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Two 22S-solanidine-type steroidal alkaloids from Fritillaria anhuiensis
Fitoterapia 81 (2010) 81–84
Contents lists available at ScienceDirect
Fitoterapia j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f i t o t e
Two 22S-solanidine-type steroidal alkaloids from Fritillaria anhuiensis Qing Yao Shou a, Qing Tan a, Zheng Wu Shen a,b,⁎ a b
School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, People's Republic of China Basilea Pharmaceutical China Ltd., Haimen, Jiangsu 226100, People's Republic of China
a r t i c l e
i n f o
Article history: Received 19 March 2009 Accepted in revised form 23 July 2009 Available online 18 August 2009 Keywords: Fritillaria anhuiensis Solanidine Steroidal alkaloid
a b s t r a c t Phytochemical investigation of the fresh bulbs of Fritillaria anhuiensis S. C. Chen et S. E. Yin, resulted in the isolation of a known steroidal alkaloid solanidine of (22S,25S)-solanid-5-en-3βol (1), which has never yet been found as a natural substance, and of a new steroidal alkaloid (22S,25S)-solanid-5,20(21)-dien-3β-ol. Compounds 1 and 2 were the first solanidine-type alkaloids with 22-S configuration discovered from nature. Their structures were elucidated based on spectroscopic analysis, including 1D and 2D NMR experiments. © 2009 Elsevier B.V. All rights reserved.
1. Introduction
2. Experimental
Fritillaria anhuiensis S. C. Chen et S. E. Yin is mainly distributed in Dabie mountains in Anhui province of China. The bulbs of the plant have been used in traditional Chinese medicine to treat diseases such as cough, sputum, and asthma [1]. Previous chemical investigation revealed four iso-steroidal alkaloids identified as verticinone, pemisine, isoverticine and Wanpeinine A [2]. Further phytochemical studies on the fresh bulbs of this species led to the isolation of solanidine [3] of (22S,25S)-solanid-5-en-3β-ol (1), which has never yet been found as a natural substance, and of (22S,25S)-solanid-5,20 (21)-dien-3β-ol (2). Compounds 1 and 2 were the first solanidine-type alkaloids with 22-S configuration discovered from nature. Herein we reported the isolation and structure elucidation of these two steroidal alkaloids. Although compound 1 had been synthesized from tomatid-5-en-3β-ol [4], we reported the spectroscopic data for the first time.
2.1. General
⁎ Corresponding author. School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, People's Republic of China. Tel.: +86 513 82198001; fax: +86 513 82198003. E-mail address: [email protected] (Z. Wu Shen). 0367-326X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2009.08.017
Optical rotation were measured with a Perkin-Elmer 341MC polarimeter; IR spectrum was acquired using a Shimadzu FTIR-8400S Spectrometer; LR-EIMS were obtained with a MAT-95 spectrometer, HR-EIMS with a Q-TOF Micro LC-MS-MS spectrometer; NMR spectrum were acquired on
Fig. 1. Structures of 1, 2 and solanidine.
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2.3. Extraction and isolation
Table 1 NMR spectroscopic data of compounds 1, 2 and solanidine. No.
