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New monoterpenes from stalks and infructescence of Sibiraea leavigata
Phytochemistry Letters 18 (2016) 1–4
Contents lists available at ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Short communication
New monoterpenes from stalks and infructescence of Sibiraea leavigata Jian-Qiang Zhaoa,d,1, Yan-Ming Wangb,d,1, Jun-Jiang Lvc,* , Shuo Wanga , Li-Juan Meia , Yan-Duo Taoa,* a
Key Laboratory of Tibetan Medicine Research, Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining 810008, PR China Key Laboratory of Chemistry of Northwestern Plant Resources of CAS and Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 730000, PR China c Chemistry and Chemical Engineering College, Chongqing University, Chongqing 400030, PR China d University of the Chinese Academy of Sciences, Beijing 100049, PR China b
A R T I C L E I N F O
Article history: Received 13 May 2016 Received in revised form 16 August 2016 Accepted 26 August 2016 Available online xxx Keywords: Monoterpenes Sibiraea leavigata Cytotoxic activity
A B S T R A C T
Chemical investigation of the ethanol extract of the stalks and infructescence of Sibiraea leavigata led to the isolation of two new monoterpenes named (4R)-2-(2-hydroxy-4-methyl-3-pentenyl)furan-2(5H)one (1) and (2R,4R)-2-(2-hydroxyethyl)-4-(2-methyl-1-propenyl)furan-5H-2-one (2) along with eight known phenylpropanoids (3–10). Their structures were established on the basis of the interpretation of spectroscopic data and electronic circular dichroism (ECD) calculations. In addition, all of these isolates were evaluated for their cytotoxic activity. The results showed that compound 3 displayed moderate cytotoxicity with IC50 values ranging from 10.8 to 49.2 mg mL1 against five cell lines. While 1 showed selective promotion effects on proliferation of gastric cancer MGC803 and RSC96 cell lines. ã 2016 Published by Elsevier Ltd on behalf of Phytochemical Society of Europe.
1. Introduction Liu-cha (Salix tea) is a traditional Tibetan medicine mainly used to cure pyrexia and plague by folks, and the leaf of which is the same as Salix leaf, so it was named as Liu-cha (Salix tea). Two plants from the genus Sibiraea, Sibiraea leavigata and Sibiraea angustata, were recorded as the original materials of Liu-cha (Duan et al., 2010; Wu et al., 2012). Most phytochemical and bioactive researches on Liu-cha were focused on S. angustata, however rare study was carried out on S. leavigata. Terpenoids were reported as the major components of S. angustata (Ito et al., 2009; Wang et al., 2013; Li et al., 2010). Biological researches showed that extracts of S. angustata exhibited hypolipidemic and antitumor activities (Xie et al., 2014), and improved liver function in rats with nonalcoholic fatty liver disease (Ma et al., 2010). Sibiraea laevigata (L.) Maxim, a shrub up to 1.5 m height, is mainly growing in the open forests, slopes, meadows, and stream sides in S Gansu (Min Xian, Xigu), E Qinghai (Haiyan Xian, Xining Shi), E Xizang (Suo Xian) of China and also distributes in
* Corresponding authors. E-mail addresses: [email protected] (J.-J. Lv), [email protected] (Y.-D. Tao). The authors contributed equally to this paper.
1
Kazakhstan, Russia (Siberia) and SE Europe (Bosnia, Croatia) (Editorial Committee of Chinese flora, 1974). The stalks and infructescence of S. leavigata have been utilized medicinally as qiregulating drug and dispelling the wind heat in traditional Tibetan medicine (Duan et al., 2010). Chemical and pharmacological studies on the stalks and infructescence of S. leavigata have been poorly conducted. As a part of our research for new bioactive secondary metabolites from Tibetan medicine, two new monoterpenes, (4R)-2-(2-hydroxy-4-methyl-3-pentenyl)furan-2(5H)one (1) and (2R,4R)-2-(2-hydroxyethyl)-4-(2-methyl-1-propenyl) furan-5H-2-one (2), were isolated from the stalks and infructescence of S. leavigata, together with eight known phenylpropanoids. The structures of new compounds with their absolute stereochemistry were elucidated on basis of comprehensive spectroscopic analysis and computational method. All of the isolates were tested for their cytotoxic activity against four tumour cell lines and RSC96 cell line. The results obtained are discussed herein. 2. Results and discussion Repeated column chromatography over silica gel, preparative and semi-preparative HPLC of the ethanol extract from the stalks and infructescence of S. leavigata, led to the isolation of two new monoterpenes, named (4R)-2-(2-hydroxy-4-methyl-3-pentenyl)
http://dx.doi.org/10.1016/j.phytol.2016.08.016 1874-3900/ã 2016 Published by Elsevier Ltd on behalf of Phytochemical Society of Europe.
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J.-Q. Zhao et al. / Phytochemistry Letters 18 (2016) 1–4
Fig. 1. Chemical structures of compounds 1 and 2 from stalks and infructescence of S. leavigata.
