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A new quaternary protoberberine alkaloid isolated from Dicranostigma leptopodum (Maxim) Fedde ab
c
a
a
Mei Zhong , Yu-Xi Ma , Jun-Xi Liu & Duo-Long Di a
Key Laboratory of Chemistry of Northwestern Plant Resources and Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 73000, P.R. China b
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Graduate University of Chinese Academy Sciences, Beijing 100049, P.R. China c
College of Chemistry and Chemistry Engineering, Xiamen University, Xiamen 361005, P.R. China Published online: 05 Feb 2014.
To cite this article: Mei Zhong, Yu-Xi Ma, Jun-Xi Liu & Duo-Long Di (2014) A new quaternary protoberberine alkaloid isolated from Dicranostigma leptopodum (Maxim) Fedde, Natural Product Research: Formerly Natural Product Letters, 28:8, 507-510, DOI: 10.1080/14786419.2013.879586 To link to this article: http://dx.doi.org/10.1080/14786419.2013.879586
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, 2014 Vol. 28, No. 8, 507–510, http://dx.doi.org/10.1080/14786419.2013.879586
A new quaternary protoberberine alkaloid isolated from Dicranostigma leptopodum (Maxim) Fedde Mei Zhongab, Yu-Xi Mac, Jun-Xi Liua* and Duo-Long Dia
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a
Key Laboratory of Chemistry of Northwestern Plant Resources and Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 73000, P.R. China; bGraduate University of Chinese Academy Sciences, Beijing 100049, P.R. China; cCollege of Chemistry and Chemistry Engineering, Xiamen University, Xiamen 361005, P.R. China (Received 8 November 2013; final version received 26 December 2013) Phytochemical investigation of the whole plants of Dicranostigma leptopodum (Maxim) Fedde has led to the isolation of two quaternary protoberberine alkaloids 5-hydroxy-coptisine (1) and berberrubine (2). This type of alkaloids was isolated from the genus Dicranostigma for the first time and the new compound structure (1) was elucidated by various spectroscopic methods including 2D NMR techniques (gCOSY, HMQC and HMBC) and HR-ESI-MS. Keywords: Papaveraceae; Dicranostigma leptopodum; quaternary protoberberine; alkaloids; 5-hydroxy-coptisine
1. Introduction The genus Dicranostigma (Papaveraceae) has been used as a folk medicine for the treatment of tonsillitis and hepatitis, and an anti-inflammatory in China for a long time, and several alkaloids have been isolated from those plants (FRPS 1999). Especially, isocorydine has been abundantly extracted from Dicranostigma leptopodum (Maxim) Fedde by our team (Dang et al. 2009; Liu et al. 2011, 2013; Zhang et al. 2011; Bo et al. 2013). Isocorydine has been demonstrated to not only inhibit cell proliferation in hepatocellular carcinoma cell lines by inducing G2/M cell cycle arrest and apoptosis, but also target drug-resistant cellular side population through PDCD4-related apoptosis in hepatocellular carcinoma. It might be a potential therapeutic drug for chemotherapeutic treatment of hepatocellular carcinoma (Lu et al. 2012; Sun et al. 2012). Therefore, our group selected isocorydine as the starting material, modified the chemical structure and screened for potent antitumour candidates; however, the results were not ideal. The extracts from D. leptopodum (Maxim) Fedde including alkaloids have been confirmed to have significant outcomes in inhibiting the growth and progression of the tumour (Zhang et al. 2010). As a continuation of our investigation to discover cytotoxic compounds from natural sources, we selected D. leptopodum (Maxim) Fedde for screening the target compound, which is mainly distributed in the northwest of China. Although, the chemical constituents of this plant have been reported previously, a new quaternary protoberberine alkaloids 5-hydroxy-coptisine (1) and berberrubine (2) have been isolated (Figure 1). In this article, the isolation and structural elucidation of 1 are reported. 2. Results and discussion Compound 1 was obtained as a yellow amorphous powder with m.p. 258– 2598C, ½a20 D þ 7:58 (c 1.00, MeOH) which gave a positive visualisation to the Dragendorff’s test. Its molecular
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
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M. Zhong et al.
