New Clerodane Diterpenes from Tinospora sagittata var. yunnanensis Zhi-Yong Jiang 1, Wen-Juan Li 1, Li-Xiang Jiao 1, Jun-Ming Guo 1, Kai Tian 1, Chun-Tao Yang 1, Xiang-Zhong Huang 1, 2 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 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
Abstract !
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 2D NMR data. The cytotoxic and antifouling activities of compounds 1–6 were evaluated.
Key words Tinospora sagittata var. Yunnanensis · Menispermaceae · clerodane 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 investigations on the genus Tinospora showed that the clerodane diterpenes possess anti-inflammatory, antibacterial, and antifeedant activity [2–6]. In our recent study on the bioactivity of the Tinospora plants, the 95 % ethanolic extract of T. sagittata var. yunnanensis showed cytotoxicity on HeLa, K562, HL60, and HepG2 cells in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid (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. yunnanensis was carried out. Our preceding research resulted in the isolation of nine compounds [7, 8]. During the subsequent investigation of T. sagittata var. yunnanensis, four new clerodane diterpenes, named sagittatayunnanosides A–D (1–4), were isolated from the 95 % ethanol extract, together with two known ones, tinospinoside 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. amphitrite. Herein, we present the isolation and structural elucidation, as well as cytotoxic and antifouling activities of the new compounds. Compound 1 was obtained as a colorless powder. The positive ESI‑MS gave a quasi-molecular ion peak at m/z 511, in accordance
with the molecular formula C26H38O10 as revealed by the HRESIMS at m/z 533.2349 [M + Na]+ (calcd. for C26H38O10Na+, 533.2362). The IR spectrum showed the absorptions for hydroxyl (3454 cm−1), carbonyl (1736 cm−1), and olefinic functions " Table 1), two olefinic (1656 cm−1). In the 1H NMR spectrum (l proton signals at δ H 6.59 (1H, dd, J = 3.6, 3.6 Hz, H-3) and 7.42 (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 methanol liberated the D-glucose, which was identified by TLC compar" Table 2) spectrum ison with an authentic sample. The 13C NMR (l 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 C 177.1 (C-16) and 171.7 (C-18) were displayed. Analyses of the NMR data suggested compound 1 was a diterpene glycoside featuring a clerodane skeleton. However, compound 1 contained an α-substituted butenolide ring, which was determined by the characteristic 1H-1H COSY " Fig. 1) cross-peak due to the broad singlet at δ 7.42 (1H, brs, (l C H-14) coupled with a two proton broad singlet at δH 4.83 (2H, brs, " Fig. 2) of H-14 H-15) [11], as well as the HMBC correlations (l 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-dihydroxyneocleroda3,13-dien-16,15 : 18,19-diolide [13] showed the existence of an extra glucose at C-17 in compound 1. The additional β-D-glucopyranosyl 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 H2 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 " Fig. 3) for H-10/H-19, H-19/H-8 and H-8/H-12 indicated that (l 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 " Fig. 1 and named sagittatayunnanoside A (1). as shown in l 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 13 C NMR spectrum showed typical resonances [δH 7.60 (brs, H16), 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-substituted 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 " Ta3.76 (s); δC 52.3 (q)], and two β-D-glucopyranosyl moieties (l 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 epitinophylloloside [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]
Jiang Z-Y et al. New Clerodane Diterpenes …
Planta Med 2014; 80: 419–425
419
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
Letters
Letters
Table 1
1
H NMR (400 MHz) data (δ) of compounds 1–4 in CD3OD, δ in ppm, J in Hz.
Position 1 2 3 6 7 8 10 11 12 14 15 16 17 19 20 OMe Glc1′ 2′ 3′ 4′ 5′ 6′ Glc1′′ 2′′ 3′′ 4′′ 5′′ 6′′
1
2
3
4
2.03–2.09 (1H, m) 1.90–1.96 (1H, m) 2.26–2.28 (m)
2.68 (1H, m) 2.09–2.14 (m, overlapped) 4.46 (1H, dd, 7.6, 13.2)
2.08–2.12 (1H, m) 1.92–1.98 (1H, m) 2.28–2.34 (m)
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) 4.83 (2H, brs) – 4.10 (1H, dd, 9.6, 3.2) 3.13–3.19 (m, overlapped) 1.27 (3H, s) 0.86 (3H, s) –
6.52 (1H, d, 3.2) 2.60 (1H, dd, 10.8, 2.8) 1.31 (1H, m) 1.73 (1H, overlapped) 1.37 (1H, m) 2.74 (1H, dd, 11.2, 3.2) 1.73 (1H, brd, 8.0) 2.09–2.14 (m, overlapped) 1.93 (1H, dd, 11.9, 13.6) 5.50 (1H, dd, 11.9, 6.4)
2.05–2.09 (1H, m) 1.91–1.96 (1H, m) 1.15 (m, overlapped) 1.77 (m, overlapped) 6.79 (1H, brs) 2.73 (1H, brd, 11.6) 1.15 (m, overlapped) 2.28–2.35 (m, overlapped)
6.52 (1H, d, 2.4) 7.54 (1H, d, 2.0) 7.60 (1H, br.s) –
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) 4.87 (2H, brs)
6.64 (1H, dd, 3.2,3.2) 2.80 (1H, brd, 12.0) 1.14 (m, overlapped) 1.58–1.60 (m, overlapped) 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) – –
1.16 (3H, s) 0.92 (3H, s) 3.76 (3H, s)
4.10 (1H, brd, 9.6) 3.14–3.19 (m, overlapped) 1.27 (3H, s) 0.85 (3H, s) –
1.28 (3H, s) 1.04 (3H, s) –
4.22 (1H, d, 8.0) 3.13–3.19 (m, overlapped) 3.36 (1H, t, 8.8) 3.27 (1H, m) 3.27 (1H, m) 3.85 (1H, dd, 12.4, 1.6) 3.66 (1H, dd, 12.0, 5.0)
4.32 (1H, d, 7.6) 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) 3.72 (1H, m)
4.21 (1H, d, 7.6) 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) 3.68–3.75 (m, overlapped)
5.46 (1H, d, 8.4) 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) 3.69 (1H, dd, 12.0, 3.6)
