Tinospinosides D, E, and Tinospin E, further clerodane diterpenoids from Tinospora sagittata

Article abstract of DOI:10.1248/cpb.c12-00482

Chemical investigation on the roots of Tinospora sagittata resulted in the isolation of three novel cis-clerodane diterpenoids, tinospinosides D, E, and tinospin E, together with two known compounds, columbin and columbin glucoside, and their structures w

Full text of DOI:10.1248/cpb.c12-00482

1324
Note
Chem. Pharm. Bull. 60(10) 1324–1328 (2012)
Vol. 60, No. 10
Tinospinosides D, E, and Tinospin E, Further Clerodane Diterpenoids
from Tinospora sagittata
a
,a
a,b
c
d
a
Chao Huang, Wei Li,* Fenghua Ma, Qin Li, Yoshihisa Asada, and Kazuo Koike
a
Faculty of Pharmaceutical Sciences, Toho University; 2–2–1 Miyama, Funabashi, Chiba 274–8510, Japan:
b
c
Heilongjang Institute for Food and Drug Control; Harbin 150001, P.R. China: Laboratory Center, The Fourth
d
Affiliated Hospital, China Medical University; Shenyang 110001, P.R. China: and Faculty of Pharmaceutical
Sciences, Tokyo University of Science; 2641 Yamazaki, Noda, Chiba 278–8510, Japan.
Received May 28, 2012; accepted July 17, 2012
Chemical investigation on the roots of Tinospora sagittata resulted in the isolation of three novel
cis-clerodane diterpenoids, tinospinosides D, E, and tinospin E, together with two known compounds,
columbin and columbin glucoside, and their structures were determined by extensive spectroscopic analyses,
chemical reactions and computer-assisted calculations. The inhibitory activity of the isolated compounds and
their chemical derivatives on nitric oxide production in lipopolysaccharide and interferon-γ activited J774.1
macrophage-like cells was also evaluated.
Key words Tinospora sagittata; Menispermaceae; clerodane diterpenoid; nitric oxide
1
13
The genus Tinospora (Menispermaceae) has about thirty
H- and C-NMR spectroscopic data also indicated the pres-
species, mainly distributing in the tropic area of Asia and ence of two carbonyl moieties at δ 173.5 and 174.4, as well
C
Africa, and many of the species have been used in traditional as a β-glucopyranosyl moiety with the anomeric resonances at
medicines, e.g., T. codifolia has been used in Ayurveda (In- δ 5.64 (1H, d, J=7.1Hz) and δ 100.5, respectively (Tables 1,
H
C
1)
dian traditional medicines) as a hepatoprotectant ; the stem 2). The β-glucopyranosyl moiety was determined to be in the
of T. sinensis has been used in Traditional Chinese Medicine D-form by GLC analysis of its trimethylsilylthiazolidine deriv-
TCM) in the treatment of rheumatism and muscle pain; the ative after acid hydrolysis of 1. The H- and C-NMR spec-
1
13
(
root of T. capillipes and T. sagittata has been used in TCM to troscopic data of 1 showed resemble B ring and C ring res-
relieve sore throat, expel superficial infection, and stop diar- onances, but different A ring resonances in comparison with
2
)
7)
rhea. The main chemical constituents of Tinospora species tinospinoside C. Namely, resonances of an epoxy moiety at
have been reported to be diterpenoids, which has 12-furano δC 54.8 (C-2) and 53.8 (C-3), and an oxyganated quaternary
3
–5)
cis-clerodane skeleton as their characteristic structure.
