Subscriber access provided by Macquarie University
Bioactive Constituents, Metabolites, and Functions
Phytochemicals accumulation in Sanhua plum (Prunus salicina L.) during fruit development and their potential use as antioxidants Quan Li, Xiaoxiao Chang, Hong Wang, Charles Brennan, and Xinbo Guo J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05087 • Publication Date (Web): 12 Feb 2019 Downloaded from http://pubs.acs.org on February 14, 2019
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 32
Journal of Agricultural and Food Chemistry
1
Phytochemicals accumulation in Sanhua plum (Prunus salicina L.) during fruit
2
development and their potential use as antioxidants
3 4
Authors: Quan Li†, Xiao-Xiao Chang‡, Hong Wang†, Charles Stephen Brennan§, Xin-
5
Bo Guo†, *
6 7
Affiliations:
8
† School of Food Science and Engineering, South China University of Technology,
9
Overseas Expertise Introduction Center for Discipline Innovation of Food Nutrition
10
and Human Health (111 Center), Guangzhou, 510640, China
11
‡ Institute of Fruit Tree Research, Guangdong Academy of Agricultural Sciences;
12
Key laboratory of South Subtropical Fruit Biology and Genetic Resource Utilization
13
(MOA); Guangdong Province Key Laboratory of Tropical and Subtropical Fruit Tree
14
Research, Guangzhou, 510640, China.
15
§ Department of Wine, Food Molecular Biosciences, Lincoln University, Lincoln 7647,
16
New Zealand
17 18
Corresponding authors:
19
* Xinbo Guo, Tel & Fax: (+86) 20-87113848; E-mail:
[email protected] 20
1
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
21
ABSTRACT
22
This study followed the flesh reddening of Sanhua plum from the surface to the center
23
during fruit development. Five key stages were identified based on colour changes
24
during fruit ripening full-green (FG), red-appeared (RA), half-red (HR), full-red (FR)
25
and purple-red (PR). Fruits were collected and analyzed for phytochemicals and
26
antioxidant properties. Concurrently, the transcript levels of genes associated with
27
phenolic, flavonoid and anthocyanin production were investigated. The titratable acid
28
(TA) content of Sanhua plum decreased during development, while total soluble sugar
29
(TSS) content increased. In addition, both the total phenolic content (TPC) and total
30
flavonoid content (TFC) decreased during development, while anthocyanin content
31
increased. The polyphenol oxidase (PPO) activity peaked at PR stage. The maximum
32
antioxidant activity in vitro was observed at FG stage, while cellular antioxidant activity
33
peaked at PR stage.
34 35
KEYWORDS
36
Prunus salicina L.; gene expression; phytochemicals; antioxidant activity; fruit
37
development
2
ACS Paragon Plus Environment
Page 2 of 32
Page 3 of 32
Journal of Agricultural and Food Chemistry
38
INTRODUCTION
39
Sanhua plum (Prunus salicina L.) is widely cultivated in southern China and belongs
40
to the Rosaceae family (1). Plums are consumed as fresh fruits, juice, fermented wine,
41
and preserves (1). Consumers are attracted to the plum by its red flesh, characteristic
42
taste and excellent nutritional properties (2). Previous research has shown that the
43
antioxidant activity of the plum could be equivalent to 129.71 ±1.36 μmol ASA/g FW
44
of ABTS in mature fruits of Sanhua plum (2). Other studies on the antioxidant
45
properties of plums have shown that mature fruits of Sanhua plum have a total
46
anthocyanin content of 8.79 ± 0.04 mg CGE/g DW (1), a total phenolic content of
47
102.43 ± 2.83 mg GAE/100 g FW, a FRAP value of 14.79 ± 0.20 μmol Fe(II)/g FW
48
and a TEAC value of 7.23 ± 0.02 μmol Trolox/g FW (1, 3). However, variations of
49
phytochemicals and antioxidant activity during Sanhua plum development have not
50
been described systematically.
51
The characteristic taste of a plum is formed gradually during the maturation and
52
linked to a progressively bright red flesh, and as such increasing attention has rested on
53
the dynamic changes of phytochemicals and antioxidant properties during maturation.
54
In this study, Sanhua plum fruits of five maturation stages distinguished by flesh color
55
ranging from full-green to purple-red were collected and analyzed for the dynamic
56
variations of gene expressions, nutritional parameters, antioxidant compounds, PPO
57
enzyme activity and antioxidant activities.
