Fitoterapia 97 (2014) 247–252
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Fitoterapia journal homepage: www.elsevier.com/locate/fitote
Steroidal esters from Ferula sinkiangensis Guangzhi Li a, Xiaojin Li b, Li Cao a, Liangang Shen a, Jun Zhu b, Jing Zhang a, Junchi Wang a, Lijing Zhang 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 14 May 2014 Accepted in revised form 19 June 2014 Available online 28 June 2014 Keywords: Ferula sinkiangensis Steroidal esters Organic acid glycoside Lignin compounds Cytotoxic activities
a b s t r a c t Two new steroidal esters with an unusual framework, Sinkiangenorin A and B, a new organic acid glycoside, Sinkiangenorin C, and four known lignin compounds were isolated from the seeds of Ferula sinkiangensis. The structures of these compounds were established by spectroscopic analysis and single-crystal X-ray diffraction. All of the isolated compounds were tested against Hela, K562 and AGS human cancer cell lines. Sinkiangenorin C showed cytotoxic activity against AGS cells with an IC50 of 36.9 μM. © 2014 Elsevier B.V. All rights reserved.
Chemical compounds studied in this article: Arctigenin (PubChem CID: 64981) Matairesinol (PubChem CID: 119205) Secroisolariciresinol (PubChem CID: 6336781) Lariciresinol (PubChem CID: 332427)
1. Introduction Ferula sinkiangensis is an important member of the genus Ferula. It mainly distributes in the Xinjiang Region of China, and has been used in traditional remedies for stomach disorders and rheumatoid arthritis [1]. Modern pharmacological studies have established the antiulcerative [2], antibacterial [3], anti-inflammatory and immunopharmacological activities [4] of this plant. Previous studies have focused on the root, resin, and volatile oils of F. sinkiangensis, whereas the constituent studies of seeds of F. sinkiangensis have nearly not been reported [5]. In our study, two new steroidal esters, Sinkiangenorin A (1) and B (2), and a new organic acid glycoside, Sinkiangenorin C (3), together with four known lignin compounds (4–7) were isolated from the seeds of F. sinkiangensis (Fig. 1). In this ⁎ Corresponding author. Tel./fax: +86 10 57833299. E-mail address: [email protected] (J. Si).
http://dx.doi.org/10.1016/j.fitote.2014.06.016 0367-326X/© 2014 Elsevier B.V. All rights reserved.
paper, we describe the structure elucidation of the three new compounds and their antineoplastic activity. 2. Experimental 2.1. General experimental procedures Melting points were determined without correction on a Fisher-Johns melting point apparatus (Fisher-Johns Scientific Company). Optical rotations were measured in MeOH at 20 °C on a Perkin-Elmer 341 digital polarimeter. UV spectra were obtained on a Shimadzu UV2550 spectrometer, and IR spectra were recorded on a FTIR-8400S spectrometer. 1D and 2D NMR spectra were performed on Bruker AV Ш 600 spectrometers operating at 600 MHz for 1H and 150 MHz for 13C. Chemical shifts are expressed in δ (ppm) and tetramethylsilane (TMS) was used as the internal reference. HRESIMS spectra were measured on a Thermo Scientific
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Fig. 1. Structures of compounds 1–7.
LTQ-Obitrap XL (Thermo Scientific, Bremen, Germany). Silica gel (100–200 mesh, Qingdao Haiyang Chemical Co., Ltd., Qingdao, P. R. China) and Sephadex LH-20 (Pharmacia Biotech, Sweden) were used for column chromatography. Precoated silica gel plates (F254, 0.25 mm, Qingdao Haiyang Chemical Co., Ltd., Qingdao, P. R. China) were used for TLC analysis. Semi-preparative HPLC was carried out on a K1001 analytic LC instrument (Beijing Chuangxintongheng Science & Technology CO., LTD) equipped with two K-501 pumps, a K-2600 UV detector, and a column of YMC-Pack ODS-A (250 × 10 mm, S-5 μm, 12 nm). HPLC analysis was performed on a Waters 2489 pump equipped with a Waters UV/visible detector and a Thermo Syncronis aQ column (250 × 4.6 mm, 5 μm). All solvents used for column chromatography were at least analytical grade (Beijing Chemical Works, Beijing, P. R. China), and solvents used for HPLC were HPLC grade (Fisher Scientific).
2.2. Plant material The seeds of F. sinkiangensis were collected in July 2008 from Yili state, Xinjiang Uygur Autonomous Region of China, and were identified 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.
2.3. Extraction and isolation The seeds of F. sinkiangensis (4.2 kg) were crushed and refluxed with 95% EtOH (3 × 15 L) three times for 2 h. The combined EtOH extracts were evaporated under reduced pressure to yield a viscous residue (400 g), which was suspended in water and partitioned by petroleum ether (b.p. 60–90 °C) and dichloromethane (4 × 5 L), respectively. The dichloromethane extract (132 g) was fractionated into ten fractions (Fr. A–G) by silica gel chromatography, eluting with CHCl3–MeOH (40:1 to 0:1, v/v). Fr. B (3.0 g) was subjected to silica gel column chromatography and eluted with CHCl3– MeOH (300:1 to 0:1, v/v), to give twenty fractions (Fr. B-1–20). Fr.B-13 (0.6 g) was further chromatographed over a Sephadex LH-20 column (2.5 × 150 cm) eluting with MeOH, yielding ten fractions (Fr. B-13-1–10). Fr. B-13-5 (45.0 mg) and Fr. B-13-6 (30 mg) were purified by semi-preparative HPLC to obtain compound 1 (19.0 mg, MeOH–H2O, 58:42,flow rate: 2.0 mL/min, tR = 19 min) and compound 2 (7.0 mg, MeOH– H2O, 62:38, flow rate: 2.0 mL/min, tR = 21 min). Fr.B-19 (0.7 g) was subjected to chromatography on a silica gel column (100–200 mesh, 2.5 × 50 cm) eluting with a gradient of CHCl3–MeOH (20:1 to 0:1, v/v) to give thirty fractions (Fr.B-19-1–30). Fr.B-19-17–19 was subjected to preparative scale chromatography eluting with CHCl3–MeOH–H2O (7.5:2.5:1). Finally, compound 3 was obtained (9 mg, Rf = 0.6). Fr.B-19-(25–27) were each separated by semi-preparative liquid chromatography with a gradient of 30% MeOH–H2O to
G. Li et al. / Fitoterapia 97 (2014) 247–252
MeOH on an YMC-Pack C18 column. Finally, 4 (23 mg, tR 23 min) and 5 (30 mg, tR 33 min) were obtained in fraction Fr.B-19–25 using a MeOH–H2O (47:53) system. Compounds 6 (16.6 mg, tR 33.7 min) and 7 (14.5 mg, tR 37.6 min) were obtained in fraction Fr.B-19–27 using a MeOH–H2O (52:48) system.
