Lignan Glycosides from the Twigs of Chaenomeles sinensis and Their Biological Activities

Article abstract of DOI:10.1021/acs.jnatprod.5b00090

Phytochemical investigation of the twigs of Chaenomeles sinensis led to the isolation and identification of six new lignan glycosides, chaenomiside A-F (1-6), along with five known ones (7-11). Their chemical structures were determined by spectroscopic methods, including NMR, MS, ECD, and GC/MS analyses. All the isolated compounds (1-11) were tested for their inhibitory effects on nitric oxide (NO) production in lipopolysaccharide-activated murine microglial cells and the secretion of nerve growth factor (NGF) in a C6 rat glioma cell line. Compound 6 significantly reduced NO levels in the murine microglia BV2 cells with an IC₅₀ value of 21.3 μM, and compounds 1, 3, and 6 were potent stimulants of NGF release with stimulation levels of 151.74 ± 6.77%, 144.31 ± 7.49%, and 167.61 ± 18.5%, respectively.

Full text of DOI:10.1021/acs.jnatprod.5b00090

Note
pubs.acs.org/jnp
Lignan Glycosides from the Twigs of Chaenomeles sinensis and Their
Biological Activities
Chung Sub Kim,† Lalita Subedi,‡ Sun Yeou Kim,‡,§ Sang Un Choi,⊥ Ki Hyun Kim,† and Kang Ro Lee*,†
†Natural Products Laboratory, School of Pharmacy, Sungkyunkwan University, Suwon 440-746, Republic of Korea
§
‡Gachon Institute of Pharmaceutical Science and College of Pharmacy, Gachon University, Incheon 406-799, Republic of Korea
⊥Korea Research Institute of Chemical Technology, Daejeon 305-343, Republic of Korea
S
* Supporting Information
ABSTRACT: Phytochemical investigation of the twigs of
Chaenomeles sinensis led to the isolation and identification of
six new lignan glycosides, chaenomiside A−F (1−6), along
with five known ones (7−11). Their chemical structures were
determined by spectroscopic methods, including NMR, MS,
ECD, and GC/MS analyses. All the isolated compounds (1−
11) were tested for their inhibitory effects on nitric oxide
(NO) production in lipopolysaccharide-activated murine
microglial cells and the secretion of nerve growth factor
(NGF) in a C6 rat glioma cell line. Compound 6 significantly
reduced NO levels in the murine microglia BV2 cells with an
IC50 value of 21.3 μM, and compounds 1, 3, and 6 were potent
stimulants of NGF release with stimulation levels of 151.74 6.77%, 144.31 7.49%, and 167.61 18.5%, respectively.
Chaenomeles sinensis Koehne (Rosaceae) is a woody plant
widely distributed in eastern Asian countries, including Korea,
Japan, and China. The fruits of this plant have been used in
Korean traditional medicine to treat throat diseases, diarrhea,
inflammatory diseases, and dry beriberi, which is caused by
damage to the nervous system.1−3 Pharmacological activities
have been identified in the components and/or extract of the
fruit of C. sinensis.4−7 Antiviral activity against influenza A virus
and antidiabetic, antiacetylcholinesterase, antihyperglycemic,
antihyperlipidemic, and antioxidant effects in streptozotocin-
induced diabetic rats have been reported from the extract of C.
sinensis fruit.4−7 Also, tissue factor inhibitory flavonoids and
triterpenoids and antipruritic flavonoids were isolated from the
same sources.1−3 However, research to isolate anti-inflamma-
tory and/or neuroprotective agents from the twigs of C. sinensis
has not been conducted.
activities. In this study, we report the isolation and structure
elucidation of bioactive components from the twigs of C.
sinensis as well as their antineuroinflammatory and neuro-
protective activities.
The 80% aqueous methanolic extract from the twigs of C.
sinensis was sequentially partitioned with n-hexane, CHCl3,
EtOAc, and n-BuOH. Repeated chromatographic purification of
the EtOAc fraction yielded six new lignan glycosides,
chaenomiside A−F (1−6), and five known ones, (7R,8S)-
dihydrodehydrodiconiferyl alcohol 9-O-β-D-glucopyranoside
(7),8 (7S,8R)-dihydrodehydrodiconiferyl alcohol 9-O-α-L-rham-
noside (8),9 (+)-9′-O-(β-D-glucopyranosyl)lyoniresinol (9),10
(−)-9′-O-(α-L-rhamnopyranosyl)lyoniresinol (10),11 and avi-
culin (11),12 which were identified by comparing their
spectroscopic data and specific rotations with reported data.
