Phenolic constituents isolated from Senna tora sprouts and their neuroprotective effects against glutamate-induced oxidative stress in HT22 and R28 cells

Article abstract of DOI:10.1016/j.bioorg.2021.105112

The consumption of sprouts has been steadily increasing due to their being an excellent source of nutrition. It is known that the bioactive constituents of legumes can be increased after germination. In this study, the extract from Senna tora sprouts is shown to exhibit improved radical scavenging activities and better neuroprotective effects in HT22 hippocampal neuronal (HT22) and R28 retina precursor (R28) cells than those from seeds due to an increased content of phenolic constituents, especially compounds 1 and 3–6. A phytochemical investigation of S. tora sprouts resulted in the isolation of two new naphthopyrone glycosides (1–2) with 27 previously reported compounds. Their structures were determined via interpreting spectroscopic data. Compounds 1 and 3–6 were found to possess radical scavenging activities and neuroprotective effects against oxidative stress in both neuronal cells. Hence, Senna tora sprouts and their constituents may be developed as natural neuroprotective agents via antioxidative effects.

Full text of DOI:10.1016/j.bioorg.2021.105112

Bioorganic Chemistry 114 (2021) 105112
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Phenolic constituents isolated from Senna tora sprouts and their
neuroprotective effects against glutamate-induced oxidative stress in HT22
and R28 cells
a
,
b
,
1
a
,
1
a
a
a
Jaeyoung Kwon
, Hoseong Hwang , Baskar Selvaraj , Jung Hwan Lee , Woongbi Park ,
c
d
a
a
a
Seung Mok Ryu , Dongho Lee , Jin-Soo Park , Hyoung Seok Kim , Jae Wook Lee ,
e,
*
a,*
Dae Sik Jang , Hak Cheol Kwon
a
KIST Gangneung Institute of Natural Products, Korea Institute of Science and Technology (KIST), Gangneung 25451, Republic of Korea
Division of Bio-Medical Science & Technology, KIST School, University of Science and Technology (UST), Gangneung 25451, Republic of Korea
Herbal Medicine Resources Research Center, Korea Institute of Oriental Medicine (KIOM), Naju 58245, Republic of Korea
Department of Plant Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 02841, Republic of Korea
Department of Life and Nanopharmaceutical Sciences, College of Pharmacy, Kyung Hee University, Seoul 02447, Republic of Korea
b
c
d
e
A R T I C L E I N F O
A B S T R A C T
Keywords:
The consumption of sprouts has been steadily increasing due to their being an excellent source of nutrition. It is
known that the bioactive constituents of legumes can be increased after germination. In this study, the extract
from Senna tora sprouts is shown to exhibit improved radical scavenging activities and better neuroprotective
effects in HT22 hippocampal neuronal (HT22) and R28 retina precursor (R28) cells than those from seeds due to
an increased content of phenolic constituents, especially compounds 1 and 3–6. A phytochemical investigation of
S. tora sprouts resulted in the isolation of two new naphthopyrone glycosides (1–2) with 27 previously reported
compounds. Their structures were determined via interpreting spectroscopic data. Compounds 1 and 3–6 were
found to possess radical scavenging activities and neuroprotective effects against oxidative stress in both
neuronal cells. Hence, Senna tora sprouts and their constituents may be developed as natural neuroprotective
agents via antioxidative effects.
Sprout
Senna tora
Fabaceae
Naphthopyrone
Antioxidant
Neuroprotective effect
1
. Introduction
Reactive oxygen species (ROS) are oxygen-containing reactive mol-
diseases.
Sprouts, commonly grown from any plant or vegetable seed, have
long been used as an ingredient in oriental diets. Recently, their popu-
larity has been steadily increasing in the world as consumers seek
inexpensive, natural, and healthy foods [5,6]. Sprouts have been known
to be a rich source of various nutrients including health-promoting
phytochemicals with antioxidants properties. Several reports have
shown that these phytochemicals are increased after germination,
demonstrating that sprouts can provide several health benefits beyond
basic nutrition [7–9].
Senna tora (L.) Roxb., an annual shrub belonging to the family
Fabaceae, grows up to 30–90 cm tall with pinnate leaves and is widely
cultivated in Asian countries [10]. Immature leaves are edible, and in
particular, ripe seeds, Cassiae semen, have long been used to treat
dizziness and headache, and to improve eye health as a roasted tea [11].
ecules naturally produced in cellular metabolism. These molecules are
constantly generated and eliminated in normal cellular physiology and
play a critical role in the regulation of signal transduction pathways [1].
However, the balance between the generation and elimination of ROS is
somehow disturbed due to endogenous and exogenous factors [1].
Glutamate is a critical excitatory neurotransmitter in several nervous
systems, but excessive glutamate leads to the accumulation of intracel-
lular ROS by blocking the cysteine uptake into neuronal cells through
the cystine/glutamate antiporter, resulting in neuronal damages by
oxidative stress [2,3]. These damages commonly lead to acute and
chronic neuronal diseases [4]. Therefore, discovering antioxidants such
as phenolic compounds represents promising strategies for these
*
Corresponding authors.
