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Food Chemistry 167 (2015) 468–474

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Content variations of triterpenic acid, nucleoside, nucleobase,


and sugar in jujube (Ziziphus jujuba) fruit during ripening
Sheng Guo a, Jin-ao Duan a,⇑, Dawei Qian a, Yuping Tang a, Dawei Wu a,
Shulan Su a, Hanqing Wang a,b, Yunan Zhao c
a
Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, National and Local Collaborative Engineering Center of Chinese Medicinal
Resources Industrialization and Formulae Innovative Medicine, and Jiangsu Key Laboratory for High Technology Research of TCM Formulae,
Nanjing University of Chinese Medicine, Nanjing 210023, China
b
College of Pharmacy, Ningxia Medical University, Yinchuan 750004, China
c
Research Center of Basic Medical College, Nanjing University of Chinese Medicine, Nanjing 210023, China

a r t i c l e i n f o a b s t r a c t

Article history: Jujube (Ziziphus jujuba) fruit is widely consumed as food and traditional Chinese medicine in Asian
Received 4 April 2014 countries due to its potential effects for human health. To facilitate selection of the maturity stage pro-
Received in revised form 25 June 2014 viding optimum health benefits, jujube fruits were analysed at six stages of growth (S1–6) for triterpenic
Accepted 2 July 2014
acids, nucleosides, nucleobases, and sugars by UHPLC–MS/MS or HPLC–ELSD methods. The content levels
Available online 11 July 2014
of most triterpenic acids and sugars increased with ripening, and reached the highest at S5 and S6,
respectively. The accumulation of the cyclic nucleotides (cAMP and cGMP) was mainly in the later stage
Keywords:
of ripening (S5–6). Therefore, if taking triterpenic acids as the major quality indicator, S5 should be the
Ziziphus jujuba
Triterpenic acid
ideal time to harvest jujube fruit, and the full ripen stage (S6) maybe the best choice when taking sugars
Nucleoside and cyclic nucleotides as the most important components.
Sugar Ó 2014 Elsevier Ltd. All rights reserved.
Ripening stage
Accumulation of the active compounds
Suitable harvest time

1. Introduction (Chen et al., 2013; Pawlowska, Camangi, Bader, & Braca, 2009),
nucleosides (Guo, Duan, Qian, Wang, et al., 2013), phenolic acids
Jujube (Ziziphus jujuba Mill.), a thorny Rhamnaceous plant, is (Du et al., 2013; Wang, Liu, Zheng, Fan, & Cao, 2011), cerebrosides,
indigenous to China and widely distributed in the temperate and sugars (Li, Ai, Yang, Liu, & Shan, 2013), and amino acids (Choi, Ahn,
subtropical areas of the northern hemisphere, especially the inland Kozukue, Levin, & Friedman, 2011; Guo, Duan, Qian, Tang, et al.,
region of north China (Gao, Wu, & Wang, 2013). Its fruit, named 2013).
dazao in China, is widely consumed in Asian countries as a food Since all above metabolites significantly contribute to the taste,
and food additive due to its high nutritional value (Li, Fan, Ding, nutritional and potential functional values of jujube fruits, knowl-
& Ding, 2007). It has also been used as a traditional Chinese edge of the composition and concentration of these bioactive com-
medicine (TCM) for thousands of years with its numerous benefi- pounds in jujube products can benefit consumers. For this purpose,
cial effects for human health. Recent studies showed that jujube in previous studies, these compounds have been determined in
fruits have multiple bioactivities, such as anticancer (Plastina jujube fruits from various cultivars and collected from different
et al., 2012), anti-inflammatory (Yu et al., 2012), hepatoprotective cultivation regions (Chen et al., 2013; Choi et al., 2011; Gao, Wu,
(Shen et al., 2009), gastrointestinal protective (Huang, Yen, Sheu, & Yu, et al., 2012; Guo, Duan, Qian, Tang, et al., 2013; Guo, Duan,
Chau, 2008), antioxidant (Cheng, Zhu, Cao, & Jiang, 2012), anti- Tang, Zhu, et al., 2010). In addition, it is known that the bioactive
insomnia, immunostimulating (Li, Shan, Liu, Fan, & Ai, 2011) and compounds can be both synthesised and degraded as the plant
neuroprotective effects (Yoo et al., 2010). Phytochemical studies matures; thus there is a need to define the contents at different
revealed that jujube fruits contain various constituents, including stages of maturity. Choi et al. (2012) reported the changes of flavo-
triterpenic acids (Lee, Min, Lee, Kim, & Kho, 2003), flavonoids noid, free amino acid and protein contents during eight stages of
growth, and the results showed that the individual metabolites
among the different stages of growth varied widely. In addition,
⇑ Corresponding author. Tel./fax: +86 25 8581 1116. ascorbic acid and phenolic acids were also investigated in the
E-mail address: dja@njutcm.edu.cn (J.-a. Duan). jujube samples (Wang, Cheng, Cao, & Jiang, 2013).

