Original Research Article

Natural Product Communications

Volume 19(5): 1–8
Analysis of Glucosinolates and Phenolic Content © The Author(s) 2024

in Sprouts of 7 Brassica rapa Subspecies

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DOI: 10.1177/1934578X241258568
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Leonel Tarcisio da Cristina Bungala1,2, Bao Van Nguyen3, Chanung Park1,

Ramaraj Sathasivam1, Gwonjeong Bok4, Jong Seok Park5,
and Sang Un Park1,3

Abstract
Objective: Brassica rapa is an important source of phytochemicals essential for plant defense and human health. To better understand
their metabolic characteristics, this study aimed to investigate the glucosinolates and phenolic content from sprouts of 7 B. rapa sub-
species. Methods: Glucosinolates and phenolic compounds were extracted using previously reported procedures, with slight mod-
ifications. Glucosinolates were identified based on their high-performance liquid chromatography peak area ratios and quantified
based on the peak areas, retention time, and response factor and phenolic compounds were identified based on retention times and
spiking tests and quantified concerning a corresponding calibration curve. Results: Statistical analysis showed that Choy sum (31.82 ±
2.3 mg/g DW) and Bok choy (31.73 ± 1.49 mg/g DW) presented the highest total glucosinolates content, and the lowest content was
observed in Turnip rape (21.75 ± 1.67 mg/g DW). Between the contents of individual glucosinolates, progoitrin presented the highest
content in Bok choy (17.84 ± 1.24 mg/g DW). The highest concentration of total phenolic compounds was found in the sprouts of
Chinese cabbage (2.7 ± 0.05 mg/g DW), whereas the lowest concentration was found in Choy sum sprouts (2.17 ± 0.05 mg/g DW).
The highest individual phenolic content was observed for benzoic acid (0.96 ± 0.03 mg/g DW) and rutin (0.85 ± 0.03 mg/g DW),
which were highest in mizuna and Chinese cabbage, respectively. Conclusion: Subspecies may influence the levels of glucosinolates
and polyphenols in vegetables. The results of our study showed the distinctive metabolic signatures embedded in the 7 B. rapa subspecies.

Keywords
organic compounds, secondary metabolites, Brassica rapa, sprouts, HPLC

Received: October 16th, 2023; Accepted: May 8th, 2024.

Introduction turnip, Bok choi,12,13 Chinese cabbage,14 and mizuna,15 and

found that the significant phenolic compounds are hydroxy-
Brassica spp. are classified in the Brassicaceae family, and most cinnamic acids.
of them can generate phytochemicals, such as glucosinolates
(GSLs) and phenolic compounds.1 For this reason, these
crops are considered agriculturally and economically important. 1
Department of Crop Science, Chungnam National University, Daejeon,
They can be consumed as vegetables, condiments, or oilseeds.2 Republic of Korea
B. rapa and its subspecies greatly enhance plant defense 2
Cereal Crop Sector, Mozambique Institute of Agricultural Research, Central
and human health.3 According to epidemiological studies, Regional Center, Chimoio, Mozambique
3
Brassica vegetables are associated with a lower risk of develop- Department of Smart Agriculture Systems, Chungnam National University,
Daejeon, Republic of Korea
ing diseases.4,5 There are many variations in B. rapa growth 4
Department of Bio-AI Convergence, Chungnam National University, Daejeon,
habits, leaf morphology, and the type of vegetable grown. Republic of Korea
This species has various uses, including as a vegetable and 5
Department of Horticultural Science, Chungnam National University,
an oilseed.6,7 Compared to most Brassica species, B. rapa has a Daejeon, Republic of Korea
broad global distribution.8 The B. rapa chemical structure
Corresponding Authors:
and its contents determine the bioavailability and activity of Jong Seok Park, Department of Horticultural Science, Chungnam National
dietary phytochemicals. GSLs and phenolic compounds University, 99 Daehak-ro, Yuseong-gu, Daejeon 34134, Republic of Korea.
have been the primary targets of previous B. rapa studies. Email: jongseok@cnu.ac.kr
For example, various B. rapa varieties were examined by Liu Sang Un Park, Department of Crop Science, Chungnam National University,
et al and Zou et al,9,10 for their GSL profiles. Additionally, 99 Daehak-ro, Yuseong-gu, Daejeon 34134, Republic of Korea.
Chihoub et al11 have examined phenolic compounds in Email: supark@cnu.ac.kr

