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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 bicyclic sesquiterpenes from the stems of Kadsura heteroclita a
b
a
c
b
Wei Su , Jian-Ping Zhao , Jian Hu , Min Yang , Melissa Jacob , a
a
a
a
Xiong Cai , Rong Zeng , Sheng-Huang Chen , Hui-Yong Huang , b
c
Ikhlas Khan , De-An Guo & Wei Wang
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a
TCM and Ethnomedicine Innovation & Development Laboratory, School of Pharmacy, Hunan University of Chinese Medicine, Changsha 410208, P.R. China b
Research Institute of Pharmaceutical Sciences, National Center for Natural Products Research, University of Mississippi, Oxford, MS 38677, USA c
Shanghai Research Center for Modernization of Traditional Chinese Medicine, National Engineering Laboratory for TCM Standardization Technology, Shanghai Institute of Materia Medica, CAS, Shanghai 201203, P.R. China Published online: 30 Apr 2014.
To cite this article: Wei Su, Jian-Ping Zhao, Jian Hu, Min Yang, Melissa Jacob, Xiong Cai, Rong Zeng, Sheng-Huang Chen, Hui-Yong Huang, Ikhlas Khan, De-An Guo & Wei Wang (2014) Two new bicyclic sesquiterpenes from the stems of Kadsura heteroclita, Natural Product Research: Formerly Natural Product Letters, 28:15, 1197-1201, DOI: 10.1080/14786419.2014.910664 To link to this article: http://dx.doi.org/10.1080/14786419.2014.910664
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Natural Product Research, 2014 Vol. 28, No. 15, 1197–1201, http://dx.doi.org/10.1080/14786419.2014.910664
Two new bicyclic sesquiterpenes from the stems of Kadsura heteroclita
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Wei Sua, Jian-Ping Zhaob, Jian Hua, Min Yangc, Melissa Jacobb, Xiong Caia, Rong Zenga, Sheng-Huang Chena*, Hui-Yong Huanga, Ikhlas Khanb, De-An Guoc and Wei Wanga* a TCM and Ethnomedicine Innovation & Development Laboratory, School of Pharmacy, Hunan University of Chinese Medicine, Changsha 410208, P.R. China; bResearch Institute of Pharmaceutical Sciences, National Center for Natural Products Research, University of Mississippi, Oxford, MS 38677, USA; c Shanghai Research Center for Modernization of Traditional Chinese Medicine, National Engineering Laboratory for TCM Standardization Technology, Shanghai Institute of Materia Medica, CAS, Shanghai 201203, P.R. China
(Received 29 January 2014; final version received 29 March 2014) Two new bicyclic sesquiterpenes, namely 6a,9a-dihydroxycadinan-4-en-3-one (1) and guai-3-en-10a-ol (2), together with one known sesquiterpene were isolated from the stems of Kadsura heteroclita, which is a Tujia ethnomedicine called ‘Xue Tong’. Their structures were elucidated by using spectroscopic analyses and by comparing the NMR data with those reported previously. Keywords: Kadsura heteroclita; Schisandraceae; sesquiterpene; Xue Tong; Tujia ethnomedicine
1. Introduction In China, many minority nationalities have their own ethnomedicine. Tujia ethnomedicine is one of the gems of Chinese folk medicine. Tujia people have developed their own ethnomedicine to prevent and treat diseases over thousands of years. Kadsura heteroclita (Roxb) Craib (Schisandraceae) is a plant mainly distributed in the southwest of China. The stems of this plant, called ‘Xue Tong’ by the Tujia minority people, are used in Xiangxibei of Hunan Province for the treatment of rheumatoid arthritis and traumatic injuries (Xu et al. 2008; Wang 2013). Previous studies indicated that dibenzocyclooctane lignans, lanostane and cycloartane triterpenoids are the main constituents contained in this plant (Han et al. 1992; Chen et al. 2006; Lu & Chen 2006; Wang, Liu, Han, et al. 2006; Wang, Liu, Liu, et al. 2006; Wang, Liu, Ma, et al. 2006; Wang, Ma, et al. 2006). Nineteen sesquiterpenes were detected from the essential oil of K. heteroclita by GS –MS (Li, Bai, et al. 2011a), but only two sesquiterpenes have been isolated and reported so far (Wang, Ma, et al. 2006d). In this article, we describe the isolation and characterisation of two new sesquiterpenes, 6a,9a-dihydroxycadinan-4-en-3-one (1) and guai-3-en-10a-ol (2) together with a known sesquiterpene cryptomeridol (Ando et al. 1994). 2. Results and discussion 6a,9a-Dihydroxycadinan-4-en-3-one (1) (Figure 1) was isolated as a rhombus crystal in CHCl3 – MeOH. Its molecular formula of C15H22O3 was determined from the HR-ESI-MS (m/z 251.16373 [M þ H]þ, calcd. 251.16472) analysis in the positive mode. The IR spectrum of 1
*Corresponding authors. Email: [email protected]; [email protected] q 2014 Taylor & Francis
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Figure 1. Structures of 1 and 2.
