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New eudesmane-type sesquiterpenoid from Solanum lyratum with cytotoxic activity ab
a
a
a
Xiu-Ping Nie , Fang Yao , Xi-Dian Yue , Gui-Sheng Li & Sheng-Jun a
Dai a
School of Pharmaceutical Science, Yantai University, Yantai 264005, P.R. China b
Yantai University Affiliated Hospital, Yantai 264005, P.R. China Published online: 21 Mar 2014.
Click for updates To cite this article: Xiu-Ping Nie, Fang Yao, Xi-Dian Yue, Gui-Sheng Li & Sheng-Jun Dai (2014) New eudesmane-type sesquiterpenoid from Solanum lyratum with cytotoxic activity, Natural Product Research: Formerly Natural Product Letters, 28:9, 641-645, DOI: 10.1080/14786419.2014.891199 To link to this article: http://dx.doi.org/10.1080/14786419.2014.891199
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 &
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Natural Product Research, 2014 Vol. 28, No. 9, 641–645, http://dx.doi.org/10.1080/14786419.2014.891199
New eudesmane-type sesquiterpenoid from Solanum lyratum with cytotoxic activity Xiu-Ping Nieab, Fang Yaoa, Xi-Dian Yuea, Gui-Sheng Lia and Sheng-Jun Daia* a
School of Pharmaceutical Science, Yantai University, Yantai 264005, P.R. China; Yantai University Affiliated Hospital, Yantai 264005, P.R. China
Downloaded by [Nova Southeastern University] at 21:35 08 January 2015
b
(Received 13 September 2013; final version received 2 February 2014) In our continuing effort to discover more new cytotoxic sesquiterpenoids from Solanum lyratum, one new eudesmane-type sesquiterpenoid (1, 3-keto-eudesm-9b,11-diol, named lyratol G), together with one known eudesmane-type sesquiterpenoid (2, 1bhydroxy-1,2-dihydro-a-santonin), was obtained. The structure of the new sesquiterpenoid was elucidated on the basis of integrated spectroscopic techniques, mainly HRFAB-MS, 1D and 2D NMR (1H– 1H COSY, HMQC, HMBC and ROESY). In vitro, two sesquiterpenoids were found to exhibit significant cytotoxicity against three cancer cell lines (P-388, HONE-1 and HT-29), and gave IC50 values in the range of 3.1– 6.9 mM. Keywords: Solanum lyratum; Solanaceae; sesquiterpenoid; lyratol G; cytotoxic activity
1. Introduction The genus Solanum, which is one of the largest genera of the Solanaceae, is widely distributed in tropical and temperate zones and is a rich source of active secondary metabolites. Many species belonging to this genus constantly draw the attention of numerous chemical researchers due to their biologically active constituents, including steroids, steroidal alkaloids and their glycosides (Zhou et al. 2006; Lu et al. 2011). Solanum lyratum, commonly known as ‘Bai-Ying’ in traditional Chinese medicine and ‘Back-MoDeung’ in traditional Korea medicine, respectively, is a perennial herb and has been used as a anti-anaphylactic (Kang et al. 1998), anti-inflammatory (Zhang et al. 2012), anti-tumour (Hsu et al. 2008), immunomodulatory (Yang et al. 2010) and antioxidant (Kuo et al. 2009) agent. In previous phytochemical studies on S. lyratum, 10 new sesquiterpenoids were separated and most of them exhibited significant cytotoxic activities (Ren et al. 2009; Yue et al. 2012; Yao et al. 2013a, 2013b). As part of our ongoing search for more new bioactive compounds, a further separation of an extract from S. lyratum was performed, and led to the isolation of one new eudesmane-type sesquiterpenoid (1, 3-keto-eudesm-9b,11-diol, named lyratol G) and one known one (2, 1b-hydroxy-1,2-dihydro-a-santonin), and their structures were elucidated by using detailed spectral data. In addition, two sesquiterpenoids were screened for cytotoxicity against P-388, HONE-1 and HT29 cancer cells. Herein, we report on the isolation, structural elucidation and the evaluation of cytotoxic effects of two eudesmane-type sesquiterpenoids.