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2 Natural Product Communications

GSLs are primarily found in cruciferous plants, including the Table 1. Shoot Length, Root Length, and Fresh Weight of 7 Brassica
widely consumed Brassicaceae family.16,17 Recent research has rapa Subspecies Sprout Determined 10 DAS in a Growth Chamber.
demonstrated the positive impacts on GSLs, which have Brassica rapa Shoot Root Fresh
direct antibacterial capabilities, regulatory roles in inflammation, subspecies length (cm) length (cm) weight (mg)
plant stress response, phase I metabolism, and antioxidant Chinese cabbage 2.6 ± 0.30d 6.4 ± 0.73d 110.8 ± 11.8ab
activities.18 Turnip 6.2 ± 0.37a 8.5 ± 0.57abc 101.0 ± 10.5ab
Among plant secondary metabolites, phenolic compounds Bok choy 4.4 ± 0.42c 6.9 ± 1.2bcd 122.4 ± 26.7a
are a large group of metabolites present in vegetables and Choy sum 5.0 ± 0.17bc 7.6 ± 0.51abcd 85.8 ± 13.2bc
other higher plant organs,19 fruit,20,21 spices,22 grains,23 Turnip rape 6.6 ± 0.61a 9.2 ± 1.7a 101.2 ± 22.1ab
legumes, and nuts,24 playing an essential role in plant quality, Wutacai 4.4 ± 0.21c 6.6 ± 0.7cd 64.6 ± 9.87c
Mizuna 5.5 ± 0.39b 8.7 ± 1.5ab 80.2 ± 15.23bc
color, flavor, and stress tolerance. As part of the human diet,
plant phenolics also play a crucial role due to their biological Different letters in the row indicate a statistically significant difference between
properties, which include antioxidant,25 antimicrobial,26 anti- the means when Duncan’s multiple range test is used (P < .05).
inflammatory,27 anticarcinogenic activities,28 and diseases asso-
ciated with oxidative stress.29 It is recommended that humans
consume plant-based foods high in phenolic compounds to
improve their health.30 Nonetheless, the variety of colors,
flavors, and tastes in B. rapa suggests a much more complex
metabolism, possibly including a whole new array of com-
pounds and biochemical processes. Brassica crops are com-
monly used for various side dishes in many countries. To
understand the metabolic characteristics, in the present study,
we detected and quantified the GSLs and phenolic compound
content across the sprouts of 7 B. rapa subspecies for the compar-
ative analysis of these 2 phytochemical profiles. Furthermore,
GSLs and phenolic compound profiling in these B. rapa subspe-
cies will improve our understanding of the distribution pattern of
GSLs and phenolics in whole plant tissues. We found variations
in the composition and the total concentration of GSLs and phe-
nolic compounds on these B. rapa subspecies.