E-mail addresses: dsjang@khu.ac.kr (D.S. Jang), hkwon@kist.re.kr (H.C. Kwon).
These authors contributed equally to this work.
1
https://doi.org/10.1016/j.bioorg.2021.105112
Received 26 February 2021; Received in revised form 16 June 2021; Accepted 18 June 2021
Available online 23 June 2021
0
045-2068/© 2021 Published by Elsevier Inc.

J. Kwon et al.
Bioorganic Chemistry 114 (2021) 105112
Table 1
Varian 500 MHz, Bruker Avance III 400 MHz, and Bruker Avance III HD
800 MHz NMR spectrometers. HRESIMS data were obtained on Bruker
MicrOTOF-Q II and AB Sciex TripleTOF 5600 mass spectrometers. On-
line HPLC-ABTS screening was performed on an Agilent 1200 HPLC
4
NMR data for compounds 1 and 2 in methanol‑d .
Position
1
2
δ
C
δ
H
, mult (J in Hz)
δ
C
H
δ , mult (J in Hz)
system with a YMC Pack ODS-A column (5
μm, 150 × 4.6 mm). Pre-
2
3
4
170.1
105.8
184.1
102.5
160.9
106.2
154.7
170.0
110.7
184.5
109.8
157.2
parative HPLC was performed using Gilson 321, YMC LC-Forte/R, and
Jasco LC-2000Plus HPLC systems with Phenomenex Luna C18 (2) (10
6.12, s
6.36, s
μ
m, 250 × 21.2 mm), Phenomenex Luna C18(2) (10
μm, 250 × 10 mm),
4
5
5
6
6
7
8
9
9
1
1
1
1
1
2
3
4
4
5
6
a
a
a
and Waters Delta-Pak C18 (15
μ
m, 300 × 30 mm) columns. Column
chromatography was performed using Merck silica gel. TLC was carried
out on a Merck pre-coated silica gel plate.
106.6
142.5
100.7
163.1
102.2
6.90, s
111.9
161.3
97.0
6.84, d (2.0)
6.79, d (2.0)
2.2. Plant materials
6.92, s
7.18, s
a
139.8
101.4
152.4
The seeds of Senna tora, cultivated in Goryeong, Republic of Korea,
were purchased from a herbal market in Yeongcheon, Republic of Korea.
The seeds were sterilized with 20 mg/mL of calcium hypochlorite for 15
min, rinsed thoroughly, and then soaked in distilled water for 4 h.
0
157.7
106.4
157.2
20.5
0a
0b
1
′
′
′
′
19.4
2.41, s
2.59, s
150.9
122.4
132.4
120.6
137.1
102.9
156.2
102.1
75.2
5.15, d (8.0)
3.69, m
Sterilized seeds were transferred to a tray and cultured in a growth
chamber at 22 ± 3 ◦C, and distilled water was supplied every day. A
78.5
3.53, t (8.8)
3.41, t (8.8)
fluorescent lamp, a 385 nm LED, a 465 nm LED, a 645 nm LED, and a
780 nm LED were used to investigate the effects of light conditions on
producing compounds compared to dark conditions. After 7 d, the
71.6
′
a
′
′
6.34, br s
77.1
68.9
3.68, m
4.05, dd (11.2, 1.6)
◦
sprouts were collected and dried at 30 C for 1 d. A voucher specimen
3
.63, dd (11.2, 6.8)
′
′
′
(STS) was maintained at the KIST Gangneung Institute of Natural
Products, Korea Institute of Science and Technology.
7
8
8
9
1
1
1
2
3
4
102.8
156.1
108.4
208.1
31.4
6.97, d (2.0)
a
′
2
.3. Extraction and isolation
′
′
0
2.63, s
1
16.1
1.98, s
′
′
′
′
′
′
′
′
The dried seeds (3.5 g) of S. tora were ground and extracted with
103.1
73.5
5.12, dd (8.0, 3.5)
3.58, t (8.5)
3.51, m
111.1
78.2
80.5
75.1
4.95, d (2.4)
3.90, d (2.4)
ethanol (35.0 mL) for 7 d. The extract was evaporated to obtain a dark
brown extract (117.3 mg). The dried sprouts (698.4 g) of S. tora culti-
vated in dark conditions were ground and extracted with ethanol (EtOH)
(2 × 5.5 L) for 7 d. After evaporation, the obtained extract (66.7 g) was
partitioned using deionized water (0.6 L) with ethyl acetate (EtOAc) (4
× 1.8 L) followed by n-buthanol (n-BuOH) (4 × 1.8 L). The EtOAc layer
76.7
69.7
3.48, t (9.0)
3.99, d (9.6)
3
.74, d (9.6)
′
′
′
′
5
6
77.4
61.0
3.55, dd (5.0, 2.0)
3.98, dd (12.0, 2.0)
65.8
56.1
3.58, br s
3
.78, dd (12.0, 5.0)
OCH
3
54.8
3.75, s
3.93, s
(
23.5 g) was chromatographed on a silica gel column (n-hexane/chlo-
roform/methanol, 1:1:0 to 0:1:1) to obtain twenty fractions (F
–F20). F
1.4 g) was isolated by preparative HPLC (prep-HPLC) (acetonitrile/
water, 3:2 to 1:0, flow rate 10.0 mL/min) to obtain 25 fractions
1
8
(
Phytochemical investigations of this plant have revealed the presence of
phenolic constituents, such as anthraquinones, naphthopyrone glyco-
sides, and naphthalene glycosides, which have been found to have a
broad range of biological activities, including antitumor, antigenotoxic,
anti-inflammatory, and antihepatotoxic effects [10,12–15].
