Physicochemical characteristics, polyphenol compositions and antioxidant potential of pomegranate juices from 10 Chinese cultivars and the environmental factors analysis

Accepted Manuscript Physicochemical characteristics, polyphenol compositions and antioxidant potential of pomegranate juices from 10 Chinese cultivars and the environmental factors analysis Xuan Li, Humaira Wasila, Linwei Liu, Tian Yuan, Zhongmei Gao, Beita Zhao, Imran Ahmad PII: DOI: Reference:

S0308-8146(14)01905-0 http://dx.doi.org/10.1016/j.foodchem.2014.12.003 FOCH 16852

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

27 July 2013 30 November 2014 2 December 2014

Please cite this article as: Li, X., Wasila, H., Liu, L., Yuan, T., Gao, Z., Zhao, B., Ahmad, I., Physicochemical characteristics, polyphenol compositions and antioxidant potential of pomegranate juices from 10 Chinese cultivars and the environmental factors analysis, Food Chemistry (2014), doi: http://dx.doi.org/10.1016/j.foodchem. 2014.12.003

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Physicochemical characteristics, polyphenol compositions and antioxidant

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potential of pomegranate juices from 10 Chinese cultivars and the

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environmental factors analysis

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Xuan Li a, Humaira Wasila a, Linwei Liu*a, Tian Yuan a, Zhongmei Gao a, Beita Zhao

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Imran Ahmad b

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a

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Shaanxi, China

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b

College of Food Science and Engineering, Northwest A&F University, Yangling 712100,

College of Horticulture, Northwest A&F University, Yangling 712100, Shaanxi, China

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Running Title

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Pomegranate juice polyphenol and environmental effects analysis

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*Corresponding author, Contact information of corresponding author.

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Linwei Liu: Tel: +86 029 87092486; E-mail address: [email protected]

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a

,

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ABSTRACT

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Various pomegranate cultivars are grown in several regions of China, but comparative

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study of their juice polyphenols, antioxidant potentials, and health benefits are few. In the

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present study, physicochemical characteristics, polyphenol compositions, and antioxidant

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potentials of pomegranate aril juices from 10 cultivars in 4 Chinese regions were investigated.

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The results showed that the soluble solid content, reducing sugar content, titratable acid

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content of them were 13.97~16.30°Brix, 62.82~110.70g/L, 2.657~36.62g/L respectively. The

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total polyphenols, flavonoids, tannin and anthocyanin concentrations were 3.15~7.43 mg

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GaE/mL, 0.045~0.335 mg QuE/mL, 0.540~2.531 mg TaE/mL, and 0.004~0.160 mg CyE/mL

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respectively. Sugar acid ratio, titratable acid content, total flavonoid concentration, and

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DPPH· scavenging capacity were affected mainly by sweet and sour cultivar type, while

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soluble solid content and total anthocyanin concentration were affected by environment.

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Temperature in maturity period and latitude of growing region significantly effected on

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polyphenol and antioxidant potential levels of pomegranate juice.

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Key words: Pomegranate; sweet; sour; polyphenols; antioxidant capacity; environment

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1. Introduction

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Pomegranate (Punica granatum L.) is one of the oldest health promoting fruits. It

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originated in the Middle East, and has spread to Mediterranean, China and tropical and

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subtropical countries around the world (Fadavi, Barzegar, Azizi, & Bayat, 2005). There are

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over 1000 cultivars of pomegranate, classified by appearance and characteristics of fruit,

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flower and tree (Sarkhosh, Zamani, Fatahi, & Ebadi, 2006). Generally, there are two varieties,

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ornamental and edible pomegranate. The edible one is further divided into sweet and sour

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types according to the taste of the juice (Hasnaoui, Mars, Ghaffari, Trifi, Melgarejo, &

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Hernandez, 2011).

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Attention on pomegranates and their products (juice, jelly, jam etc.) by both consumers

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and researchers has increased because their consumption has been found to have several

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medical benefits, such as prevention and treatment of cancer, cardiovascular disease, diabetes,

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Alzheimer's, arthritis and colitis (Fuhrman, Volkova, & Aviram, 2005; Jurenka & Julie, 2008;

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Viuda-Martos, Fernández-López, & Pérez-Álvarez, 2010; Landete, 2011).

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Pomegranate aril, the edible part of the fruit, is rich in sugars, organic acids, vitamins,

52

minerals and polyphenols (Tehranifar, Zarei, Nemati, Esfandiyari, & Vazifeshenas, 2010).

53

Polyphenol is the major antioxidant and health functional factor found in pomegranate aril

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and juice, and it mainly consists of ellagitannin (punicalagin), gallic acid, ellagic acid,

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anthocyanins, catechins, caffeic acid, and quercetin (Viuda-Martos, et al. 2010). The

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abundance of these compounds depends on cultivar type, climate, and growing region

57

(Melgarejo, Salazar, & Artes, 2000; Poyrazoğlu, Gökmen & Artιk, 2002). Until now, juice

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polyphenols of many pomegranate cultivars in Iran, Turkey, the United States, Italy and South

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Africa have been studied. However, a profile comparison of polyphenol composition and

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antioxidant potential in sweet and sour pomegranates in different areas of China has not been

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done prior to this study. Moreover, environmental conditions, regarded as the important factor

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in synthesis and metabolism of polyphenols (Hättenschwiler, & Vitousek 2000), having effect

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on juice polyphenol composition and antioxidant potential is rarely considered.

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In the present study, 10 pomegranate cultivars from 4 growing regions of China were

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studied for aril juices‟ physicochemical characteristics (soluble solid content, reducing sugar

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content, titratable acid, and sugar acid ratio), polyphenol composition, and antioxidant

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potential (total reducing capacity, DPPH• radical scavenging capacity, ABTS•+ radical

68

scavenging capacity and superoxide anion radical scavenging capacity). Meanwhile, the

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relevance of environmental factors (longitude, latitude, altitude, temperature, precipitation and

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insolation) to juice polyphenol composition and antioxidant potential were analyzed.

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Furthermore, the effect of sweet and sour type (T), growing environment (E) and their

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interaction (T×E) on aril juices‟ physicochemical characteristics, polyphenol composition

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and antioxidant potential were analyzed by comparing the contribution rates of T, E, and T×

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E that were calculated by the variances percentage of these aspects in 10 juices. These results

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would provide new information to pomegranate growers, producers, researchers and

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consumers.

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2. Materials and methods

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2.1 Materials and chemicals

79

2.1.1 Pomegranates

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The tested pomegranates (Punica granatum L. Punicaceae) were 10 commercial

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cultivars from Xinjiang (XJ), Yunnan (YN), Shandong (SD), and Shaanxi (SX) of China. In

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each site, there are one kind of sour pomegranate (SSL) and at least one kind of sweet

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pomegranate (TSL). We procured SSL and TSL from Xinjiang (XJ-SSL and XJ-TSL), SSL

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and TSL from Shandong (SD-SSL and SD-TSL), SSL and TSL (including JPT and SBT) from

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Shaanxi (SX-SSL, SX-JPT, and SX-SBT), and SSL and TSL (including SZ and LZ) from

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Yunnan (YN-SSL, YN-LZ, and YN-SZ). The Latin names of these pomegranate varieties are

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XJ-SSL (P. granatum L. cv. Kashisuan), XJ-TSL (P. granatum L. cv. Kashitian), SD-SSL (P.

