Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 73(3): 111-120, 2020 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.13128/caryologia-880 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: V. Ventura de Souza, M. Sossai Spadeto, R. Abreu Guedes, W.R. Clarindo, C.R. de Carvalho, J.A. Severi, T. da Silva Souza (2020) Toxic- ity of Aristolochia decoction: a relevant herbal in folk medicine. Caryologia 73(3): 111-120. doi: 10.13128/caryologia-880 Received: March 13, 2020 Accepted: July 14, 2020 Published: December 31, 2020 Copyright: © 2020 V. Ventura de Souza, M. Sossai Spadeto, R. Abreu Guedes, W.R. Clarindo, C.R. de Carvalho, J.A. Severi, T. da Silva Souza. This is an open access, peer-reviewed article published by Firenze University Press (http://www.fupress.com/caryologia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distri- bution, and reproduction in any medi- um, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. Toxicity of Aristolochia decoction: a relevant herbal in folk medicine Victor Ventura de Souza1, Micheli Sossai Spadeto2, Roselena Abreu Guedes4, Wellington Ronildo Clarindo1,2, Carlos Roberto de Carval- ho3, Juliana Aparecida Severi4, Tatiana da Silva Souza1,2,* 1 Departamento de Biologia, Centro de Ciências Exatas, Naturais e da Saúde, Universi- dade Federal do Espírito Santo, Alegre – ES, Brasil 2 Programa de Pós-Graduação em Genética e Melhoramento, Centro de Ciências Agrárias e Engenharias, Universidade Federal do Espírito Santo, Alegre – ES, Brasil 3 Departamento de Biologia Geral, Universidade Federal de Viçosa, Viçosa – MG, Brasil 4 Departamento de Farmácia, Centro de Ciências Exatas, Naturais e da Saúde, Universi- dade Federal do Espírito Santo, Alegre – ES, Brasil *Corresponding author. E-mail: tatianas.souza@hotmail.com Abstract. Ethnopharmacology studies report the use of Aristolochia (Aristolochi- aceae) species as medicinal plants in various parts of the world. However, the acids aristolochic (AAs), secondary metabolites present in all species of Aristolochia, have cytogenotoxic activity and they are a potent carcinogen to rodents and humans. The aim of the current research was to perform to initial screening for the toxicity of Aris- tolochia labiata and Aristolochia triangularis decoctions through germination and growth rate, flow cytometry, mitotic index and cytogenetics analysis in Allium cepa. The decoctions were prepared from 2, 4, 8, 16 and 32 g L-1. Decoctions at concentra- tions 4 g to 16 g L-1 significantly reduced the germination rate of Allium cepa. Seeds exposed to 32 g L-1 decoctions did not germinate. All decoctions reduced the growth rate of onion seedlings. Decoctions at 4 g L-1 to 16 g L-1 inhibited mitotic index. High- est concentrations of decoctions (8 g L-1 and 16 g L-1 for Aristolochia labiata; 16 g L-1 for Aristolochia triangularis) showed statistically significant increase in frequency of Allium cepa nuclei in the G0/G1 phase. Both decoctions induced the formation of het- eropycnotic nuclei. Qualitative phytochemical prospecting of decocts were performed and alkaloids secondary compounds were the largest presence in both species, indi- cating that the AAs may be related to the observed toxicity. Caution is recommended in the consumption of decoctions from Aristolochia labiata and Aristolochia triangula- ris stems. Keywords: Aristolochic acid, cytogenotoxicity, flow cytometry, heteropycnotic nuclei, Allium cepa test. INTRODUCTION Large part of the population of developing countries, especially tradi- tional communities, depends on herbal medicine for primary health care 112 Victor Ventura de Souza et al. (WHO, 1978). The use of medicinal plants has been stimulated by Brazilian government aiming at the sus- tainable use of Brazilian biodiversity and the improve- ment of the public health system (Brasil, 2006). Ethnopharmacology studies report the traditional knowledge on the use of Aristolochia (Aristolochiace- ae) species