This article was downloaded by: [University of Otago] On: 16 July 2015, At: 00:57 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: 5 Howick Place, London, SW1P 1WG
Journal of Asian Natural Products Research Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ganp20
Antitumor quinazoline alkaloids from the seeds of Peganum harmala a
b
c
c
Chun-Hua Wang , Hong Zeng , Yi-Hai Wang , Chuan Li , Jun c
c
ac
Cheng , Zhi-Jun Ye & Xiang-Jiu He a
School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, China b
Xinjiang Production & Construction Corps Key Laboratory of Protection and Utilization of Biological Resources in Tarim Basin, Tarim 843300, China
Click for updates
c
School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, China Published online: 13 Jul 2015.
To cite this article: Chun-Hua Wang, Hong Zeng, Yi-Hai Wang, Chuan Li, Jun Cheng, Zhi-Jun Ye & Xiang-Jiu He (2015) Antitumor quinazoline alkaloids from the seeds of Peganum harmala, Journal of Asian Natural Products Research, 17:5, 595-600, DOI: 10.1080/10286020.2015.1042373 To link to this article: http://dx.doi.org/10.1080/10286020.2015.1042373
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 Otago] at 00:57 16 July 2015
Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Journal of Asian Natural Products Research, 2015 Vol. 17, No. 5, 595–600, http://dx.doi.org/10.1080/10286020.2015.1042373
Antitumor quinazoline alkaloids from the seeds of Peganum harmala Chun-Hua Wanga, Hong Zengb, Yi-Hai Wangc, Chuan Lic, Jun Chengc, Zhi-Jun Yec and Xiang-Jiu Hea,c* a School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, China; bXinjiang Production & Construction Corps Key Laboratory of Protection and Utilization of Biological Resources in Tarim Basin, Tarim 843300, China; cSchool of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, China
Downloaded by [University of Otago] at 00:57 16 July 2015
(Received 31 January 2015; final version received 14 April 2015) A phytochemical study on the methanol extracts from the seeds of Peganum harmala L. led to a new quizonaline alkaloid (S)-vasicinone-1-O-b-D -glucopyranoside (1) and four known ones, (R)-vasicinone-1-O-b-D -glucopyranoside (2), (S)-vasicinone (3), vasicine (4), and deoxyvasicinone (5). Their structures were elucidated by spectroscopic analysis including IR, HR-ESI-MS, 1D and 2D NMR, and specific rotation as well as by comparison of the data with those in the literature. All of the alkaloids were screened for antiproliferative activity against human gastric cancer cells MCG-803 with MTT method. Compounds 1 and 3 exhibited moderate inhibitory activity. Keywords: Zygophyllaceae; Peganum harmala; quizonaline alkaloids; (S)-vasicinone-1-O-b-d-glucopyranoside; antiproliferative activity
1.
Introduction
Peganum harmala L. belongs to the family of Zygophyllaceae, which is widely distributed in Central Asia, North Africa, and Middle East. In China, it mainly distributed in Northwestern, such as Xinjiang, Inner Mongolia [1,2]. The whole plant of P. harmala is a traditional medicinal herb which has been widely used for a long time to treat apoplexia, asthma [3], jaundice, and lumbago [4]. Recent years of researches carried out on the chemical composition have revealed that b-carboline and quinazoline alkaloids are important ingredients accounting for many pharmacological and therapeutic effects such as analgesic [5], antibacterial [6], antiparasitic activity [7], strong reversible inhibition activity of monoamine oxidase [8], cytotoxicity and antitumor activities [9 – 11], etc. Therefore, its of great significance to carry out further
research on the chemical composition of P. harmala, considering its traditional usage and the remarkable pharmacological activities, especially antitumor activity. In our present investigation, a new quinazoline alkaloid, along with four known ones (Figure 1), was isolated from the extracts of P. harmala. In addition, a preliminary screening for the antiproliferative activity against human gastric cancer cells of all the alkaloids was conducted.
2.
Compound 1 was obtained as a white amorphous powder. Its molecular formula C17H20N2O7 was established on the basis of the HR-ESI-MS at m/z 365.1635 [M þ H]þ and 13C NMR data. The IR spectrum showed absorption bands at 1673 cm21 suggesting the presence of
*Corresponding author. Email: [email protected] q 2015 Taylor & Francis
Results and discussion
Downloaded by [University of Otago] at 00:57 16 July 2015
596
C.-H. Wang et al.
