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Phenolic and Flavonoids Analysis of Pomegranate Peel Extracts and their

Antiniflammatory and Antioxidant Activities

Article in International Journal of Pharmaceutical Quality Assurance · May 2019

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International Journal of Pharmaceutical Quality Assurance 2019; 10(1); 60-65

ISSN 0975 9506

Research Article

Phenolic and Flavonoids Analysis of Pomegranate Peel Extracts and

their Antiniflammatory and Antioxidant Activities
Qabaha K1, Al-Rimawi F2*, Nusseibeh S3, Abbadi J4, Abu-Lafi S5
1
Department of Medical Laboratory Sciences, Faculty of Allied Health Sciences, Arab American University. Jenin,
Palestine
2
Chemistry Department, Faculty of Science and Technology, Al-Quds University, P.O. Box 20002, Jerusalem, Palestine
3
Centre for Chemical and Biological Analysis, Al-Quds University, Jerusalem, Palestine
4
Biology Department, Faculty of Science and Technology, Al-Quds University, P.O. Box 20002, Jerusalem, Palestine
5
Faculty of Pharmacy, Al-Quds University, P. O. Box 20002, Abu-Dies, Palestine

Received: 7th Jan, 19; Revised: and Accepted: 20th Feb, 19; Available Online: 25th Mar, 2019

ABSTRACT
An in-vitro evaluation of the anti-inflammatory and antioxidant activities of pomegranate peel extract from Palestine
were investigated. In parallel, the total phenolic content (TPC) and the total flavonoids content (TFC) were measured.
The antioxidant activities were determined spectrophotometrically by DPPH, FRAP, CUPRAC and the ABTS methods.
The phenolic and flavonoid contents were separated and partially identified using HPLC and LC-MS. In-vitro inhibitory
effect of the extract on production of Interlukin-6 (Il-6) and Tumor Necrosis Factor-α (TNF-α) by Lipopolysacaride
(LPS)-induced polymorphonuclear Cells (PMNCs) was evaluated. Pomegranate peel extract was found to have strong
antiinflamatory activity as revealed by the reduction in the levels of IL-6 and TNF-alfa. It was also found that it is rich in
phenoloic and flavonoids that enhanced its reducing activity and free radical scavenging ability.

Keywords: Pomegranate peel, extract, anti-inflammatory, antioxidant activity, Phenolic compounds, flavonoids.

INTRODUCTION released from many types of bacteria and cause systemic

Herbal plants contain active ingredients that help in infection16. Tumor Necrosis Factor (TNF-α) and
recurring from many diseases including many infectious Interlukin-6 (IL-6) are pro-inflammatory cytokines that
and chronic ones. Many of the active ingredients were are released upon exposure to LPS from many cells as T
reported to act as antiinflammatory, animicrobial and free cells, Monocytes, B-cells and others. Excessive release of
radicals scavenging agents. Such ingredients may include such cytokines may lead to many inflammatory diseases
phenolics, anthocyanins, carotenoids, and thiols1. The as asthma, rheumatoid arthritis, atherosclerosis, and
pomegranate tree (Punica granatum L.) is native in others17-19. Levels of Il-6 and TNF-α produced by LPS
Palestine and has been used to treat dysentery, diarrhea, stimulated poly morpho nucleated cells are measured
and intestinal parasites. Around half of the fruit weight using Enzyme linked Immuno Sorbant Assay (ELIZA).
belongs to its peel and the rest are its edible part Antiinflamtory effect was examined by measuring the
consisting of seeds and arils parts2-4. Its peel was used level of IL-6 and TNF-α by the mono nucleated white
traditionally for treatment of ulcer and inflammation, and blood cells upon effect of LPS with different
it has been showed antioxidant and bacterial activities2-4. concentrations. Levels of the cytokines were measured
Several studies have demonstrated that the pomegranate using Enzyme Linked Immune Sorbent Assay method.
peel is a good source of bioactive compounds such as As part of our ongoing research about the medical effect
catechin, ellagitannins, epicatechin, rutin, and many of the plant extracts, antioxidant, antiinflammatory of
others. Such bioactive compounds are responsible for pomegranate peel extracts were investigated along with
many biological activities as antimicrobial, antioxidant, their phenolic and flavonoids content as well as analysis
antiinflamatory5-7. Additionally, other studies have shown of the active phenolic and flavonoid compounds present
the therapeutic effect of pomegranate fruit, juice, and peel in the extracts by the use of HPLC-PDA and UHPLC-
for treatment of lung cancer, esophagus, breast, PDA-ESi-MS techniques.
cardiovascular disorders and breast cancer. It has free
radical scavenging activities which mostly related to its MATERIALS AND METHODS
phytochemical components8-15. Plant Material and Extraction
Inflammation is part of a self-protection used by human Pomegranate fruit was purchased from Jenin market and
body against pathogens and also helps in healing injured then peeled off. The peel was dried in the shade at room
tissues. Lipopolysaccharide (LPS) is an endotoxin temperature for 3 weeks then grinded to be like a powder.

