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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
New abietane-type diterpene glycosides from the roots of Tripterygium wilfordii a
a
a
Hong-Mei Li , Da-Wu Wan & Rong-Tao Li a
Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500, China Published online: 15 Jan 2015.
Click for updates To cite this article: Hong-Mei Li, Da-Wu Wan & Rong-Tao Li (2015): New abietane-type diterpene glycosides from the roots of Tripterygium wilfordii, Journal of Asian Natural Products Research, DOI: 10.1080/10286020.2014.1001379 To link to this article: http://dx.doi.org/10.1080/10286020.2014.1001379
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Journal of Asian Natural Products Research, 2015 http://dx.doi.org/10.1080/10286020.2014.1001379
New abietane-type diterpene glycosides from the roots of Tripterygium wilfordii Hong-Mei Li, Da-Wu Wan and Rong-Tao Li* Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500, China
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(Received 18 September 2014; final version received 18 December 2014) Two new abietane diterpene glycosides, wilfordosides A (1) and B (2), were isolated from the roots of Tripterygium wilfordii. The structures of compounds 1 and 2 were established using spectroscopic methods including extensive 1D and 2D NMR analysis, in combination with chemical reactions. Keywords: Tripterygium wilfordii; abietane diterpene glycosides; wilfordosides A and B
1.
Introduction
Plants of the genus Tripterygium (Celastraceae) have been used in traditional Chinese medicine for the treatment of inflammation and rheumatoid arthritis for hundreds of years [1]. Tripterygium wilfordii Hook f. (Leigongteng in Chinese), widely distributed and used popularly in China and East Asia [2,3], has been used as a Chinese herb to treat diseases such as cancer, chronic nephritis, hepatitis, systemic lupus erythematosus, ankylosing spondylitis, rheumatoid arthritis, autoimmune diseases, and a variety of skin disorders [4,5]. A series of alkaloids, diterpenoids, sesquiterpenes, glycosides, and several other constituents have been isolated from T. wilfordii [1,6]. Among them, the diterpenoids and triterpenoids were active anti-inflammatory/immunomodulating natural products and some compounds were reported to possess anticancer activities [6 – 10]. In order to discover more structurally interesting and bioactive secondary metabolites from the genus Tripterygium, a phytochemical investigation on T. wilfordii was carried out, which led to the isolation of two new
abietane diterpene glycosides, wilfordosides A (1) and B (2) (Figure 1). Based on the literature mentioned above, compounds 1 and 2 were evaluated for their anti-inflammatory, anti-cancer, and antiHIV-1 activities, unfortunately the results failed to meet expectations. Herein, this paper deals with the isolation, structural determination, and bioactivities of the two new compounds.
2.
Compound 1 was isolated as a white amorphous powder. A HR-EI-MS measurement established the molecular formula C27H38O10 (m/z 522.2469, [M]þ), requiring nine degrees of unsaturation, which was subsequently confirmed by the observation of the fragment at m/z 545 [M þ Na]þ in the positive ESI-MS of 1. The 1H NMR spectrum (Table 1) displayed a methoxy signal at dH 3.66 (s), along with three methyls including two tertiary ones [dH 2.11 (s, Me-19) and 1.17 (s, Me-20)], an oxygenated methylene [dH 3.49 (dd, J ¼ 13.0, 3.4 Hz, Ha-16) and 3.61 (dd, J ¼ 13.0, 5.3 Hz, Hb-16)], and an aromatic methine (dH 6.44, s, H-12). Besides
*Corresponding author. Email: [email protected] q 2015 Taylor & Francis
Results and discussion
2
H.-M. Li et al. 17 12
HO
13
11
17 12
HO
11
13
1
10
2 3
O
18
OH HO HO
O
9 6
8
14
7
4
16
2 3
OCH3
O
1
9 10
4
5 6
19
OH
HO HO 1
14
8
O
7
H 18
H
O OH
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5
19
OH 16
20
15
20
15
OH O
2
OH
Figure 1. Structures of compounds 1 and 2.
