Fitoterapia 101 (2015) 188–193
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Amaryllidaceae alkaloids from the bulbs of Lycoris radiata with cytotoxic and anti-inflammatory activities Zhi-Ming Liu a,b, Xiao-Yun Huang c, Mao-Rong Cui b, Xiao-De Zhang b, Zhao Chen b, Ben-Shou Yang c, Xiao-Kun Zhao a,⁎ a b c
Second Xiangya Hospital, Central South University, No. 139 Middle Renmin Road, Changsha, Hunan 410011, China The First People Hospital of Qujing, Qujing 655000, Yunnan Province, China Qujing Medical College, Qujing 655000, Yunnan Province, China
a r t i c l e
i n f o
Article history: Received 24 November 2014 Accepted in revised form 2 January 2015 Accepted 6 January 2015 Available online 14 January 2015 Keywords: Lycoris radiata Amaryllidaceae Alkaloids Cytotoxicity Anti-inflammatory activity
a b s t r a c t Four new Amaryllidaceae alkaloids, (+)-1-hydroxy-ungeremine (1), (+)-6β-acetyl-8-hydroxy9-methoxy-crinamine (2), (+)-2-hydroxy-8-demethyl-homolycorine-α-N-oxide (3), (+)-Nmethoxylcarbonyl-2-demethyl-isocorydione (4), together with two known compounds, (+)6β-acetyl-crinamine (5) and 8-demethyl-homolycorine-α-N-oxide (6) were isolated from the ethanol extract of the bulbs of Lycoris radiata. Structural elucidation of all the compounds were performed by spectral methods such as 1D and 2D (1H–1H COSY, HMQC, and HMBC) NMR spectroscopy, in addition to high resolution mass spectrometry. All the isolated alkaloids were in vitro evaluated for their cytotoxic activities against eight tumor cell lines (BEN-MEN-1, CCFSTTG1, CHG-5, SHG-44, U251, BGC-823, HepG2 and SK-OV-3) and anti-inflammatory activities against Cox-1 and Cox-2. As a result, alkaloids 1 and 4 exhibited significant cytotoxic activities against all tested tumor cell lines except against BEN-MEN-1. Additionally, alkaloids 1 and 4 possessed selective inhibition of Cox-2 comparable with the standard drug NS-398 (N 90%). © 2015 Published by Elsevier B.V.
1. Introduction The genus Lycoris (Amaryllidaceae) is mainly distributed in the temperate wood lands of eastern Asia, particularly in China and Japan [1,2]. Lycoris contains various types of alkaloids with a wide range of biological activities [3–7]. Amaryllidaceae alkaloids affect the central nervous system and have acetylcholinesteraseinhibitory, analgesic, anti-inflammatory, antiviral, antimalarial, antitumor, or antineoplastic activity [8–14]. Lycoris radiata, a perennial monocot, is endemic in China, Japan and Korea [15]. It is commonly known as Shi Shuan and used in China as a traditional folk medicine, from which more than ten indole alkaloids have been isolated [16,17]. The previous phytochemical studies revealed that L. radiata ⁎ Corresponding author at: The First People Hospital of Qujing, Qujing 655000, Yunnan Province, China. Tel.: +86 731 85295888; fax: +86 731 85533525. E-mail address: [email protected] (Z.-M. Liu).
http://dx.doi.org/10.1016/j.fitote.2015.01.003 0367-326X/© 2015 Published by Elsevier B.V.
contained crinine, galanthamine, lycorine, homolycorine and montanine type alkaloids [18,19]. Amaryllidaceae alkaloids with diverse structural architectures are all biogenetically derived fromnorbelladine or its derivatives, which are produced in plants from aromatic aldehydes and tyramine [9]. The present studies on chemical constituents of the EtOH extract of L. radiata led to the isolation of four new Amaryllidaceae alkaloids, (+)-1-hydroxy-ungeremine (1), (+)-6β-acetyl-8-hydroxy-9-methoxy-crinamine (2), (+)-2hydroxy-8-demethyl-homolycorine-α-N-oxide (3), (+)-Nmethoxylcarbonyl-2-demethyl-isocorydione (4) as well as two known compounds, (+)-6β-acetyl-crinamine (5) and 8-demethyl-homolycorine-α-N-oxide (6) (Fig. 1). In this paper, we describe the isolation and structure elucidation on the basis of spectroscopic methods of the new compounds. Furthermore, all the alkaloids were evaluated in vitro for their cytotoxic and anti-inflammatory properties.
Z.-M. Liu et al. / Fitoterapia 101 (2015) 188–193
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Fig. 1. Structures of compounds 1–6.
2. Experimental 2.1. General Optical rotations were determined with a JASCO P2000 digital polarimeter (Tokyo, Japan). Ultraviolet (UV) and infrared (IR) spectra were obtained on JASCO V-650 and JASCO FT/IR4100 spectrophotometers (Tokyo, Japan), respectively. The NMR spectra were measured in CDCl3 on a Bruker AM-600 spectrometer (Fällanden, Switzerland). Chemical shifts were reported using residual CDCl3 (δH 7.26 and δC 77.0 ppm) and CD3OD (δH 3.30 and δC 49.0 ppm) as internal standard. High resolution ESI-MS spectra were obtained on a LTQ Orbitrap XL (Thermo Fisher Scientific, Waltham, MA, USA) spectrometer. Silica gel 60 (230–400 mesh, Merck, Darmstadt, Germany), LiChroprep RP-18 (Merck, 40–63 μm), and Sephadex LH-20 (Amersham Pharmacia Biotech, Roosendaal, The Netherlands) were used for column chromatography (CC). HPLC separation was performed on an instrument consisting of a Waters 600 controller, a Waters 600 pump, and a Waters 2487 dual λ absorbance detector, with a Prevail (250 × 10 mm i.d.) preparative column packed with C18 (5 μm). Precoated silica gel plates (Merck, Kieselgel 60 F254, 0.25 mm) and precoated RP-18 F254s plates (Merck) were used for analytical thin-layer chromatography analyses. 