Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 76(1): 77-86, 2023 Firenze University Press www.fupress.com/caryologiaCaryologia International Journal of Cytology, Cytosystematics and Cytogenetics ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-2092 Citation: Ali Bouzekri, Meryem Nas- sar, Souheila Slimani, Zohra Chek- roud (2023). Cytogenetic effects of Tribu- lus terrestris L. on meristematic cells of Allium cepa L. and Vicia faba L.. Caryologia 76(1): 77-86. doi: 10.36253/ caryologia-2092 Received: March 22, 2023 Accepted: June 17, 2023 Published: September, 19, 2023 Copyright: © 2023 Ali Bouzekri, Mery- em Nassar, Souheila Slimani, Zohra Chekroud. This is an open access, peer-reviewed article published by Firenze University Press (http://www. fupress.com/caryologia) and distrib- uted under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, pro- vided 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 AB: 0000-0002-1161-6957 MN: 0000-0002-1161-6957 SS: 0000-0002-2771-3727 ZC: 0000-0002-9845-6027 Cytogenetic effects of Tribulus terrestris L. on meristematic cells of Allium cepa L. and Vicia faba L. Ali Bouzekri1,2,*, Meryem Nassar1,2, Souheila Slimani1,2, Zohra Chek- roud1,2 1 Department of Biology, BP 26, University of 20 August 1955, El Hadaiek-Skikda route 21000, Algeria 2 Laboratory of Research in Biodiversity Interaction, Ecosystem and Biotechnology “LRIBEB”, University of 20 August 1955 BP 26, El Hadaiek-Skikda route 21000, Algeria *Corresponding author. E-mail: alibouzekri21@gmail.com Abstract. Tribulus terrestris is a plant of the Zygophyllaceae family frequently used worldwide to treat various diseases due to the therapeutic effects of its pharmacologi- cal components. This study examines the cytotoxic and genotoxic effects of T. terrestris using two plant models, Allium cepa and Vicia faba. Extracts of 0.00625, 0.0125, 0.025, 0.05 and 0.1mg/mL were tested on meristematic cells of A. cepa and V. faba roots. This assessment includes the study of root growth, structure and coloration, as well as the determination of the mitotic index (MI) and chromosomal aberrations (CAs) as accu- rate indicators of toxicity. Our results showed a significant decrease in the mean length of roots treated with 0.025, 0.05 and 0.1 mg/ml for A. cepa and 0.1 mg/ml for V. faba. Cytotoxicity and genotoxicity results showed a significant decrease in MI from 0.025 mg/ml in A. cepa and from 0.05 mg/ml in V. faba, and this decrease in MI is linked to the increase in concentration and treatment time with T. terrestris. Furthermore, a significant increase in CAs was observed in A. cepa and V. faba from the 0.025 mg/ml concentration. The significant reduction in MI and CAs abundance suggests the geno- toxicity of T. terrestris. Therefore, T. terrestris is a medicinal plant that should be used with caution, appropriately and based on essential therapeutic needs. Keywords: Tribulus terrestris, Allium cepa, Vicia faba, cytotoxicity, genotoxicity. INTRODUCTION Recently, there has been a rapid increase in the use of dietary supple- ments derived from bioactive compounds of plant origin (Izzo et al. 2016). Herbal medicines are often used because many people believe that all that is natural is not toxic or harmful to health. This is a mistaken belief, as many therapeutic plants have high toxicity and harmful effects on human health (Proença da Cunha et al. 2012). Many studies examine the biological effects of extracts from different plants for their potential therapeutic use. Howev- er, there is little data available on the cyto-genotoxic effects of most plants. https://doi.org/10.36253/caryologia-2092 https://doi.org/10.36253/caryologia-2092 https://doi.org/10.36253/caryologia-2092 https://orcid.org/0000-0002-1161-6957 https://orcid.org/0000-0002-1161-6957 https://orcid.org/0000-0002-2771-3727 https://orcid.org/0000-0002-9845-6027 78 Ali Bouzekri et al. Therefore, there is a need for research on these plants to assess their potential cytotoxic and genotoxic effects (Chukwujekwu and Van Staden 2014). Previous stud- ies using different bioassays have revealed significant cytotoxic and genotoxic effects in different plants. Abu- dayyak et al. (2015) reported