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ORIGINAL ARTICLE
Year : 2014  |  Volume : 10  |  Issue : 37  |  Page : 73-76  

Acridone alkaloids with cytotoxic and antimalarial activities from Zanthoxylum simullans Hance


1 College of Pharmacy, South-Central University for Nationalities, China
2 Institute of Yunnan Baiyao Group Co. Ltd, China

Date of Submission03-Dec-2012
Date of Decision23-Jan-2013
Date of Web Publication7-Feb-2014

Correspondence Address:
Xinzhou Yang
College of Pharmacy, South-Central University for Nationalities, 708 Minzu Road, Wuhan 430074
China
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Source of Support: The work was fi nancially supported by the Talented Faculty Foundation of South-Central University for Nationalities (YZ211002 to X.Z. Yang) and National Natural Science Foundation of China (81102798 to X.Z. Yang), Conflict of Interest: None


DOI: 10.4103/0973-1296.126669

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   Abstract 

Background: Zanthoxylum simullans Hance is a popular natural spice belonging to the Rutaceae family and it is one of the common prescribed herbs in traditional Chinese medicine. Materials and Methods: The chemical constituents were mainly isolated and purified by silica gel column chromatography and semi-preparative High Performance Liquid Chromatography. Their structures were identified by comparing the spectral data with those reported in the literature. Cytotoxic activities for the isolated acridone alkaloids were evaluated against two prostate cancer cell lines PC-3M and Lymph Node Carcinoma of Prostate (LNCaP), and their antimalarial activities were tested against two different strains of the parasite Plasmodium falciparum 3D7, and Dd2. Results: The root bark MeOH extract of Z. simullans Hance afforded β-sitosterol, 4-methoxy benzoic acid, daucosterol, and five acridone alkaloids, normelicopidine, normelicopine, melicopine, melicopidine, and melicopicine. All five acridone alkaloids were isolated from this plant for the first time and exhibited certain cytotoxic and antimalarial activities in vitro. Conclusion: Normelicopidine was the most active against PC-3M, LNCaP and Dd2 with IC 50 values of 12.5, 21.1, and 18.9 ug/mL respectively.

Keywords: Acridone alkaloids, antimalarial, cytotoxity, Zanthoxylum simullans Hance


How to cite this article:
Wang C, Wan J, Mei Z, Yang X. Acridone alkaloids with cytotoxic and antimalarial activities from Zanthoxylum simullans Hance. Phcog Mag 2014;10:73-6

How to cite this URL:
Wang C, Wan J, Mei Z, Yang X. Acridone alkaloids with cytotoxic and antimalarial activities from Zanthoxylum simullans Hance. Phcog Mag [serial online] 2014 [cited 2019 Oct 21];10:73-6. Available from: http://www.phcog.com/text.asp?2014/10/37/73/126669


   Introduction Top


The genus Zanthoxylum, family Rutaceae comprises about 250 species of deciduous, evergreen trees and shrubs distributed world-wide around the tropical and subtropical areas. It is economically important because of their alimentary, industrial, and medicinal applications. [1] Zanthoxylum simullans Hance, a popular natural spice, has long been prescribed in traditional Chinese medicine for the treatment of stomach ache, toothache, intestinal worms, eczema, and pruritus. [2] Extensive phytochemical investigations have been carried out on this plant, along with the study of its biological activities, especially, antiplatelet aggregation. [3],[4],[5],[6],[7] Most alkaloids isolated from this plant exhibited strong antiplatelet aggregation activities, except for zanthosimuline, a new acridone alkaloid found in the root bark. It has demonstrated a general cytotoxic response when evaluated against a variety of human cancel cell lines and P388 cells. [4] In this paper, we conducted a search for more bioactive constituents from Z. simullans Hance.


   Materials and Methods Top


Plant material

The root barks of Z. simullans Hance were collected in September 2009 at Jingxi county, Guangxi province, China and identified by Dr. Jingquan Yuan, Associate Professor of Guangxi Medicinal Herb Garden. A voucher specimen (No. 20090018Z) has been deposited in the Herbarium of South-Central University for Nationalities, Wuhan, P. R. China.

