Arch. Pharm. Res. DOI 10.1007/s12272-014-0397-2
RESEARCH ARTICLE
Alkaloids from the bulbs of Lycoris longituba and their neuroprotective and acetylcholinesterase inhibitory activities Yun-Yun Zhu • Xue Li • Heng-Yi Yu • Yu-Fang Xiong • Peng Zhang • Hui-Fang Pi Han-Li Ruan
•
Received: 11 January 2014 / Accepted: 8 April 2014 Ó The Pharmaceutical Society of Korea 2014
Abstract Three novel alkaloids (1–3), together with nineteen known ones (4–22), were isolated from the bulbs of Lycoris longituba. Their structures were elucidated on the basis of extensive spectroscopic analyses, which belong to several Amaryllidaceae alkaloid skeletons. Among them, the harmane-type alkaloids (the new compound 1 and the known compounds 5, 6 and 7) were found for the first time from Lycoris genus. The isolates were tested for their neuroprotective activities against CoCl2, H2O2 and Ab25–35-induced SH-SY5Y cell injuries, and the majority of them exhibited neuroprotective activities of different degrees. The acetylcholinesterase (AChE) inhibitory activities of the isolated alkaloids were also evaluated, while compounds 12, 14–20 and 22 exhibited extremely significant AChE inhibitory activities. Keywords Lycoris longituba Alkaloid Neuroprotective activity AChE inhibitory activity
Introduction The Amaryllidaceae family consists of about sixty genera, whose eight hundred species are widely distributed in the
Y.-Y. Zhu X. Li H.-Y. Yu P. Zhang H.-F. Pi H.-L. Ruan (&) Faculty of Pharmacy, Tongji Medical College of Huazhong University of Science and Technology, Hangkong Road 13, Wuhan 430030, People’s Republic of China e-mail: [email protected] Y.-F. Xiong Department of Biochemistry, Tongji Medical College of Huazhong University of Science and Technology, Hangkong Road 13, Wuhan 430030, People’s Republic of China
tropics and warm-temperate regions of the world (Kornienko and Evidente 2008). Most plants of Amaryllidaceae are known to produce structurally unique alkaloids, which showed diverse bioactivities such as antiviral, acetylcholinesterase (AChE) inhibitory, antibacterial, antifungal, antineoplastic and antimalarial effects (Ieven et al. 1979; Louw et al. 2002; Sener et al. 2003; Elgorashi et al. 2004; Jin 2005; Jokhadze et al. 2007). Especially, galanthamine and lycorine types exhibited significant cholinesterase inhibitory effect, which enhances cognitive functions of Alzheimer’s patients (Lopez et al. 2002). AChE inhibitors are considered to be one of the most effective approaches for treating AD (Bartus et al. 1982; Perry 1986; Senol et al. 2010). The genus Lycoris from Amaryllidaceae consists of about 20 species all over the world, with 17 (including one variety) of which distributing in China. Recently, our group reported the chemical constituents and neuroprotective activities of several lycoris plants (Li et al. 2013; Jin et al. 2014; Wu et al. 2014). As a continuation of our work, another lycoris plant, Lycoris longituba, was studied in this paper. Lycoris longituba mainly grows in Jiangsu province (McNulty et al. 2007) and the bulbs of which have been traditionally used for the treatment of sore throats, furuncle carbuncle swollen in skin and scrofula (He et al. 2011). Previously, 12 alkaloids were isolated from the bulbs of L. longituba (Liang et al. 2010, Zhao et al. 2011), but the pharmacological studies on this plant have never been reported before. To discover potentially bioactive compounds, we further investigated the bulbs of L. longituba collected in Baohua Mountain, Jiangsu province. Accordingly, three new alkaloids (1–3): lycolongirine A (1), lycolongirine B (2), lycolongirine C (3), together with nineteen known ones (4–22), incartine (4) (Berkov et al. 2007), norharmane (5) (Abramovitch and Spenser 1964;
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Y.-Y. Zhu et al.
11
H3C
10 8 8a N 9a
7
HO 1
O 1
H3CO 10
N
4b
5
4a
1
3
H
11 11b
9
O
3a
10a
8
H N
8
6a
7
6
8a
7
9a
N
8
2
6
4b
5
4a
6
4
10
H3CO H3CO
9
11c
H
7a
8
N 7
4
10
9
1
O
9
10 10b 10a
O
8
7
H3CO
9
7a
8
OMe 11 OH
3
H
3a 11c
5
6
7
O 4
N
N
6a
O
O
9
7a
8
N 6a 7
4
12
OH
OH
3
3
1 10b
4a 12 11
10
R3
R1
N R2
9
7 8
1 10b
4a 12 11
10
R
O
9
10 10b
O
8
R1
N R2 6
9
5
7
OMe 11 OH 3
4a
H
N
6a
7
6
OH
3
4
O
4
OCH3
6
6
R
11c
13a
2
4
3a
N
1
10a
CH3
6
11
3
H
2
10b 4a
6
O N
2
1
11 11b 10 11a H
3
10 9 10a
8
5
9
1
O
4
N
OCH3 HO
2
4
2
3
H
8
3
O
3a 11c
8 7a
9
Cl
OH 13b
O
4
7
4a
H
6a
3
10a
8
5
11a
2
11 11b 10 11a H
10b 4a
O
11 11b
OH H3CO 10
3
H
4
1
2
10
2
O
N
1
O
3 3a
H
11b 11a
1
9a
4a
OCH3 2 OH
3
2
1
OH HO
7
1
11
4b
5
5 R=H 6 R=CH3
HO
8a
7
3
H N
HO
3
O 1
4a
N H CH2Cl
HOH2C R
2
4
2
1
OCH3
OCH3
11 4b 12
H O HN 5 OCH H3CO 9 8 7a 3 OCH 3 7
4
10
OH
11c 4
11a
3
6
2
O
12
4a
13a
O
7
11
4
2 13 1
10
9
13b
H
N CH3
6a
6
OR 8
8
14 R=OCH3 R1=free R2=CH3 R3=H 19 R=OCH3 R1=CH2Cl R2=CH3 15 R=OCH3 R1=free R2=H R3=H 20 R=OH R1=free R2=CH3 16 R=OCH3 R1=CH2Cl R2=CH3 R3=H 17 R=OCH3 R1=free R2=CH3 R3=OH 18 R=OH R1=free R2=CH3 R3=H
21 R=OH 22 R=H
Fig. 1 The structures of compounds 1–22
Balkau and Heffernen 1973), harmane (6) (Hiroko et al. 1993), perlolyrine (7) (Li et al. 2011, 2012), lycorine (8) (Tsuda et al. 1979), hippamine (9) (Evidente et al. 1984), N-chloromethyl narcissidine (10) (Suau et al. 1990), trisphaeridine (11) (Alonso et al. 2010), N-methylcrinasiadine (12) (Cowden et al. 1994), (?)-haemanthidine (13a), (-)haemanthidine (13b) (Pabuccuoglu et al. 1989), galanthamine (14) (Kihara et al. 1994), N-norgalanthamine (15) (Treu and Jordis 2001), N-chloromethyl galanthamine (16)
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(Matusch et al. 1994), 11b-hydroxy galanthamine (17) (Andrade et al. 2011), sanguinine (18) (Kihara et al. 1994), N-chloromethyl lycoramine (19) (Matusch et al. 1994), Odemethyllycoramine (20) (Wang et al. 2010), tazettine (21) (Roberts et al. 1971), deoxytazettine (22) (Roberts et al. 1971) were isolated (Fig. 1). The isolated alkaloids were tested for their neuroprotective activities against CoCl2, H2O2 and Ab25–35 induced SH-SY5Y cell injury. Compounds 11, 16, 17 and 22 exhibited significant
Alkaloids from the bulbs of Lycoris longituba
neuroprotective effects against CoCl2-induced SH-SY5Y cell injury, and compounds 1, 2 and 22 showed significant neuroprotective effects against H2O2-induced SH-SY5Y cell death, while compounds 16, 18 and 19 had similar activities against Ab25–35-induced SH-SY5Y cell damage. The isolated alkaloids were also tested for their acetylcholiesterase (AChE) inhibitory activities. And the compounds 12, 14–20 and 22 exhibited extremely significant AChE inhibitory activities.
Materials and methods General 1
H (400 MHz), 13C (100 MHz), and 2D-NMR spectra were recorded on a Bruker AM-400 spectrometer with tetramethylsilane (TMS) as an internal standard. Chemical shifts are in ppm (d), relative to TMS and scalar coupling constants (J) reported in Hertz and used deuterated dimethyl sulfoxide (DMSO-d6), methanol (CH3OH-d4) and chloroform (CHCl3-d), pyridine (pyridine-d5) as solvents. Electrospray ionization (ESI) mass spectra were acquired in the positive ion mode on an LCQ DECAXP instrument (Thermo Finnigan, San Jose, CA, USA) equipped with an ion trap mass analyzer. HR-ESI–MS were obtained in the negative ion mode on Thermo LTQ-Orbitrap XL. IR spectra were obtained on a NicoletTM 380 spectrometer from Thermo Electron; in cm-1. UV spectra were taken on a Cary-50 spectrophotometer. Optical rotations were acquired on a PerkinElmer PE-341L polarimeter. TLC plates were HSGF254 SiO2 from Yantai Jiangyou Silica Gel Development Co., Ltd., China. Column chromatography (CC) silica gel (SiO2; 200–300 mesh; Qingdao Haiyang Chemical Co., Ltd., Qingdao, China). All other Organic solvents were of analytical reagent grade from Traditional Chinese medicine group. Plant material The fresh bulbs of L. longituba were collected in the Baohua Mountain, Zhenjiang city of Jiangsu Province, China, in September 2011 and identified by Prof. Shulan He of Zhongshan botanical garden. A voucher specimen (20110909) has been deposited in the Herbarium of Materia Medica Faculty of Pharmacy, Tongji Medical College of Huazhong University of Science and Technology, Wuhan, China.
atmosphere with 5 % CO2 incubator. The samples were solved and diluted with PBS to required concentrations. The cell viability was assessed using a MTT assay. About 9 9 103 cells in 1 mL of logarithmic phase were seeded onto a 96-well microplate. 24 Hours later, each hole with 11.5 lL sample to be tested in the normal group, while 11.5 lL PBS in control group. After 2 h, CoCl2, H2O2 or Ab25–35 was added to the normal group and control group. 15 lL of MTT solution (5 mg/mL) was added to all of the holes after the cells were cultured at 37 °C for 24 h. After continuing to culture for 4 h, the medium was removed and DMSO (100 lL/well) was added. Finally, the reduced MTT was assayed at 570 nm using a multi-function microplate reader. AChE inhibition assay AChE from electric eel (type VIS), acetylthiocholine iodide and 5, 5-dithio-bis (2-nitrobenzoic acid) (DTNB) was purchased from Sigma. AChE inhibitory activities of the isolated compounds were determined using Ellman’s colorimetric method (Ellman et al. 1961) as modified by Eldeen et al. (2005). 25 lL of 15 mM ATCl in water, 125 lL of 3 mM DTNB in Buffer C (50 mM Tris–HCl, pH 8.0, 0.1 M NaCl and 0.02 M MgCl2. 6H2O), 50 lL Buffer B (50 mM Tris–HCl, pH 8.0, 0.1 % bovine serum albumin) and 25 lL of the samples (6.25, 12.5, 25, 50 lM, 100 Lm) were added to the wells in a 96 wells microplate. Absorbance corresponding to spontaneous hydrolysis was read at 405 nm every 45 s for 5 times. Thereafter, 25 lL of 0.20 U/mL AChE was added to each well and absorbance was consecutively read again every 45 s for 9 times. Galanthamine served as the positive control. Any increase in absorbance due to the spontaneous hydrolysis of the substrate was corrected by subtracting the absorbance before adding the enzyme from the absorbance after adding the enzyme. The percentage inhibition was calculated using the equation inhibition ð%Þ ¼ Acontrol Asample = Acontrol 100. Where Asample is the absorbance of the sample and Acontrol is the absorbance of the blank (Buffer A). AChE inhibition values are means of three individual determinations each performed in triplicate. AChE inhibitory activity expressed as IC50 values were obtained from the logarithmic non-linear regression curve derived from the plotted data using GraphPad Prism software package for Windows (GraphPad version 5.0, San Diego, USA).
