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Journal of Asian Natural Products Research Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ganp20
A new cytotoxic benzophenanthridine isoquinoline alkaloid from the fruits of Macleaya cordata a
a
a
a
Hui-Liang Zou , Hong-Yu Li , Bai-Lian Liu & Guang-Xiong Zhou a
Institute of Traditional Chinese Medicine & Natural Products, Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, College of Pharmacy Jinan University, Guangzhou510632, China Published online: 11 Mar 2015.
Click for updates To cite this article: Hui-Liang Zou, Hong-Yu Li, Bai-Lian Liu & Guang-Xiong Zhou (2015): A new cytotoxic benzophenanthridine isoquinoline alkaloid from the fruits of Macleaya cordata, Journal of Asian Natural Products Research, DOI: 10.1080/10286020.2015.1016000 To link to this article: http://dx.doi.org/10.1080/10286020.2015.1016000
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, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
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Journal of Asian Natural Products Research, 2015 http://dx.doi.org/10.1080/10286020.2015.1016000
A new cytotoxic benzophenanthridine isoquinoline alkaloid from the fruits of Macleaya cordata Hui-Liang Zou, Hong-Yu Li, Bai-Lian Liu and Guang-Xiong Zhou* Institute of Traditional Chinese Medicine & Natural Products, Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, College of Pharmacy Jinan University, Guangzhou 510632, China
Downloaded by [University of Arizona] at 11:01 19 April 2015
(Received 25 October 2014; final version received 26 January 2015) A new cytotoxic benzophenanthridine isoquinoline alkaloid, named cordatine (1), together with one known alkaloid 8-methoxydihydrochelerythrine (2), was isolated from the fruits of Macleaya cordata. The structure of the new compound was elucidated by spectroscopic methods including 1D and 2D NMR, HR-ESI-MS. Both compounds indicated significant cytotoxicity against MCF-7 and SF-268 cell lines. Keywords: Macleaya cordata; benzophenanthridine alkaloid; cordatine
1.
Introduction
Macleaya cordata (Willd.) R. Br. is widely distributed in south and northwest China. In traditional Chinese medicine (TCM), the herb is mainly used for its antimicrobial, anti-fungal, anti-inflammatory, and antitumor effects. The isoquinoline alkaloid fraction from M. cordata has been used in toothpastes and mouthwashes as an anti-plaque agent. It is also an additive to animal feeds as a replacement to antibiotics for eliminating the risk of antibiotic resistance [1 – 4]. Chemically, M. cordata has been extensively studied, and shown to be a rich source of isoquinoline alkaloids with protopine and benzophenanthridine skeletons. These alkaloids have also been reported to possess pharmacological activities, such as having antimicrobial, antileukaemic, and antitumor effects. The tertiary isoquinoline alkaloids and quaternary isoquinoline alkaloids are the main effective components for these activities of M. cordata [5]. Recently, most of the study focused on the organs of the roots and stems of this herb. In contrast, only a few alkaloids have been previously reported
from the fruits of M. cordata. In our study, a new alkaloid, named cordatine, was isolated, along with a known one with same molecular formula. This paper describes the isolation and structural determination of aforementioned compounds, and the evaluation of their cytotoxic activities against MCF-7 and SF-268 human cancer cell lines.
2.
Compound 1 was obtained as a pink powder. It showed positive reaction to bismuth potassium iodide reagent. UV spectrum indicated absorption maxima at 230, 285, 320, and 350 nm in MeOH. In the IR spectrum, absorption bands for aromatic rings (1600, 1500, 1460 cm21) were observed. Its molecular formula was deduced to be C22H21NO5 from HR-ESIMS, based on the pseudo-molecular ion at m/z 380.1492 [M þ H]þ. Compound 1 was presumed to be a benzo[c ]-phenanthridine alkaloid by comparison of its UV and 13C NMR data with those of 8-methoxydihydrochelerythrine (2), obtained in our
*Corresponding author. Email: [email protected] q 2015 Taylor & Francis
Results and discussion
2
H.-L. Zou et al.