1
1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
13
H NMR a
1.20 1.45 3.98 2.78 / 5.53 1.94 1.66 1.05 / 1.60 1.29 / 1.02 1.66 3.91 1.84 0.92 1.15 2.17 1.13 2.59 1.29 1.92 1.62 2.86 1.30
2 m, 1.92 m m, 1.52 m m m brs m, 2.23 m m m m m, 1.90 m m m, 2.10 m m s s m d (6.8) m m, 1.58 m, 2.02 m m, 2.77 d (6.8)
m
m m m
b
1.18 1.45 3.95 2.73 / 5.43 1.96 1.68 1.02 / 1.61 1.48 / 1.21 1.60 4.12 3.13 0.88 1.10 / 5.33 4.25 2.34 1.28 1.88 3.08 1.02
1 m, 1.93 m m, 1.51 m m m brs m, 2.27 m m m m m, 1.93 m m m, 1.65 m m m s s brs m m m, 1.66 m m m, 2.53 m d (6.6)
Solanidine b
C NMR
a
38.4 30.5 71.9 43.5 142.5 121.9 32.7 31.6 51.1 37.7 21.7 41.4 41.9 55.5 33.2 68.4 61.7 15.5 20.2 36.3 18.6 65.9 27.4 29.0 31.0 54.4 19.5
2
b
38.3 30.5 71.9 43.4 142.5 121.7 32.6 31.6 50.7 37.7 21.8 40.2 44.4 55.1 32.9 70.6 56.9 14.9 20.2 144.6 113.1 62.7 23.0 27.2 30.2 57.6 19.4
38.0 31.4 71.4 43.7 142.1 121.3 32.1 32.5 50.7 37.1 21.4 40.2 40.7 57.9 33.7 69.4 63.4 17.1 19.7 37.0 18.6 74.9 30.1 31.4 32.8 60.3 19.7
a 1 b
H (500 MHz) and 13C (125 MHz) NMR in pyridine-d5. 1 H (400 MHz) and 13C (100 MHz) NMR in pyridine-d5.
Varian INOVA 400 or Bruker AV-500 spectrometers with TMS as internal standard; Column chromatographic (CC) separations were carried out using silica gel H60 (300–400mesh, Qingdao Haiyang Chemical Group Corporation, People's Republic of China). 2.2. Plant material The fresh bulbs of F. anhuiensis were collected from Anhui province, People's Republic of China, and authenticated by Professor Zhou Xiujia of Shanghai University of TCM. A voucher specimen had been deposited in the herbarium of the Shanghai University of TCM.
The comminuted fresh bulbs of F. anhuiensis (20 Kg) were soaked with 95% EtOH for 3 d. After solvent removal, the crude extract was suspended in distilled water and partitioned successively with chloroform. The chloroform extract was evaporated under reduced pressure to afford a brownish crude extract (96.7 g), which was applied to a silica gel column, eluting with petroleum ether containing increasing amounts of EtOAc. Repeated column chromatography yielded solanidine (51 mg), compound 1 (11 mg), compound 2 (23 mg). (22S,25S)-solanid-5-en-3β-ol (1, Fig. 1) white amorphous power; mp 143.5–144.8 °C; [α] −35.6° (c 0.11, MeOH); IR (KBr) νmax 3400, 2925, 2852, 1454, 1377, 1360 cm− 1; 1H and 13 C NMR data: see Table 1; EIMS m/z (rel. int.): 397 (19), 382 (14), 204 (23), 150 (100), 91 (22); HR-EIMS m/z 397.3349 [M]+ (calc. for C27H43NO, 397.3345). (22S,25S)-solanid-5,20(21)-dien-3β-ol (2, Fig. 1). white amorphous power; mp 152.1–153.4 °C; [α] −16.2° (c 0.21, MeOH); IR (KBr) νmax 3370, 2926, 1454, 1377, 1364, 1049 cm− 1; 1 H and 13C NMR data: see Table 1; EIMS m/z (rel. int.): 395 (42), 380 (17), 162 (100), 148 (76); HR-EIMS m/z 395.3184 [M]+ (calc. for C27H41NO, 395.3188). 3. Results and discussion Compound 1, was obtained as white amorphous powder, its molecular formula was established as C27H43NO by HREIMS at m/z 397.3349 [M]+ (calc. 397.3345), the mass spectrum of 1 also revealed the base peak at m/z 150 and a diagnostic peak at m/z 204, which correspond to the typical fragment ions of solanidine alkaloids [5]. The analysis of 1H and 13C NMR spectrum (Table 1) indicated the existence of four methyl groups [δH 0.92, δH 1.15 (3H, each, s); δH 1.13, δH 1.30 (3H, each, d, J = 6.8 Hz)], a methine bearing a hydroxyl group (δH 3.98, 1H, m; δC 71.9), and a trisubstituted double bond (δH 5.53, 1H, brs; δC 121.9, 142.5). The 13C NMR spectrum of 1 was compared with that of solanidine, they have shown a very close similarity, except the carbon signals of ring E and F of compound 1 shifted upfield, especially signals at C-22 and C-26 shifted from δC 74.9, 60.3 to δC 65.9, 54.4 respectively, which suggested the different configurations of E/F ring fusion. The relative stereochemistry of 1 was established by analysis of its NOESY spectrum (Fig. 2). δH
Fig. 2. Key NOESY correlations of 1 (H–H).
