New Butyrolactone Type Lignans from Arctii Fructus and Their Anti-inflammatory Activities

Article abstract of DOI:10.1021/acs.jafc.5b02838

Arctiidilactone (1), a novel rare butyrolactone lignan with a 6-carboxyl-2-pyrone moiety, and 11 new butyrolactone lignans (2-12) were isolated from the fruits of Arctium lappa L., together with 5 known compounds (13-17). Their structures were elucidated by interpretation of their spectroscopic data (1D and 2D NMR, UV, IR, ORD, and HRESIMS) and comparison to literature data. The absolute configurations of compounds 1-12 were determined by a combination of rotating-frame nuclear Overhauser effect spectroscopy (ROESY), circular dichroism (CD) spectroscopy, and Rh₂(OCOCF₃)₄-induced CD spectroscopy. All of the compounds were tested for their anti-inflammatory properties in terms of suppressing the production of NO in lipopolysaccharide-induced BV2 cells. Compounds 1, 6, 8, and 10 exhibited stronger anti-inflammatory effects than the positive control curcumin, particularly 1, which exhibited 75.51, 70.72, and 61.17% inhibition at 10, 1, and 0.1 μM, respectively.

Full text of DOI:10.1021/acs.jafc.5b02838

Article
pubs.acs.org/JAFC
New Butyrolactone Type Lignans from Arctii Fructus and Their Anti-
inflammatory Activities
Ya-Nan Yang,∥ Xiao-Ying Huang,∥ Zi-Ming Feng, Jian-Shuang Jiang, and Pei-Cheng Zhang*
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of
Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China
S
* Supporting Information
ABSTRACT: Arctiidilactone (1), a novel rare butyrolactone lignan with a 6-carboxyl-2-pyrone moiety, and 11 new
butyrolactone lignans (2−12) were isolated from the fruits of Arctium lappa L., together with 5 known compounds (13−17).
Their structures were elucidated by interpretation of their spectroscopic data (1D and 2D NMR, UV, IR, ORD, and HRESIMS)
and comparison to literature data. The absolute configurations of compounds 1−12 were determined by a combination of
rotating-frame nuclear Overhauser effect spectroscopy (ROESY), circular dichroism (CD) spectroscopy, and Rh2(OCOCF3)4-
induced CD spectroscopy. All of the compounds were tested for their anti-inflammatory properties in terms of suppressing the
production of NO in lipopolysaccharide-induced BV2 cells. Compounds 1, 6, 8, and 10 exhibited stronger anti-inflammatory
effects than the positive control curcumin, particularly 1, which exhibited 75.51, 70.72, and 61.17% inhibition at 10, 1, and 0.1
μM, respectively.
KEYWORDS: Arctium lappa L., Arctii Fructus, lignan, butyrolactone lignan, anti-inflammatory activity
INTRODUCTION
MATERIALS AND METHODS
■
■
General Apparatus and Chemicals. The optical rotations were
measured on a Jasco P-2000 polarimeter (Jasco Corp., Tokyo, Japan).
The UV spectra were recorded on a Jasco V650 spectrophotometer
(Jasco Corp.). The IR spectra were measured on a Nicolet 5700
spectrometer (Thermo Scientific, Waltham, MA, USA). The CD
spectra were recorded on a Jasco J-815 CD spectrometer (Jasco
Corp.). HRESIMS was recorded on an Agilent 1100 series LC-MSD
ion trap mass spectrometer (Agilent Technologies, Waldbronn,
Germany). 1D NMR and 2D NMR spectra were performed on a
Bruker Avance 500 MHz NMR spectrometer (Bruker-Biospin,
Billerica, MA, USA). Column chromatography was carried out with
Sephadex LH-20 (Pharmacia Fine Chemicals, Uppsala, Sweden) and
macroporous resin (Diaion HP-20, Mitsubishi Chemical Corp., Tokyo,
Japan). Flash chromatography was set up on Combiflash RF200
(Teledyne Isco Corp., Lincoln, NE, USA). Preparative high-perform-
ance liquid chromatography (P-HPLC) was performed on a Shimadzu
LC-6AD instrument with an SPD-20A detector (Shimadzu Corp.,
Tokyo, Japan), using a YMC-Pack ODS-A column (250 × 20 mm, 5
μm; YMC Corp., Kyoto, Japan). High-performance liquid chromatog-
raphy diode array detection (HPLC-DAD) analysis was set up on an
Agilent 1260 series system (Agilent Technologies) with an Apollo C18
column (250 × 4.6 mm, 5 μm; Alltech Corp., Lexington, KY, USA).
Plant Material. Arctii Fructus was collected in October 2011 in
Wuchang town, Heilongjiang province, China. The plant material was
identified as the fruits of A. lappa L. by Professor Lin Ma (Institute of
Materia Medica, Peking Union Medical College and Chinese Academy
of Medical Sciences, Beijing, China). A voucher specimen (ID-S-2434)
has been put in the Herbarium of the Department of Chemistry of
Natural Products, Institute of Materia Media, Chinese Academy of
Medical Sciences and Peking Union Medical College, Beijing, China.
Arctii Fructus is the dried mature fruits of Arctium lappa L.,
which is a vegetable for health care in China and Japan. It has
traditionally been used as a health tea to treat tonsillitis,
pharyngolaryngitis, and constipation in China. Modern
pharmacological studies on Arctii Fructus have revealed a
broad range of biological activities, such as anti-influenza,1
cytotoxic,2,3 antidiabetic,4 anti-inflammatory,5 and gastropro-
tective activities.6 Phytochemical studies on Arctii Fructus have
already yielded many types of chemicals, including lignans,7
phenols,8 and fatty acids.9 In our previous study on the H2O-
soluble fraction of an EtOH extract of Arctii Fructus, we
reported 12 novel 7′-hydroxy lignan glucosides and their
hepatoprotective activity.10
In our further investigation of the bioactive compounds from
Arctii Fructus, we isolated a novel rare butyrolactone lignan
with a 6-carboxyl-2-pyrone moiety, named arctiidilactone (1); a
new apolignan glucoside without an aromatic ring, named
arctiiapolignan A (2); a new sesquineolignan with a
dihydrobenzofuran unit and a butyrolactone unit, named
arctiisesquineolignan A (3); three new dibenzylbutyrolactone
lignans (4−6); and six new dibenzylbutyrolactone lignans with
an additional hydroxyl at C-7/7′ (7−12), together with five
known compounds (13−17). Their structures and absolute
configurations were characterized on the basis of spectroscopic
data, including CD and Rh2(OCOCF3)4-induced CD spectra.
In consideration of our previous report for the bioactivity of
lignan glucosides,10 the hepatoprotective activities of 1−17
were tested through in vitro assays. Furthermore, tea made
from Arctii Fructus is traditionally used to treat tonsillitis, so
the anti-inflammatory and cytotoxic activities of compounds 1−
17 are described in this paper.
Received: June 8, 2015
Revised: August 4, 2015
Accepted: August 27, 2015
© XXXX American Chemical Society
A
DOI: 10.1021/acs.jafc.5b02838
J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry
Article
Figure 1. Structures of compounds 1−17.
Extraction and Isolation. Air-dried mature fruits (100 kg) of A.
lappa L. were extracted with 80% EtOH (3 × 600 L) under reflux
three times. The solvent was evaporated under reduced pressure.
Then, the residue (4.2 kg) was suspended in H2O (10 L) and
extracted with EtOAc (3 × 10 L). The aqueous layer (400 g) was
subjected to an HP-20 macroporous adsorbent resin (4000 g) column
and eluted with H2O (20 L), 15% EtOH (20 L), 30% EtOH (20 L),
50% EtOH (20 L), and 95% EtOH (10 L), which yielded five
corresponding fractions (fractions A−E).
reverse-phase preparative HPLC with MeOH/H2O (35:65) as the
mobile phase at a flow rate of 5 mL/min to afford 8 (4 mg, 27 min).
The structures of compounds 1−17 are shown in Figure 1.
