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Cytotoxic sesquiterpene lactones from Artemisia anomala
Phytochemistry Letters 20 (2017) 177–180
Contents lists available at ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Short communication
Cytotoxic sesquiterpene lactones from Artemisia anomala a,b
c
b
b
b
c
Lu Li , Hongchun Liu , Chunping Tang , Sheng Yao , Changqiang Ke , Chenghui Xu , ⁎ Yang Yeb,d,
MARK
a
College of Pharmacy, Nanchang University, Nanchang 330006, People’s Republic of China State Key Laboratory of Drug Research, Natural Products Chemistry Department, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, People’s Republic of China c Division of Antitumor Pharmacology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, People’s Republic of China d School of Life Science and Technology, ShanghaiTech University, Shanghai 201203, People’s Republic of China b
A R T I C L E I N F O
A B S T R A C T
Keywords: Artemisia anomala Compositae Sesquiterpene lactones Structure elucidation Cytotoxicity
Two new sesquiterpene lactones, 8α-acetoxy-1,10α-epoxy-2-oxo-guaia-3,11(13)-dien-12,6α-olide (1) and 13acetoxy-1-oxo-4α-hydroxy-eudesman-2(11)-dien-12,6α-olide (2), along with six known analogs (3-8), were isolated from the whole plant of Artemisia anomala S. Moore. Their structures were elucidated by extensive analysis of spectroscopic data. Compounds 6 and 7 exhibited in vitro moderate cytotoxicity against A549 cells with IC50 values of 0.6 and 0.9 μM, and HepG2 cells with IC50 values of 5.4 and 3.0 μM, respectively.
1. Introduction The genus Artemisia belongs to the family of Compositae and comprises more than 500 species (Oberprieler et al., 2007). Many species of this genus have long been used as folk medicines in China for the treatment of diseases such as fever, malaria, hepatitis, and cancer. Artemisinin, a famous sesquiterpene isolated from A. annua, is now a well-known drug used to fight against malaria and saved millions of lives over the world (Tu, 2011). A. anomala S. Moore, known as Nan-Liu-Ji-Nu in Chinese, is a perennial herbaceous plant that grows mainly on the roadside, hillside and forest edges with a distribution in most southern areas of China. Its whole plant has been commonly used for centuries as an analgesic, homeostatic, antibiotic and wound-healing agent. In recent years, phytochemical investigations of the title plant had revealed the existence of flavonoids, coumarins, and sesquiterpene lactones, some of which exhibited remarkable antitumor (Jakupovic et al., 1987; Marco et al., 1993; Lee et al., 1998, 2002, 2003; Zan et al., 2010; Zan et al., 2012a,b,c), anti-inflammatory (Hu et al., 2012; Turak et al., 2014; Zhang et al., 2014; Chi et al., 2016) and anti-HIV-1 protease (Ma et al., 2000) activities. In our further effort to search for bioactive sesquiterpene constituents, two new sesquiterpene lactones (1 and 2) (Fig. 1), along with six known analogs (3–8), were characterized from the whole plant of A. anomala. In this paper, we describe the isolation and structural elucidation of these compounds, and their cytotoxic
activity against human tumor cell lines A549 (human lung adenocarcinoma) and HepG2 (Human hepatocellular liver carcinoma) as well. 2. Results and discussion Compound 1 possessed a molecular formula of C17H18O6 as established by the HRESIMS ion at m/z 317.1021 [M-H]− (calcd. 317.1031), implying nine degrees of unsaturation. The IR absorption band at 1765 cm−1 indicated the presence of carbonyl groups. The 1H NMR spectrum of 1 (Table 1) showed signals of three tertiary methyls at δH 1.78 (H-14), 2.13 (H-15), and 2.41 (H-2′), two oxygenated methine protons at δH 4.17 (H-6) and 5.15 (H-8), and three olefinic protons at δH 5.59 (H-13), 6.22 (H-13) and 6.23 (H-3). The 13C NMR spectrum (Table 1), in combination with HSQC and DEPT spectra, showed 17 carbon resonances ascribed to four olefinic (δC 122.1, 133.8, 135.9, 169.0), four oxygenated (δC 64.9, 65.2, 69.0, 77.5), two ester carbonyl (δC 169.6, 175.8), a ketone carbonyl (δC 200.1), three methyl (δC 18.7, 21.1, 21.2), one methylene (δC 41.2), and two methine (δC 48.5, 55.0) carbons. The aforementioned data showed high similarities to those of a known sesquiterpene lactone artemdubolide E (Huang et al., 2010), suggesting that 1 could possess a similar skeleton. Compared with artemdubolide E, additional signals of a methyl singlet at δH 2.41 and two carbon resonances at δC 175.8 and 21.1 were observed, indicative of an acetyl group present in 1. The HMBC correlations from H-8 to C1′, C-2′, C-7, and C-9 confirmed the location of the acetoxy group at C-8
⁎ Corresponding author at: State Key Laboratory of Drug Research, Natural Products Chemistry Department, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, People’s Republic of China. E-mail address: [email protected] (Y. Ye).
http://dx.doi.org/10.1016/j.phytol.2017.04.038 Received 7 February 2017; Received in revised form 24 March 2017; Accepted 13 April 2017 1874-3900/ © 2017 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
Phytochemistry Letters 20 (2017) 177–180
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Fig. 1. Structures of compounds 1–8 isolated from A. anomala. Table 1 1 H NMR (500 Hz) and No.
13
C NMR (125 Hz) data of compounds 1 and 2 in CDCl3.
