Total Phenolic, Flavonoids, Tannin Content and Antioxidant Power of Some Iranian Pomegranate Flower Cultivars (<i>Punica granatum</i> L.)

doi:10.4236/ajps.2013.49223

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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.

Email: #[email protected]

Received July 10th, 2013; revised August 10th, 2013; accepted August 25th, 2013

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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