Spectral Data. Arctiidilactone (1): white amorphous powder; [α]2D5
−28.9 (c 0.26, MeOH); CD (MeOH) λmax (Δε) 236 (−1.21), 279
(−0.34) nm; UV (MeOH) λmax (log ε) 230 (3.51), 285 (3.29), 305
(3.22) nm; IR (KBr) νmax 3364, 2922, 2851, 1765, 1712, 1640, 1515,
1465, 1391, 1263, 1238, 1157, 1077, 1024 cm−1; (+)-HRESIMS m/z
1
389.1235 [M + H]+ (calcd for C20H21O8, 389.1236). For H and 13C
NMR spectroscopic data, see Tables 1 and 2.
Fraction C (86.4 g) was submitted to flash chromatography with a
C18 column (55 × 8 cm, 20 μm), eluting with a 10-step gradient of
MeOH/H2O (2 L for one step, from 0:100 to 100:0) to provide 13
fractions (fractions C1−C13). Fraction C11 (301 mg) was separated
on a Sephadex LH-20 (100 g) column, eluting with H2O (1.2 L) to
yield four fractions (fractions C11-1−C11-4). Then fraction C11-1 (25
mg) was further purified by reverse-phase preparative HPLC, eluting
with MeOH/H2O (25:75) at a flow rate of 5 mL/min to afford 1 (5
mg, 48 min). Fraction C12 (812 mg) was separated on a Sephadex
LH-20 column (H2O, 1.5 L) and then further purified by reverse-
phase preparative HPLC, eluting with MeOH/H2O (40:60) at a flow
rate of 5 mL/min to afford 2 (27 mg, 45 min), 12 (5 mg, 51 min), and
13 (33 mg, 32 min). Fraction C13 (1.695 g) was subjected to a
Sephadex LH-20 column and eluted with H2O (2 L) to give eight
fractions (fractions C13-1−C13-8). Purification of fraction C13-3 (97
mg) with MeOH/H2O (30:70) on reverse-phase preparative HPLC at
a flow rate of 5 mL/min afforded 6 (10 mg, 37 min) and 7 (13 mg, 49
min). Fraction C13-5 (255 mg) was applied successively to
chromatography on Sephadex LH-20 (H2O, 1.2 L) to yield six
fractions (fractions C13-5-1−C13-5-6). Compound 14 (100 mg, 57
min) was obtained from fraction C13-5-3 (188 mg) on reverse-phase
preparative HPLC using MeOH/H2O (27:78) at a flow rate of 5 mL/
min. Fraction C13-8 (127 mg) was applied successively to
chromatography on Sephadex LH-20 (H2O, 1 L) to yield five
fractions (fractions C13-8-1−C13-8-5). Fraction C13-8-3 (157 mg)
was performed on reverse-phase preparative HPLC with MeOH/H2O
(30:70) at a flow rate of 5 mL/min to afford 9 (80 mg, 47 min) and 11
(10 mg, 57 min).
Fraction D (210 g) was submitted to flash chromatography with a
C18 column (55 × 8 cm, 20 μm), eluting with a 10-step gradient of
MeOH/H2O (4 L for one-step, from 0:100 to 100:0) to yield 40
fractions (fractions D1−D40). Fraction D27 (1.4 g) was separated on
a Sephadex LH-20 column and eluted with H2O (1.5 L) to give six
fractions (fractions D27-1−D27-6). Fraction D27-3 (316 mg) was
subjected to reverse-phase preparative HPLC, eluting with MeOH/
H2O (35:65) as the mobile phase at a flow rate of 5 mL/min, to afford
4 (7 mg, 23 min), 5 (6 mg, 31 min), 10 (5 mg, 44 min), and 17 (24
mg, 59 min). Fraction D13 (2.3 g) was separated into four fractions
(fractions D13-1−D13-4) on Sephadex LH-20 eluted with H2O (2 L).
Fraction D13-1 (42 mg) gave 3 (10 mg, 35 min) after purification over
a reverse-phase preparative HPLC column with MeOH/H2O (40:60)
at a flow rate of 5 mL/min. Fraction D13-2 (116 mg), with Sephadex
LH-20 (H2O, 1 L) and reverse-phase preparative HPLC with MeOH/
H2O (45:55) at a flow rate of 5 mL/min, yielded 15 (5 mg, 25 min)
and 16 (82 mg, 42 min). Fraction D13-4 (29 mg) was purified via
Arctiiapolignan A (2): white amorphous powder; [α]D25 +6.97 (c
0.18, MeOH); CD (MeOH) λmax (Δε) 227 (−4.39) nm; UV
(MeOH) λmax (log ε) 230 (3.10), 280 (2.70) nm; IR (KBr) νmax 3403,
2912, 1764, 1591, 1515, 1464, 1420, 1263, 1238, 1159, 1078, 1029
cm−1; (+)-HRESIMS m/z 451.1584 [M + Na]+ (calcd for
1
C20H28O10Na, 451.1580). For H and 13C NMR spectroscopic data,
see Tables 1 and 2.
Arctiisesquineolignan A (3): white amorphous powder; [α]2D5
+18.54 (c 0.15, MeOH); CD (MeOH) λmax (Δε) 230 (−3.55), 248
(+1.2), 275 (−0.58), 295 (+0.39) nm; UV (MeOH) λmax (log ε) 228
(3.87), 279 (3.42) nm; IR (KBr) νmax 3413, 2921, 1760, 1598, 1514,
1454, 1421, 1266, 1226, 1141, 1075, 1030 cm−1; (+)-HRESIMS m/z
883.2981 [M + Na]+ (calcd for C42H52O19Na, 883.3000). For 1H and
13C NMR spectroscopic data, see Tables 1 and 2.
Arctigenin-4-O-α-D-galactopyranosyl-(1→6)-O-β-D-glucopyrano-
side (4): white amorphous powder; [α]D25 +6.3 (c 0.07, MeOH); CD
(MeOH) λmax (Δε) 230 (−3.02), 272 (−0.34) nm; UV (MeOH) λmax
(log ε) 230 (3.12), 278 (2.69) nm; IR (KBr) νmax 3382, 2926, 1761,
1593, 1515, 1454, 1421, 1265, 1235, 1155, 1081, 1029 cm−1;
(+)-HRESIMS m/z 719.2529 [M + Na]+ (calcd for C33H44O16Na,
719.2527). For 1H and 13C NMR spectroscopic data, see Tables 1 and
2.
Arctigenin-4-O-β-D-apiofuranosyl-(1→6)-O-β-D-glucopyranoside
(5): white amorphous powder; [α]D25 −52.6 (c 0.12, MeOH); CD
(MeOH) λmax (Δε) 231 (−6.82), 274 (−0.78) nm; UV (MeOH) λmax
(log ε) 229 (3.58), 278 (3.19) nm; IR (KBr) νmax 3418, 2929, 1760,
1592, 1515, 1454, 1421, 1265, 1235, 1158, 1068, 1025 cm−1;
(+)-HRESIMS m/z 689.2430 [M + Na]+ (calcd for C32H42O15Na,
689.2421). For 1H and 13C NMR spectroscopic data, see Tables 1 and
2.
5′-Propanediolmatairesinoside (6): white amorphous powder;
[α]D25 −53.4 (c 0.11, MeOH); CD (MeOH) λmax (Δε) 229 (−6.49),
278 (−1.19) nm; UV (MeOH) λmax (log ε) 230 (3.48), 280 (3.10)
nm; IR (KBr) νmax 3426, 2928, 1758, 1597, 1513, 1461, 1422, 1267,
1228, 1157, 1072, 1030 cm−1; (+)-HRESIMS m/z 617.2207 [M +
Na]+ (calcd for C29H38O13Na, 617.2210). For 1H and 13C NMR
spectroscopic data, see Tables 1 and 2.
(7′R,8R,8′R)-Rafanotrachelogenin-4-O-β-D-glucopyranoside (7):
white amorphous powder; [α]2D5 −13.6 (c 0.12, MeOH); CD
(MeOH) λmax (Δε) 230 (−2.00), 267 (−0.22) nm; UV (MeOH)
λmax (log ε) 230 (3.41), 277 (3.08) nm; IR (KBr) νmax 3423, 2931,
1759, 1594, 1514, 1463, 1421, 1266, 1232, 1156, 1075, 1027 cm−1;
(+)-HRESIMS m/z 573.1954 [M + Na]+ (calcd for C27H34O12Na,
573.1948). For 1H and 13C NMR spectroscopic data, see Tables 2 and
3.