(
F
8.1–F8.25). Among them, F8.5 and F8.8 were found to be isotoralactone
(
3, 7.5 mg, t
R
= 36.8 min) and chrysophanol (24, 6.2 mg, t = 48.1 min),
R
respectively. F8.9 (21.6 mg) was isolated by prep-HPLC (acetonitrile/
water, 3:2 to 9:1, flow rate 8.0 mL/min) to afford physcion (25, 0.8 mg,
In screening to compare the abilities of S. tora seeds and sprouts to
t
R
= 21.9 min). F8.11 (12.1 mg) was separated by prep-HPLC (acetoni-
scavenge ROS, the extracts from sprouts showed improved activities in
′
trile/water, 3:2 to 1:0, flow rate 8.0 mL/min) to obtain chrysophanol-
2
,2 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,2-
′
1
0,10 -bianthrone (28, 2.5 mg, t
R
= 65.4 min). F12 (2.1 g) was isolated
diphenyl-1-picrylhydrazyl (DPPH) assays. Also, the extracts from plant
sprouts showed better neuroprotective activities against glutamate-
induced oxidative stress in HT22 hippocampal neuronal (HT22) and
R28 retina precursor (R28) cells. In general, antioxidative activity is
considered as one of the key factors for neuronal cell protection. Hence,
this study aimed to isolate as many components as possible observed as
peaks in the HPLC chromatogram of the S. tora sprouts extract to identify
the biologically active compounds and evaluate the possibility of the
sprout as a functional food.
by prep-HPLC (acetonitrile/water, 2:3 to 1:0, flow rate 10.0 mL/min) to
obtain 27 fractions (F12.1–F12.27). F12.11 (14.7 mg) was separated by
prep-HPLC (acetonitrile/water, 19:31, flow rate 8.0 mL/min) to afford
aurantio-obtusin (26, 0.8 mg, t
.9 mg, t
= 45.6 min). F12.17 (32.3 mg) was separated by prep-HPLC
acetonitrile/water, 13:7 to 1:0, flow rate 8.0 mL/min) to afford tor-
alactone (4, 1.2 mg, t
= 24.2 min) and emodin-physcion bianthrone
= 62.8 min). F13 (135.5 mg) was separated by prep-HPLC
acetonitrile/water, 1:9 to 1:0, flow rate 8.0 mL/min) to obtain tor-
= 71.3 min) and rubrofusarin (6, 2.6 mg, t
20.7 min). F15 (893.7 mg) was separated by prep-HPLC (acetonitrile/
water, 3:7, flow rate 8.0 mL/min) to afford cassialactone (7, 0.7 mg, t
192.4 min). F16 (1.5 g) was fractionated on a silica gel column
methylene chloride/methanol, 20:1 to 1:1) to obtain 14 fractions
F16.14). F16.3 (46.2 mg) was separated by prep-HPLC (acetonitrile/
R
= 27.3 min) and chryso-obtusin (27,
0
R
(
R
(
(
29, 0.5 mg, t
R
osachrysone (5, 0.6 mg, t
R
R
=
2
. Experimental
1
R
2
.1. General experimental procedures
=
(
(
Optical rotations were acquired on a Perkin-Elmer Model 343
F
16.1
–
polarimeter. UV spectra were measured on a Perkin-Elmer Lambda 35
UV/vis spectrophotometer. IR spectra were obtained using a Thermo
Scientific Nicolet iS50 FT-IR spectrometer. ECD spectra were obtained
via a Jasco J-1100 spectropolarimeter. NMR data were acquired on
water, 1:4 to 1:0, flow rate 8.0 mL/min) to afford 7-O-methylkaempferol
19, 1.8 mg, t = 34.7 min), and 7-
= 30.1 min), quercetin (18, 1.9 mg, t
O-methylquercetin (20, 1.0 mg, t
= 38.9 min). F16.4 (78.3 mg) was
separated by prep-HPLC (acetonitrile/water, 1:4 to 1:0, flow rate 6.5
(
R
R
R
2

J. Kwon et al.
Bioorganic Chemistry 114 (2021) 105112
Fig. 1. Radical scavenging activities of the extracts from S. tora seed and sprout assessed by DPPH (A) and on-line HPLC-ABTS (B) assays.
