Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 75(1): 99-107, 2022 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-1307 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Guadalupe Velázquez- Vázquez, Beatriz Pérez-Armendáriz, Verónica Rodríguez Soria, Anabella Handal-Silva, Luis Daniel Ortega (2022) Genotoxicity and cytotoxicity of Sam- bucus canadensis ethanol extract in meristem cells of Allium sativum. Caryologia 75(1): 99-107. doi: 10.36253/ caryologia-1307 Received: May 6, 2021 Accepted: December 17, 2021 Published: July 6, 2022 Copyright: © 2022 Guadalupe Velázquez- Vázquez, Beatriz Pérez-Armendáriz, Verónica Rodríguez Soria, Anabella Handal-Silva, Luis Daniel Ortega. This is an open access, peer-reviewed arti- cle published by Firenze University Press (http://www.fupress.com/caryo- logia) and 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. 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. ORCID GVV: 0000-0001-9879-0968 BPA: 0000-0002-4956-2480 VRS: 0000-0002-2287-8338 AHS: 0000-0002-6915-5655 LDO: 0000-0003-4672-8809 Genotoxicity and cytotoxicity of Sambucus canadensis ethanol extract in meristem cells of Allium sativum Guadalupe Velázquez-Vázquez1, Beatriz Pérez-Armendáriz1, Veróni- ca Rodríguez Soria1, Anabella Handal-Silva2, Luis Daniel Ortega1,* 1 Decanato de Ciencias Biológicas. Facultad de Biotecnología. Universidad Popular Autónoma del Estado de Puebla. 21 sur 1103 Col. Santiago. 72160. Puebla, México 2 Departamento de Biología y Toxicología de la Reproducción. Instituto de Ciencias, Ben- emérita Universidad Autónoma de Puebla. 72570 Puebla, México *Corresponding author. E-mail: : luisdaniel.ortega@upaep.mx Abstract. Sambucus canadensis is used in traditional medicine mainly in indigenous communities as an anti-inflammatory, antiviral, to treat cough, fever and other ail- ments, however, its use must be validated on scientific bases. The aim of this study was to evaluate the genotoxic and cytotoxic effect of the ethanol extract of Sambucus canadensis in meristem cells of Allium sativum with 5 treatments at concentrations of 125, 250, 500, 1000 and 1500 mg/L. Two thousand cells were counted per treatment; the mitotic index (MI) and nuclear abnormalities (NA) were evaluated. Data were ana- lyzed using variance analysis (ANOVA) and Chi square (X2) (p < 0.05). Root growth was found to be inhibited based on the concentration with statistically significant dif- ferences (p < 0.05). As the dose and exposure time of the ethanol extract increased, the MI decreased. The NA increased at the highest concentrations of 500, 1000 and 1500 mg/L and these differences were statistically significant compared to the control (p = 0.001). With the results obtained, it can be shown that the species has antiproliferative effects and genotoxic activity on the Allium sativum cell cycle, which can be extrapolat- ed to other types of eukaryotic cells. Therefore, despite being a plant with health ben- efits, moderate use and low concentrations are recommended to avoid harmful effects. Keywords: traditional medicine, chromosomal aberrations, biomodel, Allium sativum, genotoxicity, elderberry, plant extract. INTRODUCTION Medicinal plants are used by rural and urban populations in the treat- ment of numerous diseases (Chabán et al. 2019; Trap et al. 2020). In vari- ous parts of the world, they are the only source of medical care, mainly due to economic and geographical factors, customs and traditions (Marcot- ullio et al. 2018; Tedesco et al. 2017; Ullah et al. 2013). According to the World Health Organization (WHO), about 80% of the world’s population uses herbal remedies as primary health care. The use of plants to treat dis- 100 Guadalupe Velázquez-Vázquez et al. eases is still based on empirical knowledge, although they have been considered low risk compared to other synthetic drugs (De Smet 2007; Monroy et al. 2005, Newman and Cragg 2016). The scientific information available for most medicinal plants is still insufficient to guarantee their safe and efficient use (Moore et al. 2020; Pastori et al. 2013). Various studies have indi- cated the importance of evaluating their safety (Gan- jhu et al. 2015; Huang et al. 2015; Neira et al. 2018; Palatini and Komarnytsky 2019; Soliman 2010; Sousa et al. 2011, Vazirian et al. 2018) due to possible risks associated with their components, which may be poten- tially