American Journal of Plant Sciences, 2013, 4, 1815-1820
http://dx.doi.org/10.4236/ajps.2013.49223 Published Online September 2013 (http://www.scirp.org/journal/ajps)
Total Phenolic, Flavonoids, Tannin Content and
Antioxidant Power of Some Iranian Pomegranate Flower
Cultivars (Punica granatum L.)*
Mannan Hajimahmoodi1,2#, Ghazaleh Moghaddam1, Ali Mohammad Ranjbar1, Hossein Khazani3,
Naficeh Sadeghi1, Mohammad Reza Oveisi1, Behrooz Jannat4
1Department of Drug and Food Control, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran; 2Department of
Traditional, Faculty of Traditional Medicine, Tehran University of Medical Sciences, Tehran, Iran; 3Department of Biology, Tarbiat
Moalem University, Tehran, Iran; 4Ministry of Health and Medical Education, Research Center, Tehran, Iran.
Received July 10th, 2013; revised August 10th, 2013; accepted August 25th, 2013
Copyright © 2013 Mannan Hajimahmoodi et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Recently, pomegranate (Punica granatum L.) was demonstrated to be high in antioxidant activity and strong in phenolic,
flavonoid and tannin contents in its fruit, flower and also aerial part. In this paper six cultivars of Iranian pomegranate
flower including Ghojagh, Rabbab, Malas, Shishegap, Danesiah and Golnar have been investigated. The maximum
amount of total phenolic was detected in Ghojagh (25.94 mg·GAEg–1) and flavonoid showed the highest content in
Danesiah (23.06 mg·CEg–1). The lowest content of these two groups was observed in Golnar (15.19 mg·GAEg–1 and
11.46 mg·CEg–1). Measurement of tannin compounds showed that Rabbab by 2.03% and Golnar by 1.06% have the
highest and lowest amount respectively. According to the FRAP method, Ghojagh and Golnar have the highest and low-
est antioxidant values respectively (452.53 mmol·g–1 and 123.39 mmol·g–1). As a result of HPLC-DPPH method, Malas
and Danesiah have the highest and lowest antioxidant value (116.38 and 97.64 mgVEEg–1).
Keywords: Pomegranate; Flower; Antioxidant; Phenolic; HPLC
1. Introduction
Pomegranate (Punica granatum L.) which is widely cul-
tivated in Iran has been popular worldwide over the years
originated from Middle East and Iran [1,2]. The pome-
granate fruit has been commercialized and can be found
as juice, jellies, wine. The wide adoption of the pome-
granate is due to the recent studies that mentioned it that
contains a high amount of antioxidants which are benefi-
cial to our health in many ways [3-6]. Its great flavor and
health benefits have made it a great candidate for those
who search natural healthy foods [7]. Pomegranate is an
important source of bioactive compounds and different
parts of it have been used in medicine for many centuries
[3,8] and the edible parts used pharmaceutically world-
wide. In tradition medicine, the pricarp was used by
Chinese for the treatment of diarrhea, metrorrhagia, met-
rostaxis and bellyache. The flower was used as a flower
supplement to treat diabetes mellitus in Unani medicine
and the diarrhea was treatment by pomegranate fruit in
South Africa [9]. Pomegranate juice has been demon-
strated to be high in antioxidant activity and is effective
in the prevention of atherosclerosis, coronary heart dis-
ease and cancer [4,10]. There are some reports about the
presence of tannins, alkaloids, glycosides, flavonoids and
phenolic compounds as antioxidant factors in juice, peel,
pulp, and seed fractions of pomegranate [11-13]. In the
case of flower, the pomegranate flowers had a medicinal
use in Traditional Iranian Medicine and also in the cur-
rent studies [14,15]. In folk medicine the decoction of
flowers is used to stop bleeding and purging [16,17].
Pomegranate flowers (golnar) contain a variety of sec-
ondary metabolites such as poly phenols with strong an-
tioxidant activity [18]. The polyphenols in pomegranate
flowers have ellagic acid which had a marked inhibitory
effect on the occurrence and development of tumours in
mice [19]; triterpenes show antimutagenic and anticar-
cinogenic effects [20]; and oleanolic acid significantly
*The authors declare that they have no conflict of interests.
#Corresponding author.

Total Phenolic, Flavonoids, Tannin Content and Antioxidant Power of Some Iranian Pomegranate
Flower Cultivars (Punica granatum L.)