Figure 1. Structures of compounds 1 –5 isolated from P. harmala L.
amide functionality. The 1H NMR spectrum of 1 in DMSO-d6 indicated the present of four aromatic protons at dH 8.15 –8.17 (1H, m), 7.82– 7.85 (1H, m), 7.71 –7.72 (1H, m), and 7.55 –7.57 (1H, m), which revealed a typical pattern for AA0 BB0 splitting system of an orthodisubstituted benzene ring. An oxygenated methine proton signal at dH 5.18– 5.20 (1H, m) and two methylene proton signals at dH 4.07– 4.13 (1H, m), 3.98– 4.03 (1H, m), 2.49– 2.57 (1H, m), and 2.18 – 2.24 (1H, m) were observed. The anomeric proton at dH 4.74 (1H, d, J ¼ 7.8 Hz) suggested the presence of a sugar unit in compound 1. The 13C NMR spectrum showed 17 carbon signals, including a benzene ring at dC 148.6, 134.4, 127.4, 126.9, 125.9, and
120.9; seven oxygenated carbon signals at dC 101.7, 77.3, 76.9, 76.7, 73.6, 70.1, and 61.2; and two methylene carbon signals at dC 43.9 and 28.3, respectively. The 13C NMR signal at dC 101.7 was assignable to the anomeric carbon of a sugar. The 1H and 13C NMR spectra of compound 1 were similar to those of (S)-vasicinone (compound 3), except for the signals of a sugar (dC 101.7, 77.3, 76.7, 73.6, 70.1, 61.2) and the downfield-shifted C-1 signal at dC 76.9 compared with dC 71.3 of compound 3. Therefore, compound 1 was assumed to be a vasicinone glycoside. The saccharidic chain of compound 1 was determined to be D -glucosyl group by acid hydrolysis and TLC analysis, as well as its 13C NMR data, which were all in line with that of saccharidic chain hydrolysed from
Downloaded by [University of Otago] at 00:57 16 July 2015
Journal of Asian Natural Products Research
Figure 2. Key HMBC correlations of compound 1.
compound 2 and D -glucose standards. The stereo structure of the anomeric carbon was confirmed to be b-form based on its coupling constant of the anomeric proton signal at dH 4.74 (1H, d, J ¼ 7.8 Hz) in the 1 H NMR spectrum. The linkage between
Table 1.
1
the vasicinone and glucopyranosyl was established by analysis of the HMBC correlations (Figure 2) between H-10 at dH 4.74 and C-1 at dC 76.9, H-1 at dH 5.20 and C-10 at dC 101.7. The stereo structure of the chiral carbon (C-1) was determined as S-form by the specific rotation value of 2 8.43 (c ¼ 0.35, CHCl3) after enzymatic hydrolysis by emulsin, which is in accordance with those in the literature [12]. Therefore, the chemical structure of compound 1 was determined to be (S)-vasicinone-1-O-b-D -glucopyranoside, which was a new alkaloid as far as we knew. The proton and carbon signals were assigned by 1H, 13C NMR, HSQC, and HMBC experiments (Table 1). Four known compounds were identified as (R)-vasicinone-1-O-b-D -glucopyranoside (2) [13], (S)-vasicinone (3) [14], vasicine (4) [15], and deoxyvasicinone (5) [16] by comparing their spectroscopic data with the corresponding literature. All the five compounds were evaluated for their antiproliferative activity against human gastric cancer cells MCG-803 by the 3-(4,5-dimethylthiazol-2-yl)-2,5-
H and 13C NMR spectral data for compounds 1 and 2 in DMSO-d6. 1
Position 1 2 3 4 4a 5 6 7 8 8a 9a 10 20 30 40 50 60
597
2
dH (mult., J in Hz)
dC
dH (mult., J in Hz)
dC
5.18 – 5.20 (m) 2.18– 2.24 (m), 2.49 – 2.57 (m) 3.98– 4.03 (m), 4.07 – 4.13 (m) – – 8.15 – 8.17 (m) 7.53 – 7.57 (m) 7.82 – 7.85 (m) 7.71 – 7.72 (m) – – 4.74 (d, 7.8) 2.98 – 3.01 (m) 3.04 – 3.17 (m) 3.05 – 3.08 (m) 3.17 – 3.21 (m) 3.45– 3.49 (m), 3.71 – 3.74 (m)
76.9 28.3 43.9 159.9 120.9 125.9 126.9 134.4 127.4 148.6 158.2 101.7 73.6 76.7 70.1 77.3 61.2
5.27 – 5.29 (m) 2.16 – 2.22 (m), 2.46– 2.53 (m) 3.94 – 3.97 (m), 4.09– 4.13 (m) – – 8.12 – 8.14 (m) 7.51 – 7.54 (m) 7.80 – 7.84 (m) 7.67 – 7.68 (m) – – 4.43 (d, 7.8) 3.02 – 3.06 (m) 3.16 – 3.20 (m) 3.06 – 3.10 (m) 3.14 – 3.16 (m) 3.42 – 3.46 (m), 3.58– 3.62 (m)
76.5 27.1 43.6 159.9 120.7 125.9 126.9 134.5 127.2 148.7 158.1 100.7 73.0 76.2 70.0 77.1 61.0
598
C.-H. Wang et al.
Downloaded by [University of Otago] at 00:57 16 July 2015
Table 2. Antiproliferative activity against MCG-803 of the alkaloids (mean ^ SEM). Compound
IC50 (mmol/l)
1 2 3 4 5 Cisplatin
84.11 ^ 3.69 .100 94.5 ^ 10.47 .100 .100 10.22 ^ 1.43
diphenyltetrazolium bromide (MTT) method. The antiproliferative activity was shown in Table 2. Compounds 1 and 3 exhibited a moderate inhibitory activity with IC50 values of 84.11 ^ 3.69 and 94.5 ^ 10.47 mmol/l, respectively.