2.2. Plant material The bulbs of L. radiata were collected in May of 2013 from the suburb Lishui, a city of Zhejiang Province in China, and identified by one of the authors (B.S. Yang). A specimen (2013005) was deposited in the Herbarium of Shengyang Medicine College, Shengyang, China. 2.3. Extraction and isolation The dried bulbs of L. radiata (10 kg) were extracted with 80% ethanol three times under reflux for 15 h and then concentrated under reduced pressure to give a crude extract (117.2 g). The crude extract was partitioned between equal volumes of chloroform and water to provide a chloroformsoluble fraction (110.6 g) and an aqueous layer. The chloroformsoluble fraction was further fractionated through a silica gel column (200–300 mesh) using increasing volumes of acetone in petroleum ether (100:1, 50:1, 30:1, 15:1, 10:1, 7:1, 5:1, 3:1, 1:1, V/V) as eluents to give 12 fractions according to TLC analysis. Fraction 4 (2.3 g) was applied to an ODS MPLC column and eluted with MeOH–H2O to yield three subfractions
(Fr. 4-1 and 4-3). Fr. 4-2 (310 mg) was purified by preparative RP-HPLC (ODS column, 250 × 20 mm) using MeOH/H2O (25:75) as mobile phase to obtain 6 (57 mg). Fr. 4-3 (330 mg) was chromatographed on a Sephadex LH-20 column eluted with MeOH/H2O (50:50), and purified by preparative RP-HPLC (ODS column, 250 × 20 mm) using MeOH/H2O (34:66) as mobile phase to yield 3 (63 mg) and 5 (68 mg). Separation of fraction 5 (2.2 g) by silica gel column chromatography, eluted with petroleum ether-Me2CO (from 8:1 to 1:1), afforded four subfractions (Fr. 5-1 and Fr. 5-4). Fr. 5-2 (278 mg) was subjected to RP-18 (MeOH-H2O, from 2:8 to 6:4) and Sephadex LH-20 (MeOH) column chromatography to yield 4 (43 mg). Fr. 5-3 (303 mg) was repeatedly chromatographed on silica gel (chloroform–methanol gradient, from 20:1 to 10:1) and then purified on a Sephadex LH-20 column eluted with MeOH/H2O (50:50) to afford 2 (68 mg). Subfraction 6-3 (MeOH–H2O 20:80, 303 mg) was repeatedly chromatographed on silica gel (chloroform:methanol, 20:1 → 10:1) and then purified by a Sephadex LH-20 column eluted with MeOH/H2O (50:50) to afford 1 (68 mg). (+)-1-hydroxy-ungeremine (1): Yellow amorphous pow= +463.1 (c = 0.12, MeOH). UV (CDCl3) λmax(log ε): der. [a]23.3 D 375 (4.07), 308 (3.85), 254 (4.20), 212 (4.65) nm. IR (KBr) νmax 3413, 3352, 1644, 1607, 1592, 1505, 1277, 1038, and 925 cm−1. 1H NMR and 13C NMR data see Table 1. ESI-MS m/z: 282 ([M]+). HR-ESI-MS (pos.) m/z: 282.0763 ([M]+, C16H12NO+ 4. calc. 282.0761). (+)-6β-acetyl-8-hydroxy-9-methoxy-crinamine (2): Color= + 25.2 (c = 0.11, MeOH). UV (CDCl3) less oil. [a]23.3 D λmax(log ε): 292 (3.77), 239 (3.89) nm. IR (KBr) νmax 3385, 2895, 1713, 1486, 1250, 1053, and 930 cm− 1. 1H NMR and 13 C NMR data see Table 1. EI-MS m/z: 361 ([M]+). HR-ESI-MS (pos.) m/z: calc. 384.1422 ([M + Na]+, C19H23NO6Na. calc. 384.1423). (+)-2-hydroxy-8-demethyl-homolycorine-α-N-oxide (3): = +134.1 (c = 0.13, MeOH). UV (CDCl3) Colorless oil. [a]23.3 D λmax(log ε): 381 (2.73), 309 (3.558), 267 (3.75), 213 (4.55) nm. IR (KBr) νmax 2950, 1707, 1657, 1605, 1454, 1313, 1225, 1064, and 913 cm− 1. 1H NMR and 13C NMR data, see Table 1. EI-MS m/z: 333 ([M]+). HR-ESI-MS (pos.) m/z: calc. 356.1113 ([M + H]+, C17H19NO6Na. calc. 356.1110). (+)-N-methoxylcarbonyl-2-demethyl-isocorydione (4): violet = +65.2 (c = 0.16, MeOH); UV amorphous powder; [a]23.3 D (CDCl3) λmax(log ε) 305 (4.08), 285 (3.78), 216 (4.15) nm; IR (KBr) νmax 3030, 1711, 1654, 1453, and 1253 cm−1; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz) data see Table 1; EI-MS m/z 383 [M]+; HR-ESI-MS (pos.) m/z 406.0902 ([M + Na]+, C20H17NO7Na. calc. 406.0903).
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Table 1 1 H NMR data of compounds 1–4 in CDCl3 (δ in ppm and J in Hz). No.
1 1a 1b 2 3 3a 4 4a 5 6 6a 7 7a 8 9 10 10a 10b 11 11a 12 OCH2O N-CH3 1-OCH3 3-OCH3 9-OCH3 10-OCH3 CO2CH3 CO2CH3
δ 1H (Hz)
δ 13C
1
2
3
4
1
2
3
4
– – – – 7.48 (s) – – – – 8.85 (s) – 7.30 (s) – – – 7.19 (s) – – 2.66, 2.89 (m) – 4.24, 4.39 (m) 6.17 (br s) – – – – – – –
6.36 (d, 10.2) – – 6.41 (dd, 10.2, 5.0) 3.90 (m) – 2.00, 2.11 (m) 3.62 (dd, 13.8, 3.2) – 6.14 (br s) – 6.66 (s) – – – 6.84 (s) – – 3.94 (m) – 3.28, 3.45 (m) – – – 3.44 (s) 3.98 (s) – – 2.13 (s)
4.95 (d, 2.2) – – 4.28 (m) 5.79 (m) – – 4.14 (d, 10.2) – – – 7.16 (s) – – – 7.52 (s) – 3.63 (dd, 10.2, 2.1) 2.70 (m) – 3.51, 3.70 (m) – 2.97 (s) – – 3.96 (s) – – –
– – – – 6.59 (s) – 3.10 (t, 6.5)
141.8 – – 143.5 102.9 – 129.9 135.0 – 163.4 141.0 113.1 – 149.2 157.5 106.7 128.4 130.2 34.5 – 59.1 104.8 – – – – – – –
126.4 – – 132.7 72.4 – 27.6 58.2 – 88.2 125.0 112.4 – 146.5 147.1 109.0 127.0 49.9 78.3 – 58.8 101.2 – – 56.6 55.1 – 170.4 21.5
82.1 – – 67.2 118.6 – 139.3 67.0 – 166.8 117.9 121.3 – 143.3 157.0 114.1 135.3 39.7 26.5 – 71.0 – 56.2 – – 57.2 – – –
144.9 127.4 120.9 150.7 115.4 125.3 31.1 – 40.5 – 150.0 98.1 136.2 186.5 105.0 163.7 – – 178.2 117.5 – – – 62.2 – – 56.7 157.5 53.1