1 δC
1 2 3 4 5 6 7 8
64.9 200.1 133.8 169.0 48.5 77.5 55.0 69.0
9
41.2
10 11 12 13
65.2 135.9 169.6 122.1
14 15 1′ 2′
18.7 21.2 175.8 21.1
2 δH (J in Hz)
6.23 br s 3.17 br d (10.5) 4.17 dd (10.5, 10.5) 2.82 m 5.15 td (10.5, 3.0) 2.46 dd (15.0, 3.0) 2.29 dd (15.0, 10.5)
6.22 d (3.0) 5.59 d (3.0) 1.78 s 2.13 s 2.41 s
δC 200.5 125.5 152.2 70.1 57.4 79.2 168.1 22.4 35.3 45.1 119.8 171.2 54.9 18.7 24.5 170.7 20.9
δH (J in Hz)
5.90 d (10.4) 6.66 d (10.4) 2.22 d (11.8) 5.14 d (11.8) 3.15 m 2.47 m 2.32 m 1.59 m
4.82 d (12.7) 4.80 d (12.7) 1.30 s 1.64 s Fig. 2. Selected HMBC (H → C), 1H–1H COSY (HeH), ROESY(H ↔ H) correlations for compounds 1 and 2.
2.08 s
Compound 2 had a molecular formula of C17H20O6 determined by the HRESIMS ion at m/z 319.1182 [M-H]− (calcd. 319.1187), corresponding to eight degrees of unsaturation. The IR spectrum showed absorption bands for hydroxyl group (3437 cm−1), carbonyl group (1768 cm−1) and double bands (1641 cm−1). The NMR data, similar to those of the known compound 3β,13-diacetoxy-1β,4α-dihydroxyeudesm-7(11)-en-12,6α-olide (3) (Barrero et al., 2013), suggested that they shared the same skeleton. The 13C NMR spectrum of compound 2 (Table 1) revealed the characteristic signals of two olefinic carbon resonances at δC 125.5, 152.2 and a ketone carbonyl resonance at δC 200.5, which suggested the presence of an α, β-unsaturated ketone carbonyl group, rather than those of two oxygenated (δC 70.4, 76.1) and a methylene (δC 37.1) carbon reported for compound 3. In the HMBC spectrum, the long range correlations from H-3 (δH 6.66, d, J = 10.4 Hz) to C-1, C-2, C-4 and C-15, and from H-2 (δH 5.90, d, J = 10.4 Hz) to C-10, C-1, C-3, and C-4 confirmed a double bond occurring between C-2 and C-3 (Fig. 2b). The ROESY correlations of Me-15/Me-14, H-6/Me-14, H-6/Me-15, H-9β/Me-14, and H-5/H-9α
(Fig. 2a). The 1H–1H COSY spectrum implied the presence of a partial structure (C5-C9) shown as a bold line in Fig. 2a. The relative configuration of 1 was inferred from the ROESY experiment. The ROESY correlations of Me-14/H-9β, H-9β/H-8, and H-8/H-6 indicated that Me-14, H-8 and H-6 were co-facial and β-oriented, while the crosspeaks of H-7/H-9α and H-7/H-5 suggested that H-5 and H-7 were on the other face and α-oriented (Fig. 2a). The distinction of relative configuration of 1 and artemdubolide E lied in the orientation of C-14 and the epoxy functionality. The Me-14 was placed in a β-orientation in 1, which was evident from the aforementioned ROESY correlations, while Me-14 of artemdubolide E was in an α-orientation as reported (Huang et al., 2010). The different chemical shifts of C-1, C-10 and C-14 (δC 64.9, 65.2 and 18.7 in 1 vs δC 66.7, 66.7 and 21.3 in artemdubolide E) also supported that they might have different configurations on these positions. The orientation of C-14 influenced the migration of the chemical shifts of the close carbons. Therefore, compound 1 was established as 8α-acetoxy-1,10α-epoxy-2-oxo-guaia-3,11(13)-dien12,6α-olide. 178
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suggested that Me-14, Me-15, H-6 were β-oriented while H-5 was αoriented (Fig. 2b). Therefore, compound 2 was established as 13acetoxy-1-oxo-4α-hydroxy-eudesman-2(11)-dien-12,6α-olide. Six known sesquiterpene lactones, 3β,13-diacetoxy-1β,4α-dihydroxyeudesm-7(11)-en-12,6α-olide (3) (Barrero et al., 2013), eudesmaafraglaucolide (4), 5α-hydroxydehydroleucodin (5) (Zdero and Bohlmann, 1989), artanomalide D (6) (Wen et al., 2010), 8-acetylarteminolide (7), and artanomalide (8) (Lee et al., 2000), were also identified in this study. Their structures were determined by NMR and MS data analyses, and by comparison with the literature data as well. All isolates (1–8) were applied to the in vitro cytotoxicity assay against human cancer cell lines A549 and HepG2 by the Cell Counting Kit 8 (CCK-8) assay. The results showed that compounds 6 and 7 exhibited moderate inhibitory activity against A549 cells with IC50 values of 0.6 and 0.9 μM, and HepG2 cells with IC50 values of 5.4 and 3.0 μM, respectively. Previous investigations reported the cytotoxicity evaluation of some sesquiterpene monomers and dimers obtained from the genus Artemisia, showing weak or moderate cytotoxic activity with the IC50 values ranging from 1 to 50 μM. The inhibitory activity of compounds 6 and 7 and the inactivity of their regioisomer 8 are in agreement with those in literatures (Lee et al., 2002; Wen et al., 2010). Given the cytotoxicity assay results reported for analogs like arteminolide A–D, arteminolide B’-C’ (Lee et al., 1998, 2000, 2002, 2003), our findings further supported that the α-methylene-γ-lactone group of sesquiterpene lactones in guaiane- and eudesmane-type contributed significantly to the inhibitory activity against cancer cell lines (Koo et al., 2001; Lee et al., 2003; Yang et al., 2015; Chi et al., 2016). In summary, two new sesquiterpene lactones and six known ones, classified as eudesmanolides, guaianolides, and dimeric guaianolides, were isolated from A. anomala. Previous investigations revealed the existence of different families of sesquiterpene lactones with eudesmanolides and guaianolides being the most common. Our results are consistent with the previous reports and further enrich the chemical constituents of A. anomala.
