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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 apotirucallane-type triterpenoid from Atalantia buxifolia a
a
a
a
Zhi-Kai Guo , Tao Yang , Cai-Hong Cai , Wen-Hua Dong , Cui-Juan a
a
a
a
Gai , Jing-Zhe Yuan , Wen-Li Mei & Hao-Fu Dai a
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Key Laboratory of Biology and Genetic Resources of Tropical Crops, Ministry of Agriculture, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, and Hainan Key Laboratory for Research and Development of Natural Products from Li Folk Medicine, Haikou 571101, China Published online: 28 May 2015.
To cite this article: Zhi-Kai Guo, Tao Yang, Cai-Hong Cai, Wen-Hua Dong, Cui-Juan Gai, Jing-Zhe Yuan, Wen-Li Mei & Hao-Fu Dai (2015): A new apotirucallane-type triterpenoid from Atalantia buxifolia, Journal of Asian Natural Products Research, DOI: 10.1080/10286020.2015.1040775 To link to this article: http://dx.doi.org/10.1080/10286020.2015.1040775
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/10.1080/10286020.2015.1040775
A new apotirucallane-type triterpenoid from Atalantia buxifolia Zhi-Kai Guo, Tao Yang, Cai-Hong Cai, Wen-Hua Dong, Cui-Juan Gai, Jing-Zhe Yuan, Wen-Li Mei* and Hao-Fu Dai* Key Laboratory of Biology and Genetic Resources of Tropical Crops, Ministry of Agriculture, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, and Hainan Key Laboratory for Research and Development of Natural Products from Li Folk Medicine, Haikou 571101, China
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(Received 13 January 2015; final version received 8 April 2015) A new triterpenoid (1) with apotirucallane skeleton was isolated from the ethanol extract of the roots of Atalantia buxifolia (Poir.) Oliv. The complete structural assignment of the new compound was elucidated by a combination of 1D, 2D NMR (HMQC, HMBC, COSY, and NOESY), and HR-ESI-MS analysis. Moreover, this new compound was evaluated in vitro for its cytotoxic, antimicrobial and enzymes inhibitory activities. Keywords: Atalantia buxifolia; Rutaceae; triterpenoid
1.
Introduction
Atalantia buxifolia (Poir.) Oliv. (Rutaceae) is a shrub growing in South China, Vietnam, Cambodia, and Laos [1]. The roots of this plant have been used as a folk medicine by the Li people in Hainan, for treatment of malaria, chronic rheumatism, paralysis, gastric ulcer, influenza, and cough [2]. Previous phytochemical studies on this plant revealed that it mainly contained tetranorterpenoids and acridone alkaloids, together with coumarins, sesquiterpenes, triterpenoids, flavonoids, and other kinds of compounds, some of which possessed antifeedant, cytotoxic, and antimicrobial activities [3 – 7]. As our continuous identification of bioactive metabolites from the Li folk medicinal plants [8 – 11], a new triterpenoid (1) (Figure 1) possessing an apotirucallane skeleton was isolated from the ethanol extract of the roots of A. buxifolia collected in Haikou, Hainan Province, China. Apotirucallane-type triterpenoids have been found in many plants
of Simaroubaceae, Rutaceae, and Meliaceae, and many show potential cytotoxicity against tumor cell lines such as P-388 murine leukemia cells [12]. Herein, we report the details of the isolation, structural elucidation and biological activities of this new compound. 2.
Results and discussion
Compound 1, isolated as an amorphous solid, had the molecular formula C36H58O8 as determined by HR-ESI-MS ion peak at m/z 641.4028 [M þ Na]þ, and NMR data (Table 1). Its 1H NMR spectrum (in CDCl3) displayed signals of seven tertiary methyls (dH 0.86, 0.89, 0.90, 1.03, 1.21, 1.90, 2.18), a cyclopropyl methylene group (dH 0.49, 0.77, both d, J ¼ 5.0), a hydroxymethylene group (dH 3.54, 3.70, both d, J ¼ 11.5), a methoxyl group (dH 3.36), five oxygenated methine protons (dH 4.90, d, J ¼ 4.0; dH 4.68, t-like, J ¼ 2.7; dH 4.24, ddd, J ¼ 10.5, 4.9, 1.4; dH 3.75, br s; dH 3.45, dd, J ¼ 8.0, 1.5 Hz), an olefinic proton
*Corresponding author. Email: [email protected]; [email protected] First two authors contributed equally to this work. q 2015 Taylor & Francis
2
Z.-K. Guo et al. HO
25
23
O
21
24
12 19 1 5'
2
O
3 4'
1'
3' 2'
5
4
O
30
8
H 7 6
H 29
H 18 14
27
20
13 17
9 10
22
26
OH
H
H3CO
11
HO
H 16
15
OH
28
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Figure 1. Structure of compound 1.
