: Sistem Informasi Penelitian Universitas Kristen Satya Wacana J02003
Food Research International xxx (xxxx) xxx–xxx
Contents lists available at ScienceDirect
Food Research International
journal homepage: www.elsevier.com/locate/foodres
Changes in polyphenolics during maturation of Java plum (Syzygium cumini
Lam.)
Lydia Ninan Lestarioa, Luke R. Howardb,⁎, Cindi Brownmillerb, Nathan B. Stebbinsb,
Rohana Liyanagec, Jackson O. Layc
a
b
c
Department of Chemistry, Faculty of Science and Mathematics, Satya Wacana Christian University, 52-60 Diponegoro Street, Salatiga 50711, Central Java, Indonesia
Department of Food Science, University of Arkansas, 2650 North Young Avenue, Fayetteville, AR 72704, United States
University of Arkansas Statewide Mass Spectrometry Facility, 1260 West Maple Street, Fayetteville, AR 72701, United States
A R T I C L E I N F O
A B S T R A C T
Keywords:
Anthocyanins
Flavanonols
Flavonols
Hydrolysable tannins
Maturation
Syzygium cumini
Java plum (Syzygium cumini Lam.) is a rich source of polyphenolics with many purported health benefits, but the
effect of maturation on polyphenolic content is unknown. Freeze-dried samples of Java plum from seven
different maturity stages were analyzed for anthocyanin, flavonol, flavanonol and hydrolysable tannin
composition by HPLC. Anthocyanins were first detected at the green-pink stage of maturity and increased
throughout maturation with the largest increase occurring from the dark purple to black stages of maturation.
Levels of gallotannins, ellagitannins, flavonols, gallic acid and ellagic acid were highest at early stages of
maturation and decreased as the fruit ripened. For production of antioxidant-rich nutraceutical ingredients, fruit
should be harvested immature to obtain extracts rich in hydrolysable tannins and flavonols. The exceptional
anthocyanin content of black fruit may prove useful as a source of a natural colorant.
1. Introduction
The biological activities of Syzygium cumini extracts have been
attributed to the abundant and diverse array of phenolic compounds.
The fruit is rich in anthocyanins, primarily the diglucosides of
delphinidin, cyanidin, petunidin, peonidin, and malvidin (Brito et al.,
2007; Veigas, Narayan, Laxman, & Neelwarne, 2007; Faria et al., 2011;
Tavares et al., 2016), flavonols, mostly myricetin derivatives (Faria
et al., 2011; Tavares et al., 2016), and flavanonols, flavan-3-ols,
proanthocyanidins, ellagitannins and gallotannins (Tavares et al.,
2016). Anthocyanins and hydrolysable tannins are reported to be the
most abundant phenolics in the fruit followed by flavanonols, flavonols
and flavan-3-ols (Tavares et al., 2016).
Similar to other pigmented fruit, the color intensity of Java plum
fruit increases as the fruit ripens changing from green-yellow in
immature fruit to dark purple to black in fully ripe fruit. However,
there are limited reports on composition of anthocyanins and other
polyphenolics of Syzygium cumini at different stages of fruit maturity.
The objective of this study was to identify and quantify anthocyanins,
flavonols, flavanonols, and hydrolysable tannins over seven maturity
stages ranging from immature to fully ripe. This information will be useful
in development of natural colorants or anti-oxidative food additives.
Syzygium cumini Lamark fruit, known as black plum or java plum is a
small edible tropical fruit with a deep purple peel color, white to pink
colored flesh, with one seed inside. The taste is a combination of sweet,
sour and astringent. It is usually consumed in Indonesia with salt on the
fruit. The leaves, seeds, bark and fruit have long been used in India for
the treatment of diabetes (Schossler et al., 2004; Helmstadter, 2008;
Kumar et al., 2008; Ayyanar & Subash-Bubu, 2012; Tupe et al., 2015).
The health-promoting effects of Syzygium cumini extracts are thought to
due to various biological activities. Fruit extracts have anti-oxidative
activity (Benherial & Arumughan, 2007; Hassimotto, Genovese, &
Lajolo, 2005; Faria, Marques, & Mercadante, 2011; Aqil, Jeyabalan,
Munagala, Singh, & Gupta, 2016); anti-inflammatory properties (Pavan
Kumar, Prashad, Rao, Reddy, & Abhinay, 2010), antibacterial properties (Kaneria, Baravalia, Vaghasiya, & Chanda, 2009), anti-proliferative
Gupta,
Munagala,
activities
against
human
lung
(Aqil,
Jeyabalan, & Kausar, 2012) and breast cancer cells (Li et al., 2009;
Aqil et al., 2016) and pro-apoptopic effects against human breast cancer
cells (Li et al., 2009).
⁎
Corresponding author at: Department of Food Science, University of Arkansas, 2650 N. Young Ave., Fayetteville, AR 72704, United States.
E-mail address: lukeh@uark.edu (L.R. Howard).
http://dx.doi.org/10.1016/j.foodres.2017.04.023
Received 2 March 2017; Received in revised form 16 April 2017; Accepted 18 April 2017
0963-9969/ © 2017 Elsevier Ltd. All rights reserved.
Please cite this article as: Lestario, L.N., Food Research International (2017), http://dx.doi.org/10.1016/j.foodres.2017.04.023
Food Research International xxx (xxxx) xxx–xxx
L.N. Lestario et al.
2. Material and methods
side derivatives were quantified as Dpd, Cyd, Ptd, Pnd, and Mvd
glucoside equivalents using external calibration curves of a mixture of
anthocyanin glucosides ranging from 6.25 μg/mL to 200 μg/mL. Total
anthocyanins were calculated as the sum of individual anthocyanin
glycosides.
Anthocyanins were identified by HPLC/ESI-MS using conditions
described above with the HPLC interfaced to a Bruker Esquire LC/MS
ion trap mass spectrometer. Mass spectral data were collected with the
Bruker software, which also controlled the instrument and collected the
signal at 520 nm. Typical conditions for mass spectral analysis in
positive ion electrospray mode for anthocyanins included a capillary
voltage of 4000 V, a nebulizing pressure of 30.0 psi, a drying gas flow of
9.0 mL/min and a temperature of 300 °C. Data were collected in full
scan mode over a mass range of m/z 50–1000 at 1.0 s per cycle.
Characteristic ions were used for peak assignment (Table 1). For
compounds where chemical standards were commercially available,
retention times were also used to confirm the identification of
components.
2.1. Reagents and standards
A mixture of the 3-glucosides of delphinidin, cyanidin, petunidin,
peonidin, pelargonidin and malvidin was obtained from Polyphenols
Laboratories (Sandnes, Norway). Rutin, gallic acid, ellagic acid, and
formic acid were purchased from Sigma Aldrich (St. Louis, MO). HPLC
grade methanol, acetonitrile and acetone were obtained from JT Baker
Inc. (Phillipsburg, NJ, USA) and formic acid was obtained from Burdick
and Jackson (Muskegon, MI, USA).
2.2. Fruit preparation
Syzygium cumini fruit were harvested manually on November 6,
2012 from several trees located at Yogyakarta, Indonesia. On the day of
harvest, the trees had similar fruit loads with a full spectrum of fruit
from different stages of maturation. After harvest, fruit were separated
at seven different stages of maturity based on visual skin color; 1)
green-yellow, 2) green-pink, 3) pink, 4) red, 5) light purple, 6) dark
purple and 7) black. The color was located predominately in the skin of
the fruit, although some fruit had pink to light purple coloration in the
pulp. Following color sorting fruit were transported directly to the
laboratory. After washing with tap water and manually removing seeds,
the edible portions of the fruit at each maturity stage i.e. peel plus pulp,
were freeze-dried, pulverized using a coffee grinder and stored at
− 20 °C until analysis.
2.5. HPLC and HPLC/ESI-MS analysis of flavonols, flavanonols and
hydrolysable tannins
Flavonols, flavanonols, and hydrolysable tannins were analyzed on
the same Waters HPLC system described above according to the method
of Hager, Howard, and Prior (2010). Separation was performed using a
Phenomenex Aqua 5 μm C18 (250 × 4.6 mm) column (Torrance, CA).
The mobile phase ran at a flow rate of 1 mL/min was a linear gradient
of 2% acetic acid (A) and acetic acid:acetonitrile (50:50) (B) from 10%
B to 55% B for 50 min, then from 55% B to 100% B for 10 min, then
from 100% B to 10% B for 5 min. Detection wavelengths used were
254 nm for ellagic acid, 280 nm for gallotannins and galloyl-HHDPglucose and 360 nm for flavonols and flavanonols. Individual flavonols
and flavanonols and gallotannins were quantified as rutin and gallic
acid equivalents, respectively using external calibration curves of
authentic standards ranging from 6.25 μg/mL to 200 μg/mL. The
ellagitannins, G-2HHDP-glc, 2G-HHDP-glc and ellagic acid were quantified using external calibration curves of ellagic acid ranging 6.25 μg/
mL to 200 μg/mL.
