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Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
A new casbane diterpene from Euphorbia pekinensis a
Kuilong Wang , Hongli Yu a
a
abc
abc
, Hao Wu
, Xinzhi Wang
a
abc
, Yaozong
a
a
Pan , Yeqing Chen , Liping Liu , Yangping Jin & Chenchao Zhang a
School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, P.R. China b
Jiangsu Key Laboratory of Chinese Medicine Processing, Nanjing University of Chinese Medicine, Nanjing 210023, P.R. China c
Click for updates
Engineering Center of State Ministry of Education for Standardization of Chinese Medicine Processing, Nanjing University of Chinese Medicine, Nanjing 210023, P.R. China Published online: 07 Apr 2015.
To cite this article: Kuilong Wang, Hongli Yu, Hao Wu, Xinzhi Wang, Yaozong Pan, Yeqing Chen, Liping Liu, Yangping Jin & Chenchao Zhang (2015): A new casbane diterpene from Euphorbia pekinensis, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2015.1027704 To link to this article: http://dx.doi.org/10.1080/14786419.2015.1027704
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,
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Natural Product Research, 2015 http://dx.doi.org/10.1080/14786419.2015.1027704
A new casbane diterpene from Euphorbia pekinensis
Downloaded by [University of Nebraska, Lincoln] at 03:03 13 April 2015
Kuilong Wanga1, Hongli Yuabc1, Hao Wuabc*, Xinzhi Wangabc, Yaozong Pana, Yeqing Chena, Liping Liua, Yangping Jina and Chenchao Zhanga a School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, P.R. China; bJiangsu Key Laboratory of Chinese Medicine Processing, Nanjing University of Chinese Medicine, Nanjing 210023, P.R. China; cEngineering Center of State Ministry of Education for Standardization of Chinese Medicine Processing, Nanjing University of Chinese Medicine, Nanjing 210023, P.R. China
(Received 21 January 2015; final version received 1 March 2015)
A new casbane diterpenoid, referred to as pekinenin G, together with one cembrane diterpene and four known casbane diterpenoids were isolated from the roots of Euphorbia pekinensis. Their structures were elucidated on the basis of spectroscopic studies and comparison with related known compounds. The six compounds showed different cytotoxic activities against four human cancer cell lines. Keywords: Euphorbia pekinensis; casbane diterpenoid; cytotoxic activity
1. Introduction Natural products or their derivatives and mimics have contributed around 50% of current drugs, based on which new drugs are being developed (Camp et al. 2012; Grkovic et al. 2014). Diterpenoids are natural products with biological activities. Ingenol mebutate, a natural product identified from Euphorbia peplus, was later approved to treat actinic keratosis (Ogbourne & Parsons 2014). Euphorbia pekinensis, as a member of genus Euphorbia in the family Euphorbiaceae, has been used to treat gonorrhoea, oedema, migraine and warts in traditional Chinese medicine. Toxic compounds in this plant mainly comprise diterpenoids, most of which are casbane-type (Kong et al. 2002; Liang et al. 2009; Shao et al. 2011; Hou et al. 2013; Tao et al. 2013). Diterpenoids show cytotoxic activities against cancer cell lines, but the most active types have not been identified hitherto. Herein, we describe the isolation and structural elucidation of six diterpenes. Their cytotoxic activities against four human cancer cell lines (gastric cancer BGC-823, colon cancer HT-29, breast cancer MCF-7 and lung cancerA549) were assayed. *Corresponding author. Email: [email protected] q 2015 Taylor & Francis
Downloaded by [University of Nebraska, Lincoln] at 03:03 13 April 2015