– – – – – –
4.48 (1H, d, 7.6) 3.20–3.26 (m, overlapped) 3.18–3.24 (m, overlapped) 3.31–3.39 (m, overlapped) 3.50 (m, overlapped) 3.84 (1H, dd, 12.0, 2.4) 3.64 (1H, dd, 12.0, 5.6)
5.54 (1H, d, 8.0) 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) 3.68–3.75 (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" Fig. 3) between H-2/H-19/H -1 17. The ROESY correlations (l α 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 H20 was β-orientated. Consequently, the structure of compound 2 " Fig. 1 and named sagittatayunnawas deduced as depicted in l 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 exhibited absorption bands at 3448, 1707, 1652, and 1068 cm−1, corresponding to hydroxyl, ester-carbonyl, olefin, and glycosidic " Tables 1 and 2) were essentially functions. The NMR data (l identical to compound 1 except that there was one more sugar unit in compound 3. The anomeric proton signals due to the additional sugar moiety at δH 5.54 (1H, d, J = 8.0 Hz, H-2′′) in the 1H " Table 1), combined with the carbon signals asNMR spectrum (l cribable to the extra sugar at δC 95.4 (C-1′′), 73.9 (C-2′′), 78.7 (C3′′), 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
" Table 2), suggested the additional sugar moiety spectrum (l should be a β-D-glucopyranose [31]. The location of this additional sugar at C-18 was deduced by the HMBC correlation between H-2′′ (δH 5.54) and C-18 (δC 167.5). Thus, compound 3 " Fig. 1 and named sagittatayunwas characterized as shown in l 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 " Tables 1 and 2) with those of compound 1 the NMR data (l showed a high similarity with the exception that compound 4 possessed one more ester-carbonyl and one less methylene as " Table 2). The structure of compound 4 differs seen in the DEPT (l 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 " Fig. 2) from H-8 (δ HMBC correlations (l H 2.70 1H, dd, J = 12.0, 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, 1H1 " Fig. 2), and ROESY (l " Fig. 3) correlations further conH COSY (l
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
420
Letters
1
2
3
4
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 OMe Glc1′ 2′ 3′ 4′ 5′ 6′ Glc1′′ 2′′ 3′′ 4′′ 5′′ 6′′
17.6 (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) –
28.5 (t) 71.8 (d) 138.2 (d) 139.7 (s) 37.1 (s) 35.4 (t) 20.8 (t) 48.6 (d) 38.7 (s) 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)
17.5 (t) 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) –
17.4 (t) 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) –
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)
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 " Fig. 1 and named sagittatayunna4 was deduced as shown in l noside D (4). 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 HeLa, K562, HL60, and HepG2 cell lines. Results are summarized in " Table 3. It can be concluded that compounds 1 and 4 showed l superior inhibitory activity in HeLa and HL60 cells, and compounds 2 and 3 exhibited superior activity against the K562 cell line, with IC50 values of 17.6 and 14.4 µM, respectively. Compounds 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 also 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, respectively, 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 activity. It could be helpful for researchers to find anti-settlement active chemicals from a natural source. SeaNine211, a wellknown antifouling agent, was used as a positive control and had an EC50 value of 6.8 ± 1.3 µM.
Table 2 13C NMR (100 MHz) data (δ) of compounds 1–4 in CD3OD.
Materials and Methods !
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 on a Bio-Rad FTS-135 spectrometer with KBr pellets, ν in cm−1. 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: cisplatin (purity > 99 %) was purchased from Sigma-Aldrich Chemical Co., and SeaNine211 (purity > 96 %) was obtained from Rhom and Hass Co. Plant material: The roots of T. sagittata var. yunnanensis were collected in Honghe, Yunnan Province in November 2007 and identified 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. sagittata 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
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
Position
421
Letters
Fig. 1
Structures of compounds 1–6.
Fig. 2 Selected key HMBC and 1H-1H COSY correlations of compounds 1–4.
Jiang Z-Y et al. New Clerodane Diterpenes …
Planta Med 2014; 80: 419–425
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
422
Letters
Samples
1 2 3 4 5 6 95% Ethanol extract (µg/mL) Cisplatina a
Key ROESY (↔) correlations of compounds
IC50 (µM) K562
Hela
HL60
HepG2
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
11.8 ± 3.8 132.3 ± 10.4 108.5 ± 9.2 14.0 ± 5.2 87.7 ± 8.4 68.1 ± 5.7 23.1 ± 4.0 7.5
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
Table 3 Cytotoxic activities of compounds 1–6.
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 silica gel CC (10 × 120 cm, silica gel 2.0 kg, 200–300 mesh) by gradient 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 revealed 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 furnish 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 isolated 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 A (1): Colorless powder; [α]25 − 80.0 D (c = 0.10, MeOH); IR (KBr): νmax = 3454, 2978, 1736, 1670, 1656, Jiang Z-Y et al. New Clerodane Diterpenes …
Planta Med 2014; 80: 419–425
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
Fig. 3 1–4.