O 3
4 A
O 2
OH 5 B
1
O
6 N+ C
N+
O
8
OH
9 O
13 D 12 1
11
OCH3
10 O 2
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Figure 1. Chemical structures of 1 and 2. þ formula was assigned as C19 H14 NOþ 5 on the basis of the HR-ESI-MS (m/z ¼ 336.0862 [M] , þ calcd for C19 H14 NO5 : 336.0866). The UV spectrum of 1 revealed absorption maxima at 231 (log 1 ¼ 1.22), 270 (log 1 ¼ 2.52) and 355 nm (log 1 ¼ 2.61). Meanwhile, the IR (KBr) spectrum revealed characteristics bands of a hydroxylated aromatic group (3434 cm21) and aryl groups (1623.0, 1507.4, 1361.4, 1332.4 and 1050.7 cm21). The 1H NMR spectrum of 1 revealed an AB coupling system at d 7.82 (d, 1H, J ¼ 8.8 Hz, H-11), d 8.04 (d, 1H, J ¼ 8.8 Hz, H-12) and two singlet protons at d 7.81(s, 1H, H-1), d 7.15 (s, 1H, H-4) indicated four aromatic substitutions at C-2, 3, 9 and C-10 on A and D rings of the protoberberine alkaloids, and two OZCH2ZO groups substitutions could be confirmed by two methylene carbon signals (d 102.3 and d 104.6) which was assigned by a DEPT experiment. Particularly, the unusual low-field proton signals at d 10.05 (H-8) and d 9.02 (H-13) reminded us the resonance of the quaternary protoberberine alkaloids (Young et al. 2002; Dosta´la et al. 2004). The 1H and 13C NMR spectra of 1 were almost identical with coptisine, a common quaternary protoberberine alkaloid, except for the carbon signals on B ring. The methylene (d 60.8) and methane (d 62.6) groups located at B ring supported the segment to be hydroxylated at C-5; this was supported by the HMBC correlations of H-6 with C-4a, 13a and C-8 (see Figure S1). The hydroxyl group located at C-5 was b-oriented which could be confirmed by comparing the specific rotation of compound 1 with berberastine and thalidastine (Young et al. 2002; Dosta´la et al. 2004). The linkages of the abovementioned groups could be confirmed by the HMBC experiment. The HMBC correlations of H-4 with C-2, 5, 13b, H-1 with C-2, 13a and H-13 with C-13b demonstrated the attached position of A ring. The correlations of H-8 with C-6, 9, 12a, H-12 with C-10 demonstrated the position of D ring (Figure 1). Thus, the structure was proposed as 1 and named as 5-hydroxy-coptisine. The original spectra (IR, 1H NMR, 13C NMR, DEPT, 1H – 1H COSY, HMQC, HMBC and HR-ESIMS) of 1 is provided in the supplementary materials).
3. Experimental 3.1. General Melting points were obtained on an X-4 digital display micro-melting point apparatus (Taike Company Limited, Beijing, China) which were not corrected. Optical rotation was measured on an Automatic Polarimeter A21201-T (Rudolph Research Analytical Corporation, Hackettstown, NJ, USA) in MeOH solution. UV spectra were recorded on a Perkin Elmer Lambda 35 spectrophotometer (Perkin-Elmer Company, Waltham, MA, USA) in MeOH solution. IR spectra were recorded on a Nicolet NEXUS 670 FT-IR spectrometer (Thermo Nicolet Corporation, Madison, WI, USA) using KBr pellets. 1H NMR (400.16 Hz), 13C NMR (100.32 Hz) and 2D NMR spectra were recorded on a Varian INOVA-400 MHz-FTNMR spectrometer (Varian Medical Systems Incorporation, Palo Alto, CA, USA) with tetramethylsilane as the internal standard, and HR-ESI-MS, on a Bruker APEX II (Bruker Corporation, Madison, WI, USA). Circular dichroism (CD) data were observed on an Olis RSM
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1000 CD (Olis, Incorporation, Atlanta, GA, USA). Silica gel (200 –300 mesh) was used for column chromatography (CC) and silica GF254 (10 –40 mm) for thin-layer chromatography (TLC). The spots were detected on TLC under UV light or sprayed with Dragendorff’s agent.
