Chinese Journal of Natural Medicines 2015, 13(5): 0383−0389
Chinese Journal of Natural Medicines
Six new cytotoxic and anti-inflammatory 11, 20-epoxy-entkaurane diterpenoids from Isodon wikstroemioides WU Hai-Yan1, 2, WANG Wei-Guang1, DU Xue1, YANG Jin1, 2, PU Jian-Xin1*, SUN Han-Dong1* 1
State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of
Sciences, Kunming 650201, China; 2
University of Chinese Academy of Sciences, Beijing 100049, China Available online 20 May 2015
[ABSTRACT] The present study was designed to determine the chemical constituents of EtOAc extracts of the aerial parts of Isodon wikstroemioides. Compounds 1–8 were isolated and purified by normal-phase silica gel and reversed-phase C18 silica gel column chromatography and HPLC. Their structures were elucidated by extensive spectroscopic methods. Most of them were evaluated for their in vitro cytotoxicity against human cancer HL-60, SMMC-7721, A-549, MCF-7, and SW-480 cells and their inhibitory activity against nitric oxide (NO) production in LPS-activated RAW264.7 macrophages. Among the eight 11, 20-epoxy-ent-kauranoids isolated, compounds 1–6 (isowikstroemins H–M) were new diterpenoids. Compounds 1, 3, and 7 exhibited significant cytotoxicity with IC50 values ranging from (0.84 ± 0.02) to (4.09 ± 0.34) μmol·L−1, while compounds 4 and 5 showed selective cytotoxicity. In addition, compounds 1, 3, 4, and 7 exhibited inhibitory activity against nitric oxide (NO) production in LPS-activated RAW264.7 macrophages. These results provide a basis for future development of these compounds as anti-cancer and anti-inflammatory agents. [KEY WORDS] Isodon wikstroemioides; Isowikstroemins H–M; Cytotoxicity; Anti-inflammatory activity
[CLC Number] R284
[Document code] A
[Article ID] 2095-6975(2015)05-0383-07
Introduction Isodon is a cosmopolitan and important genus of the Lamiaceae family [1-2]. The Isodon species have long been used in traditional Chinese medicines [3]. The ent-kaurane diterpenoids, as the major secondary metabolites of this genus, have attracted considerable attention due to their diverse structures and interesting biological properties [4-7]. Over the past 30 years, more than 60 Isodon species have been investigated [8], and a large number of ent-kaurane diterpenoids have been isolated
[Received on] 07-Nov.-2014 [Research funding] This work was supported financially by the National Natural Science Foundation of China (Nos. 21322204 and 81172939), the NSFC-Joint Foundation of Yunnan Province (No. U1302223), the reservation-talent project of Yunnan Province (No. 2011CI043), and the West Light Foundation of the Chinese Academy of Sciences (PU JX). [*Corresponding author] Tel: 86-871-65223251, E-mail: [email protected] (PU Jian-Xin), [email protected] (SUN Han-Dong) These authors have no conflict of interest to declare. Copyright © 2015, China Pharmaceutical University. Published by Elsevier B.V. All rights reserved
and characterized by our group [8]. Isodon wikstroemioides (Hand.–Mazz.) H. Hara (Lamiaceae), a perennial herb, is primarily distributed in the northwestern regions of Yunnan Province and the western Sichuan regions in China [9]. Previous studies on this herb have led to isolation of 7, 20-epoxy-ent-kauranoids [10-11], C-20-non- oxy[12] , and C-20-oxygenatedgenated-entkauranoids [12] non-epoxy-ent-kauranoids . In our continuing research with the aim at discovering new diterpenoids with diverse structures and bioactivities, six new 11, 20-epoxy-ent- kauranoids, isowikstroemins H–M (1–6), along with two known analogues, macrocalyxin B (7) [13] and pseudoirroratin A (8) [14], have been isolated from I. wikstroemioides. In the present report, the isolation and structure elucidation of these diterpenoids are described as alongside with the cytotoxicity evaluation against five human tumor cell lines and their inhibitory activity against LPS-induced NO production in RAW 264.7 macrophages.
Results and Discussion A 70% aqueous acetone extract of the air-dried and powdered aerial parts of I. wikstroemioides (7.5 kg) was partitioned between EtOAc and H2O. The EtOAc-soluble portion
– 383 –
WU Hai-Yan, et al. / Chin J Nat Med, 2015, 13(5): 383−389
(380 g) was subjected to repeated column chromatography and semi-preparative HPLC to afford six new ent-kauranoids, isowikstroemins H–M (1–6), along with two
known analogues, macrocalyxin B (7) and pseudoirroratin A (8) (Fig. 1).
Fig. 1 Chemical structures of compounds 1–8
Isowikstroemin H (1) was obtained as white amorphous powder and gave a HREI-MS ion peak at m/z 420.214 7 ([M]+, calcd 420.214 8), which corresponded to a molecular formula of C23H32O7 with eight degrees of unsaturation. The IR spectrum indicated absorption bands for hydroxy group (3 425 cm–1), carTable 1 Position 1a 1b 2a 2b 3a 3b 5 6a 6b 7 9 12a 12b 13 14 17a 17b 18 19a 19b 20a 20b MeO AcO-3 AcO-19 a
bonyl group (1 732 cm–1), and double bond group (1 643 cm–1). The 1H NMR spectrum (Table 1) displayed characteristic signals of two methyls (δH 0.86 and 0.67), an acetyl (δH 2.01), and a methoxyl (δH 3.12), while its 13C NMR and DEPT data (Table 2) exhibited 23 carbon resonances includ-
1
H NMR data of compounds 1–6 in pyridine-d5 (J in Hz) 1a 2.54, m 1.49, m 1.87, m 1.68, m
2b 3.30, overlap 1.66, overlap 1.90, m 1.69, overlap
4.76, s
4.79, s
2.15, br d (12.4) 2.07, m 1.93, d (12.4) 4.86, br d (12.0) 2.21, s 2.89, dd (14.1, 9.1) 1.76, d (14.1) 3.24, d (9.1) 5.24, s 6.22, s 5.43, s 0.86, s
2.24, br d (12.2) 2.12, overlap 2.03, overlap 4.94, m 2.40, s 3.20, overlap 2.17, d (14.1) 3.28, overlap 5.46, s 6.21, s 5.38, s 0.89, s
0.67, s
0.71, s
4.03, d (8.8) 3.97, d (8.8) 3.12, s 2.01, s
4.22, d (8.6) 4.12, d (8.6)
3a 2.69, m 1.01, overlap 1.54, overlap 1.37, m 1.58, overlap 0.97, overlap 1.60, overlap 2.24, m 2.04, overlap 4.73, m 2.12, s 2.86, dd (14.1, 9.1) 1.75, d (14.1) 3.22, d (9.1) 5.21, s 6.23, s 5.43, s 0.93, s 4.00, overlap 3.87, d (11.0) 3.98, overlap
4b 3.42, m 1.15, m 1.59, overlap 1.37, m 1.60, overlap 1.00, overlap 1.68, br d (12.5) 2.30, overlap 2.14, overlap 4.80, m 2.31, s 3.18, dd (14.1, 9.0) 2.16, d (14.1) 3.26, d (9.0) 5.42, s 6.22, s 5.38, s 0.96, s 4.06, d (11.0) 3.95, d (11.0) 4.25, d (8.3) 4.09, d (8.3)
3.10, s 2.00, s 1.98, s
Recorded at 500 MHz. bRecorded at 400 MHz
– 384 –
1.99, s
5b 2.55, m 1.51, m 1.92, overlap 1.72, m
6b 3.30, m 1.67, overlap 1.93, overlap 1.71, overlap
4.80, s
4.80, s
2.19, br d (12.2) 2.08, overlap 1.94, overlap 4.95, d (11.7) 2.24, s
2.25, br d (12.1) 2.11, d (12.1) 1.99, overlap 4.97, dd (11.6, 2.9) 2.39, s
4.19, s
4.37, s
3.58, s 5.30, s 6.38, s 5.62, s 0.88, s
3.66, s 5.47, s 6.32, s 5.53, s 0.89, s
0.69, s
0.70, s
4.24, d (8.6) 4.07, d (8.6) 3.45, s 2.02, s
4.34, d (8.3) 4.08, d (8.3) 2.02, s
WU Hai-Yan, et al. / Chin J Nat Med, 2015, 13(5): 383−389
ing one methoxyl (δC 47.4), one carbonyl carbon (δC 207.9), one exocyclic double bond (δC 152.5 and 117.1), one acetoxyl (δC 170.3 and 21.0), two methyls (δC 27.1 and 21.6), five methylenes (δC 69.4, 40.7, 33.5, 28.1, and 24.6), six methines (δC 77.0, 74.7, 73.8, 61.9, 45.2, and 44.1), and four quaternary carbons (δC 106.2, 57.4, 49.9, and 37.7). Based on these data, compound 1 was initially presumed as a pentacyclic diterpenoid. The 1H–1H COSY (Fig. 2) correlations of H2-1/H2-2/H-3, H-5/H2-6/H-7, and H2-12/H-13/H-14 were observed; HMBC correlations of H-5 (δH 2.15) with C-7, C-18, and C-20, of H-9 (δH 2.21) with C-1, C-11, and C-15, and of H-13 (δH 3.24) with C-8, C-11, and C-15 suggested that compound 1 was an ent-kaurane diterpenoid [8]. The key HMBC correlations of H-9, H2-12, H2-20, and the proton of methoxy group with C-11 (δC 106.2) indicated that compound 1 was a 11, 20-epoxy-ent- kaurane diterpenoid with an acetal moiety at C-11. An acetoxy group was located at C-3, and two hydroxy groups were located at C-7 and C-14, respectively, on the basis of molecular formula and the HMBC correlations of H-3 (δH 4.76) with acetyl carbonyl carbon, of H-7 (δH 4.86) with C-8 and C-14, and of H-14 (δH 5.24) with C-9, C-12, and C-16.