as medicinal plants in various parts of the world (Heinrich et al., 2009; Michl et al., 2013). In Bra- zil, Albuquerque et al. (2007) verified that Aristolochia labiata (flowers, leaves and whole plant) is used by tra- ditional communities in the caatinga region to relieve or cure menstrual colic and uterine inf lammations. Decoction, infusion and maceration of leaves and stems of Aristolochia triangularis Cham. have traditionally been used to treat gynecology and urinary, gastro-intes- tinal, respiratory and musculoskeletal and joint diseases (Araujo & Lemos, 2015; Bolson et al., 2015). The ethno- botanical survey showed that the Aristolochia triangu- laris is used as tea for the cure of diseases and/or cul- turally defined symptoms. Also, the species is used for the cure of spiritual diseases in the form of baths (Silva, 2008). Araujo and Lemos (2015) and Bolson et al. (2015) document that Aristolochia triangularis had the highest use values among the species cited by residents of tradi- tional communities in the Northeast and South of Bra- zil, respectively. Some medicinal plant compounds have their toxi- cological properties well documented in the literature. Despite the therapeutic effects of Aristolochia species, these plants present the Aristolochic acids (AAs) which are nitrophenanthrenes carboxylic acids. Aristolochic acids I (AAI) and II (AAII) present known cyto- and genotoxic activity and they are a potent carcinogen to rodents and humans (Chang et al., 2007; Slade et al., 2009; Hwang et al., 2012; Bunel et al., 2016; Youl et al., 2020), causing a specific nephropathy associated with renal cancer (Li et al., 2018; Sborchia et al., 2019). As a consequence of their toxic effects, the consumption of Aristolochia species and its derivatives is prohibited in many countries such as Australia, Canada and United Kingdom (IARC, 2002; Neinhuis et al., 2005). However, in Brazil its consumption is not regulated, being com- mon to find dry parts of the plant in popular markets (Silva, 2008), pharmacies and stores of natural products, ready to be prepared. AAs toxicity becomes clinically significant after long periods of ingestion (Yamani et al., 2015). Nevertheless, the knowledge concerning about toxic effect of decoctions of Aristolochia species is very scarce (Amat et al., 2002). Allium cepa test is commonly used to evaluate the toxic potential of medicinal plants and their metabolites (Akinboro & Bakare, 2007; Oloyede et al., 2009). The germination rate of the seeds and the final growth of the seedlings are used to evaluate the phytotoxicity of the several compounds (Macedo et al., 2008). Besides, Alli- um cepa test has also been accomplished to detect chro- mosomal abnormalities generated during the cell cycle (Vicentini et al., 2001). This analysis is possible because Allium cepa has few chromosomes (2n = 16) with rela- tively large total length (Grant 1982). Still, Allium cepa test shows a similar sensitivity to other systems, like human lymphocytes (Fiskesjö 1985), and a correlation of 82% to carcinogenicity tests in rodents (Rank & Nielsen, 1994). Flow cytometry (FCM) has broadly contributed to improve knowledge on the plants and animals’ cell cycle and has been employed in the biomedical field, pharma- cology, oncology and ploidy level determination (Jayat & Ratinaud, 1993). Currently, FCM applied in plants repre- sent a very powerful tool to detect the cytotoxicity and DNA damage caused by drugs and environmental con- taminants (Citterio et al., 2002; Monteiro et al., 2010; Andrade-Vieira et al., 2012). Considering the wide use of Aristolochia labiata and Aristolochia triangularis as medicinal plants, this study was conducted to analyze the toxicogenetic effects of their decoctions in root meristematic cells of Allium cepa. From the preliminary results obtained in this study, renal and testicular histopathology on mice is ongoing. These data are expected to help elucidate the effects of