Brazilian Journal of Biological Sciences, 2018, v. 5, No. 10, p. 471-479. ISSN 2358-2731 https://doi.org/10.21472/bjbs.051023
Genotoxicity assessment of Schinopsis brasiliensis Engl. (Sapindales: Anacardiaceae) in somatic cells of Drosophila melanogaster (Meigen, 1830) (Diptera: Drosophilidae)
Silmara de Moraes Pantaleão¹, Jéssica Adele dos Santos¹, Thalyta Linacher¹, Clisiane Carla de Souza Santos², Charles dos Santos Estevam² and Neila Coelho da Costa³
¹Universidade Federal de Sergipe. Centro de Ciências Biológicas e da Saúde, Laboratório de Genética e Conservação de Recursos Naturais, Departamento de Biologia, São Cristóvão, Sergipe, Brasil. Email: [email protected]. ²Universidade Federal de Sergipe, Centro de Ciências Biológicas e da Saúde, Departamento de Fisiologia, Universidade Federal de Sergipe, São Cristóvão, Sergipe, Brasil. ³Universidade Federal de Goiás, Departamento de Ciências Biológicas, Campus Catalão, Catalão, Goiás Brasil.
Abstract. Schinopsis brasiliensis Engl. (Sapindales: Anacardiaceae) has been used in the Brazilian folk medicine to Received treat several illnesses. However, the phytochemical profile of May 5, 2018 S. brasiliensis as well as its genotoxic potential are poorly understood, which compromises population safety regarding the Accepted August 19, 2018 medicinal use of this plant species. In this study, we analyzed the genotoxic effects of S. brasiliensis using the Somatic Mutation Released and Recombination Test (SMART) of Drosophila melanogaster. August 31, 2018 Larvae from both standard cross (ST) and high bioactivation capacity cross (HB) were exposed to different concentrations of Full Text Article the hydroetanolic extract and ethyl acetate fraction of S. brasiliensis. We analyzed wings from D. melanogaster according to the type and number of mutant hair. Ours results suggested no genotoxic activity of S. brasiliensis in D. melanogaster somatic cells.
Keywords: Genotoxicity; Baraúna; Caatinga; Tradicional medicine; SMART.
0000-0001-6870-4360 Silmara de Moraes Pantaleão 0000-0002-0098-7445 Jéssica Adele dos Santos 0000-0001-6940-6891 Thalyta Linacher
ISSN 2358-2731/BJBS-2018-0026/5/10/23/471 Braz. J. Biol. Sci. http://revista.rebibio.net 472 Pantaleão et al.
0000-0001-7599-0983 Clisiane Carla de Souza Santos 0000-0003-3306-1611 Charles dos Santos Estevam 0000-0002-4449-8248 Neila Coelho da Costa
Introduction studies suggested that some species from the genus Schinopsis have a high Many plant species from Brazilian concentration of phenolic compounds Caatinga have shown pharmacological such as tannins, flavonoids, flavanones, potential, such as Erythrina velutina and gallic acid (Marín-Martinez et al., (Vasconcelos et al., 2007), Tabebuia 2009; Sánchez-Martína et al., 2010; aurea (Reis et al., 2014), and Ziziphus Saraiva et al., 2011). Analyzing the joazeir (Ribeiro et al., 2013). That fact has phytochemical profile of S. brasiliensis, raised a lot of attention for plants used in Almeida et al. (2010) noticed the the folk medicine regarding their presence of tannins, quinones, and properties and safety (Varanda, 2006; triterpens, while Santos et al. (2014) Albuquerque and Oliveira, 2007; Silva et showed that the ethyl acetate fraction al., 2012). However, when considering from the bark of S. brasiliensis contains the number of native species from aurones, catechins, chalcones, and Caatinga that have been used by local saponins (Santos et al., 2014). On one communities, there are still few studies hand, studies demonstrated that some of concerned with the composition and those compounds have protective and biological activity of most species antigenotoxic effect (Rodríguez-García, (Vicentini et al., 2001; Liporacci et al., 2013; Manzolli et al., 2015). On the other 2017). The genotoxic effects of medicinal hand, compounds such as saponins were plants are usually related to the noticed to have genotoxic effect on concentration used by the individual human lymphocytes (Kalachaveedu et al., (Corrêa et al., 2001; Sponchiado et al., 2014). In addition, the extract of 2016). Moreover, other aspects have to S. brasiliensis’ bark demonstrated be considered when analyzing the larvicide activity against Aedes aegypti genotoxic activity of plant species, such and toxic potential on Biomphalaria as the part of the plant, the age, and the glabrata (Santos et al., 2014). presence of contaminants in the plant Despite the efficacy of tissues (Saad et al., 2006). S. brasiliensis in the folk medicine, there Schinopsis brasiliensis Engl. are few studies describing its (Anacardiaceae) is an endemic tree from composition and activity on organisms Brazilian Caatinga, which occurs from the (Saraiva et al., 2009; Santos et al., 2014). State of Bahia to the State of Paraíba The lack of scientific knowledge about (Engler, 1879). The popular commum this species implies on risks to people names for this species include “baraúna”, who use S. brasiliensis as an alternative “braúna”, and “quebracho” (Prado et al., treatment for several diseases. 1995; Cardoso et al., 2015). Considering the safety involving the S. brasiliensis is used in the folk consumption of medicinal plants, we medicine to treat disturbs of the nervous found important to conduct genotoxic system (hysteria and anxiety), studies, in order to validate their use by inflammation, pain, and infection local communities. For that finality, the (Albuquerque et al., 2007; Lorenzi, 2008; Somatic Mutation and Recombination Saraiva et al., 2011). Phytochemical Test (SMART) in Drosophila
