Changes in antioxidant activity during the ripening of jujube (Ziziphus mauritiana Lamk)

Accepted Manuscript Changes in antioxidant activity during the ripening of Jujube (Ziziphus mauritiana Lamk) Zozio Suzie, Servent Adrien, Cazal Guillaume, Mbéguié-A-Mbéguié Didier, Ravion Sylvie, Pallet Dominique, Hiol Abel PII: DOI: Reference:

S0308-8146(13)01645-2 http://dx.doi.org/10.1016/j.foodchem.2013.11.022 FOCH 14972

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

6 March 2013 22 September 2013 5 November 2013

Please cite this article as: Suzie, Z., Adrien, S., Guillaume, C., Didier, A-M., Sylvie, R., Dominique, P., Abel, H., Changes in antioxidant activity during the ripening of Jujube (Ziziphus mauritiana Lamk), Food Chemistry (2013), doi: http://dx.doi.org/10.1016/j.foodchem.2013.11.022

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1

Changes in antioxidant activity during the ripening of Jujube (Ziziphus mauritiana Lamk)

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Zozio Suzie1,2., Servent Adrien2., Cazal Guillaume3., Mbéguié-A-Mbéguié Didier1,2., Ravion

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Sylvie4., Pallet Dominique2* and Hiol Abel4‡.

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1

CIRAD, UMR QUALISUD, F- 97130 Capesterre-Belle-Eau, Guadeloupe, France

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2

CIRAD, UMR QUALISUD, F-34398 Montpellier, France

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3

UNIVERSITE MONTPELLIER II, F-34095 Montpellier, France

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4

UNIVERSITE DES ANTILLES GUYANE (UAG), F-97157 Pointe à Pitre, France

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‡

Corresponding author: Professor Abel Hiol Food Sciences- Department of Biotechnological

engineering. INRA/URZ143-UAG, 97157, FR. E-mail: [email protected] phone: +

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33 692245017

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Both authors contributed equally to this work.

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1

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ABSTRACT

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Phenolic compounds from jujube fruits and related antioxidant activities were investigated

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during the ripening stages. Three different antioxidant assays, including ORAC, FRAP and

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DPPH, were monitored on crude jujube extract (CJE). Jujube fruits were additionally

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fractionated into three selective fractions F1, F2, and F3. However, only the FRAP assay gave

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the relative antioxidant activity for the three fractions. Furthermore, HPLC-ESI-MSMS (Q-Tof)

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and GC-MS were used to identify the compounds in each purified fraction. Using FRAP, F1

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mainly composed of lipids, exhibited the lowest antioxidant activity (≈0.080±0.015 mmol

21

trolox/100 g, p < 0.05). F2, rich in flavanols and flavonols, displayed 50-fold higher activity

22

(4.27±0.11 mmol trolox/100 g). Remarkably, F3 with an elevated content of condensed tannins

23

(polymeric proanthodelphinidins), exhibited the highest antioxidant activity (25.4±0.35 mmol

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trolox/100 g). The presented results showed that the phenolic profiles of the fruits were

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influenced by their developmental stage. Furthermore, during ripening, the antioxidant activity

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may be more impacted by the flavanols and condensed tannins. The purified condensed tannins

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of jujube fruits may be used as natural antioxidant extracts.

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Key words

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Jujube fruits, flavanols, flavonols, antioxidant activity, condensed tannins, thiolysis, HPLC-ESI-

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MSMS (Q-Tof), GC-MS analysis, ripening stages

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2

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1. Introduction

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Jujube fruit (Ziziphus mauritiana Lamk.), also known as “pomme-surette”, represents one of

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the most consumed fruits in the heritage of Guadeloupe (FWI). Jujube trees are distributed in

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different areas of the island, including volcanic, saline and limestone soil, but the cultivar impact

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on the fruit food applications remains unknown.

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Jujube fruits are increasingly eaten fresh or used in food products for their potential

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nutritional and medicinal value. Previously, jujube fruits have been reported in several food

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processing products, including compotes, alcoholic beverages, chutneys, pickles, cakes and

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bread, in India and in Africa (Shobha & Bharati, 2007). Fresh jujube fruits unfortunately exhibit

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rapid postharvest ripening, and may not be stored for more than 10 days under ambient

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conditions. Since the majority of the quality attributes develop during the ripening process, it has

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become essential to consider the ripening stages to better understand the phenolic profile in

45

fruits. Only few studies have been devoted to fruit quality trait changes during the ripening

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process of Z. mauritiana, whereas those on Z. jujuba have been well documented.

47

Z. mauritiana has been reported for its significant content of carbohydrates, organic acids,

48

vitamin C and minerals. The physiological relevance appears to be enhanced by the contents of

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various compounds, including triterpenoid acids, flavonoids, phenolic acids and cytokinins

50

(Pawlowska, Camangi, Bader, & Braca, 2009). Furthermore, some studies have indicated a high

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antioxidant capacity in Z. jujuba using different physiological conditions (Li, Fan, Ding, & Ding,

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2007; Zhang, Jiang, Ye, Ye, & Ren, 2010).

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The antioxidant capacity of fruits may be associated with several parameters, including the

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ripening stages and the matrix of the plant product. The complexity in the fruit matrix may lead

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to a low correlation between the results of antioxidant assays used, due to the different

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mechanisms of the antioxidants. Moreover, the method of antioxidant capacity analysis depends 3

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on the free radical generator or oxidant, and also the technology used (Zulueta, Esteve, &

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Frígola, 2009). Therefore, comparison of different antioxidant methods should provide a strong

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background for better understanding of the correlation between the bioactive compound profile

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of the fruit during ripening and the antioxidant activity. Previous bioactive investigations have

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highlighted the unclear antioxidant contribution of the various phenolic compounds in Z. jujube

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(Wu, Gao, Guo, Yu, & Wang, 2012). In this work, the main objective was to identify the

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antioxidant compounds in Z. mauritiana through fruit ripening. Therefore, we investigated the

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relationship between the identified molecules and the antioxidant activity using independent

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assays. To this end, the CJE was used in advance to choose the most appropriate assay for

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antioxidant activity within the jujube fruit extract. After several optimizations, the FRAP assay

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was designated to determine the antioxidant activity for our three fractionated extracts, including

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an apolar extract rich in lipids, fraction 1 (F1), a phenolic compounds extract, fraction 2 (F2),

69

and a condensed tannins extract, fraction 3 (F3).

