Food Chemistry 256 (2018) 171–180

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

Phytochemical profiling of the ripening of Chinese mango (Mangifera indica T

L.) cultivars by real-time monitoring using UPLC-ESI-QTOF-MS and its
potential benefits as prebiotic ingredients
⁎
Kai Hu, Abdul Ghani Dars, Qiudou Liu, Bijun Xie, Zhida Sun
College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, China

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

Keywords: Maturity has important effects on the phytochemical and biochemical characteristics of fruits. It affects the
Mango quality, nutritional value, harvest time and commercial operations. In this study, Keitt, Sensation and Xiangya
Maturity index mango cultivars in four distinct stages from southwest China were evaluated for their phytochemical profiling
Phytochemical profiling and antioxidant activities in real time. Furthermore, the biochemical characteristics indices polyphenol oxidase
UPLC-ESI-QTOF-MS
(PPO), peroxidase (POD), superoxide dismutase (SOD) and pectin methylesterase (PME) activities were de-
Prebiotic benefit
termined. Antioxidant compounds such as vitamin C, total phenolic, total flavonoid and total carotenoid content
were also analysed. A total of 34 phenolic compounds were identified and quantitatively monitored by UPLC-
ESI-QTOF-MS. Consecutive degradation of phenolic acids and its derivatives were observed upon maturity. We
found that in addition to carotenoids, phenolic acids could also be used as a measurement index of maturity in
mango. Mango juices and its phenolic extracts may be used as potential prebiotics for modulating probiotic
proliferation.

1. Introduction Mencarelli, 2002). Few studies have been conducted on biochemical

Mango (Mangifera indica L.) is one of the most popular and eco-
nomically important fruits worldwide. It is grown in many countries at
both tropical and subtropical latitudes, particularly in Asia, which has
approximately 76.39% of the world’s production (FAO 2014). Among
tropical fruits worldwide, mango production is second after bananas,
reaching 30 million tons (FAO 2013). It is consumed as a dietary sup-
plement with purported health benefits.
During fruit ripening, physiological, biochemical and molecular
changes occur that directly affect its quality traits (Osorio et al., 2013).
For example, polyphenol oxidase (PPO) was demonstrated to be in-
volved in wound healing, providing defence against stress and pathogen
attacks, and in other cellular processes such as the control of oxygen
levels in chloroplasts (Cheema & Sommerhalter, 2015). Superoxide
dismutase (SOD) and peroxidase (POD) are major antioxidant enzymes
in organisms, and can prevent oxidative damage caused by reactive
oxygen species (ROS) to maintain a relatively low level of ROS pro-
duction (Razzaq, Khan, Malik, & Shahid, 2013). Pectin methylesterase
(PME) catalyses the demethylesterification that influences the interac-
tions between cell wall components, which is relevant to mango fruit
softening and the viscosity of products (Cardarelli, Botondi, Vizovitis, &
properties of fruiting mango at different maturity stages.
Besides, the biologically active components, ascorbic acid, phenolic
compounds (Robles-Sanchez et al., 2009; Sulaiman & Ooi, 2012) and
carotenoids (Mercadante & Rodriguez-Amaya, 1998) play a consider-
able role in the nutritional values of mango, which have been verified
for their health benefit due to their prominent antioxidant activity. The
polyphenolic composition of many varieties of mango were mainly
identified as benzophenone derivatives, flavonols, xanthones and gal-
lotannins, and the content and variety of polyphenols in mango peel
were greater than in pulp. (Barreto et al., 2008; Berardini, Carle, &
Schieber, 2004; Dorta, González, Lobo, Sánchez-Moreno, & de Ancos,
2014; Schieber, Berardini, & Carle, 2003). A number of studies reported
that maturity deeply affects the phytochemicals and corresponding
antioxidant activity of fruits. For example, in mulberry fruits the con-
tent of free phenolic acids, quercetin-3-rutinoside, and 1-deoxynojir-
imycin decreased during ripening, particularly over six times that of
chlorogenic acid in the immature fruits compared to the fully matured
fruits; however, the total phenolics, anthocyanins, and antioxidant ac-
tivities increased during ripening (Lee & Hwang, 2017). In plum fruits,
levels of gallotannins, ellagitannins, flavonols, gallic acid and ellagic
acid were highest at the initial maturation and decreased as the fruit

⁎
Corresponding author.
E-mail address: sunzhida@mail.hzau.edu.cn (Z. Sun).