of China in 2010, and identified by Prof. Jin-Gui Shen from the Shanghai Institute of Materia Medica. A voucher specimen (No. 20100824) has been deposited at the Herbarium of the Shanghai Institute of Materia Medica, Chinese Academy of Sciences. 3.3. Extraction and isolation The dry whole plant of A. anomala (8.0 kg) was ground and extracted with 95% EtOH three times, 3 days each. After evaporation of the solvent under reduced pressure, the residue was suspended in H2O and partitioned with petroleum ether (PE), CHCl3, and EtOAc, successively. The CHCl3 fraction (65 g) was applied to an MCI gel column eluted with MeOH in H2O (40, 60, 80, and 95% in a step manner) to afford fractions 1–4. Fraction 4 (12 g) was subjected to silica gel CC and eluted with CH2Cl2/MeOH (40:1–1:1) to give subfractions 4A–4Q. Subfraction 4 M was subjected to chromatography on a Sephadex LH-20 column eluted with MeOH to give subfractions 4M1 to 4M6. Subfraction 4M3 (223 mg) was passed through a column of silica gel eluted with CH2Cl2/MeOH (100:1) to give subfractions 4M3A–4M3D. Subfractions 4M3 B (20 mg) was further purified by semi-preparative HPLC (eluted with a gradient of CH3CN in H2O from 50% to 75%, flow rate 3.0 mL/min, 60 min) to yield compound 7 (6 mg). Fraction 3 (14 g) was subjected to CC over silica gel and eluted with a gradient of CH2Cl2/MeOH (40:1–1:1) to give subfractions 3A to 3 M. Subfraction 3G (1.07 g) was chromatographed on a Sephadex LH20 column eluted with MeOH to give subfractions 3G1 to 3G3. Subfraction 3G3 (71 mg) was subjected to a silica gel column eluted with CH2Cl2/MeOH (150:1) to give a mixture, which was further purified by semi-preparative HPLC (eluted with a gradient of CH3CN in H2O from 35% to 60%, flow rate 3.0 mL/min, 60 min) to afford compounds 1 (2 mg) and 5 (2 mg). By a similar procedure to subfraction 3L furnished compounds 4 (20 mg), 6 (5 mg) and 8 (3 mg). Fraction 1 (6 g) was chromatographed on a Sephadex LH-20 column (eluted with MeOH) to give subfractions 1A to 1C. Subfraction 1 B (3.47 g) was subjected to silica gel CC and eluted with a gradient of CH2Cl2/MeOH (100:1–1:1) to give subfractions 1B1–1B9. Subfraction 1B2 (416 mg) was separated by silica gel CC (CH2Cl2/MeOH, 100:1) to give subfractions 1B2A to 1B2D. Subfraction 1B2C (60 mg) was purified by preparative HPLC (eluted with a gradient of CH3CN in H2O from 10% to 20%, flow rate 25.0 mL/min, 120 min) to yield compound 2 (7 mg). By a similar procedure subfraction 1B4 furnished compound 3 (38 mg).
3. Experimental 3.1. General experimental procedures Optical rotations were obtained on a RudulphAutopol VI Automatic polarimeter. IR spectra were recorded with a Nicolet Magna FTIR-750 spectrometer. Analytical HPLC and ESIMS spectra were performed on a Waters 2695 instrument coupled with a 2998 PDA, a Waters 2424 ELSD and a Waters 3100 SQDMS detector. HRESIMS were recorded on a Waters Xevo Q-Tof mass detector and an Agilent G6520 Q-TOF mass detector. NMR spectra were recorded using a BrukerAvance III for 500 M and 600 M NMR spectrometers and a Varian MR-400 for 400 M NMR spectrometer. The chemical shifts were given in δ (in ppm) with solvent resonance CDCl3 [(δH 7.26; δC 77.16)] as internal standard. Preparative HPLC was performed on a Varian PrepStar system with an Alltech 3300 ELSD using a Waters Sunfire RP C18, 5 μM, 30 × 150 mm column. Semi-preparative HPLC was performed on a Waters 2690 instrumet with a 996 PDA using a Waters Sunfire RP C18, 5 μM, 10 × 250 mm. MCI gel CHP20P (75−150 μM, Mitsubishi Chemical Industries, Tokyo, Japan), silica gel (Qingdao Marine Chemical Industrials, Qingdao, Shandong, China) and Sephadex LH-20 (Pharmacia Biotech AB, Uppsala, Sweden) were used for column chromatography. TLC was carried out on precoated silica gel 60 F254 aluminum sheets (Merck, Germany). Doxorubicin was bought from Sellleckchem. The cell lines A549 and HepG2 were obtained from the American Type Culture Collection.