423
Letters 1242, 1059 cm−1; HRESIMS (positive): m/z = 533.2349 [M + Na]+ (calcd. for C26H38O10Na+, 533.2362); 1H and 13C NMR (CD3OD) da" Tables 1 and 2. ta: l Sagittatayunnanoside B (2): Colorless powder; [α]25 − 65.7 D (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: " Tables 1 and 2. l Sagittatayunnanoside C (3): Colorless powder; [α]27.1 − 49.0 D (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" Tables 1 and 2. ta: l Sagittatayunnanoside B (4): Colorless powder; [α]27.5 − 75.8 D (c = 0.10, MeOH); IR (KBr): νmax = 3449, 2972, 1735, 1671, 1654, 1243, 1061 cm−1; HRESIMS (positive): m/z = 547.2171 (calcd. for " TaC26H36O11Na+, 547.2155); 1H and 13C NMR (CD3OD) data: l bles 1 and 2. Acidic hydrolysis: Each solution of compounds 1 and 2 (each 5 mg) in a mixture of MeOH (1.0 mL) and 10% HCl (1.0 mL) was stirred at reflux for 4 h. The hydrolysate was allowed to cool, diluted 2fold with H2O, and extracted with EtOAc (3 × 2 mL). The aqueous layer was neutralized with 2 M ammonium hydroxide and concentrated in vacuo to give a residue in which D-glucose was identified by comparison with authentic sugar samples (n-BuOH/ AcOH/H2O 4 : 1 : 5, upper layer; PhOH/H2O, 4 : 1) on TLC (sprayed with aniline phthalate reagent from Sigma-Aldrich, followed by heating). Cytotoxicity assay: Cytotoxic activities were evaluated by a modified MTT method [33] using HeLa, K562, HL60, and HepG2 cell lines. In brief, the cell suspensions (200 mL) at a density of 5 × 104 cells · mL−1 were plated in 96-well microtiter plates and incubated for 24 h at 37 °C in a humidified incubator containing 5 % CO2. The test compound solution (2 mL in DMSO) at different concentrations was added to each well and further incubated for 72 h under the same conditions. The MTT solution (20 mL) was then added to each well and incubated for 4 h. The old medium (150 mL) containing MTT was then gently replaced by DMSO and pipetted to dissolve any formazan crystals that had formed. The absorbance was then determined with a Spectra Max Plus plate reader at 540 nm. Dose-response curves were generated and the IC50 values were defined as the concentration of compound required to inhibit cell proliferation by 50 %. Cisplatin (purity > 99%), an approved agent for the treatment of many tumors, was used as the positive control. Antilarval attachment assay: The antilarval settlement activity of compounds 1–6 and SeaNine211 (purity > 96%) was determined using the larvae of the barnacle Balanus amphitrite (kindly supplied by Prof. Pei-Yuan Qian from Hong Kong University of Science and Technology) as described by Dash et al. [32]. The larvae of B. amphitrite were raised to competence according to Thiyagarajan et al. [34]. EC50 (the concentration where 50 % of the larval population was inhibited from settling in comparison with the control) values were calculated as described [35]. Four replicates were run for each sample.
Supporting information The 1D and 2D NMR spectra of the new compounds 1–4 are available as Supporting Information.
Jiang Z-Y et al. New Clerodane Diterpenes …
Planta Med 2014; 80: 419–425
Acknowledgements !
Financial support was provided by grants from the National Natural Science Foundation of China (NSFC No. 21262047, 21162041), the Natural Science Foundation of Yunnan Province (No. S2012FZ0227), the Program for Science and Technology Innovative Research Team in University of Yunnan Province (IRTSTYN), 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.
Conflict of Interest !
The authors declare no conflict of interest.
References 1 Jiangsu New Medical College. The dictionary of traditional Chinese medicine. Shanghai: Peopleʼs Publishing House; 1986: 1393–1394 2 Zhan ZJ, Zhang XY, Hou XR, Li CP, Shan WG. New diterpenoids from Tinospora capillipes. Helv Chim Acta 2009; 92: 790–794 3 Shi LM, Li RQ, Liu WH. Two new furanoid diterpenoids from Tinospora sagittata. Helv Chim Acta 2008; 91: 978–982 4 Liu XH, Hu ZL, Shi QR, Zeng HW, Shen YH, Jin HZ, Zhang WD. Anti-inflammatory and anti-nociceptive activities of compounds from Tinospora sagittata (Oliv.) Gagnep. Arch Pharm Res 2010; 33: 981–987 5 Li RW, Lin GD, Myers SP, Leach DN. Anti-inflammatory activity of Chinese medicinal vine plants. J Ethnopharmacol 2003; 85: 61–67 6 Castro A, Coll J. Neo-clerodane diterpenoids from Verbenaceae: Structural elucidation and biological activity. Nat Prod Commun 2008; 3: 1021–1031 7 Huang XZ, Cheng CM, Dai Y, Fu GM, Guo JM, Yin Y, Liang H. A novel 18norclerodane diterpenoid from the roots of Tinospora sagittata var. yunnanensis. Molecules 2010; 15: 8360–8365 8 Huang XZ, Cheng CM, Dai Y, Fu GM, Guo JM, Liang H, Wang C. A novel lignan glycoside with antioxidant activity from Tinospora sagittata var. yunnanensis. Nat