Hindawi

Journal of Food Quality

Volume 2019, Article ID 1528917, 8 pages
https://doi.org/10.1155/2019/1528917

Research Article
UPLC-DAD-QTOF/MS Analysis of Flavonoids from
12 Varieties of Korean Mulberry Fruit

O-Chul Kwon , Wan-Taek Ju, Hyun-Bok Kim, Gyoo-Byung Sung, and Yong-Soon Kim
National Institute of Agricultural Sciences, Rural Development Administration, Wanju-gun 55365, Republic of Korea

Correspondence should be addressed to Yong-Soon Kim; kaiko0214@korea.kr

Received 27 September 2018; Revised 20 November 2018; Accepted 16 December 2018; Published 8 January 2019

Academic Editor: Eduardo Puértolas

Copyright © 2019 O-Chul Kwon et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Mulberry (Morus alba L.) has been used in East Asia (Korea, China, and Japan) as a medicine because of its various phar-
macological effects including the excellent antioxidant properties of its fruit. This study analyzed extracts from 12 varieties of
Korean mulberry fruit for flavonoids using ultrahigh-performance liquid chromatography coupled with diode array detection and
quadrupole time-of-flight mass spectrometry (UPLC-DAD-QTOF/MS). Six quercetin derivatives were identified by mass
spectrometry (MS) based on the [quercetin + H]+ ion (m/z 303), while four kaempferol derivatives were identified based on the
[kaempferol + H]+ ion (m/z 287). Two new compounds (morkotin A and morkotin C, quercetin derivatives) were identified for
the first time in mulberry fruit. The total flavonoid contents of the mulberry fruits ranged from 35.0 ± 2.3 mg/100 g DW in the Baek
Ok Wang variety (white mulberry) to 119.9 ± 7.0 mg/100 g DW in the Dae Shim variety. This study has, for the first time, evaluated
the flavonoid chromatographic profiles of 12 varieties of Korean mulberry fruits in a following quali-quantitative approach, which
will contribute to improved utilization of these fruits as health foods.

