Lignans and clerodane diterpenoids from: Tinospora sinensis

Article abstract of DOI:10.1039/d0ra04917d

Sixteen lignans with diverse carbon skeletons, including tetrahydrofuran lignans (1, 13-18), 7,9′-dinorlignan (2), dibenzylbutane lignans (9-10), aryl tetrahydronaphthalene lignans (11-12), dihydrobenzofuran neolignans (19-20), 8-O-4′ neolignans (21-22), and six clerodane diterpenoid glucosides (3-8) were isolated from the 70% ethanolic extract of the stems of Tinospora sinensis (Lour.) Merr. Their structures were established by spectroscopic analyses and chemical methods. Among all these isolates, compounds 1-2 and 3-4 were determined as previously undescribed lignans and clerodane diterpenoids respectively, and compounds 9-13 and 17-22 were discovered from T. sinensis for the first time. In addition, it was the first time to report three types of lignans including 7,9′-dinorlignan, dihydrobenzofuran neolignan and 8-O-4′ neolignan from T. sinensis. The biological activities of all isolated compounds were assessed in terms of their nitric oxide production inhibitory activity in LPS stimulated RAW264.7 cells. The results showed that compound 20 exhibited a mild NO production inhibitory effect with an IC50 value of 17.43 ± 2.06 μM. This journal is

Full text of DOI:10.1039/d0ra04917d

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PAPER
Lignans and clerodane diterpenoids from Tinospora
sinensis†
Cite this: RSC Adv., 2020, 10, 28157
a
b
b
b
b
c
Chumao Wen,‡ Jun Li,‡ Xiaoshi Xu, Qingqing Li, Yitong Li, Yu Chen*
b
and Guangzhong Yang
*
0
Sixteen lignans with diverse carbon skeletons, including tetrahydrofuran lignans (1, 13–18), 7,9 -dinorlignan
2), dibenzylbutane lignans (9–10), aryl tetrahydronaphthalene lignans (11–12), dihydrobenzofuran
(
0
neolignans (19–20), 8-O-4 neolignans (21–22), and six clerodane diterpenoid glucosides (3–8) were
isolated from the 70% ethanolic extract of the stems of Tinospora sinensis (Lour.) Merr. Their structures
were established by spectroscopic analyses and chemical methods. Among all these isolates,
compounds 1–2 and 3–4 were determined as previously undescribed lignans and clerodane
diterpenoids respectively, and compounds 9–13 and 17–22 were discovered from T. sinensis for the first
0
time. In addition, it was the first time to report three types of lignans including 7,9 -dinorlignan,
Received 4th June 2020
Accepted 21st July 2020
0
dihydrobenzofuran neolignan and 8-O-4 neolignan from T. sinensis. The biological activities of all
isolated compounds were assessed in terms of their nitric oxide production inhibitory activity in LPS
stimulated RAW264.7 cells. The results showed that compound 20 exhibited a mild NO production
inhibitory effect with an IC50 value of 17.43 ꢀ 2.06 mM.
DOI: 10.1039/d0ra04917d
rsc.li/rsc-advances
that avonoids including rutin, quercetin and isorhamnetin
might be the mainly effective constituents in the crude extracts.
1
. Introduction
3
Tinospora sinensis (Lour.) Merr. is a well known medicinal plant But actually, the chemical compositions of T. sinensis were very
belonging to the family Menispermaceae, which is mainly complicated, which contained terpenoids such as sesquiterpe-
1
distributed in Tibet and southern China. The stems of T. noids, diterpenoids and triterpenoids, lignans, alkaloids,
6
sinensis have been used as Tibetan medicine, which were avonoids and so on. Among them, diterpenoids and lignans
recorded in an ancient Chinese Tibetan medicinal encyclopedia were abundant in T. sinensis. Therefore, in order to have a fully
2
“
Jingzhu Bencao”. They have been widely used for the treat- understanding of these two classes of compounds in T. sinensis,
3
ment of rheumatism, jaundice, and skin diseases for centuries.