revealed stretchings for hydroxyl groups at 3348 and 3294 cm21 and for carbonyl group at 1677 cm21. The 1H NMR spectrum revealed three methyl signals at dH 1.17 (3H, d, J ¼ 7.0 Hz), 1.81 (3H, s) and 1.99 (3H, s). The 13C NMR and DEPT NMR spectra of compound 1 indicated 15 carbon signals attributed to one carbonyl carbon (dC 198.6), four double-bond carbons (dC 113.6, 132.4, 146.5 and 154.8), two oxygenated carbons (dC 70.4 and 72.4), three methines (dC 37.0, 48.1 and 49.8), two methylenes (dC 36.2 and 36.8) and three methyl groups (dC 15.0, 15.3 and 22.5). The 1H and 13C NMR spectra of 1 were similar to those of hypocreaterpene A, which was a cadinane-type sesquiterpene isolated from a marine-derived fungus (Zhu et al. 2013). However, the chemical shifts of C-9, C-11 and C-13 of 1 had distinctive differences with those of hypocreaterpene A. Consideration of the unsaturated degrees and chemical shifts implied that a hydroxyl group should be bonded to C-9 and a double bond should be linked between C-11 and C-13. This deduction was supported by the HMBC correlations. As shown in Figure S6, the correlations from H3-12 and H2-13 to C-7 and C-11 indicated the existence of an isopropenyl unit at C-7. The HMBC correlations of H3-14 to C-1, C-9, C-10 and OH-9 to C-8 and C-10 indicated a methyl group linked to C-10 and another hydroxyl was attached to C-6. In addition, the 1H – 1H COSY correlations of H3-14/H-10/H-9/H2-8 also confirmed this (Figure S4). The NOESY experiment was performed to determine the relative configuration of 1. The observed NOESY correlations between H-7/H-9 and H-9/Me-10 indicated that H-7, H-9 and Me-10 should be positioned in b-orientation, while correlations of the proton of OH-6 with the protons of H-1, OH-9 and Me-11 revealed that H-1, OH-6 and OH-9 and the isopropenyl unit should be assigned as a-orientation (Figure S7). Furthermore, the absolute configuration of 1 has been established by the successful performance of the X-ray diffraction experiment (Figure 2). Thus, compound 1 was identified as 6a,9a-dihydroxycadinan-4-en-3-one, which is a new cadinane-type sesquiterpene. Guai-3-en-10a-ol (2) (Figure 1) was obtained as colourless oil. It had the molecular formula C15H26O based on HR-EI-MS m/z 222.1985 (calcd for 222.1984). The IR spectrum revealed absorptions corresponding to hydroxyl group at 3354 cm21. The 1H NMR spectrum indicated signals of an isopropyl group at dH 0.82 (3H, d, J ¼ 7.2 Hz), 0.87 (3H, d, J ¼ 7.2 Hz) and two methyl groups at dH 1.64 (3H, s), 1.19 (3H, s). The 13C NMR and DEPT data indicated 15 carbons including two olefinic carbons (dC 124.8 and 133.5), one oxygenated carbon (dC 72.4), four methyl carbons (dC 15.4, 21.6, 29.3 and 23.6), four methylenes (dC 19.3, 20.9, 31.2 and 34.6) and four methines (dC 26.6, 34.7, 46.1 and 43.9). On comparing these data with those of the guaiane sesquiterpenes (Lago et al. 2000; Weyerstahl et al. 2000), 2 was deduced as a guaiane skeleton with a double bond between C-3/C4 and a hydroxyl group at C-10. The planar structure of 2 was further confirmed by the HMBC spectra data (Figure S16), which indicated correlations
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Figure 2. X-ray crystallographic structure of 1.
from H-3 to C-1/C-2/C-5/C-15, from H3-12 and H3-13 to C-7/C-11 and from H3-14 to C-1/C-9/ C-10, respectively. Furthermore, the NOESY correlations of H3-14 with H-1, H-5 and H-7 with H-1, H-5 were observed (Figure S17), suggesting a-orientation for OH-10 and b-orientation for H-1, H-5 and CH3-10, respectively. Thus, compound 2 was determined as guai-3-en-10a-ol, which is a new guaiane-type sesquiterpene. The in vitro antifungal and antibacterial activities of 1, 2 and cryptomeridol were tested against Candida albicans, Candida glabrata, Candida krusei, Aspergillus fumigatus, Cryptococcus neoformans, Staphylococcus aureus, methicillin-resistant Staphylococcus, Escherichia coli, Pseudomonas aeruginosa and Mycobacterium intracellulare. They did not exhibit antimicrobial activities (IC50 . 20 mg/mL). In addition, these three compounds did not demonstrate antimalarial activity against Plasmodium falciparum D6 and W2 cells, as well as anti-leishmanial activity to inhibit Leishmania donovani in the related bioassays.