′
mL/min)
to
afford
1-[(3-methoxyl-8-hydroxyl-1-O-β-glucopyr-
hydroxymusizinyl-rubrofusarin-8 -O-β-D-glucopyranoside (1, 5.6 mg, t
R
anosylnaphthalen)-6-yl]propan-2-one (15, 2.5 mg, t
hydroxymusizin-8-O-β-glucopyranoside (16, 2.1 mg, t
torachrysone-8-O-β-glucopyranoside (8, 0.5 mg, t
R
= 22.9 min), 6-
= 221.2 min).
R
= 26.2 min), and
′
R
= 68.1 min). F16.6
2.3.1. 7-Hydroxymusizinyl-rubrofusarin-8 -O-β-D-glucopyranoside (1)
2
0
(
98.7 mg) was separated by prep-HPLC (acetonitrile/water, 17:83 to
Yellowish amorphous solid; [
α
]
D
–20.0 (c 0.05, methanol); UV
) 225 (4.32), 282 (4.23) nm; IR
1
:0, flow rate 6.5 mL/min) to obtain cassialactone-9-O-β-glucopyrano-
= 77.2 min). F16.7 (422.7 mg) was separated by prep-
HPLC (acetonitrile/water, 1:4 to 1:0, flow rate 6.5 mL/min) to obtain
cassiaside A (10, 4.1 mg, t
= 92.3 min). F17 (2.4 g) was separated by
prep-HPLC (acetonitrile/water, 1:9 to 1:0, flow rate 6.5 mL/min) to give
cassiaside B (11, 1.7 mg, t
= 197.3 min). F18 (3.7 g) was fractionated on
a silica gel column (methylene chloride/methanol, 20:1 to 1:1) to afford
4 fractions (F18.1–F18.24). F18.17 (124.8 mg), F18.18 (226.9 mg), F18.20
112.0 mg), and F18.21 (110.6 mg) were combined and isolated by prep-
HPLC (acetonitrile/water, 1:9 to 1:0, flow rate 6.5 mL/min) to afford
nepalenside A (17, 24.8 mg, t
(0.15 mM, methanol) λmax (log
ε
ν
max
(ATR) 3387, 2940, 1682, 1650, 1622, 1471, 1430, 1361, 1096 cm 1;
ꢀ
side (9, 2.0 mg, t
R
ECD (c 1.0 mM, acetonitrile) Δε + 8.4 ( 235), –0.5 ( 261), +4.2 ( 293),
1
13
R
–0.8 ( 329); H and C NMR data (methanol-d
4
), see Table 1; ESIMS
–
+
(negative) m/z 663 [M – H] ; ESIMS (positive) m/z 665 [M + H] ;
+
R
HRESIMS (positive) m/z 665.1860 [M + H] (calcd for C34
33 14
H O ,
665.1865).
2
(
2.3.2. Demethylflavasperone-10-O-β-D-glucopyranosyl-6-O-β-D-
apiofuranoside (2)
R
= 42.1 min), quercetin-3-O-neo-
85 min), kaempferol-3-O-neo-
99.3 min), quercetin-3-O-
= 104.6 min), demethylflavasperone-
0-O-β-D-glucopyranosyl-6-O-β-D-apiofuranoside (2, 1.6 mg, t = 140.2
min), demethylflavasperone-10-O-β-glucopyranoside (13, 2.3 mg, t
42.3 min), rubrofusarin-6-O-β-gentiobioside (14, 0.6 mg, t = 155.7
min), cassiaside (12, 9.3 mg, 177.6 min), and 7-
White amorphous solid; [
α
]20
D
–54.0 (c 0.05, methanol); UV (0.18
hesperidoside (22, 4.1 mg,
hesperidoside (23, 6.4 mg,
t
R
=
mM, methanol) λmax (log ) 240 (4.09), 279 (3.95), 366 (3.15) nm; IR
ε
ꢀ 1
1
13
t
R
=
ν
max (ATR) 3405, 2922, 1677, 1622, 1439, 1206, 1134 cm ; H and
C
β-glucopyranoside (21, 5.5 mg, t
R
NMR data (methanol-d
–
4
), see Table 1; ESIMS (negative) m/z 565 [M –
+
1
R
H] ; ESIMS (positive) m/z 567 [M + H] ; HRESIMS (positive) m/z
+
R
=
567.1703 [M + H] (calcd for C26
31
H O14, 567.1708).
1
R
C
t
R
=
3

J. Kwon et al.
Bioorganic Chemistry 114 (2021) 105112
Fig. 2. Neuroprotective effects of the extracts from S. tora seed and sprout against glutamate-induced oxidative stress in HT22 (A) and R28 (B) cells.
Fig. 3. HPLC chromatogram of the extracts from S. tora sprout.
2
.4. Acid hydrolysis and absolute configuration determination of sugar
linear gradients. The thiocarbamoyl-thiazolidine carboxylate de-
rivatives of the monosaccharides obtained from 1 and 2 were deter-
mined by comparing the retention times with those of the authentic
moieties
Compounds 1 and 2 (each 0.5 mg) were separately treated with 1 M
R
samples (t : D-glucose 14.6 min and D-apiose 19.3 min).
