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Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Two new ceramides from the fruit pulp of Acanthopanax senticosus (Rupr. et Maxim) Harms a
b
b
c
Zhaowei Yan , Jinping Liu , Dan Lu , Rajeshwar Narlawar , Paul c
b
Groundwater & Pingya Li a
Department of Clinical Pharmacology, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu 215006, P.R. China b
Click for updates
Institute of Frontier Medical Science of Jilin University, ChangChun, Jilin 130021, P.R. China c
Faculty of Pharmacy, University of Sydney, Sydney, NSW 2006, Australia Published online: 21 Nov 2013.
To cite this article: Zhaowei Yan, Jinping Liu, Dan Lu, Rajeshwar Narlawar, Paul Groundwater & Pingya Li (2014) Two new ceramides from the fruit pulp of Acanthopanax senticosus (Rupr. et Maxim) Harms, Natural Product Research: Formerly Natural Product Letters, 28:3, 144-149, DOI: 10.1080/14786419.2013.856908 To link to this article: http://dx.doi.org/10.1080/14786419.2013.856908
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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 & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Natural Product Research, 2014 Vol. 28, No. 3, 144–149, http://dx.doi.org/10.1080/14786419.2013.856908
Two new ceramides from the fruit pulp of Acanthopanax senticosus (Rupr. et Maxim) Harms Zhaowei Yana, Jinping Liub, Dan Lub, Rajeshwar Narlawarc, Paul Groundwaterc and Pingya Lib*
Downloaded by [George Mason University] at 12:00 01 January 2015
a
Department of Clinical Pharmacology, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu 215006, P.R. China; bInstitute of Frontier Medical Science of Jilin University, ChangChun, Jilin 130021, P.R. China; cFaculty of Pharmacy, University of Sydney, Sydney, NSW 2006, Australia (Received 25 August 2013; final version received 15 October 2013) Two new ceramides, (3S,4S,5R)-3-octadecanoylamino-4-hydroxy-5-dodecane-2,3,4,5tetrahydrofuran (1) and (3S,4S,5R)-3-[(2R)-2-hydroxyhexacosanoylamino]-4-hydroxy-5[(4E)-dodecane-4-ene]-2,3,4,5-tetrahydrofuran (2), together with eight known compounds, eleutheroside A (3), eleutheroside B (4), eleutheroside E (5), 7-hydroxy-6-methoxycoumarin (6), 6,7-dimethoxycoumarin (7), 5a,8a-epidioxyergosta-6,22-dien-3-ol (8), stigmasterol (9) and rutin (10), were isolated from the fruit pulp of Acanthopanax senticosus (Rupr. et Maxim) Harms. Their structures were elucidated by means of physicochemical properties and spectroscopic methods (1D, 2D NMR and MS). Keywords: Acanthopanax senticosus (Rupr. et Maxim) Harms; chemical constituents; new ceramides
1. Introduction The ceramides are a unique class of lipids, belonging to the family of sphingolipids, which consist of a sphingoid base and an amide-linked fatty acid chain. They play a crucial role in cell proliferation, differentiation and apoptosis (Obeid & Hannun 1995). A growing collection of evidence has indicated that ceramides are involved in a wide range including biological functions of regulating cell growth and differentiation, and participating in protein secretion and the immunological process. The anticancer activity of ceramides and their derivatives has been confirmed (Radin 2003; Reynolds et al. 2004). Acanthopanax senticosus (Rupr. et Maxim) Harms is a medicinal plant and belongs to the Araliaceae family, which is widely distributed in the Changbai mountains of Jilin Province, P.R. China (Wang et al. 2003). A. senticosus has long been used as a traditional medicine in the treatment of diabetes, tumours, hypertension and cerebrovascular diseases. At present, most of the chemical and pharmacological studies have been mainly focused on the leaves and roots of A. senticosus, and only a few reports relate to the fruit pulp. We have previously reported a new 3,4-seco-lupane-type triterpenoid from the fruit pulp of A. senticosus (Yan et al. 2010). In a continuation of our previous study and search for more new active constituents from the pulp of A. senticosus, we report here the isolation and structure elucidation of two new ceramides, (3S,4S,5R)-3-octadecanoylamino-4-hydroxy-5-dodecane-2,3,4,5-tetrahydrofuran (1) and (3S,4S,5R)-3-[(2R)-2-hydroxyhexacosanoyamino]-4-hydroxy-5-[(4E)-dodecane-4-ene]-2,3,4,5tetrahydrofuran (2), along with another eight known compounds, eleutheroside A (3),
*Corresponding author. Email: [email protected] q 2013 Taylor & Francis
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eleutheroside B (4), eleutheroside E (5), 7-hydroxy-6-methoxy-coumarin (6), 6,7-dimethoxycoumarin (7), 5a,8a-epidioxyergosta-6,22-dien-3-ol (8), stigmasterol (9) and rutin (10).
