FITOTE-02954; No of Pages 6 Fitoterapia xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Fitoterapia
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journal homepage: www.elsevier.com/locate/fitote
3Q1
Li Zhang a,1, Zequan Hua a,⁎, Yan Song b,1, Chuanwei Feng c
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a b c
Department of Oral and Maxillofacial Surgery, General Hospital of Shenyang Military Command, Shenyang, Liaoning 110016, China Department of Pharmacy, 455 Hospital of People's Liberation Army, West Huaihai Road 338, Shanghai 200052, China Department of Organic Chemistry, College of Pharmacy, Second Military Medical University, Guohe Road 325, Shanghai 200433, China
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Article history: Received 30 March 2014 Accepted in revised form 23 May 2014 Available online xxxx
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Keywords:
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Alstonia rupestris Apocynaceae Alkaloids Cytotoxic Antibacterial Antifungal
a b s t r a c t
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A chemical investigation of the 80% EtOH extract of the aerial plant of Alstonia rupestris afforded four new monoterpenoid indole alkaloids, 6,7-epoxy-8-oxo-vincadifformine (1), 11-acetyl-6,7-epoxy-8-oxo-vincadifformine (2), 11-hydroxy-14-chloro-15-hydroxyvincadifformine (3), and perakine N1,N4-dioxide (4), together with two known compounds, 11-hydroxy-6,7-epoxy-8-oxovincadifformine (5) and vinorine N1,N4-dioxide (6). Structural elucidation of all the compounds was performed by spectral methods such as 1D- and 2D-NMR, IR, UV, and HRESIMS. Alkaloids 1, 2 and 5 showed significant cytotoxicities against all the tested tumor cell lines of the head and neck squamous cell carcinoma with IC50 value less than 20 μM and antimicrobial activities against two fungi (Alternaria alternata and Phytophthora capsici). Alkaloids 4 and 6 exhibited the activity against bacterium Staphylococcus aureus. © 2014 Published by Elsevier B.V.
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1. Introduction
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The genus Alstonia, which belongs to the family Apocynaceae, comprises about 60 species and is mainly distributed in Asia and South America [1,2]. Among them, 8 species naturally occur in China [3]. Plants of the family Apocynaceae have been proven to be good sources of monoterpenoid indole alkaloids [4–8]. Monoterpenoid indole alkaloids, which originate from the condensation of tryptophan with secologanin to give strictosidine, have attracted the interest of many researchers due to their complicated structures and potent biological activities [9–15]. This type of alkaloids possesses anticancer, antibacterial, antifertility, and anti-tussive activities [16–20]. Galanthamine is a long-acting, selective, reversible and competitive acetylcholine esterase inhibitor that has been approved for use in the European Union and the United States for the treatment of Alzheimer's disease (AD). Alstonia rupestris Kerr is usually endemic in the west part of the Guangxi
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Monoterpenoid indole alkaloids from Alstonia rupestris with cytotoxic, antibacterial and antifungal activities
1
⁎ Corresponding author. Tel./fax: +86 24 28851331. E-mail address: [email protected] (Z. Hua). 1 These two authors contributed equally to this work.
15 16 17 18 19 20 21 22 23 24 25
Province of China. In the present paper, chromatographic separation of an EtOH extract of the aerial plant of A. rupestris has yielded four new monoterpenoid indole alkaloids, 6,7epoxy-8-oxo-vincadifformine (1), 11-acetyl-6,7-epoxy-8-oxovincadifformine (2), 11-hydroxy-14-chloro-15-hydroxyvincadifformine (3), and perakine N1,N4-dioxide (4), together with two known compounds, 11-hydroxy-6,7-epoxy-8oxovincadifformine (5) and vinorine N1,N4-dioxide (6) (Fig. 1). Their structures were established on the basis of their chromatographic properties, chemical and physicochemical methods. Furthermore, all the triterpenoids were evaluated for their in vitro cytotoxic, antibacterial and antifungal properties.
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2. Experimental
65
2.1. General
66
Melting points were determined using a Fisher–Johns melting point apparatus (Vernon Hills, Lake, IL, USA). Optical rotations were determined with a JASCO P2000 digital polarimeter (Tokyo, Japan). Ultraviolet (UV) and infrared (IR)
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http://dx.doi.org/10.1016/j.fitote.2014.05.018 0367-326X/© 2014 Published by Elsevier B.V.
Please cite this article as: Zhang L, et al, Monoterpenoid indole alkaloids from Alstonia rupestris with cytotoxic, antibacterial and antifungal activities, Fitoterapia (2014), http://dx.doi.org/10.1016/j.fitote.2014.05.018
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L. Zhang et al. / Fitoterapia xxx (2014) xxx–xxx
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The aerial parts of A. rupestris were collected in the Honghe, Yunnan province, China, in June 2012. A specimen (201206001), identified by one of the authors (Y. Song), was deposited in the Herbarium of Shenyang Medicine College, Shenyang, China.
92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112
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2.3. Extraction and isolation
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The aerial parts of A. rupestris (10.0 kg) were cut into small pieces and were extracted with 80% EtOH (20 L × 3) at room temperature for 24 h each time. After removal of EtOH under reduced pressure at 55 °C, the aqueous brownish syrup (1 L) was suspended in H2O (1 L) and then partitioned with chloroform (1 L × 3) to afford chloroform fraction (87.3 g). The chloroform fraction was further fractionated through a silica gel column (200–300 mesh, 10 × 80 cm, 500 g) using increasing volumes of acetone in petroleum ether (b.p. 60–90 °C) (100:1, 50:1, 30:1, 15:1, 10:1, 7:1, 5:1, 3:1, 1:1, v/v, each 3 L) as the eluent to give 8 fractions. Fraction 3 (petroleum ether–acetone 15:1, 4.1 g) was applied to an ODS MPLC column (100 g) and eluted with MeOH-H2O (20:80, 30:70, 40:60, each 500 mL) to yield four subfractions (Fr. 3-1 to Fr. 3-4). Subfraction 3-2 (MeOH-H2O, 327 mg) was purified by preparative RP–HPLC (ODS column, 250 × 20 mm) using MeOH-H2O (25:75) as mobile phase to obtain 5 (67 mg, 22.42 min). Subfraction 4-2 (MeOH-H2O, 350 mg) was chromatographed by a Sephadex LH-20 column eluting with
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77 78
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2.2. Plant material
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D
87
73 74
MeOH-H2O (50:50), and purifed by preparative RP–HPLC (ODS column, 250 × 20 mm) using MeOH-H2O (30:70) as mobile phase to yield 1 (68 mg, 23.16 min) and 2 (70 mg, 24.71 min). Subfraction 4-4 (MeOH-H2O 40:60, 99 mg) was purified by preparative RP–HPLC (ODS column, 250 × 20 mm) eluting with MeOH/H2O (22:78) to get 4 (57 mg, 24.65 min). Fraction 5 (petroleum ether–acetone 30:1, 1.4 g) was applied to an ODS column eluted with MeOH-H2O (30:70, 40:60, 50:50) to provide three subfractions (Fr. 5-1 to Fr. 5-3). Subfraction 5-2 (MeOH-H2O 20:80, 226 mg) was repeatedly chromatographed on silica gel (150 g, 60 × 2.8 cm, chloroform–methanol, 20:1 → 10:1) and then purified on a Sephadex LH-20 column eluted with MeOH-H2O (50:50) to afford 3 (78 mg, 22.33 min) and 6 (77 mg, 23.90 min). 6,7-Epoxy-8-oxo-vincadifformine (1): Colorless oil. [α]23.3 = D −97.3 (c = 0.14, MeOH). UV (CDCl3) λmax(log ε): 324 (3.68), 244 (3.79), 228 (3.80), 197 (3.75) nm. IR (KBr) νmax 3385, 1657, 1617, 1442, 1105, 750 cm−1. For NMR data see Tables 1 and 2. EI-MS m/z: 366 ([M]+). HR-ESI-MS (pos.) m/z: 389.1473 ([M + Na]+, C21H22N2O4Na. calc. 389.1477).
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spectra were obtained on JASCO V-650 and JASCO FT/IR-4100 spectrophotometers, respectively. The NMR spectra were recorded on a Varian Unity INOVA 600 FT-NMR spectrometer (Salt Lake City, UT, USA; 600 MHz for 1H; 125 MHz for 13C, respectively). 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 standards. High resolution ESIMS spectra were obtained on a LTQ Orbitrap XL (Thermo Fisher Scientific, Waltham, MA, USA) spectrometer. Silica gel 60 (Merck, Darmstadt, Germany, 230–400 mesh), LiChroprep RP-18 (Merck, 40–63 μm), and Sephadex LH-20 (Amersham Pharmacia Biotech, Roosendaal, The Netherlands) were used for column chromatography (CC). Precoated silica gel plates (Merck, Kieselgel 60 F254, 0.25 mm) and precoated RP-18 F254 plates (Merck) were used for analytical thin-layer chromatography analyses.
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Fig. 1. Structures of compounds 1–6.
113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 Q2
t1:1
Table 1 C NMR data of compounds 1–4 in CDCl3.