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
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2. Results and discussion Compound 1 was isolated and purified as a colourless viscous oil. In the HR-FAB mass spectrum, it indicated a quasi-molecular ion peak at m/z 255.1957 [M þ H]þ, corresponding to a molecular formula C15H26O3. The IR spectrum revealed absorption bands at 3410 (br) and 1697 cm21, which were in agreement with hydroxyl and carbonyl groups. The 1H NMR spectrum of 1 revealed signals corresponding to three tertiary methyl groups at dH 1.22 (3H, s, H12), 1.23 (3H, s, H-13) and 1.07 (3H, s, H-14), one secondary methyl group at dH 1.02 (3H, d, J ¼ 6.5 Hz, H-15) and an oxygenated methine at dH 3.32 (dd, J ¼ 11.7, 4.3 Hz, H-9). Detailed examination of the 1H – 1H COSY experiment revealed two spin systems. The first spin system included the signals of one methyl (dH 1.02, 3H, d, J ¼ 6.5 Hz, H-15), two methylenes (dH 1.74, m, Ha-6; 1.16, m, Hb-6; 1.83, m, Ha-8; 1.42, m, Hb-8) and four methines (dH 2.37, m, H-4; 1.20, m, H-5; 1.46, m, H-7; 3.32, dd, J ¼ 10.7, 4.3 Hz, H-9). The second spin system was traced from two methylenes (dH 2.26, m, Ha-1; 1.47, m, Hb-1; 2.48, m, Ha-2; 2.42, m, Hb-2). The 13C NMR spectrum displayed 15 carbon resonances, and the DEPT spectrum was consistent with the presence of four methyls, four methylenes, four methines (one oxygenated) and three quaternary carbons (one oxygenated and one carbonyl). Careful analyses of the above-mentioned signal patterns indicated the presence of an eudesmane-type sesquiterpenoid skeleton (Guilhon & Mu¨ller 1998). In the HMBC experiment (Figure 2), the correlations of H2-1/C-3, H2-2/C-3, H315/C-3, H-5/C-3, H-5/C-9, H3-14/C-9, H-7/C-9, H2-6/C-11, H2-8/C-11, H3-12/C-11 as well as from H3-13/C-11 clearly positioned the carbonyl group at C-3 and two hydroxyl moieties at C-9 and C-11, respectively. Based on the above-mentioned data and comprehensive 2D NMR experiments (1H – 1H COSY, HMQC and HMBC), the structure of 1 was established as shown in Figure 1. The relative configuration of 1 was determined from analyses of the 1H – 1H coupling constants and ROESY spectrum (Figure 2). The coupling constants of H-9/Ha-8 and H-9/Hb-8 were observed to be 11.7 and 4.3 Hz, respectively, suggesting that the cyclohexanone ring is adopting a chair conformation with H-9 in an axial position and C9 –OH in an equatorial position. Furthermore, the NOE correlations of H-9/H-7, H-9/H-5, H-7/H-5, H-5/H-4 and H3-14/ H3-15 indicated that H-5, H-7 and H-9 were co-facial and a-orientation, while H3-15 and H3-14 were on the opposite side of the molecular plane and thus b-orientation. Phytochemical study of this plant also resulted in the isolation of one known eudesmane-type sesquiterpenoid: 1b-hydroxy-1,2-dihydro-a-santonin. It was identified by comparing its physical and spectral data ([a ]D, UV, IR, 1H NMR, 13C NMR, MS) with reported values (Yang et al. 2006). Two isolated eudesmane-type sesquiterpenoids (1 and 2) were evaluated for their cytotoxic activities against P-388 lymphoid leukaemia, HONE-1 nasopharyngeal and HT-29 colorectal
14 1
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Figure 1. Compounds isolated from S. lyratum.