Aristolochia decoctions. MATERIALS AND METHODS Plant materials Aristolochia labiata was collected in the forest gar- den of the Universidade Federal de São João Del-Rei (UFSJ), located in the city of São João del Rei, Minas Gerais State, Brazil. Aristolochia triangularis was col- lected in the Health Ministry located in the city of Ven- da Nova do Imigrante, Espírito Santo State, Brazil. A voucher specimen of the plants has been deposited at the herbarium of UFSJ, under reference number UFSJ132 (Aristolochia labiata) and UFSJ5571 (Aristolochia trian- gularis). Plant names were checked and updated with the Royal Botanic Gardens, Kew online website www. theplantlist.org. Seeds of Allium cepa (Isla®; batch number: 774758; 90% of germination) were used as a test organism in current research because they are genetically and physi- ologically homogeneous (Leme et al., 2008). 113Toxicity of Aristolochia decoction: a relevant herbal in folk medicine Preparation of decoctions Stems of Aristolochia labiata and Aristolochia tri- angularis were boiled for 5 min in 1 L of distilled water (dH2O). Considering that 8 g L-1 is the dosage commonly used in popular medicine (Simões et al., 1995), 2, 4, 8, 16 and 32 g L-1 concentrations were prepared. Qualitative phy tochemical prospecting of both decoctions was carried out from the methodology pro- posed by Matos (1997). For this, the presence of the following metabolites was evaluated: phenols and fla- vonoids (sulfuric acid test), tannins (iron chloride III), saponins (foam index), coumarin (NaOH solution), alka- loids (Mayer and Wagner reagent), anthraquinone (ethyl ether + ammonia) and terpenoids (Liebermann–Bur- chard test). Seed germination and seedling growth rates To the test hypothesis that Aristolochia decoctions are phy totoxic (affects seed germination and seed- ling growth) to Allium cepa, onion seeds were exposed for 28 days to aqueous extracts. Culture medium MS (Murashige & Skoog, 1962) were supplemented with: 30 g of sucrose of L-1, 0.4 g of L-glutamine, 0.04 g of L-cysteine, 7 g of agar-1, pH = 5.7 ± 0.1. Culture media were autoclaved for 20 min at 121°C under 1 atm. Imme- diately after autoclaving, the decoction extracts were filter-sterilized and added to the tissue culture media. Colchicine at 0.025% was used as positive control (PC) and MS medium without addition of decocts as negative control (NC). In laminar flow, Allium cepa seeds were disinfected in 70% ethanol solution for 30 s and then with 1.5% NaOCl2 solution for 20 min, and washed three times in autoclaved dH2O for 1 min (Mursarurwa et al., 2010). Fifteen seeds were inoculated in eight flasks con- taining 15 mL of the culture medium. The inoculated seeds were maintained in a growth room with photoperiod 16/8 hours in light under 24 ± 1ºC. Seed germination (percentage of germinated seeds in each treatment after initial exposure) was recorded on the 28th day. In this time, the final length of the seed- lings was measured for each treatment using a caliper rule. Mitotic and chromosomal abnormalities Onion seeds were germinated in Petri dishes lined with moistened filter paper with the different concentra- tions of decoctions. In Addition, dH2O was used as neg- ative control (NC) and the 0.025% colchicine as positive control (PC). Germination occurred in B.O.D. at 24°C for 72 h. Allium cepa roots with 1–2 cm were excised, fixed in ethanol: glacial acetic acid (3:1, v/v) and stored at -20°C. After 24h, the roots were hydrolyzed in 1N HCl at 25°C for 20 min, and subsequently the root mer- istems were stained with 2% acetic orcein, covered with foil and crushing the material. The slides were examined under a light microscope with 100x objective. Ten slides were prepared for each treatment being analyzed 500 cells/slide. Cytotoxic potential of decoctions was evalu- ated by mitotic index (number of dividing cells/total observed cells). Genotoxic effect was measured by the chromosomal abnormalities