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melanogaster is an indicated test due to Here, we used three mutant its capacity and sensibility in the lineages of D. melanogaster (1) multiple identification of mutations and wing hairs (mwh): mwh/mwh; (2) flare-3 recombination events, besides the (flr3/In (3LR)TM3, rippsep l(3)89Aabx34e e detection of promutagens and BdS; and (3) ORR; flare-3 (ORR/ORR; procarcinogens (Graf at al., 1984; Graf flr3/In (3LR)TM3, rippsep l(3)89Aabx34e e and Van Schaik, 1992; Panchal and BdS). Considering a proportion 2:1 for Tiwari, 2017). females and males, we made two This study aimed to evaluate the different crosses: the standard cross genotoxic effects of two extracts of (ST), in which flr3 individuals were S. brasiliensis on somatic cells of females and mwh individuals were males D. melanogaster by using the Somatic (Graf et al., 1989); and the high Mutation and Recombination Test bioactivation cross (HB), in which ORR; (SMART). flr3 individuals were females and mwh individuals were males (Graf and Van Materials and methods Schaik, 1992). After the eggs posture (72 h ± Plant collection 4 h), we placed 3rd instar larvae in We collected samples of plastic recipes containing food solution S. brasiliensis in Piranhas, AL, Brazil (1.5 g of potato powder and 5 mL of one (09° 37’ 26” S e 37° 45’ 25” W). The of the four extract concentrations (0.5, species was identified and deposited at 1.0, 2.0 e 4.0 mg/mL)). The controls Federal University of Sergipe’s consisted of ultrapure water (negative herbarium, São Cristóvão, SE, Brazil. control) and 0.891 mg/mL of urethane (positive control). For each treatment, Extracts preparation we made four replicates. The larvae were The preparation of extracts submitted to chronical treatment, which consisted on keeping the plant’s bark at consisted of exposing the larvae to food room temperature until the material was solution containing the plant extract for completely dried. In sequence, we 48 h. submitted the material to maceration The emergent adults with the with ethanol (90%) for five days. We genotypes: mwh+/+ flr3 ou mwh +/+ obtained the hydroethanolic extract by TM3, BdS were separated and fixed in filtering and concentering the material ethanol (70%). The wings of D. under vacuum in rotatory evaporator (LS melanogaster were fixed on slides using LOGEN) at 45 °C. To obtain the ethyl Faure solution (30 g of gum arabic, 20 acetate fraction, we diluted the mL of glycerol, 50 g of hydrate of chloral hydroethanolic extract in methanol- and 50 mL of water), and kept on water (MeOH:H2O, 2:3). The extraction hotplate until they were completely dry. was completed by adding ethyl acetate to Mutant spots were analyzed and the solution. classified according to Graf et al. (1992).
Somatic Mutation and Survivorship assessment Recombination Test (SMART) We assessed survivorship after The use of the SMART in larvae treatment by counting the number Drosophila melanogaster to evaluate of living flies per replicate. Percent genotoxicity of medicinal plants and survival was calculated for both ST and drugs is an efficient tool due to its HB cross flies exposed to different sensibility and the genetic similarity concentrations of S. brasiliensis extracts between D. melanogaster and mammals (Figure 1). Percent survival was given by (Graf et al., 1984). [120 (the total number of larvae exposed to a concentration) - the number of adult
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flies that emerged from the same Results and discussion concentration) x 100]. Survivorship analysis of Frequency of mutant hair and D. melanogaster revealed no cytotoxic data analysis potential of extracts on The results were submitted to S. brasiliensis both ST and HB cross flies. Percent statistical analysis, according to the survival of ST cross flies varied from model of multiple decisions described by 90% to 82.5%, while percent survival of Frei and Würgler (1988). The frequency HB cross flies varied from 80% to 70%, of mutant hair found in the treatment for all HEE concentrations (Figure 1). was compared to the frequencies found Similarly, percent survival in EAF in our positive and negative control, treatments varied from 89.17% to using the binomial conditional test of 74.17%, for ST cross flies, and from 80% Kastenbaum and Bowman (1970), where to 71.67% for HB cross flies (Figure 2). = 0.05. The statistical diagnoses was positive, negative, and inconclusive (Frei andα Würgler, 1988).
