New lignans, sesquiterpenes and other constituents from twigs and leaves of Rhododendron micranthum

Article abstract of DOI:10.1016/j.fitote.2019.03.025

Rhododendron micranthum is used traditionally as a remedy for the treatment of chronic bronchitis in China. To clarify the chemical basis and provide a reference for the rational use of this medicinal plant, a phytochemical study was carried out on the twigs and leaves of R. micranthum, which afforded eight new compounds (1–8) and eight known compounds (9–16). Their structures were rigorously determined by comprehensive HRESIMS, NMR and electronic circular dichroism (ECD) analyses. The anti-inflammatory activities of these compounds were evaluated. Compounds 3, 13, and 14 suppressed the transcription of the NF-κB-dependent reporter gene in LPS-induced 293T/NF-κB-luc cells at 10 μM, while no effect on cell viability was observed.

Full text of DOI:10.1016/j.fitote.2019.03.025

Fitoterapia 135 (2019) 15–21
Contents lists available at ScienceDirect
Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
New lignans, sesquiterpenes and other constituents from twigs and leaves of
Rhododendron micranthum
1
1
Zhaoxin Zhang , Huimin Yan , Yuxun Zhu, Huanping Zhang, Lisha Chai, Li Li, Xiaojing Wang,
⁎
Yunbao Liu, Yong Li
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical
College, Beijing 100050, People's Republic of China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Rhododendron micranthum
Neolignans
Anti-inflammatory
NF-κB
Rhododendron micranthum is used traditionally as a remedy for the treatment of chronic bronchitis in China. To
clarify the chemical basis and provide a reference for the rational use of this medicinal plant, a phytochemical
study was carried out on the twigs and leaves of R. micranthum, which afforded eight new compounds (1–8) and
eight known compounds (9–16). Their structures were rigorously determined by comprehensive HRESIMS, NMR
and electronic circular dichroism (ECD) analyses. The anti-inflammatory activities of these compounds were
evaluated. Compounds 3, 13, and 14 suppressed the transcription of the NF-κB-dependent reporter gene in LPS-
induced 293T/NF-κB-luc cells at 10 μM, while no effect on cell viability was observed.
1. Introduction
κB reporter gene assay. Herein, the details of the isolation, structural
identification, and bioactivity evaluation are presented.
Plants of Rhododendron are prolific sources of bioactive natural
products, including flavonoids [1–3], lignans [4], diterpenoids [5–9],
triterpenoids [10,11], sesquiterpenoids, and many other compounds.
These compounds exhibit diverse significant activities, such as an-
algesic [8,12], antioxidant [3], and anti-inflammatory activities
2. Experimental section
2.1. General experimental procedures
[
13,14]. Among these plants, the twigs and leaves of Rhododendron
IR spectra and UV were performed on a Nicolet 5700 FT-IR spec-
trometer and a JASCO V650 spectrometer. Optical rotations were ob-
tained on a Rudolph automatic polarimeter. HRESI-MS data were ac-
quired via an Agilent 6520 Accurate-Mass Q-TOF LC/MS spectrometer.
ECD spectra were conducted on a circular dispersion spectrometer
(JASCO J-815). NMR spectra were obtained on Bruker AV600-III,
INOVA SX-600 and INOVA-500 spectrometer. Preparative HPLC was
conducted on a Shimadzu LC-6AD instrument equipped with SPD-20A
and RID-10A detecters (Kyoto, Japan) using a YMC Pack ODS-A column
(250 × 10 mm, 5 μm, Kyoto, Japan). Macroporous resin (D101 type,
The Chemical Plant of Nankai University, China), Sephadex LH-20 (GE
Chemical Corporation, USA), silica gel and GF254 TLC plates (Qingdao
Marine Chemical Factory, China) and ODS (50 μm, Merck, Germany)
were used for column chromatography (CC). Spots were detected under
micranthum (also known as zhaoshanbai) have been used as traditional
Chinese medicine for various ailments, such as chronic bronchitis, sore
swollen, menoxenia, postpartum arthralgia, and hypertension [15].
Among those diseases, chronic bronchitis, sore swollen, and postpartum
arthralgia have a close relationship with inflammation. Presently, the
extract of R. micranthum is still widely used in the treatment of chronic
bronchitis in China. However, understanding of the chemical basis of its
anti-inflammatory effects is still vague in general. Focusing on the
discovery of new anti-inflammatory compounds and revealing the
chemical basis of traditional Chinese medicine, a chemical study was
carried out on the EtOH extract of the twigs and leaves of R. mi-
cranthum. Sixteen compounds, including eleven lignans (1–5, 9–11,
1
4–16), one phenolic glycoside (6), two sesquiterpenes (7–8), and two
coumarins (12−13), were obtained. The structures of the new com-
pounds were shown in Fig. 1. Several compounds, such as compounds
UV light or spraying with 10% H
by heating.
2 4 2
SO in EtOH-H O (95:5, v/v) followed
3
, 13, and 14, showed moderate anti-inflammatory activities in an NF-
⁎
Corresponding author.
E-mail address: liyong@imm.ac.cn (Y. Li).
Z.-X. Zhang and H.-M.Yan contributed equally.
1
https://doi.org/10.1016/j.fitote.2019.03.025
Received 1 February 2019; Received in revised form 22 March 2019; Accepted 23 March 2019
Available online 25 March 2019
0367-326X/ © 2019 Elsevier B.V. All rights reserved.