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granatum L. cv. Dahongpisuan), SD-TSL (P. granatum L. cv. Damaya.),

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granatum L. cv. Suanlvzi), YN-LZ (P. granatum L. cv. Tianlvzi), YN-SZ (P. granatum L. cv.

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Tianshazi), SX-SSL (P. granatum L. cv. Lintongsuan), SX-JPT(P. granatum L. cv. Jingpitian),

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SX-SBT (P. granatum L. cv. Sanbaitian).

YN-SSL (P.

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10 Fruits of each cultivar were picked in the same day (15th of October, 2012) from 10

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trees in each orchard. Pomegranate fruits were packed and kept in the temperature of 4℃ and

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relative humility of 85% by plastic atmosphere bags in shock proof cartons, and sent to our

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laboratory by Air Express in 3 days.

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being stored in a refrigeration storage with a temperature of 4 ℃ and relative humility of

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85% for further use.

They were further cleaned and well-packaged before

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For each pomegranate cultivar, 10 pomegranate fruits were randomly divided into 3

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groups. From each group, one juice sample was obtained, and triplicate assays were

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conducted for each juice sample.

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2.1.2 Reagents and Standards

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Folin–Ciocalteu

reagent,

Folin-Dennis

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reagent,

aluminum

chloride,

potassium

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ferricyanide, ferric chloride, 2,2`-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS+),

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and 2,2-diphenyl-1-picrylhydrazyl (DPPH·) are analytical reagent grade and purchased from

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Sigma–Aldrich Co. (St. Louis, Missouri, USA). Gallic acid, punicalagin, catechin,

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chlorogenic acid, epicatechin, caffeic acid, ferulic acid, ellagic acid, and kaempferol are

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HPLC grade and purchased from Vick's biological Inc. (Chengdu, Sichuan, China). Distilled

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water was used throughout.

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2.2 Preparation of pomegranate juices and their polyphenol extracts

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Pomegranates were processed into juices in lab after one week storage at 4 ℃. The

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bottom and top of pomegranate fruits were removed before the fruit was soaked in potassium

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permanganate solution (2%) for sterilization (5min). Arils were separated from the mesocarps

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and epicarps, and squeezed by household juicer (Meidi, Guangdong, China) with seeds kept

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intact to get the aril juice. The juices were centrifuged (4000rpm 10min) by SC-2456

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Table-type centrifuge (Keda Innovation Co. LTD, Anhui, China) before storing them at -20℃

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for further use.

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Polyphenols of pomegranate juices were extracted and purified by aqueous two-phase

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system. Briefly, 10mL of pomegranate juice was evenly mixed with 6mL of acetone before

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adding 3.25g ammonium sulfate. The mixture was then treated with ultrasonic wave (30℃,

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40kHz) by KQ-600DB ultrasonic instrument (Kunshan, Jiangsu, China) for 15min. When the

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mixture separated into two phases, the supernatant was collected from the separating funnel

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and dried by RE-52AA rotary evaporators (Yarong bio-instrument Co., Shanghai, China). The

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dried substance was dissolved in 10ml methanol, and kept in -20℃ for further use.

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2.3 Determination of chemical composition and physiochemical properties

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The pH of the juice was measured using PHS-3C pH meter (INESA Scientific

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Instrument Ltd. Co., Shanghai, China). Soluble solid content (SSC) was measured using WYT

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handhold refractometer (Xingchen Company, Chengdu, China), with values being presented

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as Brix degree. Titratable acid content (TAC) was determined according to the official method

129

(AOAC 1984). Briefly, a 10mL juice sample was titrated with sodium hydroxide standard

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titration solution (0.1mol/L) to pH8.1 using a calibrated pH meter to monitor the pH. Results

131

were expressed as g citric acid per liter of sample. Total reducing sugar content (TRS) was

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determined by the standard method (AOAC 1984). Specifically, a pretreated juice sample was

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used to titrate 10mL standardized alkaline copper tartrate solution (calibrated by 1mg/mL

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glucose standard solution) with a boiling time of 2min. Results were presented as g glucose

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per liter of sample. Each measurement was conducted in triplicate.

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2.4 Determination of polyphenol composition

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2.4.1. Determination of total polyphenols concentration

138

The Folin-Ciocalteu method was used for total polyphenol concentration determination,

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based on the optimized condition by Singleton, Orthofer and Lamuela-Raventos (1999) with

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some modifications. Gallic acid was used as standard and distilled water was used as blank.

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Briefly, 0.5mL of diluted juice (2mL juice sample was diluted into 10mL by distilled water)

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was mixed with 0.5 mL of Folin-Ciocalteu reagent before adding 3mL of sodium carbonate

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solution (10%). Water was added to bring volume to 10 mL. The absorbance was read at

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760nm after 30min reaction in dark place. Results were expressed as mg GaE (gallic acid

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equivalent)/mL of pomegranate juice. Each juice sample was tested in triplicate.

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2.4.2. Determination of total flavonoid concentration

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Total flavonoid concentration was determined by the method established by Lin and

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Tang (2007). Quercetin was used as the standard and distilled water as blank. The final

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reaction mixture consisted of 0.2mL juice sample, 1.5mL of ethanol solution (95%), 0.1mL of

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aluminium chloride (10%) and potassium acetate (1mol/L) and 2.8mL distilled water. The

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absorbance was read at 415nm after 30 min reaction. Results were expressed as mg QuE

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(quercetin equivalent)/mL of pomegranate juice. Each juice sample was measured in

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triplicate.

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2.4.3. Determination of total tannin concentration

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Total tannin concentration was measured according to the previous reported method

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(Schanderl, 1970) with some modification. Tannin acid was used as standard and distilled

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water as the blank. Folin-Dennis reagent was prepared by refluxing the mixture of two

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hydrated sodium tungstate dihydrate (100 g), phosphomolybdic acid (20g), H2O (750mL)

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with phosphoric acid (50mL) in water bath for 2h, and diluted to1000mL after cooling down.

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The final reaction mixture contained 1.0mL diluted sample (2mL juice sample was diluted

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into 10mL by distilled water), 1.25mL Folin-Dennis reagent and 2.5mL sodium carbonate

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solution (10%) solution. The mixture was brought to the volume of 25mL using distilled water.

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Absorbance of the mixture was read at 700nm after 30min reaction in a dark place. The

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results were expressed as mg TaE (tannin acid equivalent)/mL of pomegranate juice. Each

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juice sample was measured in triplicate.

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2.4.4. Determination of total anthocyanin concentration

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Total anthocyanin concentration was measured by pH differential method (Giusti &

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Wrolstad, 2001). Briefly, 0.5 mL of each sample was brought to pH1.0 by 4.5mL of KCl-HCl

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solution (0.025mol/L) and to pH4.5 by 4.5mL of NaAc-HAc solution (0.4mol/L) respectively.

