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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
A new taxane diterpenoid from Taxus chinensis var. mairei a
a
a
a
Zhi-Hong Zhou , Wei Qu , Min-Zhuo Liu , Jian-Bo Sun , Meng-Hua a
a
Wang , Jing-Yu Liang & Fei-Hua Wu
b
a
Department of Natural Medicinal Chemistry, China Pharmaceutical University, #24 Tongjiaxiang, Nanjing 210009, P.R. China b
Department of Pharmacology for Chinese Materia Medica, China Pharmaceutical University, #639 Longmian, Nanjing 210009, P.R. China Published online: 28 Feb 2014.
To cite this article: Zhi-Hong Zhou, Wei Qu, Min-Zhuo Liu, Jian-Bo Sun, Meng-Hua Wang, Jing-Yu Liang & Fei-Hua Wu (2014) A new taxane diterpenoid from Taxus chinensis var. mairei, Natural Product Research: Formerly Natural Product Letters, 28:8, 530-533, DOI: 10.1080/14786419.2014.881360 To link to this article: http://dx.doi.org/10.1080/14786419.2014.881360
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 [Ams/Girona*barri Lib] at 01:47 10 October 2014
Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Natural Product Research, 2014 Vol. 28, No. 8, 530–533, http://dx.doi.org/10.1080/14786419.2014.881360
A new taxane diterpenoid from Taxus chinensis var. mairei Zhi-Hong Zhoua, Wei Qua, Min-Zhuo Liua, Jian-Bo Suna, Meng-Hua Wanga, Jing-Yu Lianga* and Fei-Hua Wub*
Downloaded by [Ams/Girona*barri Lib] at 01:47 10 October 2014
a Department of Natural Medicinal Chemistry, China Pharmaceutical University, #24 Tongjiaxiang, Nanjing 210009, P.R. China; bDepartment of Pharmacology for Chinese Materia Medica, China Pharmaceutical University, #639 Longmian, Nanjing 210009, P.R. China
(Received 4 November 2013; final version received 6 January 2014) Taxadiene (3), a new taxane diterpenoid with an unusual hydroxy substituting at C-17, and six known compounds including two taxane diterpenoids (1 and 2) and four flavonoids (4 – 7) were isolated from the whole seedling of the Taxus chinensis var. mairei. Among them, compound 7 was isolated from T. chinensis var. mairei for the first time. Structures of these compounds were elucidated on the basis of spectroscopic data and by comparison with reported literature data. Keywords: Taxus chinensis var. mairei; taxane diterpenoids; flavonoids
1. Introduction Taxus chinensis var. mairei (Taxaceae), which belongs to the genus Taxus, is widely distributed in China. Many species in genus Taxus showed significant activities in detoxication and diuretic swelling (Li et al. 2011), some branches and leaves of the species have been well used to treat kidney disease and diabetes (Li et al. 2011). Taxol, the representative composition, showed significant antitumour effect mainly due to its unique mechanism (Schiff et al. 1979). Since taxanes have been an important target for cancer therapy agents for a long time, many other important compounds such as flavonoids, lignans, steroids, phenolic acids, sesquiterpenes, nontaxane diterpenoids and inositol ethers (Li et al. 2008) have been isolated from T. chinensis var. mairei. Pharmacological studies also showed other significant activities such as anti-allergic (Koyama et al. 2006), hepatoprotective (Choi et al. 2006), anti-osteoporosis (Yin et al. 2006), hypoglycaemic (Liang 2002) and antioxidant (Veselova et al. 2007) of this plant. Because of the diversity in chemical constituents and a variety of pharmacological activities, it is important to further study T. chinensis var. mairei. In this article, we reported taxadiene (3), a new taxane together with six known compounds, including two taxane diterpenoids (1 and 2) and four flavonoids (4– 7) (Figure 1) from the whole seedling of T. chinensis var. mairei. Their structures were established on the basis of spectroscopic evidence, including 1D and 2D NMR as well as by comparison with those reported literature data.
