New eudesmenoic acid methyl esters from the seed oil of Jatropha curcas

Fitoterapia 89 (2013) 278–284

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New eudesmenoic acid methyl esters from the seed oil of Jatropha curcas Yuan-Feng Yang a,b, Jie-Qing Liu a, Zhong-Rong Li a, Yan Li a, Ming-Hua Qiu a,b,⁎ a State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Science, Kunming 650201, Yunnan, People's Republic of China b University of Chinese Academy of Science, Beijing 100049, People's Republic of China

a r t i c l e

i n f o

Article history: Received 19 March 2013 Accepted in revised form 14 June 2013 Available online 27 June 2013 Keywords: Jatropha curcas Euphorbiaceae Sesquiterpenoid Germacranolide Eudesmanolide Cytotoxicity

a b s t r a c t Three new eudesmenoic acid methyl esters (1–3), as well as five known compounds, including three germacranolides (4–6) and two eudesmanolides (7 and 8), were isolated from the seed oil of Jatropha curcas. The new compounds were elucidated by means of spectroscopic methods, including extensive NMR spectra. In addition, the structure of 8 was confirmed by a single-crystal X-ray diffraction analysis. Among the isolates, compounds 4–6 were the first reported from the genus Jatropha. Using MTS viability assay, the cytotoxicity of compounds 2–8 were evaluated against HL-60, SMMC-7721, A-549, MCF-7, and SW480 human tumor cell lines. Compounds 4 and 5 showed remarkable cytotoxicity against all the tested cell lines with IC50 values from 0.5 to 3.5 μM, and the new compound 3 displayed selective cytotoxic activity against A-549 cell with an IC50 value of 7.24 μM, but slight cytotoxicity against HL-60 and MCF-7 with IC50 values of 23.77 and 22.37 μM, respectively. © 2013 Elsevier B.V. All rights reserved.

1. Introduction The physic nut shrub, Jatropha curcas (Euphorbiaceae), has been proposed as a “miracle tree”, particularly as a substitute for diesel oil and as fuel [1,2], mainly due to the 32% oil content of its seeds [3]. The plant has been used as a traditional medicine for the treatments of diverse ailments involving from simple fevers to infectious diseases in many countries of the world [4–6]. Also, the seed oil was applied to treat diarrhea [7], as a purgative [8], and to treat rheumatic pain, skin diseases, and eczema [9]. Previous phytochemical investigations proved the presence of phorbol esters with significant tumor-promoting activity from the seed oil [10]. Originally, our group mainly focused on the chemical constituents from its roots, which have led to the identification

of jatrophadiketone (possessing a 6/6/6 tricyclic skeleton) and jatrophalone (having a rare and unique 5/6/7/3 fused ring system) [11], in addition to two sesquiterpenes [12]. With the aim of looking for more structurally and biological diverse compounds, we have chosen the seed oil of J. curcas for detailed chemical investigation. As a result, three new eudesmenoic acid methyl esters named jatrophaeudesmenes A–C (1–3), along with three known germacranolides (4–6) and two reported eudesmanolides (7 and 8), were isolated and purified (Fig. 1). Additionally, compounds 2–8 were tested for their cytotoxicity against HL-60, SMMC-7721, A-549, MCF-7, and SW480 human tumor cell lines using the MTS method. Herein, the isolation, structure elucidation, and cytotoxicity of these compounds were discussed. 2. Experimental

⁎ Corresponding author at: State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Science, Kunming 650201, Yunnan, People's Republic of China. Tel.: +86 871 65223257; fax: +86 871 65223255. E-mail address: [email protected] (M.-H. Qiu). 0367-326X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.fitote.2013.06.011

2.1. General experimental procedures IR spectra were recorded in KBr disks on a Bio-Rad FTS-135 spectrophotometer. UV spectra were determined on

