Fitoterapia 103 (2015) 222–226
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Fitoterapia journal homepage: www.elsevier.com/locate/fitote
Sesquiterpene coumarins from seeds of Ferula sinkiangensis Guangzhi Li a, Xiaojin Li b, Li Cao a, Lijing Zhang a, Liangang Shen a, Jun Zhu b, Junchi Wang a, Jianyong Si a,⁎ a b
Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100193, China Xinjiang Institute of Chinese Materia Medica and Ethical Materia Medica, Urumqi 830002, China
a r t i c l e
i n f o
Article history: Received 3 February 2015 Accepted in revised form 19 March 2015 Available online 27 March 2015 Chemical compounds studied in this article: Farnesiferl C (PubChem CID: 15559239) Umbelliprenin (PubChem CID: 1781413) Colladonin (PubChem CID: 375430) Feselol (PubChem CID: 17977) Lehmannolone Lehmannolol Feklone Episamarcandin Sinkianone Fekrynol
a b s t r a c t A new sesquiterpene coumarin with a novel sesquiterpene carbon framework, Sinkiangenorin D, and ten known sesquiterpene coumarins were isolated from the seeds of Ferula sinkiangensis. The structures of these compounds, including the relative stereochemistry, were elucidated on the basis of spectroscopic data. All of the isolated compounds were tested against the AGS, HeLa, and K562 human cancer cell lines and showed cytotoxic activities with 50% inhibitory concentration values between 12.7 and 226.6 μM. © 2015 Elsevier B.V. All rights reserved.
Keywords: Ferula sinkiangensis Sesquiterpene coumarin Sesquiterpene Cytotoxic activities
1. Introduction The genus Ferula (Umbelliferea), including more than 150 species, is widely distributed across Central Asia and the Mediterranean [1]. Some plants of the genus Ferula have been used as pharmaceutical plants in many countries for centuries [2]. Ferula sinkiangensis is an important member of this genus, which is mainly distributed in the Xinjiang Uygur Autonomous Region of China, and has long been used as a folk medicine for the treatment of rheumatoid arthritis and stomach disorders ⁎ Corresponding author. Tel./fax: +86 010 57833299. E-mail address: [email protected] (J. Si).
http://dx.doi.org/10.1016/j.fitote.2015.03.022 0367-326X/© 2015 Elsevier B.V. All rights reserved.
[3]. Chemical studies of F. sinkiangensis have been carried out since the 1980s, and sesquiterpene coumarins were reported to be one of the main components [4]. In the course of our ongoing research on the constituents of the seeds of F. sinkiangensis, a new sesquiterpene coumarin with a novel fekrynol-type sesquiterpene system, Sinkiangenorin D (1), together with ten known compounds (2–11), lehmannolol (2), lehmannolone (3), episamarcandin (4), colladonin (5), sinkianone (6), fekrynol (7), fekolone (8), feselol (9), umbelliprenin (10), and farnesiferol C (11), were isolated (Fig. 1). In this study, we describe the isolation and structural elucidation of Sinkiangenorin D, as well as the antineoplastic activity of all of the isolated compounds listed above.
G. Li et al. / Fitoterapia 103 (2015) 222–226
223
2. Experimental
2.2. Plant material
2.1. General experimental procedures
The seeds of F. sinkiangensis were collected in July 2008 from Yili state, Xinjiang Uygur Autonomous Region of China by Prof. Xiaojin Li (Xinjiang Institute of Chinese Materia Medica and Ethical Materia Medica, Urumqi, China). A voucher specimen (No. AP21020720) was deposited in the Xinjiang Institute of Chinese Materia Medica and Ethical Materia Medica.