3. Experimental 3.1. General experimental procedures Optical rotation was measured using a AutoPol IV polarimeter (Rudolph Research Analytical Inc., Hackettstown, NJ, USA) at room temperature. UV spectra were recorded by a HewlettPackard (Agilent) 8452A UV –vis spectrometer (Agilent Technologies, Santa Clara, CA, USA). IR spectra were acquired using a Bruker Tensor 27 and MIRacle ATR FT-IR spectrometers (Bruker Optics Inc., Billerica, MA, USA). HR-ESI-MS spectra were recorded on LTQ Orbitrap Velos Pro mass spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). 1D and 2D NMR spectra were obtained on an Agilent DD2-500 and a Varian Mercury plus 400 NMR spectrometers. HREIMS was determined on a LCQ mass spectrometer (Finnigan MAT Inc., San Jose, CA, USA). Silica gel H (80 – 100 mesh and 100– 200 mesh, Qingdao Marine Chemical
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Factory, Qingdao, China) was used for column chromatography and for TLC, respectively. Spots on the TLC plate were observed under UV light and visualised by spraying with 5% vanillin –H2SO4 followed by heating. Sephadex LH-20 gel was used to purify the compounds. 3.2. Plant material The stems of ‘Xue Tong’ were collected at Shimen County, Hunan province, China, in May 2012. Voucher specimens (CEL1280-KH) have been deposited in the TCM and Ethnomedicine Innovation & Development Laboratory, School of Pharmacy, Hunan University of Chinese Medicine, China. The plant was identified by Professor Wei Wang, Hunan University of Chinese Medicine.
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3.3. Extraction and isolation The stems (20 kg) were cut into small segments and extracted with 80% ethanol under reflux (2 h, 3 times, at 808C). The EtOH was evaporated under reduced pressure to yield a residue (475 g). An amount of 385 g ethanol extract was dissolved in 4000 mL aqua. The suspending solution was partitioned successively with PE, CHCl3, EtOAc and n-butanol. After evaporation of the solvent, the CHCl3 partition solution yielded a residue (145 g), which was separated by column chromatography (3.4 kg silica gel, 80 –100 mesh, a gradient system PE – EtOAc) to yield fractions 1 –8. Compound 2 (15.0 mg) was obtained by eluting out Fr.2 with PE –EtOAc (95:5 to 93:7). Fr.5 was repeatedly chromatographed on silica gel columns to yield 1 (3.5 mg). Fr.6 was repeatedly separated on silica gel and purified by using Sephadex LH-20 to afford cryptomeridol (28.0 mg). 3.4. Bioactivity assays These compounds were tested for antifungal, antibacterial, antimalarial and anti-leishmanial activities using the reported methods (Muhammad et al. 2003; Li, Babu, et al. 2011; Ilias et al. 2012). 3.5. 6a,9a-Dihydroxycadinan-4-en-3-one (1) Rhombus crystal (in CHCl3 –MeOH); ½a20 D : 250.5 (c ¼ 1.00, MeOH); UV lmax (MeOH) nm (log 1): 232.0 (6.47); IR (film): 3348, 3294, 1677, 1475, 904 cm21; HR-ESI-MS: m/z 295.1549 [M þ HCOO]2 (calcd for 295.1546). 1H NMR (500 MHz, C5D5N): 2.52 (1H, m, H-1), 2.52 (1H, m, H-2), 2.69 (1H, m, H-2), 6.94 (1H, s, H-5), 2.74 (1H, m, H-7), 2.05 (1H, m, H-8), 2.57(1H, m, H-8), 3.82 (1H, m, H-9), 2.70 (1H, m, H-10), 1.99 (3H, s, H-12), 4.93 (2H, m, H-13), 1.17 (1H, d, J ¼ 7.0 Hz, H-14), 1.81 (3H, s, H-15), 6.46 (1H, s, OH-6), 6.12 (1H, d, J ¼ 7.0 Hz, OH-9). 13C NMR (125 MHz, C5D5N): 49.8 (C-1), 36.2 (C-2), 198.6 (C-3), 132.4 (C-4), 154.8 (C-5), 72.4 (C-6), 48.1 (C-7), 36.8 (C8), 70.4 (C-9), 37.0 (C-10), 146.5 (C-11), 22.5 (C-12), 113.6 (C-13), 15.0 (C-14), 15.3 (C-15). 3.6. Guai-3-en-10a-ol (2) Colourless oil; ½a20 D : þ 38.2 (c ¼ 0.28, MeOH); UV lmax (MeOH) nm (log 1): 207.5 (1.27); IR (KBr): 3354, 2956, 2869, 1452, 1370 cm21; HR-EI-MS: m/z 222.1985 (calcd. for 222.1984). 