Y.-F. Yang et al. / Fitoterapia 89 (2013) 278–284

a Shimadzu UV-2401PC spectrophotometer. Optical rotations were performed on a JASCO P-1020 digital polarimeter. X-ray data was determined using a Bruker APEX DUO instrument. 1D and 2D NMR spectra were taken on an AVANCE Ш-600 spectrometer instrument with TMS as internal standard. Unless otherwise specified, chemical shifts (δ) are expressed in ppm with reference to the solvent signals. ESIMS was recorded on a VG Auto Spec-3000 mass spectrometer, while HREIMS was measured on an Auto Spec Premier P776 mass spectrometer instrument in positive ion mode. Semipreparative HPLC was collected on an Agilent 1100 liquid chromatograph with a ZORBAX SB-C18 column (250 × 9.4 mm, 5.0 μm) column. Column chromatography was performed with silica gel (200–300 mesh; Qingdao Marine Chemical Factory, Qingdao, People's Republic of China), Lichroprep RP-18 silica gel (40 63 μm, Merck, Darmstadt, Germany), Sephadex LH-20 (General Electric Company, Fairfield, CT); Fractions were monitored by TLC under UV light, and spots were visualized by heating silica gel plates sprayed with 10% H2SO4 in EtOH. All solvents including petroleum ether (60 − 90 °C) were distilled prior to use. 2.2. Plant material J. curcas seed oil was obtained by using the sub-critical fluid from the seeds of J. curcas, which were collected in August 2011 from Yuanmou county of Yunnan Province, People's Republic of China. The plant material was identified by Prof. Cheng-yuan Yang. A voucher specimen (KIB

279

20110804) of the seed oil was stored at − 10 °C at the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences. 2.3. Extraction and isolation The seed oil of J. curcas (5.0 kg), which was obtained from the dried and powdered seeds of J. curcas using the sub-critical fluid, was applied separately onto a normal-phase silica gel column (15 kg, 200–300 mesh, 15 × 150 cm), which had previously been packed with 5.50 kg of silica gel (200– 300 mesh). After sample application, the column was washed successively with petroleum ether (PE) (500 L), and a gradient CHCl3-MeOH system [from 80:1 (200 L) to 20:1 (800 L)], respectively. Fractions of 20 L were collected and monitored by TLC. The fraction (20:1, CHCl3-MeOH, 80 g) was fractionated by RP-18 silica gel column (400 g, 25–50 μm, 4 × 45 cm) and eluted stepwise with MeOH-H2O (from 45:55 to 75:25, flow rate 16 mL/min) to afford three fractions (Fr. A–C). Fraction A (MeOH-H2O, 45:55, v/v; 4.6 g) was submitted to chromatography over silica gel column (200 g, 4.5 × 20.0 cm) with a stepwise Me2CO-PE system (from 1:10 to 1:2) as eluent to give four subfractions: subfraction A1 was obtained using 2 L Me2CO-PE from 1:10 to 1:8; subfraction A2 was obtained using 2 L Me2CO-PE with a ratio of 1:8; subfraction A3 was obtained using 2 L Me2CO-PE with a ratio of 1:4; and subfraction A4 was obtained using 2 L Me2CO-PE with a ratio of 1:2; Further purification of subfraction A1 (80 mg) was achieved by a semipreparative HPLC on a ZORBAX SB-C18

Fig. 1. The structures of 1–8.

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column (250 × 9.4 mm, 5.0 μm). The mobile phase consisted of water (A) and methanol (B) using a gradient program of 40–45% at 0–10 min and 45–50% at 15–30 min at a flow rate of 3.0 ml/min. The detected wavelength was set at 210 nm. The retention times of the target compounds in semipreparative HPLC as follows: compound 1 (1.8 mg) at 9.7 min, 4 (3.2 mg) at 20.6 min and 5 (7.5 mg) at 22.5 min, respectively. Subfraction A2 (70 mg) was further fractionated with a Sephadex LH-20 column (20.0 g, 2.5 × 25.0 cm; MeOH), and a semipreparative HPLC (3 mL/min, UV detection at λmax = 210 nm, CH3CN/H2O, 40:60) to give compounds 2 (1.6 mg), 3 (1.4 mg) and 6 (1.5 mg) at tR = 9.3, 8.2 and 17.8 min respectively. Separation of subfraction A3 (110 mg) by semipreparative HPLC yielded compounds 7 (1.8 mg) (tR = 12.1 min) and 8 (1.6 mg) (tR = 11.2 min) using CH3CNH2O with a ratio of 50:50 at 3 mL/min. Jatrophaeudesmene A (1): White, amorphous powder; [α]25 D − 32.7 (c 0.17, CHCl3); UV (CHCl3) λmax (log ε) 241 (4.01), 204 (4.66), 193 (4.67) nm; IR (KBr) νmax 3424, 2972, 2940, 2856, 1717, 1662 cm−1; 1H and 13C NMR data see Table 1; positive ESIMS (m/z 303 [M + Na]+); HREIMS m/z 280.1683 [M]+ (calcd. 280.1675 for C16H24O4). Jatrophaeudesmene B (2): Colorless powder; [α]16 D +284.3 (c 0.16, MeOH); UV (CHCl3) λmax (log ε) 240 (4.59), 208 (4.96) nm; IR (KBr) νmax 3432, 2954, 2923, 1719, 1628, 959 cm− 1; 1H and 13C NMR data see Table 1; positive HREIMS m/z 336.3776 [M]+ (calcd. 336.1573 for C18H24O6). Jatrophaeudesmene C (3): Colorless powder; [α]16 D + 95.8 (c 0.18, CHCl3); UV (CHCl3) λmax (log ε) 240 (4.59), 209 (4.96) nm; IR (KBr) νmax 3433, 2949, 2853, 1718, 1627,