the above proton signals, a series of typical signals of sugar residue was recognized, including the anomeric proton signal at dH 5.57 (d, J ¼ 7.8 Hz, H-10 ), revealing a bconfiguration present in sugar residue on the basis of the coupling constant [11–13]. In combination with the 13C NMR data [dC 95.6 (d, C-10 ) and 62.3 (t, C-60 )], the sugar moiety of 1 was assigned as glucopyranose. Except for the signals belonging to the methoxyl and the glucosyl group, the 13C and DEPT NMR spectra (Table 1) of 1 exhibited resonances for 20 carbons: 3 methyls (dC 18.6, q, Me-17; 18.9, q, Me-19; 18.2, q, Me20), 5 methylenes including an oxygenated one (dC 68.6, t, C-16), 3 methines including an unsaturated one (dC 112.8, d, C-12), and 9 quaternary carbons containing seven unsaturated ones and an carbonyl carbon (dC 169.3, s, C-18), which were assigned to the 18 (4→3) abeo-abietane diterpene skeleton. The above NMR data of the aglycone of 1 were extremely similar to those of 16hydroxytriptobenzene H [14], except for the signals of C-3, C-4, and C-18, suggesting that the glucosyl moiety formed ester glycoside with the carboxyl group of 16-hydroxytriptobenzene H, which was confirmed by the HMBC correlation (Figure 2) from H-10 to C-18. Furthermore, the chemical shift (dC 95.6, d, C-10 ) of anomeric carbon appeared in downfield further supported the existence of ester glycoside [15]. Acid hydrolysis of 1
with 1 M HCl afforded D -glucose as sugar residue, which was confirmed by GC analysis of its corresponding trimethylsilylated L -cysteine derivative. Thus, the structure of 1 was unambiguously assigned as a glycoside derivative of 16-hydroxytriptobenzene H and named as wilfordoside A. Compound 2 was obtained as a white amorphous powder. Its molecular formula was determined to be C26H38O9 on the basis of HR-EI-MS (m/z 494.2506, [M]þ), indicating eight degrees of unsaturation. The NMR data (Table 1) showed that six of the elements of unsaturation were present as an aromatic ring, a hexose residue, and a carbonyl group, which indicated that compound 2 was tricyclic. The singlet signal of the aromatic proton (dH 6.49, s, H-12) in the 1H NMR spectrum indicated the existence of a penta-substituted benzene. Moreover, an isopropyl group [dH 3.62 (overlap, H-15), 1.13 (d, J ¼ 6.6 Hz, H-16), and 1.12 (d, J ¼ 6.6 Hz, H-17)], two tertiary methyls [dH 1.27 (s, H-18) and 1.38 (s, H-20)], a pair of hydroxylmethyl protons at dH 3.57 (d, J ¼ 11.5 Hz, Ha-19) and 4.09 (d, J ¼ 11.5 Hz, Hb-19), as well as a glucosyl anomeric proton at dH 4.49 (d, J ¼ 7.5 Hz, H-10 ) were also observed in the 1H NMR spectrum. Corresponding trimethylsilylated L -cysteine derivative of sugar residue obtained by acid hydrolysis of 2 proved the existence of D -glucose. Furthermore, the
Journal of Asian Natural Products Research
3
Table 1. 1H (400 MHz) and 13C (100 MHz) NMR spectral data of compounds 1 and 2 (CD3OD, d in ppm, J in Hz). 1 No. 1 2
Downloaded by [University of Nebraska, Lincoln] at 00:42 11 April 2015
3 4 5 6
dH
dC
dH
dC
1.40 – 1.47 (m) 3.23 (overlap) 2.49 – 2.59 (m) 2.33 – 2.42 (m)
33.4 (t)
1.73– 1.78 (m) 3.40 (overlap) 2.54 –2.60 (2H, m)
37.0 (t)
2.34 (br d, 12.7) 1.51 – 1.59 (m) 2.18 – 2.25 (m) 2.61 – 2.70 (m) 3.03 (dd, 4.9, 22.0)
7 8 9 10 11 12 13 14 15 16 17 18 19 20 10 20 30 40 50 60 -(OCH3
2
6.44 (s) 3.21 (overlap) 3.49 (dd, 13.0, 3.4) 3.61(dd, 13.0, 5.3) 1.18 (d, 7.0) 2.11 (s) 1.17 (s) 5.57 (d, 7.8) 3.37 (overlap) 3.40 (overlap) 3.38 (overlap) 3.40 (overlap) 3.85 (d, 13.2) 3.64 – 3.72 (m) 3.66 (s)
25.8 (t) 125.6 (s) 149.8 (s) 50.2 (d) 20.9 (t) 27.4 (t) 131.6 (s) 132.6 (s) 38.3 (s) 153.7 (s) 112.8 (d) 135.8 (s) 150.1 (s) 35.6 (d) 68.6 (t) 18.6 (q) 169.3 (s) 18.9 (q) 18.2 (q) 95.6 (d) 74.0 (d) 78.9 (d) 71.1 (d) 78.3 (d) 62.3 (t)
1.98 (d, 11.8) 1.50– 1.56 (m) 1.86– 1.92 (m) 2.76– 2.83 (m) 3.08 (overlap)
6.49 (s) 3.62 (overlap) 1.13 (d, 6.6)a 1.12 (d, 6.6)a 1.27 (s) 3.57 (d, 11.5) 4.09 (d, 11.5) 1.38 (s) 4.49 (d, 7.5) 3.45 (overlap) 3.38 (overlap) 3.37 (overlap) 3.12 (overlap) 3.66 (overlap) 3.78 (dd, 2.2, 11.5)
36.3 (t) 220.6 (s) 54.2 (s) 55.4 (d) 20.8 (t) 29.4 (t) 133.3 (s) 131.9 (s) 39.5 (s) 153.9 (s) 112.3 (d) 140.9 (s) 145.5 (s) 26.4 (d) 24.2 (q)b 24.3 (q)b 22.0 (q) 65.8 (t) 20.5 (q) 106.9 (d) 75.7 (d) 78.0 (d) 71.7 (d) 78.0 (d) 62.8 (t)
61.3 (q)
a,b
Assignments may be interchanged.
configuration of the glucosidic linkage was determined to be b based on the coupling constant (J ¼ 7.5 Hz) [11 – 13]. The 13C NMR and DEPT spectra of 2 showed resonances for 26 carbons: 4 methyls, 6 methenes containing two oxygenated ones, 8 methines including an unsaturated one and five oxygenated ones, together with 8 quarternary carbons including a carbonyl group (dC 220.6, s) and five aromatic ones, of which 20 were assigned to the abietanetype diterpene skeleton, and the remaining were ascribed to the glucosyl group.
