Cytotoxicity and Genotoxicity Evaluation of Zanthoxylum rhoifolium Lam and In Silico Studies of Its Alkaloids

Abstract

The alkaloids isolated from Zanthoxylum rhoifolium have demonstrated great pharmacological potential; however, the toxic profiles of these extracts and fractions are still not well elucidated. This study evaluated the toxicity of the ethanol extract (EEZR) and neutral (FNZR) and alkaloid (FAZR) fractions. Chemical characterization was performed by chromatographic methods: thin-layer chromatography (TLC) and high-performance liquid chromatography coupled with diode array detection (HPLC-DAD). The cytotoxicity of the samples was evaluated in human hepatocellular carcinoma (HepG2) cells using the cell viability method (MTT) and mutagenicity by the Allium cepa assay (ACA). Alkaloids isolated from the species were selected for toxicity prediction using preADMET and PROTOX. The molecular docking of the topoisomerase II protein (TOPOII) was used to investigate the mechanism of cell damage. In the EEZR, FNZR, and FAZR, the presence of alkaloids was detected in TCL and HPLC-DAD analyses. These samples showed a 50% inhibitory concentration (IC₅₀) greater than 400 μg/mL in HepG2 cells. In ACA, time- and concentration-dependent changes were observed, with a significant reduction in the mitotic index and an increase in chromosomal aberrations for all samples. Nuclear sprouts and a micronucleus of the positive control (PC) were observed at 10 µg/mL and in the FAZR at 30 µg/mL; a chromosomal bridge in FNZR was observed at 105 µg/mL, CP at a concentration of 40 µg/mL, and nuclear bud and mitotic abnormalities in the EEZR were observed at 170 µg/mL. The alkaloids with a benzophenanthridine were selected for the in silico study, as structural alterations demonstrated certain toxic effects. Molecular docking with topo II demonstrated that all alkaloids bind to the protein. In summary, the fractionation of Z. rhoifolium did not interfere with toxicity; it seems that alkaloids with a benzophenanthridine nucleus may be involved in this toxicity.

Keywords: Zanthoxylum rhoifolium; cytotoxicity; molecular docking; mutagenicity.

Figure 1

Benzophenanthridine and furoquinolone alkaloids isolated from Z. rhoifolium.

Figure 2

Chromatograms of Z. rhoifolium extract and fractions and their ultraviolet spectra. Chromatographic conditions: temperature: 40 °C; flow: 0.5 mL/min; volume: 20 µL; column: C18; mobile phase: deionized water, 0.1% formic acid (eluent A), and acetonitrile plus 0.1% formic acid (eluent B); reading at wavelengths of 215, 229, 290, and 320 nm. Legend: 1—ethanol extract of Z. rhoifolium (EEZR); 2—fraction of neutrals (FNZR); 3—fraction of alkaloids (FAZR); A–D: ultraviolet spectra with absorbances suggestive of chromophores present in alkaloids.

Figure 3

Determination of cell viability at different exposure times and concentrations. EEZR: Zanthoxylum rhoifolium ethanol extract; NC: negative control; CE: ethanol concentration; FNZR: neutral fraction of Zanthoxylum rhoifolium; FAZR: alkaloid fraction of Z. rhoifolium.

Figure 4

Mitotic index of the samples after 24 h at different concentrations and analysis of the cellular alterations caused by Z. rhoifolium. Mitotic index of the samples after 24 h at different concentrations: (1): prophase for the NC, telophase for the FAZR; (2): prophase and telophase for the PC, telophase and metaphase for the FAZR; (3): prophase for the FNZR; (4): prophase and telophase for the PC, telophase and metaphase for the FAZR; (5): prophase and telophase for the FAZR. Mutagenic effect at different concentrations after 24 h: (A): anaphase with a nuclear bridge and a nuclear bud for the NC after 72 h; (B): micronucleus and a nuclear bud for the PC at 10 µg/mL; (C): PC nuclear abnormalities at the concentration of 40 µg/mL; (D): C-metaphase, delay in anaphase at 80 µg/mL after 48 h for the EEZR; (E): mitotic abnormalities, T-metaphase for the EEZR at 170 µg/mL; (F): C-metaphase and a nuclear bud for the FNZR at 52.5 µg/mL; (G): chromosome-bridging anaphase for the FNZR at 105 µg/mL, (H): C-metaphase and a nuclear bud for the FAZR at 30 µg/mL.

Figure 5

Alkaloids selected for prediction studies: 1—rhoifoline A, 2—rhoifoline B, 3—dihydronitidine, 4—decarine, 5— (+/−)-6-Acetonyldihydrochelerythrine, 6—1-methoxy-7,8-dehydrorutaceacarpine, 7—magnoflorine, 8—5-methoxy-canthin-6-one, 9—zanthoxyline, 10—nitidine, 11—avicine, 12—6-acetonyldihydroavicine, 13—columbamine, 14—chelerythrine, 15—O-methylcapaurine, 16—syncarpamide, 17—bocconoline, 18—nornitidine, 19—dictamine, 20—dihydroavicine, 21—oxynitidine, 22—norfagaronine, 23—bis[6-(5,6-dihydrochelerythrinyl)], 24—dectamnine, 25—skimmianine, 26—pelitorine, 27—Z-dimethylrhoifolinate, 28—canthin-6-one, and 29—quinazoline-6-carboxylic acid.

Figure 6

Representations of the intramolecular interactions of the compounds rhoifoline A (1), rhoifoline B (2), zanthoxyline (3), and decarine (4).