O 2
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Figure 2. Key HMBC and ROESY correlations of 1.
cancer cells by using MTT assay and anti-cancer drugs, etoposide and cisplatin, as positive controls. Two sesquiterpenoids exhibited significant cytotoxicity as shown in Table 1. 3. Experimental section 3.1. General experimental procedures Optical rotations were measured on a Perkin-Elmer 241 polarimeter (PerkinElmer Corporation, Waltham, MA, USA). IR spectra were recorded on a Perkin-Elmer 683 infrared spectrometer with KBr disks (Perkin-Elmer Corporation, California, CA, USA). FAB-MS and HR-FAB-MS were recorded on an Autospec-Ultima ETOF MS spectrometer (Bruker BioSpin, Ettlingen, Germany). NMR spectra were recorded on a Varian Unity BRUKER 400 at 400 MHz (1H) and 100 MHz (13C), with tetramethylsilane as the internal standard (Bruker BioSpin, Ettlingen, Germany). Silica gel (200 – 300 mesh) for column chromatography and silica gel GF254 for preparative thin layer chromatography (TLC) were obtained from Qingdao Marine Chemical Factory, Qingdao, China. Precoated plates of silica gel GF254 were used for TLC and detected under UV light. 3.2. Plant material S. lyratum Thunb. was collected in Linyi District, Shandong Province, China, in September 2009, and identified by Professor Yan-Yan Zhao, School of Pharmaceutical Science, Yantai University. The whole plant of S. lyratum was harvested and air-dried at room temperature in the dark. A voucher specimen (YP06089) has been deposited at the herbarium of the School of Pharmaceutical Science, Yantai University. 3.3. Extraction and isolation The air-dried whole plant (20.0 kg) of S. lyratum was finely cut and extracted three times (1 h £ 3) with refluxing EtOH. The solvent was concentrated in vacuo to yield a crude extract Table 1. Cytotoxicity of compounds 1 and 2 against P-388, HONE-1 and HT29 cancer cell lines. IC50a (mM) Compounds Etoposide Cisplatinb 1 2
b
P-388
HONE-1
HT-29
2.3 ^ 0.5 2.8 ^ 0.3 3.6 ^ 0.5 3.1 ^ 0.4
1.9 ^ 0.2 2.2 ^ 0.4 4.7 ^ 0.3 4.5 ^ 0.4
2.6 ^ 0.2 2.9 ^ 0.3 5.8 ^ 0.7 6.9 ^ 0.5
a IC50 is defined as the concentration that resulted in a 50% decrease in cell number and the results are means ^ standard deviation of three independent replicates. The IC50 greater than 10 mM was considered to be no cytotoxicity. b Positive control substance.
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(1.7 kg), which was then dissolved and suspended in H2O (3.0 L) and partitioned with CHCl3 (3 £ 10 L) and EtOAc (3 £ 10 L). The CHCl3 fraction (132.7 g) was initially subjected to column (10 cm £ 120 cm) chromatography on silica gel (200 –300 mesh, 2.5 kg), eluted with cyclohexane –acetone [95:5 (5.0 L), 90:10 (5.0 L), 85:15 (6.0 L), 80:20 (5.0 L), 75:25 (6.0 L), 70:30 (5.0 L), 60:40 (4.0 L), and 50:50 (2.5 L)] to yield eight fractions. Fraction 4 (3.4 g) was separated by CC over silica gel [eluted by petroleum ether –acetone (100:0 to 70:30, v/v)] and preparative TLC [cyclohexane –acetone (7:3, v/v)] and subsequently purified on Sephadex LH20 [100 g, eluting with CHCl3 –CH3OH (10:40, v/v)] to yield compounds 1 (53 mg) and 2 (97 mg). Lyratol G (1): Colourless viscous oil, [a ]29 D : þ 32.9 (c 0.37, CHCl3). IR (KBr) nmax: 3410 (br), 1697, 1470, 1388 and 1022 cm21. FABMS m/z: 255.3 [M þ H]þ. HR-FABMS m/z: 255.1957 [M þ H]þ (Calcd for C15H26O3, 255.1960). 1H NMR (400 MHz, CDCl3) d: 1.47 (1H, m, H-1a), 2.26 (1H, m, H-1b), 2.48 (1H, m, H-2a), 2.42 (1H, m, H-2b), 2.37 (1H, m, H-4), 1.20 (1H, m, H-5a), 1.74 (1H, m, H-6a), 1.16 (1H, m, H-6b), 1.46 (1H, m, H-7a), 1.83 (1H, m, H8a), 1.42 (1H, m, H-8b), 3.32 (1H, dd, J ¼ 11.7, 4.3 Hz, H-9a), 1.22 (3H, s, H3-12), 1.23 (3H, s, H3-13), 1.07 (3H, s, H3-14), 1.02 (3H, d, J ¼ 6.5 Hz, H3-15). 13C NMR and DEPT (100 MHz, CDCl3) d: 37.3 (C-1, CH2), 37.6 (C-2, CH2), 212.7 (C-3, C), 44.9 (C-4, CH), 49.9 (C-5, CH), 25.8 (C-6, CH2), 46.7 (C-7, CH), 31.1 (C-8, CH2), 78.8 (C-9, CH), 38.6 (C-10, C), 72.3 (C-11, C), 27.0 (C-12, CH3), 27.6 (C-13, CH3), 10.3 (C-14, CH3), 11.7 (C-15, CH3).
