Fitoterapia 101 (2015) 218–223
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Fitoterapia journal homepage: www.elsevier.com/locate/fitote
Cytotoxic sesquiterpene lactone dimers isolated from Inula japonica Xing-Yu Xu, Peng Sun, De-An Guo, Xuan Liu, Jun-Hua Liu ⁎, Li-Hong Hu ⁎⁎ Shanghai Research Center for the Modernization of Traditional Chinese Medicine, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Haike Road, Shanghai 201203, PR China
a r t i c l e
i n f o
Article history: Received 1 December 2014 Accepted in revised form 9 January 2015 Available online 21 January 2015 Keywords: Inula japonica Thunb Sesquiterpene lactone dimer Biomimetic transformation Cytotoxicity
a b s t r a c t Two new sesquiterpene lactone dimers, neojaponicone B (1) and inulanolide E (2) along with five known sesquiterpene lactone dimers (3–7, resp.) were isolated from the aerial parts of Inula japonica Thunb. The chemical structures of 1 and 2 were elucidated by detailed spectroscopic analysis. The relative configuration of 2 was confirmed by biomimetic transformation from the known sesquiterpene lactone dimer inulanolide A (3). The cytotoxicities of the isolated sesquiterpene lactone dimers were evaluated against 6T-CEM and Jurkat cell lines. All compounds showed potent cytotoxicities with IC50 value of 2.2–5.9 μm. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Inula, a genus the Asteraceae family, is composed of about 100 plant species, and many of the plants in this genus have been used as herbal medicines in Asia, Africa and Europe [1,2]. About 400 sesquiterpenoids have been isolated from these species and exhibit various biological activities, especially in anti-inflammatory and anti-tumor activities [1,2]. Among these diverse sesquiterpenoids, sesquiterpene lactone dimers have been drawn attention because of their unique structures and potent anti-tumor activities [3–10]. Inula japonica Thunb., one of the plants from genus Inula, is known in China as “jinfocao”, and its aerial parts have been reported to possess diverse biological activities, such as antiinflammatory, antidiabetic, and anti-tumor activities [4,5,10–14]. Several interesting bioactive sesquiterpene lactone dimers have been isolated from I. japonica recently [4–6,9,10]. Aimed at searching for novel dimeric bioactive sesquiterpene lactones, we further researched the aerial parts of I. japonica and resulted in isolation of two new (1–2, resp.) and five known sesquiterpene ⁎ Corresponding author. Tel./fax: +86 21 20231000 2317. ⁎⁎ Corresponding author. Tel./fax: +86 21 20231965. E-mail addresses: [email protected] (J.-H. Liu), [email protected] (L.-H. Hu).
http://dx.doi.org/10.1016/j.fitote.2015.01.011 0367-326X/© 2015 Elsevier B.V. All rights reserved.
lactone dimers (3–7, resp.). All compounds showed potent cytotoxicities against 6T-CEM and Jurkat cell lines. 2. Experimental 2.1. General Optical rotations were measured with a Shenke SGW-1 automatic polarimeter. IR spectra were measured in a PerkinElmer 341 spectrometer with KBr pellets. NMR spectra were obtained at 400 and 500 MHz in CDCl3 using Varian Inova spectrometers. ESIMS and HRESIMS were carried out on an Esquire 3000 plus (Bruker Daltonics) LC–MS instrument, Esquire 6000 LC–MS instrument and a LCT Premier XE (Waters) mass spectrometer, respectively. Silica gel (200–300 mesh, Kunhai Chemical Ltd., China), MCI Gel CHP20P (75–150 μm, Mitsubishi Kasei Chemical Industries), Sephadex LH-20 (20–100 μm, Pharmacia), and C18 reversed-phase silica gel (20–45 μm, Fuji Silysia Chemical Ltd., Japan) were used for column chromatography, and pre-coated silica gel GF-254 plates (Qingdao Marine Chemical Plant) were used for TLC. Analytical HPLC was performed on Waters 2690 unit and ELSD unit equipped with Alltech 3300 evaporative light scattering detector, using ODS (AQ-C18, 4.6 × 150 mm, 5 μm, Welch Materials Inc.).
X.-Y. Xu et al. / Fitoterapia 101 (2015) 218–223
Preparative HPLC was performed on an apparatus equipped with a Varian SD-1 solvent delivery module, an NU 3000 Serials UV/VIS detector (Hanbon Sci. & Tech.). A prepacked C18 reversed-phase column (Inertsil ODS, 10 × 250 mm, 3 μm, GL Sciences Inc.) was used for semi-preparative HPLC with a binary isocratic elution (solvent A: H2O; solvent B: CH3OH; solvent C: CH3CN) and a flow rate of 10 mL/min.
Table 1 13 C NMR (100 MHz) and 1H NMR (400 MHz) data of compounds 1 and 2 in CDCl3 (δ in ppm). Position
The aerial parts of air-dried I. japonica were collected from Anhui Province, People's Republic of China, and authenticated by Prof. Li-Jiang Xuan, Shanghai Institute of Materia Medica, University of Chinese Academy of Sciences. A voucher specimen (Voucher number: XX20131001) was deposited at Shanghai Institute of Materia Medica, Academy of Sciences, Shanghai, People's Republic of China. 2.3. Extraction and isolation
2.3.1. Neojaponicone B (1) Yellow amorphous powder. [a]25 D = −14.4 (c = 0.11, MeOH). IR (KBr): 2962, 2873, 1770, 1738, 1628, 1454, 1373, 1236, 1153 and 1028 cm−1. 1H and 13C NMR: see Table 1. HREI-MS: 601.2774 ([M + Na]+, C34H42O8Na+, calc. 601.2772). 2.3.2. Inulanolide E (2) Yellow amorphous powder. [a]25 D = +109.7 (c = 0.10, MeOH). IR (KBr): 2962, 1772, 1735, 1620, 1454, 1370, 1238, 1162 and 1030 cm−1. 1H and 13C NMR: see Table 1. HR-EI-MS: 601.2781 ([M + Na]+, C34H42O8Na+, calc. 601.2772).