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2. Results and discussion Compound 1 was isolated as a yellow oil, with its molecular formula assigned by HR-ESI-MS (m/z 341 [M þ Na]þ), implying six degrees of unsaturation. IR spectrum shows hydroxyl (3420 cm21), carbonyl (1710 cm21) and olefin (1670 and 1626 cm21) groups. 13C NMR and DEPT spectra show four methyl, six methylene, five methine, and five quaternary carbon atoms, which are attributable to one carbonyl and two olefinic groups. The remaining three degrees of unsaturation are ascribed to three cyclic ring systems. In the 1H – 1H COSY spectrum, a methine proton at d0.81– 0.89 (1H, m, H-1) is coupled with a methine proton at d1.48 (1H, m, H-2) and methylene protons at d1.51 (2H, m, H-14). H-2 is further coupled with an olefinic proton at d6.22 (1H, d, H-3) and H-14 is coupled with methylene protons at d2.125 –2.158 (2H, m, H-13). The coupled proton system in this region ends with quaternary centres at C-4 and C-12, respectively. The other two coupled units were determined by H – H COSY spectrum from H-6 to H-7, and H-9 to H-11 likewise end with quaternary carbon atoms at C-5, C-8 and C-12. Location information of these units was obtained from the HMBC experiment. There are correlations between H-18 and both C-3 and C-5, H-7 and both C-5 and C9, and H-19 and C-7. Besides, H-11 and C-9, as well as H-20, H-13 and C-11 are also correlated. The above long-range correlations suggest the presence of a 14-membered macrocyclic ring. In addition, there is a substituted cyclopropyl ring, as evidenced by the presence of a quaternary carbon (d 28.4, C-15) and a gem-dimethyl functionality [dH 1.18 (s, H3-16), dH 1.21 (s, H3-17); dC 21.40 (C-16), dC 23.50 (C-17)], together with the COSY correlation between two typical cyclopropyl protons [dH 0.79 (m, H-1), dH 1.48 (m, H-2)]. Thus, 1 was a casbane-type diterpenoid. 1 H and 13C NMR spectra suggest that 4 is closely related to pekinenin A. One olefinic proton in the 1H NMR spectrum of pekinenin A is absent in that of 1, and a dd signal at 2.655 (dd, 1H) is absent in that of 2. The 13C NMR chemical shifts of C-11 and C-12 differ significantly, i.e. d125.3 and 133.4 for C-11 and C-12 respectively in pekinenin A, versus d61.15 and 59.15 for those of 1. Hence, an epoxy group exists, and there is an epoxy group between C-11 and C-12 in 1 instead of an olefinic group in pekinenin A. Relative configuration of the epoxy group was analysed by using ROESY spectroscopy. As shown in Figure S9, there are interactions between H-11 and H-13b as well as H-13b and H-1, which indicates H-11 has a b-configuration. In the ROESY spectrum, H-1 and H-3, H-1 and H-17, H-3 and H-17, and H-2 and H-16 are correlated, giving b-oriented H-1 and a-oriented H-2. Otherwise, the lack of correlation between H-18 and H-3, H-19 and H-7, suggests that the 3, 4 and 7, 8-double bond has an E-configuration. Thus, 1 was structurally confirmed as 11a,12b-epoxy-18-hydroxy-1bH, 2aH-casba-3E and 7E-dien-5-one, referred to as pekinenin G.
3. Experimental 3.1. General details NMR spectra were recorded on a Bruker DRX-400 spectrometer (Germany). HR-ESI-MS were obtained on an HPLC – MS/MS system (Triple TOFTM 5600, AB SCIEX, Singapore) equipped with an electrospray ionisation (ESI) source. Column chromatography (CC) was performed on silica gel (100 – 200 mesh and 200– 300 mesh, Marine Chemical Factory, Qingdao, China); TLC was performed on precoated silica gel plates (HSG and HSF254, Yantai Chemical Factory, Yantai, China), and spots were detected by spraying with vanillin – sulphuric acid reagent, followed by heating. Preparative HPLC was conducted under the following conditions: Waters 600 pump; Waters 2487 UV visible detector at 254 nm; Waters C18 reversed-phase column, 250 mm £ 10 mm, 5 mm.
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3.2. Plant material The roots of E. pekinensis were collected from Anguo Chinese Herbal Medicine Factory (Anguo, China) in January 2013 and authenticated by Professor Haobin Hu in Jiangsu Institute for Food and Drug Control. A voucher specimen (No. 20130108) has been deposited at the Herbarium of Nanjing University of Chinese Medicine in China.