Prod Res 2012; 26: 1876–1880 9 Li W, Huang C, Li SP, Ma FH, Li Q, Asada Y, Koike K. Clerodane diterpenoids from Tinospora sagittata (Oliv.) Gagnep. Planta Med 2012; 78: 82–85 10 Huang C, Li W, Ma FH, Li Q, Asada Y, Koike K. Tinospinosides D, E and tinospin E, further clerodane diterpenoids from Tinospora sagittata. Chem Pharm Bull 2012; 60: 1324–1328 11 Muhammad I, El-Feraly FS, Mossa JS, Ramadan AF. Terpenoids from Pulicaria glutinosa. Phytochemistry 1992; 31: 4245–4248 12 Cifuente DA, Borkowski EJ, Sosa ME, Gianello JC, Giordano OS, Tonn CE. Clerodane diterpenes from Baccharis sagittalis: insect antifeedant activity. Phytochemistry 2002; 61: 899–905 13 Ceñal JP, Giordano OS, Rossomando PC, Tonn CE. Neoclerodane diterpenes from Baccharis crispa. J Nat Prod 1997; 60: 490–492 14 Achari B, Chaudhuri C, Saha CR, Dutta PK, Pakrashi SC. A clerodane diterpene and other constituents of Clerodendron inerme. Phytochemistry 1990; 29: 3671–3673 15 Manabe S, Nishino C. Stereochemistry of cis-clerodane diterpenes. Tetrahedron 1986; 42: 3461–3470 16 Li W, Wei K, Koike K. Structure and absolute configuration of clerodane diterpene glycosides and a rearranged cadinane sesquiterpene glycosides from the stems of Tinospora sinensis. J Nat Prod 2007; 70: 1971– 1976 17 Maurya R, Manhas LR, Gupta P, Mishra PK, Singh G, Yadav PP. Amritosides A, B, C, and D: clerodane furano dietrpene glucosides from Tinospora cordifolia. Phytochemistry 2004; 65: 2051–2055 18 Martin TS, Ohtani K, Kasai R, Yamasaki K. Furanoid diterpene glucosides from Tinospora rumphii. Phytochemistry 1996; 42: 153–158 19 Maurya R, Wazir V, Tyagi Y, Kapil RS. Clerodane diterpenoids from Tinospora cordifolia. Phytochemistry 1995; 38: 659–661 20 Wazir V, Maurya R, Kapil RS. Cordioside, a clerodane furano diterpene glucoside from Tinospora cordifolia. Phytochemistry 1995; 38: 447– 449
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
424
Letters 34 Thiyagarajan V, Harder T, Qiu JW, Qian PY. Energy content at metamorphosis and growth rate of the early juvenile barnacle Balanus amphitrite. Mar Biol 2003; 143: 543–554 35 Xu Y, He HP, Schulz S, Liu X, Fusetani N, Xiong HR, Xiao X, Qian PY. Potent antifouling compounds produced by marine Streptomyces. Bioresour Technol 2010; 101: 1331–1336 received revised accepted
September 25, 2013 January 22, 2014 February 14, 2014
Bibliography 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 Correspondence 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: + 86 8 71 65 91 30 13 Fax: + 86 8 71 65 91 00 17 [email protected]
Jiang Z-Y et al. New Clerodane Diterpenes …
Planta Med 2014; 80: 419–425
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
21 Gangan VD, Pradhan P, Sipahimalani AT, Banerji A. Norditerpene furan glycosides from Tinospora cordifolia. Phytochemistry 1995; 39: 1139– 1142 22 Kiem PV, Minh CV, Dat NT, Kinh LV, Huang DT, Nam NH, Cuong NX, Huong HT, Lau TV. Aporphine alkaloids, clerodane diterpenes and other constituents from Tinospora cordifolia. Fitoterapia 2010; 81: 485–489 23 Bhatt RK, Hanuman JB, Sabata BK. A new clerodane derivative from Tinospora cordifolia. Phytochemistry 1988; 27: 1212–1216 24 Maurya R, Dhar KL, Handa SS. A sesquiterpene glucoside from Tinospora cordifolia. Phytochemistry 1997; 44: 749–750 25 Khan MA, Gray AI, Waterman PG. Tinosporaside, an 18-norclerodane glucoside from Tinospora cordifolia. Phytochemistry 1989; 28: 273– 275 26 Tuntiwachwuttikul P, Boonrasri N, Bremner JB, Taylor WC. Rearranged clerodane diterpenes from Tinospora baenzigeri. Phytochemistry 1999; 52: 1335–1340 27 Yonemitsu M, Fukuda N, Kimura T, Isobe R, Komori T. Studies on the constituents of the stems of Tinospora sinensis. II. Isolation and structure elucidation of two new dinorditerpene glucosides, tinosineside A and B. Liebigs Ann Chem 1995; 2: 437–439 28 Maurya R, Wazir V, Tyagi A, Kapil RS. Clerodane diterpenoids from Tinospora cordifolia. Phytochemistry 1995; 38: 659–661 29 Song CQ, Xu RS. New clerodane diterpenoids from Tinospora capillipes. Chin Chem Lett 1992; 3: 185–188 30 Itokawa H, Mizuno K, Tajima R, Takeya K. Furanoditerpene glucosides from Fibraurea tinctoria. Phytochemistry 1986; 25: 905–908 31 Jiang ZY, Zhang XM, Zhou J, Chen JJ. New triterpenoid glycosides from Centella asiatica. Helv Chim Acta 2005; 88: 297–303 32 Dash S, Jin CL, Lee OO, Xu Y, Qian PY. Antibacterial and antilarval settlement potential and metabolite profiles of novel sponge-associated marine bacteria. J Indian Microbiol Biotechnol 2009; 36: 1047–1056 33 Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983; 65: 55–63
425
Copyright of Planta Medica is the property of Georg Thieme Verlag Stuttgart and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
Abstract !