furan-2(5H)-one (1) and (2R,4R)-2-(2-hydroxyethyl)-4-(2-methyl1-propenyl)furan-5H-2-one (2), (Fig. 1) along with eight known phenylpropanoids (3–10). (4R)-2-(2-Hydroxy-4-methyl-3-pentenyl)furan-2(5H)-one (1), obtained as a colorless oil, possessed a molecular formula C10H14O3 with four double-bond equivalents (DBEs) as determined by the positive ESIMS (m/z 183 [M+H]+, 205 [M+Na]+ and 387 [2M +Na]+) and HRESIMS (m/z 387.1782 [2M+Na]+, calcd. 387.1778). The IR spectrum showed the presence of hydroxyl (3422 cm1), alkyl (2926 and 2856 cm1) groups, ester (1749 and 1206 cm1) group, and double bonds (1629 cm1). The 13C NMR (Table 1) and HSQC spectra displayed 10 carbon signals, arising from one carbonyl (dC 177.4), two double bonds (dC 136.6, 128.5, 131.6 and 150.0), two methyls (dC 25.9 and 18.2), two methylenes, including one oxygenbearing one (dC 72.1), and one oxygen-bearing methine (dC 67.2). The 1H NMR data (Table 1) showed typical resonances for two methyl groups (dH 1.72 and 1.67, each 3H, s) and two olefinic protons. The aforementioned data indicated that 1 could be a monoterpene lactone (Ito et al., 2009; Li et al., 2010). In the 1H-1H COSY spectrum of 1, two proton spin systems, H-3/H-4/H-5 and H9/H-10 were observed (Fig. 2). In HMBC spectrum, the correlations from H-3 [dH 2.50, ddd (J = 14.4, 7.5, 1.3 Hz) and 2.37, m] and H-9 (dH 7.41, m) to the carbonyl C-1 (dC 177.4), from H-4 [dH 4.58, dd (J = 14.7, 7.5 Hz)] to C-6 (dC 136.6), from H-7 (dH 1.72, s) and H-8 (dH 1.67, s) to C-5 (dC 128.5), and from H-10 [4.82, dd (J = 3.1, 1.5)] to C-1 (dC 177.4) were observed (Fig. 2). Thus, the planar structure of 1 was established as shown in Fig. 2. The absolute configuration of 1 was determined by ECD calculation. The calculated ECD spectrum was consistent with the experimental curve (Fig. 4), which suggested that the absolute configuration of compound 1 was 4R (Fig. 4). (2R,4R)-2-(2-Hydroxyethyl)-4-(2-methyl-1-propenyl)furan5H-2-one (2), a colorless oil, possessed a molecular formula C10H16O3 with three unsaturation degrees as determined by the positive ESIMS (m/z 207 [M+Na]+ and 391 [2M+Na]+) and HRESIMS (m/z 391.2096 [2M+Na]+, calcd. 391.2097). The IR spectrum showed the presence of hydroxyl (3421 cm1), alkyl (2970 and 2926 cm1)
Table 1 1 H and 13C NMR data for compounds 1 and 2 (d in ppm, in CD3OD, 600 and 150 MHz, respectively). No.
1
dC 1 2 3
177.4, s 131.6, s 34.5, t
4 5 6 7 8 9
67.2, d 128.5, d 136.6, s 25.9, q 18.2, q 150.0, d
10
72.1, t
Fig. 2. Selected HMBC (H ! C) and COSY (HH) correlations of compounds 1 and 2.
groups, ester (1763 and 1179 cm1) group, and double bonds (1630 cm1). The 13C NMR (Table 1) and HSQC spectra displayed 10 carbon signals, arising from one carbonyl (dC 181.5), one double bond (dC 124.1 and 141.0), two methyls (dC 25.9 and 18.4), three methylenes (one oxygen-bearing methylene at dC 60.6), and two methines (one oxymethine at dC 77.5). The 1H NMR data (Table 1) showed typical resonances for two methyls (dH 1.76 and 1.78, each 3H, s) and one olefinic proton. The aforementioned data indicated that 2 could also be a monoterpene lactone. In the 1H-1H COSY spectrum of 2, the proton spin system H-2/H-3/H-4/H-5/H-9/H-10 (Fig. 2) could be constructed. In HMBC spectrum, based on the correlations from H-3 (dH 2.53, m and 1.64, m) and H-9 (dH 2.08, m and 1.59, m) to the carbonyl C-1 (dC 181.5), from H-4 (dH 5.18, m) to C-6 (dC 141.0), and from H-7 (dH 1.76, s) and H-8 (dH 1.78, s) to C-5 (dC 124.1), the monoterpene moiety can be constructed. The connectivity of lactone was determined through the deshielded chemical shifts of H-4 (dH 5.18) and C-4 (dC 77.5) (Ito et al., 2009; Wang et al., 2013; Li et al., 2010). Thus, the planar structure of compound 2 was established as shown in Fig. 2. In the NOE spectrum of 2 (Fig. 3), the observed cross-peak between H-2 and H4 demonstrated that H-2 and H-4 located on the same face of the ring. Furthermore, the experimental ECD spectrum of 2 agreed well with that of the computational curve (Fig. 4), and the absolute configuration of 2 was determined to be 2R,4R. On the basis of the above evidences, the structure of 2 was deduced as shown in Fig. 1. The known compounds were identified as 3-(30 ,40 ,50 -trimethoxyphenyl)-prop-2-en-1-al (3) (Joshi et al., 2005), (E)-3-(4methoxyphenyl) acrylic acid (4) (Lorentzen et al., 2015), (E)-3-(3,4dimethoxyphenyl) acrylic acid (5) (Lorentzen et al., 2015), (E)ferulic acid (6) (Chen et al., 2015), caffeic acids (7) (Kumar et al., 2015), 3,4,5-trimethoxycinnamyl alcohol (8) (Sadik et al., 2003), dihydrosinapyl alcohol (9) (Lancefield et al., 2015), dihydroconiferylalcohol (10) (Huang et al., 2014) (Fig. 1), by comparison of their 1D spectroscopic data with those reported in the literature. Since Liu-cha (Salix tea) is a traditional Tibetan medicine mainly used to cure pyrexia and plague by folks and has great potential to develop into a healthy beverage. All of the isolates were evaluated for their cytotoxic activity. The cytotoxic activity was tested using the MTT method (Chang et al., 2004). The results showed that compound 3 displayed moderate cytotoxicity with
2
dH, multi. (J, Hz)
2.50, ddd (14.4,.7.5,1.3) 2.37, m 4.58, dd (14.7, 7.5) 5.17, m
dC 181.5, s 39.5, t 37.2, t
1.72, s 1.67, s 7.41, m
77.5, d 124.1, d 141.0, s 18.4, q 25.9, q 34.2, t
4.82, dd (3.1, 1.5)
60.6, t
dH, multi. (J, Hz) 2.86, m 2.53, m 1.64, m 5.18, m 5.22, m 1.76, s 1.78, s 2.08, m 1.59, m 3.70, dt (12.1, 6.6) 3.62, m Fig. 3. Key NOESY (H$H) correlations of compound 2.