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2. Results and discussion The fruit pulp of A. senticosus was extracted with water and partitioned successively with petroleum ether, CHCl3, EtOAc and n-BuOH. The dried petroleum ether, CHCl3 and EtOAc extracts were then separated by column chromatography, respectively, affording two new ceramides (1 and 2) and eight known compounds (3 –10). Compound 1 was isolated as a white amorphous powder (CHCl3), m.p. 92 –938C. HR-ESIMS showed a quasi-molecular ion peak [M þ Na]þ at m/z 560.49906 (calc. 560.50132), APCIMS (positive mode) also showed a quasi-molecular ion peak [M þ H]þ at m/z 538, and combining this with the NMR data, the molecular formula of 1 was established as C34H67NO3. The 1H NMR spectrum of 1 showed a resonances for an amide proton doublet at d 5.88 (1H, d, J ¼ 7.0 Hz), which was exchangeable upon addition of D2O, confirming the presence of a secondary amide function. In addition, signals for five H-atoms at d 3.45–4.32 in the 1H NMR spectrum, and C-atoms at d 70.4, 75.2 and 85.2 in the 13C NMR spectrum pointed to the presence of OH and/or other oxygenated groups. The overlapped signals at d 1.19–1.50 in the 1H NMR spectrum and the signals at d 22.7–33.4 in the 13C NMR spectrum indicated the presence of long aliphatic chains. Further spectroscopic analysis of 1 suggested the presence of a characteristic tetrahydrofuran ring which was verified by four carbon signals at d 70.4 (C-2), 51.9 (C-3), 75.2 (C-4) and 85.2 (C-5), together with the DEPT and HMBC spectra (Figure S1). Based on the analytical data, compound 1 was proposed to be the ceramide derivative with a tetrahydrofuran ring, which showed similar signal pattern to a known ceramide derivative Suillumide (Leo´n et al. 2008). When compared 1 with known compound Suillumide, both possess the same tetrahydrofuran skeleton, but the main difference lies in the nature of the alkyl chains (Leo´n et al. 2008). APCI-MS was applied to determine the length of alkyl chains in compound 1, giving a quasi-molecular ion peak at m/z 538 [M þ H]þ and characteristic fragment ion peaks at m/z 282 [M2 CH3(CH2)11(C4H5O) OH]þ, m/z 300 [M2 CH3(CH2)16 þ 2H]þ (Figure S1), indicating that the sphingoid moiety contains 16 carbons and the fatty acid moiety contains 18 carbons, respectively. The relative configurations of C-3, 4 and 5 were presumed as S, S, R, respectively, which were confirmed by the NOESY correlations between H-2b and H-5, and between H-3 and both H-4 and H-2a (Figure S1). Moreover, the relative configurations were also confirmed by comparing with the previously reported data (Leo´n et al. 2008). Based on the above data, compound 1 was elucidated as (3S,4S,5R)-3-octadecanoylamino-4-hydroxy-5-dodecane2,3,4,5-tetrahydrofuran. The complete assignment of the proton and carbon spectra was achieved using HMBC, HMQC and DEPT spectra. Compound 2 was isolated as a white amorphous powder (CHCl3), m.p. 82 –838C. HR-ESIMS showed a quasi-molecular ion peak [M þ Na]þ at m/z 686.60425 (calc. 686.60578), APCI-MS (positive mode) also showed a quasi-molecular ion peak [M þ H]þ at m/z 664, and combining this with the NMR data, the molecular formula of 2 was established as C42H81NO4. The 1H NMR spectrum of 2 showed resonances for an amide proton doublet at d 6.86 (1H, d, J ¼ 7.0 Hz), which was exchangeable upon addition of D2O, confirming the presence of a secondary amide function. When the 1H and 13 C NMR spectral data for 2 was compared with 1, it revealed that the spectral data of these compounds were very similar, with the exceptions that compound 2 has an additional hydroxyl group, an olefinic bond and a different length fatty acid moiety. HMBC, HMQC and DEPT techniques were applied to the analysis for the structure of compound 2. The HMBC spectrum (Figure S2) showed important long-range correlations between H-3/C-10 , C-5; H-4/C-2; H-5/C-3, C-200 ; N-H/C-10 ; H-20 /C-10 , C-30 , C-40 , which further
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1 O
5 2''
2
12''
4 OH
3
18'
2'
HN
1' O
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1
5''
1''
1 O
5 2''
2
12 ''
4''
4 OH
3 HN
2'
OH
1'
(CH2)16
3'
26'
O 2
Figure 1. The chemical structures of compounds 1 and 2.