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2. Materials and methods

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2.1. Chemicals and reagents

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The extraction solvents were of analytical or HPLC grade and were purchased from Carlo-Erba

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(Val de Reuil, France). Folin-Ciocalteu reagent for the determination of phenols and 2,4,6-

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tripyridyl-s-triazine (TPTZ) for spectrophotometry (det. ≥ 99.0%) were purchased from Fluka

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(Basel and Lausanne, Switzerland); 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2-2‟-azobis (2-

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amidinopropane) dihydrochloride (AAPH) (granular, 97%), fluorescein for fluorescence, free

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acid, and 6-hydroxy-2,5,7,8-tetramethyl-2-carboxylic acid (trolox) 97% were purchased from

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Sigma-Aldrich (Steinheim, Germany). All HPLC standards were acquired from Extrasynthese

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(Geney, France). Buffer salts and all other chemicals were of analytical grade from Sigma-

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Aldrich (Steinheim, Germany). 4

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2.2. Plant material

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2.2.1. General

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Two jujube fruit cultivars, Ziziphus mauritiana Lamk P3 and P5, were harvested from plants

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grown on a local farm based in the south of the island. The fruits were selected on the basis of

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their morphological differences and taste.

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For each accession above, fruits were harvested during January/February, established as the

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optimal fructification period. Within one day of harvesting, the fruits were washed with 1%

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chlorinated water and thoroughly rinsed. Then the fruits were stored for about four days in air at

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20°C in order to homogenize their internal temperature, whereupon the putative injured samples

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were discarded.

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The remaining fruits were sampled according to five developmental stages, based on both

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their size and color. Depending on the maturity, the skin colour of jujube shifts from green

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(stage 1) and yellow-green (stage 2) to yellow (stage 3), and then reaches a reddish (stage 4) to

94

brown (stage 5) colour. The samples were freeze-dried, crushed and stored at -20°C for further

95

experiments.

96

2.2.2.

Preparation of crude extract from jujube fruits (CJE)

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One gramme of powdered jujube fruits was dissolved in 50 ml of acetone/water/formic acid

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(70/28/2, v/v/v) by stirring for 1 hour at 4°C. The resulting material was centrifuged for 15 mins

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at 10,000 rpm and the supernatant, designated as the crude jujube extract (CJE), was saved for

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monitoring antioxidant assays and measuring the total phenolic content.

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2.2.3.

Selective extraction and fractionation of lyophilized jujube fruits

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Three fractions, containing different classes of identified molecules, were isolated from the

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lyophilized jujube. Six grammes of powdered jujube fruits were extracted by stirring in 150 ml

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dichloromethane/hexane/ethanol (70/29/1, v/v/v) for 1 hour. The resulting slurries were 5

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evaporated to dryness with a rotary evaporator at 35°C and re-dissolved with 5 ml of the same

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solvent mixture to obtain fraction 1 (F1). Furthermore, the dried pellets were re-extracted at 4°C

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by a different solvent containing acetone/water/formic acid (70/29/1, v/v/v) for 1 hour. After

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centrifugation as above, the supernatant was collected and the acetone was evaporated. The

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aqueous slurry was twice extracted with 150 ml of ethyl acetate, and the organic layer was

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evaporated to obtain fraction 2 (F2). After preliminary analysis, the resulting aqueous slurry was

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found to be rich in condensed tannins, and contained small water-soluble molecules, including

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sugars and amino acids. Therefore, after evaporation and centrifugation, this aqueous fraction

113

was purified on a 40 cm × 2 cm column packed with 15 ml (= 1 Bed Volume (BV)) of Sephadex

114

LH-20 (Sigma-Aldrich, Steinheim, Germany). The column was previously equilibrated with a

115

solvent mixture of ethanol/water/formic acid (70/29/1, v/v/v). Diluted samples (100 ml) were

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loaded onto the column and were washed with 3 BV of the above solvent mixture. Desorption of

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condensed tannins was achieved with 3 BV of a solvent mixture of acetone/water/formic acid

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(70/29/1, v/v/v) and fraction 3 (F3) was obtained after evaporation. The molecular compositions

119

of these three fractions, F1, F2 and F3, were assessed, and the antioxidant activity measured.

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2.3. Antioxidant activity determination

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2.3.1. Number of methods

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Three independent antioxidant activity determination methods were assessed to evaluate the

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antioxidant capacities of the CJE.

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2.3.2.

FRAP assay

125

The FRAP assays were carried out on a microplate spetrofluorimeter Infinite 200,

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(TECAN, Austria GMBH, Austria), as by Benzie & Strain (1996) with some modifications.

127

FRAP reagent was made by mixing an equivalent volume of 300 mM acetate buffer (pH3.6), 20

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mM FeCl3.6H2O with 10 mM TPTZ in 40 mM HCl. This working solution (170 µl) was warmed 6

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to 37°C directly in the 96-well plate for 5 mins, and then 30 µl of diluted extract (CJE, F1, F2

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and F3) were added for each fraction. Absorbance at 593 nm was measured after incubation at

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37°C for 30 min in the dark. The results, in triplicate, were expressed in mmol trolox equivalents

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/100 g.

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2.3.3.

DPPH radical-scavenging activity

134

The DPPH radical-scavenging activity was measured as by Mishra, Ojha, & Chaudhury

135

(2012), with some slight modifications. Different aliquots of the CJE (10 µl to 70 µl) were

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directly added to a spectrophotometer curvette containing a solution of DPPH• (60 mM, in

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methanol) with a final volume of 2.5 ml. The initial absorbance of the DPPH• solution was

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measured at 516 nm, using a UV-Visible spectrophotometer UV 2450 Shimadzu. Then, the

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decrease in absorbance was monitored immediately after addition of each CJE concentration,

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every 3 min in the dark, until the reaction reached a plateau. The percentage of DPPH radical-

141

scavenging activity at different concentrations of jujube extract was calculated from the

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absorbance value, using the following equation 1:

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DPPH radical-scavenging activity (%) =

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where A0 is associated with the absorbance of the DPPH• solution at 0 min, and At is the

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absorbance in the presence of jujube extract when the reaction reaches the plateau as indicated

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above. The percentage of residual steady-state DPPH• plotted as a function of the ratio of jujube

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extract to DPPH• (mg/mg) gave the effective concentration (EC50). Thereby, the influence of the

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concentration, expressed in mg extract/mg DPPH•, was standardized, and DPPH free radical-

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scavenging was defined as the concentration of jujube extract needed to decrease the initial

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DPPH radical concentration by 50%.

