New bis-thioglycosyl-1,1′-disulfides from Nasturtium officinale R. Br. and their anti-neuroinflammatory effect

Article abstract of DOI:10.1016/j.bioorg.2019.01.062

As a part of our continuing search for bioactive constituents from Brassicaceae family, three new bis-thioglycosides (1–3) were isolated from the 80% MeOH extract of Nasturtium officinale, together with 13 known compounds (4–16). The chemical structures of three new bis-thioglycosides (1–3) were elucidated using NMR techniques (¹H and ¹³C NMR, ¹H–¹H COSY, HSQC, and HMBC), HRESIMS, and a chemical method. All the compounds were evaluated for their inhibitory effects on nitric oxide (NO) levels in lipopolysaccharide (LPS)-stimulated murine microglia BV-2 cells. Among the isolates, compound 5 exhibited a strong inhibitory effect on NO production, and compounds 4 and 15 showed moderate inhibitory activities, suggesting the neuroprotective and anti-neuroinflammatory effects of bis-thioglycosides from N. officinale.

Full text of DOI:10.1016/j.bioorg.2019.01.062

Accepted Manuscript
New bis-thioglycosyl-1,1′-disulfides from Nasturtium officinale R. Br. and their
anti-neuroinflammatory effect
Tae Hyun Lee, Zahra Khan, Lalita Subedi, Sun Yeou Kim, Kang Ro Lee
PII:
S0045-2068(18)31190-8
DOI:
https://doi.org/10.1016/j.bioorg.2019.01.062
YBIOO 2766
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
18 October 2018
14 January 2019
27 January 2019
Please cite this article as: T. Hyun Lee, Z. Khan, L. Subedi, S. Yeou Kim, K. Ro Lee, New bis-thioglycosyl-1,1′-
disulfides from Nasturtium officinale R. Br. and their anti-neuroinflammatory effect, Bioorganic Chemistry (2019),
doi: https://doi.org/10.1016/j.bioorg.2019.01.062
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New bis-thioglycosyl-1,1′-disulfides from Nasturtium officinale R. Br.
and their anti-neuroinflammatory effect
a
b,c
b,c
b,c
a,*
Tae Hyun Lee , Zahra Khan , Lalita Subedi , Sun Yeou Kim , Kang Ro Lee
a
Natural Products Laboratory, School of Pharmacy, Sungkyunkwan University, Suwon 16419,
Republic of Korea
b
Gachon Institute of Pharmaceutical Science, Gachon University, 191 Hambakmoero,
Yeonsu-gu, Incheon 21936, Republic of Korea
c
College of Pharmacy, Gachon University, 191 Hambakmoero, Yeonsu-gu, Incheon 21936,
Republic of Korea
Correspondence to:
Prof. Dr. Kang Ro Lee - Tel: 82-31-290-7710; Fax: 82-31-290-7730; E-mail: krlee@skku.edu

Abstract
As a part of our continuing search for bioactive constituents from Brassicaceae family,
three new bis-thioglycosides (1-3) were isolated from the 80% MeOH extract of Nasturtium
officinale, together with 13 known compounds (4-16). The chemical structures of three new
1
13
1
1
bis-thioglycosides (1-3) were elucidated using NMR techniques ( H and C NMR, H- H
COSY, HSQC, and HMBC), HRESIMS, and a chemical method. All the compounds were
evaluated for their inhibitory effects on nitric oxide (NO) levels in lipopolysaccharide (LPS)-
stimulated murine microglia BV-2 cells. Among the isolates, compound 5 exhibited a strong
inhibitory effect on NO production, and compounds 4 and 15 showed moderate inhibitory
activities, suggesting the neuroprotective and anti-neuroinflammatory effects of bis-
thioglycosides from N. officinale.
Keywords: Nasturtium officinale, Brassicaceae, Bis-thioglycosides, Anti-neuroinflammation,
Microglia.