http://dx.doi.org/10.1016/j.foodchem.2014.07.013
0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
S. Guo et al. / Food Chemistry 167 (2015) 468–474 469

However, except for these compounds, no information could be 22, 2011 in 6 stages (S1–S6) of maturity, from 30 to 105 days after
available for other types of compounds contained in jujube fruits, flowering (Table 1 and Fig. S2). All the fruits were collected from
such as triterpenic acids, nucleosides and nucleobases as well as the same 5 jujube trees, and stored at 40 °C before analysis.
sugars. It was reported that the triterpenic acids in jujube fruits
have multiple biological effects, such as anti-inflammatory, antimi- 2.3. UHPLC analysis of triterpenic acids
crobial, hepatoprotective and antioxidant activities (Fujiwara et al.,
2011; Yu et al., 2012), while nucleosides and nucleobases are A total of 20–40 fresh jujube fruits with uniform size in each
involved in the regulation and modulation of various physiological growth stage were selected for analysis. The fruits were divided
processes in the body and exhibit antiplatelet aggregation, antiar- into pulp and shells with a knife. The pulp was then cut with a
rhythmic, antioxidant, antiseizure and antitumour effects, which knife to thin strips (2 mm  2 mm) and homogenised. A total of
all contribute to the jujube’s potential effects for human health 2 g of above pulp homogenates were extracted with 40 mL of
(Guo, Duan, Tang, Zhu, et al., 2010). In addition, the sugars includ- methanol in a cooled ultrasonic bath (40 kHz) for 60 min. The
ing glucose, fructose and sucrose were reported to be the main extract was followed by centrifugation at 15,000g for 10 min and
nutrients of jujube fruits and contribute to its taste. Therefore, then the supernatant was separated and transferred to a vial prior
the investigation of the above three types of compounds would to injecting into the UHPLC system.
be helpful for evaluating the nutritional and potential functional Samples were analysed using a Waters ACQUITY UPLC system
values of jujube fruits at different growth stages. (Waters, Milford, MA) coupled with a Waters Xevo TQ tandem
Thus, to facilitate selection of the maturity stage of the jujube quadrupole mass spectrometer (Micromass MS Technologies,
fruit that can provide optimum benefits as a functional food, in this Manchester, UK). An ACQUITY UPLC BEH C18 column
study, the contents of triterpenic acids, nucleosides and nucleo- (100 mm  2.1 mm, 1.7 lm; Waters) operated at 30 °C was used.
bases, and sugars in jujube fruits harvested at different stages of The injection volume was 2 lL, and the flow rate was maintained
growth were determined using ultra-high-performance liquid at 0.4 mL/min. The mobile phase was composed of A (methanol
chromatography coupled with tandem mass spectrometry mixed with equal volume of acetonitrile) and B (0.5% ammonium
(UHPLC–MS/MS) and high-performance liquid chromatography acetate in deionised water) with a gradient elution: 0–2 min,
coupled with evaporative light scattering detector (HPLC–ELSD). 76–77% A; 2–12 min, 77% A. The column eluent was directed to