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2.3.4.

(Equation 1)

ORAC assay 7

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The ORAC assay was carried out on a microplate spetrofluorimeter Infinite 200 as by the

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method of Zulueta, Esteve, & Frígola (2009), with some modifications. Fifty microlitres of

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diluted sample or trolox (standard) were added to 170 µl of 78nM fluorescein and then incubated

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for 15 min at 37°C before adding 30 µl of 178 nM AAPH. The reaction was performed at 37°C

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and the fluorescence was measured every minute for 1 h (excitation: 285 nm, emission: 520 nm).

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A calibration curve was obtained by plotting the area under the curve against trolox

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concentrations in the 0-40 µM range. ORAC value was expressed as mmol trolox equivalents

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/100 g.

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2.4. Determination of the total phenolics content

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The total phenolics content was evaluated at 760 nm, using Folin-Ciocalteu reagent

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(Singleton, Orthofer, & Lamuela-Raventós, 1999). The results, in triplicate, were expressed as

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milligrammes of catechin equivalents per 100 g of lyophilized fruits (mg CE/100 g).

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2.5. GC-MS identification of jujube fruit extracts from F1

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Molecules from F1 were identified, using a Focus GC (Thermo, Waltham, USA) equipped

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with a single split/splitless capillary injector and a Thermo TG-5ms column (30 m, 0.25 mm,

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0.25 µm). GC-MS operating conditions were set up, using helium flow-rate at 1.2 ml/min with

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1.0 ml injection volume and split ratio 100:1. The injector port temperature was set at 200°C,

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while the oven temperature (75°C) was increased to 300°C at a rate of 5°C/min. The ion source

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temperature was 200°C with the ionization mode of electronic impact at 70 eV, and the mass

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range was from m/z 30 to 650 amu. The relative percentage amounts of separated compounds

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were calculated using a computerized integrator (ICIS algorithm).

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2.6. LCMS identification

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2.6.1. General

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Molecules from F2 and F3 were identified by LCMS. The instrument consisted of an HPLC

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Waters alliance 2790 with a photodiode array detector 996 (Waters corp., Milford, USA) and a

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mass spectrometer (Micromass Q-Tof, Manchester, UK) with an ESI source. A Waters (Waters

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corp., Milford, USA) ACE reversed-phase column (C18, 5 µm, 250x4.6 mm) was used at a flow

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rate of 0.7 ml/min and 30 µl injection volume. The column oven temperature was set at 25°C.

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The mobile phase consisted of A (water/trifluoroacetic acid: 99.9:0.1, v/v) and B (acetonitrile/

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trifluoroacetic acid: 99.9:0.1, v/v) and the gradient programme was 0–4 min with 5% solvent B

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and 4–45 min with 5–35% B. Mass spectra were recorded in positive mode between 50 and 1000

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Da. The capillary and cone tensions were respectively set at 3000V and 20V.

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identification of molecules from F2, the fragmentation was made by ESI (+)-MS/MS with an

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optimized 30 eV collision energy. Under these conditions, the main fragmentation pathways

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observed arose from the cleavage of the C-ring linkage in position 1/3 or 0/3 as in the lower

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diagram in Table 2 (below). For procyanidins analysis with constitutive catechin units, the C-

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ring cleavages were expected to occur in one of the catechin units, leading to U, upper unit and

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the lower unit D, as previously reported by Abad-García, Berrueta, Garmón-Lobato, Gallo, and

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Vicente (2009).

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2.6.2.

For the

Analysis of condensed tannins from F3 by thiolytic degradation

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The aqueous purified condensed tannins were hydrolyzed as previously described (Jerez,

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Pinelo, Sineiro, & Núñez, 2006). Briefly, 2 ml of the corresponding fraction (F3) were mixed

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with 2 ml of methanol acidified by concentrated HCl (3.3 % v/v), 4 ml of phloroglucinol (50 g/l

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in methanol) and 0.5 ml of 10 g/l ascorbic acid. The reaction mixture was placed in a sealed

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Pyrex tub, heated to 85°C for 1 h, then cooled in ice. The degradation products were purified on

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a Waters (Waters Corp., Milford, USA) Symmetry reversed-phase column (C18, 4 µm, 250x4.6

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mm), using the binary solvent system described above for F2 analysis. The gradient programme 9

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was optimized with 0–7 min of 3% solvent B then 7–40 min up to 40% B. The flow rate of the

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mobile phase was 1 ml/min and the injection volume 30 µl. The peaks were monitored at 280 nm

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with PDA detection.

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The subunit composition of proanthocyanidins was obtained, based on the relative ease with

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which their interflavonoid C-C linkage bonds were cleaved. The terminal subunits were released

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as free flavan-3-ols after the thiolytic degradation, whereas electrophilic extension subunits were

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trapped by phloroglucinol to generate phloroglucinol adduct. Finally the terminal and extension

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subunits were analyzed by HPLC-MS, in order to determine the constitutive subunits of

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proanthocyanidins, as well as to evaluate the average mean degree of polymerization (mDP).

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2.6.3.

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Acid/n-butanol hydrolysis and quantification of proanthocyanidins from F3

Proanthocyanidins from

F3 were hydrolyzed as by Porter, Hrstich, and Chan (1985).

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Briefly, a 1 ml aliquot of the extract was mixed with 5 ml of the n-butanol/HCl reagent (95/5,

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v/v) and 0.1 ml of the iron reagent (i.e. 2% (w/v) ferric ammonium sulfate in 2N HCl). The tubes

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were capped and heated at 100°C for 60 min. This reaction produced acid-catalyzed oxidative

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depolymerization of the interflavan bonds in the proanthocyanidins, thus yielding red

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anthocyanidins in solution. The liberated anthocyanidins were analyzed on a Symmetry C18

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reversed-phase column (4.6 x 250 mm, 4 µm, Waters). Delphinidin and cyanidin standards were

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used to quantify the proanthocyanidins content.

