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Iranian Journal of Pharmaceutical Sciences 2021: 17 (1): 51-68 www.ijps.ir Original Article

Diversity of Phenolic Profiles in Peel of an Iranian Pomegranate Cultivar (Punica granatum L.)

Akram Taleghani*, Reza Akbari

Department of Chemistry, Faculty of Science, Gonbad Kavous University, Gonbad, Iran.

Abstract Pomegranate (Punica granatum L.) is one of the oldest known fruits native to Iran. In addition to multiple studies carried out on different parts of pomegranate, the peels are noted due to various phytochemicals in different colors and regions. In the present study, major anthocyanins and non-anthocyanins of an Iranian black pomegranate cultivar (i.e., pomegranate samples of Ghaemshahr, Iran) were analyzed using high-performance liquid chromatography-mass spectrometry. In total, 30 compounds were obtained from pomegranate peels. Among these compounds, 5 anthocyanins, 1 gallotannin, 15 ellagitannins, 4 hydroxybenzoic acids, 2 gallagyl esters, 1 dihydroflavonol, and 7 hydroxycinnamic acids were identified based on their fragmentation patterns and ultraviolet spectra. Among the varieties of Iranian pomegranates, the anthocyanins of peonidin-hexoside and cyanidin-pentoside were identified in this species for the first time. Peonidin-hexoside, caffeic acid, coumaric acid-hexoside, and galloyl-hexoside were major phenolic compounds. In addition, the antioxidant activity of different fractions was tested using DPPH free radical scavenging in vitro assay (IC50: 133.13-557.23 µg/mL). The obtained results of this study highlighted that this cultivar can be an important candidate for future new drug discoveries.

Keywords: Punica granatum, peel, anthocyanin, non-anthocyanin phenolic compounds, HPLC-MS/MS, antioxidant.

Corresponding Authors: Akram Taleghani, phytochemicals. It is native to the Department of Chemistry, Faculty of Science, Gonbad Mediterranean region and is cultivated in Kavous University, Gonbad, Iran. Tel: +98 9156872563 subtropical and tropical regions around the Email: [email protected] Cite this article as: Taleghani A, Akbari A, Diversity world, such as Iran, California, Turkey, Egypt, of Phenolic Profiles in Peel of an Iranian Pomegranate Italy, India, Chile, and Spain [1]. Pomegranate Cultivar (Punica granatum L.), 2021, 17 (1): 51-68. peels (PPs) remain as byproducts after juice extraction. At present, fruit peels have become 1. Introduction attractive due to beneficial phytochemicals and Pomegranate (Punica granatum L.) is are utilized in pharmaceutical, food, and consumed as a popular fruit and juice due to cosmetic industries [2, 3]. In PPs, the main the potential health benefits and phenolic compounds (i.e., one class of Taleghani A, et al. / IJPS 2021; 17 (1): 51-68

bioactive phytochemicals) previously reported countries, there are a limited number of studies in the literature include phenolic acids (i.e., are on the profiles of these compounds in the chlorogenic, syringic, caffeic, sinapic, ferulic, Iranian cultivar. In this paper, considering the p-coumaric, ellagic, cinnamic, and gallic structural diversity of the active compounds of acids), tannins (i.e., ellagic acid derivatives, pomegranates, a cultivar of p. granatum with such as punicalin, punicalagin, pedunculagin, black color was selected to investigate the and ellagitannins), and flavonoids (i.e., diversity of anthocyanins and non- anthocyanins, such as delphinidin, anthocyanins in PPs using the HPLC and mass pelargonidin, cyanidin, and their derivatives, spectrometry (MS) techniques. Firstly, and anthoxanthins, such as epicatechin, different fractions of PPs (i.e., methanol, catechin, and quercetin) [3-5]. Pomegranates ethanol acetone, and ethyl acetate) were have a large variety of colors from white to red evaluated for antioxidant activity using 2,2- and dark red. The red color and mild diphenyl-1-picrylhydrazyl (DPPH) free radical astringency are the characteristics of scavenging in vitro assay. Among different pomegranates related to polyphenol fractions of PPs, the methanolic and ethanolic constituents [6]. The color of PPs is due to the extracts were chosen for the characterization presence of anthocyanins (i.e., one class of of phenolic compounds due to the highest flavonoids). Cultivars with a dark red color amount of antioxidant activity. possess higher antioxidant activity than other portions [4, 7]. The identification and 2. Materials and Methods characterization of secondary metabolites in 2.1. Solvents and Reagents this fruit are important due to their role in All reagents and solvents used for HPLC- reducing the risk of diseases, such as cancer, MS separation and identification were inflammation, and cardiovascular and purchased (Merck & Co., Inc., Germany) with neurodegenerative diseases [8-10]. Previous analytical or HPLC grade. Moreover, all other studies have reported a number of methods for solvents for extraction were also purchased the identification of phenolic compounds in (Merck & Co., Inc., Germany). pomegranates [11]. Recently, particular attention has been given to high-performance 2.2. Plant Materials liquid chromatography (HPLC) with mass Iranian pomegranate fruits of unknown spectrometric detection due to the limited cultivar obtained from Ghaemshahr, Iran, were availability of reference standards and difficult washed and steamed (5 min) for enzyme identification of compounds only based on inactivation. A voucher number (803885) was ultraviolet spectra [12, 13]. Although there deposited in the Herbarium of Gonbad Kavous have been many studies carried out on the University, Gonbad, Iran. evolution of phenolic compounds in pomegranate cultivars grown in Iran and other