Fig. 2
1
H–1H COSY (
and key ROESY (H Table 2
C)
13
C NMR data of compounds 1–6 in pyridine-d5
Position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 MeO AcO-3 a
), selected HMBC (H H) correlations of compound 1
The relative configuration of compond 1 was determined by its ROESY spectral data analysis (Fig. 2). The NOEs of H-7/H-5β and H-9β, Me-18/H-5β, and H-12β/H-9β indicated their β-orientation, while the NOEs of H-14/H-12α and H2-20/Me-19α suggested their α-orientation. The NOEs of OMe/H-12β established the β-orientation of the methoxy group. In addition, H-3 showed NOEs with Me-19α instead of correlating with H-5β and H-9β, indicating the β-orientation of the acetoxy group. Thus, compound 1 was identified as 7α,14β-dihydroxy-11β-methoxy-3β-acetoxy-11,20-epoxy-entkaur-16-en-15-one. Isowikstroemin I (2) had a molecular formula of C22H30O7 based on HREI-MS ([M]+ m/z 406.199 8, Calcd. 406.199 2). Comparison of the 1H and 13C NMR data of compound 2 with that of compound 1 (Tables 1 and 2) indicated that both compounds had identical skeletons and substitution patterns, differing only in that compound 2 had a hydroxy group at C-11 rather than a methoxy group in compound 1. This conclusion was verified by the HMBC correlations of H-9, H2-12, and H2-20 with C-11 (δC 103.6) [13]. The ROESY spectrum of compound 2 indicated that the relative configurations of the stereogenic centers in compound 2 were identical to that of compound 1. Therefore, the structure of compound 2 was defined as 7α,11β, 14β- trihydroxy-3β-acetoxy-11, 20-epoxy- ent-kaur-16-en-15-one. Isowikstroemin J (3) gave the molecular formula C23H32O7, as established from the positive-ion HREI-MS at m/z 420.214 5 ([M]+, Calcd. 420.214 8), indicating eight degrees of unsaturation. Its 1D and 2D NMR data indicated that compound 3 was a 11,20-epoxy-ent-kauranoid with two hydroxy groups at C-7 and C-14, a methoxy group at C-11, and an acetoxy group at C-19. The conclusion was verified by the HMBC
1b 33.5, t 24.6, t 77.0, d 37.7, s 45.2, d 28.1, t 74.7, d 57.4, s 61.9, d 49.9, s 106.2, s 40.7, t 44.1, d 73.8, d 207.9, s 152.5, s 117.1, t 27.1, q 21.6, q 69.4, t 47.4, q 21.0, q 170.3, s
2c 33.5, t 24.6, t 77.0, d 37.7, s 45.4, d 28.2, t 74.9, d 57.7, s 62.5, d 50.2, s 103.6, s 46.1, t 44.8, d 74.1, d 208.7, s 153.1, s 116.5, t 27.1, q 21.7, q 68.8, t
3c 39.3, t 19.7, t 35.8, t 37.7, s 50.5, d 28.9, t 74.9, d 57.5, s 62.0, d 49.9, s 106.4, s 40.7, t 44.0, d 73.6, d 208.0, s 152.5, s 117.2, t 26.8, q 67.0, t 69.7, t 47.3, q
4a 39.8, t 20.2, t 36.4, t 38.2, s 51.1, d 29.5, t 75.6, d 58.2, s 63.0, d 50.7, s 104.3, s 46.5, t 45.2, d 74.4, d 209.1, s 153.6, s 117.0, t 27.2, q 67.5, t 69.6, t
21.1, q 170.4, s
20.7, q AcO-19 Recorded at 150 MHz. bRecorded at 125 MHz. cRecorded at 100 MHz
– 385 –
21.2, q
5c 33.0, t 24.5, t 76.8, d 37.6, s 45.0, d 27.9, t 74.3, d 57.8, s 61.9, d 50.0, s 105.9, s 80.2,d 56.2, d 72.3, d 207.5, s 147.4, s 120.2, t 27.0, q 21.4, q 69.8, t 49.1, q 21.0, q 170.3, s
6c 33.1, t 24.5, t 77.0, d 37.6, s 45.2, d 28.1, t 74.6, d 58.1, s 61.8, d 50.2, s 103.9, s 83.6, d 56.1, d 72.5, d 208.2, s 148.6, s 119.0, t 27.1, q 21.5, q 68.8, t 21.0, q 170.3, s
WU Hai-Yan, et al. / Chin J Nat Med, 2015, 13(5): 383−389
correlations of H-7 (δH 4.73) with C-5, C-8, and C-14, of H-14 (δH 5.21) with C-9, C-12 and C-16, of the proton of methoxy group (δH 3.10) with C-11, and of H2-19 (δH 3.87 and 4.00) with C-3, C-18, and acetyl carbonyl carbon. The ROESY data revealed that H-7 in 3 was β-oriented, while H-14, C-19, and C-20 were α-oriented, based on the correlations of H-7/H-9β, H-14/ H-12α, H2-20/H-1α, and H2-19/H2-20α. Therefore, the structure of compound 3 was defined as 7α, 14β-dihydroxy-11β- methoxy19-acetoxy-11, 20-epoxy-ent- kaur- 16-en-15-one. Isowikstroemin K (4) was assigned the molecular formula C22H30O7 from the positive-ion HREI-MS ([M]+ m/z 406.199 7, Calcd. 406.199 2). The NMR data of compound 4 closely resembled that for the compound 3, but the downfield shifts for H-1α from δH 2.69 in compound 3 to δH 3.42 in compound 4 and for C-12 from δC 40.7 in compound 3 to δC 46.5 in compound 4, caused by a hydroxy group instead of a methoxy group at C-11 in compound 4. This assumption was confirmed by the absence of the methoxy group and by the HMBC correlations of H-9, H2-12, and H2-20 with C-11 (δC 104.3) in compound 4. A ROESY experiment confirmed that compound 4 had the same configuration as compound 3. The structure of compound 4 was thus defined as 7α, 11β, 14βtrihydroxy-19-acetoxy-11,20-epoxy-ent-kaur-16-en-15-one. The molecular formula of isowikstroemin L (5) was determined as C23H32O8 from its HREI-MS ([M]+ m/z 436.2097, calcd 436.209 7). Comparison of the NMR data of compound 5 with that of compound 1 indicated that the methylene resonance at C-12 (δC 40.7) in compound 1 was replaced by an oxymethine resonance (δC 80.2) in compound 5. The assignment was verified by the HMBC correlations of H-13 and H-14 with C-12. The ROESY correlation of H-12/H-9β suggested the β-orientation of H-12. Further analysis of ROESY spectrum indicated that the orientations of the substituents at C-3, C-7, C-11, and C-14 in compound 5 were the same as that of compound 1. Thus, compound 5 was
characterized as 7α, 12α, 14β-trihydroxy-11β-methoxy-3βacetoxy-11, 20-epoxy- ent-kaur-16-en-15-one. Isowikstroemin M (6) was found by HREI-MS ([M]+ m/z 422.193 8, Calcd. 422.194 1) to possess the molecular formula C22H30O8. The 1H and 13C NMR spectra (Tables 1 and 2) of compound 6 were similar to those of compound 5 except that the C-11 methoxy resonance in compound 5 was replaced by the hydroxy resonance in compound 6. Such an assignment was confirmed by the HMBC correlations of H-9, H-12, and H2-20 with C-11 (δC 103.9) and the absence of the methoxy moiety in compound 6. Moreover, the correlations observed in the ROESY spectrum indicated that the orientations of the substituents in compound 6 are the same as in compound 5. Therefore, the structure of compound 6 was defined as 7α,11β, 12α, 14β-tetrahydroxy3β-acetoxy-11, 20-epoxy-ent-kaur-16-en-15-one. The 11, 20-epoxy-ent-kauranoids from the genus Isodon are relative scarce. Up to date, less than 10 of this type of compounds have been isolated and elucidated [13-16]. In this work, six new 11, 20-epoxy-ent-kauranoids have been reported, which enriched this structure type. Many diterpenoids isolated from the genus Isodon exhibit significant antitumor and anti-inflammatory activities [17-18]. The α, β-unsaturated ketone in D-ring is considered to be the active center, according to previous structure-activity relationship studies [19]. All isolates obtained from I. wikstroemioides in this work had α, β-unsaturated ketone in D-ring. Therefore, all of the isolates except compound 8 (which could not be tested due to sample limitations) were evaluated for their cytotoxicity against human tumor cell lines, HL-60 (acute leukemia), SMMC-7721 (hepatic cancer), A-549 (lung cancer), MCF-7 (breast cancer), and SW-480 (colon cancer) using the MTS method [20], using cis-platin and paclitaxel as positive controls. Compounds 1, 3, and 7 exhibited significant cytotoxic activity, with IC50 values ranging from (0.84 ± 0.02) to (4.09 ± 0.34) μmol·L−1, while compounds 4 and 5 showed selective cytotoxicity (Table 3).
Table 3 In vitro cytotoxic activity (IC50 in μmol·L−1) of compounds 1–8a (mean ± SEM, n = 3) Compounds
HL-60
SMMC-7721
A-549
MCF-7
SW480
1
1.48 ± 0.32
2.93 ± 0.05
2.16 ± 0.16
2.22 ± 0.13
1.14 ± 0.08
3
0.84 ± 0.02
2.21 ± 0.01
1.27 ± 0.03
1.25 ± 0.03
1.21 ± 0.08
4
12.93 ± 0.84
9.88 ± 0.56
10.94 ± 0.12
8.78 ± 0.58
2.87 ± 0.09
5
12.69 ± 0.72
15.81 ± 0.15
28.13 ± 3.26
13.50 ± 0.54
4.23 ± 0.04
7
1.13 ± 0.30
3.23 ± 0.10
4.09 ± 0.34
2.51 ± 0.05
1.72 ± 0.08
DDPb
1.75
4.47
7.59
15.69
14.99
Paclitaxelb < 0.008 < 0.008 < 0.008 a Compounds 2 and 6 were inactive (IC50 > 40 μmol·L−1). Compound 8 was not tested. controls
Considering that NO is an essential component of the host innate immune and inflammatory response to a variety of pathogens [21], all isolates except compound 8 were tested for their inhibitory activity against NO production in LPS- stimulated RAW264.7 cells by the MTT assay. As a result, compounds 1, 3, 4, and 7 showed significant inhibitory activity against NO production (Table 4). At the highest concentra-
< 0.008 < 0.008 b DDP (cis-platin) and paclitaxel were used as positive
tion, none of the compounds tested showed any obvious cytotoxicity toward RAW264.7 cells.