3.
Experimental
3.1. General experimental procedures Optical rotation was recorded using P1020 polarimeter (Jasco, Tokyo, Japan). The IR spectra were obtained on Spectrum 100 FT-IR Spectrometer (PerkinElmer, Fremont, CA, USA). The NMR spectra were recorded on a Bruker AV-500 spectrometer, using TMS as an internal standard (Bruker, Bremen, Germany). The ESI-MS were measured with ESI-Q-TOFMS Spectrometer (Waters, Milford, CT, USA). Semi-preparative HPLC was carried out on a Dynamax model HPXL Solvent Delivery System with a UV detector (Rainin, Oakland, CA, USA), using a Cosmosil 5C18-MS-II, 20ID £ 250 mm packed column (Nacalai, Nakagyo-ku, Japan). Sephadex LH-20 was product of the Pharmacia Biothech AB (Uppsala, Sweden). Silica gel column chromatography was performed with silica gel (Liangchen Silicon Material Co. Ltd., Anhui, lu´an, China). TLC was performed with silica gel GF254 (Liangchen Silicon Material Co. Ltd., Anhui, lu´an, China). MCG-803 cells were purchased from cell library of Sun Yat-sen University Laboratory Animal Center.
3.2. Plant material The dry seeds of P. harmala were collected in Kalpin County, Xinjiang, China, in 2012, and identified by Prof. Xiangjiu He, the School of Pharmacy, Guangdong Pharmaceutical University. The specimen (No. GDPU-NPR2013001) was deposited in the Department of Medicinal Chemistry, School of Pharmacy, Guangdong Pharmaceutical University.
3.3. Extraction and isolation The dried seeds of P. harmala (11 kg) were powdered and then extracted three times with 70% aqueous methanol under reflux. After filtration and evaporation of the solvent by rotary evaporator under reduced pressure, the residues (2.3 kg) were suspended in water and acidified to pH 1.0 with HCl (10%). Lipophilic impurities were removed with CHCl3 extraction and the aqueous fraction was alkalized to pH 7.0 with NH3·H2O. The total alkaloids were extracted four times with CHCl3 and n-BuOH sequentially. The n-BuOH fraction (220 g) was separated into 19 fractions (B1 – B19) by silica gel CC (Column chromatograph) (200 – 300 mesh, 100 £ 1700 mm) eluted with CHCl3/MeOH/ TEA (Triethylamine) (100:0:0.1 to 0:100:0.1, v/v/v), in increasing order of polarity. Fraction B14 (7.3 g) was subjected to RP-18 chromatography and eluted with MeOH/H2O (10:90 to 100:0) successively to yield 13 fractions (B14-1 to B14-13). Subfraction B14-8 (617.0 mg) was fractionated over Sephadex LH-20 (20 £ 1100 mm) eluted by MeOH/H2O (1:1) to yield a mixture (98.0 mg) that was further separated by HPLC using the Cosmosil 5C18-MS-II with MeOH/H2O (20:80) to isolate compounds 1 (Rt 23.7 min, 37.0 mg) and 2 (Rt 21.2 min, 18.0 mg), which is conducted with the flowrate of 4.0 ml/min and detected under 210 nm.
Downloaded by [University of Otago] at 00:57 16 July 2015
Journal of Asian Natural Products Research The CHCl3 fraction (200 g) was separated into 14 fractions (C1 – C14) by silica gel CC (200 – 300 mesh, 100 £ 1500 mm) eluted with CHCl3/MeOH/ TEA (100:0:0.1 to 0:100:0.1, v/v/v), in increasing order of polarity. Fraction C6 (4.3 g) was applied to a Sephadex LH-20 CC (20 £ 1100 mm) eluted by CHCl3/ MeOH (2:1), followed by recrystallization to obtain compound 3 (40.0 mg). Fraction C7 (10 g) was submitted to a silica gel CC eluted with a gradient solvent of CHCl3/ MeOH (80:1 to 0:1) to yield five fractions (C7-1 to C7-5). Fraction C7-2 (1 g), C7-3 (70 mg) were applied to Sephadex LH-20 (20 £ 1100 mm) column with CHCl3/ MeOH (2:1) to yield compounds 4 (32.0 mg) and 5 (15.0 mg), respectively. 3.3.1. (S)-Vasicinone-1-O-b-D glucopyranoside (1) White amorphous powder (MeOH-H2O); 20 ½a20 D 2 23 (c ¼ 0.2, MeOH); ½aD 2 8:43 (c ¼ 0.35, CHCl3) (aglycone); IR(KBr) y max: 3395, 2883, 1673, 1630, 1470, 1075 cm21; 1H and 13C NMR spectral data, see Table 1; HR-ESI-MS: m/z 365.1365 [M þ H] þ (calcd for C17H21N2O7, 365.1444). 3.3.2. (R)-Vasicinone-1-O-b-D glucopyranoside (2) White amorphous powder (MeOH-H2O); 20 ½a20 D 2 29 (c ¼ 0.2, MeOH); ½aD þ 6:1 (c ¼ 0.35, CHCl3) (aglycone); IR(KBr) y max: 3419, 2879, 1687, 1669, 1632, 1474, 1084 cm21; 1H and 13C NMR spectral data, see Table 1; HR-ESI-MS: m/z 365.1365 [M þ H] þ (calcd for C17H21N2O7, 365.1349). 3.4. Enzymatic hydrolysis for determination of the stereo structure Compounds 1 (1.0 mg) and 2 (1.0 mg) were subjected to hydrolysis for 12 h by emulsin in citrate buffer solution (458C,