1H NMR (400 MHz, CDCl3): 1.51 (1H, m, H-1), 1.95 (1H, m, H-2), 1.95 (1H, m, H-2), 5.55 (1H, dd, J ¼ 1.2, 5.6 Hz, H-3), 1.57 (1H, m, H-5), 1.33 (2H, m, H-6), 1.21(1H, m, H-7), 1.53 (1H, m, H-8), 1.58 (1H, m, H-8), 1.44 (1H, m, H-9), 2.24 (1H, m, H-9), 1.97 (1H, m, H-11), 0.82 (3H, J ¼ 7.2 Hz, H-12), 0.87 (3H, J ¼ 7.2 Hz, H-13), 1.19 (3H, s, H-14), 1.64 (3H, s, H-15). 13C NMR (100 MHz, CDCl3) data: 46.1 (C-1), 31.2 (C-2), 124.8 (C-3), 133.5 (C-4), 34.7 (C-5), 19.3 (C-6), 43.9 (C-7), 20.9 (C-8), 34.6 (C-9), 72.4 (C-10), 26.6 (C-11), 15.4 (C-12), 21.6 (C-13), 29.3 (C-14), 23.6 (C-15).
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4. Conclusions Two new bicyclic sesquiterpenes, namely 6a,9a-dihydroxycadinan-4-en-3-one (1) and guai-3en-10a-ol (2), together with cryptomeridol were isolated for the first time from the stems of Kadsura heteroclita. They did not exhibit expected positive antimicrobial, antimalarial and antileishmanial activities. Supplementary material The NMR (1D and 2D), MS, IR and UV data for compounds 1 and 2 are available online as supplementary material.
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Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 201381374062), Hunan Administration of TCM research plan key project (No. 201215), the Ministry of Education of Overseas Returnee Start-up Fund for Scientific Research (No. 2013693), Specialized Research Fund for the Doctoral Program of Higher Education (No. 20134323110004) and Hunan Science and Research Institutions Technological Innovation and Development Project (No. 2012TF1005).
References Ando M, Arai K, Kikuchi K, Isogai K. 1994. Synthetic studies of sesquiterpenes with a cis-fused decalin system, 4. J Nat Prod. 57:1189–1199. Chen M, Jia ZW, Chen DF. 2006. Heteroclitin H, a new lignan from Kadsura heteroclita. J Asian Nat Prod Res. 8:643– 648. Han GQ, Dai P, Xue R, Arison BH, Lankin DC. 1992. Dibenzocyclooctadiene lignans with platelet-activating factor (PAF) antagonist activity from Kadsura heteroclita. J Chin Pharm Sci. 1:20–26. Ilias M, Ibrahim MA, Khan SI, Jacob MR, Tekwani BL, Walker LA, Samoylenko V. 2012. Pentacyclic ingamine alkaloids, a new antiplasmodial pharmacophore from the marine sponge petrosid Ng5 Sp5. Planta Med. 78:1690–1697. Lago JHG, Brochini CB, Roque NF. 2000. Terpenes from leaves of Guarea macrophylla (Meliaceae). Phytochemistry. 55:727–731. Lu Y, Chen DF. 2006. Kadsutherins A –C: three new dibenzocyclooctane lignans from the stems of Kadsura species. Helv Chim Acta. 89:895–901. Li XC, Babu KS, Jacob MR, Khan SI, Agarwal AK, Clark AM. 2011. Natural product-based 6-hydroxy-2,3,4,6tetrahydropyrrolo[1, 2-a ] pyrimidinium scaffold as a new antifungal template. ACS Med Chem Lett. 2:391–395. Li HQ, Bai CQ, Chu SS, Zhou LG, Du SS, Liu ZL, Liu QZ. 2011. Chemical composition and toxicities of the essential oil derived from Kadsura heteroclita stems against Sitophilus zeamais and Meloidogyne incognita. J Med Plant Res. 5:4943–4948. Muhammad I, Li XC, Jacob MR, Tekwani BL, Dunbar DC, Ferreira D. 2003. Antimicrobial and antiparasitic (þ)-transhexahydrodibenzopyrans and analogues from Machaerium multiflorum. J Nat Prod. 66:804–809. Wang W. 2013. The research and development of Tujia ethnomedicine. Planta Med. 79:op-22. Wang W, Liu JZ, Han J, Xu ZR, Liu RX, Liu P, Wang WX, Ma XC, Guan SH, Guo DA. 2006. New triterpenoids from Kadsura heteroclita and cytotoxic activity. Planta Med. 72:450–457. Wang W, Liu JZ, Liu RX, Xu ZR, Yang M, Wang WX, Liu P, Sabia G, Wang XM, Guo DA. 2006. Four new lignans from the stems of Kadsura heteroclita. Planta Med. 72:284–288. Wang W, Liu JZ, Ma XC, Yang M, Wang WX, Xu ZR, Liu P, Guo DA. 2006. Three new cyclolanostane triterpenoids from the ethanol extract of the stems of Kadsura heteroclita. Helv Chim Acta. 89:1888–1893. Wang W, Ma XC, Liu P, Liu RX, Guan SH, Guo DA. 2006. Two new dibenzylbutyrolactone type lignans from the stems of Kadsura heteroclita. Nat Prod Commun. 