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DOI: 10.1055/s-2007-993750
© Georg Thieme Verlag KG Stuttgart · New York
Steroidal Alkaloids from Veratrum japonicum with Genotoxicity on Brain Cell DNA of the Cerebellum and Cerebral Cortex in Mice
Dr. Jin-Hui Wang
School of Taditional Chinese Materia Medica
Shenyang Pharmaceutical University
LiaoNing
Shenyang 110016
People’s Republic of China
Phone: +86-24-2398-6478
Email: Wjh.1972@yahoo.com.cn
Publication History
Received: July 30, 2007
Revised: October 22, 2007
Accepted: October 26, 2007
Publication Date:
11 December 2007 (online)
Abstract
A new steroidal alkaloid (1), together with three known steroidal alkaloids (2 - 4), has been isolated from the roots and rhizomes of Veratrum japonicum (Baker) Loes. f.., their structures were established as neogermine (1), germine (2), germerine (3), and neogermbudine (4) by means of physicochemical properties and spectroscopic analysis. Compounds 1 - 4 exhibited genotoxicity on brain cells in mice by using single-cell gel electrophoresis (Comet assay).
#Introduction
Veratrum, which belongs to the Liliaceae family, includes 13 species and varieties in China. Veratrum japonicum (Baker) Loes. f. is a well-known poisonous traditional medicinal plant in China, having toxic and irritant activity on the digestive tract mucosa, nucleus nervi vagi, and central nervous system [1]. The mutagenic and teratogenesis potential of steroidal alkaloids, well-known as both bioactive and toxic constituents of Veratrum species, were assayed in transgenic mice [2]. Our present study on the alkaloids of Veratrum japonicum (Baker) Loes. f. led to the isolation of a new steroidal alkaloid 1 and three known steroidal alkaloids 2 - 4 (Fig. [1]). This paper reports on the isolation and structure elucidation of the new compound 1, as well as the genotoxic effects of compounds 1 - 4 on brain cells in mice by single-cell gel electrophoresis (SCGE), a simple and sensitive technique for genotoxicity studies.

Fig. 1 Structures of compounds 1 - 4.
Materials and Methods
#Plant material
The plant material was collected from Hunan Province, China and identified by Professor Qishi Sun, School of Tradional Chinese Materia Medica, Shenyang Pharmaceutical University. A voucher specimen (No. 20 040 702) is deposited in the School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University.
#Instruments and chemicals
NMR spectra were taken on an AV-600 spectrometer from Bruker (Rheinstetten, Germany) using TMS as an internal standard. EI-mass spectra were measured on a VG-5050E mass spectrometer from VG (Quattro VG). ESI-MS was performed on an LCQ mass spectrometer from Finnigan (Silicon Valley, CA, USA). DYY-6B voltage-stabilizing and steady flow electrophoresis apparatus and DYCP-38B horizontal strip electrophoresis bath were products from Beijing Liuyi Equipment Factory (Beijing, China). Dimethyl sulfate (DMS) and dimethyl sulfoxide (DMSO) were obtained from Tianjin Fuchen Chemical Agencies (Tianjin, China).
#Extraction and isolation
The roots and rhizomes of Veratrum japonicum (Baker) Loes. f. (4.0 kg) were extracted with 95 % EtOH (36 L × 3) under reflux. Evaporation of the solvent gave a crude extract (260 g). The extract was suspended in H2O (3 L), and acidified to pH 3 with 10 %HCl (5 mL) followed by filtration, and then the filtrate was basified to pH 10 with 30 % NH4OH (30 mL) and extracted with CHCl3 (3 L × 4), to give a total alkaloid fraction (10.2 g). The CHCl3 extract (10.2 g) was subjected to column chromatography over silica gel (200 - 300 mesh; 3 × 40 cm; Qingdao Marine Chemical Group, Co.; Qingdao, China), eluted with CHCl3-acetone gradient. Fractions 20 - 25 [800 - 1000 mL; 50 mg; eluted with CHCl3-acetone (100 : 30)] were further purified by PTLC (Qingdao Marine Chemical Group, Co.) developing with CHCl3-acetone-HN(CH2CH3)2 (7 : 3:0.5) to get compound 3 (Rf = 0.63, 30 mg). Fractions 35 - 42 [1400 - 1680 mL; 55 mg; eluted with CHCl3-acetone (100 : 35)] were further purified by PTLC developing with petroleum ether-CHCl3-acetone-HN(CH2CH3)2 (3 : 3 : 4 : 0.5) to obtain compound 4 (Rf = 0.54, 35 mg). Fractions 56 - 63 [2240 - 2520 mL; 20 mg; eluted with CHCl3-acetone (100 : 42)] were further isolated by HPLC on a Venusil XBP-C18 column (200 mm × 4.6 mm, 5 μm, Agilent; Palo Alto, CA, USA) eluted with CNCH3-H2O [42 : 58, pH = 7.0 adjusted by N(CH2CH3)3] to give compound 1 (9 mg). Fractions 145 - 157 [5800 - 6280 mL; 50 mg; eluted with CHCl3-acetone (100 : 70)] were further purified by silica gel dry column chromatography (3 × 30 cm) with petroleum ether-acetone-HN(CH2CH3)2 (1 : 2 : 0.15) (315 mL) to afford compound 2 (30 mg).