confirmed that compound 2 also possessed characteristic tetrahydrofuran ring, and one hydroxyl group was attached to C-20 (Figure 1). The olefinic bond at d 129.5 and 131.2 was unambiguously assigned at C-400 and C-500 by the analysis of HMBC and HMQC spectra, and the E geometry was established according to the presence of two vicinal carbon signals at dC 32.4 and dC 32.6 (C-300 and C-600 ) (Hua et al. 2000; Seki et al. 2001). Moreover, the position of olefinic bond (C400 vC500 ) was also supported by the APCI-MS characteristic fragment ion peaks at m/z 520[M2 H2O2 CH3(CH2)6(CH)2]þ and 578 [M2 CH3(CH2)5]þ (Figure S2). The sphingoid moiety of compound 2 was also concluded to contain 16 carbons, the same as that in compound 1, while the fatty acid moiety was concluded to contain 26 carbons from the APCI-MS characteristic fragment ion peak at m/z 410 [M2 CH3(CH2)9(CH)2(C4H5O)OH]þ and m/z 496[M2 CH3(CH2)6(CH)2(CH2)3]þ. A NOESY experiment (Figure S2) confirmed the relative configuration of C-3, 4 and 5, which showed that the tetrahydrofuran ring of compound 2 also had the S, S, R configuration, the same as compound 1. The relative configuration of C-20 was determined to be R by comparing the spectral data of H20 and C-20 with the previously reported spectral data (Leo´n et al. 2008). Based on the above data, compound 2 was elucidated as (3S,4S,5R)-3-[(2R)-2-hydroxyhexacosanoylamino]-4hydroxy-5-[(4E)-dodecane-4-ene]-2,3,4,5-tetrahydrofuran. The complete assignment of the proton and carbon spectra was achieved using the HMBC, HMQC and DEPT spectra. The other eight known compounds were identified as eleutheroside A (Chen & Long. 2006), eleutheroside B (Wu et al. 1999), eleutheroside E (Jolad et al. 1980), 7-hydroxy-6methoxy-coumarin, 6,7-dimethoxycoumarin (Zhang & Kong 2006), 5a,8a-epidioxyergosta6,22-dien-3-ol (He et al. 2005), stigmasterol (Feng & Li. 2006) and rutin (Jiang et al. 2002), respectively, through an analysis of their NMR spectra and comparing them with the previously reported data.
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3. Experimental 3.1. General experimental procedures The melting points were determined on a WRS-1B Digital Point Apparatus (Shanghai, China) and are uncorrected. APCI mass spectra were recorded on a Finnigan MAT LCQ mass spectrometer (Thermo, San Jose, CA, USA). High-resolution ESI mass spectra were recorded on an API Qstar Pulsar instrument (PerkinElmer Sciex, Foster City, CA, USA). NMR spectra were obtained in CDCl3 on a Bruker Avance-500 spectrometer (Bruker, Karlsruhe, Germany), with TMS as the internal standard. Silica gel (200 –300 mesh) for chromatography was produced by Qingdao Ocean Chemical Group Co. Ltd. (Qingdao, China).
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3.2. Plant material The fruit of A. senticosus was collected in October 2007 in Jingyu County, Jilin Province, P.R. China, and was authenticated by Prof. Jingmin Zhang of Jilin University. A voucher specimen (No. 101) was deposited at the Institute of Frontier Medical Science of Jilin University. 3.3. Extraction and isolation The seeds were carefully removed from the fruit of A. senticosus, and the remaining parts were collected as the fruit pulp of A. senticosus. The fruit pulp (2 kg) was extracted with water (for 2 days each time, three times) at room temperature. The aqueous extract was combined and concentrated under reduced pressure, followed by the addition of 3 equiv. of ethanol to make the final ethanol concentration , 75% of the total volume (v/v) in order to precipitate proteins, polysaccharides and other macromolecules. The solution was filtered and the residue was removed. The collected ethanol filtrate was evaporated under reduced pressure to give an extract (700 g), which was then suspended in water, and extracted successively with petroleum ether, CHCl3, EtOAc and n-BuOH. The petroleum ether extract (27 g) was subjected to silica gel chromatography, eluting with petroleum ether/acetone (30:1 to 1:1) to give eight fractions: fractions 3, 5 and 6 were further purified by silica gel chromatography column to afford compound 8 (80 mg), compound 1 (160 mg) and compound 2 (85 mg), respectively. The CHCl3 extract (90 g) was subjected to silica gel chromatography column, eluting with petroleum ether/ acetone (10:1 to 1:1) to give four fractions: fraction 3 was purified by silica gel chromatography to afford compound 7 (82 mg) and compound 6 (94 mg). The EtOAc extract (100 g) was also subjected to silica gel chromatography column, eluting with CHCl3/MeOH (20:1 to 1:1) to give seven fractions: fractions 1, 2 and 5 – 7 were further purified by silica gel chromatography column to afford compound 9 (220 mg), compound 3 (68 mg), compound 10 (78 mg), compound 4 (90 mg) and compound 5 (88 mg), respectively. 3.4. Compound 1 (3S,4S,5R)-3-octadecanoylamino-4-hydroxy-5-dodecane-2,3,4,5-tetrahydrofuran: White amorphous powder (CHCl3), m.p. 92 –938C. APCI-MS: [M þ H]þ at m/z 538. HR-ESI-MS: [M þ Na]þ at m/z 560.49906 (calc. 560.50132). 1H NMR (500 MHz, CDCl3): d 5.88 (1H, d, J ¼ 7.0 Hz, –NH), 4.13 (1H, dd, J ¼ 9.0, 7.0 Hz, H-2a), 3.45 (1H, dd, J ¼ 9.0, 7.5 Hz, H-2b), 4.32 (1H, m, H-3), 3.92 (1H, t-like, J ¼ 5.0 Hz, H-4), 3.66 (1H, m, H-5), 2.16 (2H, t, J ¼ 8.0 Hz, H-20 ), 1.50 (2H, m, H-30 ), 1.19– 1.50 (28H, m, H-40 – 170 ), 0.82 (3H, t, J ¼ 7.0 Hz, Me-180 ), 1.49 (2H, m, H-100 ), 1.19 –1.50 (20H, m, H-200 – 1100 ), 0.82 (3H, t, J ¼ 7.0 Hz, Me-1200 ); 13C NMR (125 MHz, CDCl3): d 70.4 (C-2), 51.9 (C-3), 75.2 (C-4), 85.2 (C-5), 174.0 (C-10 ), 36.7 (C-20 ), 25.5 (C-30 ), 22.7 – 31.9 (C-40 –170 ), 14.1 (C-180 ), 33.4 (C-100 ), 22.7 –31.9 (C-200 –1100 ), 14.1 (C-1200 ).