Phenolic Profiles in Peel of Iranian Pomegranate

2.3. Extracts Preparation %DPPH scavenging = [Ac-As/Ac] × 100

At first, the peels were lyophilized and where As is the absorbance of the sample, ground with a blender. Then, each extract was while Ac is the absorbance of control. Different obtained as it will be described. Subsequently, concentrations of extracts inhibiting 50% the dried powder was separately extracted with DPPH (IC50) were calculated by the ethyl acetate, acetone, methanol, and ethanol concentration of the extracts against the for the evaluation of the antioxidant activity. inhibition (%) of DPPH.

2.4. Quantification of Antioxidant Activity 2.5. Extraction of Anthocyanins The antioxidant potential of pomegranate The peel powder (1.0 g) was sonicated in fractions (i.e., methanol, ethanol acetone, and 30 mL of methanol (containing 0.1 % HCl) for ethyl-acetate) was determined by DPPH free 30 min. The obtained hydroalcoholic extract radical scavenging assay. The DPPH reduced was centrifuged at 5,000 rpm for 30 min and by an antioxidant, which can donate hydrogen. dried in a rotary evaporator under vacuum at The changes in color were measured from 35°C [7]. deep violet to light yellow using an ultraviolet- visible light spectrophotometer. Firstly, dried 2.6. Extraction of Non-anthocyanin powder of peel was separately extracted with Polyphenolics ethyl acetate, acetone, methanol, and ethanol. The plant extract was prepared as described In order to determine the radical scavenging in a study by Zahin et al. [15]. The methanolic capacity of the different concentrations of and ethanolic extracts of P. granatum were extracts against stable DPPH, a slightly gained by a continuous stirring of the dried modified method was spectrophotometrically peels at ambient temperature for 2 days. used as it will be described [14]. Briefly, 50 μl Furthermore, at the end of the extraction, the of different concentrations of the obtained extracts were separately filtered to make extracts (35-250 µg/ml) were mixed with 50 µl methanol and ethanol fractions, and the fresh of the 0.1 mM solution of methanolic DPPH. solvent was added to the plant materials. The The 50-µL methanol was employed as a obtained extracts were concentrated using a control in the experiment. After 30 min of rotary evaporator for dryness. A crude extract incubation at 37ºC, the absorbance of the was obtained after the removal of the samples using a microplate reader was extraction solvent and stored at 4°C in the dark spectrophotometrically read at 517 nm. until further testing. A stock solution of 1 Butylated hydroxyl toluene (BHT) within the mg/ml in the water of LC-MS grade was range of 35-250 µg/ml was used as a positive prepared and 2 µl was injected for LC-MS control. The antioxidant activity as percentage analysis. inhibition was calculated using the following equation:

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2.7. High-Performance Liquid 3. Results and Discussion Chromatography-Mass Spectrometry (LC-MS) 3.1. Antioxidant Activity The method of Zahin et al. [15] was used The PPs have antioxidant activities due to a for the LC-MS analysis of the secondary good source of phenolic compounds (e.g., metabolites. The HPLC (Shimadzu, Tokyo, anthocyanins, quercetin, catechin, Japan) system coupled with a mass gallotannins, ferulic acid, ellagitannins, and spectrometer, which combines an AB SCIEX ellagic and gallic acids). The obtained results 3200 Q-TRAP with a Turbo V electrospray of the current study are in agreement with the ionization (ESI) source, was developed in the antioxidant activities of PPs reported in the present study. All the experiments were literature from different regions. The findings performed using a Thermo scientific liquid of studies have shown that methanol, acetone, chromatography system connected to ESI. The and aqueous extracts of Tunisian cultivars separation was carried out on a C18 reversed- have antioxidant activity with IC50 values of phase column (SUPELCO Analytical HS-C18, 1.9-1900 μg/mL [5, 17-21]. The range of IC50 4.6×150 mm, 3 µl, Sigma-Aldrich, USA). The values has been reported at 15-27 μg/mL for mobile phase consisted of gradient program the methanol extracts of Persian pomegranate water and methanol within the range of 20- cultivars [22-24]. The methanol and aqueous 100% over 25 min with a flow rate of 0.5 extracts of PPs collected from different mL/min. The MS was carried out in negative regions of India showed antioxidative potential ion mode using ESI source with some with IC50 values of 4.9-8730 μg/mL [15, 25- conditions (i.e., a capillary voltage of −10V, 27]. The ethyl acetate and ethanol extracts of capillary temperature of 200ºC, and Sheath two types of PPs obtained from Italy exhibited and Auxiliary Gas flow [N2]). The MS spectra antioxidant potential with IC50 of 2.430 μg/mL were acquired by full range acquisition and 94.7% radical inhibition, respectively [28, covering m/z 50-2000. MZmine analysis 29]. The methanol and aqueous fractions software package (version 2.3) was used for obtained from Egyptian, South African, and instrument control, data acquisition, and data Turkish cultivars displayed antioxidant analysis [16]. capacity (range: 71.65-96.24%) [28, 30-33]. In addition, the peels of Peruvian, Chinese, 2.8. Statistical Analysis Georgian, Spanish, and Mauritian Statistical analysis was carried out using pomegranates were reported to have radical Excel software with three replications. All the scavenging activities with Trolox equivalents obtained results were presented as of 5.20-2239 mmol TE/kg [11, 34-37]. In the mean±standard deviation. Moreover, a p-value present study, the antioxidant potential of the of 0.05 was considered statistically significant. peel extracts was measured using the DPPH method. The scavenging activity was evaluated at different concentrations (range:

Phenolic Profiles in Peel of Iranian Pomegranate

35-250 µg/ml). These fractions inhibited (e.g., cyanidin 3,5-diglucoside and delphinidin DPPH absorption (methanol: 92.28%, ethanol: 3,5-diglucoside), and hydrolyzable tannins 86.55%, acetone: 75.1%, ethyl acetate: (e.g., hydroxybenzoic acid, gallic acid, and 21.72%, and positive controls [BHT]: 88.1%; ellagic acid) were identified and quantified Figure 1). Furthermore, the extracts exhibited from the methanolic extract of South African strong activity to reduce the DPPH with IC50 cultivars [31]. Additionally, the reverse phase- values of 133.13-557.23 µg/ml (Table 1). The HPLC profile of Tunisian PPs showed the activity of methanolic and ethanolic extracts presence of ellagitannins of punicalagin and was stronger than that reported for the BHT punicalagin derivatives as major tannin with IC50 values of 141.25 µg/ml. Therefore, compounds and galloyl-HHDP-hexoside, these fractions were selected for the analysis pedunculagin I, pedunculagin II, granatin A, of phenolic compounds. The promising granatin B, bis-HHDP-gluconic acid antioxidant activity of the extracts might be (lagerstannin A), p-coumaric, and vanillic conferred by phenolic compounds identified acids [17, 21]. Polyphenols of gallic and through LC-MS analysis, mainly phenolic ellagic acids, punicalagin A, punicalagin B, acids, such as hydroxycinnamic acids and chlorogenic, ferulic, p-hydroxybenzoic, and p- ellagitannins. coumaric acid were reported in the extracts of Turkish PPs [38, 39]. Phenolic acids, 3.2. LC-ESI/MS Analysis of Extracts of P. anthocyanins, gallotannins, ellagitannins, granatum Peel gallagyl esters, and dihydroflavonols were The extracts of P. granatum peels were identified and quantified in the peel of analyzed using Liquid Chromatography Peruvian pomegranate cultivar [11]. Electrospray Ionization Mass Spectrometry Punicalagin in pomegranate different varieties (LC-ESI/MS) in a negative ion mode. In the from Pakistan was reported [32]. Punicalagin, observed LC-MS profile, deprotonated gallic and ellagic acids, punicalagin, and molecular ions of different phenolic punicalin were the main identified compounds compounds consist of flavonoids, in the peel extracts of Serbian pomegranate hydroxycinnamic acids, and phenolic acids. [28, 40]. Punicalagin was identified as the These polar compounds were characterized major tannin reported in the peel of a Brazilian based on the mass patterns and their retention pomegranate cultivar [41]. Gallagic acid, time by comparison to the data obtained from ellagic acid, gallic acid and punicalagin were the history of pomegranate different cultivars. the main phenolic compounds reported in the The obtained results of the present study are in peel of an Israelian pomegranate cultivar [35]. line with the findings of previous studies Phenolic compounds of gallagic and ellagic carried out on phenolic compounds of PPs acid derivatives, gallic acid, caffeic acid, from different regions. Flavonoids (e.g., catechin, punicalin, punicalagin, and granatin epicatechin, catechin, and rutin), anthocyanins A and B were detected in the PPs of Indian