2. Results and discussion Compound 3 was isolated as colourless needles. The molecular formula was established as C28H40O11 by HR-ESI-MS (m/z: 551.2498 [M 2 H]2, calcd for 551.2492), which was
*Corresponding authors. Email: [email protected]; [email protected] q 2014 Taylor & Francis
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Figure 1. The structures of isolated compounds.
consistent with the data from the 1H and 13C NMR spectra. 1H NMR spectrum showed the presence of four acetyl groups (dH 2.00, 3H; 2.02, 9H), three methyl groups (dH 0.83, 1.15 and 2.03) and three hydroxyl groups (dH 4.93, 5.26 and 6.03). 13C NMR and DEPT spectra indicated 28 resonance carbons ascribed to seven methyl carbon signals, four methylene carbons, eight methine carbons and nine quaternary carbon signals, which suggested that compound 3 belonged to the taxane analogues with a skeleton of taxa-4(20),11-diene (Yu et al. 2012). Three hydroxyl groups were assigned to C-5, C-10 and C-17 according to the chemical shifts (dC 73.1, 67.8 and 67.3) of the corresponding carbons, respectively. The assignments of all the direct 1H-13C bondings were made on the basis of 1H – 1H COSY and HSQC spectra. In the HMBC spectrum of 3 (Figure S10), H-2, H-7, H-9 and H-13 showed correlations with four carbonyl carbons at chemical shifts dC 169.2, 169.4, 170.2 and 170.4, which suggested that the positions of four acetoxyl groups were assigned to C-2, C-7, C-9 and C-13. Correlations of H-3 (dH 3.48) with C-4 (dC 146.2) and H-20 (dH 4.62 and 5.15) with C-5 (dC 73.1) supported the assigned position of the ethylenic bond between C-4 and C-20 to C-4. The methyl groups were attached to C-8 and C-12 by the HMBC correlation from 19-CH3 (dH 15.6) to C-8 (dC 47.0) and 18-CH3 (dH 20.8) to C-12 (dC 134.8). NOESY correlations (Figure S11) from 3-H to 5-OH, 20b-H to 5-H and 20a-H to 2-OAc supported that the relative configurations of 5-OH and 2-OAc were both of a-orientation. The correlations from 19-H to 7-OAc and from 16-H to 1b-H and 13-H on the NOESY spectrum supported b-orientation of 7-OAc and a-orientation of 13-OAc. Correlations from 17-OH to 10OH and 10-H to 9-OAc indicated that the relative configuration of 10-OH was of b-orientation
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and 9-OAc was of a-orientation. Thus, the compound was determined to be 5a, 10b, 17trihydroxy-2a, 7b, 9a, 13a-tetraacetoxy-taxa-4(20),11-diene, and was named as taxadiene. By comparing physical and spectroscopic data with literature values, the five known compounds were identified as taxol (1) (Chmurny et al. 1992), N-methyl-taxol C (2) (Barboni et al. 1994), ginkgetin (4) (Zhang et al. 2009), sciadopitysin (5) (Zhang et al. 2009), kaempferol (6) (Wang et al. 1999) and apigenin (7) (Zhang et al. 2009).