1
. Introduction
Chronic activation of microglia leading to excessive production of Nitric oxide (NO) can
induce the inflammatory signal which can further induce the oxidative stress like the
production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) that can
cause neuronal cell death and neurodegeneration [1,2]. These processes are selectively
involved in the pathologies of multiple neurodegenerative disorders like Parkinson’s disease
(
PD), amyotrophic lateral sclerosis (ALS), stroke/excitotoxicity, multiple sclerosis (MS), and
Alzheimer’s disease (AD) [3]. Finding the active phytochemicals that inhibit the NO
production could be a better alternative for the management of the aforementioned disease
conditions.
Nasturtium officinale R. Br. (Brassicaceae) is a hardy perennial herb native to Europe,
and commonly called as “Watercress” or “Cresson”, which is one of the brassica vegetables,
such as broccoli, cabbage, wasabi, and horseradish. The plant has aroused interest because of
the presence of sulfur-containing compounds and their important role in protecting human
health [4,5]. The characteristics mentioned above are speculated to be due to the some kinds
of sulfur compounds deriving from myrosinase-catalyzed hydrolysis of glucosinolates in
Brassicaceae family [6]. Glucosinolates could exert neuroprotective activity by regulating
inflammatory responses in the central nervous system [7,8]. The extract of N. officinale not
only possesses a variety of biological activities, including anti-inflammatory, antioxidant,
anticancer, hepatoprotective, and anti-hypercholesterolemia and anti-hyperlipidemia effects
[
[
9,10]. In our continuing efforts to study the bioactive constituents from Brassicaceae plants
11,12], we investigated MeOH extract of N. officinale, which was resulted in the isolation
and characterization of three new bis-thioglycosides (1-3) along with 13 known compounds
4-16). The structures of the new bis-thioglycosides (1-3) were elucidated by conventional
(

1
13
1
1
NMR ( H and C NMR, H- H COSY, HSQC, and HMBC), HRESIMS and a chemical
method. All the compounds (1-16) were evaluated for their potential inhibitory effects on
nitric oxide levels in LPS-induced murine microglia BV-2 cells.
2
. Experimental
2
.1 General experimental procedures
Optical rotations were measured on a JASCO P-1020 polarimeter. Infrared (IR) and
ultraviolet (UV) spectra were recorded on a Bruker IFS-66/S FT-IR spectrometer and a
Shimadzu UV-1601 UV visible spectrophotometer, respectively. NMR spectra were recorded
1
13
on a Bruker AVANCE III 700 MNR spectrometer at 700 MHz ( H) and 175 MHz ( C) with
chemical shifts given in ppm (δ). HRESIMS was acquired on a Waters SYNAPT G2 Q-TOF
mass spectrometer. LC/MS analysis was performed on an Agilent 1200 Series HPLC system
equipped with a diode array detector and 6130 Series ESI mass spectrometer using an
analytical Kinetex C18 100 Å column (100 x 2.1 mm, 5 µm; flow rate 0.3 mL/min;
Phenomenex, Torrance, CA, USA). The semi-preparative high performance liquid
chromatography (HPLC) was conducted utilizing systems of a Gilson 306 pump with a
Shodex refractive index detector. Low-pressure liquid chromatography (LPLC) was
performed over a LiChroprep Lobar-A Si 60 (240 x 10 mm) column with an FMI QSY-0
pump (ISCO). Open column chromatography was implemented with silica gel 60 (70-230
and 230-400 mesh; Merck, Darmstadt, Germany) and RP-C18 silica gel (230-400 mesh,
®
Merck, Germany). Ion exchange resin (Dowex 50WX8 hydrogen form, Sigma-Aldrich) was

used for alkali elimination. Thin layer chromatography (TLC) was carried out with pre-coated
silica gel F254 plates and reversed-phase (RP)-C18 254s plates. Spots were detected on TLC
under UV light or by heating after spraying with anisaldehyde-sulfuric acid.
F
2
.2 Plant Material
N. officinale was collected in Namyangju-si, Gyeonggi-do, Republic of Korea in October
2
014, and the plant was identified by one of the authors (K.R.Lee). A voucher specimen
(
SKKU-NPL 1408) was deposited in the herbarium of the School of Pharmacy,
Sungkyunkwan University, Suwon, Republic of Korea.
2
.3. Extraction and isolation
The whole plant (3.7 kg) was extracted three times with 80% aqueous MeOH at room
temperature and filtered. The MeOH extract (350 g) was suspended in distilled water (2.4 L)
and successively partitioned with hexane, CHCl , EtOAc, and BuOH, yielding 34, 7, 5, and
0 g of dried organic extracts, respectively. The BuOH soluble layer was chromatographed
over a normal-phase silica gel column chromatography eluting with mixtures of CHCl
MeOH-H O (7:1:0.1, 5:1:0.1, 3:1:0.1, and 1:1:0.1) to obtain six fractions (A-F). Fraction B
1.2 g) was applied to an RP-C18 silica gel column chromatography eluting with 20% aqueous
3
3
3
-
2
(
MeOH to give seven subfractions (B1-B7). Compounds 11 (30 mg) and 12 (5 mg) were
purified from the subfraction B4 (78 mg), using semi-preparative reversed-phase silica gel
HPLC eluting with an isocratic mixture of 27% aqueous MeOH (flow rate of 2.0 ml/min).
The subfraction B5 (111 mg) was further separated into three subfractions (B51-B53) by a
Lobar-A Si 60 (240 x 10 mm) column (CHCl
3
-MeOH-H O, 7:1:0.1). The subfraction B52 (68
2