the mass spectrometer and an ESI source operated in negative
2. Materials and methods ion mode was used in this assay. The parameters for the MS were
set as follows: capillary voltage 3.0 kV; source temperature 150 °C;
2.1. Chemicals desolvation temperature 550 °C; cone gas flow 50 L h1; desolva-
tion gas flow 1000 L h1. The analyte detection was performed by
Reference compounds of ceanothic acid, alphitolic acid, masli- using multiple reaction monitoring (MRM) or selected ion moni-
nic acid, 2a-hydroxyursolic acid, betulinic acid, oleanolic acid, toring (SIM). The cone voltage and collision energy were optimised
ursolic acid, betulonic acid, oleanonic acid, and ursonic acid were for each analyte and selected values are given in Table S1. Dwell
isolated from the fruits of Z. jujuba in our laboratory, and their time was automatically set by the software. All the analyses were
purities (>98%) and structures were determined by HPLC, MS, operated using MassLynx™ XS Software. Concentrations of the tar-
and NMR. The following standards: sucrose, D-glucose, D-fructose, get compounds were calculated from the peak areas of the sample
thymine, uracil, adenine, hypoxanthine, uridine, adenosine, and the corresponding standards. The concentrations were
xanthine, inosine, guanine, and cAMP were purchased from the expressed in microgram per gram of dry weight (DW) and used
National Institute for the Control of Pharmaceutical and Biological for comparing the results that came from different stage samples.
Products (Beijing, China). Thymidine, cytidine, guanosine, and
cGMP were obtained from Sigma (St. Louis, MO). The structures 2.4. UHPLC analysis of nucleosides and nucleobases
of these reference compounds are presented in Fig. S1.
The acetonitrile, methanol, and ammonium acetate were all of Nucleosides and nucleobases were analysed by the modified
HPLC grade and purchased from Merck (Darmstadt, Germany). procedure from our previous report (Guo, Duan, Qian, Wang,
Deionised water was purified by an EPED superpurification system et al., 2013). A total of 4 g of jujube pulp homogenates was
(Eped, Nanjing, China). Other reagent solutions were of analytical extracted with 40 mL of water in a cooled ultrasonic bath
grade (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China). (40 kHz) for 60 min. The extract was followed by centrifugation
(15,000g, 10 min), then the supernatant was separated and
2.2. Sampling of jujube fruits injected into the Waters UPLC system coupled with Waters Xevo
TQ tandem quadrupole mass spectrometer.
A local cultivar of Z. jujuba Mill., called ‘Lingwuchangzao’ which An ACQUITY UPLC BEH Amide column (2.1  100 mm, 1.7 lm)
is planted in Yinchuan (106°460 E, 38°340 N), China, was used in this equipped with an ACQUITY UPLC BEH Amide 1.7 lm VanGuard
assay. The trees were managed according to integrated cultivation pre-column was used for the analysis. The mobile phase was com-
protocols, and the fruits were harvested from July 9 to September posed of A (10 mM ammonium acetate in deionised water) and B