Ultraviolet (UV) spectra were recorded on a Shimadzu UV2550 spectrometer, and infrared (IR) spectra were obtained on an FTIR-8400S spectrometer. Melting points were measured (without correction) on a Fisher-Johns melting point apparatus (Fisher-Johns Scientific Company). Nuclear magnetic resonance (NMR) spectra were obtained using Bruker AV Ш 600 spectrometers operating at 150 MHz for 13C and at 600 MHz for 1H (chemical shift values are presented as δ values with TMS as the internal reference). High-resolution electrospray ionization mass spectrometry (HR-ESI-MS) was performed on a Thermo Scientific LTQ-Obitrap XL (Thermo Scientific; Bremen, Germany). Optical rotations were recorded in MeOH at 20 °C with a Perkin-Elmer 341 digital polarimeter. Semi-preparative high-performance liquid chromatography was performed on a K1001 analytic LC instrument (Beijing Chuangxintongheng Science & Technology Co., Ltd.,) equipped with a YMC-Pack ODS-A column (250 × 10 mm, S-5 μm, 12 nm), two K-501 pumps, and a K-2600 UV detector. Sephadex LH-20 (Pharmacia; Uppsala, Sweden) columns were used for column chromatography, and pre-coated silica gel plates (Zhi Fu Huang Wu Pilot Plant of Silica Gel Development, Yantai, PR China) were used for thin layer chromatography analysis. All solvents used were of analytical grade (Beijing Chemical Works; Beijing, PR China).
2.3. Extraction and isolation The powder of the seeds of F. sinkiangensis (4.2 kg) was refluxed with 95% EtOH (3 × 15 L) three times for 2 h to yield a viscous residue (401 g) that was suspended in water and partitioned by petroleum ether and dichloromethane (4 × 5 L), respectively. The dichloromethane viscous residue (132 g), eluted with CHCl3–MeOH (40:1 to 0:1, v/v), was fractionated into ten fractions (Fr. 1–10) by chromatography on a silica gel column (200–300 mesh, 10 × 100 cm). The ten fractions were separated by a semi-preparative liquid chromatography with a gradient of 60% MeOH–H2O to MeOH on an YMC-Pack ODS-A column (250 × 10 mm, S-5 μm, 12 nm), respectively. Finally, 1 (13 mg, tR 19.4 min) and 2 (15 mg, tR 23.5 min) were obtained in Fr. 2 using an MeOH–H2O (68:32) system; 3 (11.2 mg, tR 20.5 min), 4 (9.0 mg, tR 24.7 min), and 5 (15.0 mg, tR 27.6 min) were obtained in Fr. 3 using an MeOH–H2O (62:38) system; 6 (22.6 mg, tR 18.2 min), 7 (16.0 mg, tR 23.5 min), and 8 (12.0 mg, tR 25.7 min) were obtained in Fr. 5 using an MeOH–
Fig. 1. Structures of compounds (1–11).
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G. Li et al. / Fitoterapia 103 (2015) 222–226
(log ε) 325 (3.5) nm; IR νmax 3434, 1725, 1707, 1610, 1550, 1508, 1451, 1402, 1120 cm−1; 1H and 13C-APT see Table 1.
Table 1 1 H and 13C-APT NMR (600 MHz) data for 1 in CD3OD. δH (J, Hz) 2 3 4 5 6 7 8 9 10 1′ 1′ 2′ 3′ 4′ 5′1′ 6′ 6′ 7′ 7′ 8′ 9′ 9′ 10′ 10′ 11′ 12′ 13′ 14′ 15′ a
δc 6.18 (d, 9.6) 7.81 (d, 9.6) 7.45 (d, 8.4) 6.85 (dd, 8.4, 1.8) 6.76 (d, 1.8)
3.93 (d, 8.4) 3.74 (d,9.0) 2.96 (dd,11.4, 3.6)
1.92 (d, 12.6) 2.51 (d, 12.6) 1.25 (m) 1.57 a 1.85 a 1.62 a 1.57 a 1.32 (m) 1.41 (m) 3.55 (m) 1.13 (3H, s) 1.46 (3H, s) 1.59 (3H, s) 0.91 (3H, s)
HMBC 163.24 113.09 145.68 130.28 114.19 164.41 101.98 157.05 113.72 73.02
2.5. Cytotoxicity assay
C3′, C8′, C12′, C1′
41.95 44.33 125.56 132.16 25.51 33.02
3. Results and discussion
36.07 24.23 C2′, C4′ 31.80 63.44 23.11 20.76 20.31 16.42
The isolated compounds 1–11 were tested against the K562, HeLa, and AGS human cancer cell lines using the 3-(4,5dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) method [5]. Taxol (Cisen Pharmaceutical Co., H20057404) was used as a positive control. Briefly, freshly trypsinized cells were seeded at a density of 6 × 104 cells/mL per well in 96-well microliter plates with test compounds added from a dimethyl sulfoxide (DMSO) stock solution. After 48 h in culture at 37 °C in 5% CO2, 10 μL of MTT (4 mg/mL) was added to the attached cells and incubated for another 4 h. The supernatant was discarded and DMSO was added (200 μL) to each well. The absorbance was measured at 570 nm on a microplate reader (SpectramaxPlus 384; Molecular Devices; Sunnyvale, CA, USA).