1:109–112. Weyerstahl P, Marschall H, Splittgerber U, Wolf D. 2000. Analysis of the polar fraction of Haitian vetiver oil. Flavour Fragrance J. 15:153–173. Xu LJ, Liu HT, Peng Y, Chen SB, Chen SL, Xiao PG. 2008. Heteroclitins N–Q, new compounds from stems of Kadsura heteroclita (Roxb.) Craib. Helv Chim Acta. 91:220–226. Zhu H, Hua XX, Gong T, Pang J, Hou Q, Zhu P. 2013. Hypocreaterpenes A and B, cadinane-type sesquiterpenes from a marine-derived fungus, Hypocreales sp. Phytochem Lett. 6:392–396.
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Two new bicyclic sesquiterpenes from the stems of Kadsura heteroclita a
b
a
c
b
Wei Su , Jian-Ping Zhao , Jian Hu , Min Yang , Melissa Jacob , a
a
a
a
Xiong Cai , Rong Zeng , Sheng-Huang Chen , Hui-Yong Huang , b
c
Ikhlas Khan , De-An Guo & Wei Wang
a
a
TCM and Ethnomedicine Innovation & Development Laboratory, School of Pharmacy, Hunan University of Chinese Medicine, Changsha 410208, P.R. China b
Research Institute of Pharmaceutical Sciences, National Center for Natural Products Research, University of Mississippi, Oxford, MS 38677, USA c
Shanghai Research Center for Modernization of Traditional Chinese Medicine, National Engineering Laboratory for TCM Standardization Technology, Shanghai Institute of Materia Medica, CAS, Shanghai 201203, P.R. China Published online: 30 Apr 2014.
To cite this article: Wei Su, Jian-Ping Zhao, Jian Hu, Min Yang, Melissa Jacob, Xiong Cai, Rong Zeng, Sheng-Huang Chen, Hui-Yong Huang, Ikhlas Khan, De-An Guo & Wei Wang (2014) Two new bicyclic sesquiterpenes from the stems of Kadsura heteroclita, Natural Product Research: Formerly Natural Product Letters, 28:15, 1197-1201, DOI: 10.1080/14786419.2014.910664 To link to this article: http://dx.doi.org/10.1080/14786419.2014.910664
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
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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. 15, 1197–1201, http://dx.doi.org/10.1080/14786419.2014.910664
Two new bicyclic sesquiterpenes from the stems of Kadsura heteroclita
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Wei Sua, Jian-Ping Zhaob, Jian Hua, Min Yangc, Melissa Jacobb, Xiong Caia, Rong Zenga, Sheng-Huang Chena*, Hui-Yong Huanga, Ikhlas Khanb, De-An Guoc and Wei Wanga* a TCM and Ethnomedicine Innovation & Development Laboratory, School of Pharmacy, Hunan University of Chinese Medicine, Changsha 410208, P.R. China; bResearch Institute of Pharmaceutical Sciences, National Center for Natural Products Research, University of Mississippi, Oxford, MS 38677, USA; c Shanghai Research Center for Modernization of Traditional Chinese Medicine, National Engineering Laboratory for TCM Standardization Technology, Shanghai Institute of Materia Medica, CAS, Shanghai 201203, P.R. China
(Received 29 January 2014; final version received 29 March 2014) Two new bicyclic sesquiterpenes, namely 6a,9a-dihydroxycadinan-4-en-3-one (1) and guai-3-en-10a-ol (2), together with one known sesquiterpene were isolated from the stems of Kadsura heteroclita, which is a Tujia ethnomedicine called ‘Xue Tong’. Their structures were elucidated by using spectroscopic analyses and by comparing the NMR data with those reported previously. Keywords: Kadsura heteroclita; Schisandraceae; sesquiterpene; Xue Tong; Tujia ethnomedicine
