In vitro evaluation of the cytotoxicity of Croton lechleri Müll. Arg. (Euphorbiaceae) extract and its equivalence with quinoleic compounds and avermectin
DOI:
https://doi.org/10.33448/rsd-v11i15.36809Keywords:
Croton; Phytotherapy; Hemolysis; Medicinal Plants; Toxicity.Abstract
Species of the genus Croton L., especially Croton lechleri Müll. Arg. popularly known as "dragon blood", are used for various therapeutic purposes by the Brazilian population. In vitro assays evaluating the cytotoxicity of this plant in healthy cells are scarce. Thus, this study aims to evaluate the cellular toxicity of the ethanolic extract of C. lechleri bark, and compare it with commercially available compounds such as quinoleic compounds and avermectins. The hemolytic test was performed by adding the extract in red cells suspension in saline at a hematocrit of 10% with decreasing concentrations. The cytotoxicity assay was performed on VERO cells maintained in RPMI culture medium supplemented with fetal bovine serum, to which 7 concentrations diluted 1/2 from the stock solution of 500 µg/mL of the crude extract of C. lechleri and the drugs Chloroquine, Hydroxychloroquine and Ivermectin. Hemolysis was analyzed using a UV/VIS at intervals of 30, 60 and 120 minutes. Cytotoxicity after 72 hours by resazurin reduction assay. The results showed that the extract of C. lechleri at different concentrations and times, showed low or no toxicity to red blood cells. In the cytotoxicity assay, the extract presented CC₅₀ of 218,9 µg/mL, the CQ, HCQ and IVe of 128,5; 191,6 and 32,9 µg/mL, respectively. It was concluded that the ethanolic extract of C. lechleri bark did not present significant hemolysis and that it presented moderate cytotoxicity against VERO cells, showing that this natural product is potentially safe for popular use.
References
Almeida, F. K. V. Novais, V. P. Salvi, J. D. O. & Marson, R. F. (2019). Avaliação tóxica, citotóxica e mutagênica/genotóxica de um extrato comercial de sangue do dragão (Croton lechleri Müll. Arg.). Revista Fitos, 13(1), 29. DOI: https://doi.org/10.17648/2446-4775.2019.605
Alonso-Castro, A. J., Ortiz-Sánchez, E., Domínguez, F., López-Toledo, G., Chávez, M., Ortiz-Tello, A. D. J. & García-Carrancá, A. (2012). Antitumor effect of Croton lechleri Müll. Arg. (Euphorbiaceae). Journal of Ethnopharmacology, 140(2), 438–442. DOI: https://doi.org/10.1016/J.JEP.2012.01.009
Alonso, J. (2008). Fitomedicina: curso para profissionais da área da saúde (1st ed.). Pharmabooks.
Amaral, A. C. F. & Barnes, R. A. (1997). Alkaloids of Croton celtidifolius. Planta Medica. Botanical Journal of the Linnean Society, 141, 399–436.
Amaral, R. G., Andrade, L. N., Severino, P., Araújo, S. S., Santos, M. I. S., Dias, A. S., Moraes Filho, M. O., O’Pessoa, C., Carvalho, A. A., Thomazzi, S. M. & Estevam, C. S. (2018). Investigation of the possible antioxidant and anticancer effects of Croton argyrophyllus (Euphorbiaceae). Chemical Engineering Transactions, 64(January), 253–258. DOI: https://doi.org/10.3303/CET1864043
Ammerman, N. C., Beier-Sexton, M. & Azad, A. F. (2008). Growth and maintenance of VERO cell lines. Current Protocols in Microbiology, SUPPL. 11, 1–7. DOI: https://doi.org/10.1002/9780471729259.mca04es11
Azevedo, K., Alechandre, A., Lima, Á., Campos, C. A., Costa, J., Pereira, M. A., Leite, A., Melo, T. & Lima, A. (2008). Guia para a extração de sangue de grado (Croton lechleri Müll. Arg). Disponível em: https://ipam.org.br/wp-content/uploads/2008/04/guia_para_a_extrac%CC%A7a%CC%83o_de_sangue_de_grado_.pdf
Barratt-Due, A., Olsen, I. C., Nezvalova-Henriksen, K., Kåsine, T., Lund-Johansen, F., Hoel, H., Holten, A. R., Tveita, A., Mathiessen, A., Haugli, M., Eiken, R., Kildal, A. B., Berg, Å., Johannessen, A., Heggelund, L., Dahl, T. B., Skåra, K. H., Mielnik, P., Le, L. A. K., Aukrust, P. (2021). Evaluation of the Effects of Remdesivir and Hydroxychloroquine on Viral Clearance in COVID-19: A Randomized Trial. Annals of Internal Medicine, 174(9), 1261–1269. DOI: https://doi.org/10.7326/M21-0653
Belcavello, L., Cunha, M., Andrade, M. & Batitucci, M. (2012). Citotoxicidade e danos ao DNA induzidos pelo extrato de Zornia diphylla, uma planta medicinal Cytotoxicity and DNA damages induced by the Zornia diphylla extract , a medicinal plant. Natureza on Line, 10, 140–145.