As carbon resonance at δ 83.9 (C-4) were observed in 1, instead
C
a part of our continuing phytochemical research on Tinospora of resonances of C-2 oxyganated methine and C-3,4 olefin in
species, we have previously reported the isolation of three tinospinoside C. The epoxy moiety was assigned at C-2 and
1
1
novel clerodane diterpenoids, tinosposinensides A–C, from C-3 by sequential H– H correlation spectroscopy (COSY)
6
)
the stems of T. sinensis, and three novel clerodane diterpe- correlations between resonances of H-10, Hα,β-1, H-2, and
7
)
noids, tinospinosides A–C, from the roots of T. sagittata. In H-3, and a heteronuclear multiple bond connectivity (HMBC)
a further investigation on the roots of T. sagittata, we herein correlation from δH 2.69 (H-10) to δ 54.8 (C-2) (Fig. 2). The
C
report the isolation and structural determination of three new oxylated quaternary carbon resonance at δ 83.9 was assigned
C
clerodane diterpenoids, tinospinoside D (1), tinospin E (2), and to C-4, by HMBC correlations from δH 2.14 (H -19) and δ
3
H
tinospinoside E (3) (Fig. 1). The inhibitory activity of these
compounds and their chemical derivatives on nitric oxide
production in lipopolysaccharide (LPS) and interferon (INF)-γ
activited J774.1 macrophage-like cells was also evaluated.
Results and Discussion
The EtOAc fraction from the MeOH extract of roots of T.
sagittata was fractionated by repeated column chromatogra-
phy followed by HPLC purification to afford five compounds
1–5. The known compounds were identified to be columbin
(
4) and columbin glucoside (5) by comparing the physical and
8
,9)
spectroscopic data in literature.
2
2
Tinospinoside D (1) was isolated as a colorless solid, [α]D
−
28.2 (c=0.21, MeOH). Its molecular formula was established
1
as C H O by high resolution (HR)-FAB-MS. The H-NMR
2
7
36 13
spectroscopic data of 1 (Table 1) showed a set of characteristic
downfield resonances at δ 6.66 (1H, brd, J=1.6Hz), 7.67 (1H,
H
t, J=1.6Hz) and 7.79 (1H, brd, J=1.6Hz) for C-12 furan ring
10)
in cis-clerodane type diterpenoids from Tinospora sp. The
The authors declare no conflict of interest.
Fig. 1. Chemical Structures of Compounds 1–5, 3a and 5a
*
To whom correspondence should be addressed. e-mail: liwei@phar.toho-u.ac.jp
© 2012 The Pharmaceutical Society of Japan

October 2012
1325
1
1
Fig. 2. Key H– H COSY and HMBC Correlations for Compounds 1–3
2
.69 (H-10) to δC 83.9 (C-4). The O-β-glucopyranosyl and δH 1.23 (H -20) and 1.97 (H -11). Although the orientation of
3 β
carbonyl moieties connected to C-4 were determined by the C-8 hydroxy moiety could not have been directly concluded
HMBC correlations from δH 5.64 (Glc-H-1) to δ 83.9 (C-4), only by NOESY correlations, a good agreement of the C ring
C
and δH 4.44 (H-3) to δC 174.4 (C-18), respectively. The C-8 NMR spectroscopic data between 1 and tinospinoside C in-
hydroxy moiety was determined by HMBC correlations from dicated that they have same β-oriented C-8 hydroxy moiety.
δH 2.22, 2.03 (Hα,β-6), 2.18, 1.97 (Hα,β-11) and 1.23 (H -20) to The NOESY correlations between δH 2.14 (H -19) and 5.64
3
3
δC 74.6 (C-8).
(Glc-H-1), and δ 5.64 (Glc-H-1) and 4.44 (H-3) indicated their
H
cis Relationship of H-10 and CH -19 was elucidated by cis relationship, namely, β-oriented 2,3-epoxyl and 18-carbonyl
3
the nuclear Overhauser effect spectroscopy (NOESY) cor- moieties, and α-oriented 4-O-glucopyranosyl moiety. A clear
relation between δH 2.69 (H-10) and 2.14 (H -19), and trans 1,4-transannular correlation between δH 2.22 (H -6) and
3
β
relationship of H-10 and CH -20 by absence of the NOESY 1.23 (H -20) was also observed in the NOESY spectrum.
