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Changes in phenolic compounds and their antioxidant capacities in jujube (Ziziphus jujuba Miller) during three edible maturity stages
Accepted Manuscript Changes in phenolic compounds and their antioxidant capacities in jujube (Ziziphus jujuba Miller) during three edible maturity stages Bini Wang, Qingyuan Huang, Chandrasekar Venkitasamy, Hongkang Chai, Hui Gao, Ni Cheng, Wei Cao, Xingang Lv, Zhongli Pan PII:
S0023-6438(15)30223-1
DOI:
10.1016/j.lwt.2015.10.005
Reference:
YFSTL 5000
To appear in:
LWT - Food Science and Technology
Received Date: 10 April 2015 Revised Date:
9 September 2015
Accepted Date: 2 October 2015
Please cite this article as: Wang, B., Huang, Q., Venkitasamy, C., Chai, H., Gao, H., Cheng, N., Cao, W., Lv, X., Pan, Z., Changes in phenolic compounds and their antioxidant capacities in jujube (Ziziphus jujuba Miller) during three edible maturity stages, LWT - Food Science and Technology (2015), doi: 10.1016/j.lwt.2015.10.005. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Changes in phenolic compounds and their antioxidant capacities in jujube (Ziziphus
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jujuba Miller) during three edible maturity stages
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Bini Wang a, b, *, Qingyuan Huang a, Chandrasekar Venkitasamy b, Hongkang Chai a, Hui Gao a, Ni
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Cheng a, Wei Cao a, Xingang Lv a, Zhongli Pan b, c, **
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a
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Northwest University, Xi’an, Shaanxi 710069, China
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b
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Davis, One Shields Avenue, Davis, CA 95616, USA
Department of Food Science and Engineering, College of Chemical Engineering,
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c
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Albany, CA 94710, USA
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Department of Biological and Agricultural Engineering, University of California,
Healthy Processed Foods Research Unit, USDA-ARS-WRRC, 800 Buchanan St.,
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Abstract: This study investigated the changes in total phenolic content (TPC), total
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flavonoid content (TFC), individual phenolic compound content, DPPH radical
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scavenging activity and antioxidant capacity measured by FRAP assay of four
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phenolic fractions (free, esterified, glycosided and insoluble-bound) from jujube
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during three edible maturity stages. The maturity stages of jujubes were established as
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white maturity (WM), half-red maturity (HM) and red maturity (RM). The free
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fraction in jujube at WM stage had the highest TPC, TFC, total phenolic acid contents,
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and antioxidant capacities. The phenolic contents and their activities greatly decreased
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with the increasing maturity stage. Caffeic acid was the most predominant in all
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detected phenolic compounds at WM stage, while rutin dominated at HM and RM
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stages. Even though most of phenolic compounds with antioxidant activity in jujube
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existed at the WM stage as the free form, the insoluble-bound fractions also contained
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a large number of phenolic compounds.
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Chemical compounds studied in this article
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Gallic acid (PubChem CID:370); Protocatechuic acid (PubChem CID:72);
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p-Hydroxybenzonic acid (PubChem CID:135); Chlorogenic acid (PubChem
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CID:1794427); Caffeic acid (PubChem CID:689043); p-Coumaric acid (PubChem
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CID:637542); Ferulic acid (PubChem CID: 445858); Rosmarinic acid (PubChem CID:5281792); Ellagic acid (PubChem CID:5281855); Quercetin (PubChem CID:5280343); Syringic acid (PubChem CID: 10742); Hesperetin (PubChem
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CID:72281);
Rutin
(PubChem CID:5280805)
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Key words: Ziziphus, Insoluble-bound phenolic, Antioxidant activity, Maturity stage 2
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1. Introduction
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Jujube is the fruit of Ziziphus jujuba Miller, a thorny rhamnaceous plant widely
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cultivated in subtropical and tropical regions of Asia, especially in China, America
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and Europe. China is the largest producer contributing over 90% of the world jujube
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production and the only country exporting jujube fruits. The total annual yield of fresh
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jujube fruits in 2009 was 600 million kilograms (Lu et al., 2012) and has been
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increasing in the recent years in China. Globally, jujube is popularly consumed as
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fresh or dried fruit and has been used to prepare compotes, jams, beverages and cakes.
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The color of jujube peel changes from green to yellow, then to reddish and finally to
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red during maturation. These peel colors represent maturity stages typically called
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green fruit stage, white maturity (WM), half-red maturity (HM), and red maturity
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(RM) stage, respectively. The fruit is unsuitable for eating or processing until it ripens
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to WM stage. Production of various processed products prefers jujube at a specific
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maturity stage. For example, fresh consumption or most processed products require
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jujube at HM or RM stage, dried products require jujube at RM stage and processing
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into saccades needs jujube at WM stage. There is an increased interest in the potential
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health benefits of jujube to human beings. An ancient Chinese book on herbal
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medicine named Huangdi Neijing (475−221 BC) showed that jujube was one of five most valuable fruits (peach, pear, apricot, plum, and jujube) in China. It has a high
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nutritional value and potential health benefits including antioxidant activity (Gao, Wu,
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& Wang, 2013). It has been widely used as food, a functional food additive, and a
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traditional Chinese medicine for several thousands of years and attracted research on
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its bioactive substances, such as phenolic compounds (Du et al., 2013; Chen et al.,
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2013; Choi et al., 2011; Wang et al., 2010).
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Phenolic compounds are secondary metabolites of plants that play an important role in
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the pigmentation, growth, reproduction of plants as well as plant resistance to
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pathogens (Ghasemzadeh & Ghasemzadeh, 2011). Their content in fruit is largely
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affected by genotype (cultivar), pre-harvest environmental conditions, post-harvest
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storage conditions, processing and the degree of maturity at harvest (Shahidi & Naczk,
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2004). During fruit maturation, the phenolic compounds undergo a series of complex
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biosynthesis process, leading to the changes in their composition and content in plant
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and plant-derived foods (Prasanna, Prabha, & Tharanathan, 2007). They have
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received increased attention due to their potent antioxidant capacities and their
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remarkable health benefits in the prevention of various oxidative stresses associated
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diseases, such as cancer, cellular aging, cardiovascular diseases and inflammation
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(Dai & Mumper, 2010). Several studies revealed that the phenolic compounds and
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their bioactivities in jujube could be influenced by its maturity (Wu et al., 2012; Chen
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et al., 2013; Choi et al., 2012). Those studies evaluated the phenolic compounds in
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soluble extracts alone and ignored the insoluble-bound ones in residues, leading to the underestimation of real phenolic content of jujube and their corresponding antioxidant
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activities. In general, the phenolic compounds in food are classified as soluble
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(extractable) and insoluble (non-extractable) compounds based on the location of
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phenolic compounds in the plant together with the chemical structure of these
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substances (Reis Giada, 2013). The former can typically be extracted by organic
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solvents, while the latter is bound to cell wall polysaccharides or proteins forming
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insoluble stable complexes. Interestingly, these insoluble phenolics are also very
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important in health effects from the nutritional viewpoint and they exert their
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antioxidant effects to protect the body against oxidative stress (Liyana-Pathirana &
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Shahidi, 2006; Pérez-Jiménez & Torres, 2011). Though the insoluble phenolic
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compounds cannot be extracted by organic solvents, they may be released from the
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complexes by the action of intestinal enzymes or colonic microbiota and thereby
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transformed into small phenolics and metabolites that are subsequently absorbed (Jara
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& Josep, 2011). Therefore, the insoluble compounds should also be well studied.
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In our previous study, we found that the major fraction of phenolic acids in different
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tissues of jujube is insoluble-bound (Wang et al., 2011). However, there is no
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information available related to the effect of maturity stages on the soluble and
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insoluble phenolic compounds of jujube and their antioxidant capacities, which is
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important to ensure that jujube is harvested at right maturity with high antioxidants
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content. Furthermore, producers and industrialists also need the valuable information
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on bioactive compounds in jujube and their antioxidant activities at different maturity stages for marketing purpose. Thus, the aim of the present study was to evaluate the
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changes in phenolic compounds (in free, esterified, glycosided and insoluble-bound
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forms) and their antioxidant activities of jujube at three edible maturity stages. The
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results of this study could lead to identification of the optimal maturity stage for the
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harvest of jujube with high composition of polyphenols targeting increased
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antioxidant activities.
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2. Materials and Methods
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2.1 Samples of Jujube Fruits
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Fresh jujubes (Ziziphus jujube cv. Jishanbanzao) used in this study were obtained
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from a local farm in Jishan County, southwestern part of Shanxi Province, North
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China. No pesticide was used in the jujube crop. Jujube samples were harvested from
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July 25th to October 5th of 2014 at three edible stages of maturity determined based
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on the surface color. The three maturity stages were established as white maturity
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(WM), yellow skin color; half-red maturity (HM), representing jujube with red
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surface area of 40%-60%; and red maturity (RM), having 100% red surface area.