3.4. New compounds 8α-Acetoxy-1,10α-epoxy-2-oxo-guaia 3,11(13)-dien-12,6α-olide (1): colorless gum; [α]D20 + 64.6 (c 0.1, MeOH); UV(MeOH) λmax (log ε): 232 (4.18) nm; IR (KBr): 3443, 2925, 1780, 1768, 1669, 1610, 1239, 852 cm−1. HRESI-MS: m/z 317.1021 [M-H]− (calcd for C17H17O6, 317.1031); 1H and 13C NMR data see Table 1. 13-Acetoxy-1-oxo-4α-hydroxy-eudesman-2(11)-dien-12,6α-olide (2): colorless gum; [α]D20 −16.0 (c 0.1, MeOH); UV (MeOH) λmax (log ε): 225 (3.81) nm; IR (KBr): 3437, 2932, 1768, 1738, 1658, 1628, 1384, 1240, 1030 cm−1. HRESI-MS: m/z 319.1182 [M-H]− (calcd for C17H19O6, 319.1187); 1H and 13C NMR data see Table 1. 3.5. Bioassays for cytotoxic activity The cytotoxicity of the compounds against A549 and HepG2 cells were evaluated using the Cell Counting Kit 8 (CCK-8) assay. Cells were cultured in 96-well plates and incubated in humidified air containing 5% CO2 at 37 °C. Appropriate dilutions (10−2–102 μM) of the test compounds were added to the cultures. At the end of exposure time, 10 μl CCK8 (Dojindo, Kumamoto, Japan) was added to each well and the plates were kept in the incubator for 4 h, then measured at 450 nm using multi-well spectrophotometer (SpectraMax, Molecular Devices,
3.2. Plant material The whole plant of A. anomala was collected from Guangxi Province 179
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Arteminolide, an inhibitor of farnesyl-protein transferase from Artemisia sylvatica. J. Org. Chem. 63, 7111–7113. Lee, S.-H., Kang, H.-M., Song, H.-C., Lee, H., Lee, U.C., Son, K.-H., Kim, S.-H., Kwon, B.M., 2000. Sesquiterpene lactones, inhibitors of farnesyl-protein transferase, isolated from the flower of Artemisia sylvatica. Tetrahedron 56, 4711–4715. Lee, S.-H., Kim, H.-K., Seo, J.-M., Kang, H.-M., Kim, J.H., Son, K.-H., Lee, H., Kwon, B.-M., Shin, J., Seo, Y., 2002. Arteminolides B, C, and D, new inhibitors of farnesyl-protein transferase from Artemisia argyi. J. Org. Chem. 67, 7670–7675. Lee, S.H., Lee, M.-Y., Kang, H.-M., Han, D.C., Son, K.-H., Yang, D.C., Sung, N.-D., Lee, C.W., Kim, H.M., Kwon, B.-M., 2003. Anti-tumor activity of the farnesyl-protein transferase inhibitors arteminolides, isolated from Artemisa. Bioorg. Med. Chem. 11, 4545–4549. Ma, C.-M., Nakamura, N., Hottori, M., Zhu, S., Komatsu, K., 2000. Guaiane dimers and germacranolide from Artemisia caruifolia. J. Nat. Prod. 63, 1626–1629. Marco, J.A., Sanz-cervera, J.F., Manglano, E., Sancenon, F., Rustaiyan, A., Kardar, M., 1993. Sesquiterpene lactones from iranian Artemisia species. Phytochemistry 34, 1561–1564. Oberprieler, C., Himmelreich, S., Vogt, R., 2007. A new subtribal classification of the tribe Anthemideae (Compositae). Willdenowia 37, 89–114. Tu, Y.-Y., 2011. The discovery of artemisinin (qinghaosu) and gifts from Chinese medicine. Nat. Med. 17, 1217–1220. Turak, T., Shi, S.-P., Jiang, Y., Tu, P.-F., 2014. Dimeric guaianolides from Artemisia absinthium. Phytochemistry 105, 109–114. Wen, J., Shi, H., Xu, Z., Chang, H., Jia, C., Zan, K., Jiang, Y., Tu, P., 2010. Dimeric guaianolides and sesquiterpenoids from Artemisia anomala. J. Nat. Prod. 73, 67–70. Yang, Y.-X., Gao, S., Zhang, S.-D., Zu, X.-P., Shen, Y.-H., Shan, L., Li, H.-L., Zhang, W.-D., 2015. Cytotoxic 2, 4-linked sesquiterpene lactone dimers from Carpesium faberi exhibiting NF-kB inhibitory activity. RSC Adv. 5, 55285–55289. Zan, K., Chen, X.-Q., Fu, Q., Shi, S.-P., Zhou, S.-X., Xiao, M.-T., Tu, P.-F., 2010. 1, 10Secoguaianolides from Artemisia anomala (Asteraceae). Biochem. Syst. Ecol. 38, 431–434. Zan, K., Chai, X.-Y., Chen, X.-Q., Wu, Q., Fu, Q., Zhou, S.-X., Tu, P.-F., 2012a. Artanomadimers A–F: six new dimeric guaianolides from Artemisia anomala. Tetrahedron 68, 5060–5065. Zan, K., Chen, X.-Q., Chai, X.-Y., Wu, Q., Fu, Q., Zhou, S.-X., Tu, P.-F., 2012b. Two new cytotoxic eudesmane sesquiterpenoids from Artemisia anomala. Phytochem. Lett. 5, 313–315. Zan, K., Chen, X.-Q., Tu, P.-F., 2012c. A new 1, 10-secoguaianolide from the aerial parts of Artemisia anomala. Chin. J. Nat. Med. 10, 358–362. Zdero, C., Bohlmann, F., 1989. A vetispiranolide and other sesquiterpene lactones from Peyrousea umbellata. Phytochemistry 28, 3101–3104. Zhang, C., Wang, S., Zeng, K.-W., Cui, F.-X., Jin, H.-W., Guo, X.-Y., Jiang, Y., Tu, P.-F., 2014. Rupestonic acids B-G, No inhibitory sesquiterpenoids from Artemisia rupestris. Bioorg. Med. Chem. Lett. 24, 4318–4322.