Table 1. 1H and 13C NMR spectral data of compound 1 (300 MHz for 1H, 75 MHz for in CDCl3, J in Hz).
13
C,
Compound 1
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Position 1 2 3 4 4a 5 6 8 8a 9 10 11 12 12a 13 14 1a 15 8-OMe 9-OMe 12-OMe N– Me
1
H
7.11, 1H, s – – 7.67, 1H, s – 7.45, 1H, d, 8.6 7.75, 1H, d, 8.6 5.44, 1H, s – – 7.06, 1H, d, 8.5 7.57, 1H, d, 8.5 – – – – – 6.05, 2H, s 3.45, 3H, s 3.97, 3H, s 3.46, 3H, s 2.73, 3H, s
experiment and possessing a same molecular composition as 1 [5]. The 1H NMR spectrum of 1 exhibited signals of six aromatic protons at d 7.75 (1H, d, J ¼ 8.6 Hz), 7.67 (1H, s), 7.57 (1H, d, J ¼ 8.5 Hz), 7.45 (1H, d, J ¼ 8.6 Hz), 7.11 (1H, s), and 7.06 (1H, d, J ¼ 8.5 Hz); one ZOCH2O – group at d 6.05 (2H, s); three ZOMe signals at d 3.97, 3.46, and 3.45 (3H, s); and one CH3N – signal at d 2.73 (3H, s) (Table 1). Two protons [d 7.11(1H, s, H-1), 7.67 (1H, s, H-4)] in para positions and two pairs of protons [d 7.45 (1H, d, J ¼ 8.6 Hz, H-5)/7.75 (1H, d, J ¼ 8.6 Hz, H-6); 7.06 (1H, d, J ¼ 8.5 Hz, H-10)/7.57 (1H, d, J ¼ 8.5Hz, H-11)], respectively, in ortho position indicated the presence of the characteristic pattern of tetrasubstituted 7,8-dihydrochelerythrine. The 13C NMR spectrum of 1 showed 22 carbon signals, attributable to three methoxyl carbons (d 48.9, 54.0, 62.7), one methine carbon (d 86.4), one CH3N – carbon (d
13
C
104.8 148.1 147.4 100.6 126.8 123.7 129.1 86.4 124.5 145.0 116.7 120.2 148.9 124.9 122.9 138.0 131.1 101.2 54.0 62.7 48.9 40.6
HMBC C-4, C-5, C-4a, C-3 C-1, C-14, C-1a, C-2 C-1, C-13, C-4a, C-14 C-12a, C-14, C-1a
C-8a, C-9, C-12 C-13, C-12a, C-9
C-2, C-3 C-8 C-9 C-12
40.6), one methylenedioxy carbon (d 101.2), six aromatic methine carbons (d 100.6, 104.8, 116.7, 120.2, 123.7, 129.1) and 10 aromatic quaternary carbons (d 122.9, 124.5, 124.9, 126.8, 131.1, 138.0, 145.0, 147.4, 148.1, 148.9) (Table 1). In the HMBC spectrum, long-range correlations were observed from H-5 to C-1, C-4a, and C-13, from H-6 to C-14, C-1a, and C-12a, from H-10 to C-12, C-8a, and C-12a, and from H-11 to C-9, C-13, and C-10 (Figure 2). So, compound 1 was confirmed to be a 2,3,9,12-tetrasubstituted 7,8-dihydrochelerythrine. In addition, the proton at d 6.02 (2H, s, H-15) showed long-range correlations with C-2 and C-3, indicating that the methylenedioxy linked to C-2 and C-3. The carbon at d 86.4 was correlated with the proton at d 5.44 (1H, s, H-8) in the HSQC spectrum. Furthermore, the proton at d 3.45 (3H, s) showed HMBC correlation with C-8 (d 86.4), showing that a substituent (methoxyl) was linked to C-8.