Comparison of the 1H and 13C NMR data for 2 with those of triptonediol [16] suggested structural similarities, except for the presence of the additional glucosyl moiety and the disappearance of the methoxyl group (dC 60.7). The HMBC cross-peak (Figure 2) between the anomeric proton of glucosyl group and C-14 (dC 145.5 (s)) suggested that the glucosyl moiety in 2 substituted the methoxyl group at C-14 in triptonediol. Therefore, the structure of compound 2 was determined to be 11,19-dihydroxy-abieta-8,
4
H.-M. Li et al.
HO
HO
OH
O
OCH3 O OH HO HO
O H OH
H
O
O
HO HO
OH
OH O OH
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1
2
Figure 2. Key HMBC correlations of compounds 1 and 2.
11,13-triene-3-oxo-14-O-b-D -glucopyranoside and named as wilfordoside B. Compounds 1 and 2 were examined for their inhibition of superoxide anion generation and elastase release in FMLP/ CB-induced human neutrophils to evaluate their anti-inflammatory potential; however, both compounds at 10 mM concentration exhibited inhibition percentages of lower than 50%. In addition, compounds 1 and 2 were tested for their cytotoxic activity against a panel of human cancer cell lines (A549, MDA-MB-231, KB, and KB-VIN) and for anti-HIV-1 activity, unfortunately they were inactive toward these biological activities.
Milwaukee, USA), and HR-EI-MS were measured on a VG Auto Spec-3000 spectrometer (Micromass, Manchester, UK). Column chromatography (CC) was performed with silica gel (200 –300 mesh, Qingdao Marine Chemical Co. Ltd., Qingdao, China), MCI gel (CHP 20P, 75 –150 mm, Mitsubishi Chemical Corporation, Tokyo, Japan), Lichroprep RP-18 (43 – 63 mm, Merck, Darmstadt, Germany), and Sephadex LH-20 (Amersham Biosciences AB, Uppsala, Sweden). Fractions were monitored by TLC plates (Si gel GF254, Qingdao Marine Chemical and Industrial Factory, China), and spots were visualized by heating silica gel plates sprayed with 5% H2SO4 –EtOH.
3. Experimental 3.1 General experimental procedures
3.2 Plant material The roots of Tripterygium wilfordii were purchased from the Herb Material Market of Juhuacun, Kunming, Yunnan Province, China, in September 2011 and identified by Prof. Hai-zhou Li, Kunming University of Science and Technology. A voucher specimen (Kumst 20110901) was deposited at Laboratory of Phytochemistry, Kunming University of Science and Technology.
Optical rotations were run on a Jasco DIP370 digital polarimeter (JASCO Corporation, Tokyo, Japan). UV spectra were recorded on a Shimadzu UV-2450 spectropolarimeter (Shimadzu Corporation, Tokyo, Japan). IR spectra were acquired using a Bio-Rad FTS-135 spectrophotometer with KBr pellets (Bio-Rad Corporation, California, USA). 1D and 2D NMR spectra were recorded over Bruker AM-400 instrument with tetramethylsilane (TMS) as an internal standard (Bruker BioSpin Group, Karlsruhe, Germany). ESI-MS were obtained with an APIQstar-TOF instrument (Allen-Bradley,
3.3
Extraction and isolation
The air-dried roots of T. wilfordii (10 kg) were powdered and extracted with 70% aq. acetone (3 £ 20 l, 1 day each) at room temperature, then evaporated under reduced
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Journal of Asian Natural Products Research pressured to yield an extract, which was suspended in H2O, and successively partitioned with petroleum ether (30.0 l), EtOAc (30.0 l), and n-BuOH (30.0 l). The EtOAc extract (150 g) was chromatographed over Sephadex LH-20 successively, eluted with MeOH–H2O gradient system (0%, 30%, 60%, and 90%) and 50% acetone–H2O, to obtain four fractions, Fr. 1–4. Fr. 3 (9 g) was subjected to MCI CC, eluting with gradient MeOH–H2O (0%, 20%, 40%, 60%, 80%, and 100%) to yield eight subfractions, Fr. 3.1–3.8. Fr. 3.6 (1.2 g) was further purified over RP-18 (60% MeOH–H2O) followed by silica gel CC (CHCl3 –MeOH–H2O; 10:2:0.2) to produce compound 2 (11.0 mg). Fr. 3.7 (1.1 g) was separated by silica gel CC (CHCl3 – MeOH–H2O; 10:2:0.1) and then subjected to RP-18 (75% MeOH–H2O) to give compound 1 (11.0 mg).
3.3.1 Wilfordoside A (1) White amorphous powder. ½a24:6 D þ 96.0 (c 0.46, MeOH). UV lmax (MeOH) nm (log 1): 203.6 (4.50), 225.0 (sh), 285.8 (3.46). IR (KBr) nmax: 3423, 2962, 2935, 2878, 1710, 1625, 1412, 1353, 1224, 1073, 1027, 585 cm21. For 1H (400 MHz) and 13C (100 MHz) NMR spectral data (CD3OD), see Table 1. ESI-MS (pos.): m/z 545 ([M þ Na]þ); HR-EI-MS: m/z 522.2469 [M]þ (calcd for C27H38O10, 522.2465).
