970
CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
Cytotoxic Mannopyranosides of Indole Alkaloids from Zanthoxylum nitidum by Jiang Hu* a ) b ), Xiaodong Shi a ), Xia Mao a ), Jiangang Chen a ), and Hui Li c ) a
) College of Biological Resources and Environment Science, Qujing Normal University, Sanjiang Avenue, Unicorn District, Qujing 655011, P. R. China (phone/fax: þ 86-874-8998627; e-mail: [email protected]) b ) Institute of Characteristic Medicinal Resource of Ethnic Minorities, Qujing Normal University, Qujing 655011, P. R. China c ) College of Physics and Electronic Engineering, Qujing Normal University, Qujing 655011, P. R. China
Three new mannopyranosides of indole alkaloids, methyl 7-(b-d-mannopyranosyloxy)-1H-indole-2carboxylate (1), methyl 7-[(3-O-acetyl-b-d-mannopyranosyl)oxy]-1H-indole-2-carboxylate (2), and 2methyl-1H-indol-7-yl b-d-mannopyranoside (3), were isolated from an EtOH extract of the roots of Zanthoxylum nitidum. Their structures were identified as new compounds on the basis of the spectroscopic analyses. Bioactivity evaluation revealed that these alkaloids possess significant cytotoxicities against all the tested tumor cell lines with IC50 values of less than 30 mm.
Introduction. – Zanthoxylum nitidum (Roxb.) DC. (Rutaceae) occurs as a liane in rainforest and as a shrub in dryer habitats, and is widely distributed from India, northern Queensland (Australia), and throughout the southeastern part of China [1]. Z. nitidum has been used as detumescent to treat toothache, neuralgia, stomachache, sore throat, rheumatoid arthritis, turgescence, and venomous snake bites, etc. for more than 1,000 years in traditional Chinese medicine, and its fruit has been used as a spice [2]. As reported previously, the chemical components of Z. nitidum are mainly alkaloids [1] [3 – 5], coumarins [6], benzenoids, steroids [7], and derivatives thereof, among which alkaloids are considered as the main bioactive constituents [8] [9]. Thus, the amount of alkaloids is an important index to evaluate the therapeutic effects in clinical application for Z. nitidum. The present study, undertaken to investigate the chemical constituents of the roots of Z. nitidum, led to the isolation of three new mannopyranosides of indole alkaloids, named methyl 7-(b-d-mannopyranosyloxy)-1Hindole-2-carboxylate (1), methyl 7-[(3-O-acetyl-b-d-mannopyranosyl)oxy]-1H-indole2-carboxylate (2), and 2-methyl-1H-indol-7-yl b-d-mannopyranoside (3). Herein, we report their isolation and structure elucidation. Furthermore, all the alkaloids were evaluated for their in vitro cytotoxic activities.
2014 Verlag Helvetica Chimica Acta AG, Zrich
CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
971
Results and Discussion. – Chemistry. Compound 1 was obtained as a pale-yellow amorphous solid. The positive-ion-mode HR-ESI-MS exhibited a quasi-molecular-ion peak at m/z 354.1187 ([M þ H] þ ; calc. 354.1189) corresponding to the molecular formula C16H19NO8 . The UV spectrum exhibited absorption maxima characteristic of an indole chromophore (223, 283, and 300 nm) [10]. The 1H- and 13C-NMR spectra (Table 1) indicated the presence of a substituted indole ring and a COOMe group. In addition, six aliphatic signals of a hexose unit were detected in the 13C-NMR spectrum. Using the NH and HC(3) as starting points, 1H and 13C resonances of the aromatic core were assigned on the basis of HMQC and HMBC features (Fig.). Acid hydrolysis of 1 with 1m HCl in dioxane/H2O 1 : 1 gave an indole alkaloid and d-mannose as the carbohydrate component. The monosaccharide, including its absolute configuration, was identified by direct HPLC analysis of the hydrolysate, with detection being carried out by using an optical rotation (OR) detector. The H-atom multiplet patterns and coupling constants, as well as the H- and C-atom chemical shifts pointed to a b-dmannopyranosyl unit, linked to the aglycone. The position C(7) as the attachment point of the sugar unit was established by the HMBCs between C(7) (d(C) 143.5) of the indole alkaloid and the anomeric H-atom (d(H) 4.49 (HC(1’)) of the mannose moiety (Fig.). The HMBCs between HN(1) (d(H) 10.52) and HC(3) (d(H) 5.92) with the C-atom of the C¼O group (d(C) 163.3) indicated that the COOMe group was attached at C(2). Accordingly, 1 was determined to be methyl 7-(b-d-mannopyranosyloxy)-1Hindole-2-carboxylate.