◦
of hydrochloric acid (500 L) at 104 C for 3 h, followed by a partition
μ
with ethyl acetate (4 × 2.0 mL) to obtain each aglycone. The aqueous
2
.5. Computational methods
Electronic circular dichroism (ECD) calculations were performed
layers containing sugars were evaporated, dissolved in pyridine (100
μ
L), and treated with 0.5 mg of L-cysteine methyl ester hydrochloride at
◦
6
0
C for 1.5 h, followed by a reaction with O-tolylisothiocyanate (25
according to a previously reported method [16]. Briefly, 3D modeling
was carried out using the Chem3D program followed by conformational
◦
μ
L) at 60 C for 1.5 h and direct analysis via reverse-phased HPLC in
4

J. Kwon et al.
Bioorganic Chemistry 114 (2021) 105112
OH 5''
''
HO
3
OH
OH OH
HO
1''
6
''
O
OH O
3
''
5''
HO
HO
6'
O
HO
1''
O
O
O
8'
OH
6
'
a
8
'
O
8
10
HO
5'
4'
a
8
OH OH O
1
'
1
0a
11
OH OH O
O
4
'
6
5
4
7
10b
O
O
7
5a
4a
2
'
6a
6
2
3
'
1
1'
4a
10'
9a
2
4
10a
5
O
O
9
10
11
O
O
OH O
1
2
3
OH OH O
OH OH O
OH OH O
O
OH
O
O
O
4
5
6
Fig. 4. Structures of the isolated compounds 1–6.
′
searches via the Merck Molecular Force Field in the Spartan 14 software,
optimization using a density functional theory (DFT) calculation with
B3LYP/6–31+G(d.p) in the Gaussian 09 package, and a time-dependent
DFT calculation with CAM-B3LYP/SVP in acetonitrile. The computed
ECD spectra were visualized using SpecDis 1.64 software followed by
applying Boltzmann distribution with UV correction [17].
cells/well and incubated for 24 h. Subsequently, the cells were treated
with various concentrations of samples over 2 h, followed by the addi-
tion of glutamate (5 mM) and incubation for 22 h. The measurement of
cell viability was performed via an EZ-Cytox Assay Kit (Daeil lab service)
for 2 h. The absorbance was recorded at 450 nm using a Molecular
Devices FilterMax F5 microplate reader.
2
.6. Antioxidative activity
2.8. Neuroprotective activity against glutamate-induced oxidative stress in
R28 cells
The scavenging effects of samples for DPPH radicals were assessed
based on a previous report [18]. Briefly, DPPH (0.1 mM) in methanol
100 L) was mixed with various concentrations of samples (100 L) for
h in the dark. The absorbance was recorded at 517 nm.
An online HPLC-ABTS assay was performed as referenced in several
R28 retinal cells were purchased from Kerafast (Kerafast, Inc., Bos-
ton, MA, USA) and were cultured under the same conditions as HT22
cells. Cell viability was determined by performing an MTT assay. R28
(
μ
μ
1
4
cells were seeded in 96-well plates at a density of 1 × 10 cells/well and
reports, with modifications [19]. ABTS reagent was prepared as a
mixture containing ABTS (0.07 mM) with potassium persulfate (0.12
incubated for 24 h. Subsequently, the cells were treated with various
concentrations of samples over 2 h, followed by the addition of gluta-
mate (10 mM) plus BSO (0.5 mM) (glutamate/BSO) and incubation for
22 h. The measurement of cell viability was performed via an EZ-Cytox
Assay Kit (Daeil lab service) for 2 h. The absorbance was recorded at
450 nm.
◦
mM) and stored in darkness at 4 C for 12 h to stabilize radicals. All
samples were analyzed via HPLC (acetonitrile/water containing 0.02%
TFA, 1:9 to 1:0, flow rate 0.7 mL/min). The eluate was then sent to a T-
◦
junction and reacted with ABTS reagent in a reaction coil at 40 C. The
chromatogram was visualized at 254 nm (positive peak), as well as at
7
34 nm (negative peak), to record the decrease in ABTS radicals.
2.9. Inhibitory activity against glutamate-induced intracellular ROS
generation
2
.7. Neuroprotective activity against glutamate-induced oxidative stress in
HT22 cells
HT22 cells were added in 96-well black/clear bottom plates at 3 ×
1
03 cells/well and incubated at 37 C under 5% CO
◦
for 24 h. The
2
HT22 mouse hippocampal neuronal cells were purchased from the
indicated concentrations of compounds 1, 3, 4, 5, and 6 were added to
96-well plates, and 0.5% dimethyl sulfoxide (DMSO) was used for con-
trol. After 2 h, cells were treated with glutamate (5 mM) and incubated
for 8 h. After incubation, media were replaced with serum-free ones.