3.4. Anti-tumoural cytotoxic bioassays The viability of the cells after treating with various chemicals was evaluated using MTT assay performed as previously reported (Wang et al. 2008). Briefly, cells were plated in the appropriate media on 96-well plates, and triplicate wells were treated with media or agents. After 72- h incubation, the drug-containing medium was removed and replaced by fresh medium, and then the cells were incubated with MTT solution for 2 h. After the medium was removed, 150 mL of DMSO was added to each well. The plates were gently agitated until the colour reaction was uniform and the OD570 was determined using a microplate reader. The 50% inhibitory concentration (IC50) was defined as the concentration that reduced the absorbance of the untreated wells by 50% of the vehicle in the MTT assay.
4. Conclusion In conclusion, one new eudesmane-type sesquiterpenoid was isolated from the whole plant of Solanum lyratum. The discovery of new sesquiterpenoid is a further illustration to the diverse sesquiterpenoids from the genus Solanum. Furthermore, the new sesquiterpenoid exhibited significant cytotoxicities against P-388, HONE-1 and HT-29 cell lines in vitro.
Supplementary material Supplementary material relating to this article is available online, along side Figures S1 – S4.
Acknowledgements This study was supported by the National Natural Science Foundation of China (No. 21372189). The authors are grateful to Ms Wen-Yan Wang and Li Shen (School of Pharmaceutical Science, Yantai University) for the measurements of FAB-MS, HR-FAB-MS, UV, IR and NMR spectra. The authors also gratefully acknowledge Mr Hong-Bo Wang (School of Pharmaceutical Science, Yantai University) for the bioactivity screening.
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References Guilhon GMSP, Mu¨ller AH. 1998. Eudesmane sesquiterpenoids from Pluchea quitoc. Phytochemistry. 47:227–229. Hsu SC, Lu JH, Kuo CL, Yang JS, Lin MW, Chen GW, Su CC, Lu HF, Chung JG. 2008. Crude extracts of Solanum lyratum induced cytotoxicity and apoptosis in a human colon adenocarcinoma cell line (colo 205). Anticancer Res. 28:1045–1054. Kang B, Lee E, Hong I, Lee J, Kim H. 1998. Abolition of anaphylactic shock by Solanum lyratum Thunb. Int J Immunopharmacol. 19:729–734. Kuo WW, Huang CY, Chung JG, Yang SF, Tsai KL, Chiu TH, Lee SD, Ou HC. 2009. Crude extracts of Solanum lyratum protect endothelial cells against oxidized low-density lipoprotein-induced injury by direct antioxidant action. J Vasc Surg. 50:849–860. Lu YY, Luo JG, Kong LY. 2011. Steroidal alkaloid saponins and steroidal saponins from Solanum surattense. Phytochemistry. 72:668–673. Ren Y, Shen L, Zhang DW, Dai SJ. 2009. Two new sesquiterpenoids from Solanum lyratum with cytotoxic activities. Chem Pharm Bull. 57:408–410. Wang HB, Li HY, Zuo MX, Zhang Y, Liu H, Fang WS, Chen XG. 2008. Lx2-32c, a novel taxane and its antitumor activities in vitro and in vivo. Cancer Lett. 268:89–97. Yang JS, Wu CC, Kuo CL, Yeh CC, Chueh FS, Hsu CK, Wang CK, Chang CY, Ip SW, Hsu YM, Kuo WW, Chung JG. 2010. Solanum lyratum extract affected immune response in normal and leukemia murine animal in vivo. Hum Exper Toxicol. 29:359–367. Yang L, Dai J, Sakai JI, Ando M. 2006. Biotransformation of a- and 6b-santonin by fungus and plant cell cultures. J Asian Nat Prod Res. 8:317–326. Yao F, Song QL, Zhang L, Li GS, Dai SJ. 2013a. Three new cytotoxic sesquiterpenoids from Solanum lyratum. Phytochem Lett. 3:190–193. Yao F, Song QL, Zhang L, Li GS, Dai SJ, Solajiangxins A-C. 2013b. Three new cytotoxic sesquiterpenoids from Solanum lyratum. Fitoterapia. 89:200–204. Yue XD, Qu GW, Li BF, Xue CH, Li GS, Dai SJ. 2012. Two new C13-norisoprenoids from Solanum lyratum. J Asian Nat Prod Res. 14:486–490. Zhang DW, Yang Y, Yao F, Yu QY, Dai SJ. 2012. Solalyratins A and B, new anti-inflammatory metabolites from Solanum lyratum. J Nat Med. 66:362–366. Zhou XL, He XJ, Wang GH, Gao H, Zhou GX, Ye WC, Yao XS. 2006. Steroidal saponins from Solanum nigrum. J Nat Prod. 69:1158–1163.