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3.5. Compound 2 (3S,4S,5R)-3-[(2R)-2-hydroxyhexacosanoylamino]-4-hydroxy-5-[(4E)-dodecane-4-ene]-2, 3,4,5-tetrahydrofuran: White amorphous powder (CHCl3), m.p. 82 – 838C. APCI-MS: [M þ H]þ at m/z 664. HR-ESI-MS: [M þ Na]þ at m/z 686.60425 (calc. 686.60578). 1H NMR (500 MHz, CDCl3): d 6.86 (1H, d, J ¼ 7.0 Hz, – NH), 4.13 (1H, m, H-2a), 3.51 (1H, m, H-2b), 4.33 (1H, m, H-3), 3.94 (1H, t-like, J ¼ 5.0 Hz, H-4), 3.67 (1H, m, H-5), 4.09 (1H, m, H-20 ), 1.58 (2H, m, H-30 ), 1.19 –1.50 (44H, m, H-40 –270 ), 0.82 (3H, t, J ¼ 7.0 Hz, Me-280 ), 1.50 (2H, m, H-100 ), 1.50 –1.55 (2H, m, H-200 ), 1.97 (2H, m, H-300 ), 5.38 (1H, dt, J ¼ 15.5,6.0 Hz, H-400 ), 5.29 (1H, dt, J ¼ 15.5, 6.0 Hz, H-500 ), 1.90 (2H, m, H-600 ), 1.19 – 1.50 (10H, m, H-700 – 1100 ), 0.82 (3H, t, J ¼ 7.0, Me-1200 ); 13C NMR (125 MHz, CDCl3): d 70.2 (C-2), 51.6 (C-3), 75.2 (C-4), 85.1 (C-5), 174.5 (C-10 ), 72.3 (C-20 ), 34.9 (C-30 ), 22.7– 32.8 (C-40 – 270 ), 14.1 (C-280 ), 33.4 (C-100 ), 25.7 (C-200 ), 32.4 (C-300 ), 129.5 (C-400 ), 131.2 (C-500 ), 32.6 (C-600 ), 22.7 – 32.8 (C-700 –1100 ), 14.1 (C-1200 ).
4. Conclusions In conclusion, in this study, two new ceramides, (3S,4S,5R)-3-octadecanoylamino-4-hydroxy-5dodecane-2,3,4,5-tetrahydrofuran (1) and (3S,4S,5R)-3-[(2R)-2-hydroxyhexacosanoylamino]-4hydroxy-5-[(4E)-dodecane-4-ene]-2,3, 4,5-tetrahydrofuran (2), together with eight known compounds, eleutheroside A (3), eleutheroside B (4), eleutheroside E (5), 7-hydroxy-6methoxy-coumarin (6), 6,7-dimethoxycoumarin (7), 5a,8a-epidioxyergosta-6,22-dien-3-ol (8), stigmasterol (9) and rutin (10), were isolated from the fruit pulp of A. senticosus (Rupr. et Maxim) Harms. Especially, ceramides are reported for the first time from A. senticosus. The research results could provide an exact scientific basis for R&D of the fruit pulp of A. senticosus in the future.
Supplementary material Supplementary Figures S1 –S14 are available online.
References Chen TY, Long SJ. 2006. The chemical constituents in mangrove Kandelia candel. West China J Pharm Sci. 21:129–131. Feng CG, Li Q. 2006. Studies on chemical constituents from herb of Dracocephalum moldavica. Chin Tradit Pat Med. 28:94–98. He P, Li S, Wang SJ, Yang YC, Shi JG. 2005. Study on chemical constituents in rhizome of Pinellia ternate. Chin J Chin Mater Med. 30:671–674. Hua HM, Cheng MS, Li X. 2000. Studies on the ceramides of Linaria vulgaris Mill. Chin J Med Chem. 10:57–59. Jiang SJ, Wei F, Lu J, Lin RC, Zhang ZJ. 2002. Chemical studies on the Galeobdolon chinense. J China Pharm Univ. 33:487–488. Jolad SD, Hoffmann JJ, Cole JR, Tempesta MS, Bates RB. 1980. Cytotoxic agent from Penstemon deustus (Scrophulariaceae): isolation and stereochemistry of liriodendrin, a symmetrically substituted furofuranoid lignan diglucoside. J Org Chem. 45:1327–1329. Leo´n F, Brouard I, Torres F, Quintana J, Rivera A, Este´vez F, Bermejo J. 2008. A new ceramide from Suillus luteus and its cytotoxic activity against human melanoma cells. Chem Biodivers. 5:120– 125. Obeid LM, Hannun YA. 1995. Ceramide: a stress signal and mediator of growth suppression and apoptosis. J Cell Biochem. 58:191–198. Radin NS. 2003. Designing anticancer drugs via the Achilles heel: ceramide, allylic ketones, and mitochondria. Bioorg Med Chem. 11:2123–2142. Reynolds CP, Maurer BJ, Kolesnick RN. 2004. Ceramide synthesis and metabolisms as a target for cancer therapy. Cancer Lett. 206:169 –180.