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2.7. Statistical analysis

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The data were subjected to analysis of variance (ANOVA) using statistical software

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(Statsoft, version 7). Analyses were performed on three biological replicates and individual data

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were expressed as means ± standard deviation. The means were separated from each other by

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Tukey‟s honestly significant difference test at p < 0.05 level.

222 3. Results 10

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3.1. Variation of total phenolics content in the CJE with ripening stages

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Jujube fruits from cultivars P3 and P5 exhibited similar total phenolic patterns. However, P3

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showed a slight decrease (25%) from the 1st to the 4th ripening stage, whereas P5 exhibited a

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sharp decrease (60%) from the 2nd to the 5th ripening stage (Fig.1, A). The total phenolic content

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was clearly dependent on the ripening stage, and the highest concentrations were found within

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the more green stages, including 1 and 2.

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3.2. Distribution of antioxidant activities from CJE at different ripening stages

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The antioxidant activities of jujube cultivars P3 and P5 were examined for the five ripening

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stages through the FRAP, DPPH and ORAC assays. Using the FRAP assay, a slight decrease

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(13%) was observed during the first ripening stages of cultivar P3 (1st to 3rd), followed by a sharp

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decrease (53%) during the last stages (3rd to the 5th) (Fig.1, B). For P5, the diminution was

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observed from the 2nd ripening stage (78 %). However, with the ORAC assay (Fig.1, C), a sharp

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decrease (≈ 66%) was observed from the first to last ripening stages for cultivars P3 and P5.

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Likewise, using the DPPH assay, the antioxidant activity decreased during ripening. However,

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the difference between cultivars P3 and P5 was observed from the 3rd stage (Fig.1, D). In

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agreement with our results, a previous study has shown a link between the fully ripe jujube fruit

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(Ziziphus jujuba) and the decrease of antioxidant activity (Lu, Lou, Zheng, Hu, & Li, 2012). In

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contrast, for mango fruits, an unchanged antioxidant capacity was reported with 4 days‟ storage

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(Kim, Lounds-Singleton, & Talcott, 2009).

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3.3. Total phenolics content correlation with antioxidant activity

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Together, our results indicated that, during ripening, the total phenolics content exhibited a

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significantly positive correlation (p < 0.05) with the antioxidant activity using the three assays

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FRAP, DPPH and ORAC. However, the highest correlation (0.998 and 0.993) was established

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with the FRAP assay for cultivars P3 and P5, respectively. Similarly, in some studies, using food 11

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matrices, including apples, orange, broccoli and leeks, a positive correlation between antioxidant

248

activity and total phenolics has been reported (Michiels, Kevers, Pincemail, Defraigne, &

249

Dommes, 2012). DPPH results slightly correlate with total phenolics content (0.972 and 0.945

250

for cultivars P3 and P5, respectively). However, DPPH free radical-scavenging has been more

251

used to characterize synthetic antioxidant activity (Müller, Fröhlich, & Böhm, 2011). It should

252

be noted that it took three hours to reach the steady state for the lowest concentrations of jujube

253

extract, and we need six concentrations for reproducible EC50. Regarding ORAC antioxidant

254

capacity, only 7.8±1.1 mmol trolox/100 g was observed at the 5th stage of cultivar P3, while

255

17.9±2.0 mmol trolox/100 g (p<0.05) was determined using the FRAP method with the same

256

samples. Therefore, the FRAP assay showed an extensive scale for analysis of antioxidant

257

activity, and was selected for further characterization of jujube fruit extracts.

258

3.4. Antioxidant activity distribution on the selected extracts

259 260

The antioxidant activities of F1, F2 and F3, from each of the five ripening stages of both cultivars P3 and P5, were quantified by FRAP assay, as shown in Figure 2.

261

F1 exhibited a lower antioxidant activity at the five ripening stages (0.080 to 0.053 and

262

0.075 to 0.070 mmol trolox/100 g, p < 0.05) for cultivars P3 and P5, respectively. The extract

263

was analyzed by GC-MS for both cultivars (P3 and P5) at each ripening stage. As indicated in

264

Table 1, triacylglycerols, several sterols and at least one triterpenoid, identified as lupeol, were

265

determined as major constituents of F1. The profile of most of the identified compounds seems

266

to be affected by the developmental stage. Concerning the content of the compound within

267

cultivars P3 and P5, the variation remained unclear. However, the main compound identified for

268

cultivar P3 was ç-sitosterol, while sigmasterol became higher during the three last ripening

269

stages in cultivar P5. Similar results were found for Zizyphus spina-christi L., a species closely

12

270

related to Z. mauritiana (Nazif, 2002). The poor antioxidant activity of F1 may be related to the

271

higher concentration of lipid compounds including triacylglycerides and sterols.

272

The antioxidant activity of F2 from cultivar P3 (P3-F2) was slightly higher than that of

273

cultivar P5 (P5-F2). P3-F2 displayed a slow decrease (21%) during the first three ripening

274

stages, followed by a sharp decrease (70%) from the 3rd to the 5th ripening stages. For P5-F2, a

275

similar quick decrease (≈70%), from the 3rd to the 5th ripening stages was observed. As shown in

276

Table 2, F2 was rich in phenolic compounds, including flavonols, glycosides such as kaempferol,

277

and quercetin glycosides. Interestingly, we identified flavanols, e.g. catechin and gallocatechin,

278

but also flavanol dimers. Only flavonol glycosides and phenolic acids were reported at one

279

specific ripening stage of Z. mauritiana (Memon, Memon, Bhanger, & Luthria, 2013;

280

Pawlowska, Camangi, Bader, & Braca, 2009). Additionally, we found that F2 contained

281

prodelphinidin as a dimer of gallocatechin but also procyanidin as a dimer of catechin, as Chen,

282

Li, Maiwulanjiang, Zhang, Zhan, Lam, et al. (2013) found in Z. jujuba. Furthermore, the relative

283

quantification of each identified molecule was calculated for both cultivars, P3 and P5, at each of

284

the five ripening stages (Table 3). Interestingly, the flavonols content was higher than total

285

flavanol (monomers and dimers) during ripening of both cultivars, P3 and P5. However, for P5,

286

the difference was less marked, due to the low total flavonol content compared to P3 (3-fold

287

lower at the last stage). In contrast to flavanols, the flavonols content varied slightly between the

288

two ripening stages for both cultivars. Surprisingly, for both cultivar P3 and P5, the pattern of

289

total flavanols (monomers and dimers) was positively correlated with the high antioxidant

290

activity of F2 (Fig 2, A). This result may suggest a strong relationship between total flavanol

291

content and the antioxidant potential of the jujube fruit extract.