B
DOI: 10.1021/acs.jafc.5b02838
J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry
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1
Table 1. H NMR Data for 1 in Acetic Acid-d4 and for 2−6 in DMSO-d6 (500 MHz)
no.
1
2
3
4
5
6
2
3
5
6
7
6.81, brs
6.78, d (1.5)
6.77, d (2.0)
6.77, d (2.0)
6.79, d (1.5)
6.41, s
7.01, s
6.84, d (8.0)
6.98, d (8.5)
6.68, dd (8.5, 1.5)
2.82, m
7.04, d (8.5)
6.98, d (8.5)
6.97, d (8.5)
6.64, dd (8.5, 1.5)
2.81, m
6.73, d (8.0)
6.70, dd (8.5, 2.0)
2.85, dd (13.5, 5.5)
2.75, dd (13.5, 7.0)
2.69, m
6.70, dd (8.5, 2.0)
2.83, dd (13.5, 5.5)
2.76, dd (13.5, 7.0)
2.69, m
2.82, dd (13.5, 6.5)
2.65, dd (13.5, 8.5)
3.03, m
8
2.83, m
2.69, m
2.74, m
2.72, m
9
2.89, brdd (8.0, 5.0)
4.22, dd (8.5, 8.5)
3.89, dd (8.5, 8.5)
4.04, dd (10.5, 3.0)
3.33, dd (10.5, 3.0)
2.60, m
1′
2′
5′
6′
7′
6.73, d (1.5)
6.82, d (8.0)
6.68, dd (8.0, 1.5)
2.80, m
6.56, brs
6.62, d (1.5)
6.62, d (2.0)
6.81, d (8.0)
6.58, dd (8.0, 2.0)
2.47, m
6.48, brs
6.81, d (8.0)
6.60, brs
6.57, dd (8.0, 1.5)
2.47, overlap
6.47, brs
2.56, m
2.52, m
2.71, m
2.46, m
2.42, overlap
2.42, overlap
4.07, dd (8.5, 8.5)
3.84, dd (8.5, 8.5)
3.14, m
8′
9′
2.63, m
2.44, overlap
4.11, dd (8.5, 8.5)
3.87, dd (8.5, 8.5)
2.47, overlap
4.10, dd (8.5, 8.5)
3.88, dd (8.5, 8.5)
4.75, d (7.5)
3.24, m
2.50, overlap
4.09, dd (8.5, 8.5)
3.88, dd (8.5, 8.5)
4.79, d (7.5)
3.22, m
4.46, dd (8.0, 9.0)
4.08, dd (8.0, 9.5)
1″
2″
3.95, d (7.5)
2.93, m
6.94, d (2.0)
3.64, m
3.57, m
3″
3.01, m
3.25, m
3.23, m
3.64, m
3.57, m
4″
5″
6″
3.04, m
3.14, m
3.08, m
3.10, m
7.04, d (8.5)
3.48, m
3.43, m
3.63 dd (11.5, 5.5)
3.43, m
6.83, dd (8.5, 2.0)
3.62, overlap
3.54, overlap
3.82, dd (9.5, 4.5)
3.43, m
7″
8″
9″
5.46, d (4.5)
3.42, m
3.68, m
3.59, m
1‴
2‴
3‴
4‴
4.87, d (7.5)
3.22, m
4.64, d (3.0)
3.59, m
4.80, d (2.0)
3.72, m
4.82, d (7.5)
3.23, m
3.27, m
3.60, m
3.24, m
3.14, m
3.64, m
3.85, d (9.5)
3.57, d (9.5)
3.33, overlap
3.14, m
5‴
6‴
3.25, m
3.57, m
3.39, m
3.24, m
3.64, overlap
3.42, overlap
4.82, d (7.5)
3.22, m
3.64, overlap
3.43, m
1″″
2″″
3″″
4″″
5″″
6″″
3.27, m
3.14, m
3.25, m
3.64, overlap
3.43, m
3-CH3O
4-CH3O
3′-CH3O
4′-CH3O
3″-CH3O
3.81, s
3.81, s
3.74, s
3.70, s
3.70, s
3.69, s
3.70, s
3.71, s
3.71, s
3.73, s
3.72, s
3.68, s
3.70, s
3.69, s
3.68, s
(7′S,8R,8′R)-Rafanotrachelogenin-4-O-β-D-glucopyranoside (8):
white amorphous powder; [α]2D5 −19.2 (c 0.23, MeOH); CD
(MeOH) λmax (Δε) 232 (−4.42), 283 (+1.15) nm; UV (MeOH)
λmax (log ε): 228 (3.04), 278 (2.68) nm; IR (KBr) νmax 3423, 2931,
1759, 1514, 1463, 1421, 1266, 1232, 1156, 1075, 1027 cm−1;
(+)-HRESIMS m/z 573.1956 [M + Na]+ (calcd for C27H34O12Na,
573.1948). For 1H and 13C NMR spectroscopic data, see Tables 2 and
3.
(7S,8S,8′R)-4,7-Dihydroxy-3,3′,4′-trimethoxyl-9-oxo dibenzylbu-
tyrolactone lignan-4-O-β-D-glucopyranoside (9): white amorphous
powder; [α]D25 −48.5 (c 0.21, MeOH); CD (MeOH) λmax (Δε) 230
(−55.97), 278 (−5.38) nm; UV (MeOH) λmax (log ε) 230 (3.48), 278
(3.04) nm; IR (KBr) νmax 3390, 2936, 1757, 1595, 1515, 1454, 1419,
C
DOI: 10.1021/acs.jafc.5b02838
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Journal of Agricultural and Food Chemistry
Article
Table 2. 13C NMR Data for 1 in Acetic acid-d4 and for 2−12 in DMSO-d6 (125 MHz)
no.
1
2
3
4
5
6
7
8
9
10
11
12
1
131.7
112.9
149.2
147.8
112.3
121.9
37.3
132.3
114.3
149.1
145.8
115.6
121.7
33.9
132.5
114.2
149.2
145.7
116.4
121.8
34.3
132.4
114.2
149.1
145.7
115.8
121.9
34.2
132.3
114.3
149.0
145.7
115.5
121.8
33.8
132.0
114.3
149.0
145.7
115.5
121.8
34.2
131.9
114.2
148.9
145.7
115.3
121.8
34.8
137.1
110.3
149.0
145.9
115.3
117.9
70.5
134.2
110.1
147.8
145.8
115.4
118.1
70.4
136.6
111.2
148.8
146.1
115.1
118.7
71.0
136.6
111.2
148.8
146.0
115.1
118.7
71.8
2
161.5
118.7
155.8
112.5
148.2
161.9
34.4
3
4
5
6
7
8
38.0
46.0
46.0
46.0
45.9
43.2
42.8
52.8
52.8
52.5
52.5
9
44.0
71.5
178.9
178.9
178.9
179.0
179.1
179.3
178.1
178.0
176.6
176.6
10
1′
2′
3′
4′
5′
6′
7′
8′
9′
1″
2″
3″
4″
5″
6″
7″
8″
9″
1‴
2‴
3‴
4‴
5‴
6‴
179.2
130.5
111.5
149.0
147.8
112.2
120.8
37.9
66.9
46.0
132.2
113.1
144.0
146.5
129.5
117.3
37.5
131.5
112.8
149.1
147.8
112.3
120.9
37.4
131.5
112.8
149.1
147.8
112.4
120.8
37.4
128.8
110.4
147.7
143.1
128.5
121.1
37.5
136.4
110.1
149.0
148.4
112.0
118.3
72.4
135.9
110.0
149.0
148.3
111.8
118.4
72.8
131.4
112.6
149.2
147.6
112.2
120.7
38.9
131.6
112.6
149.0
147.6
112.1
120.6
38.8
131.8
113.0
149.1
147.8
112.7
121.0
38.1
130.1
113.3
148.0
145.4
115.9
121.3
38.2
177.7
41.7
41.4
41.2
41.2
41.2
46.3
45.4
36.1
35.8
39.5
39.5
72.0
71.2
71.2
71.2
71.1
68.0
68.9
72.1
71.8
71.8
71.0
104.3
73.9
77.3
70.4
77.1
61.4
135.8
110.9
149.4
146.6
115.7
118.4
87.2
101.1
73.7
100.8
73.7
44.3
100.6
73.7
100.6
73.7
100.7
73.8
100.6
73.7
100.6
73.7
62.2
77.5
77.3
62.2
77.5
77.5
77.5
77.5
77.4
70.5
70.4
70.1
70.2
70.2
70.1
70.1
75.4
76.0
77.3
77.4
77.4
77.4
77.4
66.8
68.1
61.1
61.2
61.2
61.1
61.0
53.8
63.5
100.7
73.7
99.1
70.0
69.4
68.8
71.3
61.0
109.8
76.4
79.2
73.8
63.7
100.7
73.7
77.4
70.1
77.3
61.1
77.5
70.1
77.3
61.1
1″″
2″″
100.5
73.7
3″″
77.4
4″″
70.1
5″″
77.3
6″″
61.1
3-CH3O
4-CH3O
3′-CH3O
4′-CH3O
3″-CH3O
55.0
54.9
55.9
56.0
56.2
55.9
56.1
56.2
56.1
55.9
55.8
55.8
55.9
55.9
56.0
56.1
56.1
56.1
55.8
56.1
55.9
55.9
56.1
56.0
55.9
56.0
56.1
55.9
55.7
56.0
55.9
56.1
1264, 1234, 1141, 1077, 1028 cm−1; (+)-HRESIMS m/z 573.1949 [M
1265, 1235, 1156, 1076, 1028 cm−1; (+)-HRESIMS m/z 573.1950 [M
+ Na]+ (calcd for C27H34O12Na, 573.1948). For H and 13C NMR
1
1
+ Na]+ (calcd for C27H34O12Na, 573.1948). For H and 13C NMR
spectroscopic data, see Tables 2 and 3.