2.4. Cytotoxicity assay in vitro The cytotoxic activities of the isolated compounds were determined using the revised MTT method [20,21] against BEN-MEN-1 (meningioma), CCF-STTG1 (astrocytoma), CHG-5 (glioma), SHG-44 (glioma), U251 (glioma), HL-60 (human myeloid leukemia), SMMC-7721 (hepatocellular carcinoma), and W480 (colon cancer). Doxorubicin was used as the positive control. Cancer cells (4 × 103 cells) suspended in 100 μL/well of DMEM medium containing 10% fetal calf serum were seeded onto a 96-well culture plate. After 24 h pre-incubation at 37 °C in a humidified atmosphere of 5% CO2/95% air to allow cellular attachment, various concentrations of test solution were added and cells were incubated for 48 h under the above conditions. At the end of the incubation, 10 μL of tetrazolium reagent was added into each well followed by further incubation at 37 °C for 4 h. The supernatant was decanted, and DMSO (100 μL/well) was added to allow formosan solubilization. The concentrations of the assayed compounds were 0.04, 0.2, 1.0, 5, 25, and 125 μM, respectively. The optical density (OD) of each well was detected using a microplate reader at 550 nm and for correction at 595 nm. Each determination represented the average mean of six replicates. The 50% inhibition concentration (IC50 value) was determined by non-linear regression with GraphPad Prism software version 4.0 (GraphPad Software, Inc., San Diego, CA, USA) and was used as criteria to judge the cytotoxicity. All the IC50 results represent an average of a minimum of three experiments and were expressed as means ± standard deviation (SD). All cell lines were purchased from the Cell Bank of the Shanghai Institute of Biochemistry & Cell Biology,
3.45 (t, 6.5) – – 6.95 (s) – – 5.91 (s) – – – – – – – – 3.56 (s) – – 3.69 (s) – 3.72 (s)
Chinese Academy of Sciences (Shanghai, China). Other reagents were purchased from Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China). 2.5. Anti-inflammatory assay in vitro The anti-inflammatory activities were determined according to a literature method with minor modifications [22]. The reaction system was incubated at 25 °C for 5 min, by sequential addition of the buffer, heme, test compounds, and Cox-1 or Cox-2 into the system followed by mixing with TMPD and arachidonic acid. The absorbance value was recorded at a wavelength of 590 nm after another 15 min of incubation at 25 °C. SC-560 and NS-398 were used as positive controls, which gave the inhibition of Cox-1 (63.20%) and Cox-2 (97.13%) respectively (Table 3). All cell lines were purchased from the Cell Bank of Shanghai Institute of Biochemistry & Cell Biology, Chinese Academy of Sciences (Shanghai, China). 3. Results and discussion Compound 1 was obtained as a yellow amorphous powder. The HR-ESI-MS displayed a pseudomolecular ion at m/z 282.0763 [M]+ (calcd for C16H12NO+ 4 , 282.0761) consistent with a molecular formula of C16H12NO+ 4 , corresponding to 12° of unsaturation. Its UV absorption at λmax 375, 308, 254, and 212 nm showed an extended chromophore and a methylenedioxyl substituted benzene ring. The IR absorption bands at 3413, 3352, 1644, 1607 and 925 cm−1 indicated OH groups and phenyl functions. The 1H NMR spectrum of 1 exhibited one aromatic singlet protons at δH
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7.48 (H-3), two singlets for two para-located aromatic protons at δH 7.30 (H-7) and 7.19 (H-10), a methylenedioxy signal at δH 6.17 and a downfield singlet corresponding to the proton of an iminium salt (δH 8.85) [23]. The 13C NMR spectrum showed 16 carbon signals [OCH 2O × 1, CH2 (sp3) × 2, CH (sp2) × 4 and C (sp2) × 9, Table 1]. Its NMR data (Table 1) were similar to those of ungeremine, which revealed that 1 possessed an Amaryllidaceae alkaloid with an imine moiety located between N-5 and C-6 (δC 163.4) [9]. The imine moiety between N-5 and C-6 (δC 163.2) in 1 was supported by HMBCs of δH 8.85 (H-6) with δC 135.0 (C-4a), 113.1 (C-7), 128.4 (C-10a) and 59.1 (C-12) (Fig. 2). Compared of NMR data of compounds 1 and ungeremine, the only significant difference was the presence of one more hydroxyl group at C-1 in alkaloid 1, which was confirmed by HMBC correlations from H-3 (δH 7.48, s) to C-1 (δC 141.8). Therefore, compound 1 was identified as (+)-1-hydroxy-ungeremine. Compound 2 was obtained as a colorless oil. The EI-MS afforded a quasimolecular ion peak at m/z 361, and its HR-ESIMS revealed the [M + Na]+ peak at m/z 384.1422 (calcd. for C19H23NO6Na. 384.1423) consistent with a molecular formula of C19H23NO6, corresponding to 9° of unsaturation. The IR absorption bands at 3385 and 1713 cm− 1 are ascribable to the OH group and the ester C_O group respectively. The 1H NMR spectrum exhibited two singlets at δH 6.66 (s) and 6.84 (s) assigned to two para-positioned aromatic protons, two olefinic signals at δH 6.36 and 6.41 assigned to the H-1 and H-2, and one singlets at δH 2.13 (s) ascribed to the Me of the acetoxyl group. The 13C NMR spectrum displayed 19 carbon resonances including a phenyl (δC 109.0, 112.4, 125.0, 127.0, 146.5 and 147.1), one Ac (δC 21.5 and 170.4), two methoxy (δC 55.1 and 56.6), three oxygenated methins (δC 72.4, 78.3 and 88.2), two olefinic carbons (δC 126.4 and 132.7), two CH2 (sp3), one CH (sp3) and a quaternary carbon (sp3) (Table 1). The above data resembled those of (+)-6β-acetylcrinamine (5) which was isolated from the same plant [24]. The significant difference was that the methylenedioxy between C-8 and C-9 in 4 was replaced by a hydroxyl at C-8 and a methoxy group at C-9 in 2, which was confirmed by the HMBC correlations of the methoxy group (δH 3.98) and H-7 (δH 6.66) with C-9 (δC 147.1). Another methoxy group was positioned at C-3 by the HMBC correlations of the signal at δH 3.44 with C-3 (δC 72.4). The ROESY correlations of H-4α/H-3 and H-11/H-4α suggested both H-3 and H-11 to be β-orientation, and the ROESY correlation of H-6/H-12α indicated α-orientation for H-6 (Fig. 2). Accordingly, the structure of 2 was established as (+)-6β-acetyl-8-hydroxy9-methoxy-crinamine.
