Journal of Integrative Agriculture 2022, 21(1): 261–272

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RESEARCH ARTICLE

Effect of harvest time on the chemical composition and antioxidant

capacity of Gannan navel orange (Citrus sinensis L. Osbeck ‘Newhall’)
juice

ZHANG Jun1, 2, ZHANG Jing-yi1, SHAN You-xia2, GUO Can1, HE Lian1, ZHANG Lin-yan1, LING Wei1,
LIANG Yan1, ZHONG Ba-lian1

1
National Engineering Research Center of Navel Orange, Gannan Normal University, Ganzhou 341000, P.R.China
2
South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, P.R.China

Abstract
The present study investigates the chemical composition and antioxidant capacity of juice from the Gannan navel
orange, which is harvested at one- to two-week intervals during the ripening period. The total soluble solid (TSS), total
polyphenol content (TPC), total flavonoid content (TFC), sucrose and hesperidin contents gradually increase with the
ripening of the fruit, followed by slight declines at the late maturity stage. Contrary to these observations, the contents of
titratable acid (TA), vitamin C (Vc), and limonin trend downward throughout the ripening period. However, the contents
of fructose, glucose, and narirutin fluctuate throughout the harvest time. Three in vitro antioxidant assays consistently
indicate that the harvest time exerts no significant influence (P>0.01) on the antioxidant capacity. Furthermore, principal
component analysis (PCA) and Pearson’s correlation test are performed to provide an overview of the complete dataset.
This study provides valuable information for evaluating the fruit quality and determining when to harvest the fruit in order
to meet the preferences of consumers. Meanwhile, our observations suggest that the fruits subjected to juice processing
should be harvested at the late maturity stage to alleviate the “delayed bitterness” problem without compromising the
antioxidant capacity and the flavonoid content in the juice.

Keywords: navel orange, antioxidant, harvest time, chemical composition

1. Introduction

Gannan navel orange (Citrus sinensis Osbeck ‘Newhall’)