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cultivars using HPLC-MS analysis [15, 24, 25, reported that Ardestan, Saveh, and Kerman 29, 42]. Pande and Akoh (2009) were reported pomegranates contained 5 anthocyanins, caffeic, p-coumaric, ferulic acids and catechin mainly cyanidin 3, 5-diglucoside, cyaniding 3- in the peel of Georgian pomegranate [36]. glucoside, pelargonidin 3, 5-diglucoside, Punicalagins (in alpha and beta forms) and pelargonidin 3-glucoside, and delphinidin 3- ellagic acid were identified from the glycoside; however, cyanidin-pentoside and methanolic extracts of six Spanish peonidin hexoside were not detected [43]. This pomegranate cultivars [37]. In addition, gallic variation could be due to the differences in and ellagic acids, punicalagin, punicalin, 2,3- fruit development stages. Anthocyanins’ (S)-HHDP-D-glucose, castalagin corilagin, profiles in the peel demonstrate that cyanidin- punicalin, punicalagin, and granatin A and B pentoside, peonidin hexoside, and myricetin- were detected by HPLC-MS analysis in the hexoside have been the first reports to date PPs of Indian and Serbian cultivars [40, 42]. In among the Iranian cultivars. The total the present study, 35 metabolites (i.e., chromatograms of the LC-MS analysis of the anthocyanin and non-anthocyanin) were extracts containing anthocyanins are illustrated identified and are reported in tables 2 and 3. in Fig.2. In addition, the chromatogram of the compound of peonidin hexoside as the highest 3.2.1. Anthocyanins intensity is depicted in Fig.3. Data Scan of LC-MS showed that 3, 5- diglucoside derivatives were difficult to detect 3.2.2. Non-anthocyanins due to their low concentrations and only 3- The qualitative identification of non- glucoside derivatives of cyanidin delphinidin anthocyanin polyphenolics in PPs is shown in and pelargonidin as cyanidin 3-glucoside, Table 3. Among the polyphenolics, 30 and 26 delphinidin 3-glucoside, and pelargonidin 3- compounds were characterized in the glucoside were recorded. The identified methanolic and ethanolic extracts, anthocyanins, such as peonidin hexoside, respectively, as hydrolyzable tannins, such as cyanidin-pentoside, pelargonidin 3-glucoside, ellagitannins, gallotannins, and gallagyl esters, delphinidin 3-glucoside, and cyanidin 3- in addition to hydroxycinnamic acids, glucoside at m/z 464, 420, 434, 465, and 450 hydroxybenzoic acids, and dihydroflavonols respectively, were in high-level base on mass based on comparing their mass spectrometric data (Table 2). Furthermore, a flavonol, data to previous data on other pomegranate namely myricetin hexoside, with M+ ion at m/z varieties. According to the results, the 481 was detected in the analysis of the methanolic and ethanolic extracts had almost anthocyanins of the peel extract. However, similar constituents. Compounds of there were differences between the present protocatechuic acid-derivative, study and previously reported studies dihydrokaempferol-hexoside, ellagic acid- regarding other Iranian ecotypes. Zarezadeh hexoside, valoneic acid bilactone, ellagic acid