index (CA). Mutagenicity analysis was performed by micronuclei (MN) frequency. Flow cytometry The meristems of Allium cepa root after germination for 72 hours in a Petri dish were fixed in ethanol: gla- cial acetic acid (3: 1, v / v) were stored for 24 hours at -20 ° C, transferred to 70% ethanol and stored at -20°C. FCM analyzes were performed with 5 replicates for each decoction concentration and negative control (dH2O), being three root meristems for each replicate. Root api- cal meristems were excised, washed 3 times for 10 min in dH2O. Nuclei isolation and staining were performed according to the protocol proposed by Silva et al. (2010). Nuclei suspensions were analyzed by flow cytometry PAS® Partec flow cytometer (Partec® GmbH, Munster, Germany) after calibration of the equipment. The fre- quency of nuclei in G0/G1, S and G2/M phases was meas- ured to verify the cytotoxicity of the decoctions in each specific cell cycle period. Statistical analyses Statistical analyses were performed using the Bioestat 5.3 software. The Shapiro-Wilk test was used to verify the normality of the samples. As normality crite- ria were not satisfied, the non-parametric Kruskal-Wallis test with subsequent Dunn test (p<0.05) was performed. RESULTS Qualitative phytochemical analysis of decoctions is shown in Table 1. Decoctions presented phenols (detected by sulfuric acid) and alkaloids (detected by Mayer and Wagner reagents) as secondary metabolites. Since the alkaloid secondary compounds were the largest presence in both species, other metabolites were not detected. 114 Victor Ventura de Souza et al. Table 2 shows the effects of the decoctions of Aris- tolochia labiata and Aristolochia triangularis on seed germination and seedlings of Allium cepa. Seeds of the control group germinated satisfactorily. Decoctions of both species at concentrations 4, 8 and 16 g L-1 sig- nificantly reduced the germination rate of Allium cepa. Seeds exposed to 32 g L-1 decoctions did not germinate. All decoctions concentrations reduced the onion seed- lings growth rate (Table 2, Figure 1A, 1B). Cell proliferation rate was 13.34% in the control group (Table 3). Decoctions at 2.0 g L-1 did not pro- moted significant reduction of mitotic index. Decoc- tions at 4, 8 and 16 g L-1 inhibited cell proliferation in Allium cepa (Table 3). Changes in Allium cepa cell cycle Table 1. Qualitative phytochemical analysis of Aristolochia labiata and Aristolochia triangularis Reagents Metabolite class Aristolochia labiata Aristolochia triangularis Sulfuric acid Phenols + + Sulfuric acid Flavonoides - - Iron chloride III Tannins - - Foam index Saponins - - NaOH solution Coumarin - - Mayer reagent Alkaloids +++ +++ Wagner reagent Alkaloids + + Ethyl ether + ammonia Anthraquinone - - Lieberman Burchard Terpernóides - - (-) absence; (+) presense Table 2. Effects of decoctions of to Aristolochia labiata and Aris- tolochia triangularis on seed germination and seedling growth of Allium cepa Species Samples Germination (%) Seedling growth (cm) Aristolochia labiata MS 87.5 8.48 ±4.21 Colchicine 0.025% 75.0 0.93± 0.25* 2 g l-1 87.5 1.46 ±0.92* 4 g l-1 62.5* 1.44± 0.37* 8 g l-1 62.5* 0.88±0.32* 16 g l-1 50.0* 0.67± 0.22* 32 g l-1 NG NG Aristolochia triangularis MS 87.5 8.48 ±4.21 Colchicine 0.025% 75.0 0.93± 0.25* 2 g l-1 75.0 0.53±0.19* 4 g l-1 50.0* 0.82 ±0.14* 8 g l-1 50.0* 0.45±0.50* 16 g l-1 50.0* 0.40±0.43* 32 g l-1 NG NG MS: negative control -culture medium (Murashige and Skoog, 1962). Colchicine 0.025%: positive control. NG: did not germinate. * Significant difference in relation to negative control (p <0.05) - Kruskal-Wallis test. Figure 1. Seedlings of Allium cepa after 28 days of exposure to the decoctions of (A) Aristolochia labiata and (B) Aristolochia triangularis. NC = negative control - culture medium (Murashige and Skoog, 1962); PC = positive control (0.025% colchicine). 