spectroscopic data, see Tables 2 and 3.
(7R,8S,8′R)-4,7,4′-Trihydroxy-3,3′-dimethoxyl-9-oxo dibenzylbu-
tyrolactone lignan-4-O-β-D-glucopyranoside (12): white amorphous
powder; [α]D25 −37.5 (c 0.35, MeOH); CD (MeOH) λmax (Δε) 227
(−0.89), 275 (−0.36) nm; UV (MeOH) λmax (log ε) 230 (3.27), 280
(2.90) nm; IR (KBr) νmax 3392, 2917, 1755, 1601, 1514, 1454, 1426,
1270, 1226, 1156, 1075, 1031 cm−1; (+)-HRESIMS m/z 559.1795 [M
(7S,8S,8′R)-4,7-Dihydroxy-3,3′,4′-trimethoxyl-9-oxo dibenzylbu-
tyrolactone lignan (10): white amorphous powder; [α]D25 −44.3 (c
0.11, MeOH); CD (MeOH) λmax (Δε) 234 (−8.20), 285 (−0.37) nm;
UV (MeOH) λmax (log ε) 231 (2.98), 279 (2.97) nm; IR (KBr) νmax
3387, 2921, 1760, 1595, 1516, 1464, 1423, 1265, 1237, 1154, 1026
cm−1; (+)-HRESIMS m/z 411.1416 [M + Na]+ (calcd for
1
+ Na]+ (calcd for C26H32O12Na, 559.1791). For H and 13C NMR
1
C21H24O7Na, 411.1420). For H and 13C NMR spectroscopic data,
spectroscopic data, see Tables 2 and 3.
see Tables 2 and 3.
Enzymatic Hydrolysis of 2−9, 11, and 12. Compound 2 (3.0 mg)
was dissolved in 0.1 M sodium citrate buffer (5.0 mL), and then
cellulase (3.0 mg, Sigma-Aldrich, St. Louis, MO, USA) was added. The
reaction mixture was maintained at 40 °C for 5 h. The solution was
then transferred to a liquid−liquid extractor and extracted with EtOAc
(7R,8S,8′R)-4,7-Dihydroxy-3,3′,4′-trimethoxyl-9-oxo dibenzylbu-
tyrolactone lignan-4-O-β-D-glucopyranoside (11): white amorphous
powder; [α]D25 −30.4 (c 0.13, MeOH); CD (MeOH) λmax (Δε) 227
(−3.11), 276 (−1.00) nm; UV (MeOH) λmax (log ε) 229 (3.51), 279
(3.07) nm; IR (KBr) νmax 3410, 2920, 1758, 1594, 1514, 1464, 1420,
D
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1
Table 3. H NMR Data for 7−12 in DMSO-d6 (500 MHz)
no.
7
8
9
10
11
12
2
5
6
7
6.65, d (1.5)
6.60, d (2.0)
6.97, d (1.5)
6.90, d (1.5)
6.73, d (8.5)
6.79, dd (8.5, 1.5)
5.08, brs
6.90, d (1.5)
6.89, d (1.5)
7.01, d (8.0)
6.95, d (8.0)
6.89, d (8.0)
7.03, d (8.5)
7.00, d (8.5)
6.56, dd (8.0, 1.5)
2.70, dd (13.5, 6.5)
2.53, dd (13.5, 5.5)
2.83, m
6.48, dd (8.0, 2.0)
2.82, overlap
6.86, dd (8.5, 1.5)
5.10, t (4.0)
6.78, dd (8.5, 1.5)
4.83, t (4.0)
6.77, dd (8.0, 1.5)
4.81, t (3.5)
2.63, dd (15.5, 7.5)
2.82, overlap
8
2.66, dd (6.5, 2.5)
6.45, d (2.0)
6.75, d (8.5)
6.39, dd (8.5, 2.0)
2.32, dd (13.5, 9.0)
2.05, dd (13.5, 6.0)
2.78, m
2.61, dd (7.0, 3.0)
6.42, d (1.5)
2.75, dd (7.5, 4.0)
6.73, d (2.0)
6.84, d (8.0)
6.66, dd (8.0, 2.0)
2.79, dd (11.5, 4.5)
2.61, m
2.74, m
2′
5′
6′
7′
6.78, d (1.5)
6.82, d (2.0)
6.69, d (1.5)
6.66, d (8.0)
6.54, dd (8.0, 1.5)
2.74, m
6.88, d (8.0)
6.86, d (8.0)
6.71, d (8.5)
6.74, dd (8.0, 1.5)
4.36, brs
6.75, dd (8.0, 2.0)
4.60, d (5.0)
6.38, dd (8.5, 1.5)
2.28, dd (13.5, 9.5)
1.98, dd (13.5, 5.5)
2.78, m
2.60, m
8′
9′
2.47, overlap
4.14, t (8.5)
4.00, t (8.5)
4.83, d (7.5)
3.23, m
2.50, overlap
4.00, d (7.5)
2.61, m
2.60, m
4.15, t (8.5)
3.89, dd (8.5, 3.0)
4.87, d (7.5)
3.24, m
4.12, t (8.5)
3.95, t (8.5)
3.84, t (8.5)
4.87, d (7.5)
3.23, m
3.95, t (8.5)
3.82, dd (8.5, 6.5)
4.85, d (7.5)
3.23, m
3.87, dd (8.5, 6.5)
1″
2″
3″
4″
5″
6″
4.82, d (7.5)
3.23, m
3.26, m
3.28, m
3.28, m
3.27, m
3.27, m
3.14, m
3.14, m
3.16, m
3.15, m
3.15, m
3.24, m
3.23, m
3.28, m
3.27, m
3.27, m
3.65, overlap
3.43, m
3.65, overlap
3.44, m
3.65, overlap
3.46, m
3.64, dd (11.5, 4.5)
3.43, m
3.64, dd (11.5, 4.5)
3.43, m
3-CH3O
4-CH3O
3′-CH3O
4′-CH3O
3.69, s
3.67, s
3.65, s
3.67, s
3.71, s
3.71, s
3.72, s
3.71, s
3.70, s
3.73, s
3.72, s
3.68, s
3.66, s
3.72, s
3.71, s
3.70, s
3.72, s
Figure 2. Key HMBC correlations in compounds 1−3 and 7.