toxic, mutagenic, carcinogenic or teratogenic (Abdelmigid 2013; Bratu et al. 2012; Prasansuklab et al. 2020). Among the medicinal plants with high curative potential is Sambucus canadensis (L.) Bolli. A native of Mexico belonging to the Adoxaceae family, it is com- monly known as elderberry. This species has been used medicinally in indigenous communities as a bacteri- cide, anti-inflammatory, against flu, cough, dysentery, fever, as well as in uses related to rituals for pregnant women (Álvarez-Quiroz et al. 2017; Lee and Finn 2007; Sánchez-González et al. 2008; Wu et al. 2004). Its anti- microbial, antiviral, antioxidant and chemopreventive activities, among others (Sidor et al. 2014; Tedesco et al. 2017; Thole et al. 2006) have been associated with the components present in the species such as triter- penes, tannins and various types of flavonoids such as anthocyanins (Abdelmigid 2013; Ozgen 2010; Vujosevic et al. 2004), however, the presence of these compounds could also cause harmful effects such as nausea, vomit- ing and diarrhea, such as in the case of Sambucus nigra species, whose consumption in pregnant and lactating women, as well as in children and teenagers under 18 years of age, should be avoided (EMA/HMPC Europe- an Medicines Agency 2012). Information on toxicology, cy totoxicity and genotoxicity of Sambucus canaden- sis leaves is limited (Knudsen and Kaack 2015; Lee and Finn 2007; Schmitzer et al. 2012). It is important therefore to carry out studies to evaluate chromosomal damage and alterations of the mitotic cycle. Accord- ing to Hister et al. (2017); Nefic et al. (2013); Pinho et al. (2010); Souza et al. (2010); Tedesco et al. (2015), the Allium sp. biomodel is a widely used, efficient, fast, low-cost method, with extrapolatable results since ani- mal and plant chromosomes have similar structures. The aim of this study was to evaluate the cytotoxic and genotoxic effect of Sambucus canadensis on meristem cells of Allium sativum. MATERIALS AND METHODS Plant material The Sambucus canadensis (L.) Bolli. plant was col- lected in San Miguel Eloxochitlan in the Sierra Negra zone of Puebla, Mexico, coordinates 18°30’32”N and 96°55’22”W. The plant material was identified using tax- onomic techniques and one specimen was deposited in the Arboretum of the University of Puebla Botanic Gar- den (JB-BUAP) with the ID: 83771. Preparation of Sambucus canadensis extract The leaves of Sambucus canadensis (L.) Bolli. were used. The extract was obtained by macerating 750g the dry leaves of seven plants with 4L of 96% ethanol with double filtering. The extracts were vacuum filtered with Whatman No. 4 paper, the supernatant was concentrat- ed on a Buchi® rotary vapor under reduced pressure at 35 ± 15 °C and the ethanol extract evaporated in vacuo. Later, different concentrations of the extract were made, specifically, 125 mg/L, 250 mg/L, 500 mg/L, 1000 mg/L and 1500 mg/L. Phytochemical tests were carried out for the qualitative identification of the different metabolite groups, each test was performed in triplicate (Carvajal et al. 2009; Patil and Bhise 2015). Fourier-transform mid- infrared  spectroscopy (FTIR) from 4000 to 600 cm-1 was used to obtain information about the functional groups present in the plant using a Bruker spectrometer at a resolution of 4 cm-1. The analyses were done in the High Technology Service Center (CESAT-UPAEP). Allium sativum bioassay Meristem cells from the roots of A. sativum were used to evaluate the nuclear abnormalities (NA) and the mitotic index (MI) in the concentrations (125, 250, 500, 1000, 1500 mg/L); water was used as a control. Five repe- titions were performed on each concentration with bulbs of uniform size (3 cm in diameter). The control bulbs were kept in water. The other bulbs were transferred to the different concentrations for 120 hours. At the end of exposure, the length of the roots and stem were measured and examined to detect visible morphological anomalies: changes in consistency and root color and the pres- ence of hooks or twists in the roots as a sign of general toxicity (Çelik and Aslantürk 2010). Subsequently, the meristem zone of the garlic roots (2 mm) was cut and placed on a slide, hydrolyzed in 1N HCl for 10 minutes, 101Genotoxicity and cytotoxicity of Sambucus canadensis ethanol extract in meristem cells of Allium sativum then washed with distilled water, and stained with ace- tic