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Jujubes were picked up randomly from different parts of several trees of the same
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species and were free from visible blemishes and disease. Jujubes were frozen and
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stored in airtight polyethylene bags at -18oC in a freezer until they were analyzed.
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2.2 Chemicals
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Gallic acid, protocatechuic acid, p-hydroxybenzoic acid, syringate, caffeic acid,
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p-coumaric acid, ferulic acid, chlorogenic acid, rosmarinic acid, ellagic acid, rutin, hesperetin, 2,2-diphenyl-1-picryl-hydrazyl (DPPH), and 2,4,6-tripyridyl-s-triazine
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(TPTZ) were purchased from Sigma-Aldrich (Steinheim, Germany). All other
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chemicals were also analytical grade and were obtained from Xi’an Chemical Co.
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(Xi’an, China). HPLC grade methanol was purchased from Merck (Darmstadt,
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Germany). Analytical grade acetic acid was supplied by Beijing Reagent Co. Ltd
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(Beijing, China) and HPLC grade water was purified by Milli-Q system (Millipore
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Bedford, MA, USA).
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2.3 Preparation of Crude Jujube Extracts
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The frozen jujube fruits were thawed and cleaned with tap water. The seeds were
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removed from jujubes and the edible portion was homogenized in a blender for 1 min.
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A sample of 100g of homogenized jujube was lyophilized, milled and sieved through
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a standard sieve of 100 mesh. The powdered samples (2 g) of jujube at different
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maturity stages were extracted with 15 mL of 80 % (v/v) aqueous methanol at room
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temperature. The solution was sonicated for 30 min and then centrifuged at 2000g for
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10 min to collect supernatant. The supernatant extraction was repeated for three times
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and the accumulated supernatants were used for the fractionation of free phenolic
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compounds, soluble glycosides and esters of phenolic compounds. The residue left
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after the extraction of supernatant was saved for the determination of insoluble -bound
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phenolic compounds.
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2.4 Fractionation of Free and Bound Phenolic Compounds
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Phenolic compounds in crude extracts were fractionated into free and bound forms following our previously established methods (Wang et al., 2011) as described below.
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The crude extract (supernatants) obtained after the methanol extraction from jujube as
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described in the previous step was evaporated under vacuum at 35 oC to about 10 mL.
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The aqueous suspension was acidified to pH 2 using 6 M hydrochloric acid (HCl),
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and extracted for five times with ethyl acetate at a solvent to water phase ratio of 1: l.
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The ethyl acetate extracts were referred to as the free phenolic compounds (F1). The
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aqueous phase remained after the ethyl acetate extraction was treated by alkaline
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hydrolysis
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ethylenediaminetetraacetic acid (EDTA) and 1% ascorbic acid) under nitrogen for 4 h
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at room temperature. After acidification to pH 2 with 6 M HCl, phenolic compounds
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(F2) released from soluble esters were extracted from the hydrolysate for five times
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using the procedure as described above. Following this, the aqueous phase remained
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after the F2 separation was hydrolyzed with 5 mL 6M HCl for 30 min at 85 oC under
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nitrogen. Phenolic compounds (F3) released from soluble glycosides were separated
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from the hydrolysate for five times following the procedure as described above. The
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residues from the 80% methanol extractions were hydrolyzed directly with 8 mL of 4
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M NaOH (containing 10 mM EDTA and 1% ascorbic acid) under the same conditions
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as the ester. After acidification to pH 2 using 6 M HCl, phenolic compounds (F4)
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released from methanol-insoluble ester-bound phenolics were extracted from the
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hydrolysate for five times as described above. Extraction was done for triplicate
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sodium
hydroxide
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samples. Each of the phenolic fractions, obtained as described above, was dehydrated with anhydrous sodium sulfate, filtered, and evaporated to dryness under vacuum at 35 oC. The dry residues were dissolved into 5 mL of methanol, and these solutions
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were used for the determination of phenolic contents and antioxidant activities as
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described below.
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2.5 Determination of Total Phenolic Contents
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Total phenolic contents (TPC) in each fraction of jujube extract were determined
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according to a previously described laboratory procedure of the modified
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Folin-Ciocalteu colorimetric method (Wang et al., 2011). TPC was evaluated at 760
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nm by adding Folin-Ciocalteu reagent to the sample. The average value of triplicate
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data was expressed as the gallic acid equivalents in mg per 100 gram dry weight (mg
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GAE/100 g DW).
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2.6 Determination of Total Flavonoids Content
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Total flavonoids content (TFC) in each fraction of jujube extract was measured
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colorimetrically at 510 nm following a previously reported method (Jia et al., 1999).
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TFC was expressed as rutin equivalents in mg per 100 gram dry weight (mg RE /100
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g DW). The absorbance was measured for triplicate samples.
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2.7 HPLC-ECD Analysis
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The phenolic compounds in the four phenolic fractions from jujube were separated
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and quantified using a HPLC fitted with an electrochemical detector (ECD) as
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described by Wang et al. (2011) with minor revision. HPLC analysis of phenolic compounds were carried out using an Agilent 1100 HPLC System (Agilent, USA)
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equipped with a vacuum degasser, a quaternary solvent delivery pump, a manual
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chromatographic valve, a thermostated column compartment, and a HP1049A
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programmable ECD (HP, USA). A Zorbax SB-C18 column (150 ×4.6 mm, 5.0 µm)
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connected with a Zorbax SB-C18 guard column (20 × 4.0 mm, 5 µm). The mobile
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phase adopted was methanol (A) and 0.15 % aqueous formic acid (B) (v/v) using a
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linear gradient elution of 5-8 % A at 0-6 min, 8-15 % A at 6-10 min, 15-35 % A at
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10-15 min, 35-55 % A at 15-20 min, 55-65 % A at 20-25 min and 65-80 % A at 25-30
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min. The flow-rate was kept at 1.0 mL min-1 at all times. The column was operated at
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30 oC and the injection volume was 10.0 µL. The electrochemical detector was set at
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800 mV in the oxidative mode. Re-equilibration duration was set as 6 min by using
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the starting condition before injection of the next individual sample. Quantification of
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phenolic acids was carried out by an external standard method using calibration
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curves. The amount of each phenolic acid was expressed as microgram per gram dry
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weight (µg/g DW).
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2.8 Radical DPPH Scavenging Activity
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Scavenging activity on DPPH free radicals by each phenolic acid fraction was
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assessed according to the method reported by Wang et al. (2011).
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2.9 Ferric Reducing Antioxidant Power (FRAP)
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FRAP assay of each sample was performed following a previously described
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laboratory procedure (Wang et al., 2011). 2.10 Statistical Analysis
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The results presented in the tables are the mean value±SD (standard deviation) from
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three replicates. Data analysis was carried out using SAS software, version 8.1.
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Statistically significant difference between the samples was evaluated by the Tukey’s
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test. Difference at p < 0.05 was considered to be significant. The correlation analysis
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between phenolics and antioxidant activity was made using standard Pearson
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correlation.
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3. Results and Discussion
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3.1 Total Phenolics of Jujube at Three Edible Maturity Stages
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The TPCs in four phenolic fractions of jujube at three edible maturity stages are
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shown in Table 1. The maturity stages had a significant influence on the TPCs in
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jujube. The TPC in each phenolic fraction significantly decreased (p < 0.05) with the
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increase in maturity stages. All fractions extracted from WM stage were observed to
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have several folds (1.8-12 folds) higher amounts of TPC than those at RM stage. The
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sum of TPC in four fractions of jujube sharply decreased from 1515.35 mg GAE/100
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g DW at WM stage to 362.68 mg GAE/100g DW at RM stage (p < 0.05). Previous
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studies (Wu et al., 2012; Zozio et al., 2014) reported only the TPC values of the
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soluble extracts obtained from jujube during ripening and ignored the TPC of
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insoluble extracts. The TPC of the soluble extracts decreased with maturity stage,
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which was in agreement with those reported previously for jujube and other fruits
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(Zozio et al., 2014; Wang et al., 2013; Wu et al., 2012; Kondo et al., 2002; Gruz et al., 2011). However, the quantities of TPC obtained from this study were lower than the
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previously published results (Wang et al., 2011; Wu et al., 2012). This might be
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mainly attributed to the differences in the cultivar and sources of the materials, as well
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as the regional differences (Gull et al., 2012). In the four phenolic fractions, F1 was
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the major fraction constituting 57.1% and 42.5% of the sum of the TPC at WM and
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HM stages of jujube, respectively. The F2 and F4 dominated at RM stage, comprising
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29.0% and 28.4% of TPC, respectively. These results indicated that the TPC was
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clearly dependent on maturity stages, and the maturation process increased the bound
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phenolics content.