292

F3 exhibited a higher antioxidant activity than did F1 and F2, for both jujube cultivars, P3

293

and P5, at the five ripening stages. At stage 1, cultivar P3 exhibited 25.4 mmol of trolox/100 g, 13

294

while, at the same stage, only 4.27 mmol of trolox/100 g was detected for F2. Although the

295

decrease was more pronounced in P5, a sharp decrease in antioxidant activity for both cultivars,

296

P3 and P5, was observed from stage 3 (Fig 2, B).

297

Our purified condensed tannins in F3 were analyzed by HPLC-MS before hydrolysis. As

298

expected, the detection of a large wide peak suggested the presence of polymeric

299

proanthocyanidins (data not shown). Depolymerization, using phloroglucinol, indicated that both

300

cultivars, P3 and P5, exhibited dissimilar profiles of mDP values during ripening (Table 4

301

supplementary data). In detail, P3 exhibited constant values during ripening, except for the 3rd

302

ripening stage. In contrast, the mDP of cultivar P5 exhibited a decrease during ripening.

303

Nevertheless, the mDP value of cultivar P3 was higher than that of P5. Similar high mDP values

304

were found in Lotus corniculatus (Meagher, Lane, Sivakumaran, Tavendale, & Fraser, 2004).

305

The treatment of purified tannins in F3 with butanol/Hcl/Fe showed that delphinidin was the

306

predominant anthocyanin in cultivars P3 and P5, respectively 90% and 80%) (Fig.3). This result

307

indicates that the condensed tannins may be polymeric proanthodelphinidins. Likewise, cultivar

308

P3 exhibited a higher proanthodelphinidins content than did cultivar P5. Furthermore, the

309

proanthodelphinidins content profile was similar to that observed for the antioxidant activity of

310

F3.

311

4. Discussion

312

In this study, the antioxidant activity of jujube fruits was investigated using three independent

313

antioxidant methods, including ORAC assay based on hydrogen atom transfer, while FRAP and

314

DPPH assays were based on electron transfer reactions. Although the detailed sequence of the

315

process remain unknown, several mechanisms, including reducing capacity, prevention of chain

316

initiation, binding of transition metal ion catalysts, prevention of continued hydrogen abstraction

14

317

and radical-scavenging, may explain the antioxidant activity. Therefore, combined assays are

318

needed for antioxidant determination from any plant matrix.

319

The ORAC assays have been one of the most commonly used for evaluating the antioxidant

320

capacity. However, our results on jujube fruits extract exhibited a worse correlation between

321

ORAC antioxidant capacity and the total phenolics content than did the FRAP value. In fact, the

322

ORAC assay has been reported as unsuitable for lipophilic antioxidant compounds (Huang, Ou,

323

Hampsch-Woodill, Flanagan, & Deemer, 2002). Whereas the lipid content in jujube fruits was

324

low as expected, a small quantity including vegetable sterols, was found in F1. Nevertheless, the

325

potential antioxidant activity of lipids remains unclear. In previous studies, a synergistic

326

antioxidant effect between vitamin C and α-tocopherol was highlighted (Zhu, Huang, & Chen,

327

2000). Finally, the ORAC assay clearly failed to accurately take into account the antioxidant

328

behaviour of molecules in a hydrophobic/hydrophilic heterogeneous matrix (Laguerre, López-

329

Giraldo, Lecomte, Baréa, Cambon, Tchobo, et al., 2008). In addition, although the antimicrobial

330

activity was not tested in this study, fatty acid extract from Z. spina-christi L. was reported to be

331

active against Bacillus subtilis, E. coli and Streptococcus pyogenes (Nazif, 2002).

332

In contrast to the ORAC assay, the FRAP assay gave a wide scale of values and was more

333

sensitive, even with the low antioxidant activity observed during the last stage of ripening.

334

Furthermore, after fractionation of the antioxidant compounds, the FRAP method remained

335

compatible for our three fractions F1, F2 and F3. Even if the determination of EC50 by the DPPH

336

assay was lengthy, the results obtained complied with the FRAP profile.

337

For F2, the slight variation of flavonols content observed during the ripening of cultivars P3

338

and P5 strongly indicated their poor contribution to the antioxidant activity. Furthermore, for

339

both cultivars, P3 and P5, the pattern of total flavanols (Table 3) showed a positive correlation

340

with that of the antioxidant activity assessed by FRAP (Fig.2, A). Flavanol and particularly 15

341

procyanidin dimer contents seem to conform to the same trend as the antioxidant activity during

342

the ripening of cultivars P3 and P5. Moreover, it has been shown that glycosylation onto

343

flavonoid aglycones leads to a decrease in the antioxidant capacity (Heo, Kim, Chung, & Kim,

344

2007).

345

In order to understand the structure-activity relationships of proanthocyanidins in jujube,

346

depolymerization was applied in the presence of a nucleophile, for phloroglucinol used in F3.

347

The method was shown to be efficient for ascertaining the structure of procyanidins, as

348

previously reported (Jerez, Touriño, Sineiro, Torres, & Núñez, 2007). The profile after thiolysis

349

indicated that gallocatechin (GC) and epigallocatechin (EpiGC) were the main terminal unit in

350

both jujube cultivars, and that the extension units contained gallocatechin (GC), catechin (Cat)

351

and epicatechin (EpiCat). However, the proportion of each differed with the jujube cultivar: GC

352

and Cat are higher in cultivar P3 than in P5, whereas EpiCat is lower in cultivar P3 than in P5,

353

for the extension units. The dissimilarity has also been found in the terminal units, where the

354

proportion of EpiCat was higher in cultivar P5 than in P3.

355

Regarding the mDP, a constant value was observed for cultivar P3 during the ripening stages,

356

except for the 3rd ripening stage, whereas it decreased for cultivar P5. Although the mDP value

357

of cultivar P3 was higher than that of P5 during jujube ripening, mDP was not clearly related to

358

the antioxidant activity. Similarly, a contrast in correlation between the antioxidant capacity and

359

mDP has already been reported (Jerez, Touriño, Sineiro, Torres, & Núñez, 2007; Zhou, Lin, Wei,

360

& Tam, 2011).