Z. Zhang, et al.
Fitoterapia 135 (2019) 15–21
Fig. 1. Structures of compounds 1–8 isolated from the twigs and leaves of R. micranthum.
2
.2. Plant material
t
t
t
R
R
R
= 25.5 min); with MeCN-H
= 28.0 min); and with MeCN-H
= 79.1 min). E30 (11.9 g) was separated using ODS CC and
O (10:90, 30:70, 50:50, 60:40,
70:30 and 100:0, v/v) to yield 5 fractions (E30 -E30 ).
Then, E30 (4.3 g) was purified by preparative HPLC and
semipreparative HPLC with MeCN-H O (15:85, v/v) to yield 15
(44.0 mg, t = 12.2 min). E30 was separated with MeCN-H
25:75, v/v) to yield 2 (8.0 mg, t = 41.2 min); with MeCN-H O (20:80,
v/v) to yield 5 (2.2 mg, t = 46.5 min), 3 (7.5 mg, t = 57.6 min) and
0 (14.3 mg, t = 46.3 min); and with MeCN-H O (21:79, v/v) to yield
11 (3.9 mg, t was purified with MeCN-H
(14:86, v/v) to yield 14 (36.0 mg, t = 41.9 min).
2
O (14:86, v/v) to yield 13 (8.8 mg,
2
O (13:87, v/v) to yield 16 (70.0 mg,
The twigs and leaves of R. micranthum were collected in August
014 from Shandong Province, China, and were authenticated by Prof.
5 1 3
G L M
2
eluted with a step gradient of MeOH/H
2
Peng Wan (Shandong Traditional Chinese Medicine University). A
voucher specimen (ID-S-2586) was deposited in the herbarium at the
Department of Medicinal Plants, Institute of Materia Medica, Chinese
Academy of Medical Sciences.
G
5
L
1
M
3
O
1
5 1 3 5
G L M O
5 1 3 3
G L M O
2
R
G
5
L
1
M
4
2
O
(
R
2
2
.3. Extraction and isolation
R
R
1
R
2
The dried twigs and leaves of R. micranthum (107.5 kg) were ground
R
= 26.0 min). E30
G
5
L
1
M
5
2
O
and extracted in EtOH-H
reflux. The extract residue (12 kg) was suspended in H
then partitioned sequentially with petroleum ether, CH Cl
n-butanol. The EtOAc portion (2.7 kg) was then separated using mac-
roporous resin CC and eluted with a mixture of EtOH-H O (30:70,
0:40, 95:5, v/v).
The 30% EtOH fraction (1.1 kg) was separated using silica gel CC,
which was eluted with a mixture of CH Cl :MeOH (100:1–1:1, v/v) to
obtain 9 fractions (E30 -E30 ). Then, E30 (31 g) was separated
using silica gel CC and eluted with a mixture of CH Cl :MeOH
2
O (95:5, v/v) twice (2 h each time) under
O (12L) and
, EtOAc, and
R
The 60% EtOH fraction (288 g) was separated using silica gel CC
and eluted with a mixture of CH Cl :MeOH (100:1–1:1, v/v) to obtain 9
fractions (E60 -E60 ). E60 , E60 , E60 , and E60 were then all
separated via Sephadex LH-20 CC. Fraction E60 was separated by
Sephadex LH-20 CC and eluted with MeOH−H
three fractions (E60 − E60 ). E60
parative HPLC and semipreparative HPLC with MeCN-H
to yield 7 (8.4 mg, t = 67.9 min). E60 was purified by preparative
HPLC and semipreparative HPLC with MeCN-H O (20:80, v/v) to yield
8 (6.9 mg, t = 31.2 min).
2
2
2
2
2
G
1
G
9
G
2
G
3
G
6
G
8
G
3
2
6
2
O (60:40, v/v), yielding
3 2
G L was purified by pre-
G
3
L
1
G
3
L
3
2
O (25:75, v/v)
2
2
G
1
G
9
G
4
R
8 2
G L
2
2
2
(
100:1–1:1, v/v) to obtain 10 fractions (E30
3 g) was separated via Sephadex LH-20 CC, preparative HPLC and
O (19:81, v/v), to yield 1
(60 g) was purified on a Sephadex LH-
0 CC to obtain 3 fractions (E30 - E30 ). Next, E30 was
further purified by MCI CC to yield 6 fractions (E30 -E30 ).
Subsequently, E30 was dissolved with MeOH and filtered, and
G
4
g
1
-E30G
4
g
10). E30
G
4
g
9
R
(
D
Rhodomicranthoside A (1): white powder; [α] [20] + 147.7 (c
semipreparative HPLC, with MeCN-H
30.6 mg, t = 28.9 min). E30
2
0.56, MeOH); IR(KBr) νmax 3419, 2937, 1675, 1615, 1498, 1459, 1354,
1310, 1239, 1196, 1122, 1089, 975, 943, 917, 890, 868, 835, 806,
624 cm ; for H and C NMR spectroscopic data, see Tables 1 and 2;
(
2
R
G
3
−
1
1
13
G
3
L
1
G
3
L
3
G
3
L
1
[M + Na]+ (calcd
for
G
3
L
M
1 1
G
3
L
M
1 6
HRESI-MS(positive):
34NaO12, 573.1942).