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3.2 Total Flavonoids in Jujube at Three Maturity Stages
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Most previous studies revealed that jujube contained considerable amounts of
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flavonoids including rutin (Pawlowska et al., 2009; Zhang et al., 2010; Wu et al.,
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2012; Du et al., 2013; Gao et al., 2012), and mainly examined TFC in ethanol or
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methanol extracts of jujube. Flavonoids has also been found in the insoluble
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(non-extractable) fraction and associated with dietary fiber in tomato peel and roselle
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tea (Arranz et al., 2010; Kapasakalidis, Rastall, & Gordon, 2009; Navarro-Gonzalez et
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al., 2011; Sáyago-Ayerdi et al., 2007). Little information has been reported on TFC in
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the insoluble fraction in jujube.
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In the present study, the TFC in soluble and insoluble fractions of jujube at three
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maturity stages were determined (Table 2). Results showed that the TFC in all four
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phenolic fractions of jujube rapidly decreased with the progress of maturity. The rapidest decrease in TFC occurred for F1, and the TFC was significantly reduced by 6
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folds from WM to RM stage (p < 0.05). The sum of TFC in four fractions exhibited a
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continuously decreasing trend similar to TPC and decreased from 1692.66 mg RE/100
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g DW at WM stage to 483.47 mg RE/100 g DW at RM stage, which was consistent
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with those reported for pear-jujube (Wu et al., 2012). The TFC values obtained for the
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soluble fraction of jujube (422.43 -1525.43 mg RE/ 100 g DW) were within the
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ranges reported by Zhang et al. (2010), from 276.43 to 1851.96 mg QE/ 100 g DW
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and by Gao et al. (2012), from 62.0 to 284.9 mg RE/ 100 g FW in ethanol or methanol
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extracts of different jujube cultivars. With the increase of jujube maturity, the
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percentage of TFC in F1 continuously decreased from 65.7% to 34.6%, while the TFC
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in F4 continuously increased from 16.3% to 36.7%. As jujube became fully ripen
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(RM stage), F1 and F4 were the major fractions constituting 34.6% and 36.9% of the
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sum of TFC, respectively. Therefore, the TFC in the insoluble fraction (F4) cannot be
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ignored, and further research is required to identify the association or binding of these
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flavonoids with other compounds of jujube.
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3.3 Individual Phenolic Compounds
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Thirteen phenolic compounds, including hydroxybenzoic acids (gallic, protocatechuic,
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p-hydroxybenzoic and syringate), hydroxycinnamic acids (caffeic, p-coumaric, and
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ferulic), other phenolic acids (chlorogenic, rosmarinic, and ellagic) and flavonoid
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(rutin, quercetin and hesperetin) were well separated and quantified by HPLC-ECD.
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But syringate and hesperetin were not found in jujube at all three maturity stages. The distribution of the phenolic compounds in the four fractions obtained from jujube at
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the three maturity stages is presented in Table 3. The results showed that free phenolic
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compounds were the most abundant at all of the maturity stages.
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Free phenolic compounds comprised of 80.0%, 53.5% and 85.9% of the total phenolic
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compounds in jujube at WM, HW and RW, respectively (Fig.1). The content of free
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phenolic compounds in jujube was much higher at WM (1698.29 µg/g DW) than
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those at HM and RM stages (794.50 and 1165.66 µg/g DW, respectively). Rutin was
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reported as a common component in jujube extracts (Pawlowska et al., 2009), and was
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the most predominant phenolic compound at any maturity stages in this work. The
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levels of rutin decreased from 925.83 µg/g DW (WM) to 602.04 µg/g DW (HM), and
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then increased to 670.63 µg/g DW (RM). This change is consistent with the results
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reported by Wu et al. (2012), but the obtained values in this research were more than
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10 times higher. Choi et al. (2012) also found rutin content continuously decreased as
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the fruits matured, and epicatechin was the most prevalent flavonoid during the
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maturity stages which was not observed in our study. Caffeic acid was the dominant
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phenolic compounds at WM stage, while chlorogenic acid, an ester of caffeic acid,
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was mostly present in the free form at RM stage. The p-coumaric acid was detected
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only at HM stage and ferulic acid was detected only at RM stage. In addition,
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quercetin, gallic and rosmarinic acids were not detected at any maturity stages.
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Phenolic compounds released from soluble esters constituted from 3.4% (at RM) to
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18.6% (at HM) of the total quantified phenolic compounds in jujube (Fig.1). At HM stage, the total content of phenolic compounds released from soluble esters was
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275.92 µg/g DW, which was 2-fold higher than that at WM stage and 5-fold higher
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than that at RM stage. The p-coumaric acid was the principal phenolic presented as
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soluble esters at WM stage, whereas rutin dominated at RM stage. Each of rutin and
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chlorogenic acid constituted for more than 36% of the total phenolic compounds
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content at HM stage. However, gallic and rosmarinic acids were not detected in this
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fraction at any maturity stages.
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Glycosided phenolic compounds ranged from 3.8% (at RM) to 24.6% (at HM) of the
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total phenolic compounds presented in jujube (Fig.1). The total contents of glycosided
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phenolics in jujube at WM and HM stage were 260.02 µg/g DW and 365.88 µg/g DW,
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respectively, which were about 5-fold and 7-fold higher than that at RM (52.08 µg/g
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DW), respectively. Glycosided gallic acid presented mostly at WM stage and HM
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stage and was not detected at RM stage. The percentage of glycosided gallic acid at
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WM and HM were 76.4% and 56.8% of the total glycosided phenolics, respectively.
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Though chlorogenic and protocatechuic acids were the principal phenolics at RM,
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they were at very low levels of 15.15 µg/g DW and 10.89 µg/g DW, respectively.
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The insoluble-bound phenolic compounds constituted 3.5%, 3.3%, and 6.8% of total
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phenolics in jujube at WM, HW and RW stages, respectively (Fig.1). Insoluble-bound
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p-coumaric acid was one of the major phenolic compounds in jujube found at the
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three maturity stages, and represented 23.0%, 39.1% and 35.7% of the total
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insoluble-bound phenolics at WM, HM and RM stages, respectively. Furthermore, ellagic acid mainly presented in insoluble-bound form and caffeic acid was the major
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phenolic compounds at WM stage, while p-coumaric and chlorogenic acids
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dominated this fraction at RM stage.
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The total content of quantified phenolic compounds in the four fractions from jujube
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continuously decreased from 2123.72µg/g DW to 1356.80 µg/g DW as the fruits
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matured. The total detected phenolic acid and flavonoid contents decreased by 38.7%
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and 30.1% from WM to HM stage, respectively, and then slightly decreased (by 9.8%
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and 8.6%) as the fruit ripening progressed. Moreover, both contents were almost
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equal at any maturity stage.
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The level of total individual phenolic compound contents in jujube at different
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maturity stages was calculated as a sum of individual phenolic compound present in
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all four fractions. Apparently rutin was the dominant phenolic compound in jujube at
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any maturity stage, comprising 44.5%, 51.2% and 51.9% of the total phenols at WM,
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HM and RM stages, respectively (Fig.2). Caffeic and chlorogenic acids were the
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principal phenolic compounds in jujube with 31.3% and 30.8% of the total phenols at
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WM and RM stages, respectively, while gallic acid was also in a large quantity at HM
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stage.
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3.4 DPPH Radical Scavenging Activity
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The scavenging capabilities against DPPH radicals of the four phenolic fractions from
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jujube at three maturity stages are presented in Fig.3. Overall, during the maturity process the DPPH radical scavenging activities decreased from WM to RM stage,
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with very high scavenging activity for F1 and F4 at each maturity stage and low
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activity in F3. The F1 and F4 at WM stage, F1 at HM stage and F2 at RM stage
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showed the highest scavenging activity (about 90%) in the all phenolic fractions, in
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spite of low TPC and TFC in these fractions except for F1 at WM stage. The results
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might be due to other phytochemicals with high scavenging activity in the fractions.
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An unknown peak in the fractions (not shown) was found and considered as one such
334
compound responsible for high scavenging activity, which needs to further
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investigated. Similar results of decrease in DPPH radical scavenging activity with the
336
increasing maturity stage were also reported by others (Lu et al., 2012; Zozio et al.,
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2014).