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Absorbance of equilibrated reaction mixture solutions (~25 ℃ , 15min standing) were

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measured at 510nm and at 700nm respectively using a UV-VIS spectrophotometer at ambient

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temperature with distilled water as blank. Results were expressed as mg CyE

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(cyanidin-3-glucoside equivalent)/mL juice. Each juice sample was tested in triplicate.

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2.4.5. Analysis of polyphenol monomers

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HPLC analysis was conducted to evaluate the punicalagin, gallic acid, and other

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polyphenol concentrations in pomegranate juices. HPLC system (Shimadzu, Japan) was

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equipped with auto-sampler, SPD-M20A diode array detector (280nm, 30℃), and C18

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reversed phase column with 250×21.1 mm i.d., 5µm particle size and 125 Å pore size

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(Promosil Agela Technologies, USA). The mobile phase consisted of methanol (solvent B)

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and 1% acetic acid solution (solvent A). The mobile phase flowed at a speed of 0.8mL /min.

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The gradient procedure was optimized as follows: the solvent B was run from 15% to 25% in

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first 15min, 25% for 10min, from 25% to 75% in the following 40min and returned to 15% in

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15min, keeping another 5min at last. The injection volume of standard or sample solution was

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15 μL. Standard solution was prepared by quantitatively dissolving polyphenol standards into

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methanol. Pomegranate polyphenol sample solution was prepared as mentioned above (in the

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2.2). Both of them were filtered through a 0.22um membrane filter before injection. Each

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sample was measured in triplicate. The individual polyphenol compound of the sample was

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identified by retention time and quantified by peak area.

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2.5 Determination of antioxidant potential

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2.5.1. Assay of total reducing capacity

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Total reducing capacity (TRC) was assayed using the previous reported method

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(Ferreira, Baptista, Vilas-Boas, & Barros, 2007). Specifically, 1.0 mL of diluted sample (2mL

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juice sample was diluted into 10 mL by distilled water), 2.5mL of sodium phosphate buffer

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(0.2 mol/L, pH 6.6) and potassium ferrocyanide solution

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water bath for 20min. When trichloroacetic acid solution (10%, 2.5mL) was added and mixed,

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then 2.5mL of the reaction solution was mixed with 2.5mL of distilled water and 1.0mL of

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ferric trichloride solution (0.1%), vortexed and kept still for 10min. Absorbance was read at

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700nm. Distilled water was used as the blank. Gallic acid and ascorbic acid were used as

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references. The results were expressed as mg GaE (gallic acid equivalent)/L juice and mg VcE

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(ascorbic acid equivalent)/L juice respectively. Each juice sample was tested in triplicate.

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2.5.2. Assays of free radical scavenging capacity

(1 %) were mixed and kept at 50℃

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DPPH radical scavenging capacity (DRSC) was conducted according to the method

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described by Villano, Fernández-Pachón, Moyá, Troncoso, and García-Parrilla (2007) with

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some modification. Briefly, 1.0mL of diluted sample (2mL juice sample was diluted into

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10mL by distilled water) was mixed with 4.0mL of DPPH• ethanolic solution. Absorbance at

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520nm was recorded after 10min. An ethanol solution instead of DPPH• working solution was

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used as the control and distilled water instead of sample as the blank.

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ABTS radical scavenging capacity (ARSC) was determined by the method established

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by Re, Pellegrini, Proteggente, Pannala, Yang, and Rice-Evans (1999). Briefly, the final

210

reaction mixture consisted of 50uL diluted sample (2mL juice sample was diluted into 10 mL

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by distilled water) and 4.0mL ABTS•+ working solution (5mL of 7mmol/L ABTS solution

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reacts with 88uL of 140mmol/L potassium persulfate solution, and then the reaction solution

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was diluted with ethanol to the absorbance of 0.700±0.002 at 734nm). The absorbance of the

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final reaction mixture was measured at 734 nm. 4.0 mL of ethanol instead of ABTS•+ working

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solution was used as the control and distilled water instead of sample as the blank.

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Superoxide anion scavenging capacity (SASC) was tested using the method described by

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Zhang, Chen, Li, Pei, and Liang (2010) with some modification. Specifically, 5.6 mL of

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Tris–HCl buffer solution (0.05mol/L, pH 8.2) mixed with 0.5mL diluted sample (2 mL juice

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sample was diluted into 10 mL by distilled water), followed by the addition of 0.4ml of

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pyrogallol solution (0.06mol/L). The mixture was incubated at 37℃ for 4min and the

221

reaction was stopped by adding 3 drops of hydrochloric acid (10mol/L). The mixture was then

222

brought to volume of 10mL with distilled water before reading absorbance at 320nm. Distilled

223

water instead of diluted juice was used as the blank, and 0.4mL of distilled water instead of

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pyrogallol solution as the control.

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For the three free radical scavenging capacity assays, the scavenging capacity (SC) was

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calculated by using the following equation: SC= [1-(Asample–Acontrol)/( Ablank–Acontrol)]×100.

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and the analytical gallic acid and ascorbic acid were used as references. The results were

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expressed as mg GaE (gallic acid equivalent)/L juice and mg VcE (ascorbic acid equivalent)/L

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juice respectively. Each juice sample was tested in triplicate.

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2.6 Environment information

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Geographic information, including longitude, latitude and altitude of each location, was

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provided by National Geomatics center of China. Meteorological data came from China

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meteorological Data Sharing Service System, containing precipitation (millimeter), insolation

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(hours) and temperature during pomegranate maturity and harvest time (September and

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October) in 2012. Among them, the overall average temperature is the daily average

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temperature in the period of the pomegranate maturity and harvest and the temperature

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difference is the difference of the daily average absolute high temperature and daily average

238

absolute low temperature. The detailed environmental information is listed in Table1.

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2.7 Statistical analysis

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DPS (Version Rel.6.55, for Windows, 1997) was used for statistical analysis. Correlation

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analyses were performed using a two-tailed Pearson‟s correlation test. Difference between

242

means was compared by Turkey‟s HSD post hoc test. General linear model (GLM) was used

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with cultivar type (2 levels) and growing environment (4 levels) as fixed factors.

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3. Results and discussion

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3.1 Physicochemical characteristics of pomegranate juices from 10 Chinese cultivars

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Physicochemical characteristics of pomegranate juices from 10 Chinese cultivars are listed in Table2.

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The SSC level of pomegranate juices ranged from 13.97 to 16.30°Brix, This is close to

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13.80 ~16.57°Brix of 15 cultivars in Georgia reported by Rajasekar, Akoh, Martino, and

250

MacLean (2012), and 11.37~ 15.07°Brix of 20 cultivars in Iran reported by Tehranifar et al.