Received 7 July, 2020 Accepted 16 August, 2020
Correspondence ZHANG Jun, Tel/Fax: +86-797-8393068, E-mail:
tzhangjun@gnnu.edu.cn; LIANG Yan, E-mail: zjzzf2011@gmail.
com
© 2022 CAAS. Published by Elsevier B.V. This is an open
access article under the CC BY-NC-ND license (http://
creativecommons.org/licenses/by-nc-nd/4.0/).
doi: 10.1016/S2095-3119(20)63395-0
is widely cultivated in Ganzhou (25.83°N, 114.93°E) of
Jiangxi Province, China. Currently, Ganzhou is the largest
navel orange production area in China, with an annual
output of more than 1 million tons (GGC 2020). Due to its
beautiful appearance, delicious taste, and high nutritional
value, Gannan navel orange is designated as a national
geographic indication fruit and enjoys great popularity
among the consumers in China.
262 ZHANG Jun et al. Journal of Integrative Agriculture 2022, 21(1): 261–272
The orange is one of the most widely consumed fruits
worldwide for its unique flavor and nutrition. Organic
acids and sugars are among the primary components
of oranges, and their concentrations largely affect the
characteristic taste and organoleptic quality of orange
juice. Sucrose, fructose and glucose are the major sugar
components in orange juice, and their concentrations
are usually determined by high-performance liquid
chromatography (HPLC) or gas chromatography (GC)
analysis (Kelebek et al. 2009). Phenolic compounds,
especially flavonoids (hesperidin and narirutin), are
present in large amounts in orange juice, and have shown
a wide range of health beneficial properties (Sdiri et al.
2012; Wang et al. 2016). In recent years, tremendous
attention has been paid to the antioxidant potential of
orange juice (Giuffrè et al. 2017; Hunlun et al. 2019;
Katariya et al. 2020). The phenolic compounds present in
orange juice were reported to be strongly correlated with
the antioxidant capacity determined by the 1,1-diphenyl-2-
picrylhydrazyl free radical (DPPH) and ferric ion reducing
antioxidant power (FRAP) assays (Floegel et al. 2011;
Kumar et al. 2019). However, some studies assumed
that the total antioxidant capacity of orange juice was
mainly attributed to Vc, giving rise to some differing
opinions regarding which compounds might be the major
contributors (Gardner et al. 2000; Zulueta et al. 2007).
The maturity level is an important factor responsible
for the quality of the fruits. The Valencia oranges
(C. sinensis) harvested at the late maturity stage had a
higher sugar/acid ratio (Bai et al. 2009). The contents
of total soluble solid (TSS), titratable acid (TA), and Vc
of three grapefruit cultivars varied with different harvest
times (Muhtaseb 2007). Therefore, it might be interesting
to explore how the maturity level influences orange
fruit quality, which might provide valuable information
regarding when the orange fruit should be harvested to
meet the preferences of consumers.
Eating the fresh fruit is the major consumption method
of Gannan navel orange at present. One of the major
difficulties in commercial juice production from Gannan
navel orange is the “delayed bitterness” problem, which
is mainly caused by the gradual formation of the bitter
compound limonin from the non-bitter precursor limonate
A-ring lactone under acidic conditions (Kimball and
Norman 1990; Raithore et al. 2016). Our previous study
revealed that the limonin content in the juice decreased
with the increment of postharvest storage time of the
fruit (Zhang et al. 2018). It has been reported that the
limonin contents in both Hamlin (C. sinensis) and Valencia
oranges decreased with the ripening of the fruit (Bai et al.
2016). To the best of our knowledge, there are no reports
in terms of whether the harvest time influences the limonin
content in Gannan navel orange.
Gannan navel orange is usually harvested from late
October to late December, covering a span of around
two months. A literature survey indicated that only
limited research regarding the fruit quality of Gannan
navel orange has ever been conducted, mainly focusing
on the physicochemical properties such as TSS and TA
(Hao et al. 2011). The contents and types of functional
compositions in the Gannan navel orange, as well as the
antioxidant capacity of its juice, remain largely unknown.
Therefore, the present study comprehensively investigated
the fruit quality of Gannan navel orange during its ripening
period and explored how the harvest time influenced the
physicochemical properties (TSS, TA, sucrose, fructose
and glucose), bioactive components (total polyphenol
content (TPC), total flavonoid content (TFC), Vc,
hesperidin, narirutin and limonin), and antioxidant capacity
(DPPH, 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic
acid) diammonium salt (ABTS) and FRAP) of the Gannan
navel orange juice.
2. Materials and methods
2.1. Chemicals
Vitamin C, 2,6-dichloroindophenol sodium salt, gallic acid,
quercetin, 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic
acid) diammonium salt (ABTS, 98%), 1,1-diphenyl-2-
picrylhydrazyl (DPPH free radical, 95%), 6-hydroxy-
2,5,7,8-tetramethylchromane-2-carboxylic acid (trolox,
97%), 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ), Folin-
Ciocalteu reagent, D-sucrose, D-glucose, and D-fructose
were purchased from Macklin Biochemical (Shanghai,
China). Ammonium persulphate ((NH4)2S2O8), sodium
carbonate (Na 2 CO 3 ), iron(III) chloride hexahydrate
(FeCl3∙6H2O), acetic acid (CH3COOH) and sodium acetate
(CH3COONa), were of analytical grade and purchased
from Sigma-Aldrich (Shanghai, China). Hesperidin and
narirutin were obtained commercially from Shanghai
Yuanye Bio-Technology Co., Ltd., (Shanghai, China). N,
N-dimethylformamide (DMF) was of analytical grade and
obtained from Damao Chemical Reagent Factory (Tianjin,
China). Methanol and acetonitrile were of HPLC grade
and obtained from Anaqua Chemicals Supply (Houston,
USA). Deionized water was obtained from a Milli-Q
Gradient A10 system (Millipore, Billerica, USA).
2.2. Fruit materials
Newhall navel oranges were harvested from an orchard
 ZHANG Jun et al. Journal of Integrative Agriculture 2022, 21(1): 261–272
at the National Engineering Research Center of Navel
Orange located in Ganzhou of Jiangxi Province, China.
Five healthy Newhall navel orange trees (8 years old)
grafted on trifoliate orange (Poncirus trifoliata (L.)
Raf.) and growing under the same climate and farming
conditions were selected for sample collection. The
orange trees were planted on a red-yellow loam soil
(pH 5.39±0.3) and spaced at 4 m and 3 m between and
along the rows, respectively. During the experiment,
seedcake was applied at 50.0 kg/tree in March and the