(dH 5.77, s-like), and four hydroxyl groups (dH 2.91, s; dH 2.63, d, J ¼ 8.0; dH 2.49, br s; dH 2.49, br s). The 13C NMR and DEPT135 spectra indicated the presence of 8 methyls, 10 methylenes, 10 methines, and 8 quaternary carbons (one at dC 166.5 was assigned to be an ester carbonyl carbon). These data suggested that 1 could be an apotirucallane-type triterpenoid with a Table 1. Position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
1
senecioyl ester side chain. HSQC spectrum of 1 allowed all protons to be assigned to their respective carbons, and the gross planar structure of 1 was initially deduced by comprehensive analysis of its 1H – 1H COSY and HMBC spectra. The 1H – 1H couplings from H2-2 to H2-1 and H-3, from H2-6 to H-5 and H-7, from H2-11 to H-9 and H2-12, were observed in the COSY
H (500 MHz) and 13C (125 MHz) NMR spectroscopic data of 1 (in CDCl3).
dH (J in Hz)
dC
dH (J in Hz)
Position
1.34– 1.37 (1H, m, overlap) 33.9 21 1.17– 1.21 (1H, m) 1.90– 1.92 (1H, m) 22.8 22 1.59– 1.62 (1H, m) 4.68 (1H, t-like, J ¼ 2.7) 77.0 23 36.3 24 1.92– 2.08 (1H, m, overlap) 41.3 25 1.63– 1.66 (1H, m) 24.2 26 1.56– 1.59 (1H, m, overlap) 3.75 (1H, br s) 74.2 27 39.0 28 1.33– 1.35 (1H, m, overlap) 43.9 29 37.3 30 1.33– 1.35 (2H, m, overlap) 16.1 10 1.81– 1.85 (2H, m) 25.6 20 28.5 30 36.4 40 1.88– 1.90 (1H, m) 25.9 50 1.55– 1.57 (1H, m, overlap) 1.63– 1.73 (2H, m) 26.3 21 – OMe 1.99– 2.02 (1H, m) 48.4 24-OH 0.77 (1H, d, J ¼ 5.0) 13.7 25-OH 0.49 (1H, d, J ¼ 5.0) 0.90 (3H, s) 15.7 7-OH 2.02– 2.18 (1H, m) 49.2 26-OH
4.90 (1H, d, J ¼ 4.0) 1.94 – 1.98 (1H, m, overlap) 1.74 – 1.76 (1H, m) 4.24 (1H, ddd, J ¼ 10.5, 4.9, 1.4) 3.45 (1H, dd, J ¼ 8.0, 1.5) 3.70 (1H, 3.54 (1H, 1.21 (3H, 0.86 (3H, 0.89 (3H, 1.03 (3H,
d, J ¼ 11.5) d, J ¼ 11.5) s) s) s) s)
5.77 (1H, s-like) 1.90 (3H, s) 2.18 (3H, s) 3.36 (3H, s) 2.63 (1H, d, J ¼ 8.0) 2.91 (1H, s) 2.49 (1H, br s, overlap) 2.49 (1H, br s, overlap)
dC 109.5 33.1 75.7 75.4 74.2 67.9 20.7 27.7 21.9 19.4 166.5 117.0 155.8 27.4 20.3 55.9
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Journal of Asian Natural Products Research spectrum. Another spin system from H2-15 to H-24 was also confirmed by the COSY experiment (Figure 2). The hydroxyl group at dH 2.63 attached to C-24 (dC 75.4) and the one at dH 2.49 attached to C-26 (dC 67.9) were identified by their couplings with H-24 (dH 3.45) and H2-26 (dH 3.70 and 3.54), respectively. Key HMBC correlations from H3-28 and H3-29 to C-3, C-4, and C-5; from H3-19 to C-1, C-5, C-9 and C-10; from H3-30 to C-7, C-8 and C-14; from H-9 to C-8; from H2-11 to C-13; from H2-18 to C-8, C-12, C-14, C-15 and C-17; from H-17 to C-13; from H2-16 to C-14; and from H-24 to C-25, C-26 and C-27 were observed in the HMBC spectrum. The senecioyl ester side chain was also confirmed by HMBC correlations from H3-40 and H3-50 to C-20 , C-30 , and from H320 to C-10 . The HMBC correlation from H-3 to C-10 secured the connectivity of C-3 (dC 77.0) to C-10 (dC 166.5) through an ester linkage. The methoxyl group was located at C-21 by the 3J HMBC correlation from the methoxyl protons at dH 3.36 to C-21. The HMBC correlation from H-21 to C-23 supported the connectivity of C-23 to C-21 through an ether bridge, which was also supported by the downfield chemical shift of C-21 (dC 109.5). Further analyses of the HMBC spectrum and 13C NMR data
3