1 δC
Part A
2.2. Plant material
The aerial parts of air-dried I. japonica (25 kg) were extracted with 50% EtOH three times each for 2 d at room temperature. After removal of the EtOH under reduced pressure, the aqueous suspension was passed through AB-8 macroporous adsorption resin and sequentially eluted with H2O, 50% EtOH, and 90% EtOH, respectively. 90% EtOH elution was concentrated under vacuum to yield a syrup (120 g), and this crude residue was separated by silica gel column (PE/ Acetone, 5:1–1:2) to afford four fractions (Fr.A–Fr.D). Fr.B (12 g) was subjected to MCI Gel CHP20P (CH3CN/H2O, 1:1–19:1) to afford Fr.B-1–Fr.B-5, respectively. Subfractions Fr.B-1–Fr.B-4 were further purified by preparative HPLC (CH3CN/H2O). Subfraction Fr.B-1 (360 mg) gave 1 (18 mg, CH3CN/H2O, 7:3), Fr.B-2 (560 mg) gave 2 (69 mg) and 5 (109 mg, CH3CN/H2O, 3:2–9:1), Fr.B-3 (580 mg) gave 4 (90 mg, CH3CN/ H2O, 1:1–3:2), and Fr.B-4 (330 mg) gave 6 (25 mg, CH3CN/H2O, 7:3). Subfraction Fr.B-5 was chromatographed on silica gel column (PE/Acetone, 4:1) to afford 3 (1.2 g). Fr.C (20 g) was further purified by MCI Gel CHP20P (CH3CN/H2O, 1:1–19:1) and RP-18 column (CH3CN/H2O, 2:1) to yield 7 (18 mg).
219
Part B
1a 1b 2a 2b 3a 3b 4 5 6 7 8 9 10 11 12 13a 13b 14 15 1′ 2′ 3′ 4′ 5′ 6′a 6′b 7′ 8′ 9′a 9′b 10′ 11′ 12′ 13′ a 13′ b 14′ 15′ 1″ 2″ 1‴ 2‴
2 δH (J in Hz)
δC
δH (J in Hz)
64.6 4.01, m 4.01, m 27.4 1.63, m 1.63, m 34.0 1.57, m 1.63, m 34.3 2.93, m 145.8 125.7 6.98, d (1.5) 136.2 125.3 6.91, dd (7.9, 1.5) 130.8 7.12, d (7.9) 134.9 46.9 3.43, m 172.3 28.7 1.79, m 1.95, m 19.4 2.29, s 21.4 1.21, d (6.7) 141.4 77.5 5.73, dd (7.4, 3.4) 46.7 2.93, m 92.0 139.7 23.5 2.03, m 3.14, m 47.1 2.71, m 81.1 4.25, ddd (12.0, 9.7, 2.4) 37.6 1.95, m 2.35, m 27.0 2.70, m 139.2 169.9 120.2 6.25, d (3.3)
64.6 3.98, m 3.98, m 26.9 1.60, m 1.48, m 33.5 1.60, m 1.60, m 34.4 2.94, m 145.3 124.4 7.21, d (2.1) 139.6 124.3 7.11, dd (8.0, 2.1) 130.0 7.04, d (8.0) 133.7 60.5 177.8 42.6 2.85, m 2.34, m 19.0 2.25, s 21.5 1.20, d (5.6) 61.1 82.3 4.29, s 55.0 3.74, s 135.3 138.8 26.0 2.08, m 2.94, m 45.7 2.69, m 82.7 4.19, ddd (12.8, 9.8, 3.1) 35.9 2.08, m 2.34, m 29.7 2.15, m 139.6 170.1 119.1 6.23, d (3.3)
5.64, d (3.3)
5.47, d (3.3)
18.8 28.6 171.3 21.1 170.9 20.9
1.12, d (7.3) 1.53, s 2.02, s 2.08, s
16.6 14.2 170.6 20.1 171.4 20.9
0.96, d (7.1) 1.81, s 1.19, s 1.99, s
cells were plated in 96-well plates at a density of 3000 cells/ well in 90 μL of medium per well overnight. Each cell line was exposed to compounds 1–7 at various concentrations in triplicate for 72 h, with paclitaxel as positive controls. After the incubation, 20 μL CellTiter 96® Aqueous One Solution Reagent (Promega) was added to each well and then incubated at 37 °C for 3 h. Then plates were detected by 492 nm on a spectrophotometric plate reader (Tecan GENios).