compound fertilizer was applied at 10.0 kg/tree in July.
A drip irrigation system was used to irrigate the plants in
relation to the environmental temperature and based on
the demand for water by the trees. The orchard is located
in the area of subtropical monsoon climate with an annual
average air temperature of 20°C and an average annual
rainfall of about 1 320 mm. Seven batches of fruits were
collected at one- to two-week intervals from Oct. 25 to Dec.
26, 2018. Each batch of fruit contained 50 fruits (10 from
each tree) of similar size (each fruit weight 250.0±15.0 mg)
and color determined by using a colorimeter (Konica
Minolta CM-5, Osaka, Japan), which were collected from
different parts of the crown. All fruits were sent to the
laboratory and juiced immediately by using a domestic
semi-automatic juicer (Model 4161, De’Longhi Braun
Household GmbH, Budapest, Hungary). Fresh juice
was subjected to centrifugation (Eppendorf centrifuge
5430R, 1 000×g, at 4°C) for 5 min and the supernatant
was transferred into sterile plastic Falcon tubes (50 mL)
and stored in the refrigerator at –80°C for less than one
week before analysis. The juice samples were thawed
quickly under running tap water and directly subjected to
TSS, TA, Vc, TPC, TFC and antioxidant assays, while for
the other experiments, additional steps were needed to
prepare the samples based on the analytical methods.
2.3. TSS analysis
TSS of juice was determined by using the hand-
held refractometer Atago N1 (Atago Co., Ltd., Tokyo,
Japan) and expressed as ºBrix at 20°C. Briefly, the
instrument was subjected to zero-set using deionized
water. After removing the water, one drop of juice was
added and the measurement was recorded as TSS (ºBrix).
2.4. TA and vitamin C analysis
TA was determined by titrating 20 mL of juice with a
0.1 mol L –1 sodium hydroxide aqueous solution up to
pH 8.1 according to a previous method and expressed as
grams of citric acid equivalent per 100 g of juice (Giuffrè
2019). Vitamin C was determined by the titration method,
263
using 2,6-dichlorophenolindophenol dye according to
previous literature (Stinco et al. 2012). The result was
expressed as mg ascorbic acid per 100 g of juice.
2.5. HPLC analysis of sucrose, glucose and fruc-
tose
The sucrose, glucose and fructose in the orange juice
were detected by HPLC according to a previous method
with some modifications (Kelebek et al. 2009). The juice
was diluted ten-fold with 80% methanol/water, followed
by centrifugation (Eppendorf centrifuge 5430R, 2 000×g,
at 4°C) for 10 min. The supernatant was then filtrated
through a 0.22 μm size hydrophilic polytetrafluoroethylene
syringe filter to yield the juice sample for HPLC analysis.
An Agilent 1260 system (Agilent Technology, CA, USA)
consisting of an RI detector and an Agilent ZORBAX NH2
(250 mm length, 4.6 mm i.d., 5 µm particle size) was
employed for sugar analysis. The mobile phase was an
isocratic solution (70% acetonitrile aqueous) at a flow
rate of 1.0 mL min–1. The injection volume was fixed at
10 μL, and the temperature for both column and detector
was kept at 40°C. D-sucrose (tR=8.84 min), D-glucose
(tR=7.26 min), and D-fructose (tR=6.61 min) were identified
by comparisons of their retention times with those of
the corresponding standards and the concentration of
each sugar was quantified by the external calibration
method. The calibration curves of sucrose (y=191 728x+
1 106.3, R 2=0.9996), glucose (y=139 960x+887.85, R 2
=0.9999) and fructose (y=182 405x+6 141.9, R2 =0.9994)
were constructed by plotting the peak areas versus the
concentrations (from 0.1 to 5.0 g L–1) of the corresponding
standard compound.
2.6. TPC analysis
TPC was determined spectrophotometrically with Folin-
Ciocalteu reagent according to our previous report (Guo
et al. 2020). Briefly, pre-diluted juice (20 μL, 20-fold
dilution with deionized water), deionized water (60 μL),
and pre-diluted Folin-Ciocalteu reagent (15 μL, 2-fold
dilution with deionized water) were sequentially added
into a 96-well plate and mixed well. After slightly shaking
the plate on a spectrophotometer (Tecan Spark 10M,
Männedorf, Switzerland) for 4 min at room temperature,
75 μL sodium carbonate aqueous solution (2%, w/v)
was added, followed by slight shaking for another 5 min.
After standing stationary for 15 min at room temperature,
the absorbance was then measured at 750 nm by a
spectrophotometer (Tecan Spark 10M, Männedorf,
Switzerland). Deionized water instead of juice was used
as a blank control. TPC was calculated from the linear
264 ZHANG Jun et al. Journal of Integrative Agriculture 2022, 21(1): 261–272
calibration curve of gallic acid, which was constructed by
plotting the absorbance values versus the concentrations
of gallic acid. The calibration curve (y=0.0056x–0.012,
R2=0.9998) was linear within the range of 6.25–100 mg L–1.
TPC was expressed as µmol gallic acid equivalents per
mL juice (µmol GAE mL–1).
2.7. TFC analysis
TFC was estimated by the aluminum chloride method
with minor modifications (Guo et al. 2020). To a flask
containing 500 μL juice, was sequentially added 500 μL
sodium nitrite (5%, w/v), 500 μL aluminium chloride (10%,
w/v) and 500 μL sodium hydroxide (1 mol L–1) aqueous
solution at an interval of 5 min and the solution was mixed
well upon each addition. After standing stationary for
15 min at room temperature, the reaction mixture was
diluted with deionized water to a final volume of 10 mL.
Deionized water instead of juice was used as a blank
control. The absorbance of the reaction mixture was
measured at 415 nm by a UV-vis photospectrometer
(Model 2450, Shimadzu Co., Ltd., Kyoto, Japan).
TFC was calculated from the linear calibration curve
of quercetin, which was constructed by plotting the
absorbance values versus the amounts of quercetin
and expressed as µmol of quercetin equivalents
per mL juice (µmol QE mL –1). The calibration curve
(y=0.0013x+0.0031, R 2=0.9991) was linear within the
range of 31.25–500 mg.
2.8. HPLC analysis of hesperidin and narirutin
Hesperidin and narirutin in the juice were analyzed
by our previous method (Zhang et al. 2020). Briefly,
the juice was subjected to 10-fold dilution with
20% dimethylformamide (DMF)/water, followed by
centrifugation (Eppendorf centrifuge 5430R, 2 000×g, at
4°C) for 10 min. The supernatant was then filtered through
a 0.22-μm pore-size hydrophobic polytetrafluoroethylene
syringe filter to yield the juice sample for HPLC
analysis. An Agilent 1260 System (Agilent Technology,
CA, USA), which consisted of a UV detector, a binary
gradient pump, and a sunfire™ C18 reverse-phase