Korean Cell Line Bank (Seoul, South Korea) and were cultivated in low-
glucose Dulbecco’s modified eagle’s medium (DMEM) with 10% fetal
◦
bovine serum (FBS) and penicillin/streptomycin (100 U/mL) at 37
C
′
′
under 5% CO
2
with humidified air. Cell viability was determined by a 3-
Cells were treated with 2 ,7 -dichlorodihydrofluorescein diacetate (5
M) for staining. After 30 min, serum-free media (50 L, twice) were
added for washing, and cells were analyzed by an Operetta image
(
4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
μ
μ
3
assay. HT22 cells were plated in 96-well plates at a density of 3 × 10
5

J. Kwon et al.
Bioorganic Chemistry 114 (2021) 105112
Fig. 5. NOESY (A) and HMBC (B) correlations for compound 1 and measured and calculated ECD spectra of compound 1 (C).
analysis system.
2.11. Statistical analysis
4
R28 cells were added in 96-well plates at 1 × 10 cells/well and
◦
incubated at 37 C under 5% CO
2
for 24 h. Cells were treated with
Statistical differences were evaluated using one-way ANOVA fol-
lowed by Tukey-HSD post hoc test for multiple group comparisons.
indicated concentrations of five compounds, followed by the treatment
of glutamate/BSO after 2 h. Incubation and image analysis of R28 cells
were carried out under the same conditions as in the case of HT22 cells.
3. Results and discussion
+
2
.10. Inhibitory activity against glutamate-induced Ca2 influx
3.1. Antioxidative and neuroprotective activities of S. tora seed and
sprout
3
HT22 cells were added in 96-well plates at 3 × 10 cells/well and
◦
incubated at 37 C under 5% CO
2
for 24 h. Cells were treated with
The antioxidative activities of the extracts from S. tora seeds and
sprouts were evaluated as preliminary screening using DPPH and online
HPLC-ABTS assays. The extract from the sprouts was found to be
approximately 2 times more active than that from the seeds in the DPPH
assay (Fig. 1A), and the online HPLC-ABTS assay showed similar results
(Fig. 1B). From the comparison between chromatograms of the extracts
from the sprouts and seeds, it was inferred that this was due to the newly
produced or increased amounts of phenolic compounds that were eluted
after 15 min (Fig. 1B). To investigate whether these compounds were
changed or stably produced during germination and growth processes,
sprouts were cultivated by controlling light conditions (fluorescent
lamp, 385 nm LED, 465 nm LED, 645 nm LED, and 780 nm LED) for
seven days. No distinct effects of these light conditions on producing
target compounds were observed. Hence, the dark condition was
indicated concentrations of compounds 1, 3, 4, 5, and 6, and 0.5%
DMSO was used for control. After 2 h, cells were treated with glutamate
(
5 mM) and incubated for 8 h, followed by the replacement of media
with HBSS and the addition of Flu-3 (2 M) for staining. After 15 min,
HBSS (50 L, twice) was added for washing, and cells were analyzed by
an Operetta image analysis system.
μ
μ
4
R28 cells were added in 96-well plates at 1 × 10 cells/well and
◦
incubated at 37 C under 5% CO
2
for 24 h. Cells were treated with five
compounds, followed by the treatment of glutamate/BSO after 2 h. In-
cubation and image analysis of R28 cells were performed under the same
conditions as in the case of HT22 cells.
6

J. Kwon et al.
Bioorganic Chemistry 114 (2021) 105112
Fig. 6. Neuroprotective effects of all isolated compounds against glutamate-induced oxidative stress in HT22 (A) and R28 (B) cells.
+
selected for large cultivation since these compounds were the most
produced (Fig. S1). Then an evaluation of the neuroprotective effects
was carried out, which was closely related to the antioxidative activity.
The neuroprotective effects of the extracts from S. tora seeds and
sprouts, against glutamate-induced oxidative stress in HT22 cells and
glutamate/BSO-induced oxidative stress in R28 cells, were evaluated.
The extract from the sprouts was found to be more active than that from
the seeds in both cells (Fig. 2).
