BHT(butylated hydroxytoluene) 5.1±0.05
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15. Discussion
• Reducing activity test proved that the Methanol extracts of Pomegranate
seed and Pomegranate defatted seed had the highest reducing strength.
• The results showed the highest extraction efficiencies were for Hexane and
Acetone solvents in extraction of seed and defatted seed respectively.
• Results of radical scavenging activity were similar to reducing activity
results.
• The order of antioxidant capacity of Pomegranate seed and Pomegranate
defatted seed were found to be Methanol > Water > Acetone > Butanol >
Ethyl acetate > Hexane
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16. • It is concluded Pomegranate seed functionally and nutritionally has a wide
application in food, pharmaceutical and cosmetic industries.
• The Methanol extracts of Pomegranate seed had the highest antioxidant
and antiradical activity.
• Results also confirm that addition of Pomegranate seed Methanol extract
to antioxidant free oil led to increase of oxidative stability.
• The Methanol extract had a higher antioxidant efficiency than seed and
defatted seed extracts.
• It can be concluded Pomegranate seed, which possesses high levels of
polyphenols, can be one of the sources of the natural antioxidants.
Conclusion
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References
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Pomegranate juice consumption reduces oxidative stress, atherogenic modifications to LDL, and
platelet aggregation: studies in humans and in atherosclerotic apolipoprotein E-deficient mice.
American Journal of Clinical Nutrition, 71, 1062–1076.
• Ben, N. C., Ayed, N., & Metche, M. (1996). Quantitative determination of the polyphenolic content
of pomegranate peel. Zeitschrift fur Lebensmittel-Untersuchung Und -Forschung, 203, 374–378.
• Benzie, I. F. F., & Strain, J. J. (1996). The ferric reducing ability of plasma (FRAP) as a measure of
‘‘antioxidant power’’: the FRAP assay. Anal ytical Biochem istry, 239, 70–76.
• Chisolm, G. M., & Steinberg, D. (2000). The oxidative modification hypothesis of atherogenesis:
an overview. Free Radical and Biological Medicine, 28, 1815–1826.
• Gil, M. I., Tomas-Barberan, F. A., Hess-Pierce, B., Holcroft, D. M., & Kader, A. A. (2000).
Antioxidant activity of pomegranate juice and its relationship with phenolic composition and
processing. Journal of Agricultural and Food Chemistry, 48, 4581–4589.
• Jia, Z., Tang, M., & Wu, J. (1999). The determination of flavonoid contents in mulberry and their
scavenging effects on superoxide radicals. Food Chemistry, 64, 555–559.

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• Longtin, R. (2003). The pomegranate: natures power fruit?. Journal of National Cancer Institute,
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• Murthy, K. N. C., Jayaprakasha, G. K., & Singh, R. P. (2002). Studies on antioxidant activity of
pomegranate peel extract using in vivo models. Journal of Agricultural and Food Chemistry, 50,
4791–4795.
• Scalbert, A., & Williamson, G. (2000). Dietary intake and bioavailability of polyphenols. Journal
of Nutrition, 130, 2073s–2085s.
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• Rice-Evans, C. A., Miller, N. J., & Paganga, G. (1996). Structureantioxidant activity relationship of
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