58
MATERIALS AND METHODS
59
Materials 3
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 4 of 32
60
Fruits of Sanhua plum (Prunus salicina L.) were collected at five developmental
61
stages [70 (FG), 80 (RA), 100 (HR), 120 (FR), and 130 (PR) days after full bloom
62
(DAFB)] from the Agriculture Demonstration Base located in Fengkai County
63
(Zhaoqing, Guangdong Province, China). Fruits were selected disease-free, similar in
64
shape at each growing stage (Figure 1). All samples were frozen in liquid nitrogen
65
immediately and stored at -80 °C until analyzed.
66
Reagent and chemicals
67
Folin-Ciocalteu
reagent,
dichlorofluorescin
diacetate,
2,
2'-Azobis
(2-
68
methylpropionamidine) dihydrochloride, cyanidin chloride, pelargonidin chloride, (-)-
69
epicatechin, myricetin and hesperetin were purchased from Sigma Aldrich (Mo, USA).
70
Trifluoroacetic acid, methanol and acetonitrile of HPLC grade were purchased from
71
ANPEL Scientific Instrument (Shanghai) Co., Ltd. Williams medium E (WME),
72
phosphate-buffered saline (PBS), Trypsin-EDTA solution were purchased from
73
GIBCO (Life Technologies, Grand Island, NY). Human liver cancer cell line HepG2
74
(ATCC ® HB-8065) was purchased from ATCC company (Manassas, VA, USA).
75
Other chemicals and reagents used in this work were of analytical grade.
76
Real time quantitative PCR (RT-qPCR) analysis
77
Total RNA was extracted and analyzed by the Beijing Genomics Institute (BGI,
78
Guangdong Province, China). Reverse transcription cDNA was conducted with
79
Fastking gDNA dispelling RT Supermix kit as per the manufacturer’s instructions (Tian
80
Gen, Beijing, China). RT-qPCR was conducted with Super Real PreMix Plus SYBR
81
(Green) referring to the manufacturer’s instructions (Tian Gen, Beijing, China). The 4
ACS Paragon Plus Environment
Page 5 of 32
Journal of Agricultural and Food Chemistry
82
nucleotide sequences of primers used in RT-qPCR are presented in Table 1. GAPDH
83
was regarded as the reference gene. The 2−ΔΔCt method based on three technical
84
replicates was used to analyze the transcript levels.
85
Determination of total soluble sugar (TSS), titratable acid (TA), total phenolic
86
content (TPC) and total flavonoid content (TFC)
87
TSS was determined by the anthrone method (4). TSS was determined from the
88
standard curve prepared using glucose and expressed as g glucose equivalents per 100
89
g fresh weight (g GE/100g FW).
90
TA was measured according to the AOAC 962.12 method (AOAC, 2012) with an
91
Automatic Potentiometric Titrator (TITRALAB TIM840, USA) and expressed as g
92
malic acid equivalents per 100 g fresh weight (g MAE/100g FW).
93 94
TPC was measured by Folin-Ciocalteu method (5) and expressed as mg gallic acid equivalents per 100 g fresh weight (mg GAE/100 g FW).
95
TFC (total flavonoids include flavones, flavonols, flavonones, flavononols,
96
isoflavonoids, flavanols, and anthocyanins) was measured by the sodium
97
orohydride/chloranil colorimetric method (5) and expressed as mg catechin equivalents
98
per 100 g fresh weight (mg CE/100 g FW).
99
Determination of phytochemicals
100
Phytochemicals were analyzed by HPLC technique with a Waters 2998 Photodiode
101
Array Detector with a C18 column (250 x 4.6 mm, 5μm) maintained at 35°C, as of
102
previous experimental analysis. (6, 7). Briefly, analysis of phenolic compounds was
103
performed at 280 nm wavelength with 1.0 mL/min of the mobile phases (A: 0.1% 5
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
104
trifluoroacetic acid in water, B: acetonitrile) in gradient elution as follows: 0–5 min (90%
105
A), 5–20 min (90–75% A), 20–25 min (75–65% A), 25–31 min (65–42% A), 31–34
106
min (42-40% A), 34–40 min (40–10% A), 40–50 min (10–90% A), 50–60 min (90–90%
107
A). Additionally, analysis of anthocyanins was performed at 520 nm with 1.0 mL/min
108
of the mobile phases (A: 0.1% trifluoroacetic acid in water, and B: methanol) as
109
following: 0–3 min (90% A), 3–5 min (90-60% A), 5–25 min (60-30% A), 25–27 min
110
(30-90%A), 27–30 min (90% A). The values are presented as mg per 100 g fresh weight
111
(mg/100 g FW).