1. Introduction [14]. Chu et al. [15] have previously identified apigenin,

Mulberry belongs to the Moraceae family, which includes 37
genera and over 1100 species [1] and has long been used as a
traditional medicine and food in Korea, China, and Japan
[2–4]. Mulberries are reported to exhibit numerous phar-
macological activities, such as antidiabetic [5], antioxidant
[6], anticancer [7], antistress [8], immunomodulatory [9],
and hepatoprotective [10] activities. Therefore, mulberries
should be considered as important potential sources of
bioactive compounds that can be used for the prevention or
treatment of many different medical conditions.
In Korea and other countries, mulberry fruits are used in
many commercial products, such as frozen desserts, ice
cream, jam, juice, marmalade, paste, pulp, tea, and wine
[11, 12]. In particular, mulberry fruit juice has been used as a
folk remedy for treating aphtha, asthma, colds, coughs,
diarrhea, dyspepsia, edema, fevers, headache, hypertension,
and wounds [13]. Mulberry fruits contain a variety of
chemical components, including anthocyanins, sugars, or-
ganic acids, free amino acids, vitamins, and micronutrients
caffeic acid, chlorogenic acid, gallic acid, luteolin, morin,
rutin, kaempferol, and quercetin in mulberry fruit. These
components and compounds have the potential to benefit
human health because of their significant biological
activities.
Flavonoids are a large group of polyphenolic compounds
composed of six subclasses: anthocyanins, flavan-3-ols,
flavanones, flavones, flavonols, and isoflavones [16]. Fla-
vonoids are present in mulberry fruit [11] and have bi-
ological and pharmacological activities that include
antioxidative, anti-inflammatory, antiviral, antidiabetic,
antihyperlipidemic, and antiobesity effects [17–19]. Lee et al.
[20] and Chen et al. [21] identified and quantified antho-
cyanins and flavonoids in several mulberry varieties. Paw-
lowska et al. [11] used high-performance liquid
chromatography (HPLC) to analyze the profile of flavonoids
in Morus nigra and Morus alba fruits and identified the
compounds using nuclear magnetic resonance spectroscopy
and electrospray ionization mass spectrometry (ESI-MS).
They also found that the red pigment of M. nigra fruit
2 Journal of Food Quality
contained four anthocyanins, which were identified as
cyanidin 3-O-glucoside, cyanidin 3-O-rutinoside, pelargo-
nidin 3-O-glucoside, and pelargonidin 3-O-rutinoside using
HPLC-photodiode array detection-ESI-MS analysis.
This study investigated the flavonoids from 12 varieties
of Korean mulberry fruits using a quali-quantitative ap-
proach to establish their nutrient profiles and to promote the
further development of Korean mulberry resources for use
in food and medicine.
2. Materials and Methods
2.1. Materials and Reagents. Mulberry fruits of 12 varieties
were collected from the Sericulture and Apiculture Division
(Department of Agricultural Biology, Rural Development
Administration, Jeon-Ju, Republic of Korea). The fruit
samples were cleaned, dried in a lyophilizer, and pulverized
before storage at −18°C until required for analysis. HPLC-
grade solvents (acetonitrile, methanol, and water) and
formic acid were purchased from Fisher Scientific (Fair
Lawn, NJ, USA) and Junsei Chemical (Tokyo, Japan), re-
spectively. Galangin (Sigma Aldrich, St. Louis, MO, USA)
was used as internal standard for the flavonoids.
2.2. Sample Preparation and Extraction. Extracts were taken
prepared from the samples as described by Kim et al. [22]
with few modifications. The sample of powdered fruit
sample (1 g) was mixed with 10 mL of methanol:water:for-
mic acid (50 : 45 : 5, v/v/v) solution, containing 100 ppm of
galangin (internal standard). The mixture was vortexed,
stirred with shaking for 5 min at 200 rpm, and then
centrifuged at 2000 g at 10°C for 15 min. The supernatant
was filtered through a syringe filter (0.45 μm, PTFE;
Whatman, Maidstone, UK), and 0.5 mL of flavonoid extract
was diluted with water to a final volume of 5 mL. The
flavonoid-containing crude extract was further extracted
and purified by solid-phase extraction using a Sep-Pak C18
cartridge (Waters, Milford, MA, USA). The cartridge was