column (150 mm length, 4.6 mm i.d., 3.5 μm particle
size), was employed for the analysis of hesperidin
and narirutin. The mobile phase consisted of
acetonitrile (A) and deionized water (B) at a flow rate of
1.0 mL min–1. The gradient profile was as follows: 0–
40 min, 10–25% A; 40–45 min, 25–50% A; 45–60 min,
50–65% A; 60–65 min, 65–90% A; 65–75 min, 90% A;
75–80 min, 90–10% A. The chromatogram was monitored
at 280 nm with an injection volume of 20 μL and column
temperature at 30°C. Both hesperidin and narirutin
were identified by comparisons of their retention times
and UV spectra with those of the standard compounds.
The concentration of each compound was quantified
by the external calibration curves (y=17.585x+1.8909,
R2=0.9999; y=20.448x+0.5933, R2=0.9996, for hesperidin
and narirutin, respectively), which were constructed by
plotting the peak areas versus the concentrations (from 1.0
to 100.0 mg L–1) of the standard compounds.
2.9. HPLC analysis of limonin
The determination of limonin content was performed
according to our previous method with some minor
modifications (Zhang et al. 2018). Juice (20 mL) was
acidified by adding 1.0 mol L–1 hydrochloric acid aqueous
to pH 2.0, followed by heat treatment at 50°C for
10 min. After cooling down to room temperature,
deionized water (15 mL) was added. The juice solution
was then extracted with dichloromethane (40 mL×3). The
combined dichloromethane extract was concentrated by
rotary evaporator under reduced pressure at 50°C to give
a residue, which was dissolved and diluted with methanol
to a final volume of 5.0 mL. The methanol solution was
then filtered through a 0.22-μm pore-size hydrophobic
polytetrafluoroethylene syringe filter to yield the juice
sample for HPLC analysis. An Agilent 1260 Infinity
System (Agilent Technology, CA, USA), which consisted
of a UV detector, a binary gradient pump, and a
sunfire™ C18 reverse-phase column (150 mm length,
4.6 mm i.d., 3.5 μm particle size), was employed. The
mobile phase consisted of acetonitrile (A) and deionized
water (B) at a flow rate of 1.0 mL min–1. The gradient
profile was as follows: 0–30 min, 10–70% A; 30–40 min,
70–90% A; 40–50 min, 90% A; 50–60 min, 10% A. The
detector wavelength was 210 nm with an injection volume
of 20 μL and column temperature at 30°C. The limonin
was identified by comparisons of its retention time and
UV spectrum with those of the standard compound. The
limonin content was quantified by the external calibration
method. The external calibration curve of limonin
(y=30 271x+5 851, R2=0.9995) was linear within the range
of 1.0 to 100.0 mg L–1, and constructed by plotting the
peak areas versus the concentrations of the standard
compound.
2.10. DPPH assay
The DPPH assay was performed according to a
previous method with minor modifications (Fiore et al.
2005). DPPH was freshly prepared in methanol at a
concentration of 0.1 mmol L–1. The juice was subjected
 ZHANG Jun et al. Journal of Integrative Agriculture 2022, 21(1): 261–272
to sequential two-fold dilution with deionized water to
obtain various concentrations of juice samples. A juice
sample (50 μL) was added to a 96-well plate, followed
by the addition of DPPH solution (150 μL). Deionized
water (50 μL) instead of a juice sample was added in
the control well, and methanol (150 μL) in place of the
DPPH solution was used as a blank. The plate was kept
stationary and placed in the dark for 20 min at 37°C,
then the absorption values were recorded at 517 nm
by a microplate reader (Tecan Spark 10M, Männedorf,
Switzerland). The inhibitory rate against the DPPH radical
was calculated according to the formula: Inhibition (%)=[1–
(A treated–A blank)/A control]×100, where the A treated represents
the average absorption of wells containing both DPPH
and juice sample, Ablank is the average absorption of wells
containing no DPPH, and Acontrol is the average absorption
of wells containing only DPPH. A series of concentrations
of trolox aqueous solution (6.25–100 mg L–1) was used for
the construction of a calibration curve (y=0.0069x+0.2756,
R2=0.9986). The results were expressed as µmol trolox
equivalent antioxidant capacity per mL of juice (µmol
TEAC mL–1) by a comparison with the calibration curve of
trolox.
2.11. ABTS assay
ABTS was determined according to a previous method
with some modifications (Arena et al. 2001). An aqueous
solution of ABTS (7 mmol L –1 ) was incubated with
ammonium persulphate aqueous solution (2.45 mmol L–1)
at a volume ratio of 1:1 in the dark at room temperature
for 12 h to generate the ABTS radical solution, which was
diluted with deionized water to an absorbance of 0.70±0.02
at 734 nm. Various concentrations of two-fold diluted juice
solutions (50 mL) were added into the wells of a 96-well
plate, followed by the addition of ABTS radical solution
(200 mL). Deionized water (50 μL) instead of a juice
sample was added in the control well, and water (200 μL)
in place of the ABTS radical solution was used as a
blank. The plate was steadily shaken for 10 min, then
the absorbance was measured at 734 nm by a microplate
reader (Tecan Spark 10M, Männedorf, Switzerland). The
scavenging ability against ABTS radical was calculated
according to the formula: Inhibition (%)=[1–(A treated –
Ablank)/Acontrol]×100, where Atreated represents the average
absorption of wells containing both ABTS and a juice
sample, Ablank is the average absorption of wells containing
no ABTS, and Acontrol is the average absorption of wells
containing only ABTS. The results were expressed as
µmol trolox equivalent antioxidant capacity per mL of juice
(µmol TEAC mL–1) by a comparison with the calibration
curve (y=23.97x+0.2024, R2=0.9989) of trolox, which was
265
constructed by plotting the inhibitory percentages versus
the concentrations (1.56–25.0 mg L–1) of trolox.
2.12. FRAP assay
The FRAP assay was carried out according to the
literature with a minor modification (Giuffrè 2019). A fresh
FRAP solution was prepared by mixing TPTZ solution
(10 mmol L–1) in 40 mmol L–1 hydrochloric acid, acetate
buffer (0.1 mmol L–1, pH 3.6), and ferric chloride (20 mmol
L–1) solution in 40 mmol L–1 hydrochloric acid, at a volume
ratio of 1:10:1. Various concentrations of two-fold diluted
juice solutions (50 µL) were added into a 96-well plate,
followed by the addition of freshly prepared FRAP reagent
(200 µL). The plate was then steadily shaken at room
temperature for 10 min on a spectrophotometer (Tecan
Spark 10M, Männedorf, Switzerland), followed by the
measurement of absorbance at 593 nm by the same
spectrophotometer. The results were compared to the