Rhodomicranthoside B (2): white powder; [α] [20]
m/z 573.1938
G
3
L
1
M
5
27
C H
the solid was determined to be 12 (333.7 mg). The mother solution was
separated via preparative HPLC and semipreparative HPLC with MeCN-
H
H
D
– 52.6 (c 0.29,
MeOH); IR(KBr) νmax 3389, 2921, 1676, 1615, 1505, 1458, 1368, 1276,
−1
2
O (19:81, v/v) to yield 4 (2.3 mg, t
O (21:79, v/v) to yield 9 (2.3 mg, t
R
= 37.3 min) and with MeCN-
= 26.8 min). E30 (306.6 g)
1240, 1202, 1126, 1047, 982, 944, 913, 863, 807, 721, 646 cm ; for
1
13
2
R
G
5
H and C NMR spectroscopic data, see Tables 1 and 2; HRESI-MS
(positive): m/z 515.1889 [M + Na]+ (calcd for
515.1888).
was further separated using Sephadex LH-20 CC to obtain 2 fractions
- E30 ). E30 (37.7 g) was then separated via MCI CC
and eluted with a step gradient of MeOH/H O (10:90, 30:70, 50:50,
fractions (E30
was purified by preparative HPLC and semi-
O (38:62, v/v) to yield 6 (26.3 mg,
25
C H32NaO10,
(
E
30
G
5
L
1
G
5
L
2
G
5
L
1
Rhodomicranthoside C (3): white powder; [α] [20]
D
– 22.8 (c 0.40,
2
6
E
0:40, 70:30 and 100:0, v/v) to yield
). E30
5
5 1 1
G L M -
MeOH); IR(KBr) νmax 3370, 2933, 1678, 1616, 1597, 1510, 1455, 1431,
−1
30
G
5
L
M
1 5
G
5
L
1
M
2
1339, 1206, 1120, 1048, 982, 910, 880, 835, 742, 723, 655 cm ; for
1
13
preparative HPLC, with MeOH-H
2
H and C NMR spectroscopic data, see Tables 1 and 2; HRESI-MS
16

Z. Zhang, et al.
Fitoterapia 135 (2019) 15–21
Table 1
1
H NMR spectroscopic data for compounds 1–6 in pyridine-d
5
(δ
H
in ppm, J in Hz).
No.
1a
2b
3b
4a
5a
6b
2
3
5
6
7
8
9
–
–
7.38 s
–
7.30 s
7.30 s
5.43 dd (2.7, 8.1)
4.37 m
4.17 dd (2.5, 12.5)
3.91 m
6.81 dd (2.2, 8.3)
7.10 d (8.3)
–
7.17 s
–
–
7.24 d (2.8)
–
–
7.24 d (2.8)
5.76 br s
4.89 m
4.66 ddd (2.4, 5.3, 11.9)
4.25 m
6.65 d (2.9)
–
–
7.13 d (1.5)
–
–
7.13 d (1.5)
6.10 d (7.2)
4.06 m
4.34 m
4.31 m
6.89 s
–
–
6.98 s
6.98 s
–
4.95 s
–
–
6.52 s
–
5.17 s
2.71 t (7.3)
7.17 s
5.51 d (8.1)
4.36 m
4.26 m
3.93 m
6.63 d (2.0)
–
(b) 4.44 dd (7.1, 9.5)
(a) 3.82 m
2’
3’
4’
5’
6’
7’
–
–
–
–
8.14 d (8.7)
7.16 d (8.7)
–
7.16 d (8.7)
8.14 d (8.7)
–
–
–
–
–
–
–
6.60 s
(b) 3.35 dd (7.3, 17.3)
7.05 d (2.2)
2.68 m
6.79 d (2.0)
2.73 m
6.65 d (2.9)
2.73 m
7.04 s
2.73 m
(a) 3.04 d (17.2)
8
9
’
’
2.55 dd (1.2, 7.4)
4.52 dd (3.0, 12.0)
1.97 m
3.89 m
2.02 m
3.96 m
1.98 m
3.92 m
1.98 m
3.93 m
–
–
3
.92 d (12.0)
3.53 dt (6.1, 9.5)
5.27 br s
4.57 m
4.52 m
4.29 br t (9.5)
4.22 m
3.56 dt (6.1, 9.5)
5.29 br s
4.59 dd (1.7, 3.4)
4.53 dd (3.4, 9.1)
4.30 t (9.2)
4.22 m
3.54 m
5.29 br s
4.58 m
4.52 m
4.30 td (2.2, 9.2)
4.21 m
3.54 m
1”
2”
3”
4”
5”
4.78 d (7.5)
4.08 dd (7.4, 8.9)
4.19 t (8.7)
4.25 ddd (5.2, 8.4, 10.1)
4.33 dd (5.2, 11.3)
5.29 br s
4.60 dt (1.6, 3.3)
4.54 dd (3.5, 9.2)
4.30 m
5.76 d (7.6)
4.43 dd (7.6, 8.9)
4.37 t (8.8)
4.28 t (9.8)
4.13 ddd (2.2, 6.1, 9.8)
4.23 m
3
.71 d (11.3)
6”
–
1.66 dd (3.2, 6.1)
1.66 d (6.1)
1.66 dd (2.7, 6.1)
1.66 d (6.1)
5.14 dd (2.2, 11.7)
4
.90 dd (6.1, 11.7)
2
3
4
5
6
3
5
-OCH
-OCH
-OCH
-OCH
-OCH
3
3
3
3
3
3.86 s
–
3.64 s
–
–
–
–
–
–
3.73 s
–
–
–
3.73 s
–
–
3.81 s
–
3.81 s
–
3.77 d (2.8)
–
3.77 d (2.8)
–
3.79 d (2.8)
3.79 d (2.8)
3.71 d (1.5)
–
3.71 d (1.5)
–
3.87 d (1.5)
–
3.77 d (2.7)
–
–
–
–
–
’-OCH
’-OCH
3
3
4.14 s
3.69 s
3.86 s
a
b
Recorded at 600 MHz.