activated by washing with 2 mL of methanol and then
conditioned with 2 mL of water. The diluted flavonoid ex-
tract was loaded onto the Sep-Pak C18 cartridge, and the
impurities were removed by washing with 2 mL of water. The
total flavonoid mixture was eluted from the Sep-Pak car-
tridge using 3 mL of methanol. The purified extract was
concentrated by evaporation under a stream of nitrogen gas
and then redissolved in 0.5 mL of methanol:water:formic
acid (50 : 45 : 5, v/v/v) solution without internal standard
(galangin) before instrumental analysis. All experimental
samples were analyzed in triplicate.
2.3. Analysis Conditions for UPLC-DAD-QTOF/MS. An
ultra-high-performance liquid chromatography (UPLC)
system with a diode array detector (DAD) set at 280 and
320 nm coupled to a quadrupole time-of-flight mass spec-
trometer (QTOF/MS, Waters) was used for analysis. The
ultraviolet-vis spectra were collected in the range from 210 to
600 nm. The chromatographic conditions and equipment
were as follows: column: Luna Omega 1.6 μm C18,
150 × 2.1 mm (Phenomenex, Torrance, CA, USA); pre-
column: SecurityGuard ULTRA Cartridges for UHPLC C18
for 2.1 mm i.d. column (Phenomenex); mobile phase: solvent
A (0.5% formic acid in water) and solvent B (0.5% formic
acid in acetonitrile); flow rate 0.3 mL/min; column tem-
perature 30°C; total running time 60 min; injection volume
5 μL; and gradient condition: 0–2 min 7% (B), 24 min 15%
(B), 40 min 30% (B), 48–50 min 60% (B), 53–54 min 90% (B),
and 55–60 min 7% (B). The mass analysis conditions were as
follows: sampling cone voltage 40 V; ion source temperature
120°C; desolvation temperature 400°C; desolvation gas flow
1000 L/h; cone gas 30 L/h; capillary voltage 3500 V; and mass
range m/z 50–800 using positive ion electrospray.
3. Results and Discussion
3.1. Isolation and Identification of Flavonoid Compounds in
Mulberry Fruits. Mulberries contain bioactive components,
such as alkaloids and flavonoids [23–25] and when dried
possess antioxidant, antimicrobial, antidiabetic, antiobesity,
and anti-inflammatory properties [26–34]. Qualitative
analysis by the HPLC-DAD-ESI-MS/MS method has pre-
viously been used to identify six nonanthocyanin phenolics
from two mulberry varieties: 4-caffeoylquinic acid, chloro-
genic acid, protocatechuic acid, quercetin, rutin, and taxi-
folin. Three others were also tentatively identified [35]: 3,5-
dicaffeoylquinic acid, taxifolin-hexoside, and kaempferol-
hexoside.
In the present study, flavonoids from the fruits of 12
mulberry varieties (Morus alba L.) were analyzed by UPLC-
DAD-QTOF/MS. Ten mulberry flavonoids were identified
on the chromatogram between 5 and 20 min (Figure 1). The
chemical structures of the individual flavonoids were de-
termined by analysis of fragment ion patterns. Six com-
pounds (Peaks 1, 2, 4, 5, 6, and 9) were identified as quercetin
derivatives by MS based on the presence of m/z 303
[quercetin + H]+. Four other compounds (Peaks 3, 7, 8, and
10) were assigned as kaempferol derivatives based on the
presence of m/z 287 [kaempferol + H]+. The MS spectra of
the flavonoids isolated from the mulberry fruit are shown in
Figure 2. The following flavonoid compounds were isolated
and identified from the mulberry fruits: Peak 1, quercetin 3-
O-rutinoside-7-O-glucoside (morkotin A); Peak 2, quercetin
3,7-di-O-glucoside; Peak 3, kaempferol 3,7-di-O-glucoside;
Peak 4, quercetin 3-O-rutinoside (rutin); Peak 5, quercetin
3-O-glucoside (isoquercitrin); Peak 6, quercetin 3-O-(6″-O-
malonyl)glucoside; Peak 7, kaempferol 3-O-rutinoside
(nicotiflorin); Peak 8, kaempferol 3-O-glucoside (astraga-
lin); Peak 9, quercetin 3-O-(2″-O-malonyl)glucoside
(morkotin C); and Peak 10, kaempferol 3-O-(6″-O-
malonyl)glucoside (Table 1).
The major peak of the mulberry fruit (Peak 4) generated
the ion fragments of m/z 633 [M + Na]+, 611 [M + H]+, 465
[M + H−Rham]+, 449 [M + H−Glu]+, and 303
+
[quercetin + H] and was identified as quercetin 3-O-
rutinoside (rutin) (Figure 2(a)). Peak 7 generated the ion
fragments of m/z 617 [M + Na]+, 595 [M + H]+, 449
[M + H−Rham]+, 433 [M + H−Glu]+, and 287
+
[kaempferol + H] and was identified as kaempferol 3-O-
Journal of Food Quality 3