mg) was purified by semi-preparative reversed-phase silica gel HPLC (28% aqueous MeOH,
flow rate of 2.0 ml/min), yielding compound 13 (10 mg). The subfraction B6 (198 mg) was
loaded on a Lobar-A Si 60 (240 x 10 mm) column (CHCl
3
-MeOH-H O, 6:1:0.1) to obtain
2
five subfractions (B61-B65). Compound 2 (6 mg) was purified from the subfraction B64 (34
mg) with semi-preparative reversed-phase silica gel HPLC (38% aqueous MeOH, flow rate
of 2.0 ml/min). Fraction C (1.4 g) was separated over an RP-C18 silica gel column (230-400
mesh) eluting with 25% aqueous MeOH to yield six subfractions (C1-C6). The subfraction
C4 (488 mg) was subjected to separation on a silica gel (CHCl
3
-MeOH-H O, 3.5:1:0.1 →
2
1
:1:0.1) to afford five subfractions (C41-C45). The subfraction C42 (35 mg) was purified by
semi-preparative reversed-phase silica gel HPLC (47% aqueous MeOH, flow rate of 2.0
ml/min) to acquire compound 14 (13 mg). Compounds 1 (10 mg) and 3 (8 mg) were purified
from the subfraction C5 (136 mg) using semi-preparative reversed-phase silica gel HPLC (47%
aqueous MeOH, flow rate of 2.0 ml/min). Fraction D (2.6 g) was further divided into eight
subfractions (D1-D8) by passage over an RP-C18 silica gel column chromatography (230-400
mesh) eluting with 25% aqueous MeOH. Compounds 4 (3 mg), 15 (6 mg), and 16 (5 mg)
were purified from the subfraction D2 (51 mg), using semi-preparative reversed-phase silica
gel HPLC (24% aqueous MeOH, flow rate of 2.0 ml/min). The CHCl
3
soluble layer was
subjected to a silica gel open column (hexane-CHCl -MeOH, 10:2:1 → 1:1:1), which resulted
3
in six subfractions (A-F). The subfraction C (1.0 g) was isolated using an RP-C18 silica gel
column chromatography eluting with 50% aqueous MeOH to give eight subfractions (C1-C8).
The subfraction C1 (485 mg) was separated on a normal-phase silica gel column
chromatography (CHCl -MeOH, 60:1 → 1:1) to yield five subfractions (C11-C15).
3
Compounds 7 (18 mg) and 8 (6 mg) were purified from the subfraction C11 (36 mg), using
semi-preparative reversed-phase silica gel HPLC with 40% aqueous MeOH (flow rate of 2.0
ml/min). Fraction D (1.1 g) was implemented to an RP-C18 silica gel column chromatography

eluting with 45% aqueous MeOH to obtain seven subfractions (D1-D7). The subfraction D1
217 mg) was further separated on a silica gel column chromatography (CHCl -MeOH, 60:1
1:1), which resulted in five subfractions (D11-D15). The subfraction D12 (85 mg) was
(
3
→
purified by semi-preparative reversed-phase silica gel HPLC with 40% aqueous MeOH (flow
rate of 2.0 ml/min), yielding compounds 9 (5 mg), 10 (61 mg), and 11 (8 mg). The EtOAc
soluble layer (4.0 g) was separated over a silica gel column chromatography (hexane-EtOAc,
7
:1, 5:1, 3:1, and 1:1) to acquire six fractions (A-F). Fraction B (2.1 g) was subjected to an
RP-C18 silica gel column chromatography eluting with 53% aqueous MeOH to acquire ten
sub-fractions (B1-B10). The subfraction B5 (487 mg) was applied to a silica gel column
chromatography (CHCl
B56). Compound 5 (5 mg) was isolated from the subfraction B52 (69 mg) by Sephadex LH-
0 column chromatography (100% MeOH) and purified with semi-preparative reversed-
3
-MeOH-H O, 5:1:0.1 → 1:1:0.1) to afford six subfractions (B51-
2
2
phase silica gel HPLC with an isocratic solvent system of 25% aqueous acetonitrile (flow rate
of 2.0 ml/min).
2
.3.1. 6-O-Hydrocinnamoyl-bis(1-deoxy-1-thio-β-
D
-glucopyranosyl)-1,1′-disulfide (1)
Yellowish gum; ꢀꢁ ꢃꢂ ꢄ -11.8 (c = 1.0, MeOH); IR (KBr) vmax 3726, 3336, 2941, 2830,
-1
1
1
553, 1445, 1108, 1022 cm ; UV (MeOH) λmax (log ε) 212, 337; H MNR (700 MHz) data,
1
3
see Table 1; C NMR (175 MHz) data, see Table 2; HRESIMS (positive-ion mode) m/z:
+
5
45.1119 [M + Na] (calcd. for C21
H
30
O
11
2
S , 545.1127).
2
.3.2. 3-O-Hydrocinnamoyl-bis(1-deoxy-1-thio-β-
D
-glucopyranosyl)-1,1′-disulfide (2)
Yellowish gum; ꢀꢁ ꢃꢂ ꢄ -4.2 (c = 1.0, MeOH); IR (KBr) vmax 3763, 3370, 2944, 2832,