Recorded at 500 MHz.
(
positive): m/z 575.2104 [M + Na]+ (calcd for
C
27
H
36NaO12
,
2.4. Determination of absolute configuration of sugar moieties
575.2099).
Rhodomicranthoside D (4): white powder; [α] [20]
D
– 44.8 (c 0.36,
Each compound (1.5 mg) was added to 2 N HCl (2 mL) and refluxed
for 12 h at 90 °C. The solution was extracted three times with EtOAc.
The water layer was dried to yield a residue. Then, the residue was
dissolved in pyridine (1 mL), and L-cysteine methyl hydrochloride
(2 mg) was added. The mixture was refluxed for 2 h at 60 °C, dried by
nitrogen and heated for 0.5 h at 80 °C. Next, N-trimethylsilylimidazole
(1 mL) was added, and the mixture was heated for 2 h at 60 °C. Finally,
MeOH); IR(KBr) νmax 3393, 2921, 2850, 1677, 1646, 1508, 1464, 1424,
−
1
1
327, 1276, 1207, 1126, 1050, 983, 911, 808, 721, 647 cm ; for 1
H
1
3
and C NMR spectroscopic data, see Tables 1 and 2; HRESI-MS (posi-
+
tive): m/z 584.2493 [M + Na] (calcd for C28
Rhodomicranthoside E (5): white powder; [α] [20]
MeOH); IR(KBr) νmax 3387, 2921, 2850, 1678, 1646, 1615, 1518, 1501,
H
40NaO13, 584.2469).
D
– 21.5 (c 0.33,
1
7
464, 1427, 1326, 1272, 1207, 1124, 1050, 983, 952, 911, 837, 807,
2
the mixture was added to H O (2 mL) and partitioned with n-hexane
−1
1
13
21, 645 cm ; for H and C NMR spectroscopic data, see Tables 1
(2 mL) three times. The organic layer was combined and concentrated
and used for GC analysis. Preparation of the other compounds was
conducted in the same way. The conditions of the GC experiments were
as follows: capillary CC, HP-5 (60 m × 0.25 mm, with a 0.25 μm film,
Dikma); detector, FID; injector temperature, 300 °C; detector tempera-
ture, 300 °C; initial temperature, 200 °C, increased to 280 °C at the rate
of 10 °C/min, sustained for 35 mins, decreased to 200 °C at the rate of
+
and 2; HRESI-MS (positive): m/z 559.216 [M + Na] (calcd for
36NaO11, 559.215).
Rhodomicranthoside F (6): white powder; [α] [20]
MeOH); IR(KBr) νmax 3431, 3228, 2962, 2928, 1692, 1598, 1508, 1460,
27
C H
D
– 63.6 (c 0.33,
1
1
425, 1377, 1361, 1330, 1314, 1281, 1240, 1167, 1125, 1080, 1040,
−1
017, 962, 903, 879, 850, 831, 816, 769, 693, 621, 601, 531 cm ; for
1
13
H and C NMR spectroscopic data, see Tables 1 and 2; HRESI-MS
2
40 °C/min and then sustained for 1 min; carrier gas, N . In the GC
(
positive): m/z 489.1379 [M + Na]+ (calcd for
C
22
H
26NaO11
,
chromatogram, the retention times of the derivatives of standard D-
glucose, D-xylose, and L-rhamnose were 29.6 min, 20.9 min and
23.5 min, respectively.
489.1367).
D
Rhodomicranthin I (7): yellow oil; [α] [20] – 9.0 (c 1.23, MeOH);
IR(KBr) νmax 3388, 2933, 2874, 1706, 1460, 1379, 1073, 1041,
6
MS (positive): m/z 277.1772 [M + Na] (calcd for C15
−1
1
13
23 cm ; for H and C NMR spectroscopic data, see Table 3; HRESI-
2.5. NF-κB reporter gene assay
+
H
26NaO
3
,
2
77.1774).
For the cytotoxicity assay, the cytotoxicity of compounds on 293T
Rhodomicranthoside G (8): white powder; [α] [20]
D
– 25.4 (c 0.59,
cells was determined via an MTT assay. 293T cells were placed in 96-
well plates (5 × 10 [3]/well) overnight. The cells were then treated
with 10 μM of compounds for 24 h. An equal concentration of the sol-
vent vehicle (DMSO, 0.5%) was included as a control. Subsequently,
0 μL of MTT (5 mg/mL in sterile PBS) was added to each well for an
additional 4 h incubation period. Finally, the medium was removed,
MeOH); IR(KBr) νmax 3382, 2923, 1676, 1426, 1379, 1202, 1136, 1079,
−
1
1
13
1
043, 898, 839, 801, 722, 630 cm ; for H and C NMR spectroscopi+c
data, see Table 3; HRESI-MS (positive): m/z 439.2298 [M + Na]
calcd for C21 36NaO , 439.2302).