Table 1. 1H- and 13C-NMR Data of Compounds 1 – 3 (500 and 125 MHz, resp., in CD3OD). d in ppm, J in Hz. Position
1 d( H)
HN(1) 2 3 3a 4 5 6 7 7a C¼O MeO 2-Me 1’ 2’ 3’ 4’ 5’ 6’ Ac
2 d(C )
10.52 (s) 5.92 (s) 7.45 (d, J ¼ 8.6) 7.10 (dd, J ¼ 8.6, 10.0) 7.34 (d, J ¼ 10.0)
3.94 (s) – 4.49 (d, J ¼ 1.8) 3.89 (dd, J ¼ 1.8, 2.0) 3.55 (dd, J ¼ 1.8, 8.8) 3.49 (dd, J ¼ 8.8, 9.0) 3.29 – 3.31 (m) 3.85, 3.65 (dd, J ¼ 5.0, 12.2) –
3
d( H )
d(C )
10.54 (s) 129.0 97.0 128.3 115.5 121.3 116.0 143.5 128.7 163.3 56.2 – 102.2 71.6 74.4 68.4 77.5 62.5 – –
5.93 (s) 7.46 (d, J ¼ 8.6) 7.10 (dd, J ¼ 8.6, 10.0) 7.36 (d, J ¼ 10.0)
3.95 (s) – 4.51 (d, J ¼ 1.8) 4.37 (dd, J ¼ 1.8, 2.3) 4.28 (dd, J ¼ 2.3, 9.0) 3.99 (dd, J ¼ 8.8, 9.0) 3.30 – 3.32 (m) 3.84, 3.65 (dd, J ¼ 5.0, 12.2) 2.14 (s)
d( H)
d(C )
10.57 (s) 129.1 97.0 128.4 115.4 121.4 116.2 143.8 128.7 163.4 56.1 – 101.8 67.9 76.6 64.3 78.2 62.6 170.6, 20.9
5.12 (s) 7.11 (d, J ¼ 8.6) 6.81 (dd, J ¼ 8.6, 10.0) 7.00 (d, J ¼ 10.0)
– 2.31 (s) 4.47 (d, J ¼ 2.0) 3.88 (dd, J ¼ 2.0, 2.2) 3.52 (dd, J ¼ 2.2, 9.0) 3.46 (dd, J ¼ 8.6, 9.0) 3.25 – 3.27 (m) 3.83, 3.62 (dd, J ¼ 5.0, 12.2) –
135.4 101.5 127.5 115.1 122.0 115.7 144.1 127.6 – – 20.3 102.3 71.8 74.6 68.5 77.7 62.7 – –
972
CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
Figure. Key HMBCs (H ! C) of compounds 1 and 3
Compound 2 was obtained as a pale-yellow amorphous powder. The EI-MS indicated a molecular weight corresponding to the peak at m/z 395, and its HR-ESI-MS exhibited the [M þ Na] þ peak at m/z 418.1113 (calc. 418.1114), corresponding to the molecular formula C18H21NO9 . The general features of its IR and NMR spectra closely resembled those of 1, except for the presence of an additional Ac group. The AcO group was at C(3’) based on HMBCs of HC(3’) (d(H) 4.28 (dd, J ¼ 2.3, 9.0)) with the Ac C-atom (d(C) 170.6). Therefore, compound 2 was elucidated as methyl 7-[(3-Oacetyl-b-d-mannopyranosyl)oxy]-1H-indole-2-carboxylate. Compound 3, a yellow amorphous solid, exhibited a quasi-molecular-ion peak, in the HR-MS, at m/z 310.1292 ([M þ H] þ ; calc. 310.1291), which corresponded to the molecular formula C15H19NO6 . The UV spectrum showed absorption maxima at 223, 283, and 300 nm indicating the presence of an indole chromophore. The only 13C-NMR difference between 3 and 1 was that the COOMe moiety in 1 was replaced by the signals for a Me group, which was further confirmed by the HMBC experiments (Fig.). Correspondingly, the resonances of C(2) and C(3) were shifted downfield to 135.4 and 101.5, respectively. The HMBCs between C(7) (d(C) 144.1) of the indole alkaloid and the anomeric H-atom (d(H) 4.47) of the mannose unit were observed (Fig.). Consequently, compound 3 was unambiguously determined as 2-methyl-1H-indol-7-yl b-d-mannopyranoside. Biology. The in vitro cytotoxic activities of the isolated alkaloids were evaluated against eight tumor cell lines by using the revised 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl-2H-tetrazolium bromide (MTT) method as described in the Exper. Part (Table 2). All alkaloids showed significant cytotoxicities (IC50 < 30 mm), with alkaloid 3 possessing a higher cytotoxic activity (IC50 9.21 – 12.75 mm) than alkaloids 1 (IC50
Table 2. Cytotoxicities ( IC50 [mm] ) of Compounds 1 – 3 against Eight Human Tumor Cell Lines Compound
1 2 3 Doxorubicin a ) a
Cell lines A-549
BGC-823
HCT15
HeLa
HepG2
MCF-7
SGC-7901
SK-MEL-2
18.11 23.78 11.41 0.01
19.32 25.31 11.97 0.01
20.47 26.95 11.12 0.06
20.88 26.78 10.45 0.05
19.20 23.47 10.95 0.03
20.21 27.11 9.78 0.01
21.04 28.12 9.21 0.06
20.47 26.77 12.75 0.01
) Positive control.
CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
973
18.11 – 21.04 mm) and 2 (IC50 23.47 – 28.12 mm). The results indicated that the Me group at C(2) might effectively enhance the cytotoxic activities for this type of alkaloids. Experimental Part 1. General. All solvents were distilled before use. TLC: SiO2 GF254 (10 – 40 mm; Qingdao Marine Chemical Factory, Qingdao, China). Column chromatography (CC): silica gel (SiO2 ; 200 – 300 mesh, 10 – 40 mm; Qingdao Marine Chemical Factory, Qingdao, China), Sephadex LH-20 (Amersham Pharmacia Biotech, Sweden), MCI Gel CHP20P (75 – 150 mm; Mitsubishi Kasei Chemical Industries), and C18 reversed-phase SiO2 (20 – 45 mm; Fuji Silysia Chemical Ltd.). HPLC (anal. and prep.): Shimadzu model LC-8A on YMC-pack, R&D ODS column (250 4.6 mm, 250 20 mm) and UV detector Shimadzu SPD-10AVP. M.p.: Tempo melting-point apparatus; uncorrected. Optical rotations: JASCO-20C digital polarimeter. UV Spectra: Hewlett-Packard-8452A diode-array spectrophotometer; lmax (log e) in nm. IR Spectra: PerkinElmer 577 spectrometer; ˜n in cm 1. 1H- and 13C-NMR spectra: Bruker AM-400 spectrometer; d in ppm rel. to Me4Si as internal standard, J in Hz. MS: VG AutoSpec-3000 mass spectrometer; in m/z (rel. %). HR-ESI-MS: API QSTAR Pulsar-1 mass spectrometer; in m/z. 2. Plant Material. The roots of Zanthoxylum nitidum were collected in Guangxi Province, China, in August of 2011, and identified by one of the authors (J. C.). A voucher specimen (ZN20110801) was deposited with the Herbarium of the College of Biological Resources and Environment Science, Qujing Normal University, Qujing, Yunnan Province, P. R. China. 3. Extraction and Isolation. The air-dried and powdered root wood (25 kg) of Z. nitidum was refluxed with 80% EtOH (30 l). After removal of EtOH under reduced pressure, the aq. brownish syrup (6 l) was adjusted to pH 2 with 2% HCl and filtered. The filtrate was adjusted to pH 9 by adding 20% NaOH, and then extracted with CHCl3 to afford total alkaloid content. The CHCl3 extract (65 g) was subjected to CC (SiO2 ; gradient CHCl3/MeOH) to afford 18 fractions, Frs. A1 – A18. Fr. A13 (CHCl3 , 5.24 g) was seperated by CC (RP-18 gel; MeOH/H2O from 70 to 95%) to yield three subfractions, Frs. A13a – A13c. Fr. A13b (879 mg) were purified by HPLC to afford 2 (128 mg) and 3 (115 mg). Fr. A13c (997 mg) was separated by repeated CC (Sephadex LH-20; CHCl3/MeOH 1 : 1 and MeOH) to yield 1 (74.3 mg). Methyl 7-(b-d-Mannopyranosyloxy)-1H-indole-2-carboxylate (1). Pale-yellow amorphous solid. M.p. 152 – 1738. UV (EtOH): 223 (3.93), 283 (3.45), 300 (3.78). 1H- and 13C-NMR: Table 1. EI-MS: 353 (2, M þ ), 301 (10), 180 (7), 138 (5), 105 (100), 77 (61), 65 (9), 51 (17). ESI-MS: 354.1 ([M þ H] þ ). HREI-MS: 354.1187 ([M þ H] þ , C16H20NO þ8 ; calc. 354.1189). Methyl 7-[(3-O-Acetyl-b-d-mannopyranosyl)oxy]-1H-indole-2-carboxylate (2). Pale-yellow amorphous solid. M.p. 148 – 1668. UV (EtOH): 224 (3.85), 284 (3.37), 300 (3.92). 1H- and 13C-NMR: Table 1. EI-MS: 395 (2, M þ ), 352 (18), 180 (10), 105 (100), 65 (9), 51 (17). ESI-MS: 354.1 ([M þ H] þ ). HR-EIMS: 418.1113 ([M þ Na] þ , C18H21NNaO þ9 ; calc. 418.1114). 2-Methyl-1H-indol-7-yl b-d-Mannopyranoside (3). Yellow amorphous solid. M.p. 113 – 1248. UV (EtOH): 223 (3.89), 283 (3.51), 300 (3.81). 1H- and 13C-NMR: Table 1. HR-EI-MS: 310.1292 ([M þ H] þ , C15H20NO þ6 ; calc. 310.1291). 4. Acid Hydrolysis of 1. A soln. of 1 (10 mg) in 1m HCl (dioxane/H2O 1 : 1, 4 ml) was heated at 958 for 2 h under an air atmosphere. After cooling, the mixture was neutralized by passing through an Amberlite IRA-93ZU (Organo, Tokyo, Japan) column and subjected to CC (SiO2 ; CHCl3/MeOH 19 : 1, 9 : 1, and 1 : 1) to give an aglycone and a sugar fraction (2.3 mg). HPLC Analysis of the sugar fraction under the following conditions revealed the presence of d-mannose. Column, Capcell Pak NH2UG80 (4.6 mm i.d. 250 mm, 5 mm, Shiseido), detector: Shodex OR-2 (Showa-Denko, Tokyo, Japan); solvent, MeCN/H2O 17 : 3; flow rate: 1.0 ml/min; tR [min], 14.21 (d-mannose, positive optical rotation). 5. Cytotoxicity Assay in vitro. The revised MTT method was used for in vitro evaluation of the cytotoxic potential of the isolated compounds against eight cultured human tumor cell lines [11]. The cell lines, A-549 (human lung cancer), BGC-823 cells (human gastric carcinoma), HCT15 (human colon cancer), HeLa cells (human cervical cancer), HepG2 cells (human hepatocellular carcinoma), MCF-7 cells (human breast cancer), SK-MEL-2 (human skin cancer), and SGC-7901 cells (human gastric adenocarcinoma) were cultured on RPMI-1640 medium supplemented with 10% fetal bovine serum,