U.S.A). The inhibition rate was calculated as (1-A450 treated/A450 vehicle control) × 100%. The cytotoxicity of compounds was expressed as an IC50, determined by the Logit method. Doxorubicin was used as a positive control. Acknowledgements Financial supports from the National Science and Technology Major Project “Key New Drug Creation and Manufacturing Program” (2015ZX09103002), the National Natural Science Funds of China (81673327), the Youth Innovation Promotion Association CAS, and the Chinese Academy of Sciences (GJHZ1678, GJHZ1739) are gratefully acknowledged. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytol.2017.04.038. References Barrero, A.F., Herrador del Pino, M.M., Portero, A.G., Buron, P.A., Arteaga, J.F., Alquezar, J.B., Diaz, C.E., Coloma, A.G., 2013. Terpenes and polyacetylenes from cultivated Artemisia granatensis boiss (Royal chamomile) and their defensive properties. Phytochemistry 94, 192–197. Chi, J., Li, B.-C., Dai, W.-F., Zhang, M., 2016. Highly oxidized sesquiterpenes from Artemisia austro-yunnanensis. Fitoterapia 115, 182–188. Hu, Z.H., Zhang, P., Huang, D.B., Wu, J.Z., 2012. New guaianolides from Artemisia anomala. J. Asian Nat. Prod. 14, 111–114. Huang, Z.S., Pei, Y.H., Liu, C.M., Lin, S., Tang, J., Huang, D.S., Song, T.F., Lu, L.H., Gao, Y.P., Zhang, W.D., 2010. Highly oxygenated guaianolides from Artemisia dubia. Planta Med. 76, 1710–1716. Jakupovic, J., Chen, Z.L., Bohlmann, F., 1987. Artanomaloide, a dimeric guaianolide and phenylalanine derivatives from Artemisia anomala. Phytochemistry 26, 2777–2779. Koo, T.-H., Lee, J.-H., Park, Y.-J., Hong, Y.-S., Kim, H.-S., Kim, K.-W., Lee, J.-J., 2001. A sesquiterpene lactone, costunolide, from Magnolia grandiflora inhibits NF-jB by targeting IjB phosphorylation. Planta Med. 67, 103–107. Lee, S.-H., Kim, M.-J., Bok, S.H., Lee, H., Kwon, B.-M., Shin, J., Seo, Y., 1998.
180
Contents lists available at ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Short communication
Cytotoxic sesquiterpene lactones from Artemisia anomala a,b
c
b
b
b
c
Lu Li , Hongchun Liu , Chunping Tang , Sheng Yao , Changqiang Ke , Chenghui Xu , ⁎ Yang Yeb,d,
MARK
a
College of Pharmacy, Nanchang University, Nanchang 330006, People’s Republic of China State Key Laboratory of Drug Research, Natural Products Chemistry Department, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, People’s Republic of China c Division of Antitumor Pharmacology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, People’s Republic of China d School of Life Science and Technology, ShanghaiTech University, Shanghai 201203, People’s Republic of China b
A R T I C L E I N F O
A B S T R A C T
Keywords: Artemisia anomala Compositae Sesquiterpene lactones Structure elucidation Cytotoxicity
Two new sesquiterpene lactones, 8α-acetoxy-1,10α-epoxy-2-oxo-guaia-3,11(13)-dien-12,6α-olide (1) and 13acetoxy-1-oxo-4α-hydroxy-eudesman-2(11)-dien-12,6α-olide (2), along with six known analogs (3-8), were isolated from the whole plant of Artemisia anomala S. Moore. Their structures were elucidated by extensive analysis of spectroscopic data. Compounds 6 and 7 exhibited in vitro moderate cytotoxicity against A549 cells with IC50 values of 0.6 and 0.9 μM, and HepG2 cells with IC50 values of 5.4 and 3.0 μM, respectively.
1. Introduction The genus Artemisia belongs to the family of Compositae and comprises more than 500 species (Oberprieler et al., 2007). Many species of this genus have long been used as folk medicines in China for the treatment of diseases such as fever, malaria, hepatitis, and cancer. Artemisinin, a famous sesquiterpene isolated from A. annua, is now a well-known drug used to fight against malaria and saved millions of lives over the world (Tu, 2011). A. anomala S. Moore, known as Nan-Liu-Ji-Nu in Chinese, is a perennial herbaceous plant that grows mainly on the roadside, hillside and forest edges with a distribution in most southern areas of China. Its whole plant has been commonly used for centuries as an analgesic, homeostatic, antibiotic and wound-healing agent. In recent years, phytochemical investigations of the title plant had revealed the existence of flavonoids, coumarins, and sesquiterpene lactones, some of which exhibited remarkable antitumor (Jakupovic et al., 1987; Marco et al., 1993; Lee et al., 1998, 2002, 2003; Zan et al., 2010; Zan et al., 2012a,b,c), anti-inflammatory (Hu et al., 2012; Turak et al., 2014; Zhang et al., 2014; Chi et al., 2016) and anti-HIV-1 protease (Ma et al., 2000) activities. In our further effort to search for bioactive sesquiterpene constituents, two new sesquiterpene lactones (1 and 2) (Fig. 1), along with six known analogs (3–8), were characterized from the whole plant of A. anomala. In this paper, we describe the isolation and structural elucidation of these compounds, and their cytotoxic
activity against human tumor cell lines A549 (human lung adenocarcinoma) and HepG2 (Human hepatocellular liver carcinoma) as well. 2. Results and discussion Compound 1 possessed a molecular formula of C17H18O6 as established by the HRESIMS ion at m/z 317.1021 [M-H]− (calcd. 317.1031), implying nine degrees of unsaturation. The IR absorption band at 1765 cm−1 indicated the presence of carbonyl groups. The 1H NMR spectrum of 1 (Table 1) showed signals of three tertiary methyls at δH 1.78 (H-14), 2.13 (H-15), and 2.41 (H-2′), two oxygenated methine protons at δH 4.17 (H-6) and 5.15 (H-8), and three olefinic protons at δH 5.59 (H-13), 6.22 (H-13) and 6.23 (H-3). The 13C NMR spectrum (Table 1), in combination with HSQC and DEPT spectra, showed 17 carbon resonances ascribed to four olefinic (δC 122.1, 133.8, 135.9, 169.0), four oxygenated (δC 64.9, 65.2, 69.0, 77.5), two ester carbonyl (δC 169.6, 175.8), a ketone carbonyl (δC 200.1), three methyl (δC 18.7, 21.1, 21.2), one methylene (δC 41.2), and two methine (δC 48.5, 55.0) carbons. The aforementioned data showed high similarities to those of a known sesquiterpene lactone artemdubolide E (Huang et al., 2010), suggesting that 1 could possess a similar skeleton. Compared with artemdubolide E, additional signals of a methyl singlet at δH 2.41 and two carbon resonances at δC 175.8 and 21.1 were observed, indicative of an acetyl group present in 1. The HMBC correlations from H-8 to C1′, C-2′, C-7, and C-9 confirmed the location of the acetoxy group at C-8
⁎ Corresponding author at: State Key Laboratory of Drug Research, Natural Products Chemistry Department, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, People’s Republic of China. E-mail address: [email protected] (Y. Ye).