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Seki M, Kayo A, Mori K. 2001. Synthesis of (2S,3R,11S,12R,2000 R,11000 S,12000 R)-plakoside A, a prenylated and immunosuppressive marine galactosphingolipid with cyclopropane-containing alkyl chains. Tetrahedron Lett. 42:2357–2360. Wang ZC, Shan QY, Ma AD, Bao YY. 2003. High-performance liquid chromatography combined with mass spectrum analysis for identifying the antifatigue components in Acanthopanax senticosus Harms. J First Mil Med Univ. 23:355–357. Wu LJ, Ruan LJ, Zheng J, Wang FF, Ding FL, Gong HP. 1999. Studies on chemical constituents from Acanthopanax senticosus (Rupr. et Maxim) Harms. Acta Pharm Sin. 34:839–841. Yan ZW, Liu JP, Lu D, Groundwater P, Li PY. 2010. A new 3,4-seco-lupane-type triterpenoid from the pulp of Acanthopanax senticosus (Rupr. et Maxim) Harms. Nat Prod Res. 24:1523– 1527. Zhang Y, Kong LY. 2006. Studies on the constituents of Citrus medica L.var. sarcodactylis Swingle. Mod Chin Med. 8:16–17.
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Two new ceramides from the fruit pulp of Acanthopanax senticosus (Rupr. et Maxim) Harms a
b
b
c
Zhaowei Yan , Jinping Liu , Dan Lu , Rajeshwar Narlawar , Paul c
b
Groundwater & Pingya Li a
Department of Clinical Pharmacology, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu 215006, P.R. China b
Click for updates
Institute of Frontier Medical Science of Jilin University, ChangChun, Jilin 130021, P.R. China c
Faculty of Pharmacy, University of Sydney, Sydney, NSW 2006, Australia Published online: 21 Nov 2013.
To cite this article: Zhaowei Yan, Jinping Liu, Dan Lu, Rajeshwar Narlawar, Paul Groundwater & Pingya Li (2014) Two new ceramides from the fruit pulp of Acanthopanax senticosus (Rupr. et Maxim) Harms, Natural Product Research: Formerly Natural Product Letters, 28:3, 144-149, DOI: 10.1080/14786419.2013.856908 To link to this article: http://dx.doi.org/10.1080/14786419.2013.856908
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.
Downloaded by [George Mason University] at 12:00 01 January 2015
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 & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Natural Product Research, 2014 Vol. 28, No. 3, 144–149, http://dx.doi.org/10.1080/14786419.2013.856908
Two new ceramides from the fruit pulp of Acanthopanax senticosus (Rupr. et Maxim) Harms Zhaowei Yana, Jinping Liub, Dan Lub, Rajeshwar Narlawarc, Paul Groundwaterc and Pingya Lib*
Downloaded by [George Mason University] at 12:00 01 January 2015
a
Department of Clinical Pharmacology, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu 215006, P.R. China; bInstitute of Frontier Medical Science of Jilin University, ChangChun, Jilin 130021, P.R. China; cFaculty of Pharmacy, University of Sydney, Sydney, NSW 2006, Australia (Received 25 August 2013; final version received 15 October 2013) Two new ceramides, (3S,4S,5R)-3-octadecanoylamino-4-hydroxy-5-dodecane-2,3,4,5tetrahydrofuran (1) and (3S,4S,5R)-3-[(2R)-2-hydroxyhexacosanoylamino]-4-hydroxy-5[(4E)-dodecane-4-ene]-2,3,4,5-tetrahydrofuran (2), together with eight known compounds, eleutheroside A (3), eleutheroside B (4), eleutheroside E (5), 7-hydroxy-6-methoxycoumarin (6), 6,7-dimethoxycoumarin (7), 5a,8a-epidioxyergosta-6,22-dien-3-ol (8), stigmasterol (9) and rutin (10), were isolated from the fruit pulp of Acanthopanax senticosus (Rupr. et Maxim) Harms. Their structures were elucidated by means of physicochemical properties and spectroscopic methods (1D, 2D NMR and MS). Keywords: Acanthopanax senticosus (Rupr. et Maxim) Harms; chemical constituents; new ceramides
1. Introduction The ceramides are a unique class of lipids, belonging to the family of sphingolipids, which consist of a sphingoid base and an amide-linked fatty acid chain. They play a crucial role in cell proliferation, differentiation and apoptosis (Obeid & Hannun 1995). A growing collection of evidence has indicated that ceramides are involved in a wide range including biological functions of regulating cell growth and differentiation, and participating in protein secretion and the immunological process. The anticancer activity of ceramides and their derivatives has been confirmed (Radin 2003; Reynolds et al. 2004). Acanthopanax senticosus (Rupr. et Maxim) Harms is a medicinal plant and belongs to the Araliaceae family, which is widely distributed in the Changbai mountains of Jilin Province, P.R. China (Wang et al. 2003). A. senticosus has long been used as a traditional medicine in the treatment of diabetes, tumours, hypertension and cerebrovascular diseases. At present, most of the chemical and pharmacological studies have been mainly focused on the leaves and roots of A. senticosus, and only a few reports relate to the fruit pulp. We have previously reported a new 3,4-seco-lupane-type triterpenoid from the fruit pulp of A. senticosus (Yan et al. 2010). In a continuation of our previous study and search for more new active constituents from the pulp of A. senticosus, we report here the isolation and structure elucidation of two new ceramides, (3S,4S,5R)-3-octadecanoylamino-4-hydroxy-5-dodecane-2,3,4,5-tetrahydrofuran (1) and (3S,4S,5R)-3-[(2R)-2-hydroxyhexacosanoyamino]-4-hydroxy-5-[(4E)-dodecane-4-ene]-2,3,4,5tetrahydrofuran (2), along with another eight known compounds, eleutheroside A (3),
*Corresponding author. Email: [email protected] q 2013 Taylor & Francis
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eleutheroside B (4), eleutheroside E (5), 7-hydroxy-6-methoxy-coumarin (6), 6,7-dimethoxycoumarin (7), 5a,8a-epidioxyergosta-6,22-dien-3-ol (8), stigmasterol (9) and rutin (10).