974
CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
100 U/ml benzyl penicillin, and 100 mg/ml streptomycin in 25-cm2 culture flasks at 378 in humidified atmosphere with 5% CO2 . For the cytotoxicity tests, cells in exponential growth stage were harvested from the culture by trypsin digestion and centrifuging at 180 g for 3 min, then resuspended in fresh medium at a cell density of 5 104 cells per ml. The cell suspension was dispensed into a 96-well microplate at 100 ml per well, and incubated in humidified atmosphere with 5% CO2 at 378 for 24 h, and then treated with the compounds at various concentrations (0, 1, 10, and 100 mm). After 48 h of treatment, 50 ml of 1 mg/ml MTT soln. was added to each well, and further incubated for 4 h. The cells in each well were then solubilized with DMSO (100 ml for each well), and the optical density (OD) was recorded at 595 nm. All drug doses were tested with doxorubicin as positive control in triplicate, and the IC50 values were derived from the mean OD values of the triplicate tests vs. drug concentration curves. Concentrations that inhibit cell growth by 50% (IC50) compared to non-treated cells were determined by non-linear regression with Graphpad Prism software version 4.0 (GraphPad Software, Inc., San Diego, CA). This work was supported by the grants from the Scientific Planning Project of the Applied Basic Research of Yunnan Province (S2012FZ0005), the Key Projects of Scientific Research Foundation of the Department of Education of Yunnan Province (2013Z095), the Key Projects in Scientific Research of Qujing Normal University (2011ZD003), and the Developing Key Subject of Ecology of Qujing Normal University.
REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9] [10]
[11]
D. Y. Kong, A. I. Gray, T. G. Hartley, P. G. Waterman, Biochem. Syst. Ecol. 1996, 24, 87. J. J. Chen, Y. H. Lin, S. H. Day, T. L. Hwang, I. S. Chen, Food. Chem. 2011, 125, 282. J. Hu, W. D. Zhang, R. H. Liu, C. Zhang, Y. H. Shen, H. L. Li, Chem. Biodiversity 2006, 3, 990. J. Hu, W. D. Zhang, Y. H. Shen, C. Zhang, L. Xu, R. H. Liu, B. Wang, X. K. Xu, Biochem. Syst. Ecol. 2007, 35, 114. C. H. Yang, M. J. Cheng, M. Y. Chiang, Y. H. Kuo, C. J. Wang, I. S. Chen, J. Nat. Prod. 2008, 71, 669. J. Shen, X. Zhang, Z. Tang, S. Peng, L. Ding, Chin. Herb. Med. 2004, 35, 619. J. Hu, W. D. Zhang, R. H. Liu, C. Zhang, Y. H. Shen, X. K. Xu, China J. Chin. Mater. Med. 2006, 31, 1689. Z. Dvora´k, R. Vrzal, P. Maurel, J. Ulrichova´, Chem.-Biol. Interact. 2006, 159, 117. D. Li, B. Zhao, S. P. Sim, T. K. Li, A. Liu, L. F. Liu, E. J. LaVoie, Bioorg. Med. 2003, 11, 521. F. Tillequin, S. Michel, E. Seguin, Tryptamine-Derived Indole Alkaloids, in Methods in Plant Biochemistry: Alkaloids and Sulfur Compounds, Eds. P. M. Dey, J. B. Harborne, Academic Press, London, 1993, Vol. 8, p. 309. J. J. Chen, H. C. Hung, P. J. Sung, I. S. Chen, W. L. Kuo, Phytochemistry 2011, 72, 523. Received December 8, 2013
CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
Cytotoxic Mannopyranosides of Indole Alkaloids from Zanthoxylum nitidum by Jiang Hu* a ) b ), Xiaodong Shi a ), Xia Mao a ), Jiangang Chen a ), and Hui Li c ) a
) College of Biological Resources and Environment Science, Qujing Normal University, Sanjiang Avenue, Unicorn District, Qujing 655011, P. R. China (phone/fax: þ 86-874-8998627; e-mail: [email protected]) b ) Institute of Characteristic Medicinal Resource of Ethnic Minorities, Qujing Normal University, Qujing 655011, P. R. China c ) College of Physics and Electronic Engineering, Qujing Normal University, Qujing 655011, P. R. China
Three new mannopyranosides of indole alkaloids, methyl 7-(b-d-mannopyranosyloxy)-1H-indole-2carboxylate (1), methyl 7-[(3-O-acetyl-b-d-mannopyranosyl)oxy]-1H-indole-2-carboxylate (2), and 2methyl-1H-indol-7-yl b-d-mannopyranoside (3), were isolated from an EtOH extract of the roots of Zanthoxylum nitidum. Their structures were identified as new compounds on the basis of the spectroscopic analyses. Bioactivity evaluation revealed that these alkaloids possess significant cytotoxicities against all the tested tumor cell lines with IC50 values of less than 30 mm.
