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A new phenylpropanoid from the roots of Euphorbia nematocypha ab
ab
a
a
a
Hai-Yang Jia , Zhi-Xin Liao , Fu-Yue Liu , Ling Wu , Chen Xu & Bo Zuo
a
a
Department of Pharmaceutical Engineering, School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, PR China b
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Jiang su Province Hi-Tech Key Laboratory for Bio-medical Research, Southeast University, Nanjing 211189, PR China Published online: 25 Nov 2014.
To cite this article: Hai-Yang Jia, Zhi-Xin Liao, Fu-Yue Liu, Ling Wu, Chen Xu & Bo Zuo (2015) A new phenylpropanoid from the roots of Euphorbia nematocypha, Natural Product Research: Formerly Natural Product Letters, 29:7, 650-655, DOI: 10.1080/14786419.2014.980256 To link to this article: http://dx.doi.org/10.1080/14786419.2014.980256
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Natural Product Research, 2015 Vol. 29, No. 7, 650–655, http://dx.doi.org/10.1080/14786419.2014.980256
A new phenylpropanoid from the roots of Euphorbia nematocypha Hai-Yang Jiaab, Zhi-Xin Liaoab*, Fu-Yue Liua, Ling Wua, Chen Xua and Bo Zuoa a Department of Pharmaceutical Engineering, School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, PR China; bJiang su Province Hi-Tech Key Laboratory for Bio-medical Research, Southeast University, Nanjing 211189, PR China
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(Received 11 September 2014; final version received 21 October 2014)
A phytochemical investigation of the ethanolic extracts of the dried roots of Euphorbia nematocypha resulted in the isolation of a new phenylpropanoid, 16-O-caffeoyl-16hydroxylhexadecanoic acid (1), together with 23 known compounds (2 – 24). Their structures were elucidated on the basis of spectroscopic data. Compound 1 was first to be isolated as a caffeic acyl long chain alkyl acid from the family Euphorbiaceae. The new isolated phenylpropanoid showed potent cytotoxic activities against the MCF-7 and HeLa cell lines. Keywords: Euphorbia nematocypha; 16-O-caffeoyl-16-hydroxylhexadecanoic acid; phenylpropanoid; eunicellan antiproliferative
1. Introduction Euphorbia has attracted considerable attention for its rich source of terpenoids with diverse structures and showed amazing physiological and pharmacological properties in vivo or in vitro (Jassbi 2006; Shi, Min, et al. 2008; Shi, Su, et al. 2008). Several reports on the phytochemical investigation of Euphorbia nematocypha (mainly distributed in China) or Euphorbia jolkini (mainly distributed in Japan) have obtained some diterpenoids (Uemura & Hirata 1972, 1974; Uemura et al. 1976; Zhao et al. 1995; He et al. 2008; Maru et al. 2013; Chen, You et al. 2014; Chen, Chen et al. 2014; Huang et al. 2014), triterpenoids (Cao et al. 1992), ellagitannins (Lee et al. 2004) and gallotannin (Park et al. 2010). To ascertain its chemical constituent and medicinal value, the ethanolic extract of the dried roots of E. nematocypha was investigated. 2. Results and discussion Compound 1 was obtained as yellow powder. The molecular formula of 1 was deduced to be C25H38O6 as the HR-ESI-MS spectrum showed the [M þ Na]þ ion at m/z 457.2578 (calcd for [C25H38O6 þ Na]þ: 457.2566). The IR (KBr) spectrum showed hydroxyl and carbonyl absorptions at 3448 and 1691 cm21, and absorptions at 2923 and 2852 cm21 suggested the