a phytochemical reinvestigation of T. sinensis was undertaken,
Previous studies demonstrated that the crude extracts of T. resulting in the isolation of twenty-two compounds, including
sinensis had potent immunomodulatory and anti-inammatory sixteen lignans (1–2, 9–22) and six clerodane diterpenoid
4,5
activities. However, due to the complexities and diversities of glucosides (3–8). Among them, compound 1 was a previously
its chemical constituents, the effective constituents and their undescribed tetrahydrofuran lignan, and compound 2 was
0
molecular mechanisms for its anti-arthritis effect are still poorly
a
previously undescribed 7,9 -dinorlignan glucoside.
6,7
understood. According to the previous report, the EtOAc Compounds 3–4 were previously undescribed clerodane diter-
fraction of ethanolic extract from the stems of T. sinensis penoid glucosides. In this paper, we described structure eluci-
exhibited an anti-arthritis effect, which might be attributed to dations of compounds 1–4 and their NO production inhibitory
inhibiting the production of pro-inammatory cytokines and activities of all isolated compounds 1–22.
down-regulating the MAPK signaling pathway, and suggested
2
. Result and discussion
a
College of Biomedical Engineering, South-Central University for Nationalities, Wuhan
4
30074, P. R. China
Compound 1 was obtained as amorphous powder. It exhibited
b
School of Pharmaceutical Sciences, South-Central University for Nationalities, Wuhan the molecular formula C21
H
O
by HR-ESI-MS (m/z 413.1569
26
7
4
30074, P. R. China. E-mail: yanggz888@126.com; Fax: +86 27 6784 1196; Tel: +86 27
784 1196
+
[
M + Na] , calcd for C21
H
26
O
7
Na, 413.1571), indicating 9 degrees
6
c
1
of unsaturation. The H NMR spectra (Table 1) of 1 showed two
sets of AMX type trisubstituted aromatic proton signals at dH
College of Chemistry and Material Sciences, South-Central University for
Nationalities, Wuhan 430074, P. R. China. E-mail: chenyuwh888@126.com
6
.89 (1H, d, J ¼ 1.8 Hz), 6.88 (1H, d, J ¼ 8.4 Hz), 6.84 (1H, dd, J ¼
8.4, 1.8 Hz); 6.93 (1H, d, J ¼ 1.8 Hz), 6.76 (1H, d, J ¼ 8.4 Hz) and
6.80 (1H, dd, J ¼ 8.4,1.8 Hz), three methoxy groups at d 3.85,
†
1
‡
Electronic supplementary information (ESI) available. See DOI:
0.1039/d0ra04917d
These authors contributed equally to this work.
H
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1
13
0
0
Table 1 H-NMR and C NMR data of compounds 1–2 in CD
in ppm, J in Hz)
3
OD (d 1 was assigned as to be 7S, 7 R, 8R, 8 S which was identical to
tanegool. Therefore, the structure of compound 1 was estab-
0
lished as 4 -O-methylated tanegool and named as tinosporine A.
1
2
Compound 2 had the molecular formula of C H O as
assigned by HR-ESI-MS (m/z 441.1518 [M + Na] , calcd
41.1520). The acid hydrolysis experiment revealed 2 contained
2
2
26 8
a
H
b
C
a
H
b
+
No.