*Author for Correspondence: [email protected]

 Qabaha et al. / Phenolic and Flavonoids…

Table 1: Effects of Pomegranate peel extracts and LPS warmed at 37◦C and mixed with 40 µl of the extract and
on PMNCs viability. the reaction mixtures were later incubated at 37◦C.
Contents % Viability Absorbance at 593 nm was read with reference to a
PMNCs only 95.5±1.2 reagent blank containing distilled water which was also
PMNCs with LPS 93.93±1.3 incubated at 37 ◦C for up to 1 hour instead of 4 min,
PMNCs with LPS and 300 µg/ml 90.1±1.0 which was the original time applied in FRAP assay.
Pomegranate peel extract Aqueous solutions of known Fe+2 concentrations in the
range of 2-5 mM were used for calibration, and results
Then, fifty grams of the above-mentioned powder was were expressed as mmol Fe+2 /g.
mixed with 500 ml of 96% Ethanol and kept on a shaker Cupric reducing antioxidant power (CUPRAC assay)
for 5 days. The extract was filtered through filter paper The cupric ion reducing antioxidant capacity of the
(Whatman blue ribbon No. 41). The filtrate solution was extracts was determined according to the method of Apak
dried completely under vacuum using rotary evaporator et al. (2008)21. 100 µl of sample extract was mixed with
(BUCHI Brand) at 50°C. 1ml each of 10 mM of cupper chloride solution, 7.5 mM
Chemicals and reagents of neocuproine alcoholic solution (99.9% ethanol), and 1
2,4,6-tripyridyl- S-triazine (TPTZ), hydrochloric acid M (pH 7.0) of ammonium acetate buffer solution, and 1
37% (w/w), sodium hydroxide, ferric chloride trihydrate, ml of distilled water to make final volume 4.1 ml. After
ferrous sulfate heptahydrate, potassium persulphate, 30 min, the absorbance was recorded at 450 nm against
sodium acetate, sodium carbonate, sodium nitrite, the reagent blank. Standard curve was prepared using
aluminum chloride, methanol, folin-ciocalteu reagent, different concentrations of Trolox. The results were
Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2- expressed as µmol Trolox/g.
carboxylic acid), gallic acid, cupper chloride, Free radical scavenging activity using DPPH (DPPH
neocuproine, 99.9% ethanol, ammonium acetate, DPPH, assay)
methanol, ABTS (2,2-azino-di-(3-ethylbenzothialozine- DPPH assay is based on the measurement of the
sulphonic acid)), and potassium persulphate were all scavenging ability of antioxidants towards the stable
obtained from Sigma-Aldrich, Germany. All chemicals DPPH radical. A 3.9 mL aliquot of a 0.0634 mM of
and reagents were of analytical grade. The acetonitrile DPPH solution in methanol (95%) was added to 100 µl of
and water solvents were of an HPLC grade from Sigma. each extract. The mixture was vortexed for 5-10 sec. The
Phenolic and flavonoids standards: Vanillic acid, Ferulic change in the absorbance of the sample extract was
acid, Syringic acid, trans-cinnamic acid, Catechin, p- measured at515 nm for 30 min till the absorbance reached
coumaric acid, Sinapic acid, 4-Hydroxyphenylacetic acid, a steady state. Methanol (95%) was used as a blank.
Rutin hydrate, Caffeic acid, Quercetin, Gallic acid, 3,4- Standard curve was prepared using different
dihydroxyphenylacetic acid, chlorogenic acid, Taxifolin, concentrations of Trolox. The results were expressed as
Luteolin 7-glucoside, Apigenin 7-glucoside, Luteolin, µmol Trolox/g.
Quercetin 3-D-galactose were from Sigma-Aldrich, Free radical scavenging activity using ABTS (ABTS
Germany. assay)
Regents preparation A modified procedure using ABTS (2, 2-azino-di-(3-
FRAP reagent was prepared according to Benzie and ethylbenzothialozine-sulphonic acid)) as described by
Strain (1999)20 by the addition of 2.5 mL of a 10 mM Pellegrini et al. (1999)22 was used. The ABTS stock
tripydyltriazine (TPTZ) solution in 40 mM HCl plus 2.5 solution (7 mM) was prepared through reaction of 7 mM
mL of 20mM FeCl3.6H2O and 25 mL of 0.3M acetate ABTS and 2.45 mM of potassium persulphate as the
buffer at pH 3.6. Acetate buffer (0.3M) was prepared by oxidant agent. The working solution of ABTS+˙ was
dissolving 16.8 g of acetic acid and 0.8g of sodium obtained by diluting the stock solution in 99.9% ethanol
hydroxide in 1000 mL of distilled water. to give an absorption of 0.70 ± 0.02 at 734 nm. 200 µl
HPLC and UHPLC Instrumentation systems sample extract was added to 1800 µl of ABTS+˙ solution
The analytical HPLC is Waters Alliance (e2695 and absorbance readings at 734 nm were taken at 30 °C
separations module), equipped with 2998 Photo diode exactly 10 min after initial mixing (A). The radical-
Array (PDA). Data acquisition and control were carried scavenging activity of the test samples was expressed as
out using Empower 3 chromatography data software Trolox equivalent antioxidant capacity TEAC (µmol
(Waters, Germany). The chromatography was performed Trolox/g sample).
under reverse phase conditions using a TSQ Quantum Total phenolic content (Folin-Ciocalteau assay)
Access MAX (Thermo Scientific, San Jose, CA, USA) Total phenolics were determined using Folin-Ciocalteau
which includes a Dionex Pump with degasser module, an reagents. Z. spina-christi plant extracts or gallic acid
Accela PDA detector and an Accela Autosampler. standard (40 µl) were mixed with 1.8 mL of Folin-
Measurement of Antioxidant Activity Ciocalteu reagent (prediluted 10-fold with distilled water)
FRAP assay and allowed to stand at room temperature for 5 min, and
The antioxidant activity of the extracts was determined then 1.2 mL of sodium bicarbonate (7.5%, w/v) was
using a modified method of the assay of ferric added to the mixture. After standing for 60 min at room
reducing/antioxidant power (FRAP) of Benzie and Strain temperature, absorbance was measured at 765 nm.
(1999)20. Freshly prepared FRAP reagent (3.0 mL) was Aqueous solutions of known gallic acid concentrations in