3.3.2 Wilfordoside B (2) White amorphous powder. ½a24:6 þ 62.6 D (c 0.16, MeOH). UV lmax (MeOH) nm (log 1): 203.4 (4.64), 225.0 (sh), 285.0 (3.48). IR (KBr) nmax: 3427, 2961, 2929, 2873, 1679, 1611, 1462, 1418, 1343, 1235, 1074, 1036, 624, 573 cm21. For 1H (400 MHz) and 13C (100 MHz) NMR spectral data (CD3OD), see Table 1. ESIMS (neg.): m/z 493[M –H]2, 330; HR-EIMS: m/z 494.2506 [M] þ (calcd for C26H38O9, 494.2516).
5
3.4 Acid hydrolysis and GC analysis Compounds 1 and 2 (1 mg, each) were hydrolyzed with 1 M HCl (1 ml) under 90–1008C in a screw-capped vial for 8 h. The mixture was filtered after being cooled to 2–48C, and the filtered liquor was evaporated to dryness under a vacuum to obtain the residue. Then the residue was dissolved in 0.5 ml of pyridine containing L cysteine methyl ester (10 mg/ml) and reacted at 608C for 1 h. Then the solution (0.5 ml) of trimethylsilyl imidazole in pyridine (10 mg/ ml) was added to the above mixture, and it was heated at 608C for another 1 h. After centrifugation, the supernatant was directly analyzed by GC (30QC2/AC-5 quartz capillary column, 30 m £ 0.32 mm) with the following conditions: column temperature, 180–2808C at 38C/min; carrier gas N2, 1 ml/min; injection and detector temperature, 2508C; injection volume, 4 ml; split ratio, 1:50. The standards were prepared following the same procedure. Under these conditions, the retention time of D -glucoside derivative was 18.29 min. During co-injection studies, identical retention times were observed between the different hydrolysates and authentic standards. 3.5
Biological activities
3.5.1 Preparation of human neutrophils and measurement of superoxide anion generation and elastase release The preparation of human neutrophils and measurement of superoxide anion generation and elastase release were carried out following the method in the literature [17]. 3.5.2
Cytotoxicity analysis (SRB assay)
Cytotoxicity was determined by the sulforhodamine B (SRB) colorimetric assay according to our protocol [18]. Briefly, cells [A549 (lung carcinoma), MDA-MB231 (triple-negative breast cancer), KB (epidermoid carcinoma of the nasopharynx), and KB-VIN (MDR line overexpressing P-glycoprotein), 3–5 £ 103 cells/well]
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were seeded in 96-well plates filled with RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) containing various concentrations of compounds, and incubated for 72 h. At the end of the exposure period, the attached cells were fixed with cold 50% trichloroacetic acid for 30 min followed by staining with 0.04% SRB (Sigma Chemical Co.) for 30 min. The bound SRB was solubilized in 10 mM Tris – base and the absorbance was measured at 515 nm on a Microplate Reader EL £ 800 (Bio-Tek Instruments, Winnooski, VT) with a Gen5 software. All results were representative of three or more experiments. 3.5.3 HIV-1NL4-3 replication inhibition assay in MT-4 lymphocytes A previously described HIV-1 infectivity assay was used [19,20]. A 96-well microtiter plate was used to set up the HIV-1NL4-3 replication screening assay. NL4-3 variants at a multiplicity of infection of 0.01 were used to infect MT4 cells. Culture supernatants were collected on day 4 PI for the p24 antigen capture using an ELISA kit from ZeptoMetrix Corporation (Buffalo, NY). The 50% inhibition concentration (IC50) was defined as the concentration that inhibits HIV-1NL4-3 replication by 50%. Funding The authors gratefully acknowledge the financial support for this research work by the National Natural Science Foundation of China [grant no. 21262021].
Disclosure statement No potential conflict of interest was reported by the authors.
References [1] L. Guo, L. Duan, K. Liu, E.H. Liu, and P. Li, J. Pharm. Biomed. Anal. 95, 220 (2014).
