New clerodane diterpenes from tinospora sagittata var yunnanensis

Article abstract of DOI:10.1055/s-0034-1368252

Four new clerodane diterpenes, namely sagittatayunnanosides A-D (1-4), were isolated from the roots of Tinospora sagittata var. yunnanensis, together with two known compounds, tinospinoside C (5) and tinospinoside E (6). The structures of the four new compounds were well elucidated by extensive analyses of the MS, IR, and 1D and 2DNMR data. The cytotoxic and antifouling activities of compounds 1-6 were evaluated. Georg Thieme Verlag KG Stuttgart. New York.

Full text of DOI:10.1055/s-0034-1368252

Letters 419
with the molecular formula C26H38O10 as revealed by the HRE-
SIMS at m/z 533.2349 [M + Na]+ (calcd. for C26H38O10Na+,
New Clerodane Diterpenes from
533.2362). The IR spectrum showed the absorptions for hydroxyl
(3454 cm−1), carbonyl (1736 cm−1), and olefinic functions
Tinospora sagittata var. yunnanensis
1
(1656 cm−1). In the H NMR spectrum (l Table 1), two olefinic
"
Zhi-Yong Jiang1, Wen-Juan Li1, Li-Xiang Jiao1, Jun-Ming Guo1,
Kai Tian1, Chun-Tao Yang1, Xiang-Zhong Huang1,2
proton signals at δ 6.59 (1H, dd, J = 3.6, 3.6 Hz, H-3) and 7.42
H
(1H, brs, H-14) were observed, as well as two methyls at δH 1.27
(3H, s, H-19) and 0.86 (3H, s, H-20). The anomeric proton signal at
δH 4.22 (1H, d, J = 8.0 Hz) suggested the presence of a β-linked
sugar moiety. Hydrolysis of compound 1 with 10% HCl in metha-
nol liberated the D-glucose, which was identified by TLC compar-
1
Key Laboratory of Chemistry in Ethnic Medicinal Resources,
State Ethnic Affairs Commission & Ministry of Education, School of
Chemistry and Biotechnology, Yunnan University of Nationalities,
Kunming, Yunnan, P.R. China
2
ison with an authentic sample. The 13C NMR (l Table 2) spectrum
"
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,
P.R. China
exhibited 26 carbon signals, of which two pairs of double bonds
at δC 140.2 (C-3), 140.0 (C-4), 134.8 (C-13), and 147.7 (C-14) and
two carbonyl signals at δ 177.1 (C-16) and 171.7 (C-18) were
C
displayed. Analyses of the NMR data suggested compound 1 was
a diterpene glycoside featuring a clerodane skeleton.
Abstract
!
However, compound 1 contained an α-substituted butenolide
Four new clerodane diterpenes, namely sagittatayunnanosides
A–D (1–4), were isolated from the roots of Tinospora sagittata
var. yunnanensis, together with two known compounds, tinospi-
noside C (5) and tinospinoside E (6). The structures of the four
new compounds were well elucidated by extensive analyses of
the MS, IR, and 1D and 2D NMR data. The cytotoxic and antifoul-
ing activities of compounds 1–6 were evaluated.