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3. Experimental 3.1. General experimental procedures Mass spectra were obtained on an MS Agilent 1100 Series LC/MSD Trap mass spectrometer (ESI-MS) and HR-ESI-MS was done on an Agilent 6520B ESI spectrometer (Agilent Technologies, Santa Clara, CA, USA), respectively. NMR spectra were recorded on Bruker ACF-300 NMR (Bruker Corporation, Switzerland) with TMS as internal standard. Optical rotation was tested by a WXG-4 spectropolarimeter (Shanghai Jingke Analytical Instrument Ltd, Shanghai, China). All solvents used were of analytical grade (Tianjin Chemical Co., Ltd, Tianjin, China). Chromatographic silica gel (200 – 300 mesh) and polyamide (100 – 200 mesh) were purchased from Qingdao Marine Chemical Co., Ltd (Qingdao, China). Sephadex LH-20 was purchased from Amersham Pharmacia Biotech, Uppsala, Sweden. TLC analysis was performed on silica gel GF254 produced by Qingdao Marine Chemical Co., Ltd. 3.2. Plant material The whole seedlings of T. chinensis var. mairei were collected from Mingxi County of Fujian Province in China in July 2011 and were identified by Wen-Jian Li, Senior Engineer in Southern Pharmaceutical Company, China. A voucher specimen (no. 20110708) has been deposited in the Department of Natural Medicinal Chemistry, China Pharmaceutical University, Nanjing, China. 3.3. Extraction and isolation The 80% ethanol extract (147.3 g) of T. chinensis var. mairei was suspended with water (10 L), and then partitioned sequentially with petroleum ether (10 L), EtOAc (10 L) and n-BuOH (10 L), respectively. The EtOAc extract (116.5 g) was subjected to column chromatography over silica gel and eluted with a gradient of CH2Cl2 – MeOH (from 100:1 to 1:1) to yield five fractions (Frs 1 –5). Fraction 2 was further subjected to silica gel column eluted with petroleum ether –EtOAc (from 5:4 to 1:1) to affford compounds 1 (15 mg) and 2 (18 mg). Fraction 3 was further subjected to silica gel column eluted with petroleum ether–EtOAc (from 4:1 to 1:1) to afford compounds 3 (5 mg), 4 (9 mg) and 5 (8 mg). A yellow powder obtained from fraction 3 was further subjected to Sephadex LH-20 (MeOH) to afford compounds 6 (5 mg) and 7 (3 mg). 3.4. Spectral data þ Compound 3: colourless needles; ½a25 D ¼ 234:28(CDCl3); ESI-MS m/z: 570.0 [M þ NH4] , 2 2 1 587.5 [M þ Cl] ; HR-ESI-MS m/z: 551.2498 [M 2 H] (calcd for C28H39O11, 551.2492); H NMR (300 MHz, DMSO-d6) dH: 1.92 (1H, m, H-1), 5.32 (1H, d, J ¼ 5.5 Hz, H-2), 3.48 (1H, d, J ¼ 5.5 Hz, H-3), 4.04 (1H, br s, H-5), 1.74 (1H, br d, J ¼ 10.0 Hz, H-6a), 1.55 (1H, dd, J ¼ 12.7, 5.0 Hz, H-6b), 5.47 (1H, dd, J ¼ 10.0, 5.0 Hz, H-7), 5.50 (1H, d, J ¼ 10.0 Hz, H-9), 4.89 (1H, dd, J ¼ 10.0, 6.0 Hz, H-10), 5.58 (1H, overlap, H-13), 2.50 (1H, m, H-14a), 1.49 (1H, dd, J ¼ 14.7, 6.3 Hz, H-14b), 1.15 (3H, s, H-16), 4.66 (1H, d, J ¼ 11.8 Hz, H-17a), 3.32 (1H, m, H-17b), 2.03 (3H, s, H-18), 0.83 (3H, s, H-19), 5.15 (1H, s, H-20a), 4.62 (1H, s, H-20b), 4.93
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(1H, s, OH-5), 6.03 (1H, d, J ¼ 6.0 Hz, OH-10), 5.26 (1H, d, J ¼ 6.0 Hz, OH-17), 2.02 (9H, s, 3 £ OAc-7, 9, 13), 2.00 (3H, s, OAc-2). 13 C NMR (75 MHz, DMSO-d6) dC: 44.8 (C-1), 68.9 (C-2), 39.7 (C-3), 146.2 (C-4), 73.1 (C-5), 37.7 (C-6), 69.5 (C-7), 47.0 (C-8), 78.3 (C-9), 67.8 (C-10), 136.0 (C-11), 134.8 (C-12), 69.1 (C13), 27.8 (C-14), 43.3 (C-15), 25.9 (C-16), 67.3 (C-17), 20.8 (C-18), 15.6 (C-19), 114.0 (C-20), 170.4, 170.2, 169.4, 169.2 (4 £ OAc-2, 7, 9, 13), 21.3, 20.9, 20.9, 21.1 (4 £ OAc-2, 7, 9, 13). Supplementary material Supplementary material relating to this article is available online, alongside Figures S1 – S12.