Table 1 1 H and 13C NMR spectra data of 1−3 (600 MHz for 1H and 150 MHz for No

1 2α 2β 3α 3β 4 5 6 7 8 9α 9β 10 11 12 13a 13b 14 15a 15b

1

a

13

C

79.2 (d) 32.7 (t) 124.0 (d) 132.5 145.0 121.5 48.7 66.5 44.6

(s) (s) (d) (d) (d) (t)

41.7 40.1 178.4 11.7

(s) (s) (s) (q)

18.0 (q) 20.3 (q)

3.43 2.27 2.14 5.46 – – 5.31 2.59 3.79 2.11 1.38 – 3.01 – 1.07

(dd, 10.3, 5.8) (dt, 17.7, 5.6) (m) (d, 4.6)

(d, 2.4) (d, 9.2) (m) (dd, 12.3, 3.8) (t, 12.2) (qd, 7.1, 3.3) (d, 7.1)

0.94 (s) 1.74 (br. s)

The cytotoxicity of compounds 2–8 was tested against breast cancer (MCF-7), hepatocellular carcinoma (SMMC-7721), human myeloid leukemia (HL-60), lung cancer (A-549), and colon cancer (SW480) human cell lines using an MTS assay, with cisplatin (Sigma, USA) as the positive control.

C, δ in ppm, J in Hz). 3c

13

H

2.4. Cytotoxicity assay

b

2 1

13

960 cm−1; 1H and 13C NMR data see Table 1; positive HREIMS m/z 336.3737 [M]+ (calcd. 336.1573 for C18H24O6). Crystallographic data for compound 8: C20H26O5, M = 346.41, monoclinic, a = 6.8508(9) Å, b = 11.7242(15) Å, c = 11.5995(15) Å, α = 90.00°, β = 103.111(2)°, γ = 90.00°, V = 907.4(2) Å3, T = 100(2) K, space group P21, Z = 2, μ(MoKα) = 0.090 mm−1, 9572 reflections measured, 4919 independent reflections (Rint = 0.0229). The final R1 values were 0.0525 (I N 2σ(I)). The final wR(F2) values were 0.1335 (I N 2σ(I)). The final R1 values were 0.0601 (all data). The final wR(F2) values were 0.1415 (all data). The goodness of fit on F2 was 1.051. Flack parameter = 0.6(10). The crystal structure of 8 was solved by direct method SHELXS-97 (Sheldrick, G. M. University of Gottingen: Gottingen, Germany, 1997) and full-matrix least-squares calculations. Crystallographic data for the structure of 8 have been deposited in the Cambridge Crystallographic Data Centre (deposition number: CCDC 928790). Copies of these data can be obtained free of charge via the Internet at www.ccdc.cam.ac.uk/conts/ retrieving.html (or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB21EZ, U.K.; fax (+44) 1223-336-033; or deposit @ccdc.cam.ac.uk).