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 Qabaha et al. / Phenolic and Flavonoids…

Table 2: Effect of pomegranate peel extract on production of IL-6 and TNF alpha by PMNCs.
IL6 amount (pg/ml) TNF alpha amount (pg/ml)
Average STD Average STD
Cells only 106.7 1.2 64.3 1.7
Cells with LPS 640.3 2.1 987.3 5.2
Cells with LPS and 75 µ g extract 371 2.9 669 2.9
Cells with LPS and 150 µ g extract 160 2.9 314.3 3.3
Cells with LPS and 300 µ g extract 63.3 1.2 77.7 2.1

Table 3: Total phenolic content (TPC as mg Gallic acid/g DW *), total flavonoids contents (TFC as mg catechin/g
DW), FRAP (mmol Fe+2/g DW), CUPRAC (µmol Trolox/g DW), DPPH (µmol Trolox/g DW), and ABTS (µmol
Trolox/g DW) of pomegranate peel extract.
TPC** (mg/g) TFC (mg/g) FRAP (mmol/g) CUPRAC (µmol/g) DPPH (µmol/g) ABTS (µmol/g)
33.5± 1.5 8.3± 0.9 12.4 ± 0.4 3756 ±35 225 ± 6 46.2 ±1.5
*
DW: Dry weight
**
Results are expressed as an average of three samples.