251

(2010), but lower than 15.77~19.56°Brix of 6 cultivars in Iran reported by Zarei, Azizi, and

252

Bashiri-Sadr (2010) and 16.0~19.0°Brix of 13 cultivars in Turkey reported by Poyrazoğlu et

253

al. (2002). However, compared to 14.0°Brix, the minimum value proposed by Association of

254

the Industry of Juice and Nectars (AIJN) provisional reference guideline for pomegranate

255

juice, only YN-LZ and SXJPT juices were not met. SSC level of most pomegranate juices in

256

present research had significant difference (p<0.05), which is consistent with the results

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reported by Rajasekar et al. (2012). It means SSC of pomegranate juice is influenced by

258

multiple factors such as cultivar, growing region and maturity. Moreover, Xinjiang and

259

Shandong sweet pomegranate juices had higher SSC than sour ones, whereas Shaanxi and

260

Yunnan sweet pomegranate had lower SSC than sour ones. It means that SSC levels of

261

pomegranate juices were closely related to cultivar type, and partly related to growing regions.

262

SSC levels of Shandong pomegranate juices were significantly higher than that of Xinjiang

263

(p<0.05). SSC levels of Shaanxi and Yunnan pomegranate juices were significantly lower

264

(p<0.05) and statistically equal with each other (p<0.05).

265

Reducing sugar content (RSC) of the 10 pomegranate juices ranged from 62.82 g/L to

266

110.7g/L, which was lower than 126.1g/L (average value) of Spainish pomegranate juices

267

reported by Melgarejo et al. (2000), 150g/L (average value) of Tunisia pomegranate juices

268

reported by Elfalleh et al. (2009) and 154g/L (average value) of Ganesh cultivar pomegranate

269

juice reported by Kulkarni and Aradhya (2005). Table 2 also showed that sweet pomegranate

270

juices had higher reducing sugar content than sour ones except those in Yunnan sour

271

pomegranate juice had highest RSC among the 10 juices, which is partly consistent with the

272

previous results reported by Melgarejo et al. (2000). Moreover, Xinjiang pomegranate juices

273

had lower reducing sugar contents than that of Yunnan, Shaanxi and Shandong pomegranate

274

juices. It means that high altitude may be unfavorable to the production of reducing sugar in

275

pomegranate juices. What is more, reducing sugar content difference among juices of the 10

276

cultivars was mainly caused by different maturity level that closely related to the

277

environmental conditions during maturation period. This supposition is also supported by a

278

report (Kulkarni & Aradhya, 2005) that showed fully matured pomegranates developed the

13

279

highest total sugar which might be attributed to hydrolysis of starch into simple sugar.

280

Titratable acid content (TAC) of the 10 Chinese pomegranate juices varied from 2.657 to

281

36.62g/L (citric acid). It is higher than 1.3~29.7g (citric acid)/L of Georgia pomegranate

282

juices reported by Rajasekar et al. (2012), 1.0~24.0 g (malic acid)/L of Tunisian pomegranate

283

juices reported by Zaouay, Mena, Garcia-Viguera, and Mars (2012), and 3.3 to 24.4 g (citric

284

acid)/L of Iran pomegranate juices reported by Tehranifar et al. (2010). TAC of pomegranate

285

juices from the 10 cultivars were significantly different (p<0.05). Specifically, sour

286

pomegranate juices had much higher TAC than sweet ones, such as Xinjiang sour

287

pomegranate juice was about 6.4 times higher than sweet one, Shandong sour pomegranate

288

juice was 4.7 times higher than sweet one, Yunnan sour pomegranate juice was 4.7 and 3.7

289

times higher than two sweet ones, and Shaanxi sour pomegranate juice was 8.0 to13.0 times

290

higher than two sweet ones. It means that sweet and sour cultivar types is the key determinant

291

of titratable acid content of pomegranate juice, and growing environment also play a role on

292

it.

293

SSC: TAC is generally used to quantify the juice taste (Rajasekar et al., 2012) and as a

294

maturity index (Zaouay et al., 2012). Because total sugar content (TSC) is more accurate than

295

SSC to explain sweet degree of the juice, sugar-acid ratio (SAR) defined as TSC: TAC, was

296

used in this paper to indicate the sweet-sour feature of juice. As shown in Table 2, SAR value

297

ranged from 15.98 to 37.08 in sweet pomegranate juices, and ranged from 1.48 to 5.78 in sour

298

pomegranate juices. This is one reason for palatable sweet pomegranate juice widely used in

299

juice products while sour pomegranate juice mainly used as medicinal fruit in China. SAR

300

values were significantly (p<0.05) different among sweet pomegranate juices, while the SAR

14

301

value difference of sour pomegranate juices is not significant (p>0.05). It means that taste is

302

more variable among the juices of sweet pomegranate cultivars than that of sour pomegranate

303

cultivars.

304

3.2 Polyphenol composition of pomegranate juices from 10 Chinese cultivars

305

Total polyphenols concentration (TPC) of the 10 pomegranate juices varied from 3.15 to

306

7.43 mg GaE/mL, which fall into the range of 2.602~10.086 mg GaE/mL of Turkish

307

commercial pomegranate juices reported by Tezcan, Gültekin-Özgüven, Diken, Özçelik, and

308

Erim (2009), and 2.376~9.304 mg GaE/mL of Iran pomegranate juices reported by

309

Mousavinejad, Emam-Djomeh, Rezaei, and Khodaparast (2009), but higher than 2.083~3.436

310

mg GaE/mL of Turkish pomegranate aril juices reported by Çam, Hışıl, and Durmaz (2009),

311

and is about 10 times higher than 0.272~0.849 mg GaE/mL of Georgia pomegranate aril

312

juices reported by Rajasekar et al. (2012). It indicates that total polyphenol level of

313

pomegranate juice vary greatly among different cultivars and different growing regions in the

314

world. As Table 2 showed, the highest TPC was found in SD-TSL pomegranate juice,

315

sequentially followed by XJ-SSL, SX-SSL, SX-SBT, SD-SSL, XJ-TSL, SX-JPT, YN-SSL,

316

YN-SZ and YN-LZ. It can be deduced from the sequence that total polyphenol concentration

317

of pomegranate juice is related to growing environment and cultivar, but not related to sweet

318

and sour types.

319

Flavonoids ubiquitously exist in plants, known to possess antiviral, anti-inflammatory,

320

antitumor and antioxidant potentials and thought to be a kind of phytoestrogens. Although

321

luteolin, kaempferol, and quercetin had been detected in pomegranate peel extract, no

322

flavonoid was detected in pomegranate juice from any Iranian cultivars by Mousavinejad et 15

323

al. (2009). While in the present work, total flavonoids were detected in the 10 pomegranate

324

aril juices and total flavonoid concentration (TFC) of them were in the range of 0.045~0.335

325

mg QuE /mL. SDTSL still ranked in first place, followed by that of SX-JPT, SX-SBT,

326

YN-SSL, XJ-TSL, YN-SZ, SD-SSL, YN-LZ, SX-SSL and XJ-SSL. Xinjiang, Shandong and

327

Shaanxi sweet pomegranate juices had higher TFC than their sour counterparts, but Yunnan

328

sweet pomegranates had lower TFC than their sour cultivar. These indicate that TFC of

329

pomegranate aril juice is closely related to sweet and sour cultivar and growing region.

330

Moreover, it is also affected by pomegranate maturity level as report by Kulkarni et al.

331

(2005).