115Toxicity of Aristolochia decoction: a relevant herbal in folk medicine are also shown in Table 4 and representative histograms are shown in Figure 2. According to FCM analyses, decoctions of both species promoted a concentration- dependent increase in frequency of Allium cepa nuclei in the G0/G1 phase. This increase was statistically signifi- cant for the highest concentrations of decoction (8 and 16 g L-1 for Aristolochia labiata; 16 g L-1 for Aristolochia triangularis). Thus, the frequency of nuclei in the S and G2/M phase tended to decreased, parking cells in inter- phase. Aristolochia labiata decoctions at 8 and 16 g L-1 caused a greater number of cell damage; the highest dose Table 3. Mitotic index (% percentage ± standard deviation) of Allium cepa root meristem cells exposed to decoctions of Aristolochia labiata and Aristolochia triangularis Species Samples Mitotic index % Number of cells in each cell cycle phase Interphase Prophase Metaphase Anaphase Telophase Aristolochia labiata Distilled water 13.34 ± 4.72 4333 335 126 137 69 Colchicine 0.025% 10.52 ± 3.61 4286 343 149 150 72 2 g l-1 6.92 ± 3.56 4691 75 144 81 9 4 g l-1 1.02 ± 0.86* 4949* 9* 21* 15* 6* 8 g l-1 1.10 ± 2.30* 4941* 11* 29* 13* 6* 16 g l-1 0.76 ± 0.68* 4960* 12* 6* 15* 7* 32 16 g l-1 NG NG NG NG NG NG Aristolochia triangularis Distilled water 13.34 ± 4.72 4319 339 128 142 72 Colchicine 0.025% 10.52 ± 3.61 4286 343 149 150 72 2 g l-1 8.38 ± 3.18 4951* 15 14 14 06 4 g l-1 2.20 ± 3.94* 4888* 51* 24* 24* 13* 8 g l-1 3.34 ± 3.69* 4803* 69* 73* 53* 02* 16 g l-1 3.84 ± 2.14* 4961* 16* 06* 11* 06* 32 16 g l-1 NG NG NG NG NG NG Distilled water: negative control. Colchicine 0.025%: positive control. NG: did not germinate.* Significant difference in relation to negative control (p <0.05) - Kruskal-Wallis test. Figure 2. Histogram representative of the meristem of Allium cepa. A) Histogram representing the negative control. Note the presence of the G0/G1 peak (channel 100) G2/M (channel 200), and between the particles at different times is S phase of the cell cycle, showing that the negative control has particles at all stage of G0/M. B) His- togram representing the treatment of 16 g l -1 of Aristolochia labiata, observe particles in the peak (channel 100) demonstrating that the cells are in G0/G1, but there is no peak at (channel 200), which con- firms the no progression of nuclei for the G2/M phase, indicating that the extracts prevent cell proliferation. Table 4. Frequency (%) of Allium cepa nuclei in cell cycle phases after treatment with Aristolochia labiata and Aristolochia triangula- ris decocts Species Samples %G0/G1 %S %G2/M Aristolochia labiata Distilled water 70.08 ± 6.91 18.37 ± 4.03 11.00 ± 3.62 2 g l-1 88.96 ± 1.90 8.08 ± 1.36 3.00 ± 0.93 4 g l-1 91.34 ± 2.93 6.46 ± 2.63 2.00 ± 1.07 8 g l-1 97.16 ± 2.91* 2.57 ± 2.81* 0.27 ± 0.60* 16 g l-1 99.85 ± 0.32* 0.14 ± 0.32* 0.00 ± 0.00* 32 g l-1 NG NG NG Aristolochia triangularis Distilled water 74.08 ± 4.52 11.02 ± 4.17 14.89 ± 1.93 2 g l-1 84.72 ± 2.83 8.82 ± 1.86 6.46 ± 4.54 4 g l-1 86.23 ± 3.61 8.12 ± 1.47 5.63 ± 3.24 8 g l-1 87.18 ± 4.03 7.22 ± 2.84 5.59 ± 1.48 16 g l-1 92.28 ± 2.09* 4.34 ± 1.17* 3.38 ±1.24* 32 g l-1 NG NG NG Distilled water: negative control. NG: did not germinate. * Signifi- cant difference in relation to negative control (p <0.05) - Kruskal- Wallis test. 116 Victor Ventura de Souza et al. reached a level of absence of core peak in G2/M phase. Aristolochia triangularis decoction at 16 g L-1 significant- ly reduced the number of nuclei in S and G2/M phase. Due to severe cytotoxicity, chromosomal abnormali- ties and micronuclei were not observed. Heteropycnotic nuclei (Figure 3) were analyzed separately. Decoctions of both species at 4, 8 and 16 g L-1 significantly increased the frequency of this abnormality. Aristolochia trian- gularis decoction at 2 g L-1 also induced heteropycnotic nuclei on the Allium cepa root cells (Table 5). DISCUSSION The genus Aristolochia presents a wide range of physiologically active compounds