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Acknowledgements We acknowledge the financial support by the National Natural Science Foundation of China (81072538) and National New Drug Innovation Major Project of China (2011ZX09307-002-02).
References Barboni L, Gariboldi P, Torregiani E, Appendino G, Gabetta B, Bombardelli E. 1994. Taxol analogues from the roots of Taxus media. Phytochemistry. 36:987–990. Chmurny GN, Hilton BD, Brobst S, Look SA, Witherup KM, Beutler JA. 1992. 1H- and 13C-NMR assignments for taxol, 7-epi-taxol, and cephalomannine. J Nat Prod. 55:414–423. Choi SK, Oh HM, Lee SK, Jeong DG, Ryu SE, Son KH, Han DC, Sung ND, Baek NI, Kwon BM. 2006. Biflavonoids inhibited phosphatase of regenerating liver-3 (PRL-3). Nat Prod Res. 20:341–346. Koyama J, Morita I, Kobayashi N, Hirai K, Simamura E, Nobukawa T, Kadota S. 2006. Antiallergic activity of aqueous extracts and constituents of Taxus yunnanensis. Biol Pharm Bull. 29:2310–2312. Li CF, Huo CH, Zhang ML, Shi QW. 2008. Chemistry of Chinese yew, Taxus chinensis var. mairei. Biochem Syst Ecol. 36:266–282. Li XX, Zhang CC, Xiong YK. 2011. Identification research of Taxus wallichiana Zucc. var. mairei. J Chin Med Mater. 34:538–540. Liang JY. A natural compound useful for treating diabetes its preparation and use. EP 1559703 B1, 2002-10-17. Schiff PB, Fant J, Horwitz SB. 1979. Promotion of microtubule assembly in vitro by taxol. Nature. 277:665–667. Veselova MV, Fedoreev SA, Vasilevskaya NA, Denisenko VA, Gerasimenko AV. 2007. Antioxidant activity of polyphenols from the Far-east plant Taxus cuspidata. Pharm Chem J. 41:88– 93. Wang JR, Duan JA, Zhou RH. 1999. Chemica constituents from the bark of Cercidiphyllum japonicum. Acta Bot Sin. 41:209–212. Yin J, Tezuka Y, Subehan, Shi L, Nobukawa M, Nobukawa T, Kadota S. 2006. In vivo anti-osteoporotic activity of isotaxiresinol, a lignan from wood of Taxus yunnanensis. Phytomedicine. 13:37–42. Yu JH, Wang YB, Qian H, Zhao YP, Liu BT, Fu CX. 2012. Polyprenols from the needles of Taxus chinensis var. mairei. Fitoterapia. 83:831–837. Zhang N, Lu JC, Wang J, Han L, Zhang JJ. 2009. Isolation and identification of chemical constituents from the needles of Taxus media. J Sheng Pharm Univ. 26:434–472.
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
A new taxane diterpenoid from Taxus chinensis var. mairei a
a
a
a
Zhi-Hong Zhou , Wei Qu , Min-Zhuo Liu , Jian-Bo Sun , Meng-Hua a
a
Wang , Jing-Yu Liang & Fei-Hua Wu
b
a
Department of Natural Medicinal Chemistry, China Pharmaceutical University, #24 Tongjiaxiang, Nanjing 210009, P.R. China b
Department of Pharmacology for Chinese Materia Medica, China Pharmaceutical University, #639 Longmian, Nanjing 210009, P.R. China Published online: 28 Feb 2014.