https://doi.org/10.1016/j.foodchem.2018.02.014
Received 14 September 2017; Received in revised form 31 January 2018; Accepted 3 February 2018
0308-8146/ © 2018 Published by Elsevier Ltd.
K. Hu et al.
ripened (Lestario et al., 2017). In the pulp and peel of two apple cul-
tivars, there was a reduction in the total and individual phenolic
compounds and total antioxidant activity as development proceeded
(Stanger, Steffens, Soethe, Moreira, & do Amarante, Cassandro, 2017).
For mango fruits, extensive studies on maturity were performed ad-
dressing its physicochemical properties, e.g. colour, texture, pH, ti-
tratable acidity, total soluble solids, total phenolics, total carotenoids
and vitamin C (Corrales-Bernal, Maldonado, Urango, Franco, & Rojano
2014; Manthey & Perkins-Veazie, 2009; Robles-Sanchez et al., 2009;
Sulaiman & Ooi, 2012). However, limited information about phyto-
chemical profile is available during mango ripening. In this study, 34
phenolic compounds were identified and separated into four groups,
and their variations of content during ripening were monitored in real
time.
The International Scientific Association for Probiotics and Prebiotics
(ISAPP) recently issued a new consensus statement that: phenolics and
phytochemicals are within the scope of prebiotics. Plant polyphenols
constitute a class of compounds that can meet the criteria of prebiotics
(Gibson et al., 2017). However, evidence for these emerging prebiotics
is scarce relative to fructans and galactans (Roberfroid et al., 2010) and
additional comprehensive studies determining their health benefits are
required to fulfil the prebiotic status. Thus far, no literature involving
the prebiotic effect of mango phytochemicals is available. The mango is
a climacteric fruit. Therefore, harvesting of the mango is a decisive key
step for transportation and fruit quality, as well as fruit processing.
Hence, the objectives of this study were to evaluate the activities of
PPO, POD, SOD and PME; vitamin C; total phenolic content (TPC); total
flavonoid content (TFC); total carotenoid content (TCC); and anti-
oxidant activity of three major mango cultivars at four different ma-
turity stages in China. Subsequently, secondary metabolites (phenolics
and its derivatives) were identified using UPLC-Q-TOF-MS and its po-
tential prebiotic activity was measured.
2. Materials and methods
2.1. Mango samples
Three cvs. Keitt, Sensation and Xiangya were harvested approxi-
mately every month during the four-month period from July to October
from Lijiang city of Yunnan Province, located at 26.63 °N and 101.24
°E, the highest latitude region of China where the mango is cultivated.
Each plot, consisting of 40 fruits, were obtained from different trees at
different times and were separated into four stages of maturation:
stages 1, 2, 3 and 4. According to the physical property analyses of
colour and texture, the apparent features of mangoes at the four ma-
turation stages were described as follows: stage 1, high firmness and
white pulp or light yellow near the kernel; stage 2, medium firmness
and light yellow pulp; stage 3, slightly soft texture and yellow pulp; and
stage 4, very soft texture and intensely yellow or orange pulp. Stages 1
and 2 were recognized as green mango, whereas stages 3 and 4 were
recognized as ripe mango. All fruits were acquired from the southern
part of the crown in the same plantation to ensure similar growth
condition. All samples were analysed immediately after harvesting for
activities of PPO, POD, SOD and PME. The remaining mangoes were
washed; and peeled, and the pulp and peel were instantly frozen in
liquid nitrogen and; then stored at −20 °C until further analysis within
one week.
2.2. Chemicals
Catechol, Folin–Ciocalteu reagent and guaiacol were obtained from
Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China), Biosharp
(Hefei, China) and Macklin (Shanghai, China), respectively. Pectin, 2,
6-dichloroindophenol sodium (DCIPS), gallic acid and mangiferin were
purchased from Shanghai Yuanye Bio-technology Co., Ltd. (Shanghai,
China). The 1, 1-diphenyl-2-picrylhydrazyl radical (DPPH), 2,2′-
172
Food Chemistry 256 (2018) 171–180
azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt
(ABTS), vanillic acid, ellagic acid and quercetin were obtained from
Sigma–Aldrich Co. (Shanghai, China). The SOD reagent kit was pur-
chased from Nanjing Jiancheng Bioengineering Institute (Nanjing,
China). Lactobacillus acidophilus (DSM13241) and Lactobacillus rham-
nosus (ATCC53103) were purchased from Chr. Hansen A/S (Hoersholm,
Denmark).
2.3. PPO extraction and assay
A total of 20 g pulp was exactly weighed and; then mixed with
20 mL of 0.2 M sodium phosphate buffer (pH 6.5) containing 4%
polyvinyl polypyrrolidone (PVPP), using a high-speed homogenizer.
After resting for 1 h at 4 °C, the crude extract was centrifuged at
10,000 rpm for 10 min at 4 °C. The supernatant was collected and used
for the PPO activity assay. The substrate consisting of 2.5 mL of 0.2 M
sodium phosphate buffer (pH 6.5) containing 70 mM catechol was in-
cubated at 30 °C for 10 min. Then, 0.5 mL extract was added to initiate
the reaction. Meanwhile, the absorbance was recorded with a spectro-
photometer (UV-1800, Shimadzu Suzhou Instruments Mfg. Co., Ltd.,
Suzhou, China) every 15 s for 3 min at the wavelength of 420 nm. One
unit of PPO activity was defined as the change in absorbance of
0.001 min−1 mL−1 of enzyme (Palma-Orozco, Marrufo-Hernandez,
Sampedro, & Najera, 2014).
2.4. POD extraction and assay
POD was extracted using the same method as PPO. The substrate,
consisting of a mixture of 3 mL of 0.2 M sodium phosphate buffer (pH
6.5) containing 1% guaiacol and 0.2 mL 1.5% hydrogen peroxide, was
incubated at 30 °C for 10 min. Then 0.1 mL of extract was added to
initiate the reaction. Meanwhile, the absorbance was recorded every
15 s for 3 min at a wavelength of 470 nm. One unit of POD activity was
defined as the change in absorbance of 0.001 min−1 mL−1 of enzyme
(Razzaq et al., 2013).
2.5. SOD extraction and assay
SOD crude extract was acquired by thoroughly mixing the pulp with
0.05 M sodium phosphate buffer (pH 7.5) at a proportion of 1: 3 (w/v)
using a miniature mortar in an ice bath. Then the crude extract was
centrifuged at 8000 rpm for 10 min at 4 °C. The supernatant was col-
lected and used for the enzyme activity assay. The SOD activity was
detected according to the instructions of the SOD test kit provided by
Nanjing Jiancheng Bioengineering Institute.
2.6. PME extraction and assay
The PME activity assay was conducted with a pH meter (Mettler
Toledo Co., Ltd., Shanghai, China), according to the titrimetric method
provided by Kaushik, Nadella, & Rao (2015) with some modifications.
Briefly, the puree was obtained by mixing the pulp and 0.05 M Tris
acetate (pH 7.5) at a proportion of 2: 1 (w/v) in a blender. The sub-
strate, consisting of 30 mL of 0.5% pectin solution containing 0.1 M
NaCl, was incubated at 30 °C until the reaction finished. Solutions of
2 N and 0.05 N NaOH were used to adjust the pH of the freshly prepared
pectin solution to 7.5. A total of 5 g of the puree was added to the
substrate to initiate the reaction. Then, the pH was immediately ad-