Materials and Methods General experimental procedures The optical rotations were measured in MeOH with Horiba SEPA-300 (Horiba, Japan) and JASCO P-1020 polarimeters
– 386 –
WU Hai-Yan, et al. / Chin J Nat Med, 2015, 13(5): 383−389
Table 4 Inhibitory activity against NO production in LPSactivated RAW264.7 cells of compounds 1–8a (mean ± SEM, n = 3)
a
Compound
IC50 /(μmol·L−1)
1
2.05 ± 0.21
3
1.09 ± 0.06
4
8.32 ± 0.19
5
20.91 ± 2.98
7
1.85 ± 0.2
MG-132a
0.13 ± 0.01
MG-132 was used as positive controls
(Jasco, Japan). The UV spectra were recorded using a Shimadzu UV-2401A spectrophotometer (Shimadzu, Japan). The IR spectra were obtained on a Tenor 27 FT-IR spectrometer using KBr pellets (Bruker, Germany). The NMR spectra were recorded on Bruker AM-400, DRX-500, and DRX-600 spectrometers (Bruker, Germany) using TMS as the internal standard. All chemical shifts (δ) are expressed in ppm relative to the solvent signals. HREIMS was performed on an API QSTAR TOF spectrometer (Waters, America). Column chromatography (CC) was performed with silica gel (100− 200 mesh and 200−300 mesh; Qingdao Marine Chemical, Inc., Qingdao, China), Lichroprep Rp-18 gel (40–63 μm, Merck, Darmstadt, Germany), and MCI gel (75–150 μm, Mitsubishi Chemical Corporation, Tokyo, Japan). Thin-layer chromatography was performed on precoated TLC plates (200–250 μm thickness, silica gel 60 F254, Qingdao Marine Chemical, Inc. Qingdao, China), and spots were visualized by UV light (254 nm) or by spraying heated silica gel plates with 10% H2SO4 in EtOH. Preparative HPLC was performed on a Shimadzu LC-8A preparative liquid chromatograph with a Shimadzu PRC-ODS (K) column (Shimadzu, Japan). Semi-preparative HPLC was performed on an Agilent 1100 liquid chromatograph with a Zorbax SB-C18 (9.4 mm × 25 cm, 5μm) column (Agilent, America). Plant material The aerial parts of I. wikstroemioides were collected in the Ranwu District of Sichuan Province, China, in July 2011 and identified by Prof. LI Xi-Wen at the Kunming Institute of Botany. A voucher specimen (KIB 20110939) has been deposited in the Herbarium of the Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China. Extraction and isolation The air-dried and powdered aerial parts of I. Wikstroemioides (7.5 kg) were extracted with 70% aqueous acetone (14 L) three times (three days each) at room temperature and filtered. The filtrates were evaporated under reduced pressure and then partitioned with EtOAc. The EtOAc-soluble portion (380 g) was subjected to silica gel CC (100–200 mesh, 11 cm × 120 cm, 2 kg), eluted with CHCl3/acetone (1 : 0–0 : 1 gradient system), to give seven fractions. The fractions were individually decolorized on MCI gel and eluted with 90%
MeOH/ H2O, to yield fractions A–G. Fraction C (CHCl3/acetone, 8 : 2; 19 g), brown gum, was subjected to Rp-18 column chromatography (8 cm × 50 cm, MeOH/H2O 27 : 73 to 60 : 40 gradient) to provide three fractions, C1–C3. Fraction C3 (2 g) was separated by preparative HPLC (CH3CN/H2O 34 : 66) to afford 17 fractions (C3-1– C3-17). C3-17 (200 mg) was submitted to semi-preparative HPLC (5 µm, 9.4 mm × 250 mm, flow rate 3 mL·min−1, UV detection at λmax = 210, 254, and 280 nm, eluted with MeOH/ H2O 60 : 40, tR = 14 and 20 min) to yield compounds 1 (19 mg) and 3 (7 mg), respectively. Fraction C2 (15 g) was separated into five subfractions (C2-1–C2-5) using RP-18 CC (6 cm × 40 cm, MeOH/H2O 25 : 75 to 40 : 60 gradient). C2-5 (5 g) was separated by preparative HPLC (6 cm × 29 cm, CH3CN/H2O 34 : 66) to afford seven fractions (C2-5-1– C2-5-7). C2-5-7 (40 mg) was subjected to semi-preparative HPLC (5 µm, 9.4 mm × 250 mm, flow rate 3 mL·min−1, UV detection at λmax = 210, 254, and 280 nm, eluted with CH3CN/H2O 40 : 60, tR = 12 min) to yield compound 4 (4 mg). Compounds 2 (17 mg) and 7 (10 mg) were isolated from fraction C2-5-5 (180 mg) by semi-preparative HPLC (5 µm, 9.4 mm × 250 mm, flow rate 3 mL·min−1, UV detection at λmax = 210, 254, and 280 nm, eluted with CH3CN/H2O 32 : 68, tR = 8 and 23 min, respectively). C2-4 (9 g) was subjected to Rp-18 CC (6 cm × 40 cm, CH3CN/H2O 35 : 65) to afford two fractions (C2-4- 1–C2-4-2). C2-4-2 (1.3 g) was applied to preparative HPLC (6 cm × 29 cm, CH3CN/H2O 30 : 70), and then by semi- preparative HPLC (5 µm, 9.4 mm × 250 mm, flow rate 3 mL·min−1, UV detection at λmax = 210, 254, and 280 nm, eluted with MeOH/H2O 58 : 42, tR = 16 min) to yield 8 (2 mg). Fraction D (CHCl3/acetone, 7 : 3; 50 g), brown gum, was subjected to silica gel CC (9 cm × 80 cm, 200–300 mesh, 1 kg), and eluted with CHCl3/MeOH (80 : 1) to afford seven fractions (D1–D7). D1 (870 mg) was separated by preparative HPLC (6 cm × 29 cm, CH3CN/H2O 34 : 66) into five fractions D1-1–D1-5; D1-2 (120 mg) was submitted to semi-preparative HPLC (5 µm, 9.4 mm × 250 mm, flow rate 3 mL·min−1, UV detection at λmax = 210, 254, and 280 nm, eluted with CH3CN/H2O 32 : 68) and then preparative TLC to obtain compounds 5 (11 mg) and 6 (9 mg). Isowikstroemin H (1): White amorphous powder; [α] 26 : D –76 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 231 (3.90), 196 (3.59) nm; IR (KBr) νmax 3 425, 2 941, 1 732, 1 643, 1 246, 1 094 cm–1; 1H and 13C NMR data, see Tables 1 and 2; positiveion ESIMS: m/z 443 [M + Na]+ (100); positive-ion HREI- MS [M]+ m/z 420.214 7 (Calcd. for C23H32O7, 420.214 8). Isowikstroemin I (2): White amorphous powder; [α] 26 : D –65 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 231 (3.85), 197 (3.57) nm; IR (KBr) νmax 3 423, 2 939, 1 729, 1 643, 1 245, 1 059 cm–1; 1H and 13C NMR data, see Tables 1 and 2; positive-ion ESI-MS: m/z 429 [M + Na]+ (100); positiveion HREI-MS [M]+ m/z 406.199 8 (Calcd. for C22H30O7, 406.199 2).