Results
Phenotype of B. rapa Subspecies Sprouts
After 10 days, the 7 B. rapa plants were harvested, and shoot,
root length (RL), and fresh weight (FW) were measured. For
shoot length (SL), a high value was found in turnip rape, and
a low value was observed in Chinese cabbage. A similar trend
was achieved for RL. However, Bok choy showed the highest
value for FW, whereas the lowest value was found in choy
sum (Table 1). A difference in plant development among the
subspecies was observed, and it was clear that the Chinese
cabbage presented the worst phenotypic characteristics than
the others, namely leaf development and plant height (Figure 1).
GSL Content in B. rapa Sprouts
high-performance liquid chromatography (HPLC) analysis
identified 7 GSLs, namely 4 aliphatic and 3 indolic GSLs, from
all 7 B. rapa subspecies. The concentrations of total GSLs in
all 7 B. rapa showed a statistical difference (Table 2). For glu-
cobrassicanapin content, HPLC analysis found that a high
content was achieved in Bok choy, whereas a low concentration
was observed in turnip (1.96 ± 0.14 and 0.23 ± 0.03 mg/g DW,
Figure 1. The phenotype of the 7 Brassica subspecies observed 10
DAS in grown pots (A), and after washed and cleaned (B).
Abbreviation: 1. Chinese cabbage, 2. Turnip, 3. Bok choy, 4. Choy sum,
5. Turnip rape, 6. Wutacai, 7. Mizuna.
respectively). For progoitrin content, the highest and lowest con-
centrations were found in Bok choy (17.84 ± 1.24 mg/g DW)
and mizuna (4.37 ± 0.09 mg/g DW), respectively. High concen-
trations of gluconapoleiferin were observed in wutacai sprouts
(1.15 ± 0.15 mg/g DW), and the lowest concentrations were
observed in mizuna sprouts (0.03 ± 0.00 mg/g DW). For gluco-
napin, HPLC analysis showed that the highest values were found
in mizuna (13.41 ± 0.69 mg/g DW), and the lowest values were
observed in turnip (0.16 ± 0.01 mg/g DW). Glucobrassicin con-
centrations were high in turnip sprouts (1.46 ± 0.09 mg/g DW),
and a low concentration was observed in Chinese cabbage sprouts
(0.76 ± 0.05 mg/g DW). The highest 4-methoxyglucobrassicin
content was achieved in the choy sum sprouts (5.48 ± 0.15 mg/g
DW), whereas the lowest content was observed in Bok choy
da Cristina Bungala et al 3

Table 2. The Concentration of Total Glucosinolates in Sprouts of 7 Brassica rapa Subspecies analyzed 10 DAS.
Concentration (mg/g dry wt.)
Glucosinolates Bok choy Wutacai Mizuna Turnip rape Choy sum Chinese cabbage Turnip
Progoitrin 17.84 ± 1.24 a
11.64 ± 1.35 b
4.37 ± 0.09 d
10.37 ± 1.41 bc
9.33 ± 1.04 c
8.70 ± 0.97c
9.30 ± 0.37c
Gluconapoleiferin 0.73 ± 0.07c 1.15 ± 0.15a 0.03 ± 0.00f 0.57 ± 0.09d 0.13 ± 0.02f 0.30 ± 0.04e 9.30 ± 0.37c
Gluconapin 4.50 ± 0.25d 2.06 ± 0.17e 13.41 ± 0.69a 1.70 ± 0.20e 11.66 ± 0.63b 10.17 ± 0.89c 0.16 ± 0.01f
Glucobrassicanapin 1.96 ± 0.14a 1.18 ± 0.14b 0.54 ± 0.07d 0.95 ± 0.09c 1.25 ± 0.06b 1.82 ± 0.12a 0.23 ± 0.03e
Glucobrassicin 0.60 ± 0.08d 0.60 ± 0.08d 1.04 ± 0.11b 0.84 ± 0.12c 1.06 ± 0.09b 0.76 ± 0.05cd 1.46 ± 0.09a
4-Methoxyglucobrassicin 2.89 ± 0.16c 4.51 ± 0.35b 4.73 ± 0.29b 4.49 ± 0.11b 5.48 ± 0.15a 4.59 ± 0.27b 4.28 ± 0.2b
Neoglucobrassicin 3.22 ± 0.06bc 6.06 ± 0.26a 3.58 ± 0.17b 2.84 ± 0.14cd 2.92 ± 0.52cd 2.94 ± 0.20cd 2.60 ± 0.12d
Total 31.73 ± 1.49a 27.2 ± 2.13b 27.7 ± 1.13b 21.75 ± 1.67c 31.82 ± 2.3a 29.28 ± 2.02ab 18.95 ± 0.63c
Different letters in the row indicate a statistically significant difference between the means when Duncan’s multiple range test is used (P < .05).