justed to 7 with 2 N NaOH and re-adjusted to pH 7.5 with 0.05 N NaOH.
The mixture was titrated with 0.05 N NaOH to maintain the pH at 7.5
for 10 min, and the consumption of NaOH was recorded. The PME was
calculated using the formula,
K. Hu et al.
PME activity (mmol·kg −1·min−1 )
(0.05NNaOH )(Titre volume of NaOH , mL)
= × 103
(10 min)(Weight of puree, g ) (1)
2.7. Determination of vitamin C content
A total of 5 g pulp was mixed with a few mL of 2% oxalic acid
(3–5 mL). It was then; ground for 5 min in a small glass grinder and
washed three times with 2% oxalic acid. The washing solution was
collected and centrifuged at 8000 rpm at 25 °C for 10 min. The super-
natant was diluted to a final volume of 25 mL for further use. The vi-
tamin C content was determined using a simplified method reported by
Sulaiman and Ooi (2012). Briefly, the diluted solution (25 mL) was ti-
trated with 0.1‰ 2,6-dichlorophenolindophenol sodium (DCIPS) until
the solution turned to a light pink colour that persisted for 15 s. Cali-
bration of the 0.1‰ DCIPS solution was performed with 1 mg/mL as-
corbic acid. The results were calculated and expressed as mg of ascorbic
acid equivalents (AAE) per 100g of fresh weight (FW).
2.8. Determination of total carotenoid content
Thawed pulp (3 g) was exactly weighed and thoroughly ground with
a small volume of 80% acetone several times in ice bath away from
light until the grinding solution became colourless. All solutions were
combined and centrifuged at 8000 rpm for 10 min. The supernatant was
diluted to an appropriate concentration and the absorption was mea-
sured using a spectrophotometer at wave lengths of 445, 644 and
662 nm. The TCC was calculated using the method described by Fadeel
(1962) and expressed as μg per g of FW.
2.9. Phenolic compound extraction
For extraction, the homogenized pulp and peel were mixed with
80% ethanol at a ratio of 1: 3 and 1: 10 respectively, and subjected to
ultrasonic treatment for 30 min at 40 °C. It was then centrifuged at
10,000 rpm at 4 °C for 10 min. The supernatant was collected and the
residues were re-extracted using the same procedure to ensure max-
imum extraction. The two supernatants were combined for TPC, and
TFC and antioxidant activity analyses. The pooled extract was further
evaporated to dryness at 40 °C by rotary evaporation under reduced
pressure. The residue was dissolved in chromatographic grade acet-
onitrile and filtered through a 0.22 μm nylon membrane for identifi-
cation and quantification of phenolic compounds.
2.10. Determination of total phenolic and flavonoid content
TPC was determined using the Folin–Ciocalteu method described by
Hana et al. (2010). The absorbance was recorded at 725 nm. A standard
curve was obtained with gallic acid and the results were expressed as
mg gallic acid equivalent (GAE) per 100 g FW. TFC was measured by a
method developed previously (Moohuchin et al., 2015). The absor-
bance was recorded at 415 nm. Quercetin was used as a standard, and
the results were expressed as mg of quercetin equivalent (QE) per 100 g
FW.
2.11. DPPH and ABTS free-radical scavenging assay
The DPPH free-radical scavenging capacity of mango pulp and peel
extracts were measured following the method described by Liu et al.
(2013). A standard curve was obtained by determining the vitamin C
concentration, and the results were expressed as mg VC equivalent per
100 g FW. The ABTS assay was performed according the method of
Miller, Riceevans, Davies, Gopinathan, & Milner (1993). Similarly, vi-
tamin C was as a standard and the results were expressed as mg VC
equivalent per 100 g FW.
173
Food Chemistry 256 (2018) 171–180
2.12. Quantification of major phenolic compounds
Quantification analysis was performed on an Acquity Ultra
Performance Liquid Chromatography (UPLC) system (Waters, Milford,
MA, USA), equipped with a C18 column (2.1 × 100 mm, 1.7 μm,
Waters, Milford, MA, USA) at 40 °C. The mobile phase consisted of 0.1%
formic acid water (A) and ACN (B), using the following gradients:
0–28 min: 2–50% B, 28–28.5 min: 50–100% B, 28.5–30.5 min: 100% B,
30.5–32 min: 100–2% B, 32–34 min: 2% B. The flow rate was 0.3 mL/
min, and the injected volume was 10 μL. A standard calibration curve of
different concentrations of each phenolic compound (gallic acid, val-
linic acid, ellagic acid, quercetin and mangiferin) was plotted according
to the peak area. The results were calculated and expressed as milli-
grams of phenolic compound per 100 g of fresh weight (mg/100 g FW)
(Rojas-Garbanzo et al., 2016).
2.13. Identification of phenolic compounds
The above UPLC system was coupled to a G2-XS QT of MS (Waters,
Milford, MA, USA) to analyse the phenolic compounds of the extracts
following a previous method (Wang et al., 2017). The scan range was
from m/z 100 to 2000 with a scan time of 0.3 s. The source temperature
was set to 120 °C with a cone gas flow of 50 L/h. The desolvation gas
flow was set to 800 L/h at a temperature of 400 °C. The capillary was set
at 1 kV for ESI− mode with the cone voltage at 40 V.
2.14. Prebiotic activity assay
Mango juice, phenolic extracts and gallic acid were used to de-
termine the potential capacity to support the growth of Lactobacillus
acidophilus. Mango juice was extracted from cv. Keitt in stage 3 and
filtered. Phenolic extracts from three cultivars at stage 3 were obtained
from 50 g pulp as the method described in step 2.9 and then purified
with a 20 cm3 C18 cartridge and brought to a final volume of 2.5 mL.
Then, 10 mg/mL gallic acid was prepared using distilled water. A
mixture of gallic acid and mangiferin was obtained with 10 mg/mL
gallic acid containing 0.1 mg/mL mangiferin. All samples were filter-
sterilized and added to 96-well plates to obtain different final con-
centration of 1, 1/2, 1/4, 1/8, 1/16, 1/32, 1/64 and 1/128 of the initial
value. The active Lactobacillus acidophilus and Lactobacillus rhamnosus
were transferred to MRS broth medium to yield a concentration with
1 × 106 cfu/mL of the probiotics. The cultures were incubated at 37 °C
for 22 h in an aerobic chamber (DH5000BII, Tianjin Taisite Instrument
Co., Ltd., Tianjin, China). The viability of the bacteria was determined
spectrophotometrically at 600 nm.
2.15. Statistical analysis
All tests were performed in triplicate unless otherwise stated and
data were presented as the means ± the standard deviation. Analysis
of variance (ANOVA) and principal component analysis were conducted
using the software Microcal Origin 9.0 (Microcal Software, Inc.,
Northampton, USA). Differences with a P value of < 0.05 were con-
sidered significant.
3. Results and discussion
3.1. Biochemical index analyses
The activity of PPO, POD, SOD and PEM in mango and other fruits is
an important biochemical index for evaluating the maturity and quality
during the storage and processing of fruit. In this study, as shown in
Table 1, the ripe mango possessed higher PPO and POD activities
compared to the green mango. The increase of POD activity in the ripe
mango compared to green mango may be because the ripe fruit gen-
erates more ROS; therefore, POD decomposes H2O2 by oxidizing
K. Hu et al. Food Chemistry 256 (2018) 171–180

