Plant Physiology and Biochemistry 126 (2018) 117–125

Contents lists available at ScienceDirect

Plant Physiology and Biochemistry

journal homepage: www.elsevier.com/locate/plaphy

Research article

Metabolomic characterization of pitaya fruit from three red-skinned T

cultivars with different pulp colors
Qingzhu Huaa, Canbin Chena, Noemi Tel Zurb, Huicong Wanga, Jingyu Wua, Jianye Chena,
Zhike Zhanga, Jietang Zhaoa, Guibing Hua, Yonghua Qina,∗
a
State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources/Guangdong Provincial Key Laboratory of Postharvest Science of Fruits and
Vegetables/Key Laboratory of South China Horticultural Crop Biology and Germplasm Enhancement, Ministry of Agriculture, College of Horticulture, South China
Agricultural University, Guangzhou 510642, China
b
French Associates Institute for Agriculture and Biotechnology of Drylands, The J. Blaustein Institutes for Desert Research, Ben-Gurion University of the Negev, Sede Boqer
84990, Israel

A R T I C L E I N F O A B S T R A C T

Keywords: Pitaya is a new fruit crop, whose exotically colored fruits have excellent nutritional and antioxidant properties.
Hylocereus In this study, the primary metabolite profiles of three pitaya cultivars i.e. 'Guanhuahong' (red peel with red
Fruit maturation pulp), 'Guanhuabai' (red peel with white pulp) and 'Guanhuahongfen' (red peel with pink pulp) were investigated
Metabolite profiling using GC-MS and Ultraviolet–visible spectroscopy. In the fruit pulp, levels of starch, organic acids, and inositol
GC-MS
decreased as the fruit matured. Glucose, fructose, sucrose and sorbitol contents increased gradually during fruit
maturation and reached their highest levels in the pulp at the mature stage. Citramalic acid was identified for the
first time in the pulp of Hylocereus species. Higher levels of total phenols, flavonoids and antioxidant activities
were detected in the peel than in the pulp during fruit maturation of all three cultivars. The finding of higher
levels of total phenols and flavonoids in the pitaya peel than in the pulp at the mature stage suggests that pitaya
peels are a good source of natural phenols and flavonoids.

1. Introduction Betalains are important secondary metabolites with desirable health

properties, which are conferred by their antioxidant, free radical
Pitayas, also known as dragon fruit, are species of vine cactus – scavenging (Wu et al., 2006; Tenore et al., 2012), antitumor and other
belonging to the genus Hylocereus (Berger) Britton and Rose (Cactaceae) biological activities (Song et al., 2016a,b; Stintzing et al., 2004; Strack
– that are native to Mexico, Central America and South America. Today, et al., 2003; Osorio-Esquivel et al., 2011). Additionally, pitaya seed oil
several Hylocereus species are cultivated as a fruit crop in many tropical contains significant concentrations of unsaturated fatty acids, such as
and subtropical areas worldwide. The fruits are prized not only for their linoleic and linolenic acids (Ariffin et al., 2009). This natural oil thus
exotic appearance and striking colors but also for their health properties finds application in therapeutic and cosmetic preparations for skin, hair
(Kim et al., 2011; Song et al., 2016a,b) and nutritional value, being a and nail care (Ariffin et al., 2009; Darmstadt et al., 2002; Kamairudin
source of polyphenols, vitamin C, sugars, organic acids, phytalbumin, et al., 2014). As a result of the above-described commercial value of
amino acids and pigments (flavonoids and betalains) (Suh et al., 2014; pitaya species as crop plants and the excellent nutritional properties of
Esquivel et al., 2007a,b). A particular advantage of growing pitayas as a the fruit, these species are attracting increasing attention the world over
crop in dry regions is their potential to contribute to agricultural de- (Yolanda and Jose, 2012).
velopment in those regions by virtue of their greater tolerance to long- A number studies have been undertaken to ascertain the physico-
term drought stress and poor soils. chemical characteristics of ripe fruit, with most focusing on analyses of
The genus Hylocereus comprises 14 species, all characterized by nutritional compositions and antioxidant and antiproliferative activities
triangular stems and fruits with broad scales (Tel-Zur et al., 2011). The (Menezes Cordeiro et al., 2015; Song et al., 2016c). However, little
colors of the fruit peel and pulp are conferred by pigments, particularly information is available on the physicochemical changes taking place
the water-soluble nitrogen-containing betalains, which include the red- during fruit growth and development, which is vital in any program
violet betacyanins and the yellow betaxanthin (Stintzing et al., 2002). designed to develop superior cultivars. In the current study, this lack of

∗
Corresponding author.
E-mail address: [email protected] (Y. Qin).