Bettega, P. V. C., Czlusniak, G. R., Piva, R., Namba, E. L., Ribas, C. R., Grégio, A. M. T. & Rosa, E. A. R. (2011). Fitoterapia: dos canteiros ao balcão da farmácia. Archives of Oral Research, 7(1), 89–97. DOI: https://doi.org/10.7213/AOR.V7I1.23149
Buonfrate, D., Chesini, F., Martini, D., Roncaglioni, M. C., Ojeda Fernandez, M. L., Alvisi, M. F., de Simone, I., Rulli, E., Nobili, A., Casalini, G., Antinori, S., Gobbi, M., Campoli, C., Deiana, M., Pomari, E., Lunardi, G., Tessari, R. & Bisoffi, Z. (2022). High-dose ivermectin for early treatment of COVID-19 (COVER study): a randomised, double-blind, multicentre, phase II, dose-finding, proof-of-concept clinical trial. International Journal of Antimicrobial Agents, 59(2). DOI https://doi.org/10.1016/J.IJANTIMICAG.2021.106516
Brasil (2014). Agencia Nacional de Vigilância Sanitária – ANVISA. Resolução RDC nº 26, de maio de 2014: Dispões sobre o registro de medicamentos fitorerápicos e o registro e a notificação de produtos tradicionais fitoterápicos. Diário Oficial da União, Seção1(1). 1-34. Disponível em: hhtps://bvms.saude.gov.br/bvs/saudelegis/anvisa/2014/rdc0026_13_05_2014.pdf
Cai, Y., Chen, Z. P. & Phillipson, J. D. (1993a). Diterpenes from Croton lechleri Müll. Arg. Phytochemistry, 32(3), 755–760. DOI: https://doi.org/10.1016/S0031-9422(00)95166-5
Cai, Y., Chen, Z. P. & Phillipson, J. D. (1993b). Clerodane diterpenoids from Croton lechleri Müll. Arg. . Phytochemistry, 34(1), 265–268. DOI: https://doi.org/10.1016/S0031-9422(00)90816-1
Calvo-Calle, J. M., Moreno, A., Eling, W. M. C. & Nardin, E. H. (1994). In Vitro Development of Infectious Liver Stages of P. yoelii and P. berghei Malaria in Human Cell Lines. Experimental Parasitology, 79(3), 362–373. DOI: https://doi.org/10.1006/EXPR.1994.1098
Carvalho, E. B. Borges, É. L., Carlos, L. M. B., Silva, M. A. M., Magalhães, S. M. M., Gomes, F. V. B. A. F., Carvalho, M. J. C., Quixadá, A. T. S. & Pitombeira, M. H. S. (2007). Efeito da bomba de infusão de soluções sobre o grau de hemólise em concentrados de hemácias. Revista Brasileira de Hematologia e Hemoterapia, 29(2), 149–152. DOI: https://doi.org/10.1590/S1516-84842007000200013
Caruzo, M.B.R, Secco, R.S., Medeiros, D., Rijna, R., Torres, D.S.C., Santos, R.F.D., Pereira, A.P.N., Rossine, Y., Lima, L.R., Muniz Filho., Valduga, E. Croton in Flora e Funga do Brasil. Jardim Botânico do Rio de Janeiro. Disponível em: <https://floradobrasil.jbrj.gov.br/FB22688>. Acesso em: 27 out. 2022.