2.4. Cytotoxicity assay
2.5. Biomimetic transformation of 2 from 3
The two human tumor cell lines: 6T-CEM and Jurkat were cultured in RPMI-1640 (Hyclone), supplemented with 10% fetal bovine serum (Hyclone), penicillin (100 units/mL) and streptomycin (100 μg/mL) at 37 °C in a humidified atmosphere with 5% CO2. Cell cytotoxicity was determined using the CellTiter 96® Aqueous One Solution Cell Proliferation Assay kit. Briefly,
To a solution of 3 (20 mg, 0.034 mmol) in THF (0.5 mL), BF3 · EtO2 (21 μL, 0.17 mmol) was added. After being stirred at room temperature for 6 h, CH2Cl2 (10 mL) and saturated NH4Cl (10 mL) were added, the layers were separated and the organic portions were washed with brine, dried over Na2SO4, filtered, and concentrated. The crude product was chromatographed on
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X.-Y. Xu et al. / Fitoterapia 101 (2015) 218–223
Fig. 1. Chemical structures of compounds 1–7.
silica gel column (PE/Acetone, 6:1) to afford 2 as white amorphous powder (12 mg, 62%). 3. Result and discussion The dried aerial parts of I. japonica were extracted with 50% ethanol. The combined extract was subjected to chromatographic separation on AB-8 macroporous adsorption resin with H2O, 50% EtOH, and 90% EtOH. Repeated chromatography of the 90% EtOH fraction resulted in the isolation of dimeric sesquiterpene lactones (1–7, resp., Fig. 1) including two novel compounds (1– 2). Five known compounds were identified as inulanolide A (3)
[3], japonicone Q (4) [10], japonicone N (5) [6], japonicone S (6) [10] and japonicone A (7) [4] by detailed NMR and MS analyses. Compound 1 (18 mg, 0.000072%, Fig. 1) was obtained as yellow amorphous powder. The molecular formula, C34H42O8, with thirteen double-bond equivalents (DBEs), was established by high-resolution positive-ion-mode electrospray ionization mass spectrometry [HR-ESI(+)-MS] at m/z 601.2774 [M + Na]+ (calcd for C34H42O8Na+, 601.2772). The IR spectrum showed characteristic absorption bands for carbonyl (1770 and 1738 cm−1) and olefinic bond (1628 cm−1) moieties. In accordance with the molecular formula, the 13C and DEPT NMR spectra showed the presence of eleven quaternary C-atoms, ten
Fig. 2. Key HBMC and ROESY correlations of 1.
X.-Y. Xu et al. / Fitoterapia 101 (2015) 218–223
221
Scheme 1. Plausible biosynthetic pathway for compound 1.
CH, seven CH2, and six CH3 groups. In the 1H NMR spectrum of 1, the characteristic aromatic proton signals of an ABX system at δH 7.12 (1H, d, J = 7.9 Hz, H-9), 6.91 (1H, dd, J = 1.5, 7.9 Hz, H-8), and 6.98 (1H, d, J = 1.5 Hz, H-6), and two acetoxyl groups at δH 2.02 (3H, s, H-2″) and 2.08 (3H, s, H-2‴) were observed. The 13C NMR signals indicated the presence of one exocyclic double bond (δC 120.2), four ester carbonyls (δC 169.9, 170.9, 171.3 and 172.3), one oxygen-bearing methylene (δC 64.6), two oxygen-bearing methines (δC 77.5 and 81.1), and one oxygen-bearing carbon (δC 92.0). Careful analysis of the NMR spectra of 1, including 2D NMR, allowed the 1H and 13C NMR signals (Table 1) to be assigned
to two different sesquiterpene units designated as moieties A and B (Fig. 2). Compared with the spectral data of known sesquiterpenes isolated from this plant [4–6,9,10], the 1H and 13C NMR of part B were suggestive of the presence of a guaianolide unit. Detailed comparison with known compound neojaponicone A [6] disclosed that the main difference was the disappearance of one oxygen-bearing methylene and the presence of an additional CH3 group. The location of the additional CH3 group was assigned by HMBC correlations (Fig. 2) and ROESY correlations (Fig. 2). Thus, the structure of 1 was identified as in Fig. 1, and named neojaponicone B.
Fig. 3. Key HBMC and ROESY correlations of 2.
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X.-Y. Xu et al. / Fitoterapia 101 (2015) 218–223
Table 2 Conditions screen for the biomimetic transformation of compound 2 from 3.
Entry
Conditions
t (°C)
Time (h)
Yield (%)
1 2 3
HOAc HCl (2 N) BF3 · OEt2
r.t. r.t. r.t.
6 6 6
Trace a 20 a 62 b
a b
Detected by LC–MS. Isolated yields.
Table 3 The IC50 values of compounds 1–7 against Jurkat and 6T-CEM cell lines. Compd.
IC50 (μm)a Jurkat
1 2 3 4 5 6 7 Paclitaxel a
5.9 5.5 5.8 3.3 2.5 4.5 3.1 0.15
± ± ± ± ± ± ± ±
6T-CEM 0.1 0.1 0.2 0.1 0.1 0.2 0.2 0.02
4.4 4.6 4.3 2.7 2.4 3.3 2.2 0.043
± ± ± ± ± ± ± ±
0.2 0.1 0.2 0.1 0.1 0.1 0.1 0.003
These data represent mean values ± SD (n = 3).