Table 1
Polyphenol oxidase (PPO), peroxidase (POD), superoxide dismutase (SOD) and pectin methylesterase (PME) activities in mango fruit of three cultivars in four maturities.a

Cultivars Ripenessb PPO (unit) POD (unit) SOD (unit/g FW) SOD/POD PME (mmol/kg/min)

Keitt 1 133.33 ± 26.66d 94.44 ± 8.39h 1294.98 ± 180.25fg 13.89 ± 3.12a 0.67 ± 0.05c
2 133.33 ± 13.34d 312.22 ± 18.95fg 1149.19 ± 237.31g 3.69 ± 0.83c 0.89 ± 0.04a
3 324.44 ± 40.73c 110.00 ± 21.86h 1363.58 ± 151.67efg 12.90 ± 3.90a 0.42 ± 0.03e
4 404.44 ± 46.82c 1005.55 ± 23.41d 2006.78 ± 186.86bc 2.00 ± 0.21d 0.09 ± 0.02g

Sensation 1 195.55 ± 76.69d 454.45 ± 13.47e 1805.25 ± 361.42bcd 3.99 ± 0.91bc 0.58 ± 0.03d
2 702.22 ± 30.06b 235.56 ± 12.62g 1380.74 ± 270.55efg 5.91 ± 1.42b 0.74 ± 0.04b
3 657.78 ± 77.84b 2115.55 ± 72.44a 1590.85 ± 141.70def 0.75 ± 0.05f 0.54 ± 0.04d
4 1597.78 ± 97.15a 1273.33 ± 12.02c 2559.94 ± 227.59a 2.01 ± 0.19d 0.05 ± 0.01gh