338
3.5 Antioxidant Activity
339
The antioxidant activities of the four phenolic fractions from jujube at the three
340
ripening stages were quantified by FRAP assay, as shown in Fig. 4. The FRAP values
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in the four fractions mostly displayed the same trend similar to TPC and decreased
342
from WM to RM stage, which was in accordance with the results reported previously
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(Wu et al., 2012; Zozio et al., 2014; Choi et al., 2012). The sharp increase in FRAP
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values observed in F4 at HM stage might be due to the increased content of other
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antioxidants at this maturity stage. It was also found that F4 showed higher
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antioxidant activities than F2 and F3 because they had more total phenols, total
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flavonoids as well as phenolic compounds. These results suggested that the insoluble-bound phenolics present in the residues of jujube (F4) constituted an
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important portion of the phenolic compounds in jujube with non-negligible
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antioxidant capacity. In our previous study (Wang et al., 2011), F4 in jujube pulp was
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also found to be the strongest antioxidant activity by DPPH and FRAP assays.
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Therefore, the composition of phenolic compounds of this fraction needs to be
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investigated further, which might result in the identification of some new antioxidant
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components with high bioactivities.
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3.6 Correlations
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The correlations between the antioxidant activities measured by DPPH and FRAP
357
assays and the TPC, TFC and sum of phenolic contents by HPLC of each fraction are
358
reported in Table 4 along with Pearson product-moment correlation coefficients (r)
359
and p values. The data from Table 4 were used to explore the relationship of phenolics
360
in each fraction from jujube at different maturity stages and their antioxidant activities.
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The DPPH radical scavenging activity was correlated significantly with TFC and the
362
antioxidant activity measured by FRAP assay (p < 0.05), but not significantly with the
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TPC and the sum of detected phenolic content by HPLC (p > 0.05). The FRAP value
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was significantly and positively correlated to the TPC and TFC (p < 0.05 and p < 0.01,
365
respectively), suggesting that these phenolic compounds might be responsible for a
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large proportion of the antioxidant activity. The results are well in agreement with
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previously reported findings (Xue et al., 2009; Zhang et al., 2010; Choi et al., 2011;
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Wang et al., 2011). Moreover, the TPC had significant and positive correlation (p < 0.01) with the TFC. This indicated that the flavonoids might be responsible for the
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most of the phenolics in the samples. However, the total detected phenolic content by
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HPLC was not significantly correlated with all others (p > 0.05).
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4 Conclusions
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The data obtained from this study suggested that there were great changes in TPC,
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TFC, individual phenolics, and antioxidant activity of free, esterified, glycosided, and
375
insoluble-bound phenolic fractions of jujube during its maturity. Both TPC and TFC
376
decreased with the increasing maturity from WM to RM stage. All quantified
377
phenolic acids in the four fractions were mainly in jujube at WM stage and greatly
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decreased with the increase of maturity; while rutin was mostly present at HM and
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RM stages. The antioxidant activity determined by FRAP was significantly correlated
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with TPC, TFC and DPPH radical scavenging activity. The phenolic fractions at WM
381
stage exhibited the highest antioxidant activity. In addition, the insoluble-bound
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phenolic fractions from jujube contained a large number of phenolic compounds and
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showed high activities and therefore they should not be ignored. Consequently, WM
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stage was considered as a proper harvesting period for jujube to have high antioxidant
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ingredients and for development of potential natural antioxidants. But further studies
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are still needed to identify the unknown antioxidant substances and the major phenolic
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compounds present in jujube at different maturities.
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Acknowledgements
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This work is financially supported by the National Natural Science Foundation of China (31101325) and the Science & Technology Project of Shaanxi Province (2012K02-06).
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FIGURE CAPTIONS:
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Fig. 1 Phenolic compound contents in four fractions in jujube at three maturity stages.
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Fig. 2 Total contents of individual phenolic compound in four fractions from jujube at
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three maturity stages.
Fig. 3 DPPH radical scavenging activities of each phenolic fraction from jujube at three maturity stages.
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Fig. 4 FRAP values of each phenolic fraction from jujube at three maturity stages.
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Fig. 1 Phenolic compound contents in four fractions of jujube at three maturity stages.
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Fig. 2 Total contents of individual phenolic compounds in four fractions of jujube at
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three maturity stages.
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Fig. 3 DPPH radical scavenging activities of four phenolic fractions from jujube at three maturity stages.
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Different lower case letters correspond to significant differences for maturity stages in one group
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at p < 0.05.
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Fig. 4 FRAP values of four phenolic fractions of jujube at three maturity stages.
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Different lower case letters correspond to significant differences for maturity stages in one group
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at p < 0.05.
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Table 1 TPC (mg GAE/100g DW) in four phenolic fractions of jujube during maturity Phenolic fractions Maturity stages
Esterified (F2)
Glycosided (F3)
Insoluble-bound (F4)
White maturity (WM)
864.73± 3.27a
190.30 ± 2.44a
197.83 ± 5.92a
262.49 ± 5.22a
Half-red maturity (HM)
259.00± 7.14b
75.31 ± 3.11b
122.11 ± 6.79b
152.83 ± 3.06b
Red maturity (RM)
71.67 ± 1.99c
105.13 ± 5.67b
82.78 ± 6.16c
103.10 ± 2.81c
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Free (F1)
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Table 2 TFC (mg Rutin/100 g DW) in four phenolic fractions of jujube during maturity Phenolic fractions
Maturity stages
F1
White maturity (WM)
F2
1002.00± 10.60
a
274.94 ±15.46
F3 a
F4
167.23 ± 22.97
a
248.49 ± 6.27a
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Half-red maturity (HM)
956.39± 9.17b
125.72 ± 7.97b
137.88 ± 14.10a
258.69 ± 6.90a
Red maturity (RM)
167.45 ±18.79c
77.42 ± 11.93c
61.05±7.31b
177.56 ± 7.63b
Different lower case letters correspond to significant differences at p < 0.05.
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Table 3 Composition of phenolic compounds in jujube during maturity (µg/g DW) Phenolics
White Maturity F1
F2
F3
Gallic
0.00
0.00
198.66
Protocatechuic
6.57
7.89
5.37
p-hydroxybenzoic
71.35
0.90
7.21
Caffeic
Half-red Maturity F4
Red Maturity
F1
F2
F3
F4
0.00
0.00
51.57
207.86
8.41
7.30
8.22
6.89
21.01
2.16
4.41
12.54
1.36
2.60
5.39
F1
F2
F3
F4
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Phenolic acids 0.00
0.00
0.00
0.00
9.72
1.62
10.89
3.18
0.14
0.34
2.83
1.29
10.18
1.05
15.56
138.68
1.82
0.93
5.61
0.00
34.70
7.64
16.94
18.08
10.64
7.55
19.10
Ferulic
0.00
0.70
6.29
0.00
0.00
1.86
8.85
0.00
Chlorogenic
47.55
15.42
21.39
10.58
8.01
100.00
39.52
1.26
366.53
Rosmarinic acid
0.00
0.00
0.63
0.03
0.00
0.00
6.62
0.00
0.00
Ellagic acid
9.11
3.56
5.94
18.04
6.93
0.00
13.08
2.06
772.46
73.33
254.18
72.86
192.46
174.14
308.03
925.83
17.73
0.00
0.86
602.04
101.74
51.14
Flavonoids Rutin Quercetin ∑TP
9.52
2.87
17.52
0.00
2.50
5.23
32.92
41.84
1.65
0.00
6.33
10.42
15.15
26.27
0.00
0.90
0.07
0.00
0.00
1.51
0.00
43.99
495.03
26.06
39.38
87.58
4.79
670.63
20.59
8.07
4.71
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∑TPA
76.79
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637.88
p-coumaric
0.00
0.63
0.00
0.00
0.00
0.04
6.71
0.00
0.00
0.12
4.63
0.00
1698.29
91.69
260.02
73.72
794.50
275.92
365.88
48.78
1165.66
46.77
52.08
92.29
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Table 4 Correlation coefficients (r) of phenolics and antioxidant activity TFC 0.799**
TPC TFC DPPH FRAP ** p < 0.01; *p < 0.05.
DPPH 0.565 0.654*
HPLC 0.336 0.536 0.180 0.431
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FRAP 0.871* 0.836** 0.581*
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Highlights: 1. The phenolic composition in jujube and the antioxidant activities changed during
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three edible maturity stages. 2. The phenolic contents and their activities greatly decreased with the increasing maturity stage.
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3. Hydrolyzing extracted insoluble-bound phenolics cannot be ignored.
