Iridoid Glycosides from the Twigs of Sambucus williamsii var. coreana and Their Biological Activities

Article abstract of DOI:10.1021/acs.jnatprod.7b00410

Six new iridoid glycosides, sambucusides A-F (1-6), and two known derivatives (7 and 8) were isolated from a methanol extract of the twigs of Sambucus williamsii var. coreana. Their chemical structures were elucidated by spectroscopic methods, including NMR (¹H and ¹³C NMR, ¹H-¹H COSY, HMQC, HMBC, and NOESY) and HRMS. All isolated compounds (1-8) were evaluated for their antiproliferative activities against four human cancer cell lines (A549, SK-OV-3, SK-MEL-2, and Bt549). Their effects on nitric oxide (NO) production in lipopolysaccharide (LPS)-activated BV-2 cells and their neuroprotective effects through induction of nerve growth factor (NGF) in C6 glioma cells were also examined. Compounds 2, 3, and 5 showed cytotoxic effects (IC₅₀ 1.3-8.7 μM) against the SK-MEL-2 and Bt549 cell lines and inhibitory effects on NO production (IC₅₀ of 0.9, 1.3, and 1.2 μM, respectively). Compounds 2, 4, and 8 exhibited NGF-releasing effects (147.0 ± 5.8%, 158.7 ± 5.2%, and 152.6 ± 7.3%, respectively).

Full text of DOI:10.1021/acs.jnatprod.7b00410

Article
pubs.acs.org/jnp
Iridoid Glycosides from the Twigs of Sambucus williamsii var. coreana
and Their Biological Activities
Won Se Suh,† Chung Sub Kim,† Lalita Subedi,‡,§ Sun Yeou Kim,‡,§ Sang Un Choi,⊥ and Kang Ro Lee*,†
†Natural Products Laboratory, School of Pharmacy, Sungkyunkwan University, Suwon 16419, Republic of Korea
‡Gachon Institute of Pharmaceutical Science, Gachon University, Incheon 21936, Republic of Korea
§College of Pharmacy, Gachon University, #191, Hambakmoero, Yeonsu-gu, Incheon 21936, Republic of Korea
⊥Korea Research Institute of Chemical Technology, Daejeon 34114, Republic of Korea
S
* Supporting Information
ABSTRACT: Six new iridoid glycosides, sambucusides A−F (1−6), and two known derivatives (7 and 8) were isolated from a
methanol extract of the twigs of Sambucus williamsii var. coreana. Their chemical structures were elucidated by spectroscopic
1
methods, including NMR (1H and 13C NMR, H−1H COSY, HMQC, HMBC, and NOESY) and HRMS. All isolated
compounds (1−8) were evaluated for their antiproliferative activities against four human cancer cell lines (A549, SK-OV-3, SK-
MEL-2, and Bt549). Their effects on nitric oxide (NO) production in lipopolysaccharide (LPS)-activated BV-2 cells and their
neuroprotective effects through induction of nerve growth factor (NGF) in C6 glioma cells were also examined. Compounds 2,
3, and 5 showed cytotoxic effects (IC50 1.3−8.7 μM) against the SK-MEL-2 and Bt549 cell lines and inhibitory effects on NO
production (IC50 of 0.9, 1.3, and 1.2 μM, respectively). Compounds 2, 4, and 8 exhibited NGF-releasing effects (147.0 5.8%,
158.7 5.2%, and 152.6 7.3%, respectively).
Sambucus williamsii var. coreana Nakai (Caprifoliaceae) is a
deciduous shrub, widely distributed throughout Korea and
mainland China. The stems and roots of this species have been
used in Korean folk medicine for treating bone fractures,
rheumatism, neuralgia, and osteoporosis.1−3 Previous phyto-
chemical investigations have led to the isolation of terpenes,
fatty acids, phenolic compounds, iridoid glycosides, and lignans
from this source.3−8 Recently, it has been reported that an
extract of S. williamsii was found to increase bone mass and
bone strength in ovariectomized rats.9 In addition, some lignans
isolated from S. williamsii can inhibit the proliferation of
osteoblast-like UMR106 cells.3 However, few studies have been
conducted to isolate potential anticancer, anti-inflammatory,
and neuroprotective components from S. williamsii var. coreana.
As part of a search for bioactive constituents from Korean
medicinal plants,10−12 the CHCl3 and EtOAc layers of a
S. williamsii var. coreana MeOH twig extract were studied based
on the cytotoxic activity against A549, SK-OV-3, SK-MEL-2,
and Bt549 cells in a sulforhodamine B (SRB) bioassay. They
also decreased nitric oxide (NO) levels in lipopolysaccharide
(LPS)-activated BV2 murine microglia cells. Some lignan
derivatives have been isolated from this plant, and their
biological activities have been reported previously.8 Herein, the
isolation of six new iridoid glycosides, sambucusides A−F (1−
6), and two known derivatives (7 and 8) and their cytotoxic,
anti-inflammatory, and neuroprotective activities are reported.