C

1

75.0 (d) 33.7 (t)

3.90 2.53 2.29 5.40

13

H

124.4 (d) 133.8 50.1 73.6* 62.9* 66.4* 45.4

(s) (d) (d) (s) (d) (t)

40.0 139.7* 166.8* 130.5*

(s) (s) (s) (t)

12.3 (q) 23.4 (q)

– 2.42 5.93 – 5.01 1.61 3.06 – – – 6.48 5.93 1.31 1.78

(dd, 9.7, 7.0) (d, 17.8) (m) (s)

(d, 10.8) (overlap) (br. s) (t, 11.9) (dd, 12.6, 4.5)

(s) (overlap) (s) (s)

C

1

78.3 (d) 31.4 (t)

3.47 1.56 1.85 2.30 2.05 – 2.00 5.53 – 4.35 1.18 2.34 – – – 6.30 5.71 0.86 4.48 4.82

H

34.7 (t) 143.6 51.9 69.9* 59.7* 65.9* 44.6

(s) (d) (d) (s) (d) (t)

41.4 136.9* 166.5* 131.2*

(s) (s) (s) (t)

12.6 (q) 108.5 (t)

(d, 11.4, 4.6) (ddd, 25.4, 12.7, 4.9) (m) (m) (ddd, 18.5, 9.7, 4.8) (d, 10.3) (br. s) (br. s) (t, 11.8) (m)

(s) (s) (s) (s) (s)

Note: a12-COOCH3 (δC 62.4, q; δH 3.70, s); Recorded in MeOD. b 6-OOCCH3 (δC 171.1, s), 6-OOCCH3 (δC 22.1, q; δH 1.98, s); 12-COOCH3 (δC 51.9, q; δH 3.62, s); Recorded in pyridine-d5. c 6-OOCCH3 (δC 170.9, s), 6-OOCCH3 (δC 21.3, q; δH 1.99, s), 12-COOCH3 (δC 52.2, q; δH 3.79, s); Recorded in CDCl3, and the NMR data of 3 were obtained at 0 °C. *Where 13C NMR signal was a broad singlet peak.

Y.-F. Yang et al. / Fitoterapia 89 (2013) 278–284

The cell lines were obtained from Shanghai cell bank in China and were cultured in RPMI-1640 or DMEM medium (Hyclone, USA), supplemented with 10% fetal bovine serum (Hyclone®, USA) at 37 °C in a humidified atmosphere containing 5% CO2. The viability of cells was determined by performing colorimetric measurements of the amount of soluble formazan formed in living cells according to the reduction of MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2(4-sulfophenyl)-2H-tetrazoliuminner salt). In brief, 100 μL of adherent cells were seeded in 96-well plates for 24 h before drug addition, while suspended cells were grown just before drug addition at a concentration of 1 × 105 cells/mL. Each cell line was exposed to the assayed compound dissolved in dimethyl sulfoxide (DMSO) at different concentrations in triplicate at 37 °C for 48 h. At the end of the incubation period, both MTS (20 μL) and culture solution (100 μL) were added to each well after removal of 100 μL of the medium, and then the incubation was continued for 4 h at 37 °C. The optical density of the lysate was measured at 490 nm using a microplate reader (model 680; Bio-Rad, Inc., USA). The IC50 values of each compound were calculated by Reed and Muench's method. 3. Results and discussion J. curcas seed oil, obtained from the seed of J. curcas by the sub-critical fluid, was separated into a non-polar and a polar fraction by silica gel column chromatography with petroleum ether and then a gradient CHCl3-MeOH system (from 80:1 to 20:1). The 20:1 CHCl3-MeOH fraction was dried under reduced pressure, and then subjected to successive chromatography on RP-18, silica gel, Sephadex LH-20, and then purified by HPLC to afford three new eudesmenoic acid methyl esters, jatrophaeudesmenes A–C (1–3), and five known compounds, including three germacranolides (4–6) and two eudesmanolides (7 and 8). Jatrophaeudesmene A (1) was obtained as a white powder. The molecular formula C16H24O4, indicating five degrees of unsaturation, was deduced from the HREIMS at m/z 280.1683 [M]+ (calcd. 280.1675). The IR spectrum of 1 showed bands characteristic of hydroxyl (3424 cm−1), carbonyl (1717 cm−1) and olefinic bond (1662 cm−1). In the 1H NMR spectrum (Table 1), one tertiary methyl signal at δH 0.94 (3H, s), one allic methyl at δH 1.74 (3H, br. s), one secondary methyl at δH 1.07 (3H, d, J = 7.1 Hz), one methoxyl signal at δH 3.70 (3H, br. s), two olefinic proton signals at δH 5.46 (1H, d, J = 4.6 Hz) and