[2] X.L. Tao, L.S. Davis, and P.E. Lipsky, Arthritis Rheum. 34, 1274 (1991). [3] X.L. Tao and P.E. Lipsky, Rheum. Dis. Clin. North Am. 26, 29 (2000). [4] Z.Q. Ma, Y.J. Li, L.P. Wu, and X. Zhang, Ind. Crops Prod. 52, 642 (2014). [5] A.M. Brinker, J. Ma, P.E. Lipsky, and I. Raskin, Phytochemistry. 68, 732 (2007). [6] J.Z. Xu, J. Lu, F. Sun, H.Z. Zhu, L.J. Wang, X.Y. Zhang, and Z.J. Ma, Phytochemistry. 72, 1482 (2011). [7] Y.T. Gong, B. Xue, J. Jiao, L.M. Jing, and X.M. Wang, J. Neurochem. 107, 779 (2008). [8] N. Lin, C.F. Liu, C. Xiao, H.W. Jia, K. Imada, H. Wu, and A. Ito, Biochem. Pharmacol. 73, 136 (2007). [9] R. Kannaiyan, M.K. Shanmugam, and G. Sethi, Cancer Lett. 303, 9 (2011). [10] G.Q. Li, D. Liu, Y. Zhang, Y.Y. Qian, H. Zhang, S.Y. Guo, M. Sunagawa, T. Hisamitsu, and Y.Q. Liu, PLoS One. 8, e68905 (2013). [11] L. Zhang, Z.X. Zhao, C.Z. Lin, C.C. Zhu, and L. Gao, Phytochem. Lett. 5, 104 (2012). [12] L.R. Wen, J.R. He, D. Wu, Y.M. Jiang, K.N. Prasad, M.M. Zhao, S. Lin, G.X. Jiang, W. Luo, and B. Yang, J. Funct. Foods. 8C, 26 (2014). [13] H.G. Jin, A.R. Kim, H.J. Ko, S.K. Lee, and E.R. Woo, Chem. Pharm. Bull. 62, 288 (2014). [14] G.Z. Yang and Y.C. Li, Helv. Chim. Acta. 85, 168 (2002). [15] L. Faiella, A. Temraz, T. Siciliano, N.D. Tommasi, and A. Braca, Phytochem. Lett. 5, 297 (2012). [16] H.Q. Duan, Y. Takaishi, H. Momota, Y. Ohmoto, T. Taki, Y.F. Jia, and D. Li, J. Nat. Prod. 62, 1522 (1999). [17] S.C. Yang, P.J. Chung, C.M. Ho, C.Y. Kuo, M.F. Hung, Y.T. Huang, W.Y. Chang, Y.W. Chang, K.H. Chan, and T.L. Hwang, J. Immunol. 190, 6511 (2013). [18] Z.J. Zhang, J. Tian, L.T. Wang, M.J. Wang, X. Nan, L. Yang, Y.Q. Liu, S.L. Morris-Natschke, and K.H. Lee, Bioorg. Med. Chem. 22, 204 (2014). [19] C.B. Zhu, L. Zhu, S. Holz-Smith, T.J. Matthews, and C.H. Chen, Proc. Natl. Acad. Sci. 98, 15227 (2001). [20] K. Qian, R.Y. Kuo, C.H. Chen, L. Huang, S.L. Morris-Natschke, and K.H. Lee, J. Med. Chem. 53, 3133 (2010).
Journal of Asian Natural Products Research Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ganp20
New abietane-type diterpene glycosides from the roots of Tripterygium wilfordii a
a
a
Hong-Mei Li , Da-Wu Wan & Rong-Tao Li a
Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500, China Published online: 15 Jan 2015.
Click for updates To cite this article: Hong-Mei Li, Da-Wu Wan & Rong-Tao Li (2015): New abietane-type diterpene glycosides from the roots of Tripterygium wilfordii, Journal of Asian Natural Products Research, DOI: 10.1080/10286020.2014.1001379 To link to this article: http://dx.doi.org/10.1080/10286020.2014.1001379
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 Nebraska, Lincoln] at 00:42 11 April 2015
Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Journal of Asian Natural Products Research, 2015 http://dx.doi.org/10.1080/10286020.2014.1001379
New abietane-type diterpene glycosides from the roots of Tripterygium wilfordii Hong-Mei Li, Da-Wu Wan and Rong-Tao Li* Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500, China
Downloaded by [University of Nebraska, Lincoln] at 00:42 11 April 2015
(Received 18 September 2014; final version received 18 December 2014) Two new abietane diterpene glycosides, wilfordosides A (1) and B (2), were isolated from the roots of Tripterygium wilfordii. The structures of compounds 1 and 2 were established using spectroscopic methods including extensive 1D and 2D NMR analysis, in combination with chemical reactions. Keywords: Tripterygium wilfordii; abietane diterpene glycosides; wilfordosides A and B
1.
Introduction
Plants of the genus Tripterygium (Celastraceae) have been used in traditional Chinese medicine for the treatment of inflammation and rheumatoid arthritis for hundreds of years [1]. Tripterygium wilfordii Hook f. (Leigongteng in Chinese), widely distributed and used popularly in China and East Asia [2,3], has been used as a Chinese herb to treat diseases such as cancer, chronic nephritis, hepatitis, systemic lupus erythematosus, ankylosing spondylitis, rheumatoid arthritis, autoimmune diseases, and a variety of skin disorders [4,5]. A series of alkaloids, diterpenoids, sesquiterpenes, glycosides, and several other constituents have been isolated from T. wilfordii [1,6]. Among them, the diterpenoids and triterpenoids were active anti-inflammatory/immunomodulating natural products and some compounds were reported to possess anticancer activities [6 – 10]. In order to discover more structurally interesting and bioactive secondary metabolites from the genus Tripterygium, a phytochemical investigation on T. wilfordii was carried out, which led to the isolation of two new
abietane diterpene glycosides, wilfordosides A (1) and B (2) (Figure 1). Based on the literature mentioned above, compounds 1 and 2 were evaluated for their anti-inflammatory, anti-cancer, and antiHIV-1 activities, unfortunately the results failed to meet expectations. Herein, this paper deals with the isolation, structural determination, and bioactivities of the two new compounds.
2.
