Steroids from the Roots of Cynanchum stauntonii

• Abstract
• Introduction
• Materials and Methods
• General experimental procedures
• Plant material
• Extraction and isolation
• Isolates
• X-ray crystal analysis of stauntonine (1)
• Evaluation of vasodilating activity
• Results and Discussion
• Acknowledgements
• References

Abstract

A chemical investigation of the roots of Cynanchum stauntonii has resulted in the characterization of a new hydroperoxide with a 13,14 : 14,15-disecopregnane-type skeleton, named stauntonine (1), together with three related compounds, anhydrohirundigenin (2), anhydrohirundigenin monothevetoside (3), and glaucogenin-C mono-D-thevetoside (4). Their structures were established by spectroscopic methods, including X-ray crystallographic diffraction analysis of stauntonine that confirmed its relative stereochemistry. The compound 1 showed dose-dependent relaxation on aortic rings with endothelium contracted by phenylepherine or KCl

Introduction

Species such as Cynanchum stauntonii (Decne.) Schltr. ex Levl. (Asclepiadaceae) are traditionally used in China as antitussives and expectorants [1]. The main chemical constituents isolated from the genus of Cynanchum are compounds with the 13,14 : 14,15-disecopregnane-type skeleton [2], [3]. However, previous chemical investigation on the title plant has only led to the isolation of β-sitosterol and hancockinol [4]. Recently, we have studied this plant to discover new natural products. In this paper, we describe the isolation and structure elucidation of four steroids (1 - 4) from the roots of C. stauntonii; one of which has been characterized as a new hydroperoxide (1). As a part of our search for vasodilating substances in natural products, the vasodilating activity of 1 is described.[*]

Materials and Methods

General experimental procedures

Melting points were determined on an XT₄ - 100x melting point apparatus and are uncorrected. Optical rotations were determined on a Perkin-Elmer 241 automatic polarimeter in MeOH at 25 °C. IR spectra were recorded on a Nicolet Impac 400 FTIR spectrophotometer. ¹H-NMR (500 MHz, CDCl₃), ¹³C-NMR (125 MHz, CDCl₃) spectra, and 2D NMR spectra (¹H-¹H COSY, HMQC, HMBC, NOESY) were taken on a Varian Inova-500 NMR spectrometer using tetramethylsilane as internal standard. FAB, ESI and EI-MS were measured on an AutoSpec Ultima-Tof mass spectrometer at 70 eV. The single X-ray crystallography was determined using an MAC DIP-2030K. Silica gel (200 - 300 mesh, Qingdao Marine Chemical Group Co., Qingdao, P. R. China) was used for column chromatography (CC).

Plant material

The roots of C. stauntonii were obtained from Ding Xian market, Hebei Province, P. R. China, in November 2002 and identified by the authors and by comparison with the authentic samples in the Herbarium of the institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College. A voucher specimen (No. 337 - 02) is deposited in the Department of Natural Medicinal Chemistry, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.

Extraction and isolation

The air-dried, powdered roots (5 kg) of C. stauntonii were extracted three times under reflux with 95 % EtOH. The combined EtOH extract was evaporated under reduced pressure to yield a brownish viscous residue (450 g). The residue was dissolved in 80 % aqueous ethanol (ca. 3000 mL) and the solution was extracted three times with petroleum ether (60 - 90 °C) (1500 mL each). Evaporation of the aqueous layer under reduced pressure yielded a brown residue which was dissolved in water (ca. 2500 mL) again and then extracted three times with EtOAc (1000 mL each). The combined EtOAc solution was washed three times with aqueous 5 % NaHCO₃ (1000 mL each), then three times with water (800 mL each) and dried with anhydrous Na₂SO₄. After removal of the organic solvent under reduced pressure, 27 g of residue were obtained as a brown gummy solid. The residue was chromatographed over silica gel (5 × 80 cm, 200 - 300 mesh, 600 g) eluted with a mixture of petroleum ether (60 - 90 °C)/EtOAc of increasing polarity to obtain 110 fractions (7 ∼ 8 mL/min; 250 mL for each; petroleum ether /EtOAc,10 : 1 × 10 frs, 10 : 2 × 18 frs, 10 : 3 × 13 frs, 10 : 4 × 26 frs, 10 : 5 × 43 frs) and finally with MeOH (3000 mL). After evaporation of each eluate, compound 1 (133 mg) was obtained from frs 18 to 21, compound 2 (43 mg) from frs 31 to 32, and 4 (80 mg) and 3 (33 mg) from frs 43 and 45, respectively, all as amorphous powder. Compound 1 was recrystallized from EtOAc to give prisms (103 mg), and 4 gave colorless needles (58 mg).