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3.2. Plant material The whole plants of D. leptopodum (Maxim) Fedde were collected at Pinliang of Gansu Province in May 2012 and identified by Prof. Zhi-Gang Ma at the Lanzhou University, Lanzhou, China. A voucher specimen (no. ZY201202) has been deposited at the Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, China. 3.3. Extraction and isolation The dried plants of D. leptopodum (Maxim) Fedde were pulverised and extracted at room temperature with 95% ethanol (3 £ 7 d). The solvent was removed under reduced pressure in a rotary evaporator to give a residue (200.0 g). The collected residues were suspended in water (1000 mL) and the suspension was extracted with the same volumes of petroleum ether (60–908C), chloroform and n-BuOH, successively. The n-BuOH extract (30 g) was subjected to CC over silica gel (200–300 mesh, 400 g) eluted with a gradient of CHCl3 –MeOH–H2O (gradient from 10:1:0.1 to 7:3:1) according to the differences in composition indicated by TLC; four crude fractions F1 (5 g), F2 (3 g), F3 (2 g) and F4 (4 g) were obtained. Compounds 1 (15.0 mg) and 2 (20.0 mg) were isolated from F4 with CHCl3 –MeOH–H2O (7:3:1) on CC over silica gel, respectively. 3.4. Compounds isolated 5-hydroxy-coptisine (1): yellow amorphous powder; m.p. 258– 2598C; ½a20 D þ 7:58 (c 1.00, MeOH); UV: lmax(log 1) ¼ 231 (1.22), 270 (2.52), 355 (2.61) nm; IR (KBr): nmax ¼ 3434.9, 2910.1, 2351.1, 1623, 1507, 1473.8, 1361.4, 1332.4, 1050.7, 617.4 cm21), HR-ESI-MS: 1 m/z ¼ 336.0862 [M]þ (calcd for C19 H14 NOþ 5 : 336.0866); H NMR (400 MHz, DMSO-d6): d ¼ 7.81 (1H, s, H-1), d ¼ 7.15 (1H, s, H-4), d ¼ 4.82 (1H, dd, J ¼ 2.0, 11.2 Hz, H-5), d ¼ 5.03 (2H, dd, J ¼ 2.0, 11.2 Hz, H-6), d ¼ 10.05 (1H, s, H-8), d ¼ 7.82 (1H, d, J ¼ 8.8 Hz, H-11), d ¼ 8.04 (1H, d, J ¼ 8.8 Hz, H-12), d ¼ 9.02 (1H, s, H-13), d ¼ 6.19 (2H, d, J ¼ 4.0 Hz, H-2, 3-CH2-), d ¼ 6.54 (2H, d, J ¼ 4.0 Hz, H-9, 10-CH2 –); 13C NMR (100 MHz, DMSO-d6): d ¼ 105.2 (C-1), 148.6 (C-2), 149.8 (C-3), 108.0 (C-4), 132.2 (C-4a), 62.6 (C-5), 60.8 (C-6), 145.2 (C-8), 111.8 (C-8a), 143.9 (C-9), 147.3 (C-10), 121.2 (C-11), 121.8 (C-12), 132.3 (C-12a), 121.1 (C-13), 136.3 (C-13a), 119.7 (C-13b), 102.3 (C-2, 3-CH2 –), 104.6 (C-9, 10-CH2-). Berberrubine (2): yellow amorphous powder; HR-ESI-MS: m/z ¼ 332.1072 [M]þ (calcd for 1 C19 H16 NOþ 4 : 322.1074); H NMR (400 MHz, DMSO-d6): d ¼ 7.56 (1H, s, H-1), d ¼ 7.03 (1H, s, H-4), d ¼ 3.15 (1H, t, J ¼ 2.0, 11.2 Hz, H-5), d ¼ 4.89 (2H, t, J ¼ 2.0, 11.2 Hz, H-6), d ¼ 9.96 (1H, s, H-8), d ¼ 7.87 (1H, d, J ¼ 8.4 Hz, H-11), d ¼ 8.01 (1H, d, J ¼ 8.4 Hz, H-12), d ¼ 8.81 (1H, s, H-13), d ¼ 6.51 (2H, d, J ¼ 4.0 Hz, H-2, 3-CH2 – ), d ¼ 3.86(3H, s, H-10CH3O – ); 13C NMR (100 MHz, DMSO-d6): d ¼ 111.4 (C-1), 146.5 (C-2), 146.8 (C-3), 111.6 (C4), 126.8 (C-4a), 25.9 (C-5), 55.4 (C-6), 144.5 (C-8), 119.1 (C-8a), 143.6 (C-9), 150.6 (C-10), 112.1 (C-11), 121.8 (C-12), 132.5 (C-12a), 120.8 (C-13), 137.2 (C-13a), 120.3 (C-13b), 104.4 (C-2, 3-CH2 –), 55.9 (C-10-CH2 –). 4. Conclusion The quaternary protoberberine alkaloids, 5-hydroxy-coptisine (1) and berberrubine (2), were isolated from the genus Dicranostigma for the first time. The extracts from D. leptopodum
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(Maxim) Fedde have been confirmed to express significant antitumour activity, and quaternary protoberberine alkaloids have been confirmed to possess cytotoxic activities such as apoptosis and inhibition of telomerase (Grycova et al. 2007). Therefore, we predict that the quaternary protoberberine alkaloids should be the material basis for its antitumour effects, although this plant possessed a limited content. Supplementary material Supplementary material relating to this article is available online, alongside Figures S1 – S9.