Flavonols, flavanonols and hydrolysable tannins were identified by
HPLC/ESI-MS using the same HPLC conditions described. Mass spectral
analysis was performed in negative ion electrospray mode and signals
were collected at 280 or 360 nm. Characteristic ions were used for peak
assignment (Table 1). For compounds where chemical standards were
commercially available, retention times were also used to confirm the
identification of components.
2.3. Polyphenolic extraction
Samples of freeze dried fruit (1 g) from each maturity stage were
weighed in 50 mL plastic centrifuge tubes and homogenized for 1 min
in 20 mL of extraction solution containing methanol/water/formic acid
(60:37:3, v/v/v) using a IKA T18 basic, Ultra Turrax Tissuemizer
(Tekmar-Dohrman Corp., Mason, OH). After rinsing the tissuemizer
with 5 mL of extraction solution, the homogenates were centrifuged at
2100 xg, 21 °C for 10 min (Allegra, TMX-22R), and filtered through
Miracloth (CalBiochem, La Jolla, CA). The extractions were repeated,
and the filtrates were pooled in a 100 mL volumetric flask. The residues
were re-extracted with 20 mL of extraction solution containing acetone/water/acetic acid (AWA) (70:29.5:0.5, v/v/v). All the filtrates
were pooled and adjusted to a final volume of 100 mL with AWA
solution and stored in 120 mL sealed plastic vials at 4 °C prior to
analysis. Extracts (25 mL) were placed in 50 mL plastic vials and dried
using a SpeedVac concentrator (Model SC 210A, ThermoSavant,
Holbrook, NY). Samples for anthocyanin analysis were re-suspended
in 1 mL of 3% formic acid with sonication for 10 min, while samples for
analysis of flavonols and other phenolics were re-suspended in 2 mL of
50% methanol with sonication. All samples were passed through
0.45 μm PTFE syringe filters (Varian, Inc. Palo Alto, CA) prior to
HPLC analysis.
2.6. Statistical analysis
The effect of maturation on anthocyanin, flavonol, flavanonol and
hydrolysable tannin contents were determined by one-way analysis of
variance (ANOVA) using JMP 8.0 (Cary, NC). Differences between
means (n = 3) were determined by Student's t-test. (α = 0.05).
2.4. HPLC and HPLC/ESI-MS analysis of anthocyanins
3. Results and discussion
The HPLC analysis of anthocyanins was conducted using the method
of Cho, Howard, Prior, and Clark (2004). Samples (50 μL) were
analyzed using a Waters HPLC system equipped with a model 600
pump, a model 717 Plus autosampler and a model 996 photodiode
array detector. Separation was carried out using a 4.6 mm × 250 mm
Symmetry® C18 column (Waters Corp, Milford, MA, USA) preceded by a
3.9 mm × 20 mm Symmetry® C18 guard column. The mobile phase was
a linear gradient of 5% formic acid (A) and methanol (B) from 2% B to
60% B for 60 min at 1 mL/min, then from 60% B to 2% B for 5 min at
the same flow rate. The system was equilibrated for 20 min at the initial
gradient prior to each injection. A wavelength of 520 nm was used for
peak detection. Individual anthocyanin monoglucosides and digluco-
3.1. Anthocyanin composition of Syzygium cumini fruit
Six anthocyanins were identified in Syzygium cumini extracts by
HPLC-PDA (Fig. 1A) and HPLC-PDA-MS; delphinidin-3,5-O-diglucoside
(m/z 627/303); cyanidin-3,5-O-diglucoside (m/z 611/287); petunidin3,5-O-diglucoside (m/z 641/317); peonidin-3,5-O-diglucoside (m/z
625/301), delphinidin-3-O-glucoside (m/z 465/303) and malvidin3,5-O-diglucoside (m/z 655/331). Five of the six major anthocyanins
identified were in the form of 3,5-O-diglucoside, which agrees with
previous findings (Brito et al., 2007; Sari, Wijaya, Sajuthi, & Supratman,
2012; Li et al., 2009; Faria et al., 2011; Santos et al., 2013, Tavares
2
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L.N. Lestario et al.
et al., 2016). Delphinidin was the only anthocyanin identified in the
form of 3-O-glucoside. Four additional anthocyanins cyanidin-3-Oglucoside, petunidin-3-O-glucoside; malvidin-3-O-glucoside, and delphinidin-acetyl-O-diglucoside previously reported by Faria et al. (2011)
and Tavares et al. (2016) in Syzygium cumini fruit were not detected in
our samples. The discrepancy between our results and those of Faria
et al. (2011) and Tavares et al. (2016) may be due to varietal difference,
variation in environmental growing conditions, or differences in
extraction protocol.
Table 1
Chromatographic and spectroscopic properties obtained by HPLC-DAD-MS of phenolic
compounds from Syzygium cumini.
Peak
Compound
tR (min)
λmax
(nm)
[M-H]a or
[M]+ b (m/z)
(m/z)
1
Delphindin-3,5-Odiglucoside
Cyanidin-3,5-Odiglucoside
Petunidin-3,5-Odiglucoside
Peonidin-3,5-Odiglucoside
Delphindin-3-Oglucoside
Malvidin-3,5-Odiglucoside
Galloyl glucoside
Dihydromyricetin
diglucoside
Methyldihydromyricetin
diglucoside
Dimethyldihydromyricetin
diglucoside
Myricetin-3-Oglucoside
Myricetin-3-Opentoside
Myricetin-3-Orhamnoside
Gallic acid
Vescalagin
23.21
519
627b
465,303
25.59
512
611b
449,287
27.21
523
641b
479,317
28.18
524
625b
29.47
518
465b
463,
301
303
30.48
527
655b
493,331
4.8
10.8
275
337
663
643
16.1
338
657
331
481,
355
495
20.0
338
671
509,
347
30.8
356
479
316
33.8
355
449
316
34.2
347
463
316
6.8
9.2
NDa
933
633
2
3
3.2. Changes in anthocyanin composition during maturation
4
The anthocyanin composition of Syzygium cumini fruit extracts
during maturation is presented in Table 2. As expected no anthocyanins
were detected at the green-yellow of maturity, but low levels of
cyanidin-3,5-O-diglucoside, delphindin-3,5-O-diglucoside, malvidin3,5-O-diglucoside and peonidin-3,5-O-diglucoside were detectable at
the green-pink stage of maturity. Levels of the two minor anthocyanins
in Syzygium cumini fruit, delphinidin-3-O-glucoside and peonidin-3,5-Odiglucoside were not detectable until the light purple stage of maturity.
The anthocyanin content increased sharply when the fruit ripened to
dark purple. These results confirm those of Patel and Rao (2014) who
reported a large increase in total anthocyanins in Syzygium cumini as
fruit fully ripened. Anthocyanin content has also been reported to
increase sharply in fully ripe fruit such as Vaccinium species (Prior et al.,
1998), blackberries (Siriwoharn, Wrolstad, Finn, & Pereira, 2004) and
lychee (Rivera-Lopez, Ordorica-Falomir, & Wesche-Ebeling, 1999).
Throughout fruit maturation, delphinidin-3,5-O-diglucoside was the
predominant anthocyanin accounting for 37–48% of total anthocyanins, followed by petunidin-3,5-O-diglucoside (29–33%) and malvidin3,5-O-diglucoside (19–27%), while cyanidin-3,5-O-diglucoside (3%),
delphinidin-3-O-glucoside (2–3%) and peonidin-3,5-O-diglucoside
(1–2%) were minor constituents. A similar distribution of anthocyanins
in Syzygium cumini fruit was reported by Faria et al. (2011) and Tavares
et al. (2016).
The total anthocyanin content of fully ripe black fruit in this
research, 1318.4 mg/100 g DW was higher than a previously reported
value of 777 mg/100 g DW (Brito et al., 2007). This difference may be
due to the advanced stage of ripened fruit (black) analyzed in our study
and is consistent with a previous study that reported over-ripe blackberries to have much higher levels of total anthocyanins than ripe
berries (Siriwoharn et al., 2004).