S0023-6438(15)30223-1
DOI:
10.1016/j.lwt.2015.10.005
Reference:
YFSTL 5000
To appear in:
LWT - Food Science and Technology
Received Date: 10 April 2015 Revised Date:
9 September 2015
Accepted Date: 2 October 2015
Please cite this article as: Wang, B., Huang, Q., Venkitasamy, C., Chai, H., Gao, H., Cheng, N., Cao, W., Lv, X., Pan, Z., Changes in phenolic compounds and their antioxidant capacities in jujube (Ziziphus jujuba Miller) during three edible maturity stages, LWT - Food Science and Technology (2015), doi: 10.1016/j.lwt.2015.10.005. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Changes in phenolic compounds and their antioxidant capacities in jujube (Ziziphus
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jujuba Miller) during three edible maturity stages
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Bini Wang a, b, *, Qingyuan Huang a, Chandrasekar Venkitasamy b, Hongkang Chai a, Hui Gao a, Ni
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Cheng a, Wei Cao a, Xingang Lv a, Zhongli Pan b, c, **
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a
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Northwest University, Xi’an, Shaanxi 710069, China
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b
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Davis, One Shields Avenue, Davis, CA 95616, USA
Department of Food Science and Engineering, College of Chemical Engineering,
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c
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Albany, CA 94710, USA
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Department of Biological and Agricultural Engineering, University of California,
Healthy Processed Foods Research Unit, USDA-ARS-WRRC, 800 Buchanan St.,
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Abstract: This study investigated the changes in total phenolic content (TPC), total
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flavonoid content (TFC), individual phenolic compound content, DPPH radical
15
scavenging activity and antioxidant capacity measured by FRAP assay of four
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phenolic fractions (free, esterified, glycosided and insoluble-bound) from jujube
17
during three edible maturity stages. The maturity stages of jujubes were established as
18
white maturity (WM), half-red maturity (HM) and red maturity (RM). The free
19
fraction in jujube at WM stage had the highest TPC, TFC, total phenolic acid contents,
20
and antioxidant capacities. The phenolic contents and their activities greatly decreased
21
with the increasing maturity stage. Caffeic acid was the most predominant in all
22
detected phenolic compounds at WM stage, while rutin dominated at HM and RM
23
stages. Even though most of phenolic compounds with antioxidant activity in jujube
24
existed at the WM stage as the free form, the insoluble-bound fractions also contained
25
a large number of phenolic compounds.
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Chemical compounds studied in this article
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Gallic acid (PubChem CID:370); Protocatechuic acid (PubChem CID:72);
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p-Hydroxybenzonic acid (PubChem CID:135); Chlorogenic acid (PubChem
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CID:1794427); Caffeic acid (PubChem CID:689043); p-Coumaric acid (PubChem
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CID:637542); Ferulic acid (PubChem CID: 445858); Rosmarinic acid (PubChem CID:5281792); Ellagic acid (PubChem CID:5281855); Quercetin (PubChem CID:5280343); Syringic acid (PubChem CID: 10742); Hesperetin (PubChem
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CID:72281);
Rutin
(PubChem CID:5280805)
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Key words: Ziziphus, Insoluble-bound phenolic, Antioxidant activity, Maturity stage 2
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1. Introduction
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Jujube is the fruit of Ziziphus jujuba Miller, a thorny rhamnaceous plant widely
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cultivated in subtropical and tropical regions of Asia, especially in China, America
40
and Europe. China is the largest producer contributing over 90% of the world jujube
41
production and the only country exporting jujube fruits. The total annual yield of fresh
42
jujube fruits in 2009 was 600 million kilograms (Lu et al., 2012) and has been
43
increasing in the recent years in China. Globally, jujube is popularly consumed as
44
fresh or dried fruit and has been used to prepare compotes, jams, beverages and cakes.
45
The color of jujube peel changes from green to yellow, then to reddish and finally to
46
red during maturation. These peel colors represent maturity stages typically called
47
green fruit stage, white maturity (WM), half-red maturity (HM), and red maturity
48
(RM) stage, respectively. The fruit is unsuitable for eating or processing until it ripens
49
to WM stage. Production of various processed products prefers jujube at a specific
50
maturity stage. For example, fresh consumption or most processed products require
51
jujube at HM or RM stage, dried products require jujube at RM stage and processing
52
into saccades needs jujube at WM stage. There is an increased interest in the potential
53
health benefits of jujube to human beings. An ancient Chinese book on herbal
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medicine named Huangdi Neijing (475−221 BC) showed that jujube was one of five most valuable fruits (peach, pear, apricot, plum, and jujube) in China. It has a high
56
nutritional value and potential health benefits including antioxidant activity (Gao, Wu,
57
& Wang, 2013). It has been widely used as food, a functional food additive, and a
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traditional Chinese medicine for several thousands of years and attracted research on
59
its bioactive substances, such as phenolic compounds (Du et al., 2013; Chen et al.,
60
2013; Choi et al., 2011; Wang et al., 2010).
61
Phenolic compounds are secondary metabolites of plants that play an important role in
62
the pigmentation, growth, reproduction of plants as well as plant resistance to
63
pathogens (Ghasemzadeh & Ghasemzadeh, 2011). Their content in fruit is largely
64
affected by genotype (cultivar), pre-harvest environmental conditions, post-harvest
65
storage conditions, processing and the degree of maturity at harvest (Shahidi & Naczk,
66
2004). During fruit maturation, the phenolic compounds undergo a series of complex
67
biosynthesis process, leading to the changes in their composition and content in plant
68
and plant-derived foods (Prasanna, Prabha, & Tharanathan, 2007). They have
69
received increased attention due to their potent antioxidant capacities and their
70
remarkable health benefits in the prevention of various oxidative stresses associated
71
diseases, such as cancer, cellular aging, cardiovascular diseases and inflammation
72
(Dai & Mumper, 2010). Several studies revealed that the phenolic compounds and
73
their bioactivities in jujube could be influenced by its maturity (Wu et al., 2012; Chen
74
et al., 2013; Choi et al., 2012). Those studies evaluated the phenolic compounds in
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soluble extracts alone and ignored the insoluble-bound ones in residues, leading to the underestimation of real phenolic content of jujube and their corresponding antioxidant
77
activities. In general, the phenolic compounds in food are classified as soluble
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(extractable) and insoluble (non-extractable) compounds based on the location of
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phenolic compounds in the plant together with the chemical structure of these
80
substances (Reis Giada, 2013). The former can typically be extracted by organic
81
solvents, while the latter is bound to cell wall polysaccharides or proteins forming
82
insoluble stable complexes. Interestingly, these insoluble phenolics are also very
83
important in health effects from the nutritional viewpoint and they exert their
84
antioxidant effects to protect the body against oxidative stress (Liyana-Pathirana &
85
Shahidi, 2006; Pérez-Jiménez & Torres, 2011). Though the insoluble phenolic
86
compounds cannot be extracted by organic solvents, they may be released from the
87
complexes by the action of intestinal enzymes or colonic microbiota and thereby
88
transformed into small phenolics and metabolites that are subsequently absorbed (Jara
89
& Josep, 2011). Therefore, the insoluble compounds should also be well studied.
90
In our previous study, we found that the major fraction of phenolic acids in different
91
tissues of jujube is insoluble-bound (Wang et al., 2011). However, there is no
92
information available related to the effect of maturity stages on the soluble and
93
insoluble phenolic compounds of jujube and their antioxidant capacities, which is
94
important to ensure that jujube is harvested at right maturity with high antioxidants
95
content. Furthermore, producers and industrialists also need the valuable information
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on bioactive compounds in jujube and their antioxidant activities at different maturity stages for marketing purpose. Thus, the aim of the present study was to evaluate the
98
changes in phenolic compounds (in free, esterified, glycosided and insoluble-bound
99
forms) and their antioxidant activities of jujube at three edible maturity stages. The
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results of this study could lead to identification of the optimal maturity stage for the
101
harvest of jujube with high composition of polyphenols targeting increased
102
antioxidant activities.
103
2. Materials and Methods
104
2.1 Samples of Jujube Fruits
105
Fresh jujubes (Ziziphus jujube cv. Jishanbanzao) used in this study were obtained
106
from a local farm in Jishan County, southwestern part of Shanxi Province, North
107
China. No pesticide was used in the jujube crop. Jujube samples were harvested from
108
July 25th to October 5th of 2014 at three edible stages of maturity determined based
109
on the surface color. The three maturity stages were established as white maturity
110
(WM), yellow skin color; half-red maturity (HM), representing jujube with red
111
surface area of 40%-60%; and red maturity (RM), having 100% red surface area.
112
Jujubes were picked up randomly from different parts of several trees of the same
113
species and were free from visible blemishes and disease. Jujubes were frozen and
114
stored in airtight polyethylene bags at -18oC in a freezer until they were analyzed.