Compound 1 was isolated as a white amorphous powder. A HR-EI-MS measurement established the molecular formula C27H38O10 (m/z 522.2469, [M]þ), requiring nine degrees of unsaturation, which was subsequently confirmed by the observation of the fragment at m/z 545 [M þ Na]þ in the positive ESI-MS of 1. The 1H NMR spectrum (Table 1) displayed a methoxy signal at dH 3.66 (s), along with three methyls including two tertiary ones [dH 2.11 (s, Me-19) and 1.17 (s, Me-20)], an oxygenated methylene [dH 3.49 (dd, J ¼ 13.0, 3.4 Hz, Ha-16) and 3.61 (dd, J ¼ 13.0, 5.3 Hz, Hb-16)], and an aromatic methine (dH 6.44, s, H-12). Besides
*Corresponding author. Email: [email protected] q 2015 Taylor & Francis
Results and discussion
2
H.-M. Li et al. 17 12
HO
13
11
17 12
HO
11
13
1
10
2 3
O
18
OH HO HO
O
9 6
8
14
7
4
16
2 3
OCH3
O
1
9 10
4
5 6
19
OH
HO HO 1
14
8
O
7
H 18
H
O OH
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5
19
OH 16
20
15
20
15
OH O
2
OH
Figure 1. Structures of compounds 1 and 2.
the above proton signals, a series of typical signals of sugar residue was recognized, including the anomeric proton signal at dH 5.57 (d, J ¼ 7.8 Hz, H-10 ), revealing a bconfiguration present in sugar residue on the basis of the coupling constant [11–13]. In combination with the 13C NMR data [dC 95.6 (d, C-10 ) and 62.3 (t, C-60 )], the sugar moiety of 1 was assigned as glucopyranose. Except for the signals belonging to the methoxyl and the glucosyl group, the 13C and DEPT NMR spectra (Table 1) of 1 exhibited resonances for 20 carbons: 3 methyls (dC 18.6, q, Me-17; 18.9, q, Me-19; 18.2, q, Me20), 5 methylenes including an oxygenated one (dC 68.6, t, C-16), 3 methines including an unsaturated one (dC 112.8, d, C-12), and 9 quaternary carbons containing seven unsaturated ones and an carbonyl carbon (dC 169.3, s, C-18), which were assigned to the 18 (4→3) abeo-abietane diterpene skeleton. The above NMR data of the aglycone of 1 were extremely similar to those of 16hydroxytriptobenzene H [14], except for the signals of C-3, C-4, and C-18, suggesting that the glucosyl moiety formed ester glycoside with the carboxyl group of 16-hydroxytriptobenzene H, which was confirmed by the HMBC correlation (Figure 2) from H-10 to C-18. Furthermore, the chemical shift (dC 95.6, d, C-10 ) of anomeric carbon appeared in downfield further supported the existence of ester glycoside [15]. Acid hydrolysis of 1
with 1 M HCl afforded D -glucose as sugar residue, which was confirmed by GC analysis of its corresponding trimethylsilylated L -cysteine derivative. Thus, the structure of 1 was unambiguously assigned as a glycoside derivative of 16-hydroxytriptobenzene H and named as wilfordoside A. Compound 2 was obtained as a white amorphous powder. Its molecular formula was determined to be C26H38O9 on the basis of HR-EI-MS (m/z 494.2506, [M]þ), indicating eight degrees of unsaturation. The NMR data (Table 1) showed that six of the elements of unsaturation were present as an aromatic ring, a hexose residue, and a carbonyl group, which indicated that compound 2 was tricyclic. The singlet signal of the aromatic proton (dH 6.49, s, H-12) in the 1H NMR spectrum indicated the existence of a penta-substituted benzene. Moreover, an isopropyl group [dH 3.62 (overlap, H-15), 1.13 (d, J ¼ 6.6 Hz, H-16), and 1.12 (d, J ¼ 6.6 Hz, H-17)], two tertiary methyls [dH 1.27 (s, H-18) and 1.38 (s, H-20)], a pair of hydroxylmethyl protons at dH 3.57 (d, J ¼ 11.5 Hz, Ha-19) and 4.09 (d, J ¼ 11.5 Hz, Hb-19), as well as a glucosyl anomeric proton at dH 4.49 (d, J ¼ 7.5 Hz, H-10 ) were also observed in the 1H NMR spectrum. Corresponding trimethylsilylated L -cysteine derivative of sugar residue obtained by acid hydrolysis of 2 proved the existence of D -glucose. Furthermore, the
Journal of Asian Natural Products Research
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Table 1. 1H (400 MHz) and 13C (100 MHz) NMR spectral data of compounds 1 and 2 (CD3OD, d in ppm, J in Hz). 1 No. 1 2
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3 4 5 6
dH
dC
dH
dC
1.40 – 1.47 (m) 3.23 (overlap) 2.49 – 2.59 (m) 2.33 – 2.42 (m)
33.4 (t)
1.73– 1.78 (m) 3.40 (overlap) 2.54 –2.60 (2H, m)
37.0 (t)
2.34 (br d, 12.7) 1.51 – 1.59 (m) 2.18 – 2.25 (m) 2.61 – 2.70 (m) 3.03 (dd, 4.9, 22.0)
7 8 9 10 11 12 13 14 15 16 17 18 19 20 10 20 30 40 50 60 -(OCH3
2
6.44 (s) 3.21 (overlap) 3.49 (dd, 13.0, 3.4) 3.61(dd, 13.0, 5.3) 1.18 (d, 7.0) 2.11 (s) 1.17 (s) 5.57 (d, 7.8) 3.37 (overlap) 3.40 (overlap) 3.38 (overlap) 3.40 (overlap) 3.85 (d, 13.2) 3.64 – 3.72 (m) 3.66 (s)
25.8 (t) 125.6 (s) 149.8 (s) 50.2 (d) 20.9 (t) 27.4 (t) 131.6 (s) 132.6 (s) 38.3 (s) 153.7 (s) 112.8 (d) 135.8 (s) 150.1 (s) 35.6 (d) 68.6 (t) 18.6 (q) 169.3 (s) 18.9 (q) 18.2 (q) 95.6 (d) 74.0 (d) 78.9 (d) 71.1 (d) 78.3 (d) 62.3 (t)
1.98 (d, 11.8) 1.50– 1.56 (m) 1.86– 1.92 (m) 2.76– 2.83 (m) 3.08 (overlap)
6.49 (s) 3.62 (overlap) 1.13 (d, 6.6)a 1.12 (d, 6.6)a 1.27 (s) 3.57 (d, 11.5) 4.09 (d, 11.5) 1.38 (s) 4.49 (d, 7.5) 3.45 (overlap) 3.38 (overlap) 3.37 (overlap) 3.12 (overlap) 3.66 (overlap) 3.78 (dd, 2.2, 11.5)
36.3 (t) 220.6 (s) 54.2 (s) 55.4 (d) 20.8 (t) 29.4 (t) 133.3 (s) 131.9 (s) 39.5 (s) 153.9 (s) 112.3 (d) 140.9 (s) 145.5 (s) 26.4 (d) 24.2 (q)b 24.3 (q)b 22.0 (q) 65.8 (t) 20.5 (q) 106.9 (d) 75.7 (d) 78.0 (d) 71.7 (d) 78.0 (d) 62.8 (t)
61.3 (q)
a,b
Assignments may be interchanged.