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2.33(1H, m, H-2), 2.71(2H, dd, J = 7.8, 1.2Hz, H-3), 3.25 (1H, m, H-1′), 1.41 (3H, d, J = 6.0 Hz, 2′-CH3), 1.26 (3H, S, 5-CH3), 1.27 (3H, S,4-CH3), 4.46 (1H, d, J = 9.6Hz, H-1″), 3.82 (1H, dd, J = 12, 2.4Hz, H-6″), 3.64 (1H, dd, J = 12, 2.4Hz, H-6″), 3.11– 3.34 (4H, m, H-2-5″); 13C-APT (150 MHz, CD3OD) δ: 180.1 (C-1, C), 53.4 (C-2, CH), 31.8 (C-3, CH2), 80.2 (C-1′, CH), 78.6 (C-4, C), 22.9 (C-2′, CH3), 24.6 (C-5, CH3), 24.9 (CH3-4), 98.6 (C-1″, CH), 75.3 (C-2″, CH), 77.8 (C-3″, CH), 71.9 (C-4″, CH), 78.4 (C-5″, CH), 63.2 (C-6″, CH2).
2.3.1. Sinkiangenrin A (1) Colorless crystals (MeOH): mp 217–218 °C; IR νmax 3406, 1739, 1731, 1703, 1597, 1249, 626 cm−1; UV (MeOH) λmax (log ε) 289 (2.95) nm; (+) HRESIMS [M + Na]+ m/z 443.2056 (calcd for C23H32O7Na+ 443.2046); [α]20 D = −6.0 (c 0.1, MeOH); 1H and 13C-APT see Table 1.
2.4. Cytotoxicity assay The cytotoxicity of compounds 1–7 were assessed using the MTT method against Hela, K562, and AGS human cancer cell lines. All the cells were cultured in DMEM medium (GIBCO Invitrogen Corp., Carlsbad, CA) and supplemented with 10% fetal bovine serum (Sijiqing, Hangzhou, China). Cells were cultured at a density of 6 × 104 cells/mL per well in a 96-well plate. Five different concentrations (0–50 μM) of each compound dissolved in dimethyl sulfoxide (DMSO) were added to each well. Taxol (Cisen Pharmaceutical Co., H20057404) was used as a positive control, and each concentration was tested in triplicate. After incubation at 37 °C in 5% CO2 for 48 h, 10 μL of MTT (4 mg/mL) was added to each well and cultured for another 4 h. Then the supernatant was discarded and DMSO was added (200 μL) to each well. The absorbance was recorded on a microplate reader
2.3.2. Sinkiangenrin B (2) Colorless crystals (MeOH): mp 280– °C; IR νmax 3453, 1738, 1456, 1260, 800 cm−1; UV (MeOH) λmax (log ε) 312 (2.75) nm; (+) HRESIMS [M + H]+ m/z 403.2119 (calcd 403.2121); [α]20 D = + 1.5 (c 0.04, MeOH); 1H and 13C-APT see Table 1. 2.3.3. Sinkiangenrin C (3) White amorphous powder: (−) HR-ESI-MS m/z 355.1160 (calcd for C14H27O10 [M + H2O]−, 355.1160); [α]20 D = −7.0 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 202 (2.01) nm; IR (film) νmax 1739 cm−1; 1H NMR (600 MHz, CD3OD) δ:
Table 1 1 H and 13C-APT NMR (600 MHz) data for 1 and 2 in DMSO-d6. 1 δH (J, Hz) 1a 1b 2a 2b 3 3-OH 4-OH 4 5 6 7a 7b 8 9 11 11-OH 12 13 14 15a 15b 16a 16b 17 18 19 20 21 1′ 2′ a
2.245 (m) 1.793 (m) 1.816a 1.542 (m) 3.891 (brs) 5.489 (brs) 15.790 (s)
3.250 (d, 16.2) 1.750a 2.072 (d, 11.4) 3.347a 4.813 (d, 4.20) 2.201 (t, 13.2) 1.736 (dd, 13.2, 4.20)
1.599 (dd, 12.6, 4.20) 1.800a 2.315 (m) 1.863 (dd, 13.2, 4.20) 1.939 (d, 4.8) 5.033 (m) 1.094 (s) 0.944 (s) 1.123 (d, 6.0) 1.772a
Overlapping signals.