demonstrated that the other two hydroxyl groups at dH 2.49 and 2.91 could be placed on C-7 and C-25 respectively, which completed the planar structure of an apotirucallane-type triterpenoid. The relative stereochemistry of new compound 1 could be determined by NOESY correlations and comparing 13C NMR data with those of known triterpenoids. The NOE correlations between H-3 and H3-29, H3-29 and H3-19, H3-29 and H-6b (dH 1.56 – 1.59), H-6b (dH 1.56 –1.59) and H3-19, H319 and H3-30, H3-30 and H-6b (dH 1.56– 1.59), H-6b (dH 1.56– 1.59) and H-7, H3-30 and H-7, H3-30 and H-17, H3-28 and H-5, H-5 and H-6a (dH 1.63– 1.66), H-5 and H9, and H-9 and H-18a (dH 0.77) (Figure 3) indicated that H-5, H-9, OH-7, and 14,18cyclopropane ring were on the same plane and of a-orientation, while H-3, H-17, Me19, and Me-30 were of b-orientation. The NOE correlations between H-17 and H-21, H-18b (dH 0.49) and H-20, H-20 and H-23, and H-23 and 21-OMe demonstrated that H-20, H-23 and 21-OMe were on the same plane, while the H-21 was on the other side. By comparing the chemical shifts of the C-21, C-24 and C-25 with those of the apotirucallane-type triterpenoids reported in the literature [12], the configurations at C-21, C-24 and C-25 were tentatively HO
HO
O OH H3CO
HMBC
O
H O
C
OH 1H-1HCOSY
Figure 2. Key HMBC and 1H – 1H COSY correlations of 1.
4
Z.-K. Guo et al. OH HO
H
O OH
H H H H
OCH3 H
H H H
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O
H
H
OH
Hb Ha
O
Figure 3. Selected NOE correlations of 1.
determined to be R, S, R, respectively. Therefore, the structure of 1 was elucidated as shown in Figure 1. Compound 1 was evaluated in vitro for its cytotoxic activities against human hepatic carcinoma cell line SEL-7420, gastric cell line SGC-7721, and leukemia cell line K-562, but no potential activity was detected (IC50 . 50 mM). It also showed no acetylcholinesterase (AChE) and a-glucosidase inhibitory activities at a concentration of 50 mg/ml, and no antimicrobial activities against plant pathogenic bacteria (Xanthomonas axonopodis pv. manihotis CCXanHNO3, X. axonopodis pv. manihotis CCXanGX30, and Pseudomonas solanacearum), and fungi (Colletotrichum gloeosporioides, Corynespora cassiicola) at a concentration of 20 mg/ml.
Nicolet 380 FT-IR instrument (Thermo, Pittsburgh, PA, USA). NMR spectra were measured by Bruker AvanceIII 500 NMR spectrometer (Bruker, Zurich, Switzerland) at 500 MHz for 1H NMR and at 125 MHz for 13C NMR. The chemical shifts were given in d (ppm) and referenced to the solvent signal (CDCl3, dH 7.26, dC 77.0) and tetramethylsilane (TMS) as the internal standard, and coupling constants (J) are reported in Hz. HR-ESI-MS data were recorded on an Agilent 6210 LC-TOF-MS spectrometer (Agilent, Palo Alto, CA, USA). Silica gel (200 – 300 mesh; Qingdao Marine Chemical Factory, Qingdao, China), ODS gel (20 –45 mm, Fuji Silysia Chemical Ltd, Greenville, NC, USA), and Sephadex LH-20 gel (Pharmacia Biotech, Uppsala, Sweden) were used for column chromatography (CC).
3. Experimental 3.1 General experimental procedures
3.2
Optical rotation was obtained on a Rudolph Autopol III automatic polarimeter (Rudolph Research Analytical, Hackettstown, NJ, USA). UV spectrum was recorded by a Shimadzu UV-2550 spectrometer (Beckman, Brea, CA, USA), and IR spectrum (KBr) was conducted on a
Plant material
The roots of A. buxifolia were collected in Haikou, Hainan Province, China, in April 2011, and identified by Associate Professor Zheng-Fu Dai of the Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, where a voucher specimen (AB201104) has been deposited [8].