Introduction. – Zanthoxylum nitidum (Roxb.) DC. (Rutaceae) occurs as a liane in rainforest and as a shrub in dryer habitats, and is widely distributed from India, northern Queensland (Australia), and throughout the southeastern part of China [1]. Z. nitidum has been used as detumescent to treat toothache, neuralgia, stomachache, sore throat, rheumatoid arthritis, turgescence, and venomous snake bites, etc. for more than 1,000 years in traditional Chinese medicine, and its fruit has been used as a spice [2]. As reported previously, the chemical components of Z. nitidum are mainly alkaloids [1] [3 – 5], coumarins [6], benzenoids, steroids [7], and derivatives thereof, among which alkaloids are considered as the main bioactive constituents [8] [9]. Thus, the amount of alkaloids is an important index to evaluate the therapeutic effects in clinical application for Z. nitidum. The present study, undertaken to investigate the chemical constituents of the roots of Z. nitidum, led to the isolation of three new mannopyranosides of indole alkaloids, named methyl 7-(b-d-mannopyranosyloxy)-1Hindole-2-carboxylate (1), methyl 7-[(3-O-acetyl-b-d-mannopyranosyl)oxy]-1H-indole2-carboxylate (2), and 2-methyl-1H-indol-7-yl b-d-mannopyranoside (3). Herein, we report their isolation and structure elucidation. Furthermore, all the alkaloids were evaluated for their in vitro cytotoxic activities.
2014 Verlag Helvetica Chimica Acta AG, Zrich
CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
971
Results and Discussion. – Chemistry. Compound 1 was obtained as a pale-yellow amorphous solid. The positive-ion-mode HR-ESI-MS exhibited a quasi-molecular-ion peak at m/z 354.1187 ([M þ H] þ ; calc. 354.1189) corresponding to the molecular formula C16H19NO8 . The UV spectrum exhibited absorption maxima characteristic of an indole chromophore (223, 283, and 300 nm) [10]. The 1H- and 13C-NMR spectra (Table 1) indicated the presence of a substituted indole ring and a COOMe group. In addition, six aliphatic signals of a hexose unit were detected in the 13C-NMR spectrum. Using the NH and HC(3) as starting points, 1H and 13C resonances of the aromatic core were assigned on the basis of HMQC and HMBC features (Fig.). Acid hydrolysis of 1 with 1m HCl in dioxane/H2O 1 : 1 gave an indole alkaloid and d-mannose as the carbohydrate component. The monosaccharide, including its absolute configuration, was identified by direct HPLC analysis of the hydrolysate, with detection being carried out by using an optical rotation (OR) detector. The H-atom multiplet patterns and coupling constants, as well as the H- and C-atom chemical shifts pointed to a b-dmannopyranosyl unit, linked to the aglycone. The position C(7) as the attachment point of the sugar unit was established by the HMBCs between C(7) (d(C) 143.5) of the indole alkaloid and the anomeric H-atom (d(H) 4.49 (HC(1’)) of the mannose moiety (Fig.). The HMBCs between HN(1) (d(H) 10.52) and HC(3) (d(H) 5.92) with the C-atom of the C¼O group (d(C) 163.3) indicated that the COOMe group was attached at C(2). Accordingly, 1 was determined to be methyl 7-(b-d-mannopyranosyloxy)-1Hindole-2-carboxylate.