Xiangya 1 382.22 ± 36.72c 364.44 ± 18.96ef 1702.34 ± 74.27cde 4.69 ± 0.46b 0.44 ± 0.02e
2 100.00 ± 6.67d 404.44 ± 31.51ef 1376.45 ± 100.47efg 3.41 ± 0.19c 0.42 ± 0.04e
3 628.89 ± 45.38b 1485.56 ± 106.73b 1672.32 ± 202.17cdef 1.13 ± 0.14e 0.20 ± 0.03f
4 715.56 ± 55.91b 2144.44 ± 168.54a 2131.14 ± 104.77b 1.00 ± 0.06e 0.03 ± 0.01h

a
Data are presented as means ± the standard deviation. Means with different letters within a column are significantly different at p < 0.05.
b
Ripeness stages 1, 2, 3 and 4 represented mangoes with high firmness, medium firmness, and slightly and very soft texture, respectively.

phenolic compounds to protect fruit against oxidative damage. In ad-
dition, PPO and POD are responsible for the unpleasant flavour and
colour change due to the oxidation of phenolic compounds (Cheema &
Sommerhalter, 2015; Robinson, Loveys, & Chacko, 1993). However, the
activity of PPO and POD in cv. Keitt was the lowest in experimental
mango cultivars. Therefore, cv. Keitt is likely to suffer less quality loss
during handling or processing.
SOD, as a major member of the antioxidant system, together with
POD and catalase, is responsible for balancing the production and re-
moval of ROS (Jimenez et al., 2002). As shown in Table 1, SOD activ-
ities of all experimental cultivars at the beginning of maturation were
lower than at the green stage, and then significantly increased at final
maturation stage. Similar findings were found in tomato fruit by
Jimenez et al. (2002). The SOD/POD value represents the partial an-
tioxidant capacity of the plant tissue. SOD/POD values (see in Table 1)
were generally higher in green mango than ripe mango, suggesting its
greater contribution to antioxidant capacity in the green mango.
The PME activities of experimental mangoes are shown in Table 1
and were significantly decreased during the maturation process. The
high initial activity and the following linear decline of PME indicated
the initial demethylesterification during cell wall degradation
(Cardarelli et al., 2002). Cvs. Sensation and Xiangya had relatively
higher PPO, POD, and SOD activities, as well as lower PME activities
compared to cv. Keitt. This indicated that cvs. Sensation and Xiangya
may maintain better resistance against pathogen attacks and oxidative
damage and delayed softening during ripening. Thus, this is helpful to
prolong the shelf life of fruit during storage.
3.2. Quantitative analysis of active constituents (Vitamin C, total phenolic,
total flavonoid and total carotenoid) in pulp and peel of mango
As shown in Table 2, vitamin C contents in pulps showed a dramatic
decrease during ripening, which were consistent with the findings of
Sulaiman and Ooi (2012) and Robles-Sanchez et al. (2009). The content
of vitamin C was significantly higher than the reported average levels of
cvs. Tommy Atkins, Keitt and Kent in ripe pulps (Manthey & Perkins-
Veazie, 2009). Moreover, cv. Keitt had significantly higher vitamin C
content compared to the other two cultivars in ripe stages, likely giving
it better colour and flavour retention during handling and processing
because of its direct inhibition of PPO action (Palma-Orozco et al.,
2014; Robinson et al., 1993).
As indicated in Table 2, the content of TPC and TFC in the peel is
abundant. However, this is closely related to varieties and habitats.
General decreases in TPC and TFC were observed for experimental
mango pulps during ripening. Compared with pulps, peels of the ex-
perimental three cultivars were observed with approximately 8–25
times higher TPC and TFC values. Corrales-Bernal et al. (2014) also
reported that the green mango has 45% more flavonoids than mature
mango.
In mango, the carotenoids are mainly β-carotene, followed by vio-
laxanthin esters (Petry & Mercadante, 2016). Interestingly, the car-
otenoid constitution of mango varies depending on the cultivar, cli-
matic or geographic effects and stage of maturity, as well as storage
conditions and degree of fruit processing (Pott, Breithaupt, & Carle,
2003). As shown in Table 2, TCC increased with maturity, which could
be a ripening index and harvest indicator. Cvs. Sensation and Xiangya
exhibited significantly higher TCC values in peels than pulps, but cv.
Keitt showed an adverse result. Ajila, Bhat, and Prasada Rao (2007)
reported that TCC was 4–8 times higher in ripe mango peel than raw
mango peel, and the present study showed about 3–8 times higher in
ripe mango peel. This indicated that the carotenoid accumulation at
maturity has significant potential for providing health benefits and
mango processing.
3.3. Determination of antioxidant activities
As shown in Table 2, the extracts from the three mango cultivars at
four maturation stages had significantly differently values for DPPH
free radical-scavenging activities, ranging from 63.13 to 235.83 mg
VCE/100 g FW in pulp extracts and from 1215.10 to 2784.68 mg VCE/
100 g FW in peel extracts. Its value for Keitt in stage 1 was highest of all
extracts, whether it was peel or pulp. Generally, green mango pulp had
higher DPPH free radical-scavenging activity than ripe mango pulp,
which closely coincided with the trend of TPC as a fruit ripening index,
as shown in Table 2. Green Chokanan pulp was also reported to have
higher scavenging activity than that the ripe pulp (Sulaiman & Ooi,
2012). Mango peel extracts scavenged approximately 10–40 times more
DPPH free radical than pulp extracts, indicating that mango peel as a
by-product could be a rich resource of antioxidants. Similar behaviour
was found when ABTS free radical was performed. Correlation analysis
indicated that TPC was significantly correlated with antioxidant capa-
city, suggesting phenolic compounds were leading contributors to an-
tioxidants in the extract. In the present study we detected relatively
high VC content from mango samples (Table 2), but there was a low
correlation between VC and antioxidant capacity. Possibly, the solvent
system and extraction procedure caused a considerable loss of VC.
3.4. Identification and quantitative analysis of phenolic compounds
An ultra-high-performance liquid chromatography in combination
with quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOF-
MS) method was established and successfully applied to characterize
the constituents of polyphenol in the ethanol (80%) extract of mango
flesh and peel. The extracts of Keitt peel and flesh at stage 1 produced

174
 K. Hu et al.

Table 2
Vitamin C (VC), total phenolic content (TPC), total flavonoid content (TFC), total carotenoid content (TCC) and antioxidant activities in pulp and peel of three mango cultivars in four maturities.a

Cultivars Ripenessf Pulp

VCb (mgAAE/100 g) TPCc (mgGAE/100 g) TFCd (mgQE/100 g) TCCe (μg/g) DPPH (mgVC equivalent/100 g FW)

