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Two new alkaloids from the bulbs of Lycoris sprengeri a
a
a
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Wen-Ming. Wu , Yun-Yun. Zhu , Hao-Ran. Li , Heng-Yi. Yu , Peng. a
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Zhang , Hui-Fang. Pi & Han-Li. Ruan
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Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, Faculty of Pharmacy, Tongji Medical College of Huazhong University of Science and Technology, Wuhan, 430030, China Published online: 06 Dec 2013.
To cite this article: Wen-Ming. Wu, Yun-Yun. Zhu, Hao-Ran. Li, Heng-Yi. Yu, Peng. Zhang, Hui-Fang. Pi & Han-Li. Ruan (2014) Two new alkaloids from the bulbs of Lycoris sprengeri, Journal of Asian Natural Products Research, 16:2, 192-199, DOI: 10.1080/10286020.2013.864639 To link to this article: http://dx.doi.org/10.1080/10286020.2013.864639
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Journal of Asian Natural Products Research, 2014 Vol. 16, No. 2, 192–199, http://dx.doi.org/10.1080/10286020.2013.864639
Two new alkaloids from the bulbs of Lycoris sprengeri Wen-Ming. Wu, Yun-Yun. Zhu, Hao-Ran. Li, Heng-Yi. Yu, Peng. Zhang, Hui-Fang. Pi and Han-Li. Ruan*
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Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, Faculty of Pharmacy, Tongji Medical College of Huazhong University of Science and Technology, Wuhan 430030, China (Received 11 July 2013; final version received 7 November 2013) Two new alkaloids, lycosprenine (1) and 2a-methoxy-6-O-methyllycorenine (2), along with 22 known alkaloids (3 – 23b), were isolated from the bulb of Lycoris sprengeri. Their structures were elucidated on the basis of spectral analysis and by comparison of the spectroscopic data with those of known compounds. Selected compounds (1 – 3 and 6– 9) were tested for their neuroprotective activities against H2O2-, CoCl2- and Ab25 – 35induced SH-SY5Y cell injury, most of which exhibited neuroprotective effects of different degrees. Keywords: Amaryllidaceae; Lycoris sprengeri; alkaloids; neuroprotective
1.
Introduction
Plants of the family Amaryllidaceae include about 60 genera and 800 species, which are widely distributed in the tropics and warm temperate regions of the world [1]. Most Amaryllidaceae plants are known to produce unique alkaloids, which have diverse bioactivities such as acetylcholinesterase inhibitory, analgesic, antibacterial, antifungal, antimalarial, antitumor, antiviral activities, and so on [2]. Especially, galantamine and lycorine types exhibited significant usefulness in healing Alzheimer’s disease [3]. Lycoris sprengeri Comes ex Baker is mainly distributed in the lower reaches of the Yangtze River, and its bulbs have been used as traditional Chinese medicine for the treatment of various diseases, such as sore throats, furuncle carbuncle swollen in skin and scrofula [4,5]. However, the chemical constituents of the plant have never been studied before. In order to discover the bioactive constituents from L. sprengeri, we
investigated the bulbs of L. sprengeri. Accordingly, two new alkaloids, lycosprenine (1) and 2a-methoxy-6-O-methyllycorenine (2), together with 22 known alkaloids, O-methyllycorenine (3) [6,7], lycorenine (4) [8], homolycorine (5) [9], tortuosine (6) [10], haemanthamine (7) [11], hippadine (8) [12], N-isopentylcrinasiadine (9) [2], crinasiadine (10) [13], 5,6-dihydrobicolorine (11) [14], trisphaeridine (12) [15], narcissidine (13) [16], N-(chloromethyl)narcissidine (14) [17], galantamine (15) [18], lycoramine (16) [7], lycosinine B (17) [19], tazettine (18) [20], montabuphine (19) [21], lycorine (20) [22], pluviine (21) [23], galanthine (22) [18], (þ)-haemanthidine (23a), and (–)haemanthidine (23b) [24], were isolated and identified (Figure 1). In this paper, we describe their structural elucidation as well as the neuroprotective activities [25] of the selected compounds 1–3 and 6–9.
*Corresponding author. Email: [email protected] q 2013 Taylor & Francis
Journal of Asian Natural Products Research 12
OCH3 1
H3CO
10
H3CO 9
3 3a
11b
11
8
N
7a
7
6
5
O
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1
O
H3CO H3CO 8
10 9
OCH3
4
2 R4 10a 10b 1 6a R3 6 O
O
O N
H3CO R2 2: R1=OCH3 R2=HR3 =OCH3 R4=H 3: R1=OCH3 R2=R3=R4=H R2=R3=R4=H 4: R1=OH 5: R1+R2=OR3=R4=H
O N
O 9: R=CH2CH2CH(CH3)2 10: R=H OCH3 OH
HO
H H3CO
N
12 OH
N CH3
O H
N CH3
O
N -
Cl 14
13
OH
OCH3 OH
HO
H3CO
N
O
CH3
11
H3CO
H3CO
7
6
O
R
H N
O
-
Cl
O N
O
O 8
H3CO
7 R 1
O N
OMe OH
3
4a 4
11a
11
H3C N
2
193
Cl
H3CO
H3CO 15
16
OCH3 H3C N
OH H N CH3
H3CO
O H
H3CO
OH
O 17
O
N H 19
O
O
R2 N
20: R1=R2=OCH2O R3=R4=OH 21: R1=R2=OCH3 R3=OH R4=H 22: R1=R2=OCH3 R3=OH R4=OCH3
OMe OH
OMe OH
R3
R1
OCH3
O
18 R4
H
O
O
H N OH 23a
O
H N OH 23b
Figure 1. The structures of compounds 1 – 23b.
