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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 periplogenin cardenolide from the seeds of Antiaris toxicaria a
a
a
a
a
Xiu-Li Wu , Yu-Ling Wu , Hou-Gang Li , He-Tao Liu , Xue-Yan Fu , b
a
a
Rui-Qin Cui , Jian-Huan Wang , Cheng Liu & Jing Chen
a
a
College of Pharmacy, Ningxia Medical University, Yinchuan 750004, China b
College of Traditional Chinese Medicine, Ningxia Medical University, Yinchuan 750004, China Published online: 06 Mar 2014.
Click for updates To cite this article: Xiu-Li Wu, Yu-Ling Wu, Hou-Gang Li, He-Tao Liu, Xue-Yan Fu, Rui-Qin Cui, Jian-Huan Wang, Cheng Liu & Jing Chen (2014) A new periplogenin cardenolide from the seeds of Antiaris toxicaria, Journal of Asian Natural Products Research, 16:4, 418-421, DOI: 10.1080/10286020.2014.885506 To link to this article: http://dx.doi.org/10.1080/10286020.2014.885506
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 [UNAM Ciudad Universitaria] at 15:46 19 December 2014
Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Journal of Asian Natural Products Research, 2014 Vol. 16, No. 4, 418–421, http://dx.doi.org/10.1080/10286020.2014.885506
A new periplogenin cardenolide from the seeds of Antiaris toxicaria Xiu-Li Wua, Yu-Ling Wua, Hou-Gang Lia, He-Tao Liua, Xue-Yan Fua, Rui-Qin Cuib, Jian-Huan Wanga, Cheng Liua and Jing Chena* a
College of Pharmacy, Ningxia Medical University, Yinchuan 750004, China; bCollege of Traditional Chinese Medicine, Ningxia Medical University, Yinchuan 750004, China
Downloaded by [UNAM Ciudad Universitaria] at 15:46 19 December 2014
(Received 4 September 2013; final version received 16 January 2014) A new periplogenin cardenolide, periplogulcoside (1), together with three known cardenolides, was isolated from the seeds of Antiaris toxicaria. The structure of the new compound was characterized as periplogenin-3-O-b-D -glucopyranosyl-(1 ! 4)b-D -glucopyranoside (1) by spectroscopic methods including 1D and 2D NMR, HRTOF-MS, and CD spectrometry, and the known compounds were identified by comparison of their NMR and HR-TOF-MS data with those reported in the literature. Compound 1 showed significant cytotoxicity against Hela and HepG-2 cell lines. Keywords: Antiaris toxicaria; seeds; periplogulcoside; cytotoxicity
1. Introduction Antiaris toxicaria (Pers.) Lesch. (Moraceae) is well known as “upas tree” and widespread over tropic rain forest. It is used as arrow and dart poisons throughout southeastern Asia, from Burma to Indonesia [1 – 2]. The genus Antiaris (Moraceae) comprises four species, of which only A. toxicaria is distributed in China, mainly in Guangxi, Guangdong, Yunnan, and Hainan provinces [3]. Previous studies on latex, stems, barks, and seeds of this plant had led to the isolation of cardenolides [4 – 10]. In our previous work on screening for cytotoxic agents, the ethanolic extract of the seeds of A. toxicaria showed inhibitory activity toward Hela and HepG-2 cell lines. In this paper, we report the isolation and structural elucidation of a new compound, periplogulcoside (1), along with three known compounds. Compound 1 exhibited significant cytotoxicity against Hela and HepG-2 cell lines in vitro by MTT method with IC50 values of 0.005 and 0.203 mM, respectively.