General

Melting points were determined on a Kofler melting point apparatus and are uncorrected. Utlraviolet (UV) spectra were determined on a Shimadzu UV-250 spectrometer. 1 H and 13 C NMR (Carbon-13 Nuclear Magnetic Resonance) spectra were recorded on a Bruker DRX-400 spectrometer with Tetramethylsilane (TMS) as an internal standard. The chemical shifts (δ) were reported in ppm, and coupling constants (J) were given in Hz. The ESI-MS and EI-MS (Electrospray Ionization Mass Spectrometry and Electron Impact Mass Spectrometry) data were recorded on the Quadrupole Time-of-flight (Q-TOF) Mass Spectrometry Micro Liquid Chromatography-Tandem Mass Spectrometry (LC-MS-MS) and Finnigan MAT-95 mass spectrometers. Semi-preparative HPLC system: Two PrepStar SD-1 solvent delivery modules, a ProStar UV-Vis 320 detector and a ProStar 701 Fraction Collector (Varian, Walnut Creek, CA, USA); a Sunfire RP-18 column (5 μm; 250 mm × 20 mm, waters) for semi-preparative isolation. The following types of silica gel were used: 300-400 mesh for column chromatography and GF 254 for Thin Layer Chromatography (TLC) (Qing Dao Hai Yang Chemical Group Co.). Sephadex LH 20 (GE Healthcare) was also used for column chromatography. Cell lines PC-3M, LNCaP and HEK293 were purchased from the American Type Culture Collection. Parasite strains Dd2 and 3D7 were from the Queensland Institute of Medical Research.

Extraction

Air-dried root barks of Z. simullans Hance (200 g) were ground and then extracted by maceration at room temperature with MeOH in a 1 L conical flask mounted on a shaking table (3 × 0.6 L, 4 h each). The solvents were evaporated at reduced pressure to yield 18.6 g MeOH extract.

Isolation

The MeOH extract was subjected to CC over silica gel (500 g) eluted gradiently with CH 2 Cl 2 -MeOH (25:1-1:2) to afford fractions 1-7. Fraction 2 (2.1 g) was rechromatographed on silica gel (120 g) with CH 2 Cl 2 -MeOH (20:1-10:1) to give compounds 1 (135 mg), 2 (27 mg) and 5 (87 mg). The crude alkaloids (0.9 g) derived from fraction 3 (1.6 g) after filtration over Sephadex LH 20 with MeOH were further submitted to semi-preparative HPLC eluted with a linear gradient of 10-70% aq. MeOH in 0.5% TFA at 9.0 mL/min for 120 min to yield successively compounds 4 (12 mg, t R 32.5 min), 5 (39 mg, t R 36.1 min), 6 (5 mg, t R 47.7 min), 7 (16 mg, t R 64.6 min), 8 (78 mg, t R 68.9 min).

Compound 1, colorless crystal (MeOH), mp, 140-142 ° C; Electron Impact Mass Spectrometry (EIMS) m/z 414 [M] + , 396, 381, 354, 329, 303, 273, 255; 1 H-NMR (CDCl 3 ): δ 5.33 (1H, d, J = 5.0 Hz, H-6), 3.50 (1H, m, H-3), 0.99 (3H, s, H-19), 0.92 (3H, d, J = 8.1 Hz, H-21), 0.84 (3H, t, J = 7.7 Hz, H-29), 0.82 (3H, d, J = 8.7 Hz, H-27), 0.80 (3H, d, J = 8.7 Hz, H-26), 0.66 (3H, s, 18-Me).

Compound 2, colorless crystal (EtOH), mp, 183-185 ° C; EIMS m/z 152 [M] + , 135, 122, 109, 92, 84, 56; 1 H-NMR (CDCl 3 ): δ 8.42 (2H, dd, J = 8.6, 2.0 Hz, H-2, H-6), 7.06 (2H, dd, J = 8.6, 2.0 Hz, H-3, H-5), 3.68 (3H, s, OMe); 13 C-NMR (CDCl 3 ): 125.1 (C-1), 132.3 (C-2, C-6), 114.1 (C-3, C-5), 163.6 (C-4), 168.8 (C-7), 55.4 (OMe).