-1
1
1
707, 1449, 1118, 1025 cm ; UV (MeOH) λmax (log ε) 214, 341; H MNR (700 MHz) data,
1
3
see Table 1; C NMR (175 MHz) data, see Table 2; HRESIMS (positive-ion mode) m/z:
+
5
45.1133 [M + Na] (calcd. for C21
H
30
O
11
2
S , 545.1127).
2
.3.3. 2-O-Hydrocinnamoyl-bis(1-deoxy-1-thio-β-
D
-glucopyranosyl)-1,1′-disulfide (3)
Yellowish gum; ꢀꢁ ꢃꢂ ꢄ -17.8 (c = 1.0, MeOH); IR (KBr) vmax 3722, 3368, 2945, 2830,
-1
1
1
390, 1029 cm ; UV (MeOH) λmax (log ε) 210, 338; H MNR (700 MHz) data, see Table 1;
1
3
C NMR (175 MHz) data, see Table 2; HRESIMS (positive-ion mode) m/z: 545.1119 [M +
+
Na] (calcd. for C21
H
30
O
11
2
S , 545.1127).
2
2
.3.4. β-
D
-Glucopyranoside-6-O-β- -glucopyranosyl-1-[3-(4-hydroxy-3,5-dimethoxyphenyl)-
D
propanate] (15)
Yellowish gum; ꢀꢁ ꢃꢂ ꢄ -1.9 (c = 1.0, MeOH); IR (KBr) νmax 3346, 2943, 2831, 1682,
-1
1
1
590, 1419, 1033 cm ; UV (MeOH) λmax (log ε) 211, 239, 329 nm; H MNR (700 MHz) data,
1
3
see Table 1; C NMR (175 MHz) data, see Table 2; ESIMS (positive-ion mode) m/z: 549 [M
+
+
H] .
2
.4. Alkaline hydrolysis of compounds 1, 2, and 3
Compound 1 (1.0 mg) was treated with 0.1 N KOH in H
2
O at room temperature for 1h.
®
The hydrolysate was passed through an ion exchange column (Dowex 50WX8 hydrogen
form, Sigma-Aldrich) to remove KOH, and partitioned with CHCl
3
and H O (1 ml, 3 times).
2

1
a (0.4 mg) and 1b (0.3 mg) were obtained from the H
2
O and CHCl
3
layers, respectively,
which were identified as bis(1-deoxy-1-thio-β-
D
-glucopyranosyl)-1,1′-disulfide (1a) [13] and
1
hydrocinnamic acid (1b) [14] by H NMR and MS data. Compounds 2 and 3 were also
hydrolyzed by using aforementioned method and identified by MS data and co-TLC (CHCl
3
-
MeOH-H O, 2:1:0.1) with 1a and 1b.
2
2
.5. Measurement of nitric oxide production and cell viability in LPS-stimulated BV-2 cells
Murine microglia BV-2 cells were maintained in Dulbecco’s modified Eagle’s medium
(
(
DMEM) supplemented with 5% fetal bovine serum (FBS) and 1% penicillin-streptomycin
PS). After 24h, the BV-2 cells were pretreated with compounds (1-16) for 30 min and
stimulated with 100 ng/mL LPS for another 24 h. Nitrite, a soluble oxidation product of NO,
was measured in the culture media by the Griess reaction. The supernatant (50 µL) was
harvested and mixed with an equal volume of Griess reagent (1% sulfanilamide, 0.1% N-1-
naphthylethylenediamine dihydrochloride in 5% phosphoric acid). The absorbance at 540 nm
was measured after 10 min using a microplate reader. Cell viability was assessed by 3-[4,5-
G
dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay. N -monomethyl-
L
-
arginine (NMMA, Sigma, St, Louis, MO, USA) that is a well-known nitric oxide synthesis
NOS) inhibitor was tested as a positive control [15].
(