configuration of the glucosidic linkage was determined to be b based on the coupling constant (J ¼ 7.5 Hz) [11 – 13]. The 13C NMR and DEPT spectra of 2 showed resonances for 26 carbons: 4 methyls, 6 methenes containing two oxygenated ones, 8 methines including an unsaturated one and five oxygenated ones, together with 8 quarternary carbons including a carbonyl group (dC 220.6, s) and five aromatic ones, of which 20 were assigned to the abietanetype diterpene skeleton, and the remaining were ascribed to the glucosyl group.
Comparison of the 1H and 13C NMR data for 2 with those of triptonediol [16] suggested structural similarities, except for the presence of the additional glucosyl moiety and the disappearance of the methoxyl group (dC 60.7). The HMBC cross-peak (Figure 2) between the anomeric proton of glucosyl group and C-14 (dC 145.5 (s)) suggested that the glucosyl moiety in 2 substituted the methoxyl group at C-14 in triptonediol. Therefore, the structure of compound 2 was determined to be 11,19-dihydroxy-abieta-8,
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HO
HO
OH
O
OCH3 O OH HO HO
O H OH
H
O
O
HO HO
OH
OH O OH
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1
2
Figure 2. Key HMBC correlations of compounds 1 and 2.
11,13-triene-3-oxo-14-O-b-D -glucopyranoside and named as wilfordoside B. Compounds 1 and 2 were examined for their inhibition of superoxide anion generation and elastase release in FMLP/ CB-induced human neutrophils to evaluate their anti-inflammatory potential; however, both compounds at 10 mM concentration exhibited inhibition percentages of lower than 50%. In addition, compounds 1 and 2 were tested for their cytotoxic activity against a panel of human cancer cell lines (A549, MDA-MB-231, KB, and KB-VIN) and for anti-HIV-1 activity, unfortunately they were inactive toward these biological activities.
Milwaukee, USA), and HR-EI-MS were measured on a VG Auto Spec-3000 spectrometer (Micromass, Manchester, UK). Column chromatography (CC) was performed with silica gel (200 –300 mesh, Qingdao Marine Chemical Co. Ltd., Qingdao, China), MCI gel (CHP 20P, 75 –150 mm, Mitsubishi Chemical Corporation, Tokyo, Japan), Lichroprep RP-18 (43 – 63 mm, Merck, Darmstadt, Germany), and Sephadex LH-20 (Amersham Biosciences AB, Uppsala, Sweden). Fractions were monitored by TLC plates (Si gel GF254, Qingdao Marine Chemical and Industrial Factory, China), and spots were visualized by heating silica gel plates sprayed with 5% H2SO4 –EtOH.
3. Experimental 3.1 General experimental procedures
3.2 Plant material The roots of Tripterygium wilfordii were purchased from the Herb Material Market of Juhuacun, Kunming, Yunnan Province, China, in September 2011 and identified by Prof. Hai-zhou Li, Kunming University of Science and Technology. A voucher specimen (Kumst 20110901) was deposited at Laboratory of Phytochemistry, Kunming University of Science and Technology.
Optical rotations were run on a Jasco DIP370 digital polarimeter (JASCO Corporation, Tokyo, Japan). UV spectra were recorded on a Shimadzu UV-2450 spectropolarimeter (Shimadzu Corporation, Tokyo, Japan). IR spectra were acquired using a Bio-Rad FTS-135 spectrophotometer with KBr pellets (Bio-Rad Corporation, California, USA). 1D and 2D NMR spectra were recorded over Bruker AM-400 instrument with tetramethylsilane (TMS) as an internal standard (Bruker BioSpin Group, Karlsruhe, Germany). ESI-MS were obtained with an APIQstar-TOF instrument (Allen-Bradley,
3.3
Extraction and isolation
The air-dried roots of T. wilfordii (10 kg) were powdered and extracted with 70% aq. acetone (3 £ 20 l, 1 day each) at room temperature, then evaporated under reduced
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Journal of Asian Natural Products Research pressured to yield an extract, which was suspended in H2O, and successively partitioned with petroleum ether (30.0 l), EtOAc (30.0 l), and n-BuOH (30.0 l). The EtOAc extract (150 g) was chromatographed over Sephadex LH-20 successively, eluted with MeOH–H2O gradient system (0%, 30%, 60%, and 90%) and 50% acetone–H2O, to obtain four fractions, Fr. 1–4. Fr. 3 (9 g) was subjected to MCI CC, eluting with gradient MeOH–H2O (0%, 20%, 40%, 60%, 80%, and 100%) to yield eight subfractions, Fr. 3.1–3.8. Fr. 3.6 (1.2 g) was further purified over RP-18 (60% MeOH–H2O) followed by silica gel CC (CHCl3 –MeOH–H2O; 10:2:0.2) to produce compound 2 (11.0 mg). Fr. 3.7 (1.1 g) was separated by silica gel CC (CHCl3 – MeOH–H2O; 10:2:0.1) and then subjected to RP-18 (75% MeOH–H2O) to give compound 1 (11.0 mg).