Compound 3, white powder; ESIMS m/z 599 [M + Na] + , 576 [M] + , 413 [M-glu-1] + ; EIMS m/z 414 [M] + , 396, 381, 354, 329, 303, 273, 255, 231, 213; 1 H-NMR (DMSO-d 6 ): δ 5.34 (1H, m, H-6), 4.92 (1H, d, J = 8.0 Hz, Glu, H-1'), 3.50 (1H, m, H-3), 0.99 (3H, s, H-19), 0.92 (3H, d, J = 8.1 Hz, H-21), 0.84 (3H, t, J = 7.7 Hz, H-29), 0.82 (3H, d, J = 8.7 Hz, H-27), 0.80 (3H, d, J = 8.7 Hz, H-26), 0.66 (3H, s, 18-Me); 13 C-NMR (DMSO-d 6 ): 36.7 (C-1), 29.3 (C-2), 76.8 (C-3), 38.2 (C-4), 140.3 (C-5), 120.1 (C-6), 31.3 (C-7, C-8), 50.5 (C-9), 36.1 (C-10), 22.5 (C-11), 41.7 (C-12), 45.0 (C-13), 56.1 (C-14), 24.8 (C-15), 27.7 (C-16), 55.3 (C-17), 11.6 (C-18), 19.0 (C-19), 35.4 (C-20), 18.8 (C-21), 33.0 (C-22), 25.3 (C-23), 49.5 (C-24), 28.5 (C-25), 19.6 (C-26), 12.9 (C-27), 23.8 (C-28), 12.0 (C-29), 100.6 (Glu, C-1'), 73.3 (C-2'), 76.8 (C-3'), 69.9 (C-4'), 76.6 (C-5'), 60.9 (C-6').

Compound 4, orange needles (CHCl 3 ), mp, 208-210 ° C; UV (EtOH) λmax (nm): 219, 250, 272, 304, 322, 393; ESI-MS: m/z 621 [2M + Na] + , 599 [2M + H] + , 300 [M + H] + ; 1 H-NMR (DMSO-d 6 , 400 MHz): 14.7 (1H, s, OH), 8.32 (1H, d, J = 8.9 Hz, H-8), 7.79 (1H, dd, J = 8.7, 7.6 Hz, H-6), 7.76 (1H, d, J = 8.7 Hz, H-5), 7.64 (1H, d, J = 8.9, 7.6 Hz, H-7), 6.05 (2H, s, O-CH 2 -O), 4.03 (3H, s, OCH 3 ), 3.86 (3H, s, N-CH 3 ); 13 C-NMR (DMSO-d 6 , 100 MHz): 175.9 (C-9), 145.1 (C-3), 144.4 (C-5a), 137.3 (C-4a), 136.6 (C-1), 135.4 (C-2), 133.5 (C-6), 130.1 (C-4), 126.3 (C-8), 124.1 (C-8a), 122.1 (C-7), 117.1 (C-5), 113.5 (C-1a), 102.7 (O-CH 2 -O), 61.2 (4-OMe), 42.0 (N-Me).

Compound 5, red needles (CHCl 3 ), mp, 234-236 ° C; UV (EtOH) λmax (nm): 222, 250, 277, 313, 423; ESI-MS: m/z 322 [M + Na] + , 300 [M + H] + ; EIMS m/z: 299 [M] + (83), 285 (8), 284 (100), 254 (31), 170 (16), 158 (31), 115 (17); 1 H-NMR (DMSO-d 6 , 400 MHz): 14.9 (1H, s, OH), 8.37 (1H, d, J = 7.9 Hz, H-8), 7.82 (1H, dd, J = 8.4, 7.7 Hz, H-6), 7.78 (1H, d, J = 7.7 Hz, H-5), 7.67 (1H, d, J = 8.4, 7.9 Hz, H-7), 5.99 (2H, s, O-CH 2 -O), 4.01 (3H, s, OCH 3 ), 3.97 (3H, s, N-CH 3 ); 13 C-NMR (DMSO-d 6 , 100 MHz): 176.2 (C-9), 147.9 (C-1), 143.5 (C-3), 144.1 (C-5a), 133.9 (C-6), 133.1 (C-2), 132.1 (C-4), 127.5 (C-4a), 127.3 (C-8), 122.5 (C-8a), 121.8 (C-7), 116.2 (C-5), 115.1 (C-1a), 102.4 (O-CH 2 -O), 61.1 (2-OMe), 40.6 (N-Me).