Compound 1 was obtained as a colorless gum. The
molecular formula was determined to be C27H36O11 from the
13C NMR data and the [M − H]− ion in the negative ion
In the continuing search for new anti-inflammatory
substances from Korean medicinal plants, we isolated and
identified a potential neuroprotective agent from the twigs of C.
sinensis. The EtOAc layer of the methanolic extract exhibited
significant NGF induction activity in C6 cells (226.7 11.9%;
1
HRFABMS. The H NMR spectrum displayed the presence of
two 1,2,3,5-tetrasubstituted aromatic rings [δH 6.77 (1H, s, H-
6′), 6.73 (1H, s, H-2′), and 6.69 (2H, s, H-2 and H-6)], one
oxygenated methine [δH 5.50 (1H, d, J = 6.6 Hz, H-7)], two
oxygenated methylenes [δH 3.92 (1H, dd, J = 9.8, 7.9 Hz, H-
9a), 3.77 (1H, dd, J = 9.8, 5.1 Hz, H-9b), and 3.59 (2H, t, J =
6.6 Hz, H-9′)], one methine [δH 3.67 (1H, m, H-8)], two
methylenes [δH 2.66 (2H, t, J = 7.5 Hz, H-7′) and 1.85 (2H, m,
H-8′)], three methoxy groups [δH 3.89 (3H, s, 3′-OCH3), 3.85
positive control Zingiberis Rhizoma, 148.4
6.1%). The
column chromatographic purification of the most active EtOAc
layer led to the isolation of six new lignain glycosides,
chaenomiside A−F (1−6), along with five known ones (7−
11). The chemical structures of isolated compounds were
determined by their NMR spectroscopic data (1H and 13C
1
NMR, H−1H COSY, DEPT, HMQC, and HMBC), MS and
ECD spectra, and hydrolysis. All isolates (1−11) were
evaluated for their anti-inflammatory and neuroprotective
Received: January 28, 2015
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.5b00090
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Note
1
Table 1. 13C (175 MHz) and H (700 MHz) NMR
Assignments of Compounds 1−3 in Methanol-d4
1
2
3
δH (J in
Hz)
δH (J in
Hz)
pos.
δC
δC
δC
δH (J in Hz)
1
2
3
4
5
6
134.0
133.8
133.5
104.3 6.69, s
149.5
104.4 6.69, s
149.5
113.5 6.79, overlap
149.2
136.6
136.6
146.0
149.5
149.5
116.4 6.74, d (8.0)
104.3 6.69, s
104.4 6.69, s
122.2 6.67, dd
(8.0, 1.7)
7a
7b
89.8 5.50, d
(6.6)
89.7 5.49, d
(6.5)
34.2 2.91, dd
(13.7, 5.1)
2.56, dd
(13.7,
10.9)
(6H, s, 3-OCH3 and 5-OCH3)], and one α-rhamnopyranosyl
unit [δH 4.77 (1H, d, J = 1.5 Hz, H-1″), 3.85 (1H, overlap, H-
2″), 3.63 (1H, dd, J = 9.4, 3.4 Hz, H-3″), 3.56 (1H, dq, J = 9.4,
6.1 Hz, H-5″), 3.40 (1H, t, J = 9.4 Hz, H-4″), and 1.28 (1H, d, J
= 6.1 Hz, H-6″)]. The 13C NMR spectrum revealed 24 peaks
for 27 carbons, including 12 aromatic carbons for two aromatic
rings [δC 149.5 (C-3 and C-5), 136.6 (C-4), 134.0 (C-1), and
104.3 (C-2 and C-6), and 147.6 (C-4′), 145.4 (C-3′), 137.2 (C-
1′), 129.6 (C-5′), 117.8 (C-6′), and 114.4 (C-2′)] and one
anomeric carbon [δC 101.8 (C-1″)]. These NMR data of 1
(Table 1) were similar to those of 8 with a major difference
being the one aromatic ring of 8, indicating the presence of a
methoxy group at C-5 in 1. The planar structure of 1 was
determined through 2D NMR analysis, including 1H−1H
COSY, HMQC, and HMBC spectra (Figure 1). The HMBC