OR1
O
6
R1
A
H
H
H
R2
H
A
H
H
R3
H
H
A
H
2′′
7′′
4
HO 2′
1′
5
3′
1′′
HO
O 1
1
2
3
3′′
4′′
S
OH
OH
9′′
O
8
′′
R O
2
2
S
3
O
A
=
OR3
5′
4′
6′′
6
′
5
′′
4
(=1a)
OH
hydrocinnamoyl group (1b)
OCH3
CN
S
HO
OH
OH
O O
S
O O
S
N
O
N
N
H
OH
5
6
7
O
S
O
S
O
S
N
N
N
8
9
10
O
O
H CO
3
H CO
3
H CO
OH
H
3
O
OH
O
OH
OH
O
O
O
O
HO
HO
OH
OH
OCH3
OCH3
OH
OCH3
OH
HO
HO
HO
HO
1
1
12
13
OH
O 1′′
HO
HO
OH
O
OH
O
7
OH
HO
HO
2
O 1′
H CO
OR1
3
1
HO
O
O
9
O
HO
3
O
OCH3
OR2
8
O
OH
4
6
O
HO
OH
5
HO
HO
OCH3
R1
Sinapoyl Sinapoyl
Sinapoyl
R2
14
5
Sinapoyl group
1
6
1
H
Fig. 1. Structures of compounds 1-16.

3
. Results and discussion
3
.1. Structure elucidation of isolated compounds
Compound 1 was obtained as a yellowish gum. The molecular formula of 1 was
+
determined as C21
HRESIMS spectra (calcd. for C21
displayed the presence of hydrocinnamic acid moiety [14] [δ
H
30
O
11
S
2
based on the positive ion peak at m/z: 545.1119 [M + Na] in the
1
13
H
30
O11NaS
2
, 545.1127). The H and C spectra of 1
7.17 (2H, m, H-3′′ and H-5′′),
.13 (2H, m, H-2′′ and H-6′′), 7.07 (1H, t, J =7.2 Hz, H-4′′), 2.85 (2H, t, J=7.6, H-7′′), and
.59 (2H, t, J=7.7, H-8′′); δ 174.7 (C-9′′), 142.1 (C-1′′), 129.6 (C-2′′ and C-6′′), 129.7 (C-3′′
4.31 (1H, d, J=9.7
Hz, H-1), 3.45 (1H, t, J=9.2 Hz, H-2), 3.30 (1H, d, J=8.4 Hz, H-3), 3.20 (1H, m, H-4), 3.37
1H, dd, J=6.2, 2.0 Hz, H-5), 4.32 (1H, dd, J=12.0, 2.0 Hz, H-6a), 4.10 (1H, dd, J=12.0, 6.2
H
7
2
C
and C-5′′), 127.4 (C-4′′), 37.0 (C-7′′), and 32.1(C-8′′)], and two sugars [δ
H
(
Hz, H-6b), 4.31 (1H, d, J=9.4 Hz, H-1′), 3.34 (1H, t, J=9.1 Hz, H-2′), 3.27 (1H, d, J=8.9 Hz,
H-3′), 3.21 (1H, m, H-4′), 3.23 (1H, m, H-5′), 3.78 (1H, m, H-6′a), and 3.59 (1H, dd, J=11.9,
5
6
.2 Hz, H-6′b); δ
C
91.67 (C-1), 73.08 (C-2), 79.58 (C-3), 71.43 (C-4), 79.72 (C-5), 65.02 (C-
), 91.31 (C-1′), 73.43 (C-2′), 79.51 (C-3′), 71.25 (C-4′), 82.52 (C-5′), and 62.99 (C-6′)]. The
3
3
large coupling constants [9.7 ( JH-1/H-2) and 9.4 ( JH-1′/H-2′) Hz] of anomeric proton signals of
two sugars implied that 1 was to be thioglycoside and β-form [11]. Two sugars were to be
connected through a disulfide bond on the basis of the anomeric carbon chemical shifts (δC-1
9
1.7 and δC-1′ 91.3) [16]. Thus, 1 was suggested as a bis-thioglycoside containing the
hydrocinnamic acid. The position of hydrocinnamic acid in sugar moiety was identified to be
at C-6 based on the HMBC cross-peak between H-6 [δ 4.32 (1H, dd, J=12.0, 2.0 Hz, H-6a)
and 4.10 (1H, dd, J=12.0 Hz, 6.2, H-6b)] to C-9′′ [δ 174.7] (Fig. 2). Thus, the structure of 1
was inferred as 6-O-hydrocinnamoyl-bis(1-deoxy-1-thio-β- -glucopyranosyl)-1,1′-disulfide.
H
C
D