Compound 6, light yellow needles (CHCl 3 ), mp, 176-178 ° C; UV (EtOH) λmax (nm): 215, 251, 271, 302, 404; EIMS m/z: 313 [M] + (75), 299 (28), 298 (100), 284 (21), 280 (10), 270 (50), 268 (13), 255 (10), 254 (8), 169 (8); 1 H-NMR (DMSO-d 6 , 400 MHz): δ H 8.53 (1H, dd, J = 9.0 Hz, H-8), 7.90 (1H, dd, J = 8.7, 7.7 Hz, H-6), 7.75 (1H, d, J = 8.7 Hz, H-5), 7.58 (1H, d, J = 9.0, 7.7 Hz, H-7), 6.08 (2H, s, O-CH 2 -O), 4.01, 3.98 (2 CH.H, s, 2OCH 3 ), 3.97 (3H, s, N-CH 3 ); 13 C-NMR (DMSO-d 6 , 100 MHz): 176.1 (C-9), 149.8 (C-1), 143.8 (C-3), 143.1 (C-5a), 134.1 (C-6), 133.6 (C-2), 131.9 (C-4), 127.1 (C-4a), 127.0 (C-8), 122.9 (C-8a), 121.6 (C-7), 115.5 (C-5), 114.3 (C-1a), 102.3 (O-CH 2 -O), 62.4 (1-OMe), 61.4 (2-OMe), 39.2 (N-Me).

Compound 7, light yellow prismatic crystal (EtOEt), mp, 121-122 ° C; UV (EtOH) λmax (nm): 221, 252, 276, 302, 325, 399; ESI-MS: m/z 649 [2M + Na] + , 336 [M + Na] + , 314 [M + H] + ; MS m/z: 313 [M] + (100), 298 (59), 285 (25), 280 (28), 271 (13), 270 (74), 269 (9), 268 (25), 255 (16), 253 (10), 252 (50), 240 (9), 224 (14), 212 (7); 1 H-NMR (DMSO-d 6 , 400 MHz): δ H 8.26 (1H, d, J = 8.7 Hz, H-8), 7.89 (1H, dd, J = 8.5, 7.4 Hz, H-6), 7.81 (1H, d, J = 8.5 Hz, H-5), 7.63 (1H, d, J = 8.7, 7.4 Hz, H-7), 6.02 (2H, s, O-CH 2 -O), 4.05, 3.84 (2 CH.H, s, 2OCH 3 ), 3.82 (3H, s, N-CH 3 ); 13 C-NMR (DMSO-d 6 , 100 MHz): 176.3 (C-9), 145.6 (C-3), 144.7 (C-5a), 137.6 (C-1), 137.2 (C-4a), 135.7 (C-2), 133.3 (C-6), 129.4 (C-4), 126.1 (C-8), 123.8 (C-8a), 121.7 (C-7), 116.9 (C-5), 113.9 (C-1a), 103.1 (O-CH 2 -O), 61.4 (1-OMe), 61.1 (4-OMe), 42.2 (N-Me).

Compound 8, yellow prismatic crystal (Acetone), mp 133-134 ° C; UV λmax (nm): 203 (sh), 218, 268, 310 (sh), 397; EIMS m/z: 329 [M] + , 314 (100), 270, 256, 228, 157, 149, 143, 77; 1 H-NMR (DMSO-d 6 , 400 MHz): δ H 8.41 (1H, d, J = 8.9 Hz, H-8), 7.85 (1H, dd, J = 8.4, 7.7 Hz, H-6), 7.73 (1H, d, J = 8.4 Hz, H-5), 7.59 (1H, d, J = 8.9, 7.7 Hz, H-7), 4.11, 4.02, 3.97, 3.80 (4 80.H, s, 4OCH 3 ), 3.91 (3H, s, NCH3); 13 C-NMR (DMSO-d 6 , 100 MHz): 176.4 (C-9), 152.6 (C-1), 149.7 (C-3), 144.9 (C-5a), 139.4 (C-4), 138.5 (C-4a), 136.7 (C-2), 134.1 (C-6), 126.7 (C-8), 123.5 (C-8a), 121.9 (C-7), 117.2 (C-5), 114.2 (C-1a), 62.2 (1-OMe), 61.9 (2-OMe), 61.9 (3-OMe), 62.1 (4-OMe), 42.1 (N-Me).