Neogermine (1): white amorphous solid; m. p. 188 - 190 °C; [α]D 20: -13.8 (c 0.003, CHCl3); IR (KBr): ν = 3275, 2890, 2760 cm-1; ESI-MS: m/z = 462 pseudomolecular ion peak [M+H]+; EI-MS: m/z (rel. int. %) = 461 ([M]+, 4.23), 112 (100.00), 98 (25.20), 69 (13.33), 55 (18.37); 1H-NMR (600 MHz, CDCl3) and 13C-NMR (150MHz, CDCl3) data, see Table [1].
Position | 1 | ||
1H, m, J (Hz) | 13C | HMBCC | |
1 | 1.43 m, 1.57 m | 32.2 | C-2, 3, 5, 10 |
2 | 1.53 m, 1.87 m | 26.9 | C-1, 3, 4 |
3 | 4.30 m | 69.0 | C-1, 2, 4, 5 |
4 | 3.98 m | 87.8 | C-2, 3, 5, 10 |
5 | 2.03 m | 45.7 | C-1, 4, 6, 7, 9, 10, 19 |
6 | 1.91 m | 35.2 | C-4, 5, 7, 8, 10 |
7 | 4.54 (d, J = 6.0 Hz) | 74.7 | C-4, 5, 6, 9, 14 |
8 | 1.76 (d, J = 13.2 Hz) | 52.8 | C-6, 9, 10, 11, 14 |
9 | 2.50 m | 40.6 | C-1, 7, 8, 10, 11, 14, 19 |
10 | 32.1 | ||
11 | 1.93 m | 29.0 | C-8, 9, 12, 13, 14 |
12 | 1.43 m | 48.5 | C-8, 9, 13, 18 |
13 | 1.73 m | 35.9 | C-12 |
14 | 78.0 | ||
15 | 1.64 dd (13.0, 2.8), 2.34 dd (13.0, 2.8) | 40.0 | C-8, 14, 16, 17 |
16 | 4.39 brs | 66.6 | C-13, 14, 15, 17, 20 |
17 | 0.98 m | 49.0 | C-13, 16, 18, 20 |
18 | 1.69 m, 2.76 brd (9.0) | 61.3 | C-13, 17, 22 |
19 | 0.96 s | 22.0 | C-1, 5, 9, 10 |
20 | 72.9 | ||
21 | 1.20 s | 20.2 | C-17, 20, 22 |
22 | 1.69 m | 69.8 | C-21 |
23 | 1.51 m,1.67 m | 18.3 | C-24 |
24 | 0.93 m | 28.9 | C-23 |
25 | 1.91 m | 27.3 | C-27 |
26 | 2.30 brd (10.8), 2.66 brd (10.8) | 61.6 | C-18, 22, 24, 27 |
27 | 1.07 d (7.2) | 17.0 | C-24, 25, 26 |
Animals
Male Swiss mice (20 ± 2 g), obtained from the Experimental Animal Center of the Shenyang Pharmaceutical University, were used in the present study. The animals were housed under standard conditions (22 ± 2 °C temperature, 50 ± 10 % relative humidity, 12h:12 h light/dark cycles). Food and water were available ad libitum. All animal use procedures were in accordance with the Regulations of Experimental Animal Administration issued by the State Committee of Science and Technology of People’s Republic of China, 1988.