trolox calibration curve (y=19.875x–0.0077, R2=0.9999),
which was constructed by plotting the absorbance values
versus the concentrations (3.12–100.0 mg L–1) of trolox
and expressed as µmol trolox equivalent antioxidant
capacity per mL of juice (µmol TEAC mL–1).
2.13. Statistical analysis
At least three independent experiments were
performed for each analysis and data were expressed
as mean±standard deviation (SD). The analysis of
one way variance (ANOVA) was used to compare the
means, and determine significant differences (P<0.01).
Statistical analysis of the data was done using the SPSS
Software (version 13, SPSS, Chicago, USA). PCA and
Pearson’s correlation analysis were performed in R
language (R Core Team 2017).
3. Results and discussion
3.1. Effects of harvest time on TSS and TA
The TSS is an important factor reflecting the maturity level
of fruit, which is normally reported as grams of glucose
equivalent per 100 g juice and recorded as °Brix (Giuffrè
2019). In the present study, the TSS of Gannan navel
orange showed an incremental tendency from Oct. 25
(11.41 °Brix) to Dec. 19 (13.15 °Brix) (Table 1). However,
there was a slight decline of TSS on Dec. 26 (12.79 °Brix)
when compared to that on Dec. 19 (13.15 °Brix),
indicating that the TSS might have reached a maximal
level around Dec. 19. Contrary to the TSS, the TA
showed a declining trend during the whole ripening period
266 ZHANG Jun et al. Journal of Integrative Agriculture 2022, 21(1): 261–272
Table 1 Physicochemical properties of juice from the Gannan navel orange harvested at different time1)
Harvest time TSS (g 100 g–1) TA (g 100 g–1)
Oct. 25 11.41±0.10 E 0.69±0.02 A
Nov. 8 11.93±0.04 D 0.61±0.02 B
Nov. 22 12.26±0.05 C 0.56±0.01 C
Nov. 29 12.20±0.10 C 0.51±0.03 CD
Dec. 5 12.64±0.03 B 0.49±0.01 D
Dec. 19 13.15±0.07 A 0.50±0.01 D
Dec. 26 12.79±0.07 B 0.48±0.01 D
1)
TSS, total soluble solid; TA, titratable acid.
The data are reported as mean±standard deviation from four replicates. The different capital letters within the same column indicate a
significant statistical difference (P<0.01).
from Oct. 25 (0.69 g 100 g–1) to Dec. 26 (0.48 g 100 g–1).
A significant difference (P<0.01) was found in the TA
contents among fruits harvested at the early ripening
period, from 0.69 (Oct. 25) to 0.61 (Nov. 8), and then
declining to 0.56 g 100 g–1 (Nov. 22). At the late ripening
period (from Dec. 5 to Dec. 26), TA only decreased
slightly, from 0.49 (Dec. 5) to 0.48 g 100 g–1 (Dec. 26),
showing no significant difference (P>0.01) between them.
The TSS/TA ratio is an important parameter related to
the quality characteristics of citrus fruits (Giuffrè 2019).
Due to the increment trend of TSS versus the declining
tendency of TA during the harvesting period, the TSS/
TA ratio of Gannan navel orange gradually increased
from Oct. 25 (16.53) to Dec. 26 (26.64) (Table 1). Similar
to our observations, the TSS and TA of Gannan navel
orange harvested on Dec. 20 were reported as 13.5 °Brix
and 0.75 g 100 g–1, respectively (Zeng et al. 2012). It
has been reported that the TSS of Valencia oranges
(C. sinensis L. Osbeck ‘Valencia’) produced in Florida
remained almost constant (10.3 to 11.0 °Brix) regardless
of the harvesting time (Bai et al. 2009), which is different
from the trend in the present study, and could be mainly
ascribed to the differences in both citrus varieties and
climatic conditions.
3.2. Effects of harvest time on the contents of fruc-
tose, glucose, and sucrose
Fructose, glucose, and sucrose are the primary sugar
components in orange juice. As shown in Table 1,
Gannan navel orange contains a high amount of sucrose,
ranging from 53.39 to 67.46 g L–1, followed by glucose
(25.73–29.49 g L –1) and fructose (23.14–25.29 g L –1)
with a content ratio of about 1:1. The sucrose content
displayed an increasing tendency along with the ripening
of the fruit, from 53.39 g L–1 on Oct. 25 to 67.46 g L–1 on
Dec. 19, and then it slightly decreased to 66.42 g L–1 on
Dec. 26, achieving an increasing percentage of about
26% across the whole maturity period. The sucrose
content exhibited a significant difference (P<0.01)
TSS/TA Fructose (g L–1) Glucose (g L–1) Sucrose (g L–1)
16.53 23.14±0.41 A 26.29±0.81 AB 53.39±1.88 D
19.55 23.58±0.93 A 27.31±1.53 AB 58.78±1.07 C
21.89 22.93±0.43 A 25.79±1.12 B 61.56±0.82 BC
23.92 24.19±1.63 A 27.88±1.38 AB 61.55±1.47 BC
25.79 24.11±0.75 A 25.73±0.37 B 64.84±1.51 AB
26.30 25.29±0.49 A 29.49±0.16 A 67.46±0.70 A
26.64 24.40±0.85 A 28.09±0.09 AB 66.42±0.36 A
between the first two batches of fruits harvested at the
early maturity stage on Oct. 25 (53.39 g L–1) and Nov. 8
(58.78 g L–1), while the growth rate gradually decreased
for fruits harvested in the late ripening period, showing
no significant differences between the adjacent batches
(P>0.01). There is no obvious increasing or declining
tendency for either the fructose or glucose contents during
the whole harvesting period, which fluctuated between
23.14 and 25.29 g L–1, and between 25.79 and 29.49 g
L –1, respectively. The ratio of sucrose, glucose, and
fructose contents was about 2:1:1 in the present study,
being consistent with those reported previously (Lee and
Coates 2000; Kelebek et al. 2009). The Kozan orange
(C. sinensis L. Osbeck ‘Kozan’) contained sucrose
(59.34 g L–1), glucose (32.30 g L–1) and fructose (28.55 g
L –1 ) as its main sugar compositions (Kelebek et al.
2009), which was in agreement with the present result.
The content of sucrose in Valencia orange (C. sinensis
L. Osbeck ‘Valencia’) increased from 49.0 to 56.0 g L–1
over the ripening period from February to May, while both
fructose and glucose remained stable at around 20 g L–1
(Bai et al. 2016). This tendency was consistent with our
present observations.
3.3. Effects of harvest time on TPC, TFC and Vc
TPC showed a gradually increasing trend over the
harvest period from Oct. 25 (4.39 µmol GAE mL –1) to
Dec. 19 (5.12 µmol GAE mL–1) (Table 2). This trend was
also applicable to TFC, which increased from 1.90 µmol
QE mL–1 on Oct. 25 to 2.30 µmol QE mL–1 on Dec. 19,
for a percentage increase of about 21%. However, both
the TPC and TFC declined to 4.79 µmol GAE mL–1 and
2.16 µmol QE mL–1 on Dec. 26, respectively, which might
be ascribed to the low metabolic ability of senescent fruit
cells for producing phenolic and flavonoid compounds
(Romani 1978). Contrary to the change tendencies
of both TFC and TPC, the content of Vc exhibited a
declining trend during the whole ripening period, from
around 61.10–62.54 mg 100 g–1 for the early harvest fruits
 ZHANG Jun et al. Journal of Integrative Agriculture 2022, 21(1): 261–272 267