Fig. 2. Key HMBC (
) and ROESY (
191
Compound 3 was obtained as a colorless oil. Its positive HR-ESI-MS spectrum showed a quasimolecular ion peak at m/z 356.1113 [M + H]+, consistent with the molecular formula C17H19NO6, accounting for 9° of unsaturation. The IR absorption bands at 1707 and 1657 cm−1 indicated the existence of ketones, while the UV absorption bands at 267 and 213 nm suggested a conjugated moiety. The lH NMR spectrum showed singlet signals for two aryl protons (δH 7.16 and 7.52), one olefinic proton (δH 5.79), one methoxy (δH 3.96), two vicinal methylenes [δH 2.70 (H-11) and 3.51, 3.70 (H-12)], and one oxygenated methane (δH 4.28). The 13C NMR spectrum displayed one O-CH3, one N-CH3, two CH2 and seven CH groups (including four sp3 carbons and three sp2 carbons), and six sp2 quaternary carbons. The NMR data were almost identical with those of 8-demethyl-homolycorine-α-N-oxide (6) which was isolated from the same source. The characteristic downfield signals of the carbon resonances for C-4a (δC 67.0), C-12 (δC 71.0), and N-CH3 (δC 56.2) indicated that 5 was a derivative of homolycorine-α-N-oxide [9]. Compared with 9-demethoxyhomolycorine-α-N-oxide, the significant difference was the presence of one more hydroxyl group at C-2 in alkaloid 3, which was confirmed by HMBC correlations from H-2 (δH 4.28, s) to C-4 (δC 139.3) and C-10b (δC 39.7) together with the downfield shift of signals for C-2 to δC 67.2. The methoxyl was positioned at C-9, and the methyl at N-5, respectively, based on the HMBC correlations of the proton signal of methyoxy (δH 3.96) with C-9 (δC 157.0), and of the methyl signal (δH 2.97) with C-4a and C-12, respectively (Fig. 2). The ROESY correlations of N-CH3/H-4a and H-2/H-4a suggested α-orientation of the N-oxide and the hydroxyl group at C-2. Thus, the structure of 3 was assigned the name (+)-2hydroxy-8-demethyl-homolycorine-α-N-oxide. Compound 4 was obtained as a violet amorphous powder. Its positive HR-ESI-MS spectrum showed a quasimolecular ion peak at m/z 406.0902 [M + Na]+, consistent with the molecular formula C20H17NO7 (calcd for C20H17NO7Na 406.0903), accounting for 13° of unsaturation. Its UV absorption at λmax 305, 285, and 216 nm suggested an aporphine alkaloid skeleton [25]. Its 13C NMR spectrum showed 20 carbon signals [COOCH3 × 1, OCH3 × 2, CH2 (sp3) × 2, CH (sp2) × 3 and C (sp2) × 11, Table 1]. The lH NMR spectrum showed singlet signals for three aryl protons (δH 6.95, 6.59, and 5.91), two OMe (δH 3.69 and 3.56), one N-COOCH3 (δH 3.72), and two triplets for two vicinal coupled methylenes [δH 3.10 (2H, t, J = 6.5, H-4) and 3.45 (2H, t, J = 6.5, H-5)]. The IR spectrum exhibited a conjugated carbonyl absorption (1654 cm− 1), and the 13C NMR spectrum displayed two carbonyl signals at δC 178.2 and 186.5. These spectral data were similar to
) correlations of compounds 1–4.
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Table 2 Cytotoxicity of compounds 1–6 against seven human tumor cell lines (IC50, μM)a. Cell lines
1 2 3 4 5 6 Doxorubicin
BEN-MEN-1
CCF-STTG1
CHG-5
SHG-44
U251
– – – – – – 0.018
10.3 ± 0.9 29.4 ± 4.1 83.2 ± 13.7 9.2 ± 1.3 27.1 ± 5.1 – 0.025
10.2 ± 1.6 29.4 ± 5.3 – 9.7 ± 0.9 30.0 ± 4.4 93.0 ± 21.1 0.022
9.4 ± 1.3 27.1 ± 3.2 – 10.8 ± 1.4 28.3 ± 2.7 – 0.033
11.8 ± 17.4 ± – 11.3 ± 15.8 ± – 0.028
0.7 2.1 1.6 1.7
HL-60
SMMC-7721
W480
10.8 ± 1.6 8.6 ± 1.4 – 12.8 ± 1.6 7.3 ± 1.1 ± 0.9 –
10.5 ± 68.2 ± 86.2 ± 10.0 ± 63.2 ± 85.0 ± 0.038
11.6 ± 1.1 53.5 ± 12.4 – 9.4 ± 0.9 51.1 ± 10.9 – 14.1
0.9 12.3 17.4 0.7 11.8 16.2
(–) IC50 N 100 μM.
1,4-dicarbonyl signals in N-norbulbodione [25]. The HMBC correlations (Fig. 2) of δH 3.72 (3H, s, OMe) and 3.45 (2H, t, J = 6.5, H-5) with δC 157.5 positioned the carbamate group at N-6. The positions of two methoxys were assigned based on HMBC spectrum (Fig. 2). The HMBC of OCH3 (δH 3.56) with C-1 (δC 144.9) and OCH3 (δH 3.69) with C-10 (δC 163.7) positioned two methoxys at C-1 and C-10 respectively (Fig. 2), which was further supported by the ROESY correlations of H-9 (δH 5.91) with the signal of the OCH3 (δH 3.69). Therefore, compound 4 was identified as (+)-N-methoxylcarbonyl-2demethyl-isocorydione. All these compounds were in vitro evaluated for their cytotoxic potential against eight human tumor cell lines, BEN-MEN-1 (meningioma), CCF-STTG1 (astrocytoma), CHG-5 (glioma), SHG-44 (glioma), U251 (glioma), HL-60 (human myeloid leukemia), SMMC-7721 (hepatocellular carcinoma), and W480 (colon cancer) using the modified MTT method. The in vitro cytotoxic activities of these compounds against human cell lines are summarized in Table 2. Among the tested compounds, lycorine-type alkaloid 1 and aporphine-type alkaloid 4 exhibited the most potent cytotoxic potential against all tested tumor cell lines, with IC50 values of 9.4–11.8 and 9.2–12.8 μM, except against BEN-MEN-1. Crinine-type alkaloids 2 and 5 showed significant cytotoxicities against HL-60 (IC50 b 10 μM), and moderate cytotoxicities against astrocytoma and glioma cell lines, CCF-STTG1, CHG-5, SHG44 and U251 (10 μM b IC50 ≤ 30 μM). Homolycorine-type alkaloids 3 and 6 had no cytotoxic activities (IC50 N 80 μM). The compounds 1–6 were tested in vitro for their antiinflammatory activities. The results of the anti-inflammatory assay were summarized in Table 3. Among the assayed compounds, only alkaloids 1 and 4 displayed selective inhibition of Cox-2 (N90%).
Table 3 Evaluation of anti-inflammatory activity of compounds 1–6a.
1 2 3 4 5 6 SC-560 NS-398 a
COX-1
COX-2
36.7 12.9 b0 45.5 12.9 13.4 63.2
96.5 27.6 22.3 93.2 27.6 45.3 97.1
Percent inhibition (all compounds and reference drugs concentration: 100 μM).