classified in five main categories: terpenes, phenols, alkaloids, flavonoids and lignoids (Pacheco et al., 2009). Phytochemical analy- ses indicated mainly the presence of alkaloids in both decoctions studied and in a lower extent, phenols. Phe- nols bring advantages to the plant, as they are related to attraction of pollinators and protection against her- bivory among others (Piesik et al., 2011). Species of Aris- tolochiaceae are rich in alkaloids (Schmeiser et al., 2001), including AAs that have attracted intense research inter- est because of cyto- and genotoxic properties of AAI and AAII (Wu et al., 2005; Chang et al., 2007; Slade et al., 2009; Bastek et al., 2019). The amounts of AAI and AAII in decoctions were not determined. However, as the AAs are slightly soluble in water (O’Neil, 2001), we believe that possibly they are present in decoctions, which is the way by which peo- ple consume species of Aristolochia. Hwang et al. (2012) quantified AAs in aqueous extracts of Aristolochia man- shuriensis Kom. The genotoxicity of the extracts was detected by bacterial reverse mutation assay and micro- nucleus in mice bone marrow erythrocytes. According to the authors, the genotoxicity of Aristolochia manshu- riensis is directly related to the AAs. Allium cepa test has been used as first cytogenotoxic screening of medicinal plant extracts (Dias & Takahashi, 1994; Fachinetto et al., 2007; Meneguetti et al., 2014; Mendes et al., 2012) because the results are reliable and similar to those performed with in mammals (Rank & Nielsen, 1994), contributing to the safe use of these herbs (Mendes et al., 2012). Studies concerning toxicological activity of Aris- tolochia extracts are very scarce, in spite of their use in several countries (Amat et al., 2002). In the current research, the toxic effects of decoctions of Aristolochia labiata and Aristolochia triangularis on Allium cepa were evaluated. Decoctions of both species at 32 g L-1 were phytotoxic because prevented the germination of onion seeds. The other concentrations tested promoted delayed germination and growth and mitodepressive Figure 3. Heteropicnotic nucleus (arrow) in Allium cepa meris- tematic root cells after exposure to the decoctions of Aristolochia labiata. 1000x magnification. Table 5. Percentage of chromosomal alterations (CA) and heterop- ycnotic nucleus observed in Allium cepa cells exposed to decoction of Aristolochia labiata and Aristolochia triangularis Species Samples %CA %Heteropycnotic nucleus Aristolochia labiata Distilled water 0.04 ± 0.12 0.00 ± 0.00 Colchicine 0.025% 5.88 ± 2.81* 0.00 ± 0.00 2 g l-1 0.12 ± 0.16 0.00 ± 0.00 4 g l-1 0.00 ± 0.00 8.84 ± 1.53* 8 g l-1 0.00 ± 0.00 15.8 ± 13.03* 16 g l-1 0.00 ± 0.00 29.60 ± 29.02* 32 g l-1 NG NG Aristolochia triangularis Distilled water 0.04 ± 0.12 0.00 ± 0.00 Colchicine 0.025% 5.88 ± 2.81* 0.00 ± 0.00 2 g l-1 0.00 ± 0.00 13.4 ± 7.95* 4 g l-1 0.00 ± 0.00 17.2 ± 19.57* 8 g l-1 0.00 ± 0.00 17.1 ± 13.02* 16 g l-1 0.00 ± 0.00 19.12 ± 22.2* 32 g l-1 NG NG Distilled water: negative control. Colchicine 0.025%: positive con- trol. NG: did not germinate. * Significant difference in relation to negative control (p <0.05) - Kruskal-Wallis test. 117Toxicity of Aristolochia decoction: a relevant herbal in folk medicine effects on Allium cepa root cells. Akinboro and Bakare (2007) also documented a relationship between mac- roscopic and microscopic parameters for Allium cepa root cells exposed to toxic aqueous extracts herbs. The growth inhibition of the seedlings was always accompa- nied by the reduction of the number of cells in division. Corroborating our results, Gatti et al. (2004) showed that extracts of Aristolochia esperanzae Kuntze delayed seed germination and root growth of Lactuca sativa L. and Raphanus sativus L. According to Baličević et al. (2015), extracts of Aristolochia clematitis reduced the germination and growth of Tripleurospermum inodo- rum L. (weeds), and the concentration of 