Cytotoxic Benzophenanthridine and Furoquinoline Alkaloids from Zanthoxylum buesgenii(Rutaceae)
© Sandjo et al.; licensee Chemistry Central Ltd. 2014
Received: 5 August 2014
Accepted: 7 October 2014
Published: 21 October 2014
Zanthoxylum buesgenii is a shrub used in Sierra Leone as remedy to cure venereal diseases, arthritis, and rheumatism whereas leaves and barks are employed to treat leprosy and to relieve pain. In South West Region of Cameroon, the plant locally called "Mbem" by Lewoh-Lebang community, is orally given to patients as aphrodisiac decoction and to increase sperm count. Previous chemical studies on Zanthoxylum species reported the identification of lignans, coumarins, diterpenes, sesquiterpenes, steroids, alkaloids and benzopropanoids. Besides, structurally diverse compounds belonging to these classes of secondary metabolites have been reported as trypanocidal, antileishmanial, antimycobacterial and cytotoxic metabolites.
We therefore investigated the alkaloidal constituents of Z. buesgenii. In the course of the study, two benzophenanthridines [1-methoxy-12-methyl-12,13-dihydro-[1,3]dioxolo[4',5':4,5]benzo[1,2-c]phenanthridine-2,13-diol (1) and isofagaridine (2)] were identified among them one new. Alongside, three known furoquinolines [maculine (3), kokusaginine (4) and teclearverdoornine (5)] were also obtained and their structures were established on the basis of their NMR data and by comparison with those previously reported. Furthermore, the cytotoxicities of metabolites (1-4) isolated in substantial amount were evaluated against a series of multidrugs-resistant cancer cell lines. While compounds 2-4 showed selective cytotoxicities, compound 1 displayed activities against all cancer cells.
Formerly named Fagara buesgenii, Zanthoxylum buesgenii is a shrub or small tree of about 4 m height with leaves about 20 to 75 cm long ,. In Sierra Leone, roots are used as remedy to cure venereal diseases, arthritis, and rheumatism whereas leaves and barks are employed to treat leprosy and to relieve pain . In South West Region of Cameroon, Z. buesgenii locally called "mbem" by Lewoh-Lebang community, is orally given to patients as aphrodisiac decoction and to increase sperm count . Previous chemical studies on Zanthoxylum species reported the identification of lignans, coumarins, diterpenes, sesquiterpenes, steroids, alkaloids  and benzopropanoids . Interestingly, alkaloids represent the largest group of secondary metabolites obtained from the genus Zanthoxylum with structurally diverse scaffolds including oxoaporphines , aporphines, quinolinones, furoquinolines , indolopyridoquinazolines, β-carbolines, and benzophenanthridines ,. Besides bioactivities such as trypanocidal , antileishmanial , antimycobacterial  effects, most of these alkaloids have shown from moderate to significant cytotoxicity against several cancer cell lines -. Therefore, we investigated the alkaloidal constituents of Z. buesgenii. In the course of the study, two benzophenanthridines were identified among them one new. Alongside, three known furoquinolines were also obtained.
We herein report the structure elucidation of the new compound and the cytotoxic potentiality of the identified secondary metabolites against a series of multidrugs-resistant cancer cell lines.
Results and discussion
A Dragendorff reagent-guided isolation of the aerial part of Zanthoxylum buesgenii yielded five alkaloids identified as benzophenanthridines (1 and 2) and furoquinolines (3-5).
NMR data of compound 1 (C 5 D 5 N, 400 MHZ)
δH(multi,J = Hz)
7.41, d (8.4)
7.82, d (8.4)
8.02, d (8.5)
7.62, d (8.5)
6.04, d (1.2)
6.08, d (1.2)
The known compounds (Figure 3) were identified as isofagaridine 2, maculine 3, kokusaginine 4 and teclearverdoornine 5 based on their NMR data and by comparison with those previously reported.
Cytotoxicity of the studied compounds towards sensitive and drug-resistant cancer cell lines and normal cells as determined by the resazurin assay
Isolated compounds, doxorubicin and IC50values (δM)
0.24 ± 0.01
0.30 ± 0.04
89.09 ± 6.22
49.81 ± 5.04
0.20 ± 0.06
31.58 ± 3.48
20.37 ± 2.16
63.09 ± 3.75
44.56 ± 3.92
195.12 ± 14.30
30.14 ± 4.12
41.38 ± 3.44
62.01 ± 7.24
1.10 ± 0.28
65.01 ± 5.37
113.98 ± 9.82
7.83 ± 0.47
HCT116 (p53 +/+ )
42.46 ± 3.22
87.08 ± 7.55
119.88 ± 13.14
1.41 ± 0.29
HCT116 (p53 +/+ )
62.34 ± 4.41
4.06 ± 0.07
60.55 ± 7.29
105.19 ± 9.16
70.08 ± 6.40
1.06 ± 0.15
61.84 ± 4.68
115.30 ± 13.78
6.11 ± 0.57
22.37 ± 1.97
26.69 ± 3.15
90.77 ± 8.86
3.83 ± 0.94
The purification of the aerial part of Z. buesgenii monitored by TLC and Dragendorff reagent as alkaloids indicator led to the isolation of one new benzophenanthridine (buegenine, 1) along with four known metabolites namely a benzophenanthridine (isofagaridine, 2) and three furoquinolines (maculine 3, kokusaginine 4, and teclearverdoornine 5). Compounds (1-4) in substantial amount were evaluated for cytotoxicity activities and the obtained secondary metabolites showed from moderate to strong bioactivities. The observed activities corroborated those previously reported on similar benzophenanthridine alkaloids - indicating that compounds 1 and 2 can be chemically explored to develop other chemotherapeutic agents.