Journal of Asian Natural Products Research 5
R1 12
10
1
2
O
3
O
respectively. Meanwhile, the data of compound 2 were 21.45 and 4.28 mM, respectively. The IC50 values of positive control vincristine were 37.84 and 48.37 mM, respectively.
13
11
R
1a
6
4a 14 4 7
12a 8a 8
3
N
9
OMe OMe
2 R=OMe, R1=H
1 R=H, R1=OMe
Downloaded by [University of Arizona] at 11:01 19 April 2015
Figure 1. Structures of compounds 1 –2.
Methyl protons at d 3.97 (3H, s) and 3.46 (3H, s) were, respectively, correlated with C-9 and C-12, reasoning that two methoxyl groups were connected respectively to C-9 and C-12. From the evidence aforementioned, 1 was a new compound, named cordatine. Structure of compound 1 is shown in Figure 1. 1D NMR and 2D NMR data of 1 are listed in Table 1. The Key HMBC correlations were indicated in Figure 2. In addition, the known compound, 8-methoxydihydrochelerythrine (2) [6], was also isolated from the extract and identified by comparing its NMR and ESIMS data with those in the literature. Cytotoxic activities of compounds 1 and 2 against MCF-7 and SF-268 cell lines were evaluated using the MTT method [7]. The samples of compounds 1 and 2 both gave a single peak in HPLC analyses, and the purity of both was more than 98%. The bioassay result showed that compound 1 possessed the significant inhibitory activity against MCF-7 cell line with an IC50 value of 34.78 mM, and SF-268 cell line with an IC50 value of 11.79 mM, 1
5
O 12
1a
6
2
O 15
13
11
12a 8a 8
10
4a 14
N
4
3
O
3.
Experimental
3.1. General experimental procedures Melting points were determined on an X-5 micro-MP apparatus (Huayan Corporation, Shanghai, China) and are uncorrected. UV spectra were recorded on a JASCO V-550 UV/VIS spectrometer (JASCO Corporation, Tokyo, Japan). The IR spectra were recorded on a Nicolet Impact 410-FTIR instrument (Thermo, San Jose, CA, USA) in KBr pellets. HR-ESI-MS were acquired using Agilent 6210 LC/MSD TOF mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). 1D and 2D NMR spectra were recorded in CDCl3 using Bruker AV300 spectrometer (Bruker Instrument, Inc., Zurich, Switzerland) with tetramethylsilane (TMS) as the internal standard, and the chemical shifts were expressed in d values (ppm). Reversed-phase HPLC was performed on an Ultimatee XB-C18 column (5 mm, 4.6 £ 250 mm, Welch, Potamac, MA, USA) in Analysis and Materials XBC18 (5 mm, 10 £ 250 mm) for semipreparative purification. Open column chromatography was performed on silica gel (300 – 400 mesh, Qingdao, Haiyang Chemical Group Corporation, Qingdao, China), and ODS (50 mm, YMC, Tokyo, Japan). Sephadex LH-20 (25 – 100 mm) was obtained from Pharmacia (Uppsala, Sweden). HSGF254 silica gel thin-layer chromatography (TLC) plates (0.2 mm thickness, 200 £ 200 mm, Qingdao Marine Chemical, China) were used for routine analysis of fractions.
7
9
O
O
Figure 2. Key HMBC (H ! C) correlations of 1.
3.2. Plant material The experimental material was collected from San Yun Moutain, Jixi county, Anhui province, China, in October 2011. The
4
H.-L. Zou et al.
original plant was authenticated as Macleaya cordata, by Prof. GX Zhou at Office of Teaching and Research in Pharmacognosy, college of pharmacy, Jinan University. A specimen has been deposited in the office.
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3.2.1.