2. Results and discussion Compound 1 was obtained as white amorphous solids. The molecular formula of 1 was established as C18H17NO4 by positive HR-ESI-MS from the [M þ H]þ ion peak at m/z 312.1219 [M þ H]þ, indicating 11 degrees of unsaturation. The 1H NMR spectrum of 1 revealed the presence of three methoxyl groups at d 3.92 (3H, s), 4.04 (3H, s), and 4.08 (3H, s),
two methylenes at d 3.40 (2H, t, J ¼ 8.0 Hz) and 4.48 (2H, t, J ¼ 8.0 Hz), and four aromatic protons at d 6.94 (1H, s), 7.23 (1H, s), 7.44 (1H, s), and 7.94 (1H, s). The 13C NMR and DEPT spectra of 1 displayed 18 carbon signals, which were resolved as three methoxyl carbons (dC 56.3, 56.4, and 56.4), two methylene carbons (d 27.6 and 46.9), four sp2 methine carbons (d 102.9, 103.1, 109.1, and 112.5),
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Table 1. 1H (400 MHz) and 13C NMR (100 MHz) spectral data of compound 1 in CDCl3 (d in ppm, J in Hz) Position
dC
1 2 3 3a 4 5 7 7a 8 9 10 11 11a 11b 11c 2-OCH3 9-OCH3 10-OCH3
102.9 (CH) 157.1 (C) 112.5 (CH) 132.4 (C) 27.6 (CH2) 46.9 (CH2) 159.3 (C) 121.8 (C) 109.1 (CH) 149.9 (C) 152.9 (C) 103.1 (CH) 128.4 (C) 116.8 (C) 134.1 (C) 56.4 (CH3) 56.4 (CH3) 56.3 (CH3)
dH (multiplicity) 7.23 (s, 1H) 6.94 (s, 1H) 3.40 (t, J ¼ 8.0, 2H) 4.48 (t, J ¼ 8.0, 2H) 7.94 (s, 1H) 7.44 (s, 1H)
3.92 (s, 3H) 4.04 (s, 3H) 4.08 (s, 3H)
and nine quaternary carbons (d 116.8, 121.8, 128.4, 132.4, 134.1, 149.9, 152.9, 157.1, and 159.3) including a carbonyl carbon (d 159.3). The spectral characteristics of 1 (Table 1) were very similar to those of tortuosine (6) [10], except that the olefinic proton in tortuosine at position C-7 was replaced by a carbonyl group in 1. The position of the carbonyl group at C-7 (d 159.3) was supported by HMBC correlations from H-5 to C-7 and from H-8 to C-7. In addition, the structure of 1 was further confirmed by the following key HMBC correlations: H-5/C-3a, C-11c; H-8/C-10, C-11a; H-3/C-4, C-1, C-11c
(Figure 2). From the above several pieces of evidence, the structure of 1 was established as 2,9,10-trimethoxy-4H-pyrrolo[3,2,1-de]phenanthridin-7(5H)-one, named lycosprenine. Compound 2 was obtained as white needles, with ½a20 D þ 182.80. The molecular formula of 2 was determined to be C20H27NO5 by positive HR-ESI-MS at m/z 362.1957 [M þ H]þ. The 1H NMR spectrum of 2 (Table 2) showed an NZCH3 signal at d 2.10 (3H, s), four methoxyl signals at d 3.44 (3H, s), 3.57 (3H, s), 3.88 (3H, s), and 3.89 (3H, s), two aromatic proton signals at d 6.78 (1H, s, H-7) and 6.94 (1H, s, H-10), and an olefinic proton signal at d 5.62 (1H, brs, H-3). Accordingly, the 13C NMR, DEPT, and HSQC spectra of 2 revealed the presence of an NZCH3 (d 44.5), four methoxyl groups (d 55.5, 56.0, 56.3, and 57.3), two methylenes [dC 28.4 (C-11) and 57.0 (C-12)], eight methines [d 41.2 (C-10b), 67.7 (C-4a), 69.7 (C-1); 78.6 (C-2), 98.8 (C-6), 109.8 (C-7), 112.9 (C-10), and 117.1 (C-3)], respectively. The above NMR data of 2 (Table 2) were very similar to those of O-methyllycorenine (3) [6], except that 2 had an additional methoxyl group (dH 3.44, s; dC 57.3) on C-2 which was supported by the HMBC correlations between ZOCH3 (dH 3.44, s, 3H) and C-2 (dC 78.6) as well as between H2 (dH 3.78, brs) and ZOCH3 (dC 57.3) in 2 (Figure 2). A series of 2D NMR experiments including 1H – 1H COSY, HSQC and HMBC allowed the assignment of the planar
OCH3 2
3
12
11
H3C N
4
H3CO
10
11
H3CO 9 8
11b
3a 4
11a
N6
7a
5
H3CO
10 10a 9
H3CO 8 6a 7 2
1
H 2
10b
O HMBC:
3
4a
1
1
OCH3
O 6
OCH3
COSY:
Figure 2. The key COSY and HMBC correlations of compounds 1 and 2.
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Table 2. 1H (400 MHz) and 13C NMR (100 MHz) spectral data of compound 2 in CDCl3 (d in ppm, J in Hz) Position
dC
1 2 3 4 4a 6 6a 7 8 9 10 10a 10b 11 12a 12b 2-OCH3 6-OCH3 8-OCH3 9-OCH3 N-CH3
69.7 (CH) 78.6 (CH) 117.1 (CH) 146.3 (C) 67.7 (CH) 98.8 (CH) 125.8 (C) 109.8 (CH) 148.6 (C) 148.7 (C) 112.9 (CH) 129.8 (C) 41.2 (CH) 28.4 (CH2) 57.0 (CH2) 57.0 (CH2) 57.3 (CH3) 55.5 (CH3) 56.3 (CH3) 56.0 (CH3) 44.5 (CH3)
dH (multiplicity) 4.26 (brs, 1H) 3.78 (brs, 1H) 5.62 (brs, 1H) 2.71 (d, J ¼ 9.1, 1H) 5.51 (s, 1H) 6.78 (s, 1H)
6.94 (s, 1H) 2.52 (dd, J ¼ 9.1, 1H) 2.54 (m, 1H) 3.19 (m, 1H) 2.25 (dt, J ¼ 9.4, 1H) 3.44 (s, 3H) 3.57 (s, 3H) 3.89 (s, 3H) 3.88 (s, 3H) 2.10 (s, 3H)
structure of 2 (Figure 2). The small coupling constant between H-1 and H-10b indicated the cis-junction of the B and C rings. While the larger coupling constant (9.1 Hz) of H-4a and H-10b suggested their trans-diaxial relationship. The NOESY correlations of H1, H-10b, 2-OCH3, and 6-OCH3 reflected the same orientation (Figure 3). Finally, the structure of 2 was confirmed to be 2amethoxy-6-O-methyllycorenine, which was further supported by its positive specific rotation ½a20 D þ 182.80 (CHCl3). Compounds 1– 3 and 6 – 9 were tested for their neuroprotective activities against H 2O 2-, CoCl 2- and Ab25 – 35-induced SH-SY5Y cell death. Compounds 3 and 8 exhibited significant neuroprotective effects against H2O2-induced SH-SY5Y cell death (Table 3). Compounds 1, 3, and 6 showed obvious neuroprotective effects against CoCl2-induced SH-SY5Y cell injury (Table 4). While compounds 1– 3, 6, and 8 exhibited apparent neuroprotec-
Figure 3. The key NOESY correlations of compound 2.
tive effects against Ab25 – 35-induced SHSY5Y cell injury (Table 5). 3.