C3′, C8′, C1′, C4′, C5′ C4′, C6′ C2′, C7′
Overlapping signals.
H2O (57:48) system; 9 (15.6 mg, tR 18.7 min) was obtained in Fr. 6 using an MeOH–H2O (53:47) system; and 10 (18.6 mg, tR 20.7 min) and 11 (10.6 mg, tR 23.7 min) were obtained in Fr. 8 using an MeOH–H2O (49:51) system.
2.4. Sinkiangenorin D (1) Sinkiangenorin D (1) is a colorless transparent solid: (+) HR-ESI-MS m/z 407.2179 (calcd for C24H32O5Na [M + Na]+, 407.2301); [α]20D −46.7 (c 0.075, MeOH); UV (MeOH) λmax
The EtOH extracts of the seeds of F. sinkiangensis were subjected to silica gel column chromatography; the preparative liquid chromatograph yielded a novel sesquiterpene coumarin (1) and ten known sesquiterpene coumarins (2–11). The structure of 1 was established by spectroscopic analysis. Compounds 2–11 were identified by comparing their spectroscopic data with published data for lehmannolol (2) [3], lehmannolone (3) [3], episamarcandin (4), colladonin (5), sinkianone (6) [6], fekrynol (7), fekolone (8), feselol (9), umbelliprenin (10), and farnesiferol C (11)8. Compound 1 was a colorless, transparent, solid material with a molecular formula of C24H32O5 deduced by HR-ESI-MS analysis ([M + Na]+ C24H32O5Na, calcd m/z 407.2179, obsd m/z 407.2301), which corresponds to 9° of unsaturation. The UV spectrum of 1 exhibited an absorption maximum at 325 nm, which is consistent with the presence of a coumarin moiety. The 1H NMR spectrum (Table 1) showed four methyl group signals at δH 0.91 (s, 15′-CH3), 1.13 (s, 12′-CH3), 1.46 (s, 13′CH3), and 1.59 (s, 14′-CH3). The heteronuclear single-quantum coherence (HSQC) and 13C-APT NMR spectra established that
Fig. 2. Key 2D NMR correlations of 1.
G. Li et al. / Fitoterapia 103 (2015) 222–226
225
Fig. 3. Proposed biogenetic pathway for Sinkiangenorin D.
1 possessed a coumarin moiety (δ:164.41, 163.24, 157.05, 145.68, 130.28, 114.19, 113.72, 113.09, 101.98) and a sesquiterpene moiety containing four methyl groups at δC 16.42, 20.31, 20.76, 23.11; an olefinic bond at δC 125.56, 132.16; and an oxymethine group at δC 63.4. Taking into account the 2° of unsaturation for the sesquiterpene moiety, these results indicated that 1 is a sesquiterpene coumarin with a monocyclic sesquiterene moiety. Comprehensive analysis of the two-dimensional NMR data (Fig. 2) led to the identification of the planar structure of the sesquiterpene moiety of 1. The HSQC and 1H-1H COSY spectra showed two spin systems (H-3′/H-9′/H-10′/H-11′, H-6′/H-7′/ H-8′/H-15′), which established the presence of three partial structures (C-3′–C-9′–C-10′–C-11′, C-6′–C-7′–C8′–C-15′) as shown in Fig. 2. In the HMBC spectrum, the correlations from 12′-CH3 to C-3′, C-8′, C-1′; H-1′ to C-3′, C-8′, C-12′; 15′-CH3 to C-2′, C-7′; 13′-CH3 to C-4′, C-5′; 14′-CH3 to C-4′, C-6′; and H-9′ to C-2′, C-4′ indicated the presence of a 7-membered ring system. The correlations from H-1' to C-7 enabled the identification of the position of an ether linkage linking the coumarin to the sesquiterpene moiety. The relative configuration of all of the stereogenic centers in the sesquiterpene moiety was determined from the nuclear Overhauser effect spectroscopy (NOESY) data (Fig. 2). The
Table 2 In vitro antiproliferative activity of compounds 1–11. Compound
IC50 (μM) HeLa
1 2 3 4 5 6 7 8 9 10 11 Taxolb a b
a
20.4 ± 1.3 226.6 ± 1.0 81.1 ± 1.4 – – 77.9 ± 0.7 142.7 ± 3.2 – – 202.2 ± 1.2 86.9 ± 1.1 5.6 ± 0.01
K562
AGS
81.1 ± 1.0 – 136.9 ± 1.3 112.9 ± 0.6 141.6 ± 1.5 – – – – 141.6 ± 1.1 – 8.5 ± 0.15
104.8 ± 1.2 26.0 ± 0.9 – 83.8 ± 1.4 85.5 ± 2.2 – – 75.4 ± 2.1 – 12.7 ± 0.8 101.6 ± 1.3 3.5 ± 0.04
Values represent mean ± SD of triplicate experiments. Positive control substance.