RESULTS AND DISCUSSION
■
The 80% MeOH extract from the twigs of S. williamsii var.
coreana was fractionated to yield n-hexane-, CHCl3-, EtOAc-,
and n-BuOH-soluble fractions, and then each fraction was
evaluated for inhibition of NO production in LPS-activated BV-
2 cells and a nerve growth factor (NGF) release effect in C6
glioma cells. Among them, the EtOAc-soluble fraction showed
moderate NO production inhibitory (IC50 values 72.7 μg/mL)
and significant NGF release activities (196.1 8.0%). Thus, the
most active EtOAc-soluble fraction was investigated and
Received: May 12, 2017
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.7b00410
J. Nat. Prod. XXXX, XXX, XXX−XXX

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Chart 1
1
Figure 1. H−1H COSY (bold) and HMBC (arrow) correlations of 1, 3, 4, and 6.
Figure 2. Key NOESY correlations (yellow dashed) of 1 and 6. Some protons were removed for a clearer presentation.
resulted in the isolation of six new iridoid glycosides (1−6),
together with two known iridoid derivatives (7 and 8).
ring [δH 7.48 (2H, d, J = 8.6 Hz, H-2‴ and 6‴) and 6.84 (2H,
d, J = 8.6 Hz, H-3‴ and 5‴)], a trans-olefinic moiety [δH 7.67
(1H, d, J = 16.0 Hz, H-7‴) and 6.38 (1H, d, J = 16.0 Hz, H-
8‴)], an anomeric proton of glucose [δH 4.60 (1H, d, J = 8.0
Hz, H-1″)], an isobutyl group [δH 2.21 (2H, m, H-2′), 2.06
(1H, m, H-3′), and 0.96 (6H, d, J = 6.7 Hz, H-4′, and 5′)], and
two oxymethylenes [δH 4.12 and 4.26 (each 1H, d, J = 11.5 Hz,
H-11) and 3.38 and 3.36 (each 1H, m, H-10)]. The 13C NMR
spectrum exhibited 30 carbon signals, indicating an iridoid
moiety (δC 140.9, 115.4, 91.7, 82.6, 69.9, 69.6, 46.0, 37.3, 36.3,
Compound 1 was obtained as a yellowish gum with a
negative optical rotation ([α]2D5 −121.5). Its molecular formula
was determined to be C30H40O13 based on its molecular ion
peak [M + Na]+ at m/z 631.2360 (calcd for C30H40O13Na,
631.2367) in the positive-ion HRESIMS. The IR spectrum of 1
showed the presence of hydroxy group (3450 cm−1) and ester
carbonyl group (1725 cm−1) absorption. The 1H NMR
spectrum of 1 showed signals for a 1,4-disubstituted aromatic
B
DOI: 10.1021/acs.jnatprod.7b00410
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and 29.8), an isovaleryl ester moiety [δC 173.3, 44.3, 26.8, and
22.8 (×2)], a glucopyranosyl unit (δC 101.5, 78.1, 76.2, 75.3,
71.8, and 62.8), and a trans-coumaroyl group [δC 168.4, 161.4,
HMQC, and HMBC experiments (Figure 1) and was assigned
as 1-(2-methylbutyroyl)-8-hydroxy-2″-O-trans-p-coumaroyl-
dihydropenstemide.
1
146.9, 131.3 (×2), 127.2, 117.0 (×2), 115.0]. The H and 13C
Compound 4 (sambucuside D) was isolated as a yellowish
gum, and its molecular formula was established as C32H42O14
on the basis of its 13C NMR data and the molecular ion peak
[M − H]− at m/z 649.2491 (calcd for C32H41O14, 649.2491) in
NMR spectra of 1 were similar to those of viburtinoside II,13
except for the presence of a methylene [δH 1.63 (1H, m) and
1.52 (1H, m)] at C-7 with the absence of an oxymethine
carbon (δC 78.9) and an acetyl group (δH 2.01). This was
1
the HRFABMS. The H and 13C NMR data of 4 were quite
1
supported by the H−1H COSY cross-peak between CH2-7/
similar to those of 1, except for the presence of an additional
acetyl group [δC 172.9, 20.9; δH 2.01 (3H, s)]. In the HMBC
spectrum, a correlation from H-10 [δH 3.97 (1H, d, J = 11.0
Hz) and 3.85 (1H, d, J = 11.0 Hz)] to an acetyl carbonyl
carbon (δC 172.9) was used to assign the additional acetyl
group at C-10 (Figure 1). The relative configuration of 4 was
established as being the same as that of 1 based on the coupling
constant between H-1 and H-9 (5.7 Hz for 4; 5.0 Hz for 1) and
the NOESY cross-peaks of H-5/H-9 and H-10, H-9/H-10, and
H-10/H-3′ of 4 (Figure 2). Thus, the structure of 4 was
established as 1-(2-methylbutyroyl)-8-hydroxy-10-acetyl-2″-O-
trans-p-coumaroyldihydropenstemide.
CH2-6. The 3-methylbutyroyl group was located at C-1 based
on the HMBC correlation from H-1 [δH 6.10 (1H, d, J = 5.0
Hz)] to C-1′ (δC 173.3). The location of the glucose unit was
determined to be at C-11 by analysis of the HMBC data
showing a correlation from H-11 to C-1″. The HMBC cross-
peak from H-2″ [δH 4.87 (1H, overlap)] to C-1‴ (δC 168.4)
also indicated a coumaroyl group to be present at C-2″ of the
glucose unit. The planar structure of 1 was confirmed from the
1H−1H COSY, HMQC, and HMBC spectra (Figure 1). The
relative configuration of 1 was established by analyzing the
NOESY and 13C NMR data. In the NOESY spectrum (Figure
2), correlations of H-5/H-9 and H-10 and of H-9/H-10
indicated that H-5, H-9, and CH2-10 are all β-oriented.