orcein. The slides were fixed with the squash method sealing the edges with resin. The samples were analyzed with a Leica DM1000 LED fluorescence optical micro- scope with a Jenoptik ProGres C10 digital camera. Some 2000 meristem cells were counted for each treatment. In the stages of mitosis (interphase, prophase, metaphase, anaphase and telophase), the cellular alterations were counted: chromosomal breakage, bridges, lagging chro- mosomes, strays, among others. The values obtained were used to calculate the mitotic indices (MI) and the percentage of cellular alterations (CA) with the following formulae: MI = Numberofcells ∈ mitosis ÷ Totalcells × 100 CA = Nunberofcellswithabnormalchromosomes ÷ Total- cells × 100 The results of the number of roots and length of roots and stem, and the mitotic index were analyzed with the ANOVA (Bonciu et al., 2018). The differences were evaluated with Dunnett’s post hoc test. The nuclear abnormalities were evaluated with the Chi-squared test (X2) using the Minitab 8.1 statistics program. Values of p < 0.05 were considered significant differences. RESULTS The phytochemical tests of the Sambucus canaden- sis extract revealed the presence of alkaloids, flavonoids, saponins and tannins. The infrared spectrum tests (FTIR) on the extract showed different frequencies of stretching and bending; the stretching frequencies of the O-H bond at 3350 cm-1 is associated with phenol groups; the involvement in hydrogen bonding produces a widening of the band. The C-H bond stretching vibra- tions corresponding to methyl and methylene groups appear in the 3000–2850 cm-1 range and the bands in the fingerprint region are due to the bending vibra- tions at 1386 cm-1 for methyl and 716 cm-1 for ethyl: the stretching vibrations of the carbonyl bond, C=O, appears in the 1750–1680 cm-1 range related to the pres- ence of flavonoids. Similarly, a conjugated double bond C=C of the aromatic rings appears in the 1600–1450 cm-1 range, characteristic of the basic structure of flavo- noids (Figure 1). The length of Allium sativum roots and stem at the 1000 and 1500 mg/L concentrations were 7.66 mm and 0.72 mm, respectively, showing statistically significant differences compared to the control (p = 0.000). There were no significant differences with the other treat- ments (p > 0.05). The stem length at a concentration of 1500 mg/L (2.36 mm) showed a statistically significant difference compared to the stem length of the control group (p = 0.000). It can be seen that the average length and number of roots decreased depending on the con- centration (Table 1). In terms of morphology, at con- centrations of 125 and 250 mg/L no differences were observed compared to the control, however, at concen- trations of 500 and 1000 mg/L the roots appeared yel- low, at 1500 mg/L the sparse roots were brown and stiff. Likewise, effects on the stem such as twisting and color change were observed, mainly at concentrations of 1000 and 1500 mg/L. Regarding the results of the mitotic index, Table 2 shows a decrease in the MI as concentration increases; the number of dividing cells (prophase, metaphase, ana- phase and telophase) differed between concentrations, however, at 125, 250 and 500 mg/L there were no signifi- cant differences compared to the control (p > 0.05). At a concentration of 1500 mg/L a statistically significant dif- ference was observed in the mitotic index compared to the control. The nuclear abnormalities in Allium sativum are shown in Table 3. Concentrations of 500, 1000 and 1500 mg/L of the ethanol extract had the highest number, concentrations of 125 and 250 mg/L a lesser amount. The number of cells in mitosis with anomalies was related to the increase in concentration. Figure 2 shows abnormalities such as breakage, chromosome loss, bridg- es, chromosomes with inactivated centromere, among others. Figure 1. FTIR spectra of Sambucus canadensis in the range of 4000 to 600 cm-1. 