As suggested by Zhang et al. [6], neojaponicone B (1) is presumably biosynthesized from a 1,10-secoeudesmane sesquiterpene (1-A) and a guaianolide sesquiterpene lactone (1B) as proposed in Scheme 1. Firstly, the lactone ring opening
and dehydration of 1,10-secoeudesmane sesquiterpene (1-A) give the intermediate 1-C. Subsequently, 1 is formed by the endo [4 + 2] cycloaddition of intermediate 1-C and guaianolide sesquiterpene lactone (1-B). Compound 2 (69 mg, 0.00028%, Fig. 1) was obtained as yellow amorphous powder. The molecular formula, C34H42O8, with thirteen double-bond equivalents (DBEs), was established by high-resolution positive-ion-mode electrospray ionization mass spectrometry [HR-ESI(+)-MS] m/z 601.2781 [M + Na]+ (calcd for C34H42O8Na+, 601.2772). The IR spectrum showed characteristic absorption bands for carbonyl (1772 and 1735 cm−1) and olefinic bond (1620 cm−1) moieties. In accordance with the molecular formula, thirty carbon resonances were resolved in the 13C NMR spectrum and were classified by distortionless enhancement by polarization transfer (DEPT) NMR experiments with the aid of the HSQC spectrum as six methyls, seven methylenes, ten methines, and eleven quaternary carbons, of which the signals of four ester carbonyls, ten olefinic carbons, one oxygen-bearing methylene, and two oxygen-bearing methines were typical. These data suggested that 2 was a dimeric sesquiterpene lactone. Extensive analyses of 1D and 2D NMR spectra of 2 indicated that it was similar to inulanolide D, obtained from the aerial parts of Inula britannica var. chinensis (Rupr.) Regel [3]. The most striking difference between compound 2 and inulanolide D was the presence of an additional acetyl group substituted on OH group at δC 20.9 and 171.4, and δH 1.99 (3H, s, H-2‴), which could be confirmed from 1D NMR, HSQC and HMBC experiments (Fig. 3). These data suggested that 2 was the 1-O-acetyl inulanolide D. In order to establish the relative stereochemistry of compound 2, we carefully analyzed the structures of the compound 2 and known dimeric sesquiterpene lactones isolated from this plant, and found that 2 might be derived from the known dimeric sesquiterpene lactone 3 by dehydration, lactone ring opening, and second dehydration as illustrated in Scheme 2. Based on this hypothesis, we investigated the
Scheme 2. Plausible biosynthetic pathway for compound 2.
X.-Y. Xu et al. / Fitoterapia 101 (2015) 218–223
potentially biomimetic transformation of 2 from 3. At the outset, we attempted to conduct dehydration and lactone ring opening by HOAc in THF at room temperature for 6 h (Table 2, entry 1), and trace amounts of 2 could be detected by LC–MS. Encouraged by the result, we further investigated the best reaction conditions by using 2N HCl (Table 2, entry 2) or BF3 · OEt2 (Table 2, entry 3). Gratefully, the desired 2 could be obtained by BF3 · OEt2 in 62% yield (Table 2, entry 3). Therefore, the structure of 2 was identified as in Fig. 1, and named inulanolide E. Compounds 1–7 were evaluated for their in vitro growth inhibitory effects against two human leukemia cells (6T-CEM and Jurkat), with paclitaxel as positive control. All compounds exhibited significant cytotoxicities (IC50 b 6 μm) against both cell lines (Table 3). 4. Conclusion In this paper, two new sesquiterpene lactone dimers, along with five known sesquiterpene lactone dimers were isolated from the aerial parts of I. japonica Thunb.. The relative configuration of 2 was confirmed by biomimetic transformation from inulanolide A (3). All seven compounds (1–7, resp.) showed potent cytotoxicities with IC50 value of 2.2–5.9 μm against 6T-CEM and Jurkat cell lines, suggesting that these sesquiterpene lactone dimers might be potential anticancer chemotherapy agents. Acknowledgements The work was supported by the National Natural Science Foundation of China (81273397 and 81402811), the Chinese National Science & Technology Major Project “Key New Drug Creation and Manufacturing Program” (2013ZX09508104), and the China Postdoctoral Science Foundation (2014M550255). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/http://dx.doi.org/10.1016/j.fitote.2012.09. 026.
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References [1] Zhao YM, Zhang ML, Shi QW, Kiyota H. Chemical constituents of plants from the genus Inula. Chem Biodivers 2006;3:371–84. [2] Wang GW, Qin JJ, Cheng XR, Shen YH, Shan L, Jin HZ, et al. Inula sesquiterpenoids: structural diversity, cytotoxicity and anti-tumor activity. Expert Opin Investig Drugs 2014;23:317–45. [3] Jin HZ, Lee DH, Lee JH, Lee K, Hong YS, Choung DH, et al. New sesquiterpene dimers from Inula britannica inhibit NF-κB activation and NO and TNF-α production in LPS stimulated RAW264.7 cells. Planta Med 2006;72:40–5. [4] Qin JJ, Jin HZ, Fu JJ, Hu XJ, Wang Y, Yan SK, et al. Japonicones A–D, bioactive dimeric sesquiterpenes from Inula japonica Thunb. Bioorg Med Chem Lett 2009;19:710–3. [5] Qin JJ, Jin HZ, Zhu JX, Fu JJ, Hu XJ, Liu XH, et al. Japonicones E–L, dimeric sesquiterpene lactones from Inula japonica Thunb. Planta Med 2010;76: 278–83. [6] Qin JJ, Wang LY, Zhu JX, Jin HZ, Fu JJ, Liu XF, et al. Neojaponicone A, a bioactive sesquiterpene lactone dimer with an unprecedented carbon skeleton from Inula japonica. Chem Commun (Camb) 2011;47:1222–4. [7] Qin JJ, Huang Y, Wang D, Cheng XR, Zeng Q, Zhang SD, et al. Lineariifolianoids A–D, rare unsymmetrical