that T. terrestris L. had estrogenic and genotoxic activities in rat kidney cell lines exposed to this plant. Another study by Kumar et al. (2013) on the effect of Tinospora cordifolia in A. cepa meristematic cells showed a significant cytotoxic and genotoxic at high concentrations. In addition, Results obtained from the genotoxic study of Ayubi et al. (2021) showed that the hydro-alcoholic extract of Z. multi- flora had no genotoxic effect. Bocayuva Tavares et al. ( 2021) reported that Seed proteins extract of S. saponar- ia causes cytotoxic and genotoxic effects in the human liver cancer cell line. In another study to evaluate the genotoxic effects of the plant Angelica keiskei, Maronpot (2015) demonstrated that it’s not genotoxic in Chinese hamster ovary cells. Researchers have long been interested in the geno- toxic and cytotoxic effects of natural substances such as plant extracts. Higher plants, including A. cepa and V. faba, are used frequently to assess the genotoxic- ity of these environmental pollutants (Leme and Marin- Morales 2009). Furthermore, A. cepa and V. faba tests were ideal for evaluating chromosome damage and mitotic cycle disruptions due to their excellent chro- mosome characteristics, including large and fewer in number with a stable karyotype (Fiskesjo 1985). Moreo- ver, this test method has demonstrated high sensitivity, which depends on a quick response in root development dynamics and simple detection of the endpoints associ- ated with genotoxicity (Firbas and Amon 2014). Geno- toxic endpoints include changes in MI values compared to controls (Akgündüz et al. 2020), morphological and number chromosome modifications expressed as CAs (Bonciu et al. 2018), and the frequency of Micronuclei as a simple quantitative characteristic (Bonciu et al. 2018; Younis et al. 2019). T. terrestris is an annual plant from the Zygophyl- laceae family. It has mainly cultivated in the Mediterra- nean and subtropical areas (Zhu et al. 2017). The T. ter- restris extract is one of the natural therapeutic products that is used most frequently. This extract has shown sev- eral pharmacological activities, most of which are linked to diverse flavonoid and terpenoid components. These activities include antioxidants, antimicrobial, antibacte- rial, antitoxic, antiapoptotic molecules, platelet aggre- gation inhibitors, and anti-inflammation (Almasi et al. 2017). Also, these activities treat cardiovascular diseases, tumors, diabetes, respiratory diseases, and reproductive dysfunction (Qureshi et al. 2014). The widespread dis- tribution of T. terrestris, its high content of active com- pounds (especially sterol saponins, as well as flavonoids, terpenoids, tannins, phenol, alkaloids and carboxylic acids), and the prevalence with which it is used in tra- ditional medicine, all highlight the importance of ana- lysing the phytopharmacological characteristics of the plant (Stefănescu et al. 2020), and due to the potential toxic effect of its active compounds, an assessment of a potential cytotoxic and genotoxic effect is essential for its use is safe and effective (Celik 2012). Although the therapeutic effects of this plant have been studied by sev- eral researchers, but the evaluation of the potential cyto- genotoxic effects of T. terrestris on meristematic cells has not yet been studied. Thus, the present work aimed to evaluate the cyto- toxic and genotoxic effects of T. terrestris methanolic extract on A. cepa and V. faba roots by assessing the root growth, structure and color as well as the MI and CAs. MATERIALS AND METHODS Plant methanolic extract preparation 600 g of T. terrestris aerial parts (Trunk, branches and leaves) were obtained from a local medicinal plant market (Setif-Algeria). The plant material was identi- fied by Dr. Sakhraoui Nora (a botanist) and then dried in the dark and powdered with a domestic mixer. 