Table 3. The Concentration of Total Phenolic Compounds in Sprouts of 7 Brassica rapa Subspecies analyzed 10 DAS.
Concentration (mg/g dry wt.)
Phenolic compound Bok choy Wutacai Mizuna Turnip rape Choy sum Chinese cabbage Turnip
Gallic acid 0.02 ± 0.00 a
0.02 ± 0.00 a
0.01 ± 0.00 b
0.02 ± 0.00a
0.02 ± 0.00a
0.01 ± 0.00b
0.02 ± 0.00a
4-hydroxybenzoic acid 0.08 ± 0.00cd 0.08 ± 0.01cd 0.07 ± 0.00de 0.09 ± 0.02bc 0.06 ± 0.00e 0.18 ± 0.00a 0.10 ± 0.00b
Catechin hydrate 0.18 ± 0.02a 0.17 ± 0.01a 0.16 ± 0.03a 0.10 ± 0.03b 0.10 ± 0.01b 0.07 ± 0.00b 0.09 ± 0.00b
Chlorogenic acid 0.08 ± 0.00ab 0.07 ± 0.01abc 0.07 ± 0.01abc 0.06 ± 0.00c 0.07 ± 0.00c 0.08 ± 0.01a 0.07 ± 0.00bc
Caffeic acid 0.03 ± 0.00cd 0.04 ± 0.00b 0.04 ± 0.00b 0.02 ± 0.00d 0.03 ± 0.00c 0.04 ± 0.00b 0.05 ± 0.00a
Epicatechin 0.38 ± 0.03ab 0.30 ± 0.03de 0.36 ± 0.02bc 0.32 ± 0.03cde 0.34 ± 0.02bcd 0.29 ± 0.03e 0.41 ± 0.01a
p-coumaric acid 0.14 ± 0.02cd 0.14 ± 0.00c 0.12 ± 0.01d 0.12 ± 0.00d 0.16 ± 0.01b 0.18 ± 0.01a 0.12 ± 0.00d
Ferulic acid 0.05 ± 0.01d 0.09 ± 0.01a 0.06 ± 0.01cd 0.06 ± 0.00d 0.07 ± 0.00b 0.07 ± 0.00bc 0.07 ± 0.01b
Benzoic acid 0.66 ± 0.10b 0.55 ± 0.01c 0.96 ± 0.03a 0.66 ± 0.02b 0.60 ± 0.03bc 0.44 ± 0.07d 0.90 ± 0.01a
Rutin 0.41 ± 0.02d 0.41 ± 0.01cd 0.30 ± 0.03f 0.45 ± 0.01bc 0.34 ± 0.00e 0.85 ± 0.03a 0.48 ± 0.03b
Trans-cinnamic acid 0.02 ± 0.00a 0.02 ± 0.00a 0.02 ± 0.00a 0.02 ± 0.00a 0.02 ± 0.00a 0.02 ± 0.00a 0.02 ± 0.00a
Quercetin 0.12 ± 0.01e 0.18 ± 0.00c 0.20 ± 0.02b 0.15 ± 0.01d 0.17 ± 0.01cd 0.28 ± 0.00a 0.13 ± 0.01e
Kaempferol 0.18 ± 0.00b 0.16 ± 0.01b 0.19 ± 0.01a 0.20 ± 0.02a 0.20 ± 0.01a 0.18 ± 0.01b 0.19 ± 0.00a
Total 2.34 ± 0.10cd 2.24 ± 0.02cd 2.55 ± 0.02b 2.27 ± 0.05cd 2.17 ± 0.05d 2.7 ± 0.05a 2.63 ± 0.07ab
Different letters in the row indicate a statistically significant difference between the means when Duncan’s multiple range test is used (P < .05).