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Journal of Asian Natural Products Research 3.3 Extraction and isolation Air-dried roots of A. buxifolia (8.4 kg) were powdered, and then extracted three times with EtOH (95%) under reflux. After evaporation, the residue of the EtOH extract was suspended in H 2O and partitioned with petroleum ether, EtOAc, and n-BuOH, respectively. The EtOAc fraction (183.0 g) was fractionated by silica gel CC eluted with petroleum ether – acetone (1:1, v/v). The subfractions were combined under TLC monitoring to obtain subfractions A – G. Subfraction F (49.4 g) was separated by silica gel CC eluted with a gradient of CHCl3 – MeOH (1:0 to 0:1, v/v) to yield 13 subfractions (Fr.F1 – Fr.F13). Fr.F4 (7.4 g) was subjected to chromatography over a silica gel column with petroleum ether – acetone (5:1, v/v) to give nine fractions (Fr.F41– Fr.F4-9). Fr.F4-6 (363.4 mg) was separated by repeated silica gel CC using petroleum ether – EtOAc (2:1, v/v) as eluent, and purified by Sephadex LH-20 CC with CHCl3 – MeOH (1:1, v/v) to afford compound 1 (6.8 mg). 3.3.1
Compound 1
Amorphous colorless solid; ½a28 D 2 66.4 (c 0.1, CHCl3); UV (MeOH) lmax (log e ): 219 (4.15) nm; IR (KBr) nmax: 3452, 2924, 2855, 1644, 1384, 1018 cm21; 1H and 13C NMR spectroscopic data, see Table 1; HRESI-MS: m/z 641.4028 [M þ Na]þ (calcd for C36H58O8Na, 641.4024). 3.4 Biological assays Cytotoxic activities against human hepatic carcinoma cell line SEL-7420, gastric cell line SGC-7721, and leukemia cell line K562, were evaluated in vitro by the MTT methods [13,14]. Antimicrobial activities against a variety of plant pathogenic bacteria (X. axonopodis pv. manihotis CCXanHNO3, X. axonopodis pv. manihotis CCXanGX30, and P. solanacearum), fungi (C. gloeosporioides, C. cassiicola), and
5
human pathogenic Staphylococcus aureus and Candida albicans, and the acetylcholinesterase (AChE), a-glucosidase inhibitory activities were tested in accordance with the primary literatures [15–17]. Funding This work was financially co-supported by the National Support Science and Technology Subject [grant number 2013BAI11B04]; Major Technology Project of Hainan Province [grant number ZDZX2013008-4], [grant number ZDZX2013023-1]; National Natural Science Foundation of China [grant number 41406083]; Special Fund for Agro-Scientific Research in the Public Interest [grant number 201403117].
Disclosure statement No potential conflict of interest was reported by the authors.
References [1] Guangdong Institute of Botany, Flora Hainanica (Science Press, Beijing, 1974), Vol. 3. [2] H.F. Dai, Records of Li Folk Medicine (China Science and Technology Press, Beijing, 2008), Vol. 1. [3] T.S. Wu, M. Niva, and H. Furukawa, Phytochemistry 23, 595 (1984). [4] Y.Q. Gu and Y. Han, Acta Pharm. Sin. 21, 792 (1986). [5] T.S. Wu, Y.L. Leu, Y.Y. Chan, P.L. Wu, C.S. Kuoh, S.J. Wu, and Y. Wang, Phytochemistry 45, 1393 (1997). [6] T.S. Wu and M. Chen, Chem. Pharm. Bull. 48, 85 (2000). [7] T.S. Wu, C.M. Chen, and W. Lin, J. Nat. Prod. 64, 1040 (2001). [8] T. Yang, Y.B. Zeng, Z.K. Guo, W.J. Zuo, S.S. Ma, S.S. Li, W.L. Mei, and H.F. Dai, J. Asian Nat. Prod. Res. 14, 581 (2012). [9] Y.Y. Yang, W. Yang, W.J. Zuo, Y.B. Zeng, S.B. Liu, W.L. Mei, and H.F. Dai, J. Asian Nat. Prod. Res. 15, 899 (2013). [10] X.J. Chen, W.L. Mei, W.J. Zuo, Y.B. Zeng, Z.K. Guo, X.Q. Song, and H.F. Dai, J. Asian Nat. Prod. Res. 15, 67 (2013). [11] H.D. Deng, W.L. Mei, H. Wang, Z.K. Guo, W.H. Dong, H. Wang, S.P. Li, and H.F. Dai, J. Asian Nat. Prod. Res. 16, 1024 (2014).
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[12] K. Mitsui, M. Maejima, H. Saito, H. Fukaya, Y. Hitotsuyanagi, and K. Takeya, Tetrahedron 61, 10569 (2005). [13] T. Mosmann, J. Immunol. Methods 65, 55 (1983). [14] Z.K. Guo, T. Wang, Y. Guo, Y.C. Song, R.X. Tan, and H.M. Ge, Planta Med. 77, 2057 (2011).
[15] D.L. Yang, W.L. Mei, H. Wang, and H.F. Dai, Z. Naturforsch. 65, 757 (2010). [16] Z. Zhang, L. Liao, J. Moore, T. Wu, and Z. Wang, Food Chem. 113, 160 (2009). [17] G.L. Ellman, K.D. Courtney, JR. Valentino Andres, and R.M. Featherstone, Biochem. Pharmacol. 7, 88 (1961).