Downloaded by [University of Nebraska, Lincoln] at 03:03 13 April 2015
3.3. Extraction and isolation Dried powdered roots (6.2 kg) of E. pekinensis were extracted three times with 95% ethanol for 2 h each time. After filtration and removal of the solvent under reduced pressure, the 95% ethanol extract was suspended in water and partitioned with petroleum ether (PE), dichloromethane and ethyl acetate (EtOAc) successively. The PE extract (220 g) was subjected to CC (silica gel, gradient of PE – EtOAc) to afford fractions1 – 5 (PE – EtOAc15:1, 10:1, 5:1, 3:1, 1:1). Fraction 3 was subjected to CC again to afford fractions A – G, and fraction E was subjected to preparative TLC and developed with PE – EtOAc (2:1) to afford a yellow oil, that was further subjected to preparative reversed-phase HPLC (methanol – water 80:20, v/v, flow rate10 mL/ min, wavelength 275 nm) to afford compound 1 (16 mg). Fraction 5 was subjected to Sephadex LH-20 (dichloromethane –methanol 1:1, v/v) again to obtain compound 2 (10 mg), other four known diterpenes 3 – 6 (Figure 1) were obtained from fractions 1 and 2, respectively. 11a,12b-epoxy-18-hydroxy-1bH,2aH-casba-3E,7E-dien-5-one (1, Figure 1), yellow oil, UV lmax (MeOH): 273 nm; IR: 3420, 2833, 1710, 1665, 1626, 1459, 1378, 1253, and1038 cm21;
Figure 1. Chemical structures of compounds 1 –6.
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Table 1. Cytotoxicity data for compounds against four cancer cell lines. IC50 Compound
BGC823
A549
HT-29
MCF-7
42.7 53.9 15.6 25.1 54.8 12.1
40.8 88.8 21.9 35.1 90.2 15.6
47.8 70.1 25.1 30.2 110.7 11.3
48.5 70.5 22.3 32.3 87.9 21.2
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1 2 3 4 5 6
HR-ESI-MS m/z: 341.2086 [M þ Na] þ (calcd for C20H31O2 þ , [M þ Na] þ 341.2324); 1H NMR (400 MHz) and 13C NMR (400 MHz) d (ppm). 1 H NMR (CDCl3, 400 MHz): d 0.81 –0.89, m, 1H, C1-H; 1.18, s, 3H, (CH3); 1.21, s, 3H, (CH3); 1.27, s, 3H, (CH3); 1.48, m, 1H, C2-H; 1.51, m, 2H, C14-H; 1.72, s, 3H, (CH3); 1.94, m, 2H, C10-H; 2.27, m, 2H, C9-H; 2.66, dd, J ¼ 6.4, 1H, C11-H; 2.94, d, J ¼ 10.1, 1H, C6-H; 3.76, d, J ¼ 16.4, 1H, C6-H; 4.34, dd, J ¼ 8.4, 2H, C18-H; 5.31, 1H, d, J ¼ 9.8; 6.22, 1H, d, J ¼ 14.8, C3-H. 13 C NMR (CDCl3, 400 MHz): d (C-1) 38.43; (C-2) 31.84; (C-3) 151.3; (C-4) 136.36; (C-5) 201.5; (C-6) 40.81; (C-7) 122.77; (C-8) 134.45; (C-9) 36.54; (C-10) 24.56; (C-11) 61.15; (C-12) 59.15; (C-13) 38.18; (C-14) 25.67; (C-15) 29.45; (C-16) 21.40; (C-17) 23.50; (C-18) 57.27; (C19) 15.64; (C-20) 15.19. 3.4. MTT assays The in vitro cytotoxicity of the isolated diterpenoids against four human cancer cell lines was investigated by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide] colorimetric method. The human cancer cell lines were grown in HyClone’s modified 1640 medium containing 10% foetal bovine serum and cultivated in humidified incubators (5% CO2 and 378C). Afterwards, the cells were seeded in 96-well plates and cultivated for 12 h and then treated with compounds of eight concentrations (1.9, 3.9, 7.8, 15.6, 31.2, 62.5, 125, 250 mmM) for 48 h. Then, 20 mL of MTT (5 mg/mL) was added to each well, and the cells were incubated for additional 4 h. Then, DMSO (150 mL per well) was added to dissolve the formazan crystals. Absorbance was measured at 490 nm by enzyme immunoassay instrument. 4. Conclusions We evaluated the cytotoxic activities of compound 1 – 6 against four human cancer cell lines (Table 1). As indicated by the difference between the activities of compounds 1 and 3, the epoxy group may significantly decrease the cytotoxic activity. Moreover, the hydroxyl or carbonyl group at C-5 may increase the activity, based on the outcomes of compounds 2, 4 and 6. As to compounds 5 and 2, lacking the cyclopropane ring may decrease the cytotoxic activity. Supplementary material Experimental details relating to this paper are available online. Disclosure statement No potential conflict of interest was reported by the authors.