3
3
correlation between δH 2.69 (H-10) and 1.23 (H -20) (Fig. Compound 1 adopting a unique all boat conformation for its
3
3). This conclusion was further supported by an overall ob- tricyclic ring system was also indicated by energy calculation
servation of the NOESY correlations between H -20/H -6, of the computer generated conformers using Discovery Studio
3
β
H -7, and H -19/H -6, H -7. The β-orientation of furan ring at Ver. 2.1 software, in which, a total energy for the conformer
β
3
α
α
C-12 was deduced from NOESY correlations between δ 1.23 with boat-boat-boat ring conformation was calculated lower
H
(
H -20) and 7.79 (H-16), δH 2.18 (H -11) and 6.41 (H-12), and than that with chair-chair-boat conformation. This fact can
3
α
1
Table 1. H-NMR Spectroscopic Data (δ) of Compounds 1–3 and 5a (500MHz in Pyridine-d5)
Position
1
1
2
3
5a
5.24 (d, 5.1)
2.29 (ddd, 15.5, 7.5, 3.2)
5.41 (d, 5.0)
5.37 (d, 5.0)
a)
2
.20
2
3
6
3.64 (dt, 4.6, 3.2)
4.44 (d, 4.6)
6.43 (dd, 7.8, 5.0)
6.53 (dd, 7.8, 1.4)
2.74 (dd, 15.8, 8.1)
2.35 (dd, 15.8, 3.5, β)
7.27 (dd, 8.1, 3.5)
6.50 (dd, 8.2, 5.0)
7.37 (dd, 8.2, 1.1)
2.97 (dd, 16.1, 8.1)
2.41 (dd, 15.2, 3.0, β)
7.19 (dd, 8.1, 3.0)
6.36 (dd, 8.0, 5.1)
6.49 (dd, 8.0, 1.8)
2.08 (brdd, 13.4, 7.1)
1.78 (ddd, 13.4, 6.6, 5.8, β)
2.84 (m)
a)
2.22 (β)
2
.03 (dt, 12.8, 2.7)
7
8
2.76 (m, β)
a)
2
.22
1.86 (m, β)
2.79 (dd, 10.5, 3.4)
1.58 (s)
1
1
0
1
2.69 (m)
2.08 (s)
2.07 (s)
2.18 (dd, 13.3, 8.5)
2.41 (d, 14.7)
2.13 (dd, 14.7, 11.5, β)
5.35 (d, 11.5)
6.72 (t, 0.9)
7.65 (t, 1.6)
7.78 (dd, 1.3, 0.7)
1.23 (s)
2.41 (d, 14.6)
2.11 (dd, 14.6, 11.5 β)
5.33 (d, 11.5)
6.68 (t, 0.9)
7.63 (d, 1.6)
7.72 (t, 0.7)
1.39 (s)
2.27 (dd, 14.2, 2.6, β)
2.18 (dd, 14.2, 11.7)
5.67 (dd, 11.7, 2.6)
6.72 (dd, 1.9, 0.7)
7.67 (t, 1.6)
1
.97 (dd, 13.3, 8.5, β)
1
1
1
1
1
2
2
4
5
6
9
0
6.41 (t, 8.5)
6.66 (brd, 1.6)
7.67 (t, 1.6)
7.79 (brd, 1.6)
2.14 (s)
7.84 (t, 0.7)
1.36 (s)
1.23 (s)
1.24 (s)
1.23 (s)
1.42 (s)
OCH3
Glc-1
Glc-2
Glc-3
Glc-4
Glc-5
Glc-6
3.77 (s)
5.64 (d, 7.1)
5.40 (d, 7.3)
4.24 (t, 7.3)
a)
4.26
a)
a)
4.25
4.27
4.15 (t, 8.5)
4.27 (t, 7.1)
3.93 (ddd, 8.5, 6.5, 2.5)
4.48 (dd, 11.5, 2.5)
4.02 (m)
4.54 (dd, 12.0, 1.3)
4.34 (dd, 12.0, 5.7)
4
.22 (dd, 11.5, 6.5)
a) Overlapped resonances.