– 387 –
WU Hai-Yan, et al. / Chin J Nat Med, 2015, 13(5): 383−389
Isowikstroemin J (3): White amorphous powder; [α] 25D : –162 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 231 (3.98) nm; IR (KBr) νmax 3 427, 2 932, 1 732, 1 644, 1 242, 1 096 cm–1; 1 H and 13C NMR data, see Tables 1 and 2; positive-ion ESI-MS: m/z 443 [M + Na]+ (100); positive-ion HREI-MS [M]+ m/z 420.214 5 (Calcd. for C23H32O7, 420.214 8). Isowikstroemin K (4): White amorphous powder; [α] 26 : D –134 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 230 (3.86), 196 (3.59) nm; IR (KBr) νmax 3 396, 2 928, 1 729, 1 647, 1 253, 1 035 cm–1; 1H and 13C NMR data, see Tables 1 and 2; positiveion ESI-MS: m/z 429 [M + Na]+ (100); positive-ion HREI-MS [M]+ m/z 406.199 7 (Calcd. for C22H30O7, 406.199 2). : Isowikstroemin L (5): White amorphous powder; [α] 26 D –26 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 214 (3.71) nm; IR (KBr) νmax 3 432, 2 937, 1 729, 1 634, 1 206, 1 060 cm–1; 1 H and 13C NMR data, see Tables 1 and 2; positive-ion ESI-MS: m/z 459 [M + Na]+ (100); positive-ion HREI-MS [M]+ m/z 436.209 7 (Calcd. for C23H32O8, 436.209 7). Isowikstroemin M (6): White amorphous powder; [α] 26 : D –38 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 229 (3.73), 213 (3.73) nm; IR (KBr) νmax 3 425, 2 947, 1 729, 1 646, 1 245, 1 060 cm–1; 1H and 13C NMR data, see Tables 1 and 2; positiveion ESI-MS: m/z 445 [M + Na]+ (100); positive-ion HREI-MS [M]+ m/z 422.193 8 (Calcd. for C22H30O8, 422.194 1). Macrocalyxin B (7): White amorphous powder; 1H NMR (in pyridine-d5): δ 6.24 (1H, s, H-17a), 5.44 (1H, s, H-17b), 5.20 (1H, s, H-14α), 4.88 (1H, d, J = 10.9 Hz, H-7β), 4.03 (1H, d, J = 8.6 Hz, H-20α), 3.92 (1H, d, J = 8.6 Hz, H-20α), 3.23 (1H, d, J = 8.9 Hz, H-13α), 2.88 (1H, dd, J = 14.1, 9.1 Hz, H-12a), 2.66 (1H, m, H-1a), 2.17 (1H, s, H-9β), 2.14–2.04 (2H, overlapped, H-5β, H-6a), 1.77 (1H, d, J = 14.1 Hz, H-12b), 1.65 (1H, br d, J = 10.9 Hz, H-6b), 1.57–1.45 (2H, overlapped, H-2a, H-2b), 1.36 (1H, m, H-3a), 1.07–1.01 (2H, overlapped, H-3b, H-1b), 0.92 (3H, s, H3-19); 13C NMR (in pyridine-d5): δ 38.1 (C-1), 18.7 (C-2), 31.7 (C-3), 50.5 (C-4), 43.2 (C-5), 31.2 (C-6), 73.7 (C-7), 57.4 (C-8), 61.4 (C-9), 48.6 (C-10), 106.3 (C-11), 40.7 (C-12), 44.0 (C-13), 73.7 (C-14), 207.6 (C-15), 152.3 (C-16), 117.5 (C-17), 205.2 (C-18), 13.6 (C-19), 69.4 (C-20). All data above were in agreement with that of macrocalyxin B [13]. Pseudoirroratin A (8): White amorphous powder; 1H NMR (in pyridine-d5): δ 6.22 (1H, s, H-17a), 5.43 (1H, s, H-14α), 5.38 (1H, s, H-17b), 4.84 (1H, d, J = 12.1 Hz, H-7β), 4.16 (1H, d, J = 8.6 Hz, H-20α), 4.06 (1H, d, J = 8.6 Hz, H-20α), 3.37 (1H, m, H-1a), 3.27 (1H, d, J = 8.9 Hz, H-13α), 3.20 (1H, dd, J = 14.1, 9.1 Hz, H-12a), 2.29 (1H, s, H-9β), 2.20–2.13 (2H, overlapped, H-12b, H-6a), 1.96 (1H, br q, H-6b), 1.58–1.48 (2H, overlapped, H-2a, H-5β), 1.38 (1H, m, H-2b), 1.22 (1H, m, H-3a), 1.16–1.09 (2H, overlapped, H-3b, H-1b), 0.81 (3H, s, H3-18), 0.63 (3H, s, H3-19); 13C NMR (in pyridine-d5): δ 39.6 (C-1), 20.6 (C-2), 41.8 (C-3), 34.5 (C-4),
51.5 (C-5), 29.2 (C-6), 75.3 (C-7), 58.2 (C-8), 63.0 (C-9), 50.8 (C-10), 104.0 (C-11), 46.7 (C-12), 45.3 (C-13), 74.5 (C-14), 209.2 (C-15), 153.6 (C-16), 116.9 (C-17), 32.8 (C-18), 21.7 (C-19), 69.6 (C-20). All data above were in agreement with that of pseudoirroratin A [14]. Cytotoxicity assay The MTS colorimetric assay was used to evaluate each compound’s activity against cancer cells. The following five human tumor cell lines were used: A549 lung cancer cell line, HL-60 human myeloid leukemia cell line, MCF-7 breast cancer cell line, SMMC-7721 human hepatocarcinoma cell line, and SW-480 human pancreatic carcinoma cell line. A549, HL-60, and SMMC-7721 cells were cultured in RPMI-1640 (Hyclone, Logan, UT, USA), MCF-7 and SW-480 cells were cultured in DMEM medium (Hyclone, Logan, UT, USA). All cells supplemented with 10% fetal bovine serum (Hyclone) at 37 °C in a humidified atmosphere with 5% CO2. The cell viability was assessed by conducting colorimetric measurements of the amount of insoluble formazan formed in living cells based on the reduction of 3-(4, 5-dimethylthiazol-2-yl)5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) (Promega) [20]. Briefly, 100 μL of suspended adherent cells (1 × 105 cells/mL) was seeded into each well of a 96-well cell culture plate and allowed to adhere for 12 h before drug treatment. Each tumor cell line was exposed to each of the test compounds at concentrations of 0.064, 0.32, 1.6, 8, and 40 μmol·L−1 in triplicate for 48 h; cis-platin (concentrations of 0.064, 0.32, 1.6, 8, and 40 μmol·L−1) (Sigma) was used as a positive control. After the 48 h incubation, MTS was added to each well, and the incubation was continued for 4 h at 37 °C. The cells were lysed with 100 μL of 20% SDS− 50% DMF after removal of the medium. The optical density of the lysate was measured at 490 nm with a microtiter plate reader (Bio-Rad 680, Thermo, America). The IC50 value of each compound was calculated using Reed and Muench’s method [22]. Analysis of nitric oxide production in RAW264.7 macrophages The RAW264.7 cells were seeded in 96-well cell culture plates (2 × 105 cells/well) and then treated with each of the test compounds at concentrations of 0.04, 0.2, 1, 5, and 25 μmol·L−1, followed by the stimulation with LPS (1 μg·mL−1) (Sigma) for 18 h. The NO production in the supernatant was assessed by Griess reagents (Sigma). The absorbance at 550 nm was measured with a 2104 Envision Multilabel plate reader (Perkin-Elmer Life Sciences, Inc., Boston, MA, USA). MG-132 (Sigma) was used as a positive control [23]. The viability of RAW264.7 cells was evaluated by the MTT assay [20] simultaneously to exclude the interference of the cytotoxicity of the test compounds. The absorbance was read at 595 nm.
References [1]
– 388 –
Flora Reipublicae Popularis Sinicae Tomus [M]. Beijing: Science Press, 1977: 428.
WU Hai-Yan, et al. / Chin J Nat Med, 2015, 13(5): 383−389
[2] [3] [4]
[5]
[6] [7]
[8]
[9]
[10] [11]
[12]
[13]
Flora Yunnanica Tomus [M]. Beijing: Science Press, 1977: 758. The Pharmacopoecia of People’s Republic of China (1977) [S]. Ding CY, Zhang YS, Zhou J, et al. Novel nitrogen-enriched oridonin analogues with thiazole-fused A-ring: protecting groupfree synthesis, enhanced anticancer profile, and improved aqueous solubility [J]. J Med Chem, 2013, 56(12): 5048-5058. Lu P, Gu ZH, Zakarian A. Total synthesis of maoecrystal V: early-stage C–H functionalization and lactone assembly by radical cyclization [J]. J Am Chem Soc, 2013, 135(39): 14552-14555. Zhao W, Pu JX, Sun HD, et al. Cytotoxic diterpenoids from Isodon adenolomus [J]. Chin J Nat Med, 2011, 9(4): 253-258. Liu X, Xue YB, Sun HD, et al. Three new ent-kaurane diterpenoids from Isodon rubescens and their cytotoxicity [J]. Chin J Nat Med, 2012, 10(6): 464-470. Sun HD, Huang SX, Han QB. Diterpenoids from Isodon species and their biological activities [J]. Nat Prod Rep, 2006, 23: 673-698. Delectis Florae Reipublicae Popularis Sinicae Agendae Academiae Sinicae Edita. Flora Reipublicae Popularis Sinica [M]. Beijing: Science Press, 1977, 66: 454. Wu SH, Zhang HJ, Sun HD, et al. Diterpenoids from Isodon wikstroemioides [J]. Phytochemistry, 1993, 34(4): 1099-1102. Wu HY, Pu JX, Sun HD, et al. Cytotoxic and anti-inflammatory ent-Kaurane diterpenoids from Isodon Wikstroemioides [J]. Fitoterapia, 2014, 98: 192-198. Wu HY, Pu JX, Sun HD, et al. Cytotoxic ent-kaurane diterpenoids from Isodon wikstroemioides [J]. J Nat Prod, 2014, 77(4): 931-941. Wang XR, Wang HP, Fujita T, et al. Structures of macrocalyxin B, F, G, and H, and maoyerabdosin from Isodon macrocalyx [J]. Phytochemistry, 1995, 38(4): 921-926.
[14] Zhang HJ, Sun HD, Fong HHS, et al. Pseudoirroratin A, a new cytotoxic ent-kaurene diterpene from Isodon pseudoirrorata [J]. J Nat Prod, 2002, 65(2): 215-217. [15] Liu X, Wu JZ, Sun HD, et al. Three new 11, 20-epoxyent-kauranoids from Isodon rubescens [J]. Archi Pharma Res, 2012, 35(12): 2147-2151. [16] Zhan R, Pu JX, Sun HD, et al. ent-Atisane and ent-kaurane diterpenoids from Isodon rosthornii [J]. Fitoterapia, 2013, 88: 76-81. [17] Lou HY, Zhang XM, Gao L. In vitro and in vivo antitumor activity of oridonin nanosuspension [J]. Inter J Pharma, 2009, 379(1): 181-186. [18] Li L, Sun HD, Leung PS, et al. Eriocalyxin B-induced apoptosis in pancreatic adenocarcinoma cells through thiol-containing antioxidant systems and downstream signaling pathways [J]. Curr Mol Med, 2014, 14(5): 673-689. [19] Fujita E, Nagao Y, Kuroda H, et al. The antitumor and antibacterial activity of the Isodon diterpenoids [J]. Chem Pharm Bull, 1976, 24(9): 2118-2127. [20] Monks A, Scudiero D, Vistica D, et al. Feasibility of a high-flux anticancer drug screen using a diverse panel of cultured human tumor cell lines [J]. J Natl Cancer Inst 1991, 83(11): 757-766. [21] McCartney-Francis NL, Song X, Wahl SM, et al. Selective inhibition of inducible nitric oxide synthase exacerbates erosive joint disease [J]. J Immunol 2001, 166(4): 2734-2740. [22] Reed LJ, Muench H. A simple method of estimating fifty percent endpoints [J]. Am J Hyg, 1938, 27(3): 493-497. [23] Fan JT, Su J, Peng YM, et al. Rubiyunnanins C-H, cytotoxic cyclic hexapeptides from Rubia yunnanensis inhibiting nitric oxide production and NF-κB activation[J]. Bioorg Med Chem, 2010, 18(23): 8226-8234.
Cite this article as: WU Hai-Yan, WANG Wei-Guang, DU Xue, YANG Jin, PU Jian-Xin, SUN Han-Dong. Six new cytotoxic and anti-inflammatory 11, 20-epoxy-ent-kaurane diterpenoids from Isodon wikstroemioides [J]. Chinese Journal of Natural Medicines, 2015, 13(5): 383-389.