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
New eudesmane-type sesquiterpenoid from Solanum lyratum with cytotoxic activity ab
a
a
a
Xiu-Ping Nie , Fang Yao , Xi-Dian Yue , Gui-Sheng Li & Sheng-Jun a
Dai a
School of Pharmaceutical Science, Yantai University, Yantai 264005, P.R. China b
Yantai University Affiliated Hospital, Yantai 264005, P.R. China Published online: 21 Mar 2014.
Click for updates To cite this article: Xiu-Ping Nie, Fang Yao, Xi-Dian Yue, Gui-Sheng Li & Sheng-Jun Dai (2014) New eudesmane-type sesquiterpenoid from Solanum lyratum with cytotoxic activity, Natural Product Research: Formerly Natural Product Letters, 28:9, 641-645, DOI: 10.1080/14786419.2014.891199 To link to this article: http://dx.doi.org/10.1080/14786419.2014.891199
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 &
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Natural Product Research, 2014 Vol. 28, No. 9, 641–645, http://dx.doi.org/10.1080/14786419.2014.891199
New eudesmane-type sesquiterpenoid from Solanum lyratum with cytotoxic activity Xiu-Ping Nieab, Fang Yaoa, Xi-Dian Yuea, Gui-Sheng Lia and Sheng-Jun Daia* a
School of Pharmaceutical Science, Yantai University, Yantai 264005, P.R. China; Yantai University Affiliated Hospital, Yantai 264005, P.R. China
Downloaded by [Nova Southeastern University] at 21:35 08 January 2015
b
(Received 13 September 2013; final version received 2 February 2014) In our continuing effort to discover more new cytotoxic sesquiterpenoids from Solanum lyratum, one new eudesmane-type sesquiterpenoid (1, 3-keto-eudesm-9b,11-diol, named lyratol G), together with one known eudesmane-type sesquiterpenoid (2, 1bhydroxy-1,2-dihydro-a-santonin), was obtained. The structure of the new sesquiterpenoid was elucidated on the basis of integrated spectroscopic techniques, mainly HRFAB-MS, 1D and 2D NMR (1H– 1H COSY, HMQC, HMBC and ROESY). In vitro, two sesquiterpenoids were found to exhibit significant cytotoxicity against three cancer cell lines (P-388, HONE-1 and HT-29), and gave IC50 values in the range of 3.1– 6.9 mM. Keywords: Solanum lyratum; Solanaceae; sesquiterpenoid; lyratol G; cytotoxic activity
1. Introduction The genus Solanum, which is one of the largest genera of the Solanaceae, is widely distributed in tropical and temperate zones and is a rich source of active secondary metabolites. Many species belonging to this genus constantly draw the attention of numerous chemical researchers due to their biologically active constituents, including steroids, steroidal alkaloids and their glycosides (Zhou et al. 2006; Lu et al. 2011). Solanum lyratum, commonly known as ‘Bai-Ying’ in traditional Chinese medicine and ‘Back-MoDeung’ in traditional Korea medicine, respectively, is a perennial herb and has been used as a anti-anaphylactic (Kang et al. 1998), anti-inflammatory (Zhang et al. 2012), anti-tumour (Hsu et al. 2008), immunomodulatory (Yang et al. 2010) and antioxidant (Kuo et al. 2009) agent. In previous phytochemical studies on S. lyratum, 10 new sesquiterpenoids were separated and most of them exhibited significant cytotoxic activities (Ren et al. 2009; Yue et al. 2012; Yao et al. 2013a, 2013b). As part of our ongoing search for more new bioactive compounds, a further separation of an extract from S. lyratum was performed, and led to the isolation of one new eudesmane-type sesquiterpenoid (1, 3-keto-eudesm-9b,11-diol, named lyratol G) and one known one (2, 1b-hydroxy-1,2-dihydro-a-santonin), and their structures were elucidated by using detailed spectral data. In addition, two sesquiterpenoids were screened for cytotoxicity against P-388, HONE-1 and HT29 cancer cells. Herein, we report on the isolation, structural elucidation and the evaluation of cytotoxic effects of two eudesmane-type sesquiterpenoids.