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Fitoterapia journal homepage: www.elsevier.com/locate/fitote
Amaryllidaceae alkaloids from the bulbs of Lycoris radiata with cytotoxic and anti-inflammatory activities Zhi-Ming Liu a,b, Xiao-Yun Huang c, Mao-Rong Cui b, Xiao-De Zhang b, Zhao Chen b, Ben-Shou Yang c, Xiao-Kun Zhao a,⁎ a b c
Second Xiangya Hospital, Central South University, No. 139 Middle Renmin Road, Changsha, Hunan 410011, China The First People Hospital of Qujing, Qujing 655000, Yunnan Province, China Qujing Medical College, Qujing 655000, Yunnan Province, China
a r t i c l e
i n f o
Article history: Received 24 November 2014 Accepted in revised form 2 January 2015 Accepted 6 January 2015 Available online 14 January 2015 Keywords: Lycoris radiata Amaryllidaceae Alkaloids Cytotoxicity Anti-inflammatory activity
a b s t r a c t Four new Amaryllidaceae alkaloids, (+)-1-hydroxy-ungeremine (1), (+)-6β-acetyl-8-hydroxy9-methoxy-crinamine (2), (+)-2-hydroxy-8-demethyl-homolycorine-α-N-oxide (3), (+)-Nmethoxylcarbonyl-2-demethyl-isocorydione (4), together with two known compounds, (+)6β-acetyl-crinamine (5) and 8-demethyl-homolycorine-α-N-oxide (6) were isolated from the ethanol extract of the bulbs of Lycoris radiata. Structural elucidation of all the compounds were performed by spectral methods such as 1D and 2D (1H–1H COSY, HMQC, and HMBC) NMR spectroscopy, in addition to high resolution mass spectrometry. All the isolated alkaloids were in vitro evaluated for their cytotoxic activities against eight tumor cell lines (BEN-MEN-1, CCFSTTG1, CHG-5, SHG-44, U251, BGC-823, HepG2 and SK-OV-3) and anti-inflammatory activities against Cox-1 and Cox-2. As a result, alkaloids 1 and 4 exhibited significant cytotoxic activities against all tested tumor cell lines except against BEN-MEN-1. Additionally, alkaloids 1 and 4 possessed selective inhibition of Cox-2 comparable with the standard drug NS-398 (N 90%). © 2015 Published by Elsevier B.V.
1. Introduction The genus Lycoris (Amaryllidaceae) is mainly distributed in the temperate wood lands of eastern Asia, particularly in China and Japan [1,2]. Lycoris contains various types of alkaloids with a wide range of biological activities [3–7]. Amaryllidaceae alkaloids affect the central nervous system and have acetylcholinesteraseinhibitory, analgesic, anti-inflammatory, antiviral, antimalarial, antitumor, or antineoplastic activity [8–14]. Lycoris radiata, a perennial monocot, is endemic in China, Japan and Korea [15]. It is commonly known as Shi Shuan and used in China as a traditional folk medicine, from which more than ten indole alkaloids have been isolated [16,17]. The previous phytochemical studies revealed that L. radiata ⁎ Corresponding author at: The First People Hospital of Qujing, Qujing 655000, Yunnan Province, China. Tel.: +86 731 85295888; fax: +86 731 85533525. E-mail address: [email protected] (Z.-M. Liu).
http://dx.doi.org/10.1016/j.fitote.2015.01.003 0367-326X/© 2015 Published by Elsevier B.V.
contained crinine, galanthamine, lycorine, homolycorine and montanine type alkaloids [18,19]. Amaryllidaceae alkaloids with diverse structural architectures are all biogenetically derived fromnorbelladine or its derivatives, which are produced in plants from aromatic aldehydes and tyramine [9]. The present studies on chemical constituents of the EtOH extract of L. radiata led to the isolation of four new Amaryllidaceae alkaloids, (+)-1-hydroxy-ungeremine (1), (+)-6β-acetyl-8-hydroxy-9-methoxy-crinamine (2), (+)-2hydroxy-8-demethyl-homolycorine-α-N-oxide (3), (+)-Nmethoxylcarbonyl-2-demethyl-isocorydione (4) as well as two known compounds, (+)-6β-acetyl-crinamine (5) and 8-demethyl-homolycorine-α-N-oxide (6) (Fig. 1). In this paper, we describe the isolation and structure elucidation on the basis of spectroscopic methods of the new compounds. Furthermore, all the alkaloids were evaluated in vitro for their cytotoxic and anti-inflammatory properties.
Z.-M. Liu et al. / Fitoterapia 101 (2015) 188–193
189
Fig. 1. Structures of compounds 1–6.
2. Experimental 2.1. General Optical rotations were determined with a JASCO P2000 digital polarimeter (Tokyo, Japan). Ultraviolet (UV) and infrared (IR) spectra were obtained on JASCO V-650 and JASCO FT/IR4100 spectrophotometers (Tokyo, Japan), respectively. The NMR spectra were measured in CDCl3 on a Bruker AM-600 spectrometer (Fällanden, Switzerland). Chemical shifts were reported using residual CDCl3 (δH 7.26 and δC 77.0 ppm) and CD3OD (δH 3.30 and δC 49.0 ppm) as internal standard. High resolution ESI-MS spectra were obtained on a LTQ Orbitrap XL (Thermo Fisher Scientific, Waltham, MA, USA) spectrometer. Silica gel 60 (230–400 mesh, Merck, Darmstadt, Germany), LiChroprep RP-18 (Merck, 40–63 μm), and Sephadex LH-20 (Amersham Pharmacia Biotech, Roosendaal, The Netherlands) were used for column chromatography (CC). HPLC separation was performed on an instrument consisting of a Waters 600 controller, a Waters 600 pump, and a Waters 2487 dual λ absorbance detector, with a Prevail (250 × 10 mm i.d.) preparative column packed with C18 (5 μm). Precoated silica gel plates (Merck, Kieselgel 60 F254, 0.25 mm) and precoated RP-18 F254s plates (Merck) were used for analytical thin-layer chromatography analyses. 2.2. Plant material The bulbs of L. radiata were collected in May of 2013 from the suburb Lishui, a city of Zhejiang Province in China, and identified by one of the authors (B.S. Yang). A specimen (2013005) was deposited in the Herbarium of Shengyang Medicine College, Shengyang, China. 