– 389 –
Chinese Journal of Natural Medicines
Six new cytotoxic and anti-inflammatory 11, 20-epoxy-entkaurane diterpenoids from Isodon wikstroemioides WU Hai-Yan1, 2, WANG Wei-Guang1, DU Xue1, YANG Jin1, 2, PU Jian-Xin1*, SUN Han-Dong1* 1
State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of
Sciences, Kunming 650201, China; 2
University of Chinese Academy of Sciences, Beijing 100049, China Available online 20 May 2015
[ABSTRACT] The present study was designed to determine the chemical constituents of EtOAc extracts of the aerial parts of Isodon wikstroemioides. Compounds 1–8 were isolated and purified by normal-phase silica gel and reversed-phase C18 silica gel column chromatography and HPLC. Their structures were elucidated by extensive spectroscopic methods. Most of them were evaluated for their in vitro cytotoxicity against human cancer HL-60, SMMC-7721, A-549, MCF-7, and SW-480 cells and their inhibitory activity against nitric oxide (NO) production in LPS-activated RAW264.7 macrophages. Among the eight 11, 20-epoxy-ent-kauranoids isolated, compounds 1–6 (isowikstroemins H–M) were new diterpenoids. Compounds 1, 3, and 7 exhibited significant cytotoxicity with IC50 values ranging from (0.84 ± 0.02) to (4.09 ± 0.34) μmol·L−1, while compounds 4 and 5 showed selective cytotoxicity. In addition, compounds 1, 3, 4, and 7 exhibited inhibitory activity against nitric oxide (NO) production in LPS-activated RAW264.7 macrophages. These results provide a basis for future development of these compounds as anti-cancer and anti-inflammatory agents. [KEY WORDS] Isodon wikstroemioides; Isowikstroemins H–M; Cytotoxicity; Anti-inflammatory activity
[CLC Number] R284
[Document code] A
[Article ID] 2095-6975(2015)05-0383-07
Introduction Isodon is a cosmopolitan and important genus of the Lamiaceae family [1-2]. The Isodon species have long been used in traditional Chinese medicines [3]. The ent-kaurane diterpenoids, as the major secondary metabolites of this genus, have attracted considerable attention due to their diverse structures and interesting biological properties [4-7]. Over the past 30 years, more than 60 Isodon species have been investigated [8], and a large number of ent-kaurane diterpenoids have been isolated
[Received on] 07-Nov.-2014 [Research funding] This work was supported financially by the National Natural Science Foundation of China (Nos. 21322204 and 81172939), the NSFC-Joint Foundation of Yunnan Province (No. U1302223), the reservation-talent project of Yunnan Province (No. 2011CI043), and the West Light Foundation of the Chinese Academy of Sciences (PU JX). [*Corresponding author] Tel: 86-871-65223251, E-mail: [email protected] (PU Jian-Xin), [email protected] (SUN Han-Dong) These authors have no conflict of interest to declare. Copyright © 2015, China Pharmaceutical University. Published by Elsevier B.V. All rights reserved
and characterized by our group [8]. Isodon wikstroemioides (Hand.–Mazz.) H. Hara (Lamiaceae), a perennial herb, is primarily distributed in the northwestern regions of Yunnan Province and the western Sichuan regions in China [9]. Previous studies on this herb have led to isolation of 7, 20-epoxy-ent-kauranoids [10-11], C-20-non- oxy[12] , and C-20-oxygenatedgenated-entkauranoids [12] non-epoxy-ent-kauranoids . In our continuing research with the aim at discovering new diterpenoids with diverse structures and bioactivities, six new 11, 20-epoxy-ent- kauranoids, isowikstroemins H–M (1–6), along with two known analogues, macrocalyxin B (7) [13] and pseudoirroratin A (8) [14], have been isolated from I. wikstroemioides. In the present report, the isolation and structure elucidation of these diterpenoids are described as alongside with the cytotoxicity evaluation against five human tumor cell lines and their inhibitory activity against LPS-induced NO production in RAW 264.7 macrophages.
Results and Discussion A 70% aqueous acetone extract of the air-dried and powdered aerial parts of I. wikstroemioides (7.5 kg) was partitioned between EtOAc and H2O. The EtOAc-soluble portion
– 383 –
WU Hai-Yan, et al. / Chin J Nat Med, 2015, 13(5): 383−389
(380 g) was subjected to repeated column chromatography and semi-preparative HPLC to afford six new ent-kauranoids, isowikstroemins H–M (1–6), along with two
known analogues, macrocalyxin B (7) and pseudoirroratin A (8) (Fig. 1).
Fig. 1 Chemical structures of compounds 1–8
Isowikstroemin H (1) was obtained as white amorphous powder and gave a HREI-MS ion peak at m/z 420.214 7 ([M]+, calcd 420.214 8), which corresponded to a molecular formula of C23H32O7 with eight degrees of unsaturation. The IR spectrum indicated absorption bands for hydroxy group (3 425 cm–1), carTable 1 Position 1a 1b 2a 2b 3a 3b 5 6a 6b 7 9 12a 12b 13 14 17a 17b 18 19a 19b 20a 20b MeO AcO-3 AcO-19 a
bonyl group (1 732 cm–1), and double bond group (1 643 cm–1). The 1H NMR spectrum (Table 1) displayed characteristic signals of two methyls (δH 0.86 and 0.67), an acetyl (δH 2.01), and a methoxyl (δH 3.12), while its 13C NMR and DEPT data (Table 2) exhibited 23 carbon resonances includ-
1
H NMR data of compounds 1–6 in pyridine-d5 (J in Hz) 1a 2.54, m 1.49, m 1.87, m 1.68, m
2b 3.30, overlap 1.66, overlap 1.90, m 1.69, overlap
4.76, s
4.79, s
2.15, br d (12.4) 2.07, m 1.93, d (12.4) 4.86, br d (12.0) 2.21, s 2.89, dd (14.1, 9.1) 1.76, d (14.1) 3.24, d (9.1) 5.24, s 6.22, s 5.43, s 0.86, s
2.24, br d (12.2) 2.12, overlap 2.03, overlap 4.94, m 2.40, s 3.20, overlap 2.17, d (14.1) 3.28, overlap 5.46, s 6.21, s 5.38, s 0.89, s
0.67, s
0.71, s
4.03, d (8.8) 3.97, d (8.8) 3.12, s 2.01, s
4.22, d (8.6) 4.12, d (8.6)
3a 2.69, m 1.01, overlap 1.54, overlap 1.37, m 1.58, overlap 0.97, overlap 1.60, overlap 2.24, m 2.04, overlap 4.73, m 2.12, s 2.86, dd (14.1, 9.1) 1.75, d (14.1) 3.22, d (9.1) 5.21, s 6.23, s 5.43, s 0.93, s 4.00, overlap 3.87, d (11.0) 3.98, overlap
4b 3.42, m 1.15, m 1.59, overlap 1.37, m 1.60, overlap 1.00, overlap 1.68, br d (12.5) 2.30, overlap 2.14, overlap 4.80, m 2.31, s 3.18, dd (14.1, 9.0) 2.16, d (14.1) 3.26, d (9.0) 5.42, s 6.22, s 5.38, s 0.96, s 4.06, d (11.0) 3.95, d (11.0) 4.25, d (8.3) 4.09, d (8.3)
3.10, s 2.00, s 1.98, s
Recorded at 500 MHz. bRecorded at 400 MHz
– 384 –
1.99, s
5b 2.55, m 1.51, m 1.92, overlap 1.72, m
6b 3.30, m 1.67, overlap 1.93, overlap 1.71, overlap
4.80, s
4.80, s
2.19, br d (12.2) 2.08, overlap 1.94, overlap 4.95, d (11.7) 2.24, s
2.25, br d (12.1) 2.11, d (12.1) 1.99, overlap 4.97, dd (11.6, 2.9) 2.39, s
4.19, s
4.37, s
3.58, s 5.30, s 6.38, s 5.62, s 0.88, s
3.66, s 5.47, s 6.32, s 5.53, s 0.89, s
0.69, s
0.70, s
4.24, d (8.6) 4.07, d (8.6) 3.45, s 2.02, s
4.34, d (8.3) 4.08, d (8.3) 2.02, s
WU Hai-Yan, et al. / Chin J Nat Med, 2015, 13(5): 383−389
ing one methoxyl (δC 47.4), one carbonyl carbon (δC 207.9), one exocyclic double bond (δC 152.5 and 117.1), one acetoxyl (δC 170.3 and 21.0), two methyls (δC 27.1 and 21.6), five methylenes (δC 69.4, 40.7, 33.5, 28.1, and 24.6), six methines (δC 77.0, 74.7, 73.8, 61.9, 45.2, and 44.1), and four quaternary carbons (δC 106.2, 57.4, 49.9, and 37.7). Based on these data, compound 1 was initially presumed as a pentacyclic diterpenoid. The 1H–1H COSY (Fig. 2) correlations of H2-1/H2-2/H-3, H-5/H2-6/H-7, and H2-12/H-13/H-14 were observed; HMBC correlations of H-5 (δH 2.15) with C-7, C-18, and C-20, of H-9 (δH 2.21) with C-1, C-11, and C-15, and of H-13 (δH 3.24) with C-8, C-11, and C-15 suggested that compound 1 was an ent-kaurane diterpenoid [8]. The key HMBC correlations of H-9, H2-12, H2-20, and the proton of methoxy group with C-11 (δC 106.2) indicated that compound 1 was a 11, 20-epoxy-ent- kaurane diterpenoid with an acetal moiety at C-11. An acetoxy group was located at C-3, and two hydroxy groups were located at C-7 and C-14, respectively, on the basis of molecular formula and the HMBC correlations of H-3 (δH 4.76) with acetyl carbonyl carbon, of H-7 (δH 4.86) with C-8 and C-14, and of H-14 (δH 5.24) with C-9, C-12, and C-16.