unsaturation, based on the peak at m/z 665.1860 [M + H] in the
HRESIMS. In particular, the fragments in the positive mode at m/z 503,
489, 461, and 272 suggested the presence of glucose and additional
naphthalene or naphthopyrone derivatives, including compound 6. The
ꢀ 1
IR spectrum suggested that hydroxy groups (3387 cm ), carbonyl
ꢀ 1
groups (1622 and 1650 cm ), and aromatic rings (2940 and 1583
cm ) were present. The 1H NMR data (Table 1) suggested the charac-
ꢀ 1
teristic pattern of a naphthopyrone skeleton, which exhibited five
methine signals (δ
olefinic regions, three methyl signals (δ
methoxy signal (δ 3.75). Furthermore, the signals for a typical
β-glucose moiety were observed (δ 5.12, 3.98, 3.78, 3.58, 3.55, 3.51,
and 3.48). The C NMR data (Table 1) revealed the presence of three
methyl carbons, one methoxy carbon, one methylene carbon, 10
methine carbons, including one olefinic, four aromatic, and five
oxygenated carbons, two carbonyl carbons, eight oxygenated tertiary
carbons, and nine quaternary carbons. The complete assignment of these
NMR data demonstrated the structure of 1 as consisting of isolated 6 and
H
7.18, 6.97, 6.92, 6.34, and 6.12) in the aromatic and
H
2.63, 2.41, and 1.98), and a
3
.2. Phytochemical investigation
H
H
To explore these phenolic constituents, various chromatographic and
13
spectroscopic methods for separation, isolation, and structural elucida-
tion were applied. Two new naphthopyrone glycosides (1 and 2) were
obtained from the EtOAc layer of EtOH extract of S. tora sprouts, along
with 27 known compounds, namely isotoralactone (3) [20], toralactone
(
(
4) [21], torosachrysone (5) [20], rubrofusarin (6) [22], cassialactone
7) [23], torachrysone-8-O-β-glucopyranoside (8) [24], cassialactone-9-
1
6 units, and the presence of two quaternary carbon signals at C-7 and C-
O-β-glucopyranoside (9) [25], cassiasides A–C (10–12) [21,26], deme-
thylflavasperone-10-O-β-glucopyranoside (13) [27], rubrofusarin-6-O-
β-gentiobioside (14) [21], 1-[(3-methoxyl-8-hydroxyl-1-O-β-glucopyr-
anosylnaphthalen)-6-yl]propan-2-one (15) [28], 6-hydroxymusizin-8-
O-β-glucopyranoside (16) [24], nepalenside A (17) [29], quercetin (18)
′
4
(δ
C
111.9 and 120.6) suggested that these components were unsym-
′
metrically dimerized through a 7–4 linkage. This dimerization was
further evidenced by the NOESY correlation of H-11 and 8-OCH
′
3
(
Fig. 5A). The position of the β-glucose moiety was deduced to be
′
′′
′
attached at C-8 according to a HMBC cross-peak of H-1 and C-8 (δ
C
[
30], 7-O-methylkaempferol (19) [31], 7-O-methylquercetin (20) [32],
1
56.1), and the comprehensive interpretation of the 2D NMR data,
including HMBC correlation (Fig. 5B), confirmed the planar structure of
. The β-glucose unit was determined to have a D-form by comparison
quercetin-3-O-β-glucopyranoside (21) [30], quercetin-3-O-neo-
hesperidoside (22) [33], kaempferol-3-O-neohesperidoside (23) [34],
chrysophanol (24) [35], physcion (25) [35], aurantio-obtusin (26) [36],
1
with an authentic standard sample via the HPLC method, conducted as
referenced in a previous report (Fig. S15, Supplementary material) [38].
The axial configuration between the two components was determined
using the exciton chirality method using ECD. The ECD spectra of 1
′
chryso-obtusin (27) [36], chrysophanol-10,10 -bianthrone (28) [22],
and emodin-physcion bianthrone (29) [37] by comparing the NMR and
MS data with data reported in the literature (Fig. 3 and S17, Supporting
Information) (see Fig. 4).
showed a positive Cotton effect (CE) at 293 nm (Δ
ε + 4.2) and a negative
Compound 1 was isolated as a yellowish and amorphous solid having
CE at 261 nm (Δ –0.5). This positive exciton chirality was due to
ε
32
the molecular formula C34H O14, consistent with 19 degrees of
7

J. Kwon et al.
Bioorganic Chemistry 114 (2021) 105112
Fig. 7. Neuroprotective effects of compounds 1 and 3–6 against glutamate-induced oxidative stress in HT22 cell (*P < 0.05, **P < 0.01 and ***P < 0.001) (A) and
against glutamate-induced oxidative stress in R28 cell (*P < 0.05, **P < 0.01 and ***P < 0.001) (B).
couplings between 1B
transitions of the naphthopyrone chromophores
glucopyranoside.
b
and demonstrated that the two axes are configured in a clockwise screw,
suggesting an (aS)-configuration. This result was supported by the ECD
data of previously reported naphthopyrones [39]. Furthermore, the ECD
calculation method using two possible models (aS and aR) was applied
to clarify our result. The calculated ECD data of the aS model was closely
fitted well with the experimental ECD data (Fig. 5C). Consequently,
30
Compound 2 was found to have the molecular formula C26H O14 via
HRESIMS. The complete assignment of the 1D NMR data (Table 1)
demonstrated its structure to be superimposable on that of 13, except for
the presence of an additional β-apiose moiety. The position of the
′
β-apiose moiety was deduced to be connected at C-6 of a β-glucose
1
3
moiety, which was indirectly suggested by the downfield shift of the
C
′
′
compound 1 was assigned as 7-hydroxymusizinyl-rubrofusarin-8 -O-β-D-
NMR signal at C-6 (δ
C
68.9). Both sugar units were demonstrated to
8

J. Kwon et al.
Bioorganic Chemistry 114 (2021) 105112
2
+
Fig. 8. Inhibitory effects of compounds 1 and 3–6 against glutamate-induced intracellular ROS generation (A) and Ca influx (B) in HT22 cell (*P < 0.05, **P <
.01, ***P < 0.001, and ****P < 0.0001).