Table 2 Bioactive components of juice from the Gannan navel orange harvested at different time1)
TPC TFC Vc Narirutin Hesperidin Limonin
Harvest time
(µmol GAE mL–1) (µmol QE mL–1) (mg 100 g–1) (mg L–1) (mg L–1) (mg L–1)
Oct. 25 4.39±0.06 C 1.90±0.03 D 61.10±0.43 A 257.27±4.28 BC 478.21±17.14 D 13.98±0.51 A
Nov. 8 4.58±0.08 BC 2.13±0.02 BC 62.54±0.25 A 294.91±5.29 A 533.93±21.73 D 13.29±0.69 A
Nov. 22 4.49±0.03 BC 2.05±0.02 C 61.84±0.64 A 277.03±5.29 ABC 670.13±12.92 C 9.94±0.55 B
Nov. 29 4.63±0.11 BC 2.13±0.04 BC 57.78±0.99 B 292.70±20.43 AB 792.51±34.20 AB 5.21±0.34 C
Dec. 5 4.76±0.08 B 2.22±0.03 AB 51.33±0.24 CD 259.15±9.79 ABC 745.28±46.89 BC 4.42±0.34 CD
Dec. 19 5.12±0.16 A 2.30±0.04 A 52.85±0.36 C 249.58±3.17 C 839.62±22.73 A 3.59±0.25 D
Dec. 26 4.79±0.08 B 2.16±0.02 B 50.75±0.35 D 289.37±12.18 AB 827.63±7.61 AB 3.35±0.16 D
1)
TPC, total polyphenol content; TFC, total flavonoid content; Vc, vitamin C; GAE, gallic acid equivalent; QE, quercetin equivalent.
The data are reported as mean±standard deviation from three replicates. The different capital letters within the same column indicate a
significant statistical difference (P<0.01).