4 (major flavonol)
Dae dang sang ISTD (galangin) PDA
1.5e–1 350 nm
5
AU

1.0e–1 7
2 3 8
1
5.0e–2

0.0
–0.00 2.50 5.00 7.50 10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50 40.00

PDA
180–12 4 (major flavonol) 5
ISTD (galangin) 350 nm
1.5e–1

6, 7
1.0e–1
AU

2 3 8 10
1
5.0e–2 9

0.0
–0.00 2.50 5.00 7.50 10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50 40.00
Time
Figure 1: LC chromatograms of flavonoids in samples of Korean mulberry fruits (Morus alba L.).

Peak 4. quercetin 3-O-rutinoside (rutin) Peak 7. kaempferol 3-O-rutinoside (nicotiflorin)

303 1: TOF MS ES + 2.01e5
100 100 287 1: TOF MS ES + 2.17e5
MW: 610 MW: 594

303 (quercetin aglycone + H)+ 287 (kaempferol aglycone + H)+

OH
OH OH
HO O HO O
611 (M + H)+ OH OH
O O OH
O O
O O OH
465 (M + H – Rham)+ 611
OH O HO OH O OH OH
(%)

OH O OH O
(%)

OH HO
OH
Glucose (m/z 162) rhamnose (m/z 146) Glucose (m/z 162) rhamnose (m/z 146)
465 rutinose (m/z 308) rutinose (m/z 308)
595 (M + H)+
449 (M + H – Rham)+
304
617 (M + Na)+
+
633 (M + Na) 288 595
449
+
449 (M + H – Glu) 617
508 633 433 (M + H – Glu)+
466
305 618
449 467 509 634 450
301 635665 717 757 775 919 935 951 317
371 409 433 451 487 527528 900
235 252 267 325345 401409447 510 562 577 697 1155 853 1045 1193 211 244 281 309 318 492 569
619 734 773
633 679 701 755 795851 911 978 9811032
0 100 866 919 1122 1164 1190
200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150
m/z m/z

(a) (b)
Peak 1. quercetin 3-O-rutinoside-7-O-glucoside (morkotin A) Peak 9. quercetin 3-O-(2″-O-malonyl)glucoside (morkotin C)
100 303 1: TOF MS ES + 4.04e4 100 303 1: TOF MS ES + 8.03e4
MW: 772 MW: 550
OH
Glucose (m/z 162) OH OH
303 (quercetin aglycone + H)
+ 303 (quercetin aglycone + H)+ OH
O O O
HO HO O
OH OH
HO O O
HO OH
O
OH O O O
HO OH O OH
OH OH
773 Glucose (m/z 162) rhamnose (m/z146) OH O O O OH
rutinose (m/z 308) OH
+ O Glucose (m/z 162)
773 (M + H)
(%)
(%)

611 (M + H – Glu)+ OH
Malonic acid (m/z 86)

795 (M + Na)+
465 (M + H – Rut)+ 627 (M + H – Rham)+ 551 (M + H)+
573 (M + Na)+
774 304
304 611 795
406 573
465 441
407 612 665 796 570574
333 363 476 305 1166
401 407449 489509 518564 575 627 666 797 201 273 341 359395 413 442457 545 589 663
1139 1193
223 285281 345 628 681721 752 811857 884 927 944 1015 1059 1073 1179 1198 231 281 525 658 679 739 812 828845 891 902 943 10071021 1089 1123
0 0
200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150
m/z m/z

(c)

Figure 2: Mass spectra of flavonoids detected in extracts of Korean mulberry fruits. (a) Glycoside derivative of quercetin; (b) glycoside
derivative of kaempferol; (c) MS spectra of two new flavonoids detected from extracting Korean mulberry fruits.

rutinoside (nicotiflorin) (Figure 2(b)). In analysis of flavo- time in Prunus tomentosa (Korean cherry, sweet cherry, and
noid glycosides by UPLC-DAD-QTOF/MS, Jang et al. [36] cherry). In the current study, morkotin A (MS fragments of
reported that rutin and nicotiflorin were detected for the first m/z 795 [M + Na]+, 773 [M + H]+, 627 [M + H−Rham]+, 611
4 Journal of Food Quality