5
6
7
8
9
10
11
12
13
14
15
16
3G-glc (1)
b
22.0
271
< 240,
270sh
284
b
17
18
3G-glc (2)
G-2HHDP-glcb
23.1
26.6
278
< 240
635
935
19
2G-HHDP-glcb
27.4
274
785
20
21
22
23
4G-glc (1)b
4G-glc (2)b
Ellagic acid
5G-glc (1)b
31.0
32.8
35.6
36.5
279
279
254, 365
282
787
787
301
939
38.1
279
939
41.9
277
1091
24
5G-glc (2)
25
6G-glc
b
3.3. Flavonol and flavanonol composition of Syzygium cumini fruit
Three flavanonols detected in Syzygium cumini fruit were dihydromyricetin diglucoside (m/z 643/481), methyl-dihydromyricetin diglucoside (m/z 657/495), dimethyl-dihydromyricetin diglucoside (m/z
671/509), additionally, three flavonols myricetin-3-O-hexoside (m/z
479/316), myricetin-3-O-pentoside (m/z 449/316), and myricetin-3-Orhamnoside (m/z 463/316) were detected (Fig. 1B). These compounds
were previously reported by Faria et al. (2011) in addition to myricetin
aglycone, which was not detected in our samples. Tavares et al. (2016)
identified nine flavonols in skin and pulp extracts of Syzygium cumini
fruit, besides the three flavonols identified in our study they also
detected myricetin-3-O-glucuronide, myricetin-3-O-galactoside, laricitrin-3-O-galactoside, laricitrin-3-O-glucoside, syringetin-3-O-galactoside, and syringetin-3-O-glucoside. They also reported that most of
the flavonols present in the fruit were located in the skin.
631,
425
481,
463
483
785,
633,
301
635,
483,
301
617
617
787,
401
787,
617,
465
939,
787,
461
a
Identification was confirmed with an authentic standard.
3G-glc (1) = trigalloyl glucose (isomer 1), 3G-glc (2) = trigalloyl glucose (isomer 2),
G-2HHDP-glc = galloyl dihexahydroxydiphenoyl glucose, 2G-HHDP-glc = digalloyl dihexahydroxydiphenoyl glucose, 4G-glc (1) = tetragalloyl glucose (isomer 1), 4G-glc (2)
= tetragalloyl glucose (isomer 2), 5G-glu (1) = pentagalloyl glucose (isomer 1), 5G-glu
(2) = pentagalloyl glucose (isomer 2), 6G-glu = hexagalloyl glucose.
b
tin diglucoside, and dimethyl-dihydromyricetin diglucoside were not
detectable until later stages of maturation (light to dark-purple), but
concentrations increased markedly as the fruit fully ripened, with the
highest concentration at the black stage of maturation. For the
flavonols, myricetin-3-O-hexoside was present at low concentration in
immature fruit (green-yellow to light-purple), but increased in concentration as the fruit fully ripened, with the highest concentration present
at the black stage of maturation. Conversely, myricetin-3-O-pentoside
and myricetin-3-O-rhamnoside were present at the highest concentration in immature fruit (green-yellow to green-pink) and concentrations
declined as the fruit fully ripened. Due to the fluctuation in individual
3.4. Changes in flavonol and flavanonol composition during maturation
The changes in the concentrations of the three flavonols and three
flavanonols detected during maturation are presented in Table 3.
Concentrations of the compounds varied in response to maturation.
The flavanonols dihydromyricetin diglucoside, methyl-dihydromyrice3
Food Research International xxx (xxxx) xxx–xxx
L.N. Lestario et al.
Fig. 1. HPLC chromatograms of anthocyanins (A) in Java plum at black stage of maturation (Abs 520 nm), flavonols and flavanonols (B) at yellow-green stage of maturation (Abs
360 nm), and hydrolysable tannins (C) at yellow-green stage of maturation (Abs 280 nm). Refer to Table 1 for peak identification.
Table 2
Anthocyanin composition (mg/100 g FW) of Syzygium cumini fruit at different maturity stages.
Stage of maturity
Green-yellow
1
Green-pink
f2
Pink
3
Cyanidin-3,5-O-diglucoside
Delphinidin-3,5-Odiglucoside
Delphinidin-3-O-glucoside
ND
ND
0.76 ± 0.03 (3%)
13.8f ± 0.58 (48%)
ND
ND
Malvidin-3,5-O-diglucoside
ND
5.4f ± 0.12 (19%)
Petunidin-3.5-Odiglucoside
Peonidin-3,5-O-diglucoside
ND
8.5f ± 0.35 (30%)
ND
Total anthocyanins
ND
1
2
3
Red
e
Light purple
d
1.22 ± 0.03 (3%)
20.5e ± 0.14
(42%)
ND
2.58 ± 0.22 (3%)
35.4d ± 1.50
(37%)
ND
ND
11.7e ± 0.23
(24%)
15.4e ± 0.07
(32%)
ND
25.9d ± 1.01
(27%)
30.9d ± 1.37
(33%)
ND
28.5f ± 1.08
48.8e ± 0.09
94.8d ± 4.09
c
6.7 ± 0.2 (3%)
104.5c ± 1.33
(39%)
8.6c ± 1.17
(3%)
62.8c ± 7.32
(24%)
80.9c ± 2.19
(30%)
4.1c ± 0.14
(2%)
267.6c ± 11.9
Dark purple
b
Black
12.8 ± 0.56 (3%)
199.8b ± 5.17
(43%)
11.0b ± 0.19 (2%)
35.6a ± 0.69 (3%)
583.5a ± 12.9
(44%)
30.8a ± 0.82 (2%)
98.7b ± 1.76
(21%)
139.5b ± 3.59
(30%)
5.9b ± 0.05 (1%)
268.3a ± 3.80
(20%)
385.6a ± 6.60
(29%)
14.6a ± 0.38 (1%)
467.7b ± 11.2
1318.4 ± 24.7
Non-detectable.
Mean values ± standard error of the mean (n = 3). Values within columns with different letters are significantly different (P < 0.05).
Values within parentheses represent percentage of total anthocyanins.
ries (Kosar, Kafkas, Paydas, & Baer, 2004).
flavonols during maturation, fruit at the green-pink stage of maturation
had the highest concentration of total flavonols, while fruit at the lightpurple and black stages of maturation had the lowest concentrations. In
studies involving other fruit, total flavonol content was reported to
decline during maturation of pomegranate (Fawole, Opara, & Theron,
2013) and blueberries (Castrejon, Eichholz, Rohn, Kroh & HuyskensKeil, 2008), but levels remained stable during maturation of strawber-
3.5. Hydrolysable tannin composition of Syzygium cumini fruit
Eight gallotannins were identified in Syzygium cumini fruit (Fig. 1C);
galloyl glucose (m/z 331), two isomers each of trigalloyl glucose (m/z
635), tetragalloyl glucose (m/z 787), and pentagalloyl glucose (m/z
4
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L.N. Lestario et al.
Table 3
Flavanonol and flavonol contents (mg/100 g DW) of Syzygium cumini fruit at different maturity stages.
Stage of
maturity
Flavanonols
Dihydromyricetin
diglucoside
Methyldihydromyricetin
diglucoside
Dimethyl
dihydromyricetin
diglucoside
Total
flavanonols
Flavonols
Myricetin-3Ohexoside
2.0 ± 0.04b
Myricetin-3Opentoside
3.2 ± 0.06bc
Myricetin-3Orhamnoside
6.8 ± 0.13de
Total
flavonols
12.0 ± 0.18 cd
a
b
Greenyellow
Green-pink
Pink
Red
Light
purple
Dark
purple
Black
NDa
ND
ND
ND
ND
0.11b ±
0.01b
ND
ND
ND
ND
0.41 ±
0.001b
0.49 ±
0.01b
2.8 ±
0.24a
2.0 ±
0.15a
ND
ND
ND
ND
0.70 ±
0.01c
0.98 ±
0.02b
2.9 ±
0.07a
ND
ND
ND
ND
1.1 ±
0.01b
1.6 ±
0.05b
7.7 ±
0.47a
0.72 ±
0.01e
1.0 ± 0.06cde
0.8-
3 ± 0.09de
1.3 ±
0.05c
1.1 ±
0.11 cd
3.2 ±
0.32a
4.94 ±
0.09a
5.7 ± 0.28a
3.-
1 ± 0.47bc
3.8 ±
0.32b
2.6 ±
0.26c
2.3 ±
0.32c
10.6 ±
0.22b
12.5 ± 0.56a
7.-
7 ± 1.00 cd
9.2 ±
0.55bc
5.9 ±
0.38e
4.2 ±
0.43f
16.2 ±
0.18b
19.2 ± 0.62a
11.-
6 ± 1.03 cd
14.3 ±
0.69bc
9.6 ±
0.63d
9.7 ±
0.96d
Non-detectable.