3.3.1 Wilfordoside A (1) White amorphous powder. ½a24:6 D þ 96.0 (c 0.46, MeOH). UV lmax (MeOH) nm (log 1): 203.6 (4.50), 225.0 (sh), 285.8 (3.46). IR (KBr) nmax: 3423, 2962, 2935, 2878, 1710, 1625, 1412, 1353, 1224, 1073, 1027, 585 cm21. For 1H (400 MHz) and 13C (100 MHz) NMR spectral data (CD3OD), see Table 1. ESI-MS (pos.): m/z 545 ([M þ Na]þ); HR-EI-MS: m/z 522.2469 [M]þ (calcd for C27H38O10, 522.2465).
3.3.2 Wilfordoside B (2) White amorphous powder. ½a24:6 þ 62.6 D (c 0.16, MeOH). UV lmax (MeOH) nm (log 1): 203.4 (4.64), 225.0 (sh), 285.0 (3.48). IR (KBr) nmax: 3427, 2961, 2929, 2873, 1679, 1611, 1462, 1418, 1343, 1235, 1074, 1036, 624, 573 cm21. For 1H (400 MHz) and 13C (100 MHz) NMR spectral data (CD3OD), see Table 1. ESIMS (neg.): m/z 493[M –H]2, 330; HR-EIMS: m/z 494.2506 [M] þ (calcd for C26H38O9, 494.2516).
5
3.4 Acid hydrolysis and GC analysis Compounds 1 and 2 (1 mg, each) were hydrolyzed with 1 M HCl (1 ml) under 90–1008C in a screw-capped vial for 8 h. The mixture was filtered after being cooled to 2–48C, and the filtered liquor was evaporated to dryness under a vacuum to obtain the residue. Then the residue was dissolved in 0.5 ml of pyridine containing L cysteine methyl ester (10 mg/ml) and reacted at 608C for 1 h. Then the solution (0.5 ml) of trimethylsilyl imidazole in pyridine (10 mg/ ml) was added to the above mixture, and it was heated at 608C for another 1 h. After centrifugation, the supernatant was directly analyzed by GC (30QC2/AC-5 quartz capillary column, 30 m £ 0.32 mm) with the following conditions: column temperature, 180–2808C at 38C/min; carrier gas N2, 1 ml/min; injection and detector temperature, 2508C; injection volume, 4 ml; split ratio, 1:50. The standards were prepared following the same procedure. Under these conditions, the retention time of D -glucoside derivative was 18.29 min. During co-injection studies, identical retention times were observed between the different hydrolysates and authentic standards. 3.5
Biological activities
3.5.1 Preparation of human neutrophils and measurement of superoxide anion generation and elastase release The preparation of human neutrophils and measurement of superoxide anion generation and elastase release were carried out following the method in the literature [17]. 3.5.2
Cytotoxicity analysis (SRB assay)
Cytotoxicity was determined by the sulforhodamine B (SRB) colorimetric assay according to our protocol [18]. Briefly, cells [A549 (lung carcinoma), MDA-MB231 (triple-negative breast cancer), KB (epidermoid carcinoma of the nasopharynx), and KB-VIN (MDR line overexpressing P-glycoprotein), 3–5 £ 103 cells/well]
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were seeded in 96-well plates filled with RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) containing various concentrations of compounds, and incubated for 72 h. At the end of the exposure period, the attached cells were fixed with cold 50% trichloroacetic acid for 30 min followed by staining with 0.04% SRB (Sigma Chemical Co.) for 30 min. The bound SRB was solubilized in 10 mM Tris – base and the absorbance was measured at 515 nm on a Microplate Reader EL £ 800 (Bio-Tek Instruments, Winnooski, VT) with a Gen5 software. All results were representative of three or more experiments. 3.5.3 HIV-1NL4-3 replication inhibition assay in MT-4 lymphocytes A previously described HIV-1 infectivity assay was used [19,20]. A 96-well microtiter plate was used to set up the HIV-1NL4-3 replication screening assay. NL4-3 variants at a multiplicity of infection of 0.01 were used to infect MT4 cells. Culture supernatants were collected on day 4 PI for the p24 antigen capture using an ELISA kit from ZeptoMetrix Corporation (Buffalo, NY). The 50% inhibition concentration (IC50) was defined as the concentration that inhibits HIV-1NL4-3 replication by 50%. Funding The authors gratefully acknowledge the financial support for this research work by the National Natural Science Foundation of China [grant no. 21262021].
Disclosure statement No potential conflict of interest was reported by the authors.
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