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
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presence of methylene. The 1H NMR (600 MHz, DMSO-d6) spectrum showed 1,3,4trisubstituted aromatic protons at dH 7.04 (1H, d, J ¼ 2.0 Hz), 7.00 (1H, dd, J ¼ 8.2, 2.0 Hz) and 6.77 (1H, d, J ¼ 8.2 Hz) and dH 9.12 (s), 9.58 (s) could be thought of as two substituted hydroxy signals in the benzene ring; two doublet signals at dH 7.47 (1H, d, J ¼ 15.9 Hz) and 6.26 (1H, d, J ¼ 15.9 Hz) indicated a trans-double bond and there also may be a hydroxy signal in carboxyl group at dH 11.97(s). The 13C NMR (125 MHz, DMSO-d6) spectrum displayed 10 signals in the low field region (from dC 114.46 to dC 174.95), these signals could be attributable to a benzene ring, a double bond and two carbonyl groups. Other signals in the high-field region (from dC 24.97 to dC 64.16) exhibited the characteristics of a series of methylene, whose number was calculated to be 15 according to the molecular weight (Wang et al. 2010). Therefore, three fragments were obtained, a caffeic acyl, carboxyl and a 15 methylene group, and further they were integrated into 16-O-caffeoyl-16-hydroxylhexadecanoic acid with the aid of 2D NMR (HSQC, HMBC and 1H – 1H COSY) spectra. The structure and the assignment of 1D NMR signals of 1 are shown in Figure 1 and Table S1. The known compounds (2 – 24) were b-amyrin acetate (2) and b-amyrin (3) (Huang et al. 2009), c-taraxasterol (4) (Eglseer et al. 1997), betulinic acid (5) (Ramadan et al. 2009), scopoletin (6) (Thuong et al. 2010), ellagic acid (7), 3-O-methylellagic acid (8) and 3,30 -di-Omethylellagic acid (9) (Silva et al. 2008), (E)-ferulic acid hexacosyl ester (10) (Mensah et al. 1992), 3,30 -di-O-methylellagic acid-40 -O-b-D -glucopyranoside (11) (Pakulski & Budzianowski 1996), eicosyl caffeate (12) (Jayaprakasam et al. 2006), sucrose (13) (Kato et al. 2008), chiricanines A (Loset et al. 2001), b-sitosterol (15) (El-Alfy et al. 2012), magdalenic acid (16) (Pinto et al. 1997), jolkinolide A (17) and jolkinolide E (18) (Lal et al. 1990), jolkinolide B (19) and 17-hxdroxyjolkinolide A (20) (Che et al. 1999), 3(R)-3-hydroxy-atis-16-ene-2,14-dione (21) (Abad et al. 2007), ent-atis-3b,16a,17-triol (22) (Satti et al. 1988), ent-2-hydroxy-atis-1,16(17)dien-3,14-dione (23) (Appendino et al. 2000) and (2 )-(5E,12E,2S,3S,4S,9S,11S,15R)-15benzoyloxylathyra-5,12-dien-3-ol-14-one (24) (Tian et al. 2011). All the above structures were identified by spectroscopic analysis and by comparing their spectral data with those reported in the literature. Magdalenic acid (2), which was a more common diterpene of marine eunicellan carbon skeleton (Kennard et al. 1968; Ortega et al. 1997) (also known as cladiellan), was first reported to
Figure 1. Key 1H – 1H COSY (a) and HMBC (b) correlations of compound 1.
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Table 1. The 50% inhibiting concentration values (IC50) (mmol/L) of compound 1. 50% Inhibiting concentration (IC50) (mmol/L) Sample
MCF-7
HeLa
1 Matrinea
20.2 ^ 1.2 10.3 ^ 0.47
27.8 ^ 1.4 17.6 ^ 0.76
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a
Positive control.