Castro, I. B., Câmara, G. B., Pontes, J. F., Viana, D. L., Souza, R. P., Silva Nobrega, E. D., Lira, R. B. B. & Barbosa, L. S. L. T. (2020). Estratégias nutricionais no tratamento do diabetes mellitus: revisão bibliográfica. Research, Society and Development, 9(2), 26. DOI: https://doi.org/10.33448/rsd-v9i2.2193
Doyno, C., Sobieraj, D. M. & Baker, W. L. (2020). Toxicity of chloroquine and hydroxychloroquine following therapeutic use or overdose. Clinical Toxicology, 59(1), 12–23. DOI: https://doi.org/10.1080/15563650.2020.1817479
Eisenbrand, G., Pool-Zobel, B., Baker, V., Balls, M., Blaauboer, B. J., Boobis, A., Carere, A., Kevekordes, S., Lhuguenot, J. C., Pieters, R., & Kleiner, J. (2002). Methods of in vitro toxicology. Food and Chemical Toxicology, 40(2–3), 193–236. DOI: https://doi.org/10.1016/S0278-6915(01)00118-1
Fernandes, A. F. C. (2019). Caracterização físico-química da droga vegetal, estufo fitoquímico e farmacológico de Cnidoscolus quercifolius Pohl (Euphorbiaceae) [Disseratação de mestrado, Universidade Estadual da Paraíba-UEPB].
Gil, A.C. Como elaborar projetos de pesquisa. 5. Ed. São Paulo: Editora Atlas, 2010.
Gupta, S., Vohra, S., Sethi, K., Gupta, S., Bera, B. C., Kumar, S. & Kumar, R. (2022). In vitro anti-trypanosomal effect of ivermectin on Trypanosoma evansi by targeting multiple metabolic pathways. Tropical Animal Health and Production, 54(4), 240. DOI: https://doi.org/10.1007/S11250-022-03228-1
Hill, A., Garratt, A., Levi, J., Falconer, J., Ellis, L., McCann, K., Pilkington, V., Qavi, A., Wang, J. & Wentzel, H. (2021). Retracted: Meta-analysis of Randomized Trials of Ivermectin to Treat SARS-CoV-2 Infection. Open Forum Infectious Diseases, 8(11). DOI: https://doi.org/10.1093/OFID/OFAB358
International Standard Organization. (2009). Biological evaluation of medical devices—part 5: tests for in vitro cytotoxicity (ISO 10993-5:2009). (3ª ed.). ISO.
Kory, P., Meduri, G. U., Varon, J., Iglesias, J. & Marik, P. E. (2021). Review of the Emerging Evidence Demonstrating the Efficacy of Ivermectin in the Prophylaxis and Treatment of COVID-19. American Journal of Therapeutics, 28, 299–318. Disponivel em: www.americantherapeutics.com
Li, R., Morris-Natschke, S. L. & Lee, K. H. (2016). Clerodane diterpenes: Sources, structures, and biological activities. In Natural Product Reports (Vol. 33, Issue 10, pp. 1166–1226). Royal Society of Chemistry. DOI: https://doi.org/10.1039/c5np00137d
Lopes, T. V., Félix, S. R., Schons, S. V. & Nobre, M. O. (2013). Dragon’s blood (Croton lechleri Müll. Arg.): an update on the chemical composition and medical applications of this natural plant extract. A review. Revista Brasileira de Higiene e Sanidade Animal, 7(2), 167–191. DOI: https://doi.org/10.5935/1981-2965.20130016
Lorenzi, h. & Matos, F.J.A. Plantas medicinais no Brasil: nativas e exóticas. 3. Ed. Nova Odessa-SP. Jardim Botânico Plantarum. 2021. p. 576.
Markowicz-Piasecka, M., Huttunen, K. M., Mikiciuk-Olasik, E., & Sikora, J. (2018). Biocompatible sulfenamide and sulfonamide derivatives of metformin can exert beneficial effects on plasma haemostasis. Chemico-Biological Interactions, 280, 15–27. DOI: https://doi.org/10.1016/J.CBI.2017.12.005
Milanowski, D. J., K Winter, R. E., F Elvin-Lewis, M. P. & Lewis, W. H. (2002). Geographic Distribution of Three Alkaloid Chemotypes of Croton lechleri Müll. Arg. DOI: https://doi.org/10.1021/np000270v
Navarro, M., Camprubí, D., Requena-Méndez, A., Buonfrate, D., Giorli, G., Kamgno, J., Gardon, J., Boussinesq, M., Mu ~ Noz, J. & Krolewiecki, A. (2020). Safety of high-dose ivermectin: a systematic review and meta-analysis. Journal of Antimicrobial Chermotherapy, 75, 827–834. DOI: https://doi.org/10.1093/jac/dkz524
Oliveira, V.B., Zuchetto, M., Oliveira, C.F., Paula, C.S., Duarte, A. F. F., Miguel, M. D. & Miguel, O. G. (2016). Efeito de diferentes técnicas extrativas no rendimento, atividade antioxidante, doseamentos totais e no perfil por clae-dad de dicksonia sellowiana (presl.). Hook, dicksoniaceae. Revista Brasileira de Plantas Medicinais, 18(1 suppl 1), 230–239. Disponível em: https://www.scielo.br/j/rbpm/a/zKMxjg3c3hJV8wqSfsPBGYJ/?lang=pt.