Journal of Asian Natural Products Research Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ganp20
A new apotirucallane-type triterpenoid from Atalantia buxifolia a
a
a
a
Zhi-Kai Guo , Tao Yang , Cai-Hong Cai , Wen-Hua Dong , Cui-Juan a
a
a
a
Gai , Jing-Zhe Yuan , Wen-Li Mei & Hao-Fu Dai a
Click for updates
Key Laboratory of Biology and Genetic Resources of Tropical Crops, Ministry of Agriculture, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, and Hainan Key Laboratory for Research and Development of Natural Products from Li Folk Medicine, Haikou 571101, China Published online: 28 May 2015.
To cite this article: Zhi-Kai Guo, Tao Yang, Cai-Hong Cai, Wen-Hua Dong, Cui-Juan Gai, Jing-Zhe Yuan, Wen-Li Mei & Hao-Fu Dai (2015): A new apotirucallane-type triterpenoid from Atalantia buxifolia, Journal of Asian Natural Products Research, DOI: 10.1080/10286020.2015.1040775 To link to this article: http://dx.doi.org/10.1080/10286020.2015.1040775
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 [New York University] at 03:20 20 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 http://dx.doi.org/10.1080/10286020.2015.1040775
A new apotirucallane-type triterpenoid from Atalantia buxifolia Zhi-Kai Guo, Tao Yang, Cai-Hong Cai, Wen-Hua Dong, Cui-Juan Gai, Jing-Zhe Yuan, Wen-Li Mei* and Hao-Fu Dai* Key Laboratory of Biology and Genetic Resources of Tropical Crops, Ministry of Agriculture, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, and Hainan Key Laboratory for Research and Development of Natural Products from Li Folk Medicine, Haikou 571101, China
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(Received 13 January 2015; final version received 8 April 2015) A new triterpenoid (1) with apotirucallane skeleton was isolated from the ethanol extract of the roots of Atalantia buxifolia (Poir.) Oliv. The complete structural assignment of the new compound was elucidated by a combination of 1D, 2D NMR (HMQC, HMBC, COSY, and NOESY), and HR-ESI-MS analysis. Moreover, this new compound was evaluated in vitro for its cytotoxic, antimicrobial and enzymes inhibitory activities. Keywords: Atalantia buxifolia; Rutaceae; triterpenoid
1.
Introduction
Atalantia buxifolia (Poir.) Oliv. (Rutaceae) is a shrub growing in South China, Vietnam, Cambodia, and Laos [1]. The roots of this plant have been used as a folk medicine by the Li people in Hainan, for treatment of malaria, chronic rheumatism, paralysis, gastric ulcer, influenza, and cough [2]. Previous phytochemical studies on this plant revealed that it mainly contained tetranorterpenoids and acridone alkaloids, together with coumarins, sesquiterpenes, triterpenoids, flavonoids, and other kinds of compounds, some of which possessed antifeedant, cytotoxic, and antimicrobial activities [3 – 7]. As our continuous identification of bioactive metabolites from the Li folk medicinal plants [8 – 11], a new triterpenoid (1) (Figure 1) possessing an apotirucallane skeleton was isolated from the ethanol extract of the roots of A. buxifolia collected in Haikou, Hainan Province, China. Apotirucallane-type triterpenoids have been found in many plants
of Simaroubaceae, Rutaceae, and Meliaceae, and many show potential cytotoxicity against tumor cell lines such as P-388 murine leukemia cells [12]. Herein, we report the details of the isolation, structural elucidation and biological activities of this new compound. 2.
Results and discussion
Compound 1, isolated as an amorphous solid, had the molecular formula C36H58O8 as determined by HR-ESI-MS ion peak at m/z 641.4028 [M þ Na]þ, and NMR data (Table 1). Its 1H NMR spectrum (in CDCl3) displayed signals of seven tertiary methyls (dH 0.86, 0.89, 0.90, 1.03, 1.21, 1.90, 2.18), a cyclopropyl methylene group (dH 0.49, 0.77, both d, J ¼ 5.0), a hydroxymethylene group (dH 3.54, 3.70, both d, J ¼ 11.5), a methoxyl group (dH 3.36), five oxygenated methine protons (dH 4.90, d, J ¼ 4.0; dH 4.68, t-like, J ¼ 2.7; dH 4.24, ddd, J ¼ 10.5, 4.9, 1.4; dH 3.75, br s; dH 3.45, dd, J ¼ 8.0, 1.5 Hz), an olefinic proton
*Corresponding author. Email: [email protected]; [email protected] First two authors contributed equally to this work. q 2015 Taylor & Francis
2
Z.-K. Guo et al. HO
25
23
O
21
24
12 19 1 5'
2
O
3 4'
1'
3' 2'
5
4
O
30
8
H 7 6
H 29
H 18 14
27
20
13 17
9 10
22
26
OH
H
H3CO
11
HO
H 16
15
OH
28
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Figure 1. Structure of compound 1.