correlations of 8′-H/12′-CH3 and 12′-CH3/H-9′ revealed that 8′-H, 12′-CH3, and H-9′ were coplanar. Considering that Sinkiangenorin D cannot crystallize and that there is no proper circular dichroism method available to determine the absolute configuration, the absolute configuration of Sikiangenorin D remains unknown. The structure of Sinkiangenorin D turned out to be different from that of common sesquiterpene and to consist of a unique skeleton. A possible biosynthetic pathway for Sinkiangenorin D is shown in Fig. 3. The naturally occurring fekrynol-type sesquiterene, also obtained from the seeds of F. sinkiangensis is perhaps the parent compound for the novel sesquiterpene moiety of Sinkiangenorin D. First, the formation of intermediate 2 could be initiated by the protonation of C4 and electron transfer reaction of the olefinic bond. Subsequently, the electrons of bond C4–C5 would transfer to C11, leading to formation of the seven-membered ring. The transfer of a methyl at C5′ and consequent proton loss would result in the formation of an olefinic bond of C4′–C5′, which would lead to the formation of this novel carbon framework. The isolated compounds 1–11 were tested for their cytotoxic activity in vitro against three human cancer cell lines (AGS, K562 and HeLa) using the MTT assay. The 50% inhibitory concentration (IC50) values were in the range of 12.7–226.6 μM and were compared to those of Taxol, which was used as the positive control. The data suggested that the isolated sesquiterpene coumarin derivatives had selective cytotoxic activity against the HeLa and AGS cancer cell lines (Table 2). Compounds 1, 2, 3, 6, 7, and 11 all displayed cytotoxicity against HeLa cells with IC50 values between 20.4 and 226.2 μM, and compounds 1, 2, 4, 5, 8, 10, and 11 showed cytotoxicity against AGS cells with IC50 values between 12.7 and 104.8 μM. Based on these biological results, it can be concluded that the sesquiterpene coumarin derivatives may play a role in the cytotoxic activity of F. sinkiangensis.
Acknowledgments This work was financially supported by the National Mega-project for Innovative Drugs (2012ZX09301-002-001; 2011ZX09307-002-01), the National Natural Science Foundation of China (81460661; 81460586), and the Program for Innovative Research Team in IMPLAD.
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Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.fitote.2015.03.022. References [1] Iranshahi M, Hosseini ST, Shahverdi AR, Molazade K, Khan SS, Ahmad VU. Diversolides A–G, guaianolides from the roots of Ferula diversivittata. Phytochemistry 2008;69:2753–7. [2] Ahmed AA, Hegazy ME, Zellagui A, Rhouati S, Mohamed TA, Sayed AA, et al. Ferulsinaic acid, a sesquiterpene coumarin with a rare carbon skeleton from Ferula species. Phytochemistry 2007;68:680–6.