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t1:2
No.
1
2
3
4
t1:3
2 3 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 CO2CH3 CO2CH3 COCH3 COCH3
166.5, s 165.1, s 43.6, t 42.0, t 56.8, s 137.3, s 122.9, d 120.6, d 127.8, d 108.7, d 143.2, s 51.2, d 57.1, d 89.1, s 22.4, t 7.3, q 26.2, t 40.6, s 63.3, d 51.2, q 168.3, s – –
166.1, s 164.7, s 43.3, t 41.7, t 56.6, s 131.8, s 121.0, d 112.6, d 151.3, s 104.3, d 143.1, s 50.9, d 56.9, d 88.8, s 22.1, t 7.1, q 26.2, t 40.5, s 63.2, d 51.2, q 168.1, s 20.1, q 169.8, s
166.2, s 54.3, t 50.6, t 43.9, t 54.3, s 129.7, s 121.5, d 104.8, d 159.9, s 96.6, d 144.2, s 59.3, d 75.6, d 92.5, s 26.5, t 8.0, q 22.7, t 44.2, s 69.5, d 50.8, q 168.6, s – –
147.2, s 68.6, d 67.3, d 33.3, t 58.2, s 133.1, s 126.3, d 132.4, d 128.6, d 117.5, d 149.4, s 26.4, t 26.2, d 50.8, d 78.3, d 14.4, q 66.5, d 48.6, d 201.5, d – – 20.7, q 171.1, s
t1:4 t1:5 t1:6 t1:7 t1:8 t1:9 t1:10 t1:11 t1:12 t1:13 t1:14 t1:15 t1:16 t1:17 t1:18 t1:19 t1:20 t1:21 t1:22 t1:23 t1:24 t1:25 t1:26
Please cite this article as: Zhang L, et al, Monoterpenoid indole alkaloids from Alstonia rupestris with cytotoxic, antibacterial and antifungal activities, Fitoterapia (2014), http://dx.doi.org/10.1016/j.fitote.2014.05.018
L. Zhang et al. / Fitoterapia xxx (2014) xxx–xxx t2:1 t2:2
3
Table 2 1 H NMR data of compounds 1–4 in CDCl3 (δ in ppm and J in Hz). No.
1
2
3
4
N1-H 3 5 6a 6b 9 10 11 12 14a 14b 15 16 17 18 19 20 21 CO2CH3 CH3
8.96 (s) – – 3.21, 4.42 (m) 1.74, 2.02 (m) 7.60 (dd, 7.8, 1.8) 6.79 (dt, 7.8, 1.8) 7.06 (dt, 7.8, 1.8) 6.75 (dd, 7.8, 1.8) 3.61 (d, 4.0) – 3.48 (d, 4.0) – 1.85, 2.68 (d, 15.8) 0.81 (t, 7.0) 1.11, 1.29 (m) – 3.61 (s) 3.82 (s) –
8.93 (s) – – 3.19, 4.41 (m) 1.71, 1.98 (m) 7.16 (d, 8.0) 6.71 (dd, 8.0, 2.0) – 6.75 (d, 2.0) 3.59 (d, 4.0) – 3.45 (d, 4.0) – 1.84, 2.65 (d, 15.8) 0.77 (t, 7.0) 1.08, 1.27 (m) – 3.61 (s) 3.80 (s) 2.05 (s)
8.94 (s) 3.26 (m) – 2.66, 2.95 (m) 1.68, 2.07 (m) 6.98 (d, 7.6) 6.33 (dd, 7.6, 1.8) – 6.35 (d, 7.6) 4.16 (m) – 3.89 (d, 6.2) – 2.69, 2.82 (d, 15.2) 0.70 (t, 7.2) 0.89, 1.17 (m) – 2.77 (s) 3.78 (s) –
– 5.18 (d, 9.0) 4.29 (dd, 6.6, 5.2) 2.61 (dd, 12.8, 6.6) 2.92 (dd, 12.8, 5.2) 7.76 (dd, 8.2) 7.61 (dt, 8.2, 2.0) 7.64 (dt, 8.2, 2.0) 7.81 (dd, 8.2) 2.16 (m) 2.66 (m) 3.01 (m) 5.12 (m) 1.41 (d, 8.0) 4.17 (d, 7.2) 2.73 (m) 2.77 (m) 9.83 (d, 7.0) – 2.21 (s)
145 146 147 148 149 150 151 152
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2.4. Cytotoxicity assay in vitro
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The isolated compounds 1–6 were subjected to cytotoxic evaluation against 7 head and neck squamous cell carcinomas (Hep-2, SCL-1, CAL-27, UMSCC-1, Detroit-562, SCC-PKU, and TCA-83) employing the revised MTT method [21]. Doxorubicin was used as the positive control. All tumor cell lines were cultured on RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 U mL−1 penicillin and 100 μg/mL streptomycin in 25 cm2 culture flasks at 37 °C in humidified atmosphere with 5% CO2. For the cytotoxicity tests, cells in exponential growth stage were harvested from culture by trypsin digestion and centrifuging at 180 ×g for 3 min, and then resuspended in fresh medium at a cell density of 5 × 104 cells per milliliter. The cell suspension was dispensed into a 96-well microplate at 100 μL per well, and incubated in humidified atmosphere with 5% CO2 at 37 °C for 24 h, and then treated with the compounds at various concentrations (0, 1, 10, 100 μM). After 48 h of treatment, 50 μL of 1 mg/mL MTT solution was added to each well, and
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143 144
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141 142
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139 140
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137 138
N C
135 136
11-Acetyl-6,7-epoxy-8-oxo-vincadifformine (2): Colorless oil. [α]23.3 = − 68.4 (c = 0.10, MeOH). UV (CDCl3) D λmax(log ε): 325 (3.78), 245 (3.83), 228 (3.85), 198 (3.79) nm. IR (KBr) νmax 3405, 740, 1655, 1620, 1438, 1103 cm−1. For NMR data see Tables 1 and 2. EI-MS m/z: 424 ([M]+). HR-ESI-MS (pos.) m/z: 447.1533 ([M + Na]+, C23H24N2O6Na. calc. 447.1532). 11-Hydroxy-14-chloro-15-hydroxy-vincadifformine (3): Colorless oil. [α]23.3 = −140.4 (c = 0.10, MeOH). UV (CDCl3) D λmax(logε) 326 (3.85), 244 (3.76), 228 (3.50), 223 (3.82), 198 (3.44) nm. IR (KBr) νmax 3428, 2945, 1670, 1615, 1270, 1113, 755 cm−1. For NMR data see Tables 1 and 2. EI-MS m/z: 403 ([M − 1]+), 405 ([M + 1]+). HR-ESI-MS (pos.) m/z: 404.1584 ([M − 1 + H]+, C21H26ClN2O4. calc. 404.1581), 406.1852 ([M + 1 + H]+, C21H26ClN2O4. calc. 406.1581). Perakine N1,N4-dioxide (4): Colorless oil. [α]23.3 = − 19.3 D (c = 0.17, MeOH). UV (CDCl3) λmax(logε) 265 (3.88), 221 (3.88), 196 (3.78) nm. IR (KBr) νmax 3425, 2967, 1743, 1645, 1225, 1030, 753 cm−1. For NMR data see Tables 1 and 2. EI-MS m/z: 382 ([M]+). HR-ESI-MS (pos.) m/z: 405.1423 ([M + Na]+, C21H22N2O5Na. calc. 405.1426).
U
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F
t2:3 t2:4 t2:5 t2:6 t2:7 t2:8 t2:9 t2:10 t2:11 t2:12 t2:13 t2:14 t2:15 t2:16 t2:17 t2:18 t2:19 t2:20 t2:21 t2:22 t2:23
Fig. 2. Key HMBCs (
) and 1H–1H COSY (
) correlations of compounds 1 and 4.