2.3. Extraction and isolation The dried bulbs of L. radiata (10 kg) were extracted with 80% ethanol three times under reflux for 15 h and then concentrated under reduced pressure to give a crude extract (117.2 g). The crude extract was partitioned between equal volumes of chloroform and water to provide a chloroformsoluble fraction (110.6 g) and an aqueous layer. The chloroformsoluble fraction was further fractionated through a silica gel column (200–300 mesh) using increasing volumes of acetone in petroleum ether (100:1, 50:1, 30:1, 15:1, 10:1, 7:1, 5:1, 3:1, 1:1, V/V) as eluents to give 12 fractions according to TLC analysis. Fraction 4 (2.3 g) was applied to an ODS MPLC column and eluted with MeOH–H2O to yield three subfractions
(Fr. 4-1 and 4-3). Fr. 4-2 (310 mg) was purified by preparative RP-HPLC (ODS column, 250 × 20 mm) using MeOH/H2O (25:75) as mobile phase to obtain 6 (57 mg). Fr. 4-3 (330 mg) was chromatographed on a Sephadex LH-20 column eluted with MeOH/H2O (50:50), and purified by preparative RP-HPLC (ODS column, 250 × 20 mm) using MeOH/H2O (34:66) as mobile phase to yield 3 (63 mg) and 5 (68 mg). Separation of fraction 5 (2.2 g) by silica gel column chromatography, eluted with petroleum ether-Me2CO (from 8:1 to 1:1), afforded four subfractions (Fr. 5-1 and Fr. 5-4). Fr. 5-2 (278 mg) was subjected to RP-18 (MeOH-H2O, from 2:8 to 6:4) and Sephadex LH-20 (MeOH) column chromatography to yield 4 (43 mg). Fr. 5-3 (303 mg) was repeatedly chromatographed on silica gel (chloroform–methanol gradient, from 20:1 to 10:1) and then purified on a Sephadex LH-20 column eluted with MeOH/H2O (50:50) to afford 2 (68 mg). Subfraction 6-3 (MeOH–H2O 20:80, 303 mg) was repeatedly chromatographed on silica gel (chloroform:methanol, 20:1 → 10:1) and then purified by a Sephadex LH-20 column eluted with MeOH/H2O (50:50) to afford 1 (68 mg). (+)-1-hydroxy-ungeremine (1): Yellow amorphous pow= +463.1 (c = 0.12, MeOH). UV (CDCl3) λmax(log ε): der. [a]23.3 D 375 (4.07), 308 (3.85), 254 (4.20), 212 (4.65) nm. IR (KBr) νmax 3413, 3352, 1644, 1607, 1592, 1505, 1277, 1038, and 925 cm−1. 1H NMR and 13C NMR data see Table 1. ESI-MS m/z: 282 ([M]+). HR-ESI-MS (pos.) m/z: 282.0763 ([M]+, C16H12NO+ 4. calc. 282.0761). (+)-6β-acetyl-8-hydroxy-9-methoxy-crinamine (2): Color= + 25.2 (c = 0.11, MeOH). UV (CDCl3) less oil. [a]23.3 D λmax(log ε): 292 (3.77), 239 (3.89) nm. IR (KBr) νmax 3385, 2895, 1713, 1486, 1250, 1053, and 930 cm− 1. 1H NMR and 13 C NMR data see Table 1. EI-MS m/z: 361 ([M]+). HR-ESI-MS (pos.) m/z: calc. 384.1422 ([M + Na]+, C19H23NO6Na. calc. 384.1423). (+)-2-hydroxy-8-demethyl-homolycorine-α-N-oxide (3): = +134.1 (c = 0.13, MeOH). UV (CDCl3) Colorless oil. [a]23.3 D λmax(log ε): 381 (2.73), 309 (3.558), 267 (3.75), 213 (4.55) nm. IR (KBr) νmax 2950, 1707, 1657, 1605, 1454, 1313, 1225, 1064, and 913 cm− 1. 1H NMR and 13C NMR data, see Table 1. EI-MS m/z: 333 ([M]+). HR-ESI-MS (pos.) m/z: calc. 356.1113 ([M + H]+, C17H19NO6Na. calc. 356.1110). (+)-N-methoxylcarbonyl-2-demethyl-isocorydione (4): violet = +65.2 (c = 0.16, MeOH); UV amorphous powder; [a]23.3 D (CDCl3) λmax(log ε) 305 (4.08), 285 (3.78), 216 (4.15) nm; IR (KBr) νmax 3030, 1711, 1654, 1453, and 1253 cm−1; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz) data see Table 1; EI-MS m/z 383 [M]+; HR-ESI-MS (pos.) m/z 406.0902 ([M + Na]+, C20H17NO7Na. calc. 406.0903).
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Table 1 1 H NMR data of compounds 1–4 in CDCl3 (δ in ppm and J in Hz). No.
1 1a 1b 2 3 3a 4 4a 5 6 6a 7 7a 8 9 10 10a 10b 11 11a 12 OCH2O N-CH3 1-OCH3 3-OCH3 9-OCH3 10-OCH3 CO2CH3 CO2CH3
δ 1H (Hz)
δ 13C
1
2
3
4
1
2
3
4
– – – – 7.48 (s) – – – – 8.85 (s) – 7.30 (s) – – – 7.19 (s) – – 2.66, 2.89 (m) – 4.24, 4.39 (m) 6.17 (br s) – – – – – – –
6.36 (d, 10.2) – – 6.41 (dd, 10.2, 5.0) 3.90 (m) – 2.00, 2.11 (m) 3.62 (dd, 13.8, 3.2) – 6.14 (br s) – 6.66 (s) – – – 6.84 (s) – – 3.94 (m) – 3.28, 3.45 (m) – – – 3.44 (s) 3.98 (s) – – 2.13 (s)
4.95 (d, 2.2) – – 4.28 (m) 5.79 (m) – – 4.14 (d, 10.2) – – – 7.16 (s) – – – 7.52 (s) – 3.63 (dd, 10.2, 2.1) 2.70 (m) – 3.51, 3.70 (m) – 2.97 (s) – – 3.96 (s) – – –
– – – – 6.59 (s) – 3.10 (t, 6.5)
141.8 – – 143.5 102.9 – 129.9 135.0 – 163.4 141.0 113.1 – 149.2 157.5 106.7 128.4 130.2 34.5 – 59.1 104.8 – – – – – – –
126.4 – – 132.7 72.4 – 27.6 58.2 – 88.2 125.0 112.4 – 146.5 147.1 109.0 127.0 49.9 78.3 – 58.8 101.2 – – 56.6 55.1 – 170.4 21.5
82.1 – – 67.2 118.6 – 139.3 67.0 – 166.8 117.9 121.3 – 143.3 157.0 114.1 135.3 39.7 26.5 – 71.0 – 56.2 – – 57.2 – – –
144.9 127.4 120.9 150.7 115.4 125.3 31.1 – 40.5 – 150.0 98.1 136.2 186.5 105.0 163.7 – – 178.2 117.5 – – – 62.2 – – 56.7 157.5 53.1