361

The acid-butanol assay is a colorimetric reaction based on an acid-catalyzed oxidative

362

depolymerization of condensed tannins to yield anthocyanidins. Delphinidins and cyanidins

363

were released from the condensed tannins in F3 for both cultivars, P3 and P5, but delphinidins

364

were predominant. Interestingly, the patterns of the anthocyanidins content for cultivars P3 and 16

365

P5 (Fig.3) were similar to that found for the antioxidant activity of condensed tannins in F3

366

(Fig.2, B), revealing a positive correlation coefficient (0.99; p< 0.05). This assay has proved to

367

be a useful diagnostic tool, giving accurate quantification of proantocyanindins.

368

Finally, F3, rich in condensed tannins, exhibited a better antioxidant capacity than did F1 and

369

F2 for both cultivars P3 and P5. Our results suggested that polymeric proanthodelphinidins may

370

make the greatest contribution to the antioxidant capacity of jujube. A similar study on

371

condensed tannins from grape demonstrated that the best antioxidant activity was found with

372

oligomeric and polymeric tannins, in contrast to monomers, i.e. catechins. (Spranger, Sun,

373

Mateus, Freitas, & Ricardo-da-Silva, 2008). In fact, it appears that extensive conjugation

374

between 3-OH and B-ring catechol groups, together with abundant linkages, endow a polymer

375

with significant radical-scavenging properties by increasing the stability of its radical (Haenen,

376

Arts, Bast, & Coleman, 2006; Heim, Tagliaferro, & Bobilya, 2002).

377

On the other hand, the sum of antioxidant capacity of F1, F2 and F3 did not reach the

378

antioxidant capacity of the global extract. F3 showed the strongest antioxidant contribution

379

(55%), F2 8 % and F1 less than 1 % (p<0.05), suggesting that about 37 % of the antioxidant

380

activity was not recovered.

381

The discrepancy may be due to the lack of known antioxidant phytochemicals, including

382

polysaccharides, enzymes, tocopherol, pigments, and vitamin C, and phenolic acids were most

383

likely disrupted during the fractionation. The elevated antioxidant capacity of the CJE may be

384

related to each component indicated above, but also to the synergistic effects between them.

385

Previously, antioxidant activity was associated with polysaccharides from soluble fractions of

386

Ziziphus. Interestingly, the scavenging activity was more effective in the presence of uronic

387

acid,. (Li, Liu, Fan, Ai, & Shan, 2011). Additionally phenolic acids with high antioxidant

388

capacity have been identified in Ziziphus jujuba (Wang, Liu, Zheng, Fan, & Cao, 2011; Zhang, 17

389

Jiang, Ye, Ye, & Ren, 2010). Previously,Kumar, Yadav, Jain, and Malhotra (2011) have

390

observed an antioxidative system, including superperoxide dismutase (SOD), peroxidase (POD)

391

and catalase (CAT) during the initial ripening stages of Ziziphus mauritiana in storage.

392

Furthermore, the antioxidant molecules of jujube fruit may provide a synergistic interaction

393

leading to the high antioxidant activity found in the CJE. Many synthetic antioxidants, including

394

propyl gallate, butylated hydroxyanisole and tertbutyl-hydroquinone, have been used for drugs as

395

well as in cosmetic applications. However, some recent studies have highlighted genotoxicity

396

and cytotoxicity in synthetic antioxidants, but also their interactions with other antioxidants, and

397

therefore potential risks to human health. Together, our data and other data suggest that further

398

research is needed to understand the change of bioactive molecules in jujube during ripening,

399

and the variation of antioxidant capacity, coupled with extraction methods, in order to improve

400

the potential applications of jujube fruits for functional food development.

401

5. Conclusion

402

Jujube fruit (Ziziphus mauritiana Lamk.) was found to have variable contents of

403

phytochemicals and elevated antioxidant activity tested separately by the ORAC, FRAP and

404

DPPH assays. FRAP assay proved to be an efficient method for the evaluation of the antioxidant

405

activity of jujube, as well as the acid-butanol assay, for condensed tannins. Based on the ripening

406

stages, the major constituents with potential antioxidant capacity were identified, using both LC-

407

MS and GC-MS analysis. Interestingly, the results showed that condensed tannins, and

408

specifically polymeric proanthodelphinidins, exhibited the highest antioxidant contribution in

409

both jujube cultivars. It should be noted that, during ripening, the antioxidant capacity variation

410

was more affected by the decrease in the total flavanols than the flavonols. However, despite a

411

higher content of flavonols and condensed tannins for cultivars P3 than P5, the antioxidant

412

activities measured on the crude extracts showed no significant difference. In addition to post18

413

harvest investigations, further studies may determine the bioavailability and the physiological

414

relevance of the elucidated constituents found in jujube fruits.

415 416

The results of our study suggest that the antioxidant potential of jujube fruits should be strongly considered for functional and nutritive applications.

417 418

Abbreviations

419

HPLC-ESI-MSMS (Q-Tof): High Performance Liquid Chromatography-Electrospray Ionization-

420

Tandem Mass Spectrometry (Quadrupole-Time-of-Flight); LC-MS: Liquid Chromatography-

421

Mass Spectrometry; GC-MS: Gas Chromatography- Mass Spectrometry; CJE: crude jujube

422

extract; ORAC: Oxygen Radical Absorbance Capacity; FRAP: Ferric Reducing Antioxidant

423

Power; DPPH: 2,2-diphenyl-1-picrylhydrazyl; C: Catechin; Cyan: Cyanidin; Dph: Delphinidin;

424

EC: Epicatechin; GC: Gallocatechin; EPG: Epigallocatechin; EC50: Effective Concentration;

425

F1: Fraction 1; F2: Fraction 2; F3: Fraction 3; mDP: mean degree of polymerization

426 427

Acknowledgements

428

Suzie Zozio was supported by a grant from “Région Guadeloupe”. The study is an output

429

from a research project funded by the European Union FP7 245 – 025, called African Food

430

Tradition Revisited by Research (AFTER - http://www.after-fp7.eu/). The authors are grateful

431

for the funding provided for this work.