Table 1: Ten flavonoids isolated from Korean mulberry fruits (Morus alba L.) and their mass spectrometric data.
Peak
Aglycones Glycosides Acylations Individual flavonols MW Fragment ions (m/z)
number
8 Kaempferol 3-O-glucoside (astragalin) 448 471, 449, 287
Mono Mal
Kaempferol (m/z 10 Kaempferol 3-O-(6″-O-malonyl)glucoside 534 557, 535, 287
287) 7 Kaempferol 3-O-rutinoside (nicotiflorin) 594 617, 595, 449, 287
Di
3 Kaempferol 3,7-di-O-glucoside 610 633, 611, 449, 287
5 Quercetin 3-O-glucoside (isoquercitrin) 464 487, 465, 303
6 Quercetin 3-O-(6″-O-malonyl)glucoside 464 573, 551, 465, 303
Mono Mal
Quercetin 3-O-(2″-O-malonyl)glucoside
9 550 573, 551, 303
Quercetin (m/z (morkotin C)a
303) 4 Quercetin 3-O-rutinoside (rutin) 610 633, 611, 465, 449, 303
Di
2 Quercetin 3,7-di-O-glucoside 626 649, 627, 465, 303
Quercetin 3-O-rutinoside-7-O-glucoside 795, 773, 627, 611, 465,
Tri 1 772
(morkotin A)a 303
All samples were analyzed in positive ion mode ([M + H]+) using UPLC-DAD-QTOF/MS. aNew flavonoid identified in mulberry fruit.

[M + H−Glu]+, 465 [M + H−Rut]+, and 303
+
[quercetin + H] ) and morkotin C (MS fragments of m/z 573
[M + Na]+, 551 [M + H]+, and 303 [quercetin + H]+) were
newly identified in extracts of Korean mulberry fruit
(Figure 2(c)). Ju et al. [37] reported seven new quercetin
(morkotin A, B, and C) and kaempferol (moragrol A, B, C,
and D) compounds by UPLC-DAD-QTOF/MS analysis of
flavonoids from mulberry leaves. However, the quercetin
derivatives morkotin A and morkotin C have not previously
been reported in mulberry fruit. Although quercetin and
kaempferol are reported to exhibit a range of biological
activities, including anti-inflammatory, anticancer, anti-
obesity, antioxidant, antihypercholesterolemic, and anti-
atherosclerotic activity [38–40], the biological activities of
morkotin A and morkotin B are unclear at this time. As
newly identified components of mulberry fruit, these
compounds need further investigation to determine their
beneficial effects to human health.
3.2. Flavonoid Contents of Mulberry Fruit. The contents of
the ten flavonoids isolated from the 12 varieties of mulberry
fruit are shown in Table 2. The total flavonoid contents of the
mulberry fruit ranged from 35.0 ± 2.3 mg/100 g DW in the
Baek Ok Wang variety (white mulberry) to 119.9 ± 7.0 mg/
100 g DW in the Dae Shim variety. The total flavonoid
contents (0.06–6.54 mg CE (catechin equivalents)/100 g
DW) determined in five other Korean mulberry varieties
(Pachungsipyung, Whazosipmunja, Suwonnosang, Jasan,
and Mocksang) by Bae and Suh [41] were lower than those
found in the present study. Butkhup et al. [42] reported that
the total flavonoid content of eight major mulberry varieties
from Thailand (Nakhonratchasima 60, Buriram 60,
Chumphon, Wavee, Chiang Mai, Pikultong, Kam-