correlation of H-1″ to C-9 indicated that the rhamnose unit was
linked to the oxygen at C-9, and the J value of the anomeric
proton (J = 1.5 Hz) confirmed it as α-rhamnose.13 Enzymatic
hydrolysis of 1 afforded the aglycone (7S,8R)-3,3′,5-trime-
thoxy-4′,7-epoxy-8,5′-neolignan-4,9,9′-triol (1a) and L-rham-
nose ([α]D25 +9.0), which was identified by co-TLC
confirmation and GC/MS analysis.14,15 The identification of
8
53.0 3.67, m
70.6 3.92, dd
53.3 3.65,
44.2 2.78, m
overlap
70.8 4.02, dd
(9.2, 5.2)
3.63,
overlap
9a
9b
74.0 4.03, dd
(8.4, 6.5)
(9.8, 7.9)
3.77, dd
(9.8, 5.1)
3.73, dd
(8.4, 6.7)
1′
2′
3′
4′
5′
6′
7′
137.2
137.3
135.7
114.4 6.73, s
145.4
114.3 6.77, s
145.4
110.8 6.93, d (1.3)
149.2
147.6
147.6
147.3
129.6
129.7
116.2 6.80, overlap
120.1 6.81, overlap
84.9 4.80, d (6.8)
117.8 6.77, s
118.0 6.76, s
33.0 2.66, t
(7.5)
33.0 2.66, t
(7.6)
8′
35.9 1.85, m
36.0 1.85, m
51.6 2.49, quin
(6.8)
9′a
62.3 3.59, t
(6.6)
62.3 3.59, t
(6.6)
67.0 3.82, overlap
9′b
3-OCH3
3.66, overlap
57.0 3.85, s
57.0 3.85, s
56.9 3.89, s
57.0 3.85, s
57.0 3.85, s
56.9 3.88, s
56.6 3.86, s
5-OCH3
3′-
56.6 3.87, s
OCH3
1
the aglycone was done by comparing its H NMR, MS, and
1″
2″
3″
4″
5″
6″
101.8 4.77, d
(1.5)
102.4 4.77, d
(1.5)
102.3 4.69, d (1.7)
72.4 3.82, overlap
72.7 3.66, overlap
74.0 3.39, t (9.5)
optical rotation data.16 The C-7/C-8 relative configuration was
assigned as trans from the small coupling constant (6.6 Hz, 1;
7.4 Hz, trans-form; 8.4 Hz, cis-form).17 The absolute
configuration of 1 was confirmed as 7S and 8R from the
negative Cotton effect at 222 nm and positive ones at 246 and
289 nm as seen in the electronic circular dichroism (ECD)
spectrum (Figure S31).18 Thus, the structure of 1 was
established as (7S,8R)-3,5,3′-trimethoxy-4′,7-epoxy-8,5′-neo-
lignan-4,9,9′-triol 9-O-α-L-rhamnopyranoside, and this com-
pound was named chaenomiside A.
72.3 3.85,
overlap
72.3 3.85,
overlap
72.6 3.63, dd
(9.4, 3.4)
72.6 3.67,
overlap
74.0 3.40, t
(9.4)
74.0 3.41,
overlap
70.4 3.56, dq
(9.4, 6.1)
70.3 3.62,
overlap
70.4 3.59, dq
(9.5, 6.2)
18.2 1.28, d
(6.1)
18.2 1.27, d
(6.2)
18.2 1.27, d (6.2)
Compound 2 was obtained as a colorless gum with the same
molecular formula of C27H36O11 as 1. The NMR data of 2 were
similar to those of 1 except for the slightly shifted peaks of H-9
of 2 [δH 4.02 (1H, dd, J = 9.2, 5.2 Hz, H-9a) and 3.63 (1H,
overlap, H-9b)], compared to those of 1 [δH 3.92 (1H, dd, J =
9.8, 7.9 Hz, H-9a) and 3.77 (1H, dd, J = 9.8, 5.1 Hz, H-9b)],
suggesting that 2 was a stereoisomer of 1. A full NMR analysis
demonstrated that the planar structure of 2 was the same as 1.
The ECD spectra of 1 and 2 were opposite from 220 to 300 nm
(positive Cotton effects at 231 nm and negative ones at 258
and 293 nm), indicating that the aglycone moieties of 1 and 2
were enantiomeric. Thus, the structure of 2 was determined as
(7R,8S)-3,5,3′-trimethoxy-4′,7-epoxy-8,5′-neolignan-4,9,9′-triol
9-O-α-L-rhamnopyranoside and named chaenomiside B.