1326
Vol. 60, No. 10
be interpreted as that C5–C4 bond with large substitutions to be H-7 by the HMBC correlation from δH 7.27 (H-7) to
of β-D-glucopyranosyl moiety and carbonyl methyl ester at C-17 lactone carbonyl carbon at δ 166.8 (Fig. 2). The HMBC
C
C-4, preferentially taking a more stable bowsprit orientation correlation from δ 5.41 (H-1) to carbonyl carbon at δ 175.3,
H
C
toward B ring, consequently resulting A ring in boat form. Al- suggested the lactone between C-1 and C-4. The hydroxylated
though the absolute configurations of the asymmetric centers quaternary carbon C-4 was assigned by HMBC correlations
in 1 were not determined, it was assumed to have the same from δH 1.23 (H -19) and 6.43 (H-2) to its carbon resonance
3
absolute configurations in the diterpene backbone with those at δC 81.3. By these analyses, the gross structure of 2 was
structure and biosynthesis related cis-clerodane diterpenoids elucidated.
7
)
13
isolated in this study and in a previous study.
In comparison to the C-NMR spectroscopic data of 2
Tinospin E (2) and tinospinoside E (3) were both isolated (Table 2), a glycosylation shift (+5.2ppm) was observed at
as colorless solid. The molecular formulas were established C-4 in 3. The β-D-glucopyranosyl moiety at C-4 was further
as C H O and C H O , respectively by the HR-FAB-MS determined by the HMBC correlation from δH 5.40 (Glc-H-1)
2
0
20
6
26 30 11
data. Compounds 2 and 3 were also cis-clerodane type diter- to δ 86.5 (C-4).
C
penoids, which were indicated by the typical C-12 furan ring
On acid hydrolysis of 3, it afforded a pair of compounds.
1
downfield resonances in the H-NMR spectrum (Table 1). The One was 2, and the other was identified to be fibleucin (3a), a
1
13
superimposable H- and C-NMR spectroscopic data of 3 and known compound which was firstly isolated from Fibraurea
11)
2
, except for resonances of an additional β-glucopyranosyl chloroleuca MIERS, and its structure was well established by
12)
moiety in 3 suggested that 3 was a β-glucopyranoside of 2 X-ray analysis. Compound 2 have very similar but different
1
13
(
Tables 1, 2). Thus, structural elucidation of 2 was carried out
H- and C-NMR spectroscopic data in comparison with 3a.
Compound 2 has same relative configurations for A ring and
first.
1
In the H-NMR spectrum of 2, in addition to the B ring as 3a, since same NOESY correlations between H-3/
2-oxymethine proton resonance at δH 5.35, another oxyme- H -19, H-10/H -19, H -6/H -20, and H -6/H -19 were observed
1
3
3
β
3
α
3
thine proton at δ 5.41 was also observed, which was assigned (Fig. 3). The opposite H-12 orientations of 2 and 3a were
H
1
1
to be H-1 by the H– H COSY correlation between δ 5.41 deduced from their different NOESY correlations between
H
1
1
(
H-1) and 2.08 (H-10). In the H– H COSY spectrum, H-1 H-12/H-10 for 2, and H-12/H -20 for 3a. Thus, the structure
3
was showed to be correlated with a pair of coupled olefin of 2 was determined to be with a β-orientated 12-furan ring.
proton resonances at δH 6.43 (H-2) and δH 6.53 (H-3), sug- It is necessary to note the production of a pair of
gesting presence of an olefin moiety at C-2 and C-3. Another 12-epimeric compounds, columbin (4) and a new compound
downfield olefin proton resonance at δH 7.27 was assigned 5a after acid hydrolysis of columbin glucoside (5). The
13
a)
Table 2.