Phenolic Profiles in Peel of Iranian Pomegranate derivative, and galloyl-HHDP-DHHDP- extracts are illustrated in Figure 2. hexoside (granatin B) in the methanolic extract Furthermore, the chromatograms of the were not detected in the ethanolic extract. compounds of caffeic acid derivative in the Moreover, ellagic acid derivative, bis-HHDP- methanolic extract and galloyl-hexoside in the hexoside (pedunculagin I), and castalagin ethanolic extract as the highest intensity are derivative in the ethanolic extract were not depicted in Fig. 4. Moreover, Table 4 tabulates detected in the methanolic extract. The the structures of anthocyanin and some non- comparison of the results of the presents study anthocyanin polyphenolics in the pomegranate and previous data shows that the compounds peel. of galloyl-hexoside, ellagic acid- deoxyhexoside, ellagic acid derivative, ellagic 4. Conclusion acid dihexoside, ellagic acid derivative, In this study, the application of HPLC-MS protocatechuic acid, protocatechuic acid- was reported for the characterization of the derivative, vanillic acid-hexoside, caffeic acid phenolic compounds of one black hexoside, caffeic acid hexoside derivative, pomegranate cultivar obtained from Iran. caffeic acid derivative, 5-O-caffeoylquinic Firstly, the antioxidant activity of different acid, ferulic acid-hexoside, coumaric acid, and fractions of PPs (P. granatum) was evaluated dihydrokaempferol-hexoside reported in using DPPH free radical scavenging in vitro previous studies in the juice and mesocarp of assay, and two methanolic and ethanolic this plant, were also observed in the peel extracts were selected for the analysis of extracts. Some of the identified compounds in phenolic compounds due to their high radical methanolic extract, including caffeic acid scavenging. Approximately 35 metabolites derivative, coumaric acid-hexoside, caffeic (i.e., anthocyanins and non-anthocyanins) acid hexoside, 5-O-caffeoylquinic acid, were identified from the peel extracts. coumaric acid, HHDP hexoside, Differences in anthocyanin compounds protocatechuic acid-derivative, ellagic acid profiles were observed among different Iranian derivatrive, and galloyl-hexoside, were in high pomegranate varieties. These fractions are rich levels at m/z 299, 325, 341, 353, 163, 481, in ellagitannins and ellagic acid, as a group of 425, 441, and 331, respectively. In addition, phenolics responsible for confirming chemical compounds in ethanolic extract, antioxidant properties; therefore, they could be including galloyl-hexoside, caffeic acid standardized and selected with efficacy and derivative, coumaric acid, coumaric acid- safety in the animal model system and hexoside, HHDP hexoside, and 5-O- pharmaceutical industry. In addition, the caffeoylquinic acid, were in high levels at m/z optimization, detection, and separation of 331, 299, 163, 325481, and 353, respectively. phenolic compounds, especially anthocyanins, The total chromatograms of the LC-MS in pure forms is difficult, and further studies analysis of the methanolic and ethanolic are required in this regard.