500 g of T. terrestris powder was dissolved in 4L of 96% hydro-methanolic solution (80%) for 24 hours to obtain a methanolic extract. The solution obtained was then double filtered using a Whatman No.1 paper filter. The filtrate obtained was then evaporated in a rotavapor (RE- 100 pros) at 45oC to obtain a final dry residue. During processing, the dry residue was dissolved in distilled water to prepare the different concentrations of 0.00625, 0.0125, 0.025, 0.05 and 0.1 mg/mL for the different treat- ments. Plants assay and application concentrations Healthy onion bulbs (A. cepa, 2n = 16) and V. faba seeds were obtained from a local market. Both species were kept in tap water until their roots reached 1.5-2 cm. Then they were divided into six groups, one serving as a control, while the other five were given different concen- trations (0.00625, 0.0125, 0.025, 0.05 and 0.1 mg/mL) of T. terrestris at 24°C. Each concentration was tested on a minimum of three bulbs, and seven seeds with solutions changed daily. Using a ruler, root length was measured 79Cytogenetic effects of Tribulus terrestris on meristematic cells of Allium cepa L. and Vicia faba L. at 24 h, 48 h, 72 h, 96 h, and 120 h. Other indicators of toxicity, including root structure and color changes, are also assessed. Genotoxicity assessment To investigate the potential effect of genotoxicity, A. cepa bulbs and V. faba seeds with a root length of 1.5-2 cm were exposed to T. terrestris at different concentra- tions (0.00625, 0.0125, 0.025, 0.05 and 0.1mg/mL) for 12 h and 24 h. After treatment, the bulbs and seeds were washed thoroughly and then placed in an ethanol/acetic acid solution (3v/1v) for 24 h before storage in 70% eth- anol at 4°C. After a brief wash with distilled water, the root tips were hydrolysed in 1 N HCl solution for 5 min at 60°C and stained with Schiff’s reagent for 20 min. The slides were prepared following the method of Sharma and Sharma (2014). After the first pre-treatment, the root tips were carefully washed in distilled water several times. Then they were subjected to hydrolysis in a 1 N HCl solution for five minutes at a temperature between 60°C and 70°C. Then the apical 2 mm were crushed in a 45% acetic acid solution. The apical meristems were ana- lysed at 40x magnification after being crushed in a drop of 45% acetic acid. The mitotic index and chromosome aberration index were calculated according to the meth- ods of Fiskesjo (1985). using at least 1000 cells per slide and five slides for each concentration. The following formulae (Akwu et al. 2019) were used to determine the proportion of chromosomal aberrations (CAs), the mitotic index (MI), and the chromosomal aberration frequency (AF) in the cells: Mitotic index (MI) (%) = !"#$%&'()*+",-./.-.&01*%%2 !"#$%&'()*+",1*%%2")2*+/*- x 100 1 Chromosome Aberrations (CAs) (%) = !"#$%&'()*+",$)&"($%1*%%2 !"#$%&'()*+",1*%%2")2*+/*- x 100 2 Aberrations type frequency (AF) (%) = 3'()*+",$#45*",$)*++$*%% !"#$%&'()*+",$)*++$*%%2 x 100 3 4 Statistical data analysis Graph pad prism 9.2.0 (Graph Pad Software, LLC, CA, USA) was used for statistical analysis of root length, MI (%), and ACs (%). Data Results were compared sta- tistically using one-way ANOVA and Dunnett’s multi- ple comparison test. All values were expressed as mean ± SD and were determined statistically significant when P<0.05. RESULTS The changes in root length, form and color of roots are illustrated in Table 1 (A. cepa) and Table 2 (V. faba) following different treatment with T. terrestris compared to the control. It was observed that the inhibition effect of T. terrestris increased with the increase in concen- tration and duration of treatment. Therefore, control showed highest root elongation, with mean lengths of 10.77 ± 0.06 cm and 10.48 ± 0.05 cm after 120 h in A. cepa and V. faba, respectively. In other hand, throughout the five-day treatment, the mean root lengths of A. cepa were unaffected by the 0.00625 mg/mL and 0.0125 mg/mL concentrations and 0.00625, 0.0125, 0.025, and 0.05 mg/mL concentrations in V. faba. However, A. cepa roots exposed to the 0.025, 0.05, and 0.1 mg/mL concentrations range showed a sig- nificant decrease in root growth with values of 4.71, 3.92, and 3.16 cm, respectively. Moreover, a significant decrease (P<0.05) in root length was seen in V. faba roots treated with the 0.1 mg/mL concentration (3.33 cm) compared to the control. Simultaneously, the inhibi- tory effect of T. terrestris increased positively with con- centration and time of exposure, whose