sprouts (2.89 ± 0.16 mg/g DW). The neoglucobrassicin content
was highest in wutacai (6.06 ± 0.26 mg/g DW) and lowest in
turnip (2.60 ± 0.12 mg/g DW).
Phenolic Compounds in B. rapa Subspecies
Concerning the phenolic compound concentration, 13 phenolic
compounds, namely 5 hydroxycinnamic acids, 3 hydroxyben-
zoic acids, 3 flavonols, and 2 flavan-3-ol, were identified by
HPLC analysis of the sprouts of all 7 B. rapa subspecies. The
phytochemical composition of phenolic compounds can vary
considerably from species to species and even from one crop
to another. The total concentrations of phenolic compounds
of the 7 B. rapa subspecies were submitted to ANOVA, and
the statistical analysis of concentrations of the 13 detected com-
pounds showed a significant difference (Table 3). The individual
phenolic compound with the highest concentration in all 7 B.
rapa subspecies was benzoic acid, followed by rutin, and the
phenolic compounds gallic acid and trans-cinnamic acid
showed the lowest concentrations. For the benzoic acid
concentration, mizuna sprouts (0.96 ± 0.01 mg/g DW)
showed the highest content of all analyzed subspecies. HPLC
analysis found the same content (0.02 ± 0.00 mg/g DW) for
all 7 B. rapa subspecies for the trans-cinnamic acid content.
The same results were found for gallic acid content, except in
mizuna and Chinese cabbage sprouts (0.01 ± 0.00 mg/g DW).
It appears that benzoic acid regulates growth and stress by reg-
ulating metabolism. Rutin is a flavonoid glycoside found in
many vegetables, while gallic acid and trans-cinnamic are more
present in other plant species than vegetables. Among all 7 B.
rapa subspecies, the highest concentration of total phenolic
compounds was found in Chinese cabbage sprouts (2.7 ± 0.05
mg/g DW), and a low concentration was found in Choy sum
sprouts (2.17 ± 0.05 mg/g DW).
Discussion
Brassicas have a great diversity of biotic and abiotic stress adap-
tations because of their immense genetic and morphological
diversity.31 Brassica genera have a wide variety of morphological
4 Natural Product Communications
characteristics that can be used to study domestication and arti-
ficial selection.32–35 The morphological characteristics of the 7
subspecies of B. rapa in the present study indicate variations.
These findings align with those made by Pino del Carpio et al
and Tanhuanpää et al,36,37 who discovered that B. rapa has
divergent morphologic, genetic, and geographic patterns.
However, some studies have shown that geographically
distant crops have close morphological similarities.
A plant species contains an average of 4 distinct GSLs.38
These results agree with the findings in the present study,
where 4 GSLs (progoitrin, gluconapin, 4-methoxyglucobrassi-
cin, and neoglucobrassicin) were found in higher amounts.
Among the 7 GSLs determined through HPLC analysis, the
highest concentrations were achieved for progoitrin, followed
by gluconapin. The Brassica subspecies with the highest total
concentrations of GSLs were Bok choy and Choy sum
sprouts. Our results differ from those by Bhandari et al,39
who compared 12 GSL profiles and concentrations in
9-day-old Brassica sprouts, including 2 B. rapa subspecies.
Gluconapin, glucobrassicin, and gluconasturtiin were the most
common GSL in all examined crops. Previous studies reported
that the significant GLSs found in turnip were progoitrin and
gluconapin, whereas, in Chinese cabbage, gluconapoleiferin
and gluconapin were the major GSLs.40 Likewise, according
to Yang et al,41 compared with other B. rapa subspecies,
turnip contains more aliphatic GSLs. In addition, Yang and
Quiros42 reported that turnip had a higher GSL content than
Bok choy, Chinese cabbage, and rapeseed. These results differ
from those found in the present study, where higher concentra-
tions of progoitrin were found in Bok choy, followed by
wutacai.
The phenolic compounds and GSLs in Brassicas are also
major bioactive components strongly associated with vegeta-
bles’ taste, color, and species characteristics.43 A study identi-
fied 74 phenolic compounds in 12 Brassica vegetables and