432

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24

Table 1 Relative abundance (%) of lipids and triterpene identified in F1 by CG-MS (SI > 850) from cultivars P3 and P5 Ripening stages MOLECULES Triacylglycerols Palmitin, 2-monoLinolein, 2-monoOlein, 2-monoStearin,2-monoSterols/Tocopherols Campesterol Stigmasterol ç-Sitosterol α1-Sitosterol dl-α-Tocopherol Triterpene Lupeol nd: not detectable

1

Cultivar P3 2 3 4

5

1

Cultivar P5 2 3 4

5

1.29 1.23 5.98 2.32

3.24 1.23 8.07 2.20

1.98 0.90 4.36 2.42

3.66 0.99 3.57 2.07

2.36 0.56 1.56 1.86

1.06 0.39 3.11 Nd

1.21 0.34 2.21 Nd

0.88 0.41 1.98 Nd

0.64 0.49 2.01 Nd

1.60 0.49 2.21 Nd

2.22 6.04 54.1 0.41 1.79

1.90 3.84 25.4 1.23 1.60

1.53 4.73 35.4 1.53 1.65

1.35 3.21 16.8 1.41 1.24

0.39 2.22 12.4 0.99 1.00

1.47 14.5 24.2 2.81 0.38

1.19 13.2 15.3 3.12 0.34

0.69 21.6 7.20 6.58 0.20

0.47 19.2 5.18 5.39 0.21

0.52 18.1 4.19 4.08 4.08

0.54

2.08

1.30

1.41

0.99

nd

nd

nd

nd

nd

Table 2 Tentative identification of flavonoids and derivatives i n jujube cultivars P3 and P5. The Table shows the m/z value of main product ion detected from the ESI (+) MS/MS product spectra of [M+H] at collision energy 30 eV at different Retention Times (R.T). Fragments and nomenclature pathways of the O-glycosides and aglycones studied are indicated in the lower diagram.

1

RT (min) 11.47

[M+H] + m/z 611

2

14.54

307

139 : [1,3A0]+; 181:[ 0,4B-H2O]+; 223 : [Cleavage A ring]+ ; 151: [1,2A- H2O] +; 163 :[0,4B- 2H2O] +; 195: [Cleavage A ring – CO]+;

Gallocatechin b

3

15.30

611

287:[U (1,3 A)]+ [D (1,2A)-H2O]+; 443:[U (1,3 A)]+; 425:[D (1,2A)-H2O]+ 139:[ D (1,3A)]+; 127: [1,4A +2H]+; 151: [D (1,2A)-H2O]+

Prodelphinidin B dimer isomer b (Epi) gallocatechin - (epi) gallocatechin

4

17.96

579

287:[U (1,3 A)]+ [D (1,2A)-H2O]+; 443:[U (1,3 A)]+; 425:[D (1,2A)-H2O]+ 139:[ D (1,3A)]+; 127: [1,4A +2H]+; 151: [D (1,2A)-H2O]+

Procyanidin B dimer b (Epi)catechin - (epi)catechin

5

19.56

307

139 : [1,3A0]+; 181:[ 0,4B-H2O]+; 223 : [Cleavage A ring]+ ; 151: [1,2A- H2O] +; 163 :[0,4B- 2H2O] +; 195: [Cleavage A ring – CO]+;

Epigallocatechin b

6

20.32

579

287:[U (1,3 A)]+ [D (1,2A)-H2O]+; 443:[U (1,3 A)]+; 425:[D (1,2A)-H2O]+ 139:[ D (1,3A)]+; 127: [1,4A +2H]+; 151: [D (1,2A)-H2O]+

Procyanidin B dimer isomer b (Epi)catechin - (epi)catechin

7

21.12

291

139 : [1,3A0]+; 123:[1,2B]+ ; 207 : [Cleavage A ring]+; 147:[0,4B- 2H2O] + ; 165:[0,4B- H2O]+ ; 179:[Cleavage A ring - CO]+; 273: [M+H- H2O]+

Catechin a

8

24.84

291

139 : [1,3A0]+; 123:[1,2B]+ ; 207 : [Cleavage A ring]+; 147:[0,4B- 2H2O] + ; 165:[0,4B- H2O]+ ; 179:[Cleavage A ring - CO]+; 273: [M+H- H2O]+

Epicatechin a

9

30.31

611

303 : Y0, 465 : Z1

Rutin (Quercetin-3-O-rutinoside) a

10

31.09

611

319 : Y0, 465 : Z1

Myricitin dirhamnoside b

No

Fragments ion (m/z) 287:[U (1,3 A)]+ [D (1,2A)-H2O]+; 443:[U (1,3 A)]+; 425:[D (1,2A)-H2O]+ 139:[ D (1,3A)]+; 127: [1,4A +2H]+; 151: [D (1,2A)-H2O]+

Tentative identification Prodelphinidin B dimer b (Epi)gallocatechin - (epi)gallocatechin

11

31.92

465

319 : Y0

Myricitin rhamnoside b

12

32.32

465

303 : Y0

13

34.12

595

14

34.90

493

303 : Y0, 449 : Z1, 137 : [0,3A0]+, 153 : [1,3A0]+, 165 :[ 0,3B0]+ MS3 303 = 153 : [1,3A0]+; 137 : [0,3A0]+, 285 : [Y0 - H2O]+ 303 : Y0

Isoquercetin (Quercetin-3-Oglucopyranoside) a Quercetin dirhamnoside b, a

15

35.36

585

287 : Y0, 439 : Z1

Kaempherol dideoxyhexoside b

16

36.84

585

287 : Y0, 439 : Z1

Other Kaempherol dideoxyhexoside b

17

40.29

553

287 : Y0

Kaempherol derivative b

18

41.02

787

623 : Y2, 449 : Z1, 303 : Y0

Quercetin hexoside rhamnoside b

19

41.82

553

287 : Y0

Kaempherol derivative b

20

44.27

551

287: Y0

Kaempferol derivative b

a

Identified by comparison with standard compound

b

Identified by the retention times, UV spectra and fragment ion

U is the upper unit of procyanidins and prodelphinidin dimers; D is the lower unit

.