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2.2 Chemicals
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Gallic acid, protocatechuic acid, p-hydroxybenzoic acid, syringate, caffeic acid,
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p-coumaric acid, ferulic acid, chlorogenic acid, rosmarinic acid, ellagic acid, rutin, hesperetin, 2,2-diphenyl-1-picryl-hydrazyl (DPPH), and 2,4,6-tripyridyl-s-triazine
119
(TPTZ) were purchased from Sigma-Aldrich (Steinheim, Germany). All other
120
chemicals were also analytical grade and were obtained from Xi’an Chemical Co.
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(Xi’an, China). HPLC grade methanol was purchased from Merck (Darmstadt,
122
Germany). Analytical grade acetic acid was supplied by Beijing Reagent Co. Ltd
123
(Beijing, China) and HPLC grade water was purified by Milli-Q system (Millipore
124
Bedford, MA, USA).
125
2.3 Preparation of Crude Jujube Extracts
126
The frozen jujube fruits were thawed and cleaned with tap water. The seeds were
127
removed from jujubes and the edible portion was homogenized in a blender for 1 min.
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A sample of 100g of homogenized jujube was lyophilized, milled and sieved through
129
a standard sieve of 100 mesh. The powdered samples (2 g) of jujube at different
130
maturity stages were extracted with 15 mL of 80 % (v/v) aqueous methanol at room
131
temperature. The solution was sonicated for 30 min and then centrifuged at 2000g for
132
10 min to collect supernatant. The supernatant extraction was repeated for three times
133
and the accumulated supernatants were used for the fractionation of free phenolic
134
compounds, soluble glycosides and esters of phenolic compounds. The residue left
135
after the extraction of supernatant was saved for the determination of insoluble -bound
136
phenolic compounds.
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2.4 Fractionation of Free and Bound Phenolic Compounds
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Phenolic compounds in crude extracts were fractionated into free and bound forms following our previously established methods (Wang et al., 2011) as described below.
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The crude extract (supernatants) obtained after the methanol extraction from jujube as
141
described in the previous step was evaporated under vacuum at 35 oC to about 10 mL.
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The aqueous suspension was acidified to pH 2 using 6 M hydrochloric acid (HCl),
143
and extracted for five times with ethyl acetate at a solvent to water phase ratio of 1: l.
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The ethyl acetate extracts were referred to as the free phenolic compounds (F1). The
145
aqueous phase remained after the ethyl acetate extraction was treated by alkaline
146
hydrolysis
147
ethylenediaminetetraacetic acid (EDTA) and 1% ascorbic acid) under nitrogen for 4 h
148
at room temperature. After acidification to pH 2 with 6 M HCl, phenolic compounds
149
(F2) released from soluble esters were extracted from the hydrolysate for five times
150
using the procedure as described above. Following this, the aqueous phase remained
151
after the F2 separation was hydrolyzed with 5 mL 6M HCl for 30 min at 85 oC under
152
nitrogen. Phenolic compounds (F3) released from soluble glycosides were separated
153
from the hydrolysate for five times following the procedure as described above. The
154
residues from the 80% methanol extractions were hydrolyzed directly with 8 mL of 4
155
M NaOH (containing 10 mM EDTA and 1% ascorbic acid) under the same conditions
156
as the ester. After acidification to pH 2 using 6 M HCl, phenolic compounds (F4)
157
released from methanol-insoluble ester-bound phenolics were extracted from the
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hydrolysate for five times as described above. Extraction was done for triplicate
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sodium
hydroxide
(NaOH)
containing
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samples. Each of the phenolic fractions, obtained as described above, was dehydrated with anhydrous sodium sulfate, filtered, and evaporated to dryness under vacuum at 35 oC. The dry residues were dissolved into 5 mL of methanol, and these solutions
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were used for the determination of phenolic contents and antioxidant activities as
163
described below.
164
2.5 Determination of Total Phenolic Contents
165
Total phenolic contents (TPC) in each fraction of jujube extract were determined
166
according to a previously described laboratory procedure of the modified
167
Folin-Ciocalteu colorimetric method (Wang et al., 2011). TPC was evaluated at 760
168
nm by adding Folin-Ciocalteu reagent to the sample. The average value of triplicate
169
data was expressed as the gallic acid equivalents in mg per 100 gram dry weight (mg
170
GAE/100 g DW).
171
2.6 Determination of Total Flavonoids Content
172
Total flavonoids content (TFC) in each fraction of jujube extract was measured
173
colorimetrically at 510 nm following a previously reported method (Jia et al., 1999).
174
TFC was expressed as rutin equivalents in mg per 100 gram dry weight (mg RE /100
175
g DW). The absorbance was measured for triplicate samples.
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2.7 HPLC-ECD Analysis
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The phenolic compounds in the four phenolic fractions from jujube were separated
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and quantified using a HPLC fitted with an electrochemical detector (ECD) as
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described by Wang et al. (2011) with minor revision. HPLC analysis of phenolic compounds were carried out using an Agilent 1100 HPLC System (Agilent, USA)
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equipped with a vacuum degasser, a quaternary solvent delivery pump, a manual
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chromatographic valve, a thermostated column compartment, and a HP1049A
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programmable ECD (HP, USA). A Zorbax SB-C18 column (150 ×4.6 mm, 5.0 µm)
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connected with a Zorbax SB-C18 guard column (20 × 4.0 mm, 5 µm). The mobile
185
phase adopted was methanol (A) and 0.15 % aqueous formic acid (B) (v/v) using a
186
linear gradient elution of 5-8 % A at 0-6 min, 8-15 % A at 6-10 min, 15-35 % A at
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10-15 min, 35-55 % A at 15-20 min, 55-65 % A at 20-25 min and 65-80 % A at 25-30
188
min. The flow-rate was kept at 1.0 mL min-1 at all times. The column was operated at
189
30 oC and the injection volume was 10.0 µL. The electrochemical detector was set at
190
800 mV in the oxidative mode. Re-equilibration duration was set as 6 min by using
191
the starting condition before injection of the next individual sample. Quantification of
192
phenolic acids was carried out by an external standard method using calibration
193
curves. The amount of each phenolic acid was expressed as microgram per gram dry
194
weight (µg/g DW).
195
2.8 Radical DPPH Scavenging Activity
196
Scavenging activity on DPPH free radicals by each phenolic acid fraction was
197
assessed according to the method reported by Wang et al. (2011).
198
2.9 Ferric Reducing Antioxidant Power (FRAP)
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FRAP assay of each sample was performed following a previously described
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laboratory procedure (Wang et al., 2011). 2.10 Statistical Analysis
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The results presented in the tables are the mean value±SD (standard deviation) from
203
three replicates. Data analysis was carried out using SAS software, version 8.1.
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Statistically significant difference between the samples was evaluated by the Tukey’s
205
test. Difference at p < 0.05 was considered to be significant. The correlation analysis
206
between phenolics and antioxidant activity was made using standard Pearson
207
correlation.
208
3. Results and Discussion
209
3.1 Total Phenolics of Jujube at Three Edible Maturity Stages
210
The TPCs in four phenolic fractions of jujube at three edible maturity stages are
211
shown in Table 1. The maturity stages had a significant influence on the TPCs in
212
jujube. The TPC in each phenolic fraction significantly decreased (p < 0.05) with the
213
increase in maturity stages. All fractions extracted from WM stage were observed to
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have several folds (1.8-12 folds) higher amounts of TPC than those at RM stage. The
215
sum of TPC in four fractions of jujube sharply decreased from 1515.35 mg GAE/100
216
g DW at WM stage to 362.68 mg GAE/100g DW at RM stage (p < 0.05). Previous
217
studies (Wu et al., 2012; Zozio et al., 2014) reported only the TPC values of the
218
soluble extracts obtained from jujube during ripening and ignored the TPC of
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insoluble extracts. The TPC of the soluble extracts decreased with maturity stage,
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which was in agreement with those reported previously for jujube and other fruits
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(Zozio et al., 2014; Wang et al., 2013; Wu et al., 2012; Kondo et al., 2002; Gruz et al., 2011). However, the quantities of TPC obtained from this study were lower than the
223
previously published results (Wang et al., 2011; Wu et al., 2012). This might be
224
mainly attributed to the differences in the cultivar and sources of the materials, as well
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as the regional differences (Gull et al., 2012). In the four phenolic fractions, F1 was
226
the major fraction constituting 57.1% and 42.5% of the sum of the TPC at WM and
227
HM stages of jujube, respectively. The F2 and F4 dominated at RM stage, comprising
228
29.0% and 28.4% of TPC, respectively. These results indicated that the TPC was
229
clearly dependent on maturity stages, and the maturation process increased the bound
230
phenolics content.