[3] Yang J-R, An Z, Li Z-H, Jing S, Qin H-L. Sesquiterpene coumarins from the roots of Ferula sinkiangensis and Ferula teterrima. Chem Pharm Bull 2006; 54:1595–8. [4] Iranshahy M, Iranshahi M. Traditional uses, phytochemistry and pharmacology of asafoetida (Ferula assa-foetida oleo-gum-resin)—a review. J Ethnopharmacol 2011;134:1–10. [5] Li G, Li X, Cao L, Shen L, Zhu J, Zhang J, et al. Steroidal esters from Ferula sinkiangensis. Fitoterapia 2014;97:247–52. [6] Sergio Rosselli AM, Bellone Gabriella, Formisano Carmen, Basile Adriana, Cicala Carla, Alfieri Alessio, et al. Antibacterial and anticoagulant activities of coumarins isolated from the flowers of Magydaris tomentosa. Planta Med 2006;72:5.
Contents lists available at ScienceDirect
Fitoterapia journal homepage: www.elsevier.com/locate/fitote
Sesquiterpene coumarins from seeds of Ferula sinkiangensis Guangzhi Li a, Xiaojin Li b, Li Cao a, Lijing Zhang a, Liangang Shen a, Jun Zhu b, Junchi Wang a, Jianyong Si a,⁎ a b
Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100193, China Xinjiang Institute of Chinese Materia Medica and Ethical Materia Medica, Urumqi 830002, China
a r t i c l e
i n f o
Article history: Received 3 February 2015 Accepted in revised form 19 March 2015 Available online 27 March 2015 Chemical compounds studied in this article: Farnesiferl C (PubChem CID: 15559239) Umbelliprenin (PubChem CID: 1781413) Colladonin (PubChem CID: 375430) Feselol (PubChem CID: 17977) Lehmannolone Lehmannolol Feklone Episamarcandin Sinkianone Fekrynol
a b s t r a c t A new sesquiterpene coumarin with a novel sesquiterpene carbon framework, Sinkiangenorin D, and ten known sesquiterpene coumarins were isolated from the seeds of Ferula sinkiangensis. The structures of these compounds, including the relative stereochemistry, were elucidated on the basis of spectroscopic data. All of the isolated compounds were tested against the AGS, HeLa, and K562 human cancer cell lines and showed cytotoxic activities with 50% inhibitory concentration values between 12.7 and 226.6 μM. © 2015 Elsevier B.V. All rights reserved.
Keywords: Ferula sinkiangensis Sesquiterpene coumarin Sesquiterpene Cytotoxic activities
1. Introduction The genus Ferula (Umbelliferea), including more than 150 species, is widely distributed across Central Asia and the Mediterranean [1]. Some plants of the genus Ferula have been used as pharmaceutical plants in many countries for centuries [2]. Ferula sinkiangensis is an important member of this genus, which is mainly distributed in the Xinjiang Uygur Autonomous Region of China, and has long been used as a folk medicine for the treatment of rheumatoid arthritis and stomach disorders ⁎ Corresponding author. Tel./fax: +86 010 57833299. E-mail address: [email protected] (J. Si).
http://dx.doi.org/10.1016/j.fitote.2015.03.022 0367-326X/© 2015 Elsevier B.V. All rights reserved.
[3]. Chemical studies of F. sinkiangensis have been carried out since the 1980s, and sesquiterpene coumarins were reported to be one of the main components [4]. In the course of our ongoing research on the constituents of the seeds of F. sinkiangensis, a new sesquiterpene coumarin with a novel fekrynol-type sesquiterpene system, Sinkiangenorin D (1), together with ten known compounds (2–11), lehmannolol (2), lehmannolone (3), episamarcandin (4), colladonin (5), sinkianone (6), fekrynol (7), fekolone (8), feselol (9), umbelliprenin (10), and farnesiferol C (11), were isolated (Fig. 1). In this study, we describe the isolation and structural elucidation of Sinkiangenorin D, as well as the antineoplastic activity of all of the isolated compounds listed above.
G. Li et al. / Fitoterapia 103 (2015) 222–226
223
2. Experimental
2.2. Plant material
2.1. General experimental procedures
The seeds of F. sinkiangensis were collected in July 2008 from Yili state, Xinjiang Uygur Autonomous Region of China by Prof. Xiaojin Li (Xinjiang Institute of Chinese Materia Medica and Ethical Materia Medica, Urumqi, China). A voucher specimen (No. AP21020720) was deposited in the Xinjiang Institute of Chinese Materia Medica and Ethical Materia Medica.