(
H
8
2
17

Z. Zhang, et al.
Fitoterapia 135 (2019) 15–21
Table 2
1
3C NMR spectroscopic data for compounds 1–6 in pyridine-d
5
C
(δ in ppm).
No.
1a
2b
3b
4a
5a
6b
1
2
3
4
5
6
7
8
9
125.7, C
148.3, C
137.6, C
147.9, C
102.6, CH
153.6, C
31.9, CH
42.6, CH
129.2, C
112.5, CH
149.3, C
149.2, C
117.0, CH
122.0, CH
77.6, CH
80.3, CH
128.2, C
106.6, CH
149.7, C
138.6, C
149.7, C
106.6, CH
77.8, CH
80.2, CH
133.9, C
105.9, CH
149.4, C
137.1, C
149.4, C
105.9, CH
74.3, CH
88.4, CH
130.8, C
105.3, CH
149.8, C
138.1, C
149.8, C
105.3, CH
89.2, CH
55.6, CH
135.0, C
154.5, C
105.5, CH
140.3, C
105.5, CH
154.5, C
64.8, CH
2
–
–
72.8, CH
126.9, C
124.5, C
147.4, C
139.3, C
148.7, C
2
62.0, CH
135.6, C
2
61.9, CH
134.8, C
2
61.7, CH
138.7, C
2
64.6, CH
135.6, C
2
1
2
3
4
5
6
7
8
9
1
2
3
4
5
6
2
3
4
5
6
3
5
’
’
’
’
’
’
’
’
’
”
”
”
”
”
”
122.2, C
122.2, CH
117.6, CH
143.2, C
145.1, C
117.9, CH
106.0, CH
150.0, C
133.0, C
145.7, C
110.5, CH
106.9, CH
154.3, C
135.3, C
154.3, C
106.9, CH
113.9, CH
145.2, C
147.9, C
133.2, C
118.0, CH
132.9, CH
116.5, CH
163.9, C
116.5, CH
132.9, CH
167.0, C
–
107.4, CH
30.1, CH
34.8, CH
80.9, CH
2
32.5, CH
32.3, CH
67.2, CH
2
2
2
33.0, CH
32.3, CH
67.3, CH
2
2
2
33.5, CH
32.2, CH
67.2, CH
2
2
2
33.1, CH
32.7, CH
67.3, CH
2
2
2
2
–
106.3, CH
75.6, CH
79.1, CH
71.6, CH
102.1, CH
72.8, CH
73.4, CH
74.5, CH
70.3, CH
102.2, CH
72.9, CH
73.4, CH
74.5, CH
70.3, CH
102.2, CH
72.8, CH
73.4, CH
74.4, CH
70.3, CH
102.2, CH
72.9, CH
73.4, CH
74.5, CH
70.3, CH
105.3, CH
76.5, CH
78.8, CH
72.2, CH
76.2, CH
67.7, CH
–
60.1, CH
–
2
3
3
19.1, CH
3
19.1, CH
–
56.8, CH
–
56.8, CH
–
56.4, CH
3
3
3
3
19.1, CH
–
56.8, CH
–
56.8, CH
–
3
3
3
19.1, CH
–
56.8, CH
–
56.8, CH
–
56.7, CH
3
3
3
3
65.1, CH
2
-OCH
-OCH
-OCH
-OCH
-OCH
3
3
3
3
3
–
–
56.9, CH
3
–
–
–
56.4, CH
56.4, CH
3
–
–
–
–
–
–
56.9, CH
3
’-OCH
’-OCH
3
3
61.6, CH
56.3, CH
3
3
56.6, CH
56.6, CH
3
3
–
–
–
–
a
b
Recorded at 150 MHz.
Recorded at 125 MHz.
Table 3
pathway, a luciferase assay was performed to analyze the transcription
1
H and 13C NMR data of compounds 7, 8 in pyridine-d
5
(δ
H
C
and δ in ppm).
of an NF-κB-dependent reporter gene as described previously. 293T/
5
7a
7b
8a
8b
NF-κB-luc cells were seeded into 96-well plates at a density of 5 × 10
No.
per well. After 24 h incubation, cells were pretreated with compounds
(10 μM) for 30 min before LPS (500 ng/mL) stimulation. An equal
concentration of the solvent vehicle (DMSO, 0.5%) was always included
as a control. After 6 h of stimulation, the medium was removed, and the
cells were lysed with lysis buffer in the luciferase assay system.
Afterward, the cell lysate was mixed with the luciferase assay reagent,
and the luminescence of firefly luciferase was immediately quantified
using a microplate reader.
