Cytotoxic Benzophenanthridine and Furoquinoline Alkaloids from Zanthoxylum buesgenii (Rutaceae)

Background 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. Results 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. Conclusions The observed activities corroborate those previously reported on similar benzophenanthridine alkaloids indicating that compounds 1 and 2 can chemically be explored to develop other chemotherapeutic agents. Graphical abstract Cytotoxic Benzophenanthridine and Furoquinoline Alkaloids from Zanthoxylum buesgenii (Rutaceae). Electronic supplementary material The online version of this article (doi:10.1186/s13065-014-0061-4) contains supplementary material, which is available to authorized users.


Background
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 [1,2]. 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 [2]. 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 [3]. Previous chemical studies on Zanthoxylum species reported the identification of lignans, coumarins, diterpenes, sesquiterpenes, steroids, alkaloids [4] and benzopropanoids [5]. Interestingly, alkaloids represent the largest group of secondary metabolites obtained from the genus Zanthoxylum with structurally diverse scaffolds including oxoaporphines [6], aporphines, quinolinones, furoquinolines [4], indolopyridoquinazolines, β-carbolines, and benzophenanthridines [4,7]. Besides bioactivities such as trypanocidal [8], antileishmanial [9], antimycobacterial [10] effects, most of these alkaloids have shown from moderate to significant cytotoxicity against several cancer cell lines [11][12][13]. 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 multidrugsresistant cancer cell lines.
The relative configuration at C-6 could not be established by using NMR information although the methoxy proton (δ H 4.24) showed NOE contact ( Figure 2) with the hemiaminal proton (δ H 6.54) which in turn revealed similar interactions with the NCH 3 group. Likewise, the NCH 3 group correlated with the aromatic proton H-4 at δ H 7.89 while H-1 had spatial correlations with H-12 and H-11 showed similar contact with H-10.

Biological assay
Compound 1 displayed cytotoxicity towards all the nine tested cancer cell lines with IC 50 values below or around 65 μM while other metabolites showed selective activities. The activities of compounds 2-4 were observed on 8/9, 2/9, and 6/9 of the tested cancer cell lines, respectively ( Table 2). The lowest IC 50 values of 0.24 μM and 0.30 μM were obtained with compounds 1 and 2, respectively towards the leukemia CCRF-CEM cancer cell line. The activities of compounds 1-4 were better than that of doxorubicin towards the resistant CEM/ ADR5000 cell line (Table 2). Compound 1 can therefore be considered as a potential cytotoxic candidate agent to fight malignant diseases. Interestingly, compound 1 was active on both sensitive and resistant cell lines. Meanwhile, all tested compounds were generally less toxic on normal AML12 hepatocytes. However, compound 1 was generally less active than the reference drug, doxorubicin but could inspire synthesis of more cytotoxic analogues. This assumption is supported by recent studies showing the ability of some benzophenanthridines to induce apoptosis in colon carcinoma cancer cells HCT116 [12]. Besides, sanguinarine structurally related to compounds 1 and 2 has been previously reported as apoptosis inducer in KB [19], AsPC-1, BxPC-3 [20], U937 [21], and MDA-MB-231 [22] cancer cells via different mechanisms.

Conclusions
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 [19][20][21][22] indicating that compounds 1 and 2 can be chemically explored to develop other chemotherapeutic agents.

Plant collection
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.

Cytotoxicity assay
The resazurin reduction assay [23] 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 × 10 4 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 × 10 4 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 H 2 O 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. IC 50 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 [24].