d
d
d
d
C
4
1
2
3
4
5
6
7
8
9
134.8
111.1
149.1
147.2
116.1
120.3
85.1
131.1
131.2
116.1
1
0
a D-glucose moiety. Its H-NMR displayed two sets of AA'BB type
aromatic proton signals at d
6.93 (d, 1.8)
7.15 (d, 8.4)
6.78 (d, 8.4)
H
7.15 (2H, d, J ¼ 8.4 Hz), 6.78 (2H,
157.8 d, J ¼ 8.4 Hz), 6.95 (2H, d, J ¼ 8.4 Hz), 6.67 (2H, d, J ¼ 8.4 Hz),
6.76 (d, 8.4)
6.80 (dd, 8.4, 1.8)
4.62 (d, 7.8)
1.88 (m)
6.78 (d, 8.4)
7.15 (d, 8.4)
116.1 one olenic proton signal at d 5.94 (1H, t, J ¼ 7.8 Hz), one
H
131.2
oxygenated methylene at dH 4.34 (1H, d, J ¼ 12.0 Hz), 4.54 (1H,
d, J ¼ 12.0 Hz), and a set of b-glucopyranosyl proton signals with
53.7
62.3
138.1
74.7
the resonance for the anomeric proton at d
H
4.36 (1H, d, J ¼ 7.8
3.29 (m)
3
4.54 (d, 12.0)
4.34 (d, 12.0)
13
.21 (dd, 11.4, 6.0)
Hz). Apart from the glucopyranosyl signals, the remaining C-
0
1
137.6
111.7
150.5
150.1
112.7
120.7
76.6
133.3 NMR and DEPT spectra of 2 (Table 1) exhibited 16 carbon
0
2
6.89 (d, 1.8)
6.95 (d, 8.4)
6.67 (d, 8.4)
130.4
116.3
156.7
signals, including two para-substituted benzene rings, one
double bond and two methylenes respectively. The above-
mentioned NMR data were similar to those of squadinorligno-
116.3
130.4 side, except for the chemical shi of C-9 shiing downeld
0
3
0
4
0
5
6.88 (d, 8.4)
6.67 (d, 8.4)
6.95 (d, 8.4)
3.26 (m)
0
9
6
6.84 (dd, 8.4, 1.8)
4.52 (d, 8.4)
0
7
35.2
130.7
from d 67.9 in the latter to d 74.7 in 2, which suggested that
C C
0
8
2.55 (m)
50.8
71.5
5.94 (t, 7.8)
both of them had the same aglycon and 9-OH of the aglycon of
squadinorlignoside was glucosylated in 2, instead of 4-OH of
the aglycon being glycosylated in squadinorlignoside. This
0
9
4.25 (dd, 9.0, 4.2)
.93 (dd, 9.0, 7.8)
3
0
0
0
0
0
0
0
0
0
0
0
0
1
2
3
4
5
6
4.36 (d, 7.8)
3.19 (m)
3.20 (m)
3.29 (m)
3.33 (m)
102.9
75.2
78.1
71.7
78.2
62.8
conclusion was further conrmed by the HMBC correlations of
00
2 (Fig. 2) from H
2
-9 to C-1 (d
C
102.9). Accordingly, the structure
of 2 was elucidated as showed in Fig. 1 and named as tinodi-
norlignoside A.
3.83 (dd, 12.0, 2.4)
Compound 3 was isolated as white amorphous powder, and
3.64 (dd, 12.0, 6.0)
3
3
4
-OMe
3.85 (s)
3.81 (s)
3.80 (s)
56.6
56.5
56.5
46
its molecular formula was exhibited to be C38H O17 by HR-ESI-
MS (m/z 797.2619 [M + Na] , calcd 797.2627), suggesting 16
0
+
-OMe
-OMe
0
degrees of unsaturation. The acid hydrolysis experiment indi-
a
b
1
Recorded at 600 MHz. Recorded at 150 MHz.