599
pH 5.0) respectively. The aglycone was extracted with CHCl3 for three times and then CHCl3 was removed. The aglycones were then dissolved in CHCl 3 and prepared to solutions (0.35 g/100 ml). Optical rotations of solutions were detected within 30 min. The aglycone of compound 1 was determined to be SVasicinone on the base of its optical rotation with ½a20 D 2 8:43 (c ¼ 0.35, CHCl3), which is in accordance with data in literature. The aglycone of compound 2 was determined to be R-vasicinone with ½a20 (c ¼ 0.35, CHCl 3) D þ 6:1 accordingly. 3.5 Antiproliferative assay Antiproliferative activities of the compounds against human gastric cancer cells MCG-803 were evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) method [17]. Cisplatin was used as the positive control. Acknowledgments The authors are thankful to Prof. Dr. Hao Gao, Jinan University, for his generous help in the measurement of optical rotation.
Disclosure statement No potential conflict of interest was reported by the authors.
Funding This work was financially supported by the National Natural Science Foundation of China [grant number U1203103]; the key project of Xinjiang Production & Construction Corps Key Laboratory of Protection and Utilization of Biological Resources in Tarim Basin [grant number BRZD1205].
References [1] B. Liu, J. Chin. Med. Mater. 34, 1721 (2011). [2] S.S. Wu and H.L. Zhang, Xinjiang J. Tradit. Chin. Med. 29, 61 (2011).
Downloaded by [University of Otago] at 00:57 16 July 2015
600
C.-H. Wang et al.
[3] J.H. Deng, H. Xu, and X.F. Liu, J. Med. Pharm. Chin. Minor. 14, 45 (2008). [4] Z.T. Zheng, C.H. Zhang, Z.J. Cheng, X.M. Zhang, L. Wang, and X.Y. Wang, J. Planar Chromatogr. 24, 470 (2011). doi:10.1556/JPC.24.2011.6.3. [5] L. Farouk, A. Laroubi, R. Aboufatima, A. Benharref, and A. Chait, J. Ethnopharmacol. 115, 449 (2008). doi:10.1016/ j.jep.2007.10.014. [6] A. Al-Shamma, S. Drake, D.L. Flynn, L.A. Mitscher, Y.H. Park, G.S.R. Rao, A. Simpson, J.K. Swayze, T. Veysoglu, and S.T.S. Wu, J. Nat. Prod. 44, 745 (1981). doi:10.1021/np50018a025. [7] A.J. Tanweer, N. Chand, U. Saddique, C.A. Bailey, and R.U. Khan, Parasitol. Res. 113, 2951 (2014). doi:10.1007/ s00436-014-3957-y. [8] T. Herraiz, D. Gonza´lez, C. Ancı´n-Azpilicueta, V.J. Ara´n, and H. Guille´n, Food Chem. Toxicol. 48, 839 (2010). doi:10. 1016/j.fct.2009.12.019. [9] Z.R. Fan, X.S. Yao, L.H. Gu, J.F. Wang, J. Wang, A.G. He, B.G. Zhang,
[10] [11] [12] [13] [14]
[15] [16]
[17]
X.H. Wang, and H. Wang, J. Shengyang Coll. Pharm. 10, 136 (1993). A. Daoud, J. Song, F. Xiao, and J. Shang, Eur. J. Pharmacol. 724, 219 (2014). doi:10.1016/j.ejphar.2013.12.038. M.A. El Gendy, V. Somayaji, and A.O. El-Kadi, Planta Med. 76, 671 (2010). doi:10.1055/s-0029-1240633. A. Kamal, V. Devaiah, N. Shankaraiah, and K.L. Reddy, Synlett 47, 9025 (2006). S.M. Jain and C.K. Atal, Indian J. Chem. Sect. B 26B, 585 (1987). X.Y. Zheng, Z.J. Zhang, G.X. Chou, T. Wu, X.M. Cheng, C.H. Wang, and Z.T. Wang, Arch. Pharm. Res. 32, 1245 (2009). doi:10.1007/s12272-009-1910-x. X.L. Tian, D.J. Sun, and N.S. Du, J. Xinjiang Med. Univ. 26, 113 (2003). T.M. Potewar, S.A. Ingale, and K.V. Srinivasan, ARKIVOC 2008, 100 (2008). doi:10.3998/ark.5550190.0009. e11. J.J. Luo, X.J. Ma, X.L. Yin, Y.J. Chen, and S.R. Sun, Biotechniques 20, 32 (2010).