Through the chemical shift of C-9 (δC 46.0) and the
hydroxymethyl group located at C-8, it was determined to
have a β-configuration.14 Acid hydrolysis of 1 afforded glucose
Compound 5 (sambucuside E) was obtained as a yellowish
gum, and its molecular formula was established as C32H42O14
using HRESIMS, which showed a positive-ion peak [M + Na]+
1
at m/z 673.2470 (calcd for C32H42O14Na 673.2472). The H
and 13C NMR data of 5 were similar to those of 4, except that a
trans-coumaroyl moiety of 4 was replaced with a cis-coumaroyl
moiety in 5. This was supported by the cis-coupling constant
(12.9 Hz) for H-7‴ and H-8‴ of 5. The structure of 5 was
confirmed from the 1H−1H COSY, HMQC, and HMBC
spectra obtained (Figure 1), as 1-(2-methylbutyroyl)-8-
hydroxy-10-acetyl-2″-O-cis-p-coumaroyldihydropenstemide.
Compound 6 was obtained as a colorless gum, and its
molecular formula was determined to be C21H34O12 based on
1
and trans-p-coumaric acid. The H NMR data of a trans-p-
coumaric acid unit corresponded to previously reported data.15
D-Glucose was identified by TLC comparison and GC analysis
after derivatization with an authentic sample.16 The anomeric
configuration of D-glucose was determined to be β based on the
J value (8.0 Hz) of the anomeric proton in D-glucose.17 Thus,
the structure of 1 was established as 8-hydroxy-2″-O-trans-p-
coumaroyldihydropenstemide, and it was named sambucuside
A.
1
the negative-ion HRFABMS and 13C NMR data. The H and
13C NMR spectra of 6 were similar to those of supensolide F,20
except for signals assigned to the sugar unit [δC 101.5 (C-1″),
72.5 (C-2″), 73.1 (C-3″), 75.5 (C-4″), 69.1 (C-5″), and 63.4
(C-6″) for 6; δC 103.6 (C-1″), 75.3. (C-2″), 78.2 (C-3″), 71.8
(C-4″), 78.0 (C-5″), and 62.9 (C-6″) for supensolide F]. This
indicated that 6 has a galactopyranose moiety instead of a
glucopyranose moiety as in supensolide F. The HMBC
correlation from H-1″ [δH 4.68 (1H, d, J = 8.0 Hz)] to C-11
(δC 69.9) showed that the galactopyranose unit was located at
C-11. Acid hydrolysis of 6 afforded D-galactose, which was
confirmed by co-TLC with an authentic sample and GC
analysis.16 The relative configuration of 6 was confirmed by the
NOESY spectrum, in which correlations between CH2-10/H-5
and H-9 and H-9/H-5 suggested that H-10, H-9, and H-5 are
all β-oriented. The orientation of H-7 could be expected to be
α-oriented, based on the correlation between H-7/H-1. Thus,
the structure of 6 (sambucuside F) was established as 7β,8β-
dihydroxydihydropenstemide.
Compound 2 was obtained as a yellowish gum. The
molecular formula of 2 was established as being C30H40O13
from the HRFABMS. It showed a negative ion [M − H]− at m/
1
z 607.2385 (calcd for C30H39O13 607.2385). The H and 13C
NMR data of 2 were very similar to those of 1, except for the
chemical shifts of H-7‴ and H-8‴ (δH 6.89 and 5.82 for 2; δH
7.67 and 6.38 for 1). Their relatively small J values (12.9 Hz for
2; 16.0 Hz for 1) indicated that the p-coumaroyl moiety is in
the cis-form.18 The structure of 2 was confirmed using the
1H−1H COSY, HMQC, and HMBC data (Figure 1). D-
Glucopyranose was identified in a similar manner to that
described for 1. The structure of 2 (sambucuside B) was
established as 8-hydroxy-2″-O-cis-p-coumaroyl-
dihydropenstemide.
Compound 3 was obtained as a yellowish gum. Its molecular
formula was determined as C30H40O13 from the molecular ion
peak [M + Na]+ at m/z 631.2368 (calcd for C30H40O13Na,
1
631.2367) in the positive-ion HRESIMS. The H and 13C
The two known compounds were identified as 8-methoxy-
10-methylene-2,9-dioxatricyclo(4,3,1,03,7)decane (7)21 and 4-
hydroxy-8-methoxy-10-methylene-2,9-dioxatricyclo(4,3,1,03,7)-
decane (8)22 by comparison with NMR data in the literature.
The cytotoxic activities of compounds 1−8 against the A549
(non-small-cell lung adenocarcinoma), SK-OV-3 (ovary malig-
nant ascites), SK-MEL-2 (skin melanoma), and Bt549 (invasive
ductal carcinoma) cell lines were evaluated using an SRB
bioassay. Compounds 2, 3, and 5 showed cytotoxic activities
against SK-MEL-2 and Bt549 of the human cancer cell lines
tested, with IC50 values ranging from 1.3 to 8.7 μM (Table 3).