value increased from 26.84% (0.006125 mg/mL) to 62.77% (0.1 mg/mL) after 120 h of exposure. Furthermore, it was remarked that A. cepa roots were more sensitive to T. terrestris than V. faba roots (62.77% and 55.92% inhibition, respectively). Concerning the morphology of the roots, structural and color modifications were observed, particularly in V. faba roots treated from the 0.05 mg/mL concentra- tion, which appeared slimy to slimy dark brown com- pared to the control. However, after 120 h of treatment, roots treated to the 0.05 and 0.1 mg/mL concentrations showed necrosis. Figure 1 shows the effect of different T. terrestris concentrations on A. cepa and V. faba mitotic index. Meristematic cells of these two plants that are treated with different concentrations of T. terrestris showed a significant decrease in MI compared to the control. Our result showed that the control has the highest MI in both A. cepa and V. faba (59.26 ± 0.88% and 59.90 ± 0.40%, respectively) (12 h), and (60.74 ± 0.45% and 60.14 ± 0.48%, respectively) (24 h). In addition, Cell division was unaffected by the concentrations of 0.00625, 0.0125, and 0.025 mg/mL in A. cepa and 0.00625 and 0.0125 mg/ mL in V. faba. In contrast, the values were as high as the control (12 h and 24 h) in A. cepa (60.80 ± 1.12%, and 60.24 ± 1.00%, respectively) and V. faba (59.88 ± 0.32% and 58.14 ± 0.35%, respectively). However, cytotoxic 80 Ali Bouzekri et al. Ta bl e 1. Th e ch an ge in m ea n ro ot le ng th (c m ) o f A . c ep a aft er a pp lic at io n of d iff er en t c on ce nt ra tio ns (% ) o f T . t er re str is at d iff er en t e xp os ur e tim es . Tr ea tm en ts (m g/ m l) N r M ea n ro ot le ng th (c m ) a nd in hi bi tio n (% ) a ffe ct ed b y T. te rr es tr is tr ea tm en t i n A . c ep a. 00 h 24 h 48 h 72 h 96 h 12 0 h M ea n of lo ng th ± S D Fo rm a nd co lo r M ea n ± SD M ea n ± SD In % M ea n ± SD In % M ea n ± SD In % M ea n ± SD In % M ea n ± SD In % C on tr ol 0. 00 62 5 0. 01 25 0. 02 5 0. 05 0. 1 15 15 15 15 15 15 1. 78 ± 0 .0 8 1. 87 ± 0 .0 7 1. 87 ± 0 .0 7 1. 71 ± 0 .0 7 1. 86 ± 0 .0 5 1. 69 ± 0 .0 2 4. 18 ± 0 .0 3 4. 01 ± 0 .0 4 3. 83 ± 0 .0 7 3. 56 ± 0 .0 4 2. 98 ± 0 .0 3 2. 24 ± 0 .0 6 - 4. 07 8. 38 14 .8 4 28 .7 1 46 .4 2 5. 65 ± 0 .0 2 5. 02 ± 0 .0 3 4. 55 ± 0 .0 6 4. 20 ± 0 .1 1 3. 3 ± 0. 04 2. 95 ± 0 .0 5 - 11 .1 6 19 .4 7 25 .6 7 41 .6 0 47 .7 9 7. 32 ± 0 .0 5 5. 84 ± 0 .0 4 5. 09 ± 0 .0 4 4. 93 ± 0 .0 6 3. 97 ± 0 .0 3 3. 01 ± 0 .0 8 - 20 .2 2 30 .4 7 32 .6 6 45 .7 7 58 .8 8 9. 57 ± 0 .0 9 7. 03 ± 0 .0 7 6. 66 ± 0 .0 7 5. 35 ± 0 .0 4 4. 45 ± 0 .0 9 3. 59 ± 0 .0 5 - 26 .5 5 30 .4 1 44 .1 0 53 .5 1 62 .4 9 10 .7 7 ± 0. 06 6. 91 ± 0 .0 3 5. 09 ± 0 .0 3 4. 71 ± 0 .0 3 4. 30 ± 0 .0 4 4. 19 ± 0 .0 4 - 26 .8 4 31 .3 0 48 .5 7 54 .1 4 62 .7 7 7. 49 ± 2 .7 1 5, 95 ± 1, 54 5. 50 ± 1 .4 8 4. 71 ± 0 .8 2* 3. 92 ± 0 .8 0* * 3. 16 ± .6 7* ** St ra ig ht , w hi te St ra ig ht , w hi te St ra ig ht , w hi te St ra ig ht , w hi te Sl im y, w hi te Sl im y, w hi te D at a ar e sh ow n as m ea n ± SD , N r: N um be r of r oo ts , I n (% ): th e in hi bi tio n pe rc en ta ge , * p < 0 .0 5, * * p < 0. 01 a nd * ** p < 0 .0 01 v er su s co nt ro l g ro up u sin g on e- w ay A N O VA ; D un - ne tt’ s t es t. Ta bl e 2. Th e ch an ge in m ea n ro ot le ng th (c m ) o f V . f ab a aft er a pp lic at io n of d iff er en t c on ce nt ra tio ns (% ) o f T . t er re str is at d iff er en t e xp os ur e tim es . Tr ea tm en ts (m g/ m l) N r M ea n ro ot le ng th (c m ) a nd in hi bi tio n (% ) a ffe ct ed b y T. te rr es tr is tr ea tm en t i n V. fa ba 00 h 24 h 48 h 72 h 96 h 12 0 h M ea n of lo ng th ± S D Fo rm a nd co lo r M ea n ± SD M ea n ± SD In % M ea n ± SD In % M ea n ± SD In % M ea n ± SD In % M ea n ± SD In % C on tr ol 0. 00 62 5 0. 01 25 0. 02 5 0. 05 0. 1 7 7 7 7 7 7 1. 78 ± 0 .0 8 1. 79 ± 0 .0 7 1. 77 ± 0 .0 2 1. 74 ± 0 .0 6 1. 80 ± 0 .0 3 1. 81 ± 0 .0 6 3. 38 ± 0 .0 4 3. 20 ± 0 .0 4 3. 01 ± 0 .0 6 2. 89 ± 0 .0 7 2. 32 ± 0 .0 6 2. 22 ± 0 .0 7 - 5. 53 10 .9 5 14 .5 0 31 .3 7 34 .3 2 4. 78 ± 0 .0 6 4. 27 ± 0 .0 5 4. 17 ± 0 .0 2 3. 68 ± 0 .0 4 2. 98 ± 0 .0 3 2. 58 ± 0 .1 0 - 10 .6 7 12 .7 7 23 .0 2 37 .6 6 46 .0 3 6. 24 ± 0 .0 4 5. 01 ± 0 .0 4 4. 77 ± 0 .0 3 4. 