cated that 3 had a D-glucose moiety. The H-NMR spectra (Table
2
) of 3 displayed the presence of a 1,2,4-trisubstituted aromatic
moiety [dH 7.28 (d, J ¼ 1.2 Hz), 7.15 (d, J ¼ 8.4, Hz), 7.19 (d, J ¼
.4, 1.2 Hz)], one methoxy group [d 3.82 (s)], three singlet
8
H
3
.81 and 3.80, two oxygenated methines [dH 4.52 (1H, d, J ¼ 8.4 methyls [dH 1.95 (s), 1.90 (s), 1.08 (s)], a trans double bond [dH
Hz), 4.62 (1H, d, J ¼ 7.8 Hz)], two methines [d 1.88 (1H, m), 2.55 6.42 (d, J ¼ 16.2 Hz), 7.64 (d, J ¼ 16.2 Hz)], an anomeric sugar
H
(1H, m)], two oxygenated methylenes [d
H
4.25 (1H, dd, J ¼ 9.0, proton [4.95 (d, J ¼ 7.2 Hz)], and the typical furan moiety signals
13
4
.2 Hz), 3.93 (1H, dd, J ¼ 9.0, 7.8 Hz); 3.29 (1H, m), 3.21 (1H, dd, [d 6.52 (br s), 7.52 (t, J ¼ 1.2 Hz), 7.64 (br s)]. The C NMR
H
J ¼ 11.4, 6.0 Hz)], which were characteristic proton signals of spectra and DEPT (Table 2) of 3 showed 38 carbon resonances,
0
13
a 7,9 epoxy-tetrahydrofuran lignan. The C-NMR and DEPT including one lactone carbonyl signal at dC 174.5, one 3-
spectra (Table 1) of 1 revealed 21 carbon signals, which were substituted furan moiety, two quaternary carbons, six methines,
assigned as two 1,2,4-trisubstituted benzene rings, four four methylenes and one methyl at d 15.3, which implied that 3
C
methines, two oxygenated methylenes and three methoxy possessed a clerodane diterpenoid core. Besides, the two
groups. Detailed analysis of the NMR data of 1 suggested that 1 carbonyl groups at d 172.3, d 171.3 and two methyls at d 1.95,
C
C
H
8
was structurally related to tanegool. The signicant difference
H
d 1.90 in the NMR spectrum suggested the presence of two
between them was that 1 had one more methoxy group than acetyl groups in 3. Apart from these signals, the remaining
0
tanegool, suggesting that 1 was 4 -O-methylated of tanegool signals displayed a trans-feruloyl and glucopyranosyl group. The
which was further conrmed by the HMBC correlation (Fig. 2) of abovementioned data were closely similar to those of tinosine-
0
3 H C
(d 3.80) with C-4 (d 150.1). The relative conguration of noside D, except for the presence of one more acetyl group
10
OCH
1
was determined by the ROESY spectrum (Fig. 3). The ROESY compared to tinosinenoside D. The HMBC correlation of 3
0
0
correlations of H-7/H -9/H-8 , H-8/H-7 indicated that H-7, H -9 (Fig. 2) from H-1 (dH 5.11) to the carbonyl group of OAc (dC
2
2
0
0
and H-8 were on the same side, H-8 and H-7 were on the other 171.3), from H-2 (dH 5.31) to the carbonyl group of OAc (dC
side which were consistent with those of tanegool. Compund 1 172.3) indicated that 3 could be a 1-acetylated of tinosinenoside
and tanegool had the similar chromophores. By comparison of D. The relative conguration of 3 was revealed by the coupling
the ECD of 1 with those of tanegool, they displayed the same constants and the ROESY spectrum (Fig. 3). The coupling
8
Cotton effects in the CD spectra. The absolute conguration of constants of H-1/H-2 (J1,2 ¼ 3.0 Hz) and H1/H-10 (J1,10 ¼ 11.4 Hz)
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Fig. 1 Structures of compounds 1–22.
suggested H-1, H-2 and H-10 were axial, equatorial and axial oriented, and H-12 was assigned to be a-oriented by the corre-
position respectively. The ROESY correlations of H-1/H-5 (d lation of H-12 (d 5.70)/H-10. The correlation of H-4 (d 4.85)/H-
.99)/H -20 (d 1.08) indicated that H-1, H-5 and H -20 were b- 6b (d
H
H
H
1
3
H
3
H
1.58) indicated H-4 was b-oriented. Consequently, the
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1
1
Fig. 2 Key HMBC and H– H COSY correlations of compounds 1–4.
structure of 3 was established as depicted in Fig. 1 and named
as 1-acetyltinosinenoside D.