Please cite this article as: Zhang L, et al, Monoterpenoid indole alkaloids from Alstonia rupestris with cytotoxic, antibacterial and antifungal activities, Fitoterapia (2014), http://dx.doi.org/10.1016/j.fitote.2014.05.018
155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171
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3. Results and discussion
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3.1. Chemistry
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Compound 1 was obtained as a colorless oil. The HRESIMS displayed a pseudomolecular ion at m/z 389.1473 [M + Na]+ (calcd for C21H22N2O4Na, 389.1477) consistent with a molecular formula of C21H22N2O4, corresponding to 12° of unsaturation. The IR spectrum exhibited absorptions at 3385, 1657 and
199 200 201 202 Q3 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219
225 226 227
C
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E
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R
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R
191 192
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C
187 188
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F
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All compounds (purity N 90%) were screened for their antimicrobial activity in vitro using the disk-diffusion method as described in the literature with minor modifications [22]. Strains including two species of bacteria [Staphylococcus aureus (ATCC-25923), Mycobacterium tuberculosis (ATCC-25177/H37Ra)] and five species of fungi [Gibberella pulicaris (KZN 4207), Alternaria alternata (TX-8025), Colletotrichum nicotianae (SACC-1922), Phytophthora capsici (KACC-40157), Gonatopyricularia amomi (MB-9671)] were used. Rifampicin and nystatin were used as positive controls for antibacterial and antifungal activities, respectively. A disk containing only DMSO was used as the negative control. Medium used in the antimicrobial activity included nutrient agar medium (S. aureus), Dorset egg medium (M. tuberculosis) and Sabouraud dextrose broth (SDB) agar medium (five species of fungi). To each agar plate, an inoculum containing 107 bacteria/mL or a 0.5 optical density of the McFarland Scale was incorporated. The plates were solidified and sterile filter paper disks (6-mm diameter) were done on each one. Solution of each compound (5 mM) in DMSO, antibacterial agents (rifampicin 5 μM/mL), antifungal agents (nystatin 10 μM/mL), and control vehicles (DMSO) was added into too. The plates were aerobically incubated at 37 °C for S. aureus for 18 h, for the five species of fungi for 24 h and for M. tuberculosis for 15–45 days, and four assays under identical conditions were carried out for each one. The diameter of the inhibition zone was measured for testing of antibacterial and antifungal activities. Experiments were performed in triplicate, and the results are presented as the mean values of the diameters of the inhibitory zones from three runs. The MIC values of the most active compounds, in the previous experiment, were determined using the dilution method in 96-hole plates. The diameters of the inhibitory zones and the MIC value were used as criteria to judge the antimicrobial activity (active: the diameters of the inhibitory zones ≥16 mm, MIC ≤ 5 mM; moderately active: the diameters of the inhibitory zones are visible, MIC N 5 mM; not active: the diameters of the inhibitory zones are invisible). All strains of bacteria and fungi were purchased from Shanghai Institute of Biochemistry & Cell Biology, Chinese Academy of Sciences (Shanghai, China).
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177 178
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2.5. Antimicrobial activity bioassay
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P
180
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1617 cm−1 that indicated the presence of a β-anilinoacrylate chromophore, corresponding to the carbon signals for an acrylate double bond at δC 166.5 (C-2) and 89.1 (C-16). Its 13 C NMR spectrum showed 21 carbon signals [OCH3 × 1, CH3 (sp3) × 1, CH2 (sp3) × 4, CH (sp3) × 3, C (sp3) × 2, CH (sp2) × 4 and C (sp2) × 6, Table 1]. The 1H NMR spectrum exhibited four aromatic proton signals [δH 7.60 and 6.75 (each, 1H, dd, J = 7.8, 1.8 Hz), 6.79 and 7.06 (each, 1H, dt, J = 7.8, 1.8 Hz)] ascribed to an ortho-disubstituted benzene ring, two characteristic proton signals due to an oxirane ring at δH 3.61 and 3.48 (each, d, J = 4.0), and a NH signal at δH 8.96. The 14,15-epoxy group was established by the HMBC correlations of δH 3.48 (H-15) with δC 165.1 (C-3), 26.2 (C-19) and 63.3 (C-21) and of δH 3.61 (H-14) with δC 40.6 (C-20) (Fig. 2). These data suggested that the structure of 1 was almost identical with 5 [3]. The distinct difference was that the methoxy at C-11 in 5 was absent in 1, which was supported by the observation of upfield chemical shift of C-11 from δC 156.4 in 5 to δC 127.8 in 1. The relative configuration of 1 was confirmed by a NOESY experiment and comparison of the NMR data of 1 with those of compound 5. According to NOESY data and its negative specific rotation ([α]23.3 = −68.4), the D α-orientation of the 14,15-epoxy group was determined by the correlation between H-15 and H-17β (Cao et al., 2012). Thus, the structure of 1 was established as 6,7-epoxy-8oxovincadifformine. Compound 2, a colorless oil, exhibited a molecular formula of C23H24N2O6, based on the HRESIMS spectrum which showed a pseudomolecular ion at m/z 447.1533 [M + Na]+ (calcd. 447.1532). Comparing the 1H and 13C NMR data of 2 with those of compound 1, the data were almost identical. The only significant difference was the presence of one more OAc group in compound 2. The OAc group was positioned at C-11 based on the NOE correlations of H-12/H-N1 and the presence of the ABX-system [δH 7.16 (d, J = 8.0 Hz, H-9), 6.71 (dd, J = 8.0, 2.0 Hz, H-10), 6.75 (d, J = 2.0 Hz, H-12)]. The stereochemistry of 2 was expected to be the same as 5 on the basis of the NOESY data. Therefore, 2 was identified as 11-acetyl-6,7-epoxy-8-oxo-vincadifformine. Compound 3 exhibited two isotopic peaks at m/z 404.1584 [M − 1 + H]+ (calc. 404.1581) and 406.1852 [M + 1 + H]+ (calc. 406.1581) in its HRESIMS, accounting for a molecular formula of C21H25ClN2O4, suggesting 10° of unsaturation. The existence of the chlorine atom was identified by the appropriate 13C NMR chemical shift at δC 59.3 (d, C-14) and EIMS analysis. The EI mass spectrometry showed two quasimolecular-ion peaks at m/z 403 (41.35, [M − 1]+) and 405 (13.79, [M + 1]+) with ratio of relative intensity approximating 3:1. The NMR data were similar to those of compound 1, except for a chlorine atom and a hydroxy group in 3 taking the place of the 14,15-epoxy group in 1 and 2. The 1H–1H COSY correlations of proton signals at δH 4.16 (H-14) with δH 3.26 (H-3) and δH 3.89 (H-15) indicated the location of the chlorine atom at C-14 (δC 59.3) and a hydroxy group at C-15 (δC 75.6) respectively, which was further supported by the HMBC correlations of H-3 and H-19 with C-15. Additionally, the three ABX-system protons at δH 6.98 (d, J = 7.6 Hz, H-9), 6.33 (dd, J = 7.6, 1.8 Hz, H-10), and 6.35 (d, J = 7.6 Hz, H-12) indicated the presence of the OH group at C-11 in 3. The α-orientation of H-14 and the β-orientation of H-15 were deduced from the NOESY correlations of H-14/H-21 and H-15/
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further incubated for 4 h. The cells in each well were then solubilized with DMSO (100 μL for each well) and the optical density (OD) was recorded at 570 nm. All drug doses were tested in triplicate and the IC50 values were derived from the mean OD values of the triplicate tests versus drug concentration curves. All cell lines were purchased from the Cell Bank of the Shanghai Institute of Biochemistry & Cell Biology, Chinese Academy of Sciences (Shanghai, China).
D
172 173
L. Zhang et al. / Fitoterapia xxx (2014) xxx–xxx
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Please cite this article as: Zhang L, et al, Monoterpenoid indole alkaloids from Alstonia rupestris with cytotoxic, antibacterial and antifungal activities, Fitoterapia (2014), http://dx.doi.org/10.1016/j.fitote.2014.05.018
228 229 230 231
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L. Zhang et al. / Fitoterapia xxx (2014) xxx–xxx
Cell lines
t3:4 t3:5 t3:6 t3:7 t3:8 t3:9 t3:10 t3:11
1 2 3 4 5 6 Doxorubicin
t3:12
a
SCL-1
CAL-27
UMSCC-1
Detroit-562
SCC-PKU
TCA-83
10.3 12.9 52.7 44.1 16.3 47.8 18.3
11.3 12.3 51.8 40.8 15.7 51.5 14.7
9.2 10.8 49.0 44.8 14.8 44.8 22.0
12.0 12.7 59.4 50.7 17.2 49.1 31.7
10.7 11.3 54.3 48.9 14.7 53.2 24.9
13.7 12.9 59.7 47.0 11.2 43.6 35.4
13.0 14.9 59.5 40.1 15.5 48.2 15.9
3.2. Cytotoxic activity
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All these compounds were in vitro evaluated for their cytotoxic potential against seven tumor cell lines by using the revised MTT method as described in the experimental part. The results are summarized in Table 3. The alkaloids isolated from the family Amaryllidaceae have been reported to show strong cytotoxic activities against various types of cancer and bacteria cell lines, which were agreed well with our results [5,6,13]. Alkaloids 1, 2, and 5 exhibited significant cytotoxicity against all tested cell lines of the head and neck squamous cell carcinoma (Hep-2, SCL-1, CAL-27, UMSCC-1, Detroit-562, SCC-PKU, and TCA-83) with IC50 values less than
305 306 307 308
312 313 314 315 316 317 318 319 320 321 t4:1 t4:2
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303 304
R
301 302
R
299 300
O
297 298
N C
295 296
U
293 294
3.3. Antimicrobial assay in vitro
324
All compounds were tested for their antimicrobial activities by using the disk diffusion method by measuring the inhibition zones and for the most active compounds, minimum inhibitory concentration (MIC) values were also determined (Table 4). The results showed that alkaloids 1, 2 and 5 exhibited significant activity against two fungi (A. alternata and P. capsici) with MIC values of 0.66–0.99 mM, 0.87–1.10 mM and 1.53–1.64 mM, respectively, and moderate activity of the bacterium S. aureus. Compounds 4 and 6 showed potent antibacterial activities against S. aureus, while 3 had no activity. Additionally, 4 possessed higher antibacterial activities against S. aureus than 6, indicating that the formyl group on C-20 might enhance the activities for these monoterpenoid indole alkaloids.