#Dosage and treatment
Mice were randomly divided into fourteen groups of 4 mice in each group, which included control, positive, and compounds 1 - 4 groups. The animals in the compounds 1 - 4 groups were treated by gavage with compounds 1 - 4 at 0.2, 1.0, and 2.0 mg/kg every day for 7 consecutive days. The control groups and the positive groups were treated with distilled water for 7 days. At the last day, the positive groups received an i. p. injection of DMS at 20 mg/kg. Animals were sacrificed by decapitation 1 hour after the last treatment. After sacrifice, the brain was removed and immediately dissected on ice. The brain was separated into two regions: cerebellum and cerebral cortex. The brain regions were minced, suspended to 1 mL/g in chilled homogenizing buffer containing PBS (NaCl, 8.01 g; KCl, 0.2 g; Na2HPO4, 2.9 g; KH2PO4, 0.2 g/L), and gently homogenized manually. To obtain nuclei, the homogenate was centrifuged at 500 × g for 5 min and the precipitate was resuspended in chilled homogenizing buffer for the comet assay. The comet assay was performed under alkaline conditions essentially as reported previously [3], according to the previous reports [4], [5], [6], [7] with a slight modification.
#Statistical analysis
Data were expressed as mean ± S.E.M. calculated from four mice of every group. Statistical comparisons were made by means of one-way analysis of variance (ANOVA), followed by the Fisher’s least significant difference (LSD) test (SPSS13.0 software, SPSS, USA).
#Results and Discussion
Compound 1 was obtained as a white amorphous solid, m. p. 188 - 190 °C. In the IR spectrum, 1 showed absorptions at 3275, 2890, and 2765 cm-1 [8]. Its molecular formula was established as C27H43NO5 by analysis of the ESI-MS, EI-MS and NMR spectra. The ESI-MS of 1 gave a quasimolecular ion peak [M + H]+ at m/z = 462, and the EI-MS showed a molecular ion peak [M]+ at m/z = 461(4.23) as well as fragment ion peaks at m/z = 112 (100.00), 98 (25.20), 69 (13.33), and 55 (18.37). Thus, the base peak at m/z = 112 (100.00) suggested the presence of a cevine-type steroidal alkaloid and a hydroxy group at C-20 [9], [10]. The 1H-NMR spectrum exhibited three methyl groups at δ H = 0.96 (3H, s, H-19), 1.07 (3H, d, J = 7.2 Hz, H-27), and 1.20 (3H, s, H-21), and four oxymethine protons at δ H = 4.54 (1H, d, J = 6.0 Hz, H-7), 4.39 (1H, br s, H-16), 4.30 (1H, m, H-3), and 3.98 (1H, m, H-4). The 13C-NMR and DEPT spectra indicated the presence of three methyls (δ C = 17.0, 20.2, and 22.0), nine methylenes (δ C = 18.3, 26.9, 28.9, 29.0, 32.2, 35.2, 40.0, 61.3, and 61.6), twelve methines (δ C = 27.3, 35.9, 40.6, 45.7, 48.5, 49.0, 52.8, 66.6, 69.0, 69.8, 74.7, and 87.8), and three quaternary carbons (δ C = 32.1, 72.9, and 78.0). All methyl groups and oxymethines were used as starting points to assign the other H- and C-atom signals on the basis of the HMBC, HMQC, and 1H-1H COSY correlations (see Table [1]). In the HMBC spectrum of 1, the correlations between H-4 (δ H = 3.98) and C-2 (δ C = 26.9), C-3 (δ C = 69.0), C-5 (δ C = 45.7), C-10 (δ C = 32.1), as well as between H-7 (δ H = 4.54) and C-4 (δ C = 87.8), C-5 (δ C = 45.7), C-6 (δ C = 35.2), C-9 (δ C = 40.6), C-14 (δ C = 78.0), suggested oxygenation at C-4 and C-7. The long-range correlation between H-7 (δ H = 4.54) and C-4 (δ C = 87.8) in the HMBC plot, in combination with its quasimolecular ion peak [M + H]+ at m/z = 462 in the ESI-MS, indicated the presence of an ether linkage between C-4 and C-7. The relative configuration of the ether linkage between C-4 and C-7 was established by a NOE experiment (Fig. [2]). Therefore, based on the above spectral data and comparison with those of germine [11], compound 1 was determined to be 4α,7α-oxa-5, 6-dihydro-14α-hydroxyveramarine, named as neogermine.