Mean values ± standard error of the mean (n = 3). Values within columns with different letters are significantly different (P < 0.05).
concentrations declined precipitously as the fruit ripened. An exception
was galloyl glucose, which was present at low concentration in greenyellow fruit, but increased with further ripening reaching a maximum
concentration at the red stage, followed by a major decline as the fruit
fully ripened. Changes in concentration of the phenolic acid gallic acid
during maturation also varied from the hydrolysable tannins as
concentration remained high until the red stage of maturity and then
declined markedly as the fruit fully ripened from red to black.
Hexagalloyl glucose (6G-glu) was not detectable from the light purple
to black stages of maturity and vescalagin was only detected in the
green-yellow stage. Total gallotannin content was highest at the red
stage of maturation (370.5 mg/100 g DW) due to the abundance of
galloyl glucose and gallic acid, but content decreased to 93.7 mg/100 g
DW when the fruit fully ripened to the black stage. Total ellagitannin
content was highest in immature green-yellow fruit (26.1 mg/100 g
DW) and decreased throughout maturation reaching a low of 2.9 mg/
100 g DW at the fully ripe black stage. The loss of gallotannins during
939), and hexagalloyl glucose (m/z 1091) in addition to gallic acid.
Additionally, three ellagitannins, galloyl-diHHDP-glucose (m/z 935),
digalloyl-HHDP-glucose (m/z 785) and vescalagin (m/z 933) as well as
free ellagic acid (m/z 301) were identified in the fruit extracts. These
compounds were previously identified in Syzygium cumini skin and pulp
extracts using HPLC-DAD-ESI—MS/MS (Tavares et al., 2016) in addition to a number of other gallotannins and ellagitannins that were not
detected in our samples. According to Tavares et al. (2016) most of the
gallotannins and ellagitannins are present in much higher amounts in
the skin as opposed to the pulp.
3.6. Changes in hydrolysable tannin composition during maturation
The hydrolysable tannin content of Syzygium cumini fruit from seven
different stages of maturity are presented in Table 4. All of the
hydrolysable tannins (gallo- and ellagitannins) were present at the
highest concentration in immature (green-yellow) fruit, and their
5
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L.N. Lestario et al.
Table 4
Hydrolysable tannin content (mg/100 g DW) of Syzygium cumini fruit at different maturity stages.
Stage of
maturity
Greenyellow
Green-pink
Pink
Gallotannins
Galloyl
glucose
4.9a ± 0.19d
114.1 ±
7.86c
21.39 ±
0.92d
154.7 ±
2.2a
107.6 ± 5.7c
Gallic acid
93.2 ± 2.18c
3G-glc (1)
5.6 ± 0.14d
3G-glc (2)
4.9 ± 0.12de
4G-glc (1)
11.7 ± 0.08d
4G-glc (2)
2.2 ± 0.08ab
5G-glc (1)
4.4 ± 0.12d
5G-glc (2)
2.2 ± 0.06d
6G-glc
ND
Total gallotannins
232.1 ± 5.4c
Ellagitannins
Vescalagin
G-2HHDPglc
1.5 ± 0.03 cd
2G-HHDPglc
0.97 ± 0.02de
Ellagic acid
1.8 ± 0.04bc
Total ellagitannins
4.3 ± 0.03d
a
b
38.3 ±
1.0d
13.3 ±
0.09a
13.4 ±
0.29a
25.57 ±
0.74a
3.7 ±
0.19e
37.85 ±
1.31a
12.8 ±
0.37d
2.89 ±
0.14a
2.3 ±
1.19ab
15.4 ±
1.2a
0.8 ±
0.09e
10.6 ±
0.15a
0.8 ±
0.05e
0.47 ±
0.01a
ND
265.7 ±
5.3c
Red
Light
purple
Dark
purple
Black
142.-
6 ± 12.1b
185.9 ±
14.0a
115.9 ± 4.4c
150.9 ±
9.37a
125.-
7 ± 6.63b
150.9 ±
7.16a
86.3 ± 9.62c
10.3 ± 1.96b
9.-
5 ± 0.98bc
7.0 ±
0.92 cd
5.8 ±
0.30d
17.2 ± 0.83b
12.8-
9 ± 1.13c
6.7 ±
1.08d
3.1 ±
0.13e
24.9 ± 1.37b
17.-
9 ± 2.40c
10.8 ±
1.21d
9.0 ±
0.79d
2.1 ± 0.16ab
2.-
5 ± 0.21ab
1.5 ±
0.06b
1.6 ±
0.11ab
10.3 ± 0.29b
7.-
9 ± 1.28c
4.6 ±
0.49d
3.9 ±
0.11d
4.8 ± 0.16b
3.-
5 ± 0.43c
2.4 ±
0.14d
2.3 ±
0.13d
0.2 ± 0.05b
0.2-
5 ± 0.04b
0.1 ±
0.01c
NDb
335.0 ±
11.3ab
322.-
8 ± 24.1b
370.5 ±
17.1a
228.4 ± 14.7c
ND
ND
ND
ND
ND
2.5 ± 0.21b
2.-
1 ± 0.43bc
2.3 ±
0.27b
2.6 ±
0.11b
3.1 ± 0.09b
1.-
5 ± 0.13c
1.0 ±
0.19d
0.95 ± 0.07de
1.9 ± 0.15bc
2.-
4 ± 0.19b
2.2 ±
0.40bc
1.7 ±
0.15 cd
7.4 ± 0.39b
6.-
0 ± 0.69c
5.6 ±
0.47c
5.2 ±
0.33 cd
93.7 ±
1.09d
1.0 ± 0.05a
7.5 ± 0.08a
1.2 ±
0.13d
5.6 ± 0.11a
0.63 ±
0.08e
11.9 ±
0.33a
1.1 ±
0.15d
26.1 ±
0.42a
2.9 ±
0.28e
ND
Mean values ± standard error of the mean (n = 3). Values within columns with different letters are significantly different (P < 0.05).
Non-detectable.
6
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L.N. Lestario et al.
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the pancreatic duct. Brazilian Journal of Veterinary Research and Animal Science, 41,
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S., Garcia-Romero, E., ... Hermosin-Gutierrez, I. (2016). Comprehensive study of the
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skeels). Food Research International, 82, 1–13.
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maturation are consistent with a previous study on Syzygium cumini
fruit, where total tannins measured using a casein precipitation method
decreased from 669.4 mg/100 g FW in immature fruit (green-purple) to
185.8 mg/100 g FW in ripe mature fruit (Brandão, Sena, TEShiMA,
David, & Assis, 2011). Additionally, the gallotannin content of pomegranate juice prepared from immature fruit and mature-green mangoes
had 2-fold higher levels of gallotannins than fully ripe fruit (Fawole
et al., 2013; Kim, Brecht, & Talcott, 2007). Loss of extractable tannins
during fruit ripening is associated with loss of astringency. It is thought
that tannins polymerize during ripening and readily bind to proteins
and cell wall polysaccharides, which greatly reduces their extractability
and astringency (Goldstein & Swain, 1963). The loss of ellagic acid
during maturation agrees with previous studies reporting loss of ellagic
acid in many strawberry genotypes during berry maturation (Kosar
et al., 2004; Gasperroti et al., 2013).
4. Conclusions
Anthocyanins were first detected at the green-pink stage of maturity
and increased throughout maturation with the largest increase occurring from the dark purple to black stages of maturation. Although
present at low concentrations, the levels of three flavanonols detected
also showed the largest increase from the dark purple to black stages of
maturation. Levels of gallotannins, flavonols, ellagitannins and ellagic
acid were highest at early stages of maturation (green-yellow and
green-pink) and decreased as the fruit ripened. For production of
nutraceutical ingredients fruit should be harvested immature to obtain
extracts rich in gallotannins, ellagitannins, ellagic acid, gallic acid and
flavonols. In contrast, fruit should be harvested at the fully ripe black
stage to obtain extracts rich in anthocyanins for possible use as a
natural colorant.
Appendix A. Abbreviations used
All abbreviations used in this study are listed as follows:
High performance liquid chromatography (HPLC), mass spectrometry (MS), electrospray ionization (ESI), photodiode array detector
(DAD), polytetrafluoroethylene (PTFE), trolox equivalents (TE), trigalloyl glucose isomer one ((3G-glc (1)), trigalloyl glucose isomer two
((3G-glc (2)), tetragalloyl glucose isomer one ((4G-glc (1)), tetragalloyl
glucose isomer two ((4G-glc (2)), pentagalloyl glucose isomer one ((5Gglc (1)), pentagalloyl glucose isomer two ((5G-glc (2)), hexagalloyl
glucose (6G-glc), galloyl dihexahydroxydiphenoyl glucose (G-2HHDPglc), digalloyl dihexahydroxydiphenoyl glucose (2G-HHDP-glc), nondetectiale (ND).