Table 1
Summary of the jujube fruit samples harvested at different stages of growth.

Sample No. Collecting date Days after flowering Weight (g)/fruit (mean ± SD, n = 5) Water (%)
S1 July 9 30 2.4 ± 0.2 82.8
S2 July 24 45 6.3 ± 0.6 85.4
S3 August 7 59 9.5 ± 0.8 84.2
S4 August 22 74 12.4 ± 0.6 84.6
S5 September 6 89 18.8 ± 1.2 82.2
S6 September 22 105 21.5 ± 1.6 75.1
470 S. Guo et al. / Food Chemistry 167 (2015) 468–474

(2 mM ammonium acetate in acetonitrile) with a gradient elution In addition, all these triterpenic acids have carboxyl groups
(0–6 min, 10% A; 6–8 min, 10–40% A; 8–10 min: 40% A). The flow which can form a stable negative ion, and the sensitivity and inten-
rate of the mobile phase was 0.3 mL/min, and the column temper- sity for these compounds obtained from the negative ion mode
ature was maintained at 30 °C. The injection volume was 2 lL. were higher than those from the positive ion mode. Given the
Mass spectrometry was performed using an ESI source operated above facts, the ESI- mode was selected in the study. When the
in positive ion mode and the parameters were set as in Guo, proper transition for the MS/MS detection of the analyte was
Duan, Qian, Wang, et al. (2013). The selected values of cone voltage selected, we found that all these compounds analysed in this study
and collision energy for each target compound are given in have a deprotonated molecule [MH] with abundant intensities,
Table S2. All the analyses were operated by MassLynx™ XS Soft- which were selected as the precursor ions. The abundances of
ware. For quantification, integrated chromatographic peak areas product ions for these triterpenic acids, except for ceanothic, are
from each jujube sample were compared to peak areas of known too low to be detected by MRM. Thus, SIM was used for their quan-
amounts of standard sample. The concentrations were expressed titative determination. For ceanothic acid, an abundant ion at
in micrograms per gram of DW. [MH2OCOOH], which corresponds to the neutral loss of formic
acid by a rearrangement, was detected, and this ion was selected as
2.5. HPLC analysis of sugars the product ion for MRM. The values of cone voltage and collision
energy were optimised by IntelliStart software (Table S1). In this
The content levels of sugars (sucrose, glucose, and fructose) in assay, some analytes are isomers and have the same MS character-
the samples were analysed by a modified procedure of a reported istics, such as m/z at 471 for alphitolic acid, maslinic acid and 2a-
LC method (Godin, Agneessens, Gerin, & Delcarte, 2011). A total of hydroxyursolic acid, m/z at 455 for betulinic acid, oleanolic acid
2 g of jujube pulp homogenates was extracted with 100 mL of and ursolic acid, m/z at 453 for betulonic acid, oleanonic acid and
water in a cooled ultrasonic bath (40 kHz) for 60 min. Then the ursonic acid. For these compounds, they could influence each other
extract was centrifuged (15,000g, 10 min) and the supernatant even using specific MS/MS detection. Thus, the baseline separation
was separated and transferred to vials. for these analytes should be obtained. Our previous study showed
Samples were analysed on a Waters 2695 Alliance HPLC system. that the mobile phase of aqueous methanol with 0.3% acetic acid
For each analysis, 10 lL of sample solution were used. A Carbo Sep and 0.15% triethylamine was suitable for the separation of these
CHO-682 Pb column (7.8  300 mm, 7 lm; Transgenomic, Inc., triterpenic acids by HPLC (Guo, Duan, Tang, Yang, et al., 2010).
Omaha, NE) with deionised water as the mobile phase was used However, the above mobile phase could not obtain good separation
for separation of the sugars. The flow rate and the column temper- for these target compounds, especially for oleanonic acid and
ature were optimised as 0.4 mL/min and 65 °C, respectively. A ursonic acid, in the present UHPLC analysis. Therefore, we
Waters 2424 ELSD was used for identification and the drift tube investigated the mobile phase system again and found that good
temperature and nitrogen flow-rate were set at 80 °C and 2.7 separation could be obtained when the methanol mixed with an
L/min, respectively. Quantification was performed on the basis of equal volume of acetonitrile was selected as the organic mobile
linear calibration plots of the logarithm of peak areas versus the phase. After optimisation, mobile phases A (methanol:acetonitrile,
logarithm of concentrations. The concentrations were expressed 1:1) and B (0.5% ammonium acetate in deionised water) were used.
in grams per 100 g of DW. Typical chromatograms of the reference compounds and the sam-
ple are presented in Fig. S3.
The proposed UHPLC–TQ-MS method for quantitation of trite-
2.6. Statistical analysis
rpenic acids was validated to determine the linearity, limit of
detection (LOD) and quantitation (LOQ), intra-day and inter-day
All experiments were performed at least three times and the
precisions, stability, and accuracy. The results are summarised in
results were reported as mean ± SD. Data were statistically
Tables S3 and S4. As shown in Table S3, the LODs and LOQs of this
evaluated by one-way ANOVA analysis with the aid of SPSS 16.0
method were in a range from 0.58 to 7.98 ng/mL and from 2.32 to
software (SPSS, Chicago, IL). When significant differences were
27.9 ng/mL, which revealed that the sensitivity of this proposed
found, the least significant difference (LSD) test was used to
method is much higher than HPLC–UV (0.29–0.68 lg/mL for LOD)
determine differences among means.
(Guo et al., 2009) and HPLC–ELSD (2.05–7.03 lg/mL for LOD)
(Guo, Duan, Tang, Yang, et al., 2010) methods, and is of similar sen-
3. Results and discussion sitivity to the method using pre-column derivatisation coupled
with HPLC-FLD for determination of triterpenic acids (Li, You,
3.1. Triterpenic acid content of jujube fruit et al., 2011; Li, Zhang, et al., 2011).