ring, which was determined by the characteristic 1H-1H COSY
"
(l Fig. 1) cross-peak due to the broad singlet at δC 7.42 (1H, brs,
H-14) coupled with a two proton broad singlet at δH 4.83 (2H, brs,
"
H-15) [11], as well as the HMBC correlations (l Fig. 2) of H-14
with C-13, 15, and C-16, and H-15 with C-13, 14, and C-16. The
α-substituted butenolide ring moiety was assigned to be attached
at C-12 by the HMBC correlations of H-14 with C-12, and H-12
with C-13. Comparison of the NMR data of compound 1 with
those of marrubiagenine [12] and 1α,7α-dihydroxyneocleroda-
3,13-dien-16,15:18,19-diolide [13] showed the existence of an
extra glucose at C-17 in compound 1. The additional β-D-gluco-
pyranosyl moiety linked at C-17 was further supported by the
long-range HMBC correlation between H-1′ (δH 4.22) and C-17
(δC 72.7). The C-19 resonance at δC 33.7 (q) established the A/B
cis-fusion [12,14,15]. In parallel, the HMBC correlations from H-
2 to C-3 and H-3 to C-18 established the location of a double bond
at C-3(4) and carboxylic acid (C-18) at C-4. In order to further
characterize the relative configurations of C-5, 8, 9, and C-10, a
ROESY experiment was conducted. The clear ROESY correlations
Key words
Tinospora sagittata var. Yunnanensis · Menispermaceae · clero-
dane diterpenes · cytotoxicity · antifouling activity
Supporting information available online at
http://www.thieme-connect.de/ejournals/toc/plantamedica
Tinospora sagittata var. yunnanensis (S.Y. Hu) H.S. Lo, a perennial
indeciduous creeping vine belonging to the Menispermaceae
family, is mainly distributed in Yunnan and Guangxi Provinces,
China. Its roots have widely been used for the treatment of sore
throats, diarrhea, and superficial infections [1]. Previous investi-
gations on the genus Tinospora showed that the clerodane diter-
penes possess anti-inflammatory, antibacterial, and antifeedant
activity [2–6]. In our recent study on the bioactivity of the Tino-
spora plants, the 95% ethanolic extract of T. sagittata var. yunna-
nensis showed cytotoxicity on HeLa, K562, HL60, and HepG2 cells
in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbro-
mid (MTT) bioassay and moderate anti-settlement activity
against the barnacle Balanus amphitrite (Balanidae). In order to
find the active components in this medicinal plant, an extensive
phytochemical investigation of the roots of T. sagittata var. yun-
nanensis was carried out. Our preceding research resulted in the
isolation of nine compounds [7,8]. During the subsequent inves-
tigation of T. sagittata var. yunnanensis, four new clerodane diter-
penes, named sagittatayunnanosides A–D (1–4), were isolated
from the 95% ethanol extract, together with two known ones, ti-
nospinoside C (5) [9] and tinospinoside E (6) [10]. All six isolates
were assayed for their cytotoxicity in HeLa, K562, HL60, and
HepG2 cell lines and anti-settlement activity against B. amphi-
trite. Herein, we present the isolation and structural elucidation,
as well as cytotoxic and antifouling activities of the new com-
pounds.
"
(l Fig. 3) for H-10/H-19, H-19/H-8 and H-8/H-12 indicated that
H-10, H-19, and H-8 were on the same side of the molecular
plane, and tentatively assuming an α-orientation according to
the previously isolated diterpenoids from the Tinospora genus
[2–4,16–26]. Finally, the structure of compound 1 was elucidated
"
as shown in l Fig. 1 and named sagittatayunnanoside A (1).
Compound 2 was isolated as a colorless powder. Its molecular
formula was determined as C33H48O17 by the positive HRESIMS
at m/z 739.2780 (calcd. for C33H48O17Na+, 739.2789). The 1H and
13C NMR spectrum showed typical resonances [δH 7.60 (brs, H-
16), 7.54 (d, J = 2.0 Hz, H-15), 6.52 (d, J = 2.4 Hz, H-14); δC 141.4
(d, C-16), 145.0 (d, C-15), 109.9 (d, C-14)] attributable to a β-sub-
stituted furan ring at C-12 in the clerodane-type diterpenoids
[27,28], as well as a trisubstituted olefin [δH 6.52 (d, J = 3.2 Hz,
H-3); δC 139.7 (s, C-4), 138.2 (d, C-3)], an ester methyl group [δH
"
3.76 (s); δC 52.3 (q)], and two β-D-glucopyranosyl moieties (l Ta-
bles 1 and 2). The D-glucose was also identified by TLC analysis of
the acid hydrolyzate and comparison with an authentic sugar
sample. Its NMR data were highly similar to those of epitinophyl-
loloside [29] and tinophylloloside [30], except that compound 2
had one more β-D-glucopyranose unit. The HMBC correlation
from H-1′′ (δH 4.48 1H, d, J = 7.6 Hz) to C-6′ (δC 70.0) illustrated
that the additional β-D-glucopyranose was attached to the C-6′
of the inner glucopyranose. The cleavage of the lactone between
C-17 and C-12 in compound 2 differed from tinophylloloside [30]
Compound 1 was obtained as a colorless powder. The positive
ESI‑MS gave a quasi-molecular ion peak at m/z 511, in accordance
Jiang Z-Y et al. New Clerodane Diterpenes… Planta Med 2014; 80: 419–425

420 Letters
Table 1 1H NMR (400 MHz) data (δ) of compounds 1–4 in CD3OD, δ in ppm, J in Hz.