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
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2. Results and discussion Compound 1 was isolated and purified as a colourless viscous oil. In the HR-FAB mass spectrum, it indicated a quasi-molecular ion peak at m/z 255.1957 [M þ H]þ, corresponding to a molecular formula C15H26O3. The IR spectrum revealed absorption bands at 3410 (br) and 1697 cm21, which were in agreement with hydroxyl and carbonyl groups. The 1H NMR spectrum of 1 revealed signals corresponding to three tertiary methyl groups at dH 1.22 (3H, s, H12), 1.23 (3H, s, H-13) and 1.07 (3H, s, H-14), one secondary methyl group at dH 1.02 (3H, d, J ¼ 6.5 Hz, H-15) and an oxygenated methine at dH 3.32 (dd, J ¼ 11.7, 4.3 Hz, H-9). Detailed examination of the 1H – 1H COSY experiment revealed two spin systems. The first spin system included the signals of one methyl (dH 1.02, 3H, d, J ¼ 6.5 Hz, H-15), two methylenes (dH 1.74, m, Ha-6; 1.16, m, Hb-6; 1.83, m, Ha-8; 1.42, m, Hb-8) and four methines (dH 2.37, m, H-4; 1.20, m, H-5; 1.46, m, H-7; 3.32, dd, J ¼ 10.7, 4.3 Hz, H-9). The second spin system was traced from two methylenes (dH 2.26, m, Ha-1; 1.47, m, Hb-1; 2.48, m, Ha-2; 2.42, m, Hb-2). The 13C NMR spectrum displayed 15 carbon resonances, and the DEPT spectrum was consistent with the presence of four methyls, four methylenes, four methines (one oxygenated) and three quaternary carbons (one oxygenated and one carbonyl). Careful analyses of the above-mentioned signal patterns indicated the presence of an eudesmane-type sesquiterpenoid skeleton (Guilhon & Mu¨ller 1998). In the HMBC experiment (Figure 2), the correlations of H2-1/C-3, H2-2/C-3, H315/C-3, H-5/C-3, H-5/C-9, H3-14/C-9, H-7/C-9, H2-6/C-11, H2-8/C-11, H3-12/C-11 as well as from H3-13/C-11 clearly positioned the carbonyl group at C-3 and two hydroxyl moieties at C-9 and C-11, respectively. Based on the above-mentioned data and comprehensive 2D NMR experiments (1H – 1H COSY, HMQC and HMBC), the structure of 1 was established as shown in Figure 1. The relative configuration of 1 was determined from analyses of the 1H – 1H coupling constants and ROESY spectrum (Figure 2). The coupling constants of H-9/Ha-8 and H-9/Hb-8 were observed to be 11.7 and 4.3 Hz, respectively, suggesting that the cyclohexanone ring is adopting a chair conformation with H-9 in an axial position and C9 –OH in an equatorial position. Furthermore, the NOE correlations of H-9/H-7, H-9/H-5, H-7/H-5, H-5/H-4 and H3-14/ H3-15 indicated that H-5, H-7 and H-9 were co-facial and a-orientation, while H3-15 and H3-14 were on the opposite side of the molecular plane and thus b-orientation. Phytochemical study of this plant also resulted in the isolation of one known eudesmane-type sesquiterpenoid: 1b-hydroxy-1,2-dihydro-a-santonin. It was identified by comparing its physical and spectral data ([a ]D, UV, IR, 1H NMR, 13C NMR, MS) with reported values (Yang et al. 2006). Two isolated eudesmane-type sesquiterpenoids (1 and 2) were evaluated for their cytotoxic activities against P-388 lymphoid leukaemia, HONE-1 nasopharyngeal and HT-29 colorectal
14 1
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OH
O H 15
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12 1
Figure 1. Compounds isolated from S. lyratum.