Paixão, M. S. (2018). Avaliação da toxicidade do extrato hidroalcoólico da entrecasca de Croton aryrophyllus Kunth. [Disseração de mestrado, Universidade Federal de Pernambuco-UFPE].
Papazisis, G., Siafis, S., Cepatyte, D., Giannis, D., Stamoula, E., Tzachanis, D. & Egberts, T. (2021). Safety profile of chloroquine and hydroxychloroquine: a disproportionality analysis of the FDA Adverse Event Reporting System database. European Review for Medical and Pharmacological Sciences, 25(19), 6003–6012. DOI: https://doi.org/10.26355/EURREV_202110_26878
Pereira, C. T., Pereira, M. E. T., Simão, K. L. A., Alves, M. S., Simão, B. L. A., Medeiros, M. A. A. Teles, Y. C. F., Anjos, R. M. Oliveira, H. M. B. F. Oliveira, V. F. Medeiros, C. I. S., Sousa, A. P. & Filho, A. A. O. (2020). Análise da citotoxicidade do extrato metanólico de Psidium guineense Swartz em células sanguíneas humanas. Research, Society and Development, 9(6), e61963093–e61963093. DOI: https://doi.org/10.33448/RSD-V9I6.3093
Pires, I. F. B., de Sousa, A. A., Lima, C. de A., Costa, J. D., Feitosa, M. H. A. & Costa, S. de M. (2017). Plantas medicinais: cultivo e transmissão de conhecimento em comunidade cadastrada na Estratégia Saúde da Família. Revista Brasileira de Pesquisa Em Saúde/Brazilian Journal of Health Research, 18(4), 37–45. DOI: https://doi.org/10.21722/rbps.v18i4.16729
Podsiedlik, M., Markowicz-Piasecka, M. & Sikora, J. (2020). Erythrocytes as model cells for biocompatibility assessment, cytotoxicity screening of xenobiotics and drug delivery. Chemico-Biological Interactions, 332, 109305. DOI: https://doi.org/10.1016/J.CBI.2020.109305
Rangel, M., Malpezzi, E. L. A., Susini, S. M. M. & Freitas, J. C. (1997). Hemolytic activity in extracts of the diatom Nitzschia. Toxicon. 35(2), 305-309. DOI: https://doi.org/10.1016/S0041-0101(96)00148-1
Reddy, C. S. S. S., Subramanyam, M. V. V., Vani, R. & Asha Devi, S. (2007). In vitro models of oxidative stress in rat erythrocytes: Effect of antioxidant supplements. Toxicology in Vitro, 21(8), 1355–1364. DOI: https://doi.org/10.1016/j.tiv.2007.06.010
Riss, T. L., Moravec, R. A., Niles, A. L., Duellman, S., Benink, H. A., Worzella, T. J. & Minor, L. (2016). Cell Viability Assays. Assay Guidance Manual, 1–25. Disponível em: http://www.ncbi.nlm.nih.gov/pubmed/23805433
Rogero, S. O., Lugão, A. B., Ikeda, T. I. & Cruz, Á. S. (2003). Teste in vitro de citotoxicidade: estudo comparativo entre duas metodologias. Materials Research, 6(3), 317–320. DOI: https://doi.org/10.1590/s1516-14392003000300003
Sá, L. L. F. Silva, H. V. A. Nogueira, N. C., Oliveira, K. B. V. & Brito, M. R. M. (2018). Regulamentação de fitoterápicos no Brasil e perfil daqueles vendidos em uma farmácia de manipulação de Teresina-PI. Boletim Informativo Geum, 9(3), 1–9.
Salatino, A., Salatino, M. L. F. & Negri, G. (2007). Traditional uses, chemistry and pharmacology of Croton species (Euphorbiaceae). Journal of the Brazilian Chemical Society, 18(1), 11–33. DOI: https://doi.org/10.1590/S0103-50532007000100002
Santos, A. P. de A. Pacheco, S. G. A. & Teles, C. B. G. (2015). Efeito leishmanicida in vitro do látex de Croton lechleri Müll. Arg. (Euphorbiaceae). Revista de Ciencias Farmaceuticas Basica e Aplicada, 36(3), 413–418.