(dH 5.77, s-like), and four hydroxyl groups (dH 2.91, s; dH 2.63, d, J ¼ 8.0; dH 2.49, br s; dH 2.49, br s). The 13C NMR and DEPT135 spectra indicated the presence of 8 methyls, 10 methylenes, 10 methines, and 8 quaternary carbons (one at dC 166.5 was assigned to be an ester carbonyl carbon). These data suggested that 1 could be an apotirucallane-type triterpenoid with a Table 1. Position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
1
senecioyl ester side chain. HSQC spectrum of 1 allowed all protons to be assigned to their respective carbons, and the gross planar structure of 1 was initially deduced by comprehensive analysis of its 1H – 1H COSY and HMBC spectra. The 1H – 1H couplings from H2-2 to H2-1 and H-3, from H2-6 to H-5 and H-7, from H2-11 to H-9 and H2-12, were observed in the COSY
H (500 MHz) and 13C (125 MHz) NMR spectroscopic data of 1 (in CDCl3).
dH (J in Hz)
dC
dH (J in Hz)
Position
1.34– 1.37 (1H, m, overlap) 33.9 21 1.17– 1.21 (1H, m) 1.90– 1.92 (1H, m) 22.8 22 1.59– 1.62 (1H, m) 4.68 (1H, t-like, J ¼ 2.7) 77.0 23 36.3 24 1.92– 2.08 (1H, m, overlap) 41.3 25 1.63– 1.66 (1H, m) 24.2 26 1.56– 1.59 (1H, m, overlap) 3.75 (1H, br s) 74.2 27 39.0 28 1.33– 1.35 (1H, m, overlap) 43.9 29 37.3 30 1.33– 1.35 (2H, m, overlap) 16.1 10 1.81– 1.85 (2H, m) 25.6 20 28.5 30 36.4 40 1.88– 1.90 (1H, m) 25.9 50 1.55– 1.57 (1H, m, overlap) 1.63– 1.73 (2H, m) 26.3 21 – OMe 1.99– 2.02 (1H, m) 48.4 24-OH 0.77 (1H, d, J ¼ 5.0) 13.7 25-OH 0.49 (1H, d, J ¼ 5.0) 0.90 (3H, s) 15.7 7-OH 2.02– 2.18 (1H, m) 49.2 26-OH
4.90 (1H, d, J ¼ 4.0) 1.94 – 1.98 (1H, m, overlap) 1.74 – 1.76 (1H, m) 4.24 (1H, ddd, J ¼ 10.5, 4.9, 1.4) 3.45 (1H, dd, J ¼ 8.0, 1.5) 3.70 (1H, 3.54 (1H, 1.21 (3H, 0.86 (3H, 0.89 (3H, 1.03 (3H,
d, J ¼ 11.5) d, J ¼ 11.5) s) s) s) s)
5.77 (1H, s-like) 1.90 (3H, s) 2.18 (3H, s) 3.36 (3H, s) 2.63 (1H, d, J ¼ 8.0) 2.91 (1H, s) 2.49 (1H, br s, overlap) 2.49 (1H, br s, overlap)
dC 109.5 33.1 75.7 75.4 74.2 67.9 20.7 27.7 21.9 19.4 166.5 117.0 155.8 27.4 20.3 55.9
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Journal of Asian Natural Products Research spectrum. Another spin system from H2-15 to H-24 was also confirmed by the COSY experiment (Figure 2). The hydroxyl group at dH 2.63 attached to C-24 (dC 75.4) and the one at dH 2.49 attached to C-26 (dC 67.9) were identified by their couplings with H-24 (dH 3.45) and H2-26 (dH 3.70 and 3.54), respectively. Key HMBC correlations from H3-28 and H3-29 to C-3, C-4, and C-5; from H3-19 to C-1, C-5, C-9 and C-10; from H3-30 to C-7, C-8 and C-14; from H-9 to C-8; from H2-11 to C-13; from H2-18 to C-8, C-12, C-14, C-15 and C-17; from H-17 to C-13; from H2-16 to C-14; and from H-24 to C-25, C-26 and C-27 were observed in the HMBC spectrum. The senecioyl ester side chain was also confirmed by HMBC correlations from H3-40 and H3-50 to C-20 , C-30 , and from H320 to C-10 . The HMBC correlation from H-3 to C-10 secured the connectivity of C-3 (dC 77.0) to C-10 (dC 166.5) through an ester linkage. The methoxyl group was located at C-21 by the 3J HMBC correlation from the methoxyl protons at dH 3.36 to C-21. The HMBC correlation from H-21 to C-23 supported the connectivity of C-23 to C-21 through an ether bridge, which was also supported by the downfield chemical shift of C-21 (dC 109.5). Further analyses of the HMBC spectrum and 13C NMR data