2038C; UV (MeOH) lmax: 230, 285, 320, 350 nm; IR (KBr) nmax: 1600, 1500, 1460, 1279, 1259, 1042, 993, 878, 598 cm21; 1H NMR (300 MHz, CDCl3) and 13C NMR (75 MHz, CDCl 3) spectral data, see Table 1; HR-ESI-MS: m/z 380.1492 [M þ H] þ (calcd for C22H 22NO 5, 380.1497).
Extraction and isolation
The dried fruits of Macleaya cordata (15 kg) were extracted with 85% alcohol for three times under reflux. The extracts were combined and concentrated under reduced pressure to about 1200 g. Then, the extract was suspended in distilled water and partitioned successively with petroleum ether and ethyl acetate to afford 100 and 150 g of extracts, respectively. Then, the pH value of the water layer was regulated to pH 9– 10 with NH3·H2O, and the basic solution was extracted with ethyl acetate. Crude alkaloid extract (200 g) was obtained after removing the solvent under reduced pressure, and 300 g of watersoluble residue was retained. The crude alkaloid extract (200 g) was subjected to silica gel chromatography eluting with gradient chloroform – acetone systems (100:0 ! 100:2 ! 100:3 ! 20:1 ! 9:1 ! 8:2 ! 7:3 ! 6:4 ! 1:1 ! 0:100) to afford 15 fractions (Fr-1– 15) after combination of fractions by TLC pattern. Fr-10 (8 g) was subjected to column chromatography with silica gel, eluting with petroleum ether – EtOAc (100:1, 100:2, 100:4, 100:7, 100:10, 100:20, 100:30, 100:50, 1:1, 1:2) to afford 10 subfractions (Sfr-1 to Sfr-10). Sfr-5 (60 mg) was purified on Sephadex LH-20 column eluting with CHCl3:MeOH (1:1) to obtain compound 1 (20 mg). Compound 2 (18 mg) was obtained from Sfr-6 (70 mg) after purified with Sephadex LH-20 (CHCl3:CH3OH – 1:1). 3.2.2. Cordatine Pink powder; positive reaction to bismuth potassium iodide reagent; m.p. 202 –
3.4.
Cytotoxic assays
Two human cancer cell lines were used in the MTT assay. Cell viability was measured by MTT colorimetric method. MCF-7 and SF-268 cell lines were obtained from the College of Medicine, Jinan University (Guangzhou, China). All the cell lines were cultured in RPMI-1640 media containing 10% (v/v) newborn calf serum (NBCS), 100 units/ml penicillin, and 100 mg/ml streptomycin at 378C in humidified atmosphere of 5% CO2 and 95% air. The positive drug of this test is vincristine. Five concentrations (12.5, 25, 50, 100, 200, and 400 mg/ml) of compound 1 were set for the test. In brief, cells were seeded at 8000 cells/100 ml/well in 96-well plates for 24 h, and then treated with two natural compounds and vincristine at different concentrations for 72 h. Subsequently, 12 ml of MTT dye (5 mg/ml in phosphate buffered saline) was added to each well, and the plates were incubated at 378C for 4 h. The surviving cells converted MTT to formazan crystals which were dissolved in 100 ml/well of DMSO, generating a blue-purple color. The absorbance was monitored at 570 nm using a microplate spectrophotometer.
Disclosure statement No potential conflict of interest was reported by the authors.
Funding We gratefully acknowledge the financial support from the Chinese National S&T Special
Journal of Asian Natural Products Research Project on Major New Drug Innovation (2011ZX09307-002-01).
References
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[1] F. Feng, F.Z. Ye, C.L. Li, W.Y. Liu, and N. Xie, Chin. J. Nat. Med 10, 0378 (2012). [2] Z.B. Hu, Y. Xu, S.C. Feng, and G.J. Fan, Acta. Pharm. Sin 14, 535 (1979). [3] L. Ouyang, X.L. Su, D.S. He, Y.Y. Chen, M. Ma, Q.J. Xie, and S.Z. Yao, J. Sep. Sci 33, 2026 (2010).