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2. Results and discussion The fruit pulp of A. senticosus was extracted with water and partitioned successively with petroleum ether, CHCl3, EtOAc and n-BuOH. The dried petroleum ether, CHCl3 and EtOAc extracts were then separated by column chromatography, respectively, affording two new ceramides (1 and 2) and eight known compounds (3 –10). Compound 1 was isolated as a white amorphous powder (CHCl3), m.p. 92 –938C. HR-ESIMS showed a quasi-molecular ion peak [M þ Na]þ at m/z 560.49906 (calc. 560.50132), APCIMS (positive mode) also showed a quasi-molecular ion peak [M þ H]þ at m/z 538, and combining this with the NMR data, the molecular formula of 1 was established as C34H67NO3. The 1H NMR spectrum of 1 showed a resonances for an amide proton doublet at d 5.88 (1H, d, J ¼ 7.0 Hz), which was exchangeable upon addition of D2O, confirming the presence of a secondary amide function. In addition, signals for five H-atoms at d 3.45–4.32 in the 1H NMR spectrum, and C-atoms at d 70.4, 75.2 and 85.2 in the 13C NMR spectrum pointed to the presence of OH and/or other oxygenated groups. The overlapped signals at d 1.19–1.50 in the 1H NMR spectrum and the signals at d 22.7–33.4 in the 13C NMR spectrum indicated the presence of long aliphatic chains. Further spectroscopic analysis of 1 suggested the presence of a characteristic tetrahydrofuran ring which was verified by four carbon signals at d 70.4 (C-2), 51.9 (C-3), 75.2 (C-4) and 85.2 (C-5), together with the DEPT and HMBC spectra (Figure S1). Based on the analytical data, compound 1 was proposed to be the ceramide derivative with a tetrahydrofuran ring, which showed similar signal pattern to a known ceramide derivative Suillumide (Leo´n et al. 2008). When compared 1 with known compound Suillumide, both possess the same tetrahydrofuran skeleton, but the main difference lies in the nature of the alkyl chains (Leo´n et al. 2008). APCI-MS was applied to determine the length of alkyl chains in compound 1, giving a quasi-molecular ion peak at m/z 538 [M þ H]þ and characteristic fragment ion peaks at m/z 282 [M2 CH3(CH2)11(C4H5O) OH]þ, m/z 300 [M2 CH3(CH2)16 þ 2H]þ (Figure S1), indicating that the sphingoid moiety contains 16 carbons and the fatty acid moiety contains 18 carbons, respectively. The relative configurations of C-3, 4 and 5 were presumed as S, S, R, respectively, which were confirmed by the NOESY correlations between H-2b and H-5, and between H-3 and both H-4 and H-2a (Figure S1). Moreover, the relative configurations were also confirmed by comparing with the previously reported data (Leo´n et al. 2008). Based on the above data, compound 1 was elucidated as (3S,4S,5R)-3-octadecanoylamino-4-hydroxy-5-dodecane2,3,4,5-tetrahydrofuran. The complete assignment of the proton and carbon spectra was achieved using HMBC, HMQC and DEPT spectra. Compound 2 was isolated as a white amorphous powder (CHCl3), m.p. 82 –838C. HR-ESIMS showed a quasi-molecular ion peak [M þ Na]þ at m/z 686.60425 (calc. 686.60578), APCI-MS (positive mode) also showed a quasi-molecular ion peak [M þ H]þ at m/z 664, and combining this with the NMR data, the molecular formula of 2 was established as C42H81NO4. The 1H NMR spectrum of 2 showed resonances for an amide proton doublet at d 6.86 (1H, d, J ¼ 7.0 Hz), which was exchangeable upon addition of D2O, confirming the presence of a secondary amide function. When the 1H and 13 C NMR spectral data for 2 was compared with 1, it revealed that the spectral data of these compounds were very similar, with the exceptions that compound 2 has an additional hydroxyl group, an olefinic bond and a different length fatty acid moiety. HMBC, HMQC and DEPT techniques were applied to the analysis for the structure of compound 2. The HMBC spectrum (Figure S2) showed important long-range correlations between H-3/C-10 , C-5; H-4/C-2; H-5/C-3, C-200 ; N-H/C-10 ; H-20 /C-10 , C-30 , C-40 , which further
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1 O
5 2''
2
12''
4 OH
3
18'
2'
HN
1' O
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1
5''
1''
1 O
5 2''
2
12 ''
4''
4 OH
3 HN
2'
OH
1'
(CH2)16
3'
26'
O 2
Figure 1. The chemical structures of compounds 1 and 2.