Fig. 2
1
H–1H COSY (
and key ROESY (H Table 2
C)
13
C NMR data of compounds 1–6 in pyridine-d5
Position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 MeO AcO-3 a
), selected HMBC (H H) correlations of compound 1
The relative configuration of compond 1 was determined by its ROESY spectral data analysis (Fig. 2). The NOEs of H-7/H-5β and H-9β, Me-18/H-5β, and H-12β/H-9β indicated their β-orientation, while the NOEs of H-14/H-12α and H2-20/Me-19α suggested their α-orientation. The NOEs of OMe/H-12β established the β-orientation of the methoxy group. In addition, H-3 showed NOEs with Me-19α instead of correlating with H-5β and H-9β, indicating the β-orientation of the acetoxy group. Thus, compound 1 was identified as 7α,14β-dihydroxy-11β-methoxy-3β-acetoxy-11,20-epoxy-entkaur-16-en-15-one. Isowikstroemin I (2) had a molecular formula of C22H30O7 based on HREI-MS ([M]+ m/z 406.199 8, Calcd. 406.199 2). Comparison of the 1H and 13C NMR data of compound 2 with that of compound 1 (Tables 1 and 2) indicated that both compounds had identical skeletons and substitution patterns, differing only in that compound 2 had a hydroxy group at C-11 rather than a methoxy group in compound 1. This conclusion was verified by the HMBC correlations of H-9, H2-12, and H2-20 with C-11 (δC 103.6) [13]. The ROESY spectrum of compound 2 indicated that the relative configurations of the stereogenic centers in compound 2 were identical to that of compound 1. Therefore, the structure of compound 2 was defined as 7α,11β, 14β- trihydroxy-3β-acetoxy-11, 20-epoxy- ent-kaur-16-en-15-one. Isowikstroemin J (3) gave the molecular formula C23H32O7, as established from the positive-ion HREI-MS at m/z 420.214 5 ([M]+, Calcd. 420.214 8), indicating eight degrees of unsaturation. Its 1D and 2D NMR data indicated that compound 3 was a 11,20-epoxy-ent-kauranoid with two hydroxy groups at C-7 and C-14, a methoxy group at C-11, and an acetoxy group at C-19. The conclusion was verified by the HMBC
1b 33.5, t 24.6, t 77.0, d 37.7, s 45.2, d 28.1, t 74.7, d 57.4, s 61.9, d 49.9, s 106.2, s 40.7, t 44.1, d 73.8, d 207.9, s 152.5, s 117.1, t 27.1, q 21.6, q 69.4, t 47.4, q 21.0, q 170.3, s
2c 33.5, t 24.6, t 77.0, d 37.7, s 45.4, d 28.2, t 74.9, d 57.7, s 62.5, d 50.2, s 103.6, s 46.1, t 44.8, d 74.1, d 208.7, s 153.1, s 116.5, t 27.1, q 21.7, q 68.8, t
3c 39.3, t 19.7, t 35.8, t 37.7, s 50.5, d 28.9, t 74.9, d 57.5, s 62.0, d 49.9, s 106.4, s 40.7, t 44.0, d 73.6, d 208.0, s 152.5, s 117.2, t 26.8, q 67.0, t 69.7, t 47.3, q
4a 39.8, t 20.2, t 36.4, t 38.2, s 51.1, d 29.5, t 75.6, d 58.2, s 63.0, d 50.7, s 104.3, s 46.5, t 45.2, d 74.4, d 209.1, s 153.6, s 117.0, t 27.2, q 67.5, t 69.6, t
21.1, q 170.4, s
20.7, q AcO-19 Recorded at 150 MHz. bRecorded at 125 MHz. cRecorded at 100 MHz
– 385 –
21.2, q
5c 33.0, t 24.5, t 76.8, d 37.6, s 45.0, d 27.9, t 74.3, d 57.8, s 61.9, d 50.0, s 105.9, s 80.2,d 56.2, d 72.3, d 207.5, s 147.4, s 120.2, t 27.0, q 21.4, q 69.8, t 49.1, q 21.0, q 170.3, s
6c 33.1, t 24.5, t 77.0, d 37.6, s 45.2, d 28.1, t 74.6, d 58.1, s 61.8, d 50.2, s 103.9, s 83.6, d 56.1, d 72.5, d 208.2, s 148.6, s 119.0, t 27.1, q 21.5, q 68.8, t 21.0, q 170.3, s
WU Hai-Yan, et al. / Chin J Nat Med, 2015, 13(5): 383−389
correlations of H-7 (δH 4.73) with C-5, C-8, and C-14, of H-14 (δH 5.21) with C-9, C-12 and C-16, of the proton of methoxy group (δH 3.10) with C-11, and of H2-19 (δH 3.87 and 4.00) with C-3, C-18, and acetyl carbonyl carbon. The ROESY data revealed that H-7 in 3 was β-oriented, while H-14, C-19, and C-20 were α-oriented, based on the correlations of H-7/H-9β, H-14/ H-12α, H2-20/H-1α, and H2-19/H2-20α. Therefore, the structure of compound 3 was defined as 7α, 14β-dihydroxy-11β- methoxy19-acetoxy-11, 20-epoxy-ent- kaur- 16-en-15-one. Isowikstroemin K (4) was assigned the molecular formula C22H30O7 from the positive-ion HREI-MS ([M]+ m/z 406.199 7, Calcd. 406.199 2). The NMR data of compound 4 closely resembled that for the compound 3, but the downfield shifts for H-1α from δH 2.69 in compound 3 to δH 3.42 in compound 4 and for C-12 from δC 40.7 in compound 3 to δC 46.5 in compound 4, caused by a hydroxy group instead of a methoxy group at C-11 in compound 4. This assumption was confirmed by the absence of the methoxy group and by the HMBC correlations of H-9, H2-12, and H2-20 with C-11 (δC 104.3) in compound 4. A ROESY experiment confirmed that compound 4 had the same configuration as compound 3. The structure of compound 4 was thus defined as 7α, 11β, 14βtrihydroxy-19-acetoxy-11,20-epoxy-ent-kaur-16-en-15-one. The molecular formula of isowikstroemin L (5) was determined as C23H32O8 from its HREI-MS ([M]+ m/z 436.2097, calcd 436.209 7). Comparison of the NMR data of compound 5 with that of compound 1 indicated that the methylene resonance at C-12 (δC 40.7) in compound 1 was replaced by an oxymethine resonance (δC 80.2) in compound 5. The assignment was verified by the HMBC correlations of H-13 and H-14 with C-12. The ROESY correlation of H-12/H-9β suggested the β-orientation of H-12. Further analysis of ROESY spectrum indicated that the orientations of the substituents at C-3, C-7, C-11, and C-14 in compound 5 were the same as that of compound 1. Thus, compound 5 was
characterized as 7α, 12α, 14β-trihydroxy-11β-methoxy-3βacetoxy-11, 20-epoxy- ent-kaur-16-en-15-one. Isowikstroemin M (6) was found by HREI-MS ([M]+ m/z 422.193 8, Calcd. 422.194 1) to possess the molecular formula C22H30O8. The 1H and 13C NMR spectra (Tables 1 and 2) of compound 6 were similar to those of compound 5 except that the C-11 methoxy resonance in compound 5 was replaced by the hydroxy resonance in compound 6. Such an assignment was confirmed by the HMBC correlations of H-9, H-12, and H2-20 with C-11 (δC 103.9) and the absence of the methoxy moiety in compound 6. Moreover, the correlations observed in the ROESY spectrum indicated that the orientations of the substituents in compound 6 are the same as in compound 5. Therefore, the structure of compound 6 was defined as 7α,11β, 12α, 14β-tetrahydroxy3β-acetoxy-11, 20-epoxy-ent-kaur-16-en-15-one. The 11, 20-epoxy-ent-kauranoids from the genus Isodon are relative scarce. Up to date, less than 10 of this type of compounds have been isolated and elucidated [13-16]. In this work, six new 11, 20-epoxy-ent-kauranoids have been reported, which enriched this structure type. Many diterpenoids isolated from the genus Isodon exhibit significant antitumor and anti-inflammatory activities [17-18]. The α, β-unsaturated ketone in D-ring is considered to be the active center, according to previous structure-activity relationship studies [19]. All isolates obtained from I. wikstroemioides in this work had α, β-unsaturated ketone in D-ring. Therefore, all of the isolates except compound 8 (which could not be tested due to sample limitations) were evaluated for their cytotoxicity against human tumor cell lines, HL-60 (acute leukemia), SMMC-7721 (hepatic cancer), A-549 (lung cancer), MCF-7 (breast cancer), and SW-480 (colon cancer) using the MTS method [20], using cis-platin and paclitaxel as positive controls. Compounds 1, 3, and 7 exhibited significant cytotoxic activity, with IC50 values ranging from (0.84 ± 0.02) to (4.09 ± 0.34) μmol·L−1, while compounds 4 and 5 showed selective cytotoxicity (Table 3).
Table 3 In vitro cytotoxic activity (IC50 in μmol·L−1) of compounds 1–8a (mean ± SEM, n = 3) Compounds
HL-60
SMMC-7721
A-549
MCF-7
SW480
1
1.48 ± 0.32
2.93 ± 0.05
2.16 ± 0.16
2.22 ± 0.13
1.14 ± 0.08
3
0.84 ± 0.02
2.21 ± 0.01
1.27 ± 0.03
1.25 ± 0.03
1.21 ± 0.08
4
12.93 ± 0.84
9.88 ± 0.56
10.94 ± 0.12
8.78 ± 0.58
2.87 ± 0.09
5
12.69 ± 0.72
15.81 ± 0.15
28.13 ± 3.26
13.50 ± 0.54
4.23 ± 0.04
7
1.13 ± 0.30
3.23 ± 0.10
4.09 ± 0.34
2.51 ± 0.05
1.72 ± 0.08
DDPb
1.75
4.47
7.59
15.69
14.99
Paclitaxelb < 0.008 < 0.008 < 0.008 a Compounds 2 and 6 were inactive (IC50 > 40 μmol·L−1). Compound 8 was not tested. controls
Considering that NO is an essential component of the host innate immune and inflammatory response to a variety of pathogens [21], all isolates except compound 8 were tested for their inhibitory activity against NO production in LPS- stimulated RAW264.7 cells by the MTT assay. As a result, compounds 1, 3, 4, and 7 showed significant inhibitory activity against NO production (Table 4). At the highest concentra-
< 0.008 < 0.008 b DDP (cis-platin) and paclitaxel were used as positive
tion, none of the compounds tested showed any obvious cytotoxicity toward RAW264.7 cells.