Ultraviolet (UV) spectra were recorded on a Shimadzu UV2550 spectrometer, and infrared (IR) spectra were obtained on an FTIR-8400S spectrometer. Melting points were measured (without correction) on a Fisher-Johns melting point apparatus (Fisher-Johns Scientific Company). Nuclear magnetic resonance (NMR) spectra were obtained using Bruker AV Ш 600 spectrometers operating at 150 MHz for 13C and at 600 MHz for 1H (chemical shift values are presented as δ values with TMS as the internal reference). High-resolution electrospray ionization mass spectrometry (HR-ESI-MS) was performed on a Thermo Scientific LTQ-Obitrap XL (Thermo Scientific; Bremen, Germany). Optical rotations were recorded in MeOH at 20 °C with a Perkin-Elmer 341 digital polarimeter. Semi-preparative high-performance liquid chromatography was performed on a K1001 analytic LC instrument (Beijing Chuangxintongheng Science & Technology Co., Ltd.,) equipped with a YMC-Pack ODS-A column (250 × 10 mm, S-5 μm, 12 nm), two K-501 pumps, and a K-2600 UV detector. Sephadex LH-20 (Pharmacia; Uppsala, Sweden) columns were used for column chromatography, and pre-coated silica gel plates (Zhi Fu Huang Wu Pilot Plant of Silica Gel Development, Yantai, PR China) were used for thin layer chromatography analysis. All solvents used were of analytical grade (Beijing Chemical Works; Beijing, PR China).
2.3. Extraction and isolation The powder of the seeds of F. sinkiangensis (4.2 kg) was refluxed with 95% EtOH (3 × 15 L) three times for 2 h to yield a viscous residue (401 g) that was suspended in water and partitioned by petroleum ether and dichloromethane (4 × 5 L), respectively. The dichloromethane viscous residue (132 g), eluted with CHCl3–MeOH (40:1 to 0:1, v/v), was fractionated into ten fractions (Fr. 1–10) by chromatography on a silica gel column (200–300 mesh, 10 × 100 cm). The ten fractions were separated by a semi-preparative liquid chromatography with a gradient of 60% MeOH–H2O to MeOH on an YMC-Pack ODS-A column (250 × 10 mm, S-5 μm, 12 nm), respectively. Finally, 1 (13 mg, tR 19.4 min) and 2 (15 mg, tR 23.5 min) were obtained in Fr. 2 using an MeOH–H2O (68:32) system; 3 (11.2 mg, tR 20.5 min), 4 (9.0 mg, tR 24.7 min), and 5 (15.0 mg, tR 27.6 min) were obtained in Fr. 3 using an MeOH–H2O (62:38) system; 6 (22.6 mg, tR 18.2 min), 7 (16.0 mg, tR 23.5 min), and 8 (12.0 mg, tR 25.7 min) were obtained in Fr. 5 using an MeOH–
Fig. 1. Structures of compounds (1–11).
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G. Li et al. / Fitoterapia 103 (2015) 222–226
(log ε) 325 (3.5) nm; IR νmax 3434, 1725, 1707, 1610, 1550, 1508, 1451, 1402, 1120 cm−1; 1H and 13C-APT see Table 1.