Q.Y. Shou et al. / Fitoterapia 81 (2010) 81–84
Fig. 3. Major mass fragments of compound 2.
Fig. 4. Key HMBC correlations of 2 (H–C).
2.59 (H-22) showed a clear cross peak with δH 0.92 (H-18), confirming the configuration of H-22 is β orientated. The NOESY correlations were also observed between H-14/H-16, H-16/H-17, H-17/H-21, H-16/H-27, which indicated 16α-H, 17α-H, 21α-Me and 27α-Me. Based on all of the spectroscopic evidences, the structure of compound 1 was established as (22S, 25S)-solanid-5-en-3β-ol. Compound 2, was obtained as white amorphous power, The HR-EIMS exhibited an ion peak at m/z 395.3184 [M]+ (calc.
83
395.3188), corresponding to the molecular formula C27H41NO. The mass spectrum of 2 revealed the base peak at m/z 162 and a fragment ion peak at m/z 148 (Fig. 3). The 1H and 13C NMR spectrum of 2 indicated the presence of three methyl groups [δH 0.88, δH 1.10 (3H each, s); δH 1.02, (3H, d, J = 6.6 Hz)], a methine bearing a hydroxyl group (δH 3.95, 1H, m; δC 71.9), a trisubstituted double bond (δH 5.43, 1H, brs; δC 121.7, 142.5) as well as a terminal olefin (δH 5.33, 2H, brs; δC 113.1, 144.6). Comparing the NMR spectral data of 2 with that of 1, the major differences were the presence of characteristic signals of the terminal olefin and the absence of a methyl group. The position of the terminal olefin was confirmed located at C-20 by the HMBC correlations between olefinic proton (δH 5.33) and C-17 (δC 56.9), C-22 (δC 62.7) (Fig. 4). The NOESY spectrum revealed the same configurations at C-22 as compound 1 with β orientated H-22. Thus, compound 2 was characterized as (22S, 25S)-solanid-5, 20(21)-dien-3β-ol. These two steroidal alkaloids were the first solanidine-type alkaloids with 22-S configuration discovered from nature. This type of steroidal alkaloids was suggested biogenetically derived from cholesterol [6]. Based on the hypothetic biosynthesis of solanidine, we speculated there might be more than one pathway involved into the biosynthesis of solanidine type alkaloids. They were formed either from teinemine [7] (Pathway 1) or from (20,22)-dehydrogenated solanidine (Pathway 2). The (20,22)-dehydrogenated solanidine might also be the precursor of compound 2 which can be formed through 1, 3sigmatropic rearrangement. Thus compound 1 could be obtained from the hydrogenation of compound 2 (Scheme 1.).
Scheme 1. Proposed biogenesis of 1 and 2.
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Q.Y. Shou et al. / Fitoterapia 81 (2010) 81–84
Acknowledgement
References
This work was supported by a grant from the Ministry of Personnel of the People's Republic of China “2006 returnee's research funding”.
[1] Li AR. Chin Tradit Herb Drugs 1995;26:218. [2] Li QH, Wu ZH. Acta Pharm Sin 2007;42:58. [3] Kaneko K, Katsuhara T, Kitamura Y, Nishizawa M, Chen YP, Hsu HY. Chem Pharm Bull 1988;36:4700. [4] Schreiber K, Roensch H. Tetrahedron 1965;21:645. [5] Radeglia R, Adam G, Ripperger H. Tetrahedron Lett 1977;11:903. [6] Heftmann E. Phytochemistry 1983;22:1843. [7] Kaneko K, Tanaka MW, Takahashi E, Mitsuhashi H. Phytochemistry 1977;16:1620.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.fitote.2009.08.017.