1816
enhanced acute glucose-stimulated insulin secretion at
basal and stimulatory glucose concentrations inpancreatic
b-cell. Such effects may contribute to the antidiabetic
properties [21]. The bright colour of pomegranate flow-
ers and arils is due to anthocyanins [22]; however, only
one anthocyanin compound (i.e. pelargonidin-3,5-diglu-
coside) has yet been identified in pomegranate flowers
using HPLC [23], whereas in pomegranate juice, prince-
pally cyanidin-3-O-glucoside, cyanidin-3,5-di-O-glucoside,
delphinidin-3-O-glucoside, delphinidin-3,5-di-O-glucoside,
pelargonid in-3-O-glucoside, and pelargonidin-3,5-di-O-
glucoside, have been reported [24, 25]. Oleanolic acid,
ursolic acid and gallic acid, active components contained
in pomegranate flower [26], have long been recognized
to have antihyperlipidemic properties [27,28]. It is known
that the amount of organic acids, phenolic compounds,
sugars, water-soluble vitamins, and minerals of all parts
of pomegranates are different in various researches which
may be attributed to their cultivar origins [29,30].
Therefore, in this study, the contents of total phenolic,
flavonoids, and tannins of the some Iranian pomegranate
flower cultivars and their antioxidant activity were inves-
tigated through the FRAP and HPLC-DPPH methods.
2. Materials and Methods
2.1. Sample Preparation
Six cultivars of pomegranate’s flower were obtained from
Agricultural Research Center, Yazd, Iran. The flowers
varieties (Malas, Shishegap, Danesiah, Rabbab, Ghojagh
and Golnar) were harvested during May 2012 from dif-
ferent mature trees which randomly selected. Flowers
were desiccated in shade and room temperature. Then,
different flower cultivars were grounded separately by
mortar. 0.5 g of each cultivar was shaken with methanol
80% for 2 hours and centrifuged in 10,000 rpm [Hereus-
Germany] then the extracts were separated and stored in
4˚C [9].
According to the tannin determination, 3 g of each
dried flower powder was extracted with deionized dis-
tilled water in 250 mL volumetric flask during 4 hours at
room temperature and then the sample was filtered [31].
2.2. Total Phenolic Content
Total phenolics contents were determined according to
the Folin-Ciocalteu method with slight modifications
[32]. The extract (200 μL) was mixed with 1.5 mL of
Folin-Ciocalteu reagent [previously diluted 10 times with
double distilled water] and allowed to stand at room tem-
perature for 5 min. 1.5 mL sodium bicarbonate solution
[60 g·L–1] was added to the mixture and after incubation
for 90 min at room temperature, the absorbance level was
measured at 725 nm using a UV-Visible spectropho-
tometer (GBC, Cintra 40). Total phenolic were quantified
by calibration curve obtained from measuring the absorb-
ance of the known concentrations of gallic acid standard
solutions [10 - 150 μg·mL–1 in 80% methanol]. The re-
sults were calculated as gallic acid equivalent (GAE) per
one gram dry powder and reported as mean value ± stan-
dard deviation (SD).
2.3. Total Flavonoid Content
Total flavonoid content was measured by the aluminum
chloride colorimetric method [33]. An aliquot (1 mL) of
each extract was added to 10 mL volumetric flask con-
taining 4 mL of double distilled water. Then 0.3 mL
NaNO2 5% was added to the flask and after 5 min, 0.3
mL AlCl3 [10%] was also added. At 6th min, 2 mL
NaOH (1 M) was added and the total volume was made
up to 10 mL with double distilled water. The solution
was mixed completely and the absorbance level was
measured versus prepared reagent blank at 510 nm. Total
flavonoid content was expressed as mg catechin equiva-
lents (CE) per one gram dry powder. The total flavonoid
assay was measured three times for each pomegranate
extract. 1 mL of standard solution (catechin: 5 - 100 mg/
L) was used to construct calibration curve.
2.4. Antioxidant Assay (FRAP Method)
The FRAP (Ferric reducing antioxidant power) assay was
described initially by Benzie and Strain [34]. The princi-
ple of this method is based on the reduction of the ferric-
tripyridyl triazine complex to its ferrous colored form in
the presence of antioxidants. Briefly, the FRAP reagent
contained 5 mL TPTZ (2,4,6-tripyridyl-S-triazine, 10
mmol·L–1) solution in 40 mmol·L–1 HCl plus 5 mL FeCl3
[20 mol·L–1] and 50 mL of Acetate buffer (0.3 mol·L–1).
It was prepared freshly and set at 37˚C. Aliquots of 50
μL sample supernatant were mixed with 1.5 mL FRAP
reagent and the absorbance of reaction mixture at 593 nm
was measured spectrophotometrically after incubation at
37˚C for 10 min. To construct the calibration curve five
concentrations of FeSO4·7H2O (100 - 1000 mmol·L–1)
were used and the absorbencies were measured as sample
solution. The values were expressed as the concentration
of antioxidants having a ferric reducing ability equivalent
to that of 1 mmol·L–1 FeSO4. All the measurements were
taken in triplicate and expressed as mean value ± RSD.