be isolated from the family Euphorbiaceae and secondly reported from the terrestrial plant (Pinto et al. 1997) in our work. The in vitro cytotoxic activities of compound 1 against the MCF-7and HeLa cell lines cells were tested. Matrine was used as a positive control. In the test, IC50 values of higher than 40 mmol/L were defined as inactive. The novel phenylpropanoid showed cytotoxic activities against human breast cancer cell line MCF-7, with IC50 values in the range of 20.2 –27.8 mmol/L (Table 1). Cytotoxic activities against MCF-7 cells were higher than those against HeLa cells. 3. Experimental 3.1. General experimental procedures IR spectra were recorded on a NICOLET IR200 FT-IR spectrophotometer. 1H NMR and 13C NMR spectra were scanned on a Bruker Avance DRX-600 spectrometer at 600 MHz (1H) and 125 MHz (13C). HR-ESI-MS was conducted on an Agilent Technologies 6224 TOF LC-MS apparatus. Column chromatography (CC) was performed using silica gel (200 –300 mesh, Qingdao Marine Chemical, Inc.). TLC was conducted on silica gel GF254 plates (10 – 40 mm, Qingdao Marine Chemical, Inc.). Petroleum ether, ethyl acetate, n-BuOH and other reagents were purchased from Nanjing Wanqing Reagent, Inc. Spots were visualised by UV light as well as by spraying with 10% H2SO4 – EtOH followed by heating. Tumour cells were incubated in an HF-212UV CO2 incubator and observed under an OLYMPUS CKX41 inverted microscope. Optical density values were read on a BIO-RAD Model 680 microplate reader. 3.2. Plant material The roots of E. nematocypha were collected in Gande County, Tibetan Autonomous Prefecture of Golog, Qinghai Province, China, in September 2012, and identified by Prof. Hong-fa Sun of Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining, China. A voucher specimen (No. 12-09-02) has been deposited at the Natural Products Chemistry Laboratory of Prof. Zhi-xin Liao, Southeast University, Nanjing, China. 3.3. Extraction and isolation The air-dried roots of the E. nematocypha (8.5 kg) were percolated with 95% ethanol (30.0 L £ 4) at room temperature. After removal of the solvent under reduced pressure, the ethanol extract (660 g) was suspended in water (2500 mL), partitioned sequentially with petroleum ether (1500 mL £ 4), EtOAc (2000 mL £ 5) and n-BuOH (1000 mL £ 4) to yield crude petroleum ether (110 g), EtOAc (165 g) and n-BuOH (120 g) extracts, respectively. The petroleum ether extract was isolated by repeated silica gel CC using petroleum ether – EtOAc (from 50:1 to 1:2, v/v) to yield 2 (1532 mg), 3 (46 mg), 4 (56 mg), 6 (133 mg), 12 (72 mg), 14 (233 mg), 16 (86 mg), 17 (61 mg), 19 (109 mg) and 21 (126 mg). The EtOAc extract was also subjected to silica gel CC using a gradient system of petroleum ether – EtOAc to give 1 (74 mg), 5 (43 mg), 9 (66 mg), 10
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(29 mg), 15 (73 mg), 18 (241 mg), 20 (56 mg), 22 (44 mg), 23 (133 mg) and 24 (176 mg). The nBuOH extract fraction was fractionated by CC over silica gel by the gradient system of increasing polarity (chloroform –methanol) to afford 7 (71 mg), 8 (109 mg), 11 (121 mg) and 13 (68 mg).
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3.4. 16-O-caffeoyl-16-hydroxyldodecanoic acid (1) Yellow powder; m.p. 122 – 1238C; IR (KBr) vmaxcm21: 3448, 2923, 2852, 1691; HR-ESI-MS: m/ z 457.2578[M þ Na]þ (Calcd for [C25H38O6 þ Na]þ: 457.2566); 1H NMR (600 MHz, DMSOd6) dH (ppm): 11.97 (1H, COOH-1), 2.18 (2H, t, J ¼ 7.4 Hz, H-2), 1.48 (2H, m, H-3), 1.23 (20H, s, H-(4-13)), 1.33 (2H, m, H-14), 1.61 (2H, m, H-15), 4.10 (2H, t, J ¼ 6.7 Hz, H-16), 7.04 (1H, d, J ¼ 2.0 Hz, H-20 ), 9.58 (1H, OH-30 ), 9.12 (1H, m, OH-40 ), 6.77 (1H, d, J ¼ 8.2 Hz, H-50 ), 7.00 (1H, dd, J ¼ 8.2, 2.0 Hz, H-60 ), 7.47 (1H, d, J ¼ 15.9 Hz, H-70 ), 6.26 (1H, d, J ¼ 15.9 Hz, H-80 ); 13 C NMR (125 MHz, DMSO-d6) dC (ppm): 174.95 (C-1), 34.13 (C-2), 24.97 – 29.51 (from C-3 to C-15), 64.16 (C-16), 125.97 (C-10 ), 114.46 (C-20 ), 146.03 (C-30 ), 148.83 (C-40 ), 115.27 (C-50 ), 121.76 (C-60 ), 145.45 (C-70 ), 116.17(C-80 ), 167.05 (C-90 ).