1
2
3
–
74.9, C
73.9, CH
39.8, CH
–
74.9, C
73.9, CH
39.2, CH
4.63 m
2.28 m
2
2.51 m
2.60 m
6.12 d (3.7)
–
4.52 m
2.15 m
2.02 m
2.58 m
2.50 m
5.97 d (3.4)
–
2.28 m
1.96 m
1.91 m
1.81 m
2.34 dd (7.6, 11.5)
2.21 m
0.96 d (2.5)
0.98 d (2.5)
1.44 s
4.54 m
3.91 m
4.93 d (7.8)
4.07 m
4.26 m
4.24 m
3.97 m
4.57 m
4
2
2
.04 m
4
5
6
7
8
42.7, CH
40.5, CH
124.6, CH
148.5, C
40.1, CH
40.2, CH
124.3, CH
148.8, C
2.33 m
25.6, CH
2
25.6, CH
2
2
.00 m
1.95 m
.84 m
9
43.8, CH
2
43.7, CH
2
1
3. Results and discussion
10
11
12
13
14
15
2.43 dd (7.3, 11.5)
2.24 m
0.99 s
1.00 s
1.48 s
62.2, CH
38.2, CH
21.8, CH
22.1, CH
23.4, CH
64.3, CH
61.3, CH
38.2, CH
21.8, CH
22.1, CH
23.5, CH
72.2, CH
Compound 1 was obtained as a white powder, and the molecular
formula was determined by analysis of the positive HRESIMS data (m/z
573.1938, calcd for C27H34NaO12, 573.1942), indicating an index of
3
3
3
2
3
3
3
2
4.18 dd (5.6, 10.5)
hydrogen deficiency (IHD) of 11. The IR spectrum of this compound
4
–
–
–
–
–
–
.11 dd (6.0, 10.6)
−1
showed signals typical of hydroxy groups (3419 cm ) and aromatic
1’
2’
3’
4’
5’
6’
–
–
–
–
–
–
105.6, CH
75.8, CH
79.1, CH
72.3, CH
79.0, CH
rings (1615, 1498, 1459 cm ). The 1H NMR data (Table 1) of 1
−1
showed characteristic signals for four aromatic methoxy groups (δ
4.14, 3.86, 3.69 and 3.64), two aromatic protons (δ 6.60 and 6.52),
4.78). The C NMR data (Table 2) dis-
played 27 carbon resonances, including four methyl groups, four me-
thylenes, nine methines (two aromatic methines at δ 107.4 and 102.6
and an anomeric carbon of sugar at δ 106.3), and 10 quaternary car-
bons. The COSY and HSQC spectra showed the presence of the fol-
lowing fragments (Fig. 2): –CH(O)-CH(OH)-CH(OH)-CH(OH)-CH (O)-,
–CH -CH-CH -, –CH-CH-CH -, and –CH-CH-. The HMBC correlation
(Fig. 2) between H –9′ and C-6, and the additional IHD suggested the
H
H
13
and an anomeric proton (δ
H
63.4, CH
2
.40 dd (5.4, 11.7)
C
a
Recorded at 500 MHz.
Recorded at 125 MHz.
C
b
2
1
00 μL of DMSO was added to each well, and the absorbance (A) was
detected at 490 nm using a microplate reader;
2
2
2
2
To determine the effect of compounds on the NF-κB signaling
presence of an oxygen bridge between C-9′ and C-6. Additionally, the
correlation between the anomeric proton (H-1″) and C-9 placed the
18

Z. Zhang, et al.
Fitoterapia 135 (2019) 15–21
Fig. 2. 1He H COSY (blue bold lines), key HMBC correlations (black arrows), and NOESY correlations (blue arrows) for compound 1. (For interpretation of the
1
references to colour in this figure legend, the reader is referred to the web version of this article.)
pentose moiety at C-9. The pentose moiety was determined to be in a β-
configuration due to the relatively large coupling constant of J1”,2″
well as the NOESY correlations of H-8/H-6 and H-8/H-2, indicated that
H-7 and H-8 were in the trans-configuration. A positive Cotton effect at
238 nm in the ECD spectrum suggested the S configuration of C-8 [21].
Accordingly, compound 3 was determined to be (7R, 8S)-3,5,3′-tri-
methoxy-5′,7-epoxy-9,4′-epoxyneolignan- 4,8-diol-9’-O-α-L-rhamnoside
and was named rhomicranoside C.
(
7.5 Hz). Acid hydrolysis and GC analysis of 1 confirmed that the sugar
moiety was D-xylose. The torsion angle between the two aromatic rings
was approximately 90°. C-1 and C-9′ were supposed to be on the same
side of the six-membered ring (ring B in Fig. 2), which allowed the
seven-membered ring to exist stably. Therefore, H-8′ and H-7 should be
on the other side of the ring (α-side). This assignment was confirmed by
The HRESIMS data of 4 showed a pseudomolecular positive ion at
m/z 584.2493 [M + Na]+ (calcd for 584.2469), indicating a molecular
the NOE correlations between H-7/H-(2-OCH
NOE correlations of H-7’b/H-9a, H-8’/H-7’b, and H-8’/H-9a indicated
that H-7’b, CH –9, and H-8′ were on the same side (α-side) of ring B
3
) and H-7/H-(3’-OCH
3
).
40
formula of C28H O13. The NMR data of 4 were similar to the data of
(7R,8R)-4-hydroxy-9’-O-(α-L- rhamnopyranosyl)-3,3′,5′-trimethoxy-8-
O-4′-neoligan [22], except for an additional methoxy group. The HMBC
correlations from H-(CH O-3/5) to C-3/5 placed the additional
3
methoxy group at C-5. In the HMBC spectrum, the correlation of H-1″
and C-9′ suggested that the rhamnose unit was attached at C-9′. Fur-
2
while H-8 and H-7’a were on the other side (β-side). Accordingly, the
relative configuration was determined. Contrary to (+)-ovafolinin B-9’-
O-β-D-glucopyranoside [16], compound 1 showed a negative Cotton
effect at 282 nm, which indicated the 7R configuration of 1 [16–20].