332

Tannin has been known having good antioxidant, anti-inflammatory and anti-proliferative

333

activities as studied by Seeram, N.P. et al. (2005). Tannin level in pomegranate juice can be a

334

good indicator for its nutritional quality. Table 2 shows that total tannin concentration (TTC)

335

of pomegranate juices from the ten cultivars varied from 0.54 to 2.531 mg TaE/mL. It was

336

similar to 0.640~2.699 mg/mL of the 4 pomegranate juices from “wonderful” cultivar

337

reported by Gil, Tomás-Barberán, Hess-Pierce, Holcroft, and Kader, (2000), but higher than

338

0.15~0.32 mg/mL of the pomegranate juices from 8 cultivars of Iran reported by

339

Mousavinejad et al. (2009). These differences may be caused by the difference of cultivar

340

type, climate, edaphic condition, maturity, and especially tannin determination method among

341

these researches. TTC of the 10 pomegranate juices in the present work were significantly

342

different with each other. TTC sequential order of 10 pomegranate juices was consistent with

343

that of TPC, which means tannins were the main polyphenols in pomegranate juice, and TTC

344

level and TPC level of the juice were affected by similar factors, including growth

16

345

environment, cultivar, and maturity etc.

346

The appealing color of pomegranate juice, an important index for juice quality and

347

popularity, is mainly attributed to anthocyanin concentration. And healthy functions of

348

anthocyanin have been widely recognized and studied (Stinzing, & Carle 2004). Total

349

anthocyanin concentration (TAC) of the 10 pomegranate juices varied from 0.004 to 0.160 mg

350

CyE/mL. The data is lower than 0.004~0.419 mg CyE /mL of Georgia pomegranate aril juices

351

reported by Rajasekar et al.(2012), 0.081~0.369 mg CyE /mL of Turkey pomegranate aril

352

juices reported by Çam et al. (2009), and 0.055~0.301 mg CyE /mL of Iran pomegranate aril

353

juices reported by Tehranifar et al.( 2010). It indicates that the considerable variation of total

354

anthocyanin level of pomegranate juice around the world was related to the differences of

355

cultivars and growing environment. Total anthocyanin concentration of the 10 cultivars were

356

in the order of XJ-TSL>XJ-SSL>SX-SSL>SX-JPT>SD-SSL=YN-SZ≈YN-SSL>SD-TSL>

357

YN-LZ>SX-SBT. Significant differences (p<0.05) were observed in most of the 10

358

pomegranate juices except SD-SSL, YN-SZ and YN-SSL. These may be caused by different

359

cultivars and different maturity level of the pomegranate, since it has been proved that

360

maturity degree greatly effects the total anthocyanin concentration of pomegranate arils

361

(Fawole et al. 2013, & Kulkarni et al. 2005).

362

3.3 Polyphenol monomers of pomegranate juices from 10 Chinese cultivars

363

Polyphenol profiles of 10 pomegranate juices are showed in HPLC chromatogram in Fig2.

364

The measured concentrations of punicalagin, gallic acid, catechin, chlorogenic acid, caffeic

365

acid, epicatechin, ferulic acid, ellagic acid, and kaempferol in the 10 pomegranate juices were

366

listed in Table 3. Punicalagin concentration of the 10 pomegranate juices ranged from 298.99

17

367

to 1042.93ug/mL, which was the primary polyphenol in pomegranate juices of the ten

368

cultivars. This result is similar to the conclusion of Fischer, Carle, and Kammerer (2011), and

369

Gil et al. (2000) that punicalagin and other hydrolyzable tannins were the richest polyphenol

370

compounds in pomegranate aril juice. Concentrations of the phenolic acids in each juice

371

tested in the present work were all significantly different with each other. They were in the

372

order of chlorogenic acid>gallic acid>caffeic acid>ferulic acid>ellagic acid. The

373

inconsistencies in concentration of individual phenolic acid in various cultivars was also

374

found by other researchers (Gundogdu, & Yilmaz 2012; Fischer, Carle, & Kammerer 2011;

375

Lansky & Newman 2007; Gil et al. 2000). These differences were caused not only by

376

differences of cultivar, growing region and maturity level of the tested pomegranate, but also

377

by the difference in analytical methods used. In addition, some flavonoid compounds had

378

been detected in all the 10 juices, which are in agreement with previous work (Wang, Ding,

379

Liu, Xiang, & Du 2010). Flavonoid concentrations of the juices were significantly different

380

with each other; among which epicatechin and catechin were the dominant flavonoid,

381

followed by kaempferol. It has been reported that pomegranate juice anthocyanins consist of

382

65 constituents and some adducts, including some unusual cyanidin, pelargonidin, delphinidin,

383

and pelargonidin in “wonderful” pomegranate (Sentandreu, Cerdán-Calero, & Sendra 2013).

384

While anthocyanin monomers of 10 pomegranate juices were not discussed in present work

385

since anthocyanin standards were insufficient.

386

3.4 Antioxidant potential of pomegranate juices from 10 Chinese cultivars

387

Four in vitro assays were used by complementary means to evaluate the antioxidant

388

potential of pomegranate juices from 10 cultivars, as shown in Figure 1. TRC of the 10

18

389

pomegranate juices were in the order of SD-TSL>XJ-SSL>SX-SBT>SX-SSL>SD-SSL>XJ-TSL≈

390

YN-SSL>SX-JPT>YN-SZ>YN-LZ. DRSC were in the order of SD-TSL≈XJ-SSL>SD-SSL≈SX-SBT≈

391

SX-SSL>YN-SSL>XJ-TSL>SX-JPT>YN-SZ>YN-LZ. ARSC were in the order of SD-TSL>XJ-SSL>

392

SX-SSL>SD-SSL≈SX-SBT>YN-SSL≈XJ-TSL>SX-JPT>YN-SZ>YN-LZ. SASC were in the order of

393

XJ-SSL>SD-TSL>SX-SSL>SD-SSL>XJ-TSL ≈YN-LZ ≈ YN-SZ ≈ YN-SSL>SX-SBT ≈ SX-JPL. These

394

sequential orders indicate that Shandong and Xinjiang pomegranate juices had higher

395

antioxidant potentials than Shaanxi and Yunnan pomegranate juices. Moreover, antioxidant

396

potential sequential orders of the 10 pomegranate aril juices were similar to their total

397

polyphenol concentration sequential order as shown in 3.2. This means antioxidant potential

398

of pomegranate juice mainly relies on its polyphenol levels.

399

TRC of the 10 pomegranate juices were significantly different (p<0.05) except XJ-TSL

400

and YN-SSL had same TRC level. DRSC of pomegranate juices from SD-TSL, SD-SSL,

401

XJ-SSL, SX-SBT, SX-SSL and YN-SSL had no significant difference (p>0.05) , while other 5

402

cultivars were significantly lower (p<0.05) than them. ARSC of the 10 pomegranate juices

403

were not significantly different (p>0.05) except SD-TSL, XJ-SSL had significantly higher

404

ARSC and Yunnan sweet ones had significantly lower ARSC. SASC of the pomegranate

405

juices from 3 Yunnan cultivars were not significantly different (p>0.05) from each other.