O 2
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H H
H O
H
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H
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H
H
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Figure 2. Key HMBC and ROESY correlations of 1.
cancer cells by using MTT assay and anti-cancer drugs, etoposide and cisplatin, as positive controls. Two sesquiterpenoids exhibited significant cytotoxicity as shown in Table 1. 3. Experimental section 3.1. General experimental procedures Optical rotations were measured on a Perkin-Elmer 241 polarimeter (PerkinElmer Corporation, Waltham, MA, USA). IR spectra were recorded on a Perkin-Elmer 683 infrared spectrometer with KBr disks (Perkin-Elmer Corporation, California, CA, USA). FAB-MS and HR-FAB-MS were recorded on an Autospec-Ultima ETOF MS spectrometer (Bruker BioSpin, Ettlingen, Germany). NMR spectra were recorded on a Varian Unity BRUKER 400 at 400 MHz (1H) and 100 MHz (13C), with tetramethylsilane as the internal standard (Bruker BioSpin, Ettlingen, Germany). Silica gel (200 – 300 mesh) for column chromatography and silica gel GF254 for preparative thin layer chromatography (TLC) were obtained from Qingdao Marine Chemical Factory, Qingdao, China. Precoated plates of silica gel GF254 were used for TLC and detected under UV light. 3.2. Plant material S. lyratum Thunb. was collected in Linyi District, Shandong Province, China, in September 2009, and identified by Professor Yan-Yan Zhao, School of Pharmaceutical Science, Yantai University. The whole plant of S. lyratum was harvested and air-dried at room temperature in the dark. A voucher specimen (YP06089) has been deposited at the herbarium of the School of Pharmaceutical Science, Yantai University. 3.3. Extraction and isolation The air-dried whole plant (20.0 kg) of S. lyratum was finely cut and extracted three times (1 h £ 3) with refluxing EtOH. The solvent was concentrated in vacuo to yield a crude extract Table 1. Cytotoxicity of compounds 1 and 2 against P-388, HONE-1 and HT29 cancer cell lines. IC50a (mM) Compounds Etoposide Cisplatinb 1 2
b
P-388
HONE-1
HT-29
2.3 ^ 0.5 2.8 ^ 0.3 3.6 ^ 0.5 3.1 ^ 0.4
1.9 ^ 0.2 2.2 ^ 0.4 4.7 ^ 0.3 4.5 ^ 0.4
2.6 ^ 0.2 2.9 ^ 0.3 5.8 ^ 0.7 6.9 ^ 0.5
a IC50 is defined as the concentration that resulted in a 50% decrease in cell number and the results are means ^ standard deviation of three independent replicates. The IC50 greater than 10 mM was considered to be no cytotoxicity. b Positive control substance.