sesquiterpenoid dimers comprised of xanthane and guaiane framework units from Inula lineariifolia. RSC Adv 2012;2:1307. [8] Qin JJ, Jin HZ, Huang Y, Zhang SD, Shan L, Voruganti S, et al. Selective cytotoxicity, inhibition of cell cycle progression, and induction of apoptosis in human breast cancer cells by sesquiterpenoids from Inula lineariifolia Turcz. Eur J Med Chem 2013;68:473–81. [9] Ye J, Qin JJ, Su J, Lin S, Huang Y, Jin HZ, et al. Identification and structural characterization of dimeric sesquiterpene lactones in Inula japonica Thunb. by high-performance liquid chromatography/electrospray ionization with multi-stage mass spectrometry. Rapid Commun Mass Spectrom 2013;27:2159–69. [10] Zhu JX, Qin JJ, Jin HZ, Zhang WD. Japonicones Q–T, four new dimeric sesquiterpene lactones from Inula japonica Thunb. Fitoterapia 2013;84: 40–6. [11] Yang C, Wang CM, Jia ZJ. Sesquiterpenes and other constituents from the aerial parts of Inula japonica. Planta Med 2003;69:662–6. [12] Shan JJ, Yang M, Ren JW. Anti-diabetic and hypolipidemic effects of aqueous-extract from the flower of Inula japonica in alloxan-induced diabetic mice. Biol Pharm Bull 2006;29:455–9. [13] Qin JJ, Jin HZ, Zhu JX, Fu JJ, Zeng Q, Cheng XR, et al. New sesquiterpenes from Inula japonica Thunb. with their inhibitory activities against LPSinduced NO production in RAW264.7 macrophages. Tetrahedron 2010;66: 9379–88. [14] Qin JJ, Zhu JX, Zhang WD, Zhu Y, Fu JJ, Liu XH, et al. A new ent-kaurane type diterpenoid glycoside from Inula japonica Thunb. Arch Pharm Res 2009; 32:1369–72.
Contents lists available at ScienceDirect
Fitoterapia journal homepage: www.elsevier.com/locate/fitote
Cytotoxic sesquiterpene lactone dimers isolated from Inula japonica Xing-Yu Xu, Peng Sun, De-An Guo, Xuan Liu, Jun-Hua Liu ⁎, Li-Hong Hu ⁎⁎ Shanghai Research Center for the Modernization of Traditional Chinese Medicine, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Haike Road, Shanghai 201203, PR China
a r t i c l e
i n f o
Article history: Received 1 December 2014 Accepted in revised form 9 January 2015 Available online 21 January 2015 Keywords: Inula japonica Thunb Sesquiterpene lactone dimer Biomimetic transformation Cytotoxicity
a b s t r a c t Two new sesquiterpene lactone dimers, neojaponicone B (1) and inulanolide E (2) along with five known sesquiterpene lactone dimers (3–7, resp.) were isolated from the aerial parts of Inula japonica Thunb. The chemical structures of 1 and 2 were elucidated by detailed spectroscopic analysis. The relative configuration of 2 was confirmed by biomimetic transformation from the known sesquiterpene lactone dimer inulanolide A (3). The cytotoxicities of the isolated sesquiterpene lactone dimers were evaluated against 6T-CEM and Jurkat cell lines. All compounds showed potent cytotoxicities with IC50 value of 2.2–5.9 μm. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Inula, a genus the Asteraceae family, is composed of about 100 plant species, and many of the plants in this genus have been used as herbal medicines in Asia, Africa and Europe [1,2]. About 400 sesquiterpenoids have been isolated from these species and exhibit various biological activities, especially in anti-inflammatory and anti-tumor activities [1,2]. Among these diverse sesquiterpenoids, sesquiterpene lactone dimers have been drawn attention because of their unique structures and potent anti-tumor activities [3–10]. Inula japonica Thunb., one of the plants from genus Inula, is known in China as “jinfocao”, and its aerial parts have been reported to possess diverse biological activities, such as antiinflammatory, antidiabetic, and anti-tumor activities [4,5,10–14]. Several interesting bioactive sesquiterpene lactone dimers have been isolated from I. japonica recently [4–6,9,10]. Aimed at searching for novel dimeric bioactive sesquiterpene lactones, we further researched the aerial parts of I. japonica and resulted in isolation of two new (1–2, resp.) and five known sesquiterpene ⁎ Corresponding author. Tel./fax: +86 21 20231000 2317. ⁎⁎ Corresponding author. Tel./fax: +86 21 20231965. E-mail addresses: [email protected] (J.-H. Liu), [email protected] (L.-H. Hu).
http://dx.doi.org/10.1016/j.fitote.2015.01.011 0367-326X/© 2015 Elsevier B.V. All rights reserved.
lactone dimers (3–7, resp.). All compounds showed potent cytotoxicities against 6T-CEM and Jurkat cell lines. 2. Experimental 2.1. General Optical rotations were measured with a Shenke SGW-1 automatic polarimeter. IR spectra were measured in a PerkinElmer 341 spectrometer with KBr pellets. NMR spectra were obtained at 400 and 500 MHz in CDCl3 using Varian Inova spectrometers. ESIMS and HRESIMS were carried out on an Esquire 3000 plus (Bruker Daltonics) LC–MS instrument, Esquire 6000 LC–MS instrument and a LCT Premier XE (Waters) mass spectrometer, respectively. Silica gel (200–300 mesh, Kunhai Chemical Ltd., China), MCI Gel CHP20P (75–150 μm, Mitsubishi Kasei Chemical Industries), Sephadex LH-20 (20–100 μm, Pharmacia), and C18 reversed-phase silica gel (20–45 μm, Fuji Silysia Chemical Ltd., Japan) were used for column chromatography, and pre-coated silica gel GF-254 plates (Qingdao Marine Chemical Plant) were used for TLC. Analytical HPLC was performed on Waters 2690 unit and ELSD unit equipped with Alltech 3300 evaporative light scattering detector, using ODS (AQ-C18, 4.6 × 150 mm, 5 μm, Welch Materials Inc.).