Statistical analysis Neuroprotective activity assay SH-SY5Y cells were cultured in DMEM high glucose medium containing 10 % FBS at 37 °C in a humidified
Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by post hoc multiple comparisons using the Newman–Keuls Multiple Comparison
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Y.-Y. Zhu et al. Table 1 1H NMR (400 MHz) and and 2 in CH3OH-d4
13
Compound 1 position
dC
1
150.5
dH (mult., J, Hz)
2a
C NMR (100 MHz) Data for 1
138.6
8.18 (1H, d, 5.3)
3a 116.1
4a
132.5
7.95 (1H, d, 5.3)
4b
123.3
5
123.6
8.13 (1H, d, 8.0)
6
121.9
7.23 (1H, t, 8.0)
7
130.8
7.53 (1H, t, 8.0)
7a 8
Compound 3
dH (mult., J, Hz)
position
dC
62.0
4.70 (1H, brd, 11.2)
1
118.9
5.90 (1H, brs)
53.0
3.71 (1H, ddd, 14.8, 4.1, 1.7)
2
78.3
3.54 (1H, m)
3.63 (1H, brd, 14.8)
3 4a
66.8 28.1
4.20 (1H, m) 2.63 (1H, dt, 11.2, 4.3)
8a
125.5
5.81 (1H, brd, 1.5)
4b
69.1
4.60 (1H, brs)
82.1
3.61 (1H, dd, 5.7, 2.8)
73.9
4.43, 4.38 (each 1H, d, 10.8)
130.5 114.2
7.62 (1H, d, 8.0)
115.4
6.94 (1H, s)
143.7
9
148.6
9a
135.2
10
72.6
5.33 (1H, q, 6.6)
150.1
11
24.5
1.64 (3H, d, 6.6)
114.5
7.36 (1H, s)
11a
133.5
11b
46.5
3.21 (1H, dd, 1.7, 11.2)
11c
74.2
4.00 (1H, brs)
(C-5)OCH3
58.1
3.45 (3H, s)
(C-7)OCH3
58.3
3.37 (3H, s)
(C-9)OCH3
56.6
3.82 (3H, s)
(C-10)OCH3
56.4
3.88 (3H, s)
method. The data are expressed as mean ± SEM of three assays. Extraction and isolation The fresh bulbs of L. Longituba (45 kg) were cut into pieces and extracted exhaustively with ethanol (95 %) for five times (2 h each time) at 55 °C in multi-function extraction tank. The solvent was evaporated under vacuum and the liquid extract was acidified to pH 3–4 by HCl and then extracted with CH2Cl2 for five times to afford 850 g of acid extract. After adjusting the pH value of the remaining parts to neutral by addition of NaOH, 69 g neutral components were resulted. The last part was alkalified to pH 10–11 and 13–14 successively, produce 152 g and 135 g alkaline substances, respectively. The four CH2Cl2 fractions of different pH were put together and concentrated to afford a residue, which was subjected to silica gel (200–300 mesh), and eluted with a gradient system of CH2Cl2/CH3OH (1:0, 70:1, 50:1, 30:1, 20:1, 10:1, 8:1, 6:1,
123
C NMR (100 MHz) Data for 3
dC
143.2
4
13
Compound 2
2b 3
Table 2 1H NMR (400 MHz) and in CH3OH-d4
dH (mult., J, Hz)
2.02 (1H, td, 11.9, 2.4)
4a
72.3
4b
144.1
6a
66.2
6b
4.59 (1H, brd, 11.6) 4.96 (1H, d, 15.1), 5.12 (1H, d, 15.1)
6a
119.1
7
107.7
8
149.5
9
149.6
10
108.1
10a
129.2
11
45.3
4.09 (1H, brd, 3.1)
12a
64.4
4.00 (1H, d, 10.2)
103.1
5.96 (2H, d, 0.9)
12b (C-8, 9)OCH2O
6.76 (1H, s)
6.84 (1H, s)
3.84 (1H, ddd, 1.7, 3.1, 10.2)
(C-2)OCH3
58.2
3.44 (3H, s)
N–CH2Cl
66.5
5.61, 5.59 (each 1H, d, 10.1)
4:1, 1:1, 0:1, V/V) to yield fractions A–I according to TLC analysis. The fraction A was chromatographed over a series of silica gel and Sephadex LH-20 (CH2Cl2/CH3OH 1:1) to afford compound 11 (31 mg) and compound 12 (25 mg). Fraction B was separated into four parts (B-1 to B-4) by silica gel. The part B-2 was again subjected to silica gel to give six subfractions (B-2-1 to B-2-6). Among them, subfractions B-2-3 and B-2-4 were further purified with Sephadex LH-20 (MeOH) and then high performance liquid chromatography (HPLC) to yield the new compound 1 (12.7 mg) and compound 5 (12.6 mg), respectively. Subfraction B-2-5 was obtained as compound 6 (16.7 mg). The part B-3 was subjected to Sephadex LH-20 (MeOH) and then silica gel again to get four subfractions (B-3-1 to B-3-4). Subfraction B-3-2 was produced compound 7 (15.6 mg) and 14 (10.2 mg) by HPLC. Subfraction B-3-3 was afforded compound 21 (15 g). Fraction C was resulted in compound 22 (19.7 mg). The Fractions D–I were applied on Sephadex LH-20 (CH2Cl2/CH3OH 1:1 or 0:1), TLC and HPLC to produce compound 3 (29.7 mg), 4 (18.0 mg) and the others. Lycolongirine A (1) light yellow powder; [a]20 D ? 9.35°(c 0.42, CH3OH); UV k max (CH3OH) nm (log e):
Alkaloids from the bulbs of Lycoris longituba Fig. 2 Key correlations of compound 1
H3C O N N
1
H-1H-COSY
Key NOESY correlations
HMBC H to C Fig. 3 Key correlations of compound 2
HO
H3CO
OH
HN H3CO
OCH3
OCH3
1
H-1H-COSY
Key NOESY correlations
HMBC H to C
235 (0.71), 289 (0.33), 340 (0.09); IR (KBr) t max cm-1: 3089, 2970, 2838, 1625, 1588, 1430, 1371, 1221, 740; 1HNMR and 13C-NMR data: see Table 1; Positive HR-ESI– MS m/z: 211.0868 [M ? H]? (calc. for C13H11N2O?, 211.0871). Lycolongirine B (2) yellow oil; [a]20 D ? 25.71°(c 1.39, CH3OH); UV k max (CH3OH) nm (log e): 206 (1.42), 236 (0.36), 282 (0.13); IR (KBr) t max cm-1: 3471, 2932, 2829, 1606, 1517, 1464, 1370, 1285, 1188, 1111, 937; 1H-NMR and 13C-NMR data: see Table 1; Positive HR-ESI–MS m/z: 366.1906 [M ? H]? (calc. for C19H28NO6?, 366.1917). Lycolongirine C (3) colorless needles; [a]20 D ? 26.68° (c 1.13, CH3OH); UV k max (CH3OH) nm (log e): 212 (2.21), 244 (0.41), 291 (0.52); IR (KBr) t max cm-1: 3383, 3027, 2959, 2826, 1636, 1507, 1488, 1247, 937; 1H-NMR and 13C-NMR data: see Table 2; Positive HR-ESI–MS m/z: 350.1152 [M]? (calc. for C18H21ClNO4?, 350.1154). Results and discussion Compound 1 was isolated as light yellow powder, [a]20 D ? 9.35°(c 0.42, CH3OH). Its molecular formula was established as C13H10N2O by HR-ESI–MS from the
[M ? H]? ion peak at m/z 211.0868 [M ? H]? (calc. 211.0871), indicating ten degrees of unsaturation. The 1HNMR spectrum of 1 exhibited the presence of a 1, 2-substituted benzene at dH 8.13 (1H, d, J = 8.0 Hz, H-5), 7.62 (1H, d, J = 8.0 Hz, H-8), 7.53 (1H, t, J = 8.0 Hz, H-7) and 7.23 (1H, t, J = 8.0 Hz, H-6), an ortho-positioned pyridine at dH 8.18 (1H, d, J = 5.3 Hz, H-3) and 7.95 (1H, d, J = 5.3 Hz, H-4), a methyl at dH 1.64 (3H, d, J = 6.6 Hz) and an O-methine at dH 5.33 (1H, q, J = 6.6 Hz, H-10) (Table 1). In turn, the 13C-NMR and DEPT spectra of 1 showed 13 carbon signals [CH3 9 1, CH (sp3) 9 1, CH (sp2) 9 6 and C (sp2) 9 5, Table 1]. The existence of an –OCHCH3 moiety was supported by the 1H–1H–COSY spectrum (Fig. 2). The NMR spectral characteristics of 1 were very similar to those of harmane (Hiroko et al. 1993), except that the methine carbon (C-10) on the –OCHCH3 linked with N-9, wherever the oxygen of the –OCHCH3 linked to C-1 to form a five-membered ring in 1. The above conclusion was confirmed by the HMBC experiment (Fig. 2). The stereochemical structure of 1 was speculated as the form showed in Fig. 2, which was supported by its positive specific rotation ([a]20 D ? 9.35°) (Bandini et al. 2008; Stanley and Hartwig 2009). According to references (Bandini et al. 2008; Stanley and Hartwig
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Y.-Y. Zhu et al.