the range of 10 - 500 mg/L were used for calibration. was maintained at 35°C. The chromatographic separation
Results were expressed as mg gallic acid equivalents was achieved using the same HPLC linear gradient
(GAE)/ g sample. program using formic acid instead of acetic acid at a
Total flavonoid content constant flow rate of 0.4 mL/min over a total run time of
The determination of total flavonoids was performed 70 min. The samples were detected by a TSQ Quantum
according to the colorimetric assay of Kim et al. (2003) 23. Access Max mass spectrometer using Electron Spray
Distilled water (4 mL) was added to 1 mL of the extract ionization (ESI) and full scan acquisition. Air was
in a test tube. Then, 0.3 mL of 5% sodium nitrite solution produced (SF 2 FF compressor, Atlas Copco, Belgium).
was added, followed by 0.3 mL of 10% aluminum Purified nitrogen was used as source and exhaust gases.
chloride solution. Test tubes were incubated at ambient Samples of the crude extracts were prepared at a
temperature for 5 minutes, and then 2 mL of 1 M sodium concentration of 5 mg/ml by dissolving 50 mg of crude
hydroxide were added to the mixture. Immediately, the extract in 10 ml of respective solvent (water, 80%
volume of reaction mixture was made to 10 mL with ethanol, or 100% ethanol).
distilled water. The mixture was thoroughly mixed using Cell Culture
test tube shaker and the absorbance of the pink color Polymorphonuclear cells were isolated from a freshly
developed was determined at 510 nm. Aqueous solutions transfused 5 ml whole blood. The blood was mixed with
of known catechin concentrations in the range of 50 - 100 an equal amount of phosphate buffered saline (PBs) in a
mg/L were used for calibration and the results were 1:1 ratio in a sterile condition. Three ml of Histopaque
expressed as mg catechin equivalents (CEQ)/ g sample. were pipetted in 15 ml conical tube. The mixture of the
Chromatographic conditions PBS and the blood was added gently to the tube and then
The HPLC analytical experiments of the crude ethanol spun for 20 min at 400 G. The PMNCs were aspirated
extracts were run on ODS column of Waters (XBridge, and then washed 3 times with 10 ml PBS in a 12 ml
4.6 ID x 150 mm, 5 μm) connected to guard column of conical tube at 100 G for 10 minutes. Supernatant was
Xbridge ODS, 20 mm x 4.6mm ID, 5 μm. The mobile discarded and the cells were isolated and used in our
phase is a mixture of 0.5% acetic acid solution (A) and study.
acetonitrile (B) run in a linear gradient mode. A 100% The isolated PMNCs were cultured in Roswell Park
(A) was descended to 70% (A) in 40 minutes then to 40% Memorial Institute (RBMI) medium that was enriched
(A) in 20 minutes and then to 10% (A) in 2 minutes and with 100-μg ml-1 streptomycin, 100 Uml-1 penicillin,
stayed for 6 minutes and back to the initial conditions in 2 and with heat-inactivated 10% Fetal Bovine Serum
minutes. The HPLC system was equilibrated for 5 (FBS). The mixtures were incubated into 12-well tray at
minutes with the initial acidic water mobile phase (100 % 37°C in 5% CO2 incubator for 24 hours. One ml of the
A) before injecting next sample. All the samples were mixture contains 1 million of the cells and was incubated
filtered with a 0.45 m PTFE filter. The PDA in one well. Different concentrations of the pomegranate
wavelengths range was from 210-500 nm. The flow rate extract were added to lipo poly saccharide (1 μg/ well)
was 1 ml/min. Injection volume was 20 l and the stimulated cells.
column temperature was set at 25◦C. Samples were Cytotoxicity Test
filtered through 0.45 m micro porous disposable filter. The trypan blue exclusion test was used to evaluate the
The UHPLC chromatographic separations were run on a cytotoxicity of the pomegranate extract as illustrated by
Kinetex™ (Phenomenex, Torrance, CA, USA) through Avelar-Freitas et al. 201414. Trypan blue dye of an acid
C8 column (2.6 µm particle size, 100 Å pore size, 100 x azo exclusion medium was prepared in 0.4 % dilution,
2.1 mm), protected by a SecurityGuard™ (Phenomenex, then mixed with an equal amount of the cell suspension
Torrance, CA, USA) cartridge C8 column (2.1 mm ID). with 300 µ gram/1 ml of the pomegranate extract. After 2
The injection volume was 10 μL, the oven temperature minutes, Hemocytometer was used to count the cells.