Experimental
3.1 General experimental procedures Optical rotations were measured with an automatic polarimeter (PE-341LC, PerkinElmer Co., Waltham, MA, USA). Melting points were determinned on Beijing Tech X-5 microscopic melting point apparatus (Taike Corp., Beijing, China). UV spectra were obtained on a Shanghai Spectrum 765PC spectrophotometer (Shanghai Spectrum Instruments Co., Ltd, Shanghai, China). IR spectra were recorded on a Perkin-Elmer spectrum (Perkin-Elmer Co.) one FT-IR spectrometer. HR-ESIMS were obtained on a Thermo Scientific LTQ-Qrbitrap XL mass spectrometer (Thermo Fisher Scientific, Inc., Waltham, MA, USA). 1D and 2D NMR spectra were recorded on a Bruker AM-400 spectrometer (Bruker Analytische Messtechnik GmbH, Karlsruse, Germany) with TMS as the internal standard. Silica gel (200 – 300 mesh; Qingdao Marine Chemical, Inc., Qingdao, China) and Sephadex LH-20 gel (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) were used for column chromatography (CC). Thin-layer chromatography was carried out on silica gel 60
Note: The activity was quantified in terms of OD570, shown as a percentage of the value with no inhibitor, taken as 100%. All data were analyzed by one-way ANOVA followed by Dunnett’s “t” test (n ¼ 3) with a kind of statistical software PASW statistics 18.0, *p , 0.05, **p , 0.01, ***p , 0.001.
56.03 ^ 0.62 42.48 ^ 0.76 56.06 ^ 0.62 45.99 ^ 0.40 54.48 ^ 0.15 54.48 ^ 0.15 45.99 ^ 0.40 56.30 ^ 0.42 38.78 ^ 0.61 61.65 ^ 0.25* 17.96 ^ 0.69*** 48.51 ^ 0.30 57.27 ^ 0.62 42.66 ^ 0.15 59.08 ^ 0.23 43.33 ^ 0.50 60.97 ^ 0.21* 42.31 ^ 0.46** 51.39 ^ 0.23 58.87 ^ 0.12*** 47.61 ^ 0.51 1 2 3 6 7 8 9
57.27 ^ 0.81 40.37 ^ 0.70 59.42 ^ 0.10 44.41 ^ 0.21 57.20 ^ 0.42* 65.63 ^ 0.20*** 44.41 ^ 0.32
62.34 ^ 0.30* 45.10 ^ 0.50 68.86 ^ 0.38** 38.63 ^ 0.49*** 59.71 ^ 0.50** 64.71 ^ 0.36*** 49.23 ^ 0.62*
59.76 ^ 0.50* 46.09 ^ 0.23 63.11 ^ 0.61** 25.94 ^ 0.93** 50.37 ^ 0.32 55.10 ^ 0.59 46.41 ^ 0.74
350 mM H2O2 12.5 Compound
6.25
25
50
100
W.-M. Wu et al.
Test concentrations (mM)
Table 3. Cell viabilities (%) of compounds 1 – 3 and 6 –9 in different concentrations in H2O2-induced SH-SY5Y cell injuries.
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F254 over glass plates (Qingdao Marine Chemical, Inc.) using various solvent systems. HPLC was carried out on an Agilent 1260 system using YMC-Pack ODS-A C18 5 mm (250 mm £ 10 mm i.d.) columns (Agilent Technologies, Inc., Santa Clara, CA, USA) for the purpose of analysis and semi-preparation. 3.2
Plant material
The bulbs of L. sprengeri were collected from Taizhou City of Zhejiang Province of China in July 2011, and authenticated by Mr Xiaogang Wang. A voucher specimen (No. 20110829) has been deposited in the Faculty of Pharmaceutical Sciences, Tongji Medical College of Huazhong University of Science and Technology, Wuhan, China. 3.3 Extraction and isolation Air-dried, powdered bulbs (17 kg) of L. sprengeri were extracted with 95% EtOH (10 times, each 3 h) at the temperature of 558C and concentrated in vacuo to yield a crude extract (2000 g). The extract was suspended in water (20 l) and extracted with CH2Cl2 to yield CH2Cl2 fraction (60 g). Then the aqueous phase was adjusted to pH 10 –12 with NaOH and then extracted with CH2Cl2 to give an alkaloid residue (170 g). The alkaloid residue (170 g) was subjected to silica gel CC eluted with CH2Cl2 – MeOH (1:0 to 0:1) gradient as the mobile phase to provide seven fractions A – G. Fraction A (2 g) was subjected to silica gel column, eluting with a gradient of CH2Cl2 – MeOH (1:0 to 0:1) to provide four subfractions A1, A2, A3, and A 4. Subfraction A1 (0.6 g) was subjected to silica gel CC eluted with CH2Cl2 – MeOH ¼ 280:1 to yield compounds 12 (20 mg) and 11 (15 mg). Subfraction A2 (0.7 g) was further separated by semi-preparative HPLC (90:10 MeOH – H2 O mobile phase) to give
Note: The activity was quantified in terms of OD570, shown as a percentage of the value with no inhibitor, taken as 100%. All data were analyzed by one-way ANOVA followed by Dunnett’s “t” test (n ¼ 3) with a kind of statistical software PASW statistics 18.0, *p , 0.05, **p , 0.01, ***p , 0.001.
51.60 ^ 0.44 69.80 ^ 0.50 51.60 ^ 0.44 58.08 ^ 0.26 58.33 ^ 0.40 58.33 ^ 0.40 58.08 ^ 0.26 58.24 ^ 0.32** 73.74 ^ 0.35** 61.49 ^ 0.30** 64.87 ^ 0.35*** 28.75 ^ 0.35 62.33 ^ 0.23** 53.37 ^ 0.20 62.81 ^ 0.55** 76.50 ^ 0.47** 67.33 ^ 0.59*** 66.87 ^ 0.10*** 33.67 ^ 0.40 61.73 ^ 0.13* 58.92 ^ 0.23 63.01 ^ 0.26*** 68.20 ^ 0.51 65.65 ^ 0.42*** 63.58 ^ 0.55** 31.16 ^ 0.15 60.14 ^ 0.31* 58.83 ^ 0.21 1 2 3 6 7 8 9
62.00 ^ 0.40*** 70.10 ^ 0.53 64.64 ^ 0.15** 65.93 ^ 0.55** 34.85 ^ 0.36 61.92 ^ 0.21** 62.04 ^ 0.25***
64.23 ^ 0.44*** 73.78 ^ 0.40** 71.18 ^ 0.42*** 71.31 ^ 0.66** 36.08 ^ 0.53 63.00 ^ 0.46** 64.39 ^ 0.12***
100 50 25 12.5 6.25 Compound
Test concentrations (mM)
Table 4. Cell viabilities (%) of compounds 1 – 3 and 6 –9 in different concentrations in CoCl2-induced SH-SY5Y cell injuries.