3.1.1. Establishment of the analytical method 3.1.2. Changes of triterpenic acid content
In previous study, we have established methods such as HPLC– Due to the fact that only the pulp of jujube fruit is mainly con-
UV and HPLC–ELSD for the analysis of triterpenic acids in jujube sumed, the pulp of fruit was used in this assay to investigate the
samples (Guo, Duan, Tang, Yang, et al., 2010; Guo et al., 2009, content variations of the target compounds. The triterpenic acid
2011). However, the sensitivities of these methods were relative contents in the pulp of jujube fruit at different grown stages are
low and insufficient to detect trace target compounds in the pres- listed in Table 2. In general, the total contents of triterpenes exhib-
ent analysis. Thus, a more sensitive approach, UHPLC–MS/MS, was ited an increasing trend as the fruits matured, especially after stage
used; 10 triterpenic acid including ceanothic acid, alphitolic acid, S3, and reached the highest (6130 lg/g) at S5, then dropped to
maslinic acid, 2a-hydroxyursolic acid, betulinic acid, oleanolic 4740 lg/g at stage S6. As for the different types of triterpenic acids
acid, ursolic acid, betulonic acid, oleanonic acid and ursonic acid analysed in the assay, the variation trends of their contents were
were analysed in this assay. Considering the low polarity of the different. For example, as shown in Fig. 1A–C, the contents of
above compounds and the high content of water in the juice lupane-type (alphitolic acid, betulinic acid and betulonic acid)
samples, methanol and ethanol were compared for the extraction and ceanothane-type (ceanothic acid) compounds increased dur-
efficiency of the target compounds. The results showed that meth- ing ripening except for betulonic acid; while for the oleanane-type
anol was suitable for the samples because it could provide highest (maslinic acid, oleanolic aicd and oleanonic acid) and ursane-type
values for the contents of the 10 markers. compounds (2a-hydroxyursolic acid, ursolic acid and ursonic acid),
S. Guo et al. / Food Chemistry 167 (2015) 468–474 471

Table 2
Contents (lg/g of DW, mean ± SD, n = 3) of the investigated triterpenic acids in samples at different ripening stages.

Analyte Ripening stages


S1a S2 S3 S4 S5 S6
Ceanothic acid 5.0 ± 0.2ab 1.8 ± 0.1b 3.6 ± 0.1c 2.3 ± 0.1d 15.4 ± 0.5e 35.0 ± 1.1f
Alphitolic acid 44.1 ± 1.5a 51.4 ± 1.8b 195 ± 5.4c 598.5 ± 16.3d 639 ± 10.8e 687 ± 21.5f
Maslinic acid 62.1 ± 2.1a 101 ± 2.5b 225 ± 6.1c 410.1 ± 11.2d 506 ± 7.9e 354 ± 8.3f
2a-Hydroxyursolic acid 5.3 ± 0.2a 8.4 ± 0.3b 31.9 ± 1.3c 129.3 ± 2.5d 228 ± 4.6e 183 ± 4.2f
Betulinic acid 12.7 ± 0.3a 14.8 ± 0.5b 63.4 ± 2.1c 344.6 ± 5.9d 756 ± 22.3e 785 ± 18.5f
Oleanolic acid 16.9 ± 0.5a 24.7 ± 1.0b 132 ± 4.5c 424.0 ± 10.1d 534 ± 12.6e 360 ± 10.7f
Ursolic acid ndc a nd a 4.3 ± 0.2b 65.6 ± 1.2c 310 ± 9.0d 177 ± 3.7e
Betulonic acid 3.5 ± 0.1a 3.4 ± 0.1a 25.7 ± 0.9b 425 ± 9.8c 1400 ± 35.1d 1320 ± 26.4e
Oleanonic acid 15.3 ± 0.6a 12.5 ± 0.4b 86.2 ± 1.4c 842 ± 20.6d 1030 ± 19.3e 493 ± 12.6f
Ursonic acid nd a nd a nd a nd a 710 ± 20.6b 350 ± 12.5c
Total 166 ± 5.4a 218 ± 7.7b 767 ± 24.6c 3240 ± 66.2d 6126 ± 130.9e 4740 ± 104f
a
The sample No. is same as Table 1.
b
Values followed by the same letter in the same row are not significantly different (p < 0.05).
c
Not detected.

Table 3
Contents (lg/g of DW, mean ± SD, n = 3) of the investigated nucleosides and nucleobases in samples with different ripening stages.