http://dx.doi.org/10.1016/j.phytol.2017.04.038 Received 7 February 2017; Received in revised form 24 March 2017; Accepted 13 April 2017 1874-3900/ © 2017 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
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Fig. 1. Structures of compounds 1–8 isolated from A. anomala. Table 1 1 H NMR (500 Hz) and No.
13
C NMR (125 Hz) data of compounds 1 and 2 in CDCl3.
1 δC
1 2 3 4 5 6 7 8
64.9 200.1 133.8 169.0 48.5 77.5 55.0 69.0
9
41.2
10 11 12 13
65.2 135.9 169.6 122.1
14 15 1′ 2′
18.7 21.2 175.8 21.1
2 δH (J in Hz)
6.23 br s 3.17 br d (10.5) 4.17 dd (10.5, 10.5) 2.82 m 5.15 td (10.5, 3.0) 2.46 dd (15.0, 3.0) 2.29 dd (15.0, 10.5)
6.22 d (3.0) 5.59 d (3.0) 1.78 s 2.13 s 2.41 s
δC 200.5 125.5 152.2 70.1 57.4 79.2 168.1 22.4 35.3 45.1 119.8 171.2 54.9 18.7 24.5 170.7 20.9
δH (J in Hz)
5.90 d (10.4) 6.66 d (10.4) 2.22 d (11.8) 5.14 d (11.8) 3.15 m 2.47 m 2.32 m 1.59 m
4.82 d (12.7) 4.80 d (12.7) 1.30 s 1.64 s Fig. 2. Selected HMBC (H → C), 1H–1H COSY (HeH), ROESY(H ↔ H) correlations for compounds 1 and 2.
2.08 s
Compound 2 had a molecular formula of C17H20O6 determined by the HRESIMS ion at m/z 319.1182 [M-H]− (calcd. 319.1187), corresponding to eight degrees of unsaturation. The IR spectrum showed absorption bands for hydroxyl group (3437 cm−1), carbonyl group (1768 cm−1) and double bands (1641 cm−1). The NMR data, similar to those of the known compound 3β,13-diacetoxy-1β,4α-dihydroxyeudesm-7(11)-en-12,6α-olide (3) (Barrero et al., 2013), suggested that they shared the same skeleton. The 13C NMR spectrum of compound 2 (Table 1) revealed the characteristic signals of two olefinic carbon resonances at δC 125.5, 152.2 and a ketone carbonyl resonance at δC 200.5, which suggested the presence of an α, β-unsaturated ketone carbonyl group, rather than those of two oxygenated (δC 70.4, 76.1) and a methylene (δC 37.1) carbon reported for compound 3. In the HMBC spectrum, the long range correlations from H-3 (δH 6.66, d, J = 10.4 Hz) to C-1, C-2, C-4 and C-15, and from H-2 (δH 5.90, d, J = 10.4 Hz) to C-10, C-1, C-3, and C-4 confirmed a double bond occurring between C-2 and C-3 (Fig. 2b). The ROESY correlations of Me-15/Me-14, H-6/Me-14, H-6/Me-15, H-9β/Me-14, and H-5/H-9α
(Fig. 2a). The 1H–1H COSY spectrum implied the presence of a partial structure (C5-C9) shown as a bold line in Fig. 2a. The relative configuration of 1 was inferred from the ROESY experiment. The ROESY correlations of Me-14/H-9β, H-9β/H-8, and H-8/H-6 indicated that Me-14, H-8 and H-6 were co-facial and β-oriented, while the crosspeaks of H-7/H-9α and H-7/H-5 suggested that H-5 and H-7 were on the other face and α-oriented (Fig. 2a). The distinction of relative configuration of 1 and artemdubolide E lied in the orientation of C-14 and the epoxy functionality. The Me-14 was placed in a β-orientation in 1, which was evident from the aforementioned ROESY correlations, while Me-14 of artemdubolide E was in an α-orientation as reported (Huang et al., 2010). The different chemical shifts of C-1, C-10 and C-14 (δC 64.9, 65.2 and 18.7 in 1 vs δC 66.7, 66.7 and 21.3 in artemdubolide E) also supported that they might have different configurations on these positions. The orientation of C-14 influenced the migration of the chemical shifts of the close carbons. Therefore, compound 1 was established as 8α-acetoxy-1,10α-epoxy-2-oxo-guaia-3,11(13)-dien12,6α-olide. 178
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suggested that Me-14, Me-15, H-6 were β-oriented while H-5 was αoriented (Fig. 2b). Therefore, compound 2 was established as 13acetoxy-1-oxo-4α-hydroxy-eudesman-2(11)-dien-12,6α-olide. Six known sesquiterpene lactones, 3β,13-diacetoxy-1β,4α-dihydroxyeudesm-7(11)-en-12,6α-olide (3) (Barrero et al., 2013), eudesmaafraglaucolide (4), 5α-hydroxydehydroleucodin (5) (Zdero and Bohlmann, 1989), artanomalide D (6) (Wen et al., 2010), 8-acetylarteminolide (7), and artanomalide (8) (Lee et al., 2000), were also identified in this study. Their structures were determined by NMR and MS data analyses, and by comparison with the literature data as well. All