Keitt 1 163.94 ± 1.45d 97.59 ± 0.42a 60.31 ± 2.05b 3.51 ± 0.57h 235.83 ± 8.32a
2 175.79 ± 0.42c 79.71 ± 0.32b 50.29 ± 3.19c 10.62 ± 0.41f 182.23 ± 5.67c
3 116.87 ± 1.55f 73.66 ± 0.69c 31.82 ± 1.89f 18.24 ± 0.60d,e 150.54 ± 0.64d
4 46.87 ± 1.41i 59.43 ± 1.89e 44.04 ± 1.42d 40.42 ± 4.19b 106.84 ± 4.47f

Sensation 1 176.03 ± 0.21c 23.58 ± 0.51h 49.74 ± 0.90c 6.59 ± 0.40g 63.87 ± 1.51i
2 282.17 ± 2.51a 65.21 ± 2.84d 102.63 ± 4.10a 9.39 ± 0.34f 118.53 ± 3.44e
3 57.94 ± 1.88h 58.47 ± 0.45e 23.82 ± 2.07g 16.90 ± 0.26e 103.01 ± 11.07f
4 29.34 ± 0.78j 37.55 ± 1.37g 62.06 ± 1.72b 49.04 ± 2.77a 71.42 ± 6.45g,h

Xiangya 1 160.35 ± 1.77e 96.15 ± 0.25a 50.89 ± 2.77c 9.73 ± 0.24f 216.37 ± 4.47b
2 204.90 ± 0.78b 38.59 ± 0.87g 35.68 ± 1.52e 15.94 ± 0.32e 63.13 ± 4.95i
3 71.00 ± 0.57g 38.83 ± 0.12g 23.59 ± 0.83g 20.29 ± 0.30d 77.17 ± 5.91g
4 30.84 ± 0.78j 41.15 ± 1.00f 41.51 ± 0.50d 34.56 ± 0.24c 74.93 ± 3.87g

Cultivars Pulp Peel

ABTS (mgVC equivalent/100 g FW) VC (mgAAE/100 g) TPC (mgGAE/100 g) TFC (mgQE/100 g) TCC (μg/g) DPPH (mgVC equivalent/100 g FW) ABTS (mgVC equivalent/100 g FW)

175
Keitt 103.43 ± 2.26a 100.50 ± 3.17f 1207.02 ± 8.28a 833.93 ± 42.39d 3.42 ± 0.41k 2784.68 ± 33.61a 2884.32 ± 52.10a
86.88 ± 2.91b 108.94 ± 2.62e 669.96 ± 14.09f 784.30 ± 13.60d,e 9.36 ± 0.60j 1848.89 ± 79.20e,f 1574.88 ± 35.35e
78.66 ± 2.62c 148.69 ± 2.69d 602.62 ± 8.44h 749.37 ± 20.88e,f 15.33 ± 0.69h 1501.16 ± 121.49h 1144.88 ± 3.06 h
54.50 ± 1.71e 46.96 ± 1.71h 641.90 ± 25.45g 957.09 ± 36.48c 26.97 ± 0.35e 1637.27 ± 47.89g 1452.78 ± 40.20f

Sensation 20.79 ± 2.94i 47.45 ± 2.48h 300.07 ± 4.51j 953.42 ± 22.29c 17.79 ± 0.34g 1290.60 ± 68.60i 484.85 ± 41.23j
63.00 ± 2.66d 559.87 ± 3.01a 821.40 ± 5.46b 1693.32 ± 33.54a 22.50 ± 0.28f 2602.84 ± 81.40b 2160.59 ± 10.62b
49.02 ± 2.76f 227.82 ± 3.54c 679.58 ± 6.06e,f 583.01 ± 19.37h 34.33 ± 0.41d 1826.56 ± 39.37f 1491.71 ± 38.28f
47.07 ± 1.46f 268.07 ± 2.99b 604.22 ± 19.29h 1140.00 ± 21.42b 61.82 ± 0.58a 1475.64 ± 41.51h 1302.37 ± 45.15g

Xiangya 105.46 ± 3.99a 28.82 ± 0.78i 723.67 ± 6.36d 684.11 ± 38.89g 10.71 ± 0.39j 2012.65 ± 45.00d 1700.51 ± 21.45d
42.73 ± 2.27g 21.37 ± 2.80j 795.82 ± 12.73c 694.22 ± 46.98f,g 13.35 ± 0.33i 2314.66 ± 33.51c 1992.48 ± 39.13c
41.85 ± 2.79g 89.32 ± 3.17g 694.01 ± 3.67e 515.91 ± 37.91i 44.74 ± 0.94b 1958.42 ± 76.76d,e 1645.66 ± 33.15d
32.29 ± 0.61h 27.70 ± 1.51i 368.52 ± 1.39i 665.73 ± 39.03g 61.23 ± 0.82a 1215.10 ± 59.08i 622.87 ± 77.25i

a
Data are presented as means ± the standard deviation and expressed on flesh weight basis. Means with different letters within a column are significantly different at p < 0.05.
b
VC expressed as mg of ascorbic acid per 100 g of fresh weight (FW).
c
TPC expressed as mg of gallic acid per 100 g of fresh weight (FW).
d
TFC expressed as mg of quercetin per 100 g of fresh weight (FW).
e
TCC expressed as mg per g of fresh weight (FW).
f
Ripeness stages 1, 2, 3 and 4 represented mangoes with high firmness, medium firmness, and slightly and very soft texture, respectively.
Food Chemistry 256 (2018) 171–180
K. Hu et al. Food Chemistry 256 (2018) 171–180

Table 3
Phenolic compounds identified in the pulp and peel extracts of three mango cultivars in four different maturities by UPLC-ESI-QTOF-MS.

Compound RTa (min) Calculated [M−H]− m/z Observed [M−H]− m/z Error (ppm) Ion fragment Identity Formula