The remaining sixteen known compounds were identied as
10
10
tinosinenoside D (5), tinosinenoside F (6), tinosinenoside E
10
11
Compound 4 shared the same molecular formula of (7), tinosinenoside K (8), 2-(4-hydroxy-3,5-dimethoxybenzyl)-
12
38 46
C H O17 as 3, which was determined by HR-ESI-MS at m/z 3-(4-hydroxy-3-methoxybenzyl)butane-1,4-diol (9), secoisolar-
+
13
13
7
97.2624 [M + Na] (calcd for C38
H
46
O
17Na, 797.2627) and
C
iciresinol (10), 1,2,3,4-tetrahydro-7-hydroxy-1-(4-hydroxy-3,5-
NMR data. Its NMR data (Table 2) exhibited close similarities to dimethoxyphenyl)-6,8-dimethoxy-2,3-naphthalenedimethanol
14
15
those of compound 3, except for the absence of a trans double (11), isolariciresinol (12), rel-(2R,3S,4R,5S)-tetrahydro-2,5-bis-
16
bond signal and the presence of a cis double bond signal [dH (4-hydroxy-3-methoxyphenyl)-3,4-furandimethanol (13), syrin-
1
3
17
18
6
.93 (d, J ¼ 13.2 Hz), 5.93 (d, J ¼ 13.2 Hz); d 144.8, 118.6]. So it garesinol (14),
medioresinol (15),
pinoresinol (16),
C
18
19
suggested that the trans-feruloyl group at C-4 in 3 was lirioresinol-A (17), epipinoresinol (18), dehydrodiconiferyl
substituted by a cis-feruloyl group in 4, which was further alcohol (19), simulanol (20), threo-guaiacylglycerol-b-O-4 -
conrmed by the HMBC spectrum (Fig. 2). The relative stereo- coniferyl ether (21), erythro-guaiacylglycerol-b-O-4 -coniferyl
chemistry of 4 was determined by the ROESY correlations, ether (22) based on the comparison of their NMR data with
which was consistent with those of 3. Thus, the structure of 4 those reported in the literatures.
was dened and named as 1-acetyltinosinenoside E.
20
21
0
22
0
22
Fig. 3 Key ROESY correlations of compounds 1 and 3.
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1
13
Table 2 H-NMR and C NMR data of compounds 3–4 in CD
in ppm, J in Hz)
3
OD (d diterpenoid glucosides might not be the effective constituents
of T. sinensis in the treatment of inammation.
3
4
3
. Experimental
a
H
b
C
a
H
b
No.
d
d
d
d
C
3
.1 General experimental procedures
Optical rotations were recorded on an Autopol IV polarimeter
Rudolph Research Analytical, Hackettstown, NJ, USA). UV
1
2
3
5.11 (dd, 11.4, 3.0)
5.31 (q, 3.0)
2.32 (overlapped)
74.6
70.3
32.1
5.13 (dd, 11.4, 3.0)
5.30 (q, 3.0)
2.36 (overlapped)
1.99 (overlapped)
4.91 (overlapped)
2.01 (m)
74.4
70.3
32.1
(
1.98 (overlapped)
spectra were obtained on a UH5300 UV-VIS Double Beam
spectrophotometer (Hitachi Co., Tokyo, Japan). NMR spectra
were measured with a Bruker AVANCE IIITM 600 MHz spec-
trometer (Bruker, Ettlingen, Germany) in C D N using tetra-
5 5
methylsilane (TMS) as an internal reference standard. Chemical
4
5
6
4.85 (overlapped)
1.99 (m)
1.58 (m)
72.8
39.0
26.5
72.5
38.8
26.7
1.58 (m)