2.4. Cytotoxicity assay in vitro The cytotoxic activities of the isolated compounds were determined using the revised MTT method [20,21] against BEN-MEN-1 (meningioma), CCF-STTG1 (astrocytoma), CHG-5 (glioma), SHG-44 (glioma), U251 (glioma), HL-60 (human myeloid leukemia), SMMC-7721 (hepatocellular carcinoma), and W480 (colon cancer). Doxorubicin was used as the positive control. Cancer cells (4 × 103 cells) suspended in 100 μL/well of DMEM medium containing 10% fetal calf serum were seeded onto a 96-well culture plate. After 24 h pre-incubation at 37 °C in a humidified atmosphere of 5% CO2/95% air to allow cellular attachment, various concentrations of test solution were added and cells were incubated for 48 h under the above conditions. At the end of the incubation, 10 μL of tetrazolium reagent was added into each well followed by further incubation at 37 °C for 4 h. The supernatant was decanted, and DMSO (100 μL/well) was added to allow formosan solubilization. The concentrations of the assayed compounds were 0.04, 0.2, 1.0, 5, 25, and 125 μM, respectively. The optical density (OD) of each well was detected using a microplate reader at 550 nm and for correction at 595 nm. Each determination represented the average mean of six replicates. The 50% inhibition concentration (IC50 value) was determined by non-linear regression with GraphPad Prism software version 4.0 (GraphPad Software, Inc., San Diego, CA, USA) and was used as criteria to judge the cytotoxicity. All the IC50 results represent an average of a minimum of three experiments and were expressed as means ± standard deviation (SD). All cell lines were purchased from the Cell Bank of the Shanghai Institute of Biochemistry & Cell Biology,
3.45 (t, 6.5) – – 6.95 (s) – – 5.91 (s) – – – – – – – – 3.56 (s) – – 3.69 (s) – 3.72 (s)
Chinese Academy of Sciences (Shanghai, China). Other reagents were purchased from Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China). 2.5. Anti-inflammatory assay in vitro The anti-inflammatory activities were determined according to a literature method with minor modifications [22]. The reaction system was incubated at 25 °C for 5 min, by sequential addition of the buffer, heme, test compounds, and Cox-1 or Cox-2 into the system followed by mixing with TMPD and arachidonic acid. The absorbance value was recorded at a wavelength of 590 nm after another 15 min of incubation at 25 °C. SC-560 and NS-398 were used as positive controls, which gave the inhibition of Cox-1 (63.20%) and Cox-2 (97.13%) respectively (Table 3). All cell lines were purchased from the Cell Bank of Shanghai Institute of Biochemistry & Cell Biology, Chinese Academy of Sciences (Shanghai, China). 3. Results and discussion Compound 1 was obtained as a yellow amorphous powder. The HR-ESI-MS displayed a pseudomolecular ion at m/z 282.0763 [M]+ (calcd for C16H12NO+ 4 , 282.0761) consistent with a molecular formula of C16H12NO+ 4 , corresponding to 12° of unsaturation. Its UV absorption at λmax 375, 308, 254, and 212 nm showed an extended chromophore and a methylenedioxyl substituted benzene ring. The IR absorption bands at 3413, 3352, 1644, 1607 and 925 cm−1 indicated OH groups and phenyl functions. The 1H NMR spectrum of 1 exhibited one aromatic singlet protons at δH
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7.48 (H-3), two singlets for two para-located aromatic protons at δH 7.30 (H-7) and 7.19 (H-10), a methylenedioxy signal at δH 6.17 and a downfield singlet corresponding to the proton of an iminium salt (δH 8.85) [23]. The 13C NMR spectrum showed 16 carbon signals [OCH 2O × 1, CH2 (sp3) × 2, CH (sp2) × 4 and C (sp2) × 9, Table 1]. Its NMR data (Table 1) were similar to those of ungeremine, which revealed that 1 possessed an Amaryllidaceae alkaloid with an imine moiety located between N-5 and C-6 (δC 163.4) [9]. The imine moiety between N-5 and C-6 (δC 163.2) in 1 was supported by HMBCs of δH 8.85 (H-6) with δC 135.0 (C-4a), 113.1 (C-7), 128.4 (C-10a) and 59.1 (C-12) (Fig. 2). Compared of NMR data of compounds 1 and ungeremine, the only significant difference was the presence of one more hydroxyl group at C-1 in alkaloid 1, which was confirmed by HMBC correlations from H-3 (δH 7.48, s) to C-1 (δC 141.8). Therefore, compound 1 was identified as (+)-1-hydroxy-ungeremine. Compound 2 was obtained as a colorless oil. The EI-MS afforded a quasimolecular ion peak at m/z 361, and its HR-ESIMS revealed the [M + Na]+ peak at m/z 384.1422 (calcd. for C19H23NO6Na. 384.1423) consistent with a molecular formula of C19H23NO6, corresponding to 9° of unsaturation. The IR absorption bands at 3385 and 1713 cm− 1 are ascribable to the OH group and the ester C_O group respectively. The 1H NMR spectrum exhibited two singlets at δH 6.66 (s) and 6.84 (s) assigned to two para-positioned aromatic protons, two olefinic signals at δH 6.36 and 6.41 assigned to the H-1 and H-2, and one singlets at δH 2.13 (s) ascribed to the Me of the acetoxyl group. The 13C NMR spectrum displayed 19 carbon resonances including a phenyl (δC 109.0, 112.4, 125.0, 127.0, 146.5 and 147.1), one Ac (δC 21.5 and 170.4), two methoxy (δC 55.1 and 56.6), three oxygenated methins (δC 72.4, 78.3 and 88.2), two olefinic carbons (δC 126.4 and 132.7), two CH2 (sp3), one CH (sp3) and a quaternary carbon (sp3) (Table 1). The above data resembled those of (+)-6β-acetylcrinamine (5) which was isolated from the same plant [24]. The significant difference was that the methylenedioxy between C-8 and C-9 in 4 was replaced by a hydroxyl at C-8 and a methoxy group at C-9 in 2, which was confirmed by the HMBC correlations of the methoxy group (δH 3.98) and H-7 (δH 6.66) with C-9 (δC 147.1). Another methoxy group was positioned at C-3 by the HMBC correlations of the signal at δH 3.44 with C-3 (δC 72.4). The ROESY correlations of H-4α/H-3 and H-11/H-4α suggested both H-3 and H-11 to be β-orientation, and the ROESY correlation of H-6/H-12α indicated α-orientation for H-6 (Fig. 2). Accordingly, the structure of 2 was established as (+)-6β-acetyl-8-hydroxy9-methoxy-crinamine.