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Funding This work was supported by the National Natural Science Foundation of China [grant number 81202915]; and A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). We are grateful to the Analytical Center of Nanjing University of Chinese Medicine for identification of the measurements of HR-ESI-MS.
Note 1. These authors contributed equally to this work.
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References Camp D, Davis RA, Campitelli M, Ebdon J, Quinn RJ. 2012. Drug-like properties: guiding principles for the design of natural product libraries. J Nat Prod. 75:72–81. doi:10.1021/np200687v. Grkovic T, Pouwer RH, Vial M-L, Gambini L, Noe¨l A, Hooper JNA, Wood SA, Mellick GD, Quinn RJ. 2014. NMR fingerprints of the drug-like natural-product space identify iotrochotazinea: a chemical probe to study Parkinson’s disease. Angew Chem Int Ed. 53:6070– 6074. doi:10.1002/anie.201402239. Hou P, Zeng Y, Ma B, Bi K, Chen X. 2013. A new cytotoxic cembrane diterpene from the roots of Euphorbia pekinensis Rupr. Fitoterapia. 90:10–13. doi:10.1016/j.fitote.2013.07.004. Kong L-Y, Li Y, Wu X-L, Min Z-D. 2002. Cytotoxic diterpenoids from Euphorbia pekinensis. Planta Med. 68:249–252. doi:10.1055/s-2002-23132. Liang QL, Dai CC, Jiang JH, Tang YP, Duan JA. 2009. A new cytotoxic casbane diterpene from Euphorbia pekinensis. Fitoterapia. 80:514–516. doi:10.1016/j.fitote.2009.06.014. Ogbourne SM, Parsons PG. 2014. The value of nature’s natural product library for the discovery of New Chemical Entities: the discovery of ingenol mebutate. Fitoterapia. 98:36–44. doi:10.1016/j.fitote.2014.07.002. Shao F-G, Bu R, Zhang C, Chen C-J, Huang J, Wang J-H. 2011. Two new casbane diterpenoids from the roots of Euphorbia pekinensis. J Asian Nat Prod Res. 13:805–810. doi:10.1080/10286020.2011.596828. Tao W-W, Duan J-A, Tang Y-P, Yang N-Y, Li J-P, Qian Y-F. 2013. Casbane diterpenoids from the roots of Euphorbia pekinensis. Phytochemistry. 94:249–253. doi:10.1016/j.phytochem.2013.06.009.
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
A new casbane diterpene from Euphorbia pekinensis a
Kuilong Wang , Hongli Yu a
a
abc
abc
, Hao Wu
, Xinzhi Wang
a
abc
, Yaozong
a
a
Pan , Yeqing Chen , Liping Liu , Yangping Jin & Chenchao Zhang a
School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, P.R. China b
Jiangsu Key Laboratory of Chinese Medicine Processing, Nanjing University of Chinese Medicine, Nanjing 210023, P.R. China c
Click for updates
Engineering Center of State Ministry of Education for Standardization of Chinese Medicine Processing, Nanjing University of Chinese Medicine, Nanjing 210023, P.R. China Published online: 07 Apr 2015.