Position
1
2
3
4
1
2.03–2.09 (1H, m)
1.90–1.96 (1H, m)
2.26–2.28 (m)
2.68 (1H, m)
2.05–2.09 (1H, m)
1.91–1.96 (1H, m)
1.15 (m, overlapped)
1.77 (m, overlapped)
6.79 (1H, brs)
2.08–2.12 (1H, m)
1.92–1.98 (1H, m)
2.28–2.34 (m)
2.09–2.14 (m, overlapped)
4.46 (1H, dd, 7.6, 13.2)
2
3
6
6.59 (1H, dd, 3.6, 3.6)
2.72 (1H, dd, 10.8, 3.6)
1.12 (m, overlapped)
1.79–1.82 (1H, m)
1.12 (m, overlapped)
1.76–1.78 (1H, m)
1.53 (1H, brd.6.0)
1.79–1.81 (1H, m)
1.68–1.69 (1H, m)
2.37 (m, overlapped)
2.21 (m, overlapped)
7.42 (1H, brs)
6.52 (1H, d, 3.2)
6.64 (1H, dd, 3.2,3.2)
2.80 (1H, brd, 12.0)
2.60 (1H, dd, 10.8, 2.8)
1.31 (1H, m)
2.73 (1H, brd, 11.6)
1.15 (m, overlapped)
2.28–2.35 (m, overlapped)
1.14 (m, overlapped)
1.58–1.60 (m, overlapped)
7
1.73 (1H, overlapped)
1.37 (1H, m)
8
10
11
2.74 (1H, dd, 11.2, 3.2)
1.73 (1H, brd, 8.0)
1.77 (m, overlapped)
1.54 (1H, brd.5.6)
1.77 (m, overlapped)
1.63–1.69 (1H, m)
2.33 (m, overlapped)
2.20 (m, overlapped)
7.42 (1H, brs)
2.70 (1H, dd, 12.0, 3.0)
1.59 (1H, brd.6.0)
1.84–1.89 (1H, m)
1.65–1.68 (1H, m)
2.49 (1H, m)
2.20 (1H, m)
7.51 (1H, brs)
4.83 (2H, brs)
–
2.09–2.14 (m, overlapped)
1.93 (1H, dd, 11.9, 13.6)
5.50 (1H, dd, 11.9, 6.4)
12
14
15
16
17
6.52 (1H, d, 2.4)
7.54 (1H, d, 2.0)
7.60 (1H, br.s)
–
4.83 (2H, brs)
4.87 (2H, brs)
–
4.10 (1H, dd, 9.6, 3.2)
3.13–3.19 (m, overlapped)
1.27 (3H, s)
4.10 (1H, brd, 9.6)
3.14–3.19 (m, overlapped)
1.27 (3H, s)
–
19
20
OMe
Glc-
1′
1.16 (3H, s)
0.92 (3H, s)
3.76 (3H, s)
1.28 (3H, s)
1.04 (3H, s)
–
0.86 (3H, s)
0.85 (3H, s)
–
–
4.22 (1H, d, 8.0)
4.32 (1H, d, 7.6)
4.21 (1H, d, 7.6)
5.46 (1H, d, 8.4)
2′
3.13–3.19 (m, overlapped)
3.36 (1H, t, 8.8)
3.20–3.26 (m, overlapped)
3.31–3.39 (m, overlapped)
3.31–3.39 (m, overlapped)
3.20–3.26 (m, overlapped)
4.15 (1H, brd, 10.0)
3.14–3.19 (m, overlapped)
3.35–3.39 (m, overlapped)
3.26–3.28 (m, overlapped)
3.26–3.28 (m, overlapped)
3.85 (1H, brd, 12.0)
3.24 (1H, dd, 8.8, 8.4)
3.33–3.36 (m, overlapped)
3.33–3.36 (m, overlapped)
3.36–3.42 (m, overlapped)
3.79 (1H, brd, 11.6)
3′
4′
3.27 (1H, m)
5′
3.27 (1H, m)
6′
3.85 (1H, dd, 12.4, 1.6)
3.66 (1H, dd, 12.0, 5.0)
3.72 (1H, m)
3.68–3.75 (m, overlapped)
3.69 (1H, dd, 12.0, 3.6)
Glc-
1′′
2′′
3′′
4′′
5′′
6′′
–
–
–
–
–
–
4.48 (1H, d, 7.6)
5.54 (1H, d, 8.0)
–
–
–
–
–
–
3.20–3.26 (m, overlapped)
3.18–3.24 (m, overlapped)
3.31–3.39 (m, overlapped)
3.50 (m, overlapped)
3.35–3.39 (m, overlapped)
3.35–3.39 (m, overlapped)
3.35–3.39 (m, overlapped)
3.26–3.28 (m, overlapped)
3.85 (1H, brd, 12.0)
3.84 (1H, dd, 12.0, 2.4)
3.64 (1H, dd, 12.0, 5.6)
3.68–3.75 (m, overlapped)
"