To cite this article: Zhi-Hong Zhou, Wei Qu, Min-Zhuo Liu, Jian-Bo Sun, Meng-Hua Wang, Jing-Yu Liang & Fei-Hua Wu (2014) A new taxane diterpenoid from Taxus chinensis var. mairei, Natural Product Research: Formerly Natural Product Letters, 28:8, 530-533, DOI: 10.1080/14786419.2014.881360 To link to this article: http://dx.doi.org/10.1080/14786419.2014.881360
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 [Ams/Girona*barri Lib] at 01:47 10 October 2014
Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Natural Product Research, 2014 Vol. 28, No. 8, 530–533, http://dx.doi.org/10.1080/14786419.2014.881360
A new taxane diterpenoid from Taxus chinensis var. mairei Zhi-Hong Zhoua, Wei Qua, Min-Zhuo Liua, Jian-Bo Suna, Meng-Hua Wanga, Jing-Yu Lianga* and Fei-Hua Wub*
Downloaded by [Ams/Girona*barri Lib] at 01:47 10 October 2014
a Department of Natural Medicinal Chemistry, China Pharmaceutical University, #24 Tongjiaxiang, Nanjing 210009, P.R. China; bDepartment of Pharmacology for Chinese Materia Medica, China Pharmaceutical University, #639 Longmian, Nanjing 210009, P.R. China
(Received 4 November 2013; final version received 6 January 2014) Taxadiene (3), a new taxane diterpenoid with an unusual hydroxy substituting at C-17, and six known compounds including two taxane diterpenoids (1 and 2) and four flavonoids (4 – 7) were isolated from the whole seedling of the Taxus chinensis var. mairei. Among them, compound 7 was isolated from T. chinensis var. mairei for the first time. Structures of these compounds were elucidated on the basis of spectroscopic data and by comparison with reported literature data. Keywords: Taxus chinensis var. mairei; taxane diterpenoids; flavonoids
1. Introduction Taxus chinensis var. mairei (Taxaceae), which belongs to the genus Taxus, is widely distributed in China. Many species in genus Taxus showed significant activities in detoxication and diuretic swelling (Li et al. 2011), some branches and leaves of the species have been well used to treat kidney disease and diabetes (Li et al. 2011). Taxol, the representative composition, showed significant antitumour effect mainly due to its unique mechanism (Schiff et al. 1979). Since taxanes have been an important target for cancer therapy agents for a long time, many other important compounds such as flavonoids, lignans, steroids, phenolic acids, sesquiterpenes, nontaxane diterpenoids and inositol ethers (Li et al. 2008) have been isolated from T. chinensis var. mairei. Pharmacological studies also showed other significant activities such as anti-allergic (Koyama et al. 2006), hepatoprotective (Choi et al. 2006), anti-osteoporosis (Yin et al. 2006), hypoglycaemic (Liang 2002) and antioxidant (Veselova et al. 2007) of this plant. Because of the diversity in chemical constituents and a variety of pharmacological activities, it is important to further study T. chinensis var. mairei. In this article, we reported taxadiene (3), a new taxane together with six known compounds, including two taxane diterpenoids (1 and 2) and four flavonoids (4– 7) (Figure 1) from the whole seedling of T. chinensis var. mairei. Their structures were established on the basis of spectroscopic evidence, including 1D and 2D NMR as well as by comparison with those reported literature data.