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[4] M. Stiborova, J. Vostalova, A. Zdarilova, J. Ulrichova, J. Hudecek, K. Tschirner, and V. Simanek, Biol. Pap. Med. Fac. Univ. Pala. Olom. Czec. Repu 152, 35 (2008). [5] A.J. Deng and H.L. Qin, Phytochemistry 71, 816 (2010). [6] L. Xu, S.L. Niu, Z.L. Wu, X. Liu, and F. Shi, Chin. Tradit. Herb Drug 40, 538 (2009). [7] T. Mosmann, J. Immunol. Methods 65, 55 (1983).
Journal of Asian Natural Products Research Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ganp20
A new cytotoxic benzophenanthridine isoquinoline alkaloid from the fruits of Macleaya cordata a
a
a
a
Hui-Liang Zou , Hong-Yu Li , Bai-Lian Liu & Guang-Xiong Zhou a
Institute of Traditional Chinese Medicine & Natural Products, Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, College of Pharmacy Jinan University, Guangzhou510632, China Published online: 11 Mar 2015.
Click for updates To cite this article: Hui-Liang Zou, Hong-Yu Li, Bai-Lian Liu & Guang-Xiong Zhou (2015): A new cytotoxic benzophenanthridine isoquinoline alkaloid from the fruits of Macleaya cordata, Journal of Asian Natural Products Research, DOI: 10.1080/10286020.2015.1016000 To link to this article: http://dx.doi.org/10.1080/10286020.2015.1016000
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, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
Downloaded by [University of Arizona] at 11:01 19 April 2015
Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Journal of Asian Natural Products Research, 2015 http://dx.doi.org/10.1080/10286020.2015.1016000
A new cytotoxic benzophenanthridine isoquinoline alkaloid from the fruits of Macleaya cordata Hui-Liang Zou, Hong-Yu Li, Bai-Lian Liu and Guang-Xiong Zhou* Institute of Traditional Chinese Medicine & Natural Products, Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, College of Pharmacy Jinan University, Guangzhou 510632, China
Downloaded by [University of Arizona] at 11:01 19 April 2015
(Received 25 October 2014; final version received 26 January 2015) A new cytotoxic benzophenanthridine isoquinoline alkaloid, named cordatine (1), together with one known alkaloid 8-methoxydihydrochelerythrine (2), was isolated from the fruits of Macleaya cordata. The structure of the new compound was elucidated by spectroscopic methods including 1D and 2D NMR, HR-ESI-MS. Both compounds indicated significant cytotoxicity against MCF-7 and SF-268 cell lines. Keywords: Macleaya cordata; benzophenanthridine alkaloid; cordatine
1.
Introduction
Macleaya cordata (Willd.) R. Br. is widely distributed in south and northwest China. In traditional Chinese medicine (TCM), the herb is mainly used for its antimicrobial, anti-fungal, anti-inflammatory, and antitumor effects. The isoquinoline alkaloid fraction from M. cordata has been used in toothpastes and mouthwashes as an anti-plaque agent. It is also an additive to animal feeds as a replacement to antibiotics for eliminating the risk of antibiotic resistance [1 – 4]. Chemically, M. cordata has been extensively studied, and shown to be a rich source of isoquinoline alkaloids with protopine and benzophenanthridine skeletons. These alkaloids have also been reported to possess pharmacological activities, such as having antimicrobial, antileukaemic, and antitumor effects. The tertiary isoquinoline alkaloids and quaternary isoquinoline alkaloids are the main effective components for these activities of M. cordata [5]. Recently, most of the study focused on the organs of the roots and stems of this herb. In contrast, only a few alkaloids have been previously reported
from the fruits of M. cordata. In our study, a new alkaloid, named cordatine, was isolated, along with a known one with same molecular formula. This paper describes the isolation and structural determination of aforementioned compounds, and the evaluation of their cytotoxic activities against MCF-7 and SF-268 human cancer cell lines.
2.