isolates (1–8) were applied to the in vitro cytotoxicity assay against human cancer cell lines A549 and HepG2 by the Cell Counting Kit 8 (CCK-8) assay. The results showed that compounds 6 and 7 exhibited moderate inhibitory activity against A549 cells with IC50 values of 0.6 and 0.9 μM, and HepG2 cells with IC50 values of 5.4 and 3.0 μM, respectively. Previous investigations reported the cytotoxicity evaluation of some sesquiterpene monomers and dimers obtained from the genus Artemisia, showing weak or moderate cytotoxic activity with the IC50 values ranging from 1 to 50 μM. The inhibitory activity of compounds 6 and 7 and the inactivity of their regioisomer 8 are in agreement with those in literatures (Lee et al., 2002; Wen et al., 2010). Given the cytotoxicity assay results reported for analogs like arteminolide A–D, arteminolide B’-C’ (Lee et al., 1998, 2000, 2002, 2003), our findings further supported that the α-methylene-γ-lactone group of sesquiterpene lactones in guaiane- and eudesmane-type contributed significantly to the inhibitory activity against cancer cell lines (Koo et al., 2001; Lee et al., 2003; Yang et al., 2015; Chi et al., 2016). In summary, two new sesquiterpene lactones and six known ones, classified as eudesmanolides, guaianolides, and dimeric guaianolides, were isolated from A. anomala. Previous investigations revealed the existence of different families of sesquiterpene lactones with eudesmanolides and guaianolides being the most common. Our results are consistent with the previous reports and further enrich the chemical constituents of A. anomala.
of China in 2010, and identified by Prof. Jin-Gui Shen from the Shanghai Institute of Materia Medica. A voucher specimen (No. 20100824) has been deposited at the Herbarium of the Shanghai Institute of Materia Medica, Chinese Academy of Sciences. 3.3. Extraction and isolation The dry whole plant of A. anomala (8.0 kg) was ground and extracted with 95% EtOH three times, 3 days each. After evaporation of the solvent under reduced pressure, the residue was suspended in H2O and partitioned with petroleum ether (PE), CHCl3, and EtOAc, successively. The CHCl3 fraction (65 g) was applied to an MCI gel column eluted with MeOH in H2O (40, 60, 80, and 95% in a step manner) to afford fractions 1–4. Fraction 4 (12 g) was subjected to silica gel CC and eluted with CH2Cl2/MeOH (40:1–1:1) to give subfractions 4A–4Q. Subfraction 4 M was subjected to chromatography on a Sephadex LH-20 column eluted with MeOH to give subfractions 4M1 to 4M6. Subfraction 4M3 (223 mg) was passed through a column of silica gel eluted with CH2Cl2/MeOH (100:1) to give subfractions 4M3A–4M3D. Subfractions 4M3 B (20 mg) was further purified by semi-preparative HPLC (eluted with a gradient of CH3CN in H2O from 50% to 75%, flow rate 3.0 mL/min, 60 min) to yield compound 7 (6 mg). Fraction 3 (14 g) was subjected to CC over silica gel and eluted with a gradient of CH2Cl2/MeOH (40:1–1:1) to give subfractions 3A to 3 M. Subfraction 3G (1.07 g) was chromatographed on a Sephadex LH20 column eluted with MeOH to give subfractions 3G1 to 3G3. Subfraction 3G3 (71 mg) was subjected to a silica gel column eluted with CH2Cl2/MeOH (150:1) to give a mixture, which was further purified by semi-preparative HPLC (eluted with a gradient of CH3CN in H2O from 35% to 60%, flow rate 3.0 mL/min, 60 min) to afford compounds 1 (2 mg) and 5 (2 mg). By a similar procedure to subfraction 3L furnished compounds 4 (20 mg), 6 (5 mg) and 8 (3 mg). Fraction 1 (6 g) was chromatographed on a Sephadex LH-20 column (eluted with MeOH) to give subfractions 1A to 1C. Subfraction 1 B (3.47 g) was subjected to silica gel CC and eluted with a gradient of CH2Cl2/MeOH (100:1–1:1) to give subfractions 1B1–1B9. Subfraction 1B2 (416 mg) was separated by silica gel CC (CH2Cl2/MeOH, 100:1) to give subfractions 1B2A to 1B2D. Subfraction 1B2C (60 mg) was purified by preparative HPLC (eluted with a gradient of CH3CN in H2O from 10% to 20%, flow rate 25.0 mL/min, 120 min) to yield compound 2 (7 mg). By a similar procedure subfraction 1B4 furnished compound 3 (38 mg).