The molecular formula of compound 3 was determined to be
C26H34O10 from positive ion HRFABMS and 13C NMR data.
The 1H and 13C NMR spectra were similar to those of
lariciresinol,19,20 except for the presence of α-rhamnose signals
[δH 4.69 (1H, d, J = 1.7 Hz, H-1″), 3.82 (1H, overlap, H-2″),
3.66 (1H, overlap, H-3″), 3.59 (1H, dq, J = 9.5, 6.2 Hz, H-5″),
3.39 (1H, t, J = 9.5 Hz, H-4″), and 1.27 (1H, d, J = 6.2 Hz, H-
6″); δC 102.3 (C-1″), 74.0 (C-4″), 72.7 (C-3″), 72.4 (C-2″),
70.4 (C-5″), and 18.2 (C-6″)] and a deshielded signal of C-9′
(3, δC 67.0; lariciresinol, δC 60.5). The HMBC correlation from
H-1″ to C-9′ showed that the sugar unit was located at C-9′.
Enzymatic hydrolysis of 3 afforded (−)-lariciresinol, identified
by the 1H NMR, FABMS, and negative specific rotation {[α]2D5
−18.0 (c 0.05, MeOH)},19,20 and L-rhamnose, which was
confirmed by GC/MS analysis.14,15 The relative configuration
of 3 was confirmed by NOESY correlations of H-2′/H-8 and
H-8′, and the 8S,7′R,8′S configuration of 3 was deduced from
B
DOI: 10.1021/acs.jnatprod.5b00090
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Note
structure of 3 (chaenomiside C) as (−)-lariciresinol 9′-O-α-L-
rhamnopyranoside.
1
Inspection of the H and 13C NMR data of 4 (Table 2)
revealed that the two aromatic rings [δH 6.52 (2H, s, H-2 and
H-6) and 3.86 (6H, s, 3-OCH3 and 5-OCH3), and 6.65 (2H, s,
H-2′ and H-6′) and 3.87 (6H, s, 3′-OCH3 and 5′-OCH3); δC
149.5 (C-3 and C-5), 135.0 (C-4), 132.7 (C-1), and 107.0 (C-2
and C-6), and 149.4 (C-3′ and C-5′), 136.2 (C-4′), 134.9 (C-
1′), and 104.5 (C-2′ and C-6′)] are symmetrical 1,2,3,5-
tetrasubstituted moieties. This was supported by 1H−1H
COSY, HMQC, and HMBC spectra. The sugar analysis and
determination of stereochemistry of 4 were performed by the
same method as for 3. The ECD spectrum of 4 showed a
similar pattern of Cotton effects (positive at 224 and 282 nm
and negative at 246 nm) to those of 3, which confirmed the
same absolute configuration as 3. Thus, the structure of 4 was
determined as (8S,7′R,8′S)-5,5′-dimethoxylariciresinol 9′-O-α-
L-rhamnopyranoside and named chaenomiside D.
1
The H and 13C NMR data of 5 resembled those of 4, but
1
Figure 1. Key correlations of H−1H COSY (bold lines) and HMBC
there were slight shifts in the signals of H-9′ [5, δH 3.89 (1H,
overlap, H-9′a) and 3.69 (1H, overlap, H-9′b); 4, δH 4.00 (1H,
dd, J = 9.9, 6.7 Hz, H-9′a) and 3.54 (1H, dd, J = 9.9, 6.5 Hz, H-
9′b)], indicating that 5 could be a stereoisomer of 4. The planar
structure of 5 was determined by 2D NMR (1H−1H COSY,
HMQC, and HMBC) spectroscopic analysis. The ECD spectra
of 4 and 5 were opposite from 220 to 285 nm (negative CEs at
(arrows) of 1, 3, 4, and 6.
the positive Cotton effects at 231 and 277 nm and negative one
at 246 nm as seen in the ECD spectrum (Figure S32).21 An
extensive NMR analysis including 2D NMR data confirmed the
1
Table 2. 13C (175 MHz) and H (700 MHz) NMR Assignments of Compounds 4−6 in Methanol-d4
4
5
6
pos.