J.-Q. Zhao et al. / Phytochemistry Letters 18 (2016) 1–4
3
Fig. 4. Calculated and experimental ECD curves of 1 and 2.
IC50 values values of 10.8, 12.4, 29.9, 49.2 and 32.2 mg mL1 against MGC803, A549, HCT116, HepG2 and RSC96 cell lines, respectively. While 1 showed selective promotion effects at a concentration of 100 mg mL1 on proliferation of gastric cancer MGC803 and RSC96 cell lines, respectively. Other isolates showed no cytotoxic activity at a concentration of 100 mg mL1. 3. Materials and methods 3.1. General experimental procedures Optical rotations were measured with a HORIBA SEPA-300 high-sensitive polarimeter. IR spectra were measured on a Bio-Rad FTS-135 series spectrometer with KBr pellets. UV spectra were recorded on a Shimadzu UV2401A ultraviolet-visible spectrophotometer. CD spectra were tested on a Chirascan Circular Dichroism spectrometer. HRESIMS spectra were run on an API QSTAR Pulsar-1 spectrometer and an Agilent 6230 Accurate-Mass spectrometer, respectively. NMR spectra measured in CDCl3 and CD3OD solution and recorded on a Bruker Avance III-600 spectrometer, using TMS as an internal standard. Chemical shifts were reported in units of d (ppm) and coupling constants (J) were expressed in Hz. Column chromatography (CC) were carried out over silica gel (200– 300 mesh, Qingdao Marine Chemical Factory) and Sephadex LH-20 (25–100 mm, Pharmacia Fine Chemical Co., Ltd. Uppsala, Sweden). Pre-coated silica gel plates (Qingdao Haiyang Chemical Co.) were used for TLC. Detection was done under UV light (254 nm and 365 nm) and by spraying the plates with 10% sulfuric acid followed by heating. An Agilent series 1200 (Agilent Technologies) was used for HPLC. A Hanbon preparative HPLC (Hanbon Sci & Tech, Jiangsu) and C18-HC column (10 mm, 100 Å, 250 mm 20 mm, Acchrom, Beijing, China) were used for semi-preparative HPLC separations. 3.2. Plant material The stalks and infructescence of Sibiraea leavigata were collected in Huzhu, Qinghai Province, People’s Republic of China, in August 2011. The identification of the plant material was verified by Professor Lijuan Mei (Norhtwest Institute of Plateau Biology, Chinese Academy of Science). A voucher specimen (No. 0317608) has been deposited in the Key Laboratory of Tibetan Medicine Research. 3.3. Extraction and isolation The air-dried and powdered stalks and infructescence of S. leavigata (9.5 kg) were extracted three times with ethanol under reflux at 70 C. After removal of the organic solvent, the ethanol extract (1150 g) was suspended in water and then extracted
successively with petroleum ether, EtOAc and n-BuOH. The EtOAc fraction (195 g) was subjected to a silica gel column chromatography (CC), eluting with CHCl3:Me2CO (10:1–1:10), to give five fractions (Fr.1-5). Fr.2 (31 g) was applied to CC over silica gel (petroleum ether:EtOAc, 20:1–1:1) to give five fractions (Fr.2a-2e). Fr.2b (1.2 g) was applied to repeated CC over silica gel (petroleum ether:EtOAc, 10:1–1:1) and Sephadex LH-20 (CHCl3:MeOH, 1:1), followed by semi-preparative HPLC (C18-HC column, ACN: H2O = 25%) to afford 1 (4.0 mg). In the same case, 2 (2.3 mg), 3 (13.5 mg) and 4 (26.1 mg) were yielded from Fr.2c (2.2 g). Fr.3 (27 g) was applied to CC over silica gel (petroleum ether:EtOAc, 20:1-1:1) to give seven fractions (Fr.3a–3 g). Fr. 3b (970 mg) was subjected to a Sephadex LH-20 (CC), eluting with CHCl3:MeOH (1:1), to give 9 (10.7 mg). Fr.3c (2.5 g) was applied to repeated CC over silica gel (petroleum ether:EtOAc, 10:1-1:1) and Sephadex LH-20 (CHCl3: MeOH, 1:1), followed by semi-preparative HPLC (C18-HC column, ACN:H2O = 23%) to afford 5 (11.7 mg) and 6 (7.4 mg). In the same case, 7 (2.1 mg), 8 (15.0 mg) and 10 (32.1 mg) were yielded from Fr. 3d (3.1 g). 3.3.1. (4R)-2-(2-hydroxy-4-methyl-3-pentenyl)furan-2(5H)-one (1) colorless oil, [a]D25.6 +10.78 (c 0.24, MeOH); UV (MeOH) lmax nm (log e): 204 (4.28), 275 (3.00); CD (MeOH, c = 7.6 104 mol L1); lmax nm (De): 202 (2.4), 250 (0.4); IR (KBr) nmax: 3422, 2926, 2856, 1749, 1629, 1206 cm1; NMR spectral data see Table 1; MS (ESI): 205 [M+Na]+, 387 [2M+Na]+, HRMS (ESI): calcd. for C20H28O6Na+ 387.1778; found 387.1782 [2M+Na]+. NMR and ECD spectra of 1 are available as Supporting information. 3.3.2. (2R,4R)-2-(2-hydroxyethyl)-4-(2-methyl-1-propenyl)furan5H-2-one (2) colorless oil, [a]D25.7 44.95 (c 0.13, MeOH); UV (MeOH) lmax nm (log e): 202 (4.16), 273 (2.45); CD (MeOH, c = 3.9 104 mol L1); lmax nm (De): 198 (26.3), 218 (3.7); IR (KBr) nmax: 3421, 2972, 2926, 1763, 1630, 1179 cm1; NMR spectral data see Table 1; MS (ESI): 207 [M+Na]+, 391 [2M+Na]+, HRMS (ESI): calcd. for C20H32O6Na+ 391.2091; found 391.2096 [2M+Na]+. NMR and ECD spectra of 2 are available as Supporting information. 3.4. Computational methods The structure models of compounds 1 and 2 were constructed based on NMR analysis in the paper. The conformation analysis was carried out using Monte Carlo searching with MMFFs to generate 5 and 8 conformers for 1 and 2, respectively. All the calculations were run with Gaussian ‘09 (Gaussian 09, 2009). The resulted conformers were re-optimized using DFT at the B3LYP-/6-311G(d, p) level. The free energies and vibrational frequencies were calculated at the same level to confirm their stability, and no imaginary