102 Guadalupe Velázquez-Vázquez et al. DISCUSSION The test with Allium spp. is a suitable biomodel for identifying the cytotoxic and genotoxic effects of differ- ent plants (Bagatini et al. 2007; Lubini et al. 2008; Trapp et al. 2020). The study of raw extracts is important since traditional medicine uses part of the plant structure (leaves, stem, root) or the whole plant, without sepa- rating its components. Furthermore, it has been shown that different bioactive compounds act synergistically (Tallarida 2011) and that a combination of compounds exhibits a greater effect than individual compounds, suggesting that the effects of some plants are the result of the interaction of their components (Lamy et al. 2018). In this study, the qualitative analyses (phytochemi- cal tests; FTIR) of the plant showed the presence of alkaloids, tannins, saponins and flavonoids. The spec- troscopy used provides important information about functional groups as well as being an accessible and useful technique in the chemical and structural analy- sis of plants (Günzler and Gremlich 2002; Heredia- Guerrero et al. 2014). The functional group associ- ated with the signals reported in the evaluated spectra (FTIR) is flavonoids, which present inhibitory activity against diverse fungi and bacteria species. The metabo- lites reported (alkaloids, tannins, saponins, flavonoids) showed antimicrobial, antioxidant and antiviral activ- Table 1. Number and length of roots, and stem length of A. sativum exposed to the ethanol extract of Sambucus canadensis. Treatment mg/L Number of roots (x) (δ) Length of roots (x) (δ) Stems length (x) (δ) 125 13.2 ± 2.66 15.82 ± 724 25.52 ± 17.54 250 9.84 ± 4.94 11.47 ± 6.12 18.76 ± 10.81 500 11.20 ± 6.81 19.76 ± ±12.95 20.8 ± 12.62 1000 7.36 ± 3.68 8.66 ± 10.32* 17.6 ± 8.43 1500 0.92 ± 0.90 * 0.72 ± 0.42 * 2.36 ± 0.46* Control 13.4 ± 7.96 25.84 ± 19.79 36.3 ± 44.25 Values are mean ± S.E, One way ANOVA (*) are not significantly different p <0.05. Table 2. Allium sativum merismatic cell numbers in the different cell cycle phases, and index mitotic extract of Sambucus canadensis. Treatment mg/L Interphase Prophase Metaphase Anaphase Telophase Cells in division Mitotic Index (%) Control 3831 1988 81 59 41 2169 36,1 125 4020 1840 66 39 35 1980 33 250 4356 1571 29 23 21 1644 27,4 500 4306 1580 58 34 32 1704 14,3 1000 4558 1330 36 15 21 1402* 12,0 1500 5398 556 17 13 16 602* 5,03 *p <0.05 in One Way ANOVA. Table 3. Cellular abnormalities observed in Allium sativum exposed to the ethanolic extract of Sambucus canadensis. Treatments mg/L Control 125 250 500 1000 1500 Number of cells in division 2169 1980 1644 1704 1442 602 Bridges - - 1 4 37 25 Chromosome fragments - - 2 - 4 5 Binucleate 2 3 24 99 40 30 Chromosome lagging and disoriented 2 1 1 7 7 4 Sticky chromosome - 1 - 1 5 2 Vagrant chromosome - - - 2 3 4 Trinucleated - - - 10 5 20 Total cells aberrations 4a 5a 28b 123b 101b 90b Cells aberration (%) 0,2 0,3 1,7* 7,2* 7,0b 15,0b *The chi-square test. Significant difference p <0.05. 103Genotoxicity and cytotoxicity of Sambucus canadensis ethanol extract in meristem cells of Allium sativum ity, among others. Flavonoids in particular exhibit important pharmacological activities, in addition to being eff ective in chemoprevention and chemotherapy (Paduch et al. 2007; Perveen 2018). Th e evaluation of the S. canadensis extract with the Allium sativum test allowed us to determine the eff ects on root and stem growth as well as morphology; the highest concentra- tions,1000 and 1500 mg/L, signifi cantly inhibited root and stem growth compared to the control. Th e mitotic index (MI) decreased signifi cantly as the concentration of Sambucus canadensis increased, matching the results reported for other species of Sambucus sp. (Tedesco et al. 2017; Th ole 2006). Other authors have reported that plant extracts such as P. leiocarpa and P. myriantha (Lubini et al. 2008), Campomanesia xanthocarpa (Pas- tori et al. 2013), Vernonanthura polyanthes (Almeida et al. 2020), Amaranthus spinosus (Prajitha and Th op- pil, 2016), Achyrocline satureioides (Fachinetto et al. 2007), Luehea divaricata (Frescura et al. 2012) caused a reduction in the mitotic index when increasing the concentration, which may be an indication of antipro- liferative activity such as that reported by Bagatini et al. (2009), Knoll et al. (2006). Th e results obtained in this study may be associated with the plant components; in this sense, the fl avonoids found in the FTIR analysis may inhibit or stimulate the cellular cycle. Tedesco et al. (2017) found that Sambucus australis has fl avonoids such as rutin, kaempferol and quercetin, among others, to which diff erent pharmacological eff ects have been attributed, including antiproliferative and anticancer action. One study developed by Lee and Finn, (2007) reported that Sambucus canadensis presents