(5.0 mL). The organic layer was evaporated under reduced pressure
and purified using reversed-phase preparative HPLC with MeOH/
H2O (45:55) as the mobile phase to afford 2a (1.1 mg, 43 min). The
aqueous layer was evaporated and dried under reduced pressure to
yield a monosaccharide residue. This residue was purified by
preparative silica gel thin-layer chromatography (PTLC) [CHCl3/
MeOH/H2O (7:3:1)] to afford glucose (Rf = 0.2) with positive optical
rotation ([α]D25 +36.5). Hydrolysis of 3−9, 11, and 12 and sugar
identification were performed according to the procedure described
for 2. The D-galactose (Rf = 0.16, [α]2D5 + 62.8) and D-apiose (Rf = 0.5,
[α]D25 +8.7) were confirmed by TLC analysis [CHCl3/MeOH/H2O
(7:3:1)] in comparison with standard samples.
Rh2(OCOCF3)4-Induced CD Experiments of Compounds 7a and
8a. Compound 7a (0.2 mg) was dissolved in dried chloroform (1
mL). The CD spectrum of 7a in the 450−300 nm region was
measured. Then, Rh2(OCOCF3)4 (2 mg) was added in the solution,
and the mixture was stirred at room temperature for 10 min. The CD
spectrum of the reaction mixture in the 450−300 nm region was also
measured. On the basis of the bulkiness rule for secondary alcohols,11
a negative Cotton effect at 340 nm (the E band) in the
Rh2(OCOCF3)4-induced CD spectrum (Figure S49, Supporting
Information) indicated the 7′R configuration of 7a. Similarly, the 7′S
configuration of 8a was confirmed.
Anti-inflammatory Effects of Compounds. Compounds 1−17
were tested for anti-inflammatory activity on BV2 cells.12,13 After the
BV2 cells were pre-incubated for 24 h at 37 °C under a 5% CO2
atmosphere in a 96-well plate, the cells were treated with various
concentrations of the test compounds (1 × 10−5, 1 × 10−6, and 1 ×
10−7 M), followed by stimulation with LPS for 24 h. The production
of NO was determined by measuring the concentration of nitrite in the
culture supernatant. NaNO2 was used to generate a standard curve.
The absorbances at 540 nm were measured. Curcumin was utilized as
a positive control.
RESULTS AND DISCUSSION
■
Structure Elucidation of the New Compounds.
Compound 1, a white amorphous powder, had the molecular
formula C20H20O8 (m/z 389.1235 [M + H]+ in HRESIMS),
which indicated 11 degrees of unsaturation. The IR spectrum
exhibited absorption bands attributable to hydroxyl groups
(3364 cm−1), carbonyl groups (1765, 1712, and 1640 cm−1),
and aromatic rings (1515 and 1465 cm−1). In the H NMR
1
spectrum (Table 1), ABX-system aromatic proton signals [δH
6.73 (1H, d, J = 1.5 Hz, H-2), 6.82 (1H, d, J = 8.0 Hz, H-5),
and 6.68 (1H, dd, J = 8.0, 1.5 Hz, H-6)] and two proton signals
that displayed as a broad singlet [δH 6.41 (1H, s, H-3) and 7.01
E
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(1H, s, H-5)] were observed in the downfield region. In
addition, two oxygenated methylene protons at δH 4.46 (1H,
dd, J = 8.0, 9.0 Hz, H-9′a) and 4.08 (1H, dd, J = 8.0, 9.5 Hz, H-
9′b), four methylene protons at δH 2.83 (1H, m, H-8a), 2.69
(1H, m, H-8b), 2.80 (1H, m, H-7′a), and 2.71 (1H, m, H-7′b),
two methine protons at δH 2.89 (1H, brdd, J = 8.0, 5.0 Hz, H-
9) and 2.63 (1H, m, H-8′), and two methoxy groups at δH 3.81
(3H, s) and 3.81 (3H, s) were also observed. The 13C NMR
spectrum (Table 2) of 1 displayed 20 carbon signals, including
two C6−C3 units and two methoxy groups. On the basis of the
aforementioned data, compound 1 was determined as a
butyrolactone lignan similar to arctigenin, except that NMR
resonances for the 6-carboxyl-2-pyrone moiety in 1 were
observed in place of the resonances for the 4-hydroxy-3-
methoxyphenyl unit in arctigenin. This interpretation was
verified by the HMBC and 13C NMR spectra. The correlations
from H-8′ to C-8, C-9, C-10, C-1′, C-7′, and C-9′ and from H-
9 to C-4, C-8, C-10, C-7′, C-8′, and C-9′ indicated the presence
of a benzylbutyrolactone moiety in 1. Detailed analysis of the
13C NMR spectrum, together with the remaining five degrees of
unsaturation, implied the existence of a 6-carboxyl-2-pyrone
moiety; this moiety was verified by the HMBC correlations
(Figure 2) from H-3 to C-2 and C-5 and from H-5 to C-6 and
C-7. The connection between C-8 and C-4 was also determined
by the key correlations from H-8 to C-3, C-4, and C-5. The
previous study indicated that the relative configuration of H-9
and H-8′ could be determined by the ΔδH9′a−H9′b values
(ΔδH9′a−H9′b ≥ 0.2 for trans, ΔδH9′a−H9′b ≈ 0 for cis).14,15
Therefore, the trans configuration of H-9 and H-8′ was
established by the H-9′a (δH 4.46) and H-9′b (δH 4.08) signals
C42H52O19 on the basis of the positive HRESIMS ion (m/z
883.2981 [M + Na]+). The 1H NMR spectroscopy data (Table
1) of 3 revealed two sets of ABX-system aromatic rings [δH
6.78 (1H, d, J = 1.5 Hz, H-2), 6.98 (1H, d, J = 8.5 Hz, H-5),
6.68 (1H, dd, J = 8.5, 1.5 Hz, H-6), 6.94 (1H, d, J = 2.0 Hz, H-
2″), 7.04 (1H, d, J = 8.5 Hz, H-5″), 6.83 (1H, dd, J = 8.5, 2.0
Hz, H-6″)] and a 1,3,4,5-tetrasubstituted aromatic ring [δH 6.56
(1H, brs, H-2′), 6.60 (1H, brs, H-6′)]. Additionally, two
oxymethylene protons [δH 4.11 (1H, dd, J = 8.5, 8.5 Hz, H-
9′a), 3.87 (1H, dd, J = 8.5, 8.5 Hz, H-9′b), 3.68 (1H, m, H-
9″a), 3.59 (1H, m, H-9″b)], an oxymethine proton [δH 5.46
(1H, d, J = 4.5 Hz, H-7″)], two methylene protons [δH 2.82
(2H, m, H-7), 2.56 (1H, m, H-7′a), 2.46 (1H, m, H-7′b)],
three methine protons [δH 2.74 (1H, m, H-8), 2.44 (1H,
overlap, H-8′), 3.42 (1H, m, H-8″)], three methoxyl protons
[δH 3.70 (3H, s, 3-OCH3), 3.73 (3H, s, 3′-OCH3), 3.72 (3H, s,
3″-OCH3)], and two glucopyranosyl anomeric protons [δH
4.87 (1H, d, J = 7.5 Hz, H-1‴), 4.82 (1H, d, J = 7.5 Hz, H-
1″″)] were observed in the upfield region. The 13C NMR
spectrum (Table 2) of 3 displayed 42 carbon signals, 12 of
which were assigned to two glucose units; the remaining 30
carbons were assigned to three C6−C3 units and three
methoxy groups. These data revealed that 3 was a
sesquineolignan.17 In the HMBC spectrum (Figure 2), the
characteristic correlations from H-7 (δH 2.82) to C-1, C-2, C-9,
and C-8′, from H-7′ (δH 2.56, 2.46) to C-1′, C-2′, C-9′, and C-
8, and from H-7″ (δH 5.46) to C-4′, C-5′, C-1″, and C-9″
suggested the presence of a dihydrobenzofuran unit and a
benzylbutyrolactone unit in compound 3. The HMBC
correlation peaks of three methoxy groups at δH 3.70, 3.73,
and 3.72 with C-3, C-3′, and C-3″ confirmed the linkage
positions of these methoxy groups. Two glucose units were
determined to be located at C-4 and C-4″ on the basis of the
correlations of H-1‴ with C-4 and H-1″″ with C-4″ in the
HMBC spectrum. The chemical shifts of H-9′ (δH 4.11 and
3.87) indicated the trans configuration of H-8/H-8′. Moreover,
the correlation peaks of H-7″ with H-9″ and of H-8″ with H-2″
and H-6″ in the ROESY spectrum (Figure 3) indicated the
1
in the H NMR spectrum. This result, combined with the
negative Cotton effect at 230 nm in the CD spectrum,
suggested that the absolute configurations of C-9 and C-8′ in 1
were 9R and 8′R, respectively.16 Consequently, 1 was identified
as in Figure 1 and named arctiidilactone.