325 326 Q5
References
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P
310
291 292
20 μM, while no cytotoxicity was detected for other alkaloids 322 (IC50 ≥ 40 μM). 323
C
309
H-17β, respectively. Accordingly, compound 3 was identified as 11-hydroxy-14-chloro-15-hydroxy-vincadifformine. Compound 4 was obtained as a colorless oil. Its positive HRESIMS spectrum showed a quasimolecular ion peak at m/z 405.1423 [M + Na]+, consistent with the molecular formula C21H22N2O5, accounting for 12° of unsaturation. The 13C NMR and DEPT spectra displayed signals of two Me, two CH2, and twelve CH groups, together with five quaternary C-atoms. The NMR signals at δH 7.76 (dd, J = 8.0, 2.0 Hz, H-9), 7.61 (dt, J = 8.0, 2.0 Hz, H-10), 7.64 (dt, J = 8.0, 2.0 Hz, H-11), and 7.81 (dd, J = 8.0, 2.0 Hz, H-12) are assigned to an ortho-disubstituted benzene ring. The 1D and 2D-NMR data were closely related to those of 6 [3]. The only significant difference was that the carboxy group at C-20 in 6 was replaced by a formyl group in compound 4, which was confirmed by the HMBC correlations of the proton signal of the formyl group [δH 9.83 (1H, d, J = 7.0)] with C-15 and C-19 (Fig. 2). The NOESY analysis showed that the relative configurations of the C-3, C-5, C-15, C-16, and C-17 were in good agreement with those of compounds 4 and 6. Therefore, compound 4 was identified as perakine N1,N4-dioxide.
R O O
Doxorubicin activities are expressed as IC50 values in nM, and those of compounds 1–6 are expressed as IC50 values in μM. (−) IC50 N 100 μM.
D
289 290
Hep-2
F
Compounds
E
t3:3
Table 3 Cytotoxicity of compounds 1–6 against seven human tumor cell lines (IC50, μM)a.
T
t3:1 t3:2
5
[1] Narine LL, Maxwell AR. Monoterpenoid indole alkaloids from Palicourea crocea. Phytochem Lett 2009;2:34–6. [2] Tan SJ, Lim KH, Subramaniam G, Kam TS. Macroline-sarpagine and macroline-pleiocarpamine bisindole alkaloids from Alstonia angustifolia. Phytochemistry 2013;85:194–202. [3] Cao P, Liang Y, Gao X, Li XM, Song ZQ, Liang GB. Monoterpenoid indole alkaloids from Alstonia yunnanensis and their cytotoxic and antiinflammatory activities. Molecules 2012;17:13631–41. [4] Lim SH, Low YY, Tan SJ, Lim KH, Thomas NF, Kam TS. Perhentidines A–C: macroline–macroline bisindoles from Alstonia and the absolute configuration of perhentinine and macralstonine. J Nat Prod 2012;75:942–50. [5] Lim SH, Tan SJ, Low YY, Kam TS. Lumutinines A–D, linearly fused macroline–macroline and macroline–sarpagine bisindoles from Alstonia macrophylla. J Nat Prod 2011;74:2556–62. [6] Tan SJ, Choo YM, Thomas NF, Robinson WT, Komiyama K, Kam TS. Unusual indole alkaloid–pyrrole, –pyrone, and –carbamic acid adducts from Alstonia angustifolia. Tetrahedron 2010;66:7799–806.
Table 4 Antimicrobial and antifungal activities (zones of inhibition/and MIC mM, n = 3) of compounds 1–6.
t4:3
Compounds
S.aureus
M. tuberculosis
G. pulicaris
A. alternata
C. nicotianae
P. capsici
G. amomi
t4:4 t4:5 t4:6 t4:7 t4:8 t4:9 t4:10 t4:11
1 2 3 4 5 6 Rifampicin Nystatin
15.72 16.33 – 23/0.49 14.91 19/0.83 25/0.003 –
– – – – – – 22/0.003 –
– – – – – – – 20/0.008
19/0.66 20/0.87 – – 19/1.53 – – 17/0.007
– – – – – – – 21/0.006
18/0.99 17/1.10 – – 16/1.64 – – 18/0.061
– – – – – – – 19/0.010
t4:12
No activity.
Please cite this article as: Zhang L, et al, Monoterpenoid indole alkaloids from Alstonia rupestris with cytotoxic, antibacterial and antifungal activities, Fitoterapia (2014), http://dx.doi.org/10.1016/j.fitote.2014.05.018
327 328 329 330 331 332 333 334 335 336 337 338
340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356
[17]
[18]
[19] [20] [21]
[22]
F
[16]
O
[15]
spatulata. Revision of the C-20 configuration of scholaricine. J Nat Prod 2010;73:1891–7. Hutchinson CR. Tetrahedron report number 105: camptothecin: chemistry, biogenesis and medicinal chemistry. Tetrahedron 1981;37:1047–65. Jagetia GC, Baliga MS. Evaluation of anticancer activity of the alkaloid fraction of Alstonia scholaris (Sapthaparna) in vitro and in vivo. Phytother Res 2006;20:103–9. Kam TS, Choo YM, Komiyama K. Unusual spirocyclic macroline alkaloids, nitrogenous derivatives, and a cytotoxic bisindole from Alstonia. Tetrahedron 2004;60:3957–66. Kam TS, Tan SJ, Ng SW, Komiyama K. Bipleiophylline, an unprecedented cytotoxic bisindole alkaloid constituted from the bridging of two indole moieties by an aromatic spacer unit. Org Lett 2008;10:3749–52. Khan MR, Omoloso AD, Kihara M. Antibacterial activity of Alstonia scholaris and Leea tetramera. Fitoterapia 2003;74:736–40. Chen WM, Yan YP, Liang XT. Alkaloids from roots of Alstonia yunnanensis. Planta Med 1983;49:62. Yu JO, Liao ZX, Lei JC, Hu XM. Antioxidant and cytotoxic activities of various fractions of ethanol extract of Dianthus superbus. Food Chem 2007;104:1215–9. Zhang W, Hu JF, Lv WW, Zhao QC, Shi GB. Antibacterial, antifungal and cytotoxic isoquinoline alkaloids from Litsea cubeba. Molecules 2012;17:12950–60.
N
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O
R
R
E
C
T
E
D
P
[7] Arai H, Hirasawa Y, Rahman A, Kusumawati I, Zaini NC, Sato S, et al. Alstiphyllanines E–H, picraline and ajmaline-type alkaloids from Alstonia macrophylla inhibiting sodium glucose cotransporter. Bioorg Med Chem 2010;18:2152–8. [8] Hirasawa Y, Arai H, Zaima K, Oktarina R, Rahman A, Ekasari W, et al. Alstiphyllanines A–D, indole alkaloids from Alstonia macrophylla. J Nat Prod 2009;72:304–7. [9] Arai H, Hirasawa Y, Rahman A, Kusumawati I, Zaini NC, Sato S, et al. Alstiphyllanines E–H, picraline and ajmaline-type alkaloids from Alstonia macrophylla inhibiting sodium glucose cotransporter. Bioorg Med Chem 2010;18:2152–8. [10] Lim SH, Low YY, Tan SJ, Lim KH, Thomas NF, Kam TS. Perhentidines A–C: macroline–macroline bisindoles from Alstonia and the absolute configuration of perhentinine and macralstonine. J Nat Prod 2012;75:942–50. [11] Hirasawa Y, Arai H, Zaima K, Oktarina R, Rahman A, Ekasari W, et al. Alstiphyllanines A–D, indole alkaloids from Alstonia macrophylla. J Nat Prod 2009;72:304–7. [12] Koyama K, Hirasawa Y, Nugroho AE, Hosoya T, Hoe TC, Chan KL, et al. Alsmaphorazines A and B, novel indole alkaloids from Alstonia pneumatophora. Org Lett 2010;12:4188–91. [13] Ku WF, Tan SJ, Low YY, Komiyama K, Kam TS. Angustilobine and andranginine type indole alkaloids and an uleine-secovallesamine bisindole alkaloid from Alstonia angustiloba. Phytochemistry 2011;72:2212–8. [14] Tan SJ, Low YY, Choo YM, Abdullah Z, Etoh T, Hayashi M, et al. Strychnan and secoangustilobine A type alkaloids from Alstonia
U
357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 405
L. Zhang et al. / Fitoterapia xxx (2014) xxx–xxx
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6
Please cite this article as: Zhang L, et al, Monoterpenoid indole alkaloids from Alstonia rupestris with cytotoxic, antibacterial and antifungal activities, Fitoterapia (2014), http://dx.doi.org/10.1016/j.fitote.2014.05.018
382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404
Contents lists available at ScienceDirect
Fitoterapia
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journal homepage: www.elsevier.com/locate/fitote
3Q1
Li Zhang a,1, Zequan Hua a,⁎, Yan Song b,1, Chuanwei Feng c
4 5 6
a b c
Department of Oral and Maxillofacial Surgery, General Hospital of Shenyang Military Command, Shenyang, Liaoning 110016, China Department of Pharmacy, 455 Hospital of People's Liberation Army, West Huaihai Road 338, Shanghai 200052, China Department of Organic Chemistry, College of Pharmacy, Second Military Medical University, Guohe Road 325, Shanghai 200433, China
11 12 13 14
Article history: Received 30 March 2014 Accepted in revised form 23 May 2014 Available online xxxx
26
Keywords:
27 28 29 30 31 32 34 33
Alstonia rupestris Apocynaceae Alkaloids Cytotoxic Antibacterial Antifungal
a b s t r a c t
D
i n f o
A chemical investigation of the 80% EtOH extract of the aerial plant of Alstonia rupestris afforded four new monoterpenoid indole alkaloids, 6,7-epoxy-8-oxo-vincadifformine (1), 11-acetyl-6,7-epoxy-8-oxo-vincadifformine (2), 11-hydroxy-14-chloro-15-hydroxyvincadifformine (3), and perakine N1,N4-dioxide (4), together with two known compounds, 11-hydroxy-6,7-epoxy-8-oxovincadifformine (5) and vinorine N1,N4-dioxide (6). Structural elucidation of all the compounds was performed by spectral methods such as 1D- and 2D-NMR, IR, UV, and HRESIMS. Alkaloids 1, 2 and 5 showed significant cytotoxicities against all the tested tumor cell lines of the head and neck squamous cell carcinoma with IC50 value less than 20 μM and antimicrobial activities against two fungi (Alternaria alternata and Phytophthora capsici). Alkaloids 4 and 6 exhibited the activity against bacterium Staphylococcus aureus. © 2014 Published by Elsevier B.V.