a high quantity of anthocyanins and polyphenols which have a potent antioxidant eff ect, perhaps also explaining the inhibition of cellular division in Allium sativum. In the same way, the phenolic components of the species have been associated with a more potent anticancer activity than Sambucus nigra (Th ole et al. 2006). Figure 2. Allium sativum cells exposed to the ethanolic extract of Sambucus canadensis a) sticky chromosome b) bridges c) sticky and lag- ging chromosome with bridges d) lagging and sticky chromosome e) abnormal anaphase f ) lagging chromosome g) vagrant chromosome and lagging chromosome, h) bridge and vagrant chromosome, i) bridges. 104 Guadalupe Velázquez-Vázquez et al. The main chromosomal aberrations found in this study include the formation of bridges, which, accord- ing to Türkoğlu (2007), are produced due to the fusion of chromosomes or chromatids as a result of chromo- somal stickiness or due to unequal translocation. Lag- ging chromosomes moving to both sides of the poles without being fused by the spindle apparatus can also induce bridges. Another aberration found in the results of this investigation were sticky chromosomes formed by the free movement of chromosomes, which can pro- duce chromosomal breakage and may lead to the loss of genetic material (Dutta et al. 2018). Stray chromosomes advance ahead of the chromosome group towards the poles resulting in an unequal distribution of chromo- somes in daughter cells (Sondhi et al. 2018). Similarly, Fachinetto and Tedesco (2009) attribute various chromosomal anomalies, bridges, binucleated cells, among others, to the components of the plants. Along these lines, Bagatini et al. (2009); Toloza et al. (2006), indicate that the genotoxic and antiproliferative activity presented by some plant extracts are the result of the interactions of their different chemical compo- nents. In this regard, Amado et al. (2020) reported that the Smilax brasiliensis extract and the rutin and querce- tin fractions, which have also been found in the spe- cies Sambucus sp. cause genotoxic effects. It has been suggested that, if extracts cause damage to plant cell chromosomes, they may also be potentially harmful for mammalian cell chromosomes (Feretti et al. 2007). According to the results, no abnormalities in the A. sativum root were found at low concentrations of 125 mg/L and 250 mg/L. However, in concentrations of 500, 1000 and 1500 mg/L, a considerable number of alterations, such as bridges, chromosome breaks and strays were found, suggesting that the extract presents a genotoxic effect at high concentrations. The results match those reported by Bratu (2012) which indicate that at low concentrations the species Sambucus nigra presents no mutagenic effects. According to Ifeoluwa et al. (2013) and Sabini et al. (2011), some plants induce cytotoxicity but not mutagenic effects. Generally speak- ing, the frequency of aberrations increases significantly as the concentration increases, suggesting that Sambucus canadensis leaf extract presents clastogenic effects, which agrees with the reports of Bidau et al. (2004); Çelik and Aslantürk (2010) and Mattana et al. (2014) on the effects of herbal plants. Sambucus canadensis is a plant that should be used with caution not only because it is used for curing dis- eases but also because it is used by pregnant women (Velazquez-Vázquez et al. 2019) and could be harmful to their health. The use of plants before and after pregnan- cy may cause conditions from vomiting, infection and gastrointestinal problems to placental retention, uterine hypotonia, cervical tear, miscarriage, uterine bleeding and others. Since few studies exist on the safety and efficacy of the use of herbal plants during pregnancy, a situation which exposes both the mother and the fetus, it is recom- mended that they are not used during pregnancy unless such use is supported by scientific studies which validate their safety (Ahmed et al. 2017; Frawley et al. 2015; Hall et al. 2011; Illamola et al. 2020; Nergard et al. 2015). CONCLUSION The results of this study suggest that the ethanol extract from Sambucus canadensis induces antiprolif- erative effects. It was also found that in concentrations higher than 500 mg/L the extract affects root growth, cellular division and chromosomal changes in the cells of Allium sativum. 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