1 1.600pb 331.0665 331.0732 20.24 169.0218 mono-galloyl-glucose C13H16O10

2 2.215f c 169.0137 169.0212 44.38 125.0321, 107.0185 gallic acid C7H6O5
2.219p 169.0222 50.29 125.0334, 107.0223
3 2.920f 299.0767 299.0831 21.40 137.0323 hydroxybenzoic acid hexoside C13H16O8
2.878p 299.0832 21.73 137.0330
4 3.582f 331.0665 331.0724 17.82 169.0218, 152.0324 iso-mono-galloyl-glucose C13H16O10
3.582p 331.0731 19.94 169.0224,125.0331
5 3.656f 331.0665 331.0728 19.03 169.0220 iso-mono-galloyl-glucose C13H16O10
6 4.287f 299.0767 299.0825 19.39 137.0328 hydroxybenzoic acid hexoside C13H16O8
7 5.403f 137.0239 137.0318 57.65 93.0433 hydroxybenzoic acid C7H6O3
5.382p 137.0325 62.76 93.0430
8 5.660f 163.0395 163.0476 49.68 119.0581 coumaric acid C9H8O3
5.660p 163.0476 49.68 119.0585
9 6.065p 359.0767 359.0720 13.10 197.0003, 160.8070 rosmarinic acid C18H16O8
10 6.660f 289.0712 289.0778 22.83 245.0344, 179.0657, 125.0324 catechin C15H14O6
11 6.770f 193.0501 193.0578 39.89 149.0327, 134.0456 ferulic acid C10H10O4
12 6.801f 575.1037 575.1031 −1.04 303.0570, 193.0220 maclurin-mono-O-galloyl-glucoside C26H24O15
6.853p 575.1039 0.35 303.0569, 193.0219
13 7.200p 167.0344 167.0415 42.51 122.9901 vanillic acid C8H8O4
14 8.283f 281.1389 281.1453 22.76 dihydrophaseic acid C15H22O5
15 8.321f 421.0771 421.0806 8.31 331.0510, 301.0406 mangiferin C19H18O11
8.252p 421.0799 6.65 331.0519, 301.0408
16 8.353f 727.1147 727.1062 −11.69 575.1044 maclurin-di-O-galloyl-glucoside C33H28O19
8.353p 727.1080 −9.21 575.1025
17 8.946f 787.0994 787.0914 −10.16 635.0902, 617.0795 tetra-O-galloyl-glucose C34H28O22
8.989p 787.0914 −10.16 635.0864, 617.0757
18 9.577f 573.0880 573.0878 −0.35 421.0774, 301.0415, 331.0497 mangiferin gallate C26H22O15
9.624p 573.0878 −0.35 421.0816, 301.0466, 331.0522 mangiferin gallate C26H22O15
19 9.772f 711.1197 711.1197 0.00 559.1125, 389.0872 iriflophenone di-O-galloyl-glucose C33H28O18
9.783p 711.1161 −5.06 559.1092, 389.0916
20 10.061p 573.0880 573.0869 −1.92 421.0810, 313.0618, 301.0405 mangiferin gallate C26H22O15
21 10.208f 787.0994 787.0883 −14.10 635.0845, 617.0749 tetra-O-galloyl-glucose C34H28O22
10.198p 787.0920 −9.40 635.0869, 617.0772, 465.0709
22 10.271f 300.9984 301.0041 18.94 257.0132, 229.0313 ellagic acid C14H6O8
10.309p 301.0052 22.59 257.0155, 229.0210
23 10.398f 787.0994 787.0881 −14.36 635.0836, 617.0749, 465.0670 tetra-O-galloyl-glucose C34H28O22
10.461p 787.0883 −14.10 635.0845, 617.0750, 465.0705 C34H28O22
24 10.719f 787.0994 787.0950 −5.59 635.0822, 617.0732 tetra-O-galloyl-glucose C34H28O22
25 10.766f 463.0877 463.0891 3.02 301.0380, 300.0319, 271.0310 quercetin-3-O-hexoside C21H20O12
10.818p 463.0891 3.02 301.0380, 300.0319,271.0311 C21H20O12
26 10.892p 787.0994 787.0920 −9.40 635.0875, 617.0773, 465.0713 tetra-O-galloyl-glucose C34H28O22
27 11.029f 463.0877 463.0891 3.02 301.0380, 300.0319, 271.0311 quercetin-3-O-hexoside C21H20O12
11.092p 463.0900 4.97 301.0396, 300.0332, 271.0313 C21H20O12
28 11.292p 281.1389 281.1448 20.99 dihydrophaseic acid C15H22O5
29 11.423f 433.0771 433.0775 0.92 301.0393, 300.0331, 271.0317 quercetin-3-O-pentoside C20H18O11
30 11.660f 433.0771 433.0785 3.23 301.0058, 300.0325, 271.0345 quercetin-3-O-pentoside C20H18O11
31 11.702p 939.1104 939.1035 −7.35 787.0917, 769.0826, 617.0768 penta-O-galloyl-glucose C41H32O26
32 11.896f 433.0771 433.0796 5.77 301.0416,300.0322, 271.0322 quercetin-3-O-pentoside C20H18O11
33 13.311f 939.1104 939.0914 −20.23 787.0915, 769.0782, 617.0750 penta-O-galloyl-glucose C41H32O26
34 12.722f 1091.1213 1091.1016 −18.05 939.0955, 769.0737 hexa-O-galloy-glucose C48H36O30
35 15.556p 1091.1213 1091.0859 −32.44 939.0941, 769.0803 hexa-O-galloy-glucose C48H36O30

a
RT, retention time.
b
RTp, retention time in mango peel sample.
c
RTf, retention time in mango flesh sample.

similar phenolic profiles with other cultivars in different maturation
stages. A representative total ion chromatogram of the peel and flesh
extracts of Keitt at stage 1 is shown in Fig. 1S (supplementary material).
A total of 34 compounds were identified or tentatively identified as
phenolic acids and its derivatives, mangiferin and its derivatives (in-
cluding maclurin formed by ring-opening reaction), gallotannins and
quercetin derivatives by characteristic diagnostic fragment ions and
comparing mass spectrometric data with the literatures (Table 3). Here,
we reported the presence of rosmarinic acid in mango fruits for the first
time (compound 9).
3.4.1. Phenolic acids and their derivatives
Compound 1 gave an ion [M−H]− at m/z 331.0732, and yielded a
characteristic peak ion at m/z 169.0218 after losing a glucose
(162.0514 Da) moiety. Compounds 4 and 5 shared a similar precursor
ion and fragment ions with compound 1 and were identified as mono-
galloyl-glucose and its isomers (Berardini et al., 2004; Ramirez,
Zambrano, Sepulveda, & Simirgiotis, 2014). Compound 2 produced a
[M−H]− ion at m/z 169.0212 and diagnostic ions at m/z 125.0321 and
107.0185 corresponding to losses of CO2 (43.9891 Da) and H2O
(18.0136 Da) and was identified as gallic acid (Rojas-Garbanzo et al.,
2016). Compound 7 was tentatively assigned hydroxybenzoic acid be-
cause it produced a ion [M−H]− at m/z 137.0325 and a characteristic
ion at m/z 93.0430 by losing a CO2 (43.9895 Da) group. Compound 3
gave a ion [M−H]− at m/z 299.0832, generating a characteristic ion
[M−H]− at m/z 137.0330 by the loss of hexose (162.0502 Da), which
was similar to quasi-molecular ion of compound 7. Therefore, com-
pound 3 and 6 were tentatively identified as hydroxybenzoic acid
hexoside and its isomer. Compound 8 was tentatively identified as
coumaric acid, because it generated ion [M−H]− at m/z 163.0476,