NMR data of 3 were quite similar to those of 1, except for the
presence of the 2-methylbutyroyl group, rather than a 3-
methylbutyroyl group at C-1 [δH 2.38 (1H, m, H-2′), 1.64 (1H,
overlap, H-3′a), 1.49 (1H, overlap, H-3′b), 0.92 (3H, t, J = 7.5
Hz, H-4′), and 1.13 (3H, d, J = 7.0 Hz), δC 176.9 (C-1′), 42.3
(C-2′), 27.9 (C-3′), 11.9 (C-4′), and 16.8 (C-5′) for 3; δH 2.21
(1H, dd, J = 7.1, 1.9 Hz, H-2′), 2.06 (1H, m, H-3), and 0.96
(6H, t, J = 6.7 Hz, H-4′, 5′), δC 173.3 (C-1′), 44.3 (C-2′), 26.8
(C-3′), and 22.8 (C-4′,5′) for 1].19 This was supported by the
HMBC cross-peaks of H-5′/C-1′, C-2′, and C-3′. The structure
1
of 3 (sambucuside C) was confirmed using H−1H COSY,
C
DOI: 10.1021/acs.jnatprod.7b00410
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1
Table 1. H NMR [ppm, mult, (J in Hz)] Data of Compounds 1−6 in Methanol-d4 (700 MHz)
position
1
2
3
4
5
6
1
6.10, d (5.0)
6.36, brs
6.09, d (4.9)
6.35, brs
6.09, d (5.1)
6.36, brs
6.07, d (5.7)
6.36, brs
6.11, d (5.1)
6.36, brs
6.17, d (4.9)
6.38 brs
3
5
2.64, m
2.62, m
2.65, m
2.63, m
2.62, m
3.06, m
6a
6b
7α
7β
9
1.74, m
1.73, m
1.74, m
1.80, m
1.74, m
2.02, dd (7.8, 4.1)
1.69, m
1.70, m
1.70, m
1.68, m
1.68, m
1.52, m
1.56, m
1.51, overlap
1.63, overlap
2.14, dd (9.1, 5.0)
3.38, overlap
3.34, overlap
4.26, d (11.5)
4.13, d (11.5)
2.38, m
1.53, overlap
1.58, overlap
2.09, overlap
3.97, d (11.0)
3.85, d (11.0)
4.29, d (11.4)
4.09, d (11.4)
2.20, brd (7.1)
2.06, overlap
1.57, m
4.00, brt (3.9)
1.63, m
1.64, m
1.62, m
2.14, dd (9.0, 5.0)
3.37, overlap
3.34, overlap
4.26, d (11.5)
4.12, d (11.5)
2.21, dd (7.1, 1.9)
2.06, m
2.03, overlap
3.38, overlap
3.34, overlap
4.26, d (11.4)
4.09, d (11.4)
2.20, dd (7.2, 1.8)
2.07, m
1.99, dd (9.3, 5.1)
3.94, d (11.0)
3.88, d (11.0)
4.28, d (11.3)
4.07, d (11.3)
2.20, brd (7.2)
2.07, overlap
2.36, dd (9.8, 4.9)
3.79, d (11.2)
3.68, d (11.2)
4.28, d (11.5)
4.10, d (11.5)
2.23, brd (7.3)
2.10, m
10a
10b
11a
11b
2′
3′
1.64, overlap
1.49, overlap
0.92, t (7.5)
1.13, d (7.0)
4.59, d (8.0)
4.86, overlap
3.64, m
4′
5′
1″
2″
0.96, d (6.7)
0.96, d (6.7)
4.60, d (8.0)
4.85, overlap
3.65, m
0.97, d (6.7)
0.97, d (6.7)
4.53, d (8.0)
4.83, overlap
3.58, m
0.96, d (6.7)
0.96, d (6.7)
4.58, d (8.0)
4.86, overlap
3.64, m
0.96, d (6.7)
0.96, d (6.7)
4.52, d (8.0)
4.83, overlap
3.59, m
0.99, d (6.7)
0.99, d (6.7)
4.68, d (8.0)
3.33, overlap
4.06, m
3″
4″
3.42, m
3.42, m
3.42, m
3.42, m
3.40, m
3.49, m
5″
3.38, overlap
3.93, dd (12.0, 2.0)
3.72, dd (12.0, 5.8)
7.48, d (8.6)
6.84, d (8.6)
7.67, d (16.0)
6.38, d (16.0)
3.34, overlap
3.91, dd (11.9, 2.1)
3.72, dd (11.9, 5.8)
7.75, d (8.7)
6.77, d (8.7)
6.89, d (12.9)
5.82, d (12.9)
3.37, overlap
3.93, dd (11.9, 1.9)
3.72, dd (12.0, 5.8)
7.47, d (8.6)
6.81, d (8.6)
7.67, d (15.9)
6.37, d (15.9)
3.37, m
3.35, overlap
3.91, dd (12.0, 2.2)
3.72, dd (12.0, 5.9)
7.77, d (8.7)
6.75, d (8.7)
6.89, d (12.9)
5.82, d (12.9)
2.05, s
3.70, overlap
3.86, overlap
3.67, overlap
6″a
6″b
2‴,6‴
3‴,5‴
7‴
3.93, dd (11.9, 2.2)
3.73, dd (12.0, 5.8)
7.48, d (8.6)
6.83, d (8.6)
7.67, d (15.9)
6.38, d (15.9)
2.01, s
8‴
CH3CO
In particular, compound 3 displayed quite potent cytotoxic
activity against four human cancer cell lines (A549, SK-OV-3,
SK-MEL-2, and Bt549 cells), with IC50 values of 3.8, 1.9, 1.4,
and 1.3 μM, respectively. Interestingly, although compounds 1
and 3 have similar structures except for the functional group at
C-1, only compound 3 was active, suggesting that the presence
of the 2-methylbutyroyl group at C-1 plays an important role in
mediating the cytotoxicity against the cancer cell lines tested.