X.-Y. Xu et al. / Fitoterapia 101 (2015) 218–223
Preparative HPLC was performed on an apparatus equipped with a Varian SD-1 solvent delivery module, an NU 3000 Serials UV/VIS detector (Hanbon Sci. & Tech.). A prepacked C18 reversed-phase column (Inertsil ODS, 10 × 250 mm, 3 μm, GL Sciences Inc.) was used for semi-preparative HPLC with a binary isocratic elution (solvent A: H2O; solvent B: CH3OH; solvent C: CH3CN) and a flow rate of 10 mL/min.
Table 1 13 C NMR (100 MHz) and 1H NMR (400 MHz) data of compounds 1 and 2 in CDCl3 (δ in ppm). Position
The aerial parts of air-dried I. japonica were collected from Anhui Province, People's Republic of China, and authenticated by Prof. Li-Jiang Xuan, Shanghai Institute of Materia Medica, University of Chinese Academy of Sciences. A voucher specimen (Voucher number: XX20131001) was deposited at Shanghai Institute of Materia Medica, Academy of Sciences, Shanghai, People's Republic of China. 2.3. Extraction and isolation
2.3.1. Neojaponicone B (1) Yellow amorphous powder. [a]25 D = −14.4 (c = 0.11, MeOH). IR (KBr): 2962, 2873, 1770, 1738, 1628, 1454, 1373, 1236, 1153 and 1028 cm−1. 1H and 13C NMR: see Table 1. HREI-MS: 601.2774 ([M + Na]+, C34H42O8Na+, calc. 601.2772). 2.3.2. Inulanolide E (2) Yellow amorphous powder. [a]25 D = +109.7 (c = 0.10, MeOH). IR (KBr): 2962, 1772, 1735, 1620, 1454, 1370, 1238, 1162 and 1030 cm−1. 1H and 13C NMR: see Table 1. HR-EI-MS: 601.2781 ([M + Na]+, C34H42O8Na+, calc. 601.2772).
1 δC
Part A
2.2. Plant material
The aerial parts of air-dried I. japonica (25 kg) were extracted with 50% EtOH three times each for 2 d at room temperature. After removal of the EtOH under reduced pressure, the aqueous suspension was passed through AB-8 macroporous adsorption resin and sequentially eluted with H2O, 50% EtOH, and 90% EtOH, respectively. 90% EtOH elution was concentrated under vacuum to yield a syrup (120 g), and this crude residue was separated by silica gel column (PE/ Acetone, 5:1–1:2) to afford four fractions (Fr.A–Fr.D). Fr.B (12 g) was subjected to MCI Gel CHP20P (CH3CN/H2O, 1:1–19:1) to afford Fr.B-1–Fr.B-5, respectively. Subfractions Fr.B-1–Fr.B-4 were further purified by preparative HPLC (CH3CN/H2O). Subfraction Fr.B-1 (360 mg) gave 1 (18 mg, CH3CN/H2O, 7:3), Fr.B-2 (560 mg) gave 2 (69 mg) and 5 (109 mg, CH3CN/H2O, 3:2–9:1), Fr.B-3 (580 mg) gave 4 (90 mg, CH3CN/ H2O, 1:1–3:2), and Fr.B-4 (330 mg) gave 6 (25 mg, CH3CN/H2O, 7:3). Subfraction Fr.B-5 was chromatographed on silica gel column (PE/Acetone, 4:1) to afford 3 (1.2 g). Fr.C (20 g) was further purified by MCI Gel CHP20P (CH3CN/H2O, 1:1–19:1) and RP-18 column (CH3CN/H2O, 2:1) to yield 7 (18 mg).
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Part B
1a 1b 2a 2b 3a 3b 4 5 6 7 8 9 10 11 12 13a 13b 14 15 1′ 2′ 3′ 4′ 5′ 6′a 6′b 7′ 8′ 9′a 9′b 10′ 11′ 12′ 13′ a 13′ b 14′ 15′ 1″ 2″ 1‴ 2‴
2 δH (J in Hz)
δC
δH (J in Hz)
64.6 4.01, m 4.01, m 27.4 1.63, m 1.63, m 34.0 1.57, m 1.63, m 34.3 2.93, m 145.8 125.7 6.98, d (1.5) 136.2 125.3 6.91, dd (7.9, 1.5) 130.8 7.12, d (7.9) 134.9 46.9 3.43, m 172.3 28.7 1.79, m 1.95, m 19.4 2.29, s 21.4 1.21, d (6.7) 141.4 77.5 5.73, dd (7.4, 3.4) 46.7 2.93, m 92.0 139.7 23.5 2.03, m 3.14, m 47.1 2.71, m 81.1 4.25, ddd (12.0, 9.7, 2.4) 37.6 1.95, m 2.35, m 27.0 2.70, m 139.2 169.9 120.2 6.25, d (3.3)
64.6 3.98, m 3.98, m 26.9 1.60, m 1.48, m 33.5 1.60, m 1.60, m 34.4 2.94, m 145.3 124.4 7.21, d (2.1) 139.6 124.3 7.11, dd (8.0, 2.1) 130.0 7.04, d (8.0) 133.7 60.5 177.8 42.6 2.85, m 2.34, m 19.0 2.25, s 21.5 1.20, d (5.6) 61.1 82.3 4.29, s 55.0 3.74, s 135.3 138.8 26.0 2.08, m 2.94, m 45.7 2.69, m 82.7 4.19, ddd (12.8, 9.8, 3.1) 35.9 2.08, m 2.34, m 29.7 2.15, m 139.6 170.1 119.1 6.23, d (3.3)
5.64, d (3.3)
5.47, d (3.3)
18.8 28.6 171.3 21.1 170.9 20.9
1.12, d (7.3) 1.53, s 2.02, s 2.08, s
16.6 14.2 170.6 20.1 171.4 20.9
0.96, d (7.1) 1.81, s 1.19, s 1.99, s
cells were plated in 96-well plates at a density of 3000 cells/ well in 90 μL of medium per well overnight. Each cell line was exposed to compounds 1–7 at various concentrations in triplicate for 72 h, with paclitaxel as positive controls. After the incubation, 20 μL CellTiter 96® Aqueous One Solution Reagent (Promega) was added to each well and then incubated at 37 °C for 3 h. Then plates were detected by 492 nm on a spectrophotometric plate reader (Tecan GENios).