3.5. In vitro cytotoxic assay Compound 1 was tested for its cytotoxicity against the MCF-7 and HeLa cell lines by the MTT method, and matrine was used as a positive control. First, the cells (5 £ 104 cells/well) were seeded into a 96-well micro-plate for 24 h. Then, the different concentrations of the samples were added to the wells in sextuplicates and incubated for 48 h. After the incubation period, 20 mL of the MTT solution (2.5 mg mL21 in PBS) was added to each well and the cells were further incubated for 4 h. Subsequently, removal of the supernatant and 150 mL of DMSO were added to dissolve formazan. Finally, the absorbance was measured at 490 nm using an ELISA reader.
4. Conclusion 16-O-caffeoyl-16-hydroxylhexadecanoic acid (1), together with 23 known compounds (2 –24), was isolated from the dried roots of E. nematocypha. The structure of the new compound was identified on the basis of spectroscopic analysis. Compound 1 was first to be isolated as a caffeic acyl long chain alkyl acid from the family Euphorbiaceae. The isolated phenylpropanoid exhibited potent cytotoxic activities against the MCF-7 and HeLa cell lines.
Supplementary material The original spectra of IR, 1D and 2D NMR, and HR-ESI-MS data of the new compound (1) are available online, alongside Figures S1 –S7 and Table S1.
Acknowledgements We are grateful to Prof. Hong-fa Sun of Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining, China, for the identification of the plant material.
Funding This work was funded by the State Key Laboratory Cultivation Base for Chemistry and Molecular Engineering of Medicinal Resources, Ministry of Science and Technology of China [grant number CMEMR2014-B04].
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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 phenylpropanoid from the roots of Euphorbia nematocypha ab
ab
a
a
a
Hai-Yang Jia , Zhi-Xin Liao , Fu-Yue Liu , Ling Wu , Chen Xu & Bo Zuo
a
a
Department of Pharmaceutical Engineering, School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, PR China b
Click for updates
Jiang su Province Hi-Tech Key Laboratory for Bio-medical Research, Southeast University, Nanjing 211189, PR China Published online: 25 Nov 2014.
To cite this article: Hai-Yang Jia, Zhi-Xin Liao, Fu-Yue Liu, Ling Wu, Chen Xu & Bo Zuo (2015) A new phenylpropanoid from the roots of Euphorbia nematocypha, Natural Product Research: Formerly Natural Product Letters, 29:7, 650-655, DOI: 10.1080/14786419.2014.980256 To link to this article: http://dx.doi.org/10.1080/14786419.2014.980256
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
Downloaded by [University of Sussex Library] at 09:41 26 March 2015
Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Natural Product Research, 2015 Vol. 29, No. 7, 650–655, http://dx.doi.org/10.1080/14786419.2014.980256
A new phenylpropanoid from the roots of Euphorbia nematocypha Hai-Yang Jiaab, Zhi-Xin Liaoab*, Fu-Yue Liua, Ling Wua, Chen Xua and Bo Zuoa a Department of Pharmaceutical Engineering, School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, PR China; bJiang su Province Hi-Tech Key Laboratory for Bio-medical Research, Southeast University, Nanjing 211189, PR China
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(Received 11 September 2014; final version received 21 October 2014)
A phytochemical investigation of the ethanolic extracts of the dried roots of Euphorbia nematocypha resulted in the isolation of a new phenylpropanoid, 16-O-caffeoyl-16hydroxylhexadecanoic acid (1), together with 23 known compounds (2 – 24). Their structures were elucidated on the basis of spectroscopic data. Compound 1 was first to be isolated as a caffeic acyl long chain alkyl acid from the family Euphorbiaceae. The new isolated phenylpropanoid showed potent cytotoxic activities against the MCF-7 and HeLa cell lines. Keywords: Euphorbia nematocypha; 16-O-caffeoyl-16-hydroxylhexadecanoic acid; phenylpropanoid; eunicellan antiproliferative