Journal of Asian Natural Products Research Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ganp20
Antitumor quinazoline alkaloids from the seeds of Peganum harmala a
b
c
c
Chun-Hua Wang , Hong Zeng , Yi-Hai Wang , Chuan Li , Jun c
c
ac
Cheng , Zhi-Jun Ye & Xiang-Jiu He a
School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, China b
Xinjiang Production & Construction Corps Key Laboratory of Protection and Utilization of Biological Resources in Tarim Basin, Tarim 843300, China
Click for updates
c
School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, China Published online: 13 Jul 2015.
To cite this article: Chun-Hua Wang, Hong Zeng, Yi-Hai Wang, Chuan Li, Jun Cheng, Zhi-Jun Ye & Xiang-Jiu He (2015) Antitumor quinazoline alkaloids from the seeds of Peganum harmala, Journal of Asian Natural Products Research, 17:5, 595-600, DOI: 10.1080/10286020.2015.1042373 To link to this article: http://dx.doi.org/10.1080/10286020.2015.1042373
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 Otago] at 00:57 16 July 2015
Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Journal of Asian Natural Products Research, 2015 Vol. 17, No. 5, 595–600, http://dx.doi.org/10.1080/10286020.2015.1042373
Antitumor quinazoline alkaloids from the seeds of Peganum harmala Chun-Hua Wanga, Hong Zengb, Yi-Hai Wangc, Chuan Lic, Jun Chengc, Zhi-Jun Yec and Xiang-Jiu Hea,c* a School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, China; bXinjiang Production & Construction Corps Key Laboratory of Protection and Utilization of Biological Resources in Tarim Basin, Tarim 843300, China; cSchool of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, China
Downloaded by [University of Otago] at 00:57 16 July 2015
(Received 31 January 2015; final version received 14 April 2015) A phytochemical study on the methanol extracts from the seeds of Peganum harmala L. led to a new quizonaline alkaloid (S)-vasicinone-1-O-b-D -glucopyranoside (1) and four known ones, (R)-vasicinone-1-O-b-D -glucopyranoside (2), (S)-vasicinone (3), vasicine (4), and deoxyvasicinone (5). Their structures were elucidated by spectroscopic analysis including IR, HR-ESI-MS, 1D and 2D NMR, and specific rotation as well as by comparison of the data with those in the literature. All of the alkaloids were screened for antiproliferative activity against human gastric cancer cells MCG-803 with MTT method. Compounds 1 and 3 exhibited moderate inhibitory activity. Keywords: Zygophyllaceae; Peganum harmala; quizonaline alkaloids; (S)-vasicinone-1-O-b-d-glucopyranoside; antiproliferative activity
1.
Introduction
Peganum harmala L. belongs to the family of Zygophyllaceae, which is widely distributed in Central Asia, North Africa, and Middle East. In China, it mainly distributed in Northwestern, such as Xinjiang, Inner Mongolia [1,2]. The whole plant of P. harmala is a traditional medicinal herb which has been widely used for a long time to treat apoplexia, asthma [3], jaundice, and lumbago [4]. Recent years of researches carried out on the chemical composition have revealed that b-carboline and quinazoline alkaloids are important ingredients accounting for many pharmacological and therapeutic effects such as analgesic [5], antibacterial [6], antiparasitic activity [7], strong reversible inhibition activity of monoamine oxidase [8], cytotoxicity and antitumor activities [9 – 11], etc. Therefore, its of great significance to carry out further
research on the chemical composition of P. harmala, considering its traditional usage and the remarkable pharmacological activities, especially antitumor activity. In our present investigation, a new quinazoline alkaloid, along with four known ones (Figure 1), was isolated from the extracts of P. harmala. In addition, a preliminary screening for the antiproliferative activity against human gastric cancer cells of all the alkaloids was conducted.
2.
Compound 1 was obtained as a white amorphous powder. Its molecular formula C17H20N2O7 was established on the basis of the HR-ESI-MS at m/z 365.1635 [M þ H]þ and 13C NMR data. The IR spectrum showed absorption bands at 1673 cm21 suggesting the presence of
*Corresponding author. Email: [email protected] q 2015 Taylor & Francis
Results and discussion
Downloaded by [University of Otago] at 00:57 16 July 2015
596
C.-H. Wang et al.