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Acknowledgements This work was supported by the ‘West Light Program’, ‘Build Coalitions of the National Academy of Sciences’ of the Chinese Academy of Sciences and the Science and Technology Program of Gansu (no. 1304FKCA062).
References Bo LN, Zhao JX, Liu JX, Li WG, Liu YW. 2013. Antitumor activity of isocorydione in vitro and vivo. Chin Pharmacol Bull. 29:832–836. Dang Y, Gong HF, Liu JX, Yu SJ. 2009. Alkaloid from Dicranostigma leptopodum (Maxim) Fedde. Chin Chem Lett. 20:1218–1220. Dosta´la J, Man S, Secˇka´rˇova´ P, Hulova´ D, Dommissef R. 2004. Berberine and coptisine free bases. J Mol Struct. 687:135–142. Grycova L, Dosta J, Marek R. 2007. Quaternary protoberberine alkaloids. Phytochemistry. 68:150– 175. Liu YJ, Liu JX, Di DL, Li M, Fen Y. 2013. Structural and mechanistic bases of the anticancer activity of natural aporphinoid alkaloids. Curr Top Med Chem. 13:2116–2126. Liu DH, Zhang TC, Liu JX, Di DL. 2011. Chemical constituents of alkaloids from Dicranostigma leptopodum. Chin Tradit Herb Drugs. 42:1505–1508. Lu P, Sun HF, Zhang LX, Li JJ. 2012. Isocorydine targets the drug-resistant cellular side population through PDCD4related apoptosis in hepatocellular carcinoma. Mol Med. 18:1136–1146. Sun HF, Hou HL, Lu P, Li JJ. 2012. Isocorydine inhibits cell proliferation in hepatocellular carcinoma cell lines by inducing G2/M cell cycle arrest and apoptosis. PLoS One. 7:e36808. Wu ZY, Zhuang X, Su ZY. 1999. Flora Republican Popularis Sinicae. 32. Beijing: Science Press; p. 61. Young WJ, Jin WJ, Miran K, In KC, Weontae L. 2002. NMR studies on antitumor drug candidates berberine and berberrubine. Bull Korean Chem Soc. 23:391–394. Zhang WH, Lv MH, Hai J, Wang QP. 2010. Dicranostigma leptopodum (maxim) Fedde induced apoptosis in SMMC7721 human hepatoma cells and inhibited tumor growth in mice. Nat Sci. 2:457–463. Zhang TC, Ye HL, Liu JX, Di DL. 2011. Study on semi-synthetic transforming technology for the natural product of isocorydione. Acta Pharmcol Sin. 46:1471–1475.
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
A new quaternary protoberberine alkaloid isolated from Dicranostigma leptopodum (Maxim) Fedde ab
c
a
a
Mei Zhong , Yu-Xi Ma , Jun-Xi Liu & Duo-Long Di a
Key Laboratory of Chemistry of Northwestern Plant Resources and Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 73000, P.R. China b
Click for updates
Graduate University of Chinese Academy Sciences, Beijing 100049, P.R. China c
College of Chemistry and Chemistry Engineering, Xiamen University, Xiamen 361005, P.R. China Published online: 05 Feb 2014.