1. Introduction Euphorbia has attracted considerable attention for its rich source of terpenoids with diverse structures and showed amazing physiological and pharmacological properties in vivo or in vitro (Jassbi 2006; Shi, Min, et al. 2008; Shi, Su, et al. 2008). Several reports on the phytochemical investigation of Euphorbia nematocypha (mainly distributed in China) or Euphorbia jolkini (mainly distributed in Japan) have obtained some diterpenoids (Uemura & Hirata 1972, 1974; Uemura et al. 1976; Zhao et al. 1995; He et al. 2008; Maru et al. 2013; Chen, You et al. 2014; Chen, Chen et al. 2014; Huang et al. 2014), triterpenoids (Cao et al. 1992), ellagitannins (Lee et al. 2004) and gallotannin (Park et al. 2010). To ascertain its chemical constituent and medicinal value, the ethanolic extract of the dried roots of E. nematocypha was investigated. 2. Results and discussion Compound 1 was obtained as yellow powder. The molecular formula of 1 was deduced to be C25H38O6 as the HR-ESI-MS spectrum showed the [M þ Na]þ ion at m/z 457.2578 (calcd for [C25H38O6 þ Na]þ: 457.2566). The IR (KBr) spectrum showed hydroxyl and carbonyl absorptions at 3448 and 1691 cm21, and absorptions at 2923 and 2852 cm21 suggested the
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
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presence of methylene. The 1H NMR (600 MHz, DMSO-d6) spectrum showed 1,3,4trisubstituted aromatic protons at dH 7.04 (1H, d, J ¼ 2.0 Hz), 7.00 (1H, dd, J ¼ 8.2, 2.0 Hz) and 6.77 (1H, d, J ¼ 8.2 Hz) and dH 9.12 (s), 9.58 (s) could be thought of as two substituted hydroxy signals in the benzene ring; two doublet signals at dH 7.47 (1H, d, J ¼ 15.9 Hz) and 6.26 (1H, d, J ¼ 15.9 Hz) indicated a trans-double bond and there also may be a hydroxy signal in carboxyl group at dH 11.97(s). The 13C NMR (125 MHz, DMSO-d6) spectrum displayed 10 signals in the low field region (from dC 114.46 to dC 174.95), these signals could be attributable to a benzene ring, a double bond and two carbonyl groups. Other signals in the high-field region (from dC 24.97 to dC 64.16) exhibited the characteristics of a series of methylene, whose number was calculated to be 15 according to the molecular weight (Wang et al. 2010). Therefore, three fragments were obtained, a caffeic acyl, carboxyl and a 15 methylene group, and further they were integrated into 16-O-caffeoyl-16-hydroxylhexadecanoic acid with the aid of 2D NMR (HSQC, HMBC and 1H – 1H COSY) spectra. The structure and the assignment of 1D NMR signals of 1 are shown in Figure 1 and Table S1. The known compounds (2 – 24) were b-amyrin acetate (2) and b-amyrin (3) (Huang et al. 2009), c-taraxasterol (4) (Eglseer et al. 1997), betulinic acid (5) (Ramadan et al. 2009), scopoletin (6) (Thuong et al. 2010), ellagic acid (7), 3-O-methylellagic acid (8) and 3,30 -di-Omethylellagic acid (9) (Silva et al. 2008), (E)-ferulic acid hexacosyl ester (10) (Mensah et al. 1992), 3,30 -di-O-methylellagic acid-40 -O-b-D -glucopyranoside (11) (Pakulski & Budzianowski 1996), eicosyl caffeate (12) (Jayaprakasam et al. 2006), sucrose (13) (Kato et al. 2008), chiricanines A (Loset et al. 2001), b-sitosterol (15) (El-Alfy et al. 2012), magdalenic acid (16) (Pinto et al. 1997), jolkinolide A (17) and jolkinolide E (18) (Lal et al. 1990), jolkinolide B (19) and 17-hxdroxyjolkinolide A (20) (Che et al. 1999), 3(R)-3-hydroxy-atis-16-ene-2,14-dione (21) (Abad et al. 2007), ent-atis-3b,16a,17-triol (22) (Satti et al. 1988), ent-2-hydroxy-atis-1,16(17)dien-3,14-dione (23) (Appendino et al. 2000) and (2 )-(5E,12E,2S,3S,4S,9S,11S,15R)-15benzoyloxylathyra-5,12-dien-3-ol-14-one (24) (Tian et al. 2011). All the above structures were identified by spectroscopic analysis and by comparing their spectral data with those reported in the literature. Magdalenic acid (2), which was a more common diterpene of marine eunicellan carbon skeleton (Kennard et al. 1968; Ortega et al. 1997) (also known as cladiellan), was first reported to
Figure 1. Key 1H – 1H COSY (a) and HMBC (b) correlations of compound 1.