https://doi.org/10.1016/j.plaphy.2018.02.027
Received 9 September 2017; Received in revised form 2 January 2018; Accepted 26 February 2018
Available online 02 March 2018
0981-9428/ © 2018 Elsevier Masson SAS. All rights reserved.
Q. Hua et al. Plant Physiology and Biochemistry 126 (2018) 117–125

information was addressed by characterizing three new red-skinned and Qiu, 2006) with minor modifications. Briefly, 100 μL and 500 μL
fruit cultivars with pulp of different colors. The findings constitute aliquots of the 80% (v/v) methanolic extracts from the peel and pulp
fundamental information about the physicochemical changes taking were used in the assay, respectively. A mixture of 1.9 mL of 80% (v/v)
place in the different pitaya cultivars with different pulp colors and may methanol and 300 μL 5% NaNO2 was added to each aliquot, followed by
find application in the development of a highly reliable 'maturity index' storage at room temperature for 6 min. Thereafter, 300 μL of 10% Al
that can be used for taking harvest decisions. (NO3)3 and 2 mL of 4% NaOH were added to each mixture. After
10 min, absorbance was determined at 510 nm with an Infinite M200
2. Materials and methods spectrophotometer (Tecan Co.). Rutin was used as the reference com-
pound, and values were expressed as mg of rutin equivalent (RE)/g of
2.1. Plant material fresh weight. The standard solution of rutin was diluted to 30, 60, 90,
120 and 150 μg/mL and used for constructing a calibration curve.
Three new red-skinned fruit cultivars, i.e., 'Guanhuahong' (red peel
with red pulp), 'Guanhuabai' (red peel with white pulp) and 2.5. Determination of total antioxidant activity
'Guanhuahongfen' (red peel with pink pulp) were selected from
'Conghuazhixing' [the hybrid offspring of H. monacanthus (Lem.) Britton Total antioxidant activity was determined using Total Antioxidant
and Rose and H. undatus (Haw.) Britton and Rose] through seedling Capacity Assay Kit with ABTS and FRAP detection capabilities, ac-
selection. All three cultivars are self-compatible and have good com- cording to the manufacturer's instructions with minor modifications.
mercial potential in terms of their large fruit size and excellent fruit Briefly, 20 μL of peroxidase solution, 10 μL of 80% methanol (as a
quality, high yields and long storage life. The cultivars were planted in blank) and 10 μL of each extract were added to each well of a 96-well
the same orchard of Dalingshan Forest Park, Dongguan City, microplate and mixed lightly. Thereafter, 70 μL of the ABTS working
Guangdong Province, China. Fruits were harvested from mid-July to solution was mixed lightly into each well. The plates were stored at
mid-August 2014 at five critical stages of maturity development, i.e., room temperature for 30 min, and the absorbance at 414 nm was read
black seed stage (on the 19th day after flowering; DAF), three pulp with a microplate reader (Infinite M200, Tecan Co.). Total antioxidant
coloration stages (on the 23rd, 25th and 27th DAF) and at the mature activity was calculated according to the standard curve constructed
stage (on the 29th DAF) (Fig. 1). During this period, temperatures with a Trolox standard solution. For the FRAP method, 5 μL of 80%
ranged from 27 to 36 °C (Fig. S1). The time from full flowering to fruit methanol solution (as a blank), 5 μL of each extract and 180 μL of FRAP
harvest was 28–30 days. Five uniformly sized fruits were randomly solution were added to each well of a 96-well microplate, and the mi-
selected from each cultivar. A section (including peel and pulp) ap- croplates were maintained at 37 °C for 5 min. The absorbance was read
proximately 1 cm thick was sliced from each whole fruit at its equator at 593 nm with a microplate reader (Infinite M200, Tecan Co.).
and then divided into four equal parts. Peel and pulp fractions from the FeSO4.7H2O was used to construct a standard curve, and data were
section were separated, frozen quickly in liquid nitrogen and stored at expressed as the concentration of FeSO4 (mM).
−80 °C until used for metabolite analyses.
2.6. Assay of total starch content
2.2. Chemical reagents
Total starch content was measured using a Starch Content Assay Kit,
Methanol, acetone, Folin-Ciocalteu reagent (FCR), gallic acid, rutin, with some modifications. Starch was separated from the soluble sugars
Na2CO3, NaNO2, Al(NO3)3, NaOH, and chloroform were purchased from with 80% ethyl alcohol and subjected to acid hydrolysis. Then, the
Sangon Biotech company (Shanghai, China). Total Antioxidant glucose was determined using anthrone colorimetry as follows: 100 mg
Capacity Assay Kits with Rapid ABTS [2, 2′-azino-bis (3-ethyl- of pulp powder was homogenized in 1 mL of reagent 1 and extracted for
benzthiazoline-6-sulfonic acid)] (S0121) and FRAP [ferric reducing 30 min at 80 °C. The mixture was centrifuged at 3000 g for 5 min at
ability of plasma] (S0116) detection capabilities were bought from 25 °C, and the precipitate was dissolved in 500 μL of distilled water and
Beyotime Biotechnology Company (Shanghai, China). A Starch Assay maintained at 95 °C for 15 min. After allowing the sample to cool,
Kit was purchased from Comin Biotechnology Company (Suzhou, 350 μL of reagent 2 was added, and the mixture was stored at 25 °C for
China). Pure pyridine, N-methyl-N-(trimethylsilyl) trifluoroacetamide 15 min, followed by gentle vortex for 3–5 times. Distilled water
(MSTFA), methoxyamine hydrochloride, and standard compounds were (850 μL) was added to the mixture, which was centrifuged at 3000 g for
obtained from Sigma Chemical Co. (St. Louis, MO, USA). 10 min at 25 °C. The supernatant was diluted 10 times with an ex-
tracting solution containing reagent 2 and distilled water (3:7, v/v).
2.3. Determination of chlorophyll content Then, 250 μL of working solution (225 mL of reagent 2, 3.75 mL of
distilled water and 21.25 mL of H2SO4) was added to 50 μL of the di-
One gram of frozen peel was ground to a power in liquid nitrogen luted supernatant. After the reaction mixture had been heated at 95 °C
and transferred to a 15 mL tube, to which 5 mL of 80% (v/v) acetone for 10 min, the sample was assayed at 620 nm with a microplate reader
containing about 0.1 g calcium carbonate were added. The mixture was (Infinite M200, Tecan Co.). Starch content was calculated according to
shaken on a vortex, sonicated for 10 min with an ultrasonic cleaner the following formula: Starch content (mg/g.fresh
(SB25-12DT, Ningbo, China), and then stored in the dark at room weight) = 10*0.578*(A+0.0295)/W (where A - absorbance; W - fresh
temperature until the residue became colorless (approximately 60 min). weight).
The extract was centrifuged at 5000 rpm for 10 min. The chlorophyll
content in the supernatant was determined spectrophotometrically at 2.7. Determination of the concentrations of sugars and organic acids
645 nm and 663 nm (Infinite M200, Tecan Co.), according to the
method of Arnon (1949). Gas chromatography-mass spectrometry (GC-MS) was used to de-
termine sugars and organic acids according to Lisec et al. (2015), with
2.4. Analyses of betalains, total phenols and flavonoids some modifications: 100 mg of pulp were ground to powder in liquid
nitrogen and then transferred into a 2 mL tube. Each sample was ex-
Betalains were determined according to the procedure of Hua et al. tracted with 1.4 mL of 70% (v/v) pre-cooled methanol (−20 °C) by
(2016). The FCR method (Singleton and Rossi, 1965) was used to assay vortexing for 10 s. Then, as an internal quantitative standard, 60 μL of
concentrations of total phenols in the peel and pulp. Levels of total ribitol (0.2 mg/mL dissolved in doubly distilled H2O) was added to each
flavonoids were determined according to the method of You et al. (You mixture and vortexed for 10 s. The mixtures were shaken at 70 °C in a