To cite this article: Kuilong Wang, Hongli Yu, Hao Wu, Xinzhi Wang, Yaozong Pan, Yeqing Chen, Liping Liu, Yangping Jin & Chenchao Zhang (2015): A new casbane diterpene from Euphorbia pekinensis, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2015.1027704 To link to this article: http://dx.doi.org/10.1080/14786419.2015.1027704
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,
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systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Natural Product Research, 2015 http://dx.doi.org/10.1080/14786419.2015.1027704
A new casbane diterpene from Euphorbia pekinensis
Downloaded by [University of Nebraska, Lincoln] at 03:03 13 April 2015
Kuilong Wanga1, Hongli Yuabc1, Hao Wuabc*, Xinzhi Wangabc, Yaozong Pana, Yeqing Chena, Liping Liua, Yangping Jina and Chenchao Zhanga a School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, P.R. China; bJiangsu Key Laboratory of Chinese Medicine Processing, Nanjing University of Chinese Medicine, Nanjing 210023, P.R. China; cEngineering Center of State Ministry of Education for Standardization of Chinese Medicine Processing, Nanjing University of Chinese Medicine, Nanjing 210023, P.R. China
(Received 21 January 2015; final version received 1 March 2015)
A new casbane diterpenoid, referred to as pekinenin G, together with one cembrane diterpene and four known casbane diterpenoids were isolated from the roots of Euphorbia pekinensis. Their structures were elucidated on the basis of spectroscopic studies and comparison with related known compounds. The six compounds showed different cytotoxic activities against four human cancer cell lines. Keywords: Euphorbia pekinensis; casbane diterpenoid; cytotoxic activity
1. Introduction Natural products or their derivatives and mimics have contributed around 50% of current drugs, based on which new drugs are being developed (Camp et al. 2012; Grkovic et al. 2014). Diterpenoids are natural products with biological activities. Ingenol mebutate, a natural product identified from Euphorbia peplus, was later approved to treat actinic keratosis (Ogbourne & Parsons 2014). Euphorbia pekinensis, as a member of genus Euphorbia in the family Euphorbiaceae, has been used to treat gonorrhoea, oedema, migraine and warts in traditional Chinese medicine. Toxic compounds in this plant mainly comprise diterpenoids, most of which are casbane-type (Kong et al. 2002; Liang et al. 2009; Shao et al. 2011; Hou et al. 2013; Tao et al. 2013). Diterpenoids show cytotoxic activities against cancer cell lines, but the most active types have not been identified hitherto. Herein, we describe the isolation and structural elucidation of six diterpenes. Their cytotoxic activities against four human cancer cell lines (gastric cancer BGC-823, colon cancer HT-29, breast cancer MCF-7 and lung cancerA549) were assayed. *Corresponding author. Email: [email protected] q 2015 Taylor & Francis
Downloaded by [University of Nebraska, Lincoln] at 03:03 13 April 2015
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K. Wang et al.
2. Results and discussion Compound 1 was isolated as a yellow oil, with its molecular formula assigned by HR-ESI-MS (m/z 341 [M þ Na]þ), implying six degrees of unsaturation. IR spectrum shows hydroxyl (3420 cm21), carbonyl (1710 cm21) and olefin (1670 and 1626 cm21) groups. 13C NMR and DEPT spectra show four methyl, six methylene, five methine, and five quaternary carbon atoms, which are attributable to one carbonyl and two olefinic groups. The remaining three degrees of unsaturation are ascribed to three cyclic ring systems. In the 1H – 1H COSY spectrum, a methine proton at d0.81– 0.89 (1H, m, H-1) is coupled with a methine proton at d1.48 (1H, m, H-2) and methylene protons at d1.51 (2H, m, H-14). H-2 is further coupled with an olefinic proton at d6.22 (1H, d, H-3) and H-14 is coupled with methylene protons at d2.125 –2.158 (2H, m, H-13). The coupled proton system in this region ends with quaternary centres at C-4 and C-12, respectively. The other two coupled units were determined by H – H COSY spectrum from H-6 to H-7, and H-9 to H-11 likewise end with quaternary carbon atoms at C-5, C-8 and C-12. Location information of these units was obtained from the HMBC experiment. There are correlations between H-18 and both C-3 and C-5, H-7 and both C-5 and C9, and H-19 and C-7. Besides, H-11 and C-9, as well as H-20, H-13 and C-11 are also correlated. The above long-range correlations suggest the presence of a 14-membered macrocyclic ring. In addition, there is a substituted cyclopropyl ring, as evidenced by the presence of a quaternary carbon (d 28.4, C-15) and a gem-dimethyl functionality [dH 1.18 (s, H3-16), dH 1.21 (s, H3-17); dC 21.40 (C-16), dC 23.50 (C-17)], together with the COSY correlation between two typical cyclopropyl protons [dH 0.79 (m, H-1), dH 1.48 (m, H-2)]. Thus, 1 was a casbane-type diterpenoid. 