Materials and Methods General experimental procedures The optical rotations were measured in MeOH with Horiba SEPA-300 (Horiba, Japan) and JASCO P-1020 polarimeters
– 386 –
WU Hai-Yan, et al. / Chin J Nat Med, 2015, 13(5): 383−389
Table 4 Inhibitory activity against NO production in LPSactivated RAW264.7 cells of compounds 1–8a (mean ± SEM, n = 3)
a
Compound
IC50 /(μmol·L−1)
1
2.05 ± 0.21
3
1.09 ± 0.06
4
8.32 ± 0.19
5
20.91 ± 2.98
7
1.85 ± 0.2
MG-132a
0.13 ± 0.01
MG-132 was used as positive controls
(Jasco, Japan). The UV spectra were recorded using a Shimadzu UV-2401A spectrophotometer (Shimadzu, Japan). The IR spectra were obtained on a Tenor 27 FT-IR spectrometer using KBr pellets (Bruker, Germany). The NMR spectra were recorded on Bruker AM-400, DRX-500, and DRX-600 spectrometers (Bruker, Germany) using TMS as the internal standard. All chemical shifts (δ) are expressed in ppm relative to the solvent signals. HREIMS was performed on an API QSTAR TOF spectrometer (Waters, America). Column chromatography (CC) was performed with silica gel (100− 200 mesh and 200−300 mesh; Qingdao Marine Chemical, Inc., Qingdao, China), Lichroprep Rp-18 gel (40–63 μm, Merck, Darmstadt, Germany), and MCI gel (75–150 μm, Mitsubishi Chemical Corporation, Tokyo, Japan). Thin-layer chromatography was performed on precoated TLC plates (200–250 μm thickness, silica gel 60 F254, Qingdao Marine Chemical, Inc. Qingdao, China), and spots were visualized by UV light (254 nm) or by spraying heated silica gel plates with 10% H2SO4 in EtOH. Preparative HPLC was performed on a Shimadzu LC-8A preparative liquid chromatograph with a Shimadzu PRC-ODS (K) column (Shimadzu, Japan). Semi-preparative HPLC was performed on an Agilent 1100 liquid chromatograph with a Zorbax SB-C18 (9.4 mm × 25 cm, 5μm) column (Agilent, America). Plant material The aerial parts of I. wikstroemioides were collected in the Ranwu District of Sichuan Province, China, in July 2011 and identified by Prof. LI Xi-Wen at the Kunming Institute of Botany. A voucher specimen (KIB 20110939) has been deposited in the Herbarium of the Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China. Extraction and isolation The air-dried and powdered aerial parts of I. Wikstroemioides (7.5 kg) were extracted with 70% aqueous acetone (14 L) three times (three days each) at room temperature and filtered. The filtrates were evaporated under reduced pressure and then partitioned with EtOAc. The EtOAc-soluble portion (380 g) was subjected to silica gel CC (100–200 mesh, 11 cm × 120 cm, 2 kg), eluted with CHCl3/acetone (1 : 0–0 : 1 gradient system), to give seven fractions. The fractions were individually decolorized on MCI gel and eluted with 90%
MeOH/ H2O, to yield fractions A–G. Fraction C (CHCl3/acetone, 8 : 2; 19 g), brown gum, was subjected to Rp-18 column chromatography (8 cm × 50 cm, MeOH/H2O 27 : 73 to 60 : 40 gradient) to provide three fractions, C1–C3. Fraction C3 (2 g) was separated by preparative HPLC (CH3CN/H2O 34 : 66) to afford 17 fractions (C3-1– C3-17). C3-17 (200 mg) was submitted to semi-preparative HPLC (5 µm, 9.4 mm × 250 mm, flow rate 3 mL·min−1, UV detection at λmax = 210, 254, and 280 nm, eluted with MeOH/ H2O 60 : 40, tR = 14 and 20 min) to yield compounds 1 (19 mg) and 3 (7 mg), respectively. Fraction C2 (15 g) was separated into five subfractions (C2-1–C2-5) using RP-18 CC (6 cm × 40 cm, MeOH/H2O 25 : 75 to 40 : 60 gradient). C2-5 (5 g) was separated by preparative HPLC (6 cm × 29 cm, CH3CN/H2O 34 : 66) to afford seven fractions (C2-5-1– C2-5-7). C2-5-7 (40 mg) was subjected to semi-preparative HPLC (5 µm, 9.4 mm × 250 mm, flow rate 3 mL·min−1, UV detection at λmax = 210, 254, and 280 nm, eluted with CH3CN/H2O 40 : 60, tR = 12 min) to yield compound 4 (4 mg). Compounds 2 (17 mg) and 7 (10 mg) were isolated from fraction C2-5-5 (180 mg) by semi-preparative HPLC (5 µm, 9.4 mm × 250 mm, flow rate 3 mL·min−1, UV detection at λmax = 210, 254, and 280 nm, eluted with CH3CN/H2O 32 : 68, tR = 8 and 23 min, respectively). C2-4 (9 g) was subjected to Rp-18 CC (6 cm × 40 cm, CH3CN/H2O 35 : 65) to afford two fractions (C2-4- 1–C2-4-2). C2-4-2 (1.3 g) was applied to preparative HPLC (6 cm × 29 cm, CH3CN/H2O 30 : 70), and then by semi- preparative HPLC (5 µm, 9.4 mm × 250 mm, flow rate 3 mL·min−1, UV detection at λmax = 210, 254, and 280 nm, eluted with MeOH/H2O 58 : 42, tR = 16 min) to yield 8 (2 mg). Fraction D (CHCl3/acetone, 7 : 3; 50 g), brown gum, was subjected to silica gel CC (9 cm × 80 cm, 200–300 mesh, 1 kg), and eluted with CHCl3/MeOH (80 : 1) to afford seven fractions (D1–D7). D1 (870 mg) was separated by preparative HPLC (6 cm × 29 cm, CH3CN/H2O 34 : 66) into five fractions D1-1–D1-5; D1-2 (120 mg) was submitted to semi-preparative HPLC (5 µm, 9.4 mm × 250 mm, flow rate 3 mL·min−1, UV detection at λmax = 210, 254, and 280 nm, eluted with CH3CN/H2O 32 : 68) and then preparative TLC to obtain compounds 5 (11 mg) and 6 (9 mg). Isowikstroemin H (1): White amorphous powder; [α] 26 : D –76 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 231 (3.90), 196 (3.59) nm; IR (KBr) νmax 3 425, 2 941, 1 732, 1 643, 1 246, 1 094 cm–1; 1H and 13C NMR data, see Tables 1 and 2; positiveion ESIMS: m/z 443 [M + Na]+ (100); positive-ion HREI- MS [M]+ m/z 420.214 7 (Calcd. for C23H32O7, 420.214 8). Isowikstroemin I (2): White amorphous powder; [α] 26 : D –65 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 231 (3.85), 197 (3.57) nm; IR (KBr) νmax 3 423, 2 939, 1 729, 1 643, 1 245, 1 059 cm–1; 1H and 13C NMR data, see Tables 1 and 2; positive-ion ESI-MS: m/z 429 [M + Na]+ (100); positiveion HREI-MS [M]+ m/z 406.199 8 (Calcd. for C22H30O7, 406.199 2).