Isolates

Stauntonine (3β-hydroxy-18-hydroperoxy-15,20α:18,20β-diepoxy-13,14 : 14,15-disecopregna- 5,12-dien-14-oic acid 16-oxy-lactone; 1): Colorless prisms, C₂₁H₂₈O₇, m. p. 173 - 175 °C (EtOAc); [α]_D ²⁵: -46.97° (c 0.132, MeOH); IR (KBr): ν_max = 3398, 3170 (OH), 2941, 1724 (C = O), 1631 (C = C), 1486, 1385, 1365, 1132, 1043 cm^-1; FAB-MS: m/z = 375 [M + H - H₂O]⁺ and 359 [M + H - H₂O₂]⁺; EI-MS: m/z = 374, 358, 340, 191, 166, 145 (100), 83; ¹H-NMR and ¹³C-NMR, see Table [1].

Anhydrohirundigenin (2): Amorphous powder, [α]_D ²⁵: -19.62° (c 0.107, MeOH) [5].

Anhydrohirundigenin mono-thevetoside (3): Amorphous powder, C₂₈H₄₀O_8, [α]_D ²⁵: -22.82° (c 0.241, MeOH); IR (KBr): ν_max = 3440 (OH), 2937, 2852, 1651(C = C), 1441, 1381, 1192, 1068, 750 cm^-1; ESI-MS: m/z = 527 [M + Na]⁺, 505 [M + H]⁺; ¹H-NMR and ¹³C-NMR, see Table [1].

Glaucogenin-C mono-D-thevetoside (4): Colorless fine needles, m. p. 185 - 187 °C; [α]_D ²⁵: + 26.10° (c 0.991, MeOH) [6].

X-ray crystal analysis of stauntonine (1)

Crystal data: C₂₁H₂₈O₇, Mr = 392.45, orthorhombic, space group P2₁2₁2₁, a = 7.926(1) Å, b = 8.254(1) Å, c = 29.791(2) Å, V = 1948.9(2) ų, Z = 4, Dc = 1.342 g/cm³, μ = 0.09 mm^-1. Intensity data were collected with an MAC DIP-2030K Image Plate diffractometer with a graphite monochromator (ω-2Θ scans, 2Θ _max = 50.0°), MoKα (λ = 0.71073 Å) radiation. A total of 1912 unique reflections were collected , of which 1905 were observed (|F|² 8σ|F|²). The structure was solved by direct methods using SHELX-86 and expanded using difference Fourier techniques, refined by the program and the NOMCSDP and full-matrix least-squares calculations. Hydrogen atoms were fixed at the calculated positions. The final indices were R_f = 0.068, R_w = 0.064(w = 1/σ|F|²). The crystallographic data have been deposited with the Cambridge Crystallographic Data Centre as Deposition No. CCDC-226 278. Copies of data can be obtained on application to the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK [fax: + 44 (1223) 336 033, or e-mail: deposit@ccdc.cam.ac.uk].

Evaluation of vasodilating activity

The vasodilating activity of stauntonine has been evaluated on isolated rat aortic rings pre-contracted by phenylephrine 10 ^{- 7} mol/L or KCl 100 mmol/L with or without endothelium. The aortic rings were mounted on a 10-mL organ bath, connected to a force transducer and a computer with BIOPAC software. The organ bath containing Krebs’s solution was bubbled with 95 % O₂ + 5 % CO₂ at 37 °C. The solution in the bath was changed every 20 min and the rings were stretched to approximate 1.0 g, and allowed to further equilibrate for 60 min. Before data collection, the rings were stimulated with phenylephrine 10 ^{- 7} mol/L two times. The cumulative concentration 10 ^{- 8} ∼ 10 ^{- 4} mol/L of stauntonine was added to the bath when the contraction was at a plateau induced by phenylephrine 10 ^{- 7} mol/L. In another series of experiments, the aortic rings were contracted by KCl 100 mmol/L. Recorded were the values of concentration and relaxation.