References
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phytochemical constituents and traditional uses. Asian Pacific Journal of Tropical
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Changes in enzymes, phenolic compounds, tannins and vitamin C in various stages of
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(2007). Anthocyanins present in selected tropical fruits: acerola, jambolao, jussara,
7
Contents lists available at ScienceDirect
Food Research International
journal homepage: www.elsevier.com/locate/foodres
Changes in polyphenolics during maturation of Java plum (Syzygium cumini
Lam.)
Lydia Ninan Lestarioa, Luke R. Howardb,⁎, Cindi Brownmillerb, Nathan B. Stebbinsb,
Rohana Liyanagec, Jackson O. Layc
a
b
c
Department of Chemistry, Faculty of Science and Mathematics, Satya Wacana Christian University, 52-60 Diponegoro Street, Salatiga 50711, Central Java, Indonesia
Department of Food Science, University of Arkansas, 2650 North Young Avenue, Fayetteville, AR 72704, United States
University of Arkansas Statewide Mass Spectrometry Facility, 1260 West Maple Street, Fayetteville, AR 72701, United States
A R T I C L E I N F O
A B S T R A C T
Keywords:
Anthocyanins
Flavanonols
Flavonols
Hydrolysable tannins
Maturation
Syzygium cumini
Java plum (Syzygium cumini Lam.) is a rich source of polyphenolics with many purported health benefits, but the
effect of maturation on polyphenolic content is unknown. Freeze-dried samples of Java plum from seven
different maturity stages were analyzed for anthocyanin, flavonol, flavanonol and hydrolysable tannin
composition by HPLC. Anthocyanins were first detected at the green-pink stage of maturity and increased
throughout maturation with the largest increase occurring from the dark purple to black stages of maturation.
Levels of gallotannins, ellagitannins, flavonols, gallic acid and ellagic acid were highest at early stages of
maturation and decreased as the fruit ripened. For production of antioxidant-rich nutraceutical ingredients, fruit
should be harvested immature to obtain extracts rich in hydrolysable tannins and flavonols. The exceptional
anthocyanin content of black fruit may prove useful as a source of a natural colorant.
1. Introduction
The biological activities of Syzygium cumini extracts have been
attributed to the abundant and diverse array of phenolic compounds.
The fruit is rich in anthocyanins, primarily the diglucosides of
delphinidin, cyanidin, petunidin, peonidin, and malvidin (Brito et al.,
2007; Veigas, Narayan, Laxman, & Neelwarne, 2007; Faria et al., 2011;
Tavares et al., 2016), flavonols, mostly myricetin derivatives (Faria
et al., 2011; Tavares et al., 2016), and flavanonols, flavan-3-ols,
proanthocyanidins, ellagitannins and gallotannins (Tavares et al.,
2016). Anthocyanins and hydrolysable tannins are reported to be the
most abundant phenolics in the fruit followed by flavanonols, flavonols
and flavan-3-ols (Tavares et al., 2016).
Similar to other pigmented fruit, the color intensity of Java plum
fruit increases as the fruit ripens changing from green-yellow in
immature fruit to dark purple to black in fully ripe fruit. However,
there are limited reports on composition of anthocyanins and other
polyphenolics of Syzygium cumini at different stages of fruit maturity.
The objective of this study was to identify and quantify anthocyanins,
flavonols, flavanonols, and hydrolysable tannins over seven maturity
stages ranging from immature to fully ripe. This information will be useful
in development of natural colorants or anti-oxidative food additives.
Syzygium cumini Lamark fruit, known as black plum or java plum is a
small edible tropical fruit with a deep purple peel color, white to pink
colored flesh, with one seed inside. The taste is a combination of sweet,
sour and astringent. It is usually consumed in Indonesia with salt on the
fruit. The leaves, seeds, bark and fruit have long been used in India for
the treatment of diabetes (Schossler et al., 2004; Helmstadter, 2008;
Kumar et al., 2008; Ayyanar & Subash-Bubu, 2012; Tupe et al., 2015).
The health-promoting effects of Syzygium cumini extracts are thought to
due to various biological activities. Fruit extracts have anti-oxidative
activity (Benherial & Arumughan, 2007; Hassimotto, Genovese, &
Lajolo, 2005; Faria, Marques, & Mercadante, 2011; Aqil, Jeyabalan,
Munagala, Singh, & Gupta, 2016); anti-inflammatory properties (Pavan
Kumar, Prashad, Rao, Reddy, & Abhinay, 2010), antibacterial properties (Kaneria, Baravalia, Vaghasiya, & Chanda, 2009), anti-proliferative
Gupta,
Munagala,
activities
against
human
lung
(Aqil,
Jeyabalan, & Kausar, 2012) and breast cancer cells (Li et al., 2009;
Aqil et al., 2016) and pro-apoptopic effects against human breast cancer
cells (Li et al., 2009).
⁎
Corresponding author at: Department of Food Science, University of Arkansas, 2650 N. Young Ave., Fayetteville, AR 72704, United States.
E-mail address: lukeh@uark.edu (L.R. Howard).
http://dx.doi.org/10.1016/j.foodres.2017.04.023
Received 2 March 2017; Received in revised form 16 April 2017; Accepted 18 April 2017
0963-9969/ © 2017 Elsevier Ltd. All rights reserved.
Please cite this article as: Lestario, L.N., Food Research International (2017), http://dx.doi.org/10.1016/j.foodres.2017.04.023
Food Research International xxx (xxxx) xxx–xxx
L.N. Lestario et al.
2. Material and methods
side derivatives were quantified as Dpd, Cyd, Ptd, Pnd, and Mvd
glucoside equivalents using external calibration curves of a mixture of
anthocyanin glucosides ranging from 6.25 μg/mL to 200 μg/mL. Total
anthocyanins were calculated as the sum of individual anthocyanin
glycosides.
Anthocyanins were identified by HPLC/ESI-MS using conditions
described above with the HPLC interfaced to a Bruker Esquire LC/MS
ion trap mass spectrometer. Mass spectral data were collected with the
Bruker software, which also controlled the instrument and collected the
signal at 520 nm. Typical conditions for mass spectral analysis in
positive ion electrospray mode for anthocyanins included a capillary
voltage of 4000 V, a nebulizing pressure of 30.0 psi, a drying gas flow of
9.0 mL/min and a temperature of 300 °C. Data were collected in full
scan mode over a mass range of m/z 50–1000 at 1.0 s per cycle.
Characteristic ions were used for peak assignment (Table 1). For
compounds where chemical standards were commercially available,
retention times were also used to confirm the identification of
components.
2.1. Reagents and standards
A mixture of the 3-glucosides of delphinidin, cyanidin, petunidin,
peonidin, pelargonidin and malvidin was obtained from Polyphenols
Laboratories (Sandnes, Norway). Rutin, gallic acid, ellagic acid, and
formic acid were purchased from Sigma Aldrich (St. Louis, MO). HPLC
grade methanol, acetonitrile and acetone were obtained from JT Baker
Inc. (Phillipsburg, NJ, USA) and formic acid was obtained from Burdick
and Jackson (Muskegon, MI, USA).
2.2. Fruit preparation
Syzygium cumini fruit were harvested manually on November 6,
2012 from several trees located at Yogyakarta, Indonesia. On the day of
harvest, the trees had similar fruit loads with a full spectrum of fruit
from different stages of maturation. After harvest, fruit were separated
at seven different stages of maturity based on visual skin color; 1)
green-yellow, 2) green-pink, 3) pink, 4) red, 5) light purple, 6) dark
purple and 7) black. The color was located predominately in the skin of
the fruit, although some fruit had pink to light purple coloration in the
pulp. Following color sorting fruit were transported directly to the
laboratory. After washing with tap water and manually removing seeds,
the edible portions of the fruit at each maturity stage i.e. peel plus pulp,
were freeze-dried, pulverized using a coffee grinder and stored at
− 20 °C until analysis.
2.5. HPLC and HPLC/ESI-MS analysis of flavonols, flavanonols and
hydrolysable tannins
Flavonols, flavanonols, and hydrolysable tannins were analyzed on
the same Waters HPLC system described above according to the method
of Hager, Howard, and Prior (2010). Separation was performed using a
Phenomenex Aqua 5 μm C18 (250 × 4.6 mm) column (Torrance, CA).