Fig. 4 Key correlations of compound 3
Table 3 Cell viabilities (%) of compounds in different concentrations in H2O2 induced SH-SY5Y cells injuries Compound
6.25 1
300 lM
Test concentration (lM) 12.5
81.50 ± 1.58
2 3
b
60.89 ± 0.10 90.41 ± 0.90b
4
81.01 ± 1.15a
25
50
100
H2O2
94.53 ± 0.15c
89.58 ± 0.82c
84.52 ± 0.35c
73.97 ± 1.80
74.39 ± 0.47
b
59.50 ± 0.31 83.63 ± 1.19a
a
57.10 ± 0.21 78.34 ± 0.61
56.14 ± 0.10a 74.26 ± 0.95
55.33 ± 0.55a 68.84 ± 0.92
51.54 ± 0.53 72.64 ± 0.25
93.72 ± 1.00c
83.25 ± 0.21b
81.56 ± 0.64b
76.86 ± 0.99
74.81 ± 0.70 51.52 ± 0.86
b
5
54.71 ± 0.85
59.97 ± 0.82
52.23 ± 0.40
51.11 ± 0.95
44.61 ± 0.68
6
78.99 ± 0.85a
89.38 ± 0.50c
91.73 ± 0.93b
81.60 ± 0.97b
70.14 ± 0.81
74.39 ± 0.47
7
55.18 ± 0.42
62.24 ± 0.25b
48.40 ± 0.62
32.19 ± 0.42
12.11 ± 0.58
51.20 ± 0.86
8
59.61 ± 0.85
58.37 ± 0.82
53.63 ± 0.46
50.11 ± 0.75
44.45 ± 1.08
67.48 ± 0.98
9
60.75 ± 0.67
82.97 ± 0.47b
95.36 ± 0.45c
94.23 ± 0.54c
84.75 ± 0.38b
63.46 ± 1.42
10
67.82 ± 0.91
72.89 ± 0.10
78.10 ± 1.14
70.08 ± 0.38
67.72 ± 0.1
72.86 ± 0.21
11
66.70 ± 0.85a
75.50 ± 0.68b
70.69 ± 0.31c
73.14 ± 0.60b
66.61 ± 0.85a
59.66 ± 0.15
12
65.70 ± 2.02
75.54 ± 1.81b
71.01 ± 0.51b
68.51 ± 1.07a
59.30 ± 0.45
59.66 ± 0.15
13
68.21 ± 1.25
78.09 ± 0.29b
67.27 ± 0.86
64.70 ± 0.72
54.19 ± 0.93
70.43 ± 0.46
14
63.70 ± 0.95a
63.85 ± 0.68a
71.69 ± 0.31b
65.74 ± 1.60b
63.31 ± 1.85a
49.66 ± 0.15
c
15
73.18 ± 0.55
77.53 ± 0.35
71.37 ± 0.78
59.90 ± 0.44
67.51 ± 0.89
70.43 ± 0.46
16
75.95 ± 0.90
93.35 ± 0.31c
87.70 ± 0.62c
83.55 ± 1.10b
79.29 ± 0.42a
74.81 ± 0.70
17
67.03 ± 0.44
73.41 ± 0.44
79.20 ± 0.15c
75.12 ± 0.40a
73.30 ± 0.36
72.86 ± 0.21
18 19
67.93 ± 0.40b 82.25 ± 0.78b
70.07 ± 0.38c 82.81 ± 0.25c
74.03 ± 0.35c 83.73 ± 0.47c
76.83 ± 0.23c 83.93 ± 0.69c
69.08 ± 0.15c 85.53 ± 0.40b
57.72 ± 0.25 72.26 ± 0.32
20
64.58 ± 0.35b
68.48 ± 0.45b
69.91 ± 0.31c
73.53 ± 0.59b
65.95 ± 0.32c
57.72 ± 0.25
b
c
b
75.69 ± 0.40
72.17 ± 0.38
80.02 ± 0.97a
72.64 ± 0.25
83.48 ± 0.62b
72.17 ± 0.38
21
68.41 ± 0.40
74.47 ± 0.59
78.03 ± 0.26
79.77 ± 0.31
22
83.03 ± 0.78b
92.91 ± 0.66c
84.48 ± 0.68b
82.14 ± 0.38c
Vitamin E
2009), for similar structures like compound 1, when the configuration of the carbon linked to the nitrogen (C-10) is R, the optical rotation is positive. Thus, 1 was assigned as (?)-(R)-1-methyl-1H-2-oxa-3,9b-diazacyclopenta [jk] fluorine, and given the name lycolongirine A. Compound 2 was obtained as yellow oil, [a]20 D ? 25.71°(c 1.39, CH3OH). The HR-ESI–MS revealed
123
quasimolecular ion peaks at m/z 366.1906 (calc. for C19H28NO6?, 366.1917) [M ? H]?, and m/z 731.3740 (calc. for C38H55N2O12?, 731.3755) [2 M ? H]?, corresponding to the molecular formula C19H27NO6 with 7 degrees of unsaturation. The 1H-NMR spectrum of 2 (Table 1) revealed two singlets for the para-oriented aromatic protons at dH 7.36 (H-11) and 6.94 (H-8), one
Alkaloids from the bulbs of Lycoris longituba Table 4 Cell viabilities (%) of compounds in different concentrations in Ab25–35 induced SH-SY5Y cells injuries Compound
1 lM
Test concentration (lM) 6.25
12.5
25
1
63.33 ± 1.08
76.52 ± 0.21c
75.14 ± 0.64b
72.85 ± 0.38
68.69 ± 0.31
67.71 ± 0.21
2
78.27 ± 0.83
74.07 ± 0.83
72.04 ± 0.45
60.43 ± 0.35
54.70 ± 0.49
74.39 ± 0.53
3
68.05 ± 0.42b
64.51 ± 0.44
63.10 ± 0.31
61.21 ± 0.32
a
50
b
100
Ab25–35
52.85 ± 0.51
58.12 ± 0.30
b
74.84 ± 0.83
74.39 ± 0.53
4
72.63 ± 0.78
78.59 ± 0.46
81.26 ± 0.61
87.13 ± 0.17
5
58.24 ± 0.42
60.99 ± 0.76
68.28 ± 1.14
72.77 ± 1.03b
62.59 ± 0.95
63.85 ± 0.97
6
68.03 ± 0.91
64.44 ± 0.71
62.51 ± 0.57
59.77 ± 0.75
54.83 ± 0.78
67.30 ± 1.14
7 8
55.84 ± 0.47 60.31 ± 0.85
60.64 ± 0.97a 57.47 ± 0.82
50.68 ± 0.35 51.73 ± 0.46
46.64 ± 0.49 48.31 ± 0.76
9.67 ± 0.15 43.45 ± 1.18
54.35 ± 0.65 67.48 ± 0.98
9
65.81 ± 0.37
75.51 ± 0.71b
73.47 ± 0.56b
72.29 ± 0.95b
68.35 ± 0.84
65.46 ± 0.49
62.80 ± 0.17
59.63 ± 0.87
49.69 ± 0.60
63.85 ± 0.97 58.12 ± 0.30
a
10
68.21 ± 0.95
63.51 ± 0.30
11
62.26 ± 0.50b
69.53 ± 0.32c
64.43 ± 0.31c
63.42 ± 0.26c
61.17 ± 0.12b
12
b
70.92 ± 0.40
73.94 ± 0.78
c
b
b
68.12 ± 0.15b
59.33 ± 0.74
13
73.55 ± 0.67c
59.29 ± 0.86
14
80.68 ± 1.40 c
72.94 ± 0.25
69.26 ± 0.36
52.73 ± 1.27
46.60 ± 0.96
43.86 ± 0.53
59.33 ± 0.74
85.94 ± 1.78c
86.90 ± 1.25c
79.26 ± 0.36
78.12 ± 0.15
77.13 ± 0.74
c
77.51 ± 0.20c
75.45 ± 0.25c
73.95 ± 0.15c
62.33 ± 0.30
15
73.20 ± 0.51
73.98 ± 0.44
16
65.00 ± 0.55b
70.13 ± 0.72c
73.38 ± 0.32b
69.15 ± 0.31b
59.55 ± 0.26a
53.92 ± 0.36
17
66.80 ± 0.31
80.13 ± 0.32c
77.60 ± 0.36c
75.43 ± 0.40b
73.50 ± 0.15c
67.71 ± 0.21
18
76.14 ± 0.98a
78.30 ± 0.38b
82.52 ± 0.68c
76.50 ± 0.06a
72.96 ± 0.55
70.25 ± 0.70
19
73.31 ± 0.21c
81.79 ± 0.36c
85.14 ± 0.21c
78.23 ± 0.21c
70.49 ± 0.35c
62.33 ± 0.30
20
68.39 ± 0.31b
78.09 ± 0.32c
72.41 ± 0.26c
70.85 ± 0.30c
66.72 ± 0.25b
63.02 ± 0.21
21
b
73.08 ± 0.49
82.86 ± 0.51
c
c
b
70.66 ± 0.35
63.02 ± 0.21
22 Vitamin E
60.38 ± 0.78a
62.49 ± 0.30c
55.25 ± 0.42 69.92 ± 0.55b
53.92 ± 0.36 63.02 ± 0.21
75.83 ± 0.15
72.92 ± 0.50
66.13 ± 0.44c
60.73 ± 0.44b
olefinic proton at dH 5.81 (1H, brd, 1.5, H-3), four methoxy groups at dH 3.88 (3H, s), 3.82 (3H, s), 3.45 (3H, s) and 3.37 (3H, s), two AB system CH2 at dH 4.43 (1H, d, J = 10.8 Hz, H-7a) and 4.38 (1H, d, J = 10.8 Hz, H-7b), dH 3.71 (1H, ddd, J = 14.8, 4.1, 1.7 Hz, H-2a) and 3.63 (1H, brd, J = 14.8 Hz, H-2b), respectively. The 13C-NMR (Table 1) and DEPT spectra of 2 displayed 19 carbon signals [OCH3 9 4, CH2 (sp3) 9 2, CH (sp3) 9 5, CH (sp2) 9 3 and C (sp2) 9 5, Table 1]. From the NMR evidences, compound 2 has one benzene and one olefinic group in the structure, needing two more rings to meet the need of the unsaturation. The above spectral characteristics of 2 were very similar to those of lycorine-type alkaloid (Tsuda et al. 1979) with one ring splitting. The HMBC spectrum revealed that a –CH2OCH3 fragment was connected on C-7a, with C-7 and N-6 splitting from the ring (Kitagawa et al. 1959). The 1H–1H–COSY spectrum of 2 revealed the presence of a CH–CH–CH (OH)–CH2–CH=C and a CH (OCH3)–CH (OH) fragments, respectively. The HMBC correlation of H-5 (dH 3.61) with 5-OCH3 (dC 58.1) suggested that the methoxy group was connected on C-5. Accordingly, the two hydroxy groups were connected on C-1 (dC 62.0) and C-4 (dC 69.1), respectively. These
substitutions were demonstrated by the 1H–1H–COSY and HMBC spectra (Fig. 3). From above analysis, the planar structure of 2 was deduced unambiguously to be the structure as shown in Fig. 3. In the NOESY spectrum (Fig. 3), the H-11c signal gave a correlation with H-4 and H-5, while showing no correlation with H-1.The relative configurations of H-11c and H-11b in 2 was assigned as aand b-orientations, respectively, which was always appeared in the lycorine-type alkaloids isolated from the genus Lycoris (Evidente et al. 1983). So H-1 was deduced as b-orientation, H-4 and H-5 was a-orientation. Thus, the structure of 2 was identified as (2R,3R,6R,7S,7aS)-7-(4,5dimethoxy-2-(methoxymethyl)phenyl)-2-methoxy-2,3,5,6,7, 7a-hexahydro-1H-indole-3,6-diol, named lycolongirine B. Compound 3, colorless needles, [a]20 D ? 26.68° (c 1.13, CH3OH). The HR-ESI–MS of 3 displayed a molecular ion at m/z 350.1152 [M]?, being consistent with a molecular formula of C18H21ClNO4? (calc. 350.1154) and corresponding to 9 degrees of unsaturation. Inspection of the 1H-NMR (Table 2) spectrum of 3 revealed the presence of 20 protons, including two singlets for the paralocated aromatic protons at dH 6.84 (H, s, H-10) and 6.76 (H, s, H-7), an olefinic proton at dH 5.90 (H, s, H-1), a