2.4. Cytotoxicity assay
2.5. Biomimetic transformation of 2 from 3
The two human tumor cell lines: 6T-CEM and Jurkat were cultured in RPMI-1640 (Hyclone), supplemented with 10% fetal bovine serum (Hyclone), penicillin (100 units/mL) and streptomycin (100 μg/mL) at 37 °C in a humidified atmosphere with 5% CO2. Cell cytotoxicity was determined using the CellTiter 96® Aqueous One Solution Cell Proliferation Assay kit. Briefly,
To a solution of 3 (20 mg, 0.034 mmol) in THF (0.5 mL), BF3 · EtO2 (21 μL, 0.17 mmol) was added. After being stirred at room temperature for 6 h, CH2Cl2 (10 mL) and saturated NH4Cl (10 mL) were added, the layers were separated and the organic portions were washed with brine, dried over Na2SO4, filtered, and concentrated. The crude product was chromatographed on
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X.-Y. Xu et al. / Fitoterapia 101 (2015) 218–223
Fig. 1. Chemical structures of compounds 1–7.
silica gel column (PE/Acetone, 6:1) to afford 2 as white amorphous powder (12 mg, 62%). 3. Result and discussion The dried aerial parts of I. japonica were extracted with 50% ethanol. The combined extract was subjected to chromatographic separation on AB-8 macroporous adsorption resin with H2O, 50% EtOH, and 90% EtOH. Repeated chromatography of the 90% EtOH fraction resulted in the isolation of dimeric sesquiterpene lactones (1–7, resp., Fig. 1) including two novel compounds (1– 2). Five known compounds were identified as inulanolide A (3)
[3], japonicone Q (4) [10], japonicone N (5) [6], japonicone S (6) [10] and japonicone A (7) [4] by detailed NMR and MS analyses. Compound 1 (18 mg, 0.000072%, Fig. 1) was obtained as yellow amorphous powder. The molecular formula, C34H42O8, with thirteen double-bond equivalents (DBEs), was established by high-resolution positive-ion-mode electrospray ionization mass spectrometry [HR-ESI(+)-MS] at m/z 601.2774 [M + Na]+ (calcd for C34H42O8Na+, 601.2772). The IR spectrum showed characteristic absorption bands for carbonyl (1770 and 1738 cm−1) and olefinic bond (1628 cm−1) moieties. In accordance with the molecular formula, the 13C and DEPT NMR spectra showed the presence of eleven quaternary C-atoms, ten
Fig. 2. Key HBMC and ROESY correlations of 1.
X.-Y. Xu et al. / Fitoterapia 101 (2015) 218–223
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Scheme 1. Plausible biosynthetic pathway for compound 1.
CH, seven CH2, and six CH3 groups. In the 1H NMR spectrum of 1, the characteristic aromatic proton signals of an ABX system at δH 7.12 (1H, d, J = 7.9 Hz, H-9), 6.91 (1H, dd, J = 1.5, 7.9 Hz, H-8), and 6.98 (1H, d, J = 1.5 Hz, H-6), and two acetoxyl groups at δH 2.02 (3H, s, H-2″) and 2.08 (3H, s, H-2‴) were observed. The 13C NMR signals indicated the presence of one exocyclic double bond (δC 120.2), four ester carbonyls (δC 169.9, 170.9, 171.3 and 172.3), one oxygen-bearing methylene (δC 64.6), two oxygen-bearing methines (δC 77.5 and 81.1), and one oxygen-bearing carbon (δC 92.0). Careful analysis of the NMR spectra of 1, including 2D NMR, allowed the 1H and 13C NMR signals (Table 1) to be assigned
to two different sesquiterpene units designated as moieties A and B (Fig. 2). Compared with the spectral data of known sesquiterpenes isolated from this plant [4–6,9,10], the 1H and 13C NMR of part B were suggestive of the presence of a guaianolide unit. Detailed comparison with known compound neojaponicone A [6] disclosed that the main difference was the disappearance of one oxygen-bearing methylene and the presence of an additional CH3 group. The location of the additional CH3 group was assigned by HMBC correlations (Fig. 2) and ROESY correlations (Fig. 2). Thus, the structure of 1 was identified as in Fig. 1, and named neojaponicone B.