2 δC
type
δH (J, Hz)
δC
type
33.6
CH2
333.6
CH2
26.7
CH2
1.521 2.080 2.380 2.587
32.3
CH2
64.4
CH
178.6 111.9 202.7 42.5
C C C CH2
48.7 57.7 37.6 65.3
C CH C CH
48.1
CH2
45.4 214.5 40.5
C C CH2
16.2
CH2
52.5 69.1 21.0 21.9 18.1 20.4 169.2
CH CH CH3 CH3 CH3 CH3 C
(td, 4.80, 13.8) (m) (m) (m)
192.8
C
141.5 132.2 120.6 138.0
C C CH CH
50.0 50.9 37.0 30.2
C CH C CH2
77.9
CH
54.1 216.7 43.3
C C CH2
20.0
CH2
45.7 71.3 19.2 19.2 18.2 20.7 169.4
CH CH CH3 CH3 CH3 CH3 C
8.312 (s)
6.590 (d, 10.2) 5.798 (d, 10.2)
2.112 (d, 9.00) 2.200 (m) 3.636 (t, 9.0) 5.335 (s, OH)
a
1.756 2.303a 1.915a 2.314a 2.424 (d, 5.40) 5.006 (qd, 1.80, 6.00) 0.811 (s) 0.959 (s) 1.149 (d, 6.60) 1.855 (s)
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at a wavelength of 570 nm (SpectramaxPlus 384; Molecular Devices, Sunnyvale, CA) [6]. 3. Results and discussion After successive stages of chromatography, the F. sinkiangensis seed extract yielded two new steroidal esters Sinkiangenrin A and B and a new organic glycoside Sinkiangenrin C, together with four known compounds. Their structures were elucidated on the basis of spectroscopic analysis and X-ray crystallographic data. Compounds 4–7 were identified by comparing their spectroscopic data with published data for arctigenin (4) [7], matairesinol (5) [7], secroisolariciresinol (6) [8] and lariciresinol (7) [9]. Compound 1 was obtained as a colorless crystal from MeOH. Its molecular formula was deduced as C23H32O7 on the basis of the [M + Na]+ ion peak at m/z 443.2056 (calcd
for C23H32O7Na+, 443.2046) in the HRESIMS, which corresponds to 8° of unsaturation. The IR absorption bands indicated the presence of carbonyl (1703 and 1739 cm−1) and hydroxyl groups (3406 cm−1). The 1H NMR spectrum (Table 1) of 1 exhibited four methyl group signals at δH 1.09 (s, CH3-19), 0.94 (s, CH3-20), 1.12 (d, CH3-21), 1.77 (s, CH3-1′) and one hydroxyl signal at δH 15.79 (OH-4). The HSQC and 13C-APT NMR spectra of 1 showed 23 carbon signals including four methyl groups at δC 21.0, 21.9, 18.1, and 20.4; two olefinic carbons at δC 178.6, 111.9 (an enol group), and three carbonyl groups at δC 202.7, 214.5, and 169.2. These results indicated that the degree of unsaturation for the rest of structure was 4, suggesting that there would be four rings in the structure of compound 1. The planar structure of 1 was determined by the interpretation of the 2D NMR data (Supporting Information, Fig. 2). The 1H-1H COSY and HSQC spectra showed three spin systems (H-1/H-2/H-3/HO-3, H-9/H-11/HO-11/H-12, H-15/
Fig. 2. Key 2D NMR correlations of 1–3.
G. Li et al. / Fitoterapia 97 (2014) 247–252
H-16/H-17/H-18/CH3-21), which suggested the presence of three partial structures (C-1-C-3, C-9-C-12, C15-C-18-C-21) as shown in Fig. 2. In the HMBC spectrum, the correlations from H-1′ and H-18 to C-2′ confirmed the presence of the ester structure; the correlations from H-3, HO-4, H-2b to C-4; H-7, H-1, HO-4, CH3-19 to C-5; HO-4, H-7 to C-6; H-9, H-7 to C-8; H-1, CH3-19 to C-9; and H-1, CH3-19 to C-10 indicated the presence of an A/B ring system. The correlations from H-9, H-7, H-15, H-12, CH3-20 to C-14; and H-12, CH3-20 to C-13 supported the presence of the B/C ring system. The presence of the C/D ring system was confirmed by the correlations from H-17 to C-13; H-15 to C-8; and H-17, H-15 to C-14. The relative configuration of 1 was determined from the NOESY data (Supporting information, Fig. 2). The correlations of HO-3/H-9 (β) and HO-11/H-9 (β) revealed that HO-3 and HO-11 were β-oriented. In addition, crosspeaks of H-11/ CH3-19; CH3-20/H-11; CH3-20/H-18; H-21/H-17 and H-9/ H-17 supported that CH3-19, CH3-20 and H-18 were α-oriented and H-17 was β-oriented. We also obtained crystals of 1 and performed an X-ray diffraction experiment using Cu Kα radiation (λ = 1.54178 Å). The X-ray crystallographic data (Supporting Information) confirmed the planar structure of 1 and allowed the assignment of the absolute configuration as 3S, 8R, 9S, 10S, 11S, 13S, 17R, and 18R [Flack parameter = 0.2(4)] [10]. The structure of 1 was similar to the published structure of oleagenin-type cardenolide, which also bears an unusual framework, although the configurations of C-8, C-9, C-10 and C-13 were different from that of oleagenin-type cardenolide [11]. Therefore, this is the first report of the isolation of this type of steroidal ester, which we have named Sinkiangenrin A. Compound 2 was obtained as colorless needles (MeOH), and analyzed for C23H30O6 by positive-ion HRESIMS. Its IR spectrum showed the presence of hydroxyl and carbonyl groups at 3453 and 1738 cm−1, respectively. The 1H NMR spectrum suggested the presence of four methyl signal at δH
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0.81, 0.96, 1.45, and 1.85; and two olefinic protons at δH 5.79 (d, J = 10.2 Hz), and 6.59 (d, J = 10.2 Hz). The 13C-APT spectrum showed 23 carbons including four methyl carbons; four olefinic carbons at δC 120.6, 132.2, 138.0, and 141.5 and three carbonyl carbons at δC 169.4, 192.8, and 216.7. The assignment of 1H NMR and 13C-APT spectroscopic data of 2 (Table 1) was based on HSQC, HMBC and 1H-1H COSY spectrum (Supporting Information). The 1H NMR and 13C-APT features of 2 were similar to those of 1, except that the hydroxyl at C-3 in compound 1 was replaced by carbonyl group in compound 2, the hydroxyl at C-11 in 1 transferred to C-12 in 2 and the carbonyl group at C-6 in compound 1 was replaced by the ethylenic bond between C-6 and C-7 in compound 2. In the HMBC spectrum of 2, correlations from H-2, H-1, HO-4 to C-3 suggested the presence of a ketone group at C-3; the correlations from H-6 to C-4, C-5, C-10, C-8 and H-7 to C-8, C-9, and C-15 confirmed the presence of a 6, 7-ethylenic bond. The correlations from H-11, CH3-20 to C-12 (δC 77.9) supported the presence of a hydroxyl at C12. The relative configuration of 2 was confirmed by NOESY data (Supporting Information, Fig. 2). The correlations of H-12/CH3-19 and H-12/CH3-20 (β) suggested that they were β-oriented. The crosspeaks of H-17(α)/ H-9 revealed that they were cofacial. The planar structure of 2 was confirmed by an X-ray diffraction experiment using Cu Kα radiation (λ = 1.54178 Å). The absolute configuration was assigned as 8S, 9S, 10S, 12R, 13R, 17R, and 18R [Flack parameter = 0.0(10)] [12], and we named this compound Sinkiangenrin B. Sinkiangenrin A (1) and B (2) feature an unusual carbon skeleton and a plausible biogenetic origin of this skeleton could be traced back to the C21-steroids as shown in Fig. 3. The rearrangement of the D-ring could be initiated by the formation of a C8–C14 pregnane epoxide, followed by the formation of a carbocation center at C-8. Subsequently, enzymatic Wagner–Meerwein rearrangements lead to the migration of the C14–15 bond to C-8 and the generation of a
Fig. 3. Proposed biogenetic pathway for and 2.