found that quercetin, kaempferol, and isorhamnetin were the
main flavonoids. In contrast, the primary hydroxycinnamic
acids were ferulic, sinapic, caffeic, and p-coumaric acids.44
According to Miean and Mohamed,45 myricetin, quercetin,
and kaempferol are significant flavonoids in the Brassicaceae
family. In the present study, among the 13 phenolic compounds
identified in the HPLC analysis, the highest total concentrations
were found in benzoic acid (a hydroxybenzoic acid), followed by
rutin (a flavonol glycoside). These results do not agree with the
findings of Kyriacou et al, Soengas et al, and Dejanovic
et al,15,46,47 who showed that Chinese cabbage, Bok choy,
turnips, and mizuna presented high amounts of kaempferol
derivatives. There is also a discrepancy between the current
findings and previous studies on Brassica microgreens, in
which quercetin and kaempferol are the main flavonols,48 and
the primary hydroxycinnamic acids include caffeic and ferulic
acids.49 In another study,3 with 14 main B. rapa groups for
metabolite profiling analysis found a higher concentration of
kaempferol, followed by quercetin and isorhamnetin. For
example, a study conducted by Romani et al50 revealed that
turnip top contains a high amount of isorhamnetin, kaempferol,
and quercetin. In a study with hairy roots of turnips, Chung
et al51 found higher levels of gentisic acid, ferulic acid, and cat-
echin. These results do not agree with those found in the
current research, which showed high levels of benzoic acid, fol-
lowed by rutin and epicatechin. In a study done by Jeon et al,12
the amount of rutin in purple Bok choy was much higher than
that of the other compounds. Our findings show that among
the 3 flavonols identified in HPLC analysis, the highest concen-
trations of rutin, followed by kaempferol and quercetin, were
observed.
Limitations of the Study
The results of this study deliver helpful information on the phy-
tochemical composition of B. rapa subspecies. Still, it is essential
to recognize that some limitations must be considered when
interpreting the results of this study. The study was limited to
evaluating the content of phenolic compounds and GSLs.
The other classes of phytochemicals that were not analyzed
are not within the scope of this research. Further studies
should address other phytochemical classes. Other chromatog-
raphy techniques, such as gas chromatography–time-of-flight
mass spectrometry, could also be performed to find specific
phytochemicals. The second limitation concerns the exclusive
identification of free forms of polyphenols that the current
study addressed and the lack of a comprehensive method for
GSL analysis, such as chromatography (gas chromatography),
liquid chromatography combined with mass spectrometry, or
another method capable of detecting more GSLs. This limita-
tion was due to the absence of this type of instrument in the lab-
oratory. The third limitation concerns the harvesting period; 10
days after sowing (DAS) may not reflect the full range of vari-
ations in B. rapa phenolic and GSLs compounds. The phyto-
chemicals detected during this period offer an idea, but more
time variation sampling could provide a complete understand-
ing of the plant’s phytochemical composition. Finally, the
limited availability of articles that only compare phenolic com-
pounds and GSLs within cultivars of B. rapa also impacted
the discussion section of this article. Studies such as the one pre-
sented here will partly overcome this limitation.
Conclusion
In conclusion, the current findings illustrate the accumulation
patterns of individual GSLs and phenolic compounds in 7 sub-
species of B. rapa sprouts. Our findings show that plants
belonging to the same species can vary the content of phyto-
chemical compounds such as GSLs and phenolic acids, and
also show that the phenotypic shape in B. rapa subspecies
after 10 DAS is not directly proportional to the total
content of GSLs and phenolic acids. Using HPLC analysis,
a total of 20 metabolites were successfully identified. The