Compound 2 was obtained as a white amorphous powder; its
molecular formula was confirmed to be C20H28O10 on the basis
of the positive HRESIMS ion observed at m/z 451.1584 [M +
1
Na]+. The H NMR spectrum (Table 1) of 2 showed three
aromatic proton signals at δH 6.81 (1H, brs, H-2), 6.84 (1H, d,
J = 8.0 Hz, H-5), and 6.73 (1H, d, J = 8.0 Hz, H-6), revealing
the presence of an ABX-system aromatic ring. Additionally, two
methoxy groups at δH 3.74 (3H, s) and 3.70 (3H, s) and a
glucopyranosyl anomeric proton at δH 3.95 (1H, d, J = 7.5 Hz,
H-1″) were also observed in the upfield region. In the 13C
NMR spectrum (Table 2), signals associated with a C3−C6−
C3 unit, two methoxy groups, and a glucopyranosyl group were
observed. All of the aforementioned spectroscopic data
indicated that 2 was a butyrolactone-type lignan, but without
an aromatic ring. The HMBC correlations (Figure 2) from H-7
to C-1, C-2, C-6, C-8, C-9, and C-2′ and from H-1′ to C-8, C-
2′, and C-3′ confirmed the benzylbutyrolactone skeleton of 2.
The linkage point of the glucose was identified by the
correlation between the anomeric proton H-1″ of glucose
and C-1′ in the HMBC experiment. The 8R,2′S configurations
of 2 were deduced from the chemical shifts of H-9 (δH 4.22,
3.89) in the H NMR spectrum (Table 1) and from the
negative Cotton effect at 227 nm in the CD spectrum. Thus, 2
was determined to be arctiiapolignan A.
The IR spectrum of compound 3 showed absorptions for a
hydroxyl group (3413 cm−1), methylene (2921 cm−1), benzene
ring (1598, 1514, and 1454 cm−1), and carbonyl group (1760
cm−1). The molecular formula of 3 was determined to be
Figure 3. Key ROESY correlations in compounds 1, 3, and 7.
trans configuration of H-7″/H-8″. In the CD spectrum, the
negative Cotton effect at 230 revealed the 8R,8′R config-
urations of the benzylbutyrolactone unit,16 and the negative
Cotton effect at 290 nm revealed the 7″R,8″S configurations of
the dihydrobenzofuran unit.18 Therefore, compound 3 was
confirmed to be arctiisesquineolignan A.
The spectroscopic data for compound 4 were similar to those
of arctiin, except for an additional galactose unit.19 The
downfield shift of C-6″ (δC 66.8) of glucose in 4 suggested that
the linkage point of the galactose was at C-6″ of glucose, which
was further confirmed by the correlations between the
anomeric proton H-1‴ of galactose and C-6″ of glucose in
the HMBC experiment. The trans configuration of H-8 and H-
8′ was established on the basis of the H-9′a (δH 4.10) and H-
9′b (δH 3.88) signals in the 1H NMR spectrum (Table 1). The
1
F
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Journal of Agricultural and Food Chemistry
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absolute configurations of C-8 and C-8′ in 4 were defined as
8R,8′R on the basis of the negative Cotton effect at 230 nm
observed in the CD spectrum. Thus, 4 was identified as Figure
1 and named arctigenin-4-O-α-D-galactopyranosyl-(1→6)-O-β-
D-glucopyranoside.
8R,8′R. Cellulase hydrolysis of 7 resulted in 7a (the aglycone)
and D-glucose. The 7′R configuration was supported by a
negative Cotton effect at 340 nm in the Rh2(OCOCF3)4-
induced CD spectrum of 7a (Figure S49, Supporting
Information).11 Consequently, 7 was identified as Figure 1
and named (7′R,8R,8′R)-rafanotrachelogenin-4-O-β-D-gluco-
pyranoside.
Compound 5 was demonstrated to have the molecular
formula C32H42O15 on the basis of HRESIMS with m/z
689.2430 [M + Na]+. The IR spectrum exhibited signals for a
hydroxyl group (3418 cm−1), a methylene (2929 cm−1),
aromatic rings (1592, 1515, and 1454 cm−1), and a carbonyl
group (1760 cm−1). Detailed analysis of the 1D NMR spectra
(Table 1) suggested that the structure of 5 was similar to that of
4. The difference between these two compounds was the
presence of an apiofuranose group20 at C″-6 in 5 instead of a
galactose group at C″-6 in 4, which was supported by the
HMBC correlation between the apiofuranose anomeric proton
H-1‴ (δH 4.80) and C-6″ (δC 68.1) in 5. The absolute
configurations of C-8 and C-8′ in 5 were also confirmed by the
same method used to confirm those in 4. The chemical shifts of
H-9′a (δH 4.09) and H-9′b (δH 3.88) verified the trans
configuration of H-8/8′. This result, combined with the
negative Cotton effect at 230 nm in the CD spectrum,
indicated the 8R,8′R configurations for 5. Thus, compound 5
was determined to be arctigenin-4-O-β-D-apiofuranosyl-(1→6)-
O-β-D-glucopyranoside.
Compound 6 had the molecular formula C29H38O13, and its
IR spectrum exhibited absorption bands that were attributed to
a hydroxyl (3426 cm−1), a methylene (2928 cm−1), a carbonyl
group (1758 cm−1), and an aromatic ring (1597, 1513, and
1461 cm−1). Comprehensive analysis of the NMR spectra
indicated that compound 6 was a dibenzylbutyrolactone lignan
glucoside with an additional C3 unit. In the HMBC spectrum,
the correlation peaks from H-2″ (δH 3.57, 3.64) and H-3″ (δH
3.57, 3.64) to C-1″ (δC 44.3) and from H-1″ (δH 3.14) to C-2″
(δC 62.2) and C-3″ (δC 62.2), together with their chemical
shifts, demonstrated the propanediol structure of the C3 unit.
In addition, the HMBC correlations from H-1″ (δH 3.14) to C-
4′ (δC 143.1), C-5′ (δC 128.5), and C-6′ (δC 121.1) indicated
that C-1″ of the propanediol moiety was connected to C-5′ of
the dibenzylbutyrolactone lignan by a C−C bond. The absolute
configurations of C-8 and C-8′ were determined by the
procedure described for 4 and 5. A negative Cotton effect at
229 nm observed in the CD spectrum, in combination with the
chemical shifts of H-9′ at δH 4.07 and 3.84, verified the 8R,8′R
configurations for 6. On the basis of the aforementioned
information, compound 6 was confirmed to be 5′-propane-
diolmatairesinoside.
Compound 8 was obtained as a white amorphous powder. Its
molecular formula was determined to be C27H34O12 on the
basis of the positive HRESIMS ion observed at m/z 573.1956
[M + Na]+. A comparison of the IR, UV, and NMR data (Table
2) of 8 with those of 7 revealed that these two compounds
possessed the same planar structure. As previously described,
the ROESY correlation of H-7′/H-8 indicated the trans
configuration of H-8/H-8′ in 8. In the CD spectrum, a
negative Cotton effect at 227 nm helped confirm the 8R,8′R
configurations for 8. The 7′S configuration was determined by a
positive Cotton effect at 340 nm in the Rh2(OCOCF3)4-
induced CD spectrum of 8a (the aglycone) (Figure S57,
Supporting Information), which was obtained by cellulase
hydrolysis of 8. Thus, compound 8 was deduced to be
(7′S,8R,8′R)-rafanotrachelogenin-4-O-β-D-glucopyranoside.