2.5. Antioxidant Assay [HPLC-DPPH Method]
The chromatographic analysis was carried out by a Kna-
uer HPLC system [Berlin, Germany] equipped with an
auto-sampler, pump and a UV–Vis detector. 20 µL of
each samples (1 mL extract was volumed to 5 mL with
methanol 80% in volumetric flask) was added to 2 mL

Total Phenolic, Flavonoids, Tannin Content and Antioxidant Power of Some Iranian Pomegranate
Flower Cultivars (Punica granatum L.)
1817
1,1-diphenyl-2-picrylhydrazyl [DPPH] solution at a con-
centration of 0.1 mmol·L–1 and mixed with 20 mL de-
ionized distilled water. Trolox (1 mg·mL–1) and deion-
ized distilled water were used as the standard of vitamin
E and blank respectively and were prepared by adding 20
µL of each one to 2 mL DPPH solution. The mixture was
shacked 20 seconds and then kept in the darkness 40 min
at room temperature. 20 µL of prepared samples which
were filtered through 0.2 µm membrane filter [Control
Biogen-Spain] was injected to the HPLC. The radical
scavenging activity of DPPH was measured at 517 nm.
All samples were analyzed in triplicate (mean ± RSD)
[35].
2.6. Tannin Assay
The analyses of tannin content in flowers were per-
formed according to the International Pharmacopoeia and
AOAC methods [36] with some modifications. 3 g of
flower powder was infused with 250 mL of deionized
double distilled water and then it was filtered through
0.45 µm (Control Biogen-Spain) sample filter. 25 mL of
the infusion was added into 1 L conical flask and then 25
mL of indigo solution [0.6%] and 750 mL deionized dis-
tilled water was added. The solution has been titrated
with 0.1 N aqueous solution of KMNO4 until the blue
colored solution changed to golden yellow one. Standard
solution of indigo carmine was prepared as following: 6
g indigo carmine was dissolved in 500 mL of deionized
distilled water by heating and after cooling 50 mL of
98% H2SO4 was added. The solution was diluted to 1 L
with deionized distilled water and then it was filtered
through 0.2 µm membrane filter. The blank test was car-
ried out by titration of the mixture of 25 mL indigo car-
mine and 775 mL double distilled water. All samples
were analyzed in duplicates. The tannin percent [%] in
the samples were calculated as follows:
T (%) = [V – V0] 0.004157 × 250 × 100/g × 25
where V is the volume of 0.1 N aqueous solution of
KMNO4 used in the titration of the sample and V0 is the
volume of 0.1 N aqueous solution of KMNO4 used in the
titration of the blank sample as mL; 0.004157 is the tan-
nins equivalent in 1 mL of 0.1 N aqueous solution of
KMNO4; g is the mass of the sample taken for the analy-
sis as gram and 250 is the volume of the volumetric
flask.
2.7. Statistical Analysis
Three replicates of each sample were used for statistical
analysis and the values were reported as mean ± RSD.
Pearson’s correlation was carried out using SPSS statis-
tical program to study the relationship between antioxi-
dant activity and total phenolic and flavonoid content.
Data were also subjected to the analysis of variance and
mean values were compared by Tukey post-hoc multi-
omparison test. Differences at p-value <0.05 were con-
sidered to be significant.
3. Results and Discussion
Pomegranate flower has been used in traditional Iranian
medicine according to its medicinal effects [14]. The
total phenolic content of pomegranate flower extracts is
expressed in term of gallic acid equivalent (the standard
curve equation: Y = 0.005X – 0.0234, r2 = 0.9975). It
was ranged from 25.94% to 15.19% mg gallic acid equi-
valents per gram of dry powder in Ghojagh and Golnar
respectively (Table 1). The total flavonoid content of
flower extracts is also expressed in terms of catechin
equivalent (the standard curve equation: y = 0.005x +
0.1478, r2 = 0.9919), ranged from 23.06% to 11.46% mg
catechin equivalents per gram of dry flower powder in
Danesiah and Golnar respectively (Table 1).
Phenolic and flavonoid contents are important in anti-
oxidant power of herbals and the analysis of their amount
in different pomegranate flower cultivars via ANOVA
shows that Ghojagh has the most amount of total phenol
and Golnar has the least ones (p-value < 0.05). In the
case of total flavonoid, Danesiah cultivar has the most
content (23.06 mgCE·g–1) and Golnar cultivar has the
Table 1. The total phenolic, flavonoid, tannin content and flavonoid-phenolic ratio of six pomegranate flower cultivars.