Fig. 2. The 1H–1H COSY (

281

5.31 (1H, d, J = 2.4 Hz), two oxymethine proton signals at δH 3.43 (dd, J = 10.3, 5.8 Hz) and 3.79 (m), were observed. The 13C NMR spectral data of 1 revealed 16 carbon signals that were resolved by DEPT experiment into three methyls, two methylenes, six methines (two olefinics and two oxygenated), and four quaternary carbons (one ester carbonyl and two olefinics). The above data suggested that the structure of 1 was an eudesmen-12-oic acid methyl ester skeleton [13], similar to 1α,6α-dihydroxyeudesma-3,11(13)-dien-12-carboxylic acid methyl ester [13a]. Detailed analysis of HSQC and 1H–1H COSY spectra of 1 allowed the establishment of two structural fragments as drawn in blue bold lines in Fig. 2. One methyl formate was evident from 1H NMR signal at δH 3.70 (br. s) and 13C NMR signals at δC 178.4 (C-12) and 62.4 (OCH3). In the HMBC spectrum of 1 (Fig. 2), the correlations from H-11, OCH3 and H3-13 to δC 178.4 (C-12) deduced the methyl formate at C-12; the strong correlations from the allic methyl at δH 1.74 to C-3, C-4, and C-5 indicated H3-14 was located at C-4 and a double bond between C-3 and C-4. The trisubstituted double bond between C-5 and C-6 was determined by the HMBC correlations from H-7, H-11, and H-3 to C-5, and from H3-14, H3-15, and H-7 to C-5, in combination with the correlation between H-6 and H-7 in the 1H–1H COSY spectrum. An oxygenated methine resonance [δC 79.2; δH 3.73 (dd, J = 10.3, 5.8 Hz)] was then assigned to C-1 by its cross peaks of H2-2, H-3, and H3-14 in the HMBC spectrum. The assignment of a hydroxyl group at C-8 was confirmed by the HMBC correlations from H-6, H-7, and H2-9 to C-8 (δC 66.5). To our knowledge, the relative configuration of H3-14 was in β-oriented in the eudesmane skeleton. In the ROESY spectrum (Fig. 2), the observed correlations of H3-14/H-9β, H-9β/H-8 suggested that 8-OH was α-oriented. In addition, the correlation of H-7 with H-9α indicated that H-7 should be α-oriented. The β-orientation of the hydroxyl group at C-1 was determined by the fact that H-1 correlated with only the α-oriented proton of H2-2 and without of H3-14 in the ROESY spectrum. Consequently, the structure of 1 was elucidated as 1β,8α-dihydroxyeudesma-3,5-dien-12-carboxylic acid methyl ester, named jatrophaeudesmene A. Jatrophaeudesmene B (2) was determined to have the molecular formula C18H24O6 by HREIMS. The IR absorptions at 1719 and 1628 cm−1 and the UV absorption at λmax 240 nm suggested that 2 contained an α,β-unsaturated ester group. The 1H NMR spectrum showed signals assignable to

), key HMBC correlations (H → C) and key ROESY correlations (

) of compound 1.