406

SASC of Xinjiang and Shaanxi sour pomegranate juices were significantly higher (p<0.05)

407

than their sweet counterparts, but SASC of Shandong sour pomegranate juice was

408

significantly lower (p<0.05) than that of sweet one. These results were consistent with the

409

results by Zaouay et al. (2012), but did not show that sour pomegranates consistently had

410

higher antioxidant potentials than sweet pomegranates.

19

411

The correlation analysis showed that, punicalagin concentration of the tested pomegranate

412

juice positively correlated with the 4 antioxidant capacites (p<0.01) and concentrations of

413

caffeic acid and ferulic acid (p<0.05). That means punicalagin is the most important

414

antioxidant in pomegranate aril juices. Catechin, caffeic acid and ferulic acid concentrations

415

were all positively correlated with ARSC (p<0.05), and caffeic acid concentration was also

416

positively correlated with DRSC (p<0.05). That means each polyphenol in pomegranate juice

417

have pertinence on certain free radical and redox system and they play the antioxidant

418

function by synergistic effect. Interestingly, kaempferol was negatively correlated with ferulic

419

acid concentration (p<0.05), and DRSC (p<0.05). None of other detected phenolic monomers

420

was correlation significant with antioxidant capacities (p>0.05). Across all examined

421

antioxidant capacities, TRC significantly correlated with DRSC, ARSC and SASC (p<0.01),

422

while ARSC significantly correlated with DRSC and SASC respectively (p<0.01).

423

3.5 The effect of environmental factors on polyphenol composition and antioxidant capacities

424

The correlation among each environmental factor and pomegranate juices polyphenol

425

compositions and antioxidant capacities were exhibited in Table 4. There were negative

426

correlations of overall average temperature with total polyphenol concentration, total tannin

427

concentration and punicalagin concentration (p<0.05). This means the lower average

428

temperature during maturity and harvest period could promote the primary polyphenols

429

accumulation in pomegranate arils. Moreover, the significant negative correlation (p<0.05)

430

among overall average temperature with TRC and DRSC suggested that lower temperature

431

during the maturity and harvest time were favorable to increase total reducing capacity and

432

DPPH radical scavenging capacity of pomegranate juice. Temperature differences positively

20

433

correlated with SASC (r=0.62, p< 0.05), indicating that formation of anti superoxide anion

434

components in pomegranate arils were related to high temperature difference during maturity

435

and harvest time. Latitude positively correlated with total polyphenol concentration, total

436

reducing capacity and DPPH radical scavenging capacity (p<0.05), while longitude negatively

437

correlated with total anthocyanin concentration (p<0.05), which indicated that Chinese

438

pomegranate grown in high latitude and low longitude region is favored to accumulate more

439

polyphenols and higher antioxidant potential in its aril juice. In addition, precipitation and

440

insolation had no apparent effects on phenolics composition and antioxidant capacities of

441

pomegranate juices. As far as we know, the regulating and controlling factors of fruit

442

polyphenols remain undetermined, because the factors, ranging from intrinsic genetic to

443

various extrinsic environmental and their interactions, for polyphenol production among

444

cultivars varied through time and space (Hättenschwiler, & Vitousek 2000).

445

3.6 The effect of cultivar type (T), environment (E) and their interaction (T×E) on

446

physicochemical characteristics, polyphenol compositions and antioxidant potential of

447

pomegranate juice

448

The effects of sweet and sour pomegranate type (T), growth environment (E) and their

449

interaction (T×E) on physiochemical characteristics, polyphenol compositions and antioxidant

450

capacities of pomegranate juice were summarized in Table 5. It can be seen that E showed

451

significant contribution to SSC and the proportions of variance were 79.45% (P<0.001).

452

52.77% variance of RSC was explained by T×E, followed by 43.70% explained by E and

453

3.52% explained by T. Moreover, T accounted for 86.17% and 94.96% (p<0.001) variation in

454

TAC and SAR, followed by E and T×E interaction. In brief, soluble solid content and

21

455

reducing sugar content of pomegranate juices was growing environment dependent, whereas

456

titratable acid content and sugar acid ratio were mainly affected by sweet and sour cultivar

457

type. It can be inferred that the 10 pomegranate fruits from 4 growing regions may have

458

different maturity level when harvested at same time, since their environment conditions were

459

different, which subsequently impacted reducing sugar content, soluble solid content and

460

titratable acid content of the pomegranate juices.

461

T×E interaction explained the largest variation of total polyphenols concentration

462

(52.95%, p<0.001) and tannins concentration (54.35%, p<0.001), followed by E (46.82%,

463

45.59%, p<0.001). However, E explained more variation of total anthocyanin concentration

464

(90.32%, p<0.001) and T explained more variation in flavonoids concentration (61.11%,

465

p<0.001). It indicates that accumulation of total polyphenols and tannins in pomegranate juice

466

were related to multi factors. Total anthocyanin level of pomegranate juice was proved to be

467

closely related to maturity degree that was mostly determined by environmental conditions in

468

present work, because the 10 pomegranate samples picked simultaneously from different

469

growing regions may have different maturity levels. Total flavonoid level was mainly

470

determined by sweet and sour cultivar type, which can be proved by the results that all sweet

471

pomegranate juice had higher total flavonoids concentration than sour counterparts except

472

Yunnan sour pomegranate juice had higher total flavonoids than that of sweet pomegranate

473

cultivars. TRC of pomegranate juice were affected more by E than T and T×E interaction,

474

whereas ARSC and SASC were more susceptible to T×E interaction than E and T.

475

Specifically, E contributed 45.25%, 45.58% and 33.97% (p<0.001) of variances to TRC,

476

ARSC and SASC respectively. T×E explained 44.44%, 54.24% and 60.76% (p<0.001) of

22

477

variances in TRC, ARSC and SASC respectively. Additionally, majority variation of DRSC

478

was attributed to T (64.25%, p<0.001), followed by E (20.00%, p<0.001) and T×E interaction

479

(15.75%, p<0.001). In brief, antioxidant capacities of pomegranate juice were affected

480

significantly by environment than sweet and sour types, implying that adjusting planting

481

condition during pomegranate mature and harvest period will improve antioxidant potential of

482

pomegranate juice.

483

Several researches have investigated the effect of cultivar, maturity, and irrigation

484

conditions on titratable acid content, soluble solid content, total polyphenol concentration,

485

total anthocyanin concentration, and antioxidant potential of pomegranate juice (Shwartz et al.

486

2009; Borochov-Neori et al. 2009; Gundogdu, et al. 2012). Their results showed that soluble

487

solid content, TSS/TA ratio and total anthocyanin concentration of the pomegranate aril juice

488

were increased through half-ripe to full-ripe stages, while titratable acid content, total

489

polyphenol concentration and antioxidant potential were decreased in the same period

490

(Fawole and Opara 2013). Deficit irrigation in the early maturity period led to an increase in

491

total polyphenol concentration and total anthocyanin concentration in pomegranate arils, but

492

led to a reduction in fruit size and yield (Mellisho et al. 2012). New results from our research

493

are as follow: low average temperature and widely variable temperature differences during

494

maturity and harvest time are key determinants for primary polyphenols production and

495

antioxidant potential improvement. One possible explanation for these results is under these

496

conditions, carbon should be preferentially allocated to the synthesis of primary metabolites,

497

the amount of which are not detrimental but promote the synthesis of carbon-based secondary

498

metabolites, primarily polyphenols. Therefore, this paper may assist pomegranate growers to

23

499

improve juice nutritional properties by selecting more suitable cultivars and adjusting better

500

growing conditions.