2. Results and discussion Compound 1 was obtained as a white powder. Its quasi-molecular ion peak at m/z 737.3362 [M þ Na]þ by HR-TOF-MS and 13C NMR data suggested the molecular formula as C35H54O15. The IR spectrum displayed absorptions for hydroxyls (3748, 3673, 3649 cm21), conjugated carbonyl (1735 cm21), and double bond (1651 cm21). The signals of one olefinic proton at d 6.13 (1H, s, H-22), and of two characteristic protons at d 5.32, 5.04 (each 1H, d, J ¼ 18.0 Hz, H-21a, H-21b) in 1H NMR spectrum, and of four corresponding carbons at d 177.3, 175.9, 118.9, and 75.1 in 13C NMR spectrum suggested that compound 1 was a cardenolide [5]. Other prominent signals including two methyls at d 1.01 (CH3-18) and 1.07 (CH3-19), and three O-substituted carbons on the skeleton at d 75.0 (C-3), 74.4 (C-5), and 86.0 (C-14) indicated that the aglycon was periplogenin [11]. Of the 35 carbon signals, 23 were assigned to the aglycon part and 12 to the sugar moiety. Acid
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
Downloaded by [UNAM Ciudad Universitaria] at 15:46 19 December 2014
Journal of Asian Natural Products Research hydrolysis of 1 afforded sugar components identified as D -glucose by TLC and GC analysis. The presence of two anomeric proton signals at d 4.99 (d, J ¼ 8.0 Hz) and 5.19 (d, J ¼ 6.5 Hz), and of two corresponding carbon signals at d 102.5 and 106.2 also indicated that 1 contained a disaccharide of two D -glucoses. The large 3 JH1 – H2 coupling constant (8.0, 6.5 Hz) suggested that anomeric configuration of the glucose unit was b. The sequence of the disaccharide moiety was determined by long-range correlations of H-1 (d 4.99) of the inner glucose and C-3 (d 75.7) of the aglycon, and H-1 (d 5.19) of the terminal glucose with C-4 (d 82.8) of the inner glucose in the HMBC spectrum. Thus, the structure of 1 was determined to be periplogenin-3-O-b- D -glucopyranosyl(1 ! 4)-b-D -glucopyranoside, and named as periplogulcoside (Figure 1). In addition, the known compounds, antialloside (2) [4], strophalloside (3) [12], and strophanthojavoside (4) [12], were also isolated and identified by comparing NMR and ESI-MS data with those in the literature. Cytotoxic activity of compound 1 was evaluated against Hela and HepG-2 cell lines using the MTT method [13]. Compound 1 gave a single peak in HPLC analyses, and the purity was more than 98%. The result showed that compound 1 possessed the strong inhibitory activity against Hela cell lines with an IC50 value of 0.005 mM, and HepG-2 cell lines with an IC50 value of 0.203 mM, respectively.
HO 4″ HO 6″ 3″ OH HO 1″
O 22
O
12
O 4′ 6′ OH HO 3′ HO
O
1 2
19 11 9
10 5
1′
O 3
4 OH 6
21
18 20 17
13 H 14
H 8
23 O
OH 7
Figure 1. Structure of compound 1.
15
419
Mitomycin C was used as a positive control with IC50 values of 10.5 and 6.2 mM, respectively. 3.
Experimental
3.1 General experimental procedures Optical rotation was determined on a Beckman DU800 spectrometer (Beckman Coulter Corporation, Danvers, MA, USA). IR spectrum (KBr) was recorded on a Bruker IFS 55 spectrometer (Bruck Corporation, Ettlingen, Germany). UV spectrum was measured with a Shimadzu UV-2200 spectrophotometer (Shimadzu Corporation, Kyoto, Japan). 1D and 2D NMR spectra were obtained on a Bruker ARX-500 spectrometer with TMS as the internal standard (Bruck BioSpin, Fa¨llanden, Switzerland). HR-TOF-MS datum was performed on a LCT Premier XE mass spectrometer (Waters Corporation, Milford, MA, USA). CD spectrum was recorded with a Jasco CD-2095-plus circular dichroism detector (JASCO Corporation, Tokyo, Japan). For column chromatography, silica gel (Qingdao Marine Chemical Industry, Qingdao, China, 60–80 and 200– 300 mesh) was used. TLC was preformed with silica gel GF254 (Qingdao Marine Chemical Industry). Semi-preparative HPLC was carried out using a system composed of a Hitachi Pump L-7110 (Hitachi, Tokyo, Japan), with a Hitachi L-7420 UV (Hitachi, Tokyo, Japan) spectrophotometric detector and a TEDAchrom YWG C18 reversed-phase column (10 mm £ 250 mm, detected at UV 210 nm; TEDAChromScience Inc., Dalian, China). GC analysis was carried out on an FL-9790 apparatus using an OV-17 column (30 m £ 0.32 mm), with an FID detector (detector temperature: 2808C; column temperature: 2208C; carrier gas: N2).