123
Y.-Y. Zhu et al. Table 5 Cell viabilities (%) of compounds in different concentrations in CoCl2 induced SH-SY5Y cells injuries Compound
265 lM
Test concentration (lM) 6.25
12.5
25
50
84.52 ± 0.21c
81.34 ± 0.55b
77.61 ± 0.61a
74.69 ± 0.57
74.10 ± 0.45
51.76 ± 0.50
39.57 ± 0.25
28.31 ± 0.21
54.44 ± 0.32
83.14 ± 0.49c
74.14 ± 0.68a
65.96 ± 0.55
70.82 ± 0.72
b
74.51 ± 0.55
73.84 ± 0.47
b
70.75 ± 0.61
68.03 ± 0.21
70.39 ± 0.42b
62.56 ± 0.91
37.73 ± 0.36
64.47 ± 0.53
80.58 ± 0.0c
76.41 ± 0.20a
72.54 ± 0.36
74.10 ± 0.45
74.26 ± 0.42 50.73 ± 0.76
49.81 ± 0.68 46.31 ± 0.86
9.50 ± 0.60 42.47 ± 0.67
64.47 ± 0.53 67.48 ± 0.98
70.76 ± 0.67c
81.12 ± 0.72c
49.32 ± 0.49
67.69 ± 1.16
58.90 ± 0.47
36.99 ± 0.46
59.71 ± 0.65
90.91 ± 0.86c
80.26 ± 1.23a
75.15 ± 0.50b
70.26 ± 0.42
68.39 ± 0.25
64.12 ± 0.12
63.25 ± 0.32
70.26 ± 0.42
48.21 ± 1.47
1
78.27 ± 0.21b
2
a
61.67 ± 0.66
54.86 ± 0.50
3
69.72 ± 0.40
71.68 ± 0.64 c
4
66.47 ± 1.00
83.14 ± 0.40
5
53.81 ± 0.76
62.68 ± 0.1
6
80.65 ± 0.21b
88.65 ± 0.42c a
c
7 8
59.56 ± 0.21 60.54 ± 0.65
68.35 ± 0.62 55.46 ± 0.52
9
55.98 ± 0.81a
65.78 ± 0.58b
66.91 ± 0.57b
c
a
10
66.39 ± 2.12
71.97 ± 0.50
11
71.86 ± 0.32
92.63 ± 087c
12
70.91 ± 0.31
82.59 ± 0.35
13
54.89 ± 0.60
51.12 ± 0.55
c
100
CoCl2
36.80 ± 0.45
34.93 ± 0.20
70.94 ± 0.15
b
14
65.18 ± 0.89
66.78 ± 0.58
73.31 ± 0.97
69.76 ± 1.67
62.12 ± 0.72
63.47 ± 1.49
15
66.50 ± 0.49
70.17 ± 1.22
86.15 ± 0.83b
70.90 ± 0.85
61.33 ± 0.65
70.49 ± 0.15
16
58.44 ± 0.42
91.46 ± 0.20c
73.94 ± 1.08a
70.63 ± 0.50a
63.55 ± 0.15
66.72 ± 0.40
17
61.11 ± 0.15
83.03 ± 0.21c
71.57 ± 0.40c
68.33 ± 0.60b
54.26 ± 0.32
59.71 ± 0.65
18
64.42 ± 0.42a
69.37 ± 0.12b
78.77 ± 0.21b
71.42 ± 0.67b
66.25 ± 0.30b
60.53 ± 0.58
19
59.75 ± 0.26
65.89 ± 0.21c
67.23 ± 0.31b
69.52 ± 0.25c
68.71 ± 0.21c
58.49 ± 0.10
20
63.31 ± 1.11
68.09 ± 0.21
b
c
78.90 ± 0.42
70.10 ± 0.56
b
a
65.10 ± 0.70
60.53 ± 0.58
21
58.25 ± 0.38
79.90 ± 0.26c
83.33 ± 0.42c
81.87 ± 0.21c
79.86 ± 0.23c
69.68 ± 0.35
22 Vitamin E
76.57 ± 0.76b
91.95 ± 0.61c
81.05 ± 0.91b
77.96 ± 0.26b
63.75 ± 1.28 78.69 ± 0.87b
70.82 ± 0.72 70.82 ± 0.72
In Tables 3, 4 and 5, 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 = 3) with a kind of statistical software PASW statistics 18.0, a for p \ 0.05, b for p \ 0.01, c for p \ 0.001
methylenedioxy group at dH 5.96 (2H, d, J = 0.9 Hz, H-13), a chloromethyl group at dH 5.61 and 5.59 (each 1H, d, J = 10.1 Hz). The 13C-NMR and DEPT spectra of 3 exhibited 18 carbon signals [CH3 9 1, CH2 (sp3) 9 5, CH (sp3) 9 4, CH (sp2) 9 3 and C (sp2) 9 5, Table 2]. The 1 H–1H–COSY and HMBC spectra of 3 (Fig. 4) revealed the presence of a –CH2CH(O)CH(O)– and a = CH– CH(O)– fragments. The above NMR data as well as the positive optical rotation of 3 are similar to those of the known compound (?) coccinine (Jin and Weinreb 1997) except that 3 has an extra chloromethyl group at dH 5.51, 5.59 (each 1H, d, J = 10.1 Hz) and dC 66.5. The HMBC (Fig. 4) correlations between the two choloromethyl protons and C-4a, C-6, C-12, as well as the deshielding chemical shifts of H-4a, H-6, H-12 in the 1H NMR and C-4a, C-6, C-12 in the 13C NMR suggested the location of the chloromethyl group at N atom (Jin and Weinreb 1997). The NOESY correlations of H-2/H-4a and H-3/H-4a suggested H-2, H-3 and H-4a to be the same a-orientation. No correlation between H-11 and H-4a proved 11-CH2 to be borientation (Pearson and Lian 1998) (Fig. 4). Finally, the
123
structure of 3 was confirmed to be (?)-N-methylchloride coccinine, given the name lycolongirine C. As galanthamine easily forms a quaternary ammonium salt ((-)-N-(choloromethyl) galanthaminium chloride) by reaction with the solvent CH2Cl2 (Matusch et al. 1994), which was also used in our separation process, compound 3 could also be an artificial product. The isolated alkaloids were tested for their neuroprotective effects against CoCl2, H2O2 and Ab25–35-induced neuronal cell death in dopaminergic neuroblastoma SHSY5Y cells. Compounds 4, 6, 11, 16, 18, 20 and 22 showed significant neuroprotective effects against all three cell injury models; compounds 1-3 and 12 exhibited significant neuroprotective activities against H2O2 (Table 3) and Ab25–35-induced cell death (Table 4); while compounds 13, 15 and 17 had similar activities against CoCl2induced cell damage (Table 5). The above results provide a preliminary indication that Amaryllidaceae alkaloids may have a big potential to neuroprotective activities against both hypoxic injury and oxidative damage in neuronal cells.
Alkaloids from the bulbs of Lycoris longituba Table 6 AChE inhibitory activity of the isolated compounds (n = 3) Compound
Percent inhibitory activity (%)
IC50 (lM)
1
38.59 % ± 3.27
241.50 ± 2.46
2
37.11 % ± 2.69
157.40 ± 3.51
3
45.05 % ± 2.46
117.60 ± 1.79
4
41.08 % ± 0.85
148.70 ± 1.46
5
45.44 % ± 1.33
164.10 ± 1.59
6
57.29 % ± 0.82
77.10 ± 1.65
7
34.76 % ± 2.80
181.00 ± 4.00
8
32.36 % ± 2.34
224.80 ± 3.01
9
42.36 % ± 2.02
194.80 ± 2.31
10
36.43 % ± 3.23
151.10 ± 2.42
11 12
33.00 % ± 0.91 84.91 % ± 0.59
190.70 ± 2.00 4.23 ± 1.13
13
38.96 % ± 0.78
208.10 ± 1.58
15
91.50 % ± 0.67
2.76 ± 0.65
16
93.51 % ± 0.87
5.55 ± 0.63
17
96.05 % ± 0.45
3.04 ± 0.61
18
92.63 % ± 0.15
5.30 ± 0.76
19
78.78 % ± 0.93
25.76 ± 1.09
20
85.63 % ± 0.61
8.13 ± 1.49 275.10 ± 1.86
21
32.22 % ± 1.58
22
89.44 % ± 0.38
8.44 ± 0.83
Galanthamine (14)
94.54 % ± 0.51
2.43 ± 0.66
IC50 value represents the concentration of compounds required to inhibit the 50 % activity of AChE. The percent inhibitory activity (%) represents inhibitory activity of compound in the concentration of 100 lM. ‘‘±’’ represents standard error of mean of these enzyme inhibition assays. Galanthamine was used as positive control
The isolates were also evaluated for their in vitro AChE inhibition according to a modified Ellman method (Eldeen et al. 2005) using galanthamine as a positive control. Compounds 12, 15-18, 20 and 22 were found to exhibit strong AChE inhibitory effects with IC50 values ranging from 2.76 to 8.44 lM (Table 6). The observed AChE inhibitory effects (Table 6) seem to be related to some structural characteristics among the different skeleton types. Only the alkaloids belonging to the galanthamine (compounds 15–20), the narciclasine (compound 12), and tazettine (compound 22) types exhibited significant AChE inhibitory activities. While the harmane-type alkaloid compound 6 (harmane) was also found to have moderate anti-AChE activity with the IC50 of 77.10 lM (Table 6). Our results indicated that except of the galantamine-type, the narciclasine and tazettine type alkaloids also have great potential of anti-AChE activity. Acknowledgments Financial supports 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 (No. 81072547, 31270394 and 30873361) are gratefully acknowledged. Conflict of interest All the authors have no conflict of interest to declare.