118
Q. Hua et al. Plant Physiology and Biochemistry 126 (2018) 117–125

Fig. 1. Changes in skin and pulp color during fruit maturation stages in the cultivars: (A) 'Guanhuabai', (B) 'Guanhuahongfen' and (C) 'Guanhuahong'. A scale plate is shown where the
black and white squares are 1 cm × 1 cm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Thermomixer Compact (Eppendorf, Germany) at 950 rpm for 10 min, cooled doubly distilled H2O (4 °C) was added, and the mixtures were
followed by centrifugation at 11,000 g for 10 min. The supernatants vortexed for 10 s. The mixtures were then centrifuged at 2200 g for
were transferred to Schott GL14 glass vials. To each supernatant, a 15 min. For each mixture, 100 μL of the upper phase (polar phase) was
mixture of 750 μL pre-cooled chloroform (at −20 °C) and 1.5 mL of pre- transferred into a new 1.5 mL tube, and the tubes were dried in a

119
Q. Hua et al. Plant Physiology and Biochemistry 126 (2018) 117–125

vacuum concentrator (Eppendorf Concentrator plus, Germany) at room

temperature. The dried samples were oximated with 40 μL of methox-
yamination reagent. One derivatization reaction without samples was
used as a control. The reaction mixtures were incubated at 37 °C in a
Thermomixer Compact (Eppendorf, Germany) at 700 rpm for 2 h. After
oximation, silylation was performed by adding 60 μL of MSTFA to each
mixture, and the mixtures were incubated at 37 °C in a Thermomixer
Compact (Eppendorf, Germany) at 700 rpm for 30 min. 80 μL of each
derivatization products were transferred into glass vials for GC-MS
analysis.
GC-MS analysis was performed using an Agilent 7890A GC system
equipped with an Agilent 7693 autosampler and Agilent 5975C-inert
MSD with Triple Axis Detector (Agilent, Atlanta, GA). A HP-5MS ca-
pillary column (5% phenyl methyl siloxane, 30 m * 0.25 mm i.d.,
0.25 μm film thickness) was used with helium gas at a constant flow of
1 mL/min. One microliter of derivatized sample was injected at 230 °C
in split mode with the split ratio adjusted to 1:20. The total flow rate of
the injection port was 24 mL/min. The following temperature regime
was used: 70 °C for 2 min, followed by 210 °C from 2 to 16 min with a
10 °C per min hold, followed by 250 °C from 16 to 24 min with a 5 °C
per min hold, followed by a 10 °C per min ramp to 330 °C from 24 to Fig. 2. Chlorophyll contents in the three cultivars in the fruit peel after flowering. The
36 min and a final holding for 3 min. The electron ionization was set at curves were drawn according to the equation: ax2 + bx + c using a binomial formula in
70 eV, and the mass spectra were recorded over the range of 50–600 m/ Microsoft Office Excel 2007. The values of a, b and c are listed in the Table below. The
z. vertical bars represent the SD of the mean for three replicates. Different letters indicate
significant differences within each pitaya cultivar at the same stage (p ≤ 0.05; Duncan's
test).
2.8. Statistical analysis