E
a r t i c l e
E
C
T
10 9
R
35
1. Introduction
37
The genus Alstonia, which belongs to the family Apocynaceae, comprises about 60 species and is mainly distributed in Asia and South America [1,2]. Among them, 8 species naturally occur in China [3]. Plants of the family Apocynaceae have been proven to be good sources of monoterpenoid indole alkaloids [4–8]. Monoterpenoid indole alkaloids, which originate from the condensation of tryptophan with secologanin to give strictosidine, have attracted the interest of many researchers due to their complicated structures and potent biological activities [9–15]. This type of alkaloids possesses anticancer, antibacterial, antifertility, and anti-tussive activities [16–20]. Galanthamine is a long-acting, selective, reversible and competitive acetylcholine esterase inhibitor that has been approved for use in the European Union and the United States for the treatment of Alzheimer's disease (AD). Alstonia rupestris Kerr is usually endemic in the west part of the Guangxi
46 47 48 49 50 51 52
O
N C
44 45
U
42 43
R
36
40 41
P
7
38 39
R O O
2
Monoterpenoid indole alkaloids from Alstonia rupestris with cytotoxic, antibacterial and antifungal activities
1
⁎ Corresponding author. Tel./fax: +86 24 28851331. E-mail address: [email protected] (Z. Hua). 1 These two authors contributed equally to this work.
15 16 17 18 19 20 21 22 23 24 25
Province of China. In the present paper, chromatographic separation of an EtOH extract of the aerial plant of A. rupestris has yielded four new monoterpenoid indole alkaloids, 6,7epoxy-8-oxo-vincadifformine (1), 11-acetyl-6,7-epoxy-8-oxovincadifformine (2), 11-hydroxy-14-chloro-15-hydroxyvincadifformine (3), and perakine N1,N4-dioxide (4), together with two known compounds, 11-hydroxy-6,7-epoxy-8oxovincadifformine (5) and vinorine N1,N4-dioxide (6) (Fig. 1). Their structures were established on the basis of their chromatographic properties, chemical and physicochemical methods. Furthermore, all the triterpenoids were evaluated for their in vitro cytotoxic, antibacterial and antifungal properties.
53
2. Experimental
65
2.1. General
66
Melting points were determined using a Fisher–Johns melting point apparatus (Vernon Hills, Lake, IL, USA). Optical rotations were determined with a JASCO P2000 digital polarimeter (Tokyo, Japan). Ultraviolet (UV) and infrared (IR)
67 68
http://dx.doi.org/10.1016/j.fitote.2014.05.018 0367-326X/© 2014 Published by Elsevier B.V.
Please cite this article as: Zhang L, et al, Monoterpenoid indole alkaloids from Alstonia rupestris with cytotoxic, antibacterial and antifungal activities, Fitoterapia (2014), http://dx.doi.org/10.1016/j.fitote.2014.05.018
54 55 56 57 58 59 60 61 62 63 64
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L. Zhang et al. / Fitoterapia xxx (2014) xxx–xxx
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The aerial parts of A. rupestris were collected in the Honghe, Yunnan province, China, in June 2012. A specimen (201206001), identified by one of the authors (Y. Song), was deposited in the Herbarium of Shenyang Medicine College, Shenyang, China.
92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112
C
E
R
90 91
R
89
2.3. Extraction and isolation
O
83 84
C
81 82
The aerial parts of A. rupestris (10.0 kg) were cut into small pieces and were extracted with 80% EtOH (20 L × 3) at room temperature for 24 h each time. After removal of EtOH under reduced pressure at 55 °C, the aqueous brownish syrup (1 L) was suspended in H2O (1 L) and then partitioned with chloroform (1 L × 3) to afford chloroform fraction (87.3 g). The chloroform fraction was further fractionated through a silica gel column (200–300 mesh, 10 × 80 cm, 500 g) using increasing volumes of acetone in petroleum ether (b.p. 60–90 °C) (100:1, 50:1, 30:1, 15:1, 10:1, 7:1, 5:1, 3:1, 1:1, v/v, each 3 L) as the eluent to give 8 fractions. Fraction 3 (petroleum ether–acetone 15:1, 4.1 g) was applied to an ODS MPLC column (100 g) and eluted with MeOH-H2O (20:80, 30:70, 40:60, each 500 mL) to yield four subfractions (Fr. 3-1 to Fr. 3-4). Subfraction 3-2 (MeOH-H2O, 327 mg) was purified by preparative RP–HPLC (ODS column, 250 × 20 mm) using MeOH-H2O (25:75) as mobile phase to obtain 5 (67 mg, 22.42 min). Subfraction 4-2 (MeOH-H2O, 350 mg) was chromatographed by a Sephadex LH-20 column eluting with
N
79 80
U
77 78
P
2.2. Plant material
75 76
D
87
73 74
MeOH-H2O (50:50), and purifed by preparative RP–HPLC (ODS column, 250 × 20 mm) using MeOH-H2O (30:70) as mobile phase to yield 1 (68 mg, 23.16 min) and 2 (70 mg, 24.71 min). Subfraction 4-4 (MeOH-H2O 40:60, 99 mg) was purified by preparative RP–HPLC (ODS column, 250 × 20 mm) eluting with MeOH/H2O (22:78) to get 4 (57 mg, 24.65 min). Fraction 5 (petroleum ether–acetone 30:1, 1.4 g) was applied to an ODS column eluted with MeOH-H2O (30:70, 40:60, 50:50) to provide three subfractions (Fr. 5-1 to Fr. 5-3). Subfraction 5-2 (MeOH-H2O 20:80, 226 mg) was repeatedly chromatographed on silica gel (150 g, 60 × 2.8 cm, chloroform–methanol, 20:1 → 10:1) and then purified on a Sephadex LH-20 column eluted with MeOH-H2O (50:50) to afford 3 (78 mg, 22.33 min) and 6 (77 mg, 23.90 min). 6,7-Epoxy-8-oxo-vincadifformine (1): Colorless oil. [α]23.3 = D −97.3 (c = 0.14, MeOH). UV (CDCl3) λmax(log ε): 324 (3.68), 244 (3.79), 228 (3.80), 197 (3.75) nm. IR (KBr) νmax 3385, 1657, 1617, 1442, 1105, 750 cm−1. For NMR data see Tables 1 and 2. EI-MS m/z: 366 ([M]+). HR-ESI-MS (pos.) m/z: 389.1473 ([M + Na]+, C21H22N2O4Na. calc. 389.1477).
T
85 86
spectra were obtained on JASCO V-650 and JASCO FT/IR-4100 spectrophotometers, respectively. The NMR spectra were recorded on a Varian Unity INOVA 600 FT-NMR spectrometer (Salt Lake City, UT, USA; 600 MHz for 1H; 125 MHz for 13C, respectively). 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 standards. High resolution ESIMS spectra were obtained on a LTQ Orbitrap XL (Thermo Fisher Scientific, Waltham, MA, USA) spectrometer. Silica gel 60 (Merck, Darmstadt, Germany, 230–400 mesh), LiChroprep RP-18 (Merck, 40–63 μm), and Sephadex LH-20 (Amersham Pharmacia Biotech, Roosendaal, The Netherlands) were used for column chromatography (CC). Precoated silica gel plates (Merck, Kieselgel 60 F254, 0.25 mm) and precoated RP-18 F254 plates (Merck) were used for analytical thin-layer chromatography analyses.