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Table 1. The 50% inhibiting concentration values (IC50) (mmol/L) of compound 1. 50% Inhibiting concentration (IC50) (mmol/L) Sample
MCF-7
HeLa
1 Matrinea
20.2 ^ 1.2 10.3 ^ 0.47
27.8 ^ 1.4 17.6 ^ 0.76
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a
Positive control.
be isolated from the family Euphorbiaceae and secondly reported from the terrestrial plant (Pinto et al. 1997) in our work. The in vitro cytotoxic activities of compound 1 against the MCF-7and HeLa cell lines cells were tested. Matrine was used as a positive control. In the test, IC50 values of higher than 40 mmol/L were defined as inactive. The novel phenylpropanoid showed cytotoxic activities against human breast cancer cell line MCF-7, with IC50 values in the range of 20.2 –27.8 mmol/L (Table 1). Cytotoxic activities against MCF-7 cells were higher than those against HeLa cells. 3. Experimental 3.1. General experimental procedures IR spectra were recorded on a NICOLET IR200 FT-IR spectrophotometer. 1H NMR and 13C NMR spectra were scanned on a Bruker Avance DRX-600 spectrometer at 600 MHz (1H) and 125 MHz (13C). HR-ESI-MS was conducted on an Agilent Technologies 6224 TOF LC-MS apparatus. Column chromatography (CC) was performed using silica gel (200 –300 mesh, Qingdao Marine Chemical, Inc.). TLC was conducted on silica gel GF254 plates (10 – 40 mm, Qingdao Marine Chemical, Inc.). Petroleum ether, ethyl acetate, n-BuOH and other reagents were purchased from Nanjing Wanqing Reagent, Inc. Spots were visualised by UV light as well as by spraying with 10% H2SO4 – EtOH followed by heating. Tumour cells were incubated in an HF-212UV CO2 incubator and observed under an OLYMPUS CKX41 inverted microscope. Optical density values were read on a BIO-RAD Model 680 microplate reader. 3.2. Plant material The roots of E. nematocypha were collected in Gande County, Tibetan Autonomous Prefecture of Golog, Qinghai Province, China, in September 2012, and identified by Prof. Hong-fa Sun of Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining, China. A voucher specimen (No. 12-09-02) has been deposited at the Natural Products Chemistry Laboratory of Prof. Zhi-xin Liao, Southeast University, Nanjing, China. 3.3. Extraction and isolation The air-dried roots of the E. nematocypha (8.5 kg) were percolated with 95% ethanol (30.0 L £ 4) at room temperature. After removal of the solvent under reduced pressure, the ethanol extract (660 g) was suspended in water (2500 mL), partitioned sequentially with petroleum ether (1500 mL £ 4), EtOAc (2000 mL £ 5) and n-BuOH (1000 mL £ 4) to yield crude petroleum ether (110 g), EtOAc (165 g) and n-BuOH (120 g) extracts, respectively. The petroleum ether extract was isolated by repeated silica gel CC using petroleum ether – EtOAc (from 50:1 to 1:2, v/v) to yield 2 (1532 mg), 3 (46 mg), 4 (56 mg), 6 (133 mg), 12 (72 mg), 14 (233 mg), 16 (86 mg), 17 (61 mg), 19 (109 mg) and 21 (126 mg). The EtOAc extract was also subjected to silica gel CC using a gradient system of petroleum ether – EtOAc to give 1 (74 mg), 5 (43 mg), 9 (66 mg), 10
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(29 mg), 15 (73 mg), 18 (241 mg), 20 (56 mg), 22 (44 mg), 23 (133 mg) and 24 (176 mg). The nBuOH extract fraction was fractionated by CC over silica gel by the gradient system of increasing polarity (chloroform –methanol) to afford 7 (71 mg), 8 (109 mg), 11 (121 mg) and 13 (68 mg).