Figure 1. Structures of compounds 1 –5 isolated from P. harmala L.
amide functionality. The 1H NMR spectrum of 1 in DMSO-d6 indicated the present of four aromatic protons at dH 8.15 –8.17 (1H, m), 7.82– 7.85 (1H, m), 7.71 –7.72 (1H, m), and 7.55 –7.57 (1H, m), which revealed a typical pattern for AA0 BB0 splitting system of an orthodisubstituted benzene ring. An oxygenated methine proton signal at dH 5.18– 5.20 (1H, m) and two methylene proton signals at dH 4.07– 4.13 (1H, m), 3.98– 4.03 (1H, m), 2.49– 2.57 (1H, m), and 2.18 – 2.24 (1H, m) were observed. The anomeric proton at dH 4.74 (1H, d, J ¼ 7.8 Hz) suggested the presence of a sugar unit in compound 1. The 13C NMR spectrum showed 17 carbon signals, including a benzene ring at dC 148.6, 134.4, 127.4, 126.9, 125.9, and
120.9; seven oxygenated carbon signals at dC 101.7, 77.3, 76.9, 76.7, 73.6, 70.1, and 61.2; and two methylene carbon signals at dC 43.9 and 28.3, respectively. The 13C NMR signal at dC 101.7 was assignable to the anomeric carbon of a sugar. The 1H and 13C NMR spectra of compound 1 were similar to those of (S)-vasicinone (compound 3), except for the signals of a sugar (dC 101.7, 77.3, 76.7, 73.6, 70.1, 61.2) and the downfield-shifted C-1 signal at dC 76.9 compared with dC 71.3 of compound 3. Therefore, compound 1 was assumed to be a vasicinone glycoside. The saccharidic chain of compound 1 was determined to be D -glucosyl group by acid hydrolysis and TLC analysis, as well as its 13C NMR data, which were all in line with that of saccharidic chain hydrolysed from
Downloaded by [University of Otago] at 00:57 16 July 2015
Journal of Asian Natural Products Research
Figure 2. Key HMBC correlations of compound 1.
compound 2 and D -glucose standards. The stereo structure of the anomeric carbon was confirmed to be b-form based on its coupling constant of the anomeric proton signal at dH 4.74 (1H, d, J ¼ 7.8 Hz) in the 1 H NMR spectrum. The linkage between
Table 1.
1
the vasicinone and glucopyranosyl was established by analysis of the HMBC correlations (Figure 2) between H-10 at dH 4.74 and C-1 at dC 76.9, H-1 at dH 5.20 and C-10 at dC 101.7. The stereo structure of the chiral carbon (C-1) was determined as S-form by the specific rotation value of 2 8.43 (c ¼ 0.35, CHCl3) after enzymatic hydrolysis by emulsin, which is in accordance with those in the literature [12]. Therefore, the chemical structure of compound 1 was determined to be (S)-vasicinone-1-O-b-D -glucopyranoside, which was a new alkaloid as far as we knew. The proton and carbon signals were assigned by 1H, 13C NMR, HSQC, and HMBC experiments (Table 1). Four known compounds were identified as (R)-vasicinone-1-O-b-D -glucopyranoside (2) [13], (S)-vasicinone (3) [14], vasicine (4) [15], and deoxyvasicinone (5) [16] by comparing their spectroscopic data with the corresponding literature. All the five compounds were evaluated for their antiproliferative activity against human gastric cancer cells MCG-803 by the 3-(4,5-dimethylthiazol-2-yl)-2,5-
H and 13C NMR spectral data for compounds 1 and 2 in DMSO-d6. 1
Position 1 2 3 4 4a 5 6 7 8 8a 9a 10 20 30 40 50 60
597
2
dH (mult., J in Hz)
dC
dH (mult., J in Hz)
dC
5.18 – 5.20 (m) 2.18– 2.24 (m), 2.49 – 2.57 (m) 3.98– 4.03 (m), 4.07 – 4.13 (m) – – 8.15 – 8.17 (m) 7.53 – 7.57 (m) 7.82 – 7.85 (m) 7.71 – 7.72 (m) – – 4.74 (d, 7.8) 2.98 – 3.01 (m) 3.04 – 3.17 (m) 3.05 – 3.08 (m) 3.17 – 3.21 (m) 3.45– 3.49 (m), 3.71 – 3.74 (m)
76.9 28.3 43.9 159.9 120.9 125.9 126.9 134.4 127.4 148.6 158.2 101.7 73.6 76.7 70.1 77.3 61.2
5.27 – 5.29 (m) 2.16 – 2.22 (m), 2.46– 2.53 (m) 3.94 – 3.97 (m), 4.09– 4.13 (m) – – 8.12 – 8.14 (m) 7.51 – 7.54 (m) 7.80 – 7.84 (m) 7.67 – 7.68 (m) – – 4.43 (d, 7.8) 3.02 – 3.06 (m) 3.16 – 3.20 (m) 3.06 – 3.10 (m) 3.14 – 3.16 (m) 3.42 – 3.46 (m), 3.58– 3.62 (m)
76.5 27.1 43.6 159.9 120.7 125.9 126.9 134.5 127.2 148.7 158.1 100.7 73.0 76.2 70.0 77.1 61.0
598
C.-H. Wang et al.
Downloaded by [University of Otago] at 00:57 16 July 2015
Table 2. Antiproliferative activity against MCG-803 of the alkaloids (mean ^ SEM). Compound
IC50 (mmol/l)
1 2 3 4 5 Cisplatin
84.11 ^ 3.69 .100 94.5 ^ 10.47 .100 .100 10.22 ^ 1.43
diphenyltetrazolium bromide (MTT) method. The antiproliferative activity was shown in Table 2. Compounds 1 and 3 exhibited a moderate inhibitory activity with IC50 values of 84.11 ^ 3.69 and 94.5 ^ 10.47 mmol/l, respectively.