3. Experimental 3.1. General experimental procedures Optical rotations were obtained on a RudulphAutopol VI Automatic polarimeter. IR spectra were recorded with a Nicolet Magna FTIR-750 spectrometer. Analytical HPLC and ESIMS spectra were performed on a Waters 2695 instrument coupled with a 2998 PDA, a Waters 2424 ELSD and a Waters 3100 SQDMS detector. HRESIMS were recorded on a Waters Xevo Q-Tof mass detector and an Agilent G6520 Q-TOF mass detector. NMR spectra were recorded using a BrukerAvance III for 500 M and 600 M NMR spectrometers and a Varian MR-400 for 400 M NMR spectrometer. The chemical shifts were given in δ (in ppm) with solvent resonance CDCl3 [(δH 7.26; δC 77.16)] as internal standard. Preparative HPLC was performed on a Varian PrepStar system with an Alltech 3300 ELSD using a Waters Sunfire RP C18, 5 μM, 30 × 150 mm column. Semi-preparative HPLC was performed on a Waters 2690 instrumet with a 996 PDA using a Waters Sunfire RP C18, 5 μM, 10 × 250 mm. MCI gel CHP20P (75−150 μM, Mitsubishi Chemical Industries, Tokyo, Japan), silica gel (Qingdao Marine Chemical Industrials, Qingdao, Shandong, China) and Sephadex LH-20 (Pharmacia Biotech AB, Uppsala, Sweden) were used for column chromatography. TLC was carried out on precoated silica gel 60 F254 aluminum sheets (Merck, Germany). Doxorubicin was bought from Sellleckchem. The cell lines A549 and HepG2 were obtained from the American Type Culture Collection.
3.4. New compounds 8α-Acetoxy-1,10α-epoxy-2-oxo-guaia 3,11(13)-dien-12,6α-olide (1): colorless gum; [α]D20 + 64.6 (c 0.1, MeOH); UV(MeOH) λmax (log ε): 232 (4.18) nm; IR (KBr): 3443, 2925, 1780, 1768, 1669, 1610, 1239, 852 cm−1. HRESI-MS: m/z 317.1021 [M-H]− (calcd for C17H17O6, 317.1031); 1H and 13C NMR data see Table 1. 13-Acetoxy-1-oxo-4α-hydroxy-eudesman-2(11)-dien-12,6α-olide (2): colorless gum; [α]D20 −16.0 (c 0.1, MeOH); UV (MeOH) λmax (log ε): 225 (3.81) nm; IR (KBr): 3437, 2932, 1768, 1738, 1658, 1628, 1384, 1240, 1030 cm−1. HRESI-MS: m/z 319.1182 [M-H]− (calcd for C17H19O6, 319.1187); 1H and 13C NMR data see Table 1. 3.5. Bioassays for cytotoxic activity The cytotoxicity of the compounds against A549 and HepG2 cells were evaluated using the Cell Counting Kit 8 (CCK-8) assay. Cells were cultured in 96-well plates and incubated in humidified air containing 5% CO2 at 37 °C. Appropriate dilutions (10−2–102 μM) of the test compounds were added to the cultures. At the end of exposure time, 10 μl CCK8 (Dojindo, Kumamoto, Japan) was added to each well and the plates were kept in the incubator for 4 h, then measured at 450 nm using multi-well spectrophotometer (SpectraMax, Molecular Devices,
3.2. Plant material The whole plant of A. anomala was collected from Guangxi Province 179
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U.S.A). The inhibition rate was calculated as (1-A450 treated/A450 vehicle control) × 100%. The cytotoxicity of compounds was expressed as an IC50, determined by the Logit method. Doxorubicin was used as a positive control. Acknowledgements Financial supports from the National Science and Technology Major Project “Key New Drug Creation and Manufacturing Program” (2015ZX09103002), the National Natural Science Funds of China (81673327), the Youth Innovation Promotion Association CAS, and the Chinese Academy of Sciences (GJHZ1678, GJHZ1739) are gratefully acknowledged. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytol.2017.04.038. References Barrero, A.F., Herrador del Pino, M.M., Portero, A.G., Buron, P.A., Arteaga, J.F., Alquezar, J.B., Diaz, C.E., Coloma, A.G., 2013. Terpenes and polyacetylenes from cultivated Artemisia granatensis boiss (Royal chamomile) and their defensive properties. Phytochemistry 94, 192–197. Chi, J., Li, B.-C., Dai, W.-F., Zhang, M., 2016. Highly oxidized sesquiterpenes from Artemisia austro-yunnanensis. Fitoterapia 115, 182–188. Hu, Z.H., Zhang, P., Huang, D.B., Wu, J.Z., 2012. New guaianolides from Artemisia anomala. J. Asian Nat. Prod. 14, 111–114. Huang, Z.S., Pei, Y.H., Liu, C.M., Lin, S., Tang, J., Huang, D.S., Song, T.F., Lu, L.H., Gao, Y.P., Zhang, W.D., 2010. Highly oxygenated guaianolides from Artemisia dubia. Planta Med. 76, 1710–1716. Jakupovic, J., Chen, Z.L., Bohlmann, F., 1987. Artanomaloide, a dimeric guaianolide and phenylalanine derivatives from Artemisia anomala. Phytochemistry 26, 2777–2779. Koo, T.-H., Lee, J.-H., Park, Y.-J., Hong, Y.-S., Kim, H.-S., Kim, K.-W., Lee, J.-J., 2001. A sesquiterpene lactone, costunolide, from Magnolia grandiflora inhibits NF-jB by targeting IjB phosphorylation. Planta Med. 67, 103–107. Lee, S.-H., Kim, M.-J., Bok, S.H., Lee, H., Kwon, B.-M., Shin, J., Seo, Y., 1998.
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