Compound 1 was obtained as a pink powder. It showed positive reaction to bismuth potassium iodide reagent. UV spectrum indicated absorption maxima at 230, 285, 320, and 350 nm in MeOH. In the IR spectrum, absorption bands for aromatic rings (1600, 1500, 1460 cm21) were observed. Its molecular formula was deduced to be C22H21NO5 from HR-ESIMS, based on the pseudo-molecular ion at m/z 380.1492 [M þ H]þ. Compound 1 was presumed to be a benzo[c ]-phenanthridine alkaloid by comparison of its UV and 13C NMR data with those of 8-methoxydihydrochelerythrine (2), obtained in our
*Corresponding author. Email: [email protected] q 2015 Taylor & Francis
Results and discussion
2
H.-L. Zou et al.
Table 1. 1H and 13C NMR spectral data of compound 1 (300 MHz for 1H, 75 MHz for in CDCl3, J in Hz).
13
C,
Compound 1
Downloaded by [University of Arizona] at 11:01 19 April 2015
Position 1 2 3 4 4a 5 6 8 8a 9 10 11 12 12a 13 14 1a 15 8-OMe 9-OMe 12-OMe N– Me
1
H
7.11, 1H, s – – 7.67, 1H, s – 7.45, 1H, d, 8.6 7.75, 1H, d, 8.6 5.44, 1H, s – – 7.06, 1H, d, 8.5 7.57, 1H, d, 8.5 – – – – – 6.05, 2H, s 3.45, 3H, s 3.97, 3H, s 3.46, 3H, s 2.73, 3H, s
experiment and possessing a same molecular composition as 1 [5]. The 1H NMR spectrum of 1 exhibited signals of six aromatic protons at d 7.75 (1H, d, J ¼ 8.6 Hz), 7.67 (1H, s), 7.57 (1H, d, J ¼ 8.5 Hz), 7.45 (1H, d, J ¼ 8.6 Hz), 7.11 (1H, s), and 7.06 (1H, d, J ¼ 8.5 Hz); one ZOCH2O – group at d 6.05 (2H, s); three ZOMe signals at d 3.97, 3.46, and 3.45 (3H, s); and one CH3N – signal at d 2.73 (3H, s) (Table 1). Two protons [d 7.11(1H, s, H-1), 7.67 (1H, s, H-4)] in para positions and two pairs of protons [d 7.45 (1H, d, J ¼ 8.6 Hz, H-5)/7.75 (1H, d, J ¼ 8.6 Hz, H-6); 7.06 (1H, d, J ¼ 8.5 Hz, H-10)/7.57 (1H, d, J ¼ 8.5Hz, H-11)], respectively, in ortho position indicated the presence of the characteristic pattern of tetrasubstituted 7,8-dihydrochelerythrine. The 13C NMR spectrum of 1 showed 22 carbon signals, attributable to three methoxyl carbons (d 48.9, 54.0, 62.7), one methine carbon (d 86.4), one CH3N – carbon (d
13
C
104.8 148.1 147.4 100.6 126.8 123.7 129.1 86.4 124.5 145.0 116.7 120.2 148.9 124.9 122.9 138.0 131.1 101.2 54.0 62.7 48.9 40.6
HMBC C-4, C-5, C-4a, C-3 C-1, C-14, C-1a, C-2 C-1, C-13, C-4a, C-14 C-12a, C-14, C-1a
C-8a, C-9, C-12 C-13, C-12a, C-9
C-2, C-3 C-8 C-9 C-12
40.6), one methylenedioxy carbon (d 101.2), six aromatic methine carbons (d 100.6, 104.8, 116.7, 120.2, 123.7, 129.1) and 10 aromatic quaternary carbons (d 122.9, 124.5, 124.9, 126.8, 131.1, 138.0, 145.0, 147.4, 148.1, 148.9) (Table 1). In the HMBC spectrum, long-range correlations were observed from H-5 to C-1, C-4a, and C-13, from H-6 to C-14, C-1a, and C-12a, from H-10 to C-12, C-8a, and C-12a, and from H-11 to C-9, C-13, and C-10 (Figure 2). So, compound 1 was confirmed to be a 2,3,9,12-tetrasubstituted 7,8-dihydrochelerythrine. In addition, the proton at d 6.02 (2H, s, H-15) showed long-range correlations with C-2 and C-3, indicating that the methylenedioxy linked to C-2 and C-3. The carbon at d 86.4 was correlated with the proton at d 5.44 (1H, s, H-8) in the HSQC spectrum. Furthermore, the proton at d 3.45 (3H, s) showed HMBC correlation with C-8 (d 86.4), showing that a substituent (methoxyl) was linked to C-8.