Table 1 1 H and 13C-APT NMR (600 MHz) data for 1 in CD3OD. δH (J, Hz) 2 3 4 5 6 7 8 9 10 1′ 1′ 2′ 3′ 4′ 5′1′ 6′ 6′ 7′ 7′ 8′ 9′ 9′ 10′ 10′ 11′ 12′ 13′ 14′ 15′ a
δc 6.18 (d, 9.6) 7.81 (d, 9.6) 7.45 (d, 8.4) 6.85 (dd, 8.4, 1.8) 6.76 (d, 1.8)
3.93 (d, 8.4) 3.74 (d,9.0) 2.96 (dd,11.4, 3.6)
1.92 (d, 12.6) 2.51 (d, 12.6) 1.25 (m) 1.57 a 1.85 a 1.62 a 1.57 a 1.32 (m) 1.41 (m) 3.55 (m) 1.13 (3H, s) 1.46 (3H, s) 1.59 (3H, s) 0.91 (3H, s)
HMBC 163.24 113.09 145.68 130.28 114.19 164.41 101.98 157.05 113.72 73.02
2.5. Cytotoxicity assay
C3′, C8′, C12′, C1′
41.95 44.33 125.56 132.16 25.51 33.02
3. Results and discussion
36.07 24.23 C2′, C4′ 31.80 63.44 23.11 20.76 20.31 16.42
The isolated compounds 1–11 were tested against the K562, HeLa, and AGS human cancer cell lines using the 3-(4,5dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) method [5]. Taxol (Cisen Pharmaceutical Co., H20057404) was used as a positive control. Briefly, freshly trypsinized cells were seeded at a density of 6 × 104 cells/mL per well in 96-well microliter plates with test compounds added from a dimethyl sulfoxide (DMSO) stock solution. After 48 h in culture at 37 °C in 5% CO2, 10 μL of MTT (4 mg/mL) was added to the attached cells and incubated for another 4 h. The supernatant was discarded and DMSO was added (200 μL) to each well. The absorbance was measured at 570 nm on a microplate reader (SpectramaxPlus 384; Molecular Devices; Sunnyvale, CA, USA).
C3′, C8′, C1′, C4′, C5′ C4′, C6′ C2′, C7′
Overlapping signals.
H2O (57:48) system; 9 (15.6 mg, tR 18.7 min) was obtained in Fr. 6 using an MeOH–H2O (53:47) system; and 10 (18.6 mg, tR 20.7 min) and 11 (10.6 mg, tR 23.7 min) were obtained in Fr. 8 using an MeOH–H2O (49:51) system.
2.4. Sinkiangenorin D (1) Sinkiangenorin D (1) is a colorless transparent solid: (+) HR-ESI-MS m/z 407.2179 (calcd for C24H32O5Na [M + Na]+, 407.2301); [α]20D −46.7 (c 0.075, MeOH); UV (MeOH) λmax
The EtOH extracts of the seeds of F. sinkiangensis were subjected to silica gel column chromatography; the preparative liquid chromatograph yielded a novel sesquiterpene coumarin (1) and ten known sesquiterpene coumarins (2–11). The structure of 1 was established by spectroscopic analysis. Compounds 2–11 were identified by comparing their spectroscopic data with published data for lehmannolol (2) [3], lehmannolone (3) [3], episamarcandin (4), colladonin (5), sinkianone (6) [6], fekrynol (7), fekolone (8), feselol (9), umbelliprenin (10), and farnesiferol C (11)8. Compound 1 was a colorless, transparent, solid material with a molecular formula of C24H32O5 deduced by HR-ESI-MS analysis ([M + Na]+ C24H32O5Na, calcd m/z 407.2179, obsd m/z 407.2301), which corresponds to 9° of unsaturation. The UV spectrum of 1 exhibited an absorption maximum at 325 nm, which is consistent with the presence of a coumarin moiety. The 1H NMR spectrum (Table 1) showed four methyl group signals at δH 0.91 (s, 15′-CH3), 1.13 (s, 12′-CH3), 1.46 (s, 13′CH3), and 1.59 (s, 14′-CH3). The heteronuclear single-quantum coherence (HSQC) and 13C-APT NMR spectra established that
Fig. 2. Key 2D NMR correlations of 1.
G. Li et al. / Fitoterapia 103 (2015) 222–226
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Fig. 3. Proposed biogenetic pathway for Sinkiangenorin D.