04 ± 0 .0 5 3. 67 ± 0 .0 6 3. 24 ± 0 .0 6 - 19 .7 2 23 .5 6 35 .2 6 41 .1 9 48 .0 8 8. 55 ± 0 .0 6 6. 77 ± 0 .0 7 6. 02 ± 0 .0 5 5. 22 ± 0 .0 8 4. 52 ± 0 .0 5 3. 99 ± 0 .0 9 - 20 .8 2 29 .6 0 38 .9 5 47 .1 4 50 .3 4 10 .4 8 ± 0. 04 8. 21 ± 0 .0 6 7. 32 ± 0 .0 6 6. 01 ± 0 .0 3 4. 99 ± 0 .0 2 4. 62 ± 0 .0 5 - 21 .6 7 30 .1 6 44 .5 6 52 .3 9 55 .9 2 6. 68 ± 2 .8 5 5. 49 ± 2 .0 0 5. 05 ± 1 .6 6 4. 36 ± 1 .2 4 3. 69 ± 1 .0 9 3. 33 ± 0 .9 8 * St ra ig ht , w hi te St ra ig ht , w hi te St ra ig ht , w hi te Sl im y, w hi te Sl im y, da rk b ro w n Sl im y, da rk b ro w n D at a ar e sh ow n as m ea n ± SD , N r: N um be r o f r oo ts , I n (% ): Th e in hi bi tio n pe rc en ta ge , * p < 0 .0 5 ve rs us c on tr ol , u se d on e- w ay A N O VA ; D un ne tt te st . 81Cytogenetic effects of Tribulus terrestris on meristematic cells of Allium cepa L. and Vicia faba L. effects were observed from the 0.025 mg/ml concentra- tion in V. faba and 0.05 mg/mL in A. cepa accompanied by a significant decrease in MI (P<0.001) (12 h and 24 h). Figures 2, 3, 4, and 5 show T. terrestris-induced aber- ration percentages and different chromosomal abnormal- ities. It was showed that the increase in CAs (%) depends on T. terrestris concentration and treatment duration. Compared to the control, no significant effect on CAs (%) was noted after treatment with the concentra- tions 0.00625 and 0.05 mg/mL (12 h and 24 h) in A. cepa and V. faba. While a significant increase (P<0.001) in ACs (%) was observed in cells treated with the concen- trations 0.025, 0.05, and 0.1 mg/mL (12 h and 24 h) com- pared to the control. The highest CAs (%) in A. cepa and V. faba were 7.5 ± 0.129% and 5.88 ± 0.16%, respectively, after 24 h treatment with T. terrestris at the 0.1 mg/mL concentra- tion, and the lowest was 0.48 ± 0.10% and 0.64 ± 0.13%, respectively at the 0.00625 mg/mL concentration (12 h). The most frequent types of CAs were multipolar in A. cepa (34.89 ± 2.30%) (12 h) and (33.97 ± 4.36%) (24 h) and in  V. faba (34.08 ± 2.23%) (12 h) and (30.96 ± 4.13%) (24 h), followed by break in A. cepa (19.89% ± 1.75%) (12 h) and (26.07 ± 3.79%) (24 h) and in V. faba  (24.20 ± 0.79%) (12h) and (24.19 ± 1.35%) (24 h), stickiness in A. cepa (17.49 ± 1.25%) (12 h) and (20.24 ± 3.58%) (24 h) and in V. faba  (20.05 ± 3.79%) (12 h) and (19.18 ± 1.77%) (24 h), vagrant in A. cepa (18.94 ± 2.27%) Alliu m ce pa Vici a f ab a Alliu m ce pa Vici a f ab a Alliu m ce pa Vici a f ab a Alliu m ce pa Vici a f ab a Alliu m ce pa Vici a f ab a Alliu m ce pa Vici a f ab a 0 20 40 60 80 Groups (12 h) M ito tic in de x (M I) (% ) Control 0.00625 mg/mL 0.0125 mg/mL 0.025 mg/mL 0.05 mg/mL 0.1 mg/mL *** *** *** *** *** A Alliu m ce pa Vici a f ab a Alliu m ce pa Vici a f ab a Alliu m ce pa Vici a f ab a Alliu m ce pa Vici a f ab a Alliu m ce pa Vici a f ab a Alliu m ce pa Vici a f ab a 0 20 40 60 80 Groups (24 h) M ito tic in de x (M I) (% ) Control 0.00625 mg/mL 0.0125 mg/mL 0.025 mg/mL 0.05 mg/mL 0.1 mg/mL *** *** *** *** *** B Figure 1. The effect of different T. terrestris concentrations (mg/mL) and exposure times on the MI (%) in A. cepa cells and V. faba cells after 12 h (A) and 24 h (B). Three bulbs and seven seeds were treated in each concentration, including the control. Five roots for each con- centration were used. At least 1000 cells on each slide and five slides for each concentration were examined. Alliu m ce pa Vici a f ab a Alliu m ce pa Vici a f ab a Alliu m ce pa Vici a f ab a Alliu m ce pa Vici a f ab a Alliu m ce pa Vici a f ab a Alliu m ce pa Vici a f ab a 0 2 4 6 8 10 Groups (12 h) C hr om os om e ab er ra tio ns (A C s) (% ) Control 0.00625 mg/mL 0.0125 mg/mL 0.025 mg/mL 0.05 mg/ml 0.1 mg/mL *** *** *** *** ** A Alliu m ce pa Vici a f ab a Alliu m ce pa Vici a f ab a Alliu m ce pa Vici a f ab a Alliu m ce pa Vici a f ab a Alliu m ce pa Vici a f ab a Alliu m ce pa Vici a f ab a 0 2 4 6 8 10 Groups (24 h) C hr om os om e ab er ra tio ns (A C s) (% ) Control 0.00625 mg/mL 0.0125 mg/mL 0.025 mg/mL 0.05 mg/mL 0.1 mg/mL *** *** *** *** ** ** B Figure 2. Percentage of CAs in A. cepa and V. faba cells induced by different concentrations of T. terrestris (mg/mL) after 12 h (A) and 24 h (B). Three bulbs and seven seeds were treated in each concentration, including the control. Five roots for each concentration were used. At least 1000 cells on each slide and five slides for each concentration were examined. 