112
Determination of polyphenol oxidase (PPO) activity, antioxidant activities in vitro
113
and cell
114 115
PPO activity was measured by a spectrophotometer at λ 420 nm (8) and expressed as 0.01△A/g FW/min.
116
Antioxidant activity in vitro was determined by the PSC assay (7) and presented as
117
micromoles of ascorbic acid (ASA) equivalents per 100 g fresh weight (μmol ASA
118
equiv./100 g FW). Cellular antioxidant activity was determined by the CAA assay (9)
119
and presented as micromoles of quercetin equivalents per 100 g fresh weight (μmol
120
QE/100 g FW).
121
Statistical analysis
122
Data is expressed as mean ± standard deviation (SD) for triplicate analysis. Result is
123
presented as ANOVA statistical analysis by using the IBM SPSS statistical software
124
21.0 (SPSS Inc., Chicago, IL), and the data is presented as significant in comparison
125
among five stages with p-value < 0.05. 6
ACS Paragon Plus Environment
Page 6 of 32
Page 7 of 32
Journal of Agricultural and Food Chemistry
126
RESULTS AND DISCUSSION
127
Variations of gene expression
128
In order to investigate the fluctuations of the expression levels of key genes
129
participating in the biosynthesis of phenolics, flavonoids and anthocyanins during fruit
130
development of Sanhua plum, RT-qPCR was conducted for C4H, CHS1, CHI, F3H,
131
DFR, LAR, ANS and ANR. Part of the phenylpropanoid biosynthesis pathway is shown
132
in Figure 2, and this illustrates the variations of the transcript levels of these genes. The
133
mean expression ratios of genes were calculated as the ratio between the relative
134
expression of those key genes at each stage and that of FG. Among these genes, C4H
135
and CHS1 showed increasing expressions. F3H and ANS exhibited higher transcript
136
levels during plum development, while both CHI and LAR showed lower levels during
137
plum development. In addition, DFR was highly expressed at HR and FR. ANR was
138
highly expressed at RA and HR while lowly expressed at FR and PR.
139
In the phenylpropanoid pathway, cinnamic acid is converted to chalcone by
140
enzymatic reactions sequentially catalyzed by C4H and CHS1. Thereon, the conversion
141
of chalcone to flavonoids is catalyzed by CHI. Other enzymes including F3H, DFR and
142
ANS (involved in the flavonoid pathway), have been shown to be required for
143
formation of anthocyanins (10). LAR catalyzes the reductions of leucocyanidin and
144
leucopelargonidin in the anthocyanin pathway into (+)-catechin and (+)-afzelechin,
145
respectively. In addition, ANR catalyzes the reductions of cyanidin and pelargonidin
146
into (-)-epicatechin and (-)-epiafzelechin, respectively (11).
147
Previous research had revealed that C4H, CHS, CHI, F3H, DFR and ANS were highly 7
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
148
expressed in blood-fleshed peach of cv. Dahongpao in the late stages of coloration
149
development (12). Anthocyanin-related genes (including CHS, DFR and ANS) were
150
mainly expressed during the fruit coloration in ‘Toyonaka’ and ‘Tokun’ strawberry (11).
151
However, decreased expressions of LAR and ANR were presented by ‘Red Heart’ plum
152
during pulp coloring (13). In our work, the variations of C4H, CHS1, F3H and ANS in
153
Sanhua plum were similar to blood-fleshed peach which also belongs to the Rosaceae
154
family. DFR was mainly expressed during the coloring stages of plum development
155
(HR and FR), while lowly expressed at PR, which was probably caused by the coloring
156
termination before PR. Additionally, the alterations of LAR and ANR in Sanhua plum
157
were similar to ‘Red Heart’ plum. The obtained data of those key genes in Sanhua plum
158
could support an insight for phytochemical accumulations.
159
Variations of TSS, TA, TPC and TFC
160
In Sanhua plum, the TA contents at the former four stages were significantly (p