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Pomegranate (Punica granatum L.) and Inter- (2010) 123(3): 878-85. Relationships With Total Phenolic, Tannin, [39] Dikmen M, Ozturk N, Ozturk Y. The Anthocyanin, and Flavonoid Contents. Food Sci. Antioxidant Potency of Punica granatum L. Fruit Peel Biotechnol. (2012) 21(2): 373-87. Reduces Cell Proliferation and Induces Apoptosis on [34] Guo C, Yang J, Wei J, Li Y, Xu J, Jiang Y. Breast Cancer. J. Med. Food (2011) 14(12): 1638-46. Antioxidant Activities of Peel, Pulp and Seed [40] Stojanović I, Šavikin K, Đedović N, Živković J, Fractions of Common Fruits as Determined by FRAP Saksida T, Momčilović M, Koprivica I, Vujičić M, Assay. Nutr. Res. (2003) 23(12): 1719-26. Stanisavljević S, Miljković Đ, Menković N. [35] Orgil O, Schwartz E, Baruch L, Matityahu I, Pomegranate Peel Extract Ameliorates Autoimmunity Mahajna J, Amir R. The Antioxidative and Anti- in Animal Models of Multiple Sclerosis and Type 1 Proliferative Potential of Non-Edible Organs of the Diabetes. J. Funct. Foods (2017) 35: 522-30. Pomegranate Fruit and Tree. Food Sci. Technol. [41] Morzelle MC, Salgado JM, Telles M, Mourelle (2014) 58(2): 571-7. D, Bachiega P, Buck HS, Araujo Viel T. [36] Pande G, Akoh CC. Antioxidant Capacity and Neuroprotective Effects of Pomegranate Peel Extract Lipid Characterization of Six Georgia-Grown after Chronic Infusion with Amyloid-Β Peptide in Pomegranate Cultivars. J. Agric. Food Chem. (2009) Mice. PloS one. (2016) 11(11). 57(20): 9427-36. [42] Singh JP, Kaur A, Shevkani K, Singh N. [37] Rosas‐ Burgos EC, Burgos‐ Hernández A, Composition, Bioactive Compounds and Antioxidant Noguera‐ Artiaga L, Kačániová M, Activity of Common Indian Fruits and Vegetables. J. Hernández‐ García F, Cárdenas‐ López JL, Food Sci. Technol. (2016) 53(11): 4056-66. Carbonell‐ Barrachina Á. Antimicrobial Activity of [43] Zarezadeh Mehrizi R, Emam-Djomeh Z, Bagh Pomegranate Peel Extracts as Affected by Cultivar. J. Khandan MS, Loni E, Akhavan H, Biabani J. Sci. Food Agric. (2017) 97(3): 802-10. Identification and Quantification of Anthocyanins in [38] Çam M, Hışıl Y. Pressurised Water Extraction of Pomegranate Peel Extract. J. Food Sci. Technol. Polyphenols from Pomegranate Peels. Food Chem. 2015;12(49): 31-40.

Phenolic Profiles in Peel of Iranian Pomegranate

Tables:

Table 1. Total antioxidant capacity of the P.granatum extracts according to the DPPH assays.

Fractions Inhibition Calibration curve IC50 (%) (µg/ml) Methanol 92.28 y = 0.3738x + 0.2342 133.13 R² = 0.9979

Ethanol 86.55 y = 0.3519x - 0.4355 143.32 R² = 0.9983 Acetone 75.1 y = 0.3093x - 1.1678 165.43 R² = 0.9955

Ethyl acetate 21.72 y = 0.0899x - 0.0952 557.23 R² = 0.9891

BHT 88.1 y = 0.365x - 1.5584 141.25 R² = 0.9917

Table 2. The anthocyanins present in pomegranant peel.

No. Name tR (min) (m/z) Relative intensity (%) 1 Pelargonidin 3- 16.14 434 2.7 glucoside 2 Cyaniding 3- 16.23 450 1.0 glucoside 3 Cyaniding- 16.84 420 48.0 pentoside 4 Peonidin hexoside 16.84 464 90.0 5 Delphinidin 3- 16.84 465 17.0 glucoside

Table 3. The non-anthocyanin phenolic compounds of the peel methanolic and ethanolic extracts of black pomegranate peel. Methanolic extract No. Classification Name tR (min) (m/z) Relative intensity (%) 1 Hydroxybenzoic acids Protocatechuic acid 22.31 153 1.7 2 Gallic acid 19.15 169 3.8 3 Vanillic acid 4- 20.21 329 15.4 hexoside 4 Protocatechuic acid-derivative 14.19 425 67.0 5 Hydroxycinnamic acids Coumaric acid 7.50 163 4.7 6 Caffeic acid derivative 27.92 299 59.0 7 Coumaric acid-hexosides 24.56 325 36.0

8 Caffeic acid hexoside 21.05 341 12.4 9 5-O-Caffeoylquinic acid 24.70 353 4.4 10 Ferulic acid-hexoside 9.75 355 1.0 11 Caffeic acid hexoside derivative 16.50 451 4.4

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derivative 12 Dihydroflavonol Dihydrokaempferol-hexoside 19.94 449 4.8 13 Gallagyl esters Gallagyl-hexoside (penicillin) 7.10 781 1.8 14 HHDP-gallagyl-hexoside (punicalagin) 14.98 1083 4.2 15 Gallotannins Galloyl-hexoside 24.70 331 49.0