82 Ali Bouzekri et al. (12 h) and (15.38 ± 3.11%) (24 h) and in V. faba  (15.07 ± 1.88%) (12 h) and (14.31 ± 2.05%) (24h), bridge in A. cepa (17.00 ± 2.82%) (12 h) and (12.80 ± 3.89%) (24 h) and in V. faba (15.73 ± 1.10%) (12 h) and (18.22 ± 3.52%) (24 h), C-mitosis in A. cepa (3.29 ± 1.63%) (12 h) and (3.52 ± 0.91%) (24 h) and in V. faba  (2.06 ± 0.98%) (12 h) and (1.89 ± 1.34%) (24 h), and M-nucleus in A. cepa (0.35 ± 0.39%) (12 h) and (0.52 ± 0.75%) (24 h) and in V. faba (0.54 ± 0.74%) (12 h) and (0.59 ± 0.49%) (24 h) (Fig. 3A, 3B, 3C, 3D). DISCUSSION This study was conducted to assess the cytotoxic and genotoxic effects of T. terrestris methanolic extract by analysing the change in root growth, morphology, and color of A. cepa and V. faba. as well as the determination of MI and different types of CAs. Our results revealed that T. terrestris treatment induced cytotoxic and geno- toxic effects manifested by inhibition of root growth, modification of root colour and structure, decrease in MI (%), and increase in CAs (%) with the appearance of various types of chromosomal aberrations. These changes are dependent on the different concentrations and duration of exposure to T. terrestris. Furthermore, Mult ipo lar Brid ge Stic kin es s Vag ran t C-m ito sis Brea k M-nu cle us 0 10 20 30 40 50 Chromosome aberration types in A. cepa cells (12 h) A be rr at io n ty pe fr eq ue nc y (% ) Control 0.00625 mg/mL 0.025 mg/mL 0.025 mg/mL 0.05 mg/mL 0.1mg/mL A Mult ipo lar Brid ge Stic kin es s Vag ran t C-m ito sis Brea k M-nu cle us 0 10 20 30 40 50 Chromosome aberration types in V. faba cells (12 h) A be rr at io n ty pe fr eq ue nc y (% ) Control 0.00625 mg/mL 0.0125 mg/mL 0.025 mg/mL 0.05mg/mL 0.1mg/mL C Mult ipo lar Brid ge Stic kin es s Vag ran t C-m ito sis Brea k M-nu cle us 0 10 20 30 40 50 Chromosome aberration types in V. faba cells (24 h) A be rr at io n ty pe fr eq ue nc y (% ) Control 0.00625 mg/mL 0.0125 mg/mL 0.025 mg/mL 0.05mg/mL 0.1mg/mL D Mult ipo lar Brid ge Stic kin es s Vag ran t C-m ito sis Brea k M-nu cle us 0 10 20 30 40 50 Chromosome aberration types in A. cepa cells (24 h) A be rr at io n ty pe fr eq ue nc y (% ) Control 0.00625 mg/mL 0.0125 mg/mL 0.025 mg/mL 0.05mg/mL 0.1mg/mL B Figure 3. Frequency of different types of CAs induced by different concentrations of T. terrestris (mg/mL) in A. cepa cells after 12 h (A) and after 24 h (B) and in V. faba cells after 12 h (C) and after 24 h (D). Three bulbs and seven seeds were treated in each concentration, includ- ing the control. Five roots for each concentration were used. At least 1000 cells on each slide and five slides for each concentration were examined. 83Cytogenetic effects of Tribulus terrestris on meristematic cells of Allium cepa L. and Vicia faba L. Figure 4. Photomicrographs showing different CAs induced by different concentrations of T. terrestris in A. cepa root tip cells: vagrant (A1) with break (A2), and bridge (A3), multipolar and bridge (B2), break (B1) and stickiness (B3), stickiness (C1, C2), c-mitosis (D1), stickiness (D2) and break (D3), stickiness (E1, E4) and multipolar (E2, E3), micro-nucleus (F1), c-mitosis (G1), stickiness (H1, H2). After 12 h and 24 h of treatment. scale bar = 0.5 μm. Figure 5. Photomicrographs showing different CAs induced by different concentrations of T. terrestris in V. faba root tip cells: stickiness (A1) and vagrant (A2), stickiness (B1, B2), multipolar (C1) and multipolar with break (C2), stickiness (D1) and vagrant (D2), multipolar with bridge (E1), break (F1, F2, F3), c-mitosis (G1) and stickiness (G2, G3), micro-nicleus (H1). After 12 h and 24 h of treatment. scale bar = 0.5 μm. 84 Ali Bouzekri et al. the results obtained on root growth and structure are in agreement with the results of Basu and Tripura (2021) on Cascabela thevetia who found a decrease in root growth, turgescence, and color change in A. cepa and V. faba treated with a high concentration of Cascabela the- vetia extract. Similar results were approved by