100 g L-1 inhib- ited the germination of seeds of these plants. Aqueous extract at 25 g L-1 of Aristolochia triangularis presented antimitotic action to meristematic cells of Allium cepa (Amat et al., 2002). Watanabe et al. (1988) documented that AAs are potent inhibitors of seed germination. FCM analyses also showed a presence of compounds in the decoctions that delayed the progression of the cell cycle. The decoctions tested caused a concentration- dependent increase of Allium cepa nuclei in G0/G1. Con- sequently, Aristolochia decoctions promoted a reduc- tion of Allium cepa nuclei in S and G2/M phases. These results could reflect the activation of G0/G1 checkpoints in response to DNA damage. Plant cells have a p53-inde- pendent control of proliferation (Pelayo et al., 2003). The checkpoint pathways transduce antimitogenic sig- nals that lead to the temporary interruption of the cycle. Thus, the repair mechanisms can act before the irrevers- ible transition to the subsequent cycle phase (Pelayo et al., 2003; Junqueira & Carneiro, 2012). Also, heterop- ycnotic nuclei were observed in response to decoctions exposure. These markers of cell death are characterized by condensation of the nucleus (Andrade-Vieira, et al., 2012), making it inoperative for failure of the enzyme synthesis (Manjo & Joris, 1995; Levin et al., 1999). The activation of cell death mechanisms is the last resource to avoid proliferation of cells containing abnormal DNA. In this way, we could infer that DNA damage was not repaired in G1, and in response, cell death pathways were activated. Some studies also documented that Aristolochia extracts and AAs promoted cell cycle arrest and cell death in mammalian cell lines. Li et al. (2006) reported that AAI may cause DNA damage and cell cycle delay in porcine proximal tubular epithelial cell lines through a wild-type p53-independent pathway, prior to apopto- sis or necrosis. Chang et al. (2007) found that cell cycle distribution determined by flow cytometry showed an increase of human urinary tract epithelium cells in the G0/G1 phase after exposure to AAs mixture (41% AA I and 56% AA II). Proteins levels that block the cell cycle (p53, p21 and p27) have increased. Additionally, there was a decrease in cyclinD1/cdk4 complex, which control proteins required for the progression of the cycle (Chang et al., 2007). Li and Wang (2013) verified that methanol extract from Aristolochia debilis Siebold & Zucc. stems inhibited proliferation of human colon cancer cells by inducing sub-G1 arrest. The authors also showed that Aristolochia debilis induced apoptosis in HT-29 cells by upregulation of Bax and corresponding downregulation of Bcl-2 expression as well as ROS production. Allium cepa test was suitable for screening initial toxicity of Aristolochia labiata and Aristolochia triangu- laris decoctions. Our studies report similar results for other test systems. In vivo research on renal and testicu- lar histopathology of decoctions in mice is ongoing. CONCLUSION Allium cepa test was used to evaluate decoctions from Aristolochia labiata and Aristolochia triangula- ris stems. Phytochemical analysis indicated mainly the presence of alkaloids in both decoctions studied. The decoctions promoted inhibition of onion seed germina- tion and seedling growth. The mitodepressive effect of both decoctions was determined by mitotic index and FCM analyses. The induction of heteropycnotic nuclei suggests that decoctions promote cell death. We sug- gest that the AAs may be related to the observed toxicity. Caution is recommended in the consumption of decoc- tions from Aristolochia labiata and Aristolochia triangu- laris stems. ACKNOWLEDGEMENTS The authors thank Fundação de Apoio à Pesquisa e Inovação do Espírito Santo for undergraduate scholar- ship. We also thank Damielle Leite Figueiredo for tech- nical support and Ariane Tonetto Vieira for editing images. 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