Fig. 3. Key HBMC and ROESY correlations of 2.
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X.-Y. Xu et al. / Fitoterapia 101 (2015) 218–223
Table 2 Conditions screen for the biomimetic transformation of compound 2 from 3.
Entry
Conditions
t (°C)
Time (h)
Yield (%)
1 2 3
HOAc HCl (2 N) BF3 · OEt2
r.t. r.t. r.t.
6 6 6
Trace a 20 a 62 b
a b
Detected by LC–MS. Isolated yields.
Table 3 The IC50 values of compounds 1–7 against Jurkat and 6T-CEM cell lines. Compd.
IC50 (μm)a Jurkat
1 2 3 4 5 6 7 Paclitaxel a
5.9 5.5 5.8 3.3 2.5 4.5 3.1 0.15
± ± ± ± ± ± ± ±
6T-CEM 0.1 0.1 0.2 0.1 0.1 0.2 0.2 0.02
4.4 4.6 4.3 2.7 2.4 3.3 2.2 0.043
± ± ± ± ± ± ± ±
0.2 0.1 0.2 0.1 0.1 0.1 0.1 0.003
These data represent mean values ± SD (n = 3).
As suggested by Zhang et al. [6], neojaponicone B (1) is presumably biosynthesized from a 1,10-secoeudesmane sesquiterpene (1-A) and a guaianolide sesquiterpene lactone (1B) as proposed in Scheme 1. Firstly, the lactone ring opening
and dehydration of 1,10-secoeudesmane sesquiterpene (1-A) give the intermediate 1-C. Subsequently, 1 is formed by the endo [4 + 2] cycloaddition of intermediate 1-C and guaianolide sesquiterpene lactone (1-B). Compound 2 (69 mg, 0.00028%, Fig. 1) was obtained as yellow amorphous powder. The molecular formula, C34H42O8, with thirteen double-bond equivalents (DBEs), was established by high-resolution positive-ion-mode electrospray ionization mass spectrometry [HR-ESI(+)-MS] m/z 601.2781 [M + Na]+ (calcd for C34H42O8Na+, 601.2772). The IR spectrum showed characteristic absorption bands for carbonyl (1772 and 1735 cm−1) and olefinic bond (1620 cm−1) moieties. In accordance with the molecular formula, thirty carbon resonances were resolved in the 13C NMR spectrum and were classified by distortionless enhancement by polarization transfer (DEPT) NMR experiments with the aid of the HSQC spectrum as six methyls, seven methylenes, ten methines, and eleven quaternary carbons, of which the signals of four ester carbonyls, ten olefinic carbons, one oxygen-bearing methylene, and two oxygen-bearing methines were typical. These data suggested that 2 was a dimeric sesquiterpene lactone. Extensive analyses of 1D and 2D NMR spectra of 2 indicated that it was similar to inulanolide D, obtained from the aerial parts of Inula britannica var. chinensis (Rupr.) Regel [3]. The most striking difference between compound 2 and inulanolide D was the presence of an additional acetyl group substituted on OH group at δC 20.9 and 171.4, and δH 1.99 (3H, s, H-2‴), which could be confirmed from 1D NMR, HSQC and HMBC experiments (Fig. 3). These data suggested that 2 was the 1-O-acetyl inulanolide D. In order to establish the relative stereochemistry of compound 2, we carefully analyzed the structures of the compound 2 and known dimeric sesquiterpene lactones isolated from this plant, and found that 2 might be derived from the known dimeric sesquiterpene lactone 3 by dehydration, lactone ring opening, and second dehydration as illustrated in Scheme 2. Based on this hypothesis, we investigated the
Scheme 2. Plausible biosynthetic pathway for compound 2.
X.-Y. Xu et al. / Fitoterapia 101 (2015) 218–223
potentially biomimetic transformation of 2 from 3. At the outset, we attempted to conduct dehydration and lactone ring opening by HOAc in THF at room temperature for 6 h (Table 2, entry 1), and trace amounts of 2 could be detected by LC–MS. Encouraged by the result, we further investigated the best reaction conditions by using 2N HCl (Table 2, entry 2) or BF3 · OEt2 (Table 2, entry 3). Gratefully, the desired 2 could be obtained by BF3 · OEt2 in 62% yield (Table 2, entry 3). Therefore, the structure of 2 was identified as in Fig. 1, and named inulanolide E. Compounds 1–7 were evaluated for their in vitro growth inhibitory effects against two human leukemia cells (6T-CEM and Jurkat), with paclitaxel as positive control. All compounds exhibited significant cytotoxicities (IC50 b 6 μm) against both cell lines (Table 3). 4. Conclusion In this paper, two new sesquiterpene lactone dimers, along with five known sesquiterpene lactone dimers were isolated from the aerial parts of I. japonica Thunb.. The relative configuration of 2 was confirmed by biomimetic transformation from inulanolide A (3). All seven compounds (1–7, resp.) showed potent cytotoxicities with IC50 value of 2.2–5.9 μm against 6T-CEM and Jurkat cell lines, suggesting that these sesquiterpene lactone dimers might be potential anticancer chemotherapy agents. Acknowledgements The work was supported by the National Natural Science Foundation of China (81273397 and 81402811), the Chinese National Science & Technology Major Project “Key New Drug Creation and Manufacturing Program” (2013ZX09508104), and the China Postdoctoral Science Foundation (2014M550255). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/http://dx.doi.org/10.1016/j.fitote.2012.09. 026.
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