Identification of the isolated compounds [Figure 1] was based on comparison of UV, MS, 1 H-NMR and 13 C-NMR data to previously reported ones as β-sitosterol 1, [8] 4-methoxy benzoic acid 2, [8] daucosterol 3 [9] and five acridone alkaloids, normelicopidine 4, [10] normelicopine 5, [11] melicopine 6, [11] melicopidine 7 [11] and melicopicine 8. [12] It is noteworthy to mention that all these acridone alkaloids were isolated from this plant for the first time.
Figure 1: Structures for compounds 4-8

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Cytotoxity assay

Compounds 4-8 were added to Falcon 384 well black/clear tissue treated assay plates containing 3000 adherent cells/well (HEK293) in an assay volume of 45 uL. The plates were incubated for 72 h at 37 ° C and 5% CO 2 . After incubation the supernatant was aspirated from the wells and 40 uL of 10% Alamar Blue added per well. Plates were incubated for a further 5-6 h and measured for fluorescence at 535 nm excitation and 590 nm emission using a VICTOR II (PerkinElmer, Waltham, MA). The % inhibition of cell proliferation was calculated using Dimethylsulfoxide (DMSO) and 10 uM 5-Fluorouracil control data. IC 50 values were obtained by plotting % inhibition against log dose using the Prizm4 graphing package and nonlinear regression with variable slope plot.

Antimalarial assay

Compounds 4-8 were incubated in the presence of 2 or 3% parasitemia (3D7 or Dd2) and 0.3% hematocrit in a total assay volume of 50 uL, for 72 h at 37 ° C and 5% CO 2 , in poly-Dlysine-coated Cell Carrier imaging plates. After incubation plates were stained with DAPI in the presence of saponin and Triton X-100 and incubated for a further 5 h at RT in the dark before imaging on the Evotec OPERA High Throughput Screening confocal imaging system (PerkinElmer, Waltham, MA). The digital images obtained were analyzed using the Perkin-Elmer Acapella spot detection software, where fluorescent spots that fulfilled the criteria established for a stained parasite were counted. The % inhibition of parasite replication was calculated using DMSO and 2 uM artemisinin control data.


   Results and Discussion Top


Cytotoxity [Table 1] of five acridone alkaloids isolated from this herb was evaluated against two prostate cancer cell lines PC-3M and LNCaP. Preliminary toxicity toward human cells was investigated using a human embryonic kidney cell line (HEK293). The results revealed that all five compounds exhibited potential cytotoxity against PC-3M and LNCaP with IC 50 value range between 12 ug/mL and 65 ug/mL, and showed no cytotoxity against HEK293 up to 100 ug/mL. Compound 4 was the most active against PC-3M and LNCaP with IC 50 values of 12.5 and 21.1 ug/mL respectively.

Antimalarial activity [Table 2] for the acridone alkaloids has also been tested against a chloroquine-sensitive (3D7) and a chloroquine-resistant (Dd2) Plasmodium falciparum strain. All Compounds displayed IC 50 values in a range of 18-42 ug/mL against 3D7 and Dd2. Compound 4 showed the strongest activity against Dd2 with IC 50 value of 18.9 ug/mL.
Table 1: Cytotoxic activities of compounds 4-8 (IC50, ug/mL; mean±SD, n=3)

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Table 2: Antimalarial and cytotoxic activity for compounds 4-8 (IC50, ug/mL; mean±SD, n=3)

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   Conclusion Top


Acridone alkaloids are abundant in the Rutaceae family, especially occurring in citrus plants, and many of them showed interesting pharmaceutical properties such as anticancer, antitumor, antiviral, antimalarial, and antiallergic activities. Our investigation on the root bark of Z. simullans Hance has led to the isolation of five acridone alkaloids, normelicopidine, normelicopine, melicopine, melicopidine, and melicopicine from this plant for the first time. All tested compounds exhibited certain cytotoxic and antimalarial activities in vitro, and compound 4 was the most active against PC-3M, LNCaP and Dd2 with IC 50 values of 12.5, 21.1 and 18.9 ug/mL respectively.


   Acknowledgments Top


The work was financially supported by the Talented Faculty Foundation of South-Central University for Nationalities (YZ211002 to X.Z. Yang) and National Natural Science Foundation of China (81102798 to X.Z. Yang).

 
   References Top

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5.Chen IS, Wu SJ, Leu YL, Tsai IW, Wu TS. Alkaloids from root bark of Zanthoxylum simulans. Phytochemistry 1996;24:217-9.  Back to cited text no. 5
    
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8.Pan ZH, Luo L, Li ZQ, Zhang LL, Dong Q, Ma GY. The chemical constituents from Pittosporum podocarpum Gagnep. J Yunnan Univ (Natural Sciences) 2012;34:450-3.  Back to cited text no. 8
    
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    Figures

  [Figure 1]
 
 
    Tables

  [Table 1], [Table 2]


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