Contents lists available at ScienceDirect
Fitoterapia j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f i t o t e
Two 22S-solanidine-type steroidal alkaloids from Fritillaria anhuiensis Qing Yao Shou a, Qing Tan a, Zheng Wu Shen a,b,⁎ a b
School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, People's Republic of China Basilea Pharmaceutical China Ltd., Haimen, Jiangsu 226100, People's Republic of China
a r t i c l e
i n f o
Article history: Received 19 March 2009 Accepted in revised form 23 July 2009 Available online 18 August 2009 Keywords: Fritillaria anhuiensis Solanidine Steroidal alkaloid
a b s t r a c t Phytochemical investigation of the fresh bulbs of Fritillaria anhuiensis S. C. Chen et S. E. Yin, resulted in the isolation of a known steroidal alkaloid solanidine of (22S,25S)-solanid-5-en-3βol (1), which has never yet been found as a natural substance, and of a new steroidal alkaloid (22S,25S)-solanid-5,20(21)-dien-3β-ol. Compounds 1 and 2 were the first solanidine-type alkaloids with 22-S configuration discovered from nature. Their structures were elucidated based on spectroscopic analysis, including 1D and 2D NMR experiments. © 2009 Elsevier B.V. All rights reserved.
1. Introduction
2. Experimental
Fritillaria anhuiensis S. C. Chen et S. E. Yin is mainly distributed in Dabie mountains in Anhui province of China. The bulbs of the plant have been used in traditional Chinese medicine to treat diseases such as cough, sputum, and asthma [1]. Previous chemical investigation revealed four iso-steroidal alkaloids identified as verticinone, pemisine, isoverticine and Wanpeinine A [2]. Further phytochemical studies on the fresh bulbs of this species led to the isolation of solanidine [3] of (22S,25S)-solanid-5-en-3β-ol (1), which has never yet been found as a natural substance, and of (22S,25S)-solanid-5,20 (21)-dien-3β-ol (2). Compounds 1 and 2 were the first solanidine-type alkaloids with 22-S configuration discovered from nature. Herein we reported the isolation and structure elucidation of these two steroidal alkaloids. Although compound 1 had been synthesized from tomatid-5-en-3β-ol [4], we reported the spectroscopic data for the first time.
2.1. General
⁎ Corresponding author. School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, People's Republic of China. Tel.: +86 513 82198001; fax: +86 513 82198003. E-mail address: [email protected] (Z. Wu Shen). 0367-326X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2009.08.017
Optical rotation were measured with a Perkin-Elmer 341MC polarimeter; IR spectrum was acquired using a Shimadzu FTIR-8400S Spectrometer; LR-EIMS were obtained with a MAT-95 spectrometer, HR-EIMS with a Q-TOF Micro LC-MS-MS spectrometer; NMR spectrum were acquired on
Fig. 1. Structures of 1, 2 and solanidine.
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Q.Y. Shou et al. / Fitoterapia 81 (2010) 81–84
2.3. Extraction and isolation
Table 1 NMR spectroscopic data of compounds 1, 2 and solanidine. No.