C-NMR Spectroscopic Data (δ) of Compounds 1–3 and 5a (125MHz in Pyridine-d5)
Position
1
2
3
5a
1
2
3
4
5
6
7
8
9
22.1
54.8
53.8
83.9
42.6
29.1
27.0
74.6
40.6
45.5
43.8
71.1
127.8
109.6
144.4
140.3
173.5
174.4
29.0
22.4
52.1
100.5
75.8
78.8
72.1
78.5
63.0
74.6
130.7
138.6
81.3
73.8
131.2
134.0
86.5
74.6
129.4
138.5
81.7
43.2
44.5
38.8
31.7
31.7
26.5
141.3
137.9
37.2
141.2
137.8
37.2
16.8
42.9
36.4
10
11
12
13
14
15
16
17
18
19
20
54.9
54.3
55.2
44.4
44.2
48.8
70.2
70.2
70.1
124.6
109.5
144.1
140.8
166.8
175.3
26.5
124.5
109.4
144.0
140.8
166.7
171.8
26.5
125.3
109.5
144.3
140.7
175.2
175.6
24.1
25.5
25.6
28.8
OCH3
Glc-1
Glc-2
Glc-3
Glc-4
Glc-5
Glc-6
101.8
75.3
78.7
71.5
78.7
62.4
a) Assignment were based on DQF-COSY, TOCSY, DEPT, NOESY, and HMBC experiments.

October 2012
1327
Fig. 3. Key NOESY Correlations for Compounds 1–3 and 5a
structure of 5a was determined to be 12-epimer of 4 by the microplate reader.
characteristic NOESY correlation between H-12/H -20 (Fig.
Plant Material Plant collection and identification of the
roots of T. sagittata were same as previously described.
Extraction and Isolation The MeOH extract and EtOAc
3
7
)
3
), and therefore was given the trivial name 12-epi-columbin.
Compounds 1, 2, 3, 4, 5 and 5a were evaluated for their
inhibitory activities on nitric oxide production in LPS and fraction were obtained by the same procedure as previ-
7
)
INFγ-activited J774.1 macrophage-like cells. Only compound ously reported. The EtOAc fraction (37.8g) was subjected
a showed nitric oxide inhibitory activity with the IC50 to silica gel column chromatography with a gradient of
value of 84µM, while the IC50 value of the positive control CHCl –MeOH–H O to give five fractions (1–5). Fraction 4
5
3
2
G
N -monomethyl-L-arginine (L-NMMA) was 43.1µM in this (10.4g) was then subject to ODS column chromatography
assay. The highest concentration (100µM) of other compounds eluting with MeOH–H O (2:3). Combination of the eluate by
2
did not reach the half maximal inhibitory concentration.
TLC afforded seven sub-fractions S1–S7. Final purification
was carried out by preparative HPLC with an ODS column
(YMC-Pack Pro C18, 150×20mm). The flow rate for HPLC
Experimental
General Optical rotations were measured with a JASCO was 5.0mL/min. In detail, from sub-fraction S3 (1.6g), with
P-2200 polarimeter in a 0.5-dm cell. IR spectra were measured 25% CH CN, compounds 3 (20mg, t 32min) and 5 (36mg, t
3
R
R
on a JASCO FT/IR-4100 fourier transform infrared spectrom- 36min) were isolated; from sub-fraction A4 (0.8g), with 30%
eter by the KBr disk method. Circular dichroism (CD) spectra CH CN, compound 1 (24mg, t 22min) was isolated; from
3
R
were recorded on a JASCO J-720W spectropolarimeter. NMR sub-fraction A6, with 30% CH CN, compounds 2 (6mg, tR
3
spectra were measured on a JEOL ECP-500 spectrometer 58min) and 4 (17mg, t 62min) were isolated.
R
1
13
with TMS as the internal reference, and the chemical shifts
Tinospinoside D (1): Colorless solid. H- and C-NMR
−
1
are expressed in δ (ppm). Electrospray ionization (ESI)-MS spectroscopic data, see Tables 1 and 2. IR (KBr) cm : 3423,
was recorded on an LCQ mass analyzer and HR-FAB-MS on 2929, 2853, 1735, 1720, 1637, 1628, 1458, 1384, 1255, 1024.