Results and Discussion

Compound 1 was isolated as a crystalline solid, m. p. 173 - 175 °C, molecular formula C₂₁H₂₈O₇. The IR spectrum displayed absorption bands at 3398 and 3170 (OH), 1724 (C = O) and 1631 (C = C) cm^-1. The FAB mass spectrum showed peaks at m/z = 375 [M + H - H₂O]⁺ and 359 [M + H - H₂O₂]⁺, suggesting the presence of a hydroperoxy group [7]. The ¹H-, ¹³C- and DEPT NMR spectral data of 1 showed close resemblance to those of glaucogenin-C mono-D-thevetoside (4) which was previously isolated from the plant of C. glaucescens Hand-Mazz [6]. The main differences were the absence of the characteristic signals for the thevetose moiety and a methylene group in the high-field region as well as the appearance of one methine group in the low field region at δ = 107.5 of the ¹³C-NMR, which showed a correlation with the ¹H-NMR signal at δ = 5.75 (1H, br s, H-18) in HMQC experiments. In addition, the double bond signals at δ = 143.1 (d) and 113.7 (s) in 4 were replaced by double bond signals at δ = 134.3 (d) and 139.5 (s) in 1 in the ¹³C-NMR spectra, which suggested that the olefinic methine carbon in 1 must not be linked directly to an oxygen atom. The HMBC experiment was used to confirm the connectivities of all carbon atoms in the molecule (Fig. [1]), and the NOESY experiment performed on 1 established its relative configurations (Fig. [2]). Full NMR assignments for 1 were obtained by careful analysis of 2D NMR data. Since the position of the hydroperoxy group was indeterminable, the structure of 1 was further studied by single-crystal X-ray diffraction analysis with its structural confirmation as in Fig. [3]. The X-ray crystallography defined the relative configuration of 1 and gave results consistent with the data of NOESY experiments. The hydroxy group at C-3, the C-19 methyl group, and the C-8 and C-18 protons are found to be in the β-orientation, and the C-9, C-16 and C-17 protons, as well as C-21 methyl group in the α-orientation. Ring A and the nine-membered ring C are in a chair conformation, ring B in a semi-chair conformation, and the five-membered rings D and E in the envelope conformation. The B/C rings are found to be trans-fused, and C/D and D/E rings cis-fused. On the basis of the above findings, the structure of 1 was assigned as 3β-hydroxy-18-hydroperoxy-15,20α:18,20β-diepoxy-13,14 : 14,15-disecopregna-5,12-dien-14-oic acid 16-oxylactone.

Compound 3 was obtained as an amorphous powder, with a molecular formula of C₂₈H₄₀O₈ based on NMR and ESI mass spectrometry data; the latter spectrum showed ion peaks at m/z = 505 [M + H]⁺, and 527 [M + Na]⁺. The IR spectrum (KBr) showed absorption bands at 3440 (OH) and 1651 (C = C) cm^-1. The ¹H-, ¹³C- and DEPT NMR spectral data of 3 were very similar with those of anhydrohirundigenin (2), which was previously isolated from the plant of C. hancockianum (Maxim.) Al. Iljinski [8]. The main difference was the appearance of the characteristic signals for a thevetose moiety [6] with the glycosidation shifts observed at C-2 (-2.4), C-3 (+ 6.4), and C-4 (-3.7) in the ¹³C-NMR for the aglycone moiety of 3, so the thevetose group is linked with the C-3 hydroxy group of 2. The sugar moiety located at C-3 was also confirmed by the correlation of the signal of H-1′ with the signal of C-3 in the HMBC spectrum. The β-configuration of the sugar was deduced from the anomeric proton at δ = 4.34 with a coupling constant of 7.5Hz in the ¹H-NMR spectrum of 3. Full NMR assignments for 3 were obtained by careful analysis of 2D NMR data. Thus, 3 is anhydrohirundigenin mono-thevetoside.

Two known compounds, anhydrohirundigenin (2) [8], glaucogenin-C mono-D-thevetoside (4) [6] were also isolated. Their structures were identified by comparison of spectroscopic data (IR, ¹H-NMR, ¹³C-NMR, MS) with the literature data. This is the first isolation and characterization of compounds 2 and 4 from C. stauntonii.

Stauntonine (1) had a dose-dependent relaxation effect on aortic rings with endothelium contracted by phenylepherine, and the IC₅₀ was 5.37 × 10 ^{- 6} mol/L. The inhibitory effect of stauntonine on aortic rings without endothelium contracted by phenylephrine was not significant in the lower concentrations (10 ^{- 8} ∼ 10 ^{- 5} mol/L) while it still exerted the relaxation effect in the high concentration (10 ^{- 4} mol/L) with a relaxation percentage 64.8±26.9 % (Table [2]). Meanwhile, stauntonine was also found to relax the aorta rings contracted by KCl in the high concentration (10 ^{- 4} mol/L), with a relaxation percentage 53.4 ± 7.3 % (Table [3]). Verapamil relaxed the aorta rings to 97.36 ± 8.51 % at 10 ^{- 6} mol/L. Compounds 2, 3, and 4 were inactive in the vasodilation tests.

Acknowledgements

Special thanks are due to Dr. F. Liang for mass spectral measurements, to Professor Y. Lu for X-ray crystal analysis. This work was supported by the Science Foundation of the State Administration of Traditional Chinese Medicine, P. R. China (02-03ZP09).