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Table 2 In vitro antiproliferative activity of compounds 1–7. Compound
1 2 3 4 5 6 7 Taxolb a b
IC50 (μM) Hela
K562
AGS
– – 215.2 ± 1.4a 105.1 ± 1.3 85.5 ± 2.2 212.7 ± 0.7 142.7 ± 3.2 5.6 ± 0.01
– – – – – – – 8.5 ± 0.15
– – 36.9 ± 1.7 78.2 ± 1.6 99.4 ± 2.5 – – 3.5 ± 0.04
Value present mean ± SD of triplicate experiments. Positive control substance.
protonated carbonyl at C-14, which is eventually deprotonated [14]. The backbone is then modified by a series of enzymecatalytic reactions to afford 1 and 2. Compound 3 was obtained as white amorphous powder (MeOH). It was assigned a molecular formula of C14H26O9 by negative-ion HRESIMS. The IR absorption bands indicated the presence of a carbonyl group (1739 cm−1). The 1H NMR spectrum showed three methyl signals [δH 1.264, (s); 1.281, (s); 1.409, (d, J = 6.0Hz)]. The 13C-APT spectrum showed 14 carbon signals including three methyl carbons at δC 22.9, 24.6, 24.9 and a carbonyl carbon at δC 180.1. Sugar proton and carbon signals in the NMR spectra of 3 were assigned by 1H–1H COSY, HSQC and HMBC spectra. The 1H–1H COSY spectra displayed a spin system (H-3/H-2/H-1′/CH3-2′) which suggested the presence of the partial structure (C-3-C-2-C-1′-CH3-2′) as shown in Fig. 2. In the HMBC spectrum (Fig. 2), long-range couplings were observed for H-3 (δH 2.71), H-2 (δH 2.33), H-1′ (δH 3.25) to C_O (δC 180.1); and H-1″ (δH 4.46) to C-4 (δC 78.6), suggesting the location of the carbonyl group and glucosidic bond at C-2 and C-4, respectively. The 13C NMR spectrum of the sugar moiety was similar to the published spectra of β-D-glucopyranose and in the 1H NMR spectrum, large coupling constants between H-1″ and H-2″ (J = 9.6 Hz) indicated that the glycone unit was β-D-glucopyranose [13]. Therefore, the structure of 3 was assigned as 2-(1-hydroxyethyl)-4-methyl pentanoic acid-4-O-β-D-glucopyranoside. This is the first reported isolation of this compound, and we have named it Sinkiangenrin C. The isolated compounds 1–7 were tested for their cytotoxic activity in vitro against three human cancer cell lines, Hela, K562 and AGS, using the MTT assay and choosing Taxol as the positive control. The data in Table 2 suggested that the isolated steroidal ester derivatives (1 and 2) had no cytotoxic activity against Hela, K562 and AGS cancer cell lines. Compounds 3–5
all displayed cytotoxicity against AGS cells with IC50 values between 36.9 and 98.3 μM, and 3–7 showed weak cytotoxicity against Hela cells with IC50 value between 85.5 and 215.2 μM. Based on our study, it can be concluded that the organic acid glycoside and lignin compounds may play a role in the cytotoxic activity of Ferula sinkiangensis. Acknowledgments This work was financially supported by the National Natural Science Foundation of China (81060265), National Mega-project for Innovative Drugs (2012ZX09301-002-001; 2011ZX09307-002-01), and Program for Innovative Research Team in IMPLAD. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.fitote.2014.06.016. References [1] Yang J, An Z, Li Z, Jing S, Qina H. Sesquiterpene coumarins from the roots of Ferula sinkiangensis and Ferula teterrima. Chem Pharm Bull 2006;54(11):1595–8. [2] Li X, Jiang L, Da P. Preparation and investigation of the pharmacodynamics of effective antiulcerative composition in Ferula sinkiangensis K.M. Shen. Sci Technol 2007;9(10):8–10. [3] Yang L, Zhao HQ, Yao G, Cai Z, Wang JM. The preliminary study of antibacterial effect of five kinds of bacterials of Ferula sinkiangensis. J Tradit Chin Vet Med 2007;54:33–4. [4] Zhang H, Hu J. Anti-inflammatory and immunopharmacological effect of Xinjiang ferula oil. Chin Pharmacol Bull 1987;5(3):288–90. [5] Wang Y, Si J, Li X, Lin J, Zhu J. Chemical constituents from the seeds of Ferula sinkiangensis. Mod Chin Med 2011;13(1):26–8. [6] Wang S, Ma G, Zhong M, Yu S, Xu X, Hu Y, et al. Triterpene saponins from Tabellae Clinopodii. Fitoterapia 2013;90:14–9. [7] Wang H, Yang J. Studies on the chemical constituent Ts of Ar Ctiumlappa L. Acta Pharm Sin 1993;28(12):911–7. [8] Zeng X, Wang G, Wu X, Li G, Ye W, Li Y. Chemical constituents from Syringa pinnatifolia. Chin Tradit Herb Drugs 2013;44(13D):1721–5. [9] Cui Y, Mu Q, Hu C. Study on the phenylpropanoids from Caragana rosea. Nat Prod Res Dev 2003;15(4):277–83. [10] Crystallographic data of compound 1 have been deposited at the Cambridge, UK., under the reference number CCDC 980109. [11] Hua Y, Liu H, Ni W, Chen C, Lu Y, Wang C, et al. 5a-Steroidal glycosides from Parepigynum funingense. J Nat Prod 2003;66(6):898–900. [12] Crystallographic data of compound 2 have been deposited at the Cambridge, UK., under the reference number CCDC 980110. [13] Li H, Yu Y, Wang Z, Dai Y, Gao H, Xiao W, et al. Iridoid and bis-iridoid glucosides from the fruit of Gardenia jasminoides. Fitoterapia 2013;88:7–11. [14] Zhang M, Zhu Y, Zhan G, Shu P, Sa R, Lei l, et al. Micranthanone A, a new diterpene with an unprecedented carbon skeleton from Rhododendron micranthum. Org Lett 2013;15(12):3094–7.