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J.-Q. Zhao et al. / Phytochemistry Letters 18 (2016) 1–4
frequencies were found. The optimized low energy conformers with energy <2 Kcal/mol were considered for ECD calculation. The TD-DFT/B3LYP/6-311G (d, p) method was applied to calculate the excited energies, oscillator strength and rotational strength. The excited energies and rotational strength were used to simulate ECD spectra of each conformer by introducing the Gaussian Function. The final ECD spectra of each compound were obtained by averaging all the simulated ECD spectra of all conformers according to their excited energies and Boltzmann distribution. 3.5. Cytotoxic activity assays Four human tumor cell lines, gastric cancer MGC803, liver cancer HepG2, lung cancer A549, and colon cancer Hct116, together with a normal cell line RSC96, were used in the cytotoxic assay. All the cells were cultured in DMEM medium (Hyclone, USA), supplemented with 10% fetal bovine serum (Hyclone, USA). The cytotoxicity assay was performed according to the MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) method in 96-well microplates (Chang et al., 2004). Briefly, adherent cells (100 mL) was seeded into each well of 96-well cell culture plates and allowed to adhere for 12 h before drug addition, while suspended cells were seeded just before drug addition with an initial density of 0.5 105–1 105 cells mL1. Each cell line was exposed to the test compounds dissolved in DMSO in triplicates for 48 h at 37 C with doxorubicin as positive control. Then, MTT (50 mL) was added to each well, and the cells were incubated for another 4 h at 37 C. After the supernatant liquor was removed, DMSO (150 mL) was added to each well. The optical density was measured at 595 nm on a microplate reader. Cell viability was detected and a cell growth curve was graphed. IC50 values were calculated by Reed and Muench’s method (Reed and Muench, 1938). Acknowledgments This work was supported by the Significant Science & Technological Project of Qinghai Province (2014-GX-A3A). We appreciate Prof. Y. J. Zhang (State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences) for her suggestions about ECD computational calculation. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. phytol.2016.08.016.
Supporting information available: 1D NMR, 2D NMR and ECD spectra of compounds 1 and 2. This material is available free of charge via the Internet. References Chang, S.L., Chang, C.L., Chiang, Y.M., Hsieh, R.H., Tzeng, C.R., Wu, T.K., Sytwu, H.K., Shyur, L.F., Yang, W.C., 2004. Polyacetylenic compounds and butanol fraction from Bidiens pilosa can modulate the differentiation of helper T cells and prevent autoimmune diabetic mice. Planta Med. 70, 1045–1051. Chen, L., Liang, Y., Song, T.F., Anjum, K., Wang, W.L., Yu, S.R., Huang, H.C., Lian, X.Y., Zhang, Z.Z., 2015. Synthesis and bioactivity of tripolinolate A from Tripolium vulgare and its analogs. Bioorg. Med. Chem. Lett. 25, 2629–2633. Duan, B.W., Li, Y., Liu, X., Yang, Y.J., 2010. Effect of polysaccharides in processed Sibiraea on immunologic function of immunosuppression mice. China J. Chin. Materia Medica 35, 1466–1469. Editorial Committee of Chinese flora, 1974. Chinese Academy of Sciences. Flora of China, vol. 36. Science Press, Beijing, pp. 68. Huang, Y.H., Zeng, W.M., Li, G.Y., Liu, G.Q., Zhao, D.D., Wang, J., Zhang, Y.L., 2014. Characterization of a new sesquiterpene and antifungal activities of chemical constituents from Dryopteris fragrans (L.) Schott. Molecules 19, 507–513. Ito, Y., Kamo, S., Sadhu, S.K., Ohtsuki, T., Ishibashi, M., Kano, Y., 2009. Structure of new monoterpene glycoside from Sibiraea angustata RCHD: and its anti-obestic effect. Chem. Pharm. Bull. 57, 294–297. Joshi, B.P., Sharma, A., Sinha, A.K., 2005. Ultrasound-assisted convenient synthesis of hypolipidemic activenatural methoxylated (E)-arylalkenes and arylalkanones. Tetrahedron 61, 3075–3080. Kumar, N., Pruthi, V., Goel, N., 2015. Structural, thermal and quantum chemical studies of p-coumaric and caffeic acids. J. Mol. Struct. 