Consequently, compound 1 was determined as (7R,8R,8′R)-ovafolinin
B-9-O-β-D-xylopyranoside and was given the trivial name rhomicrano-
side A.
thermore, the anomeric proton of the rhamnose at δ
broad single peak, indicating that the rhamnose was in the α-config-
uration. Meanwhile, H-7 (δ 5.76) showed a broad single peak, in-
H
5.29 (H-1″) was a
H
dicating that H-7 and H-8 were in the erythro-configuration [23]. Fur-
thermore, a negative Cotton effect at 247 nm was observed in the ECD
spectrum, which revealed the 8R configuration [22,24]. Thus, com-
pound 4 was determined as (7R, 8R)-4,7,9-trihydroxy-9′-O-(α-L- rham-
nopyranosyl)-3,3′,5,5′-tetramethoxy-8-O-4′- neoligan and was named
rhomicranoside D.
Compound 2 was obtained as a white powder. The molecular for-
mula was determined to be C25
32
H O10 based on its pseudomolecular
+
positive ion at m/z 515.1889 [M + Na] (calcd for 515.1888), in-
1
13
dicating an IHD of 10. The H and C NMR data of 2 consist of the
features of a lignan rhamnoside. The structure was determined by the
HSQC, HMBC and CIGAR spectra. In the CIGAR spectrum, the corre-
lations of H-7/C-5′, H-8/C-1, and H-9/C-4′ indicated the presence of a
Based on the HRESIMS peak at m/z 559.216 (calcd 559.215
[M + Na]+), the molecular formula of 5 was determined to be
seven-membered ring, in which two C
6
-C
3
moieties were connected via
27 36
C H O11. The NMR data showed that 5 was closely related to chae-
5
9
9
’-O-7 and 9-O-4′ oxygen bridges. In addition, HMBC correlations of H-
’/C-1″ and H-1”/C-9′ suggested that the rhamnose was connected to C-
′. Furthermore, the anomeric proton of the rhamnose showed a broad-
nomiside A [25]. However, HMBC correlations from H-1″ to C-9′ and
from H-9′ to C-1″ indicated that the rhamnose was attached at C-9′ in 5
instead of at C-9 in chaenomiside A. The broad single peak of the
single peak at δ
H
5.27, which indicated that the rhamnose is in the α-
H
anomeric proton at δ 5.29 (H-1″) confirmed that the rhamnose was in
configuration. The large coupling constant of J7,8 (8.1 Hz), as well as
the NOESY correlations of H-8/H-6 and H-8/H-2, indicated that H-7
and H-8 were in the trans-configuration. A positive Cotton effect at
the α-configuration. After acid hydrolysis of 5, the absolute configura-
tion of the rhamnose was determined by GC analyses. The C-7/C-8
relative configuration was assigned as the trans-form, according to the
231 nm was observed in the ECD spectrum, which suggested an 8S
2
NOESY correlations of H –9/H-7 and H-8/H-2(6). The ECD spectrum
configuration [21]. Additionally, acid hydrolysis and GC analysis of 2
confirmed that the sugar moiety was L-rhamnose. Thus, the structure of
showed two negative Cotton effects at 239 nm and 288 nm, which
suggested the 7S and 8R configuration of 5 [26]. Finally, 5 was de-
termined to have the structure (7S,8R)-3,5,3′-trimethoxy-4′,7-epoxy-
8,5′-neoligan-4,9-diol-9’-O-α-L-rhamnopyranoside and was named rho-
micranoside E.
2
was determined as (7R, 8S)-4-methoxy-5′,7-epoxy-9,4′-epox-
yneolignan- 3,8-diol-9’-O-α-L-rhamnoside and was named rhomicrano-
side B.
Compound 3, a white powder, was determined to have a molecular
Compound 6, a white powder, was determine to have a molecular
formula of C27
H
36
O
12 based on its HRESI-MS peak at m/z 575.2104
formula of C22
showed that 6 was closely related to saccharumoside B [27]. The HMBC
correlations from H-(CH O-2) to C-2 and those from H-(CH O-6) to C-6
revealed the location of the methoxy substitutions in 6. The coupling
constant of the anomeric proton (J1”,2″ = 7.6 Hz) suggested that glucose
was in the β-configuration. Acid hydrolysis of 6 gave a β-D-glucose,
26
H O11 based on the HRESIMS data. The NMR data
+
[
M + Na] (calcd for 575.2099). The NMR data of 3 were comparable
to those of 2, except for the substituents on benzene rings. According to
the HSQC and HMBC data, in 3, a hydroxy group instead of a methoxy
group was located at C-4, and three hydroxy groups at C-3, C-3′, and C-
3
3
5
were all methylated. The large coupling constant of J7,8 (8.1 Hz), as
19

Z. Zhang, et al.
Fitoterapia 135 (2019) 15–21
Fig. 4. The inhibitory effects of 2, 3, 5, 6, 9–16 (10 μM) on the transcription of
the NF-κB-dependent reporter gene in LPS-induced 293T/NF-κB-luc cells.
xylopyranoside (16) [36]. These structures were identified by com-
paring their NMR data with data from the published literature.