Analyte Ripening stages


a
S1 S2 S3 S4 S5 S6
Thymine nd acb
nd a nd a nd a nd a 3.65 ± 0.29b
Uracil nd a nd a nd a nd a nd a 8.45 ± 0.44b
Thymidine 0.73 ± 0.02a nd b 0.11 ± 0.01c 0.19 ± 0.02d nd b 0.49 ± 0.06e
Adenine 96.3 ± 2.13a 120 ± 2.76b 58.7 ± 1.76c 55.2 ± 1.01c 55.6 ± 1.33c 40.2 ± 1.19d
Hypoxanthine 2.07 ± 0.04a 0.98 ± 0.02b 0.56 ± 0.01c 1.27 ± 0.03d 5.44 ± 0.17e 2.52 ± 0.26f
Uridine 233 ± 3.57a 244 ± 5.65a 144 ± 3.21b 152 ± 4.23b 93.7 ± 2.02c 71.2 ± 2.10d
Adenosine 9.38 ± 0.24a 9.76 ± 0.26a 2.67 ± 0.07b 1.30 ± 0.06c 0.43 ± 0.05d 0.55 ± 0.05d
Xanthine 6.99 ± 0.12a 10.6 ± 0.24b 3.29 ± 0.08c 1.85 ± 0.05d 10.3 ± 2.11b 10.1 ± 2.33b
Inosine 0.63 ± 0.03a 0.76 ± 0.04b 0.23 ± 0.02c 0.20 ± 0.02c 0.32 ± 0.04d 1.10 ± 0.04e
Guanine 61.2 ± 1.45a 43.4 ± 1.56b 46.0 ± 1.72bc 48.4 ± 1.78c 57.8 ± 1.36d 23.7 ± 0.91e
Cytidine 1.21 ± 0.03a 0.82 ± 0.03b 1.48 ± 0.04c 1.54 ± 0.06c 3.93 ± 0.16d 8.93 ± 0.25e
Guanosine 34.6 ± 1.03a 46.4 ± 1.24b 27.5 ± 0.83c 25.4 ± 0.68d 23.6 ± 0.77d 34.3 ± 0.96a
cAMP 3.28 ± 0.06a 4.20 ± 0.15b 4.93 ± 0.39c 5.17 ± 0.19c 4.98 ± 0.22c 31.3 ± 1.18d
cGMP nd a nd a 1.14 ± 0.03b 1.98 ± 0.07c 3.33 ± 0.09d 16.1 ± 0.82e
Total 450 ± 6.98a 481 ± 11.16b 291 ± 6.96c 294 ± 7.93c 260 ± 7.89d 253 ± 7.05d
a
The sample No. is same as Table 1.
b
Not detected.
c
Values followed by the same letter in the same row are not significantly different (p < 0.05).

with the fruit maturing, their contents were gradually increasing nucleobases and nucleotides (Guo, Duan, Qian, Wang, et al.,
until period S5, where their contents reached a maximum, then 2013). After slight modifications of the mobile phase, this method
decreased. In addition, during the early stages of maturity from was applied to determine 14 compounds including nucleosides
S1 to S3, the levels of the compounds containing the 2,3-dihydroxy and nucleobases. The method was validated to determine the line-
group were highest of the different types of compounds. Then, arity, LOD, LOQ, intra-day and inter-day precision, stability, and
with the fruit maturing, the contents of those compounds with accuracy, and the results are summarised in Tables S5 and S6. A
3-carbonyl moieties were surging and became the abundant typical chromatogram of the sample is presented in Fig. S4. All
triterpenic acids during the late stages of maturation. above results showed that the method could be suitable for deter-
The above result indicated that the accumulation of triterpe- mination of the nucleosides and nucleobases in fruits harvested at
noids in the pulp of jujube fruits is mainly in the later stages of different growth stages.
fruit maturity. In fact, the jujube fruits in S5 have similar size to Table 3 lists the contents of individual nucleosides and nucleo-
those in S6 as shown in Fig. S2 and Table 1, and their colour begins bases in the pulp of jujube fruits harvested at six growth stages
to change from green to red. Therefore, due to the total triterpenic from the same plants. The results showed that total contents of
acid level in S5 being substantially higher than that in S6, S5 may the 14 analytes increased in the initial stage of fruit growth and
be a more appealing choice for consumers than S6, The result is in reached a peak value of 481 lg/g at S2, then decreased. Most of
accordance with the conclusions obtained on the contents of flavo- the nucleosides and nucleobases compositions were present in
noids and phenolic acids,(Chen et al., 2013; Choi et al., 2012; Wang the whole period of jujube fruit growth and development except
et al., 2013). thymine and uracil. As for the individual compounds, different var-
iation trends were exhibited for their contents with increasing
3.2. Nucleoside and nucleobase contents of jujube fruit maturity. For example, with the jujube fruit maturing, the content
of adenosine gradually reduced from 9.38 lg/g (S1) to 0.55 lg/g
In our previous study, we have established a UHPLC–MS/MS (S6), a decrease of more than 90%. A similar trend was found for
method for simultaneous determination of nucleosides, uridine and adenine (Fig. 1D), while the opposite trend was found
472 S. Guo et al. / Food Chemistry 167 (2015) 468–474