IJPQA, Volume 10, Issue 1, January 2019 – March 2019 Page 62

 Qabaha et al. / Phenolic and Flavonoids…

253.2 255.6 355.7

27.648
0.35
256.8
353.3353.3

26.251
26.770
221.4
261.5
253.2 261.5 348.5348.5
0.30
265.1 355.7

0.25

25.644
0.20 363.8
AU

477.4488.3492.0
0.15

250.00 300.00 350.00 400.00 450.00 500.00

nm

29.953
24.020
24.530
0.10

23.210
23.588
22.935
22.45522.061
21.50321.732
15.614

34.940
31.653
27.377
20.520

30.685

66.487
0.05

30.993

36.902
20.276

28.991

33.130
28.545
18.913

65.025
14.449

43.350
20.986
18.395
14.868

65.302
64.852
63.820

66.767
0.00

0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00 70.00
Minutes

Figure 1: HPLC-PDA chromatograms of crude Pomegranate peel ethanol extract at 350 nm.

255.6255.6255.6
214.3
215.5
215.5
353.3353.3
354.5
352.1

348.5

250.00 300.00 350.00 400.00 450.00 500.00

nm

Figure 2: Overlaid UV-Vis spectra of the major five peaks of Pomegranate peel ethanol extract eluted between 25.64-
29.95 minutes.

Viable cells were unstained and dead cells were stained effect on the viability of the PMNCs when compared with
blue. the control group. Table 1 represents such results. Such
Immunoassay for TNF-alpha and IL-6: results indicate that any possible reduction in the
Enzyme Linked Immunoassay was employed to measure cytokines’ production is not due to the PMNCs death.
IL-6 and TNF-alpha according to manufacturer’s Anti-inflammatory activities of the plant extracts
instructions. LPS-stimulated PMNCs after 24h, TNF-α, and IL-6
Statistical Analysis levels increased significantly. However when it was
The means of TNF-α and IL-6 concentrations were treated with pomegranate peel extracts with
compared using Paired samples t test applying SPSS concentrations ranged from 75-300 µgm/ ml, TNF-α and
version 19. Differences with P less than 0.05 were IL-6 in cell culture medium were all significantly reduced
considered significant. and the greater the reduction in their concentration, the
stronger the anti-inflammatory effect of the extract (Table
RESULTS AND DISCUSSIONS 2). Such results make the pomegranate peel extracts a
Cytotoxicity of the extracts good source candidate for anti-inflammatory drugs.
Pomegranate peel extracts at concentration of 300 µg/ ml Total Phenolic Content (TPC) and Total Flavonoid
and LPS at concentration of 1 µg/ ml have no obvious Content (TFC)

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 Qabaha et al. / Phenolic and Flavonoids…