16
3.2 Plant material The seeds of A. toxicaria (Pers.) Lesch were collected in Wanning County of Hainan Province, China, in October 2011
420
X.-L. Wu et al.
and identified by Dr Jing Chen, Associate Professor in College of Pharmacy, Ningxia Medical University, China. A voucher specimen (No. AN201111) has been deposited in College of Pharmacy, Ningxia Medical University.
Table 1. 1H and 13C NMR spectral data of 1 (500 MHz in pyridine-d5). 1 No.
dH (mult, J in Hz)
dC
1
1.35, m 2.25, m 2.05, m 1.94, m 4.45, m 1.57, m 1.94, m – 2.07, m 1.94, m 1.35, m 2.25, m 1.83, m 1.40, m – 1.76, m 1.84, m 1.64, m – – 2.07, m 1.94, m 2.06, m 1.95, m 2.79, m 1.01, s 1.07, s – 5.32, d, 18.0 5.04, d, 18.0 6.13, s – 4.99, d, 8.0 4.01, dd, 8.0, 8.0 4.30, m 4.30, m 3.93, m 4.30, m 5.19, d, 6.5 4.20, dd, 6.5, 8.0 4.30, m 4.15, m 4.01, m 4.52, m
23.2
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2
3.3 Extraction and isolation The dried and crushed seeds (1.5 kg) of A. toxicaria were extracted with 95% EtOH (3 £ 10 l, total amount 30 l) for three times at room temperature. The combined extract was evaporated in vacuo to yield the EtOH extract (32 g), which was dissolved in H2O and then extracted successively with petroleum ether (3 £ 200 ml), EtOAc (3 £ 200 ml), and n-BuOH (3 £ 200 ml). The EtOAc fraction (8.5 g), which showed potent cytotoxic activity, was subjected to silica gel CC eluted with CHCl3 –MeOH (v/v, 100:1–0:100) to obtain 20 subfractions (Fr.1–Fr.20). Fr.12 (100:40) was further separated by semi-preparative HPLC using MeOH–H2O (v/v, 68:32) at 2.5 ml/min to yield 1 (6.5 mg, tR ¼ 15.26 min), 2 (11.0 mg, tR ¼ 28.33 min), and 3 (15.5 mg, tR ¼ 40.24 min) and 4 (20.8 mg, tR ¼ 45.65 min). 3.3.1 Periplogulcoside White powder; ½a20 D þ 0:09 (c ¼ 0.02, MeOH); UV (MeOH) lmax (log 1): 221 (4.25) nm; IR (KBr) nmax: 3748, 3673, 3649, 2933, 1735, 1715, 1651, 1558, 1508, 1456, 699 cm21; CD (MeOH) D1225.0 nm: 2 7.8; for 1H NMR (C5D5N, 500 MHz) and 13C NMR (C5D5N, 125 MHz) spectral data, see Table 1; HR-TOF-MS: m/z 737.3362 [M þ Na] þ (calcd for C35H54O15Na, 737.3360). 3.4
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 10 20 30 40 50 60 100 200 300 400 500 600
27.4 75.7 37.6 74.4 35.2 25.9 42.2 40.5 42.5 27.9 41.2 51.3 86.0 34.4 28.6 52.6 17.4 18.5 177.3 75.1 118.9 175.9 102.5 76.2 79.6 82.8 78.2 63.8 106.2 76.0 79.7 72.8 78.1 63.3
Sugar analysis
Acid hydrolysis and determination of absolute configuration of sugar were carried out according to the method described previously [14,15]. The absolute
configuration of sugar was confirmed by the same retention time of its acetylated thiazolidine derivative by comparison with that of standard monosaccharide prepared
Journal of Asian Natural Products Research in a similar manner. The retention time of standard D -glucose was 9.58 min. Acknowledgments This work was financially supported by New Century Excellent Talent Support Plan (NCET10-0915) and Ningxia Nature Fund (NZ12171).