All experiments were performed in triplicate. Mean values, standard cultivars during fruit maturation (Fig. 3D). The relatively higher levels
deviations and analyses of variance (ANOVA) were calculated with of betalains in the pulp of the three cultivars on the 19th DAF (black
SPSS Statistics17 software. Multiple mean comparisons were performed seed stage) are probably due to the high content of these pigments in
using Duncan's test (p ≤ 0.05), with 95% confidence intervals for each the seeds, since the seeds of some betalain-producing plants are known
mean. to contain high concentrations of these phytochemicals (Abderrahim
et al., 2015; Caldas-Cueva et al., 2016). The higher contents of beta-
3. Results and discussion cyanins and betaxanthins in the peel than in the pulp suggests that the
peel could serve as a potential source of pigments. In summary, our
3.1. Changes in chlorophyll concentration in the peel during fruit results are consistent with the finding of Suh et al. (2014) where they
maturation reported that significant differences were observed among different
varieties and different organs of pitaya Hylocereus. Additionally, our
As shown in Fig. 2, chlorophyll contents decreased in all three findings that the coloration time of the pulp is independent of that in
cultivars as the fruit matured. The chlorophyll contents in the peels the peel (being earlier in the pulp than in the peel for 'Guanhuahong'
were significantly higher in 'Guanhuabai' and 'Guanhuahongfen' than in and 'Guanhuahongfen') are in accordance with previous studies in pi-
'Guanhuahong' before the mature stage, but at the mature stage there tayas and other cactus plants (Jamaludin et al., 2011; Coria Cayupan
were no significant differences between the three cultivars. These re- et al., 2011; Castellar et al., 2012).
sults are consistent with the findings of previous studies in red-fleshed
and yellow pitayas (Jamaludin et al., 2011; Nerd and Mizrahi, 1998).
3.3. Changes in the concentrations of total phenols in the peel and pulp
3.2. Changes in the concentration of betalains in the peel and pulp during during fruit maturation
fruit maturation
As shown in Fig. 4, there was a negative correlation between the
The contents of the two classes of betalains – betacyanins and be- concentration of total phenols in the pulp and that in the peel the
taxanthins (absorbing in the UV spectrum at 538 nm 483 nm, respec- during fruit maturation. The levels of total phenols in the peel of the
tively) – gradually increased in the peels of the three cultivars during three cultivars increased gradually during fruit maturation to a max-
fruit maturation and reached maximum levels at the fully mature stage imum at the mature stage (Fig. 4A), but in the pulp, they rose slightly
(Fig. 3A and B). The highest levels of betacyanins and betaxanthins up to the 23rd DAF and decreased thereafter (Fig. 4B), with the highest
were detected in the peel of 'Guanhuahongfen', followed by 'Guanhua- values for total phenols being 2.82, 1.84 and 1.59 mg/g FW for the pulp
hong' and 'Guanhuabai'. The concentrations of betacyanins and betax- of 'Guanhuahongfen', 'Guanhuahong' and 'Guanhuabai', respectively
anthins in the pulp of 'Guanhuahong' were significantly higher than (Fig. 4B). Higher levels of total phenols were detected in both the peel
those in 'Guanhuahongfen' and 'Guanhuabai' from the 25th DAF to 29th and the pulp of 'Guanhuahongfen' vs the other two cultivars. The con-
DAF, namely, through the stages of full coloration to the mature stage centrations of total phenols in the peel of mature fruits were higher
(Fig. 3C and D). In the 'Guanhuahong' pulp, the betacyanin con- than those in the pulp, which suggests that pitaya peels are a promising
centration increased sharply towards fruit maturity, whereas the levels source of natural phenols. Our results are in accordance with those of
in the 'Guanhuahongfen' and 'Guanhuabai' pulp remained relatively previous studies in Hylocereus showing that the levels of most betalain-
stable (Fig. 3C). Betaxanthin concentration increased in the pulp of related metabolites in the fruit peel were significantly higher than that
'Guanhuahong' towards the mature fruit stage, whereas betaxanthin of the fruit pulp (Kim et al., 2011; Wu et al., 2006).
levels in the pulp of 'Guanhuahongfen' and 'Guanhuabai' decreased
gradually, with no significant differences between the latter two

120
Q. Hua et al. Plant Physiology and Biochemistry 126 (2018) 117–125

Fig. 3. Relationship in the three cultivars between day after

pollination and (A) peel levels of betacyanin, (B) peel levels
of betaxanthin, (C) pulp levels of betacyanin, and (D) pulp
levels of betaxanthin. The curves were fitted using a bino-
mial formula in Microsoft Office Excel 2007, according to
the equation: ax2 + bx + c. The values of a, b and c are
given in the Table below. Vertical bars represent the SD of
the mean for three replicates. Different letters indicate sig-
nificant differences within each pitaya at the same stage
(p ≤ 0.05; Duncan's test).

Fig. 4. Changes in the concentrations of total phenols in the peel (A) and pulp (B) in the three pitaya cultivars during fruit maturation. Vertical bars represent the SD of the mean for three
replicates. Different letters indicate significant differences within each pitaya at the same stage (p ≤ 0.05 Duncan's test).

Fig. 5. Concentrations of total flavonoids during fruit maturation in (A) the peel and (B) the pulp in the three cultivars. Vertical bars represent the SD of the mean for three replicates.
Different letters indicate significant differences within each pitaya at the same stage (p ≤ 0.05; Duncan's test).