16 Ellagitannins Ellagic acid 26.35 301 10.0 17 Ellagic acid derivatrive 18.10 441 50.0 18 Ellagic acid derivative 26.40 443 5.8 19 Ellagic acid-deoxyhexoside 16.99 447 3.4 20 Ellagic acid-hexoside 23.21 463 4.8 21 Valoneic acid bilactone 15.01 469 19.6 22 HHDP-hexoside 28.41 481 69.0 23 Ellagic acid dihexoside 16.01 625 6.0 24 Galloyl-HHDP-hexoside 25.02 633 1.2 25 Digalloyl-HHDP- 29.84 785 10.0 hexoside(pedunculagin II) 26 Ellagic acid derivative: granatin A 24.41 799 19.5 (HHDP-DHHDP-hexoside) or lagerstannin A (bis-HHDP-gluconic acid) 27 Galloyl-bis-HHDP-hexoside 25.87 935 0.31 (casuarinin) 28 Flavogalloyl-HHDP-gluconic acid 1.64 949 45.0 (lagerstannin B) 29 Galloyl-HHDP-DHHDP-hexoside 21.34 951 5.9 (granatin B) 30 hydroxylated derivative of 28.65 967 7.0 lagerstannin B Ethanolic extract 1 Hydroxybenzoic acids Protocatechuic acid 22.29 153 2.9 2 Gallic acid 25.79 169 12.0 3 Vanillic acid 4- 11.66 329 29.0 hexoside 4 Hydroxycinnamic acids Coumaric acid 18.93 163 72.0 5 Caffeic acid derivative 28.16 299 100.0 6 Coumaric acid-hexosides 27.46 325 63.0

7 Caffeic acid hexoside 21.61 341 15.9 8 5-O-Caffeoylquinic acid 11.45 353 41.0 9 Ferulic acid-hexoside 11.31 355 78.0 10 Caffeic acid hexoside derivative 17.03 451 10.3 derivative 11 Gallagyl esters Gallagyl-hexoside (penicillin) 14.05 781 1.1 12 HHDP-gallagyl-hexoside (punicalagin) 12.51 1083 0.48 13 Gallotannins Galloyl-hexoside 24.62 331 68/0

14 Ellagitannins Ellagic acid 22.46 301 1.0 15 Ellagic acid derivatrive 19.05 441 13.0 16 Ellagic acid derivative 19.05 443 7.5 17 Ellagic acid-deoxyhexoside 22.40 447 1.5 18 Ellagic acid derivative 7.25 392 22.0 19 Bis-HHDP-hexoside (pedunculagin I) 16.13 783 1.0 20 HHDP-hexoside 27.93 481 55.0 21 Ellagic acid dihexoside 15.25 625 10.5 22 Galloyl-HHDP-hexoside 26.82 633 4.3 23 Digalloyl-HHDP- 16.45 785 1.0 hexoside(pedunculagin II)

Phenolic Profiles in Peel of Iranian Pomegranate

24 Castalagin derivative 26.52 965 5.5 25 Galloyl-bis-HHDP-hexoside 28.33 935 1.8 (casuarinin) 26 Flavogalloyl-HHDP-gluconic acid 18.37 949 15.0 (lagerstannin B)

Table 4. Chemical structure of anthocyanins and some non- anthocyanins of P.granatum peel. Anthocyanins Pelargonidin 3-glucoside Cyaniding 3-glucoside Cyaniding-pentoside

Peonidin hexoside Delphinidin 3-glucoside

Non-anthocyanins Protocatechuic acid Gallic acid Coumaric acid

5-O-Caffeoylquinic acid Gallagyl-hexoside (penicillin) HHDP-gallagyl-hexoside (punicalagin)

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Ellagic acid Valoneic acid bilactone Galloyl-bis-HHDP-hexoside (casuarinin)

Galloyl-HHDP-DHHDP-hexoside (granatin B)

Phenolic Profiles in Peel of Iranian Pomegranate

Figures:

Figure 1. Radical scavenging activity of different concentrations (35–250 µg/mL) of pomegranate peel fractions (methanol, ethanol, acetone, and ethyl acetate) by DPPH method. The activity is compared with BHT.

Figure 2. The HPLC-MS/MS total chromatogram of anthocyanins and non- anthocyanins phenolic compounds: (A) total chromatogram of acidic methanol extract; (B) total chromatogram of methanolic extract; and (C) total chromatogram of ethanolic extract.

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Figure 3. The HPLC-MS/MS analysis of peonidin hexoside.

Phenolic Profiles in Peel of Iranian Pomegranate

Figure 4. The HPLC-MS/MS analysis of methanolic and ethanolic extracts: (A) caffeic acid derivative chromatogram; and (B) galloyl-hexoside chromatogram.

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