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RESEARCH ARTICLE
Alkaloids from the bulbs of Lycoris longituba and their neuroprotective and acetylcholinesterase inhibitory activities Yun-Yun Zhu • Xue Li • Heng-Yi Yu • Yu-Fang Xiong • Peng Zhang • Hui-Fang Pi Han-Li Ruan
•
Received: 11 January 2014 / Accepted: 8 April 2014 Ó The Pharmaceutical Society of Korea 2014
Abstract Three novel alkaloids (1–3), together with nineteen known ones (4–22), were isolated from the bulbs of Lycoris longituba. Their structures were elucidated on the basis of extensive spectroscopic analyses, which belong to several Amaryllidaceae alkaloid skeletons. Among them, the harmane-type alkaloids (the new compound 1 and the known compounds 5, 6 and 7) were found for the first time from Lycoris genus. The isolates were tested for their neuroprotective activities against CoCl2, H2O2 and Ab25–35-induced SH-SY5Y cell injuries, and the majority of them exhibited neuroprotective activities of different degrees. The acetylcholinesterase (AChE) inhibitory activities of the isolated alkaloids were also evaluated, while compounds 12, 14–20 and 22 exhibited extremely significant AChE inhibitory activities. Keywords Lycoris longituba Alkaloid Neuroprotective activity AChE inhibitory activity
Introduction The Amaryllidaceae family consists of about sixty genera, whose eight hundred species are widely distributed in the
Y.-Y. Zhu X. Li H.-Y. Yu P. Zhang H.-F. Pi H.-L. Ruan (&) Faculty of Pharmacy, Tongji Medical College of Huazhong University of Science and Technology, Hangkong Road 13, Wuhan 430030, People’s Republic of China e-mail: [email protected] Y.-F. Xiong Department of Biochemistry, Tongji Medical College of Huazhong University of Science and Technology, Hangkong Road 13, Wuhan 430030, People’s Republic of China
tropics and warm-temperate regions of the world (Kornienko and Evidente 2008). Most plants of Amaryllidaceae are known to produce structurally unique alkaloids, which showed diverse bioactivities such as antiviral, acetylcholinesterase (AChE) inhibitory, antibacterial, antifungal, antineoplastic and antimalarial effects (Ieven et al. 1979; Louw et al. 2002; Sener et al. 2003; Elgorashi et al. 2004; Jin 2005; Jokhadze et al. 2007). Especially, galanthamine and lycorine types exhibited significant cholinesterase inhibitory effect, which enhances cognitive functions of Alzheimer’s patients (Lopez et al. 2002). AChE inhibitors are considered to be one of the most effective approaches for treating AD (Bartus et al. 1982; Perry 1986; Senol et al. 2010). The genus Lycoris from Amaryllidaceae consists of about 20 species all over the world, with 17 (including one variety) of which distributing in China. Recently, our group reported the chemical constituents and neuroprotective activities of several lycoris plants (Li et al. 2013; Jin et al. 2014; Wu et al. 2014). As a continuation of our work, another lycoris plant, Lycoris longituba, was studied in this paper. Lycoris longituba mainly grows in Jiangsu province (McNulty et al. 2007) and the bulbs of which have been traditionally used for the treatment of sore throats, furuncle carbuncle swollen in skin and scrofula (He et al. 2011). Previously, 12 alkaloids were isolated from the bulbs of L. longituba (Liang et al. 2010, Zhao et al. 2011), but the pharmacological studies on this plant have never been reported before. To discover potentially bioactive compounds, we further investigated the bulbs of L. longituba collected in Baohua Mountain, Jiangsu province. Accordingly, three new alkaloids (1–3): lycolongirine A (1), lycolongirine B (2), lycolongirine C (3), together with nineteen known ones (4–22), incartine (4) (Berkov et al. 2007), norharmane (5) (Abramovitch and Spenser 1964;
123
Y.-Y. Zhu et al.
11
H3C
10 8 8a N 9a
7
HO 1
O 1
H3CO 10
N
4b
5
4a
1
3
H
11 11b
9
O
3a
10a
8
H N
8
6a
7
6
8a
7
9a
N
8
2
6
4b
5
4a
6
4
10
H3CO H3CO
9
11c
H
7a
8
N 7
4
10
9
1
O
9
10 10b 10a
O
8
7
H3CO
9
7a
8
OMe 11 OH
3
H
3a 11c
5
6
7
O 4
N
N
6a
O
O
9
7a
8
N 6a 7
4
12
OH
OH
3
3
1 10b
4a 12 11
10
R3
R1
N R2
9
7 8
1 10b
4a 12 11
10
R
O
9
10 10b
O
8
R1
N R2 6
9
5
7
OMe 11 OH 3
4a
H
N
6a
7
6
OH
3
4
O
4
OCH3
6
6
R
11c
13a
2
4
3a
N
1
10a
CH3
6
11
3
H
2
10b 4a
6
O N
2
1
11 11b 10 11a H
3
10 9 10a
8
5
9
1
O
4
N
OCH3 HO
2
4
2
3
H
8
3
O
3a 11c
8 7a
9
Cl
OH 13b
O
4
7
4a
H
6a
3
10a
8
5
11a
2
11 11b 10 11a H
10b 4a
O
11 11b
OH H3CO 10
3
H
4
1
2
10
2
O
N
1
O
3 3a
H
11b 11a
1
9a
4a
OCH3 2 OH
3
2
1
OH HO
7
1
11
4b
5
5 R=H 6 R=CH3
HO
8a
7
3
H N
HO
3
O 1
4a
N H CH2Cl
HOH2C R
2
4
2
1
OCH3
OCH3
11 4b 12
H O HN 5 OCH H3CO 9 8 7a 3 OCH 3 7
4
10
OH
11c 4
11a
3
6
2
O
12
4a
13a
O
7
11
4
2 13 1
10
9
13b
H
N CH3
6a
6
OR 8
8
14 R=OCH3 R1=free R2=CH3 R3=H 19 R=OCH3 R1=CH2Cl R2=CH3 15 R=OCH3 R1=free R2=H R3=H 20 R=OH R1=free R2=CH3 16 R=OCH3 R1=CH2Cl R2=CH3 R3=H 17 R=OCH3 R1=free R2=CH3 R3=OH 18 R=OH R1=free R2=CH3 R3=H
21 R=OH 22 R=H
Fig. 1 The structures of compounds 1–22
Balkau and Heffernen 1973), harmane (6) (Hiroko et al. 1993), perlolyrine (7) (Li et al. 2011, 2012), lycorine (8) (Tsuda et al. 1979), hippamine (9) (Evidente et al. 1984), N-chloromethyl narcissidine (10) (Suau et al. 1990), trisphaeridine (11) (Alonso et al. 2010), N-methylcrinasiadine (12) (Cowden et al. 1994), (?)-haemanthidine (13a), (-)haemanthidine (13b) (Pabuccuoglu et al. 1989), galanthamine (14) (Kihara et al. 1994), N-norgalanthamine (15) (Treu and Jordis 2001), N-chloromethyl galanthamine (16)
123
(Matusch et al. 1994), 11b-hydroxy galanthamine (17) (Andrade et al. 2011), sanguinine (18) (Kihara et al. 1994), N-chloromethyl lycoramine (19) (Matusch et al. 1994), Odemethyllycoramine (20) (Wang et al. 2010), tazettine (21) (Roberts et al. 1971), deoxytazettine (22) (Roberts et al. 1971) were isolated (Fig. 1). The isolated alkaloids were tested for their neuroprotective activities against CoCl2, H2O2 and Ab25–35 induced SH-SY5Y cell injury. Compounds 11, 16, 17 and 22 exhibited significant
Alkaloids from the bulbs of Lycoris longituba
neuroprotective effects against CoCl2-induced SH-SY5Y cell injury, and compounds 1, 2 and 22 showed significant neuroprotective effects against H2O2-induced SH-SY5Y cell death, while compounds 16, 18 and 19 had similar activities against Ab25–35-induced SH-SY5Y cell damage. The isolated alkaloids were also tested for their acetylcholiesterase (AChE) inhibitory activities. And the compounds 12, 14–20 and 22 exhibited extremely significant AChE inhibitory activities.
Materials and methods General 1
H (400 MHz), 13C (100 MHz), and 2D-NMR spectra were recorded on a Bruker AM-400 spectrometer with tetramethylsilane (TMS) as an internal standard. Chemical shifts are in ppm (d), relative to TMS and scalar coupling constants (J) reported in Hertz and used deuterated dimethyl sulfoxide (DMSO-d6), methanol (CH3OH-d4) and chloroform (CHCl3-d), pyridine (pyridine-d5) as solvents. Electrospray ionization (ESI) mass spectra were acquired in the positive ion mode on an LCQ DECAXP instrument (Thermo Finnigan, San Jose, CA, USA) equipped with an ion trap mass analyzer. HR-ESI–MS were obtained in the negative ion mode on Thermo LTQ-Orbitrap XL. IR spectra were obtained on a NicoletTM 380 spectrometer from Thermo Electron; in cm-1. UV spectra were taken on a Cary-50 spectrophotometer. Optical rotations were acquired on a PerkinElmer PE-341L polarimeter. TLC plates were HSGF254 SiO2 from Yantai Jiangyou Silica Gel Development Co., Ltd., China. Column chromatography (CC) silica gel (SiO2; 200–300 mesh; Qingdao Haiyang Chemical Co., Ltd., Qingdao, China). All other Organic solvents were of analytical reagent grade from Traditional Chinese medicine group. Plant material The fresh bulbs of L. longituba were collected in the Baohua Mountain, Zhenjiang city of Jiangsu Province, China, in September 2011 and identified by Prof. Shulan He of Zhongshan botanical garden. A voucher specimen (20110909) has been deposited in the Herbarium of Materia Medica Faculty of Pharmacy, Tongji Medical College of Huazhong University of Science and Technology, Wuhan, China.
atmosphere with 5 % CO2 incubator. The samples were solved and diluted with PBS to required concentrations. The cell viability was assessed using a MTT assay. About 9 9 103 cells in 1 mL of logarithmic phase were seeded onto a 96-well microplate. 24 Hours later, each hole with 11.5 lL sample to be tested in the normal group, while 11.5 lL PBS in control group. After 2 h, CoCl2, H2O2 or Ab25–35 was added to the normal group and control group. 15 lL of MTT solution (5 mg/mL) was added to all of the holes after the cells were cultured at 37 °C for 24 h. After continuing to culture for 4 h, the medium was removed and DMSO (100 lL/well) was added. Finally, the reduced MTT was assayed at 570 nm using a multi-function microplate reader. AChE inhibition assay AChE from electric eel (type VIS), acetylthiocholine iodide and 5, 5-dithio-bis (2-nitrobenzoic acid) (DTNB) was purchased from Sigma. AChE inhibitory activities of the isolated compounds were determined using Ellman’s colorimetric method (Ellman et al. 1961) as modified by Eldeen et al. (2005). 25 lL of 15 mM ATCl in water, 125 lL of 3 mM DTNB in Buffer C (50 mM Tris–HCl, pH 8.0, 0.1 M NaCl and 0.02 M MgCl2. 6H2O), 50 lL Buffer B (50 mM Tris–HCl, pH 8.0, 0.1 % bovine serum albumin) and 25 lL of the samples (6.25, 12.5, 25, 50 lM, 100 Lm) were added to the wells in a 96 wells microplate. Absorbance corresponding to spontaneous hydrolysis was read at 405 nm every 45 s for 5 times. Thereafter, 25 lL of 0.20 U/mL AChE was added to each well and absorbance was consecutively read again every 45 s for 9 times. Galanthamine served as the positive control. Any increase in absorbance due to the spontaneous hydrolysis of the substrate was corrected by subtracting the absorbance before adding the enzyme from the absorbance after adding the enzyme. The percentage inhibition was calculated using the equation inhibition ð%Þ ¼ Acontrol Asample = Acontrol 100. Where Asample is the absorbance of the sample and Acontrol is the absorbance of the blank (Buffer A). AChE inhibition values are means of three individual determinations each performed in triplicate. AChE inhibitory activity expressed as IC50 values were obtained from the logarithmic non-linear regression curve derived from the plotted data using GraphPad Prism software package for Windows (GraphPad version 5.0, San Diego, USA).