2. Results and discussion Compound 3 was isolated as colourless needles. The molecular formula was established as C28H40O11 by HR-ESI-MS (m/z: 551.2498 [M 2 H]2, calcd for 551.2492), which was
*Corresponding authors. Email: [email protected]; [email protected] q 2014 Taylor & Francis
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Figure 1. The structures of isolated compounds.
consistent with the data from the 1H and 13C NMR spectra. 1H NMR spectrum showed the presence of four acetyl groups (dH 2.00, 3H; 2.02, 9H), three methyl groups (dH 0.83, 1.15 and 2.03) and three hydroxyl groups (dH 4.93, 5.26 and 6.03). 13C NMR and DEPT spectra indicated 28 resonance carbons ascribed to seven methyl carbon signals, four methylene carbons, eight methine carbons and nine quaternary carbon signals, which suggested that compound 3 belonged to the taxane analogues with a skeleton of taxa-4(20),11-diene (Yu et al. 2012). Three hydroxyl groups were assigned to C-5, C-10 and C-17 according to the chemical shifts (dC 73.1, 67.8 and 67.3) of the corresponding carbons, respectively. The assignments of all the direct 1H-13C bondings were made on the basis of 1H – 1H COSY and HSQC spectra. In the HMBC spectrum of 3 (Figure S10), H-2, H-7, H-9 and H-13 showed correlations with four carbonyl carbons at chemical shifts dC 169.2, 169.4, 170.2 and 170.4, which suggested that the positions of four acetoxyl groups were assigned to C-2, C-7, C-9 and C-13. Correlations of H-3 (dH 3.48) with C-4 (dC 146.2) and H-20 (dH 4.62 and 5.15) with C-5 (dC 73.1) supported the assigned position of the ethylenic bond between C-4 and C-20 to C-4. The methyl groups were attached to C-8 and C-12 by the HMBC correlation from 19-CH3 (dH 15.6) to C-8 (dC 47.0) and 18-CH3 (dH 20.8) to C-12 (dC 134.8). NOESY correlations (Figure S11) from 3-H to 5-OH, 20b-H to 5-H and 20a-H to 2-OAc supported that the relative configurations of 5-OH and 2-OAc were both of a-orientation. The correlations from 19-H to 7-OAc and from 16-H to 1b-H and 13-H on the NOESY spectrum supported b-orientation of 7-OAc and a-orientation of 13-OAc. Correlations from 17-OH to 10OH and 10-H to 9-OAc indicated that the relative configuration of 10-OH was of b-orientation
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and 9-OAc was of a-orientation. Thus, the compound was determined to be 5a, 10b, 17trihydroxy-2a, 7b, 9a, 13a-tetraacetoxy-taxa-4(20),11-diene, and was named as taxadiene. By comparing physical and spectroscopic data with literature values, the five known compounds were identified as taxol (1) (Chmurny et al. 1992), N-methyl-taxol C (2) (Barboni et al. 1994), ginkgetin (4) (Zhang et al. 2009), sciadopitysin (5) (Zhang et al. 2009), kaempferol (6) (Wang et al. 1999) and apigenin (7) (Zhang et al. 2009).
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3. Experimental 3.1. General experimental procedures Mass spectra were obtained on an MS Agilent 1100 Series LC/MSD Trap mass spectrometer (ESI-MS) and HR-ESI-MS was done on an Agilent 6520B ESI spectrometer (Agilent Technologies, Santa Clara, CA, USA), respectively. NMR spectra were recorded on Bruker ACF-300 NMR (Bruker Corporation, Switzerland) with TMS as internal standard. Optical rotation was tested by a WXG-4 spectropolarimeter (Shanghai Jingke Analytical Instrument Ltd, Shanghai, China). All solvents used were of