Downloaded by [UNAM Ciudad Universitaria] at 15:46 19 December 2014
References [1] B. Kopp, W.P. Bauer, and B. Schnuerch, J. Ethnopharmacol. 36, 57 (1992). [2] T. Shrestha, B. Kopp, and G. Bisset, J. Ethnopharmacol. 37, 129 (1992). [3] H.Y. Chen, Flora of Hainanica (Science Press, Beijing, 1965), Vol. 2, p. 384. [4] P. Muhlradt, E.K. Weiss, and T. Reichstein, Helv. Chim. Acta 47, 2164 (1964). [5] M.M. Jiang, Y. Dai, H. Gao, X. Zhang, G.H. Wang, J.Y. He, Q.Y. Hu, J.Z. Zeng, X.K. Zhang, and S. Yao, Chem. Pharm. Bull. 56, 1005 (2008). [6] H.F. Dai, Y.J. Gan, D.M. Que, and J. Wu, J. Asian Nat. Prod. Res. 11, 832 (2009).
421
[7] Y.J. Gan, W.L. Mei, Y.X. Zhao, and F. Dai, Chin. Chem. Lett. 20, 450 (2009). [8] L.S. Shi, Y.R. Liao, M.J. Su, A.S. Lee, P.C. Kuo, A.G. Damu, S.C. Kuo, H.D. Sun, K.H. Lee, and S. Wu, J. Nat. Prod. 73, 1214 (2010). [9] W.H. Dong, W.L. Mei, Y.X. Zhao, Y.B. Zeng, W.J. Zuo, H. Wang, X.N. Li, and F. Dai, Planta Med. 77, 1730 (2011). [10] M.M. Jiang, H. Gao, Y. Dai, X. Zhang, N.L. Wang, and S. Yao, Planta Med. 75, 340 (2009). [11] Z.H. Lei, S. Yanhara, T. Nohara, B.S. Tai, J.Z. Xiong, and L. Ma, Chem. Pharm. Bull. 48, 290 (2000). [12] F. Dolder, C. Tamm, and T. Reichstein, Helv. Chim. Acta 38, 1364 (1955). [13] T. Mosmann, J. Immunol. Methods 65, 55 (1983). [14] W.J. Zuo, H.F. Dai, Y.B. Zeng, H. Wang, H.Q. Chen, and J.H. Wang, J. Asian Nat. Prod. Res. 14, 308 (2012). [15] S. Hara, H. Okbae, and K. Mihashi, Chem. Pharm. Bull. 34, 1843 (1986).
Journal of Asian Natural Products Research Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ganp20
A new periplogenin cardenolide from the seeds of Antiaris toxicaria a
a
a
a
a
Xiu-Li Wu , Yu-Ling Wu , Hou-Gang Li , He-Tao Liu , Xue-Yan Fu , b
a
a
Rui-Qin Cui , Jian-Huan Wang , Cheng Liu & Jing Chen
a
a
College of Pharmacy, Ningxia Medical University, Yinchuan 750004, China b
College of Traditional Chinese Medicine, Ningxia Medical University, Yinchuan 750004, China Published online: 06 Mar 2014.