1085, 242–248. Lancefield, C.S., Ojo, O.S., Tran, F., Westwood, N.J., 2015. Isolation of functionalized phenolic monomers through selective oxidation and C-O bond cleavage of the b-O-4 linkages in lignin. Angew. Chem. Int. Ed. 54, 258–262. Li, B., Lee, J.B., Hayashi, K., Ito, Y., Sun, W.J., Wang, X.J., Kano, Y., Hayashi, T., 2010. Two new monoterpenes from Sibiraea angustata. J. Nat. Med. 64, 89–92. Lorentzen, M., Bayer, A., Sydnes, M.O., Jørgensen, K.B., 2015. Total synthesis of tubastrine and 3-dehydroxy tubastrine by microwave-assisted cross-coupling reactions. Tetrahedron 71, 8278–8284. Ma, X.Y., Wang, J.Y., Lu, Y.S., Tian, Q.S., Xia, M., Liu, X., 2010. Effects of Liucha extract on liver lipid metabolism in rats with nonalcoholic fatty liver disease. World Chin. J. Digestol. 18, 2739–2744. Reed, L.J., Muench, H., 1938. A sample method of estimating fifty percent end points. Am. J. Hyg. 27, 493–497. Sadik, G., Islam, R., Rahman, M.M., Khondkar, P., Rashidc, M.A., Sarkerd, S.D., 2003. Antimicrobial and cytotoxic constituents of Loranthus globosus. Fitoterapia 74, 308–311. Wang, Z.W., Li, B., Yu, S.S., Lai, X.W., Liu, B., Chen, J., Hayashi, T., 2013. Eight new monoterpene acylglucosides from Sibiraea angustata. Phytochem. Lett. 6, 461– 466. Wu, Y., He, H., Liu, J., 2012. Antimicrobial and antioxidant properties of the essential oid of Sibiraea angustata RCHD. Chin. Hosp. Pharm. J. 32, 1819–1820. Xie, Y.H., Yu, S.H., Yu, Y.F., Deng, H.P., Liu, H., Ma, S.L., Li, B., 2014. Studies on advances of chemical constituents and pharmacological activities of Sibiraea angustata. J. Jiangxi Univ. TCM 26, 97–100.
Contents lists available at ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Short communication
New monoterpenes from stalks and infructescence of Sibiraea leavigata Jian-Qiang Zhaoa,d,1, Yan-Ming Wangb,d,1, Jun-Jiang Lvc,* , Shuo Wanga , Li-Juan Meia , Yan-Duo Taoa,* a
Key Laboratory of Tibetan Medicine Research, Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining 810008, PR China Key Laboratory of Chemistry of Northwestern Plant Resources of CAS and Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 730000, PR China c Chemistry and Chemical Engineering College, Chongqing University, Chongqing 400030, PR China d University of the Chinese Academy of Sciences, Beijing 100049, PR China b
A R T I C L E I N F O
Article history: Received 13 May 2016 Received in revised form 16 August 2016 Accepted 26 August 2016 Available online xxx Keywords: Monoterpenes Sibiraea leavigata Cytotoxic activity
A B S T R A C T
Chemical investigation of the ethanol extract of the stalks and infructescence of Sibiraea leavigata led to the isolation of two new monoterpenes named (4R)-2-(2-hydroxy-4-methyl-3-pentenyl)furan-2(5H)one (1) and (2R,4R)-2-(2-hydroxyethyl)-4-(2-methyl-1-propenyl)furan-5H-2-one (2) along with eight known phenylpropanoids (3–10). Their structures were established on the basis of the interpretation of spectroscopic data and electronic circular dichroism (ECD) calculations. In addition, all of these isolates were evaluated for their cytotoxic activity. The results showed that compound 3 displayed moderate cytotoxicity with IC50 values ranging from 10.8 to 49.2 mg mL1 against five cell lines. While 1 showed selective promotion effects on proliferation of gastric cancer MGC803 and RSC96 cell lines. ã 2016 Published by Elsevier Ltd on behalf of Phytochemical Society of Europe.
1. Introduction Liu-cha (Salix tea) is a traditional Tibetan medicine mainly used to cure pyrexia and plague by folks, and the leaf of which is the same as Salix leaf, so it was named as Liu-cha (Salix tea). Two plants from the genus Sibiraea, Sibiraea leavigata and Sibiraea angustata, were recorded as the original materials of Liu-cha (Duan et al., 2010; Wu et al., 2012). Most phytochemical and bioactive researches on Liu-cha were focused on S. angustata, however rare study was carried out on S. leavigata. Terpenoids were reported as the major components of S. angustata (Ito et al., 2009; Wang et al., 2013; Li et al., 2010). Biological researches showed that extracts of S. angustata exhibited hypolipidemic and antitumor activities (Xie et al., 2014), and improved liver function in rats with nonalcoholic fatty liver disease (Ma et al., 2010). Sibiraea laevigata (L.) Maxim, a shrub up to 1.5 m height, is mainly growing in the open forests, slopes, meadows, and stream sides in S Gansu (Min Xian, Xigu), E Qinghai (Haiyan Xian, Xining Shi), E Xizang (Suo Xian) of China and also distributes in
* Corresponding authors. E-mail addresses: [email protected] (J.-J. Lv), [email protected] (Y.-D. Tao). The authors contributed equally to this paper.