main GSLs identified were progoitrin, Gluconapin, and
4-Methoxyglucobrassicin, and the main flavonol identified
da Cristina Bungala et al
was rutin. The main flavanol detected was epicatechin; the
primary phenolic acid was p-coumaric acid. Bok choy and
Choy sum sprouts presented a high content of total GSLs,
and Chinese cabbage and turnip presented a better content of
total phenolic compounds detected among the subspecies.
The results of the present study showed those distinctive met-
abolic signatures entrenched in these Brassica subspecies. They
will be helpful for future studies and for choosing the best sub-
species to use as sprouts containing functional compounds
among B. rapa.
More studies on bioavailability, metabolism, dietary intake,
effective dosages, and dietary guidelines are necessary to
improve health and nutrition. Researchers are also encouraged
to examine the nutritional and phytochemical composition of
sprouts of other brassicas subspecies. Furthermore, there are
still gaps in knowledge regarding metabolic profiling, antioxi-
dant activity, anthocyanin, carotenoid, chlorophyll, protein,
vitamin, and mineral categories.
Materials and Methods
Plant Material
The seeds of the 7 B. rapa subspecies were obtained from Asia
Seed Co, South Korea (Table 4). A total of 100 seeds were
counted and immersed in distilled water overnight. These
seeds were then transferred into the pots filled with vermiculite.
The experiment was arranged in a completely randomized
design with 3 replications for each subspecies. The seedlings
were germinated and grown under controlled conditions of a
temperature between 24.8 °C and 26.8 °C, a photoperiod of
16/8 h, a fluorescent light photon flux of 700 lux, and 60%
to 70% humidity in an LED plant growing chamber (Sejong
Scientific Co., Sejong, South Korea). The sprouts from each
subspecies were harvested 10 DAS. After being harvested, the
sprouts were washed and cleaned with distilled water. For
growth measurements, 10 plants from each subspecies were
selected randomly, and growth measurements were taken.
The SL and RL were measured in cm using a meter ruler. To
determine the FW, the Brassica subspecies were weighed in mg
using a balance. The non-selected sprouts were ground into a
powder by using a motor and pestle with liquid nitrogen and
then freeze-dried using a freeze-dryer machine (HyperCOOL,
Table 4. List of B. rapa Subspecies Used in the Study.
Taxon Common name
B. rapa subsp. pekinensis (Lour.) Hanelt Chinese cabbage
B. rapa subsp. rapa Turnip
B. rapa subsp. chinensis Bok choy
B. rapa subsp. chinensis (L.) Hanelt var. parachinensis Choy sum
(L. H. Bailey) Hanelt
B. rapa subsp. oleifera (DC.) Metzg. Turnip rape
B. rapa subsp. narinosa (L. H. Bailey) Hanelt Wutacai/tatsoi
B. rapa subsp. nipposinica (L. H. Bailey) Hanelt Mizuna/mibuna
5
Gyrozen Co. LTD, Gyeonggi-do, Korea). After 72 h in a
dryer machine at −40 °C, the dehydrated powder sprout
samples were used to analyze for GSLs and phenolic acid
content (Figure 2).
GSL Extraction and HPLC Analysis
With some modifications, GSLs were extracted using previ-
ously reported procedures.52 Briefly, 100 mg of a freeze-dried
powdered sample was collected in a 2 mL Eppendorf tube,
and 1.5 mL of methanol was added. For 5 min, the tubes
were placed in a water bath at 70 °C. In a new 5 mL
Eppendorf tube, supernatants were collected after centrifuga-
tion at 12 000 rpm for 10 min at 4 °C. Using the combined
supernatants from the 2 extractions described above, crude
GSL extracts were prepared from the residues. A Mini
column filled with DEAE-Sephadex A-25 (Sigma-Aldrich,
St. Louis, MO, USA) was used to load the extracts, and 75 μL
of aryl sulfatase solution was used to desulfate them. With
0.5 mL of ultrapure water, the desulfated (DS) GSLs were
eluted into 2 mL microcentrifuge tubes. A 1200 series HPLC
system attached with a photodiode array detector (Agilent
Technologies 1200 series, Seoul, South Korea) was used to
analyze DS-GSLs. The mobile phase compositions were used
as follows: 0 min (B) 0%, kept constant at (B) 0% by 2 min, con-