Click for updates To cite this article: Xiu-Li Wu, Yu-Ling Wu, Hou-Gang Li, He-Tao Liu, Xue-Yan Fu, Rui-Qin Cui, Jian-Huan Wang, Cheng Liu & Jing Chen (2014) A new periplogenin cardenolide from the seeds of Antiaris toxicaria, Journal of Asian Natural Products Research, 16:4, 418-421, DOI: 10.1080/10286020.2014.885506 To link to this article: http://dx.doi.org/10.1080/10286020.2014.885506
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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Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Journal of Asian Natural Products Research, 2014 Vol. 16, No. 4, 418–421, http://dx.doi.org/10.1080/10286020.2014.885506
A new periplogenin cardenolide from the seeds of Antiaris toxicaria Xiu-Li Wua, Yu-Ling Wua, Hou-Gang Lia, He-Tao Liua, Xue-Yan Fua, Rui-Qin Cuib, Jian-Huan Wanga, Cheng Liua and Jing Chena* a
College of Pharmacy, Ningxia Medical University, Yinchuan 750004, China; bCollege of Traditional Chinese Medicine, Ningxia Medical University, Yinchuan 750004, China
Downloaded by [UNAM Ciudad Universitaria] at 15:46 19 December 2014
(Received 4 September 2013; final version received 16 January 2014) A new periplogenin cardenolide, periplogulcoside (1), together with three known cardenolides, was isolated from the seeds of Antiaris toxicaria. The structure of the new compound was characterized as periplogenin-3-O-b-D -glucopyranosyl-(1 ! 4)b-D -glucopyranoside (1) by spectroscopic methods including 1D and 2D NMR, HRTOF-MS, and CD spectrometry, and the known compounds were identified by comparison of their NMR and HR-TOF-MS data with those reported in the literature. Compound 1 showed significant cytotoxicity against Hela and HepG-2 cell lines. Keywords: Antiaris toxicaria; seeds; periplogulcoside; cytotoxicity
1. Introduction Antiaris toxicaria (Pers.) Lesch. (Moraceae) is well known as “upas tree” and widespread over tropic rain forest. It is used as arrow and dart poisons throughout southeastern Asia, from Burma to Indonesia [1 – 2]. The genus Antiaris (Moraceae) comprises four species, of which only A. toxicaria is distributed in China, mainly in Guangxi, Guangdong, Yunnan, and Hainan provinces [3]. Previous studies on latex, stems, barks, and seeds of this plant had led to the isolation of cardenolides [4 – 10]. In our previous work on screening for cytotoxic agents, the ethanolic extract of the seeds of A. toxicaria showed inhibitory activity toward Hela and HepG-2 cell lines. In this paper, we report the isolation and structural elucidation of a new compound, periplogulcoside (1), along with three known compounds. Compound 1 exhibited significant cytotoxicity against Hela and HepG-2 cell lines in vitro by MTT method with IC50 values of 0.005 and 0.203 mM, respectively.