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demonstrated that the other two hydroxyl groups at dH 2.49 and 2.91 could be placed on C-7 and C-25 respectively, which completed the planar structure of an apotirucallane-type triterpenoid. The relative stereochemistry of new compound 1 could be determined by NOESY correlations and comparing 13C NMR data with those of known triterpenoids. The NOE correlations between H-3 and H3-29, H3-29 and H3-19, H3-29 and H-6b (dH 1.56 – 1.59), H-6b (dH 1.56 –1.59) and H3-19, H319 and H3-30, H3-30 and H-6b (dH 1.56– 1.59), H-6b (dH 1.56– 1.59) and H-7, H3-30 and H-7, H3-30 and H-17, H3-28 and H-5, H-5 and H-6a (dH 1.63– 1.66), H-5 and H9, and H-9 and H-18a (dH 0.77) (Figure 3) indicated that H-5, H-9, OH-7, and 14,18cyclopropane ring were on the same plane and of a-orientation, while H-3, H-17, Me19, and Me-30 were of b-orientation. The NOE correlations between H-17 and H-21, H-18b (dH 0.49) and H-20, H-20 and H-23, and H-23 and 21-OMe demonstrated that H-20, H-23 and 21-OMe were on the same plane, while the H-21 was on the other side. By comparing the chemical shifts of the C-21, C-24 and C-25 with those of the apotirucallane-type triterpenoids reported in the literature [12], the configurations at C-21, C-24 and C-25 were tentatively HO
HO
O OH H3CO
HMBC
O
H O
C
OH 1H-1HCOSY
Figure 2. Key HMBC and 1H – 1H COSY correlations of 1.
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Z.-K. Guo et al. OH HO
H
O OH
H H H H
OCH3 H
H H H
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O
H
H
OH
Hb Ha
O
Figure 3. Selected NOE correlations of 1.
determined to be R, S, R, respectively. Therefore, the structure of 1 was elucidated as shown in Figure 1. Compound 1 was evaluated in vitro for its cytotoxic activities against human hepatic carcinoma cell line SEL-7420, gastric cell line SGC-7721, and leukemia cell line K-562, but no potential activity was detected (IC50 . 50 mM). It also showed no acetylcholinesterase (AChE) and a-glucosidase inhibitory activities at a concentration of 50 mg/ml, and no antimicrobial activities against plant pathogenic bacteria (Xanthomonas axonopodis pv. manihotis CCXanHNO3, X. axonopodis pv. manihotis CCXanGX30, and Pseudomonas solanacearum), and fungi (Colletotrichum gloeosporioides, Corynespora cassiicola) at a concentration of 20 mg/ml.
Nicolet 380 FT-IR instrument (Thermo, Pittsburgh, PA, USA). NMR spectra were measured by Bruker AvanceIII 500 NMR spectrometer (Bruker, Zurich, Switzerland) at 500 MHz for 1H NMR and at 125 MHz for 13C NMR. The chemical shifts were given in d (ppm) and referenced to the solvent signal (CDCl3, dH 7.26, dC 77.0) and tetramethylsilane (TMS) as the internal standard, and coupling constants (J) are reported in Hz. HR-ESI-MS data were recorded on an Agilent 6210 LC-TOF-MS spectrometer (Agilent, Palo Alto, CA, USA). Silica gel (200 – 300 mesh; Qingdao Marine Chemical Factory, Qingdao, China), ODS gel (20 –45 mm, Fuji Silysia Chemical Ltd, Greenville, NC, USA), and Sephadex LH-20 gel (Pharmacia Biotech, Uppsala, Sweden) were used for column chromatography (CC).
3. Experimental 3.1 General experimental procedures
3.2
Optical rotation was obtained on a Rudolph Autopol III automatic polarimeter (Rudolph Research Analytical, Hackettstown, NJ, USA). UV spectrum was recorded by a Shimadzu UV-2550 spectrometer (Beckman, Brea, CA, USA), and IR spectrum (KBr) was conducted on a
Plant material
The roots of A. buxifolia were collected in Haikou, Hainan Province, China, in April 2011, and identified by Associate Professor Zheng-Fu Dai of the Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, where a voucher specimen (AB201104) has been deposited [8].