To cite this article: Mei Zhong, Yu-Xi Ma, Jun-Xi Liu & Duo-Long Di (2014) A new quaternary protoberberine alkaloid isolated from Dicranostigma leptopodum (Maxim) Fedde, Natural Product Research: Formerly Natural Product Letters, 28:8, 507-510, DOI: 10.1080/14786419.2013.879586 To link to this article: http://dx.doi.org/10.1080/14786419.2013.879586
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, 2014 Vol. 28, No. 8, 507–510, http://dx.doi.org/10.1080/14786419.2013.879586
A new quaternary protoberberine alkaloid isolated from Dicranostigma leptopodum (Maxim) Fedde Mei Zhongab, Yu-Xi Mac, Jun-Xi Liua* and Duo-Long Dia
Downloaded by [National Sun Yat-Sen University] at 00:35 08 January 2015
a
Key Laboratory of Chemistry of Northwestern Plant Resources and Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 73000, P.R. China; bGraduate University of Chinese Academy Sciences, Beijing 100049, P.R. China; cCollege of Chemistry and Chemistry Engineering, Xiamen University, Xiamen 361005, P.R. China (Received 8 November 2013; final version received 26 December 2013) Phytochemical investigation of the whole plants of Dicranostigma leptopodum (Maxim) Fedde has led to the isolation of two quaternary protoberberine alkaloids 5-hydroxy-coptisine (1) and berberrubine (2). This type of alkaloids was isolated from the genus Dicranostigma for the first time and the new compound structure (1) was elucidated by various spectroscopic methods including 2D NMR techniques (gCOSY, HMQC and HMBC) and HR-ESI-MS. Keywords: Papaveraceae; Dicranostigma leptopodum; quaternary protoberberine; alkaloids; 5-hydroxy-coptisine
1. Introduction The genus Dicranostigma (Papaveraceae) has been used as a folk medicine for the treatment of tonsillitis and hepatitis, and an anti-inflammatory in China for a long time, and several alkaloids have been isolated from those plants (FRPS 1999). Especially, isocorydine has been abundantly extracted from Dicranostigma leptopodum (Maxim) Fedde by our team (Dang et al. 2009; Liu et al. 2011, 2013; Zhang et al. 2011; Bo et al. 2013). Isocorydine has been demonstrated to not only inhibit cell proliferation in hepatocellular carcinoma cell lines by inducing G2/M cell cycle arrest and apoptosis, but also target drug-resistant cellular side population through PDCD4-related apoptosis in hepatocellular carcinoma. It might be a potential therapeutic drug for chemotherapeutic treatment of hepatocellular carcinoma (Lu et al. 2012; Sun et al. 2012). Therefore, our group selected isocorydine as the starting material, modified the chemical structure and screened for potent antitumour candidates; however, the results were not ideal. The extracts from D. leptopodum (Maxim) Fedde including alkaloids have been confirmed to have significant outcomes in inhibiting the growth and progression of the tumour (Zhang et al. 2010). As a continuation of our investigation to discover cytotoxic compounds from natural sources, we selected D. leptopodum (Maxim) Fedde for screening the target compound, which is mainly distributed in the northwest of China. Although, the chemical constituents of this plant have been reported previously, a new quaternary protoberberine alkaloids 5-hydroxy-coptisine (1) and berberrubine (2) have been isolated (Figure 1). In this article, the isolation and structural elucidation of 1 are reported. 2. Results and discussion Compound 1 was obtained as a yellow amorphous powder with m.p. 258– 2598C, ½a20 D þ 7:58 (c 1.00, MeOH) which gave a positive visualisation to the Dragendorff’s test. Its molecular