The mobile phase ran at a flow rate of 1 mL/min was a linear gradient
of 2% acetic acid (A) and acetic acid:acetonitrile (50:50) (B) from 10%
B to 55% B for 50 min, then from 55% B to 100% B for 10 min, then
from 100% B to 10% B for 5 min. Detection wavelengths used were
254 nm for ellagic acid, 280 nm for gallotannins and galloyl-HHDPglucose and 360 nm for flavonols and flavanonols. Individual flavonols
and flavanonols and gallotannins were quantified as rutin and gallic
acid equivalents, respectively using external calibration curves of
authentic standards ranging from 6.25 μg/mL to 200 μg/mL. The
ellagitannins, G-2HHDP-glc, 2G-HHDP-glc and ellagic acid were quantified using external calibration curves of ellagic acid ranging 6.25 μg/
mL to 200 μg/mL.
Flavonols, flavanonols and hydrolysable tannins were identified by
HPLC/ESI-MS using the same HPLC conditions described. Mass spectral
analysis was performed in negative ion electrospray mode and signals
were collected at 280 or 360 nm. Characteristic ions were used for peak
assignment (Table 1). For compounds where chemical standards were
commercially available, retention times were also used to confirm the
identification of components.
2.3. Polyphenolic extraction
Samples of freeze dried fruit (1 g) from each maturity stage were
weighed in 50 mL plastic centrifuge tubes and homogenized for 1 min
in 20 mL of extraction solution containing methanol/water/formic acid
(60:37:3, v/v/v) using a IKA T18 basic, Ultra Turrax Tissuemizer
(Tekmar-Dohrman Corp., Mason, OH). After rinsing the tissuemizer
with 5 mL of extraction solution, the homogenates were centrifuged at
2100 xg, 21 °C for 10 min (Allegra, TMX-22R), and filtered through
Miracloth (CalBiochem, La Jolla, CA). The extractions were repeated,
and the filtrates were pooled in a 100 mL volumetric flask. The residues
were re-extracted with 20 mL of extraction solution containing acetone/water/acetic acid (AWA) (70:29.5:0.5, v/v/v). All the filtrates
were pooled and adjusted to a final volume of 100 mL with AWA
solution and stored in 120 mL sealed plastic vials at 4 °C prior to
analysis. Extracts (25 mL) were placed in 50 mL plastic vials and dried
using a SpeedVac concentrator (Model SC 210A, ThermoSavant,
Holbrook, NY). Samples for anthocyanin analysis were re-suspended
in 1 mL of 3% formic acid with sonication for 10 min, while samples for
analysis of flavonols and other phenolics were re-suspended in 2 mL of
50% methanol with sonication. All samples were passed through
0.45 μm PTFE syringe filters (Varian, Inc. Palo Alto, CA) prior to
HPLC analysis.
2.6. Statistical analysis
The effect of maturation on anthocyanin, flavonol, flavanonol and
hydrolysable tannin contents were determined by one-way analysis of
variance (ANOVA) using JMP 8.0 (Cary, NC). Differences between
means (n = 3) were determined by Student's t-test. (α = 0.05).
2.4. HPLC and HPLC/ESI-MS analysis of anthocyanins
3. Results and discussion
The HPLC analysis of anthocyanins was conducted using the method
of Cho, Howard, Prior, and Clark (2004). Samples (50 μL) were
analyzed using a Waters HPLC system equipped with a model 600
pump, a model 717 Plus autosampler and a model 996 photodiode
array detector. Separation was carried out using a 4.6 mm × 250 mm
Symmetry® C18 column (Waters Corp, Milford, MA, USA) preceded by a
3.9 mm × 20 mm Symmetry® C18 guard column. The mobile phase was
a linear gradient of 5% formic acid (A) and methanol (B) from 2% B to
60% B for 60 min at 1 mL/min, then from 60% B to 2% B for 5 min at
the same flow rate. The system was equilibrated for 20 min at the initial
gradient prior to each injection. A wavelength of 520 nm was used for
peak detection. Individual anthocyanin monoglucosides and digluco-
3.1. Anthocyanin composition of Syzygium cumini fruit
Six anthocyanins were identified in Syzygium cumini extracts by
HPLC-PDA (Fig. 1A) and HPLC-PDA-MS; delphinidin-3,5-O-diglucoside
(m/z 627/303); cyanidin-3,5-O-diglucoside (m/z 611/287); petunidin3,5-O-diglucoside (m/z 641/317); peonidin-3,5-O-diglucoside (m/z
625/301), delphinidin-3-O-glucoside (m/z 465/303) and malvidin3,5-O-diglucoside (m/z 655/331). Five of the six major anthocyanins
identified were in the form of 3,5-O-diglucoside, which agrees with
previous findings (Brito et al., 2007; Sari, Wijaya, Sajuthi, & Supratman,
2012; Li et al., 2009; Faria et al., 2011; Santos et al., 2013, Tavares
2
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L.N. Lestario et al.
et al., 2016). Delphinidin was the only anthocyanin identified in the
form of 3-O-glucoside. Four additional anthocyanins cyanidin-3-Oglucoside, petunidin-3-O-glucoside; malvidin-3-O-glucoside, and delphinidin-acetyl-O-diglucoside previously reported by Faria et al. (2011)
and Tavares et al. (2016) in Syzygium cumini fruit were not detected in
our samples. The discrepancy between our results and those of Faria
et al. (2011) and Tavares et al. (2016) may be due to varietal difference,
variation in environmental growing conditions, or differences in
extraction protocol.
Table 1
Chromatographic and spectroscopic properties obtained by HPLC-DAD-MS of phenolic
compounds from Syzygium cumini.
Peak
Compound
tR (min)
λmax
(nm)
[M-H]a or
[M]+ b (m/z)
(m/z)
1
Delphindin-3,5-Odiglucoside
Cyanidin-3,5-Odiglucoside
Petunidin-3,5-Odiglucoside
Peonidin-3,5-Odiglucoside
Delphindin-3-Oglucoside
Malvidin-3,5-Odiglucoside
Galloyl glucoside
Dihydromyricetin
diglucoside
Methyldihydromyricetin
diglucoside
Dimethyldihydromyricetin
diglucoside
Myricetin-3-Oglucoside
Myricetin-3-Opentoside
Myricetin-3-Orhamnoside
Gallic acid
Vescalagin
23.21
519
627b
465,303
25.59
512
611b
449,287
27.21
523
641b
479,317
28.18
524
625b
29.47
518
465b
463,
301
303
30.48
527
655b
493,331
4.8
10.8
275
337
663
643
16.1
338
657
331
481,
355
495
20.0
338
671
509,
347
30.8
356
479
316
33.8
355
449
316
34.2
347
463
316
6.8
9.2
NDa
933
633
2
3
3.2. Changes in anthocyanin composition during maturation
4
The anthocyanin composition of Syzygium cumini fruit extracts
during maturation is presented in Table 2. As expected no anthocyanins
were detected at the green-yellow of maturity, but low levels of
cyanidin-3,5-O-diglucoside, delphindin-3,5-O-diglucoside, malvidin3,5-O-diglucoside and peonidin-3,5-O-diglucoside were detectable at
the green-pink stage of maturity. Levels of the two minor anthocyanins
in Syzygium cumini fruit, delphinidin-3-O-glucoside and peonidin-3,5-Odiglucoside were not detectable until the light purple stage of maturity.
The anthocyanin content increased sharply when the fruit ripened to
dark purple. These results confirm those of Patel and Rao (2014) who
reported a large increase in total anthocyanins in Syzygium cumini as
fruit fully ripened. Anthocyanin content has also been reported to
increase sharply in fully ripe fruit such as Vaccinium species (Prior et al.,
1998), blackberries (Siriwoharn, Wrolstad, Finn, & Pereira, 2004) and
lychee (Rivera-Lopez, Ordorica-Falomir, & Wesche-Ebeling, 1999).
Throughout fruit maturation, delphinidin-3,5-O-diglucoside was the
predominant anthocyanin accounting for 37–48% of total anthocyanins, followed by petunidin-3,5-O-diglucoside (29–33%) and malvidin3,5-O-diglucoside (19–27%), while cyanidin-3,5-O-diglucoside (3%),
delphinidin-3-O-glucoside (2–3%) and peonidin-3,5-O-diglucoside
(1–2%) were minor constituents. A similar distribution of anthocyanins
in Syzygium cumini fruit was reported by Faria et al. (2011) and Tavares
et al. (2016).
The total anthocyanin content of fully ripe black fruit in this
research, 1318.4 mg/100 g DW was higher than a previously reported
value of 777 mg/100 g DW (Brito et al., 2007). This difference may be
due to the advanced stage of ripened fruit (black) analyzed in our study
and is consistent with a previous study that reported over-ripe blackberries to have much higher levels of total anthocyanins than ripe
berries (Siriwoharn et al., 2004).