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 2D NMR data. The cytotoxic and antifouling activities of compounds 1–6 were evaluated.
Key words Tinospora sagittata var. Yunnanensis · Menispermaceae · clerodane 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 investigations on the genus Tinospora showed that the clerodane diterpenes possess anti-inflammatory, antibacterial, and antifeedant activity [2–6]. In our recent study on the bioactivity of the Tinospora plants, the 95 % ethanolic extract of T. sagittata var. yunnanensis showed cytotoxicity on HeLa, K562, HL60, and HepG2 cells in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid (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. yunnanensis was carried out. Our preceding research resulted in the isolation of nine compounds [7, 8]. During the subsequent investigation of T. sagittata var. yunnanensis, four new clerodane diterpenes, named sagittatayunnanosides A–D (1–4), were isolated from the 95 % ethanol extract, together with two known ones, tinospinoside 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. amphitrite. Herein, we present the isolation and structural elucidation, as well as cytotoxic and antifouling activities of the new compounds. Compound 1 was obtained as a colorless powder. The positive ESI‑MS gave a quasi-molecular ion peak at m/z 511, in accordance
with the molecular formula C26H38O10 as revealed by the HRESIMS at m/z 533.2349 [M + Na]+ (calcd. for C26H38O10Na+, 533.2362). The IR spectrum showed the absorptions for hydroxyl (3454 cm−1), carbonyl (1736 cm−1), and olefinic functions " Table 1), two olefinic (1656 cm−1). In the 1H NMR spectrum (l proton signals at δ H 6.59 (1H, dd, J = 3.6, 3.6 Hz, H-3) and 7.42 (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 methanol liberated the D-glucose, which was identified by TLC compar" Table 2) spectrum ison with an authentic sample. The 13C NMR (l 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 C 177.1 (C-16) and 171.7 (C-18) were displayed. Analyses of the NMR data suggested compound 1 was a diterpene glycoside featuring a clerodane skeleton. However, compound 1 contained an α-substituted butenolide ring, which was determined by the characteristic 1H-1H COSY " Fig. 1) cross-peak due to the broad singlet at δ 7.42 (1H, brs, (l C H-14) coupled with a two proton broad singlet at δH 4.83 (2H, brs, " Fig. 2) of H-14 H-15) [11], as well as the HMBC correlations (l 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-dihydroxyneocleroda3,13-dien-16,15 : 18,19-diolide [13] showed the existence of an extra glucose at C-17 in compound 1. The additional β-D-glucopyranosyl 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 H2 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 " Fig. 3) for H-10/H-19, H-19/H-8 and H-8/H-12 indicated that (l 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 " Fig. 1 and named sagittatayunnanoside A (1). as shown in l 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 13 C NMR spectrum showed typical resonances [δH 7.60 (brs, H16), 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-substituted 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 " Ta3.76 (s); δC 52.3 (q)], and two β-D-glucopyranosyl moieties (l 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 epitinophylloloside [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]
Jiang Z-Y et al. New Clerodane Diterpenes …
Planta Med 2014; 80: 419–425
419
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
Letters
Letters
Table 1
1
H NMR (400 MHz) data (δ) of compounds 1–4 in CD3OD, δ in ppm, J in Hz.
Position 1 2 3 6 7 8 10 11 12 14 15 16 17 19 20 OMe Glc1′ 2′ 3′ 4′ 5′ 6′ Glc1′′ 2′′ 3′′ 4′′ 5′′ 6′′
1
2
3
4
2.03–2.09 (1H, m) 1.90–1.96 (1H, m) 2.26–2.28 (m)
2.68 (1H, m) 2.09–2.14 (m, overlapped) 4.46 (1H, dd, 7.6, 13.2)
2.08–2.12 (1H, m) 1.92–1.98 (1H, m) 2.28–2.34 (m)
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) 4.83 (2H, brs) – 4.10 (1H, dd, 9.6, 3.2) 3.13–3.19 (m, overlapped) 1.27 (3H, s) 0.86 (3H, s) –
6.52 (1H, d, 3.2) 2.60 (1H, dd, 10.8, 2.8) 1.31 (1H, m) 1.73 (1H, overlapped) 1.37 (1H, m) 2.74 (1H, dd, 11.2, 3.2) 1.73 (1H, brd, 8.0) 2.09–2.14 (m, overlapped) 1.93 (1H, dd, 11.9, 13.6) 5.50 (1H, dd, 11.9, 6.4)
2.05–2.09 (1H, m) 1.91–1.96 (1H, m) 1.15 (m, overlapped) 1.77 (m, overlapped) 6.79 (1H, brs) 2.73 (1H, brd, 11.6) 1.15 (m, overlapped) 2.28–2.35 (m, overlapped)
6.52 (1H, d, 2.4) 7.54 (1H, d, 2.0) 7.60 (1H, br.s) –
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) 4.87 (2H, brs)
6.64 (1H, dd, 3.2,3.2) 2.80 (1H, brd, 12.0) 1.14 (m, overlapped) 1.58–1.60 (m, overlapped) 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) – –
1.16 (3H, s) 0.92 (3H, s) 3.76 (3H, s)
4.10 (1H, brd, 9.6) 3.14–3.19 (m, overlapped) 1.27 (3H, s) 0.85 (3H, s) –
1.28 (3H, s) 1.04 (3H, s) –
4.22 (1H, d, 8.0) 3.13–3.19 (m, overlapped) 3.36 (1H, t, 8.8) 3.27 (1H, m) 3.27 (1H, m) 3.85 (1H, dd, 12.4, 1.6) 3.66 (1H, dd, 12.0, 5.0)
4.32 (1H, d, 7.6) 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) 3.72 (1H, m)
4.21 (1H, d, 7.6) 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) 3.68–3.75 (m, overlapped)
5.46 (1H, d, 8.4) 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) 3.69 (1H, dd, 12.0, 3.6)
– – – – – –
4.48 (1H, d, 7.6) 3.20–3.26 (m, overlapped) 3.18–3.24 (m, overlapped) 3.31–3.39 (m, overlapped) 3.50 (m, overlapped) 3.84 (1H, dd, 12.0, 2.4) 3.64 (1H, dd, 12.0, 5.6)