The isolated compounds were evaluated for anti-inflammatory ac-
tivities. The inhibitory effects of compounds (2, 3, 5, 6, 9–16) on the
NF-κB signaling pathway are shown in Fig. 4. At a concentration of
Fig. 3. Key NOESY correlations of compound 7. (For interpretation of the re-
ferences to colour in this figure legend, the reader is referred to the web version
of this article.)
1
0 μM, several compounds, such as compounds 3, 13, and 14, sig-
which was determined by comparison with standard sugar via GC.
Therefore, compound 6 was determined to be (2,6-dimethoxy-4-hy-
droxymethyl-phenol) 1-O-β-D-(6-O-p- hydroxybenzoyl)-glucopyrano-
side and was named rhomicranoside F.
nificantly suppressed the transcription of the NF-κB-dependent reporter
gene in LPS-induced 293T/NF-κB-luc cells. In addition, these inhibitory
effects were not caused by nonspecific cytotoxicity since all tested
compounds had no effect on cell viability, as determined by an MTT
assay on 293T cells (data not shown). Among the molecules, compound
Compound 7 was obtained as yellow oil. The molecular formula was
1
3
confirmed to be C15
H
26
O
3
. Based on the C NMR and DEPT data, 15
21.8, 22.1, and
25.6, 39.8, 43.8, and 64.3), six methines (δ
8.2, 40.5, 42.7, 62.2, 73.9, and 124.6), and two quaternary carbons
74.9 and 148.5). These features consist of a (10 → 1) abeoeu-
1
3, a coumarin glycoside, was more effective than the others (with an
carbons were found, including three methyl groups (δ
C
inhibition rate of 49.6%).
2
3
3.4), four methylenes (δ
C
C
In conclusion, we demonstrated the isolation and structural de-
termination of 16 compounds from the twigs and leaves of
R. micranthum. Their structures were determined by extensive spectro-
scopic analyses. The anti-inflammatory activities of the compounds
were evaluated. Among them, compounds 3, 13, and 14 significantly
suppressed the transcription of the NF-κB-dependent reporter gene in
LPS-induced 293T/NF-κB-luc cells. R. micranthum is a traditional anti-
inflammatory Chinese medicine. However, to date, its anti-in-
flammatory components are still unclear. These experimental results
can not only expand the diversity of chemical structures but also pro-
vide evidence for revealing the correlations between chemical con-
stituents and the traditional effects.
(δ
C
desmane sesquiterpene with three oxygenated substituents and a
double bond [28,29], which was further confirmed by the HMBC cor-
relations from H
3.8, CH ). HMBC correlations from H
74.9, C), from H-3, H-4 and H-10 to C-2 (δ
64.3, CH ) placed the three hydroxy groups
3
–14 to C-1 (δ
C
74.9, C), C-10 (δ
–14, H-5, H-9 and H-10 to C-1
73.9, CH), and from H-
C
62.2, CH), and C-9 (δ
C
4
2
3
(
δ
C
C
3
, H-4 and H-5 to C-15 (δ
C
2
at C-1, C-2, and C-15, respectively. The double bond was determined to
be at C-6/7, as suggested by HMBC correlations from H-5, H-8 and H-11
to C-6 (δ
148.4, C). The planar structure of 7 was then settled. Moreover, the
NOE correlations (Fig. 3) of H-2/H-4, H-2/H-5, H-2/H-10, H-2/H –14,
and H
C 3 3
124.6, CH) and from H-5, H-9, H-11, H –12 and H –13 to C-7
(
C
δ
3
Conflict of interest
3
–14/H-5 suggested that these protons were all cofacial (β-or-
iented). Compound 7 was determined as 1α,2α,15-trihydroxy-5-epi-6-
dien-14(10 → 1) abeoeudesmane, and the compound was named rho-
micranin I.
The authors declare that they have no conflicts of interest.
Acknowledgments
Compound 8 was obtained as a white amorphous powder. Its mo-
lecular formula was C21
glycoside of 7. In the HMBC spectrum, the correlations from H-1′ to C-
5 and from H –15 to C-1′ indicated that the glycoside was at C-15. The
coupling constant of the anomeric proton (J1’,2′ = 7.8 Hz) suggested
that glucose was in the β-configuration. The D-configuration of the
glucose was then determined by GC analyses of the hydrolysate of 8.
Thus, compound 8 was tentatively determined as 1α,2α-dihydroxy-5-
epi-6-dien-14(10 → 1) abeoeudesmane 15-O-β-D-glucoside and was
named rhomicranoside G.
36 8
H O . The NMR spectra showed that 8 was the
This work was supported by grants from the National Natural
Science Foundation of China (Nos. 21572274, 21732008, and
1
2
81630094), the CAMS Innovation Fund for Medical Sciences (No. 2016-
I2M-3-012), and the Young Elite Scientists Sponsorship Program by
CAST (2016QNRC001).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.fitote.2019.03.025.
The known compounds isolated from the plant are (7R,8S)-4-hy-
droxy-9’-O–
(α-L-rhamnopyranosyl)-3,3′,5′-trimethoxy-8-O-4′-neo-
lignan (9) [22], (2S,3R)-2,3- dihydro-3-hydroxymethyl-7-methoxy-2-
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polin (13) [33], ssioriside (14) [34], (−)-secoisolariciresinol-9-O-β-D-
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