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
508
M. Zhong et al.
O 3
4 A
O 2
OH 5 B
1
O
6 N+ C
N+
O
8
OH
9 O
13 D 12 1
11
OCH3
10 O 2
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Figure 1. Chemical structures of 1 and 2. þ formula was assigned as C19 H14 NOþ 5 on the basis of the HR-ESI-MS (m/z ¼ 336.0862 [M] , þ calcd for C19 H14 NO5 : 336.0866). The UV spectrum of 1 revealed absorption maxima at 231 (log 1 ¼ 1.22), 270 (log 1 ¼ 2.52) and 355 nm (log 1 ¼ 2.61). Meanwhile, the IR (KBr) spectrum revealed characteristics bands of a hydroxylated aromatic group (3434 cm21) and aryl groups (1623.0, 1507.4, 1361.4, 1332.4 and 1050.7 cm21). The 1H NMR spectrum of 1 revealed an AB coupling system at d 7.82 (d, 1H, J ¼ 8.8 Hz, H-11), d 8.04 (d, 1H, J ¼ 8.8 Hz, H-12) and two singlet protons at d 7.81(s, 1H, H-1), d 7.15 (s, 1H, H-4) indicated four aromatic substitutions at C-2, 3, 9 and C-10 on A and D rings of the protoberberine alkaloids, and two OZCH2ZO groups substitutions could be confirmed by two methylene carbon signals (d 102.3 and d 104.6) which was assigned by a DEPT experiment. Particularly, the unusual low-field proton signals at d 10.05 (H-8) and d 9.02 (H-13) reminded us the resonance of the quaternary protoberberine alkaloids (Young et al. 2002; Dosta´la et al. 2004). The 1H and 13C NMR spectra of 1 were almost identical with coptisine, a common quaternary protoberberine alkaloid, except for the carbon signals on B ring. The methylene (d 60.8) and methane (d 62.6) groups located at B ring supported the segment to be hydroxylated at C-5; this was supported by the HMBC correlations of H-6 with C-4a, 13a and C-8 (see Figure S1). The hydroxyl group located at C-5 was b-oriented which could be confirmed by comparing the specific rotation of compound 1 with berberastine and thalidastine (Young et al. 2002; Dosta´la et al. 2004). The linkages of the abovementioned groups could be confirmed by the HMBC experiment. The HMBC correlations of H-4 with C-2, 5, 13b, H-1 with C-2, 13a and H-13 with C-13b demonstrated the attached position of A ring. The correlations of H-8 with C-6, 9, 12a, H-12 with C-10 demonstrated the position of D ring (Figure 1). Thus, the structure was proposed as 1 and named as 5-hydroxy-coptisine. The original spectra (IR, 1H NMR, 13C NMR, DEPT, 1H – 1H COSY, HMQC, HMBC and HR-ESIMS) of 1 is provided in the supplementary materials).
3. Experimental 3.1. General Melting points were obtained on an X-4 digital display micro-melting point apparatus (Taike Company Limited, Beijing, China) which were not corrected. Optical rotation was measured on an Automatic Polarimeter A21201-T (Rudolph Research Analytical Corporation, Hackettstown, NJ, USA) in MeOH solution. UV spectra were recorded on a Perkin Elmer Lambda 35 spectrophotometer (Perkin-Elmer Company, Waltham, MA, USA) in MeOH solution. IR spectra were recorded on a Nicolet NEXUS 670 FT-IR spectrometer (Thermo Nicolet Corporation, Madison, WI, USA) using KBr pellets. 1H NMR (400.16 Hz), 13C NMR (100.32 Hz) and 2D NMR spectra were recorded on a Varian INOVA-400 MHz-FTNMR spectrometer (Varian Medical Systems Incorporation, Palo Alto, CA, USA) with tetramethylsilane as the internal standard, and HR-ESI-MS, on a Bruker APEX II (Bruker Corporation, Madison, WI, USA). Circular dichroism (CD) data were observed on an Olis RSM
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1000 CD (Olis, Incorporation, Atlanta, GA, USA). Silica gel (200 –300 mesh) was used for column chromatography (CC) and silica GF254 (10 –40 mm) for thin-layer chromatography (TLC). The spots were detected on TLC under UV light or sprayed with Dragendorff’s agent.