Alkaline hydrolysis of 1 afforded both bis(1-deoxy-1-thio-β-
D
-glucopyranosyl)-1,1′-disulfide
1
(
1a) [13] and hydrocinnamic acid (1b) [14], which were verified by their H NMR and MS
data. Therefore, the structure of 1 was determined as 6-O-hydrocinnamoyl-bis(1-deoxy-1-
thio-β- -glucopyranosyl)-1,1′-disulfide.
D
Compound 2 was purified as a yellowish gum and identified the same molecular formula,
+
C
21
H
30
O
11
S
2, as 1 based on the positive ion peak at m/z: 545.1133 [M + Na] in the HRESIMS
1
13
spectra (calcd. for C21
were very similar to 1 with the main differences in signals of down-field shifted proton at H-3
4.90, 2; δ 3.30, 1) and up-field shifted carbon at C-6 (δ 62.7, 2; δ 65.0, 1), suggesting
that hydrocinnamic acid (1b) was to be attached in the other position of 1a. The HMBC
correlation of H-3 [δ 4.90 (1H, t, J=9.3 Hz, H-3)] with C-9′′ [δ 174.6] assured that the
hydrocinnamic acid (1b) was located at C-3 of 1a (Fig. 2). Thus, the structure of 2 was
determined as 3-O-hydrocinnamoyl-bis(1-deoxy-1-thio-β- -glucopyranosyl)-1,1′-disulfide.
H
30
O11NaS
2
, 545.1127). The H and C NMR spectroscopic data of 2
(
δ
H
H
C
C
H
C
D
Compound 3 was obtained as a yellowish gum. The molecular formula of 3 was also
exhibited the same molecular formula, C21
H
30
11
O S2, as 1 and identified from the positive ion
+
peak at m/z: 545.1119 [M + Na] in the HRESIMS spectra (calcd. for C21
H
30
O
11NaS ,
2
1
13
5
45.1127). Inspection of the H and C NMR spectra of 3 was suggestive of those of 1,
except for the signals of H-2 (δ 4.82, 3; δ 3.30, 1) and C-6 (δ 62.8, 3; δ 65.0, 1), which
indicated that 3 is also a positional isomer of 1. Analysis of the HMBC correlation of H-2 [δ
.82 (1H, t, J=9.6 Hz, H-2)] with C-9′′ [δ 173.7] confirmed that the hydrocinnamic acid (1b)
to be connected at C-2 of 1a (Fig. 2). Hence, the structure of 3 was determined as 2-O-
hydrocinnamoyl-bis(1-deoxy-1-thio-β- -glucopyranosyl)-1,1′-disulfide.
H
H
C
C
H
4
C
D
The thirteen reported compounds (4-16) were confirmed by comparison of their recorded
spectroscopic data as bis(1-deoxy-1-thio-β- -glucopyranosyl)-1,1′-disulfide (4) [13], indole-
D

3
-acetonitrile-4-methoxy-2-S-β-
6) [18], 9-(methylsulfonyl)nonanenitrile (7) [18], 7-(methylsulfinyl)heptanenitrile (8) [18],
-(methylsulfinyl)heptanenitrile (9) [18], 9-(methylsulfinyl)heptanenitrile (10) [18], syringin
11) [19], sinapic aldehyde 4-O-β- -glucopyranoside (12) [20], 1-sinapoyl-β-
glucopyranoside (13) [21], 1,2-di-O-E-sinapoyl-β-gentiobiose (14) [22], β-
glucopyranoside-6-O-β-
15) [23], and lycibarbarphenylpropanoid C (16) [24]. The full assignments of H and
NMR data of 15 are first reported in this study (Tables 1 and 2).
D
-glucopyranoside (5) [17], 8-(methylsulfonyl)octanonitrile
(
8
(
D
D
-
D
-
D
-glucopyranosyl-1-[3-(4-hydroxy-3,5-dimethoxyphenyl)-2propanate]
1
13
(
C

O
OH
O
HO
O
HO
O
S
OH
OH
HO
O
O
O
S
HO
HO
S
OH
OH
OH
S
OH
O
OH
OH
1
2
OH
HO
O
HO
HO
O
O
S
OH
OH
S
1
1
H- H-COSY
OH
O
HMBC
3
Fig. 2. COSY (bold line) and HMBC (arrow) correlation of compounds 1, 2, and 3.