3.4. Changes in the concentrations of total flavonoids in the peel and pulp up to the 23rd DAF and decreased thereafter (Fig. 5A). In the pulp of all
during fruit maturation three cultivars, the highest levels of total flavonoids were detected on
the 23rd DAF, after which they declined rapidly (Fig. 5B). At the mature
Total flavonoid levels in the peel of 'Guanhuahongfen' and stage, the lowest concentrations of total flavonoids were measured in
'Guanhuahong' rose gradually during fruit maturation stages in a pat- the peel and pulp of 'Guanhuabai'. For all three cultures, the levels of
tern that was similar to the patterns for betalains and total phenols, total flavonoids in the peel were higher than those in the pulp at the
with the highest concentration being detected in 'Guanhuahongfen'. In mature stage. These findings were similar to those for concentrations of
contrast, in 'Guanhuabai' the concentration of total flavonoids increased total phenols and in keeping with those of Wu et al. (2006) for red

121
Q. Hua et al. Plant Physiology and Biochemistry 126 (2018) 117–125

Fig. 6. Total antioxidant activity during fruit maturation in the (A, C) peel and (B, D) pulp of the three cultivars. (A, C) ABTS method; (B, D) FRAP method. Vertical bars represent the SD of
the mean for three replicates. Different letters indicate significant differences within each pitaya at the same stage (p ≤ 0.05; Duncan's test).

Table 1 pitaya.
P-value of correlation of total antioxidant activity and betalains, total phenols and fla-
vonoids.
3.5. Changes in total antioxidant activity in the peel and pulp during fruit
Growth Total phenols Total flavonoids Betacyanin Betaxanthin maturation

ABTS—Peel 0.000 0.000 0.000 0.000 As shown in Fig. 6, similar results and trends were obtained with the
FRAP—Peel 0.000 0.000 0.000 0.000
two methods, i.e., ABTS and FRAP, used to assay total antioxidant ac-
ABTS—Pulp 0.045 0.007 0.075 0.037
FRAP—Pulp 0.003 0.007 0.187 0.121 tivity in the pulp and peel. The agreement between the results of the
two methods suggests that both are suitable for the assay of antioxidant
*The regression method is panel Random-effects GLS regression. activity in Hylocereus. For the peel of all three cultivars, there were no
marked changes in antioxidant activity up to the 27th DAF, but from the
27th DAF to 29th DAF total antioxidant activity increased significantly
(Fig. 6A and C). In contrast, in the pulp, total antioxidant activity in-
creased markedly up to the 23rd DAF and decreased gradually there-
after (Fig. 6B and D). For the pulp, the changes in the total antioxidant
activity were in agreement with the accumulation of total flavonoids
(Fig. 5B). The total antioxidant activity in the peel was higher than that
in pulp throughout fruit maturation for all three cultivars (Fig. 6), with
the highest level being found in 'Guanhuahongfen', followed by
'Guanhuahong' and 'Guanhuabai' (Fig. 6). These results suggest that
peels can be considered as a good source of antioxidants (Wu et al.,
2006; Lourith and Kanlayavattanakul, 2013).
The findings thus indicated that the pattern of total antioxidant
activity in the peel and the pulp of the three cultivars was similar to the
patterns for betalains, total phenols and total flavonoids during fruit
maturation (Figs. 3–6, Table 1). This similarity in the patterns is in
keeping with the strong positive correlation previously reported be-
tween total antioxidant activity and betalains, total phenols and total
flavonoids in pitayas and in Opuntia (Wu et al., 2006; Osorio-Esquivel
Fig. 7. Total starch content in the pulp during fruit maturation. Vertical bars represent et al., 2011; Esquivel et al., 2007a,b).
the SD of the mean for three replicates. Different letters indicate significant differences
within each pitaya at the same stage (p ≤ 0.05; Duncan's test).
3.6. Changes in the concentration of total starch in the pulp during fruit
maturation

An investigation of total starch content in the pulp of the different

cultivars (Fig. 7) revealed that total starch initially decreased sharply in

122
Q. Hua et al. Plant Physiology and Biochemistry 126 (2018) 117–125

Fig. 8. Concentrations of major sugars in the pulp during fruit maturation. (A) glucose, (B) fructose, (C) sorbitol, (D) inositol, and (E) sucrose. Vertical bars represent the SD of the mean
for three replicates. Different letters indicate significant differences within each pitaya at the same stage (p ≤ 0.05; Duncan's test).

'Guanhuahong' and 'Guanhuahongfen' and then more gradually as fruit 3.7. Changes in concentrations and compositions of major sugars in the pulp
maturation progressed. In contrast, in 'Guanhuabai' total starch in- during fruit maturation
creased slightly up to the 23rd DAF and then decreased thereafter. In all
three cultivars, total starch was markedly higher during the immature Glucose, fructose, sorbitol, inositol and sucrose showed similar ac-
stages of fruit development. After the 27th DAF, total starch content cumulation patterns in all three cultivars (Fig. 8), with the concentra-
began to level out, with the lowest values for all three cultivars being tions of all these soluble sugars, except inositol, accumulating gradually
obtained at the mature stage (29th DAF) (Fig. 7). during fruit growth and development to a maximum at the fully mature
Starch is a temporary storage form of carbon that can be catabolized stage (Fig. 8A–C and E). For inositol, concentrations increased steeply
to produce sugars during fruit growth and development. In different up to the 23rd DAF and then decreased gradually (Fig. 8D).
fruits, starch contents vary among the different cultivars, plant tissues It has been reported that the main sugars in pitaya are glucose and
and growth stages (Li et al., 2002; Al-Madhagi et al., 2014). In the fructose (Nomura et al., 2005). In this study, average glucose contents
current study, the highest starch content was found in 'Guanghuahong', of the three cultivars accounted for 39.7% (the percentage is the ratio of
followed by 'Guanghuabai' and 'Guanghuahongfen', as was to be ex- glucose to glucose, fructose, sorbitol, inositol and sucrose), followed by
pected from the average weight per fruit, i.e., 376.7 g/fruit for 'Guan- fructose (29.3%). Sucrose concentrations remained relatively low
ghuahong', followed by 300.0 g/fruit for 'Guanghuabai' and 239.6 g/ (1.9%) during fruit maturation, which is in accordance with previous
fruit for 'Guanghuahongfen'. These results suggest that starch content is findings in Hylocereus (Suh et al., 2014; Esquivel et al., 2007a,b;
positively correlated to weight per fruit in pitaya. Nomura et al., 2005; Yang et al., 2012).