Compounds 1 and 4, possessing a trans-coumaroyl substituent
in the glucosyl unit, exhibited no cytotoxic activities. However,
compounds 2 and 5, having a cis-coumaroyl group, showed
strong cytotoxic activities to the SK-MEL-2 and Bt549 cancer
cell lines tested. These data suggest that the geometry of the
double bond in the coumaroyl group is of relevance for their
cytotoxic effects in SK-MEL-2 and Bt549 cancer cell lines.
Anti-neuroinflammatory effects of all the isolated compounds
(1−8) were evaluated by measuring NO levels in LPS-
stimulated murine microglia BV2 cells (Table 4). Among
them, compounds 2, 3, and 5 inhibited the NO level with IC50
values of 0.9, 1.3, and 1.2 μM, respectively. They showed more
potent NO-inhibitory activity than NG-monomethyl-L-arginine
(L-NMMA), the positive control (IC50 13.2 μM), without
demonstrating any cell toxicity at a concentration of 20 μM.
Neuroprotective activities of the isolated compounds (1−8)
were examined by measuring NGF secretion in C6 cells (Table
5). The level of NGF released into the medium was measured,
and cell viability was determined using an MTT assay. Among
the compounds isolated, compounds 2−4 and 8 were
stimulants of NGF release, with levels of NGF stimulated at
147.0 5.8%, 145.4 5.8%, 158.7 5.2%, and 152.6 7.3%,
respectively (146.3
without producing cytotoxic effects at a concentration of 20
μM.
7.2% for 6-shogaol, a positive control)
Iridoid glycosides with a methylbutyroyl group at C-1 and a
sugar at C-11 have been reported in species of Viburnum,
Valeriana, and Sambucus.23−25 These iridoid derivatives were
evaluated for inhibitory activity against HeLa S3 cancer cells
and induced a significant hepatoprotection as a reduction in
serum ALT and AST. The iridoid glycosides with a
methylbutytoyl group at position C-1 and with a sugar at
position C-11, as isolated from this plant source in the present
investigation, showed cytotoxic activities to the SK-MEL-2 and
Bt549 cancer cell lines, inhibition of NO levels in LPS-
stimulated murin microglia BV2 cells, and NGF secretion-
inducing activities.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured with a JASCO P-1020 polarimeter (Easton, MD, USA).
Infrared (IR) spectra were recorded using a Bruker IFS-66/S Fourier-
transform IR spectrometer (Bruker, Karlsruhe, Germany). Ultraviolet
(UV) spectra were recorded with a Shimadzu UV-1601 UV−visible
spectrophotometer (Shimadzu, Tokyo, Japan). NMR spectra were
recorded with a Bruker AVANCE III 700 NMR spectrometer at 700
MHz (1H) and 175 MHz (13C). HRESIMS data were obtained with a
Waters SYNAPT G2 mass spectrometer. HRFABMS was performed
with a JEOL JMS700 mass spectrometer. A Hewlett-Packard (HP) GC
6890 Series system equipped with a 5973 mass selective detector
system was controlled by the Enhanced ChemStation version B.01.00
software. The capillary column used for GC was an Agilent J&W HP-
5MS UI (30.0 m × 0.25 mm i.d., 0.25 μm film thickness coated 5%
diphenyl, 95% dimethylpolysiloxane). The preparative high-perform-
D
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Journal of Natural Products
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Table 2. 13C NMR (ppm, mult) Data of Compounds 1−6 in
methanol-d4 (175 MHz)
Table 4. Inhibitory Effect of Compounds 1−8 on NO
Production in LPS-Activated BV-2 Cells after 24 h of
Treatment
carbon
1
2
3
4
5
6
a
b
compound
IC50 (μM)
cell viability (%)
1
91.7
91.7
91.8
91.7
140.8
115.3
36.5
91.6
140.7
115.2
36.0
92.0
140.1
116.9
33.1
38.4
79.7
84.1
45.0
66.5
69.9
173.3
44.4
26.9
22.8
22.8
101.3
72.5
73.1
69.1
75.5
63.4
3
140.9
115.4
36.3
140.8
115.2
36.1
140.9
115.1
36.3
1
2
3
4
5
6
7
8
45.5
0.9
102.3 9.9
103.0 3.1
136.4 2.1
142.7 0.6
130.4 8.3
103.1 3.7
103.7 9.9
36.0 9.9
4
5
1.3
6
29.8
29.7
29.8
29.9
29.7
29.8
1.2
7
37.3
37.4
37.4
38.1
38.0
8
82.6
82.7
82.7
80.9
80.8
112.5
94.2
110.1
13.2
9
46.0
46.1
46.1
46.7
46.7
10
69.6
69.8
69.7
71.7
71.8
c
11
69.9
70.0
69.9
70.1
70.0
L-NMMA
120.3 6.3
a
1′
2′
3′
4′
5′
1″
2″
3″
173.3
44.3
173.4
44.4
176.9
42.3
173.2
44.4
173.3
44.4
IC50 value of each compound was defined as the concentration (μM)
that caused 50% inhibition of NO production in LPS-activated BV-2
b
26.8
26.9
27.9
27.0
26.9
cells. The cell viability after treatment with 20 μM of each compound
was determined by the MTT assay and is expressed as a percentage
(%). Results are means of three independent experiments, and the data
22.8
22.8
11.9
22.8
22.8
22.8
22.8
16.8
22.8
22.8
c
are expressed as means SD. Positive control.