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
1
132.7
107.0
149.5
135.0
149.5
107.0
34.6
132.8
107.0
149.5
135.0
149.5
107.0
34.7
133.2
107.2
149.2
134.6
149.2
107.2
36.7
2
6.52, s
6.52, s
6.32, s
3
a
4
5
6
6.52, s
6.52, s
6.32, s
7a
7b
8
2.90, dd (13.6, 5.3)
2.58, dd (13.6, 11.2)
2.80, m
2.92, dd (13.6, 5.4)
2.57, dd (13.6, 10.8)
2.80, m
2.75, dd (13.8, 6.1)
2.54, dd (13.8, 9.2)
1.95, m
44.2
73.9
44.2
74.0
44.3
63.1
9a
9b
1′
2′
3′
4′
5′
6′
7′a
7′b
8′
9′a
9′b
4.06, dd (8.4, 6.6)
3.76, dd (8.4, 6.5)
4.04, dd (8.4, 6.7)
3.76, dd (8.4, 6.9)
3.78, overlap
3.52, dd (11.0, 7.3)
134.9
104.5
149.4
136.2
149.4
104.5
85.0
135.0
104.4
149.4
136.1
149.4
104.4
85.1
133.0
107.2
149.2
134.5
149.2
107.2
37.0
6.65, s
6.65, s
6.30, s
a
6.65, s
6.65, s
6.30, s
4.84, d (6.9)
4.81, d (6.5)
2.67, dd (13.8, 6.7)
2.60, dd (13.8, 8.8)
2.11, m
51.8
66.9
2.48, m
51.7
67.1
2.51, m
41.0
69.6
4.00, dd (9.9, 6.7)
3.54, dd (9.9, 6.5)
3.86, s
3.89, overlap
3.69, overlap
3.86, s
3.85, dd (9.9, 5.6)
3.39, overlap
a
a
a
3,5-OCH3
56.9
57.0
102.3
72.4
72.7
74.0
70.5
18.2
56.9
57.0
102.3
72.4
72.7
74.0
70.4
18.2
56.7
56.8
102.5
72.6
72.8
74.0
70.3
18.2
3.77 , s
a
3′,5′-OCH3
3.87, s
3.87, s
3.76 , s
1″
2″
3″
4″
5″
6″
4.74, d (1.4)
3.86, overlap
3.69, dd (9.5, 6.3)
3.41, t (9.5)
3.64, dq (9.5, 6.2)
1.27, d (6.2)
4.71, d (1.5)
3.83, dd (3.3, 1.5)
3.67, overlap
3.40, t (9.3)
3.60, dq (9.3, 6.1)
1.27, d (6.1)
4.65, d (1.5)
3.86, dd (3.3, 1.5)
3.72, dd (9.4, 3.3)
3.42, t (9.4)
3.67, dq (9.4, 6.2)
1.28, d (6.2)
a
Exchangeable peaks.
C
DOI: 10.1021/acs.jnatprod.5b00090
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Note
223 and 271 nm and a positive CE at 244 nm), suggesting that
the aglycone moieties of 4 and 5 were enantiomeric. Thus, the
structure of 5 was determined as (8R,7′S,8′R)-5,5′-dimethox-
ylariciresinol 9′-O-α-L-rhamnopyranoside and named chaeno-
miside E.
ma), and HCT-15 (colon adenocarcinoma), using the SRB
bioassay,23 but they were inactive (IC50 > 10.0 μM).
This work shows that the twigs of C. sinensis are rich in lignan
glycosides, the main bioactive constituents with anti-inflamma-
tory and neuroprotective activities. In particular, chaenomiside
F (6), which exhibited a potent inhibitory effect on NO
production in LPS-stimulated BV-2 cells and NGF secretion
activity in C6 cells, could be useful for the development of
novel anti-inflammatory and neuroprotective agents.