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one tertiary methyl signal at δH 1.31, one allic methyl signal at δH 1.78, one acetyl methyl signal at δH 1.98, one methoxyl signal at δH 3.62, and three olefinic proton signals at δH 6.48, 5.93 and 5.40. The 13C NMR (Table 1) and HSQC data indicated the presence of four methyls (one acetyl methyl, one methoxyl), three methylenes (one olefinic), five methines (three oxygenated), and six quaternary carbons (two olefinics, one oxygenated and two carbonyls). Thus, four degrees of unsaturation were due to unsaturated bonds (two C_C and two C_O groups) and compound 2 possessed a tricyclic system. The 1H–1H COSY experiment revealed three spin systems corresponding to H-1/H2-2/H-3, H-5/H-6, and H-8/H-9. In addition, the HMBC experiment showed the correlations from H3-14 to C-1, C-5, C-9, and C-10, from H3-15 to C-3, C-4, and C-5, and from H2-13 to C-7, C-11, and C-13. Particularly, the epoxy ring located between C-7 and C-8 was confirmed by the HMBC correlations of H2-9 with C-5, C-7, and C-8, and of H2-13 with C-7 (Fig. 2). Thus, compound 2 was eudesmen-12-oic acid methyl ester with an epoxy ring. Furthermore, the weak but important HMBC correlation from H-6 to the ester group (δC 171.1) contributed to the acetoxyl substituted on C-6. The hydroxyl group linked at C-1 was determined by the HMBC correlations from H2-2, H-3, H2-9, and H3-14 to C-1. Correlation from the OCH3 proton (δH 3.62, s) to δC 166.8 (12-COOCH3) and from the exocyclic methylene protons H2-13 (δH 6.48 and 5.93) to C-7, C-11, and C-12 suggested that the methoxycarbonyl group located at C-12. In the ROESY spectrum of 2 (Fig. 3), the detected correlations of H3-14/H-9β, H-9β/H-8, H3-14/H-6 suggested that these protons should be in β-orientation. Thus, the 7,8-epoxide and 6-OAc were both in α-orientation. However, missing ROESY correlation between H3-14 and H-5 indicated characteristic of the trans-fusion of the A/B rings of decalin moiety [14] and implied that the orientation of H-5 was opposite to that of H3-14. The β-oriented hydroxyl group at C-1 was determined by the key ROESY correlation between H-5 and H-1. Accordingly, the structure of 2 was identified as 5-epi-eudesm-3,11(13)-dien-1β-hydroxy-6αacetoxyl-7α,8α-epoxy-12-carboxylic acid methyl ester, named jatrophaeudesmene B. Jatrophaeudesmene C (3), colorless powder, exhibited a molecular ion peak at m/z 336.3737 [M]+ in its HREIMS, corresponding to C18H24O6, indicating seven degrees of unsaturation. In the 13C NMR and DEPT spectra of 3, some

Fig. 3. The 1H–1H COSY (

resonances could not be recorded clearly in CDCl3 at normal temperature (+ 22 °C), while they could be recorded clearly in the same solvent at about 0 °C (see Fig. 4). All the hydrogen protons directly bonded with carbon atoms were assigned unequivocally by the results of HSQC spectrum, as described in Table 1. The presence of three methyls (one acetyl methyl, one methoxyl), five methylenes (two olefinics), four methines (three oxygenated), and six quaternary carbons (two olefinics, one oxygenated, two carbonyls) were indicated by analysis of the 1D NMR and HSQC data of 3 (Table 1). Comparison of the 1H and 13C NMR spectroscopic data of 3 with those of 2 found an overall similarity, except for the apparent different chemical shifts of C-3, C-4, and C-15. The difference between these two compounds was due to an occurrence that a trisubstituted double bond between C-3 and C-4 in 2 was converted to a disubstituted double bond between C-4 and C-15 in 3. This assumption was verified by HMBC correlations from the exomethylene protons H2-15 to C-3, C-4, and C-5. Extensive 2D NMR experiments (HSQC, HMBC and 1H–1H COSY) confirmed the planar structure of 3 as shown. The ROESY spectrum showed that 3 possessed the same ring fusion as 2. The 13C NMR signals of 7,8-epoxide group [δC 59.7 (s) and 65.9 (δH 4.35, br. s, 1H)] were superimposable over those of 2. So the relative configuration of the 7,8-epoxide was assigned to be in a α orientation. Therefore, the structure of 3 was identified as 5-epi-eudesm4(15),11(13)-dien-1β-hydroxy-6α-acetoxyl-7α,8α-epoxy12-carboxylic acid methyl ester, and named jatrophaeudesmene C. The sesquiterpenoid lactone, chrysanin (8), was isolated previously from the flowers of Chrysanthemum cinerariaefolium [15]. In our study, the X-ray crystal analysis of 8 (Fig. 5) confirmed its structure and relative configuration as shown in Fig. 1. Other four known compounds, pyrethrosin (4) [16], 8β-acetoxyanhydroverlotorin (5) [17], chamissarin (6) [18], and 6α-tigloyloxy-1β-hydroxy-4(15),11-eudesmadien-8α,12olide (7) [19], were identified by comparison with those reported. Compounds 2− 8 were evaluated for their cytotoxicity against HL-60, SMMC-7721, A-549, MCF-7, and SW480 human tumor cell lines using the MTS method; and cisplatin was used as a positive control. Results are summarized in Table 2. As can be observed, compounds 4 and 5 showed

), key HMBC correlations (H → C) and key ROESY correlations (

) of compound 2.