– 387 –
WU Hai-Yan, et al. / Chin J Nat Med, 2015, 13(5): 383−389
Isowikstroemin J (3): White amorphous powder; [α] 25D : –162 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 231 (3.98) nm; IR (KBr) νmax 3 427, 2 932, 1 732, 1 644, 1 242, 1 096 cm–1; 1 H and 13C NMR data, see Tables 1 and 2; positive-ion ESI-MS: m/z 443 [M + Na]+ (100); positive-ion HREI-MS [M]+ m/z 420.214 5 (Calcd. for C23H32O7, 420.214 8). Isowikstroemin K (4): White amorphous powder; [α] 26 : D –134 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 230 (3.86), 196 (3.59) nm; IR (KBr) νmax 3 396, 2 928, 1 729, 1 647, 1 253, 1 035 cm–1; 1H and 13C NMR data, see Tables 1 and 2; positiveion ESI-MS: m/z 429 [M + Na]+ (100); positive-ion HREI-MS [M]+ m/z 406.199 7 (Calcd. for C22H30O7, 406.199 2). : Isowikstroemin L (5): White amorphous powder; [α] 26 D –26 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 214 (3.71) nm; IR (KBr) νmax 3 432, 2 937, 1 729, 1 634, 1 206, 1 060 cm–1; 1 H and 13C NMR data, see Tables 1 and 2; positive-ion ESI-MS: m/z 459 [M + Na]+ (100); positive-ion HREI-MS [M]+ m/z 436.209 7 (Calcd. for C23H32O8, 436.209 7). Isowikstroemin M (6): White amorphous powder; [α] 26 : D –38 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 229 (3.73), 213 (3.73) nm; IR (KBr) νmax 3 425, 2 947, 1 729, 1 646, 1 245, 1 060 cm–1; 1H and 13C NMR data, see Tables 1 and 2; positiveion ESI-MS: m/z 445 [M + Na]+ (100); positive-ion HREI-MS [M]+ m/z 422.193 8 (Calcd. for C22H30O8, 422.194 1). Macrocalyxin B (7): White amorphous powder; 1H NMR (in pyridine-d5): δ 6.24 (1H, s, H-17a), 5.44 (1H, s, H-17b), 5.20 (1H, s, H-14α), 4.88 (1H, d, J = 10.9 Hz, H-7β), 4.03 (1H, d, J = 8.6 Hz, H-20α), 3.92 (1H, d, J = 8.6 Hz, H-20α), 3.23 (1H, d, J = 8.9 Hz, H-13α), 2.88 (1H, dd, J = 14.1, 9.1 Hz, H-12a), 2.66 (1H, m, H-1a), 2.17 (1H, s, H-9β), 2.14–2.04 (2H, overlapped, H-5β, H-6a), 1.77 (1H, d, J = 14.1 Hz, H-12b), 1.65 (1H, br d, J = 10.9 Hz, H-6b), 1.57–1.45 (2H, overlapped, H-2a, H-2b), 1.36 (1H, m, H-3a), 1.07–1.01 (2H, overlapped, H-3b, H-1b), 0.92 (3H, s, H3-19); 13C NMR (in pyridine-d5): δ 38.1 (C-1), 18.7 (C-2), 31.7 (C-3), 50.5 (C-4), 43.2 (C-5), 31.2 (C-6), 73.7 (C-7), 57.4 (C-8), 61.4 (C-9), 48.6 (C-10), 106.3 (C-11), 40.7 (C-12), 44.0 (C-13), 73.7 (C-14), 207.6 (C-15), 152.3 (C-16), 117.5 (C-17), 205.2 (C-18), 13.6 (C-19), 69.4 (C-20). All data above were in agreement with that of macrocalyxin B [13]. Pseudoirroratin A (8): White amorphous powder; 1H NMR (in pyridine-d5): δ 6.22 (1H, s, H-17a), 5.43 (1H, s, H-14α), 5.38 (1H, s, H-17b), 4.84 (1H, d, J = 12.1 Hz, H-7β), 4.16 (1H, d, J = 8.6 Hz, H-20α), 4.06 (1H, d, J = 8.6 Hz, H-20α), 3.37 (1H, m, H-1a), 3.27 (1H, d, J = 8.9 Hz, H-13α), 3.20 (1H, dd, J = 14.1, 9.1 Hz, H-12a), 2.29 (1H, s, H-9β), 2.20–2.13 (2H, overlapped, H-12b, H-6a), 1.96 (1H, br q, H-6b), 1.58–1.48 (2H, overlapped, H-2a, H-5β), 1.38 (1H, m, H-2b), 1.22 (1H, m, H-3a), 1.16–1.09 (2H, overlapped, H-3b, H-1b), 0.81 (3H, s, H3-18), 0.63 (3H, s, H3-19); 13C NMR (in pyridine-d5): δ 39.6 (C-1), 20.6 (C-2), 41.8 (C-3), 34.5 (C-4),
51.5 (C-5), 29.2 (C-6), 75.3 (C-7), 58.2 (C-8), 63.0 (C-9), 50.8 (C-10), 104.0 (C-11), 46.7 (C-12), 45.3 (C-13), 74.5 (C-14), 209.2 (C-15), 153.6 (C-16), 116.9 (C-17), 32.8 (C-18), 21.7 (C-19), 69.6 (C-20). All data above were in agreement with that of pseudoirroratin A [14]. Cytotoxicity assay The MTS colorimetric assay was used to evaluate each compound’s activity against cancer cells. The following five human tumor cell lines were used: A549 lung cancer cell line, HL-60 human myeloid leukemia cell line, MCF-7 breast cancer cell line, SMMC-7721 human hepatocarcinoma cell line, and SW-480 human pancreatic carcinoma cell line. A549, HL-60, and SMMC-7721 cells were cultured in RPMI-1640 (Hyclone, Logan, UT, USA), MCF-7 and SW-480 cells were cultured in DMEM medium (Hyclone, Logan, UT, USA). All cells supplemented with 10% fetal bovine serum (Hyclone) at 37 °C in a humidified atmosphere with 5% CO2. The cell viability was assessed by conducting colorimetric measurements of the amount of insoluble formazan formed in living cells based on the reduction of 3-(4, 5-dimethylthiazol-2-yl)5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) (Promega) [20]. Briefly, 100 μL of suspended adherent cells (1 × 105 cells/mL) was seeded into each well of a 96-well cell culture plate and allowed to adhere for 12 h before drug treatment. Each tumor cell line was exposed to each of the test compounds at concentrations of 0.064, 0.32, 1.6, 8, and 40 μmol·L−1 in triplicate for 48 h; cis-platin (concentrations of 0.064, 0.32, 1.6, 8, and 40 μmol·L−1) (Sigma) was used as a positive control. After the 48 h incubation, MTS was added to each well, and the incubation was continued for 4 h at 37 °C. The cells were lysed with 100 μL of 20% SDS− 50% DMF after removal of the medium. The optical density of the lysate was measured at 490 nm with a microtiter plate reader (Bio-Rad 680, Thermo, America). The IC50 value of each compound was calculated using Reed and Muench’s method [22]. Analysis of nitric oxide production in RAW264.7 macrophages The RAW264.7 cells were seeded in 96-well cell culture plates (2 × 105 cells/well) and then treated with each of the test compounds at concentrations of 0.04, 0.2, 1, 5, and 25 μmol·L−1, followed by the stimulation with LPS (1 μg·mL−1) (Sigma) for 18 h. The NO production in the supernatant was assessed by Griess reagents (Sigma). The absorbance at 550 nm was measured with a 2104 Envision Multilabel plate reader (Perkin-Elmer Life Sciences, Inc., Boston, MA, USA). MG-132 (Sigma) was used as a positive control [23]. The viability of RAW264.7 cells was evaluated by the MTT assay [20] simultaneously to exclude the interference of the cytotoxicity of the test compounds. The absorbance was read at 595 nm.
References [1]
– 388 –
Flora Reipublicae Popularis Sinicae Tomus [M]. Beijing: Science Press, 1977: 428.
WU Hai-Yan, et al. / Chin J Nat Med, 2015, 13(5): 383−389
[2] [3] [4]
[5]
[6] [7]
[8]
[9]
[10] [11]
[12]
[13]
Flora Yunnanica Tomus [M]. Beijing: Science Press, 1977: 758. The Pharmacopoecia of People’s Republic of China (1977) [S]. Ding CY, Zhang YS, Zhou J, et al. Novel nitrogen-enriched oridonin analogues with thiazole-fused A-ring: protecting groupfree synthesis, enhanced anticancer profile, and improved aqueous solubility [J]. J Med Chem, 2013, 56(12): 5048-5058. Lu P, Gu ZH, Zakarian A. Total synthesis of maoecrystal V: early-stage C–H functionalization and lactone assembly by radical cyclization [J]. J Am Chem Soc, 2013, 135(39): 14552-14555. Zhao W, Pu JX, Sun HD, et al. Cytotoxic diterpenoids from Isodon adenolomus [J]. Chin J Nat Med, 2011, 9(4): 253-258. Liu X, Xue YB, Sun HD, et al. Three new ent-kaurane diterpenoids from Isodon rubescens and their cytotoxicity [J]. Chin J Nat Med, 2012, 10(6): 464-470. Sun HD, Huang SX, Han QB. Diterpenoids from Isodon species and their biological activities [J]. Nat Prod Rep, 2006, 23: 673-698. Delectis Florae Reipublicae Popularis Sinicae Agendae Academiae Sinicae Edita. Flora Reipublicae Popularis Sinica [M]. Beijing: Science Press, 1977, 66: 454. Wu SH, Zhang HJ, Sun HD, et al. Diterpenoids from Isodon wikstroemioides [J]. Phytochemistry, 1993, 34(4): 1099-1102. Wu HY, Pu JX, Sun HD, et al. Cytotoxic and anti-inflammatory ent-Kaurane diterpenoids from Isodon Wikstroemioides [J]. Fitoterapia, 2014, 98: 192-198. Wu HY, Pu JX, Sun HD, et al. Cytotoxic ent-kaurane diterpenoids from Isodon wikstroemioides [J]. J Nat Prod, 2014, 77(4): 931-941. Wang XR, Wang HP, Fujita T, et al. Structures of macrocalyxin B, F, G, and H, and maoyerabdosin from Isodon macrocalyx [J]. Phytochemistry, 1995, 38(4): 921-926.
[14] Zhang HJ, Sun HD, Fong HHS, et al. Pseudoirroratin A, a new cytotoxic ent-kaurene diterpene from Isodon pseudoirrorata [J]. J Nat Prod, 2002, 65(2): 215-217. [15] Liu X, Wu JZ, Sun HD, et al. Three new 11, 20-epoxyent-kauranoids from Isodon rubescens [J]. Archi Pharma Res, 2012, 35(12): 2147-2151. [16] Zhan R, Pu JX, Sun HD, et al. ent-Atisane and ent-kaurane diterpenoids from Isodon rosthornii [J]. Fitoterapia, 2013, 88: 76-81. [17] Lou HY, Zhang XM, Gao L. In vitro and in vivo antitumor activity of oridonin nanosuspension [J]. Inter J Pharma, 2009, 379(1): 181-186. [18] Li L, Sun HD, Leung PS, et al. Eriocalyxin B-induced apoptosis in pancreatic adenocarcinoma cells through thiol-containing antioxidant systems and downstream signaling pathways [J]. Curr Mol Med, 2014, 14(5): 673-689. [19] Fujita E, Nagao Y, Kuroda H, et al. The antitumor and antibacterial activity of the Isodon diterpenoids [J]. Chem Pharm Bull, 1976, 24(9): 2118-2127. [20] Monks A, Scudiero D, Vistica D, et al. Feasibility of a high-flux anticancer drug screen using a diverse panel of cultured human tumor cell lines [J]. J Natl Cancer Inst 1991, 83(11): 757-766. [21] McCartney-Francis NL, Song X, Wahl SM, et al. Selective inhibition of inducible nitric oxide synthase exacerbates erosive joint disease [J]. J Immunol 2001, 166(4): 2734-2740. [22] Reed LJ, Muench H. A simple method of estimating fifty percent endpoints [J]. Am J Hyg, 1938, 27(3): 493-497. [23] Fan JT, Su J, Peng YM, et al. Rubiyunnanins C-H, cytotoxic cyclic hexapeptides from Rubia yunnanensis inhibiting nitric oxide production and NF-κB activation[J]. Bioorg Med Chem, 2010, 18(23): 8226-8234.
Cite this article as: WU Hai-Yan, WANG Wei-Guang, DU Xue, YANG Jin, PU Jian-Xin, SUN Han-Dong. Six new cytotoxic and anti-inflammatory 11, 20-epoxy-ent-kaurane diterpenoids from Isodon wikstroemioides [J]. Chinese Journal of Natural Medicines, 2015, 13(5): 383-389.
– 389 –