5.54 (1H, d, 8.0) 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) 3.68–3.75 (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" Fig. 3) between H-2/H-19/H -1 17. The ROESY correlations (l α 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 H20 was β-orientated. Consequently, the structure of compound 2 " Fig. 1 and named sagittatayunnawas deduced as depicted in l 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 exhibited absorption bands at 3448, 1707, 1652, and 1068 cm−1, corresponding to hydroxyl, ester-carbonyl, olefin, and glycosidic " Tables 1 and 2) were essentially functions. The NMR data (l identical to compound 1 except that there was one more sugar unit in compound 3. The anomeric proton signals due to the additional sugar moiety at δH 5.54 (1H, d, J = 8.0 Hz, H-2′′) in the 1H " Table 1), combined with the carbon signals asNMR spectrum (l cribable to the extra sugar at δC 95.4 (C-1′′), 73.9 (C-2′′), 78.7 (C3′′), 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
" Table 2), suggested the additional sugar moiety spectrum (l should be a β-D-glucopyranose [31]. The location of this additional sugar at C-18 was deduced by the HMBC correlation between H-2′′ (δH 5.54) and C-18 (δC 167.5). Thus, compound 3 " Fig. 1 and named sagittatayunwas characterized as shown in l 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 " Tables 1 and 2) with those of compound 1 the NMR data (l showed a high similarity with the exception that compound 4 possessed one more ester-carbonyl and one less methylene as " Table 2). The structure of compound 4 differs seen in the DEPT (l 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 " Fig. 2) from H-8 (δ HMBC correlations (l H 2.70 1H, dd, J = 12.0, 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, 1H1 " Fig. 2), and ROESY (l " Fig. 3) correlations further conH COSY (l
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
420
Letters
1
2
3
4
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 OMe Glc1′ 2′ 3′ 4′ 5′ 6′ Glc1′′ 2′′ 3′′ 4′′ 5′′ 6′′
17.6 (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) –
28.5 (t) 71.8 (d) 138.2 (d) 139.7 (s) 37.1 (s) 35.4 (t) 20.8 (t) 48.6 (d) 38.7 (s) 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)
17.5 (t) 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) –
17.4 (t) 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) –
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)
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 " Fig. 1 and named sagittatayunna4 was deduced as shown in l noside D (4). 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 HeLa, K562, HL60, and HepG2 cell lines. Results are summarized in " Table 3. It can be concluded that compounds 1 and 4 showed l superior inhibitory activity in HeLa and HL60 cells, and compounds 2 and 3 exhibited superior activity against the K562 cell line, with IC50 values of 17.6 and 14.4 µM, respectively. Compounds 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 also 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, respectively, 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 activity. It could be helpful for researchers to find anti-settlement active chemicals from a natural source. SeaNine211, a wellknown antifouling agent, was used as a positive control and had an EC50 value of 6.8 ± 1.3 µM.
Table 2 13C NMR (100 MHz) data (δ) of compounds 1–4 in CD3OD.
Materials and Methods !
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 on a Bio-Rad FTS-135 spectrometer with KBr pellets, ν in cm−1. 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: cisplatin (purity > 99 %) was purchased from Sigma-Aldrich Chemical Co., and SeaNine211 (purity > 96 %) was obtained from Rhom and Hass Co. Plant material: The roots of T. sagittata var. yunnanensis were collected in Honghe, Yunnan Province in November 2007 and identified 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. sagittata 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
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
Position
421
Letters
Fig. 1
Structures of compounds 1–6.
Fig. 2 Selected key HMBC and 1H-1H COSY correlations of compounds 1–4.
Jiang Z-Y et al. New Clerodane Diterpenes …
Planta Med 2014; 80: 419–425
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
422
Letters
Samples
1 2 3 4 5 6 95% Ethanol extract (µg/mL) Cisplatina a
Key ROESY (↔) correlations of compounds
IC50 (µM) K562
Hela
HL60
HepG2
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
11.8 ± 3.8 132.3 ± 10.4 108.5 ± 9.2 14.0 ± 5.2 87.7 ± 8.4 68.1 ± 5.7 23.1 ± 4.0 7.5
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
Table 3 Cytotoxic activities of compounds 1–6.
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 silica gel CC (10 × 120 cm, silica gel 2.0 kg, 200–300 mesh) by gradient 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 revealed 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 furnish 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 isolated 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 A (1): Colorless powder; [α]25 − 80.0 D (c = 0.10, MeOH); IR (KBr): νmax = 3454, 2978, 1736, 1670, 1656, Jiang Z-Y et al. New Clerodane Diterpenes …
Planta Med 2014; 80: 419–425
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
Fig. 3 1–4.