123
Q. Hua et al. Plant Physiology and Biochemistry 126 (2018) 117–125

Fig. 9. Concentrations of major organic acids in the pulp during fruit maturation. (A) malic acid, (B) citric acid, (C) citramalic acid, and (D) oxalic acid. Vertical bars represent the SD of
the mean for three replicates. Different letters indicate significant differences within each pitaya species at the same stage (p ≤ 0.05; Duncan's test).

3.8. Changes in concentrations and compositions of major organic acids in most common and abundant acids in fruits of many plants (Glew et al.,
the pulp during fruit maturation 2005). In our study, four organic acids were identified in the pulp of the
three cultivars – malic, citric, citramalic, and oxalic acids – with malic
The three cultivars showed similar patterns of accumulation of and citramalic acids being particularly abundant. Citramalic acid is an
malic, citric, citramalic and oxalic acids during fruit maturation. In the analog of malic acid in which the hydrogen at position 2 has been re-
pulp of 'Guanhuahong' and 'Guanhuabai', the levels of malic acid in- placed by a methyl group, and, as such, it can inhibit the accumulation
creased gradually during the pulp coloration stages and reached their of malic acid. Currently, there are only a few studies reporting the
maximum when the peels changed from green to green-yellow presence of citramalic acid in fruits (Khorassani et al., 2011; Santucci
(Fig. 9A). In the 'Guanhuahong' pulp, the concentration of citric acid et al., 2015), and the current study is the first to identify it in Hylocereus
rose steeply during the pulp coloration stages to reach a maximum on fruits. In the three cultivars, the levels of citramalic acid were higher
the 27th DAF (Fig. 9B). Indeed, after the 25th DAF, it was markedly than those of the other organic acids until the 23rd DAF; thereafter, they
higher than the concentrations in 'Guanghuabai' and 'Guanghua- showed a decreasing trend during fruit maturation (Fig. 9C).
hongfen'. In the latter two cultivars, citric acid levels were relatively
low and remained constant throughout fruit maturation (Fig. 9B). Ci-
tramalic acid content was higher than citric acid in mature pitaya fruits, 4. Conclusion
with significant differences in citramalic acid levels being observed
between the three cultivars. In 'Guanhuahong' and 'Guanhuahongfen', In the present study, we analyzed the metabolite profiling in pulp
citramalic acid showed an increasing trend up to the 23rd DAF but and peel of three pitaya cultivars with different pulp colors during fruit
decreased thereafter. In contrast, citramalic acid in 'Guanhuabai' de- maturation. Citramalic acid was identified from pitaya pulp for the first
creased from 19th to the 29th DAF (Fig. 9C). Low contents of oxalic acid time. Higher levels of total phenols, flavonoids and antioxidant activ-
were detected in all three pitaya cultivars (Fig. 9D). At the mature stage ities were detected in peels than in pulps suggesting pitaya peels are
(29th DAF), there were no significant differences in the contents of good sources for extracting betalains, flavonoids and phenols.
malic, citramalic and oxalic acids between 'Guanhuabai', 'Guanhua-
hong' and 'Guanhuahongfen' pitayas (Fig. 9).
The accumulation of sugars and the fall in the levels of organic acids Authors’ contributions
are characteristic of fruit ripening, and therefore the ratio of sugars to
acids is one of the most important indexes used to evaluate fruit quality Y.H.Q. conceived the project and designed the experiments. Q.Z.H,
(Ortiz and Takahashi, 2015). In the three cultivars, pulp sugar contents C.B.C., H.C.W., and J.Y.W. performed experiments and analyzed the
increased rapidly while levels of the major organic acids decreased after data. N.T.Z., H.C.W., J.Y.C., Z.K.Z., J.T.Z., and G.B.H. were involved in
the 25th DAF (Figs. 8 and 9). This pattern may be due to hydrolysis of the data analysis. Q.Z.H, N.T.Z. and Y.H.Q. wrote the manuscript.
starch into simple sugars and the conversion of organic acids into su-
gars, particularly glucose and fructose, as is known for other species of
Hylocereus (Suh et al., 2014; Esquivel et al., 2007a,b; Nomura et al., Conflicts of interest
2005; Yang et al., 2012). In Hylocereus species, the major compounds
that contribute to fruit acidity are citric and malic acids (Suh et al., The authors declared that they have no conflicts of interest to this
2014; Esquivel et al., 2007a,b), the latter compound being one of the work.