E
71 72
R O
Fig. 1. Structures of compounds 1–6.
113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 Q2
t1:1
Table 1 C NMR data of compounds 1–4 in CDCl3.
13
t1:2
No.
1
2
3
4
t1:3
2 3 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 CO2CH3 CO2CH3 COCH3 COCH3
166.5, s 165.1, s 43.6, t 42.0, t 56.8, s 137.3, s 122.9, d 120.6, d 127.8, d 108.7, d 143.2, s 51.2, d 57.1, d 89.1, s 22.4, t 7.3, q 26.2, t 40.6, s 63.3, d 51.2, q 168.3, s – –
166.1, s 164.7, s 43.3, t 41.7, t 56.6, s 131.8, s 121.0, d 112.6, d 151.3, s 104.3, d 143.1, s 50.9, d 56.9, d 88.8, s 22.1, t 7.1, q 26.2, t 40.5, s 63.2, d 51.2, q 168.1, s 20.1, q 169.8, s
166.2, s 54.3, t 50.6, t 43.9, t 54.3, s 129.7, s 121.5, d 104.8, d 159.9, s 96.6, d 144.2, s 59.3, d 75.6, d 92.5, s 26.5, t 8.0, q 22.7, t 44.2, s 69.5, d 50.8, q 168.6, s – –
147.2, s 68.6, d 67.3, d 33.3, t 58.2, s 133.1, s 126.3, d 132.4, d 128.6, d 117.5, d 149.4, s 26.4, t 26.2, d 50.8, d 78.3, d 14.4, q 66.5, d 48.6, d 201.5, d – – 20.7, q 171.1, s
t1:4 t1:5 t1:6 t1:7 t1:8 t1:9 t1:10 t1:11 t1:12 t1:13 t1:14 t1:15 t1:16 t1:17 t1:18 t1:19 t1:20 t1:21 t1:22 t1:23 t1:24 t1:25 t1:26
Please cite this article as: Zhang L, et al, Monoterpenoid indole alkaloids from Alstonia rupestris with cytotoxic, antibacterial and antifungal activities, Fitoterapia (2014), http://dx.doi.org/10.1016/j.fitote.2014.05.018
L. Zhang et al. / Fitoterapia xxx (2014) xxx–xxx t2:1 t2:2
3
Table 2 1 H NMR data of compounds 1–4 in CDCl3 (δ in ppm and J in Hz). No.
1
2
3
4
N1-H 3 5 6a 6b 9 10 11 12 14a 14b 15 16 17 18 19 20 21 CO2CH3 CH3
8.96 (s) – – 3.21, 4.42 (m) 1.74, 2.02 (m) 7.60 (dd, 7.8, 1.8) 6.79 (dt, 7.8, 1.8) 7.06 (dt, 7.8, 1.8) 6.75 (dd, 7.8, 1.8) 3.61 (d, 4.0) – 3.48 (d, 4.0) – 1.85, 2.68 (d, 15.8) 0.81 (t, 7.0) 1.11, 1.29 (m) – 3.61 (s) 3.82 (s) –
8.93 (s) – – 3.19, 4.41 (m) 1.71, 1.98 (m) 7.16 (d, 8.0) 6.71 (dd, 8.0, 2.0) – 6.75 (d, 2.0) 3.59 (d, 4.0) – 3.45 (d, 4.0) – 1.84, 2.65 (d, 15.8) 0.77 (t, 7.0) 1.08, 1.27 (m) – 3.61 (s) 3.80 (s) 2.05 (s)
8.94 (s) 3.26 (m) – 2.66, 2.95 (m) 1.68, 2.07 (m) 6.98 (d, 7.6) 6.33 (dd, 7.6, 1.8) – 6.35 (d, 7.6) 4.16 (m) – 3.89 (d, 6.2) – 2.69, 2.82 (d, 15.2) 0.70 (t, 7.2) 0.89, 1.17 (m) – 2.77 (s) 3.78 (s) –
– 5.18 (d, 9.0) 4.29 (dd, 6.6, 5.2) 2.61 (dd, 12.8, 6.6) 2.92 (dd, 12.8, 5.2) 7.76 (dd, 8.2) 7.61 (dt, 8.2, 2.0) 7.64 (dt, 8.2, 2.0) 7.81 (dd, 8.2) 2.16 (m) 2.66 (m) 3.01 (m) 5.12 (m) 1.41 (d, 8.0) 4.17 (d, 7.2) 2.73 (m) 2.77 (m) 9.83 (d, 7.0) – 2.21 (s)
145 146 147 148 149 150 151 152
R O O
P
153
E
D
2.4. Cytotoxicity assay in vitro
154
The isolated compounds 1–6 were subjected to cytotoxic evaluation against 7 head and neck squamous cell carcinomas (Hep-2, SCL-1, CAL-27, UMSCC-1, Detroit-562, SCC-PKU, and TCA-83) employing the revised MTT method [21]. Doxorubicin was used as the positive control. All tumor cell lines were cultured on RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 U mL−1 penicillin and 100 μg/mL streptomycin in 25 cm2 culture flasks at 37 °C in humidified atmosphere with 5% CO2. For the cytotoxicity tests, cells in exponential growth stage were harvested from culture by trypsin digestion and centrifuging at 180 ×g for 3 min, and then resuspended in fresh medium at a cell density of 5 × 104 cells per milliliter. The cell suspension was dispensed into a 96-well microplate at 100 μL per well, and incubated in humidified atmosphere with 5% CO2 at 37 °C for 24 h, and then treated with the compounds at various concentrations (0, 1, 10, 100 μM). After 48 h of treatment, 50 μL of 1 mg/mL MTT solution was added to each well, and
T
C
E
143 144
R
141 142
R
139 140
O
137 138
N C
135 136
11-Acetyl-6,7-epoxy-8-oxo-vincadifformine (2): Colorless oil. [α]23.3 = − 68.4 (c = 0.10, MeOH). UV (CDCl3) D λmax(log ε): 325 (3.78), 245 (3.83), 228 (3.85), 198 (3.79) nm. IR (KBr) νmax 3405, 740, 1655, 1620, 1438, 1103 cm−1. For NMR data see Tables 1 and 2. EI-MS m/z: 424 ([M]+). HR-ESI-MS (pos.) m/z: 447.1533 ([M + Na]+, C23H24N2O6Na. calc. 447.1532). 11-Hydroxy-14-chloro-15-hydroxy-vincadifformine (3): Colorless oil. [α]23.3 = −140.4 (c = 0.10, MeOH). UV (CDCl3) D λmax(logε) 326 (3.85), 244 (3.76), 228 (3.50), 223 (3.82), 198 (3.44) nm. IR (KBr) νmax 3428, 2945, 1670, 1615, 1270, 1113, 755 cm−1. For NMR data see Tables 1 and 2. EI-MS m/z: 403 ([M − 1]+), 405 ([M + 1]+). HR-ESI-MS (pos.) m/z: 404.1584 ([M − 1 + H]+, C21H26ClN2O4. calc. 404.1581), 406.1852 ([M + 1 + H]+, C21H26ClN2O4. calc. 406.1581). Perakine N1,N4-dioxide (4): Colorless oil. [α]23.3 = − 19.3 D (c = 0.17, MeOH). UV (CDCl3) λmax(logε) 265 (3.88), 221 (3.88), 196 (3.78) nm. IR (KBr) νmax 3425, 2967, 1743, 1645, 1225, 1030, 753 cm−1. For NMR data see Tables 1 and 2. EI-MS m/z: 382 ([M]+). HR-ESI-MS (pos.) m/z: 405.1423 ([M + Na]+, C21H22N2O5Na. calc. 405.1426).
U
133 134
F
t2:3 t2:4 t2:5 t2:6 t2:7 t2:8 t2:9 t2:10 t2:11 t2:12 t2:13 t2:14 t2:15 t2:16 t2:17 t2:18 t2:19 t2:20 t2:21 t2:22 t2:23
Fig. 2. Key HMBCs (
) and 1H–1H COSY (
) correlations of compounds 1 and 4.