501

4

Conclusion

502

In general, pomegranate juices from 10 representative Chinese cultivars had different

503

physicochemical characteristics and polyphenol compositions. Polyphenol compositions and

504

antioxidant potential of pomegranate juice significantly related to the average temperature and

505

daily temperature difference during maturity and harvest period. And they were also related to

506

the altitude, latitude and longitude of growing regions to some extent.

507

Further study is needed to build up a database for pomegranate juices that include

508

physiochemical characteristics, polyphenol compositions, antioxidant potentials and their

509

correlation to key environmental factors in different growing regions around the world. The

510

database would help producers make better products with high nutritional value, better

511

physicochemical properties and added antioxidants. This database may lead to geographic

512

pomegranate product labeling and brand identification.

513

5

Acknowledgements

514

The authors would like to acknowledge College of Food Science and Engineering,

515

Northwest A&F University for the timely equipment support and instruction. We are grateful

516

to teachers and students who contribute to this work.

517 518

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Figure caption Fig.1 Antioxidant capacities of aril juices of 10 pomegranate cultivars from 4 growing regions of China Data were expressed as gallic acid equivalent concentration (GaEC) and ascorbic acid equivalent concentration (VcEC), EC equivalent concentration (mg/L). Vertical bars represent the standard deviation (n=3); Different case letters above the bars represent the significant difference (p<0.05). TRC, total reducing capacity; DRSC, DPPH radical scavenging capacity; ARSC, ABTS radical scavenging capacity; SASC, Superoxide anion scavenging capacity. XJ, Xinjiang; SD, Shandong; YN, Yunnan; SX, Shaanxi; TSL, SZ, LZ, JPT, SBT belong to sweet pomegranate, SSL is sour pomegranate.

Fig.2 Polyphenols profiles of pomegranate juice from 10 varieties of pomegranate by HPLC analysis. HPLC chromatograms was recorded at 280 nm, compounds were identified as follow: 1) PunicalaginⅠ; 1`) PunicalaginⅡ; 2) Gallic acid; 3) Catechin; 4) Chlorogenic acid; 5) Caffeic acid; 6) Epicatechin; 7) Ferulic acid; 8) Ellagic acid; 9) Keampferol.

31

Table1. Environmental information during the mature and harvest season of the pomegranates in the four growing regions Cultivars XJ-TSL

Difference in temp. (℃) 14.2

Overall avg. temp. (℃) 16.85

Precipitation （mm） 90

Insolation (h) 5410

Altitude (m) 1338

Latitude (S) 39°28'

Longitude (O) 75°59'

XJ-SSL

14.2

16.85

90

5410

1338

39°28'

75°59'

SD-TSL

11.55

17.5

668

4436

74

34°30'

117°32'

SD-SSL

11.55

17.5

668

4436

74

34°30'

117°32'

YN-SZ

7.8

21.25

1012

3248

1302

23° 23'

103° 23'

YN-LZ

7.8

21.25

1012

3248

1302

23° 23'

103° 23'

YN-SSL

7.8

21.25

1012

3248

1302

23° 23'

103° 23'

SX-JPT

7.0

18.4

1864

1989

471.8

34°22'

109°12'

SX-SBT

7.0

18.4

1864

1989

471.8

34°22'

109°12'

SX-SSL

7.0

18.4

1864

1989

471.8

34°22'

109°12'

Environmental information include longitude, latitude, altitude; precipitation (millimeter), insolation (hours), difference of the absolute high and absolute low temperature (℃) and overall average temperature ( ℃ ) of the locations during pomegranate mature time (September and October) in 2012. XJ, Xinjiang, Kashi; SD, Shandong Zaozhuang; YN, Yunnan Mengzi; SX, Shaanxi, Lintong; TSL, SZ, LZ, JPT, SBT and SSL are names of pomegranate cultivars.

Table2. Physicochemical characteristics of aril juices of 10 pomegranate cultivars from 4 growing regions of China Cultivars

Reducing sugar (g/L) 79.32±0.84 g

Titratable acid (g/L) 5.539±0.10 e

Sugar-acid ratio

XJ-TSL

SSC (%) 15.13±0.12 c

15.98±0.29e

Total polyphenols ( GaE mg/mL ) 4.352±0.09 de

Flavonoids (QuE mg/mL) 0.118±0.00 d

Tannins (TaE mg/mL) 1.165±0.09 e

Anthocyanins (GyE mg/mL) 0.160±0.00 a

XJ-SSL

14.97±0.06 cd

62.82±0.82 h

35.60±0.26 b

2.785±0.02 fg

6.147±0.11 b

0.045±0.01 h

1.916±0.02 b

0.122±0.00 b

SD-TSL

16.30±0.17 a

102.7±0.88 b

3.891±0.13 g

26.73±0.93 c

7.429±0.12 a

0.335±0.13 a

2.531±0.08 a

0.034±0.00 f

SD-SSL

15.87±0.32 b

78.50±1.00 g

18.49±0.44 c

5.573±0.13 f

4.481±0.11 d

0.093±0.00 e

1.295±0.01 d

0.063±0.00 e

YN-SZ

14.63±0.32 d

81.52±0.58 f

2.657±0.21 i

37.08±2.92 a

3.234±0.06 g

0.099±0.00e

0.895±0.01 f

0.063±0.00 e

YN-LZ

13.97±0.46 e

93.55±0.89 d

3.328±0.16 h

20.46±1.02 d

3.151±0.05 g

0.084±0.00 f

0.540±0.02 g

0.026±0.00 g

YN-SSL

14.63±0.15 d

110.7±0.58 a

12.47±0.28 d

5.784±0.13 f

4.142±0.08 f

0.171±0.00 c

1.130±0.11 e

0.062±0.00 e

SX-JPT

13.63±0.12 e

86.59±0.83 e

4.486±0.38 f

17.14±1.43 e

4.219±0.10ef

0.259±0.00 b

1.142±0.07 e

0.107±0.00 d

SX-SBT

14.67±0.15 d

100.3±0.25 c

2.809±0.42 i

30.52±4.84 b

4.750±0.08 c

0.170±0.00 c

1.461±0.04 c

0.004±0.01 h

SX-SSL

14.87±0.12 cd

85.99±0.34 e

36.62±0.31 a

1.481±0.01 g

4.735±0.03 c

0.054±0.00 g

1.331±0.04 d

0.116±0.00 c

Data were expressed as mean ± standard deviation (n= 3). Different letters represent significant differences (p< 0.05). XJ, Xinjiang; SD, Shandong; YN, Yunnan; SX, Shaanxi; TSL, SZ, LZ, JPT, SBT belong to sweet pomegranate, SSL belongs to sour pomegranate cultivar. GaE, gallic acid equivalent; QuE, quercetin equivalent; TaE, tannin acid equivalent; CyE, cyanidin equivalent.