3.
Experimental
3.1. General experimental procedures Optical rotation was recorded using P1020 polarimeter (Jasco, Tokyo, Japan). The IR spectra were obtained on Spectrum 100 FT-IR Spectrometer (PerkinElmer, Fremont, CA, USA). The NMR spectra were recorded on a Bruker AV-500 spectrometer, using TMS as an internal standard (Bruker, Bremen, Germany). The ESI-MS were measured with ESI-Q-TOFMS Spectrometer (Waters, Milford, CT, USA). Semi-preparative HPLC was carried out on a Dynamax model HPXL Solvent Delivery System with a UV detector (Rainin, Oakland, CA, USA), using a Cosmosil 5C18-MS-II, 20ID £ 250 mm packed column (Nacalai, Nakagyo-ku, Japan). Sephadex LH-20 was product of the Pharmacia Biothech AB (Uppsala, Sweden). Silica gel column chromatography was performed with silica gel (Liangchen Silicon Material Co. Ltd., Anhui, lu´an, China). TLC was performed with silica gel GF254 (Liangchen Silicon Material Co. Ltd., Anhui, lu´an, China). MCG-803 cells were purchased from cell library of Sun Yat-sen University Laboratory Animal Center.
3.2. Plant material The dry seeds of P. harmala were collected in Kalpin County, Xinjiang, China, in 2012, and identified by Prof. Xiangjiu He, the School of Pharmacy, Guangdong Pharmaceutical University. The specimen (No. GDPU-NPR2013001) was deposited in the Department of Medicinal Chemistry, School of Pharmacy, Guangdong Pharmaceutical University.
3.3. Extraction and isolation The dried seeds of P. harmala (11 kg) were powdered and then extracted three times with 70% aqueous methanol under reflux. After filtration and evaporation of the solvent by rotary evaporator under reduced pressure, the residues (2.3 kg) were suspended in water and acidified to pH 1.0 with HCl (10%). Lipophilic impurities were removed with CHCl3 extraction and the aqueous fraction was alkalized to pH 7.0 with NH3·H2O. The total alkaloids were extracted four times with CHCl3 and n-BuOH sequentially. The n-BuOH fraction (220 g) was separated into 19 fractions (B1 – B19) by silica gel CC (Column chromatograph) (200 – 300 mesh, 100 £ 1700 mm) eluted with CHCl3/MeOH/ TEA (Triethylamine) (100:0:0.1 to 0:100:0.1, v/v/v), in increasing order of polarity. Fraction B14 (7.3 g) was subjected to RP-18 chromatography and eluted with MeOH/H2O (10:90 to 100:0) successively to yield 13 fractions (B14-1 to B14-13). Subfraction B14-8 (617.0 mg) was fractionated over Sephadex LH-20 (20 £ 1100 mm) eluted by MeOH/H2O (1:1) to yield a mixture (98.0 mg) that was further separated by HPLC using the Cosmosil 5C18-MS-II with MeOH/H2O (20:80) to isolate compounds 1 (Rt 23.7 min, 37.0 mg) and 2 (Rt 21.2 min, 18.0 mg), which is conducted with the flowrate of 4.0 ml/min and detected under 210 nm.
Downloaded by [University of Otago] at 00:57 16 July 2015
Journal of Asian Natural Products Research The CHCl3 fraction (200 g) was separated into 14 fractions (C1 – C14) by silica gel CC (200 – 300 mesh, 100 £ 1500 mm) eluted with CHCl3/MeOH/ TEA (100:0:0.1 to 0:100:0.1, v/v/v), in increasing order of polarity. Fraction C6 (4.3 g) was applied to a Sephadex LH-20 CC (20 £ 1100 mm) eluted by CHCl3/ MeOH (2:1), followed by recrystallization to obtain compound 3 (40.0 mg). Fraction C7 (10 g) was submitted to a silica gel CC eluted with a gradient solvent of CHCl3/ MeOH (80:1 to 0:1) to yield five fractions (C7-1 to C7-5). Fraction C7-2 (1 g), C7-3 (70 mg) were applied to Sephadex LH-20 (20 £ 1100 mm) column with CHCl3/ MeOH (2:1) to yield compounds 4 (32.0 mg) and 5 (15.0 mg), respectively. 3.3.1. (S)-Vasicinone-1-O-b-D glucopyranoside (1) White amorphous powder (MeOH-H2O); 20 ½a20 D 2 23 (c ¼ 0.2, MeOH); ½aD 2 8:43 (c ¼ 0.35, CHCl3) (aglycone); IR(KBr) y max: 3395, 2883, 1673, 1630, 1470, 1075 cm21; 1H and 13C NMR spectral data, see Table 1; HR-ESI-MS: m/z 365.1365 [M þ H] þ (calcd for C17H21N2O7, 365.1444). 3.3.2. (R)-Vasicinone-1-O-b-D glucopyranoside (2) White amorphous powder (MeOH-H2O); 20 ½a20 D 2 29 (c ¼ 0.2, MeOH); ½aD þ 6:1 (c ¼ 0.35, CHCl3) (aglycone); IR(KBr) y max: 3419, 2879, 1687, 1669, 1632, 1474, 1084 cm21; 1H and 13C NMR spectral data, see Table 1; HR-ESI-MS: m/z 365.1365 [M þ H] þ (calcd for C17H21N2O7, 365.1349). 3.4. Enzymatic hydrolysis for determination of the stereo structure Compounds 1 (1.0 mg) and 2 (1.0 mg) were subjected to hydrolysis for 12 h by emulsin in citrate buffer solution (458C,
599
pH 5.0) respectively. The aglycone was extracted with CHCl3 for three times and then CHCl3 was removed. The aglycones were then dissolved in CHCl 3 and prepared to solutions (0.35 g/100 ml). Optical rotations of solutions were detected within 30 min. The aglycone of compound 1 was determined to be SVasicinone on the base of its optical rotation with ½a20 D 2 8:43 (c ¼ 0.35, CHCl3), which is in accordance with data in literature. The aglycone of compound 2 was determined to be R-vasicinone with ½a20 (c ¼ 0.35, CHCl 3) D þ 6:1 accordingly. 3.5 Antiproliferative assay Antiproliferative activities of the compounds against human gastric cancer cells MCG-803 were evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) method [17]. Cisplatin was used as the positive control. Acknowledgments The authors are thankful to Prof. Dr. Hao Gao, Jinan University, for his generous help in the measurement of optical rotation.