phaengsaen, and Kamnanchul) ranged from 69.58 to
211.01 mg CE/100 g DW. Mahmood et al. [43] reported that
the total contents of flavonols (myricetin, quercetin, and
kaempferol) of four mulberry fruits (Morus laevigata, M.
macroura, M. nigra, and M. alba) at different stages of
maturity (unripe, semiripe, and fully ripe) ranged from 28.3
to 221.8 mg/100 g DW. These results for total flavonoid
contents from Butkhup et al. [42] and Mahmood et al. [43]
are similar to those found in the present study. Another
study found that the total flavonoid contents in the fruits,
leaves, and roots of two mulberry varieties (M. alba and M.
nigra) ranged between 0.8948 and 67.369 mg RE (rutin
equivalents)/g of dried extract, with the contents in the
leaves and roots being higher than those in the fruit [44].
Recently, Ju et al. [37] reported that the total contents of the
17 flavonoids isolated from the leaves of 12 mulberry va-
rieties ranged from 748.5 to 1297.9 mg/100 g DW. In ad-
dition, various flavonoid derivatives (moragrols A–D,
kaempferol 3-O-rhamnoside-7-O-glucoside, morkotin B,
and quercetin 3-O-rhamnoside-7-O-glucoside) other than
those found in mulberry fruits in this study were found in
mulberry leaves. Thus, the total flavonoid contents of
mulberry can vary according to the plant variety, the parts of
the plant, and the stages of fruit maturity.
Of the 10 flavonoids isolated from mulberry fruit (Ta-
ble 2), rutin showed the highest content (range 7.8 ± 0.5 to
67.8 ± 5.4 mg/100 g DW), being detected at a retention time
of 13.56 min (Peak 4). The highest contents of rutin were
found in the Dae Shim and Dae Dang Sang varieties
(66.1 ± 4.1 and 67.8 ± 5.4 mg/100 g DW, respectively), while
the lowest was found in the Baek Ok Wang variety (white
mulberry; 7.8 ± 0.5 mg/100 g DW). Butkhup et al. [42] re-
ported that the major flavonoid compounds in eight varieties
of mulberry fruit were (+)-catechin (309.26–750.01 mg/100 g
DW), procyanidin B1 (62.59–224.41 mg/100 g DW), quer-
cetin (5.36–58.42 mg/100 g DW), rutin (18.73–26.90 mg/
100 g DW), and (–)-epicatechin (8.47–29.21 mg/100 g DW).
Our study found that the major flavonoid in mulberry fruit
was rutin (Peak 4), at levels higher than those found by
Butkhup et al. [42]. Radojkovic et al. [44] reported that rutin
was predominant among the six compounds (gallic acid,
chlorogenic acid, ferulic acid, sinapic acid, rutin, and
quercetin) identified in the dried extracts of mulberry fruit
from two species (43.5 mg/100 g for M. alba fruit and
72.6 mg/100 g for M. nigra fruit). Katsube et al. [45] have also
reported that rutin was the main flavonol glycoside of M.
alba. Similarly, the present study also found rutin to be the
most abundant of the ten flavonoids isolated from each of
 Journal of Food Quality