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(1.7 kg), which was then dissolved and suspended in H2O (3.0 L) and partitioned with CHCl3 (3 £ 10 L) and EtOAc (3 £ 10 L). The CHCl3 fraction (132.7 g) was initially subjected to column (10 cm £ 120 cm) chromatography on silica gel (200 –300 mesh, 2.5 kg), eluted with cyclohexane –acetone [95:5 (5.0 L), 90:10 (5.0 L), 85:15 (6.0 L), 80:20 (5.0 L), 75:25 (6.0 L), 70:30 (5.0 L), 60:40 (4.0 L), and 50:50 (2.5 L)] to yield eight fractions. Fraction 4 (3.4 g) was separated by CC over silica gel [eluted by petroleum ether –acetone (100:0 to 70:30, v/v)] and preparative TLC [cyclohexane –acetone (7:3, v/v)] and subsequently purified on Sephadex LH20 [100 g, eluting with CHCl3 –CH3OH (10:40, v/v)] to yield compounds 1 (53 mg) and 2 (97 mg). Lyratol G (1): Colourless viscous oil, [a ]29 D : þ 32.9 (c 0.37, CHCl3). IR (KBr) nmax: 3410 (br), 1697, 1470, 1388 and 1022 cm21. FABMS m/z: 255.3 [M þ H]þ. HR-FABMS m/z: 255.1957 [M þ H]þ (Calcd for C15H26O3, 255.1960). 1H NMR (400 MHz, CDCl3) d: 1.47 (1H, m, H-1a), 2.26 (1H, m, H-1b), 2.48 (1H, m, H-2a), 2.42 (1H, m, H-2b), 2.37 (1H, m, H-4), 1.20 (1H, m, H-5a), 1.74 (1H, m, H-6a), 1.16 (1H, m, H-6b), 1.46 (1H, m, H-7a), 1.83 (1H, m, H8a), 1.42 (1H, m, H-8b), 3.32 (1H, dd, J ¼ 11.7, 4.3 Hz, H-9a), 1.22 (3H, s, H3-12), 1.23 (3H, s, H3-13), 1.07 (3H, s, H3-14), 1.02 (3H, d, J ¼ 6.5 Hz, H3-15). 13C NMR and DEPT (100 MHz, CDCl3) d: 37.3 (C-1, CH2), 37.6 (C-2, CH2), 212.7 (C-3, C), 44.9 (C-4, CH), 49.9 (C-5, CH), 25.8 (C-6, CH2), 46.7 (C-7, CH), 31.1 (C-8, CH2), 78.8 (C-9, CH), 38.6 (C-10, C), 72.3 (C-11, C), 27.0 (C-12, CH3), 27.6 (C-13, CH3), 10.3 (C-14, CH3), 11.7 (C-15, CH3).
3.4. Anti-tumoural cytotoxic bioassays The viability of the cells after treating with various chemicals was evaluated using MTT assay performed as previously reported (Wang et al. 2008). Briefly, cells were plated in the appropriate media on 96-well plates, and triplicate wells were treated with media or agents. After 72- h incubation, the drug-containing medium was removed and replaced by fresh medium, and then the cells were incubated with MTT solution for 2 h. After the medium was removed, 150 mL of DMSO was added to each well. The plates were gently agitated until the colour reaction was uniform and the OD570 was determined using a microplate reader. The 50% inhibitory concentration (IC50) was defined as the concentration that reduced the absorbance of the untreated wells by 50% of the vehicle in the MTT assay.
4. Conclusion In conclusion, one new eudesmane-type sesquiterpenoid was isolated from the whole plant of Solanum lyratum. The discovery of new sesquiterpenoid is a further illustration to the diverse sesquiterpenoids from the genus Solanum. Furthermore, the new sesquiterpenoid exhibited significant cytotoxicities against P-388, HONE-1 and HT-29 cell lines in vitro.
Supplementary material Supplementary material relating to this article is available online, along side Figures S1 – S4.
Acknowledgements This study was supported by the National Natural Science Foundation of China (No. 21372189). The authors are grateful to Ms Wen-Yan Wang and Li Shen (School of Pharmaceutical Science, Yantai University) for the measurements of FAB-MS, HR-FAB-MS, UV, IR and NMR spectra. The authors also gratefully acknowledge Mr Hong-Bo Wang (School of Pharmaceutical Science, Yantai University) for the bioactivity screening.
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