423
Letters 1242, 1059 cm−1; HRESIMS (positive): m/z = 533.2349 [M + Na]+ (calcd. for C26H38O10Na+, 533.2362); 1H and 13C NMR (CD3OD) da" Tables 1 and 2. ta: l Sagittatayunnanoside B (2): Colorless powder; [α]25 − 65.7 D (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: " Tables 1 and 2. l Sagittatayunnanoside C (3): Colorless powder; [α]27.1 − 49.0 D (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" Tables 1 and 2. ta: l Sagittatayunnanoside B (4): Colorless powder; [α]27.5 − 75.8 D (c = 0.10, MeOH); IR (KBr): νmax = 3449, 2972, 1735, 1671, 1654, 1243, 1061 cm−1; HRESIMS (positive): m/z = 547.2171 (calcd. for " TaC26H36O11Na+, 547.2155); 1H and 13C NMR (CD3OD) data: l bles 1 and 2. Acidic hydrolysis: Each solution of compounds 1 and 2 (each 5 mg) in a mixture of MeOH (1.0 mL) and 10% HCl (1.0 mL) was stirred at reflux for 4 h. The hydrolysate was allowed to cool, diluted 2fold with H2O, and extracted with EtOAc (3 × 2 mL). The aqueous layer was neutralized with 2 M ammonium hydroxide and concentrated in vacuo to give a residue in which D-glucose was identified by comparison with authentic sugar samples (n-BuOH/ AcOH/H2O 4 : 1 : 5, upper layer; PhOH/H2O, 4 : 1) on TLC (sprayed with aniline phthalate reagent from Sigma-Aldrich, followed by heating). Cytotoxicity assay: Cytotoxic activities were evaluated by a modified MTT method [33] using HeLa, K562, HL60, and HepG2 cell lines. In brief, the cell suspensions (200 mL) at a density of 5 × 104 cells · mL−1 were plated in 96-well microtiter plates and incubated for 24 h at 37 °C in a humidified incubator containing 5 % CO2. The test compound solution (2 mL in DMSO) at different concentrations was added to each well and further incubated for 72 h under the same conditions. The MTT solution (20 mL) was then added to each well and incubated for 4 h. The old medium (150 mL) containing MTT was then gently replaced by DMSO and pipetted to dissolve any formazan crystals that had formed. The absorbance was then determined with a Spectra Max Plus plate reader at 540 nm. Dose-response curves were generated and the IC50 values were defined as the concentration of compound required to inhibit cell proliferation by 50 %. Cisplatin (purity > 99%), an approved agent for the treatment of many tumors, was used as the positive control. Antilarval attachment assay: The antilarval settlement activity of compounds 1–6 and SeaNine211 (purity > 96%) was determined using the larvae of the barnacle Balanus amphitrite (kindly supplied by Prof. Pei-Yuan Qian from Hong Kong University of Science and Technology) as described by Dash et al. [32]. The larvae of B. amphitrite were raised to competence according to Thiyagarajan et al. [34]. EC50 (the concentration where 50 % of the larval population was inhibited from settling in comparison with the control) values were calculated as described [35]. Four replicates were run for each sample.
Supporting information The 1D and 2D NMR spectra of the new compounds 1–4 are available as Supporting Information.
Jiang Z-Y et al. New Clerodane Diterpenes …
Planta Med 2014; 80: 419–425
Acknowledgements !
Financial support was provided by grants from the National Natural Science Foundation of China (NSFC No. 21262047, 21162041), the Natural Science Foundation of Yunnan Province (No. S2012FZ0227), the Program for Science and Technology Innovative Research Team in University of Yunnan Province (IRTSTYN), 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.
Conflict of Interest !
The authors declare no conflict of interest.
References 1 Jiangsu New Medical College. The dictionary of traditional Chinese medicine. Shanghai: Peopleʼs Publishing House; 1986: 1393–1394 2 Zhan ZJ, Zhang XY, Hou XR, Li CP, Shan WG. New diterpenoids from Tinospora capillipes. Helv Chim Acta 2009; 92: 790–794 3 Shi LM, Li RQ, Liu WH. Two new furanoid diterpenoids from Tinospora sagittata. Helv Chim Acta 2008; 91: 978–982 4 Liu XH, Hu ZL, Shi QR, Zeng HW, Shen YH, Jin HZ, Zhang WD. Anti-inflammatory and anti-nociceptive activities of compounds from Tinospora sagittata (Oliv.) Gagnep. Arch Pharm Res 2010; 33: 981–987 5 Li RW, Lin GD, Myers SP, Leach DN. Anti-inflammatory activity of Chinese medicinal vine plants. J Ethnopharmacol 2003; 85: 61–67 6 Castro A, Coll J. Neo-clerodane diterpenoids from Verbenaceae: Structural elucidation and biological activity. Nat Prod Commun 2008; 3: 1021–1031 7 Huang XZ, Cheng CM, Dai Y, Fu GM, Guo JM, Yin Y, Liang H. A novel 18norclerodane diterpenoid from the roots of Tinospora sagittata var. yunnanensis. Molecules 2010; 15: 8360–8365 8 Huang XZ, Cheng CM, Dai Y, Fu GM, Guo JM, Liang H, Wang C. A novel lignan glycoside with antioxidant activity from Tinospora sagittata