Disclosure statement No potential conflict of interest was reported by the authors.
Funding This work was financially supported by the National Natural Science Foundation of China [grant number U1203103]; the key project of Xinjiang Production & Construction Corps Key Laboratory of Protection and Utilization of Biological Resources in Tarim Basin [grant number BRZD1205].
References [1] B. Liu, J. Chin. Med. Mater. 34, 1721 (2011). [2] S.S. Wu and H.L. Zhang, Xinjiang J. Tradit. Chin. Med. 29, 61 (2011).
Downloaded by [University of Otago] at 00:57 16 July 2015
600
C.-H. Wang et al.
[3] J.H. Deng, H. Xu, and X.F. Liu, J. Med. Pharm. Chin. Minor. 14, 45 (2008). [4] Z.T. Zheng, C.H. Zhang, Z.J. Cheng, X.M. Zhang, L. Wang, and X.Y. Wang, J. Planar Chromatogr. 24, 470 (2011). doi:10.1556/JPC.24.2011.6.3. [5] L. Farouk, A. Laroubi, R. Aboufatima, A. Benharref, and A. Chait, J. Ethnopharmacol. 115, 449 (2008). doi:10.1016/ j.jep.2007.10.014. [6] A. Al-Shamma, S. Drake, D.L. Flynn, L.A. Mitscher, Y.H. Park, G.S.R. Rao, A. Simpson, J.K. Swayze, T. Veysoglu, and S.T.S. Wu, J. Nat. Prod. 44, 745 (1981). doi:10.1021/np50018a025. [7] A.J. Tanweer, N. Chand, U. Saddique, C.A. Bailey, and R.U. Khan, Parasitol. Res. 113, 2951 (2014). doi:10.1007/ s00436-014-3957-y. [8] T. Herraiz, D. Gonza´lez, C. Ancı´n-Azpilicueta, V.J. Ara´n, and H. Guille´n, Food Chem. Toxicol. 48, 839 (2010). doi:10. 1016/j.fct.2009.12.019. [9] Z.R. Fan, X.S. Yao, L.H. Gu, J.F. Wang, J. Wang, A.G. He, B.G. Zhang,
[10] [11] [12] [13] [14]
[15] [16]
[17]
X.H. Wang, and H. Wang, J. Shengyang Coll. Pharm. 10, 136 (1993). A. Daoud, J. Song, F. Xiao, and J. Shang, Eur. J. Pharmacol. 724, 219 (2014). doi:10.1016/j.ejphar.2013.12.038. M.A. El Gendy, V. Somayaji, and A.O. El-Kadi, Planta Med. 76, 671 (2010). doi:10.1055/s-0029-1240633. A. Kamal, V. Devaiah, N. Shankaraiah, and K.L. Reddy, Synlett 47, 9025 (2006). S.M. Jain and C.K. Atal, Indian J. Chem. Sect. B 26B, 585 (1987). X.Y. Zheng, Z.J. Zhang, G.X. Chou, T. Wu, X.M. Cheng, C.H. Wang, and Z.T. Wang, Arch. Pharm. Res. 32, 1245 (2009). doi:10.1007/s12272-009-1910-x. X.L. Tian, D.J. Sun, and N.S. Du, J. Xinjiang Med. Univ. 26, 113 (2003). T.M. Potewar, S.A. Ingale, and K.V. Srinivasan, ARKIVOC 2008, 100 (2008). doi:10.3998/ark.5550190.0009. e11. J.J. Luo, X.J. Ma, X.L. Yin, Y.J. Chen, and S.R. Sun, Biotechniques 20, 32 (2010).