tinually raised (B) 10% until 7 min, progressively improved (B)
31% by 16 min, maintained steady at (B) 31% by 19 min,
further (B) 0% by 21 min, and then kept constant at (B) 0%
for 6 min (total 27 min). A desulpho-sinigrin (Sigma-Aldrich
Co., Ltd, St. Louis, MO, USA) was used as a standard to identify
and quantify GSLs by high HPLC peak area ratios, retention
times, and response factors. The results were carried out in
triplicate and expressed as milligrams per gram of dry weight
(mg/g DW).
Determination of Phenolic Compounds
A method previously described by Lee et al52 was used to
analyze phenolic compounds. A volume of 1.5 mL of
aqueous methanol was added to 100 mg of dried sprout
powder samples. At 25 °C, the mixture was sonicated for 1 h.
In a fresh tube, the supernatant was transferred after centrifug-
ing the crude extract for 10 min at 10 000 rpm. Two more
extractions were performed to remove the remaining sludge.
A 0.45 μm PTFE syringe filter was used to filter the collected
supernatant after centrifugation for 15 min at 10 000 rpm.
Based on a previous study performed by Lee et al,52 the
HPLC analysis conditions, system, and gradient program
were used. We used the HPLC (NS-4000, Futecs, South
Korea) and RP-HPLC columns (OptimaPak C18, 250 × 4.6
mm, 5 μm, RS Tech, Daejeon, South Korea) with a UV-vis
detector and autosampler. Solvent A: 0.2% (v/v) acetic acid,
and Solvent B: pure methanol was used for the gradient
program of the mobile phase. Following this condition,
solvent B started at 5%, followed a linear gradient to 45% for
6 Natural Product Communications
Figure 2. A roadmap for the detection and quantification of phenolic compounds and glucosinolates.
55 min, held for 10 min, and then slowly decreased to 5% for
about 30 min. Injection volume and column oven were 0.1 mL
and 30 °C, respectively, and compounds were chromatographi-
cally detected at 280 nm. Individual phenolics were identified
by comparing retention times using the spike test, and com-
pounds in the sample extracts were quantified using a calibration
curve. The pure standard solutions of gallic acid, 4-hydroxyben-
zoic acid, catechin hydrate, chlorogenic acid, caffeic acid, epicate-
chin, p-coumaric acid, ferulic acid, benzoic acid, rutin,
trans-cinnamic acid, quercetin, kaempferol (Sigma-Aldrich,
St. Louis, MO, USA) were dissolved in methanol and analyzed
the samples. The results were carried out in triplicate and
expressed as milligrams per gram of dry weight (mg/g DW).
Statistical Analyses
With statistical analysis software (SAS) at P ≤ .05, the mean
values of 3 biological replicates and their respective standard
deviations for the identified GSLs and phenolic compounds
were determined, and Duncan’s multiple range test was used
to assess cultivar differences using the results of ANOVA.
Author Contributions
JSP and SUP designed the experiments and analyzed the data. LTDCB,
BVN, CP, RS, and GB performed the experiments and analyzed the
data. LTDCB wrote the original draft of the manuscript. LTDCB
and RS review and edit the manuscript. All authors have read and
agreed to the published version of the manuscript.
Declaration of Conflicting Interests
The authors declared no potential conflicts of interest with respect to
the research, authorship, and/or publication of this article.
Funding
The authors disclosed receipt of the following financial support for the
research, authorship, and/or publication of this article: This work was
supported by Ministry of Agriculture, Food and Rural Affairs of
Republic of Korea (322077-3, Development of water supply technol-
ogy and foundation for universal use of paddy fields according to
changes in agricultural environment).
Statement of Human and Animal Rights
No human participants were included in this study, and informed
consent was not required.
Statement of Informed Consent
No human subjects are in this article, and informed consent is not
required.
ORCID iD
Sang Un Park https://orcid.org/0000-0003-2157-2246
da Cristina Bungala et al
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