confirmed that compound 2 also possessed characteristic tetrahydrofuran ring, and one hydroxyl group was attached to C-20 (Figure 1). The olefinic bond at d 129.5 and 131.2 was unambiguously assigned at C-400 and C-500 by the analysis of HMBC and HMQC spectra, and the E geometry was established according to the presence of two vicinal carbon signals at dC 32.4 and dC 32.6 (C-300 and C-600 ) (Hua et al. 2000; Seki et al. 2001). Moreover, the position of olefinic bond (C400 vC500 ) was also supported by the APCI-MS characteristic fragment ion peaks at m/z 520[M2 H2O2 CH3(CH2)6(CH)2]þ and 578 [M2 CH3(CH2)5]þ (Figure S2). The sphingoid moiety of compound 2 was also concluded to contain 16 carbons, the same as that in compound 1, while the fatty acid moiety was concluded to contain 26 carbons from the APCI-MS characteristic fragment ion peak at m/z 410 [M2 CH3(CH2)9(CH)2(C4H5O)OH]þ and m/z 496[M2 CH3(CH2)6(CH)2(CH2)3]þ. A NOESY experiment (Figure S2) confirmed the relative configuration of C-3, 4 and 5, which showed that the tetrahydrofuran ring of compound 2 also had the S, S, R configuration, the same as compound 1. The relative configuration of C-20 was determined to be R by comparing the spectral data of H20 and C-20 with the previously reported spectral data (Leo´n et al. 2008). Based on the above data, compound 2 was elucidated as (3S,4S,5R)-3-[(2R)-2-hydroxyhexacosanoylamino]-4hydroxy-5-[(4E)-dodecane-4-ene]-2,3,4,5-tetrahydrofuran. The complete assignment of the proton and carbon spectra was achieved using the HMBC, HMQC and DEPT spectra. The other eight known compounds were identified as eleutheroside A (Chen & Long. 2006), eleutheroside B (Wu et al. 1999), eleutheroside E (Jolad et al. 1980), 7-hydroxy-6methoxy-coumarin, 6,7-dimethoxycoumarin (Zhang & Kong 2006), 5a,8a-epidioxyergosta6,22-dien-3-ol (He et al. 2005), stigmasterol (Feng & Li. 2006) and rutin (Jiang et al. 2002), respectively, through an analysis of their NMR spectra and comparing them with the previously reported data.
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3. Experimental 3.1. General experimental procedures The melting points were determined on a WRS-1B Digital Point Apparatus (Shanghai, China) and are uncorrected. APCI mass spectra were recorded on a Finnigan MAT LCQ mass spectrometer (Thermo, San Jose, CA, USA). High-resolution ESI mass spectra were recorded on an API Qstar Pulsar instrument (PerkinElmer Sciex, Foster City, CA, USA). NMR spectra were obtained in CDCl3 on a Bruker Avance-500 spectrometer (Bruker, Karlsruhe, Germany), with TMS as the internal standard. Silica gel (200 –300 mesh) for chromatography was produced by Qingdao Ocean Chemical Group Co. Ltd. (Qingdao, China).
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3.2. Plant material The fruit of A. senticosus was collected in October 2007 in Jingyu County, Jilin Province, P.R. China, and was authenticated by Prof. Jingmin Zhang of Jilin University. A voucher specimen (No. 101) was deposited at the Institute of Frontier Medical Science of Jilin University. 3.3. Extraction and isolation The seeds were carefully removed from the fruit of A. senticosus, and the remaining parts were collected as the fruit pulp of A. senticosus. The fruit pulp (2 kg) was extracted with water (for 2 days each time, three times) at room temperature. The aqueous extract was combined and concentrated under reduced pressure, followed by the addition of 3 equiv. of ethanol to make the final ethanol concentration , 75% of the total volume (v/v) in order to precipitate proteins, polysaccharides and other macromolecules. The solution was filtered and the residue was removed. The collected ethanol filtrate was evaporated under reduced pressure to give an extract (700 g), which was then suspended in water, and extracted successively with petroleum