1
a
Table 1. H (700 MHz) spectral data of 1, 2, 3, and 15 in CD
3
OD ( in ppm).
Position
1
2
3
Position
15
Glc-1
4.31 (d, 9.7)
4.38 (d, 9.6)
4.52 (d, 10.0)
4.82 (t, 9.6)
3.46 (t, 8.9)
3.32 (m)
1
2
3
4
5
3.45 (t, 9.2)
3.30 (d, 8.4)
3.20, (m)
3.63 (t, 9.4)
4.90 (t, 9.3)
2
3
4
5
6.95 (s)
3.38 (t, 9.6)
3.37 (dd, 6.2, 2.0)
4.32 (dd, 12.0, 2.0)
4.10 (dd, 12.0, 6.2)
4.31 (d, 9.4)
3.34 (t, 9.1)
3.30 (dd, 5.8, 2.4)
3.79 (dd, 12.0, 1.8)
3.60 (dd, 12.1, 5.5)
4.33 (d, 9.6)
3.21 (m)
6a
3.81 (dd, 12.0, 1.8)
3.63 (dd, 12.0, 5.3)
4.33 (d, 9.6)
6
7
6.95 (s)
6d
7.75 (d, 15.8)
6.45, (d, 15.8)
Glc-1′
8
2
3
4
5
′
′
′
′
3.33 (t, 9.2)
3.41 (t, 9.2)
9
3.27 (d, 8.9)
3.21 (m)
3.27 (t, 8.7)
3.29 (m)
OCH
3
-3
-5
3.90 (s)
3.90 (s)
3.19 (m)
3.20 (m)
OCH
3
3.23 (m)
3.21 (m)
3.21 (m)
Glc-1′
2′
5.57 (d, 7.8)
3.45 (m)
6′a
3.78 (m)
3.78 (dd, 12.1, 1.8)
3.54 (dd, 12.0, 5.9)
3.76 (dd, 12.0, 1.5)
3.55 (dd, 12.0, 5.3)
6′b
3.59 (dd, 11.9, 5.2)
3′
3.36 (d, 7.8)
3.47 (m)
1
′′
′′
′′
′′
′′
′′
′′
′′
′′
4′
2
7.13 (m)
7.17 (m)
7.15 (m)
7.15 (m)
7.07 (t, 7.1)
7.15 (m)
7.15 (m)
2.86 (m)
2.61 (m)
7.15 (m)
7.15 (m)
7.07 (t, 7.1)
7.15 (m)
7.15 (m)
2.85 (m)
2.60 (m)
5′
3.61 (m)
3
4
5
6
7
8
9
6′a
6′b
Glc-1′′
2′′
4.19 (dd, 11.4, 1.9)
3.80 (dd, 11.5, 5.3)
4.36 (d, 7.8)
7.07 (t, 7.2)
7.17 (m)
7.13 (m)
3.23 (dd, 9.1, 7.9)
3.30 (d, 8.7)
2.85 (t, 7.6)
2.59 (t, 7.7)
3′′
4′′
3.48 (m)
5′′
3.27 (m)
6′′a
3.86 (dd, 11.9, 2.1)
6′′b
3.67 (dd, 12.0, 5.6)
a
1
1
δ is in ppm, and J values are in parentheses. The assignments were based on H- H COSY, HSQC,
and HMBC experiments.

13
a
Table 2. C (175 MHz) spectral data of 1, 2, 3, and 15 in CD
3
OD ( in ppm).
Position
1
2
3
Position
15
Glc-1
91.7
90.7
89.7
1
126.5
2
73.1
79.6
71.4
80.5
73.8
77.4
71.1
2
3
4
5
6
7
8
9
107.3
149.7
140.4
149.7
107.3
148.8
115.3
167.8
57.0
3
4
71.4
69.5
5
6
79.7
82.4
82.6
65.0
62.7
62.8
Glc-1′
91.3
92.4
91.8
2
3
4
5
6
′
′
′
′
′
73.4
73.6
73.3
79.7
71.5
82.6
63.1
142.1
129.5
129.7
127.4
129.7
129.5
31.9
79.5
79.7
71.3
71.4
OCH
3
-3
-5
82.5
82.8
OCH
3
57.0
63.0
63.1
Glc-1′
2′
96.0
1
2
3
4
5
6
7
8
9
′′
′′
′′
′′
′′
′′
′′
′′
′′
142.1
129.6
129.7
127.4
129.7
129.6
32.1
142.4
129.5
129.6
127.3
129.6
129.5
32.0
74.2
3′
78.0
4′
71.7
5′
78.2
6′
69.7
Glc-1′′
2′′
104.7
75.3
37.0
37.2
37.1
173.7
3′′
78.0
174.7
174.6
4′′
71.2
5
6
′′
′′
78.1
62.8
a
1
1
δ is in ppm, and J values are in parentheses. The assignments were based on H- H COSY, HSQC,
and HMBC experiments.