Journal of Asian Natural Products Research 5
R1 12
10
1
2
O
3
O
respectively. Meanwhile, the data of compound 2 were 21.45 and 4.28 mM, respectively. The IC50 values of positive control vincristine were 37.84 and 48.37 mM, respectively.
13
11
R
1a
6
4a 14 4 7
12a 8a 8
3
N
9
OMe OMe
2 R=OMe, R1=H
1 R=H, R1=OMe
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Figure 1. Structures of compounds 1 –2.
Methyl protons at d 3.97 (3H, s) and 3.46 (3H, s) were, respectively, correlated with C-9 and C-12, reasoning that two methoxyl groups were connected respectively to C-9 and C-12. From the evidence aforementioned, 1 was a new compound, named cordatine. Structure of compound 1 is shown in Figure 1. 1D NMR and 2D NMR data of 1 are listed in Table 1. The Key HMBC correlations were indicated in Figure 2. In addition, the known compound, 8-methoxydihydrochelerythrine (2) [6], was also isolated from the extract and identified by comparing its NMR and ESIMS data with those in the literature. Cytotoxic activities of compounds 1 and 2 against MCF-7 and SF-268 cell lines were evaluated using the MTT method [7]. The samples of compounds 1 and 2 both gave a single peak in HPLC analyses, and the purity of both was more than 98%. The bioassay result showed that compound 1 possessed the significant inhibitory activity against MCF-7 cell line with an IC50 value of 34.78 mM, and SF-268 cell line with an IC50 value of 11.79 mM, 1
5
O 12
1a
6
2
O 15
13
11
12a 8a 8
10
4a 14
N
4
3
O
3.
Experimental
3.1. General experimental procedures Melting points were determined on an X-5 micro-MP apparatus (Huayan Corporation, Shanghai, China) and are uncorrected. UV spectra were recorded on a JASCO V-550 UV/VIS spectrometer (JASCO Corporation, Tokyo, Japan). The IR spectra were recorded on a Nicolet Impact 410-FTIR instrument (Thermo, San Jose, CA, USA) in KBr pellets. HR-ESI-MS were acquired using Agilent 6210 LC/MSD TOF mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). 1D and 2D NMR spectra were recorded in CDCl3 using Bruker AV300 spectrometer (Bruker Instrument, Inc., Zurich, Switzerland) with tetramethylsilane (TMS) as the internal standard, and the chemical shifts were expressed in d values (ppm). Reversed-phase HPLC was performed on an Ultimatee XB-C18 column (5 mm, 4.6 £ 250 mm, Welch, Potamac, MA, USA) in Analysis and Materials XBC18 (5 mm, 10 £ 250 mm) for semipreparative purification. Open column chromatography was performed on silica gel (300 – 400 mesh, Qingdao, Haiyang Chemical Group Corporation, Qingdao, China), and ODS (50 mm, YMC, Tokyo, Japan). Sephadex LH-20 (25 – 100 mm) was obtained from Pharmacia (Uppsala, Sweden). HSGF254 silica gel thin-layer chromatography (TLC) plates (0.2 mm thickness, 200 £ 200 mm, Qingdao Marine Chemical, China) were used for routine analysis of fractions.
7
9
O
O
Figure 2. Key HMBC (H ! C) correlations of 1.