5
6
7
8
9
10
11
12
13
14
15
16
3G-glc (1)
b
22.0
271
< 240,
270sh
284
b
17
18
3G-glc (2)
G-2HHDP-glcb
23.1
26.6
278
< 240
635
935
19
2G-HHDP-glcb
27.4
274
785
20
21
22
23
4G-glc (1)b
4G-glc (2)b
Ellagic acid
5G-glc (1)b
31.0
32.8
35.6
36.5
279
279
254, 365
282
787
787
301
939
38.1
279
939
41.9
277
1091
24
5G-glc (2)
25
6G-glc
b
3.3. Flavonol and flavanonol composition of Syzygium cumini fruit
Three flavanonols detected in Syzygium cumini fruit were dihydromyricetin diglucoside (m/z 643/481), methyl-dihydromyricetin diglucoside (m/z 657/495), dimethyl-dihydromyricetin diglucoside (m/z
671/509), additionally, three flavonols myricetin-3-O-hexoside (m/z
479/316), myricetin-3-O-pentoside (m/z 449/316), and myricetin-3-Orhamnoside (m/z 463/316) were detected (Fig. 1B). These compounds
were previously reported by Faria et al. (2011) in addition to myricetin
aglycone, which was not detected in our samples. Tavares et al. (2016)
identified nine flavonols in skin and pulp extracts of Syzygium cumini
fruit, besides the three flavonols identified in our study they also
detected myricetin-3-O-glucuronide, myricetin-3-O-galactoside, laricitrin-3-O-galactoside, laricitrin-3-O-glucoside, syringetin-3-O-galactoside, and syringetin-3-O-glucoside. They also reported that most of
the flavonols present in the fruit were located in the skin.
631,
425
481,
463
483
785,
633,
301
635,
483,
301
617
617
787,
401
787,
617,
465
939,
787,
461
a
Identification was confirmed with an authentic standard.
3G-glc (1) = trigalloyl glucose (isomer 1), 3G-glc (2) = trigalloyl glucose (isomer 2),
G-2HHDP-glc = galloyl dihexahydroxydiphenoyl glucose, 2G-HHDP-glc = digalloyl dihexahydroxydiphenoyl glucose, 4G-glc (1) = tetragalloyl glucose (isomer 1), 4G-glc (2)
= tetragalloyl glucose (isomer 2), 5G-glu (1) = pentagalloyl glucose (isomer 1), 5G-glu
(2) = pentagalloyl glucose (isomer 2), 6G-glu = hexagalloyl glucose.
b
tin diglucoside, and dimethyl-dihydromyricetin diglucoside were not
detectable until later stages of maturation (light to dark-purple), but
concentrations increased markedly as the fruit fully ripened, with the
highest concentration at the black stage of maturation. For the
flavonols, myricetin-3-O-hexoside was present at low concentration in
immature fruit (green-yellow to light-purple), but increased in concentration as the fruit fully ripened, with the highest concentration present
at the black stage of maturation. Conversely, myricetin-3-O-pentoside
and myricetin-3-O-rhamnoside were present at the highest concentration in immature fruit (green-yellow to green-pink) and concentrations
declined as the fruit fully ripened. Due to the fluctuation in individual
3.4. Changes in flavonol and flavanonol composition during maturation
The changes in the concentrations of the three flavonols and three
flavanonols detected during maturation are presented in Table 3.
Concentrations of the compounds varied in response to maturation.
The flavanonols dihydromyricetin diglucoside, methyl-dihydromyrice3
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L.N. Lestario et al.
Fig. 1. HPLC chromatograms of anthocyanins (A) in Java plum at black stage of maturation (Abs 520 nm), flavonols and flavanonols (B) at yellow-green stage of maturation (Abs
360 nm), and hydrolysable tannins (C) at yellow-green stage of maturation (Abs 280 nm). Refer to Table 1 for peak identification.
Table 2
Anthocyanin composition (mg/100 g FW) of Syzygium cumini fruit at different maturity stages.
Stage of maturity
Green-yellow
1
Green-pink
f2
Pink
3
Cyanidin-3,5-O-diglucoside
Delphinidin-3,5-Odiglucoside
Delphinidin-3-O-glucoside
ND
ND
0.76 ± 0.03 (3%)
13.8f ± 0.58 (48%)
ND
ND
Malvidin-3,5-O-diglucoside
ND
5.4f ± 0.12 (19%)
Petunidin-3.5-Odiglucoside
Peonidin-3,5-O-diglucoside
ND
8.5f ± 0.35 (30%)
ND
Total anthocyanins
ND
1
2
3
Red
e
Light purple
d
1.22 ± 0.03 (3%)
20.5e ± 0.14
(42%)
ND
2.58 ± 0.22 (3%)
35.4d ± 1.50
(37%)
ND
ND
11.7e ± 0.23
(24%)
15.4e ± 0.07
(32%)
ND
25.9d ± 1.01
(27%)
30.9d ± 1.37
(33%)
ND
28.5f ± 1.08
48.8e ± 0.09
94.8d ± 4.09
c
6.7 ± 0.2 (3%)
104.5c ± 1.33
(39%)
8.6c ± 1.17
(3%)
62.8c ± 7.32
(24%)
80.9c ± 2.19
(30%)
4.1c ± 0.14
(2%)
267.6c ± 11.9
Dark purple
b
Black
12.8 ± 0.56 (3%)
199.8b ± 5.17
(43%)
11.0b ± 0.19 (2%)
35.6a ± 0.69 (3%)
583.5a ± 12.9
(44%)
30.8a ± 0.82 (2%)
98.7b ± 1.76
(21%)
139.5b ± 3.59
(30%)
5.9b ± 0.05 (1%)
268.3a ± 3.80
(20%)
385.6a ± 6.60
(29%)
14.6a ± 0.38 (1%)
467.7b ± 11.2
1318.4 ± 24.7
Non-detectable.
Mean values ± standard error of the mean (n = 3). Values within columns with different letters are significantly different (P < 0.05).
Values within parentheses represent percentage of total anthocyanins.
ries (Kosar, Kafkas, Paydas, & Baer, 2004).
flavonols during maturation, fruit at the green-pink stage of maturation
had the highest concentration of total flavonols, while fruit at the lightpurple and black stages of maturation had the lowest concentrations. In
studies involving other fruit, total flavonol content was reported to
decline during maturation of pomegranate (Fawole, Opara, & Theron,
2013) and blueberries (Castrejon, Eichholz, Rohn, Kroh & HuyskensKeil, 2008), but levels remained stable during maturation of strawber-
3.5. Hydrolysable tannin composition of Syzygium cumini fruit
Eight gallotannins were identified in Syzygium cumini fruit (Fig. 1C);
galloyl glucose (m/z 331), two isomers each of trigalloyl glucose (m/z
635), tetragalloyl glucose (m/z 787), and pentagalloyl glucose (m/z
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Table 3
Flavanonol and flavonol contents (mg/100 g DW) of Syzygium cumini fruit at different maturity stages.
Stage of
maturity
Flavanonols
Dihydromyricetin
diglucoside
Methyldihydromyricetin
diglucoside
Dimethyl
dihydromyricetin
diglucoside
Total
flavanonols
Flavonols
Myricetin-3Ohexoside
2.0 ± 0.04b
Myricetin-3Opentoside
3.2 ± 0.06bc
Myricetin-3Orhamnoside
6.8 ± 0.13de
Total
flavonols
12.0 ± 0.18 cd
a
b
Greenyellow
Green-pink
Pink
Red
Light
purple
Dark
purple
Black
NDa
ND
ND
ND
ND
0.11b ±
0.01b
ND
ND
ND
ND
0.41 ±
0.001b
0.49 ±
0.01b
2.8 ±
0.24a
2.0 ±
0.15a
ND
ND
ND
ND
0.70 ±
0.01c
0.98 ±
0.02b
2.9 ±
0.07a
ND
ND
ND
ND
1.1 ±
0.01b
1.6 ±
0.05b
7.7 ±
0.47a
0.72 ±
0.01e
1.0 ± 0.06cde
0.8-
3 ± 0.09de
1.3 ±
0.05c
1.1 ±
0.11 cd
3.2 ±
0.32a
4.94 ±
0.09a
5.7 ± 0.28a
3.-
1 ± 0.47bc
3.8 ±
0.32b
2.6 ±
0.26c
2.3 ±
0.32c
10.6 ±
0.22b
12.5 ± 0.56a
7.-
7 ± 1.00 cd
9.2 ±
0.55bc
5.9 ±
0.38e
4.2 ±
0.43f
16.2 ±
0.18b
19.2 ± 0.62a
11.-
6 ± 1.03 cd
14.3 ±
0.69bc
9.6 ±
0.63d
9.7 ±
0.96d
Non-detectable.