3
.2. Anti-inflammatory activities of the isolated compounds
In this study, we evaluted the role of the isolated compounds (1-16) in inhibiting the NO
production against LPS-activated murine microglia BV-2 cells. Among the isolates,
compound 5 significantly inhibited the production of NO in LPS-induced BV-2 cells with an
IC50 value of 21.30 µM, compared with positive control,
Additionally, compounds 4 and 15 showed moderate activities with IC50 value of 46.70 and
9.20 µM, respectively. These results make a suggestion that compound 5 exhibits better
potency than the well-known iNOS inhibitor, -NMMA.
L
-NMMA (IC50 30.06 µM).
3
L

Table 3.
Inhibitory effects of compounds 1-16 on NO production in LPS-activated BV-2 cells.
b
Cell viability (%)
a
Compound
IC (M)
5
0
2
5 M
50 M
91.01±6.72
88.89±8.15
83.44±2.76
103.92±7.72
78.32±0.48
78.72±3.09
93.93±6.97
79.30±3.9
1
2
3
4
5
6
7
8
9
6
4.89
97.09±2.13
89.80±1.31
85.02±3.20
100.51±6.82
87.24±0.90
90.79±7.09
99.64±2.16
89.32±2.64
83.32±8.50
87.56±7.46
81.62±6.82
81.43±3.20
86.42±3.02
80.70±5.07
99.22±0.74
91.59±5.63
95.47±1.49
244.34
9
2.88
6.70
1.30
4
2
126.50
98.43
>
500
205.63
80.51±2.05
89.93±5.74
73.36±4.77
87.71±5.37
74.02±0.48
73.91±0.55
84.60±4.70
86.50±8.43
95.47±1.49
1
0
1
8
2.19
1
233.12
1
1
1
1
1
2
3
4
5
6
>
500
1
46
6
2.99
9.20
3
>
500
L
-NMMA
30.06
a
IC50 value of each compound was defined as the concentration (μM) that caused 50% inhibition of
b
NO production in LPS-activated BV-2 cells; cell viability after treatment with 25 and 50 μM of each
compound was determined by MTT assay and is expressed in percentage (%). The results are
averages of three independent experiments, and the data are expressed as mean ± SD.

4
. Conclusion
In the present study, three new bis-thioglycosides (1-3), together with thirteen known
compounds (4-16) were isolated from N. officinale. Moreover, we evaluated the anti-
inflammatory activity of the compounds (1-16). Anti-inflammatory assays of the compounds
(
1-16) revealed that the compound 5 exhibited significant inhibition of NO production in
LPS-activated microglia BV-2 cells with an IC50 value of 21.30 µM, and compounds 4 and 15
showed moderate anti-inflammatory activity by inhibiting the NO levels in the cells (IC50
value of 46.70 and 39.20 µM, respectively). These results are in accordance with the previous
studies which glucosinolates and sulfur-containing compounds were associated with
neuroprotective activity, and this study showed that bis-thioglycosides are an important role
for the neuroprotective and anti-neuroinflammatory effects of N. officinale.
Acknowledgments
This research was supported by the Basic Science Research Program through the National
Research Foundation of Korea (NRF), funded by the Ministry of Education, Science and
Technology (2012R1A5A2A28671860). We are thankful to the Korea Basic Institute (KBSI)
for the measurements of MS spectra.
Appendix A. Supplementary material
Supplementary data related with this paper can be found, in the online version at

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New bis-thioglycosyl-1,1′-disulfides from Nasturtium officinale R.
Br. and their anti-neuroinflammatory effect
a
b,c
b,c
b,c
a,*
Tae Hyun Lee , Zahra Khan , Lalita Subedi , Sun Yeou Kim , Kang Ro Lee

Highlights



Three new bis-thioglycosyl-1,1′-disulfides and thirteen known compounds including
seven sulfur-containing compounds were isolated from Nasturtium officinale R. Br.
The chemical constituents of the isolates were elucidated using NMR and MS
analysis.
Compound 5 exhibited significant inhibition activity of nitric oxide (NO) production
with IC50 value of 21.30 µM.