100 80 60 40 ST (%) 20 HB 0 Percent survival survival Percent Water 0.5 1 2 4 Concentration (mg/mL) Figure 1. Survivorship curve for Drosophila melanogaster (N = 120) exposed to the hydroetanolic extract of Schinopsis brasiliensis.
100 80 60 survival 40 ST (%) 20 HB 0 Percent Water 0.5 1 2 4
Concentration (mg/mL) Figure 2. Survivorship curve for Drosophila melanogaster (N = 120) exposed to the ethyl acetate fraction of Schinopsis brasiliensis.
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Results on chronic treatment of crosses, there was no significant D. melanogaster larvae suggested no difference in the frequency of mutant genotoxic effect of S. brasiliensis extracts spots in flies treated with HEE, EAF, and at the tested concentrations. For both negative control (Table 1 and 2).
HEE and EAF had negative results which has normal metabolization activity for small single spots and large single (Graf et al., 1984). spots. For both extracts, the results on In Table 2, we sumarized the twin spots were inconclusive, fact that is results of the HB cross, which analyzed usually associated to the low frequency the activity of substances dependent on of twin spots found in negative controls. metabolization via cytocrome P450. The However, analyzing the total spots the HB cross includes the ORR lineage, which results for either HEE or EFA were is known by its resistence to DDT and negative at the standard cross (ST), high level of CYP450 enzymes (Pádua et
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al., 2013). This fact enhances fly and Waszczynskyj, 2004). Moreover, an sensibility to promutagens and antigenotoxicity study regarding the use procarcinogens, which allows the of pro-anthocyanins (condensed tannins) identification of subtances with indirect extracted from Vittis vinifera seeds activity. For the HB cross our results on suggested a protective effect induced by large single spots and twin spots were the substance on genotoxic damages inconclusive for all concentrations of caused by doxorubicin hydrochloride (an HEE and EAF. However, those results antracicline that acts on the formation of have low biological significance, free radicals by interfering the enzyme considering that the frequency of total topoisomerase II) (Rezende et al., 2009). spots we found in the HB cross flies Thus, future researches would focus on exposed to HEE and EAF were lower than the antigenotoxic potential of the frequencies found in the positive S. brasiliensis. control. Our results were statiscally non- The analysis of the trans- significant for both ST and HB crosses, heterozygous progeny from both ST and which indicates that the two extracts of HB crosses exposed to uretan showed S. brasiliensis had neither genotoxic effect that the negative results obtained in the on D. melanogaster somatic cells with treatments with HEE and EAF are not low metabolization activity, nor false-negatives. genotoxic effect on D. melanogaster Machado (2012) verified that somatic cells with high metabolization S. brasiliensis extract had on its activity, regarding the promotion of phytochemical composition, substances mutation, deletion, non-disjunction and that were able to inhibit development of mitotic recombination. Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae and Pseudomonas Conclusions aeruginosa, suggesting antimicrobial properties of S. brasiliensis. Santos et al. The results presented in our (2014) noticed that the ethyl acetate preliminary study suggest no genotoxic fraction of S. brasiliensis had larvicide effect of S. brasiliensis on activity on Aedes aegypti and toxic effect D. melanogaster. However, considering on Biomphlaria glabrata. In addition, the other organisms is a step necessary to authors classified tannins as the main guarantee the safe use of S. brasiliensis by phenolic compounds in the bark of the population or even to validate its use S. brasiliensis. It is known that the as a medicine. Additional studies would amount of tannins found in wild plants is also focus on the anti-genotoxic potential usually higher than the amount found in of S. brasiliensis in order to explore the domesticated plants (Hagerman and pharmacological potential of this species. Butler, 1991). This fact is understood as an adaptation to herbivory, seeing that Acknowledgments tannins create a negative response to predation by promoting antinutricional CSE and CCSS coordinate the and toxic effects on herbivores through extract preparation and biological the coagulation of mucoproteins and studies. NCS and SMP contributed to consequent inhibition of digestion design the study supervised the (Harborne, 1988). laboratory work execution and critical On the other hand, phenolic reading of the manuscript. JASS, TLGSR, compounds have antioxidant properties, CCSS, MRB and PS contributed in running once those substances have the ability of the laboratory work, analysis of the data donating hydrogen and electrons due to and drafted the paper. JASS contributed the presence of stable radicals that avoid in plant identification and herbarium the oxidation of compounds (Degáspari confection.
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Braz. J. Biol. Sci., 2018, v. 5, No. 10, p. 461-469.