Quercetin derivative b

Table 3 Contents of major flavanols and flavonols found in F2 of jujube cultivars P3 and P5 during the ripening. Values are expressed in catechin or quercetin equivalents (µg/g of lyophilized jujube). Catechin standard was used to quantify flavanols at 280 nm and quercetin standard for flavonols at 370 nm

1

2

Cultivar P3 3

4

5

1

2

IDENTIFIED MOLECULES Gallocatechin

166.7a

160.9 a

112.3 c

27.3 b

26.5 b

124.8 a

112.1 a

Epigallocat

71.1 a

88.6 c

58.7 a

26.1 b

15.7 b

85.4 a

Catechin

80.9 a

63.6 b

53.0 b

20.1 c

0.0 d

Epicatechin

24.3 a

19.9 a

19.4 a

7.4 b

343±28

333±19

243±22

Prodelphinidin dimers

75.9 a

78.4 a

Proanthocyanidin dimers

409 a

Ripening stages

Cultivar P5 3

4

5

50.1 c

19.2 b

0.0 b

62.3 b

87.4 a

14.1 c

0.0 d

59.3 c

41.6 a

32.3 a

0.0 b

0.0 b

0.0 b

26.6 a

34.3 a

26.3 a

0.0 b

0.0 b

81±7

42±8

296±31

250±26

196±16

33±6

0.0

37.1 b

37.2 b

0.0 c

215 a

223 b

190 a

88.4 c

0.0 d

379 a

244 b

67.9 c

0.0 d

111 a

179 a

128 a

52.1 b

0.0 c

485±33

457±22

281±15

105±12

0.0

326±16

401±23

318±18

140±9

0.0

Rutin

1667 a

1924 b

2111 c

1554 d

1773 e

4278 b

4657 a

4517 a

4306 b

2871 c

Myricitin dirhamnoside

10129 a

10043 a

11970 b

9008 c

9244 d

2090 b

2494 a

3329 c

2497 a

1661 d

Myricitin rhamnoside

5670 a

6087 b

8402 c

9252 d

5697 a

2542 b

2146 c

1916 a

1938 a

688 d

Isoquercetin

4691 a

4640 a

6330 b

5822 c

4421 d

2739 b

2414 c

2088 a

2133 a

904 d

Quercetin dirhamnoside

4057 a

4443 b

4694 c

4803 d

4240 e

2302 b

2771 a

2144 c

2893 a

1803 d

Kaempferol dideoxyhexoside

1119 a

1057 a

1454 b

2056 c

1441 b

1031 a

1199 c

635 b

972 a

646 b

Other Kaempferol dideoxyhexoside

118 a

103 a

168 a

319 b

133 a

348 a

320 a,b

68.0 c

433 b

117 c

Kaempferol derivative

90.5 a

95.2 a

238 b

325 b,c

373 c

0.0 b

157 c

81.6 d

58.2 a

55.1 a

Other Kaempferol derivative

57.1 a

57.1 a

183 b

302 c

494 a

773 a

861 a

301 b

286 b

403 c

Kaempferol derivative

69.8 a

154 b

159 b

160 b

95.2 a

0.0 b

98.0 a

102 a

81.6 a

93.9 a

TOTAL FLAVAN-3-OLS

TOTAL FLAVANOL DIMERS

TOTAL FLAVONOLS 27667±320 28603±299 35708±312 33601±333 27911±186 16102±155 17115±197 15182±212 15597±229 Means values with different lowercase letters in the same row are significantly different by Tukey’s HSD (Honestly Significant Difference) test at p < 0.05 level

9243±97

Figure 1 Antioxidant activity changes in jujube cultivars P3 and P5 during the ripening. Total phenolics (A) are expressed in mg of catechin equivalents /100 g lyophilized jujube. FRAP (B) and ORAC (C) assay are expressed in mmol of trolox per 100g of lyophilized jujube. DPPH assay (D) are expressed in Effective Concentration (EC50) [EC50 (mg extract/mg DPPH)].

Figure 2 Antioxidant activity measured by FRAP assay for F2 (A) and the F3 (B) from cultivars P3 and P5. Values are expressed in mmol of trolox /100g of lyophilized jujube.

Figure 3 Content variation of cyanidin (Cyan) and delphinidin (Dph) in F 3 from cultivars P3 and P5. Cyanidin and delphinidin were quantified by HPLC/MS, using external standards as described in Material and methods. Results are expressed in mg of delphinidins and cyanidins /100g of lyophilized jujube.

A a

5.E+3

a,b b,c

a

4.E+3

a c

b

3.E+3

d c

2.E+3 d 1.E+3

P3 P5

0.E+0 0

a

45 FRAP (mmol trolox/100g)

Total phenolic (mg catechin/ 100g)

50

6.E+3

40 a,b

35

b

30

2

3

4

c

20 10

P3 P5

5

d

0

1

2

C

4

5

14

D c

12

20

10

b

a

c

15 b

d c

e d

P3 P5

e

0 2 3 Ripening stage

4

5

EC 50 mg/mg

ORAC (mmol Trolox/100g )

3

Ripening stage

a

1

d

15

5

25

0

c

25

Ripening stage

5

b

a

0

1

10

B

b

b

c

8 6 a

4

a

b

a a

2

P3 a

0

P5

0

1

a

2 3 Ripenning stage

4

5

FRAP (mmol Trlox/ 100 g)

5

A

a

a,b

4

b

a

3

a

a

2

c

1

c

P3 P5

d d

0 0

1

2 3 Ripening stage

4

5

FRAP (mmol Trolox/ 100g)

30 a

25

B

a,b a,b

20

a

a,b

b

15

b

10 5

d

0 0

1

d

c

P3 P5 2

3

Ripening stage

4

5

35 a Anthocyanidins (mg/ 100g)

30 25

a

P3 Dph P5 Dph P3 cyan P5 cyan

a

a a

20

b b

15 10

c

c 5

d

0 0

1

2 3 Ripening stage

4

5

The highlights related to our paper entitled: Changes in antioxidant activity during the ripening of Jujube (Ziziphus mauritiana Lamk) The phenolic profile of Jujube fruits was influenced by the ripening stage. The major bioactive constituents with antioxidant capacity were identified by mass spectrometry Flavanols and condensed tannins showed more influence on the antioxidant activity extend during the ripening of jujube. The purified condensed tannins from jujube fruits may be used as natural antioxidant