1.47 (td, 13.8, 5.4)
7
2.31 (overlapped)
30.2
2.34 (overlapped)
30.3
1
.72 (td, 13.2, 5.4)
1.71 (td, 413.8, 4.2)
shis (d) have been shown in ppm and the coupling constants
8
9
1
1
76.7
40.8
36.6
37.2
76.6
40.9
36.7
37.1
(J) have been expressed in Hz. High-resolution electrospray
mass spectroscopy was performed on an Thermo Scientic Q
Exactive Orbitrap LC-MS/MS System (HR-ESI-MS) (Thermo
Scientic, Waltham, MA, USA). High-performance liquid chro-
matography (HPLC) was conducted on an Ultimate 3000 HPLC
system (Dionex Co., Sunnyvale, CA, USA) equipped with an
Ultimate 3000 pump and Ultimate 3000 Variable Wavelength
detector, as well as a semi-preparative YMC-Pack ODS-A column
0
1
2.67 (overlapped)
2.67 (overlapped)
2.54 (t, 11.4)
2.66 (t, 13.2)
1.99 (overlapped)
5.65 (dd, 13.2, 3.6)
1.99 (overlapped)
1
1
1
1
1
1
2
1
2
3
4
5
6
7
8
9
1
2
3
4
5
6
7
5.70 (dd, 12.6, 3.6)
73.6
73.4
127.1
109.7
145.5
141.4
174.5
15.3
168.1
117.4
146.9
130.4
113.0
151.1
150.2
117.4
123.5
21.5
127.0
109.7
145.4
141.4
174.6
15.4
167.7
118.6
144.8
130.9
115.4
6.52 (br s)
7.52 (t, 1.2)
7.64 (br s)
6.55 (d, 1.2)
7.53 (t, 1.2)
7.65 (br s)
(
250 ꢁ 10 mm, 5 mm), column chromatography (CC) was con-
0
1.08 (s)
1.11 (s)
ducted with silica gel (200–300 mesh and 300–400 mesh,
Qingdao Haiyang Chemical Industry Co., Ltd, Qingdao, China).
Chromatographic grade acetonitrile was purchased from Chang
Tech Enterprise Co., Ltd (Taiwan, China). RAW264.7 murine
macrophages were purchased from the cell bank of Chinese
0
0
0
0
0
0
0
0
0
6.42 (d, 16.2)
7.64 (d, 16.2)
5.93 (d, 13.2)
6.93 (d, 13.2)
7.28 (d, 1.2)
7.74 (d, 1.8)
150.1 Academy of Sciences (Shanghai, China). Dexamethasone and
149.3
116.8
126.1
21.5
lipopolysaccharides (LPS) were purchased from Sigma Chem-
ical Co. Ltd (St. Louis, MO, USA). Cell Counting Kit (CCK-8) was
purchased from Beyotime Biotechnology (Shanghai, China).
Dulbecco modied Eagle medium (DMEM) and penicillin-
streptomycin solution were purchased from GE Healthcare
Life Sciences (Logan, UT, USA). Fetal bovine serum (FBS) was
purchased from Gibco, Life Technologies (Grand Island, NY,
USA). Reagent grade dimethyl sulfoxide (DMSO) was purchased
from Vetec, Sigma Chemical Co. (St. Louis, MO, USA). The
absorbance was read on a Multiskan GO microplate reader
7.15 (d, 8.4)
7.19 (d, 8.4, 1.2)
1.90 (s)
7.14 (d, 8.4)
7.18 (d, 8.4, 1.8)
1.92 (s)
-Ac
-Ac
171.3
171.4
21.2
172.4
56.7
102.3
74.9
78.0
71.4
78.4
62.6
2
1.95 (s)
21.3
2.02 (s)
172.3
0
6
-OCH
3
3.82 (s)
4.95 (d, 7.2)
3.48 (m)
3.45 (m)
3.37 (m)
56.9
102.2
74.9
77.9
71.4
78.4
62.5
3.86 (s)
4.98 (d, 7.8)
3.50 (m)
3.48 (m)
3.40 (m)
3.44 (m)
3.69 (dd, 12.0, 5.4)
3.88 (overlapped)
00
Glc-1
0
0
0
0
0
0
0
0
0
0
2
3
4
5
6
3.42 (m)
3.66 (dd, 12.0, 5.4)
(Thermo Fisher Scientic Inc. Waltham, MA, USA).