1 possessed a coumarin moiety (δ:164.41, 163.24, 157.05, 145.68, 130.28, 114.19, 113.72, 113.09, 101.98) and a sesquiterpene moiety containing four methyl groups at δC 16.42, 20.31, 20.76, 23.11; an olefinic bond at δC 125.56, 132.16; and an oxymethine group at δC 63.4. Taking into account the 2° of unsaturation for the sesquiterpene moiety, these results indicated that 1 is a sesquiterpene coumarin with a monocyclic sesquiterene moiety. Comprehensive analysis of the two-dimensional NMR data (Fig. 2) led to the identification of the planar structure of the sesquiterpene moiety of 1. The HSQC and 1H-1H COSY spectra showed two spin systems (H-3′/H-9′/H-10′/H-11′, H-6′/H-7′/ H-8′/H-15′), which established the presence of three partial structures (C-3′–C-9′–C-10′–C-11′, C-6′–C-7′–C8′–C-15′) as shown in Fig. 2. In the HMBC spectrum, the correlations from 12′-CH3 to C-3′, C-8′, C-1′; H-1′ to C-3′, C-8′, C-12′; 15′-CH3 to C-2′, C-7′; 13′-CH3 to C-4′, C-5′; 14′-CH3 to C-4′, C-6′; and H-9′ to C-2′, C-4′ indicated the presence of a 7-membered ring system. The correlations from H-1' to C-7 enabled the identification of the position of an ether linkage linking the coumarin to the sesquiterpene moiety. The relative configuration of all of the stereogenic centers in the sesquiterpene moiety was determined from the nuclear Overhauser effect spectroscopy (NOESY) data (Fig. 2). The
Table 2 In vitro antiproliferative activity of compounds 1–11. Compound
IC50 (μM) HeLa
1 2 3 4 5 6 7 8 9 10 11 Taxolb a b
a
20.4 ± 1.3 226.6 ± 1.0 81.1 ± 1.4 – – 77.9 ± 0.7 142.7 ± 3.2 – – 202.2 ± 1.2 86.9 ± 1.1 5.6 ± 0.01
K562
AGS
81.1 ± 1.0 – 136.9 ± 1.3 112.9 ± 0.6 141.6 ± 1.5 – – – – 141.6 ± 1.1 – 8.5 ± 0.15
104.8 ± 1.2 26.0 ± 0.9 – 83.8 ± 1.4 85.5 ± 2.2 – – 75.4 ± 2.1 – 12.7 ± 0.8 101.6 ± 1.3 3.5 ± 0.04
Values represent mean ± SD of triplicate experiments. Positive control substance.
correlations of 8′-H/12′-CH3 and 12′-CH3/H-9′ revealed that 8′-H, 12′-CH3, and H-9′ were coplanar. Considering that Sinkiangenorin D cannot crystallize and that there is no proper circular dichroism method available to determine the absolute configuration, the absolute configuration of Sikiangenorin D remains unknown. The structure of Sinkiangenorin D turned out to be different from that of common sesquiterpene and to consist of a unique skeleton. A possible biosynthetic pathway for Sinkiangenorin D is shown in Fig. 3. The naturally occurring fekrynol-type sesquiterene, also obtained from the seeds of F. sinkiangensis is perhaps the parent compound for the novel sesquiterpene moiety of Sinkiangenorin D. First, the formation of intermediate 2 could be initiated by the protonation of C4 and electron transfer reaction of the olefinic bond. Subsequently, the electrons of bond C4–C5 would transfer to C11, leading to formation of the seven-membered ring. The transfer of a methyl at C5′ and consequent proton loss would result in the formation of an olefinic bond of C4′–C5′, which would lead to the formation of this novel carbon framework. The isolated compounds 1–11 were tested for their cytotoxic activity in vitro against three human cancer cell lines (AGS, K562 and HeLa) using the MTT assay. The 50% inhibitory concentration (IC50) values were in the range of 12.7–226.6 μM and were compared to those of Taxol, which was used as the positive control. The data suggested that the isolated sesquiterpene coumarin derivatives had selective cytotoxic activity against the HeLa and AGS cancer cell lines (Table 2). Compounds 1, 2, 3, 6, 7, and 11 all displayed cytotoxicity against HeLa cells with IC50 values between 20.4 and 226.2 μM, and compounds 1, 2, 4, 5, 8, 10, and 11 showed cytotoxicity against AGS cells with IC50 values between 12.7 and 104.8 μM. Based on these biological results, it can be concluded that the sesquiterpene coumarin derivatives may play a role in the cytotoxic activity of F. sinkiangensis.
Acknowledgments This work was financially supported by the National Mega-project for Innovative Drugs (2012ZX09301-002-001; 2011ZX09307-002-01), the National Natural Science Foundation of China (81460661; 81460586), and the Program for Innovative Research Team in IMPLAD.
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