Compound 9 had the same molecular formula as 8:
C27H34O12. A comparison of the NMR spectra of 9 and 8
revealed that the differences between these two compounds
were the linkage point of the alcoholic hydroxyl group. In the
HMBC spectrum, the correlation peaks from H-7 (δH 5.10) to
C-1, C-2, C-6, C-8, C-9, and C-8′ and from H-7′ (δH 2.32,
2.05) to C-1′, C-2′, C-6′, C-8′, C-9′, and C-8, together with the
chemical shifts of H-7/7′ and C-7/7′, indicated that the
hydroxyl was located at C-7. Thus, the planar structure of 9 was
elucidated as 4,7-dihydroxy-3,3′,4′-trimethoxyl-9-oxo dibenzyl-
butyrolactone lignan-4-O-β-D-glucopyranoside. The relative
configuration of H-8 and H-8′ in 9 was defined as trans on
the basis of the characteristic correlation peak of H-8′ with H-7
in the ROESY spectrum. The 8S,8′R configuration for 9 was
confirmed by the negative Cotton effects at 243 and 282 nm in
the CD spectrum. Cellulase hydrolysis of 9 resulted in 9a (the
aglycone), which was purified by preparative HPLC. The 7,8-
erythro configuration of 9a was deduced on the basis of the
1
coupling constant J7,8 (4.0 Hz) in the H NMR experiment
(Figure S66, Supporting Information).22 Thus, 9 was elucidated
to be (7S,8S,8′R)-4,7-dihydroxy-3,3′,4′-trimethoxyl-9-oxo di-
benzylbutyrolactone lignan-4-O-β-D-glucopyranoside.
A comparison of the molecular formula (C21H24O7) and
NMR data of 10 with those of 9 (Tables 2 and 3) indicated the
absence of glucose unit resonances in 10. Using the same
methods as described for 9 (Figures S74−S76, Supporting
Information), we elucidated the 7S,8S,8′R configurations of 10.
Thus, compound 10 was determined to be (7S,8S,8′R)-4,7-
dihydroxy-3,3′,4′-trimethoxyl-9-oxo dibenzylbutyrolactone li-
gnan.
Compound 11 had the same planar structure as 9, as
indicated by a comparison of their NMR spectra and molecular
formulas. The absolute configurations of C-8 and C-8′ were
established as 8S,8′R by the correlation of H-7/8′ in the
ROESY spectrum and the negative Cotton effects at 243 and
282 nm in the CD spectrum. However, in the 1H NMR
spectrum of 11a (the aglycone) (Figure S84, Supporting
Information), which was obtained by hydrolysis of 11, the
coupling constant of H-7 (J7,8 = 8.5 Hz) suggested that the
relative configuration between C-7 and C-8 was threo instead of
the erythro configuration in 9.21 Thus, the 7R configuration was
Compound 7 was obtained as a white amorphous powder
with a molecular formula of C27H34O12 on the basis of an ion
peak at m/z 573.1954 [M + Na]+ in the HRESIMS spectrum.
The spectroscopic data (Tables 2 and 3) for 7 were almost
identical to the data for rafanotrachelogenin-4-O-β-D-glucopyr-
anoside,21 which was isolated from Trachelospermum lucidum.
However, the absolute configurations of rafanotrachelogenin-4-
O-β-D-glucopyranoside had not yet been identified. To
determine the absolute configuration of C-8, C-7′, and C-8′
in compound 7, the ROESY spectrum, CD exciton chirality,
and Rh2(OCOCF3)4-induced CD spectrum were assayed. The
relative configuration of H-8 and H-8′ in 7 was defined as trans
on the basis of the characteristic correlation peaks of H-7′ with
H-8 in the ROESY spectrum (Figure 3). In combination with
the negative Cotton effect at 230 nm in the CD spectrum, the
absolute configuration of C-8 and C-8′ was confirmed as
G
DOI: 10.1021/acs.jafc.5b02838
J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry
Article
Scheme 1. Plausible Biogenetic Pathway of 1
determined and the structure of 11 was characterized as Figure
1 and named (7R,8S,8′R)-4,7-dihydroxy-3,3′,4′-trimethoxyl-9-
oxo dibenzylbutyrolactone lignan-4-O-β-D-glucopyranoside.
The molecular formula of compound 12 was determined to
be C26H32O12 on the basis of the positive HRESIMS ion
observed at m/z 559.1795 [M + Na]+. The UV, IR, and NMR
spectra of 12 were almost identical to the corresponding
spectra of 11; the difference between the compounds was that
12 contained a 4′-OH group in place of the 4′-OCH3 in 11.
The 7R,8S,8′R configuration of 12 was also confirmed by the
ROESY correlation and CD spectrum and by the coupling
constant of H-7 (J7,8 = 8.0 Hz) in 12a (the aglycone) (Figure
S93, Supporting Information). On the basis of these results, 12
was determined to be (7R,8S,8′R)-4,7,4′-trihydroxy-3,3′-
dimethoxyl-9-oxo dibenzylbutyrolactone lignan-4-O-β-D-gluco-
pyranoside.
The known compounds matairesinol-4,4′-di-O-β-D-glucopyr-
anoside (13),23 arctiin (14),24 styraxlignolide E (15),25
styraxlignolide D (16),25 and arctigenin-4-O-β-D-gentiobioside
(17)26 were identified by comparison of these physical and
spectral data with literature values. In addition, their absolute
configurations were established from NMR and CD data.
Cytotoxic Activity. All of the isolated compounds were
tested for cytotoxic activity using an MTT assay in HCT-8, Bel-
7402, BGC-823, A549, and A2780 cells.27 Compounds 1−17
exhibited no significant cytotoxicity in the cell model systems at
a concentration of 10 μM.
Hepatoprotective Activity. Given our previous bioactivity
screening results for the lignan glucosides, the hepatoprotective
activities10 of 1−17 were tested through in vitro assays.
Unfortunately, all of the tested compounds exhibited weak
hepatoprotective activities at a concentration of 10 μM.
Anti-inflammatory Activity. Compounds 1−17 were
tested for anti-inflammatory activity by suppressing the
production of NO in lipopolysaccharide-induced BV2 cells.
From the data obtained, compounds 1, 6, 8, and 10 exhibited
stronger anti-inflammatory effects than the positive control
curcumin at a concentration of 10 μM. Among them,
compound 1 demonstrated 75.51, 70.72, and 61.17% inhibition
at 10, 1, and 0.1 μM, respectively, whereas the positive control
curcumin showed 53.02, 36.74, and 0% inhibition at 10, 1, and
0.1 μM. Furthermore, compound 6 exhibited 71.59, 38.64, and
2.27% inhibition at 10, 1, and 0.1 μM, respectively. Compound
8 exhibited 87.77, 53.24, and 11.51% inhibition at 10, 1, and 0.1
μM, respectively. Compound 10 exhibited 63.31, 40.29, and
37.41% inhibition at 10, 1, and 0.1 μM, respectively.
butyrolactone unit (3); three new dibenzylbutyrolactone
lignans (4−6); six new dibenzylbutyrolactone lignans with an
additional hydroxyl at C-7/7′ (7−12); and five known
compounds (13−17). A literature search revealed that no
butyrolactone lignan with a 6-carboxyl-2-pyrone moiety has
been previously reported. A plausible biogenetic pathway of 1 is
proposed in Scheme 1. The 6-carboxyl-2-pyrone might be
formed from a 3,4-dihydroxy phenyl moiety through oxidative
cleavage, oxidation, and esterification.
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jafc.5b02838.
NMR, IR, MS, and CD spectra of compounds 1−12
(PDF)
AUTHOR INFORMATION
■
Corresponding Author
*(P.-C.Z.) Phone: +86-10-63165231. E-mail: pczhang@imm.
ac.cn. Fax: 86-10-63017757.
Author Contributions
∥Y.-N.Y. and X.-Y.H. contributed equally to this work.
Funding
This research was supported by the National Science and
Technology Project of China (No. 2012ZX09301002-002) and
the Fundamental Research Funds for the Central Institutes
(No. 2014TD03).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Ying-hong Wang for NMR measurements.