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256 mM CoCl2
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compounds 8 (6 mg, tR 13 min) and 10 (2.5 mg, tR 11.5 min). Fraction B (1.1 g) was separated into three subfractions B1, B2 and B3 by Sephadex LH-20 using CH2Cl2 – MeOH (1:1). Subfraction B2 (0.3 g) was further purified by semipreparative HPLC (60:40 MeOH – H2O mobile phase) to give compounds 16 (4 mg) and 19 (40 mg). Fraction C (8.1 g) was subjected to silica gel CC eluted with CH2Cl2 – MeOH (18:1) to provide compounds 13 (5 mg), 14 (6 g), and 17 (4 mg). Similar to fraction B, fraction D (5 g) was separated into five subfractions D1, D2, D3, D4, and D5. Then subfraction D3 (2.4 g) was separated into further four subfractions D3-1, D3-2, D3-3, and D3-4, by Sephadex LH-20 using CH2Cl2 –MeOH (1:1). Subfraction D3-3 (0.9 g) was purified by HPLC (80:20 MeOH – H2O mobile phase) to yield compounds 3 (12 mg) and 18 (25 mg). Fraction E (8 g) was subjected to silica gel CC, eluting with a gradient of CH2Cl2 – MeOH (1:0 to 0:1) to provide four subfractions E1, E2, E3, and E4. From subfraction E2 (1.8 g), compound 9 (4.5 mg) was obtained over a further silica gel column eluted with CH2Cl2 –MeOH (100:1). Subfracted E3 (1.1 g) was purified by HPLC (80:20 MeOH – H2O mobile phase) to yield compound 1 (3.5 mg, tR 10.9 min). Subfraction E4 (3.4 g) was subjected to separation on RP-C18, eluting with MeOH –H2O (30:70) to yield compounds 6 (0.6 g) and 15 (15 mg). Fraction F (7 g) was separated into four subfractions F1, F2, F3, and F4, by Sephadex LH-20 using MeOH. Subfraction F3 (2.3 g) was further purified by HPLC (60:40 MeOH – H2O mobile phase) to yield compounds 4 (20 mg, tR 9.3 min) and 22 (10 mg, tR 14.7 min). Fraction G (12 g) was subjected to silica gel column to separate into five subfractions G1, G2, G3, G4, and G5 by eluting with a gradient of CH2Cl2 –MeOH (1:0 to 0:1). Subfraction G3 (1.2 g) was purified by HPLC (80:20 MeOH – H2O, mobile phase) to yield compounds 2 (4 mg, tR 11 min) and 5 (10 mg, tR 12.3 min).
Note: The activity was quantified in terms of OD570, shown as a percentage of the value with no inhibitor, taken as 100%. All data were analyzed by one-way ANOVA followed by Dunnett’s “t” test (n ¼ 3) with a kind of statistical software PASW statistics 18.0, *p , 0.05, **p , 0.01, ***p , 0.001.
55.75 ^ 0.20 59.49 ^ 0.44 55.75 ^ 0.20 56.02 ^ 0.45 61.43 ^ 0.53 61.43 ^ 0.53 56.02 ^ 0.45 52.14 ^ 0.35 70.45 ^ 0.15** 52.40 ^ 0.15 59.71 ^ 0.38** 51.01 ^ 0.38 64.30 ^ 0.47* 50.58 ^ 0.57 54.46 ^ 0.50 71.63 ^ 0.25*** 56.60 ^ 0.75 63.23 ^ 0.45*** 51.01 ^ 0.38 64.30 ^ 0.47* 53.45 ^ 0.15 58.55 ^ 0.42* 72.36 ^ 0.40*** 58.74 ^ 0.36** 63.98 ^ 0.26** 47.85 ^ 0.31 66.56 ^ 0.15* 57.07 ^ 0.55 1 2 3 6 7 8 9
59.44 ^ 0.32** 64.70 ^ 0.71* 56.75 ^ 0.30 72.36 ^ 0.40*** 45.30 ^ 0.56 65.37 ^ 0.30* 55.91 ^ 0.46
64.93 ^ 0.53** 77.57 ^ 0.64*** 65.74 ^ 0.35*** 66.71 ^ 0.49*** 47.85 ^ 0.31 71.77 ^ 0.40*** 59.40 ^ 0.75*
1mM Ab25-35 50 25 Compound
6.25
12.5
100
W.-M. Wu et al.
Test concentrations (mM)
Table 5. Cell viabilities (%) of compounds 1 – 3 and 6 –9 in different concentrations in Ab25 – 35-induced SH-SY5Y cell injuries.
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Subfraction G4 (4.8 g) was further separated into four subfractions G4-1, G4-2, G43 and G4-4, by Sephadex LH-20 using MeOH. Subfraction G4-3 (1.1 g) was further purified by HPLC (60:40 MeOH – H2O, mobile phase) to yield compounds 7 (15 mg, tR 12 min), 20 (10 mg, tR 8 min), 21 (15 mg, tR 14 min), 23a (15 mg, tR 9 min), and 23b (15 mg, tR 9 min). 3.3.1
Lycosprenine (1)
White amorphous solids; UV (MeOH) lmax: (log 1) 205 (2.84), 230 (2.55), and 285 (2.24) nm; IR (KBr) nmax: 2969, 2922, 2864, 1736, 1598, 1514, 1459, 1389, 1261, 1022, 871, 798, 748, and 664 cm21; 1H NMR and 13C NMR (400 MHz, CDCl3) spectral data (see Table 1); HR-ESI-MS: m/z 312.1219 [M þ H] þ (calcd for C18H18NO4, 312.1236). 3.3.2 2a-Methoxy-6-O-methyllycorenine (2) White needles; ½a20 D þ 182.80 (CHCl3); m. p. 126–1288C; UV(MeOH) lmax: (log 1) 205 (4.33), 235 (3.93), and 280 (3.48) nm; IR (KBr) nmax: 2969, 2926, 2788, 2658, 2499, 2027, 1694, 1611, 1513, 1464, 1353, 1262, 1187, 958, 796, and 609 cm21; 1H NMR and 13C NMR (400 MHz, CDCl3) spectral data (see Table 2); HR-ESI-MS: m/z 362.1957 [M þ H]þ (calcd for C20H28NO5, 362.1967). 3.4 Bioassay procedure SH-SY5Y cells, which were obtained from the cell bank of Basic Medical College of Huazhong University of Science and Technology (Wuhan, China), were cultured in Dulbecco’s modified eagle medium high glucose medium containing 10% fetal calf serum at 37 8C in a humidified atmosphere with 5% CO2 incubator. The cell viability was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT)
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Journal of Asian Natural Products Research assay. About 9 £ 103 cells in 1 ml of logarithmic phase were seeded onto a 96well microplate. Twenty-four hours later, each hole with 11.5 ml sample was tested in the normal group, while 11.5 ml PBS in the control group. After 2 h, CoCl2, H2O2 or Ab25 – 35 was added to the normal group and control group. A total of 15 ml of MTT solution (5 mg/ml) were added to all the holes after the cells were cultured at 37 8C for 24 h. After continuing to culture for 4 h, the medium was removed and dimethyl sulfoxide (100 ml/well) was added. Finally, the reduced MTT was assayed at 570 nm using a multi-function microplate reader. Acknowledgments Financial support from the Ministry of Science and Technology of the People’s Republic of China (International Cooperative Project, Grant No. 2010DFA32430) and Natural Science Foundation of China (Nos 81072547, 31270394 and 30873361) is gratefully acknowledged.