Contents lists available at ScienceDirect
Fitoterapia journal homepage: www.elsevier.com/locate/fitote
Steroidal esters from Ferula sinkiangensis Guangzhi Li a, Xiaojin Li b, Li Cao a, Liangang Shen a, Jun Zhu b, Jing Zhang a, Junchi Wang a, Lijing Zhang 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 14 May 2014 Accepted in revised form 19 June 2014 Available online 28 June 2014 Keywords: Ferula sinkiangensis Steroidal esters Organic acid glycoside Lignin compounds Cytotoxic activities
a b s t r a c t Two new steroidal esters with an unusual framework, Sinkiangenorin A and B, a new organic acid glycoside, Sinkiangenorin C, and four known lignin compounds were isolated from the seeds of Ferula sinkiangensis. The structures of these compounds were established by spectroscopic analysis and single-crystal X-ray diffraction. All of the isolated compounds were tested against Hela, K562 and AGS human cancer cell lines. Sinkiangenorin C showed cytotoxic activity against AGS cells with an IC50 of 36.9 μM. © 2014 Elsevier B.V. All rights reserved.
Chemical compounds studied in this article: Arctigenin (PubChem CID: 64981) Matairesinol (PubChem CID: 119205) Secroisolariciresinol (PubChem CID: 6336781) Lariciresinol (PubChem CID: 332427)
1. Introduction Ferula sinkiangensis is an important member of the genus Ferula. It mainly distributes in the Xinjiang Region of China, and has been used in traditional remedies for stomach disorders and rheumatoid arthritis [1]. Modern pharmacological studies have established the antiulcerative [2], antibacterial [3], anti-inflammatory and immunopharmacological activities [4] of this plant. Previous studies have focused on the root, resin, and volatile oils of F. sinkiangensis, whereas the constituent studies of seeds of F. sinkiangensis have nearly not been reported [5]. In our study, two new steroidal esters, Sinkiangenorin A (1) and B (2), and a new organic acid glycoside, Sinkiangenorin C (3), together with four known lignin compounds (4–7) were isolated from the seeds of F. sinkiangensis (Fig. 1). In this ⁎ Corresponding author. Tel./fax: +86 10 57833299. E-mail address: [email protected] (J. Si).
http://dx.doi.org/10.1016/j.fitote.2014.06.016 0367-326X/© 2014 Elsevier B.V. All rights reserved.
paper, we describe the structure elucidation of the three new compounds and their antineoplastic activity. 2. Experimental 2.1. General experimental procedures Melting points were determined without correction on a Fisher-Johns melting point apparatus (Fisher-Johns Scientific Company). Optical rotations were measured in MeOH at 20 °C on a Perkin-Elmer 341 digital polarimeter. UV spectra were obtained on a Shimadzu UV2550 spectrometer, and IR spectra were recorded on a FTIR-8400S spectrometer. 1D and 2D NMR spectra were performed on Bruker AV Ш 600 spectrometers operating at 600 MHz for 1H and 150 MHz for 13C. Chemical shifts are expressed in δ (ppm) and tetramethylsilane (TMS) was used as the internal reference. HRESIMS spectra were measured on a Thermo Scientific
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G. Li et al. / Fitoterapia 97 (2014) 247–252
Fig. 1. Structures of compounds 1–7.
LTQ-Obitrap XL (Thermo Scientific, Bremen, Germany). Silica gel (100–200 mesh, Qingdao Haiyang Chemical Co., Ltd., Qingdao, P. R. China) and Sephadex LH-20 (Pharmacia Biotech, Sweden) were used for column chromatography. Precoated silica gel plates (F254, 0.25 mm, Qingdao Haiyang Chemical Co., Ltd., Qingdao, P. R. China) were used for TLC analysis. Semi-preparative HPLC was carried out on a K1001 analytic LC instrument (Beijing Chuangxintongheng Science & Technology CO., LTD) equipped with two K-501 pumps, a K-2600 UV detector, and a column of YMC-Pack ODS-A (250 × 10 mm, S-5 μm, 12 nm). HPLC analysis was performed on a Waters 2489 pump equipped with a Waters UV/visible detector and a Thermo Syncronis aQ column (250 × 4.6 mm, 5 μm). All solvents used for column chromatography were at least analytical grade (Beijing Chemical Works, Beijing, P. R. China), and solvents used for HPLC were HPLC grade (Fisher Scientific).
2.2. Plant material The seeds of F. sinkiangensis were collected in July 2008 from Yili state, Xinjiang Uygur Autonomous Region of China, and were identified 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.
2.3. Extraction and isolation The seeds of F. sinkiangensis (4.2 kg) were crushed and refluxed with 95% EtOH (3 × 15 L) three times for 2 h. The combined EtOH extracts were evaporated under reduced pressure to yield a viscous residue (400 g), which was suspended in water and partitioned by petroleum ether (b.p. 60–90 °C) and dichloromethane (4 × 5 L), respectively. The dichloromethane extract (132 g) was fractionated into ten fractions (Fr. A–G) by silica gel chromatography, eluting with CHCl3–MeOH (40:1 to 0:1, v/v). Fr. B (3.0 g) was subjected to silica gel column chromatography and eluted with CHCl3– MeOH (300:1 to 0:1, v/v), to give twenty fractions (Fr. B-1–20). Fr.B-13 (0.6 g) was further chromatographed over a Sephadex LH-20 column (2.5 × 150 cm) eluting with MeOH, yielding ten fractions (Fr. B-13-1–10). Fr. B-13-5 (45.0 mg) and Fr. B-13-6 (30 mg) were purified by semi-preparative HPLC to obtain compound 1 (19.0 mg, MeOH–H2O, 58:42,flow rate: 2.0 mL/min, tR = 19 min) and compound 2 (7.0 mg, MeOH– H2O, 62:38, flow rate: 2.0 mL/min, tR = 21 min). Fr.B-19 (0.7 g) was subjected to chromatography on a silica gel column (100–200 mesh, 2.5 × 50 cm) eluting with a gradient of CHCl3–MeOH (20:1 to 0:1, v/v) to give thirty fractions (Fr.B-19-1–30). Fr.B-19-17–19 was subjected to preparative scale chromatography eluting with CHCl3–MeOH–H2O (7.5:2.5:1). Finally, compound 3 was obtained (9 mg, Rf = 0.6). Fr.B-19-(25–27) were each separated by semi-preparative liquid chromatography with a gradient of 30% MeOH–H2O to
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MeOH on an YMC-Pack C18 column. Finally, 4 (23 mg, tR 23 min) and 5 (30 mg, tR 33 min) were obtained in fraction Fr.B-19–25 using a MeOH–H2O (47:53) system. Compounds 6 (16.6 mg, tR 33.7 min) and 7 (14.5 mg, tR 37.6 min) were obtained in fraction Fr.B-19–27 using a MeOH–H2O (52:48) system.