References

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• 3 Sugama K, Hayashi K, Mitsuhashi H, Kaneko K. Studies on the constituents of Asclepiadaceae plants. LXVI. The structures of three new glycosides, cynapanosides A, B, and C, from the Chinese drug ”Xu-Chang-Qing,” Cynanchum paniculatum Kitagawa.  Chem Pharm Bull. 1986;  34 4500-7
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• 4 Qiu S X. Studies on the chemical constituents of Cynanchum stauntonii (Decne.) Schlt. ex Levl.  Journal of Chinese Materia Medica. 1994;  19 488-9
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• 5 Kennard O, Fawcett J K, Watson D G, Ann Kerr K, Stockel K, Stocklin W. et al .Hirundigenin and anhydrohirundigenin, two natural 15-oxasteroids of plant origin. Chemical and X-ray investigation. Tetrahedron Lett 1968: 3799-804
  Search in Google Scholar
• 6 Nakagawa T, Hayashi K, Mitsuhashi H. Studies on the constituents of Asclepiadaceae plants. LIII. The structures of glaucogenin-A, -B, and -C mono-D-thevetoside from the Chinese drug ”Pai-ch’ien,” Cynanchum glaucescens Hand-Mazz.  Chem Pharm Bull. 1983;  31 870-8
  CrossrefPubMedSearch in Google Scholar
• 7 El-Feraly F S, Chan Y M. Peroxycostunolide and peroxyparthenolide: two cytotoxic germacranolide hydroperoxide from Magnolia grandiflora. Structural revision of verlotorin and artemorin. Tetrahedron Lett 1977: 1973-6
  Search in Google Scholar
• 8 Lou H X, Li X, Zhu T R. C₂₁ steroidal constituents from Cynanchum hancockianum .  Acta Pharm Sinica. 1992;  27 595-602
  PubMedSearch in Google Scholar

Dr. Hai-Lin Qin

Institute of Materia Medica

Chinese Academy of Medical Sciences and Peking Union Medical College

Beijing 100050

P. R. China

Phone: +86-010-83172503

Fax: +86-010-63017757

Email: qinhailin@imm.ac.cn

References

• 1 Xie Z W, Liu M L, Lou Z Q. Pharmacognostical studies of the Chinese drugs Pai-ch’ien and Pai-wei.  Acta Pharm Sinica. 1959;  7 175-88
  PubMedSearch in Google Scholar
• 2 Qiu S X, Zhang Z X, Zhou J. Steroidal glycosides from the root of Cynanchum versicolor .  Phytochemistry. 1989;  28 3175-8
  CrossrefPubMedSearch in Google Scholar
• 3 Sugama K, Hayashi K, Mitsuhashi H, Kaneko K. Studies on the constituents of Asclepiadaceae plants. LXVI. The structures of three new glycosides, cynapanosides A, B, and C, from the Chinese drug ”Xu-Chang-Qing,” Cynanchum paniculatum Kitagawa.  Chem Pharm Bull. 1986;  34 4500-7
  PubMedSearch in Google Scholar
• 4 Qiu S X. Studies on the chemical constituents of Cynanchum stauntonii (Decne.) Schlt. ex Levl.  Journal of Chinese Materia Medica. 1994;  19 488-9
  PubMedSearch in Google Scholar
• 5 Kennard O, Fawcett J K, Watson D G, Ann Kerr K, Stockel K, Stocklin W. et al .Hirundigenin and anhydrohirundigenin, two natural 15-oxasteroids of plant origin. Chemical and X-ray investigation. Tetrahedron Lett 1968: 3799-804
  Search in Google Scholar
• 6 Nakagawa T, Hayashi K, Mitsuhashi H. Studies on the constituents of Asclepiadaceae plants. LIII. The structures of glaucogenin-A, -B, and -C mono-D-thevetoside from the Chinese drug ”Pai-ch’ien,” Cynanchum glaucescens Hand-Mazz.  Chem Pharm Bull. 1983;  31 870-8
  CrossrefPubMedSearch in Google Scholar
• 7 El-Feraly F S, Chan Y M. Peroxycostunolide and peroxyparthenolide: two cytotoxic germacranolide hydroperoxide from Magnolia grandiflora. Structural revision of verlotorin and artemorin. Tetrahedron Lett 1977: 1973-6
  Search in Google Scholar
• 8 Lou H X, Li X, Zhu T R. C₂₁ steroidal constituents from Cynanchum hancockianum .  Acta Pharm Sinica. 1992;  27 595-602
  PubMedSearch in Google Scholar

Dr. Hai-Lin Qin

Institute of Materia Medica

Chinese Academy of Medical Sciences and Peking Union Medical College

Beijing 100050

P. R. China

Phone: +86-010-83172503

Fax: +86-010-63017757

Email: qinhailin@imm.ac.cn