1. Introduction In China, many minority nationalities have their own ethnomedicine. Tujia ethnomedicine is one of the gems of Chinese folk medicine. Tujia people have developed their own ethnomedicine to prevent and treat diseases over thousands of years. Kadsura heteroclita (Roxb) Craib (Schisandraceae) is a plant mainly distributed in the southwest of China. The stems of this plant, called ‘Xue Tong’ by the Tujia minority people, are used in Xiangxibei of Hunan Province for the treatment of rheumatoid arthritis and traumatic injuries (Xu et al. 2008; Wang 2013). Previous studies indicated that dibenzocyclooctane lignans, lanostane and cycloartane triterpenoids are the main constituents contained in this plant (Han et al. 1992; Chen et al. 2006; Lu & Chen 2006; Wang, Liu, Han, et al. 2006; Wang, Liu, Liu, et al. 2006; Wang, Liu, Ma, et al. 2006; Wang, Ma, et al. 2006). Nineteen sesquiterpenes were detected from the essential oil of K. heteroclita by GS –MS (Li, Bai, et al. 2011a), but only two sesquiterpenes have been isolated and reported so far (Wang, Ma, et al. 2006d). In this article, we describe the isolation and characterisation of two new sesquiterpenes, 6a,9a-dihydroxycadinan-4-en-3-one (1) and guai-3-en-10a-ol (2) together with a known sesquiterpene cryptomeridol (Ando et al. 1994). 2. Results and discussion 6a,9a-Dihydroxycadinan-4-en-3-one (1) (Figure 1) was isolated as a rhombus crystal in CHCl3 – MeOH. Its molecular formula of C15H22O3 was determined from the HR-ESI-MS (m/z 251.16373 [M þ H]þ, calcd. 251.16472) analysis in the positive mode. The IR spectrum of 1
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Figure 1. Structures of 1 and 2.
revealed stretchings for hydroxyl groups at 3348 and 3294 cm21 and for carbonyl group at 1677 cm21. The 1H NMR spectrum revealed three methyl signals at dH 1.17 (3H, d, J ¼ 7.0 Hz), 1.81 (3H, s) and 1.99 (3H, s). The 13C NMR and DEPT NMR spectra of compound 1 indicated 15 carbon signals attributed to one carbonyl carbon (dC 198.6), four double-bond carbons (dC 113.6, 132.4, 146.5 and 154.8), two oxygenated carbons (dC 70.4 and 72.4), three methines (dC 37.0, 48.1 and 49.8), two methylenes (dC 36.2 and 36.8) and three methyl groups (dC 15.0, 15.3 and 22.5). The 1H and 13C NMR spectra of 1 were similar to those of hypocreaterpene A, which was a cadinane-type sesquiterpene isolated from a marine-derived fungus (Zhu et al. 2013). However, the chemical shifts of C-9, C-11 and C-13 of 1 had distinctive differences with those of hypocreaterpene A. Consideration of the unsaturated degrees and chemical shifts implied that a hydroxyl group should be bonded to C-9 and a double bond should be linked between C-11 and C-13. This deduction was supported by the HMBC correlations. As shown in Figure S6, the correlations from H3-12 and H2-13 to C-7 and C-11 indicated the existence of an isopropenyl unit at C-7. The HMBC correlations of H3-14 to C-1, C-9, C-10 and OH-9 to C-8 and C-10 indicated a methyl group linked to C-10 and another hydroxyl was attached to C-6. In addition, the 1H – 1H COSY correlations of H3-14/H-10/H-9/H2-8 also confirmed this (Figure S4). The NOESY experiment was performed to determine the relative configuration of 1. The observed NOESY correlations between H-7/H-9 and H-9/Me-10 indicated that H-7, H-9 and Me-10 should be positioned in b-orientation, while correlations of the proton of OH-6 with the protons of H-1, OH-9 and Me-11 revealed that H-1, OH-6 and OH-9 and the isopropenyl unit should be assigned as a-orientation (Figure S7). Furthermore, the absolute configuration of 1 has been established by the successful performance of the X-ray diffraction experiment (Figure 2). Thus, compound 1 was identified as 6a,9a-dihydroxycadinan-4-en-3-one, which is a new cadinane-type