Fig. 2 Important NOE and HMBC correlations of 1. Key NOE correlations observed in NOESY spectra. Key HMBC correlations observed in HMBC spectra.
Compounds 2 - 4 were identified as germine, germerine, and neogermbudine, respectively, by spectroscopic analysis (1H NMR, 13C NMR, and HMBC) and comparison with published data [11], [12].
After oral administration of compounds 1 - 4, at the doses of 0.2, 1.0, and 2.0 mg/kg every day, for 7 consecutive days, DNA damages were detected in the cerebellum and cerebral cortex in mice. The values of tail moment length (μm) were significantly increased when compared with those of control groups (one-way ANOVA; p < 0.001). Furthermore, compounds 3 and 4 caused more damage to brain cells of the cerebellum and cerebral cortex than compounds 1 and 2 (Fig. [3]).

Fig. 3 Effects of compound 1 (A), compound 2 (B), compound 3 (C), and compound 4 (D) on brain cells DNA strand breaks in the cerebellum and cerebral cortex of mice. The extent of DNA damage was calculated from relative changes in tail moment length. Two hundred cells were examined in duplicate for each condition and the tail moments are expressed as mean ± S.E.M. *** P < 0.001, Veratrum japonicum (Baker)Loes. f. compared with control group; n = 4.
Acknowledgements
This work was financially supported by the Program for New Century Excellent Talents in University of Peoples Republic of China (NO. NCET-04-0289). We are grateful to Professor Qishi Sun for identification of the plant material.
- Supporting Information for this article is available online at
- Supporting Information .
References
- 1 Tang J, Li H L, Huang H Q, Zhang W D. Progress in the research on chemical constituents of Veratrum plants. Prog Pharm Sci. 2006; 30 206-12.
- 2 Crawford L, Myhr B. A preliminary assessment of the toxic and mutagenic potential of steroidal alkaloids in transgenic mice. Food Chem Toxicol. 1995; 33 91-4.
- 3 Guo L, Wang L H, Sun B S, Yang J Y, Zhao Y Q, Dong Y X. et al . Direct in vivo evidence of protective effects of grape seed procyanidin fractions and other antioxidants against ethanol-induced oxidative DNA damage in mouse brain cells. J Agric Food Chem. 2007; 55 5881-91.
- 4 Speit G, Hartmann A. The comet assay (single-cell gel test): a sensitive genotoxicity test for the detection of DNA damage and repair. Methods Mol Biol. 1999; 113 203-12.
- 5 Sasaki Y F, Saga A, Akasaka M, Nishidate E, Watanabe A M, Ohta T. et al . In vivo genotoxicity of heterocyclic amines detected by a modified alkaline single cell gel electrophoresis assay in a multiple organ study in the mouse. Mutat. Res1997; 395 57-73.
- 6 Sasaki Y F, Kawaguchi S, Kamaya A, Ohshita M, Kabasawa K, Iwama K. et al . The comet assay with 8 mouse organs: results with 39 currently used food additives. Mutat Res. 2002; 519 103-19.
- 7 Singh N P, Lai H, Khan A. Ethanol-induced single-strand DNA breaks in rat brain cells. Mutat Res. 1995; 345 191-6.
- 8 Bohlmann F. Lupinen-Alkaloide VIII. Zur Konfigurationsbestimmung von Chinolizidin-Derivaten. Chem Ber. 1958; 91 2157-67.
- 9 Cong P Z, Li S Y. Natural organic mass spectrometry. In: Cong PZ, editor. Peimine-type alkaloid. Beijing; Chinese Medical Technology Publishing House 2002: 522-5.
- 10 Liang G Y. Overview of the research on alkaloids of Veratrum plants. J Pharm. 1984; 19 309-20.
- 11 Sayed K AE, Mcchesney J D, Halim A F, Zaghloul A M, Lee I S. A study of alkaloids in Veratrum viride aiton. Int J Pharm. 1996; 34 161-73.