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Journal of Asian Natural Products Research Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ganp20
Two new alkaloids from the bulbs of Lycoris sprengeri a
a
a
a
Wen-Ming. Wu , Yun-Yun. Zhu , Hao-Ran. Li , Heng-Yi. Yu , Peng. a
a
Zhang , Hui-Fang. Pi & Han-Li. Ruan
a
a
Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, Faculty of Pharmacy, Tongji Medical College of Huazhong University of Science and Technology, Wuhan, 430030, China Published online: 06 Dec 2013.
To cite this article: Wen-Ming. Wu, Yun-Yun. Zhu, Hao-Ran. Li, Heng-Yi. Yu, Peng. Zhang, Hui-Fang. Pi & Han-Li. Ruan (2014) Two new alkaloids from the bulbs of Lycoris sprengeri, Journal of Asian Natural Products Research, 16:2, 192-199, DOI: 10.1080/10286020.2013.864639 To link to this article: http://dx.doi.org/10.1080/10286020.2013.864639
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Journal of Asian Natural Products Research, 2014 Vol. 16, No. 2, 192–199, http://dx.doi.org/10.1080/10286020.2013.864639
Two new alkaloids from the bulbs of Lycoris sprengeri Wen-Ming. Wu, Yun-Yun. Zhu, Hao-Ran. Li, Heng-Yi. Yu, Peng. Zhang, Hui-Fang. Pi and Han-Li. Ruan*
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Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, Faculty of Pharmacy, Tongji Medical College of Huazhong University of Science and Technology, Wuhan 430030, China (Received 11 July 2013; final version received 7 November 2013) Two new alkaloids, lycosprenine (1) and 2a-methoxy-6-O-methyllycorenine (2), along with 22 known alkaloids (3 – 23b), were isolated from the bulb of Lycoris sprengeri. Their structures were elucidated on the basis of spectral analysis and by comparison of the spectroscopic data with those of known compounds. Selected compounds (1 – 3 and 6– 9) were tested for their neuroprotective activities against H2O2-, CoCl2- and Ab25 – 35induced SH-SY5Y cell injury, most of which exhibited neuroprotective effects of different degrees. Keywords: Amaryllidaceae; Lycoris sprengeri; alkaloids; neuroprotective
1.
Introduction
Plants of the family Amaryllidaceae include about 60 genera and 800 species, which are widely distributed in the tropics and warm temperate regions of the world [1]. Most Amaryllidaceae plants are known to produce unique alkaloids, which have diverse bioactivities such as acetylcholinesterase inhibitory, analgesic, antibacterial, antifungal, antimalarial, antitumor, antiviral activities, and so on [2]. Especially, galantamine and lycorine types exhibited significant usefulness in healing Alzheimer’s disease [3]. Lycoris sprengeri Comes ex Baker is mainly distributed in the lower reaches of the Yangtze River, and its bulbs have been used as traditional Chinese medicine for the treatment of various diseases, such as sore throats, furuncle carbuncle swollen in skin and scrofula [4,5]. However, the chemical constituents of the plant have never been studied before. In order to discover the bioactive constituents from L. sprengeri, we
investigated the bulbs of L. sprengeri. Accordingly, two new alkaloids, lycosprenine (1) and 2a-methoxy-6-O-methyllycorenine (2), together with 22 known alkaloids, O-methyllycorenine (3) [6,7], lycorenine (4) [8], homolycorine (5) [9], tortuosine (6) [10], haemanthamine (7) [11], hippadine (8) [12], N-isopentylcrinasiadine (9) [2], crinasiadine (10) [13], 5,6-dihydrobicolorine (11) [14], trisphaeridine (12) [15], narcissidine (13) [16], N-(chloromethyl)narcissidine (14) [17], galantamine (15) [18], lycoramine (16) [7], lycosinine B (17) [19], tazettine (18) [20], montabuphine (19) [21], lycorine (20) [22], pluviine (21) [23], galanthine (22) [18], (þ)-haemanthidine (23a), and (–)haemanthidine (23b) [24], were isolated and identified (Figure 1). In this paper, we describe their structural elucidation as well as the neuroprotective activities [25] of the selected compounds 1–3 and 6–9.
*Corresponding author. Email: [email protected] q 2013 Taylor & Francis
Journal of Asian Natural Products Research 12
OCH3 1
H3CO
10
H3CO 9
3 3a
11b
11
8
N
7a
7
6
5
O
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1
O
H3CO H3CO 8
10 9
OCH3
4
2 R4 10a 10b 1 6a R3 6 O
O
O N
H3CO R2 2: R1=OCH3 R2=HR3 =OCH3 R4=H 3: R1=OCH3 R2=R3=R4=H R2=R3=R4=H 4: R1=OH 5: R1+R2=OR3=R4=H
O N
O 9: R=CH2CH2CH(CH3)2 10: R=H OCH3 OH
HO
H H3CO
N
12 OH
N CH3
O H
N CH3
O
N -
Cl 14
13
OH
OCH3 OH
HO
H3CO
N
O
CH3
11
H3CO
H3CO
7
6
O
R
H N
O
-
Cl
O N
O
O 8
H3CO
7 R 1
O N
OMe OH
3
4a 4
11a
11
H3C N
2
193
Cl
H3CO
H3CO 15
16
OCH3 H3C N
OH H N CH3
H3CO
O H
H3CO
OH
O 17
O
N H 19
O
O
R2 N
20: R1=R2=OCH2O R3=R4=OH 21: R1=R2=OCH3 R3=OH R4=H 22: R1=R2=OCH3 R3=OH R4=OCH3
OMe OH
OMe OH
R3
R1
OCH3
O
18 R4
H
O
O
H N OH 23a
O
H N OH 23b
Figure 1. The structures of compounds 1 – 23b.