Cultivar Total phenol
[mg GAE/g dry powder ± RSD] Total flavonoid [mg CE/g dry powder ± RSD] Total tannin [% ± RSD] Flavonoid/Phenolic
Ghojagh 25.94a ± 7.00 19.17b ± 4.31 1.33b ± 0.22 0.74c
Rabbab 24.57a ± 5.04 16.76c ± 2.17 2.03a ± 0.15 0.68c
Shishegap 20.60b ± 6.21 18.30b ± 2.32 1.29b ± 0.43 0.89b
Danesiah 23.48a ± 4.46 23.06a ± 3.46 1.98a ± 0.67 0.98a
Malas 18.68c ± 3.01 18.21b ± 4.49 1.47b ± 04 0.97a
Golnar 15.19d ± 2.02 11.46d ± 2.17 1.06c ± 0.11 0.75c
Values in the same column bearing different superscripts are significantly (p ≤ 0.05) different.

Total Phenolic, Flavonoids, Tannin Content and Antioxidant Power of Some Iranian Pomegranate
Flower Cultivars (Punica granatum L.)
1818
Table 2. Antioxidant power of six pomegranate flower cultivar according to the FRAP and HPLC-DPPH method.
Cultivar mmol Fe2+ equivalent/g dr y po wder ± RSD Mg vitamin E equivalent/g of dry powder ± RSD
Ghojagh 452.53a ± 25.08 109.93b ± 20.60
Rabbab 219.77c ±19.87 112.13a ± 14.40
Shishegap 219.05c ± 13.18 109.68b ± 16.48
Danesiah 200.33c ± 21.44 97.64c ± 16.69
Malas 337.04b ± 15.45 116.38a ± 20.81
Golnar 123.39d ± 17.38 107.63b ± 21.02
Values in the same column bearing different superscripts are significantly (p ≤ 0.05) different.
least content (11.46 mgCE·g–1) significantly. Analysis of
antioxidant power according to the FRAP method show
Ghojagh (452.53 mmol Fe2+·g –1) has the most antioxi-
dant power while Golnar (123.39 mmol Fe2+·g–1) has the
least ones. By the HPLC-DPPH method Malas (116.38
mgVitEE–1) have the most antioxidant effect and Dane-
siah (97.64 mgVitEE–1) has the least antioxidant power
(Table 2). The Pearson’s correlation showed no signifi-
cant correlation between total phenolic and flavonoid con-
tent and antioxidant power via both FRAP and HPLC-
DPPH methods. The total tannin content in different cul-
tivars were also compared according to the statistical cal-
culation and the results showed Rabbab and Danesiah
cultivars have the highest amount of tannin and Golnar
has the least content. The flavonoid-phenolic ratio in Ta-
ble 1 is mentioned to show the importance of flavonoids
in total phenolic content and its antioxidant activity. The
range of this ratio is between 0.98 in Danesiah and 0.68
in Rabbab.
In a prosperous in vitro and also in vivo study by Kaur
et al. [15] the pomegranate flower extract indicated a sig-
nificant antioxidant activity and it was found to exhibit a
potent protective role in acute oxidative tissue injury ani-
mal in vivo model. Also the ethanolic extract of pome-
granate flower showed 81.6% antioxidant activity in
DPPH model system.
Comparison of the pomegranate flower results with its
pulp and peel [37] showed flowers have higher amount
of the total phenol and flavonoids content, but it has less
antioxidant activity according to the FRAP method. It
can be suggested that water soluble antioxidant such as
organic acid can leads to antioxidant activity of pulp and
water insoluble component to the flower. In another
study the antioxidant activity and total phenolic content
of pomegranate flower and juice were compared and in
spite of higher amounts of phenolic compounds in flower
extracts, antioxidant activity of juices were more than
flowers indicating results as the same as this study [38].
Total phenolic and flavonoid content of peel and its an-
tioxidant activity in another research is remarkable more
than the flower and suggest the peel as a better source of
antioxidant components [37]. In Orak et al. study [39],
the DPPH scavenging activity of antioxidant in juice,
peel, and seed parts of pomegranate were investigated.
The results showed that the EC50 value of DPPH scav-
enging activities in peel extracts was 23.4-fold higher
than the juice extracts, and the seed extracts had 2.3-fold
higher than juice. Also the reducing power in peel ex-
tracts was found to be 4.7-fold higher than seed extracts
and 10.5-fold higher than the juice. The data expressed
that, in peel and pulp except that, the total polyphenol
and tannin contents, flavonoid and anthocyanin play an
important role in antioxidant activity respectively.
4. Conclusion
The antioxidants assessment suggests that the studied po-
megranate flower and its associated bioactive compounds
such as phenolic, flavonoids and tannins compounds may
possess a strong potential as a chemo preventive and pos-
sibly as new tools for preventing various human diseases.
However, further studies should focus on developing the
novel pomegranate derived products such as ready-to-eat
pomegranate flower, single-strength juices, flower ex-
tract concentrates, flower in syrup, and the frozen flower,
to benefit from these constituents throughout a healthy
life cycle.
5. Acknowledgements
This work was student thesis and supported by the grant
from the research council of Tehran University of Medi-
cal Sciences, Tehran, Iran.
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