analytical grade (Tianjin Chemical Co., Ltd, Tianjin, China). Chromatographic silica gel (200 – 300 mesh) and polyamide (100 – 200 mesh) were purchased from Qingdao Marine Chemical Co., Ltd (Qingdao, China). Sephadex LH-20 was purchased from Amersham Pharmacia Biotech, Uppsala, Sweden. TLC analysis was performed on silica gel GF254 produced by Qingdao Marine Chemical Co., Ltd. 3.2. Plant material The whole seedlings of T. chinensis var. mairei were collected from Mingxi County of Fujian Province in China in July 2011 and were identified by Wen-Jian Li, Senior Engineer in Southern Pharmaceutical Company, China. A voucher specimen (no. 20110708) has been deposited in the Department of Natural Medicinal Chemistry, China Pharmaceutical University, Nanjing, China. 3.3. Extraction and isolation The 80% ethanol extract (147.3 g) of T. chinensis var. mairei was suspended with water (10 L), and then partitioned sequentially with petroleum ether (10 L), EtOAc (10 L) and n-BuOH (10 L), respectively. The EtOAc extract (116.5 g) was subjected to column chromatography over silica gel and eluted with a gradient of CH2Cl2 – MeOH (from 100:1 to 1:1) to yield five fractions (Frs 1 –5). Fraction 2 was further subjected to silica gel column eluted with petroleum ether –EtOAc (from 5:4 to 1:1) to affford compounds 1 (15 mg) and 2 (18 mg). Fraction 3 was further subjected to silica gel column eluted with petroleum ether–EtOAc (from 4:1 to 1:1) to afford compounds 3 (5 mg), 4 (9 mg) and 5 (8 mg). A yellow powder obtained from fraction 3 was further subjected to Sephadex LH-20 (MeOH) to afford compounds 6 (5 mg) and 7 (3 mg). 3.4. Spectral data þ Compound 3: colourless needles; ½a25 D ¼ 234:28(CDCl3); ESI-MS m/z: 570.0 [M þ NH4] , 2 2 1 587.5 [M þ Cl] ; HR-ESI-MS m/z: 551.2498 [M 2 H] (calcd for C28H39O11, 551.2492); H NMR (300 MHz, DMSO-d6) dH: 1.92 (1H, m, H-1), 5.32 (1H, d, J ¼ 5.5 Hz, H-2), 3.48 (1H, d, J ¼ 5.5 Hz, H-3), 4.04 (1H, br s, H-5), 1.74 (1H, br d, J ¼ 10.0 Hz, H-6a), 1.55 (1H, dd, J ¼ 12.7, 5.0 Hz, H-6b), 5.47 (1H, dd, J ¼ 10.0, 5.0 Hz, H-7), 5.50 (1H, d, J ¼ 10.0 Hz, H-9), 4.89 (1H, dd, J ¼ 10.0, 6.0 Hz, H-10), 5.58 (1H, overlap, H-13), 2.50 (1H, m, H-14a), 1.49 (1H, dd, J ¼ 14.7, 6.3 Hz, H-14b), 1.15 (3H, s, H-16), 4.66 (1H, d, J ¼ 11.8 Hz, H-17a), 3.32 (1H, m, H-17b), 2.03 (3H, s, H-18), 0.83 (3H, s, H-19), 5.15 (1H, s, H-20a), 4.62 (1H, s, H-20b), 4.93
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(1H, s, OH-5), 6.03 (1H, d, J ¼ 6.0 Hz, OH-10), 5.26 (1H, d, J ¼ 6.0 Hz, OH-17), 2.02 (9H, s, 3 £ OAc-7, 9, 13), 2.00 (3H, s, OAc-2). 13 C NMR (75 MHz, DMSO-d6) dC: 44.8 (C-1), 68.9 (C-2), 39.7 (C-3), 146.2 (C-4), 73.1 (C-5), 37.7 (C-6), 69.5 (C-7), 47.0 (C-8), 78.3 (C-9), 67.8 (C-10), 136.0 (C-11), 134.8 (C-12), 69.1 (C13), 27.8 (C-14), 43.3 (C-15), 25.9 (C-16), 67.3 (C-17), 20.8 (C-18), 15.6 (C-19), 114.0 (C-20), 170.4, 170.2, 169.4, 169.2 (4 £ OAc-2, 7, 9, 13), 21.3, 20.9, 20.9, 21.1 (4 £ OAc-2, 7, 9, 13). Supplementary material Supplementary material relating to this article is available online, alongside Figures S1 – S12.
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Acknowledgements We acknowledge the financial support by the National Natural Science Foundation of China (81072538) and National New Drug Innovation Major Project of China (2011ZX09307-002-02).
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