1
Kazakhstan, Russia (Siberia) and SE Europe (Bosnia, Croatia) (Editorial Committee of Chinese flora, 1974). The stalks and infructescence of S. leavigata have been utilized medicinally as qiregulating drug and dispelling the wind heat in traditional Tibetan medicine (Duan et al., 2010). Chemical and pharmacological studies on the stalks and infructescence of S. leavigata have been poorly conducted. As a part of our research for new bioactive secondary metabolites from Tibetan medicine, two new monoterpenes, (4R)-2-(2-hydroxy-4-methyl-3-pentenyl)furan-2(5H)one (1) and (2R,4R)-2-(2-hydroxyethyl)-4-(2-methyl-1-propenyl) furan-5H-2-one (2), were isolated from the stalks and infructescence of S. leavigata, together with eight known phenylpropanoids. The structures of new compounds with their absolute stereochemistry were elucidated on basis of comprehensive spectroscopic analysis and computational method. All of the isolates were tested for their cytotoxic activity against four tumour cell lines and RSC96 cell line. The results obtained are discussed herein. 2. Results and discussion Repeated column chromatography over silica gel, preparative and semi-preparative HPLC of the ethanol extract from the stalks and infructescence of S. leavigata, led to the isolation of two new monoterpenes, named (4R)-2-(2-hydroxy-4-methyl-3-pentenyl)
http://dx.doi.org/10.1016/j.phytol.2016.08.016 1874-3900/ã 2016 Published by Elsevier Ltd on behalf of Phytochemical Society of Europe.
2
J.-Q. Zhao et al. / Phytochemistry Letters 18 (2016) 1–4
Fig. 1. Chemical structures of compounds 1 and 2 from stalks and infructescence of S. leavigata.
furan-2(5H)-one (1) and (2R,4R)-2-(2-hydroxyethyl)-4-(2-methyl1-propenyl)furan-5H-2-one (2), (Fig. 1) along with eight known phenylpropanoids (3–10). (4R)-2-(2-Hydroxy-4-methyl-3-pentenyl)furan-2(5H)-one (1), obtained as a colorless oil, possessed a molecular formula C10H14O3 with four double-bond equivalents (DBEs) as determined by the positive ESIMS (m/z 183 [M+H]+, 205 [M+Na]+ and 387 [2M +Na]+) and HRESIMS (m/z 387.1782 [2M+Na]+, calcd. 387.1778). The IR spectrum showed the presence of hydroxyl (3422 cm1), alkyl (2926 and 2856 cm1) groups, ester (1749 and 1206 cm1) group, and double bonds (1629 cm1). The 13C NMR (Table 1) and HSQC spectra displayed 10 carbon signals, arising from one carbonyl (dC 177.4), two double bonds (dC 136.6, 128.5, 131.6 and 150.0), two methyls (dC 25.9 and 18.2), two methylenes, including one oxygenbearing one (dC 72.1), and one oxygen-bearing methine (dC 67.2). The 1H NMR data (Table 1) showed typical resonances for two methyl groups (dH 1.72 and 1.67, each 3H, s) and two olefinic protons. The aforementioned data indicated that 1 could be a monoterpene lactone (Ito et al., 2009; Li et al., 2010). In the 1H-1H COSY spectrum of 1, two proton spin systems, H-3/H-4/H-5 and H9/H-10 were observed (Fig. 2). In HMBC spectrum, the correlations from H-3 [dH 2.50, ddd (J = 14.4, 7.5, 1.3 Hz) and 2.37, m] and H-9 (dH 7.41, m) to the carbonyl C-1 (dC 177.4), from H-4 [dH 4.58, dd (J = 14.7, 7.5 Hz)] to C-6 (dC 136.6), from H-7 (dH 1.72, s) and H-8 (dH 1.67, s) to C-5 (dC 128.5), and from H-10 [4.82, dd (J = 3.1, 1.5)] to C-1 (dC 177.4) were observed (Fig. 2). Thus, the planar structure of 1 was established as shown in Fig. 2. The absolute configuration of 1 was determined by ECD calculation. The calculated ECD spectrum was consistent with the experimental curve (Fig. 4), which suggested that the absolute configuration of compound 1 was 4R (Fig. 4). (2R,4R)-2-(2-Hydroxyethyl)-4-(2-methyl-1-propenyl)furan5H-2-one (2), a colorless oil, possessed a molecular formula C10H16O3 with three unsaturation degrees as determined by the positive ESIMS (m/z 207 [M+Na]+ and 391 [2M+Na]+) and HRESIMS (m/z 391.2096 [2M+Na]+, calcd. 391.2097). The IR spectrum showed the presence of hydroxyl (3421 cm1), alkyl (2970 and 2926 cm1)
Table 1 1 H and 13C NMR data for compounds 1 and 2 (d in ppm, in CD3OD, 600 and 150 MHz, respectively). No.
1
dC 1 2 3
177.4, s 131.6, s 34.5, t
4 5 6 7 8 9
67.2, d 128.5, d 136.6, s 25.9, q 18.2, q 150.0, d
10
72.1, t
Fig. 2. Selected HMBC (H ! C) and COSY (HH) correlations of compounds 1 and 2.