101.5
75.3
101.7
75.4
101.6
75.4
102.0
75.4
102.0
75.0
Table 5. Effects of Compounds 1−8 on NGF Secretion in C6
Cells after 24 h of Cell Treatment
76.2
76.2
76.3
76.2
76.2
4″
5″
6″
71.8
71.9
71.9
71.9
71.9
a
b
78.1
78.2
78.2
78.2
78.2
compound
NGF secretion (%)
cell viability (%)
62.8
62.8
62.8
62.8
62.8
1
2
3
4
5
6
7
8
130.4 5.0
147.0 5.8
145.4 5.8
158.7 5.2
134.1 4.4
107.4 12.0
133.4 11.4
152.6 7.3
146.3 7.2
115.0 2.1
101.7 3.1
101.3 3.4
119.7 2.7
104.0 4.2
115.7 11.3
105.6 0.9
110.7 0.5
98.8 5.2
1‴
2‴,6‴
3‴,5‴
4‴
7‴
8‴
9‴
CH3CO
CH3CO
127.2
131.3
117.0
161.4
146.9
115.0
168.4
127.4
134.3
116.1
160.8
145.8
116.2
167.2
126.9
131.4
117.3
162.1
147.1
115.0
168.5
127.2
131.4
117.1
161.6
146.9
115.3
168.4
172.9
20.9
127.5
134.4
116.2
160.8
145.7
116.3
167.2
173.0
20.9
c
6-shogaol
a
C6 cells were treated with 20 μM of each compounds. After 24 h, the
content of NGF secreted in the C6-conditioned medium was
measured by ELISA. The level of secreted NGF is expressed as a
percentage of the untreated control (set as 100%). Data are means
Table 3. Cytotoxicity of Compounds 1−8 against Four
Cultured Human Cancer Cell Lines
b
a
SD of three independent experiments performed in triplicate. The
IC50 (μM)
cell viability after treatment with 20 μM of each compound was
determined by an MTT assay and is expressed as percentages (%).
Results are means of three independent experiments, and the data are
compound
A549
SK-OV-3
SK-MEL-2
Bt549
1
2
3
4
5
6
7
8
>10
>10
3.8
>10
>10
1.9
>10
8.4
>10
8.7
c
expressed as means SD. Positive control.
1.4
1.3
detected on TLC under UV light or by heating after spraying samples
with anisaldehyde−sulfuric acid.
>10
>10
>10
>10
>10
1.1
>10
7.5
>10
5.2
>10
4.0
Plant Material. Twigs of S. williamsii var. coreana were collected
from Chungbuk Goesan, Korea, in August 2012. The plant materials
were identified by one of the authors of this study (K.R.L.). A voucher
specimen (SKKU-NPL-1214) has been deposited at the Herbarium of
School of Pharmacy, Sungkyunkwan University, Suwon, Korea.
Extraction and Isolation. Twigs of S. williamsii var. coreana (10.0
kg) were extracted with 80% aqueous MeOH under reflux and filtered.
The filtrate was evaporated under reduced pressure to obtain a MeOH
extract (280 g), which was then suspended in distilled H2O and
successively partitioned with n-hexane, CHCl3, EtOAc, and n-butanol,
yielding 16, 30, 17, and 40 g of residue, respectively. The EtOAc-
soluble fraction (20 g) was separated on an RP-C18 silica gel column
using gradient elution with 30% aqueous MeOH → 80% aqueous
MeOH, which yielded seven pooled fractions (E1−E7). Fraction E2
(5.1 g) was chromatographed on a silica gel column using CHCl3−
MeOH−H2O (10:1:0.1 → 3:1:0.1), which resulted in eight fractions
(E2a−E2h). Fractions E2a (60 mg) and E22 (69 mg) were purified by
semipreparative HPLC (2 mL/min, 40% aqueous MeOH), which
yielded compounds 7 (8 mg) and 8 (5 mg), respectively. Fraction E2g
(1.2 g) was separated over an RP-C18 silica gel column using 35%
aqueous MeOH, which gave seven additional fractions (E2ga−E2gg).
>10
>10
>10
2.9
>10
>10
>10
0.2
>10
>10
>10
1.0
b
etoposide
a
50% inhibitory concentration; the concentration of compound that
caused a 50% inhibition in cell growth. The cells tested were treated
with serial dilutions (30, 10, 3, 1, 0.3, 0.1 μM) of each compounds.