1
1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
13
H NMR a
1.20 1.45 3.98 2.78 / 5.53 1.94 1.66 1.05 / 1.60 1.29 / 1.02 1.66 3.91 1.84 0.92 1.15 2.17 1.13 2.59 1.29 1.92 1.62 2.86 1.30
2 m, 1.92 m m, 1.52 m m m brs m, 2.23 m m m m m, 1.90 m m m, 2.10 m m s s m d (6.8) m m, 1.58 m, 2.02 m m, 2.77 d (6.8)
m
m m m
b
1.18 1.45 3.95 2.73 / 5.43 1.96 1.68 1.02 / 1.61 1.48 / 1.21 1.60 4.12 3.13 0.88 1.10 / 5.33 4.25 2.34 1.28 1.88 3.08 1.02
1 m, 1.93 m m, 1.51 m m m brs m, 2.27 m m m m m, 1.93 m m m, 1.65 m m m s s brs m m m, 1.66 m m m, 2.53 m d (6.6)
Solanidine b
C NMR
a
38.4 30.5 71.9 43.5 142.5 121.9 32.7 31.6 51.1 37.7 21.7 41.4 41.9 55.5 33.2 68.4 61.7 15.5 20.2 36.3 18.6 65.9 27.4 29.0 31.0 54.4 19.5
2
b
38.3 30.5 71.9 43.4 142.5 121.7 32.6 31.6 50.7 37.7 21.8 40.2 44.4 55.1 32.9 70.6 56.9 14.9 20.2 144.6 113.1 62.7 23.0 27.2 30.2 57.6 19.4
38.0 31.4 71.4 43.7 142.1 121.3 32.1 32.5 50.7 37.1 21.4 40.2 40.7 57.9 33.7 69.4 63.4 17.1 19.7 37.0 18.6 74.9 30.1 31.4 32.8 60.3 19.7
a 1 b
H (500 MHz) and 13C (125 MHz) NMR in pyridine-d5. 1 H (400 MHz) and 13C (100 MHz) NMR in pyridine-d5.
Varian INOVA 400 or Bruker AV-500 spectrometers with TMS as internal standard; Column chromatographic (CC) separations were carried out using silica gel H60 (300–400mesh, Qingdao Haiyang Chemical Group Corporation, People's Republic of China). 2.2. Plant material The fresh bulbs of F. anhuiensis were collected from Anhui province, People's Republic of China, and authenticated by Professor Zhou Xiujia of Shanghai University of TCM. A voucher specimen had been deposited in the herbarium of the Shanghai University of TCM.
The comminuted fresh bulbs of F. anhuiensis (20 Kg) were soaked with 95% EtOH for 3 d. After solvent removal, the crude extract was suspended in distilled water and partitioned successively with chloroform. The chloroform extract was evaporated under reduced pressure to afford a brownish crude extract (96.7 g), which was applied to a silica gel column, eluting with petroleum ether containing increasing amounts of EtOAc. Repeated column chromatography yielded solanidine (51 mg), compound 1 (11 mg), compound 2 (23 mg). (22S,25S)-solanid-5-en-3β-ol (1, Fig. 1) white amorphous power; mp 143.5–144.8 °C; [α] −35.6° (c 0.11, MeOH); IR (KBr) νmax 3400, 2925, 2852, 1454, 1377, 1360 cm− 1; 1H and 13 C NMR data: see Table 1; EIMS m/z (rel. int.): 397 (19), 382 (14), 204 (23), 150 (100), 91 (22); HR-EIMS m/z 397.3349 [M]+ (calc. for C27H43NO, 397.3345). (22S,25S)-solanid-5,20(21)-dien-3β-ol (2, Fig. 1). white amorphous power; mp 152.1–153.4 °C; [α] −16.2° (c 0.21, MeOH); IR (KBr) νmax 3370, 2926, 1454, 1377, 1364, 1049 cm− 1; 1 H and 13C NMR data: see Table 1; EIMS m/z (rel. int.): 395 (42), 380 (17), 162 (100), 148 (76); HR-EIMS m/z 395.3184 [M]+ (calc. for C27H41NO, 395.3188). 3. Results and discussion Compound 1, was obtained as white amorphous powder, its molecular formula was established as C27H43NO by HREIMS at m/z 397.3349 [M]+ (calc. 397.3345), the mass spectrum of 1 also revealed the base peak at m/z 150 and a diagnostic peak at m/z 204, which correspond to the typical fragment ions of solanidine alkaloids [5]. The analysis of 1H and 13C NMR spectrum (Table 1) indicated the existence of four methyl groups [δH 0.92, δH 1.15 (3H, each, s); δH 1.13, δH 1.30 (3H, each, d, J = 6.8 Hz)], a methine bearing a hydroxyl group (δH 3.98, 1H, m; δC 71.9), and a trisubstituted double bond (δH 5.53, 1H, brs; δC 121.9, 142.5). The 13C NMR spectrum of 1 was compared with that of solanidine, they have shown a very close similarity, except the carbon signals of ring E and F of compound 1 shifted upfield, especially signals at C-22 and C-26 shifted from δC 74.9, 60.3 to δC 65.9, 54.4 respectively, which suggested the different configurations of E/F ring fusion. The relative stereochemistry of 1 was established by analysis of its NOESY spectrum (Fig. 2). δH
Fig. 2. Key NOESY correlations of 1 (H–H).