+
a JEOL JMS-700 MStation spectrometer. Preparative HPLC HR-FAB-MS m/z: 591.2064 [M+Na] (Calcd for C H O Na:
2
7
36 13
+
22
was performed on a JASCO PU-2086 HPLC system, equipped 591.2053). ESI-MS m/z: 591.3 [M+Na] . [α] −28.2 (c=0.21,
with a JASCO RI-2301 Differential Refractometer detector MeOH). CD (MeOH): [θ] (nm) −1672 (209), 4762 (235).
Tinospin E (2): Colorless solid. H- and C-NMR spectro-
gel (Silica Gel 60N, Kanto Chemical Co., Inc., Tokyo, Japan) scopic data, see Tables 1 and 2. IR (KBr) cm : 3435, 2928,
was used for column chromatography. TLC was conducted 1707, 1638, 1542, 1458, 1383, 1272, 1152, 1024. HR-FAB-MS
D
2
2
1
13
and an YMC-Pack RP-C18 column (150×20mm i.d.). Silica
−1
+
using Silica gel 60 F254 plates (E. Merck). GLC was carried m/z: 357.1325 [M+H] (Calcd for C H O : 357.1338). ESI-MS
20
21
6
+
22
D
out on a Perkin-Elmer Clarus 500 GC-MS instrument. Absor- m/z: 379.1 [M+Na] . [α]
+32.8 (c=0.22, MeOH). CD
2
2
bance for bioassay was measured using ImmunoMini NJ-2300 (MeOH): [θ] (nm) −8508 (207), 47608 (223), −23207 (247).

1328
Vol. 60, No. 10
1
13
6
Tinospinoside E (3): Colorless solid. H- and C-NMR 1×10 cells/well and allowed to adhere for 2h at 37°C in a
−
1
spectroscopic data, see Tables 1 and 2. IR (KBr) cm : 3423, humidified atmosphere containing 5% CO . Then the medium
2
1
706, 1638, 1542, 1458, 1383, 1271, 1058, 1024. HR-FAB-MS was replaced with fresh medium containing LPS (10ng/mL)
+
m/z: 541.16782 [M+Na] (Calcd for C H O Na: 541.16858). and INFγ (40U/mL) together with test compounds at various
ESI-MS m/z: 541.2 [M+Na] . [α] +28.7 (c=0.22, MeOH). concentrations, and the cells were further incubated for an-
2
6
30 11
+
22
D
2
2
CD (MeOH): [θ] (nm) −20993 (209), 33493 (227), −25623 other 48h. Nitric oxide production was determined by measur-
247). ing the accumulation of nitrite in the culture supernatant using
Acid Hydrolysis A solution of 3 (5mg) in 1M HCl (diox- Griess reagent. Briefly, 100µL of the supernatant from incu-
(
ane/H O, 1:1, 1mL) was heated at 80°C for 3h, Then treated bates were mixed with equal volume of Griess reagent (1%
2
with a Diaion HP-20 column, eluted with H O and MeOH to sulfanilamide and 0.1% naphthylene–diamide dihydrochloride
2
afford subfractions E1 and E2. Subfraction E2 was further in 2.5% H PO ) and were allowed to stand for 10min at 37°C
3
4
purified by reverse-phase HPLC with CH CN–H O (35:65) in a humidified atmosphere containing 5% CO . Cell viability
3
2
2
to give the tinospin E (2) (1.2mg) and fibleucin (1mg). Acid was determined using the mitochondrial respiration-dependent
hydrolysis of 5 were carried out using the same conditions, to MTT reduction method. Absorbance at 550nm was measured
give columbin (4) (0.5mg) and 5a (1.2mg).
using a microplate reader.
1
13
5
a: Colorless solid. H- and C-NMR spectroscopic data,
17
see Tables 1 and 2. [α] +20.3 (c=0.12, MeOH). CD (MeOH): References
D
2
2
[
θ] (nm) −32629 (205), 18344 (232).
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