sesquiterpene. Guai-3-en-10a-ol (2) (Figure 1) was obtained as colourless oil. It had the molecular formula C15H26O based on HR-EI-MS m/z 222.1985 (calcd for 222.1984). The IR spectrum revealed absorptions corresponding to hydroxyl group at 3354 cm21. The 1H NMR spectrum indicated signals of an isopropyl group at dH 0.82 (3H, d, J ¼ 7.2 Hz), 0.87 (3H, d, J ¼ 7.2 Hz) and two methyl groups at dH 1.64 (3H, s), 1.19 (3H, s). The 13C NMR and DEPT data indicated 15 carbons including two olefinic carbons (dC 124.8 and 133.5), one oxygenated carbon (dC 72.4), four methyl carbons (dC 15.4, 21.6, 29.3 and 23.6), four methylenes (dC 19.3, 20.9, 31.2 and 34.6) and four methines (dC 26.6, 34.7, 46.1 and 43.9). On comparing these data with those of the guaiane sesquiterpenes (Lago et al. 2000; Weyerstahl et al. 2000), 2 was deduced as a guaiane skeleton with a double bond between C-3/C4 and a hydroxyl group at C-10. The planar structure of 2 was further confirmed by the HMBC spectra data (Figure S16), which indicated correlations
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Figure 2. X-ray crystallographic structure of 1.
from H-3 to C-1/C-2/C-5/C-15, from H3-12 and H3-13 to C-7/C-11 and from H3-14 to C-1/C-9/ C-10, respectively. Furthermore, the NOESY correlations of H3-14 with H-1, H-5 and H-7 with H-1, H-5 were observed (Figure S17), suggesting a-orientation for OH-10 and b-orientation for H-1, H-5 and CH3-10, respectively. Thus, compound 2 was determined as guai-3-en-10a-ol, which is a new guaiane-type sesquiterpene. The in vitro antifungal and antibacterial activities of 1, 2 and cryptomeridol were tested against Candida albicans, Candida glabrata, Candida krusei, Aspergillus fumigatus, Cryptococcus neoformans, Staphylococcus aureus, methicillin-resistant Staphylococcus, Escherichia coli, Pseudomonas aeruginosa and Mycobacterium intracellulare. They did not exhibit antimicrobial activities (IC50 . 20 mg/mL). In addition, these three compounds did not demonstrate antimalarial activity against Plasmodium falciparum D6 and W2 cells, as well as anti-leishmanial activity to inhibit Leishmania donovani in the related bioassays.
3. Experimental 3.1. General experimental procedures Optical rotation was measured using a AutoPol IV polarimeter (Rudolph Research Analytical Inc., Hackettstown, NJ, USA) at room temperature. UV spectra were recorded by a HewlettPackard (Agilent) 8452A UV –vis spectrometer (Agilent Technologies, Santa Clara, CA, USA). IR spectra were acquired using a Bruker Tensor 27 and MIRacle ATR FT-IR spectrometers (Bruker Optics Inc., Billerica, MA, USA). HR-ESI-MS spectra were recorded on LTQ Orbitrap Velos Pro mass spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). 1D and 2D NMR spectra were obtained on an Agilent DD2-500 and a Varian Mercury plus 400 NMR spectrometers. HREIMS was determined on a LCQ mass spectrometer (Finnigan MAT Inc., San Jose, CA, USA). Silica gel H (80 – 100 mesh and 100– 200 mesh, Qingdao Marine Chemical
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Factory, Qingdao, China) was used for column chromatography and for TLC, respectively. Spots on the TLC plate were observed under UV light and visualised by spraying with 5% vanillin –H2SO4 followed by heating. Sephadex LH-20 gel was used to purify the compounds. 3.2. Plant material The stems of ‘Xue Tong’ were collected at Shimen County, Hunan province, China, in May 2012. Voucher specimens (CEL1280-KH) have been deposited in the TCM and Ethnomedicine Innovation & Development Laboratory, School of Pharmacy, Hunan University of Chinese Medicine, China. The plant was identified by Professor Wei Wang, Hunan University of Chinese Medicine.