0
2
+
Fig. 9. Inhibitory effects of compounds 1 and 3–6 against glutamate-induced intracellular ROS generation (A) and Ca influx (B) in R28 cell (*P < 0.05, **P < 0.01,
**P < 0.001, and ****P < 0.0001).
*
possess a D-form according to the same HPLC method (Fig. S15, Sup-
apiofuranoside.
plementary material). Consequently, compound 2 was assigned as
′
demethylflavasperone-10-O-β-D-glucopyranosyl-6 -O-β-D-
9

J. Kwon et al.
Bioorganic Chemistry 114 (2021) 105112
3
.3. Antioxidative and neuroprotective activities of the isolated
treatment of compounds 3 and 6 showed potent inhibitory effects on
2
+
compounds
Ca influx. Even though 50
exhibited potent inhibitory effects to glutamate-mediated Ca influx,
5.6 M of these compounds showed no activities (Fig. 9B). As a result,
μM and 16.7
μ
M of compounds 4 and 5
2
+
The antioxidative activities of all obtained compounds were rapidly
μ
evaluated using an online HPLC-ABTS assay with UV (254 nm, positive
cell viability results of compounds 3, 4, 5, and 6 in R28 cells correlated
+
2+
signal) and ABTS quenching (734 nm, negative signal) chromatograms.
with ROS generation’s inhibitory effects and Ca influx.
Compounds 1, 3–8, 10, 12, 14–16, and 18–22 were found to be active
due to showing relatively strong signals, whereas the others exhibited
weak or even no activities (Fig. S16, Supplementary material). Besides,
all obtained compounds were assessed for their neuroprotective effects
against glutamate-induced oxidative stress in HT22 cells and glutamate/
BSO-induced oxidative stress in R28 cells, with N-acetyl-L-cysteine
4. Conclusion
Our initial findings demonstrated that the extracts of S. tora sprouts
cultivated in the dark condition possess improved biological activities
than those of seeds. These results were due to the creation of new con-
stituents or the increased amount of existing phenolic compounds.
Hence, we identified most constituents in the HPLC chromatogram and
found the most potent neuroprotective antioxidants of S. tora (1 and
3–6). In particular, only a little has been reported on the dimerized form
of naphthopyrone and naphthalene derivatives. This is the first report
that dimerized compounds can have activities and be produced in
S. tora. Although we found that some active compounds could prevent
intracellular ROS generation and Ca2 influx, further and more exten-
sive studies, including mechanistic research, might be needed to develop
S. tora sprouts and their constituents as valuable neuroprotective natural
products.
(
NAC), used as a positive control. HT22 cells were treated with gluta-
mate (5 mM) in the absence or presence of various concentrations of the
compounds. Although cell viability was decreased to 45.7% after
exposure to glutamate, compounds 1, 3–6, 13, 16, 18, and 25 increased
the viability to over 92.1% at the lowest concentration (5.6
μ
M) of the
screening system, while NAC (1 mM) restored the viability to 101.0%
(
(
Fig. 6A). Also, R28 cells were treated with glutamate (10 mM)/BSO
0.5 mM) in the absence or presence of the compounds. Despite the
+
reduction of the viability to 68.9%, due to excitotoxicity induced by
glutamate/BSO, compounds 1, 3–6, and 24 increased cell viability to
over 88.3% at 5.6 M, whereas 1 mM of NAC restored the viability to
μ
9
4.8% (Fig. 6B). Taken together, compounds 1 and 3–6 were found to be
active in both cells (Table S1, Supplementary material) and were further
analyzed for their neuroprotective potentials using a six-point serial
Declaration of Competing Interest
concentration. An amount of 16.7
as 50.0 M of 6, exhibited more than a 105.1% neuroprotective effect in
HT22 cells (Fig. 7A). Also, 16.7 M of compounds 1, 3, and 5, as well as
0.0 M of 4 and 6, showed a greater than 84.5% neuroprotective effect
μM of compounds 1 and 3–5, as well
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
μ
μ
5
μ
in R28 cells, based on their restoration of cell viability (Fig. 7B).
The above results demonstrate that compounds 1 and 3–6, derived
from sprouts with newly produced or an increased amount of naph-
thopyrones, were commonly active in the three experiments, and can
protect neuronal HT22 and R28 cells via their antioxidative activities.
Although the exact structure–activity relationship could not be
demonstrated, in general, it was presumed that the activities of the
isolated naphthopyrones could be increased by decreasing the number
of sugar units, according to a comparison of the activities between
compounds 2 and 13, compounds 4 and 12, compounds 6, 11, and 14,
and compounds 7 and 9.
Acknowledgements
The research was financially supported by the Korea Institute of
Science
& Technology Program (2E31300) and the KHU-KIST
Converging Science and Technology - Joint Research Program.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.105112.
3
.4. Inhibitory activities of the isolated compounds against glutamate-
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