(Table 2) to around 50.75–52.85 mg 100 g–1 for the late
harvest fruits, showing a significant difference (P<0.01)
between them. In good agreement with the present
observations, the Vc of Valencia (C. sinensis L. Osbeck
‘Valencia’) and Bergamot fruits (C. bergamia Risso)
decreased throughout the harvest season (Bai et al.
2009; Giuffrè 2019). The Vc content was 58.4 mg 100 g–1
for Washington navel orange (C. sinensis L. Osbeck
‘Washington’) (Kimball and Norman 1990), and 55.37
and 55.11 mg 100 g–1 for Tarocco (C. sinensis L. Osbeck
‘Tarocco’) and Moro (C. sinensis L. Osbeck ‘Moro’)
sweet orange varieties, respectively (Rapisarda et al.
2008), which are similar to the Vc content reported in this
study. It has been reported that the TPC was 8.81 µmol
GAE mL–1 for Hamlin (C. sinensis Osbeck ‘Hamlin’) (Xu
et al. 2008), 5.66 µmol GAE mL–1 for Salustiana orange
(C. sinensis Osbeck ‘Salustiana’) (Roussos 2011), and
1.43 µmol GAE mL–1 for a commercially available orange
in the Belgian market (Kevers et al. 2007). The variations
of TPC between our present study and the previous
reports might be attributed to the differences in factors
such as the genotypes, cultivation conditions, post-
harvest processes, and maturity stages of the fruits. The
TFC in Fantastico (C. bergamia ‘Fantastico’) increased
with fruit ripening, from 361 mg L–1 in October to 678
mg L–1 in March of the next year (Giuffrè 2019). The
concentration of flavonoids in Persian lime (C. latifolia
Tanaka) increased during the whole fruit growth period,
whereas the phenolic compounds increased at first,
followed by a decline during the rest of the maturation
phase (Ledesma-Escobar et al. 2018). The tendency
of change of TFC/TPC in these reports was almost
consistent with the present observations. However,
it has been reported that in the flesh of Chachiensis
(C. reticulata Blanco ‘Chachiensis’), the vitamin C
content increased, while the TPC decreased by about
15% throughout the ripening period (Wang et al. 2016),
which was exactly the opposite of what we found in the
present study.
3.4. Effects of harvest time on hesperidin and na-
rirutin
As shown in Table 2, the hesperidin content increased
continuously from Oct. 25 (478.21 mg L –1) to Dec. 19
(839.62 mg L–1) with the ripening of fruit, then decreased
slightly to 827.63 mg L –1 on Dec. 26. There was a
significant difference in the hesperidin content between
the fruits harvested at the early and the late stages
(P<0.01), with an increasing percentage of about 73%
during the whole harvest period. The content of narirutin,
however, fluctuated between 249.58 (Dec. 19) and
294.91 (Nov. 8) mg L–1 throughout the maturity period,
exhibiting no obvious tendency of increase or decline.
The contents of hesperidin and narirutin were 192.2 and
43.0 mg L–1, respectively, in blood orange juice (C. sinensis
L. Osbeck ‘Moro’) (Giuffrè et al. 2017), 171.17 and 39.91
mg L –1, respectively, in Kozan (C. sinensis L. Osbeck
‘Kozan’) (Kelebek et al. 2009), 489.64 and 102.77
mg L–1, respectively, in Hamlin (C. sinensis L. Osbeck
‘Hamlin’) (Xu et al. 2008), and about 470–761 and
185–222 mg L–1, respectively, in the pigmented orange
juice (C. sinensis L. Osbeck) (Leuzzi et al. 2000). The
difference between our present results and those reported
previously might be attributable mainly to the different
Citrus species and sample preparation methods used.
In general, the hesperidin content was determined by
the HPLC method, whereas the sample preparation
method before HPLC analysis may have highly
influenced the results (Zhang et al. 2020). In contrast
with our present observations, the hesperidin content
in the Chachiensis (C. reticulata Blanco ‘Chachiensis’)
flesh increased with the ripening of fruit, at 896.47 µg
g–1 DW for immature fruit, 928.57 for semi-mature and
1 076.54 µg g–1 DW for mature fruit, while the narirutin
content was almost the same throughout the ripening
period (Wang et al. 2016). The content of narirutin in
Valencia orange (C. sinensis L. Osbeck ‘Valencia’)
remained constant in March and April, but decreased
268 ZHANG Jun et al. Journal of Integrative Agriculture 2022, 21(1): 261–272
from April to May (Bai et al. 2009). Naringin was a
main flavonoid in bergamot (C. bergamia Risso), and
its highest content was obtained in the fruits harvested
at the last sampling date on March 2017/2018 (Giuffrè
2019), showing a tendency of change similar to that of the
hesperidin in the present study.
3.5. Effects of harvest time on limonin content
Limonin is believed to be the primary bitterness compound
in navel orange juice, which is formed gradually from a
non-bitter precursor during juice processing (Raithore
et al. 2016). In this study, the non-bitter precursor in
navel orange juice was completely transferred to limonin
in the presence of acid and heat according to our previous
report (Zhang et al. 2018). As shown in Table 2, the
limonin content was the highest (13.98 mg L–1) in the
fruits harvested on Oct. 25, and it exhibited a declining
tendency with the ripening time, down to 3.35 mg L–1 on
Dec. 26, which represents about a 76% reduction over
the whole maturity period. The sensitivity threshold of
limonin content in the juice is suggested to be 6.0 mg
L–1, so a concentration less than that is considered to
be acceptable for the consumers (Raithore et al. 2016).
The limonin content in Gannan navel orange juice was
below 6.0 mg L–1 on Nov. 29 (5.21 mg L–1), therefore the
fruits harvested after this time should be suitable for juice
production. Similar to the present results, the limonin
concentration in the early Washington navel orange
(C. sinensis L. Osbeck ‘Washington’) juice was about 17.2
mg L–1 (Kimball and Norman 1990). It has been reported
that the limonin content in Valencia orange (C. sinensis
L. Osbeck ‘Valencia’) declined from February (0.90 mg
g –1) to May (0.53 mg g–1) in 2007, and from February
(5.6 mg g–1) to May (2.4 mg g–1) in 2012 (Bai et al. 2016).
The limonin content in the Thai tangerine (C. reticulata
Blanco) decreased with the ripening of the fruit, from 4.0
to 1.0 mg L–1 (Jungsakulrujirek and Noomhorm 1998).
The average limonin concentration in Hamlin oranges
Table 3 Antioxidant capacity of juice from the Gannan navel orange harvested at different time1)
Harvest time FRAP (µmol TEAC mL–1)
Oct. 25 3.66±0.12 A
Nov. 8 3.67±0.07 A
Nov. 22 3.73±0.12 A
Nov. 29 3.64±0.07 A
Dec. 5 3.60±0.21 A
Dec. 19 3.88±0.10 A
Dec. 26 3.75±0.06 A
1)
FRAP, ferric ion reducing antioxidant power; ABTS, 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt; DPPH,
1,1-diphenyl-2-picrylhydrazyl free radical; TEAC, trolox equivalent antioxidant capacity.
The data are reported as mean±standard deviation from four replicates. The same capital letter within the same column indicates no
significant statistical difference (P>0.01).
(C. sinensis L. Osbeck ‘Hamlin’) fell from a high of 6.2
mg L–1 in September to 1.8 mg L–1 in January of the next
year during the maturity season based on TLC (thin-layer
chromatography) analysis (Albach et al. 1981). These
reports were in agreement with our present results,
showing the limonin content in juice decreased with the
increase of the fruit maturity level. It is worth pointing out
that the values of limonin content were different between
the present study and some of the previous reports, which
might be attributed to the differences in the citrus species
and the sample preparation methods employed.
3.6. Effects of harvest time on antioxidant capacity
To comprehensively estimate the antioxidant capacity of
orange juice, the three platforms of DPPH, ABTS, and
FRAP were used in the present study. The antioxidant
capacity was expressed as µmol trolox equivalents per
mL orange juice (µmol TEAC mL –1) for each of these
three antioxidant platforms. As shown in Table 3, the
FRAP values did not tend to increase or decrease with
the ripening of the fruit, varying between 3.60 (Dec. 5)
and 3.88 µmol TEAC mL –1 (Dec. 19) throughout the
harvesting period. Consistent with the FRAP, the ABTS
value displayed fluctuations between 3.43 (Nov. 8) and
4.00 µmol TEAC mL –1 (Dec. 19) with no significant
difference between them (P>0.01). The antioxidant
potential against the DPPH radical was not significantly
different among the fruits harvested at different maturity
stages (P>0.01), ranging from 5.28 (Oct. 25) to 6.01 µmol
TEAC mL–1 (Dec. 5), similar to those observed in both
the FRAP and ATBS assays. Therefore, all three of
the antioxidant platforms consistently indicated that
the harvest time of the orange exerted no significant
influence (P>0.01) on the antioxidant capacity of Gannan
navel orange juice. Vitamin C, phenolic compounds and
flavonoids have been reported to be the most important
antioxidants in orange juice (Sdiri et al. 2012). In the
present study, the content of Vc declined, while the TPC/
ABTS (µmol TEAC mL–1) DPPH (µmol TEAC mL–1)
3.87±0.13 A 5.28±0.09 A
3.43±0.12 A 5.58±0.13 A
3.82±0.19 A 5.38±0.07 A
3.77±0.09 A 5.51±0.15 A
3.55±0.17 A 6.01±0.19 A
4.00±0.18 A 5.48±0.14 A
3.79±0.22 A 5.44±0.09 A
 ZHANG Jun et al. Journal of Integrative Agriculture 2022, 21(1): 261–272
TFC increased during the fruit ripening period, imposing
two opposite effects on the antioxidant capacity of juice,
which might be responsible for the fluctuations of the
values of FRAP, ABTS, and DPPH. The potency of
bergamot juice against the DPPH radical was highly
correlated with the content of its flavonoids, while the Vc
was found to be responsible for the antioxidant capacity
determined by the FRAP assay (Giuffrè 2019). It was
reported that the ABTS values of the ready-to-drink
orange juices available in Brazil were 0.58–3.15 µmol
TEAC mL–1 (Stella et al. 2011), and those of the freshly
squeezed and processed blood orange juices were
2.38–5.18 µmol TEAC mL–1 (Arena et al. 2001), which are
similar to the values in the present report. The DPPH and
ABTS radical scavenging capacity of seven commercial
red orange juices were 1.26–3.08 and 2.28–3.53 µmol
TEAC mL–1, respectively (Fiore et al. 2005), slightly less
than those reported in the present study, which might
be due to the deterioration of antioxidants during the
processing and storage period of the commercial orange
juice (Klimczak et al. 2007).
3.7. PCA and Pearson’s correlation analysis
PCA is a statistical method that reduces a large number of
variables into a smaller number of uncorrelated variables
and provides an overview of the complete dataset (Kawaii
et al. 1999). Two principal components (PCs) were
obtained from the orange juice data sets, accounting
for 99.9% of the cumulative percentage of the total
variation, with PC 1 and PC 2 accounting for 97.89 and
2.03% of the variance, respectively. The results of PCA
are presented as the scores and loading plots (Fig. 1),
where the contribution of a variable to PC 1 and PC 2 is
indicated by the direction and length of the vector. As
Fig. 1 The loading and score plots of the PCA describing data sets obtained from the Gannan navel orange juice.
269
shown in Fig. 1, the oranges harvested on Oct. 25, Nov. 8,
Nov. 22, and Dec. 19 were grouped very well, while those
harvested on Nov. 29, Dec. 5, and Dec. 26 overlapped
each other partially. The proximity of the early harvesting
fruits (Oct. 25, Nov. 8, and Nov. 22) to the loading
vectors involving Vc, limonin, and TA, revealed that the
orange fruits harvested at the early ripening stage were
characterized by high contents of Vc, limonin, and TA.
The loading vectors for hesperidin, sucrose and TSS were
close to the fruits harvested at the late maturity stage (Dec.
19 and Dec. 26), indicating these three were the main
chemical components in the late harvest fruits. Pearson’s
correlation analysis revealed that the hesperidin content
was highly negatively correlated with the contents of
limonin (coefficient=–0.96, P<0.01), TA (coefficient=–0.92,
P<0.01), and Vc (coefficient=–0.78, P<0.01) in fruit, while
positively correlated with TSS (coefficient=0.88, P<0.01),
sucrose (coefficient=0.90, P<0.01), TPC (coefficient=0.73,
P<0.01), and TFC (coefficient=0.74, P<0.01) (Fig. 2).
This pattern was in good agreement with the PCA results,
where the variables of hesperidin, TSS, and sucrose
had the same vector direction which was opposite to
the vector direction of limonin, TA, and Vc. As shown
in Fig. 2, DPPH demonstrated low correlations with
TPC (coefficient=0.21, P<0.01), TFC (coefficient=0.48,
P<0.01), and Vc (coefficient=–0.43, P<0.01). Similar to
DPPH, both ABTS and FRAP were poorly correlated with
TPC (coefficient=0.22, 0.42, respectively, P<0.01), TFC
(coefficient=–0.07, 0.26, respectively, P<0.01), and Vc
(coefficient=–0.14, –0.15, respectively, P<0.01). These
data indicated that the antioxidant capacity of Gannan
navel orange juice was not determined by only one of
the three factors including TPC, TFC, and Vc, which
should indicate a synergistic effect among all these
factors. Xu et al. (2008) suggested that the Vc, not the
270 ZHANG Jun et al. Journal of Integrative Agriculture 2022, 21(1): 261–272