Mean values ± standard error of the mean (n = 3). Values within columns with different letters are significantly different (P < 0.05).
concentrations declined precipitously as the fruit ripened. An exception
was galloyl glucose, which was present at low concentration in greenyellow fruit, but increased with further ripening reaching a maximum
concentration at the red stage, followed by a major decline as the fruit
fully ripened. Changes in concentration of the phenolic acid gallic acid
during maturation also varied from the hydrolysable tannins as
concentration remained high until the red stage of maturity and then
declined markedly as the fruit fully ripened from red to black.
Hexagalloyl glucose (6G-glu) was not detectable from the light purple
to black stages of maturity and vescalagin was only detected in the
green-yellow stage. Total gallotannin content was highest at the red
stage of maturation (370.5 mg/100 g DW) due to the abundance of
galloyl glucose and gallic acid, but content decreased to 93.7 mg/100 g
DW when the fruit fully ripened to the black stage. Total ellagitannin
content was highest in immature green-yellow fruit (26.1 mg/100 g
DW) and decreased throughout maturation reaching a low of 2.9 mg/
100 g DW at the fully ripe black stage. The loss of gallotannins during
939), and hexagalloyl glucose (m/z 1091) in addition to gallic acid.
Additionally, three ellagitannins, galloyl-diHHDP-glucose (m/z 935),
digalloyl-HHDP-glucose (m/z 785) and vescalagin (m/z 933) as well as
free ellagic acid (m/z 301) were identified in the fruit extracts. These
compounds were previously identified in Syzygium cumini skin and pulp
extracts using HPLC-DAD-ESI—MS/MS (Tavares et al., 2016) in addition to a number of other gallotannins and ellagitannins that were not
detected in our samples. According to Tavares et al. (2016) most of the
gallotannins and ellagitannins are present in much higher amounts in
the skin as opposed to the pulp.
3.6. Changes in hydrolysable tannin composition during maturation
The hydrolysable tannin content of Syzygium cumini fruit from seven
different stages of maturity are presented in Table 4. All of the
hydrolysable tannins (gallo- and ellagitannins) were present at the
highest concentration in immature (green-yellow) fruit, and their
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Table 4
Hydrolysable tannin content (mg/100 g DW) of Syzygium cumini fruit at different maturity stages.
Stage of
maturity
Greenyellow
Green-pink
Pink
Gallotannins
Galloyl
glucose
4.9a ± 0.19d
114.1 ±
7.86c
21.39 ±
0.92d
154.7 ±
2.2a
107.6 ± 5.7c
Gallic acid
93.2 ± 2.18c
3G-glc (1)
5.6 ± 0.14d
3G-glc (2)
4.9 ± 0.12de
4G-glc (1)
11.7 ± 0.08d
4G-glc (2)
2.2 ± 0.08ab
5G-glc (1)
4.4 ± 0.12d
5G-glc (2)
2.2 ± 0.06d
6G-glc
ND
Total gallotannins
232.1 ± 5.4c
Ellagitannins
Vescalagin
G-2HHDPglc
1.5 ± 0.03 cd
2G-HHDPglc
0.97 ± 0.02de
Ellagic acid
1.8 ± 0.04bc
Total ellagitannins
4.3 ± 0.03d
a
b
38.3 ±
1.0d
13.3 ±
0.09a
13.4 ±
0.29a
25.57 ±
0.74a
3.7 ±
0.19e
37.85 ±
1.31a
12.8 ±
0.37d
2.89 ±
0.14a
2.3 ±
1.19ab
15.4 ±
1.2a
0.8 ±
0.09e
10.6 ±
0.15a
0.8 ±
0.05e
0.47 ±
0.01a
ND
265.7 ±
5.3c
Red
Light
purple
Dark
purple
Black
142.-
6 ± 12.1b
185.9 ±
14.0a
115.9 ± 4.4c
150.9 ±
9.37a
125.-
7 ± 6.63b
150.9 ±
7.16a
86.3 ± 9.62c
10.3 ± 1.96b
9.-
5 ± 0.98bc
7.0 ±
0.92 cd
5.8 ±
0.30d
17.2 ± 0.83b
12.8-
9 ± 1.13c
6.7 ±
1.08d
3.1 ±
0.13e
24.9 ± 1.37b
17.-
9 ± 2.40c
10.8 ±
1.21d
9.0 ±
0.79d
2.1 ± 0.16ab
2.-
5 ± 0.21ab
1.5 ±
0.06b
1.6 ±
0.11ab
10.3 ± 0.29b
7.-
9 ± 1.28c
4.6 ±
0.49d
3.9 ±
0.11d
4.8 ± 0.16b
3.-
5 ± 0.43c
2.4 ±
0.14d
2.3 ±
0.13d
0.2 ± 0.05b
0.2-
5 ± 0.04b
0.1 ±
0.01c
NDb
335.0 ±
11.3ab
322.-
8 ± 24.1b
370.5 ±
17.1a
228.4 ± 14.7c
ND
ND
ND
ND
ND
2.5 ± 0.21b
2.-
1 ± 0.43bc
2.3 ±
0.27b
2.6 ±
0.11b
3.1 ± 0.09b
1.-
5 ± 0.13c
1.0 ±
0.19d
0.95 ± 0.07de
1.9 ± 0.15bc
2.-
4 ± 0.19b
2.2 ±
0.40bc
1.7 ±
0.15 cd
7.4 ± 0.39b
6.-
0 ± 0.69c
5.6 ±
0.47c
5.2 ±
0.33 cd
93.7 ±
1.09d
1.0 ± 0.05a
7.5 ± 0.08a
1.2 ±
0.13d
5.6 ± 0.11a
0.63 ±
0.08e
11.9 ±
0.33a
1.1 ±
0.15d
26.1 ±
0.42a
2.9 ±
0.28e
ND
Mean values ± standard error of the mean (n = 3). Values within columns with different letters are significantly different (P < 0.05).
Non-detectable.
6
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maturation are consistent with a previous study on Syzygium cumini
fruit, where total tannins measured using a casein precipitation method
decreased from 669.4 mg/100 g FW in immature fruit (green-purple) to
185.8 mg/100 g FW in ripe mature fruit (Brandão, Sena, TEShiMA,
David, & Assis, 2011). Additionally, the gallotannin content of pomegranate juice prepared from immature fruit and mature-green mangoes
had 2-fold higher levels of gallotannins than fully ripe fruit (Fawole
et al., 2013; Kim, Brecht, & Talcott, 2007). Loss of extractable tannins
during fruit ripening is associated with loss of astringency. It is thought
that tannins polymerize during ripening and readily bind to proteins
and cell wall polysaccharides, which greatly reduces their extractability
and astringency (Goldstein & Swain, 1963). The loss of ellagic acid
during maturation agrees with previous studies reporting loss of ellagic
acid in many strawberry genotypes during berry maturation (Kosar
et al., 2004; Gasperroti et al., 2013).
4. Conclusions
Anthocyanins were first detected at the green-pink stage of maturity
and increased throughout maturation with the largest increase occurring from the dark purple to black stages of maturation. Although
present at low concentrations, the levels of three flavanonols detected
also showed the largest increase from the dark purple to black stages of
maturation. Levels of gallotannins, flavonols, ellagitannins and ellagic
acid were highest at early stages of maturation (green-yellow and
green-pink) and decreased as the fruit ripened. For production of
nutraceutical ingredients fruit should be harvested immature to obtain
extracts rich in gallotannins, ellagitannins, ellagic acid, gallic acid and
flavonols. In contrast, fruit should be harvested at the fully ripe black
stage to obtain extracts rich in anthocyanins for possible use as a
natural colorant.
Appendix A. Abbreviations used
All abbreviations used in this study are listed as follows:
High performance liquid chromatography (HPLC), mass spectrometry (MS), electrospray ionization (ESI), photodiode array detector
(DAD), polytetrafluoroethylene (PTFE), trolox equivalents (TE), trigalloyl glucose isomer one ((3G-glc (1)), trigalloyl glucose isomer two
((3G-glc (2)), tetragalloyl glucose isomer one ((4G-glc (1)), tetragalloyl
glucose isomer two ((4G-glc (2)), pentagalloyl glucose isomer one ((5Gglc (1)), pentagalloyl glucose isomer two ((5G-glc (2)), hexagalloyl
glucose (6G-glc), galloyl dihexahydroxydiphenoyl glucose (G-2HHDPglc), digalloyl dihexahydroxydiphenoyl glucose (2G-HHDP-glc), nondetectiale (ND).
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