Structure-Activity Relationship of Benzophenanthridine Alkaloids from Zanthoxylum rhoifolium Having Antimicrobial Activity

Structure-Activity Relationship of Benzophenanthridine Alkaloids from Zanthoxylum rhoifolium Having Antimicrobial Activity Luciana de C. Tavares1, Graciane Zanon1, Andréia D. Weber1, Alexandre T. Neto1, Clarice P. Mostardeiro2, Ivana B. M. Da Cruz2,3, Raul M. Oliveira2, Vinicius Ilha1, Ionara I. Dalcol1,2, Ademir F. Morel1* 1 Center for Research in Natural Products (NPPN), Federal University of Santa Maria, Santa Maria, RS, Brazil, 2 Graduate Program in Pharmacology, Health Sciences Center, Federal University of Santa Maria, Santa Maria, RS, Brazil, 3 Graduate Program in Toxicological Biochemistry, Federal University of Santa Maria, Santa Maria, RS, Brazil Abstract Zanthoxylum rhoifolium (Rutaceae) is a plant alkaloid that grows in South America and has been used in Brazilian traditional medicine for the treatment of different health problems. The present study was designed to evaluate the antimicrobial activity of the steam bark crude methanol extract, fractions, and pure alkaloids of Z. rhoifolium. Its stem bark extracts exhibited a broad spectrum of antimicrobial activity, ranging from 12.5 to 100 mg/mL using bioautography method, and from 125 to 500 mg/mL in the microdilution bioassay. From the dichloromethane basic fraction, three furoquinoline alkaloids (1–3), and nine benzophenanthridine alkaloids (4–12) were isolated and the antimicrobial activity of the benzophenanthridine alkaloids is discussed in terms of structure-activity relationships. The alkaloid with the widest spectrum of activity was chelerythrine (10), followed by avicine (12) and dihydrochelerythrine (4). The minimal inhibitory concentrations of chelerythrine, of 1.50 mg/mL for all bacteria tested, and between 3.12 and 6.25 mg/mL for the yeast tested, show this compound to be a more powerful antimicrobial agent when compared with the other active alkaloids isolated from Z. rhoifolium. To verify the potential importance of the methylenedioxy group (ring A) of these alkaloids, chelerythrine was selected to represent the remainder of the benzophenanthridine alkaloids isolated in this work and was subjected to a demethylation reaction giving derivative 14. Compared to chelerythrine, the derivative (14) was less active against the tested bacteria and fungi. Kinetic measurements of the bacteriolytic activities of chelerythrine against the bacteria Bacillus subtilis (Gram-positive) and Escherichia coli (Gram-negative) were determined by optical density based on real time assay, suggesting that its mechanism of action is not bacteriolytic. The present study did not detect hemolytic effects of chelerythrine on erythrocytes and found a protective effect considering the decrease in TBARS and AOPP (advanced oxidized protein products) levels when compared to the control group. Citation: Tavares LdC, Zanon G, Weber AD, Neto AT, Mostardeiro CP, et al. (2014) Structure-Activity Relationship of Benzophenanthridine Alkaloids from Zanthoxylum rhoifolium Having Antimicrobial Activity. PLoS ONE 9(5): e97000. doi:10.1371/journal.pone.0097000 Editor: Mark J. van Raaij, Centro Nacional de Biotecnologia – CSIC, Spain Received December 17, 2013; Accepted April 15, 2014; Published May 13, 2014 Copyright: ß 2014 Tavares et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors thank CNPq (Conselho Nacional de Desenvolvimento Cientı́fico e Tecnológico) and FAPERGS (Fundação de Amparo a Pesquisa do Estado do Rio Grande do Sul)-PRONEX for financial support for this work. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: In previous studies of our research team on the crude extract of Z. rhoifolium, the isolation and structural elucidation of a number of alkaloids have been reported, including zanthoxyline, nitidine, oxynitidine and skimmianine [19]. We have also reported the antibacterial activity of the crude MeOH extract, aqueous extract, acid and basic fractions of Z. rhoifolium by disc diffusion method [20] and of some isolated alkaloids by the TLC bioassay (bioautography method) [21]. More recently, our research team reported the presence of several benzophenanthridine alkaloids with interesting antitumoral activities from the same source [22]. Because many diseases that are widespread among underprivileged and indigenous populations, such as fever, stomach upset and respiratory diseases, among others, can be caused by fungi and bacteria, this study investigated the antimicrobial activities of the crude methanol extract, fractions obtained from this extract and purified compounds from the stem bark of Z. rhoifolium collected in Rio Grande do Sul, Brazil, were initially screened by Introduction Many studies have underlined the importance and use of plants as a source of antimicrobial agents [1]. In this context, plants of the genus Zanthoxylum (Rutaceae), which encompasses about 250 species distributed throughout the world, have been of great importance. Among these, Zanthoxylum rhoifolium (syn. Fagara rhoifolium), native to South America (Brazil, Uruguay, Paraguay and Argentina) has been used in Brazilian traditional medicine for the treatment of many different health problems [2–4]. In French Guiana, Z. rhoifolium has been used as an antimalarial treatment [5]. These plants are known to be a rich source of natural products, mainly alkaloids and lignans. Some of these alkaloids, including the benzophenanthridines and furoquinolines, display a variety of biological activities, such as antitumor [6–11], antimicrobial [12], anti-inflammatory [13–15] and antiplasmodial [16– 18] activity. PLOS ONE | www.plosone.org 1 May 2014 | Volume 9 | Issue 5 | e97000 Antimicrobial Benzophenanthridine Alkaloids bioautography method [23,24] to select extracts, fractions and active alkaloids. The selected extracts, fractions and alkaloids were subsequently analyzed using the broth microdilution method [25,26] for determining the minimum inhibitory concentration (MIC) and the minimum bactericidal/fungicidal concentration (MBC/MFC). Chelerythrine, representing the other benzophenanthridine alkaloids isolated in this work, presented potential anti-tumor, anti-microbial and anti-inflammatory properties [27]. Chelerythrine is also a well-known protein kinase C inhibitor molecule responsible for the maintenance of erythrocyte deformability [28]. Some studies, such as that of Kim et al. [29], have suggested that chelerythrine stimulates the production of reactive oxygen species. The acid and basic fractions of the plant, obtained after acidbasic extraction of part of the methanol crude extract, exhibited significant inhibitory activity against some of the microorganism (...truncated)