(with a lactone between C-17 and C-12) and was deduced by the
relatively lower chemical shift of C-17 at δC 177.7, and finally
verified by the absent HMBC correlation between H-12 and C-
spectrum (l Table 2), suggested the additional sugar moiety
should be a β-D-glucopyranose [31]. The location of this addi-
tional sugar at C-18 was deduced by the HMBC correlation be-
tween H-2′′ (δH 5.54) and C-18 (δC 167.5). Thus, compound 3
"
17. The ROESY correlations (l Fig. 3) between H-2/H-19/Hα-1
"
and H‑19/H‑10/H-12/H‑8, together with no cross-peak for H-20/
H-10, suggested the H-2, 8, 10, and 19 were α-orientated and H-
20 was β-orientated. Consequently, the structure of compound 2
was characterized as shown in l Fig. 1 and named sagittatayun-
nanoside C (3).
Compound 4 was isolated as a colorless powder with a molecular
formula of C26H36O11 deduced by a positive HRESIMS at m/z
547.2171 (calcd. for C26H36O11Na+, 547.2155). Comparison of
the NMR data (l Tables 1 and 2) with those of compound 1
showed a high similarity with the exception that compound 4
possessed one more ester-carbonyl and one less methylene as
seen in the DEPT (l Table 2). The structure of compound 4 differs
"
was deduced as depicted in l Fig. 1 and named sagittatayunna-
noside B (2).
"
Compound 3 was obtained as a colorless powder and assigned
the molecular formula C32H48O15 based on the HRESIMS at m/z
695.2899 (calcd. for C32H48O15Na+, 695.2891). Its IR spectrum ex-
hibited absorption bands at 3448, 1707, 1652, and 1068 cm−1
,
"
corresponding to hydroxyl, ester-carbonyl, olefin, and glycosidic
functions. The NMR data (l Tables 1 and 2) were essentially
from 1 mainly in C-17, where an ester-carbonyl was linked at C-8
instead of a methylene as in compound 1. This was verified by the
HMBC correlations (l Fig. 2) from H-8 (δH 2.70 1H, dd, J = 12.0,
"
"
identical to compound 1 except that there was one more sugar
unit in compound 3. The anomeric proton signals due to the addi-
tional sugar moiety at δH 5.54 (1H, d, J = 8.0 Hz, H-2′′) in the 1H
3.0 Hz) to the additional ester-carbonyl signal δC 174.9 (s, C-17),
and H-1′ (δH 5.46 1H, d, J = 8.4 Hz) to C-17. The other HMBC, 1H-
1
"
"
"
NMR spectrum (l Table 1), combined with the carbon signals as-
H COSY (l Fig. 2), and ROESY (l Fig. 3) correlations further con-
cribable to the extra sugar at δC 95.4 (C-1′′), 73.9 (C-2′′), 78.7 (C-
3′′), 71.1 (C-4′′), 78.0 (C-5′′), 63.2 (C-6′′) appearing in the 13C NMR
Jiang Z-Y et al. New Clerodane Diterpenes… Planta Med 2014; 80: 419–425

Letters 421
Table 2 13C NMR (100 MHz) data
(δ) of compounds 1–4 in CD3OD.