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2.33(1H, m, H-2), 2.71(2H, dd, J = 7.8, 1.2Hz, H-3), 3.25 (1H, m, H-1′), 1.41 (3H, d, J = 6.0 Hz, 2′-CH3), 1.26 (3H, S, 5-CH3), 1.27 (3H, S,4-CH3), 4.46 (1H, d, J = 9.6Hz, H-1″), 3.82 (1H, dd, J = 12, 2.4Hz, H-6″), 3.64 (1H, dd, J = 12, 2.4Hz, H-6″), 3.11– 3.34 (4H, m, H-2-5″); 13C-APT (150 MHz, CD3OD) δ: 180.1 (C-1, C), 53.4 (C-2, CH), 31.8 (C-3, CH2), 80.2 (C-1′, CH), 78.6 (C-4, C), 22.9 (C-2′, CH3), 24.6 (C-5, CH3), 24.9 (CH3-4), 98.6 (C-1″, CH), 75.3 (C-2″, CH), 77.8 (C-3″, CH), 71.9 (C-4″, CH), 78.4 (C-5″, CH), 63.2 (C-6″, CH2).
2.3.1. Sinkiangenrin A (1) Colorless crystals (MeOH): mp 217–218 °C; IR νmax 3406, 1739, 1731, 1703, 1597, 1249, 626 cm−1; UV (MeOH) λmax (log ε) 289 (2.95) nm; (+) HRESIMS [M + Na]+ m/z 443.2056 (calcd for C23H32O7Na+ 443.2046); [α]20 D = −6.0 (c 0.1, MeOH); 1H and 13C-APT see Table 1.
2.4. Cytotoxicity assay The cytotoxicity of compounds 1–7 were assessed using the MTT method against Hela, K562, and AGS human cancer cell lines. All the cells were cultured in DMEM medium (GIBCO Invitrogen Corp., Carlsbad, CA) and supplemented with 10% fetal bovine serum (Sijiqing, Hangzhou, China). Cells were cultured at a density of 6 × 104 cells/mL per well in a 96-well plate. Five different concentrations (0–50 μM) of each compound dissolved in dimethyl sulfoxide (DMSO) were added to each well. Taxol (Cisen Pharmaceutical Co., H20057404) was used as a positive control, and each concentration was tested in triplicate. After incubation at 37 °C in 5% CO2 for 48 h, 10 μL of MTT (4 mg/mL) was added to each well and cultured for another 4 h. Then the supernatant was discarded and DMSO was added (200 μL) to each well. The absorbance was recorded on a microplate reader
2.3.2. Sinkiangenrin B (2) Colorless crystals (MeOH): mp 280– °C; IR νmax 3453, 1738, 1456, 1260, 800 cm−1; UV (MeOH) λmax (log ε) 312 (2.75) nm; (+) HRESIMS [M + H]+ m/z 403.2119 (calcd 403.2121); [α]20 D = + 1.5 (c 0.04, MeOH); 1H and 13C-APT see Table 1. 2.3.3. Sinkiangenrin C (3) White amorphous powder: (−) HR-ESI-MS m/z 355.1160 (calcd for C14H27O10 [M + H2O]−, 355.1160); [α]20 D = −7.0 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 202 (2.01) nm; IR (film) νmax 1739 cm−1; 1H NMR (600 MHz, CD3OD) δ:
Table 1 1 H and 13C-APT NMR (600 MHz) data for 1 and 2 in DMSO-d6. 1 δH (J, Hz) 1a 1b 2a 2b 3 3-OH 4-OH 4 5 6 7a 7b 8 9 11 11-OH 12 13 14 15a 15b 16a 16b 17 18 19 20 21 1′ 2′ a
2.245 (m) 1.793 (m) 1.816a 1.542 (m) 3.891 (brs) 5.489 (brs) 15.790 (s)
3.250 (d, 16.2) 1.750a 2.072 (d, 11.4) 3.347a 4.813 (d, 4.20) 2.201 (t, 13.2) 1.736 (dd, 13.2, 4.20)
1.599 (dd, 12.6, 4.20) 1.800a 2.315 (m) 1.863 (dd, 13.2, 4.20) 1.939 (d, 4.8) 5.033 (m) 1.094 (s) 0.944 (s) 1.123 (d, 6.0) 1.772a
Overlapping signals.