- 12 Zhao W J, Tezuka Y, Kikuchi T, Chen J, Guo Y T. Studies on the constituents of Veratrum plants II.constituents of Veratrum L. var. ussuriense. (1) structure and 1H-and 13C-nuclear magnetic resonance spectra of a new alkaloid, verussurinine, and related alkaloids. Chem Pharm Bull. 1991; 39 549-54.
Dr. Jin-Hui Wang
School of Taditional Chinese Materia Medica
Shenyang Pharmaceutical University
LiaoNing
Shenyang 110016
People’s Republic of China
Phone: +86-24-2398-6478
Email: Wjh.1972@yahoo.com.cn
References
- 1 Tang J, Li H L, Huang H Q, Zhang W D. Progress in the research on chemical constituents of Veratrum plants. Prog Pharm Sci. 2006; 30 206-12.
- 2 Crawford L, Myhr B. A preliminary assessment of the toxic and mutagenic potential of steroidal alkaloids in transgenic mice. Food Chem Toxicol. 1995; 33 91-4.
- 3 Guo L, Wang L H, Sun B S, Yang J Y, Zhao Y Q, Dong Y X. et al . Direct in vivo evidence of protective effects of grape seed procyanidin fractions and other antioxidants against ethanol-induced oxidative DNA damage in mouse brain cells. J Agric Food Chem. 2007; 55 5881-91.
- 4 Speit G, Hartmann A. The comet assay (single-cell gel test): a sensitive genotoxicity test for the detection of DNA damage and repair. Methods Mol Biol. 1999; 113 203-12.
- 5 Sasaki Y F, Saga A, Akasaka M, Nishidate E, Watanabe A M, Ohta T. et al . In vivo genotoxicity of heterocyclic amines detected by a modified alkaline single cell gel electrophoresis assay in a multiple organ study in the mouse. Mutat. Res1997; 395 57-73.
- 6 Sasaki Y F, Kawaguchi S, Kamaya A, Ohshita M, Kabasawa K, Iwama K. et al . The comet assay with 8 mouse organs: results with 39 currently used food additives. Mutat Res. 2002; 519 103-19.
- 7 Singh N P, Lai H, Khan A. Ethanol-induced single-strand DNA breaks in rat brain cells. Mutat Res. 1995; 345 191-6.
- 8 Bohlmann F. Lupinen-Alkaloide VIII. Zur Konfigurationsbestimmung von Chinolizidin-Derivaten. Chem Ber. 1958; 91 2157-67.
- 9 Cong P Z, Li S Y. Natural organic mass spectrometry. In: Cong PZ, editor. Peimine-type alkaloid. Beijing; Chinese Medical Technology Publishing House 2002: 522-5.
- 10 Liang G Y. Overview of the research on alkaloids of Veratrum plants. J Pharm. 1984; 19 309-20.
- 11 Sayed K AE, Mcchesney J D, Halim A F, Zaghloul A M, Lee I S. A study of alkaloids in Veratrum viride aiton. Int J Pharm. 1996; 34 161-73.
- 12 Zhao W J, Tezuka Y, Kikuchi T, Chen J, Guo Y T. Studies on the constituents of Veratrum plants II.constituents of Veratrum L. var. ussuriense. (1) structure and 1H-and 13C-nuclear magnetic resonance spectra of a new alkaloid, verussurinine, and related alkaloids. Chem Pharm Bull. 1991; 39 549-54.
Dr. Jin-Hui Wang
School of Taditional Chinese Materia Medica
Shenyang Pharmaceutical University
LiaoNing
Shenyang 110016
People’s Republic of China
Phone: +86-24-2398-6478
Email: Wjh.1972@yahoo.com.cn

Fig. 1 Structures of compounds 1 - 4.

Fig. 2 Important NOE and HMBC correlations of 1. Key NOE correlations observed in NOESY spectra. Key HMBC correlations observed in HMBC spectra.

Fig. 3 Effects of compound 1 (A), compound 2 (B), compound 3 (C), and compound 4 (D) on brain cells DNA strand breaks in the cerebellum and cerebral cortex of mice. The extent of DNA damage was calculated from relative changes in tail moment length. Two hundred cells were examined in duplicate for each condition and the tail moments are expressed as mean ± S.E.M. *** P < 0.001, Veratrum japonicum (Baker)Loes. f. compared with control group; n = 4.
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