Position
1
2
3
4
1
2
17.6 (t)
28.5 (t)
17.5 (t)
17.4 (t)
25.0 (t)
140.2 (d)
140.0 (s)
37.1 (s)
37.5 (t)
24.7 (d)
44.8 (d)
40.8 (s)
46.6 (d)
36.9 (t)
20.0 (t)
134.8 (s)
147.7 (d)
72.2 (t)
177.1 (s)
72.7 (t)
171.7 (s)
33.7 (q)
19.8 (q)
–
71.8 (d)
138.2 (d)
139.7 (s)
37.1 (s)
24.5 (t)
143.0 (d)
138.7 (s)
37.4 (s)
37.2 (t)
25.2 (t)
44.8 (d)
40.8 (s)
46.6 (d)
36.9 (t)
20.0 (t)
134.8 (s)
147.8 (d)
72.2 (t)
177.2 (s)
72.7 (t)
167.5 (s)
33.7 (q)
19.9 (q)
–
24.8 (t)
140.5 (d)
139.7 (s)
37.0 (s)
36.7 (t)
24.0 (t)
51.5 (d)
41.5 (s)
46.5 (d)
38.3 (t)
19.6 (t)
134.4 (s)
148.2 (d)
72.3 (t)
177.3 (s)
174.9 (s)
171.3 (s)
33.7 (q)
20.5 (q)
–
3
4
5
6
35.4 (t)
7
20.8 (t)
8
48.6 (d)
38.7 (s)
9
10
11
12
13
14
15
16
17
18
19
20
OMe
Glc-
1′
51.1 (d)
45.6 (t)
71.7 (d)
125.9 (s)
109.9 (d)
145.0 (d)
141.4 (d)
177.7 (s)
169.3 (s)
31.4 (q)
23.5 (q)
52.3 (q)
105.2 (d)
75.2 (d)
78.0 (d)
71.6 (d)
77.7 (d)
62.7 (t)
104.7 (d)
74.9 (d)
77.9 (d)
71.3 (d)
77.8 (d)
70.0 (t)
105.2 (d)
75.2 (d)
78.2 (d)
71.6 (d)
77.8 (d)
62.7 (t)
95.5 (d)
74.0 (d)
78.7 (d)
70.9 (d)
78.0 (d)
62.1 (t)
2′
3′
4′
5′
6′
Glc-
1′′
2′′
3′′
4′′
5′′
6′′
104.7 (d)
74.9 (d)
77.7 (d)
71.4 (d)
76.9 (d)
62.6 (t)
95.4 (d)
73.9 (d)
78.7 (d)
71.1 (d)
78.0 (d)
63.2 (t)
firmed the structure of compound 4. The structure of compound
4 was deduced as shown in l Fig. 1 and named sagittatayunna-
Materials and Methods
!
"
noside D (4).
General: Column chromatography (CC): silica gel (200–300
mesh; Qingdao Marine Chemical, Inc.); Lichrospher Rp-18 gel
(40–63 µ; Merck). Optical rotations were carried out on a Horiba
SEPA-300 high sensitivity polarimeter. IR spectra were measured
The known compounds 5 and 6 were identified by comparing
their NMR data with those in the literatures [9,10]. The cytotoxic
activities of compounds 1–6 were evaluated in vitro by using He-
La, K562, HL60, and HepG2 cell lines. Results are summarized in
on a Bio-Rad FTS-135 spectrometer with KBr pellets, ν in cm−1
.
"
l Table 3. It can be concluded that compounds 1 and 4 showed
MS data were obtained on a VG Auto Spec-3000 instrument.
NMR spectra were recorded on a Bruker AV – 400 (1H/13C,
400 MHz/100 MHz) spectrometer, and chemical shifts were given
in δ with TMS as the internal reference. Positive controls: cispla-
tin (purity > 99%) was purchased from Sigma-Aldrich Chemical
Co., and SeaNine211 (purity > 96%) was obtained from Rhom
and Hass Co.
superior inhibitory activity in HeLa and HL60 cells, and com-
pounds 2 and 3 exhibited superior activity against the K562 cell
line, with IC50 values of 17.6 and 14.4 µM, respectively. Com-
pounds 5 and 6 possessed moderate cytotoxic activity in K562
cells. Compound 6 was found to possess a superior inhibitory
ability in HL60 cells with an IC50 value of 13.1 µM.
The ability of compounds 1–6 to inhibit larval settlement was al-
so assessed using a B. amphitrite assay [32]. The results showed
that only compounds 1 and 4 possessed moderate activity against
B. amphitrite, with EC50 values of 28.3 5.3 and 33.8 6.7 µM, re-
spectively, indicating that the carboxyl group at C-4 was essential
for the antifouling activity for these clerodane diterpenes. This is
the first report that clerodane diterpenes possess antifouling ac-
tivity. It could be helpful for researchers to find anti-settlement
active chemicals from a natural source. SeaNine211, a well-
known antifouling agent, was used as a positive control and had
an EC50 value of 6.8 1.3 µM.