Table 1. 1H- and 13C-NMR Data of Compounds 1 – 3 (500 and 125 MHz, resp., in CD3OD). d in ppm, J in Hz. Position
1 d( H)
HN(1) 2 3 3a 4 5 6 7 7a C¼O MeO 2-Me 1’ 2’ 3’ 4’ 5’ 6’ Ac
2 d(C )
10.52 (s) 5.92 (s) 7.45 (d, J ¼ 8.6) 7.10 (dd, J ¼ 8.6, 10.0) 7.34 (d, J ¼ 10.0)
3.94 (s) – 4.49 (d, J ¼ 1.8) 3.89 (dd, J ¼ 1.8, 2.0) 3.55 (dd, J ¼ 1.8, 8.8) 3.49 (dd, J ¼ 8.8, 9.0) 3.29 – 3.31 (m) 3.85, 3.65 (dd, J ¼ 5.0, 12.2) –
3
d( H )
d(C )
10.54 (s) 129.0 97.0 128.3 115.5 121.3 116.0 143.5 128.7 163.3 56.2 – 102.2 71.6 74.4 68.4 77.5 62.5 – –
5.93 (s) 7.46 (d, J ¼ 8.6) 7.10 (dd, J ¼ 8.6, 10.0) 7.36 (d, J ¼ 10.0)
3.95 (s) – 4.51 (d, J ¼ 1.8) 4.37 (dd, J ¼ 1.8, 2.3) 4.28 (dd, J ¼ 2.3, 9.0) 3.99 (dd, J ¼ 8.8, 9.0) 3.30 – 3.32 (m) 3.84, 3.65 (dd, J ¼ 5.0, 12.2) 2.14 (s)
d( H)
d(C )
10.57 (s) 129.1 97.0 128.4 115.4 121.4 116.2 143.8 128.7 163.4 56.1 – 101.8 67.9 76.6 64.3 78.2 62.6 170.6, 20.9
5.12 (s) 7.11 (d, J ¼ 8.6) 6.81 (dd, J ¼ 8.6, 10.0) 7.00 (d, J ¼ 10.0)
– 2.31 (s) 4.47 (d, J ¼ 2.0) 3.88 (dd, J ¼ 2.0, 2.2) 3.52 (dd, J ¼ 2.2, 9.0) 3.46 (dd, J ¼ 8.6, 9.0) 3.25 – 3.27 (m) 3.83, 3.62 (dd, J ¼ 5.0, 12.2) –
135.4 101.5 127.5 115.1 122.0 115.7 144.1 127.6 – – 20.3 102.3 71.8 74.6 68.5 77.7 62.7 – –
972
CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
Figure. Key HMBCs (H ! C) of compounds 1 and 3
Compound 2 was obtained as a pale-yellow amorphous powder. The EI-MS indicated a molecular weight corresponding to the peak at m/z 395, and its HR-ESI-MS exhibited the [M þ Na] þ peak at m/z 418.1113 (calc. 418.1114), corresponding to the molecular formula C18H21NO9 . The general features of its IR and NMR spectra closely resembled those of 1, except for the presence of an additional Ac group. The AcO group was at C(3’) based on HMBCs of HC(3’) (d(H) 4.28 (dd, J ¼ 2.3, 9.0)) with the Ac C-atom (d(C) 170.6). Therefore, compound 2 was elucidated as methyl 7-[(3-Oacetyl-b-d-mannopyranosyl)oxy]-1H-indole-2-carboxylate. Compound 3, a yellow amorphous solid, exhibited a quasi-molecular-ion peak, in the HR-MS, at m/z 310.1292 ([M þ H] þ ; calc. 310.1291), which corresponded to the molecular formula C15H19NO6 . The UV spectrum showed absorption maxima at 223, 283, and 300 nm indicating the presence of an indole chromophore. The only 13C-NMR difference between 3 and 1 was that the COOMe moiety in 1 was replaced by the signals for a Me group, which was further confirmed by the HMBC experiments (Fig.). Correspondingly, the resonances of C(2) and C(3) were shifted downfield to 135.4 and 101.5, respectively. The HMBCs between C(7) (d(C) 144.1) of the indole alkaloid and the anomeric H-atom (d(H) 4.47) of the mannose unit were observed (Fig.). Consequently, compound 3 was unambiguously determined as 2-methyl-1H-indol-7-yl b-d-mannopyranoside. Biology. The in vitro cytotoxic activities of the isolated alkaloids were evaluated against eight tumor cell lines by using the revised 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl-2H-tetrazolium bromide (MTT) method as described in the Exper. Part (Table 2). All alkaloids showed significant cytotoxicities (IC50 < 30 mm), with alkaloid 3 possessing a higher cytotoxic activity (IC50 9.21 – 12.75 mm) than alkaloids 1 (IC50
Table 2. Cytotoxicities ( IC50 [mm] ) of Compounds 1 – 3 against Eight Human Tumor Cell Lines Compound
1 2 3 Doxorubicin a ) a
Cell lines A-549
BGC-823
HCT15
HeLa
HepG2
MCF-7
SGC-7901
SK-MEL-2
18.11 23.78 11.41 0.01
19.32 25.31 11.97 0.01
20.47 26.95 11.12 0.06
20.88 26.78 10.45 0.05
19.20 23.47 10.95 0.03
20.21 27.11 9.78 0.01
21.04 28.12 9.21 0.06
20.47 26.77 12.75 0.01
) Positive control.
CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
973
18.11 – 21.04 mm) and 2 (IC50 23.47 – 28.12 mm). The results indicated that the Me group at C(2) might effectively enhance the cytotoxic activities for this type of alkaloids. Experimental Part 1. General. All solvents were distilled before use. TLC: SiO2 GF254 (10 – 40 mm; Qingdao Marine Chemical Factory, Qingdao, China). Column chromatography (CC): silica gel (SiO2 ; 200 – 300 mesh, 10 – 40 mm; Qingdao Marine Chemical Factory, Qingdao, China), Sephadex LH-20 (Amersham Pharmacia Biotech, Sweden), MCI Gel CHP20P (75 – 150 mm; Mitsubishi Kasei Chemical Industries), and C18 reversed-phase SiO2 (20 – 45 mm; Fuji Silysia Chemical Ltd.). HPLC (anal. and prep.): Shimadzu model LC-8A on YMC-pack, R&D ODS column (250 4.6 mm, 250 20 mm) and UV detector Shimadzu SPD-10AVP. M.p.: Tempo melting-point apparatus; uncorrected. Optical rotations: JASCO-20C digital polarimeter. UV Spectra: Hewlett-Packard-8452A diode-array spectrophotometer; lmax (log e) in nm. IR Spectra: PerkinElmer 577 spectrometer; ˜n in cm 1. 1H- and 13C-NMR spectra: Bruker AM-400 spectrometer; d in ppm rel. to Me4Si as internal standard, J in Hz. MS: VG AutoSpec-3000 mass spectrometer; in m/z (rel. %). HR-ESI-MS: API QSTAR Pulsar-1 mass spectrometer; in m/z. 2. Plant Material. The roots of Zanthoxylum nitidum were collected in Guangxi Province, China, in August of 2011, and identified by one of the authors (J. C.). A voucher specimen (ZN20110801) was deposited with the Herbarium of the College of Biological Resources and Environment Science, Qujing Normal University, Qujing, Yunnan Province, P. R. China. 3. Extraction and Isolation. The air-dried and powdered root wood (25 kg) of Z. nitidum was refluxed with 80% EtOH (30 l). After removal of EtOH under reduced pressure, the aq. brownish syrup (6 l) was adjusted to pH 2 with 2% HCl and filtered. The filtrate was adjusted to pH 9 by adding 20% NaOH, and then extracted with CHCl3 to afford total alkaloid content. The CHCl3 extract (65 g) was subjected to CC (SiO2 ; gradient CHCl3/MeOH) to afford 18 fractions, Frs. A1 – A18. Fr. A13 (CHCl3 , 5.24 g) was seperated by CC (RP-18 gel; MeOH/H2O from 70 to 95%) to yield three subfractions, Frs. A13a – A13c. Fr. A13b (879 mg) were purified by HPLC to afford 2 (128 mg) and 3 (115 mg). Fr. A13c (997 mg) was separated by repeated CC (Sephadex LH-20; CHCl3/MeOH 1 : 1 and MeOH) to yield 1 (74.3 mg). Methyl 7-(b-d-Mannopyranosyloxy)-1H-indole-2-carboxylate (1). Pale-yellow amorphous solid. M.p. 152 – 1738. UV (EtOH): 223 (3.93), 283 (3.45), 300 (3.78). 1H- and 13C-NMR: Table 1. EI-MS: 353 (2, M þ ), 301 (10), 180 (7), 138 (5), 105 (100), 77 (61), 65 (9), 51 (17). ESI-MS: 354.1 ([M þ H] þ ). HREI-MS: 354.1187 ([M þ H] þ , C16H20NO þ8 ; calc. 354.1189). Methyl 7-[(3-O-Acetyl-b-d-mannopyranosyl)oxy]-1H-indole-2-carboxylate (2). Pale-yellow amorphous solid. M.p. 148 – 1668. UV (EtOH): 224 (3.85), 284 (3.37), 300 (3.92). 1H- and 13C-NMR: Table 1. EI-MS: 395 (2, M þ ), 352 (18), 180 (10), 105 (100), 65 (9), 51 (17). ESI-MS: 354.1 ([M þ H] þ ). HR-EIMS: 418.1113 ([M þ Na] þ , C18H21NNaO þ9 ; calc. 418.1114). 2-Methyl-1H-indol-7-yl b-d-Mannopyranoside (3). Yellow amorphous solid. M.p. 113 – 1248. UV (EtOH): 223 (3.89), 283 (3.51), 300 (3.81). 1H- and 13C-NMR: Table 1. HR-EI-MS: 310.1292 ([M þ H] þ , C15H20NO þ6 ; calc. 310.1291). 4. Acid Hydrolysis of 1. A soln. of 1 (10 mg) in 1m HCl (dioxane/H2O 1 : 1, 4 ml) was heated at 958 for 2 h under an air atmosphere. After cooling, the mixture was neutralized by passing through an Amberlite IRA-93ZU (Organo, Tokyo, Japan) column and subjected to CC (SiO2 ; CHCl3/MeOH 19 : 1, 9 : 1, and 1 : 1) to give an aglycone and a sugar fraction (2.3 mg). HPLC Analysis of the sugar fraction under the following conditions revealed the presence of d-mannose. Column, Capcell Pak NH2UG80 (4.6 mm i.d. 250 mm, 5 mm, Shiseido), detector: Shodex OR-2 (Showa-Denko, Tokyo, Japan); solvent, MeCN/H2O 17 : 3; flow rate: 1.0 ml/min; tR [min], 14.21 (d-mannose, positive optical rotation). 