2 δC
type
δH (J, Hz)
δC
type
33.6
CH2
333.6
CH2
26.7
CH2
1.521 2.080 2.380 2.587
32.3
CH2
64.4
CH
178.6 111.9 202.7 42.5
C C C CH2
48.7 57.7 37.6 65.3
C CH C CH
48.1
CH2
45.4 214.5 40.5
C C CH2
16.2
CH2
52.5 69.1 21.0 21.9 18.1 20.4 169.2
CH CH CH3 CH3 CH3 CH3 C
(td, 4.80, 13.8) (m) (m) (m)
192.8
C
141.5 132.2 120.6 138.0
C C CH CH
50.0 50.9 37.0 30.2
C CH C CH2
77.9
CH
54.1 216.7 43.3
C C CH2
20.0
CH2
45.7 71.3 19.2 19.2 18.2 20.7 169.4
CH CH CH3 CH3 CH3 CH3 C
8.312 (s)
6.590 (d, 10.2) 5.798 (d, 10.2)
2.112 (d, 9.00) 2.200 (m) 3.636 (t, 9.0) 5.335 (s, OH)
a
1.756 2.303a 1.915a 2.314a 2.424 (d, 5.40) 5.006 (qd, 1.80, 6.00) 0.811 (s) 0.959 (s) 1.149 (d, 6.60) 1.855 (s)
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at a wavelength of 570 nm (SpectramaxPlus 384; Molecular Devices, Sunnyvale, CA) [6]. 3. Results and discussion After successive stages of chromatography, the F. sinkiangensis seed extract yielded two new steroidal esters Sinkiangenrin A and B and a new organic glycoside Sinkiangenrin C, together with four known compounds. Their structures were elucidated on the basis of spectroscopic analysis and X-ray crystallographic data. Compounds 4–7 were identified by comparing their spectroscopic data with published data for arctigenin (4) [7], matairesinol (5) [7], secroisolariciresinol (6) [8] and lariciresinol (7) [9]. Compound 1 was obtained as a colorless crystal from MeOH. Its molecular formula was deduced as C23H32O7 on the basis of the [M + Na]+ ion peak at m/z 443.2056 (calcd
for C23H32O7Na+, 443.2046) in the HRESIMS, which corresponds to 8° of unsaturation. The IR absorption bands indicated the presence of carbonyl (1703 and 1739 cm−1) and hydroxyl groups (3406 cm−1). The 1H NMR spectrum (Table 1) of 1 exhibited four methyl group signals at δH 1.09 (s, CH3-19), 0.94 (s, CH3-20), 1.12 (d, CH3-21), 1.77 (s, CH3-1′) and one hydroxyl signal at δH 15.79 (OH-4). The HSQC and 13C-APT NMR spectra of 1 showed 23 carbon signals including four methyl groups at δC 21.0, 21.9, 18.1, and 20.4; two olefinic carbons at δC 178.6, 111.9 (an enol group), and three carbonyl groups at δC 202.7, 214.5, and 169.2. These results indicated that the degree of unsaturation for the rest of structure was 4, suggesting that there would be four rings in the structure of compound 1. The planar structure of 1 was determined by the interpretation of the 2D NMR data (Supporting Information, Fig. 2). The 1H-1H COSY and HSQC spectra showed three spin systems (H-1/H-2/H-3/HO-3, H-9/H-11/HO-11/H-12, H-15/
Fig. 2. Key 2D NMR correlations of 1–3.
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H-16/H-17/H-18/CH3-21), which suggested the presence of three partial structures (C-1-C-3, C-9-C-12, C15-C-18-C-21) as shown in Fig. 2. In the HMBC spectrum, the correlations from H-1′ and H-18 to C-2′ confirmed the presence of the ester structure; the correlations from H-3, HO-4, H-2b to C-4; H-7, H-1, HO-4, CH3-19 to C-5; HO-4, H-7 to C-6; H-9, H-7 to C-8; H-1, CH3-19 to C-9; and H-1, CH3-19 to C-10 indicated the presence of an A/B ring system. The correlations from H-9, H-7, H-15, H-12, CH3-20 to C-14; and H-12, CH3-20 to C-13 supported the presence of the B/C ring system. The presence of the C/D ring system was confirmed by the correlations from H-17 to C-13; H-15 to C-8; and H-17, H-15 to C-14. The relative configuration of 1 was determined from the NOESY data (Supporting information, Fig. 2). The correlations of HO-3/H-9 (β) and HO-11/H-9 (β) revealed that HO-3 and HO-11 were β-oriented. In addition, crosspeaks of H-11/ CH3-19; CH3-20/H-11; CH3-20/H-18; H-21/H-17 and H-9/ H-17 supported that CH3-19, CH3-20 and H-18 were α-oriented and H-17 was β-oriented. We also obtained crystals of 1 and performed an X-ray diffraction experiment using Cu Kα radiation (λ = 1.54178 Å). The X-ray crystallographic data (Supporting Information) confirmed the planar structure of 1 and allowed the assignment of the absolute configuration as 3S, 8R, 9S, 10S, 11S, 13S, 17R, and 18R [Flack parameter = 0.2(4)] [10]. The structure of 1 was similar to the published structure of oleagenin-type cardenolide, which also bears an unusual framework, although the configurations of C-8, C-9, C-10 and C-13 were different from that of oleagenin-type cardenolide [11]. Therefore, this is the first report of the isolation of this type of steroidal ester, which we have named Sinkiangenrin A. Compound 2 was obtained as colorless needles (MeOH), and analyzed for C23H30O6 by positive-ion HRESIMS. Its IR spectrum showed the presence of hydroxyl and carbonyl groups at 3453 and 1738 cm−1, respectively. The 1H NMR spectrum suggested the presence of four methyl signal at δH
251
0.81, 0.96, 1.45, and 1.85; and two olefinic protons at δH 5.79 (d, J = 10.2 Hz), and 6.59 (d, J = 10.2 Hz). The 13C-APT spectrum showed 23 carbons including four methyl carbons; four olefinic carbons at δC 120.6, 132.2, 138.0, and 141.5 and three carbonyl carbons at δC 169.4, 192.8, and 216.7. The assignment of 1H NMR and 13C-APT spectroscopic data of 2 (Table 1) was based on HSQC, HMBC and 1H-1H COSY spectrum (Supporting Information). The 1H NMR and 13C-APT features of 2 were similar to those of 1, except that the hydroxyl at C-3 in compound 1 was replaced by carbonyl group in compound 2, the hydroxyl at C-11 in 1 transferred to C-12 in 2 and the carbonyl group at C-6 in compound 1 was replaced by the ethylenic bond between C-6 and C-7 in compound 2. In the HMBC spectrum of 2, correlations from H-2, H-1, HO-4 to C-3 suggested the presence of a ketone group at C-3; the correlations from H-6 to C-4, C-5, C-10, C-8 and H-7 to C-8, C-9, and C-15 confirmed the presence of a 6, 7-ethylenic bond. The correlations from H-11, CH3-20 to C-12 (δC 77.9) supported the presence of a hydroxyl at C12. The relative configuration of 2 was confirmed by NOESY data (Supporting Information, Fig. 2). The correlations of H-12/CH3-19 and H-12/CH3-20 (β) suggested that they were β-oriented. The crosspeaks of H-17(α)/ H-9 revealed that they were cofacial. The planar structure of 2 was confirmed by an X-ray diffraction experiment using Cu Kα radiation (λ = 1.54178 Å). The absolute configuration was assigned as 8S, 9S, 10S, 12R, 13R, 17R, and 18R [Flack parameter = 0.0(10)] [12], and we named this compound Sinkiangenrin B. Sinkiangenrin A (1) and B (2) feature an unusual carbon skeleton and a plausible biogenetic origin of this skeleton could be traced back to the C21-steroids as shown in Fig. 3. The rearrangement of the D-ring could be initiated by the formation of a C8–C14 pregnane epoxide, followed by the formation of a carbocation center at C-8. Subsequently, enzymatic Wagner–Meerwein rearrangements lead to the migration of the C14–15 bond to C-8 and the generation of a
Fig. 3. Proposed biogenetic pathway for and 2.