231
3.2 Total Flavonoids in Jujube at Three Maturity Stages
232
Most previous studies revealed that jujube contained considerable amounts of
233
flavonoids including rutin (Pawlowska et al., 2009; Zhang et al., 2010; Wu et al.,
234
2012; Du et al., 2013; Gao et al., 2012), and mainly examined TFC in ethanol or
235
methanol extracts of jujube. Flavonoids has also been found in the insoluble
236
(non-extractable) fraction and associated with dietary fiber in tomato peel and roselle
237
tea (Arranz et al., 2010; Kapasakalidis, Rastall, & Gordon, 2009; Navarro-Gonzalez et
238
al., 2011; Sáyago-Ayerdi et al., 2007). Little information has been reported on TFC in
239
the insoluble fraction in jujube.
240
In the present study, the TFC in soluble and insoluble fractions of jujube at three
241
maturity stages were determined (Table 2). Results showed that the TFC in all four
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phenolic fractions of jujube rapidly decreased with the progress of maturity. The rapidest decrease in TFC occurred for F1, and the TFC was significantly reduced by 6
244
folds from WM to RM stage (p < 0.05). The sum of TFC in four fractions exhibited a
245
continuously decreasing trend similar to TPC and decreased from 1692.66 mg RE/100
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g DW at WM stage to 483.47 mg RE/100 g DW at RM stage, which was consistent
247
with those reported for pear-jujube (Wu et al., 2012). The TFC values obtained for the
248
soluble fraction of jujube (422.43 -1525.43 mg RE/ 100 g DW) were within the
249
ranges reported by Zhang et al. (2010), from 276.43 to 1851.96 mg QE/ 100 g DW
250
and by Gao et al. (2012), from 62.0 to 284.9 mg RE/ 100 g FW in ethanol or methanol
251
extracts of different jujube cultivars. With the increase of jujube maturity, the
252
percentage of TFC in F1 continuously decreased from 65.7% to 34.6%, while the TFC
253
in F4 continuously increased from 16.3% to 36.7%. As jujube became fully ripen
254
(RM stage), F1 and F4 were the major fractions constituting 34.6% and 36.9% of the
255
sum of TFC, respectively. Therefore, the TFC in the insoluble fraction (F4) cannot be
256
ignored, and further research is required to identify the association or binding of these
257
flavonoids with other compounds of jujube.
258
3.3 Individual Phenolic Compounds
259
Thirteen phenolic compounds, including hydroxybenzoic acids (gallic, protocatechuic,
260
p-hydroxybenzoic and syringate), hydroxycinnamic acids (caffeic, p-coumaric, and
261
ferulic), other phenolic acids (chlorogenic, rosmarinic, and ellagic) and flavonoid
262
(rutin, quercetin and hesperetin) were well separated and quantified by HPLC-ECD.
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But syringate and hesperetin were not found in jujube at all three maturity stages. The distribution of the phenolic compounds in the four fractions obtained from jujube at
265
the three maturity stages is presented in Table 3. The results showed that free phenolic
266
compounds were the most abundant at all of the maturity stages.
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Free phenolic compounds comprised of 80.0%, 53.5% and 85.9% of the total phenolic
268
compounds in jujube at WM, HW and RW, respectively (Fig.1). The content of free
269
phenolic compounds in jujube was much higher at WM (1698.29 µg/g DW) than
270
those at HM and RM stages (794.50 and 1165.66 µg/g DW, respectively). Rutin was
271
reported as a common component in jujube extracts (Pawlowska et al., 2009), and was
272
the most predominant phenolic compound at any maturity stages in this work. The
273
levels of rutin decreased from 925.83 µg/g DW (WM) to 602.04 µg/g DW (HM), and
274
then increased to 670.63 µg/g DW (RM). This change is consistent with the results
275
reported by Wu et al. (2012), but the obtained values in this research were more than
276
10 times higher. Choi et al. (2012) also found rutin content continuously decreased as
277
the fruits matured, and epicatechin was the most prevalent flavonoid during the
278
maturity stages which was not observed in our study. Caffeic acid was the dominant
279
phenolic compounds at WM stage, while chlorogenic acid, an ester of caffeic acid,
280
was mostly present in the free form at RM stage. The p-coumaric acid was detected
281
only at HM stage and ferulic acid was detected only at RM stage. In addition,
282
quercetin, gallic and rosmarinic acids were not detected at any maturity stages.
283
Phenolic compounds released from soluble esters constituted from 3.4% (at RM) to
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18.6% (at HM) of the total quantified phenolic compounds in jujube (Fig.1). At HM stage, the total content of phenolic compounds released from soluble esters was
286
275.92 µg/g DW, which was 2-fold higher than that at WM stage and 5-fold higher
287
than that at RM stage. The p-coumaric acid was the principal phenolic presented as
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soluble esters at WM stage, whereas rutin dominated at RM stage. Each of rutin and
289
chlorogenic acid constituted for more than 36% of the total phenolic compounds
290
content at HM stage. However, gallic and rosmarinic acids were not detected in this
291
fraction at any maturity stages.
292
Glycosided phenolic compounds ranged from 3.8% (at RM) to 24.6% (at HM) of the
293
total phenolic compounds presented in jujube (Fig.1). The total contents of glycosided
294
phenolics in jujube at WM and HM stage were 260.02 µg/g DW and 365.88 µg/g DW,
295
respectively, which were about 5-fold and 7-fold higher than that at RM (52.08 µg/g
296
DW), respectively. Glycosided gallic acid presented mostly at WM stage and HM
297
stage and was not detected at RM stage. The percentage of glycosided gallic acid at
298
WM and HM were 76.4% and 56.8% of the total glycosided phenolics, respectively.
299
Though chlorogenic and protocatechuic acids were the principal phenolics at RM,
300
they were at very low levels of 15.15 µg/g DW and 10.89 µg/g DW, respectively.
301
The insoluble-bound phenolic compounds constituted 3.5%, 3.3%, and 6.8% of total
302
phenolics in jujube at WM, HW and RW stages, respectively (Fig.1). Insoluble-bound
303
p-coumaric acid was one of the major phenolic compounds in jujube found at the
304
three maturity stages, and represented 23.0%, 39.1% and 35.7% of the total
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insoluble-bound phenolics at WM, HM and RM stages, respectively. Furthermore, ellagic acid mainly presented in insoluble-bound form and caffeic acid was the major
307
phenolic compounds at WM stage, while p-coumaric and chlorogenic acids
308
dominated this fraction at RM stage.
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The total content of quantified phenolic compounds in the four fractions from jujube
310
continuously decreased from 2123.72µg/g DW to 1356.80 µg/g DW as the fruits
311
matured. The total detected phenolic acid and flavonoid contents decreased by 38.7%
312
and 30.1% from WM to HM stage, respectively, and then slightly decreased (by 9.8%
313
and 8.6%) as the fruit ripening progressed. Moreover, both contents were almost
314
equal at any maturity stage.
315
The level of total individual phenolic compound contents in jujube at different
316
maturity stages was calculated as a sum of individual phenolic compound present in
317
all four fractions. Apparently rutin was the dominant phenolic compound in jujube at
318
any maturity stage, comprising 44.5%, 51.2% and 51.9% of the total phenols at WM,
319
HM and RM stages, respectively (Fig.2). Caffeic and chlorogenic acids were the
320
principal phenolic compounds in jujube with 31.3% and 30.8% of the total phenols at
321
WM and RM stages, respectively, while gallic acid was also in a large quantity at HM
322
stage.
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3.4 DPPH Radical Scavenging Activity
325
The scavenging capabilities against DPPH radicals of the four phenolic fractions from
326 327
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jujube at three maturity stages are presented in Fig.3. Overall, during the maturity process the DPPH radical scavenging activities decreased from WM to RM stage,
328
with very high scavenging activity for F1 and F4 at each maturity stage and low
329
activity in F3. The F1 and F4 at WM stage, F1 at HM stage and F2 at RM stage
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showed the highest scavenging activity (about 90%) in the all phenolic fractions, in
331
spite of low TPC and TFC in these fractions except for F1 at WM stage. The results
332
might be due to other phytochemicals with high scavenging activity in the fractions.
333
An unknown peak in the fractions (not shown) was found and considered as one such
334
compound responsible for high scavenging activity, which needs to further
335
investigated. Similar results of decrease in DPPH radical scavenging activity with the
336
increasing maturity stage were also reported by others (Lu et al., 2012; Zozio et al.,
337
2014).