176
K. Hu et al.
yielding a fragment ion at m/z 119.0581 by losing a CO2 (43.9895 Da)
group (Yasir, Sultana, & Amicucci, 2016). Compound 9 tentatively
corresponded to rosmarinic acid (molecular ion peak at m/z 359.0720),
which cleaved into two product ions at m/z 197.0003 [parent ion-
H + H2O-caffeic acid]− and 160.8070 [caffeic acid-H-H2O]− (Yasir
et al., 2016). Compound 10 produced a ion [M−H]− at m/z 289.0778
with fragment ions at m/z 245.0344 and 179.0657 corresponding to a
loss of 44.0434 Da (CO2 group) and 110.0121 Da and was tentatively
assigned to catechin (Zhao et al., 2014). Compound 11 had a ion
[M−H]− at m/z 193.0578 with fragment ions at m/z 149.0327 and
134.0456 by losing 44.0251 Da (CO2) and 59.0122 Da. It was tenta-
tively identified as ferulic acid. Compound 13 produced a [M−H]− ion
at m/z 167.0415, yielding a characteristic fragment ion at m/z
122.9901 by losing a CO2 (44.0514 Da), which was assigned vanillic
acid (Palafox-Carlos, Yahia, & González-Aguilar, 2012; Wang et al.,
2017). Compound 14 and 28 were tentatively identified as dihy-
drophaseic acid and its isomer, since they produced similar ion
[M−H]− at m/z 281.1453 (error 22.76) and 281.1448. Compound 22
generated a ion [M−H]− at m/z 301.0041, yielding fragment ions at
m/z 257.0132 and 229.0313 by successive losses of 43.9909 Da (CO2
group) and 27.9819 Da (CO group). As a result, 22 was identified as
ellagic acid (Dorta et al., 2014; Ramirez et al., 2014).
3.4.2. Mangiferin and its derivatives and/or related compounds
Compound 12 gave a [M−H]− ion at m/z 575.1031 (error −1.04)
with fragment ions 303.0570 and 193.0220 corresponding to successive
losses of 272.0461 Da [M-H-120-152]− and 110.0350 Da and was
tentatively identified as maclurin-mono-O-galloyl-glucose (Berardini
et al., 2004). Compound 16 showed an [M−H]− ion at m/z 727.1062
and a daughter MS ion at m/z 575.1044 by losing a 152 Da (galloyl
moiety) and was identified as maclurin-di-O-galloyl-glucose (Berardini
et al., 2004). Compound 15 gave a quasi-molecular ion at m/z 421.0806
(error 8.31), yielding fragment ions at m/z 331.0510 and 301.0406 by
losing 90.0296 Da and 120.0400 Da, a fragmentation behaviour typical
of C-glycosides. Therefore, 15 was identified as mangiferin (Ramirez
et al., 2014; Schieber et al., 2003). Compound 18 produced a quasi-
molecular ion at m/z 573.0878 (error -0.35) and daughter ion at m/z
421.0774 by losing a galloyl moiety (152.0104 Da), and fragment ion at
m/z 331.0497 and 301.0415 indicated an identical fragmentation be-
haviour with mangiferin. Thus 18 and 20 were mangiferin gallate
(Schieber et al., 2003). Compound 19 had a quasi-molecular ion at m/z
711.1197 (error 0), yielding fragment ions at m/z 559.1125 and
389.0872 corresponding to the successive losses of 152.0072 Da (gal-
loyl moiety) and 170.0253 Da (gallic acid). Therefore, 19 was identified
as iriflophenone di-O-galloyl-glucose (Berardini et al., 2004).
3.4.3. Gallotannins
Compound 17 showed a ion [M−H]− at m/z 787.0914 (error
−10.16) and fragment ions at m/z 635.0902 and 617.0795 corre-
sponding to losses of 152.0012 Da (galloyl moiety) and 170.0119 Da
(gallic acid) and was tentatively identified as tetra-O-galloyl-glucose
(Berardini et al., 2004). Compounds 21, 23, 24 and 26 were considered
iso-tetra-O-galloyl-glucose due to a similar quasi-molecular ions (error
−9.40, −14.36, −5.59 and −9.40, respectively) and fragmentation
manner with compound 17. Compound 31 gave a ion [M−H]− at m/z
939.1035 and fragment ion 787.0917 by losing 152.0118 Da (galloyl
moiety). Further fragmentation caused the loss of H2O (18.0091 Da)
and gallic acid (170.0149 Da), resulting in fragment ions at m/z
769.0826 and 617.0768. Hence, 31 and 33 were identified as penta-O-
galloyl-glucose and iso-penta-O-galloyl-glucose (Berardini et al., 2004).
Compound 34 generated a precursor ion at m/z 1091.1016 and
daughter ions at m/z 939.0955 and 769.0737 by losing 152.0061 Da
(galloyl moiety) and 170.0218 Da (gallic acid). Therefore, 34 and 35
were identified as hexa-O-galloy-glucose and iso-hexa-O-galloy-glucose
(Berardini et al., 2004).
177
Food Chemistry 256 (2018) 171–180
3.4.4. Quercetin derivatives
Compound 25 had a ion [M−H]− at m/z 463.0891 (error 3.02) and
yielded fragment ions at m/z 301.0380, 300.0319, and 271.0310 cor-
responding to losses of 162.0511 Da (hexose moiety), 163.0572 Da and
192.0581 Da. Therefore, 25 and 27 were assigned as quercetin-3-O-
galactoside and quercetin-3-O-glucoside (Ramirez et al., 2014). Com-
pound 29 produced a quasi-molecular ion [M−H]− at m/z 433.0775
(error 0.92), yielding fragment ions 301.0393, 300.0331 and 271.0317
by losing 132.0382 Da (pentose moiety), 133.0444 Da and
162.0458 Da. Referring to the result reported by Schieber et al. (2003),
29, 30 and 32 were identified as quercetin-3-O-xyloside, quercetin-3-O-
arabinopyranoside and quercetin-3-O-arabinofuranoside.
3.4.5. Quantitative analysis of phenolic compounds
Gallic acid, vanillic acid, ellagic acid, mangiferin and quercetin
were quantified by comparing peak areas of samples and standards by
UPLC (Table 1S, supplementary material). Gallic acid was detected in
all tested samples. In pulps, the content of gallic acid was the highest in
cv. Sensation at the fourth mature stage, whereas it was lowest in cv.
Keitt. Mango peel had significantly higher gallic acid content
(8.19–59.29 mg/100 g FW) than mango pulp. The vanillic acid content
increased during Keitt mango ripening, suggesting its possible con-
tribution as a flavouring constituent. A total of 12 mango cultivars from
Malaysia were previously analysed, and ellagic acid was not found in all
tested cultivars (Sulaiman & Ooi, 2012). However, our study detected
minute quantities of ellagic acid (0.94–1.36 mg/100 g FW) in mango
pulps. The highest content of mangiferin in pulp (9.38 mg/100 g FW)
and peel (73.17 mg/100 g FW) were measured from Keitt at stage 1 and
Xiangya at stage 3 respectively. In previous studies, 6.50–13.41 mg/
100 g FW quercetin was quantified from the 80% methanol extract of
pulp from four Chinese mango cultivars (Liu et al., 2013). However, this
study showed that quercetin was only detected in mango peel of cv.
Keitt and cv. Sensation in stage 4 and in stage 1 respectively. Quercetin
was also not detected in three Brazilian cultivars Haden, Tommy Atkins
and Palmer (Ribeiro et al., 2008). Perhaps it was distributed in plant
tissues broadly as derivatives according to the above identification of
phenolic compounds in mango.
3.5. Principal components analysis
The principal component analysis was performed to observe pos-
sible clusters (Fig. 1). First two principal components accounted for
Fig. 1. A biplot based on principal component analysis of pytochemicals and biochemical
properties in mango pulp of three cultivars in four maturities. Arrows from the origin
represented different variables PPO, POD, SOD, PME, VC, TPC, TFC, TCC, GA (gallic
acid), M (mangiferin), VA (vanillic acid), EA (ellagic acid), DPPH activity and ABTS ac-
tivity.
K. Hu et al.
Fig. 2. Variation of the peak area of phenolic groups (A) phenolic acids and its derivatives, (B) mangiferin and its derivatives, (C) quercetin derivatives and (D) gallotannins with mango
ripening. Maturation stages 1, 2, 3 and 4 represented mangoes with high firmness, medium firmness, and slightly and very soft texture, respectively. Each data point represents a mean
and vertical bars indicate the standard deviation. Means with different letters within each picture are significantly different (p < 0.05).
71.94% of the variation (PC1 = 58.83%, PC 2 = 12.72%). All samples
were grouped separately into twelve small clusters according to the
phytochemicals and biochemical properties. Moreover, the samples
were grouped into two large clusters green pulp and ripe pulp along
with PC1. Based on the loading information, the green mango pulp had
higher PME activity, TPC, TFC, VC, mangiferin, and ellagic acid con-
tents and DPPH and ABTS scavenging activities, and lower PPO, POD,
SOD and TCC. As exhibited in Fig. 2S (Supplementary material), pulp
and peel samples were thoroughly separated into two groups on PC1
and the scores of pulp samples were extremely analogous, indicating
that the largest contribution to scores was the fruit tissue type from
which the sample was taken. In addition, almost all indices were greater
in mango peel, suggesting mango peel possessed more abundant
bioactive compounds than pulp, and as a by-product, mango peel has
significant potential for health benefits.
3.6. Quantification analysis of phenolic groups with mango ripening
Based on the analysis of UPLC, as well as the distribution and
structure characteristics of phenolic compounds in the chromatogram,
the phenolic compounds in Keitt mango pulp are divided into 4 main
groups, (i) phenolic acid/derivatives (RT 0 to 8.00 min), (ii) mangi-
ferin/derivatives (RT 8.00 to 10.00 min), (iii) quercetin derivatives (RT
10.70–12.00 min) and (iv) gallotannins (RT 10.00–10.70 and 11.80 to
20.00 min) (Fig. 3S, Supplementary material). Each group was rela-
tively quantified by peak area to investigate its variation during mango
ripening. As maturity evolved, phenolic acids and its derivatives were
found to almost linearly decline as mango ripened (Fig. 2A), coinciding
with the trend in total phenolics (Table 2). A dramatic decrease of
mangiferin and its derivatives (Fig. 2B), quercetin derivatives (Fig. 2C)
178
Food Chemistry 256 (2018) 171–180
and gallotannins (Fig. 2D) were simultaneously observed in stage 2.
Then, mangiferin and its derivatives gradually increased to one-half the
level of the initial phase (stage 1), and the quercetin derivatives rose to
near the initial value, whereas gallotannins have been markedly syn-
thesized at approximate maturity (stage 3) and then decreased at the
senescence phase (stage 4). All four groups achieved maximum accu-
mulation when mango was hard green (stage 1). Phenolic acids and its
derivatives represented more than 50% of total phenolics throughout
maturation (Fig. 4S, Supplementary material), indicating that they were
dominant phenolic compounds, whereas mangiferin and its derivatives
and quercetin derivatives made up a small proportion. Furthermore,
phenolic acids and its derivatives could also be as a ripening index with
converse trend compared with TCC.
3.7. Prebiotic activity assay
In the current work, we studied the probable prebiotic activities of
mango juice and mango extracts, as well as major phenolic acid, pri-
marily gallic acid. All test samples promoted the growth of Lactobacillus
acidophilus at appropriate concentrations (Fig. 3). Mango juice at a wide
range of concentrations enhanced the proliferation of L. acidophilus up
to 25–35% compared with control. As with extracts from mango pulp,
maximum inhibition was found with an initial concentration, with cvs.
Keitt, Sensation and Xiangya inhibiting the growth of L. acidophilus to
46, 94 and 49%, respectively. At lower concentrations of < 1/4, all
extracts induced the proliferation of L. acidophilus, particularly cv.
Sensation. Gallic acid generally exerted a positive effect on the growth
of L. acidophilus except for the initial concentration (Fig. 3). This in-
dicated that gallic acid may be an important contributor in mango juice
and extracts for the proliferation of L. acidophilus. All tests showed a
K. Hu et al.
Fig. 3. Effect of mango juice and polyphenolic extracts on the proliferation of
Lactobacillus acidophilus. The values are the mean proliferations in comparison with the
control, which was normalised to 100% ± SEM (n = 3).
marked concentration-effect relationship (Fig. 3). Notwithstanding
physical mixture mangiferin and gallic acid was expected to synergize
proliferation, it did not. We found that our samples could not promote
the growth of L. rhamnosus (Fig. 5S, Supplementary material). This
indicated that mango juice and its extracts can selectively proliferate
probiotics. Parkar, Trower, and Stevenson (2013) reported that poly-
phenols were potentially able to exert beneficial effects on gut micro-
biota, but only indirectly and their study showed that original plant
compounds were ineffective, but biotransformation products had a
significant effect on proliferation of Bifidobacterium spp. The in vitro
study on the proliferation functionality of mangoes provided an in-
dication of the prebiotic benefit that may be derived in vivo by the
consumption of mango juice or its extracts. Increasing evidence in-
dicates that health benefits associated with polyphenol consumption
depend on microbial utilization and the metabolites produced, rather
than on parent compounds (Duenas et al., 2015). Hence, in vivo testing
is required for investigating metabolites of mango polyphenolics and its
modulation of the gut microbial milieu. Effects of mango products or its
phenolic extracts on gut microbiota are worth further research, which is
likely promising.
4. Conclusion
Experimental mango cultivars were cultivated in the Jinsha River
Valley, southwestern China with enough light, rich heat, and higher
diurnal temperature variation. Phytochemical and biochemical prop-
erties of mango were considerably different among different cultivars
and at different maturation stages. In addition to carotenoids, phenolic
acids could also be used as a measuring index of maturity. Mango juice
and its extracts provided potential promotion for the growth of pro-
biotics. Moreover, this may be useful in the selection of mango cultivars
and maturities and in the formulation of functional foods aimed at
superior prebiotic activity and antioxidant activities.
Acknowledgments
This work was supported by Special Fund for Agro-scientific
Research in the Public Interest.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.foodchem.2018.02.014.
179
Food Chemistry 256 (2018) 171–180
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