Please cite this article as: Zhang L, et al, Monoterpenoid indole alkaloids from Alstonia rupestris with cytotoxic, antibacterial and antifungal activities, Fitoterapia (2014), http://dx.doi.org/10.1016/j.fitote.2014.05.018
155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171
221
3. Results and discussion
222
3.1. Chemistry
223 224
Compound 1 was obtained as a colorless oil. The HRESIMS displayed a pseudomolecular ion at m/z 389.1473 [M + Na]+ (calcd for C21H22N2O4Na, 389.1477) consistent with a molecular formula of C21H22N2O4, corresponding to 12° of unsaturation. The IR spectrum exhibited absorptions at 3385, 1657 and
199 200 201 202 Q3 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219
225 226 227
C
197 198
E
195 196
R
193 194
R
191 192
O
189 190
C
187 188
N
185 186
U
183 184
F
220
All compounds (purity N 90%) were screened for their antimicrobial activity in vitro using the disk-diffusion method as described in the literature with minor modifications [22]. Strains including two species of bacteria [Staphylococcus aureus (ATCC-25923), Mycobacterium tuberculosis (ATCC-25177/H37Ra)] and five species of fungi [Gibberella pulicaris (KZN 4207), Alternaria alternata (TX-8025), Colletotrichum nicotianae (SACC-1922), Phytophthora capsici (KACC-40157), Gonatopyricularia amomi (MB-9671)] were used. Rifampicin and nystatin were used as positive controls for antibacterial and antifungal activities, respectively. A disk containing only DMSO was used as the negative control. Medium used in the antimicrobial activity included nutrient agar medium (S. aureus), Dorset egg medium (M. tuberculosis) and Sabouraud dextrose broth (SDB) agar medium (five species of fungi). To each agar plate, an inoculum containing 107 bacteria/mL or a 0.5 optical density of the McFarland Scale was incorporated. The plates were solidified and sterile filter paper disks (6-mm diameter) were done on each one. Solution of each compound (5 mM) in DMSO, antibacterial agents (rifampicin 5 μM/mL), antifungal agents (nystatin 10 μM/mL), and control vehicles (DMSO) was added into too. The plates were aerobically incubated at 37 °C for S. aureus for 18 h, for the five species of fungi for 24 h and for M. tuberculosis for 15–45 days, and four assays under identical conditions were carried out for each one. The diameter of the inhibition zone was measured for testing of antibacterial and antifungal activities. Experiments were performed in triplicate, and the results are presented as the mean values of the diameters of the inhibitory zones from three runs. The MIC values of the most active compounds, in the previous experiment, were determined using the dilution method in 96-hole plates. The diameters of the inhibitory zones and the MIC value were used as criteria to judge the antimicrobial activity (active: the diameters of the inhibitory zones ≥16 mm, MIC ≤ 5 mM; moderately active: the diameters of the inhibitory zones are visible, MIC N 5 mM; not active: the diameters of the inhibitory zones are invisible). All strains of bacteria and fungi were purchased from Shanghai Institute of Biochemistry & Cell Biology, Chinese Academy of Sciences (Shanghai, China).
O
181 182
177 178
R O
2.5. Antimicrobial activity bioassay
176
P
180
174 175
1617 cm−1 that indicated the presence of a β-anilinoacrylate chromophore, corresponding to the carbon signals for an acrylate double bond at δC 166.5 (C-2) and 89.1 (C-16). Its 13 C NMR spectrum showed 21 carbon signals [OCH3 × 1, CH3 (sp3) × 1, CH2 (sp3) × 4, CH (sp3) × 3, C (sp3) × 2, CH (sp2) × 4 and C (sp2) × 6, Table 1]. The 1H NMR spectrum exhibited four aromatic proton signals [δH 7.60 and 6.75 (each, 1H, dd, J = 7.8, 1.8 Hz), 6.79 and 7.06 (each, 1H, dt, J = 7.8, 1.8 Hz)] ascribed to an ortho-disubstituted benzene ring, two characteristic proton signals due to an oxirane ring at δH 3.61 and 3.48 (each, d, J = 4.0), and a NH signal at δH 8.96. The 14,15-epoxy group was established by the HMBC correlations of δH 3.48 (H-15) with δC 165.1 (C-3), 26.2 (C-19) and 63.3 (C-21) and of δH 3.61 (H-14) with δC 40.6 (C-20) (Fig. 2). These data suggested that the structure of 1 was almost identical with 5 [3]. The distinct difference was that the methoxy at C-11 in 5 was absent in 1, which was supported by the observation of upfield chemical shift of C-11 from δC 156.4 in 5 to δC 127.8 in 1. The relative configuration of 1 was confirmed by a NOESY experiment and comparison of the NMR data of 1 with those of compound 5. According to NOESY data and its negative specific rotation ([α]23.3 = −68.4), the D α-orientation of the 14,15-epoxy group was determined by the correlation between H-15 and H-17β (Cao et al., 2012). Thus, the structure of 1 was established as 6,7-epoxy-8oxovincadifformine. Compound 2, a colorless oil, exhibited a molecular formula of C23H24N2O6, based on the HRESIMS spectrum which showed a pseudomolecular ion at m/z 447.1533 [M + Na]+ (calcd. 447.1532). Comparing the 1H and 13C NMR data of 2 with those of compound 1, the data were almost identical. The only significant difference was the presence of one more OAc group in compound 2. The OAc group was positioned at C-11 based on the NOE correlations of H-12/H-N1 and the presence of the ABX-system [δH 7.16 (d, J = 8.0 Hz, H-9), 6.71 (dd, J = 8.0, 2.0 Hz, H-10), 6.75 (d, J = 2.0 Hz, H-12)]. The stereochemistry of 2 was expected to be the same as 5 on the basis of the NOESY data. Therefore, 2 was identified as 11-acetyl-6,7-epoxy-8-oxo-vincadifformine. Compound 3 exhibited two isotopic peaks at m/z 404.1584 [M − 1 + H]+ (calc. 404.1581) and 406.1852 [M + 1 + H]+ (calc. 406.1581) in its HRESIMS, accounting for a molecular formula of C21H25ClN2O4, suggesting 10° of unsaturation. The existence of the chlorine atom was identified by the appropriate 13C NMR chemical shift at δC 59.3 (d, C-14) and EIMS analysis. The EI mass spectrometry showed two quasimolecular-ion peaks at m/z 403 (41.35, [M − 1]+) and 405 (13.79, [M + 1]+) with ratio of relative intensity approximating 3:1. The NMR data were similar to those of compound 1, except for a chlorine atom and a hydroxy group in 3 taking the place of the 14,15-epoxy group in 1 and 2. The 1H–1H COSY correlations of proton signals at δH 4.16 (H-14) with δH 3.26 (H-3) and δH 3.89 (H-15) indicated the location of the chlorine atom at C-14 (δC 59.3) and a hydroxy group at C-15 (δC 75.6) respectively, which was further supported by the HMBC correlations of H-3 and H-19 with C-15. Additionally, the three ABX-system protons at δH 6.98 (d, J = 7.6 Hz, H-9), 6.33 (dd, J = 7.6, 1.8 Hz, H-10), and 6.35 (d, J = 7.6 Hz, H-12) indicated the presence of the OH group at C-11 in 3. The α-orientation of H-14 and the β-orientation of H-15 were deduced from the NOESY correlations of H-14/H-21 and H-15/
T
179
further incubated for 4 h. The cells in each well were then solubilized with DMSO (100 μL for each well) and the optical density (OD) was recorded at 570 nm. All drug doses were tested in triplicate and the IC50 values were derived from the mean OD values of the triplicate tests versus drug concentration curves. All cell lines were purchased from the Cell Bank of the Shanghai Institute of Biochemistry & Cell Biology, Chinese Academy of Sciences (Shanghai, China).
D
172 173
L. Zhang et al. / Fitoterapia xxx (2014) xxx–xxx
E
4
Please cite this article as: Zhang L, et al, Monoterpenoid indole alkaloids from Alstonia rupestris with cytotoxic, antibacterial and antifungal activities, Fitoterapia (2014), http://dx.doi.org/10.1016/j.fitote.2014.05.018
228 229 230 231
232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 Q4 279 280 281 282 283 284 285 286 287 288
L. Zhang et al. / Fitoterapia xxx (2014) xxx–xxx
Cell lines
t3:4 t3:5 t3:6 t3:7 t3:8 t3:9 t3:10 t3:11
1 2 3 4 5 6 Doxorubicin
t3:12
a
SCL-1
CAL-27
UMSCC-1
Detroit-562
SCC-PKU
TCA-83
10.3 12.9 52.7 44.1 16.3 47.8 18.3
11.3 12.3 51.8 40.8 15.7 51.5 14.7
9.2 10.8 49.0 44.8 14.8 44.8 22.0
12.0 12.7 59.4 50.7 17.2 49.1 31.7
10.7 11.3 54.3 48.9 14.7 53.2 24.9
13.7 12.9 59.7 47.0 11.2 43.6 35.4
13.0 14.9 59.5 40.1 15.5 48.2 15.9
3.2. Cytotoxic activity
311
All these compounds were in vitro evaluated for their cytotoxic potential against seven tumor cell lines by using the revised MTT method as described in the experimental part. The results are summarized in Table 3. The alkaloids isolated from the family Amaryllidaceae have been reported to show strong cytotoxic activities against various types of cancer and bacteria cell lines, which were agreed well with our results [5,6,13]. Alkaloids 1, 2, and 5 exhibited significant cytotoxicity against all tested cell lines of the head and neck squamous cell carcinoma (Hep-2, SCL-1, CAL-27, UMSCC-1, Detroit-562, SCC-PKU, and TCA-83) with IC50 values less than
305 306 307 308
312 313 314 315 316 317 318 319 320 321 t4:1 t4:2
E
303 304
R
301 302
R
299 300
O
297 298
N C
295 296
U
293 294
3.3. Antimicrobial assay in vitro
324
All compounds were tested for their antimicrobial activities by using the disk diffusion method by measuring the inhibition zones and for the most active compounds, minimum inhibitory concentration (MIC) values were also determined (Table 4). The results showed that alkaloids 1, 2 and 5 exhibited significant activity against two fungi (A. alternata and P. capsici) with MIC values of 0.66–0.99 mM, 0.87–1.10 mM and 1.53–1.64 mM, respectively, and moderate activity of the bacterium S. aureus. Compounds 4 and 6 showed potent antibacterial activities against S. aureus, while 3 had no activity. Additionally, 4 possessed higher antibacterial activities against S. aureus than 6, indicating that the formyl group on C-20 might enhance the activities for these monoterpenoid indole alkaloids.