2. Results and discussion Compound 1 was obtained as a white powder. Its quasi-molecular ion peak at m/z 737.3362 [M þ Na]þ by HR-TOF-MS and 13C NMR data suggested the molecular formula as C35H54O15. The IR spectrum displayed absorptions for hydroxyls (3748, 3673, 3649 cm21), conjugated carbonyl (1735 cm21), and double bond (1651 cm21). The signals of one olefinic proton at d 6.13 (1H, s, H-22), and of two characteristic protons at d 5.32, 5.04 (each 1H, d, J ¼ 18.0 Hz, H-21a, H-21b) in 1H NMR spectrum, and of four corresponding carbons at d 177.3, 175.9, 118.9, and 75.1 in 13C NMR spectrum suggested that compound 1 was a cardenolide [5]. Other prominent signals including two methyls at d 1.01 (CH3-18) and 1.07 (CH3-19), and three O-substituted carbons on the skeleton at d 75.0 (C-3), 74.4 (C-5), and 86.0 (C-14) indicated that the aglycon was periplogenin [11]. Of the 35 carbon signals, 23 were assigned to the aglycon part and 12 to the sugar moiety. Acid
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
Downloaded by [UNAM Ciudad Universitaria] at 15:46 19 December 2014
Journal of Asian Natural Products Research hydrolysis of 1 afforded sugar components identified as D -glucose by TLC and GC analysis. The presence of two anomeric proton signals at d 4.99 (d, J ¼ 8.0 Hz) and 5.19 (d, J ¼ 6.5 Hz), and of two corresponding carbon signals at d 102.5 and 106.2 also indicated that 1 contained a disaccharide of two D -glucoses. The large 3 JH1 – H2 coupling constant (8.0, 6.5 Hz) suggested that anomeric configuration of the glucose unit was b. The sequence of the disaccharide moiety was determined by long-range correlations of H-1 (d 4.99) of the inner glucose and C-3 (d 75.7) of the aglycon, and H-1 (d 5.19) of the terminal glucose with C-4 (d 82.8) of the inner glucose in the HMBC spectrum. Thus, the structure of 1 was determined to be periplogenin-3-O-b- D -glucopyranosyl(1 ! 4)-b-D -glucopyranoside, and named as periplogulcoside (Figure 1). In addition, the known compounds, antialloside (2) [4], strophalloside (3) [12], and strophanthojavoside (4) [12], were also isolated and identified by comparing NMR and ESI-MS data with those in the literature. Cytotoxic activity of compound 1 was evaluated against Hela and HepG-2 cell lines using the MTT method [13]. Compound 1 gave a single peak in HPLC analyses, and the purity was more than 98%. The result showed that compound 1 possessed the strong inhibitory activity against Hela cell lines with an IC50 value of 0.005 mM, and HepG-2 cell lines with an IC50 value of 0.203 mM, respectively.
HO 4″ HO 6″ 3″ OH HO 1″
O 22
O
12
O 4′ 6′ OH HO 3′ HO
O
1 2
19 11 9
10 5
1′
O 3
4 OH 6
21
18 20 17
13 H 14
H 8
23 O
OH 7
Figure 1. Structure of compound 1.
15
419
Mitomycin C was used as a positive control with IC50 values of 10.5 and 6.2 mM, respectively. 3.
Experimental
3.1 General experimental procedures Optical rotation was determined on a Beckman DU800 spectrometer (Beckman Coulter Corporation, Danvers, MA, USA). IR spectrum (KBr) was recorded on a Bruker IFS 55 spectrometer (Bruck Corporation, Ettlingen, Germany). UV spectrum was measured with a Shimadzu UV-2200 spectrophotometer (Shimadzu Corporation, Kyoto, Japan). 1D and 2D NMR spectra were obtained on a Bruker ARX-500 spectrometer with TMS as the internal standard (Bruck BioSpin, Fa¨llanden, Switzerland). HR-TOF-MS datum was performed on a LCT Premier XE mass spectrometer (Waters Corporation, Milford, MA, USA). CD spectrum was recorded with a Jasco CD-2095-plus circular dichroism detector (JASCO Corporation, Tokyo, Japan). For column chromatography, silica gel (Qingdao Marine Chemical Industry, Qingdao, China, 60–80 and 200– 300 mesh) was used. TLC was preformed with silica gel GF254 (Qingdao Marine Chemical Industry). Semi-preparative HPLC was carried out using a system composed of a Hitachi Pump L-7110 (Hitachi, Tokyo, Japan), with a Hitachi L-7420 UV (Hitachi, Tokyo, Japan) spectrophotometric detector and a TEDAchrom YWG C18 reversed-phase column (10 mm £ 250 mm, detected at UV 210 nm; TEDAChromScience Inc., Dalian, China). GC analysis was carried out on an FL-9790 apparatus using an OV-17 column (30 m £ 0.32 mm), with an FID detector (detector temperature: 2808C; column temperature: 2208C; carrier gas: N2).