3.2. Plant material The experimental material was collected from San Yun Moutain, Jixi county, Anhui province, China, in October 2011. The
4
H.-L. Zou et al.
original plant was authenticated as Macleaya cordata, by Prof. GX Zhou at Office of Teaching and Research in Pharmacognosy, college of pharmacy, Jinan University. A specimen has been deposited in the office.
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3.2.1.
2038C; UV (MeOH) lmax: 230, 285, 320, 350 nm; IR (KBr) nmax: 1600, 1500, 1460, 1279, 1259, 1042, 993, 878, 598 cm21; 1H NMR (300 MHz, CDCl3) and 13C NMR (75 MHz, CDCl 3) spectral data, see Table 1; HR-ESI-MS: m/z 380.1492 [M þ H] þ (calcd for C22H 22NO 5, 380.1497).
Extraction and isolation
The dried fruits of Macleaya cordata (15 kg) were extracted with 85% alcohol for three times under reflux. The extracts were combined and concentrated under reduced pressure to about 1200 g. Then, the extract was suspended in distilled water and partitioned successively with petroleum ether and ethyl acetate to afford 100 and 150 g of extracts, respectively. Then, the pH value of the water layer was regulated to pH 9– 10 with NH3·H2O, and the basic solution was extracted with ethyl acetate. Crude alkaloid extract (200 g) was obtained after removing the solvent under reduced pressure, and 300 g of watersoluble residue was retained. The crude alkaloid extract (200 g) was subjected to silica gel chromatography eluting with gradient chloroform – acetone systems (100:0 ! 100:2 ! 100:3 ! 20:1 ! 9:1 ! 8:2 ! 7:3 ! 6:4 ! 1:1 ! 0:100) to afford 15 fractions (Fr-1– 15) after combination of fractions by TLC pattern. Fr-10 (8 g) was subjected to column chromatography with silica gel, eluting with petroleum ether – EtOAc (100:1, 100:2, 100:4, 100:7, 100:10, 100:20, 100:30, 100:50, 1:1, 1:2) to afford 10 subfractions (Sfr-1 to Sfr-10). Sfr-5 (60 mg) was purified on Sephadex LH-20 column eluting with CHCl3:MeOH (1:1) to obtain compound 1 (20 mg). Compound 2 (18 mg) was obtained from Sfr-6 (70 mg) after purified with Sephadex LH-20 (CHCl3:CH3OH – 1:1). 3.2.2. Cordatine Pink powder; positive reaction to bismuth potassium iodide reagent; m.p. 202 –
3.4.
Cytotoxic assays
Two human cancer cell lines were used in the MTT assay. Cell viability was measured by MTT colorimetric method. MCF-7 and SF-268 cell lines were obtained from the College of Medicine, Jinan University (Guangzhou, China). All the cell lines were cultured in RPMI-1640 media containing 10% (v/v) newborn calf serum (NBCS), 100 units/ml penicillin, and 100 mg/ml streptomycin at 378C in humidified atmosphere of 5% CO2 and 95% air. The positive drug of this test is vincristine. Five concentrations (12.5, 25, 50, 100, 200, and 400 mg/ml) of compound 1 were set for the test. In brief, cells were seeded at 8000 cells/100 ml/well in 96-well plates for 24 h, and then treated with two natural compounds and vincristine at different concentrations for 72 h. Subsequently, 12 ml of MTT dye (5 mg/ml in phosphate buffered saline) was added to each well, and the plates were incubated at 378C for 4 h. The surviving cells converted MTT to formazan crystals which were dissolved in 100 ml/well of DMSO, generating a blue-purple color. The absorbance was monitored at 570 nm using a microplate spectrophotometer.
Disclosure statement No potential conflict of interest was reported by the authors.
Funding We gratefully acknowledge the financial support from the Chinese National S&T Special
Journal of Asian Natural Products Research Project on Major New Drug Innovation (2011ZX09307-002-01).
References
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