REFERENCES
■
(1) Liu, J. Y.; Cai, Y. Z.; Wong, R. N. S.; Lee, C. K.; Tang, S. C. W.;
Sze, S. C. W.; Tong, Y.; Zhang, Y. B. Comparative analysis of
caffeoylquinic acids and lignans in roots and seeds among various
burdock (Arctium lappa) genotypes with high antioxidant activity. J.
Agric. Food Chem. 2012, 60, 4067−4075.
(2) Ming, D. S.; Guns, E.; Eberding, A.; Towers, N. G. H. Isolation
and characterization of compounds with anti-prostate cancer activity
from Arctium lappa L. using bioactivity-guided fractionation. Pharm.
Biol. 2004, 42, 44−48.
(3) Matsumoto, T.; Hosono-Nishiyama, K.; Yamada, H. Antiprolifer-
ative and apoptotic effects of butyrolactone lignans from Arctium lappa
on leukemic cells. Planta Med. 2006, 72, 276−278.
(4) Xu, Z. H.; Wang, X.; Zhou, M.; Ma, L.; Deng, Y.; Zhang, H.;
Zhao, A.; Zhang, Y. Y.; Jia, W. The antidiabetic activity of total lignan
from Fructus Arctii against alloxan-induced diabetes in mice and rats.
Phytother. Res. 2008, 22, 97−101.
In the continued search for bioactive natural products from
Arctii Fructus, 17 butyrolactone type lignans (1−17) were
isolated, including a novel rare butyrolactone lignan with a 6-
carboxyl-2-pyrone moiety, named arctiidilactone (1); a new
apolignan glucoside without an aromatic ring (2); a new
sesquineolignan with a dihydrobenzofuran unit and a
H
DOI: 10.1021/acs.jafc.5b02838
J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry
Article
(5) Kim, J. H.; Bae, J. T.; Song, M. H.; Lee, G. S.; Choe, S. Y.; Pyo,
H. B. Biological activities of Fructus Arctii fermented with the
basidiomycete Grifola frondosa. Arch. Pharmacal Res. 2010, 33, 1943−
1951.
(6) Dos Santos, A. C.; Baggio, C. H.; Freitas, C. S.; Lepieszynski, J.;
Mayer, B. Gastroprotective activity of the chloroform extract of the
roots from Arctium lappa L. J. Pharm. Pharmacol. 2008, 60, 795−801.
(7) Park, S. Y.; Hong, S. S.; Han, X. H.; Hwang, J. S.; Lee, D.; Ro, J.
S.; Hwang, B. Y. Lignans from Arctium lappa and their inhibition of
LPS-induced nitric oxide production. Chem. Pharm. Bull. 2007, 55,
150−152.
(25) Min, B. S.; Na, M. K.; Oh, S. R.; Ahn, K. S.; Jeong, G. S.; Li, G.;
Lee, S. K.; Joung, Y.; Lee, H. K. New furofuran and butyrolactone
lignans with antioxidant activity from the stem bark of Styrax japonica.
J. Nat. Prod. 2004, 67, 1980−1984.
(26) Nishibe, S.; Hisada, S.; Inagaki, I. Lignans of Trachelospermum
liukiuense and T. foetidum. Chem. Pharm. Bull. 1973, 21, 674−675.
(27) Li, C. L.; Zhang, D. M.; Luo, Y. M.; Yu, S. S.; Li, Y.; Lu, Y. Bis-
sesquiterpenes and diterpenes from Chloranthus henryi. Phytochemistry
2008, 69, 2867−2874.
(8) Umehara, K.; Nakamura, M.; Miyase, T.; Kuroyanagi, M.; Ueno,
A. Studies on differentiation inducers VI. Lignan derivatives from
Arctium Fructus. Chem. Pharm. Bull. 1996, 44, 2300−2304.
(9) Kravtsova, S. S.; Khasanov, V. V. Lignans and fatty-acid
composition of Arctium lappa seeds. Chem. Nat. Compd. 2011, 47,
800−801.
(10) Yang, Y. N.; Huang, X. Y.; Feng, Z. M.; Jiang, J. S.; Zhang, P. C.
Hepatoprotective activity of twelve novel 7′-hydroxy lignan glucosides
from Arctii Fructus. J. Agric. Food Chem. 2014, 62, 9095−9102.
(11) Frelek, J.; Szczepek, W. J. [Rh2(OCOCF3)4] as an auxiliary
chromophore in chiroptical studies on steroidal alcohols. Tetrahedron:
Asymmetry 1999, 10, 1507−1520.
(12) Yang, S.; Tamai, R.; Akashi, S.; Takeuchi, O.; Akira, S.;
Sugawara, S.; Takada, H. Synergistic effect of muramyldipeptide with
lipopolysaccharide or lipoteichoic acid to induce inflammatory
cytokines in human monocytic cells in culture. Infect. Immun. 2001,
69, 2045−2053.
(13) Vidal, V.; Dewulf, J.; Bahr, G. M. Enhanced maturation and
functional capacity of monocyte-derived immature dendritic cells by
the synthetic immunomodulator Murabutide. Immunology 2001, 103,
479−487.
(14) Corrie, J. E. T.; Green, G. H.; Ritchie, E.; Taylor, W. C. The
chemical constituents of Australian Zanthoxylum species. V. The
constituents of Z. pluviatile Hartley; the structures of two new lignans.
Aust. J. Chem. 1970, 23, 133−145.
(15) Lopes, L. M. X.; Yoshida, M.; Gottlieb, O. R. Dibenzylbutyr-
olactone lignans from Virola sebifera. Phytochemistry 1983, 22, 1516−
1518.
(16) Ren, Y. L.; Yang, J. S. The spectroscopic features of
dibenzylbutyrolactone lignans. Chin. J. Magn. Reson. 2002, 19, 15−24.
(17) Cutillo, F.; D’Abrosca, B.; DellaGreca, M.; Fiorentino, A.;
Zarrelli, A. Lignans and neolignans from Brassica fruticulosa: effects on
seed germination and plant growth. J. Agric. Food Chem. 2003, 51,
6165−6172.
(18) Yang, Y. N.; Liu, Z. Z.; Feng, Z. M.; Jiang, J. S.; Zhang, P. C.
Lignans from the root of Rhodiola crenulata. J. Agric. Food Chem. 2012,
60, 964−972.
(19) Chakladar, S.; Cheng, L.; Choi, M.; Liu, J.; Bennet, A. J.
Mechanistic evaluation of melA α-galactosidase from Citrobacter
freundii: a family 4 glycosyl hydrolase in which oxidation is rate-
limiting. Biochemistry 2011, 50, 4298−4308.
(20) Park, J. H.; Jung, Y. J.; Jung, J. W.; Shrestha, S.; Lim, D. W.;
Han, D.; Baek, N. I. A new flavonoid glycoside from the root bark of
Morus alba L. Nat. Prod. Res. 2014, 28, 1859−1863.
(21) Kousar, F.; Noomrio, M. H.; Talpur, M. M. A.; Ahmad, V. U.;
Arfan, M. Two new lignan glycosides from Trachelospermum lucidum
(D. Don) Schum. J. Asian Nat. Prod. Res. 2008, 10, 285−288.
(22) Neidigh, K. A.; Kingston, D. G. I.; Lewis, N. G. Synthesis of
stereospecifically deuterated matairesinol, podorhizol, epipodorhizol,
and yatein. J. Nat. Prod. 1994, 57, 791−800.
(23) Ekholm, F. S.; Eklund, P.; Leino, R. A short semi-synthesis and
complete NMR-spectroscopic characterization of the naturally
occurring lignan glycoside matairesinol 4,4′-di-O-β-D-diglucoside.
Carbohydr. Res. 2010, 345, 1963−1967.
(24) Shi, Y.; Shi, R.; Liu, B.; Lu, Y.; Du, L. Isolation and elucidation
of chemical constituents with antiviral action from Yinqiaosan on
influenza virus. Chin. J. Chin. Mater. Med. 2003, 28, 47−51.
I
DOI: 10.1021/acs.jafc.5b02838
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