Optical rotation: JASCO P-2000 polarimeter; IR (KBr disc): JASCO A-302 spectrophotometer; HR-ESI-MS: JOEL MS apparatus; 1 and 2D NMR: Brüker DRX-400 mHz with TMS as internal reference. Thin layer chromatography (TLC) was performed over silica gel aluminum plates 60 F254. Silica gel 40-63 μM were used for columns chromatography (CC) separation. The melting point (m.p.) was measured by an Electro thermal IA 9000 digital melting point apparatus: uncorrected.
The aerial of Z. buesgenii was collected in Buea, South West region of Cameroon, in January 2014. Voucher specimens (BUD 0510) were deposited in the Herbarium of the Botany Department of the University of Dschang, Cameroon.
Extraction and isolation
The dried aerial part (1.8 kg) of Z. buesgenii was cut into small pieces, crushed and the powder was extracted for two days with a sufficient volume of methylene chloride (DCM)/MeOH (1:1). The solid residue was further extracted with MeOH for 24 h. Both solutions were pooled together and evaporated in vacuo to afford 50 g of crude extract. This latter was subjected to a liquid-solid extraction using successively n-hexane (hex), ethyl acetate (EA) and MeOH as the liquid part. Hex and EA fractions were pooled together based on the TLC profile to give fraction A (35 g). TLC of fractions A and B (MeOH) sprayed with Dragendorff's reagent, revealed the presence of alkaloids in A. Therefore, this latter was purified by silica gel CC eluted with hex, hex/EA (gradient) and EA yielding six sub-fractions (A1-A6). Maculine (3, 1.5 mg) was isolated from A2 eluted with hex/EA (95:5). A3 [5.2 g, hex/EA (3:1)] was further chromatographed on silica gel column eluted with hex/EA in gradient conditions. 60 sub-fractions were collected and isofagaridine (2, 3.1 mg) was filtered from the sub-fractions 10-15 eluted with hex/EA (9:1) while kokusaginine (4, 5.1 mg) was obtained from the sub-fractions 17-23 eluted with the same mixture of solvent. Compound 1 (3.7 mg) was further isolated from sub-fractions 26-33 eluted with hex/EA (85:15). A4 [10.2 g, hex/EA (1:1)] followed the same purification process under isocratic conditions of hex/EA (3:1) used as eluent to give teclearverdoornine (5, 0.7 mg). This latter (0.21 mg) was further obtained from the purification of A5 [8.7 g, hex/EA (1:3)] by using Hex/EA in the gradient condition.
Buesgenine, 1-methoxy-12-methyl-12,13-dihydro-[1,3]dioxolo[4',5':4,5]benzo[1,2-c]phenanthridine-2,13-diol (1)
Red powder, m.p. 177°C; [α]D -7 [c 0.37, CH3OH]; IR (KBr), νmax 3450, 3074, 1540, 1480, 1475, 1404, 1385, 1321, 1284, 1257, 1203, 1161, 1126, 1082 cm-1; 1H (C5D5N, 400 MHZ) and 13C (C5D5N, 100 MHZ) NMR data see Table 1 and these data have been compiled in the Additional file 1 provided as supporting information; HR-ESIMS: m/z 374.1003 [C20H17NO5 + Na]+ (calcd. 374.1004).
The resazurin reduction assay  was performed to assess the cytotoxicity of compounds and doxorubicin as control drug towards various sensitive and drug-resistant cancer cell lines, including the CCRF-CEM and CEM/ADR5000 leukemia, MDA-MB231 breast cancer cells and its resistant subline MDA-MB231/BCRP, HCT116p53 +/+ colon cancer cells and its resistant subline HCT116p53 -/- , U87MG glioblastoma cells and its resistant subline U87MG. ΔEGFR and HepG2 hepatocarcinoma cells and normal AML12 hepatocytes. The assay is based on the reduction of the indicator dye, resazurin, to the highly fluorescent resorufin by viable cells. Non-viable cells rapidly lose their metabolic capacity to reduce resazurin and, thus, do not produce fluorescent signals anymore. Briefly, adherent cells were detached by treatment with 0.25% trypsin/EDTA (Invitrogen, Darmstadt Germany) and an aliquot of 1 ± 104 cells was placed in each well of a 96-well cell culture plate (Thermo Scientific, Langenselbold, Germany) in a total volume of 200 μL. Cells were allowed to attach overnight and then were treated with different concentrations of compounds. For suspension cells, aliquots of 2 ± 104 cells per well were seeded in 96-well-plates in a total volume of 100 μL. The studied compounds were immediately added in varying concentrations in an additional 100 μL of culture medium to obtain a total volume of 200 μL/well. After 72 h, resazurin (Sigma-Aldrich, Schnelldorf, Germany) (20 μL, 0.01% w/v) in distilled H2O was added to each well and the plates were incubated at 37°C for 4 h. Fluorescence was measured on an Infinite M2000 ProTM plate reader (Tecan, Crailsheim, Germany) using an excitation wavelength of 544 nm and an emission wavelength of 590 nm. Each assay was done at least twice with six replicates each. The viability was evaluated based on a comparison with untreated cells. IC50 values represent the compound concentrations required to inhibit 50% of cell proliferation and were calculated from a calibration curve by linear regression using Microsoft Excel .
RST and SLP isolated the compounds, SLP elucidated the structure and wrote the manuscript, VK carried out the bio-assays, TE and BTN read and brought some corrections to the paper. All authors read and approved the final manuscript.
V. K. is very grateful to the Alexander von Humboldt foundation for an 18 months fellowship to visit the Department of Prof. Efferth (Johannes Gutenberg-University, Mainz, Germany) through the "Georg Foster Research Fellowship for Experienced Researcher" program for funding this work.
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