Q.Y. Shou et al. / Fitoterapia 81 (2010) 81–84
Fig. 3. Major mass fragments of compound 2.
Fig. 4. Key HMBC correlations of 2 (H–C).
2.59 (H-22) showed a clear cross peak with δH 0.92 (H-18), confirming the configuration of H-22 is β orientated. The NOESY correlations were also observed between H-14/H-16, H-16/H-17, H-17/H-21, H-16/H-27, which indicated 16α-H, 17α-H, 21α-Me and 27α-Me. Based on all of the spectroscopic evidences, the structure of compound 1 was established as (22S, 25S)-solanid-5-en-3β-ol. Compound 2, was obtained as white amorphous power, The HR-EIMS exhibited an ion peak at m/z 395.3184 [M]+ (calc.
83
395.3188), corresponding to the molecular formula C27H41NO. The mass spectrum of 2 revealed the base peak at m/z 162 and a fragment ion peak at m/z 148 (Fig. 3). The 1H and 13C NMR spectrum of 2 indicated the presence of three methyl groups [δH 0.88, δH 1.10 (3H each, s); δH 1.02, (3H, d, J = 6.6 Hz)], a methine bearing a hydroxyl group (δH 3.95, 1H, m; δC 71.9), a trisubstituted double bond (δH 5.43, 1H, brs; δC 121.7, 142.5) as well as a terminal olefin (δH 5.33, 2H, brs; δC 113.1, 144.6). Comparing the NMR spectral data of 2 with that of 1, the major differences were the presence of characteristic signals of the terminal olefin and the absence of a methyl group. The position of the terminal olefin was confirmed located at C-20 by the HMBC correlations between olefinic proton (δH 5.33) and C-17 (δC 56.9), C-22 (δC 62.7) (Fig. 4). The NOESY spectrum revealed the same configurations at C-22 as compound 1 with β orientated H-22. Thus, compound 2 was characterized as (22S, 25S)-solanid-5, 20(21)-dien-3β-ol. These two steroidal alkaloids were the first solanidine-type alkaloids with 22-S configuration discovered from nature. This type of steroidal alkaloids was suggested biogenetically derived from cholesterol [6]. Based on the hypothetic biosynthesis of solanidine, we speculated there might be more than one pathway involved into the biosynthesis of solanidine type alkaloids. They were formed either from teinemine [7] (Pathway 1) or from (20,22)-dehydrogenated solanidine (Pathway 2). The (20,22)-dehydrogenated solanidine might also be the precursor of compound 2 which can be formed through 1, 3sigmatropic rearrangement. Thus compound 1 could be obtained from the hydrogenation of compound 2 (Scheme 1.).
Scheme 1. Proposed biogenesis of 1 and 2.
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Q.Y. Shou et al. / Fitoterapia 81 (2010) 81–84
Acknowledgement
References
This work was supported by a grant from the Ministry of Personnel of the People's Republic of China “2006 returnee's research funding”.
[1] Li AR. Chin Tradit Herb Drugs 1995;26:218. [2] Li QH, Wu ZH. Acta Pharm Sin 2007;42:58. [3] Kaneko K, Katsuhara T, Kitamura Y, Nishizawa M, Chen YP, Hsu HY. Chem Pharm Bull 1988;36:4700. [4] Schreiber K, Roensch H. Tetrahedron 1965;21:645. [5] Radeglia R, Adam G, Ripperger H. Tetrahedron Lett 1977;11:903. [6] Heftmann E. Phytochemistry 1983;22:1843. [7] Kaneko K, Tanaka MW, Takahashi E, Mitsuhashi H. Phytochemistry 1977;16:1620.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.fitote.2009.08.017.