Table 2: Contents of ten flavonoids isolated from 12 varieties of Korean mulberry fruit (Morus alba L.).
Data given in units of mg/100 g dry weight
Peak number
Cheong-Il Hwan Ship Jo Saeng Su Hyang Dae Shim Cheong-Il 4X 180-11 Shim Heung Cheong Su 180-12 Dae Dang Sang Baek Ok Wang 181-18
1a 1.3 ± 0.2 1.8 ± 0.1 1.6 ± 0.1 1.6 ± 0.2 1.2 ± 0.1 0.8 ± 0.0 1.5 ± 0.0 0.6 ± 0.1 0.7 ± 0.1 2.1 ± 0.0 0.3 ± 0.0 0.7 ± 0.0
2 0.6 ± 0.0 1.0 ± 0.1 1.0 ± 0.1 0.6 ± 0.1 0.5 ± 0.0 0.4 ± 0.0 0.5 ± 0.1 0.1 ± 0.0 2.8 ± 0.3 0.8 ± 0.0 0.4 ± 0.0 0.3 ± 0.0
3 ND ND ND ND ND ND ND ND 0.5 ± 0.0 0.3 ± 0.0 ND ND
4 24.0 ± 1.5 37.9 ± 2.7 38.1 ± 1.7 66.1 ± 4.1 22.1 ± 1.2 39.0 ± 1.7 60.5 ± 5.8 24.8 ± 4.2 20.6 ± 1.9 67.8 ± 5.4 7.8 ± 0.5 24.0 ± 1.1
5 5.5 ± 0.2 7.6 ± 0.4 7.5 ± 0.2 11.0 ± 0.5 4.8 ± 0.2 5.2 ± 0.3 6.2 ± 0.7 3.0 ± 0.4 17.1 ± 1.6 8.9 ± 0.8 5.7 ± 0.4 3.4 ± 0.1
6 19.6 ± 1.2 21.2 ± 1.5 20.8 ± 0.7 26.7 ± 1.4 18.0 ± 0.9 12.9 ± 0.4 ND 5.2 ± 0.9 15.5 ± 1.3 ND 13.0 ± 0.9 6.3 ± 0.2
7 2.2 ± 0.1 2.9 ± 0.2 2.4 ± 0.1 5.3 ± 0.3 1.9 ± 0.1 1.6 ± 0.1 5.0 ± 0.5 0.5 ± 0.1 3.1 ± 0.3 4.4 ± 0.3 1.6 ± 0.1 1.3 ± 0.0
8 2.0 ± 0.1 4.1 ± 0.3 4.1 ± 0.1 3.1 ± 0.4 1.6 ± 0.0 1.9 ± 0.1 ND 1.3 ± 0.2 3.8 ± 0.2 2.2 ± 0.3 2.5 ± 0.1 4.7 ± 0.1
9a 0.9 ± 0.1 1.1 ± 0.1 1.0 ± 0.1 1.4 ± 0.1 0.8 ± 0.1 0.7 ± 0.1 ND 0.3 ± 0.1 0.7 ± 0.0 ND 0.7 ± 0.0 0.3 ± 0.0
10 2.4 ± 0.1 3.5 ± 0.3 3.1 ± 0.1 4.1 ± 0.5 2.4 ± 0.2 1.3 ± 0.0 ND 0.5 ± 0.1 3.0 ± 0.3 ND 3.0 ± 0.2 1.3 ± 0.0
Total 58.6 ± 3.1 81.1 ± 5.6 79.6 ± 2.9 119.9 ± 7.0 53.3 ± 2.7 63.8 ± 2.4 73.8 ± 6.9 36.3 ± 6.1 67.7 ± 5.6 86.5 ± 6.3 35.0 ± 2.3 42.4 ± 1.5
All samples were analyzed in positive ion mode ([M + H]+) using UPLC-DAD-QTOF/MS. Each value calculated as mean ± SD of three replicates using galangin as internal standard. Peak 1: quercetin 3-O-
rutinoside-7-O-glucoside (morkotin A); Peak 2: quercetin 3,7-di-O-glucoside; Peak 3: kaempferol 3,7-di-O-glucoside; Peak 4: quercetin 3-O-rutinoside (rutin); Peak 5: quercetin 3-O-glucoside (isoquercitrin);
Peak 6: quercetin 3-O-(6″-O-malonyl)glucoside; Peak 7: kaempferol 3-O-rutinoside (nicotiflorin); Peak 8: kaempferol 3-O-glucoside (astragalin); Peak 9: quercetin 3-O-(2″-O-malonyl)glucoside (morkotin C);
Peak 10: kaempferol 3-O-(6″-O-malonyl)glucoside; ND: not detected. aNew flavonoid identified in mulberry fruit.
5
6 Journal of Food Quality
the 12 varieties of mulberry fruit except for Baek Ok Wang
(white mulberry).
The content of quercetin 3-O-glucoside (isoquercitrin)
(Peak 5) ranged from 3.0 ± 0.4 to 17.1 ± 1.6 mg/100 g DW
with the highest content found in the 180-12 variety
(17.1 ± 1.6 mg/100 g DW) and the lowest in the Dae Shim
variety (3.0 ± 0.4 mg/100 g DW). Pawlowska et al. [11] re-
ported that the contents of quercetin 3-O-glucoside in M.
alba and M. nigra fruits were 29 and 34 mg/100 g DW,
respectively, which are higher concentrations than the levels
observed in the present study. Overall, of the ten flavonoids
assayed, the two new compounds (morkotin C and A), as
well as quercetin 3,7-di-O-glucoside, kaempferol 3,7-di-O-
glucoside, nicotiflorin, kaempferol 3-O-glucoside (astraga-
lin), and kaempferol 3-O-(6″-O-malonyl)glucoside, were
found at low levels. Kaempferol 3,7-di-O-glucoside (Peak 3)
was detected in only two varieties: 180-12 (0.5 ± 0.0 mg/100 g
DW) and Dae Dang Sang (0.3 ± 0.0 mg/100 g DW). Quer-
cetin 3-O-(6″-O-malonyl)glucoside (Peak 6) and morkotin
C (Peak 9) were not detected in the Shim Hueng and Dae
Dang Sang varieties, while astragalin (Peak 8) was not de-
tected in the Shim Hueng variety. Flavonols such as
kaempferol and quercetin are bioactive compounds, pos-
sessing antioxidant and anti-inflammatory properties [46]
and have been used for the prevention and treatment of
cancers, cardiovascular disease, neurological and metabolic
disorders, and bone diseases [39, 47]. In mulberries,
kaempferol and quercetin are present mainly as glycosides
such as rutin, isoquercitrin, astragalin, kaempferol-3-(6-O-