the stud- ies of Issa et al. (2020) on the roots of Avena fatua and Echinochloa crus-galli exposed to a high concentration of Vitex negundo. According to Wierzbicka (1988), root growth is directly related to the enzymatic activity and cell elongation of the meristematic zone. This activity promotes cell elongation and membrane release during cell differentiation (Silveira et al. 2017). Thus, the slow- ing of root growth may be due to the inhibitory effect of T. terrestris on the enzymatic activity that promotes the elongation of the meristematic region. The mitotic index (MI) is an indicator to determine the cytotoxicity induced by toxic substances (Leme and Marin-Morales 2009). The MI is also used to measure the portion of dividing and arrested cells during the cell cycle (Rojas et al. 1993). In this study, the decrease in MI after 12 h and 24 h of treatment with T. ter- restris suggested the significant cytotoxic effect of this plant. Furthermore, our results indicate that T. terrestris inhibits cell division in A. cepa and V. faba, significant- ly reducing MI at high concentrations. The cytotoxic and genotoxic effects of plants are evaluated by sev- eral studies, including Vitex negundo (Issa et al. 2020), Citrus aurantiifolia (Fagodia et al. 2017) and Plantago major (Ždralović et al. 2019). However, research inves- tigating the genotoxicity of T. terrestris on meristematic cells is scarce. The current study showed a correlation between the increase in T. terrestris concentration and the reduction of MI. According to the results of Qari and El-Assouli (2019), the aqueous extract of T. terrestris fruit can inhibit the proliferation of human lymphocytes in cul- ture. This decrease in IM could be caused by the arrest of mitotic phases or by decelerating the cytokinesis pro- cess (Kundu and Ray 2017). However, our results suggest that this inhibition is caused by the genotoxic effects of one or more components of the T. terrestris extract that can damage DNA strands in a specific way (Qari and El- Assouli 2019). In addition, Kundu and Ray (2017) found that T. terrestris fruit extract can inhibit cell division due to a DNA defect, suggesting that it could be used as an anticancer agent based on its ability to inhibit cell proliferation, its safety on the DNA molecule at lower doses, and its antioxidant component. Different types of chromosomal aberrations (CAs) were observed at all concentrations applied. There is a significant increase in CAs (%) from the concentration of 0.025 mg/mL. It also produced aberrant chromosome segregation and caused the formation of various anoma- lies such as multipolar, chromosome bridge, stickiness, vagrant, c-mitosis and micro-nucleus. Similar results were obtained by Anita Sharma et al. (2019) in A. cepa root cells treated with H. suaveolens extract. Further- more, Sabeen et al. (2020) reported that CAs are pro- duced by proteolysis and by blocking DNA synthesis. Data analysis showed that multipolar anaphases were the most common CAs. Khallef et al. (2019) suggests that mitotic spindle instabilities may produce anaphase multipolarity. In addition, Sabeen et al. (2020) suggests that CAs, like chromosomal breaks and bridges, indicate clastogenic activity. However, stickiness, which causes cell death, may be caused by excess chromosomal con- densation or inappropriate nucleoprotein biosynthesis (Sabeen et al. 2020). According to Mercykutty and Ste- phen (1980), stickiness may be caused by the depolym- erization of DNA, the partial dissolution of nucleopro- teins, the breakage and exchanges of the basic folded fib- er units of chromatids, and the stripping of the protein covering of DNA in chromosomes. This study typically found chromosome break for- mation and stickiness. Kuchy et al. (2015) suggests that chromosomal condensation or excessive nucleoprotein production can form stickiness. Another notable abnor- mality was the chromosomal vagrant. Due to spindle abnormality, vagrant chromosomes are induced, result- ing in the dissociation of an unequal distribution of chromosomes in the daughter nuclei and the generation of daughter cells with abnormally small or sized nuclei during interphase (El-Ghamery et al. 