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Table 2 In vitro antiproliferative activity of compounds 1–7. Compound
1 2 3 4 5 6 7 Taxolb a b
IC50 (μM) Hela
K562
AGS
– – 215.2 ± 1.4a 105.1 ± 1.3 85.5 ± 2.2 212.7 ± 0.7 142.7 ± 3.2 5.6 ± 0.01
– – – – – – – 8.5 ± 0.15
– – 36.9 ± 1.7 78.2 ± 1.6 99.4 ± 2.5 – – 3.5 ± 0.04
Value present mean ± SD of triplicate experiments. Positive control substance.
protonated carbonyl at C-14, which is eventually deprotonated [14]. The backbone is then modified by a series of enzymecatalytic reactions to afford 1 and 2. Compound 3 was obtained as white amorphous powder (MeOH). It was assigned a molecular formula of C14H26O9 by negative-ion HRESIMS. The IR absorption bands indicated the presence of a carbonyl group (1739 cm−1). The 1H NMR spectrum showed three methyl signals [δH 1.264, (s); 1.281, (s); 1.409, (d, J = 6.0Hz)]. The 13C-APT spectrum showed 14 carbon signals including three methyl carbons at δC 22.9, 24.6, 24.9 and a carbonyl carbon at δC 180.1. Sugar proton and carbon signals in the NMR spectra of 3 were assigned by 1H–1H COSY, HSQC and HMBC spectra. The 1H–1H COSY spectra displayed a spin system (H-3/H-2/H-1′/CH3-2′) which suggested the presence of the partial structure (C-3-C-2-C-1′-CH3-2′) as shown in Fig. 2. In the HMBC spectrum (Fig. 2), long-range couplings were observed for H-3 (δH 2.71), H-2 (δH 2.33), H-1′ (δH 3.25) to C_O (δC 180.1); and H-1″ (δH 4.46) to C-4 (δC 78.6), suggesting the location of the carbonyl group and glucosidic bond at C-2 and C-4, respectively. The 13C NMR spectrum of the sugar moiety was similar to the published spectra of β-D-glucopyranose and in the 1H NMR spectrum, large coupling constants between H-1″ and H-2″ (J = 9.6 Hz) indicated that the glycone unit was β-D-glucopyranose [13]. Therefore, the structure of 3 was assigned as 2-(1-hydroxyethyl)-4-methyl pentanoic acid-4-O-β-D-glucopyranoside. This is the first reported isolation of this compound, and we have named it Sinkiangenrin C. The isolated compounds 1–7 were tested for their cytotoxic activity in vitro against three human cancer cell lines, Hela, K562 and AGS, using the MTT assay and choosing Taxol as the positive control. The data in Table 2 suggested that the isolated steroidal ester derivatives (1 and 2) had no cytotoxic activity against Hela, K562 and AGS cancer cell lines. Compounds 3–5
all displayed cytotoxicity against AGS cells with IC50 values between 36.9 and 98.3 μM, and 3–7 showed weak cytotoxicity against Hela cells with IC50 value between 85.5 and 215.2 μM. Based on our study, it can be concluded that the organic acid glycoside and lignin compounds may play a role in the cytotoxic activity of Ferula sinkiangensis. Acknowledgments This work was financially supported by the National Natural Science Foundation of China (81060265), National Mega-project for Innovative Drugs (2012ZX09301-002-001; 2011ZX09307-002-01), and Program for Innovative Research Team in IMPLAD. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.fitote.2014.06.016. References [1] Yang J, An Z, Li Z, Jing S, Qina H. Sesquiterpene coumarins from the roots of Ferula sinkiangensis and Ferula teterrima. Chem Pharm Bull 2006;54(11):1595–8. [2] Li X, Jiang L, Da P. Preparation and investigation of the pharmacodynamics of effective antiulcerative composition in Ferula sinkiangensis K.M. Shen. Sci Technol 2007;9(10):8–10. [3] Yang L, Zhao HQ, Yao G, Cai Z, Wang JM. The preliminary study of antibacterial effect of five kinds of bacterials of Ferula sinkiangensis. J Tradit Chin Vet Med 2007;54:33–4. [4] Zhang H, Hu J. Anti-inflammatory and immunopharmacological effect of Xinjiang ferula oil. Chin Pharmacol Bull 1987;5(3):288–90. [5] Wang Y, Si J, Li X, Lin J, Zhu J. Chemical constituents from the seeds of Ferula sinkiangensis. Mod Chin Med 2011;13(1):26–8. [6] Wang S, Ma G, Zhong M, Yu S, Xu X, Hu Y, et al. Triterpene saponins from Tabellae Clinopodii. Fitoterapia 2013;90:14–9. [7] Wang H, Yang J. Studies on the chemical constituent Ts of Ar Ctiumlappa L. Acta Pharm Sin 1993;28(12):911–7. [8] Zeng X, Wang G, Wu X, Li G, Ye W, Li Y. Chemical constituents from Syringa pinnatifolia. Chin Tradit Herb Drugs 2013;44(13D):1721–5. [9] Cui Y, Mu Q, Hu C. Study on the phenylpropanoids from Caragana rosea. Nat Prod Res Dev 2003;15(4):277–83. [10] Crystallographic data of compound 1 have been deposited at the Cambridge, UK., under the reference number CCDC 980109. [11] Hua Y, Liu H, Ni W, Chen C, Lu Y, Wang C, et al. 5a-Steroidal glycosides from Parepigynum funingense. J Nat Prod 2003;66(6):898–900. [12] Crystallographic data of compound 2 have been deposited at the Cambridge, UK., under the reference number CCDC 980110. [13] Li H, Yu Y, Wang Z, Dai Y, Gao H, Xiao W, et al. Iridoid and bis-iridoid glucosides from the fruit of Gardenia jasminoides. Fitoterapia 2013;88:7–11. [14] Zhang M, Zhu Y, Zhan G, Shu P, Sa R, Lei l, et al. Micranthanone A, a new diterpene with an unprecedented carbon skeleton from Rhododendron micranthum. Org Lett 2013;15(12):3094–7.