2. Results and discussion Compound 1 was obtained as white amorphous solids. The molecular formula of 1 was established as C18H17NO4 by positive HR-ESI-MS from the [M þ H]þ ion peak at m/z 312.1219 [M þ H]þ, indicating 11 degrees of unsaturation. The 1H NMR spectrum of 1 revealed the presence of three methoxyl groups at d 3.92 (3H, s), 4.04 (3H, s), and 4.08 (3H, s),
two methylenes at d 3.40 (2H, t, J ¼ 8.0 Hz) and 4.48 (2H, t, J ¼ 8.0 Hz), and four aromatic protons at d 6.94 (1H, s), 7.23 (1H, s), 7.44 (1H, s), and 7.94 (1H, s). The 13C NMR and DEPT spectra of 1 displayed 18 carbon signals, which were resolved as three methoxyl carbons (dC 56.3, 56.4, and 56.4), two methylene carbons (d 27.6 and 46.9), four sp2 methine carbons (d 102.9, 103.1, 109.1, and 112.5),
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Table 1. 1H (400 MHz) and 13C NMR (100 MHz) spectral data of compound 1 in CDCl3 (d in ppm, J in Hz) Position
dC
1 2 3 3a 4 5 7 7a 8 9 10 11 11a 11b 11c 2-OCH3 9-OCH3 10-OCH3
102.9 (CH) 157.1 (C) 112.5 (CH) 132.4 (C) 27.6 (CH2) 46.9 (CH2) 159.3 (C) 121.8 (C) 109.1 (CH) 149.9 (C) 152.9 (C) 103.1 (CH) 128.4 (C) 116.8 (C) 134.1 (C) 56.4 (CH3) 56.4 (CH3) 56.3 (CH3)
dH (multiplicity) 7.23 (s, 1H) 6.94 (s, 1H) 3.40 (t, J ¼ 8.0, 2H) 4.48 (t, J ¼ 8.0, 2H) 7.94 (s, 1H) 7.44 (s, 1H)
3.92 (s, 3H) 4.04 (s, 3H) 4.08 (s, 3H)
and nine quaternary carbons (d 116.8, 121.8, 128.4, 132.4, 134.1, 149.9, 152.9, 157.1, and 159.3) including a carbonyl carbon (d 159.3). The spectral characteristics of 1 (Table 1) were very similar to those of tortuosine (6) [10], except that the olefinic proton in tortuosine at position C-7 was replaced by a carbonyl group in 1. The position of the carbonyl group at C-7 (d 159.3) was supported by HMBC correlations from H-5 to C-7 and from H-8 to C-7. In addition, the structure of 1 was further confirmed by the following key HMBC correlations: H-5/C-3a, C-11c; H-8/C-10, C-11a; H-3/C-4, C-1, C-11c
(Figure 2). From the above several pieces of evidence, the structure of 1 was established as 2,9,10-trimethoxy-4H-pyrrolo[3,2,1-de]phenanthridin-7(5H)-one, named lycosprenine. Compound 2 was obtained as white needles, with ½a20 D þ 182.80. The molecular formula of 2 was determined to be C20H27NO5 by positive HR-ESI-MS at m/z 362.1957 [M þ H]þ. The 1H NMR spectrum of 2 (Table 2) showed an NZCH3 signal at d 2.10 (3H, s), four methoxyl signals at d 3.44 (3H, s), 3.57 (3H, s), 3.88 (3H, s), and 3.89 (3H, s), two aromatic proton signals at d 6.78 (1H, s, H-7) and 6.94 (1H, s, H-10), and an olefinic proton signal at d 5.62 (1H, brs, H-3). Accordingly, the 13C NMR, DEPT, and HSQC spectra of 2 revealed the presence of an NZCH3 (d 44.5), four methoxyl groups (d 55.5, 56.0, 56.3, and 57.3), two methylenes [dC 28.4 (C-11) and 57.0 (C-12)], eight methines [d 41.2 (C-10b), 67.7 (C-4a), 69.7 (C-1); 78.6 (C-2), 98.8 (C-6), 109.8 (C-7), 112.9 (C-10), and 117.1 (C-3)], respectively. The above NMR data of 2 (Table 2) were very similar to those of O-methyllycorenine (3) [6], except that 2 had an additional methoxyl group (dH 3.44, s; dC 57.3) on C-2 which was supported by the HMBC correlations between ZOCH3 (dH 3.44, s, 3H) and C-2 (dC 78.6) as well as between H2 (dH 3.78, brs) and ZOCH3 (dC 57.3) in 2 (Figure 2). A series of 2D NMR experiments including 1H – 1H COSY, HSQC and HMBC allowed the assignment of the planar
OCH3 2
3
12
11
H3C N
4
H3CO
10
11
H3CO 9 8
11b
3a 4
11a
N6
7a
5
H3CO
10 10a 9
H3CO 8 6a 7 2
1
H 2
10b
O HMBC:
3
4a
1
1
OCH3
O 6
OCH3
COSY:
Figure 2. The key COSY and HMBC correlations of compounds 1 and 2.
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Table 2. 1H (400 MHz) and 13C NMR (100 MHz) spectral data of compound 2 in CDCl3 (d in ppm, J in Hz) Position
dC
1 2 3 4 4a 6 6a 7 8 9 10 10a 10b 11 12a 12b 2-OCH3 6-OCH3 8-OCH3 9-OCH3 N-CH3
69.7 (CH) 78.6 (CH) 117.1 (CH) 146.3 (C) 67.7 (CH) 98.8 (CH) 125.8 (C) 109.8 (CH) 148.6 (C) 148.7 (C) 112.9 (CH) 129.8 (C) 41.2 (CH) 28.4 (CH2) 57.0 (CH2) 57.0 (CH2) 57.3 (CH3) 55.5 (CH3) 56.3 (CH3) 56.0 (CH3) 44.5 (CH3)
dH (multiplicity) 4.26 (brs, 1H) 3.78 (brs, 1H) 5.62 (brs, 1H) 2.71 (d, J ¼ 9.1, 1H) 5.51 (s, 1H) 6.78 (s, 1H)
6.94 (s, 1H) 2.52 (dd, J ¼ 9.1, 1H) 2.54 (m, 1H) 3.19 (m, 1H) 2.25 (dt, J ¼ 9.4, 1H) 3.44 (s, 3H) 3.57 (s, 3H) 3.89 (s, 3H) 3.88 (s, 3H) 2.10 (s, 3H)
structure of 2 (Figure 2). The small coupling constant between H-1 and H-10b indicated the cis-junction of the B and C rings. While the larger coupling constant (9.1 Hz) of H-4a and H-10b suggested their trans-diaxial relationship. The NOESY correlations of H1, H-10b, 2-OCH3, and 6-OCH3 reflected the same orientation (Figure 3). Finally, the structure of 2 was confirmed to be 2amethoxy-6-O-methyllycorenine, which was further supported by its positive specific rotation ½a20 D þ 182.80 (CHCl3). Compounds 1– 3 and 6 – 9 were tested for their neuroprotective activities against H 2O 2-, CoCl 2- and Ab25 – 35-induced SH-SY5Y cell death. Compounds 3 and 8 exhibited significant neuroprotective effects against H2O2-induced SH-SY5Y cell death (Table 3). Compounds 1, 3, and 6 showed obvious neuroprotective effects against CoCl2-induced SH-SY5Y cell injury (Table 4). While compounds 1– 3, 6, and 8 exhibited apparent neuroprotec-
Figure 3. The key NOESY correlations of compound 2.
tive effects against Ab25 – 35-induced SHSY5Y cell injury (Table 5). 3.