1 H and 13C NMR spectra suggest that 4 is closely related to pekinenin A. One olefinic proton in the 1H NMR spectrum of pekinenin A is absent in that of 1, and a dd signal at 2.655 (dd, 1H) is absent in that of 2. The 13C NMR chemical shifts of C-11 and C-12 differ significantly, i.e. d125.3 and 133.4 for C-11 and C-12 respectively in pekinenin A, versus d61.15 and 59.15 for those of 1. Hence, an epoxy group exists, and there is an epoxy group between C-11 and C-12 in 1 instead of an olefinic group in pekinenin A. Relative configuration of the epoxy group was analysed by using ROESY spectroscopy. As shown in Figure S9, there are interactions between H-11 and H-13b as well as H-13b and H-1, which indicates H-11 has a b-configuration. In the ROESY spectrum, H-1 and H-3, H-1 and H-17, H-3 and H-17, and H-2 and H-16 are correlated, giving b-oriented H-1 and a-oriented H-2. Otherwise, the lack of correlation between H-18 and H-3, H-19 and H-7, suggests that the 3, 4 and 7, 8-double bond has an E-configuration. Thus, 1 was structurally confirmed as 11a,12b-epoxy-18-hydroxy-1bH, 2aH-casba-3E and 7E-dien-5-one, referred to as pekinenin G.
3. Experimental 3.1. General details NMR spectra were recorded on a Bruker DRX-400 spectrometer (Germany). HR-ESI-MS were obtained on an HPLC – MS/MS system (Triple TOFTM 5600, AB SCIEX, Singapore) equipped with an electrospray ionisation (ESI) source. Column chromatography (CC) was performed on silica gel (100 – 200 mesh and 200– 300 mesh, Marine Chemical Factory, Qingdao, China); TLC was performed on precoated silica gel plates (HSG and HSF254, Yantai Chemical Factory, Yantai, China), and spots were detected by spraying with vanillin – sulphuric acid reagent, followed by heating. Preparative HPLC was conducted under the following conditions: Waters 600 pump; Waters 2487 UV visible detector at 254 nm; Waters C18 reversed-phase column, 250 mm £ 10 mm, 5 mm.
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3.2. Plant material The roots of E. pekinensis were collected from Anguo Chinese Herbal Medicine Factory (Anguo, China) in January 2013 and authenticated by Professor Haobin Hu in Jiangsu Institute for Food and Drug Control. A voucher specimen (No. 20130108) has been deposited at the Herbarium of Nanjing University of Chinese Medicine in China.
Downloaded by [University of Nebraska, Lincoln] at 03:03 13 April 2015
3.3. Extraction and isolation Dried powdered roots (6.2 kg) of E. pekinensis were extracted three times with 95% ethanol for 2 h each time. After filtration and removal of the solvent under reduced pressure, the 95% ethanol extract was suspended in water and partitioned with petroleum ether (PE), dichloromethane and ethyl acetate (EtOAc) successively. The PE extract (220 g) was subjected to CC (silica gel, gradient of PE – EtOAc) to afford fractions1 – 5 (PE – EtOAc15:1, 10:1, 5:1, 3:1, 1:1). Fraction 3 was subjected to CC again to afford fractions A – G, and fraction E was subjected to preparative TLC and developed with PE – EtOAc (2:1) to afford a yellow oil, that was further subjected to preparative reversed-phase HPLC (methanol – water 80:20, v/v, flow rate10 mL/ min, wavelength 275 nm) to afford compound 1 (16 mg). Fraction 5 was subjected to Sephadex LH-20 (dichloromethane –methanol 1:1, v/v) again to obtain compound 2 (10 mg), other four known diterpenes 3 – 6 (Figure 1) were obtained from fractions 1 and 2, respectively. 11a,12b-epoxy-18-hydroxy-1bH,2aH-casba-3E,7E-dien-5-one (1, Figure 1), yellow oil, UV lmax (MeOH): 273 nm; IR: 3420, 2833, 1710, 1665, 1626, 1459, 1378, 1253, and1038 cm21;
Figure 1. Chemical structures of compounds 1 –6.