After 48 h, the cell survival rates were measured using an SRB assay.
Results are means of three independent experiments, and the data are
b
expressed as means SD. Positive control.
ance liquid chromatography (HPLC) system used was equipped with a
Gilson 306 pump (Middleton, WI, USA) and a Shodex refractive index
detector (New York, NY, USA). Column chromatography was
performed using silica gel 60 (70−230 and 230−400 mesh; Merck,
Darmstadt, Germany) and RP-C18 silica gel (Merck, 230−400 mesh).
Merck precoated silica gel F254 plates and reversed-phase (RP)-18 F254s
plates were used for thin-layer chromatography (TLC). Spots were
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Article
Fraction E2a (169 mg) was purified by semipreparative HPLC (2 mL/
min, 55% aqueous MeOH), which yielded compound 6 (4 mg).
Fraction E3 (2.1 g) was separated over a silica gel column using
CHCl3−MeOH−H2O (8:1:0.1 → 3:1:0.1), which gave seven
subfractions (E3a−E3g). Compounds 1 (11 mg), 2 (3 mg), and 3
(4 mg) were obtained by purifying fraction E3f (196 mg) using
semipreparative HPLC (55% aqueous MeOH). Fraction E4 (2.0 g)
was separated over a silica gel column using CHCl3−MeOH−H2O
(8:1:0.1 → 3:1:0.1), which gave five subfractions (E4a−E4e).
Compounds 4 (9 mg) and 5 (5 mg) were obtained by purifying
fraction E4b (275 mg) through semipreparative HPLC using 55%
aqueous MeOH.
Cytotoxicity Assessment. The cytotoxicity of the isolated
compounds against the cultured human tumor cell lines A549, SK-
OV-3, SK-MEL-2, and Bt549 was evaluated by an SRB method.26
These assays were performed at the Korea Research Institute of
Chemical Technology. Cells used in the current study were purchased
from the American Type Culture Collection (Manassas, VA, USA) and
maintained at the Korea Research Institute of Chemical Technology.
Each tumor cell line was inoculated into standard 96-well flat-bottom
microplates and incubated at 37 °C for 24 h in a humidified
atmosphere with 5% CO2. Attached cells were incubated with serially
diluted compounds (30, 10, 3, 1, 0.3, and 0.1 μM). After 48 h of
continuous exposure to these compounds, the culture medium was
removed and cells were fixed with 10% cold trichloroacetic acid at 4
°C for 1 h. After washing with tap water, cells were stained with 0.4%
SRB dye and incubated at room temperature for 30 min. These cells
were washed again and then solubilized with 10 mM unbuffered Tris
base solution (pH 10.5). Absorbance was measured spectrophoto-
metrically at 520 nm with a microtiter plate reader. Etoposide (≥98%;
Sigma Chemical Co., St. Louis, MO, USA) was used as a positive
control. The 50% inhibitory concentration of cell growth (IC50) was
expressed as the mean of three independent experiments standard
deviation (SD).
Measurement of NO Production and Cell Viability in LPS-
Activated BV-2 Cells. BV-2 cells, developed by Dr. V. Bocchini at the
University of Perugia (Perugia, Italy), were used for this study. The
BV-2 microglia cells were first treated with the test compounds and
incubated for 30 min. After 30 min of incubation, the treated cells were
stimulated by adding LPS (100 ng/mL) and incubated for 24 h for
treatment. Nitrite (a soluble oxidation product of NO) was measured
in the culture medium using the Griess reaction after 24 h of
treatment. 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).
After 10 min of incubation, absorbance was measured at a wavelength
of 570 nm using a microplate reader (Emax, Molecular Device,
Sunnyvale, CA, USA). Graded sodium nitrite solution was used as
standard to calculate nitrite concentration. Cell viability was measured
using the MTT assay. NG-Monomethyl-L-arginine (Sigma), a NO
synthase inhibitor, was used as a positive control.
NGF and Cell Viability Assays. C6 glioma cells were used to
measure NGF release. C6 cells were purchased from the Korean Cell
Line Bank and maintained in DMEM supplemented with 10% fetal
bovine serum (FBS) and 1% penicillin−streptomycin in a humidified
incubator with 5% CO2. The C6 cells were seeded into 24-well cell
culture plates at a density of 1 × 105 cells/well to measure NGF
content in the medium and cell viability. After 24 h, the cells were
treated with DMEM containing 2% FBS, 1% penicillin−streptomycin,
and 20 μM of each compound for 1 day. The medium supernatant was
used for NGF ELISA (R&D Systems, Minneapolis, MN, USA). Cell
viability was measured using an MTT assay. After 24 h of treatments,
cells were incubated with MTT reagent for 1 h in a humidified
incubator with 5% CO2 at 37 °C. Blue-stained viable cells were then
converted into purple-colored solution in the presence of DMSO.
Absorbance value was then measured at a wavelength of 570 nm on a
microplate reader. The positive control was 6-shogaol.
Sambucuside A (1): yellowish gum; [α]2D5 −122 (c 0.2, MeOH);
UV (MeOH) λmax (log ε) 312 (1.11), 226 (0.75), 211 (0.75) nm; IR
(KBr) νmax 3450, 2967, 2845, 1725, 1605, 1168, 1003 cm−1; 1H NMR
(700 MHz) data, see Table 1; 13C NMR (175 MHz) data, see Table 2;
HRESIMS (positive-ion mode) m/z 631.2360 [M + Na]+ (calcd for
C30H40O13Na, m/z 631.2367).