1,600 1,200
Alphitolic acid Maslinic acid

Content (μg/g DW)


Betulinic acid Oleanolic acid

Content (μg/g DW)


1,200 900
Betulonic acid Oleanonic acid
800 Ceanothic acid 600

400 300

0 0
S1 S2 S3 S4 S5 S6 S1 S2 S3 S4 S5 S6
Stage of maturity Stage of maturity

(A) (B)
800 2α-hydroxyursolic acid 300
Uridine

Content (μg/g DW)


Ursolic acid Adenine
Content (μg/g DW)

600
Ursonic acid 200

400
100
200

0 0
S1 S2 S3 S4 S5 S6 S1 S2 S3 S4 S5 S6
Stage of maturity Stages of maturity

(C) (D)
12 Hypoxanthine 80 Guanine
Adenosine Guanosine
Content (μg/g DW)

Content (μg/g DW)

9 Cytidine 60

6 40

3 20

0 0
S1 S2 S3 S4 S5 S6 S1 S2 S3 S4 S5 S6
Stages of maturity Stages of maturity

(E) (F)
40 40
cAMP Sucrose
Content (μg/g DW)

Glucose
Content (% D W)

30 cGMP 30
Fructose
20 20

10 10

0 0
S1 S2 S3 S4 S5 S6 S1 S2 S3 S4 S5 S6
Stages of maturity Stages of maturity

(G) (H)
Fig. 1. Changing tendency of selected compound levels in jujube fruits during fruit growth. Data of (A)–(C) (contents of triterpenic acids) and (D)–(G) (contents of nucleosides
and nucleobases) were determined by UHPLC–TQ-MS, data of H (the contents of sugars) was determined by HPLC–ELSD method. All these data are the mean of three
replicates.

for the levels of cytidine, which increased from 1.21 lg/g (S1) to compounds with fruit growth. For the cyclic nucleotides, cAMP
8.93 lg/g (S6) (Fig. 1E). In addition, as shown in Fig. 1F, it is inter- and cGMP, their contents showed slow increasing trends in the
esting that the changing trend of guanine contents was exactly early stages (S1–S5), as shown in Fig. 1G, then rapidly increased
opposite to that of guanosine during ripening. Due to the fact that from 4.98 lg/g (S5) to 31.3 lg/g (S6) for cAMP and 3.33 lg/g (S5)
the difference between the structures of guanine and guanosine is to 16.1 lg/g (S6) for cGMP. Results revealed that the accumulation
only a moiety of ribose, the result found above could indicate that of cyclic nucleotides in jujube fruits occurs mainly during the later
there may be structural transformation between these two stages of ripening.
S. Guo et al. / Food Chemistry 167 (2015) 468–474 473

Table 4 National Natural Science Foundation of China (No. 30672678)


Contents (% DW) of the investigated sugars in samples with different ripening stages. and the Priority Academic Program Development of Jiangsu Higher
Sample No. Contents of analyte (mean ± SD, n = 3) Total Education Institutions (ysxk-2014).
Sucrose Glucose Fructose
S1a ndb ac 9.99 ± 0.28a 7.06 ± 0.17a 17.1 ± 0.42a
S2 nd a 15.5 ± 0.41b 9.91 ± 0.32b 25.4 ± 0.75b Appendix A. Supplementary data
S3 nd a 18.6 ± 0.55c 11.7 ± 0.49c 30.3 ± 0.88c
S4 nd a 18.4 ± 0.46c 12.1 ± 0.40c 30.6 ± 0.79c Supplementary data associated with this article can be found, in
S5 13.0 ± 0.41b 21.6 ± 0.59d 13.6 ± 0.28d 48.3 ± 1.41d
the online version, at http://dx.doi.org/10.1016/j.foodchem.2014.
S6 19.0 ± 0.85c 33.0 ± 1.03e 21.3 ± 0.83e 73.2 ± 1.98e
07.013.
a
The sample No. is same as Table 1.
b
Not detected.
c
Values followed by the same letter in the same column are not significantly
different (p < 0.05). References

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