groups, ester (1763 and 1179 cm1) group, and double bonds (1630 cm1). The 13C NMR (Table 1) and HSQC spectra displayed 10 carbon signals, arising from one carbonyl (dC 181.5), one double bond (dC 124.1 and 141.0), two methyls (dC 25.9 and 18.4), three methylenes (one oxygen-bearing methylene at dC 60.6), and two methines (one oxymethine at dC 77.5). The 1H NMR data (Table 1) showed typical resonances for two methyls (dH 1.76 and 1.78, each 3H, s) and one olefinic proton. The aforementioned data indicated that 2 could also be a monoterpene lactone. In the 1H-1H COSY spectrum of 2, the proton spin system H-2/H-3/H-4/H-5/H-9/H-10 (Fig. 2) could be constructed. In HMBC spectrum, based on the correlations from H-3 (dH 2.53, m and 1.64, m) and H-9 (dH 2.08, m and 1.59, m) to the carbonyl C-1 (dC 181.5), from H-4 (dH 5.18, m) to C-6 (dC 141.0), and from H-7 (dH 1.76, s) and H-8 (dH 1.78, s) to C-5 (dC 124.1), the monoterpene moiety can be constructed. The connectivity of lactone was determined through the deshielded chemical shifts of H-4 (dH 5.18) and C-4 (dC 77.5) (Ito et al., 2009; Wang et al., 2013; Li et al., 2010). Thus, the planar structure of compound 2 was established as shown in Fig. 2. In the NOE spectrum of 2 (Fig. 3), the observed cross-peak between H-2 and H4 demonstrated that H-2 and H-4 located on the same face of the ring. Furthermore, the experimental ECD spectrum of 2 agreed well with that of the computational curve (Fig. 4), and the absolute configuration of 2 was determined to be 2R,4R. On the basis of the above evidences, the structure of 2 was deduced as shown in Fig. 1. The known compounds were identified as 3-(30 ,40 ,50 -trimethoxyphenyl)-prop-2-en-1-al (3) (Joshi et al., 2005), (E)-3-(4methoxyphenyl) acrylic acid (4) (Lorentzen et al., 2015), (E)-3-(3,4dimethoxyphenyl) acrylic acid (5) (Lorentzen et al., 2015), (E)ferulic acid (6) (Chen et al., 2015), caffeic acids (7) (Kumar et al., 2015), 3,4,5-trimethoxycinnamyl alcohol (8) (Sadik et al., 2003), dihydrosinapyl alcohol (9) (Lancefield et al., 2015), dihydroconiferylalcohol (10) (Huang et al., 2014) (Fig. 1), by comparison of their 1D spectroscopic data with those reported in the literature. Since Liu-cha (Salix tea) is a traditional Tibetan medicine mainly used to cure pyrexia and plague by folks and has great potential to develop into a healthy beverage. All of the isolates were evaluated for their cytotoxic activity. The cytotoxic activity was tested using the MTT method (Chang et al., 2004). The results showed that compound 3 displayed moderate cytotoxicity with
2
dH, multi. (J, Hz)
2.50, ddd (14.4,.7.5,1.3) 2.37, m 4.58, dd (14.7, 7.5) 5.17, m
dC 181.5, s 39.5, t 37.2, t
1.72, s 1.67, s 7.41, m
77.5, d 124.1, d 141.0, s 18.4, q 25.9, q 34.2, t
4.82, dd (3.1, 1.5)
60.6, t
dH, multi. (J, Hz) 2.86, m 2.53, m 1.64, m 5.18, m 5.22, m 1.76, s 1.78, s 2.08, m 1.59, m 3.70, dt (12.1, 6.6) 3.62, m Fig. 3. Key NOESY (H$H) correlations of compound 2.
J.-Q. Zhao et al. / Phytochemistry Letters 18 (2016) 1–4
3
Fig. 4. Calculated and experimental ECD curves of 1 and 2.
IC50 values values of 10.8, 12.4, 29.9, 49.2 and 32.2 mg mL1 against MGC803, A549, HCT116, HepG2 and RSC96 cell lines, respectively. While 1 showed selective promotion effects at a concentration of 100 mg mL1 on proliferation of gastric cancer MGC803 and RSC96 cell lines, respectively. Other isolates showed no cytotoxic activity at a concentration of 100 mg mL1. 3. Materials and methods 3.1. General experimental procedures Optical rotations were measured with a HORIBA SEPA-300 high-sensitive polarimeter. IR spectra were measured on a Bio-Rad FTS-135 series spectrometer with KBr pellets. UV spectra were recorded on a Shimadzu UV2401A ultraviolet-visible spectrophotometer. CD spectra were tested on a Chirascan Circular Dichroism spectrometer. HRESIMS spectra were run on an API QSTAR Pulsar-1 spectrometer and an Agilent 6230 Accurate-Mass spectrometer, respectively. NMR spectra measured in CDCl3 and CD3OD solution and recorded on a Bruker Avance III-600 spectrometer, using TMS as an internal standard. Chemical shifts were reported in units of d (ppm) and coupling constants (J) were expressed in Hz. Column chromatography (CC) were carried out over silica gel (200– 300 mesh, Qingdao Marine Chemical Factory) and Sephadex LH-20 (25–100 mm, Pharmacia Fine Chemical Co., Ltd. Uppsala, Sweden). Pre-coated silica gel plates (Qingdao Haiyang Chemical Co.) were used for TLC. Detection was done under UV light (254 nm and 365 nm) and by spraying the plates with 10% sulfuric acid followed by heating. An Agilent series 1200 (Agilent Technologies) was used for HPLC. A Hanbon preparative HPLC (Hanbon Sci & Tech, Jiangsu) and C18-HC column (10 mm, 100 Å, 250 mm 20 mm, Acchrom, Beijing, China) were used for semi-preparative HPLC separations. 3.2. Plant material The stalks and infructescence of Sibiraea leavigata were collected in Huzhu, Qinghai Province, People’s Republic of China, in August 2011. The identification of the plant material was verified by Professor Lijuan Mei (Norhtwest Institute of Plateau Biology, Chinese Academy of Science). A voucher specimen (No. 0317608) has been deposited in the Key Laboratory of Tibetan Medicine Research. 3.3. Extraction and isolation The air-dried and powdered stalks and infructescence of S. leavigata (9.5 kg) were extracted three times with ethanol under reflux at 70 C. After removal of the organic solvent, the ethanol extract (1150 g) was suspended in water and then extracted