325 326 Q5
References
339
P
310
291 292
20 μM, while no cytotoxicity was detected for other alkaloids 322 (IC50 ≥ 40 μM). 323
C
309
H-17β, respectively. Accordingly, compound 3 was identified as 11-hydroxy-14-chloro-15-hydroxy-vincadifformine. Compound 4 was obtained as a colorless oil. Its positive HRESIMS spectrum showed a quasimolecular ion peak at m/z 405.1423 [M + Na]+, consistent with the molecular formula C21H22N2O5, accounting for 12° of unsaturation. The 13C NMR and DEPT spectra displayed signals of two Me, two CH2, and twelve CH groups, together with five quaternary C-atoms. The NMR signals at δH 7.76 (dd, J = 8.0, 2.0 Hz, H-9), 7.61 (dt, J = 8.0, 2.0 Hz, H-10), 7.64 (dt, J = 8.0, 2.0 Hz, H-11), and 7.81 (dd, J = 8.0, 2.0 Hz, H-12) are assigned to an ortho-disubstituted benzene ring. The 1D and 2D-NMR data were closely related to those of 6 [3]. The only significant difference was that the carboxy group at C-20 in 6 was replaced by a formyl group in compound 4, which was confirmed by the HMBC correlations of the proton signal of the formyl group [δH 9.83 (1H, d, J = 7.0)] with C-15 and C-19 (Fig. 2). The NOESY analysis showed that the relative configurations of the C-3, C-5, C-15, C-16, and C-17 were in good agreement with those of compounds 4 and 6. Therefore, compound 4 was identified as perakine N1,N4-dioxide.
R O O
Doxorubicin activities are expressed as IC50 values in nM, and those of compounds 1–6 are expressed as IC50 values in μM. (−) IC50 N 100 μM.
D
289 290
Hep-2
F
Compounds
E
t3:3
Table 3 Cytotoxicity of compounds 1–6 against seven human tumor cell lines (IC50, μM)a.
T
t3:1 t3:2
5
[1] Narine LL, Maxwell AR. Monoterpenoid indole alkaloids from Palicourea crocea. Phytochem Lett 2009;2:34–6. [2] Tan SJ, Lim KH, Subramaniam G, Kam TS. Macroline-sarpagine and macroline-pleiocarpamine bisindole alkaloids from Alstonia angustifolia. Phytochemistry 2013;85:194–202. [3] Cao P, Liang Y, Gao X, Li XM, Song ZQ, Liang GB. Monoterpenoid indole alkaloids from Alstonia yunnanensis and their cytotoxic and antiinflammatory activities. Molecules 2012;17:13631–41. [4] Lim SH, Low YY, Tan SJ, Lim KH, Thomas NF, Kam TS. Perhentidines A–C: macroline–macroline bisindoles from Alstonia and the absolute configuration of perhentinine and macralstonine. J Nat Prod 2012;75:942–50. [5] Lim SH, Tan SJ, Low YY, Kam TS. Lumutinines A–D, linearly fused macroline–macroline and macroline–sarpagine bisindoles from Alstonia macrophylla. J Nat Prod 2011;74:2556–62. [6] Tan SJ, Choo YM, Thomas NF, Robinson WT, Komiyama K, Kam TS. Unusual indole alkaloid–pyrrole, –pyrone, and –carbamic acid adducts from Alstonia angustifolia. Tetrahedron 2010;66:7799–806.
Table 4 Antimicrobial and antifungal activities (zones of inhibition/and MIC mM, n = 3) of compounds 1–6.
t4:3
Compounds
S.aureus
M. tuberculosis
G. pulicaris
A. alternata
C. nicotianae
P. capsici
G. amomi
t4:4 t4:5 t4:6 t4:7 t4:8 t4:9 t4:10 t4:11
1 2 3 4 5 6 Rifampicin Nystatin
15.72 16.33 – 23/0.49 14.91 19/0.83 25/0.003 –
– – – – – – 22/0.003 –
– – – – – – – 20/0.008
19/0.66 20/0.87 – – 19/1.53 – – 17/0.007
– – – – – – – 21/0.006
18/0.99 17/1.10 – – 16/1.64 – – 18/0.061
– – – – – – – 19/0.010
t4:12
No activity.
Please cite this article as: Zhang L, et al, Monoterpenoid indole alkaloids from Alstonia rupestris with cytotoxic, antibacterial and antifungal activities, Fitoterapia (2014), http://dx.doi.org/10.1016/j.fitote.2014.05.018
327 328 329 330 331 332 333 334 335 336 337 338
340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356
[17]
[18]
[19] [20] [21]
[22]
F
[16]
O
[15]
spatulata. Revision of the C-20 configuration of scholaricine. J Nat Prod 2010;73:1891–7. Hutchinson CR. Tetrahedron report number 105: camptothecin: chemistry, biogenesis and medicinal chemistry. Tetrahedron 1981;37:1047–65. Jagetia GC, Baliga MS. Evaluation of anticancer activity of the alkaloid fraction of Alstonia scholaris (Sapthaparna) in vitro and in vivo. Phytother Res 2006;20:103–9. Kam TS, Choo YM, Komiyama K. Unusual spirocyclic macroline alkaloids, nitrogenous derivatives, and a cytotoxic bisindole from Alstonia. Tetrahedron 2004;60:3957–66. Kam TS, Tan SJ, Ng SW, Komiyama K. Bipleiophylline, an unprecedented cytotoxic bisindole alkaloid constituted from the bridging of two indole moieties by an aromatic spacer unit. Org Lett 2008;10:3749–52. Khan MR, Omoloso AD, Kihara M. Antibacterial activity of Alstonia scholaris and Leea tetramera. Fitoterapia 2003;74:736–40. Chen WM, Yan YP, Liang XT. Alkaloids from roots of Alstonia yunnanensis. Planta Med 1983;49:62. Yu JO, Liao ZX, Lei JC, Hu XM. Antioxidant and cytotoxic activities of various fractions of ethanol extract of Dianthus superbus. Food Chem 2007;104:1215–9. Zhang W, Hu JF, Lv WW, Zhao QC, Shi GB. Antibacterial, antifungal and cytotoxic isoquinoline alkaloids from Litsea cubeba. Molecules 2012;17:12950–60.
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R
R
E
C
T
E
D
P
[7] Arai H, Hirasawa Y, Rahman A, Kusumawati I, Zaini NC, Sato S, et al. Alstiphyllanines E–H, picraline and ajmaline-type alkaloids from Alstonia macrophylla inhibiting sodium glucose cotransporter. Bioorg Med Chem 2010;18:2152–8. [8] Hirasawa Y, Arai H, Zaima K, Oktarina R, Rahman A, Ekasari W, et al. Alstiphyllanines A–D, indole alkaloids from Alstonia macrophylla. J Nat Prod 2009;72:304–7. [9] Arai H, Hirasawa Y, Rahman A, Kusumawati I, Zaini NC, Sato S, et al. Alstiphyllanines E–H, picraline and ajmaline-type alkaloids from Alstonia macrophylla inhibiting sodium glucose cotransporter. Bioorg Med Chem 2010;18:2152–8. [10] Lim SH, Low YY, Tan SJ, Lim KH, Thomas NF, Kam TS. Perhentidines A–C: macroline–macroline bisindoles from Alstonia and the absolute configuration of perhentinine and macralstonine. J Nat Prod 2012;75:942–50. [11] Hirasawa Y, Arai H, Zaima K, Oktarina R, Rahman A, Ekasari W, et al. Alstiphyllanines A–D, indole alkaloids from Alstonia macrophylla. J Nat Prod 2009;72:304–7. [12] Koyama K, Hirasawa Y, Nugroho AE, Hosoya T, Hoe TC, Chan KL, et al. Alsmaphorazines A and B, novel indole alkaloids from Alstonia pneumatophora. Org Lett 2010;12:4188–91. [13] Ku WF, Tan SJ, Low YY, Komiyama K, Kam TS. Angustilobine and andranginine type indole alkaloids and an uleine-secovallesamine bisindole alkaloid from Alstonia angustiloba. Phytochemistry 2011;72:2212–8. [14] Tan SJ, Low YY, Choo YM, Abdullah Z, Etoh T, Hayashi M, et al. Strychnan and secoangustilobine A type alkaloids from Alstonia
U
357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 405
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R O
6
Please cite this article as: Zhang L, et al, Monoterpenoid indole alkaloids from Alstonia rupestris with cytotoxic, antibacterial and antifungal activities, Fitoterapia (2014), http://dx.doi.org/10.1016/j.fitote.2014.05.018
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