ether, CHCl3, EtOAc and n-BuOH. The petroleum ether extract (27 g) was subjected to silica gel chromatography, eluting with petroleum ether/acetone (30:1 to 1:1) to give eight fractions: fractions 3, 5 and 6 were further purified by silica gel chromatography column to afford compound 8 (80 mg), compound 1 (160 mg) and compound 2 (85 mg), respectively. The CHCl3 extract (90 g) was subjected to silica gel chromatography column, eluting with petroleum ether/ acetone (10:1 to 1:1) to give four fractions: fraction 3 was purified by silica gel chromatography to afford compound 7 (82 mg) and compound 6 (94 mg). The EtOAc extract (100 g) was also subjected to silica gel chromatography column, eluting with CHCl3/MeOH (20:1 to 1:1) to give seven fractions: fractions 1, 2 and 5 – 7 were further purified by silica gel chromatography column to afford compound 9 (220 mg), compound 3 (68 mg), compound 10 (78 mg), compound 4 (90 mg) and compound 5 (88 mg), respectively. 3.4. Compound 1 (3S,4S,5R)-3-octadecanoylamino-4-hydroxy-5-dodecane-2,3,4,5-tetrahydrofuran: White amorphous powder (CHCl3), m.p. 92 –938C. APCI-MS: [M þ H]þ at m/z 538. HR-ESI-MS: [M þ Na]þ at m/z 560.49906 (calc. 560.50132). 1H NMR (500 MHz, CDCl3): d 5.88 (1H, d, J ¼ 7.0 Hz, –NH), 4.13 (1H, dd, J ¼ 9.0, 7.0 Hz, H-2a), 3.45 (1H, dd, J ¼ 9.0, 7.5 Hz, H-2b), 4.32 (1H, m, H-3), 3.92 (1H, t-like, J ¼ 5.0 Hz, H-4), 3.66 (1H, m, H-5), 2.16 (2H, t, J ¼ 8.0 Hz, H-20 ), 1.50 (2H, m, H-30 ), 1.19– 1.50 (28H, m, H-40 – 170 ), 0.82 (3H, t, J ¼ 7.0 Hz, Me-180 ), 1.49 (2H, m, H-100 ), 1.19 –1.50 (20H, m, H-200 – 1100 ), 0.82 (3H, t, J ¼ 7.0 Hz, Me-1200 ); 13C NMR (125 MHz, CDCl3): d 70.4 (C-2), 51.9 (C-3), 75.2 (C-4), 85.2 (C-5), 174.0 (C-10 ), 36.7 (C-20 ), 25.5 (C-30 ), 22.7 – 31.9 (C-40 –170 ), 14.1 (C-180 ), 33.4 (C-100 ), 22.7 –31.9 (C-200 –1100 ), 14.1 (C-1200 ).
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3.5. Compound 2 (3S,4S,5R)-3-[(2R)-2-hydroxyhexacosanoylamino]-4-hydroxy-5-[(4E)-dodecane-4-ene]-2, 3,4,5-tetrahydrofuran: White amorphous powder (CHCl3), m.p. 82 – 838C. APCI-MS: [M þ H]þ at m/z 664. HR-ESI-MS: [M þ Na]þ at m/z 686.60425 (calc. 686.60578). 1H NMR (500 MHz, CDCl3): d 6.86 (1H, d, J ¼ 7.0 Hz, – NH), 4.13 (1H, m, H-2a), 3.51 (1H, m, H-2b), 4.33 (1H, m, H-3), 3.94 (1H, t-like, J ¼ 5.0 Hz, H-4), 3.67 (1H, m, H-5), 4.09 (1H, m, H-20 ), 1.58 (2H, m, H-30 ), 1.19 –1.50 (44H, m, H-40 –270 ), 0.82 (3H, t, J ¼ 7.0 Hz, Me-280 ), 1.50 (2H, m, H-100 ), 1.50 –1.55 (2H, m, H-200 ), 1.97 (2H, m, H-300 ), 5.38 (1H, dt, J ¼ 15.5,6.0 Hz, H-400 ), 5.29 (1H, dt, J ¼ 15.5, 6.0 Hz, H-500 ), 1.90 (2H, m, H-600 ), 1.19 – 1.50 (10H, m, H-700 – 1100 ), 0.82 (3H, t, J ¼ 7.0, Me-1200 ); 13C NMR (125 MHz, CDCl3): d 70.2 (C-2), 51.6 (C-3), 75.2 (C-4), 85.1 (C-5), 174.5 (C-10 ), 72.3 (C-20 ), 34.9 (C-30 ), 22.7– 32.8 (C-40 – 270 ), 14.1 (C-280 ), 33.4 (C-100 ), 25.7 (C-200 ), 32.4 (C-300 ), 129.5 (C-400 ), 131.2 (C-500 ), 32.6 (C-600 ), 22.7 – 32.8 (C-700 –1100 ), 14.1 (C-1200 ).
4. Conclusions In conclusion, in this study, two new ceramides, (3S,4S,5R)-3-octadecanoylamino-4-hydroxy-5dodecane-2,3,4,5-tetrahydrofuran (1) and (3S,4S,5R)-3-[(2R)-2-hydroxyhexacosanoylamino]-4hydroxy-5-[(4E)-dodecane-4-ene]-2,3, 4,5-tetrahydrofuran (2), together with eight known compounds, eleutheroside A (3), eleutheroside B (4), eleutheroside E (5), 7-hydroxy-6methoxy-coumarin (6), 6,7-dimethoxycoumarin (7), 5a,8a-epidioxyergosta-6,22-dien-3-ol (8), stigmasterol (9) and rutin (10), were isolated from the fruit pulp of A. senticosus (Rupr. et Maxim) Harms. Especially, ceramides are reported for the first time from A. senticosus. The research results could provide an exact scientific basis for R&D of the fruit pulp of A. senticosus in the future.
Supplementary material Supplementary Figures S1 –S14 are available online.
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