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Table 1. Cytotoxicity data for compounds against four cancer cell lines. IC50 Compound
BGC823
A549
HT-29
MCF-7
42.7 53.9 15.6 25.1 54.8 12.1
40.8 88.8 21.9 35.1 90.2 15.6
47.8 70.1 25.1 30.2 110.7 11.3
48.5 70.5 22.3 32.3 87.9 21.2
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1 2 3 4 5 6
HR-ESI-MS m/z: 341.2086 [M þ Na] þ (calcd for C20H31O2 þ , [M þ Na] þ 341.2324); 1H NMR (400 MHz) and 13C NMR (400 MHz) d (ppm). 1 H NMR (CDCl3, 400 MHz): d 0.81 –0.89, m, 1H, C1-H; 1.18, s, 3H, (CH3); 1.21, s, 3H, (CH3); 1.27, s, 3H, (CH3); 1.48, m, 1H, C2-H; 1.51, m, 2H, C14-H; 1.72, s, 3H, (CH3); 1.94, m, 2H, C10-H; 2.27, m, 2H, C9-H; 2.66, dd, J ¼ 6.4, 1H, C11-H; 2.94, d, J ¼ 10.1, 1H, C6-H; 3.76, d, J ¼ 16.4, 1H, C6-H; 4.34, dd, J ¼ 8.4, 2H, C18-H; 5.31, 1H, d, J ¼ 9.8; 6.22, 1H, d, J ¼ 14.8, C3-H. 13 C NMR (CDCl3, 400 MHz): d (C-1) 38.43; (C-2) 31.84; (C-3) 151.3; (C-4) 136.36; (C-5) 201.5; (C-6) 40.81; (C-7) 122.77; (C-8) 134.45; (C-9) 36.54; (C-10) 24.56; (C-11) 61.15; (C-12) 59.15; (C-13) 38.18; (C-14) 25.67; (C-15) 29.45; (C-16) 21.40; (C-17) 23.50; (C-18) 57.27; (C19) 15.64; (C-20) 15.19. 3.4. MTT assays The in vitro cytotoxicity of the isolated diterpenoids against four human cancer cell lines was investigated by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide] colorimetric method. The human cancer cell lines were grown in HyClone’s modified 1640 medium containing 10% foetal bovine serum and cultivated in humidified incubators (5% CO2 and 378C). Afterwards, the cells were seeded in 96-well plates and cultivated for 12 h and then treated with compounds of eight concentrations (1.9, 3.9, 7.8, 15.6, 31.2, 62.5, 125, 250 mmM) for 48 h. Then, 20 mL of MTT (5 mg/mL) was added to each well, and the cells were incubated for additional 4 h. Then, DMSO (150 mL per well) was added to dissolve the formazan crystals. Absorbance was measured at 490 nm by enzyme immunoassay instrument. 4. Conclusions We evaluated the cytotoxic activities of compound 1 – 6 against four human cancer cell lines (Table 1). As indicated by the difference between the activities of compounds 1 and 3, the epoxy group may significantly decrease the cytotoxic activity. Moreover, the hydroxyl or carbonyl group at C-5 may increase the activity, based on the outcomes of compounds 2, 4 and 6. As to compounds 5 and 2, lacking the cyclopropane ring may decrease the cytotoxic activity. Supplementary material Experimental details relating to this paper are available online. Disclosure statement No potential conflict of interest was reported by the authors.
Natural Product Research
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Funding This work was supported by the National Natural Science Foundation of China [grant number 81202915]; and A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). We are grateful to the Analytical Center of Nanjing University of Chinese Medicine for identification of the measurements of HR-ESI-MS.
Note 1. These authors contributed equally to this work.
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