Experimental
3.1 General experimental procedures Optical rotations were measured with an automatic polarimeter (PE-341LC, PerkinElmer Co., Waltham, MA, USA). Melting points were determinned on Beijing Tech X-5 microscopic melting point apparatus (Taike Corp., Beijing, China). UV spectra were obtained on a Shanghai Spectrum 765PC spectrophotometer (Shanghai Spectrum Instruments Co., Ltd, Shanghai, China). IR spectra were recorded on a Perkin-Elmer spectrum (Perkin-Elmer Co.) one FT-IR spectrometer. HR-ESIMS were obtained on a Thermo Scientific LTQ-Qrbitrap XL mass spectrometer (Thermo Fisher Scientific, Inc., Waltham, MA, USA). 1D and 2D NMR spectra were recorded on a Bruker AM-400 spectrometer (Bruker Analytische Messtechnik GmbH, Karlsruse, Germany) with TMS as the internal standard. Silica gel (200 – 300 mesh; Qingdao Marine Chemical, Inc., Qingdao, China) and Sephadex LH-20 gel (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) were used for column chromatography (CC). Thin-layer chromatography was carried out on silica gel 60
Note: The activity was quantified in terms of OD570, shown as a percentage of the value with no inhibitor, taken as 100%. All data were analyzed by one-way ANOVA followed by Dunnett’s “t” test (n ¼ 3) with a kind of statistical software PASW statistics 18.0, *p , 0.05, **p , 0.01, ***p , 0.001.
56.03 ^ 0.62 42.48 ^ 0.76 56.06 ^ 0.62 45.99 ^ 0.40 54.48 ^ 0.15 54.48 ^ 0.15 45.99 ^ 0.40 56.30 ^ 0.42 38.78 ^ 0.61 61.65 ^ 0.25* 17.96 ^ 0.69*** 48.51 ^ 0.30 57.27 ^ 0.62 42.66 ^ 0.15 59.08 ^ 0.23 43.33 ^ 0.50 60.97 ^ 0.21* 42.31 ^ 0.46** 51.39 ^ 0.23 58.87 ^ 0.12*** 47.61 ^ 0.51 1 2 3 6 7 8 9
57.27 ^ 0.81 40.37 ^ 0.70 59.42 ^ 0.10 44.41 ^ 0.21 57.20 ^ 0.42* 65.63 ^ 0.20*** 44.41 ^ 0.32
62.34 ^ 0.30* 45.10 ^ 0.50 68.86 ^ 0.38** 38.63 ^ 0.49*** 59.71 ^ 0.50** 64.71 ^ 0.36*** 49.23 ^ 0.62*
59.76 ^ 0.50* 46.09 ^ 0.23 63.11 ^ 0.61** 25.94 ^ 0.93** 50.37 ^ 0.32 55.10 ^ 0.59 46.41 ^ 0.74
350 mM H2O2 12.5 Compound
6.25
25
50
100
W.-M. Wu et al.
Test concentrations (mM)
Table 3. Cell viabilities (%) of compounds 1 – 3 and 6 –9 in different concentrations in H2O2-induced SH-SY5Y cell injuries.
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F254 over glass plates (Qingdao Marine Chemical, Inc.) using various solvent systems. HPLC was carried out on an Agilent 1260 system using YMC-Pack ODS-A C18 5 mm (250 mm £ 10 mm i.d.) columns (Agilent Technologies, Inc., Santa Clara, CA, USA) for the purpose of analysis and semi-preparation. 3.2
Plant material
The bulbs of L. sprengeri were collected from Taizhou City of Zhejiang Province of China in July 2011, and authenticated by Mr Xiaogang Wang. A voucher specimen (No. 20110829) has been deposited in the Faculty of Pharmaceutical Sciences, Tongji Medical College of Huazhong University of Science and Technology, Wuhan, China. 3.3 Extraction and isolation Air-dried, powdered bulbs (17 kg) of L. sprengeri were extracted with 95% EtOH (10 times, each 3 h) at the temperature of 558C and concentrated in vacuo to yield a crude extract (2000 g). The extract was suspended in water (20 l) and extracted with CH2Cl2 to yield CH2Cl2 fraction (60 g). Then the aqueous phase was adjusted to pH 10 –12 with NaOH and then extracted with CH2Cl2 to give an alkaloid residue (170 g). The alkaloid residue (170 g) was subjected to silica gel CC eluted with CH2Cl2 – MeOH (1:0 to 0:1) gradient as the mobile phase to provide seven fractions A – G. Fraction A (2 g) was subjected to silica gel column, eluting with a gradient of CH2Cl2 – MeOH (1:0 to 0:1) to provide four subfractions A1, A2, A3, and A 4. Subfraction A1 (0.6 g) was subjected to silica gel CC eluted with CH2Cl2 – MeOH ¼ 280:1 to yield compounds 12 (20 mg) and 11 (15 mg). Subfraction A2 (0.7 g) was further separated by semi-preparative HPLC (90:10 MeOH – H2 O mobile phase) to give
Note: The activity was quantified in terms of OD570, shown as a percentage of the value with no inhibitor, taken as 100%. All data were analyzed by one-way ANOVA followed by Dunnett’s “t” test (n ¼ 3) with a kind of statistical software PASW statistics 18.0, *p , 0.05, **p , 0.01, ***p , 0.001.
51.60 ^ 0.44 69.80 ^ 0.50 51.60 ^ 0.44 58.08 ^ 0.26 58.33 ^ 0.40 58.33 ^ 0.40 58.08 ^ 0.26 58.24 ^ 0.32** 73.74 ^ 0.35** 61.49 ^ 0.30** 64.87 ^ 0.35*** 28.75 ^ 0.35 62.33 ^ 0.23** 53.37 ^ 0.20 62.81 ^ 0.55** 76.50 ^ 0.47** 67.33 ^ 0.59*** 66.87 ^ 0.10*** 33.67 ^ 0.40 61.73 ^ 0.13* 58.92 ^ 0.23 63.01 ^ 0.26*** 68.20 ^ 0.51 65.65 ^ 0.42*** 63.58 ^ 0.55** 31.16 ^ 0.15 60.14 ^ 0.31* 58.83 ^ 0.21 1 2 3 6 7 8 9
62.00 ^ 0.40*** 70.10 ^ 0.53 64.64 ^ 0.15** 65.93 ^ 0.55** 34.85 ^ 0.36 61.92 ^ 0.21** 62.04 ^ 0.25***
64.23 ^ 0.44*** 73.78 ^ 0.40** 71.18 ^ 0.42*** 71.31 ^ 0.66** 36.08 ^ 0.53 63.00 ^ 0.46** 64.39 ^ 0.12***
100 50 25 12.5 6.25 Compound
Test concentrations (mM)
Table 4. Cell viabilities (%) of compounds 1 – 3 and 6 –9 in different concentrations in CoCl2-induced SH-SY5Y cell injuries.