successively with petroleum ether, EtOAc and n-BuOH. The EtOAc fraction (195 g) was subjected to a silica gel column chromatography (CC), eluting with CHCl3:Me2CO (10:1–1:10), to give five fractions (Fr.1-5). Fr.2 (31 g) was applied to CC over silica gel (petroleum ether:EtOAc, 20:1–1:1) to give five fractions (Fr.2a-2e). Fr.2b (1.2 g) was applied to repeated CC over silica gel (petroleum ether:EtOAc, 10:1–1:1) and Sephadex LH-20 (CHCl3:MeOH, 1:1), followed by semi-preparative HPLC (C18-HC column, ACN: H2O = 25%) to afford 1 (4.0 mg). In the same case, 2 (2.3 mg), 3 (13.5 mg) and 4 (26.1 mg) were yielded from Fr.2c (2.2 g). Fr.3 (27 g) was applied to CC over silica gel (petroleum ether:EtOAc, 20:1-1:1) to give seven fractions (Fr.3a–3 g). Fr. 3b (970 mg) was subjected to a Sephadex LH-20 (CC), eluting with CHCl3:MeOH (1:1), to give 9 (10.7 mg). Fr.3c (2.5 g) was applied to repeated CC over silica gel (petroleum ether:EtOAc, 10:1-1:1) and Sephadex LH-20 (CHCl3: MeOH, 1:1), followed by semi-preparative HPLC (C18-HC column, ACN:H2O = 23%) to afford 5 (11.7 mg) and 6 (7.4 mg). In the same case, 7 (2.1 mg), 8 (15.0 mg) and 10 (32.1 mg) were yielded from Fr. 3d (3.1 g). 3.3.1. (4R)-2-(2-hydroxy-4-methyl-3-pentenyl)furan-2(5H)-one (1) colorless oil, [a]D25.6 +10.78 (c 0.24, MeOH); UV (MeOH) lmax nm (log e): 204 (4.28), 275 (3.00); CD (MeOH, c = 7.6 104 mol L1); lmax nm (De): 202 (2.4), 250 (0.4); IR (KBr) nmax: 3422, 2926, 2856, 1749, 1629, 1206 cm1; NMR spectral data see Table 1; MS (ESI): 205 [M+Na]+, 387 [2M+Na]+, HRMS (ESI): calcd. for C20H28O6Na+ 387.1778; found 387.1782 [2M+Na]+. NMR and ECD spectra of 1 are available as Supporting information. 3.3.2. (2R,4R)-2-(2-hydroxyethyl)-4-(2-methyl-1-propenyl)furan5H-2-one (2) colorless oil, [a]D25.7 44.95 (c 0.13, MeOH); UV (MeOH) lmax nm (log e): 202 (4.16), 273 (2.45); CD (MeOH, c = 3.9 104 mol L1); lmax nm (De): 198 (26.3), 218 (3.7); IR (KBr) nmax: 3421, 2972, 2926, 1763, 1630, 1179 cm1; NMR spectral data see Table 1; MS (ESI): 207 [M+Na]+, 391 [2M+Na]+, HRMS (ESI): calcd. for C20H32O6Na+ 391.2091; found 391.2096 [2M+Na]+. NMR and ECD spectra of 2 are available as Supporting information. 3.4. Computational methods The structure models of compounds 1 and 2 were constructed based on NMR analysis in the paper. The conformation analysis was carried out using Monte Carlo searching with MMFFs to generate 5 and 8 conformers for 1 and 2, respectively. All the calculations were run with Gaussian ‘09 (Gaussian 09, 2009). The resulted conformers were re-optimized using DFT at the B3LYP-/6-311G(d, p) level. The free energies and vibrational frequencies were calculated at the same level to confirm their stability, and no imaginary
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frequencies were found. The optimized low energy conformers with energy <2 Kcal/mol were considered for ECD calculation. The TD-DFT/B3LYP/6-311G (d, p) method was applied to calculate the excited energies, oscillator strength and rotational strength. The excited energies and rotational strength were used to simulate ECD spectra of each conformer by introducing the Gaussian Function. The final ECD spectra of each compound were obtained by averaging all the simulated ECD spectra of all conformers according to their excited energies and Boltzmann distribution. 3.5. Cytotoxic activity assays Four human tumor cell lines, gastric cancer MGC803, liver cancer HepG2, lung cancer A549, and colon cancer Hct116, together with a normal cell line RSC96, were used in the cytotoxic assay. All the cells were cultured in DMEM medium (Hyclone, USA), supplemented with 10% fetal bovine serum (Hyclone, USA). The cytotoxicity assay was performed according to the MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) method in 96-well microplates (Chang et al., 2004). Briefly, adherent cells (100 mL) was seeded into each well of 96-well cell culture plates and allowed to adhere for 12 h before drug addition, while suspended cells were seeded just before drug addition with an initial density of 0.5 105–1 105 cells mL1. Each cell line was exposed to the test compounds dissolved in DMSO in triplicates for 48 h at 37 C with doxorubicin as positive control. Then, MTT (50 mL) was added to each well, and the cells were incubated for another 4 h at 37 C. After the supernatant liquor was removed, DMSO (150 mL) was added to each well. The optical density was measured at 595 nm on a microplate reader. Cell viability was detected and a cell growth curve was graphed. IC50 values were calculated by Reed and Muench’s method (Reed and Muench, 1938). Acknowledgments This work was supported by the Significant Science & Technological Project of Qinghai Province (2014-GX-A3A). We appreciate Prof. Y. J. Zhang (State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences) for her suggestions about ECD computational calculation. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. phytol.2016.08.016.
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