Fig. 2. Key HMBC (
) and ROESY (
191
Compound 3 was obtained as a colorless oil. Its positive HR-ESI-MS spectrum showed a quasimolecular ion peak at m/z 356.1113 [M + H]+, consistent with the molecular formula C17H19NO6, accounting for 9° of unsaturation. The IR absorption bands at 1707 and 1657 cm−1 indicated the existence of ketones, while the UV absorption bands at 267 and 213 nm suggested a conjugated moiety. The lH NMR spectrum showed singlet signals for two aryl protons (δH 7.16 and 7.52), one olefinic proton (δH 5.79), one methoxy (δH 3.96), two vicinal methylenes [δH 2.70 (H-11) and 3.51, 3.70 (H-12)], and one oxygenated methane (δH 4.28). The 13C NMR spectrum displayed one O-CH3, one N-CH3, two CH2 and seven CH groups (including four sp3 carbons and three sp2 carbons), and six sp2 quaternary carbons. The NMR data were almost identical with those of 8-demethyl-homolycorine-α-N-oxide (6) which was isolated from the same source. The characteristic downfield signals of the carbon resonances for C-4a (δC 67.0), C-12 (δC 71.0), and N-CH3 (δC 56.2) indicated that 5 was a derivative of homolycorine-α-N-oxide [9]. Compared with 9-demethoxyhomolycorine-α-N-oxide, the significant difference was the presence of one more hydroxyl group at C-2 in alkaloid 3, which was confirmed by HMBC correlations from H-2 (δH 4.28, s) to C-4 (δC 139.3) and C-10b (δC 39.7) together with the downfield shift of signals for C-2 to δC 67.2. The methoxyl was positioned at C-9, and the methyl at N-5, respectively, based on the HMBC correlations of the proton signal of methyoxy (δH 3.96) with C-9 (δC 157.0), and of the methyl signal (δH 2.97) with C-4a and C-12, respectively (Fig. 2). The ROESY correlations of N-CH3/H-4a and H-2/H-4a suggested α-orientation of the N-oxide and the hydroxyl group at C-2. Thus, the structure of 3 was assigned the name (+)-2hydroxy-8-demethyl-homolycorine-α-N-oxide. Compound 4 was obtained as a violet amorphous powder. Its positive HR-ESI-MS spectrum showed a quasimolecular ion peak at m/z 406.0902 [M + Na]+, consistent with the molecular formula C20H17NO7 (calcd for C20H17NO7Na 406.0903), accounting for 13° of unsaturation. Its UV absorption at λmax 305, 285, and 216 nm suggested an aporphine alkaloid skeleton [25]. Its 13C NMR spectrum showed 20 carbon signals [COOCH3 × 1, OCH3 × 2, CH2 (sp3) × 2, CH (sp2) × 3 and C (sp2) × 11, Table 1]. The lH NMR spectrum showed singlet signals for three aryl protons (δH 6.95, 6.59, and 5.91), two OMe (δH 3.69 and 3.56), one N-COOCH3 (δH 3.72), and two triplets for two vicinal coupled methylenes [δH 3.10 (2H, t, J = 6.5, H-4) and 3.45 (2H, t, J = 6.5, H-5)]. The IR spectrum exhibited a conjugated carbonyl absorption (1654 cm− 1), and the 13C NMR spectrum displayed two carbonyl signals at δC 178.2 and 186.5. These spectral data were similar to
) correlations of compounds 1–4.
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Table 2 Cytotoxicity of compounds 1–6 against seven human tumor cell lines (IC50, μM)a. Cell lines
1 2 3 4 5 6 Doxorubicin
BEN-MEN-1
CCF-STTG1
CHG-5
SHG-44
U251
– – – – – – 0.018
10.3 ± 0.9 29.4 ± 4.1 83.2 ± 13.7 9.2 ± 1.3 27.1 ± 5.1 – 0.025
10.2 ± 1.6 29.4 ± 5.3 – 9.7 ± 0.9 30.0 ± 4.4 93.0 ± 21.1 0.022
9.4 ± 1.3 27.1 ± 3.2 – 10.8 ± 1.4 28.3 ± 2.7 – 0.033
11.8 ± 17.4 ± – 11.3 ± 15.8 ± – 0.028
0.7 2.1 1.6 1.7
HL-60
SMMC-7721
W480
10.8 ± 1.6 8.6 ± 1.4 – 12.8 ± 1.6 7.3 ± 1.1 ± 0.9 –
10.5 ± 68.2 ± 86.2 ± 10.0 ± 63.2 ± 85.0 ± 0.038
11.6 ± 1.1 53.5 ± 12.4 – 9.4 ± 0.9 51.1 ± 10.9 – 14.1
0.9 12.3 17.4 0.7 11.8 16.2
(–) IC50 N 100 μM.
1,4-dicarbonyl signals in N-norbulbodione [25]. The HMBC correlations (Fig. 2) of δH 3.72 (3H, s, OMe) and 3.45 (2H, t, J = 6.5, H-5) with δC 157.5 positioned the carbamate group at N-6. The positions of two methoxys were assigned based on HMBC spectrum (Fig. 2). The HMBC of OCH3 (δH 3.56) with C-1 (δC 144.9) and OCH3 (δH 3.69) with C-10 (δC 163.7) positioned two methoxys at C-1 and C-10 respectively (Fig. 2), which was further supported by the ROESY correlations of H-9 (δH 5.91) with the signal of the OCH3 (δH 3.69). Therefore, compound 4 was identified as (+)-N-methoxylcarbonyl-2demethyl-isocorydione. All these compounds were in vitro evaluated for their cytotoxic potential against eight human tumor cell lines, BEN-MEN-1 (meningioma), CCF-STTG1 (astrocytoma), CHG-5 (glioma), SHG-44 (glioma), U251 (glioma), HL-60 (human myeloid leukemia), SMMC-7721 (hepatocellular carcinoma), and W480 (colon cancer) using the modified MTT method. The in vitro cytotoxic activities of these compounds against human cell lines are summarized in Table 2. Among the tested compounds, lycorine-type alkaloid 1 and aporphine-type alkaloid 4 exhibited the most potent cytotoxic potential against all tested tumor cell lines, with IC50 values of 9.4–11.8 and 9.2–12.8 μM, except against BEN-MEN-1. Crinine-type alkaloids 2 and 5 showed significant cytotoxicities against HL-60 (IC50 b 10 μM), and moderate cytotoxicities against astrocytoma and glioma cell lines, CCF-STTG1, CHG-5, SHG44 and U251 (10 μM b IC50 ≤ 30 μM). Homolycorine-type alkaloids 3 and 6 had no cytotoxic activities (IC50 N 80 μM). The compounds 1–6 were tested in vitro for their antiinflammatory activities. The results of the anti-inflammatory assay were summarized in Table 3. Among the assayed compounds, only alkaloids 1 and 4 displayed selective inhibition of Cox-2 (N90%).
Table 3 Evaluation of anti-inflammatory activity of compounds 1–6a.
1 2 3 4 5 6 SC-560 NS-398 a
COX-1
COX-2
36.7 12.9 b0 45.5 12.9 13.4 63.2
96.5 27.6 22.3 93.2 27.6 45.3 97.1
Percent inhibition (all compounds and reference drugs concentration: 100 μM).
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