Sambucuside B (2): yellowish gum; [α]2D5 −76 (c 0.1, MeOH); UV
(MeOH) λmax (log ε) 315 (2.40), 226 (1.80), 211 (1.81) nm; IR
1
(KBr) νmax 3702, 3410, 2970, 2824, 1600, 1032 cm−1; H NMR (700
MHz) data, see Table 1; 13C NMR (175 MHz) data, see Table 2;
HRFABMS (negative-ion mode) m/z 607.2385 [M − H]− (calcd for
C30H39O13, m/z 607.2385).
Sambucuside C (3): yellowish gum; [α]2D5 −68 (c 0.1, MeOH); UV
(MeOH) λmax (log ε) 315 (3.98), 227 (3.04), 211 (3.08) nm; IR
1
(KBr) νmax 3434, 2964, 2853, 1715, 1602, 1169, 1035, 999 cm−1; H
NMR (700 MHz) data, see Table 1; 13C NMR (175 MHz) data, see
Table 2; HRESIMS (positive-ion mode) m/z 631.2368 [M + Na]+
(calcd for C30H40O13Na, m/z 631.2367).
Sambucuside D (4): yellowish gum; [α]2D5 −95 (c 0.1, MeOH); UV
(MeOH) λmax (log ε) 313 (3.04), 227 (1.95), 210 (1.99) nm; IR
(KBr) νmax 3701, 3442, 2969, 2866, 1725, 1605, 1168, 1032, 756 cm−1;
1H NMR (700 MHz) data, see Table 1; 13C NMR (175 MHz) data,
see Table 2; HRFABMS (negative-ion mode) m/z 649.2491 [M −
H]− (calcd for C32H41O14, m/z 649.2491).
Sambucuside E (5): yellowish gum; [α]2D5 −117 (c 0.2, MeOH); UV
(MeOH) λmax (log ε) 311 (0.47), 227 (0.45), 204 (0.60) nm; IR
1
(KBr) νmax 3450, 2972, 2853, 1727, 1606, 1170, 1031, 755 cm−1; H
NMR (700 MHz) data, see Table 1; 13C NMR (175 MHz) data, see
Table 2; HRESIMS (positive-ion mode) m/z 673.2470 [M + Na]+
(calcd for C32H42O14Na, m/z 673.2472).
Sambucuside F (6): colorless gum; [α]2D5 −90 (c 0.1, MeOH); UV
(MeOH) λmax (log ε) 285 (0.34), 204 (1.42) nm; IR (KBr) νmax 3435,
1
3187, 1718, 1667, 1014, 756 cm−1; H NMR (700 MHz) data, see
Table 1; 13C NMR (175 MHz) data, see Table 2; HRFABMS
(negative-ion mode) m/z 477.1966 [M − H]− (calcd for C21H33O12,
m/z 477.1967).
Acid Hydrolysis of Compounds 1−6 and Sugar Analysis.
Each compound (2.0 mg each) was dissolved in 1 mL of 1 N HCl. The
solution was then heated at 80 °C for 2 h. This reaction mixture was
diluted with H2O and extracted with CH2Cl2. The CH2Cl2 extract was
evaporated in vacuo to yield aglycone. The H2O layer was dried in
vacuo and mixed with pyridine (0.1 mL) and L-cysteine methyl ester
hydrochloride (2.0 mg). The mixture was stirred at 60 °C for 1.5 h.
After the reaction mixture was dried in vacuo, the residue was
trimethylsilylated with 1-trimethylsilylimidazole (0.1 mL) for 2 h. The
mixture was then partitioned between n-hexane and H2O (0.3 mL).
The organic layer (1 μL) was analyzed by GC-MS under the following
conditions: capillary column, HP-5MS UI (30 m × 0.25 mm × 0.25
μm, Agilent); temperatures of the injector and detector, both at 200
°C. A temperature gradient system was used for the oven, starting at
100 °C for 1 min and increasing to 180 °C at a rate of 5 °C/min. D-
Glucose and D-galactose were treated in the same manner. D-Glucose
(1: 15.37 min, 2: 15.33 min, 3: 15.36 min, 4: 15.34 min, 5: 15.36 min)
and D-galactose (6: 14.86 min) were detected for compounds 1−6.
Identification of D-glucose (15.35 min) and D-galactose (14.85 min)
detected in each case was performed by co-injection of the hydrolysate
with standard silylated sugars.
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.7b00410.
HRMS and NMR data of 1−6 (PDF)
AUTHOR INFORMATION
■
Corresponding Author
*Tel: 82-31-290-7710. Fax: 82-31-290-7730. E-mail: krlee@
skku.edu.
F
DOI: 10.1021/acs.jnatprod.7b00410
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
ORCID
Kang Ro Lee: 0000-0002-3725-5192
Notes
The authors declare no competing financial interest.
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 (2016R1A2B2008380). We are thankful to the
Korea Basic Science Institute (KBSI) for the measurements of
mass spectra.
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DOI: 10.1021/acs.jnatprod.7b00410
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