Pomegranate peel extract was found to be rich with hydrogen donors including phenolics. The bleaching of
phenolic and flavonoids with TPC and TFC of 33.5 and DPPH solution increases linearly with increasing amount
8.3 mg/g, respectively, Table 3. of extract in a given volume.
Antioxidant activity DPPH antioxidant activity of pomegranate peel extract
There are two types of typical assays used to evaluate the was found to be 225±6 µmole Trolox/g (Table 1).
AA of plant extracts. The first category measures the Free radical scavenging activity using ABTS
potential of plant extracts to reduce ions or oxidants (by A modified procedure using ABTS (2,2-azino-di-(3-
acting as reducing agents) like ferric ion, cupric ion. The ethylbenzothialozine-sulphonic acid) was used. The
main two assays of this category are FRAP (measures the ABTS+˙ stock solution (7 mM) was prepared through
reduction potential of ferric to ferrous ion), and CUPRAC reaction of 7 mM ABTS and 2.45 mM of potassium
(measures the reduction of cupric to cuprous ion). The persulphate as the oxidant agent. The working solution of
second category of AA measures the ability of plant ABTS+˙ was obtained by diluting the stock solution in
extracts to scavenge free radicals. DPPH and ABTS ethanol to give an absorption of 0.70 ± 0.02 at λ = 734
assays (where DPPH and ABTS are stable free radicals) nm. Sample extract (100 μl) was added to 900 μl of
are the two main examples of this category. These assays ABTS+˙ solution and absorbance readings at 734 nm were
are quick and simple, and their reactions are reproducible taken at 30 °C exactly 10 min after initial mixing.
and linearly related to the concentration of the The ABTS assay measures the relative antioxidant ability
antioxidant(s) present. of extracts to scavenge the radical-cation ABTS+.
Reducing potential of plant extracts produced by the oxidation of 2,2'-azinobis-3-
FRAP assay (Ferric reducing-antioxidant power) ethylbenzothiazoline-6-sulphonate.
This method measures the ability of antioxidants to ABTS antioxidant activity of pomegranate peel extract
reduce ferric iron. It is based on the reduction of the was found to be 46.2 ±1.5 µmol Trolox/g (Table 1).
yellow complex of ferric iron and 2,3,5-triphenyl-1,3,4- HPLC-PDA profiles of pomegranate peel extract
triaza-2-azoniacyclopenta-1,4-diene chloride (TPTZ) to Pomegranate peel was extracted in ethanol solvent and
the blue ferrous form by electron-donating substances concentrated. The chromatogram of 10 l extract at 350
(such as phenolic compounds) at low pH. This reduction nm is shown in figure 1 along with overlaid UV-Vis
is monitored by measuring the change in absorption at spectra. The first eluted peaks at 14.45, 14.86 and 15.61
593 nm. Antioxidant assay was conducted by the method share the same wavelength maximum of 226.6 nm and a
developed by (Benzie and Strain 1999). FRAP values can shoulder at 274 nm. The peaks from 18.39 to 24.53
be obtained by comparing the absorption change in the minutes were also shared similar spectral pattern of two
test mixture with those obtained from increasing maxima’s of around 265 nm and 355 nm respectively.
concentrations of Fe2+. Moreover, the UV-Vis spectra of the major eluted peaks
The antioxidant test based on FRAP assay of between 25.64 to 29.95 minutes is shown in figure 2. The
pomegranate peel extract was found to be 12.4 ±0.4 first maximum wavelengths were at 255.6 nm and the
mmol Fe+2/g of dry material. second maxima was between 348-354 nm. These
Cupric reducing antioxidant power (CUPRAC) absorption patterns characteristics are of typical phenolic
Although FRAP antioxidant assay has been very popular and flavonoid compounds.
among researchers, CUPRAC assay is a relatively new Using very similar conditions to the analytical HPLC-
assay (Apak et al. 2008). It utilizes the copper(II)– PDA, a full scan of the ethanol extract was examined
neocuproine [Cu(II)–Nc] reagent as the chromogenic using UHPLC-MS in the positive and negative
oxidizing agent and is based on the cupric reducing electrospray ionization modes between the range of 200-
ability of reducing compounds to cuprous. 1200 Da. Formic acid was used instead of acetic acid at
The antioxidant test based on CUPRAC assay of 0.1% concentration. The MS results revealed the presence
pomegranate peel extract was found to be 3756 ±35 of some phenolic and flavonoids which were previously
µmole Trolox /g of dry material. identified using different spectroscopic methods.
Free radical scavenging ability of plant extracts
Free radical scavenging activity using DPPH CONCLUSIONS
DPPH is a free radical compound and has been widely Pomegranate peel extract is rich in phenolic and
used to test the free radical scavenging ability of various flavonoids contents and presented an excellent DPPH and
samples (Sakanaka et al. 200524. It is a stable free radical ABTS radical scavenging activities. It also showed
with a characteristic absorption at 517 nm that was used excellent reducing abilities as represented by FRAP and
to study the radical-scavenging effects of extracts. As CUPRAC assays. Pomegranate peel extract also showed a
antioxidants donate protons to this radical, the absorption strong reduction in the production of IL-6 and TNF-α
decreases. Antioxidants, on interaction with DPPH, either from the LPS-induced PMNCs, indicating strong
transfer an electron or hydrogen atom to DPPH, thus antiinflammatory effects. Such results make extracts of
neutralizing its free radical character (Naik et al. 2003)25. pomegranate peels promising source of antiinflammatory
The color changed from purple to yellow and the candidates to be used in the pharmacological industry.
absorbance at wavelength 517 nm decreased.
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IJPQA, Volume 10, Issue 1, January 2019 – March 2019 Page 65

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