Statistical analysis Neuroprotective activity assay SH-SY5Y cells were cultured in DMEM high glucose medium containing 10 % FBS at 37 °C in a humidified
Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by post hoc multiple comparisons using the Newman–Keuls Multiple Comparison
123
Y.-Y. Zhu et al. Table 1 1H NMR (400 MHz) and and 2 in CH3OH-d4
13
Compound 1 position
dC
1
150.5
dH (mult., J, Hz)
2a
C NMR (100 MHz) Data for 1
138.6
8.18 (1H, d, 5.3)
3a 116.1
4a
132.5
7.95 (1H, d, 5.3)
4b
123.3
5
123.6
8.13 (1H, d, 8.0)
6
121.9
7.23 (1H, t, 8.0)
7
130.8
7.53 (1H, t, 8.0)
7a 8
Compound 3
dH (mult., J, Hz)
position
dC
62.0
4.70 (1H, brd, 11.2)
1
118.9
5.90 (1H, brs)
53.0
3.71 (1H, ddd, 14.8, 4.1, 1.7)
2
78.3
3.54 (1H, m)
3.63 (1H, brd, 14.8)
3 4a
66.8 28.1
4.20 (1H, m) 2.63 (1H, dt, 11.2, 4.3)
8a
125.5
5.81 (1H, brd, 1.5)
4b
69.1
4.60 (1H, brs)
82.1
3.61 (1H, dd, 5.7, 2.8)
73.9
4.43, 4.38 (each 1H, d, 10.8)
130.5 114.2
7.62 (1H, d, 8.0)
115.4
6.94 (1H, s)
143.7
9
148.6
9a
135.2
10
72.6
5.33 (1H, q, 6.6)
150.1
11
24.5
1.64 (3H, d, 6.6)
114.5
7.36 (1H, s)
11a
133.5
11b
46.5
3.21 (1H, dd, 1.7, 11.2)
11c
74.2
4.00 (1H, brs)
(C-5)OCH3
58.1
3.45 (3H, s)
(C-7)OCH3
58.3
3.37 (3H, s)
(C-9)OCH3
56.6
3.82 (3H, s)
(C-10)OCH3
56.4
3.88 (3H, s)
method. The data are expressed as mean ± SEM of three assays. Extraction and isolation The fresh bulbs of L. Longituba (45 kg) were cut into pieces and extracted exhaustively with ethanol (95 %) for five times (2 h each time) at 55 °C in multi-function extraction tank. The solvent was evaporated under vacuum and the liquid extract was acidified to pH 3–4 by HCl and then extracted with CH2Cl2 for five times to afford 850 g of acid extract. After adjusting the pH value of the remaining parts to neutral by addition of NaOH, 69 g neutral components were resulted. The last part was alkalified to pH 10–11 and 13–14 successively, produce 152 g and 135 g alkaline substances, respectively. The four CH2Cl2 fractions of different pH were put together and concentrated to afford a residue, which was subjected to silica gel (200–300 mesh), and eluted with a gradient system of CH2Cl2/CH3OH (1:0, 70:1, 50:1, 30:1, 20:1, 10:1, 8:1, 6:1,
123
C NMR (100 MHz) Data for 3
dC
143.2
4
13
Compound 2
2b 3
Table 2 1H NMR (400 MHz) and in CH3OH-d4
dH (mult., J, Hz)
2.02 (1H, td, 11.9, 2.4)
4a
72.3
4b
144.1
6a
66.2
6b
4.59 (1H, brd, 11.6) 4.96 (1H, d, 15.1), 5.12 (1H, d, 15.1)
6a
119.1
7
107.7
8
149.5
9
149.6
10
108.1
10a
129.2
11
45.3
4.09 (1H, brd, 3.1)
12a
64.4
4.00 (1H, d, 10.2)
103.1
5.96 (2H, d, 0.9)
12b (C-8, 9)OCH2O
6.76 (1H, s)
6.84 (1H, s)
3.84 (1H, ddd, 1.7, 3.1, 10.2)
(C-2)OCH3
58.2
3.44 (3H, s)
N–CH2Cl
66.5
5.61, 5.59 (each 1H, d, 10.1)
4:1, 1:1, 0:1, V/V) to yield fractions A–I according to TLC analysis. The fraction A was chromatographed over a series of silica gel and Sephadex LH-20 (CH2Cl2/CH3OH 1:1) to afford compound 11 (31 mg) and compound 12 (25 mg). Fraction B was separated into four parts (B-1 to B-4) by silica gel. The part B-2 was again subjected to silica gel to give six subfractions (B-2-1 to B-2-6). Among them, subfractions B-2-3 and B-2-4 were further purified with Sephadex LH-20 (MeOH) and then high performance liquid chromatography (HPLC) to yield the new compound 1 (12.7 mg) and compound 5 (12.6 mg), respectively. Subfraction B-2-5 was obtained as compound 6 (16.7 mg). The part B-3 was subjected to Sephadex LH-20 (MeOH) and then silica gel again to get four subfractions (B-3-1 to B-3-4). Subfraction B-3-2 was produced compound 7 (15.6 mg) and 14 (10.2 mg) by HPLC. Subfraction B-3-3 was afforded compound 21 (15 g). Fraction C was resulted in compound 22 (19.7 mg). The Fractions D–I were applied on Sephadex LH-20 (CH2Cl2/CH3OH 1:1 or 0:1), TLC and HPLC to produce compound 3 (29.7 mg), 4 (18.0 mg) and the others. Lycolongirine A (1) light yellow powder; [a]20 D ? 9.35°(c 0.42, CH3OH); UV k max (CH3OH) nm (log e):
Alkaloids from the bulbs of Lycoris longituba Fig. 2 Key correlations of compound 1
H3C O N N
1
H-1H-COSY
Key NOESY correlations
HMBC H to C Fig. 3 Key correlations of compound 2
HO
H3CO
OH
HN H3CO
OCH3
OCH3
1
H-1H-COSY
Key NOESY correlations
HMBC H to C
235 (0.71), 289 (0.33), 340 (0.09); IR (KBr) t max cm-1: 3089, 2970, 2838, 1625, 1588, 1430, 1371, 1221, 740; 1HNMR and 13C-NMR data: see Table 1; Positive HR-ESI– MS m/z: 211.0868 [M ? H]? (calc. for C13H11N2O?, 211.0871). Lycolongirine B (2) yellow oil; [a]20 D ? 25.71°(c 1.39, CH3OH); UV k max (CH3OH) nm (log e): 206 (1.42), 236 (0.36), 282 (0.13); IR (KBr) t max cm-1: 3471, 2932, 2829, 1606, 1517, 1464, 1370, 1285, 1188, 1111, 937; 1H-NMR and 13C-NMR data: see Table 1; Positive HR-ESI–MS m/z: 366.1906 [M ? H]? (calc. for C19H28NO6?, 366.1917). Lycolongirine C (3) colorless needles; [a]20 D ? 26.68° (c 1.13, CH3OH); UV k max (CH3OH) nm (log e): 212 (2.21), 244 (0.41), 291 (0.52); IR (KBr) t max cm-1: 3383, 3027, 2959, 2826, 1636, 1507, 1488, 1247, 937; 1H-NMR and 13C-NMR data: see Table 2; Positive HR-ESI–MS m/z: 350.1152 [M]? (calc. for C18H21ClNO4?, 350.1154). Results and discussion Compound 1 was isolated as light yellow powder, [a]20 D ? 9.35°(c 0.42, CH3OH). Its molecular formula was established as C13H10N2O by HR-ESI–MS from the
[M ? H]? ion peak at m/z 211.0868 [M ? H]? (calc. 211.0871), indicating ten degrees of unsaturation. The 1HNMR spectrum of 1 exhibited the presence of a 1, 2-substituted benzene at dH 8.13 (1H, d, J = 8.0 Hz, H-5), 7.62 (1H, d, J = 8.0 Hz, H-8), 7.53 (1H, t, J = 8.0 Hz, H-7) and 7.23 (1H, t, J = 8.0 Hz, H-6), an ortho-positioned pyridine at dH 8.18 (1H, d, J = 5.3 Hz, H-3) and 7.95 (1H, d, J = 5.3 Hz, H-4), a methyl at dH 1.64 (3H, d, J = 6.6 Hz) and an O-methine at dH 5.33 (1H, q, J = 6.6 Hz, H-10) (Table 1). In turn, the 13C-NMR and DEPT spectra of 1 showed 13 carbon signals [CH3 9 1, CH (sp3) 9 1, CH (sp2) 9 6 and C (sp2) 9 5, Table 1]. The existence of an –OCHCH3 moiety was supported by the 1H–1H–COSY spectrum (Fig. 2). The NMR spectral characteristics of 1 were very similar to those of harmane (Hiroko et al. 1993), except that the methine carbon (C-10) on the –OCHCH3 linked with N-9, wherever the oxygen of the –OCHCH3 linked to C-1 to form a five-membered ring in 1. The above conclusion was confirmed by the HMBC experiment (Fig. 2). The stereochemical structure of 1 was speculated as the form showed in Fig. 2, which was supported by its positive specific rotation ([a]20 D ? 9.35°) (Bandini et al. 2008; Stanley and Hartwig 2009). According to references (Bandini et al. 2008; Stanley and Hartwig
123
Y.-Y. Zhu et al.