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256 mM CoCl2
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compounds 8 (6 mg, tR 13 min) and 10 (2.5 mg, tR 11.5 min). Fraction B (1.1 g) was separated into three subfractions B1, B2 and B3 by Sephadex LH-20 using CH2Cl2 – MeOH (1:1). Subfraction B2 (0.3 g) was further purified by semipreparative HPLC (60:40 MeOH – H2O mobile phase) to give compounds 16 (4 mg) and 19 (40 mg). Fraction C (8.1 g) was subjected to silica gel CC eluted with CH2Cl2 – MeOH (18:1) to provide compounds 13 (5 mg), 14 (6 g), and 17 (4 mg). Similar to fraction B, fraction D (5 g) was separated into five subfractions D1, D2, D3, D4, and D5. Then subfraction D3 (2.4 g) was separated into further four subfractions D3-1, D3-2, D3-3, and D3-4, by Sephadex LH-20 using CH2Cl2 –MeOH (1:1). Subfraction D3-3 (0.9 g) was purified by HPLC (80:20 MeOH – H2O mobile phase) to yield compounds 3 (12 mg) and 18 (25 mg). Fraction E (8 g) was subjected to silica gel CC, eluting with a gradient of CH2Cl2 – MeOH (1:0 to 0:1) to provide four subfractions E1, E2, E3, and E4. From subfraction E2 (1.8 g), compound 9 (4.5 mg) was obtained over a further silica gel column eluted with CH2Cl2 –MeOH (100:1). Subfracted E3 (1.1 g) was purified by HPLC (80:20 MeOH – H2O mobile phase) to yield compound 1 (3.5 mg, tR 10.9 min). Subfraction E4 (3.4 g) was subjected to separation on RP-C18, eluting with MeOH –H2O (30:70) to yield compounds 6 (0.6 g) and 15 (15 mg). Fraction F (7 g) was separated into four subfractions F1, F2, F3, and F4, by Sephadex LH-20 using MeOH. Subfraction F3 (2.3 g) was further purified by HPLC (60:40 MeOH – H2O mobile phase) to yield compounds 4 (20 mg, tR 9.3 min) and 22 (10 mg, tR 14.7 min). Fraction G (12 g) was subjected to silica gel column to separate into five subfractions G1, G2, G3, G4, and G5 by eluting with a gradient of CH2Cl2 –MeOH (1:0 to 0:1). Subfraction G3 (1.2 g) was purified by HPLC (80:20 MeOH – H2O, mobile phase) to yield compounds 2 (4 mg, tR 11 min) and 5 (10 mg, tR 12.3 min).
Note: The activity was quantified in terms of OD570, shown as a percentage of the value with no inhibitor, taken as 100%. All data were analyzed by one-way ANOVA followed by Dunnett’s “t” test (n ¼ 3) with a kind of statistical software PASW statistics 18.0, *p , 0.05, **p , 0.01, ***p , 0.001.
55.75 ^ 0.20 59.49 ^ 0.44 55.75 ^ 0.20 56.02 ^ 0.45 61.43 ^ 0.53 61.43 ^ 0.53 56.02 ^ 0.45 52.14 ^ 0.35 70.45 ^ 0.15** 52.40 ^ 0.15 59.71 ^ 0.38** 51.01 ^ 0.38 64.30 ^ 0.47* 50.58 ^ 0.57 54.46 ^ 0.50 71.63 ^ 0.25*** 56.60 ^ 0.75 63.23 ^ 0.45*** 51.01 ^ 0.38 64.30 ^ 0.47* 53.45 ^ 0.15 58.55 ^ 0.42* 72.36 ^ 0.40*** 58.74 ^ 0.36** 63.98 ^ 0.26** 47.85 ^ 0.31 66.56 ^ 0.15* 57.07 ^ 0.55 1 2 3 6 7 8 9
59.44 ^ 0.32** 64.70 ^ 0.71* 56.75 ^ 0.30 72.36 ^ 0.40*** 45.30 ^ 0.56 65.37 ^ 0.30* 55.91 ^ 0.46
64.93 ^ 0.53** 77.57 ^ 0.64*** 65.74 ^ 0.35*** 66.71 ^ 0.49*** 47.85 ^ 0.31 71.77 ^ 0.40*** 59.40 ^ 0.75*
1mM Ab25-35 50 25 Compound
6.25
12.5
100
W.-M. Wu et al.
Test concentrations (mM)
Table 5. Cell viabilities (%) of compounds 1 – 3 and 6 –9 in different concentrations in Ab25 – 35-induced SH-SY5Y cell injuries.
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Subfraction G4 (4.8 g) was further separated into four subfractions G4-1, G4-2, G43 and G4-4, by Sephadex LH-20 using MeOH. Subfraction G4-3 (1.1 g) was further purified by HPLC (60:40 MeOH – H2O, mobile phase) to yield compounds 7 (15 mg, tR 12 min), 20 (10 mg, tR 8 min), 21 (15 mg, tR 14 min), 23a (15 mg, tR 9 min), and 23b (15 mg, tR 9 min). 3.3.1
Lycosprenine (1)
White amorphous solids; UV (MeOH) lmax: (log 1) 205 (2.84), 230 (2.55), and 285 (2.24) nm; IR (KBr) nmax: 2969, 2922, 2864, 1736, 1598, 1514, 1459, 1389, 1261, 1022, 871, 798, 748, and 664 cm21; 1H NMR and 13C NMR (400 MHz, CDCl3) spectral data (see Table 1); HR-ESI-MS: m/z 312.1219 [M þ H] þ (calcd for C18H18NO4, 312.1236). 3.3.2 2a-Methoxy-6-O-methyllycorenine (2) White needles; ½a20 D þ 182.80 (CHCl3); m. p. 126–1288C; UV(MeOH) lmax: (log 1) 205 (4.33), 235 (3.93), and 280 (3.48) nm; IR (KBr) nmax: 2969, 2926, 2788, 2658, 2499, 2027, 1694, 1611, 1513, 1464, 1353, 1262, 1187, 958, 796, and 609 cm21; 1H NMR and 13C NMR (400 MHz, CDCl3) spectral data (see Table 2); HR-ESI-MS: m/z 362.1957 [M þ H]þ (calcd for C20H28NO5, 362.1967). 3.4 Bioassay procedure SH-SY5Y cells, which were obtained from the cell bank of Basic Medical College of Huazhong University of Science and Technology (Wuhan, China), were cultured in Dulbecco’s modified eagle medium high glucose medium containing 10% fetal calf serum at 37 8C in a humidified atmosphere with 5% CO2 incubator. The cell viability was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT)
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Journal of Asian Natural Products Research assay. About 9 £ 103 cells in 1 ml of logarithmic phase were seeded onto a 96well microplate. Twenty-four hours later, each hole with 11.5 ml sample was tested in the normal group, while 11.5 ml PBS in the control group. After 2 h, CoCl2, H2O2 or Ab25 – 35 was added to the normal group and control group. A total of 15 ml of MTT solution (5 mg/ml) were added to all the holes after the cells were cultured at 37 8C for 24 h. After continuing to culture for 4 h, the medium was removed and dimethyl sulfoxide (100 ml/well) was added. Finally, the reduced MTT was assayed at 570 nm using a multi-function microplate reader. Acknowledgments Financial support from the Ministry of Science and Technology of the People’s Republic of China (International Cooperative Project, Grant No. 2010DFA32430) and Natural Science Foundation of China (Nos 81072547, 31270394 and 30873361) is gratefully acknowledged.
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