Table3. Polyphenol monomers of aril juices of 10 pomegranate cultivars from 4 growing regions of China (ug/mL) Cultivars

Punicalagin

Gallic acid

Catechin

Caffeic acid

Epicatechin

Ferulic acid

Ellagic acid

Kaempferol

4.88±0.08 j

Chlorogenic acid 9.48±0.03 j

XJ-TSL

396.31±0.02 g

2.14±0.03 f

2.32±0.06 c

10.04±0.05 i

0.44±0.03 g

1.02±0.03 a

8.12±0.08 f

XJ-SSL

791.81±0.05 b

14.50±0.02 c

5.63±0.05 i

27.11±0.05 d

1.93±0.01 e

14.04±0.02 f

0.46±0.01 g

0.28±0.01 f

10.66±0.09 e

SD-TSL

1042.93±0.01 a

6.23±0.06 d

40.53±0.04 b

25.56±0.01 e

2.56±0.03 a

9.28±0.03 j

1.72±0.02 a

0.73±0.01 b

1.50±0.02 i

SD-SSL

573.31±0.07 c

3.79±0.04 e

9.70±0.05 g

40.83±0.04 b

2.44±0.05 b

35.65±0.01 a

1.27±0.01 b

0.53±0.02 d

1.67±0.02 h

YN-LZ

264.06±0.02 i

0.70±0.03 h

15.46±0.03 e

23.47±0.02 f

1.37±0.04 g

35.02±0.04 b

0.74±0.01 e

0.25±0.01 g

17.30±0.09 b

YN-SZ

149.85±0.02 j

0.74±0.01 gh

5.98±0.02 h

21.49±0.05 g

1.11±0.02 i

21.26±0.02 e

0.23±0.01 h

0.25±0.00 g

17.79±0.09 a

YN-SSL

479.39±0.01 f

2.09±0.07 f

16.55±0.05 d

44.21±0.03 a

2.19±0.03 d

22.61±0.05 d

0.73±0.02 e

0.65±0.01 c

2.65±0.01 g

SX-JPT

298.99±0.03 h

15.93±0.03 b

9.99±0.07 f

13.96±0.07 i

1.62±0.05 f

25.44±0.06 c

0.81±0.02 d

0.27±0.01 fg

16.89±0.08 d

SX-SBT

504.34±0.01 e

0.80±0.05 g

34.44±0.02 c

21.42±0.03 h

1.22±0.04 h

10.66±0.01 h

1.19±0.04 c

0.53±0.02 d

0.25±0.01 j

SX-SSL

560.83±0.01 d

17.19±0.01 a

41.23±0.01 a

32.26±0.01 c

2.20±0.02 d

12.84±0.03 g

0.59±0.01 f

0.44±0.01 e

17.08±0.08 c

Data were expressed as mean ± standard deviation (n= 3). Different letters represent significant differences (p< 0.05). XJ, Xinjiang; SD, Shandong; YN, Yunnan; SX, Shaanxi; TSL, SZ, LZ, JPT, SBT belong to sweet pomegranate, SSL belongs sour pomegranate.

Table4. Correlation between polyphenol compositions, antioxidant capacities and environmental factors Overall avg. temp. -0.67*

Precipitation

Insolation

Altitude

Latitude

Longitude

Total polyphenols

Difference in temp. 0.51

-0.28

0.39

-0.46

0.61*

0.02

Total flavonoids

-0.12

-0.10

0.20

-0.15

-0.49

0.04

0.46

Total tannin

0.47

-0.65*

-0.25

0.35

-0.49

0.58

0.06

Total anthocyanin

0.48

-0.46

-0.34

0.37

0.29

0.53

-0.66*

Punicalagin

0.50

-0.51*

-0.31

0.41

-0.47

0.52

0.08

Gallic acid

0.01

-0.44

0.24

-0.17

-0.26

0.49

-0.06

Catechin

-0.36

-0.09

0.51

-0.45

-0.59

0.07

0.55

Chlorogenic acid

-0.12

0.24

0.01

-0.02

-0.18

-0.32

0.39

Caffeic acid

0.56

-0.55

-0.43

0.51

-0.36

0.45

0.01

Epicatechin

-0.26

0.46

0.06

-0.11

0.02

-0.52

0.33

Ferulic acid

0.02

-0.30

0.12

-0.03

-0.81**

0.18

0.66**

Ellagic acid

0.47

-0.40

-0.38

0.43

-0.09

0.36

-0.18

Kaempferol

-0.32

0.34

0.23

-0.29

0.38

-0.25

-0.19

TRC

0.50

-0.70*

-0.26

0.36

-0.49

0.65*

-0.04

DRSC

0.38

-0.67*

-0.11

0.22

-0.46

0.64*

0.01

ARSC

0.39

-0.56

-0.20

0.30

-0.55

0.47

0.20

SASC

0.62*

-0.48

-0.54

0.59

-0.05

0.42

-0.28

The results were expressed as Pearson correlation coefficients (r value). ** p< 0.01*. p< 0.05 TRC, total reducing capacity; DRS, DPPH radical scavenging capacity; ARS, ABTS radical scavenging capacity; SAS, Superoxide anion scavenging capacity.

Table5. The effects of cultivar type (T), growing environment (E) and their interactions (T×E) on physicochemical characteristics, polyphenol compositions and antioxidant capacities of aril juices of 10 pomegranate cultivars from 4 growing regions of China. T

E

T×E

SSC (%)

3.19*

79.45***

17.35***

RSC(g/L)

3.52***

43.70***

52.77***

SAR

94.96***

3.94***

1.09***

TAC(g/L)

86.17***

7.79***

6.04***

Total polyphenols (mg/ml)

0.02

46.82***

52.95***

Total flavonoids (mg/ml)

61.11***

14.49***

24.40***

Total tannin (mg/ml)

0.06

45.59***

54.35***

Total anthocyanin (mg/ml)

0.00

90.32***

9.68***

TRC (mgGa/L)

10.31***

45.25***

44.44***

DRSC (mgGa/L)

64.25***

20.00**

15.75*

ARSC (mgGa/L)

0.18

45.58***

54.24***

SASC (mgGa/L)

5.38***

33.96***

60.67***

The results were expressed as the proportion of variance explained by the variable (%). *** p< 0.001; **p<0.01; *p<0.05 SSC, solid soluble content; RSC, reducing sugar content; SAR, sugar acid ratio; TAC, total acid content; TRC, total reducing capacity; DRSC, DPPH radical scavenging capacity; ARSC, ABTS radical scavenging capacity; SASC, Superoxide anion scavenging capacity; Ga, gallic acid equivalents.

Highlights 1. Physicochemical characteristic of pomegranate juices from 10 cultivars was different. 2. The correlation of phenolic compositions and antioxidant capacities was significant. 3. Environmental factors effected phenolic composition and antioxidant capacity. 4. Type and environment interaction influenced phenolic composition and antioxidant capacity.