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3.3. Extraction and isolation The stems (20 kg) were cut into small segments and extracted with 80% ethanol under reflux (2 h, 3 times, at 808C). The EtOH was evaporated under reduced pressure to yield a residue (475 g). An amount of 385 g ethanol extract was dissolved in 4000 mL aqua. The suspending solution was partitioned successively with PE, CHCl3, EtOAc and n-butanol. After evaporation of the solvent, the CHCl3 partition solution yielded a residue (145 g), which was separated by column chromatography (3.4 kg silica gel, 80 –100 mesh, a gradient system PE – EtOAc) to yield fractions 1 –8. Compound 2 (15.0 mg) was obtained by eluting out Fr.2 with PE –EtOAc (95:5 to 93:7). Fr.5 was repeatedly chromatographed on silica gel columns to yield 1 (3.5 mg). Fr.6 was repeatedly separated on silica gel and purified by using Sephadex LH-20 to afford cryptomeridol (28.0 mg). 3.4. Bioactivity assays These compounds were tested for antifungal, antibacterial, antimalarial and anti-leishmanial activities using the reported methods (Muhammad et al. 2003; Li, Babu, et al. 2011; Ilias et al. 2012). 3.5. 6a,9a-Dihydroxycadinan-4-en-3-one (1) Rhombus crystal (in CHCl3 –MeOH); ½a20 D : 250.5 (c ¼ 1.00, MeOH); UV lmax (MeOH) nm (log 1): 232.0 (6.47); IR (film): 3348, 3294, 1677, 1475, 904 cm21; HR-ESI-MS: m/z 295.1549 [M þ HCOO]2 (calcd for 295.1546). 1H NMR (500 MHz, C5D5N): 2.52 (1H, m, H-1), 2.52 (1H, m, H-2), 2.69 (1H, m, H-2), 6.94 (1H, s, H-5), 2.74 (1H, m, H-7), 2.05 (1H, m, H-8), 2.57(1H, m, H-8), 3.82 (1H, m, H-9), 2.70 (1H, m, H-10), 1.99 (3H, s, H-12), 4.93 (2H, m, H-13), 1.17 (1H, d, J ¼ 7.0 Hz, H-14), 1.81 (3H, s, H-15), 6.46 (1H, s, OH-6), 6.12 (1H, d, J ¼ 7.0 Hz, OH-9). 13C NMR (125 MHz, C5D5N): 49.8 (C-1), 36.2 (C-2), 198.6 (C-3), 132.4 (C-4), 154.8 (C-5), 72.4 (C-6), 48.1 (C-7), 36.8 (C8), 70.4 (C-9), 37.0 (C-10), 146.5 (C-11), 22.5 (C-12), 113.6 (C-13), 15.0 (C-14), 15.3 (C-15). 3.6. Guai-3-en-10a-ol (2) Colourless oil; ½a20 D : þ 38.2 (c ¼ 0.28, MeOH); UV lmax (MeOH) nm (log 1): 207.5 (1.27); IR (KBr): 3354, 2956, 2869, 1452, 1370 cm21; HR-EI-MS: m/z 222.1985 (calcd. for 222.1984). 1H NMR (400 MHz, CDCl3): 1.51 (1H, m, H-1), 1.95 (1H, m, H-2), 1.95 (1H, m, H-2), 5.55 (1H, dd, J ¼ 1.2, 5.6 Hz, H-3), 1.57 (1H, m, H-5), 1.33 (2H, m, H-6), 1.21(1H, m, H-7), 1.53 (1H, m, H-8), 1.58 (1H, m, H-8), 1.44 (1H, m, H-9), 2.24 (1H, m, H-9), 1.97 (1H, m, H-11), 0.82 (3H, J ¼ 7.2 Hz, H-12), 0.87 (3H, J ¼ 7.2 Hz, H-13), 1.19 (3H, s, H-14), 1.64 (3H, s, H-15). 13C NMR (100 MHz, CDCl3) data: 46.1 (C-1), 31.2 (C-2), 124.8 (C-3), 133.5 (C-4), 34.7 (C-5), 19.3 (C-6), 43.9 (C-7), 20.9 (C-8), 34.6 (C-9), 72.4 (C-10), 26.6 (C-11), 15.4 (C-12), 21.6 (C-13), 29.3 (C-14), 23.6 (C-15).
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4. Conclusions Two new bicyclic sesquiterpenes, namely 6a,9a-dihydroxycadinan-4-en-3-one (1) and guai-3en-10a-ol (2), together with cryptomeridol were isolated for the first time from the stems of Kadsura heteroclita. They did not exhibit expected positive antimicrobial, antimalarial and antileishmanial activities. Supplementary material The NMR (1D and 2D), MS, IR and UV data for compounds 1 and 2 are available online as supplementary material.
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Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 201381374062), Hunan Administration of TCM research plan key project (No. 201215), the Ministry of Education of Overseas Returnee Start-up Fund for Scientific Research (No. 2013693), Specialized Research Fund for the Doctoral Program of Higher Education (No. 20134323110004) and Hunan Science and Research Institutions Technological Innovation and Development Project (No. 2012TF1005).
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