DPPH

FRAP

ABTS

Hesperidin

TSS

Sucrose Correlation
1.0
TPC 0.5
0
TFC
–0.5
Fructose –1.0

Glucose

Narirutin

Vc

TA

Limonin
AP
H

TS

in

se

se

tin

Vc

TA

in
os
TS
PP

TP

TF
rid

on
to

co

iru
AB
FR

cr
pe

uc

m
D

lu

ar
Su

Li
G
Fr
es

N
H

Fig. 2 Pearson’s correlation analysis among the data sets from the Gannan navel orange juice. DPPH, 1,1-diphenyl-2-picrylhydrazyl
free radical; FRAP, ferric ion reducing antioxidant power; ABTS, 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium
salt; TSS, total soluble solid; TPC, total polyphenol content; TFC, total flavonoid content; Vc, vitamin C; TA, titratable acid.
*
, P<0.05; **, P<0.01; ***, P<0.001.

phenolic compounds, was the major contributor to the
total antioxidant capacity of citrus juice. However, some
studies reported that the phenolic compounds were
primarily responsible for the total antioxidant capacity
of citrus (Rapisarda et al. 1999; Hunlun et al. 2019).
The discrepancy between the previous and the present
observations may arise from the differences such as the
citrus varieties, fruit maturity stages, sample preparation
procedures and analytical methods used. The present
study indicated that the fruits harvested at the early
ripening time exhibited high contents of Vc and TA, which
might be potentially beneficial for human health and
desirable for some consumers who prefer oranges with a
more acid taste. Meanwhile, the early harvested fruit was
suggested to have an enhanced ability to withstand long-
distance transportation and postharvest storage (Eckert
and Eaks 1989). For the oranges harvested at the middle
stage of maturation, they accumulated more sugars
and exhibited a relatively high value of TSS/TA, thereby
making them more popular as a fresh eating fruit among
the consumers inclined to eat sweeter fruits. However,
the oranges harvested at the late maturity stage were
very suitable for juice processing due to the much lower
amount of limonin as compared to the other maturity
stages while still maintaining relatively high antioxidant
capacity and flavonoid content.
4. Conclusion
In this study, the effect of harvest time on the chemical
composition and antioxidant capacity of Gannan navel
orange juice was comprehensively investigated for the
first time. Our results indicated that the fruits harvested
during the early ripening time had high contents of TA,
Vc, and limonin, and all of them trended downward
throughout the maturity period of the fruit. The contents
of TSS, sucrose, hesperidin, TPC and TFC, however,
demonstrated increasing trends with the ripening of fruit,
followed by a slight decline at the late maturity stage.
Throughout the harvest period, the contents of fructose,
 ZHANG Jun et al. Journal of Integrative Agriculture 2022, 21(1): 261–272
glucose, and narirutin fluctuated without an apparent
tendency of either increase or decrease. Furthermore, the
harvest time exerted no obvious effect on the antioxidant
capacity of the Gannan navel orange juice, evident from
the consistent results of DPPH, ABTS, and FRAP assays
showing that there were no statistical differences (P>0.01)
in antioxidant capacity for the fruits harvested at different
maturity stages. The present study provides valuable
information for evaluating the fruit quality and determining
when to harvest the fruit to meet the preferences of
different consumers. Meanwhile, our observations
suggest that the fruits subjected to juice processing
should be harvested at the late maturity stage to alleviate
the “delayed bitterness” problem without compromising
the antioxidant capacity or the flavonoid content in the
juice.
Acknowledgements
This research work was financially supported by the
National Natural Science Foundation of China (31860091),
the Natural Science Foundation of Jiangxi Province,
China (20171BCB24011), the Open Foundation of
National Engineering Research Center of Navel Orange,
and the Research Foundation of Ganzhou, Jiangxi, China
(2017179 and 201960). The authors also thank Dr.
Kumaravel Kaliaperumal from Gannan Normal University
for proofreading this manuscript.
Declaration of competing interest
The authors declare that they have no conflict of interest.
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Executive Editor-in-Chief WANG Qiang
Managing Editor WENG Ling-yun