16
3.2 Plant material The seeds of A. toxicaria (Pers.) Lesch were collected in Wanning County of Hainan Province, China, in October 2011
420
X.-L. Wu et al.
and identified by Dr Jing Chen, Associate Professor in College of Pharmacy, Ningxia Medical University, China. A voucher specimen (No. AN201111) has been deposited in College of Pharmacy, Ningxia Medical University.
Table 1. 1H and 13C NMR spectral data of 1 (500 MHz in pyridine-d5). 1 No.
dH (mult, J in Hz)
dC
1
1.35, m 2.25, m 2.05, m 1.94, m 4.45, m 1.57, m 1.94, m – 2.07, m 1.94, m 1.35, m 2.25, m 1.83, m 1.40, m – 1.76, m 1.84, m 1.64, m – – 2.07, m 1.94, m 2.06, m 1.95, m 2.79, m 1.01, s 1.07, s – 5.32, d, 18.0 5.04, d, 18.0 6.13, s – 4.99, d, 8.0 4.01, dd, 8.0, 8.0 4.30, m 4.30, m 3.93, m 4.30, m 5.19, d, 6.5 4.20, dd, 6.5, 8.0 4.30, m 4.15, m 4.01, m 4.52, m
23.2
Downloaded by [UNAM Ciudad Universitaria] at 15:46 19 December 2014
2
3.3 Extraction and isolation The dried and crushed seeds (1.5 kg) of A. toxicaria were extracted with 95% EtOH (3 £ 10 l, total amount 30 l) for three times at room temperature. The combined extract was evaporated in vacuo to yield the EtOH extract (32 g), which was dissolved in H2O and then extracted successively with petroleum ether (3 £ 200 ml), EtOAc (3 £ 200 ml), and n-BuOH (3 £ 200 ml). The EtOAc fraction (8.5 g), which showed potent cytotoxic activity, was subjected to silica gel CC eluted with CHCl3 –MeOH (v/v, 100:1–0:100) to obtain 20 subfractions (Fr.1–Fr.20). Fr.12 (100:40) was further separated by semi-preparative HPLC using MeOH–H2O (v/v, 68:32) at 2.5 ml/min to yield 1 (6.5 mg, tR ¼ 15.26 min), 2 (11.0 mg, tR ¼ 28.33 min), and 3 (15.5 mg, tR ¼ 40.24 min) and 4 (20.8 mg, tR ¼ 45.65 min). 3.3.1 Periplogulcoside White powder; ½a20 D þ 0:09 (c ¼ 0.02, MeOH); UV (MeOH) lmax (log 1): 221 (4.25) nm; IR (KBr) nmax: 3748, 3673, 3649, 2933, 1735, 1715, 1651, 1558, 1508, 1456, 699 cm21; CD (MeOH) D1225.0 nm: 2 7.8; for 1H NMR (C5D5N, 500 MHz) and 13C NMR (C5D5N, 125 MHz) spectral data, see Table 1; HR-TOF-MS: m/z 737.3362 [M þ Na] þ (calcd for C35H54O15Na, 737.3360). 3.4
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 10 20 30 40 50 60 100 200 300 400 500 600
27.4 75.7 37.6 74.4 35.2 25.9 42.2 40.5 42.5 27.9 41.2 51.3 86.0 34.4 28.6 52.6 17.4 18.5 177.3 75.1 118.9 175.9 102.5 76.2 79.6 82.8 78.2 63.8 106.2 76.0 79.7 72.8 78.1 63.3
Sugar analysis
Acid hydrolysis and determination of absolute configuration of sugar were carried out according to the method described previously [14,15]. The absolute
configuration of sugar was confirmed by the same retention time of its acetylated thiazolidine derivative by comparison with that of standard monosaccharide prepared
Journal of Asian Natural Products Research in a similar manner. The retention time of standard D -glucose was 9.58 min. Acknowledgments This work was financially supported by New Century Excellent Talent Support Plan (NCET10-0915) and Ningxia Nature Fund (NZ12171).
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