3.85 (dd, 12.0, 1.8)
3.2 Plant material
a
b
Recorded at 600 MHz. Recorded at 150 MHz.
The stems of T. sinensis were purchased from Xining, Qinghai
province, P. R. China and identied by Associate professor
Xinqiao Liu, School of Pharmaceutical Sciences, South Central
University for Nationalities.
In addition, all the isolated compounds were assessed for
their inhibitory activities against NO production in LPS stimu-
lated RAW264.7 cells with dexamethasone as a positive control 3.3 Extraction and isolation
(
IC50 ¼ 9.58 ꢀ 3.34 mM). As a result, compound 20 exhibited
The air-dried stems of Tinospora sinensis (Lour.) Merr. (9.4 kg)
were extracted by 70% EtOH for three times (each 24 h) at room
temperature, yielding a crude extract (1.0 kg) aer evaporation,
then the extract was chromatographed over a polyamide
mild NO production inhibitory effects with IC values of 17.43
5
0
ꢀ
2.06 mM, however, the remaining compounds did not display
obvious NO production inhibitory activity (IC50 > 20 mM). These
results showed that six clerodane diterpenoid glucosides had no
obvious inhibitory activities against NO production which was
column (80–100 mesh) eluting with a gradient of EtOH–H O
2
(1 : 0, 7 : 3, 1 : 1, 3 : 7, 0 : 1) to obtain ve fractions (Fr. A–Fr. E).
10
consistent with the previous report, suggesting that clerodane
Fr. B (41.3 g) was subjected to ODS column chromatography
(
MeOH–H
2
O, 2 : 8, 4 : 6, 6 : 4, 8 : 2, 1 : 0) to afford een
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subfractions (Fr. B1–Fr. B15). Fr. B11 was chromatographed by
silica gel column eluting with a gradient of CH Cl –MeOH
4. Conclusions
2
2
(19 : 1, 9 : 1, 1 : 1), and then further puried by semi- In summary, our study on the chemical constituents of Tino-
preparative HPLC to afford compounds 1 (4.0 mg), 11 (3.0 spora sinensis has led to the isolation of a previously unde-
mg), 12 (8.7 mg), 13 (4.2 mg), 21 (29.0 mg), 22 (11.5 mg). Fr. B13 scribed tetrahydrofuran lignan, tinosporine A (1), a previously
0
was chromatographed by silica gel column eluting with undescribed 7,9 -dinorlignan glucoside, tinodinorlignoside A
2 2
a gradient of CH Cl –MeOH (19 : 1, 9 : 1, 8 : 2, 7 : 3, 1 : 1), and (2), two previously undescribed clerodane diterpenoid gluco-
then further puried by semi-preparative HPLC to afford sides, 1-acetyltinosinenoside D (3) and 1-acetyltinosinenoside E
compounds 2 (5.0 mg), 3 (9.9 mg), 4 (2.2 mg), 5 (24.8 mg), 6 (1.8 (4), together with four known clerodane diterpenoid glucosides
mg), 7 (9.0 mg), 8 (3.5 mg), 9 (4.0 mg), 10 (123.1 mg), 14 (10.3 (5–8) and fourteen known lignans (9–22). According to these
mg), 15 (1.5 mg), 16 (30.8 mg), 17 (1.4 mg), 18 (5.7 mg), 19 (8.8 structure types, these compounds might be divided into lignans
0
mg), 20 (11.0 mg).
including tetrahydrofuran lignans (1, 13–18), 7,9 -dinorlignan
(
2), dibenzylbutane lignans (9–10), aryl tetrahydronaphthalene
0
3.4 Spectroscopic data
lignans (11–12), dihydrobenzofuran neolignans (19–20), 8-O-4
neolignans (21–22), and six clerodane diterpenoid glucosides
Tinosporine A (1). Colorless powder; [a]D ¼ +77.0 (c 0.03,
(
3–8). Among them, compounds 9–13, 17–22 were discovered
MeOH); UV (MeOH) lmax (log 3): 235 (3.60), 275 (3.58) nm; ECD
ꢂ4
from the genus Tinospora for the rst time. Moreover, it was the
rst time to report three types of lignans including 7,9 -dinor-
lignan, dihydrobenzofuran neolignan and 8-O-4 neolignan
(
c 8 ꢁ 10 M, MeOH) l (q): 281 (+0.47), 236 (+1.11), 205 (+8.31).