The molecular formula of 6 was determined to be C28H40O12
from the [M − H]− ion in the negative ion HRFABMS and 13
C
NMR data. The 1H and 13C NMR spectra were similar to those
of (8S,8′S)-bisdihydrosiringenin,22 except for the presence of α-
rhamnose signals [δH 4.65 (1H, d, J = 1.5 Hz, H-1″), 3.86 (1H,
dd, J = 3.3, 1.5, H-2″), 3.72 (1H, dd, J = 9.4, 3.3, H-3″), 3.67
(1H, dq, J = 9.4, 6.2 Hz, H-5″), 3.42 (1H, t, J = 9.4 Hz, H-4″),
and 1.28 (1H, d, J = 6.2 Hz, H-6″); δC 102.5 (C-1″), 74.0 (C-
4″), 72.8 (C-3″), 72.6 (C-2″), 70.3 (C-5″), and 18.2 (C-6″)]
and a deshielded signal of C-9′ [6, δC 69.6; (8S,8′S)-
bisdihydrosiringenin, δC 61.7], indicating the location of the
sugar moiety at C-9′. This was corroborated by the HMBC
correlation of H-1″ to C-9′. Enzymatic hydrolysis of 6 yielded
the aglycone (8S,8′S)-bisdihydrosiringenin (6a), whose 1H
NMR, FABMS, and negative specific rotation {[α]2D5 +30.2 (c
0.05, MeOH)} were in good agreement with the reported
value,22 and L-rhamnose was identified by the same method as
1. Thus, the structure of 6 was established as (8S,8′S)-
bisdihydrosiringenin 9-O-α-L-rhamnopyranoside and named
chaenomiside F.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
recorded with a Jasco P-1020 polarimeter. UV spectra were acquired
on an Agilent 8453 UV−visible spectrophotometer. IR spectra were
measured with a Bruker IFS-66/S FT-IR spectrometer. ECD spectra
were recorded with a JASCO J-810 spectropolarimeter. NMR spectra
were recorded in methanol-d4 solutions on a Bruker AVANCE III 700
NMR spectrometer operating at 700 MHz (1H) and 175 MHz (13C).
HRFABMS were measured with a Waters SYNAPT G2 and a JEOL
JMS700 mass spectrometer. Semipreparative HPLC was performed on
a Gilson 306 pump equipped with a Shodex refractive index detector
at a flow rate of 2 mL/min. Silica gel 60 (Merck, 230−400 mesh) and
RP-C18 silica gel (Merck, 230−400 mesh) were used for column
chromatography. LPLC was performed over a LiChroprep Lobar-A Si
60 column (Merck, 240 mm × 10 mm i.d.) equipped with an FMI
QSY-0 pump. Sephadex LH-20 (Pharmacia Co. Ltd.) was used as a
packing material for molecular sieve column chromatography.
Plant Material. Twigs of C. sinensis were collected in Seoul, Korea,
in January 2012. A voucher specimen of the plants (SKKU-NPL 1206)
was identified by one of the authors (K.R.L.) and deposited at the
herbarium of the School of Pharmacy, Sungkyunkwan University,
Suwon, Korea.
To investigate the effect of compounds (1−11) on
neuroinflammation, we measured nitric oxide (NO) levels in
murine microglia BV2 cells stimulated by lipopolysaccharide
(LPS) (Table S1). Among the isolates, compound 6
significantly reduced NO levels in the medium with an IC50
value of 21.3 μM, which displayed more activity than the
positive control, L-NMMA (IC50 24.8 μM). Compounds 5 and
9−11 exhibited moderate activities with IC50 values ranging
from 29.8 to 40.2 μM. None of the compounds showed any
significant cellular toxicity up to 20 μM. Interestingly, although
the chemical structures of compounds 4 and 5 were quite
similar, they differed substantially with respect to their
inhibitory effect on NO production (4, IC50 199.3 μM; 5,
IC50 29.8 μM). The data suggest that the 8R,7′S,8′R-form of
the aglycone moiety in tetrahydrofuran-type lignans is
important for the NO inhibitory activity, which was supported
by the weak activity of 3 (IC50 179.7 μM), possessing an
(8S,7′R,8′S)-tetrahydrofuran moiety.