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3.4. 16-O-caffeoyl-16-hydroxyldodecanoic acid (1) Yellow powder; m.p. 122 – 1238C; IR (KBr) vmaxcm21: 3448, 2923, 2852, 1691; HR-ESI-MS: m/ z 457.2578[M þ Na]þ (Calcd for [C25H38O6 þ Na]þ: 457.2566); 1H NMR (600 MHz, DMSOd6) dH (ppm): 11.97 (1H, COOH-1), 2.18 (2H, t, J ¼ 7.4 Hz, H-2), 1.48 (2H, m, H-3), 1.23 (20H, s, H-(4-13)), 1.33 (2H, m, H-14), 1.61 (2H, m, H-15), 4.10 (2H, t, J ¼ 6.7 Hz, H-16), 7.04 (1H, d, J ¼ 2.0 Hz, H-20 ), 9.58 (1H, OH-30 ), 9.12 (1H, m, OH-40 ), 6.77 (1H, d, J ¼ 8.2 Hz, H-50 ), 7.00 (1H, dd, J ¼ 8.2, 2.0 Hz, H-60 ), 7.47 (1H, d, J ¼ 15.9 Hz, H-70 ), 6.26 (1H, d, J ¼ 15.9 Hz, H-80 ); 13 C NMR (125 MHz, DMSO-d6) dC (ppm): 174.95 (C-1), 34.13 (C-2), 24.97 – 29.51 (from C-3 to C-15), 64.16 (C-16), 125.97 (C-10 ), 114.46 (C-20 ), 146.03 (C-30 ), 148.83 (C-40 ), 115.27 (C-50 ), 121.76 (C-60 ), 145.45 (C-70 ), 116.17(C-80 ), 167.05 (C-90 ).
3.5. In vitro cytotoxic assay Compound 1 was tested for its cytotoxicity against the MCF-7 and HeLa cell lines by the MTT method, and matrine was used as a positive control. First, the cells (5 £ 104 cells/well) were seeded into a 96-well micro-plate for 24 h. Then, the different concentrations of the samples were added to the wells in sextuplicates and incubated for 48 h. After the incubation period, 20 mL of the MTT solution (2.5 mg mL21 in PBS) was added to each well and the cells were further incubated for 4 h. Subsequently, removal of the supernatant and 150 mL of DMSO were added to dissolve formazan. Finally, the absorbance was measured at 490 nm using an ELISA reader.
4. Conclusion 16-O-caffeoyl-16-hydroxylhexadecanoic acid (1), together with 23 known compounds (2 –24), was isolated from the dried roots of E. nematocypha. The structure of the new compound was identified on the basis of spectroscopic analysis. Compound 1 was first to be isolated as a caffeic acyl long chain alkyl acid from the family Euphorbiaceae. The isolated phenylpropanoid exhibited potent cytotoxic activities against the MCF-7 and HeLa cell lines.
Supplementary material The original spectra of IR, 1D and 2D NMR, and HR-ESI-MS data of the new compound (1) are available online, alongside Figures S1 –S7 and Table S1.
Acknowledgements We are grateful to Prof. Hong-fa Sun of Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining, China, for the identification of the plant material.
Funding This work was funded by the State Key Laboratory Cultivation Base for Chemistry and Molecular Engineering of Medicinal Resources, Ministry of Science and Technology of China [grant number CMEMR2014-B04].
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