124
Q. Hua et al. Plant Physiology and Biochemistry 126 (2018) 117–125

Abbreviations red-fleshed dragon fruit (Hylocereus polyrhizus) during fruit development. J. Sci. Food
Agr. 91, 278–285.
Kamairudin, N., Abd Gani, S.S., Masoumi, H.R.F., Hashim, P., 2014. Optimization of
ABTS, 2, 2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid); natural lipstick formulation based on pitaya (Hylocereus polyrhizus) seed oil using D-
ANOVA, analyses of variance; DAF, days after flowering; FCR, Folin- Optimal mixture experimental design. Molecules 19, 16672–16683.
Ciocalteu reagent; FRAP, ferric reducing ability of plasma; GC-MS, Gas Khorassani, R., Hettwer, U., Ratzinger, A., Steingrobe, B., Karlovsky, P., Claassen, N.,
2011. Citramalic acid and salicylic acid in sugar beet root exudates solubilize soil
Chromatography Mass Spectrometry. phosphorus. BMC Plant Biol. 11, 121.
Kim, H., Choi, H., Moon, J.Y., Kim, Y.S., Mosaddik, A., Cho, S.K., 2011. Comparative
Acknowledgments antioxidant and antiproliferative activities of red and white pitayas and their corre-
lation with flavonoid and polyphenol content. J. Food Sci. 76, 38–45.
Li, P.H., Dong, X.Y., Wang, Y.Z., Yan, S.P., Liu, C.L., 2002. Study on the starch metabolism
This work was supported by the Science and Technology Planning and ultrastructure of starch grain in apple fruit development process. J. Fruit Sci. 3,
Project of Guangdong Province (2014B020202010), Science and 141–144.
Lisec, J., Schauer, N., Kopka, J., Willmitzer, L., Fernie, A.R., 2015. Gas chromatography
Technology Program of Guangzhou (201704020003 and 2014Y2-
mass spectrometry-based metabolite profiling in plants. Nat. Protoc. 10, 1457.
00164), YangFan Innovative and Entrepreneurial Research Team Lourith, N., Kanlayavattanakul, M., 2013. Antioxidant and stability of dragon fruit peel
Project (2014YT02H013) and the earmarked fund for Guangdong colour. Agro Food Ind. Hi Tec. 24, 56–58.
Modern Agro-industry Technology Research System (2016LM1128). Menezes Cordeiro, M.H., Da Silva, J.M., Mizobutsi, G.P., Mizobutsi, E.H., Da Mota, W.F.,
2015. Physical, chemical and nutritional characterization of pink pitaya of red pulp.
Rev. Bras. Frutic. 37, 20–26.
Appendix A. Supplementary data Nerd, A., Mizrahi, Y., 1998. Fruit development and ripening in yellow pitaya. J. Am. Soc.
Hortic. Sci. 123, 560–562.
Nomura, K., Ide, M., Yonemoto, Y., 2005. Changes in sugars and acids in pitaya
Supplementary data related to this article can be found at http://dx. (Hylocereus undatus) fruit during development. J.Hortic. Sci. Biotech. 80, 711–715.
doi.org/10.1016/j.plaphy.2018.02.027. Ortiz, T.A., Takahashi, L.S.A., 2015. Physical and chemical characteristics of pitaya fruits
at physiological maturity. Genet. Mol. Res. 14, 14422–14439.
Osorio-Esquivel, O., Moreno, Alicia-Ortiz, Alvarez, V.B., Dorantes-Alvarez, L., Giusti,
References M.M., 2011. Phenolics, betacyanins and antioxidant activity in Opuntia joconostle
fruits. Food Res. Int. 44, 2160–2168.
Abderrahim, F., Huanatico, E., Segura, R., Arribas, S., Carmen Gonzalez, M., Condezo- Santucci, C., Brizzolara, S., Tenori, L., 2015. Comparison of frozen and fresh apple pulp
Hoyos, L., 2015. Physical features, phenolic compounds, betalains and total anti- for NMR-based metabolomic analysis. Food Anal. Method 8, 2135–2140.
oxidant capacity of coloured quinoa seeds (Chenopodium quinoa Willd.) from Singleton, V.L., Rossi, J.A., 1965. Colorimetry of total phenolics with phosphomolybdic
Peruvian Altiplano. Food Chem. 183, 83–90. phosphotungstic acid reagents. Am. J. Enol. Vitic. 16, 144–158.
Al-Madhagi, I.A.H., Hasan, S.M.Z., Ahmad, A., Yusoff, W.A., 2014. The starch status Song, H.Z., Chu, Q., Xu, D.D., Xu, Y., Zheng, X.D., 2016c. Purified betacyanins from
during growth and development of strawberry plant under tropical climatic condi- Hylocereus undatus peel ameliorate obesity and insulin resistance in high-fat-diet-fed
tion. Acta Hort. (ISHS) 1024, 115–120. mice. J. Agr. Food Chem. 64, 236–244.
Ariffin, A.A., Bakar, J., Tan, C.P., Rahman, R.A., Karim, R., Loi, C.C., 2009. Essential fatty Song, H.Z., Chu, Q., Yan, F.J., Yang, Y.Y., Han, W., Zheng, X.D., 2016a. Red pitaya be-