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3.2. Plant material The whole plants of D. leptopodum (Maxim) Fedde were collected at Pinliang of Gansu Province in May 2012 and identified by Prof. Zhi-Gang Ma at the Lanzhou University, Lanzhou, China. A voucher specimen (no. ZY201202) has been deposited at the Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, China. 3.3. Extraction and isolation The dried plants of D. leptopodum (Maxim) Fedde were pulverised and extracted at room temperature with 95% ethanol (3 £ 7 d). The solvent was removed under reduced pressure in a rotary evaporator to give a residue (200.0 g). The collected residues were suspended in water (1000 mL) and the suspension was extracted with the same volumes of petroleum ether (60–908C), chloroform and n-BuOH, successively. The n-BuOH extract (30 g) was subjected to CC over silica gel (200–300 mesh, 400 g) eluted with a gradient of CHCl3 –MeOH–H2O (gradient from 10:1:0.1 to 7:3:1) according to the differences in composition indicated by TLC; four crude fractions F1 (5 g), F2 (3 g), F3 (2 g) and F4 (4 g) were obtained. Compounds 1 (15.0 mg) and 2 (20.0 mg) were isolated from F4 with CHCl3 –MeOH–H2O (7:3:1) on CC over silica gel, respectively. 3.4. Compounds isolated 5-hydroxy-coptisine (1): yellow amorphous powder; m.p. 258– 2598C; ½a20 D þ 7:58 (c 1.00, MeOH); UV: lmax(log 1) ¼ 231 (1.22), 270 (2.52), 355 (2.61) nm; IR (KBr): nmax ¼ 3434.9, 2910.1, 2351.1, 1623, 1507, 1473.8, 1361.4, 1332.4, 1050.7, 617.4 cm21), HR-ESI-MS: 1 m/z ¼ 336.0862 [M]þ (calcd for C19 H14 NOþ 5 : 336.0866); H NMR (400 MHz, DMSO-d6): d ¼ 7.81 (1H, s, H-1), d ¼ 7.15 (1H, s, H-4), d ¼ 4.82 (1H, dd, J ¼ 2.0, 11.2 Hz, H-5), d ¼ 5.03 (2H, dd, J ¼ 2.0, 11.2 Hz, H-6), d ¼ 10.05 (1H, s, H-8), d ¼ 7.82 (1H, d, J ¼ 8.8 Hz, H-11), d ¼ 8.04 (1H, d, J ¼ 8.8 Hz, H-12), d ¼ 9.02 (1H, s, H-13), d ¼ 6.19 (2H, d, J ¼ 4.0 Hz, H-2, 3-CH2-), d ¼ 6.54 (2H, d, J ¼ 4.0 Hz, H-9, 10-CH2 –); 13C NMR (100 MHz, DMSO-d6): d ¼ 105.2 (C-1), 148.6 (C-2), 149.8 (C-3), 108.0 (C-4), 132.2 (C-4a), 62.6 (C-5), 60.8 (C-6), 145.2 (C-8), 111.8 (C-8a), 143.9 (C-9), 147.3 (C-10), 121.2 (C-11), 121.8 (C-12), 132.3 (C-12a), 121.1 (C-13), 136.3 (C-13a), 119.7 (C-13b), 102.3 (C-2, 3-CH2 –), 104.6 (C-9, 10-CH2-). Berberrubine (2): yellow amorphous powder; HR-ESI-MS: m/z ¼ 332.1072 [M]þ (calcd for 1 C19 H16 NOþ 4 : 322.1074); H NMR (400 MHz, DMSO-d6): d ¼ 7.56 (1H, s, H-1), d ¼ 7.03 (1H, s, H-4), d ¼ 3.15 (1H, t, J ¼ 2.0, 11.2 Hz, H-5), d ¼ 4.89 (2H, t, J ¼ 2.0, 11.2 Hz, H-6), d ¼ 9.96 (1H, s, H-8), d ¼ 7.87 (1H, d, J ¼ 8.4 Hz, H-11), d ¼ 8.01 (1H, d, J ¼ 8.4 Hz, H-12), d ¼ 8.81 (1H, s, H-13), d ¼ 6.51 (2H, d, J ¼ 4.0 Hz, H-2, 3-CH2 – ), d ¼ 3.86(3H, s, H-10CH3O – ); 13C NMR (100 MHz, DMSO-d6): d ¼ 111.4 (C-1), 146.5 (C-2), 146.8 (C-3), 111.6 (C4), 126.8 (C-4a), 25.9 (C-5), 55.4 (C-6), 144.5 (C-8), 119.1 (C-8a), 143.6 (C-9), 150.6 (C-10), 112.1 (C-11), 121.8 (C-12), 132.5 (C-12a), 120.8 (C-13), 137.2 (C-13a), 120.3 (C-13b), 104.4 (C-2, 3-CH2 –), 55.9 (C-10-CH2 –). 4. Conclusion The quaternary protoberberine alkaloids, 5-hydroxy-coptisine (1) and berberrubine (2), were isolated from the genus Dicranostigma for the first time. The extracts from D. leptopodum
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(Maxim) Fedde have been confirmed to express significant antitumour activity, and quaternary protoberberine alkaloids have been confirmed to possess cytotoxic activities such as apoptosis and inhibition of telomerase (Grycova et al. 2007). Therefore, we predict that the quaternary protoberberine alkaloids should be the material basis for its antitumour effects, although this plant possessed a limited content. Supplementary material Supplementary material relating to this article is available online, alongside Figures S1 – S9.
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Acknowledgements This work was supported by the ‘West Light Program’, ‘Build Coalitions of the National Academy of Sciences’ of the Chinese Academy of Sciences and the Science and Technology Program of Gansu (no. 1304FKCA062).
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