Fig. 4 Key correlations of compound 3
Table 3 Cell viabilities (%) of compounds in different concentrations in H2O2 induced SH-SY5Y cells injuries Compound
6.25 1
300 lM
Test concentration (lM) 12.5
81.50 ± 1.58
2 3
b
60.89 ± 0.10 90.41 ± 0.90b
4
81.01 ± 1.15a
25
50
100
H2O2
94.53 ± 0.15c
89.58 ± 0.82c
84.52 ± 0.35c
73.97 ± 1.80
74.39 ± 0.47
b
59.50 ± 0.31 83.63 ± 1.19a
a
57.10 ± 0.21 78.34 ± 0.61
56.14 ± 0.10a 74.26 ± 0.95
55.33 ± 0.55a 68.84 ± 0.92
51.54 ± 0.53 72.64 ± 0.25
93.72 ± 1.00c
83.25 ± 0.21b
81.56 ± 0.64b
76.86 ± 0.99
74.81 ± 0.70 51.52 ± 0.86
b
5
54.71 ± 0.85
59.97 ± 0.82
52.23 ± 0.40
51.11 ± 0.95
44.61 ± 0.68
6
78.99 ± 0.85a
89.38 ± 0.50c
91.73 ± 0.93b
81.60 ± 0.97b
70.14 ± 0.81
74.39 ± 0.47
7
55.18 ± 0.42
62.24 ± 0.25b
48.40 ± 0.62
32.19 ± 0.42
12.11 ± 0.58
51.20 ± 0.86
8
59.61 ± 0.85
58.37 ± 0.82
53.63 ± 0.46
50.11 ± 0.75
44.45 ± 1.08
67.48 ± 0.98
9
60.75 ± 0.67
82.97 ± 0.47b
95.36 ± 0.45c
94.23 ± 0.54c
84.75 ± 0.38b
63.46 ± 1.42
10
67.82 ± 0.91
72.89 ± 0.10
78.10 ± 1.14
70.08 ± 0.38
67.72 ± 0.1
72.86 ± 0.21
11
66.70 ± 0.85a
75.50 ± 0.68b
70.69 ± 0.31c
73.14 ± 0.60b
66.61 ± 0.85a
59.66 ± 0.15
12
65.70 ± 2.02
75.54 ± 1.81b
71.01 ± 0.51b
68.51 ± 1.07a
59.30 ± 0.45
59.66 ± 0.15
13
68.21 ± 1.25
78.09 ± 0.29b
67.27 ± 0.86
64.70 ± 0.72
54.19 ± 0.93
70.43 ± 0.46
14
63.70 ± 0.95a
63.85 ± 0.68a
71.69 ± 0.31b
65.74 ± 1.60b
63.31 ± 1.85a
49.66 ± 0.15
c
15
73.18 ± 0.55
77.53 ± 0.35
71.37 ± 0.78
59.90 ± 0.44
67.51 ± 0.89
70.43 ± 0.46
16
75.95 ± 0.90
93.35 ± 0.31c
87.70 ± 0.62c
83.55 ± 1.10b
79.29 ± 0.42a
74.81 ± 0.70
17
67.03 ± 0.44
73.41 ± 0.44
79.20 ± 0.15c
75.12 ± 0.40a
73.30 ± 0.36
72.86 ± 0.21
18 19
67.93 ± 0.40b 82.25 ± 0.78b
70.07 ± 0.38c 82.81 ± 0.25c
74.03 ± 0.35c 83.73 ± 0.47c
76.83 ± 0.23c 83.93 ± 0.69c
69.08 ± 0.15c 85.53 ± 0.40b
57.72 ± 0.25 72.26 ± 0.32
20
64.58 ± 0.35b
68.48 ± 0.45b
69.91 ± 0.31c
73.53 ± 0.59b
65.95 ± 0.32c
57.72 ± 0.25
b
c
b
75.69 ± 0.40
72.17 ± 0.38
80.02 ± 0.97a
72.64 ± 0.25
83.48 ± 0.62b
72.17 ± 0.38
21
68.41 ± 0.40
74.47 ± 0.59
78.03 ± 0.26
79.77 ± 0.31
22
83.03 ± 0.78b
92.91 ± 0.66c
84.48 ± 0.68b
82.14 ± 0.38c
Vitamin E
2009), for similar structures like compound 1, when the configuration of the carbon linked to the nitrogen (C-10) is R, the optical rotation is positive. Thus, 1 was assigned as (?)-(R)-1-methyl-1H-2-oxa-3,9b-diazacyclopenta [jk] fluorine, and given the name lycolongirine A. Compound 2 was obtained as yellow oil, [a]20 D ? 25.71°(c 1.39, CH3OH). The HR-ESI–MS revealed
123
quasimolecular ion peaks at m/z 366.1906 (calc. for C19H28NO6?, 366.1917) [M ? H]?, and m/z 731.3740 (calc. for C38H55N2O12?, 731.3755) [2 M ? H]?, corresponding to the molecular formula C19H27NO6 with 7 degrees of unsaturation. The 1H-NMR spectrum of 2 (Table 1) revealed two singlets for the para-oriented aromatic protons at dH 7.36 (H-11) and 6.94 (H-8), one
Alkaloids from the bulbs of Lycoris longituba Table 4 Cell viabilities (%) of compounds in different concentrations in Ab25–35 induced SH-SY5Y cells injuries Compound
1 lM
Test concentration (lM) 6.25
12.5
25
1
63.33 ± 1.08
76.52 ± 0.21c
75.14 ± 0.64b
72.85 ± 0.38
68.69 ± 0.31
67.71 ± 0.21
2
78.27 ± 0.83
74.07 ± 0.83
72.04 ± 0.45
60.43 ± 0.35
54.70 ± 0.49
74.39 ± 0.53
3
68.05 ± 0.42b
64.51 ± 0.44
63.10 ± 0.31
61.21 ± 0.32
a
50
b
100
Ab25–35
52.85 ± 0.51
58.12 ± 0.30
b
74.84 ± 0.83
74.39 ± 0.53
4
72.63 ± 0.78
78.59 ± 0.46
81.26 ± 0.61
87.13 ± 0.17
5
58.24 ± 0.42
60.99 ± 0.76
68.28 ± 1.14
72.77 ± 1.03b
62.59 ± 0.95
63.85 ± 0.97
6
68.03 ± 0.91
64.44 ± 0.71
62.51 ± 0.57
59.77 ± 0.75
54.83 ± 0.78
67.30 ± 1.14
7 8
55.84 ± 0.47 60.31 ± 0.85
60.64 ± 0.97a 57.47 ± 0.82
50.68 ± 0.35 51.73 ± 0.46
46.64 ± 0.49 48.31 ± 0.76
9.67 ± 0.15 43.45 ± 1.18
54.35 ± 0.65 67.48 ± 0.98
9
65.81 ± 0.37
75.51 ± 0.71b
73.47 ± 0.56b
72.29 ± 0.95b
68.35 ± 0.84
65.46 ± 0.49
62.80 ± 0.17
59.63 ± 0.87
49.69 ± 0.60
63.85 ± 0.97 58.12 ± 0.30
a
10
68.21 ± 0.95
63.51 ± 0.30
11
62.26 ± 0.50b
69.53 ± 0.32c
64.43 ± 0.31c
63.42 ± 0.26c
61.17 ± 0.12b
12
b
70.92 ± 0.40
73.94 ± 0.78
c
b
b
68.12 ± 0.15b
59.33 ± 0.74
13
73.55 ± 0.67c
59.29 ± 0.86
14
80.68 ± 1.40 c
72.94 ± 0.25
69.26 ± 0.36
52.73 ± 1.27
46.60 ± 0.96
43.86 ± 0.53
59.33 ± 0.74
85.94 ± 1.78c
86.90 ± 1.25c
79.26 ± 0.36
78.12 ± 0.15
77.13 ± 0.74
c
77.51 ± 0.20c
75.45 ± 0.25c
73.95 ± 0.15c
62.33 ± 0.30
15
73.20 ± 0.51
73.98 ± 0.44
16
65.00 ± 0.55b
70.13 ± 0.72c
73.38 ± 0.32b
69.15 ± 0.31b
59.55 ± 0.26a
53.92 ± 0.36
17
66.80 ± 0.31
80.13 ± 0.32c
77.60 ± 0.36c
75.43 ± 0.40b
73.50 ± 0.15c
67.71 ± 0.21
18
76.14 ± 0.98a
78.30 ± 0.38b
82.52 ± 0.68c
76.50 ± 0.06a
72.96 ± 0.55
70.25 ± 0.70
19
73.31 ± 0.21c
81.79 ± 0.36c
85.14 ± 0.21c
78.23 ± 0.21c
70.49 ± 0.35c
62.33 ± 0.30
20
68.39 ± 0.31b
78.09 ± 0.32c
72.41 ± 0.26c
70.85 ± 0.30c
66.72 ± 0.25b
63.02 ± 0.21
21
b
73.08 ± 0.49
82.86 ± 0.51
c
c
b
70.66 ± 0.35
63.02 ± 0.21
22 Vitamin E
60.38 ± 0.78a
62.49 ± 0.30c
55.25 ± 0.42 69.92 ± 0.55b
53.92 ± 0.36 63.02 ± 0.21
75.83 ± 0.15
72.92 ± 0.50
66.13 ± 0.44c
60.73 ± 0.44b
olefinic proton at dH 5.81 (1H, brd, 1.5, H-3), four methoxy groups at dH 3.88 (3H, s), 3.82 (3H, s), 3.45 (3H, s) and 3.37 (3H, s), two AB system CH2 at dH 4.43 (1H, d, J = 10.8 Hz, H-7a) and 4.38 (1H, d, J = 10.8 Hz, H-7b), dH 3.71 (1H, ddd, J = 14.8, 4.1, 1.7 Hz, H-2a) and 3.63 (1H, brd, J = 14.8 Hz, H-2b), respectively. The 13C-NMR (Table 1) and DEPT spectra of 2 displayed 19 carbon signals [OCH3 9 4, CH2 (sp3) 9 2, CH (sp3) 9 5, CH (sp2) 9 3 and C (sp2) 9 5, Table 1]. From the NMR evidences, compound 2 has one benzene and one olefinic group in the structure, needing two more rings to meet the need of the unsaturation. The above spectral characteristics of 2 were very similar to those of lycorine-type alkaloid (Tsuda et al. 1979) with one ring splitting. The HMBC spectrum revealed that a –CH2OCH3 fragment was connected on C-7a, with C-7 and N-6 splitting from the ring (Kitagawa et al. 1959). The 1H–1H–COSY spectrum of 2 revealed the presence of a CH–CH–CH (OH)–CH2–CH=C and a CH (OCH3)–CH (OH) fragments, respectively. The HMBC correlation of H-5 (dH 3.61) with 5-OCH3 (dC 58.1) suggested that the methoxy group was connected on C-5. Accordingly, the two hydroxy groups were connected on C-1 (dC 62.0) and C-4 (dC 69.1), respectively. These
substitutions were demonstrated by the 1H–1H–COSY and HMBC spectra (Fig. 3). From above analysis, the planar structure of 2 was deduced unambiguously to be the structure as shown in Fig. 3. In the NOESY spectrum (Fig. 3), the H-11c signal gave a correlation with H-4 and H-5, while showing no correlation with H-1.The relative configurations of H-11c and H-11b in 2 was assigned as aand b-orientations, respectively, which was always appeared in the lycorine-type alkaloids isolated from the genus Lycoris (Evidente et al. 1983). So H-1 was deduced as b-orientation, H-4 and H-5 was a-orientation. Thus, the structure of 2 was identified as (2R,3R,6R,7S,7aS)-7-(4,5dimethoxy-2-(methoxymethyl)phenyl)-2-methoxy-2,3,5,6,7, 7a-hexahydro-1H-indole-3,6-diol, named lycolongirine B. Compound 3, colorless needles, [a]20 D ? 26.68° (c 1.13, CH3OH). The HR-ESI–MS of 3 displayed a molecular ion at m/z 350.1152 [M]?, being consistent with a molecular formula of C18H21ClNO4? (calc. 350.1154) and corresponding to 9 degrees of unsaturation. Inspection of the 1H-NMR (Table 2) spectrum of 3 revealed the presence of 20 protons, including two singlets for the paralocated aromatic protons at dH 6.84 (H, s, H-10) and 6.76 (H, s, H-7), an olefinic proton at dH 5.90 (H, s, H-1), a