Plant material: The roots of T. sagittata var. yunnanensis were col-
lected in Honghe, Yunnan Province in November 2007 and iden-
tified by Prof. Shao-Bin Ma from Yunan University. A voucher
specimen (TSY200711) was deposited in the Key Laboratory of
Chemistry in Ethnic Medicinal Resources, State Ethnic Affairs
Commission & Ministry of Education, School of Chemistry and
Biotechnology, Yunnan University of Nationalities.
Extraction and isolation: The air-dried roots (10.0 kg) of T. sagitta-
ta var. yunnanensis were powdered and extracted with 95% EtOH
(60 L × 3, 48 hours each time) at room temperature. The extract
was concentrated under vacuum to give a residue, which was
partitioned between water, EtOAc, and n-BuOH, respectively, to
Jiang Z-Y et al. New Clerodane Diterpenes… Planta Med 2014; 80: 419–425

422 Letters
Fig. 1 Structures of compounds 1–6.
Fig. 2 Selected key HMBC and 1H-1H COSY corre-
lations of compounds 1–4.
Jiang Z-Y et al. New Clerodane Diterpenes… Planta Med 2014; 80: 419–425

Letters 423
Fig. 3 Key ROESY (↔) correlations of compounds
1–4.
Samples
IC50 (µM)
K562
Table 3 Cytotoxic activities of
compounds 1–6.
Hela
11.8 3.8
HL60
HepG2
1
86.2 8.9
17.6 4.7
14.4 5.9
149.0 12.9
28.5 4.9
29.8 4.5
30.4 5.6
11.2
16.1 5.5
93.9 8.1
57.2 5.0
20.4 6.6
51.5 5.4
13.1 5.0
28.8 5.7
12.7
124.0 10.1
110.6 13.3
83.6 9.8
122.8 11.5
138.2 9.9
120.1 9.4
42.4 6.1
10.4
2
132.3 10.4
108.5 9.2
14.0 5.2
87.7 8.4
68.1 5.7
23.1 4.0
7.5
3
4
5
6
95% Ethanol extract (µg/mL)
Cisplatina
a Positive control
provide the EtOAc fraction (250.0 g) and the n-BuOH fraction
(380.0 g). The n-BuOH fraction (280.0 g) was fractionated on a sil-
ica gel CC (10 × 120 cm, silica gel 2.0 kg, 200–300 mesh) by gra-
dient elution with CHCl3/MeOH/H2O (100:0:0, 98:2:0,
95:5:0, 90:10:0, 85:15:1, 80:20:2, 70:30:5, each in 4 L,
500 mL per fraction). According to the chemical distinction re-
vealed by TLC, seven crude fractions (A–G) were obtained. Fr. C
(15.0 g) was subjected to silica gel CC (4 × 80 cm; 300 g) and
eluted with CHCl3/MeOH (90:10) to provide Frs. C1–4. Fr. C2
(1.2 g) was performed on a silica gel CC (2 × 50 cm; 50 g, CHCl3/
MeOH 90:10; 100 mL per fraction) to afford three fractions (Frs.
C2.1–3). Fr. C2.2 (0.5 g) was chromatographed on a silica gel CC
(2 × 50 cm; 50 g) with an eluent of CHCl3/Me2CO (90:10; 100 mL
per fraction) to provide two subfractions (Fr. C2.2.1 and Fr.
C2.2.2), Fr. C2.2.2 (0.2 g) was further purified on an Rp-18 CC
(2 × 40 cm; 50 g, MeOH/H2O 80:20; 50 mL per fraction) to fur-
nish compounds 4 (purity > 97%, HPLC; Rp-18 TLC, Rf = 0.6,
MeOH/H2O 90:10; 15 mg) and 1 (purity > 96.5%, HPLC; Rp-18
TLC, Rf = 0.35, MeOH/H2O 90:10; 21 mg). Fr. C2.3 (0.3 g) was iso-
lated on an Rp-18 CC (2 × 40 cm; 50 g, MeOH/H2O 80:20) and
further purified by a silica gel column (2 × 50 cm; 60 g, CHCl3/
MeOH 85:15; 50 mL per fraction) to yield compound 5 (purity
> 98%, HPLC; 106 mg). Fr. D (35.0 g) was performed on a silica gel
CC (4 × 80 cm; 400 g) with an elution of CHCl3/MeOH/H2O
(90:10:0 to 80:20:2, 250 mL per fraction) to provide Frs. D1–5.