acids of pitaya (dragon fruit) seed oil. Food Chem. 114, 561–564. tacyanins protects from diet-induced obesity, liver steatosis and insulin resistance in
Arnon, D.I., 1949. Copper enzymes in isolated chloroplasts polyphenoloxidase in Beta association with modulation of gut microbiota in mice. J. Gastroenterol. Hepatol. 31,
Vulgaris. Plant Physiol. 24, 1–15. 1462–1469.
Caldas-Cueva, J.P., Morales, P., Ludena, F., Betalleluz-Pallardel, I., Chirinos, R., Noratto, Song, H.Z., Zheng, Z.H., Wu, J.N., Lai, J., Chu, Q., Zheng, X.D., 2016b. White pitaya
G., Campos, D., 2016. Stability of betacyanin pigments and antioxidants in ayrampo (Hylocereus undatus) juice attenuates insulin resistance and hepatic steatosis in diet-
(Opuntia soehrensii Britton and Rose) seed extracts and as a yogurt natural colorant. J. induced obese mice. PLoS One 11 e01496702.
Food Process. Pres. 40, 541–549. Stintzing, F.C., Conrad, J., Iris, K., Beifuss, U., Carle, R., 2004. Structural investigations on
Castellar, M.R., Solano, F., Obon, J.M., 2012. Betacyanin and other antioxidants pro- betacyanin pigments by LC NMR and 2D NMR spectroscopy. Phytochemistry 65,
duction during growth of Opuntia stricta (Haw.) fruits. Plant Food. Hum. Nutr. 67, 415–422.
337–343. Stintzing, F.C., Schieber, A., Carle, R., 2002. Betacyanins in fruits from red-purple pitaya,
Coria Cayupan, Y.S., Ochoa, M.J., Nazareno, M.A., 2011. Health-promoting substances Hylocereus polyrhizus (weber) Britton & rose. Food Chem. 77, 517.
and antioxidant properties of Opuntia sp. fruits. Changes in bioactive-compound Strack, D., Vogt, T., Schliemann, W., 2003. Recent advances in betalain research.
contents during ripening process. Food Chem. 126, 514–519. Phytochemistry 62, 247–269.
Darmstadt, G.L., Mao-Qiang, M., Chi, E., Saha, S.K., Ziboh, V.A., Black, R.E., Santosham, Suh, D.H., Lee, S., Heo, D.Y., Kim, Y., Cho, S.K., Lee, S., Lee, C.H., 2014. Metabolite
M., Elias, P.M., 2002. Impact of tropical oils on the skin barrier: possible implications profiling of red and white pitayas (Hylocereus polyrhizus and Hylocereus undatus) for
for neonatal health in developing countries. Acta Paediatr. 91, 546–554. comparing betalain biosynthesis and antioxidant activity. J. Agr. Food Chem. 62,
Esquivel, P., Stintzing, F.C., Carle, R., 2007a. Comparison of morphological and chemical 8764–8771.
fruit traits from different pitaya genotypes (Hylocereus sp.) grown in Costa Rica. J. Tel-Zur, N., Mizrahi, Y., Cisneros, A., Mouyal, J., Schneider, B., Doyle, J.J., 2011.
Appl. Bot. Food Qual. 81, 7–14. Phenotypic and genomic characterization of vine cactus collection (Cactaceae).
Esquivel, P., Stintzing, F.C., Carle, R., 2007b. Phenolic compound profiles and their Genet. Resour. Crop Ev. 58, 1075–1085.
corresponding antioxidant capacity of purple pitaya (Hylocereus sp.) genotypes. Z. Tenore, G.C., Novellino, E., Basile, A., 2012. Nutraceutical potential and antioxidant
Naturforsch. C. 62, 636–644. benefits of red pitaya (Hylocereus polyrhizus) extracts. J. Funct. Foods 4, 129–136.
Glew, R.H., Ayaz, F.A., Millson, M., Huang, H.S., Chuang, L.T., Sanz, C., Golding, J.B., Wu, L.C., Hsu, H.W., Chen, Y.C., Chiu, C.C., Lin, Y.I., Ho, J., 2006. Antioxidant and an-
2005. Changes in sugars, acids and fatty acids in naturally parthenocarpic date plum tiproliferative activities of red pitaya. Food Chem. 95, 319–327.
persimmon (Diospyros lotus L.) fruit during maturation and ripening. Eur. Food Res. Yang, D.F., Lin, Q.H., Xie, H.G., Cai, Q.Y., Chen, Y., Zhuo, Y.H., 2012. Soluble sugars and
Technol. 221, 113–118. organic acids of pitaya fruits during development. Fujian J. Agri. Sci. 27, 1076–1080.
Hua, Q.Z., Chen, C.J., Chen, Z., Chen, P.K., Ma, Y.W., Wu, J.Y., Zheng, J., Hu, G.B., Zhao, Yolanda, D. Ortiz-Hernandez, Jose, A. Carrillo-Salazar, 2012. Pitahaya (Hylocereus spp.):
J.T., Qin, Y.H., 2016. Transcriptomic analysis reveals key genes related to betalain a short review. Comun. Sci. 3, 220–237.
biosynthesis in pulp coloration of Hylocereus polyrhizus. Front. Plant Sci. 6, 1179. You, X.X., Qiu, N.X., 2006. Study on extraction of total flavone from Fineleaf Schizonepeta
Jamaludin, N.A., Ding, P., Hamid, A.A., 2011. Physico-chemical and structural changes of Herb by ultrasonic wave. Acta Agric. Boreali - occidentalis Sin 15, 152–155.

125