5. Cytotoxicity Assay in vitro. The revised MTT method was used for in vitro evaluation of the cytotoxic potential of the isolated compounds against eight cultured human tumor cell lines [11]. The cell lines, A-549 (human lung cancer), BGC-823 cells (human gastric carcinoma), HCT15 (human colon cancer), HeLa cells (human cervical cancer), HepG2 cells (human hepatocellular carcinoma), MCF-7 cells (human breast cancer), SK-MEL-2 (human skin cancer), and SGC-7901 cells (human gastric adenocarcinoma) were cultured on RPMI-1640 medium supplemented with 10% fetal bovine serum,
974
CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
100 U/ml benzyl penicillin, and 100 mg/ml streptomycin in 25-cm2 culture flasks at 378 in humidified atmosphere with 5% CO2 . For the cytotoxicity tests, cells in exponential growth stage were harvested from the culture by trypsin digestion and centrifuging at 180 g for 3 min, then resuspended in fresh medium at a cell density of 5 104 cells per ml. The cell suspension was dispensed into a 96-well microplate at 100 ml per well, and incubated in humidified atmosphere with 5% CO2 at 378 for 24 h, and then treated with the compounds at various concentrations (0, 1, 10, and 100 mm). After 48 h of treatment, 50 ml of 1 mg/ml MTT soln. was added to each well, and further incubated for 4 h. The cells in each well were then solubilized with DMSO (100 ml for each well), and the optical density (OD) was recorded at 595 nm. All drug doses were tested with doxorubicin as positive control in triplicate, and the IC50 values were derived from the mean OD values of the triplicate tests vs. drug concentration curves. Concentrations that inhibit cell growth by 50% (IC50) compared to non-treated cells were determined by non-linear regression with Graphpad Prism software version 4.0 (GraphPad Software, Inc., San Diego, CA). This work was supported by the grants from the Scientific Planning Project of the Applied Basic Research of Yunnan Province (S2012FZ0005), the Key Projects of Scientific Research Foundation of the Department of Education of Yunnan Province (2013Z095), the Key Projects in Scientific Research of Qujing Normal University (2011ZD003), and the Developing Key Subject of Ecology of Qujing Normal University.
REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9] [10]
[11]
D. Y. Kong, A. I. Gray, T. G. Hartley, P. G. Waterman, Biochem. Syst. Ecol. 1996, 24, 87. J. J. Chen, Y. H. Lin, S. H. Day, T. L. Hwang, I. S. Chen, Food. Chem. 2011, 125, 282. J. Hu, W. D. Zhang, R. H. Liu, C. Zhang, Y. H. Shen, H. L. Li, Chem. Biodiversity 2006, 3, 990. J. Hu, W. D. Zhang, Y. H. Shen, C. Zhang, L. Xu, R. H. Liu, B. Wang, X. K. Xu, Biochem. Syst. Ecol. 2007, 35, 114. C. H. Yang, M. J. Cheng, M. Y. Chiang, Y. H. Kuo, C. J. Wang, I. S. Chen, J. Nat. Prod. 2008, 71, 669. J. Shen, X. Zhang, Z. Tang, S. Peng, L. Ding, Chin. Herb. Med. 2004, 35, 619. J. Hu, W. D. Zhang, R. H. Liu, C. Zhang, Y. H. Shen, X. K. Xu, China J. Chin. Mater. Med. 2006, 31, 1689. Z. Dvora´k, R. Vrzal, P. Maurel, J. Ulrichova´, Chem.-Biol. Interact. 2006, 159, 117. D. Li, B. Zhao, S. P. Sim, T. K. Li, A. Liu, L. F. Liu, E. J. LaVoie, Bioorg. Med. 2003, 11, 521. F. Tillequin, S. Michel, E. Seguin, Tryptamine-Derived Indole Alkaloids, in Methods in Plant Biochemistry: Alkaloids and Sulfur Compounds, Eds. P. M. Dey, J. B. Harborne, Academic Press, London, 1993, Vol. 8, p. 309. J. J. Chen, H. C. Hung, P. J. Sung, I. S. Chen, W. L. Kuo, Phytochemistry 2011, 72, 523. Received December 8, 2013