2003). C-mitosis and micro-nucleus were rare in our results and their presence may be due to the spindle apparatus’ incapac- ity to arrange and function appropriately (Rosculete et al. 2019). T. terrestris has been traditionally used for medici- nal purposes throughout history, addressing various health issues such as impotence, rheumatism, edema, hypertension, and kidney stones (Chhatre et al. 2014). Pharmacological studies conducted on T. terrestris have demonstrated its aphrodisiac, analgesic, antibi- otic, antihyperglycemic, antihyperlipidemic, larvicidal, repellent, antioxidant, cytotoxic, immunomodulatory, hypolipidemic, anticancer, antibacterial, and antifun- gal properties (Chhatre et al., 2014). Furthermore, in order to ensure safe therapy, it is important to test the cytogenotoxicity of T. terrestris. Our current study on the cytogenetic impact of T. terrestris revealed cyto- toxic and genotoxic effects depending on the concentra- tions applied and the duration of treatment. It observed that signs of toxicity appeared from a concentration of 85Cytogenetic effects of Tribulus terrestris on meristematic cells of Allium cepa L. and Vicia faba L. 0.05 mg/mL, resulting in a significant reduction in the mitotic index and a significant increase in chromosomal aberrations, as well as a change in the shape and colour of the roots of A. cepa and V. faba, indicating necrosis. Our results underline the interest in using T. terrestris as an effective medicinal plant for various diseases, but its use must respect appropriate therapeutic doses and not be anarchic. CONCLUSION This study is the first to investigate the cytotoxic and genotoxic effects of T. terrestris on meristematic cells of A. cepa and V. faba. Decreased root growth with decreased mitotic index and increased chromosomal abnormalities resulting from treatment with T. terrestris are signs of cytotoxicity and genotoxicity. Therefore, this plant should be used with caution in traditional medi- cine. ACKNOWLEDGEMENTS The authors would like to thank all those who con- tributed to the completion of this work. Special thanks go to the Laboratory of Research in Biodiversity Inter- action, Ecosystem and Biotechnology “LRIBEB”, where this research was conducted. REFERENCES Abudayyak M, Jannuzzi AT, Özhan G, Alpertunga B. 2015. Investigation on the toxic potential of Tribulus terrestris in vitro. Pharm Biol 53(4):469-476. Akgündüz MÇ, Çavuşoğlu K. and Yalçın E. 2020. The Potential Risk Assessment of Phenoxyethanol with a Versatile Model System. Sci Rep 10: 1-10. 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Shah², Namrata Sharma¹ Comparative cytogenetics of four endemic Capoeta (Teleostei: Cyprinidae) species from Anatolia, Türkiye Sevgi Unal-Karakus1,*, Muhammet Gaffaroglu2, Muradiye Karasu-Ayata3 A karyomorphological comparison of seven species of Achillea L. from Kurdistan of Iran Fatemeh Nezhadi1, Farzad Fayaz2,*, Ezzat Karami2, Hooshmand Safari3, Abdol Rahman Rahimi2 Genotoxicity of a synthetic plant growth regulator, Forchlorfenuron (CPPU), on human lymphocytes using chromosome aberration assay Ayşe Yavuz Kocaman1,*, Berna Yakar2 Phenthoate toxicity evaluation in root meristem of Pisum sativum L. Sazada Siddiqui Karyomorphology of two subspecies of Anthemis maritima (Asteraceae) from Algeria Meryem Nassar1,4,*, Nora Sakhraoui2,4, Gianniantonio Domina3 Cytogenetic effects of Tribulus terrestris L. on meristematic cells of Allium cepa L. and Vicia faba L. Ali Bouzekri1,2,*, Meryem Nassar1,2, Souheila Slimani1,2, Zohra Chekroud1,2 New chromosomal data, karyotype asymmetry and polyploid variations of some Gundelia (Asteraceae) species from Turkey Esra Martin1, Metin Armağan2, Halil Erhan Eroğlu3*, Aslı Doğru-Koca4, Osman Tugay5, Golshan Zare6, Osman Kola7, Mahmut Miski8, Nur Tan9, Ernst Vitek10 Allelopathic and toxicological effects of Origanum vulgare L. essential oil Lejla Husić, Adisa Parić, Aner Mesic* Cytogenetic analysis in Tetragonopterus franciscoensis (Characiformes): another piece to the karyoevolutionary puzzle of tetra fishes Mauricio Barros Fernandes, Jamille de Araújo Bitencourt, Joandson Calixto dos Santos, José Henrique Galdino*, Paulo Roberto Antunes de Mello Affonso