338
3.5 Antioxidant Activity
339
The antioxidant activities of the four phenolic fractions from jujube at the three
340
ripening stages were quantified by FRAP assay, as shown in Fig. 4. The FRAP values
341
in the four fractions mostly displayed the same trend similar to TPC and decreased
342
from WM to RM stage, which was in accordance with the results reported previously
343
(Wu et al., 2012; Zozio et al., 2014; Choi et al., 2012). The sharp increase in FRAP
344
values observed in F4 at HM stage might be due to the increased content of other
345
antioxidants at this maturity stage. It was also found that F4 showed higher
346
antioxidant activities than F2 and F3 because they had more total phenols, total
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flavonoids as well as phenolic compounds. These results suggested that the insoluble-bound phenolics present in the residues of jujube (F4) constituted an
349
important portion of the phenolic compounds in jujube with non-negligible
350
antioxidant capacity. In our previous study (Wang et al., 2011), F4 in jujube pulp was
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also found to be the strongest antioxidant activity by DPPH and FRAP assays.
352
Therefore, the composition of phenolic compounds of this fraction needs to be
353
investigated further, which might result in the identification of some new antioxidant
354
components with high bioactivities.
355
3.6 Correlations
356
The correlations between the antioxidant activities measured by DPPH and FRAP
357
assays and the TPC, TFC and sum of phenolic contents by HPLC of each fraction are
358
reported in Table 4 along with Pearson product-moment correlation coefficients (r)
359
and p values. The data from Table 4 were used to explore the relationship of phenolics
360
in each fraction from jujube at different maturity stages and their antioxidant activities.
361
The DPPH radical scavenging activity was correlated significantly with TFC and the
362
antioxidant activity measured by FRAP assay (p < 0.05), but not significantly with the
363
TPC and the sum of detected phenolic content by HPLC (p > 0.05). The FRAP value
364
was significantly and positively correlated to the TPC and TFC (p < 0.05 and p < 0.01,
365
respectively), suggesting that these phenolic compounds might be responsible for a
366
large proportion of the antioxidant activity. The results are well in agreement with
367
previously reported findings (Xue et al., 2009; Zhang et al., 2010; Choi et al., 2011;
369
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Wang et al., 2011). Moreover, the TPC had significant and positive correlation (p < 0.01) with the TFC. This indicated that the flavonoids might be responsible for the
370
most of the phenolics in the samples. However, the total detected phenolic content by
371
HPLC was not significantly correlated with all others (p > 0.05).
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4 Conclusions
373
The data obtained from this study suggested that there were great changes in TPC,
374
TFC, individual phenolics, and antioxidant activity of free, esterified, glycosided, and
375
insoluble-bound phenolic fractions of jujube during its maturity. Both TPC and TFC
376
decreased with the increasing maturity from WM to RM stage. All quantified
377
phenolic acids in the four fractions were mainly in jujube at WM stage and greatly
378
decreased with the increase of maturity; while rutin was mostly present at HM and
379
RM stages. The antioxidant activity determined by FRAP was significantly correlated
380
with TPC, TFC and DPPH radical scavenging activity. The phenolic fractions at WM
381
stage exhibited the highest antioxidant activity. In addition, the insoluble-bound
382
phenolic fractions from jujube contained a large number of phenolic compounds and
383
showed high activities and therefore they should not be ignored. Consequently, WM
384
stage was considered as a proper harvesting period for jujube to have high antioxidant
385
ingredients and for development of potential natural antioxidants. But further studies
386
are still needed to identify the unknown antioxidant substances and the major phenolic
387
compounds present in jujube at different maturities.
388
Acknowledgements
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This work is financially supported by the National Natural Science Foundation of China (31101325) and the Science & Technology Project of Shaanxi Province (2012K02-06).
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References
394
Arranz, S., Silván, J. M., & Saura-Calixto, F. (2010). Nonextractable polyphenols,
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FIGURE CAPTIONS:
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Fig. 1 Phenolic compound contents in four fractions in jujube at three maturity stages.
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Fig. 2 Total contents of individual phenolic compound in four fractions from jujube at
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three maturity stages.
Fig. 3 DPPH radical scavenging activities of each phenolic fraction from jujube at three maturity stages.
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Fig. 4 FRAP values of each phenolic fraction from jujube at three maturity stages.
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Fig. 1 Phenolic compound contents in four fractions of jujube at three maturity stages.
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Fig. 2 Total contents of individual phenolic compounds in four fractions of jujube at
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three maturity stages.
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Fig. 3 DPPH radical scavenging activities of four phenolic fractions from jujube at three maturity stages.
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Different lower case letters correspond to significant differences for maturity stages in one group
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Fig. 4 FRAP values of four phenolic fractions of jujube at three maturity stages.
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Different lower case letters correspond to significant differences for maturity stages in one group
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Table 1 TPC (mg GAE/100g DW) in four phenolic fractions of jujube during maturity Phenolic fractions Maturity stages
Esterified (F2)
Glycosided (F3)
Insoluble-bound (F4)
White maturity (WM)
864.73± 3.27a
190.30 ± 2.44a
197.83 ± 5.92a
262.49 ± 5.22a
Half-red maturity (HM)
259.00± 7.14b
75.31 ± 3.11b
122.11 ± 6.79b
152.83 ± 3.06b
Red maturity (RM)
71.67 ± 1.99c
105.13 ± 5.67b
82.78 ± 6.16c
103.10 ± 2.81c
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Free (F1)
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Table 2 TFC (mg Rutin/100 g DW) in four phenolic fractions of jujube during maturity Phenolic fractions
Maturity stages
F1
White maturity (WM)
F2
1002.00± 10.60
a
274.94 ±15.46
F3 a
F4
167.23 ± 22.97
a
248.49 ± 6.27a
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Half-red maturity (HM)
956.39± 9.17b
125.72 ± 7.97b
137.88 ± 14.10a
258.69 ± 6.90a
Red maturity (RM)
167.45 ±18.79c
77.42 ± 11.93c
61.05±7.31b
177.56 ± 7.63b
Different lower case letters correspond to significant differences at p < 0.05.
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Table 3 Composition of phenolic compounds in jujube during maturity (µg/g DW) Phenolics
White Maturity F1
F2
F3
Gallic
0.00
0.00
198.66
Protocatechuic
6.57
7.89
5.37
p-hydroxybenzoic
71.35
0.90
7.21
Caffeic
Half-red Maturity F4
Red Maturity
F1
F2
F3
F4
0.00
0.00
51.57
207.86
8.41
7.30
8.22
6.89
21.01
2.16
4.41
12.54
1.36
2.60
5.39
F1
F2
F3
F4
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Phenolic acids 0.00
0.00
0.00
0.00
9.72
1.62
10.89
3.18
0.14
0.34
2.83
1.29
10.18
1.05
15.56
138.68
1.82
0.93
5.61
0.00
34.70
7.64
16.94
18.08
10.64
7.55
19.10
Ferulic
0.00
0.70
6.29
0.00
0.00
1.86
8.85
0.00
Chlorogenic
47.55
15.42
21.39
10.58
8.01
100.00
39.52
1.26
366.53
Rosmarinic acid
0.00
0.00
0.63
0.03
0.00
0.00
6.62
0.00
0.00
Ellagic acid
9.11
3.56
5.94
18.04
6.93
0.00
13.08
2.06
772.46
73.33
254.18
72.86
192.46
174.14
308.03
925.83
17.73
0.00
0.86
602.04
101.74
51.14
Flavonoids Rutin Quercetin ∑TP
9.52
2.87
17.52
0.00
2.50
5.23
32.92
41.84
1.65
0.00
6.33
10.42
15.15
26.27
0.00
0.90
0.07
0.00
0.00
1.51
0.00
43.99
495.03
26.06
39.38
87.58
4.79
670.63
20.59
8.07
4.71
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76.79
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637.88
p-coumaric
0.00
0.63
0.00
0.00
0.00
0.04
6.71
0.00
0.00
0.12
4.63
0.00
1698.29
91.69
260.02
73.72
794.50
275.92
365.88
48.78
1165.66
46.77
52.08
92.29
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Table 4 Correlation coefficients (r) of phenolics and antioxidant activity TFC 0.799**
TPC TFC DPPH FRAP ** p < 0.01; *p < 0.05.
DPPH 0.565 0.654*
HPLC 0.336 0.536 0.180 0.431
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FRAP 0.871* 0.836** 0.581*
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Highlights: 1. The phenolic composition in jujube and the antioxidant activities changed during
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three edible maturity stages. 2. The phenolic contents and their activities greatly decreased with the increasing maturity stage.
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3. Hydrolyzing extracted insoluble-bound phenolics cannot be ignored.