0
1
13
H and C NMR data see Table 1; HR-ESI-MS m/z 413.1569 [M +
0
+
Na] (calcd for C21
H
26
O
7
Na, 413.1571).
from T. sinensis, which could be regard as characteristic maker
constituents of T. sinensis. Three types lignans such as tetra-
hydrofuran, dibenzylbutane and aryl tetrahydronaphthalene
lignans and clerodane diterpenoids are the most prominent
secondary metabolism in the genus of Tinospora, such as T.
Tinodinorlignoside A (2). Colorless powder; [a]
D
¼ ꢂ11.4 (c
0
.04, MeOH); UV (MeOH) l (log 3): 220 (3.95), 280 (3.89) nm;
max
1
13
H and C NMR data see Table 1; HR-ESI-MS m/z 441.1518 [M +
Na] (calcd for C H O Na, 441.1520).
+
2
2
26 8
1-Acetyltinosinenoside D (3). Colorless powder; [a]
D
¼ +16.3
2
capillipes and T. cordifolia. In contrast, it was noteworthy that
(
3
7
c 0.65, MeOH); UV (MeOH) lmax (log 3): 240 (2.67), 295 (2.63),
1
13
the clerodane diterpenoid glucosides with a feruloyl group,
such as compounds 3–8, had been only found in T. sinensis,
which suggested that this type of compounds might be used to
distinguish T. sinensis from other species of Tinospora. These
ndings together with those previous reports imply that lignans
and clerodane diterpenoids could serve as chemotaxonomic
markers of species in series Tinospora, but also need to be
further research.
20 (2.67) nm; H and C NMR data see Table 2; HR-ESI-MS m/z
+
97.2619 [M + Na] (calcd for C38
-Acetyltinosinenoside E (4). Colorless powder; [a]
c 0.08, MeOH); UV (MeOH) l (log 3): 235 (3.55), 300
46
H O17Na, 797.2627).
1
D
¼ ꢂ43.7
(
(
7
max
1
13
3.52) nm; H and C NMR data see Table 2; HR-ESI-MS m/z
+
97.2624 [M + Na] (calcd for C H O Na, 797.2627).
38 46 17
23
3
.5 Acid hydrolysis of compounds 2–4
Compounds 2 (1.1 mg), 3 (1.2 mg) and 4 (0.9 mg) were respec-
tively dissolved in 2.0 mL of 4.0 M CF COOH, three reaction Conflicts of interest
3
ꢃ
mixtures were reuxed at 95 C for 3 h and then extracted with
EtOAc for three times (each 2 mL). The aqueous layers were
There are no conicts to declare.
evaporated to obtain the sugar fractions. The absolute cong-
uration of sugar was determined by the following method.
Pyridine (0.5 mL) and L-cysteine methyl ester hydrochloride (1.0
Acknowledgements
mg) were added to the sugar fractions and standard of D-glucose This work was nancially supported by the National Major New
ꢃ
respectively, and four reaction mixtures were reuxed at 60 C Drugs Innovation and Development (2017Z09301060) and the
for 1 h. Aer cooling, 2-methylphenyl isothiocyanate was added Special Fund for Basic Scientic Research of Central Colleges,
to four reaction mixtures, and continued to react for 1 h at South-Central University for Nationalities (CZP18004).
ꢃ
60
C. Then four reaction mixtures were subjected to HPLC
analysis (column: YMC-Pack ODS-A 250 ꢁ 4.6 mm I. D; mobile
ꢂ1
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were closely consistent with that of D-glucose (20.81 min).
Therefore, the sugars moiety in 2–4 were identied to be D-
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