var. yunnanensis. Nat Prod Res 2012; 26: 1876–1880 9 Li W, Huang C, Li SP, Ma FH, Li Q, Asada Y, Koike K. Clerodane diterpenoids from Tinospora sagittata (Oliv.) Gagnep. Planta Med 2012; 78: 82–85 10 Huang C, Li W, Ma FH, Li Q, Asada Y, Koike K. Tinospinosides D, E and tinospin E, further clerodane diterpenoids from Tinospora sagittata. Chem Pharm Bull 2012; 60: 1324–1328 11 Muhammad I, El-Feraly FS, Mossa JS, Ramadan AF. Terpenoids from Pulicaria glutinosa. Phytochemistry 1992; 31: 4245–4248 12 Cifuente DA, Borkowski EJ, Sosa ME, Gianello JC, Giordano OS, Tonn CE. Clerodane diterpenes from Baccharis sagittalis: insect antifeedant activity. Phytochemistry 2002; 61: 899–905 13 Ceñal JP, Giordano OS, Rossomando PC, Tonn CE. Neoclerodane diterpenes from Baccharis crispa. J Nat Prod 1997; 60: 490–492 14 Achari B, Chaudhuri C, Saha CR, Dutta PK, Pakrashi SC. A clerodane diterpene and other constituents of Clerodendron inerme. Phytochemistry 1990; 29: 3671–3673 15 Manabe S, Nishino C. Stereochemistry of cis-clerodane diterpenes. Tetrahedron 1986; 42: 3461–3470 16 Li W, Wei K, Koike K. Structure and absolute configuration of clerodane diterpene glycosides and a rearranged cadinane sesquiterpene glycosides from the stems of Tinospora sinensis. J Nat Prod 2007; 70: 1971– 1976 17 Maurya R, Manhas LR, Gupta P, Mishra PK, Singh G, Yadav PP. Amritosides A, B, C, and D: clerodane furano dietrpene glucosides from Tinospora cordifolia. Phytochemistry 2004; 65: 2051–2055 18 Martin TS, Ohtani K, Kasai R, Yamasaki K. Furanoid diterpene glucosides from Tinospora rumphii. Phytochemistry 1996; 42: 153–158 19 Maurya R, Wazir V, Tyagi Y, Kapil RS. Clerodane diterpenoids from Tinospora cordifolia. Phytochemistry 1995; 38: 659–661 20 Wazir V, Maurya R, Kapil RS. Cordioside, a clerodane furano diterpene glucoside from Tinospora cordifolia. Phytochemistry 1995; 38: 447– 449
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
424
Letters 34 Thiyagarajan V, Harder T, Qiu JW, Qian PY. Energy content at metamorphosis and growth rate of the early juvenile barnacle Balanus amphitrite. Mar Biol 2003; 143: 543–554 35 Xu Y, He HP, Schulz S, Liu X, Fusetani N, Xiong HR, Xiao X, Qian PY. Potent antifouling compounds produced by marine Streptomyces. Bioresour Technol 2010; 101: 1331–1336 received revised accepted
September 25, 2013 January 22, 2014 February 14, 2014
Bibliography 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 Correspondence 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: + 86 8 71 65 91 30 13 Fax: + 86 8 71 65 91 00 17 [email protected]
Jiang Z-Y et al. New Clerodane Diterpenes …
Planta Med 2014; 80: 419–425
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
21 Gangan VD, Pradhan P, Sipahimalani AT, Banerji A. Norditerpene furan glycosides from Tinospora cordifolia. Phytochemistry 1995; 39: 1139– 1142 22 Kiem PV, Minh CV, Dat NT, Kinh LV, Huang DT, Nam NH, Cuong NX, Huong HT, Lau TV. Aporphine alkaloids, clerodane diterpenes and other constituents from Tinospora cordifolia. Fitoterapia 2010; 81: 485–489 23 Bhatt RK, Hanuman JB, Sabata BK. A new clerodane derivative from Tinospora cordifolia. Phytochemistry 1988; 27: 1212–1216 24 Maurya R, Dhar KL, Handa SS. A sesquiterpene glucoside from Tinospora cordifolia. Phytochemistry 1997; 44: 749–750 25 Khan MA, Gray AI, Waterman PG. Tinosporaside, an 18-norclerodane glucoside from Tinospora cordifolia. Phytochemistry 1989; 28: 273– 275 26 Tuntiwachwuttikul P, Boonrasri N, Bremner JB, Taylor WC. Rearranged clerodane diterpenes from Tinospora baenzigeri. Phytochemistry 1999; 52: 1335–1340 27 Yonemitsu M, Fukuda N, Kimura T, Isobe R, Komori T. Studies on the constituents of the stems of Tinospora sinensis. II. Isolation and structure elucidation of two new dinorditerpene glucosides, tinosineside A and B. Liebigs Ann Chem 1995; 2: 437–439 28 Maurya R, Wazir V, Tyagi A, Kapil RS. Clerodane diterpenoids from Tinospora cordifolia. Phytochemistry 1995; 38: 659–661 29 Song CQ, Xu RS. New clerodane diterpenoids from Tinospora capillipes. Chin Chem Lett 1992; 3: 185–188 30 Itokawa H, Mizuno K, Tajima R, Takeya K. Furanoditerpene glucosides from Fibraurea tinctoria. Phytochemistry 1986; 25: 905–908 31 Jiang ZY, Zhang XM, Zhou J, Chen JJ. New triterpenoid glycosides from Centella asiatica. Helv Chim Acta 2005; 88: 297–303 32 Dash S, Jin CL, Lee OO, Xu Y, Qian PY. Antibacterial and antilarval settlement potential and metabolite profiles of novel sponge-associated marine bacteria. J Indian Microbiol Biotechnol 2009; 36: 1047–1056 33 Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983; 65: 55–63
425
Copyright of Planta Medica is the property of Georg Thieme Verlag Stuttgart and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