Fr. D2 (1.1 g) was subjected to MCI gel CC (2 × 50 cm; 80 g, MeOH/
H2O 70:30 to 80:20), followed by silica gel CC (2 × 50 cm; 50 g,
CHCl3/MeOH/H2O 85:15:1) to give compounds 2 (purity > 96%,
HPLC; 48 mg) and 3 (purity > 96.5%, HPLC; 70 mg). Fr. D3 (2.0 g)
was subjected to MCI gel CC (2 × 50 cm; 80 g, MeOH/H2O 70:30
to 80:20) and further purified by Rp-18 CC (2 × 40 cm; 50 g,
MeOH/H2O 80:20) to obtain compounds 2 (purity > 95.5%, HPLC;
10 mg, combined with compound 2 from fraction Fr. D2) and 6
(purity > 97.5%, HPLC; 117 mg).
Sagittatayunnanoside
(c = 0.10, MeOH); IR (KBr): νmax = 3454, 2978, 1736, 1670, 1656,
A
(1): Colorless powder; [α]2D5 − 80.0
Jiang Z-Y et al. New Clerodane Diterpenes… Planta Med 2014; 80: 419–425

424 Letters
1242, 1059 cm−1; HRESIMS (positive): m/z = 533.2349 [M + Na]+
Acknowledgements
(calcd. for C26H38O10Na+, 533.2362); 1H and 13C NMR (CD3OD) da-
!
"
ta: l Tables 1 and 2.
Sagittatayunnanoside
Financial support was provided by grants from the National Nat-
ural Science Foundation of China (NSFC No. 21262047,
21162041), the Natural Science Foundation of Yunnan Province
(No. S2012FZ0227), the Program for Science and Technology In-
novative Research Team in University of Yunnan Province (IRT-
STYN), the Innovation Team Project of Dai Medicine Research,
Yunnan University of Nationalities, and the Innovation Project of
Graduate (11HXYJS03) from the Chemistry and Biotechnology
School.
B
(2): Colorless powder; [α]2D5 − 65.7
(c = 0.07, MeOH); IR (KBr):
νmax = 3440, 1705, 1652, 1249,
1065 cm−1; HRESIMS (positive): m/z = 739.2780 [M + Na]+ (calcd.
for C33H48O17Na+, 739.2789); 1H and 13C NMR (CD3OD) data:
"
l Tables 1 and 2.
Sagittatayunnanoside C (3): Colorless powder; [α]2D7.1 − 49.0
(c = 0.14, MeOH); IR (KBr): νmax = 3448, 2972, 1735, 1707, 1652,
1266, 1068 cm−1; HRESIMS (positive): m/z = 695.2899 [M + Na]+
(calcd. for C32H48O15Na+, 695.2891); 1H and 13C NMR (CD3OD) da-
"
ta: l Tables 1 and 2.
Conflict of Interest
!
Sagittatayunnanoside B (4): Colorless powder; [α]2D7.5 − 75.8
(c = 0.10, MeOH); IR (KBr): νmax = 3449, 2972, 1735, 1671, 1654,
1243, 1061 cm−1; HRESIMS (positive): m/z = 547.2171 (calcd. for
The authors declare no conflict of interest.
13
"
C
26H36O11Na+, 547.2155); 1H and C NMR (CD3OD) data: l Ta-
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Supporting information
The 1D and 2D NMR spectra of the new compounds 1–4 are avail-
able as Supporting Information.
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DOI http://dx.doi.org/10.1055/s-0034-1368252
Published online March 14, 2014
Planta Med 2014; 80: 419–425
© Georg Thieme Verlag KG Stuttgart · New York ·
ISSN 0032‑0943
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Prof. Dr. Xiang-Zhong Huang
Key Laboratory of Chemistry in Ethnic Medicinal Resources
State Ethnic Affairs Commission & Ministry of Education
School of Chemistry and Biotechnology
Yunnan University of Nationalities
Jingming South Road, Chenggong New District
Kunming, Yunnan, 650500
P.R. China
Phone: + 8687165913013
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Fax: + 8687165910017
xiangzhongh@126.com
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