123
Y.-Y. Zhu et al. Table 5 Cell viabilities (%) of compounds in different concentrations in CoCl2 induced SH-SY5Y cells injuries Compound
265 lM
Test concentration (lM) 6.25
12.5
25
50
84.52 ± 0.21c
81.34 ± 0.55b
77.61 ± 0.61a
74.69 ± 0.57
74.10 ± 0.45
51.76 ± 0.50
39.57 ± 0.25
28.31 ± 0.21
54.44 ± 0.32
83.14 ± 0.49c
74.14 ± 0.68a
65.96 ± 0.55
70.82 ± 0.72
b
74.51 ± 0.55
73.84 ± 0.47
b
70.75 ± 0.61
68.03 ± 0.21
70.39 ± 0.42b
62.56 ± 0.91
37.73 ± 0.36
64.47 ± 0.53
80.58 ± 0.0c
76.41 ± 0.20a
72.54 ± 0.36
74.10 ± 0.45
74.26 ± 0.42 50.73 ± 0.76
49.81 ± 0.68 46.31 ± 0.86
9.50 ± 0.60 42.47 ± 0.67
64.47 ± 0.53 67.48 ± 0.98
70.76 ± 0.67c
81.12 ± 0.72c
49.32 ± 0.49
67.69 ± 1.16
58.90 ± 0.47
36.99 ± 0.46
59.71 ± 0.65
90.91 ± 0.86c
80.26 ± 1.23a
75.15 ± 0.50b
70.26 ± 0.42
68.39 ± 0.25
64.12 ± 0.12
63.25 ± 0.32
70.26 ± 0.42
48.21 ± 1.47
1
78.27 ± 0.21b
2
a
61.67 ± 0.66
54.86 ± 0.50
3
69.72 ± 0.40
71.68 ± 0.64 c
4
66.47 ± 1.00
83.14 ± 0.40
5
53.81 ± 0.76
62.68 ± 0.1
6
80.65 ± 0.21b
88.65 ± 0.42c a
c
7 8
59.56 ± 0.21 60.54 ± 0.65
68.35 ± 0.62 55.46 ± 0.52
9
55.98 ± 0.81a
65.78 ± 0.58b
66.91 ± 0.57b
c
a
10
66.39 ± 2.12
71.97 ± 0.50
11
71.86 ± 0.32
92.63 ± 087c
12
70.91 ± 0.31
82.59 ± 0.35
13
54.89 ± 0.60
51.12 ± 0.55
c
100
CoCl2
36.80 ± 0.45
34.93 ± 0.20
70.94 ± 0.15
b
14
65.18 ± 0.89
66.78 ± 0.58
73.31 ± 0.97
69.76 ± 1.67
62.12 ± 0.72
63.47 ± 1.49
15
66.50 ± 0.49
70.17 ± 1.22
86.15 ± 0.83b
70.90 ± 0.85
61.33 ± 0.65
70.49 ± 0.15
16
58.44 ± 0.42
91.46 ± 0.20c
73.94 ± 1.08a
70.63 ± 0.50a
63.55 ± 0.15
66.72 ± 0.40
17
61.11 ± 0.15
83.03 ± 0.21c
71.57 ± 0.40c
68.33 ± 0.60b
54.26 ± 0.32
59.71 ± 0.65
18
64.42 ± 0.42a
69.37 ± 0.12b
78.77 ± 0.21b
71.42 ± 0.67b
66.25 ± 0.30b
60.53 ± 0.58
19
59.75 ± 0.26
65.89 ± 0.21c
67.23 ± 0.31b
69.52 ± 0.25c
68.71 ± 0.21c
58.49 ± 0.10
20
63.31 ± 1.11
68.09 ± 0.21
b
c
78.90 ± 0.42
70.10 ± 0.56
b
a
65.10 ± 0.70
60.53 ± 0.58
21
58.25 ± 0.38
79.90 ± 0.26c
83.33 ± 0.42c
81.87 ± 0.21c
79.86 ± 0.23c
69.68 ± 0.35
22 Vitamin E
76.57 ± 0.76b
91.95 ± 0.61c
81.05 ± 0.91b
77.96 ± 0.26b
63.75 ± 1.28 78.69 ± 0.87b
70.82 ± 0.72 70.82 ± 0.72
In Tables 3, 4 and 5, 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 = 3) with a kind of statistical software PASW statistics 18.0, a for p \ 0.05, b for p \ 0.01, c for p \ 0.001
methylenedioxy group at dH 5.96 (2H, d, J = 0.9 Hz, H-13), a chloromethyl group at dH 5.61 and 5.59 (each 1H, d, J = 10.1 Hz). The 13C-NMR and DEPT spectra of 3 exhibited 18 carbon signals [CH3 9 1, CH2 (sp3) 9 5, CH (sp3) 9 4, CH (sp2) 9 3 and C (sp2) 9 5, Table 2]. The 1 H–1H–COSY and HMBC spectra of 3 (Fig. 4) revealed the presence of a –CH2CH(O)CH(O)– and a = CH– CH(O)– fragments. The above NMR data as well as the positive optical rotation of 3 are similar to those of the known compound (?) coccinine (Jin and Weinreb 1997) except that 3 has an extra chloromethyl group at dH 5.51, 5.59 (each 1H, d, J = 10.1 Hz) and dC 66.5. The HMBC (Fig. 4) correlations between the two choloromethyl protons and C-4a, C-6, C-12, as well as the deshielding chemical shifts of H-4a, H-6, H-12 in the 1H NMR and C-4a, C-6, C-12 in the 13C NMR suggested the location of the chloromethyl group at N atom (Jin and Weinreb 1997). The NOESY correlations of H-2/H-4a and H-3/H-4a suggested H-2, H-3 and H-4a to be the same a-orientation. No correlation between H-11 and H-4a proved 11-CH2 to be borientation (Pearson and Lian 1998) (Fig. 4). Finally, the
123
structure of 3 was confirmed to be (?)-N-methylchloride coccinine, given the name lycolongirine C. As galanthamine easily forms a quaternary ammonium salt ((-)-N-(choloromethyl) galanthaminium chloride) by reaction with the solvent CH2Cl2 (Matusch et al. 1994), which was also used in our separation process, compound 3 could also be an artificial product. The isolated alkaloids were tested for their neuroprotective effects against CoCl2, H2O2 and Ab25–35-induced neuronal cell death in dopaminergic neuroblastoma SHSY5Y cells. Compounds 4, 6, 11, 16, 18, 20 and 22 showed significant neuroprotective effects against all three cell injury models; compounds 1-3 and 12 exhibited significant neuroprotective activities against H2O2 (Table 3) and Ab25–35-induced cell death (Table 4); while compounds 13, 15 and 17 had similar activities against CoCl2induced cell damage (Table 5). The above results provide a preliminary indication that Amaryllidaceae alkaloids may have a big potential to neuroprotective activities against both hypoxic injury and oxidative damage in neuronal cells.
Alkaloids from the bulbs of Lycoris longituba Table 6 AChE inhibitory activity of the isolated compounds (n = 3) Compound
Percent inhibitory activity (%)
IC50 (lM)
1
38.59 % ± 3.27
241.50 ± 2.46
2
37.11 % ± 2.69
157.40 ± 3.51
3
45.05 % ± 2.46
117.60 ± 1.79
4
41.08 % ± 0.85
148.70 ± 1.46
5
45.44 % ± 1.33
164.10 ± 1.59
6
57.29 % ± 0.82
77.10 ± 1.65
7
34.76 % ± 2.80
181.00 ± 4.00
8
32.36 % ± 2.34
224.80 ± 3.01
9
42.36 % ± 2.02
194.80 ± 2.31
10
36.43 % ± 3.23
151.10 ± 2.42
11 12
33.00 % ± 0.91 84.91 % ± 0.59
190.70 ± 2.00 4.23 ± 1.13
13
38.96 % ± 0.78
208.10 ± 1.58
15
91.50 % ± 0.67
2.76 ± 0.65
16
93.51 % ± 0.87
5.55 ± 0.63
17
96.05 % ± 0.45
3.04 ± 0.61
18
92.63 % ± 0.15
5.30 ± 0.76
19
78.78 % ± 0.93
25.76 ± 1.09
20
85.63 % ± 0.61
8.13 ± 1.49 275.10 ± 1.86
21
32.22 % ± 1.58
22
89.44 % ± 0.38
8.44 ± 0.83
Galanthamine (14)
94.54 % ± 0.51
2.43 ± 0.66
IC50 value represents the concentration of compounds required to inhibit the 50 % activity of AChE. The percent inhibitory activity (%) represents inhibitory activity of compound in the concentration of 100 lM. ‘‘±’’ represents standard error of mean of these enzyme inhibition assays. Galanthamine was used as positive control
The isolates were also evaluated for their in vitro AChE inhibition according to a modified Ellman method (Eldeen et al. 2005) using galanthamine as a positive control. Compounds 12, 15-18, 20 and 22 were found to exhibit strong AChE inhibitory effects with IC50 values ranging from 2.76 to 8.44 lM (Table 6). The observed AChE inhibitory effects (Table 6) seem to be related to some structural characteristics among the different skeleton types. Only the alkaloids belonging to the galanthamine (compounds 15–20), the narciclasine (compound 12), and tazettine (compound 22) types exhibited significant AChE inhibitory activities. While the harmane-type alkaloid compound 6 (harmane) was also found to have moderate anti-AChE activity with the IC50 of 77.10 lM (Table 6). Our results indicated that except of the galantamine-type, the narciclasine and tazettine type alkaloids also have great potential of anti-AChE activity. Acknowledgments Financial supports 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 (No. 81072547, 31270394 and 30873361) are gratefully acknowledged. Conflict of interest All the authors have no conflict of interest to declare.
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