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Journal of Asian Natural Products Research 3.3 Extraction and isolation Air-dried roots of A. buxifolia (8.4 kg) were powdered, and then extracted three times with EtOH (95%) under reflux. After evaporation, the residue of the EtOH extract was suspended in H 2O and partitioned with petroleum ether, EtOAc, and n-BuOH, respectively. The EtOAc fraction (183.0 g) was fractionated by silica gel CC eluted with petroleum ether – acetone (1:1, v/v). The subfractions were combined under TLC monitoring to obtain subfractions A – G. Subfraction F (49.4 g) was separated by silica gel CC eluted with a gradient of CHCl3 – MeOH (1:0 to 0:1, v/v) to yield 13 subfractions (Fr.F1 – Fr.F13). Fr.F4 (7.4 g) was subjected to chromatography over a silica gel column with petroleum ether – acetone (5:1, v/v) to give nine fractions (Fr.F41– Fr.F4-9). Fr.F4-6 (363.4 mg) was separated by repeated silica gel CC using petroleum ether – EtOAc (2:1, v/v) as eluent, and purified by Sephadex LH-20 CC with CHCl3 – MeOH (1:1, v/v) to afford compound 1 (6.8 mg). 3.3.1
Compound 1
Amorphous colorless solid; ½a28 D 2 66.4 (c 0.1, CHCl3); UV (MeOH) lmax (log e ): 219 (4.15) nm; IR (KBr) nmax: 3452, 2924, 2855, 1644, 1384, 1018 cm21; 1H and 13C NMR spectroscopic data, see Table 1; HRESI-MS: m/z 641.4028 [M þ Na]þ (calcd for C36H58O8Na, 641.4024). 3.4 Biological assays Cytotoxic activities against human hepatic carcinoma cell line SEL-7420, gastric cell line SGC-7721, and leukemia cell line K562, were evaluated in vitro by the MTT methods [13,14]. Antimicrobial activities against a variety of plant pathogenic bacteria (X. axonopodis pv. manihotis CCXanHNO3, X. axonopodis pv. manihotis CCXanGX30, and P. solanacearum), fungi (C. gloeosporioides, C. cassiicola), and
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human pathogenic Staphylococcus aureus and Candida albicans, and the acetylcholinesterase (AChE), a-glucosidase inhibitory activities were tested in accordance with the primary literatures [15–17]. Funding This work was financially co-supported by the National Support Science and Technology Subject [grant number 2013BAI11B04]; Major Technology Project of Hainan Province [grant number ZDZX2013008-4], [grant number ZDZX2013023-1]; National Natural Science Foundation of China [grant number 41406083]; Special Fund for Agro-Scientific Research in the Public Interest [grant number 201403117].
Disclosure statement No potential conflict of interest was reported by the authors.
References [1] Guangdong Institute of Botany, Flora Hainanica (Science Press, Beijing, 1974), Vol. 3. [2] H.F. Dai, Records of Li Folk Medicine (China Science and Technology Press, Beijing, 2008), Vol. 1. [3] T.S. Wu, M. Niva, and H. Furukawa, Phytochemistry 23, 595 (1984). [4] Y.Q. Gu and Y. Han, Acta Pharm. Sin. 21, 792 (1986). [5] T.S. Wu, Y.L. Leu, Y.Y. Chan, P.L. Wu, C.S. Kuoh, S.J. Wu, and Y. Wang, Phytochemistry 45, 1393 (1997). [6] T.S. Wu and M. Chen, Chem. Pharm. Bull. 48, 85 (2000). [7] T.S. Wu, C.M. Chen, and W. Lin, J. Nat. Prod. 64, 1040 (2001). [8] T. Yang, Y.B. Zeng, Z.K. Guo, W.J. Zuo, S.S. Ma, S.S. Li, W.L. Mei, and H.F. Dai, J. Asian Nat. Prod. Res. 14, 581 (2012). [9] Y.Y. Yang, W. Yang, W.J. Zuo, Y.B. Zeng, S.B. Liu, W.L. Mei, and H.F. Dai, J. Asian Nat. Prod. Res. 15, 899 (2013). [10] X.J. Chen, W.L. Mei, W.J. Zuo, Y.B. Zeng, Z.K. Guo, X.Q. Song, and H.F. Dai, J. Asian Nat. Prod. Res. 15, 67 (2013). [11] H.D. Deng, W.L. Mei, H. Wang, Z.K. Guo, W.H. Dong, H. Wang, S.P. Li, and H.F. Dai, J. Asian Nat. Prod. Res. 16, 1024 (2014).
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[12] K. Mitsui, M. Maejima, H. Saito, H. Fukaya, Y. Hitotsuyanagi, and K. Takeya, Tetrahedron 61, 10569 (2005). [13] T. Mosmann, J. Immunol. Methods 65, 55 (1983). [14] Z.K. Guo, T. Wang, Y. Guo, Y.C. Song, R.X. Tan, and H.M. Ge, Planta Med. 77, 2057 (2011).
[15] D.L. Yang, W.L. Mei, H. Wang, and H.F. Dai, Z. Naturforsch. 65, 757 (2010). [16] Z. Zhang, L. Liao, J. Moore, T. Wu, and Z. Wang, Food Chem. 113, 160 (2009). [17] G.L. Ellman, K.D. Courtney, JR. Valentino Andres, and R.M. Featherstone, Biochem. Pharmacol. 7, 88 (1961).