We also evaluated the neuroprotective activities of the
isolated compounds (1−11) by determining their effects on
NGF secretion in C6 cells (Table S2). Of the tested
compounds at 20 μM, 1, 3, and 6 were potent stimulants of
NGF release with stimulation levels of 151.74 6.77%, 144.31
7.49%, and 167.61 18.5%, respectively (positive control 6-
shogaol was 141.75 9.43%), while compounds 5, 7, and 11
displayed moderate activities, with NGF secretion levels of
Extraction and Isolation. Twigs of C. sinensis (7.0 kg) were
extracted three times with 80% aqueous MeOH (each 10 L × 1 day)
under reflux and filtered. The filtrate was evaporated under vacuum to
obtain a MeOH extract (320 g), which was suspended in distilled H2O
and successively partitioned with n-hexane, CHCl3, EtOAc, and n-
BuOH, to yield 3, 15, 6, and 30 g fractions, respectively. The EtOAc-
soluble layer (6 g) was applied to a Sephadex LH-20 column with a
solvent system of 90% aqueous MeOH to yield five subfractions (E1−
E5). Fraction E1 (3.8 g) was subjected to silica gel column
chromatography (CHCl3−MeOH, 10:1) to give 10 subfractions
(E1A−E1J). Fraction E1E (400 mg) was chromatographed over a
Lobar-A RP-18 column (60% aqueous MeOH) to give five
subfractions (E1E1−E1E5), and fraction E1E1 (170 mg) was further
separated on a Lobar-A RP-18 (50% MeOH(aq)) to give five
subfractions (E1E1A−E1E1E). Compounds 4 (5 mg) and 5 (5 mg),
and 2 (5 mg) and 3 (4 mg) were acquired by purification of fractions
E1E1D (30 mg) and E1E1E (21 mg) using semipreparative HPLC
(35% MeOH(aq)), respectively. Fraction E1F (280 mg) was separated
over a Lobar-A RP-18 with a solvent system of 50% aqueous MeOH
and further purified by semipreparative HPLC (35−50% MeOH(aq))
to yield compounds 1 (6 mg), 8 (8 mg), and 10 (20 mg). Fraction
E1G (350 mg) was subjected to a Lobar-A RP-18 with a solvent
system of 40% MeOH(aq) and purified by semipreparative HPLC
(40−50% MeOH(aq)) to yield compounds 6 (2 mg) and 11 (20 mg).
Fraction E1I (120 mg) was chromatographed on a Lobar-A RP-18
with a solvent system of 50% MeOH(aq) and purified by
semipreparative HPLC (40−50% MeOH(aq)) to yield compounds
7 (10 mg) and 9 (6 mg).
124.67
7.80%, 140.04
16.06%, and 123.06
1.36%,
respectively. Among the dihydrobenzofuran-type neolignans
possessing a rhamnosyl unit at C-9 (1, 2, and 8), 1 was the
most potent stimulant of NGF release (1, 151.74 6.77%; 2,
106.85 4.25%; 8, 102.88 6.40%), which implies that both
the 7S,8R-configuration and a C-5 methoxy group were
essential for an effect on NGF release.
The antiproliferative activities of compounds 1−11 were
evaluated by determining their inhibitory effects on four human
tumor cell lines, namely, A549 (non-small-cell lung carcinoma),
SK-OV-3 (ovary malignant ascites), SK-MEL-2 (skin melano-
Chaenomiside A (1): colorless gum; [α]2D5 −6.4 (c 0.2, MeOH); IR
(KBr) νmax 3358, 2945, 2832, 1452, 1033 cm−1; UV (MeOH) λmax (log
ε) 280 (1.31), 231 (4.03) nm; CD (MeOH) λmax (Δε) 289 (+5.12),
1
246 (+7.92), 222 (−2.91) nm; H and 13C NMR data, see Table 1;
negative HRFABMS m/z 535.2174 [M − H]− (calcd for C27H35O11,
535.2179).
Chaenomiside B (2): colorless gum; [α]2D5 +10 (c 0.1, MeOH); IR
(KBr) νmax 3359, 2946, 2831, 1451, 1032 cm−1; UV (MeOH) λmax (log
D
DOI: 10.1021/acs.jnatprod.5b00090
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Note
ε) 282 (1.36), 231 (4.11) nm; CD (MeOH) λmax (Δε) 293 (−3.79),
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ASSOCIATED CONTENT
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S
* Supporting Information
(23) Nguyen, X. N.; Lee, H. Y.; Kim, N. Y.; Park, S. J.; Kim, E. S.;
Han, J. E.; Yang, H.; Kim, S. H. Magn. Reson. Chem. 2012, 50, 772−
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1D and 2D NMR data of 1−6; enzymatic hydrolysis and
bioassay procedures. The material is available free of charge via
the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: 82-31-290-7710. Fax: 82-31-290-7730. E-mail: krlee@
skku.edu.
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 (2013R1A1A2A10005315). We are thankful to the
Korea Basic Science Institute (KBSI) for acquiring the MS data.
E
DOI: 10.1021/acs.jnatprod.5b00090
J. Nat. Prod. XXXX, XXX, XXX−XXX