malonyl)-β-D-glucoside, and quercetin-3-O-(6-O-
malonyl)-β-D-glucoside, with contents varying according to
variety, cultivation, harvest period, maturity, and heat-
processing method [48].
An analysis of anthocyanins in different types of berry
showed that blackcurrants contain the highest levels
(5521 nmol/g), followed by blueberries (4810 nmol/g),
cranberries (725 nmol/g), and redcurrants (328 nmol/g).
The anthocyanins in blackcurrants were responsible for
73% of the total antioxidant capacity, whereas vitamin C
contributed only 18% [46]. In comparison, the total amount
of anthocyanins in M. nigra and M. alba, made up of fla-
vonols such as kaempferol and quercetin, was reported as
27 mg/10 g of fresh berries with cyanidin 3-O-glucoside
being the major anthocyanin (66.3%), followed by cyani-
din 3-O-rutinoside (27.8%), pelargonidin 3-O-glucoside
(4.4%), and pelargonidin 3-O-rutinoside (1.5%) [11].
Overall, the quercetin and kaempferol contents of blue-
berries, cranberries, and redcurrants are lower than those of
mulberries without anthocyanins as found in the present
study. These results show that mulberry fruit is a significant
source of flavonoids, which have protective effects on human
health, as suggested in recent epidemiological and experi-
mental studies [12, 41, 42, 44].
Mulberry can grow in various climatic and soil condi-
tions. The variation of flavonoid contents in mulberry could
be caused by various factors, such as growing conditions,
regional differences, climatic conditions, and genetic dif-
ferences. For example, phenylalanine ammonia-lyase is re-
ported as the key enzyme in the formation of flavonoids in
mulberry leaves. The expression of the phenylalanine
ammonia-lyase gene can be induced by environmental
factors such as low temperatures, and content of flavonoids
can be increased under such conditions [49]. However, there
is a general lack of studies on the variation of flavonoid
compounds in mulberry fruit caused by environmental
factors, such as growing conditions, regional differences, and
climatic conditions. In addition, there is little information
available regarding the occurrence of flavonoid compounds
in fruit according to plant variety for Korean mulberries, and
these compounds have received little attention regarding
their biological health effects on humans. Therefore, further
studies that build upon the present work are required to
highlight the bioactive properties of flavonoid compounds in
mulberry fruit and to promote further development of
valuable mulberry resources in Korea.
4. Conclusions
The contents and composition of 10 flavonoids, isolated and
identified in 12 varieties of Korean mulberry fruits, varied
considerably. An UPLC-DAD-QTOF/MS system was used
with complementary information obtained from LC spectra,
MS ions, and MS/MS fragments to identify the constituents
of the mulberry fruits. To the best of our knowledge, two out
of the 10 compounds have been identified in mulberry fruits
for the first time, so further research will be needed to
evaluate their biological activity. The quantitative analysis of
the functional flavonoids available in mulberry fruits pro-
vided by this study will help to evaluate the standards and
physiological aspects of Korean mulberry fruits and so will
encourage their cultivation and use.
Data Availability
The data used to support the findings of this study are
available from the corresponding author upon request.
Conflicts of Interest
The authors have no conflicts of interest to declare.
Acknowledgments
This study was supported by the Department of Agricultural
Biology, National Academy of Agricultural Science, Rural
Development Administration (project: PJ 01360602). We
thank Philip Creed, PhD, from Edanz Group (http://www.
edanzediting.com/ac) for editing a draft of this manuscript.
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