Y.-F. Yang et al. / Fitoterapia 89 (2013) 278–284

283

without signals at δ

C

58-67 ppm

22 °C

6-OOCCH3

6-OOCCH3 C-10

C-4 12-COOCH3

C-11

C-15

C-1

C-8 C-6

C-13

-OCH3 C- 7 C-4

C-2 C-3 C-14 0 °C

Fig. 4. 13C NMR spectra of compound 3 (600 MHz, CDCl3).

potent cytotoxicity against above cells lines with IC50 values from 0.5 to 3.5 μM. Compound 6 exhibited the equivalent inhibitory activity as the positive control, with IC50 values of 4.85, 11.15, 11.52, 11.84, and 9.09 μM against all the tested cell lines, respectively. Compound 3 displayed selective cytotoxic activity against A-549 cell with an IC50 value of 7.24 μM, but slight cytotoxicity against HL-60 and MCF-7

with IC50 values of 23.77 and 22.37 μM, respectively. Compound 2 expressed negligible cytotoxicity against HL-60 and A-549 cells. Compounds 7 and 8 were not active in this test (IC50 N 40 μM). Pyrethrosin (4), the first recognized germacrane sesquiterpene lactone [16], was previously isolated from the flowers of C. cinerariaefolium (Asteraceae) [15]. Pyrethrosin

Fig. 5. X-ray crystal structure of compound 8.

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Table 2 Cytotoxicity data of compounds 2−8 with IC50 values (μM).a Compound(s)

HL-60

SMMC-7721

A-549

MCF-7

SW480

2 3 4 5 6 7 8 Cisplatin

29.92 23.77 1.95 0.50 4.85 N40 N40 1.32

N40 N40 3.13 2.17 11.15 N40 N40 6.24

36.93 7.24 2.26 1.72 11.52 N40 N40 11.83

N40 22.37 3.28 2.98 11.84 N40 N40 15.17

N40 N40 3.30 1.81 9.09 N40 N40 12.95

a Data were obtained from triplicate experiments, and cisplatin was used as positive control.

exhibited diverse biological activities including phytotoxicity (plant growth inhibitor) [20], antibacterial [21], antifungal [22], molluscicidal [23], antimicrobial [24], and cytotoxicity [25]. Besides, it could be an activity enhancing agent, pyrethrosin itself had no insecticidal activity but displayed a potent activating effect on pyrethrin-type insecticides [26]. To the best of our knowledge, germacrane-type sesquiterpenes (4–6) were the first detected from the genus Jatropha. In addition, previous structural studies have shown that eudesmane-type sesquiterpenes are not common in the Euphorbiaceae family. Until now, only one cis-eudesmane sesquiterpene from Jatropha neopauciflora [27] and two sesquiterpenes from J. curcas have been reported [12]. Thus, J. curcas is not only a source for the production of biodiesel and for medicinal purposes, but also can be a raw material to promote the further development of pyrethrosin and its widespread applications. Conflict of interest There is no conflict of interest. Acknowledgments Thanks are due to Prof. Cheng-yuan Yang for the identification of the plant. This work was supported financially by the Key Projects of the Chinese Ministry of Science and Technology (2007BAD32B01-03 and SB2007FY400), the National Natural Science Foundation of China (81202437) and the National Knowledge Innovation Program of Chinese Academy of Sciences (KSCX2-YW-G-038). The authors thank the staff of the analytical group of the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.fitote.2013.06.011. References [1] Berchmans HJ, Hirata S. Biodiesel production from crude Jatropha curcas L. seed oil with a high content of free fatty acids. Bioresour Technol 2008;99:1716–21. [2] Lu H, Liu Y, Zhou H, Yang Y, Chen M, Liang B. Production of biodiesel from Jatropha curcas L. oil. Comput Chem Eng 2009;33:1091–6.

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