Schiar, V. P. P. Santos, D. B., Lüdtke, D. S., Vargas, F., Paixão, M. W., Nogueira, C. W., Zeni, G. & Rocha, J. B. T. (2007). Screening of potentially toxic chalcogens in erythrocytes. Toxicology in Vitro, 21(1), 139–145. DOI: https://doi.org/10.1016/j.tiv.2006.08.006
Silva, A. D. S. Melo e Silva, K., Neto, J. C. Oliveira Costa, V. C. Lunaf Pessôa, H., Tavares, J. F. Silva, M. S. & Andrade Cavalcante, F. (2016). Croton grewioides Baill. (Euphorbiaceae) Shows Antidiarrheal Activity in Mice. Pharmacognosy Research, 8(3), 202. DOI: https://doi.org/10.4103/0974-8490.181465
Sousa, A. P., Ferreira, M. D. L., Fernandes, D. A., Cordeiro, L. V., Souza, M. F. V., Pessoa, H. L. F., Filho, A. A. de O. & Sá, R. de C. S. (2021). In silico, in vitro and ex-vivo Toxicological Profiling of 5,7,4’-Trihydroxyflavone-8-C-β-Glucopyranoside - Vitexin. Revista de Ciências Farmacêuticas Básica e Aplicada, 42, 1–9. DOI: https://doi.org/10.4322/2179-443X.0709
Stefano, L., Ogburn, E. L., Ram, M., Scharfstein, D. O., Li, T., Khanal, P., Baksh, S. N., McBee, N., Gruber, J., Gildea, M. R., Goldenberg, N. A., Bennani, Y., Brown, S. M., Buckel, W. R., Clement, M. E., Mulligan, M. J., O’Halloran, J. A., Rauseo, A. M., Self, W. H., Freilich, D. (2022). Hydroxychloroquine/Chloroquine for the Treatment of Hospitalized Patients with COVID-19: An Individual Participant Data Meta-Analysis. MedRxiv. DOI: https://doi.org/10.1101/2022.01.10.22269008
Tedesco, M., Kuhn, A. W., Boligon, A. A., Laughinghouse IV, H. D., Athayde, M. L., Silva, A. C. F. & Tedesco, S. B. (2015). Chromatographic analysis, antiproliferative effect and genotoxicity of aqueous extracts of Citrus sinensis (L.) osbeck on the Allium cepa L. test system. Bioscience Journal, 31(4), 1213–1221. DOI: https://doi.org/10.14393/bj-v31n4a2015-23245
Wang, M., Cao, R., Zhang, L., Yang, X., Liu, J., Xu, M., Shi, Z., Hu, Z., Zhong, W. & Xiao, G. (2020). Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro. Cell Research, 30(3), 269. DOI: https://doi.org/10.1038/S41422-020-0282-0
Yang, J., Guo, Z., Liu, X., Liu, Q., Wu, M., Yao, X., Liu, Y., Cui, C., Li, H., Song, C., Liu, D. & Xue, L. (2020). Cytotoxicity Evaluation of Chloroquine and Hydroxychloroquine in Multiple Cell Lines and Tissues by Dynamic Imaging System and Physiologically Based Pharmacokinetic Model. Frontiers in Pharmacology, 11. DOI: https://doi.org/10.3389/FPHAR.2020.574720/FULL
Yao, X., Ye, F., Zhang, M., Cui, C., Huang, B., Niu, P., Liu, X., Zhao, L., Dong, E., Song, C., Zhan, S., Lu, R., Li, H., Tan, W. & Liu, D. (2020). In Vitro Antiviral Activity and Projection of Optimized Dosing Design of Hydroxychloroquine for the Treatment of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Clinical Infectious Diseases, 71(15), 732–739. DOI: https://doi.org/10.1093/CID/CIAA237
Yokomizo, A. Y., Antoniazzi, M. M., Galdino, P. L., Azambuja, N., Jorge, S. A. C. & Pereira, C. A. (2004). Rabies Virus Production in High VERO Cell Density Cultures on Macroporous Microcarriers. Biotechnology and Bioengineering, 85(5), 506–515. DOI: https://doi.org/10.1002/bit.10917
Zevallos-Pollito, P. A. & Filho, M. T. (2007). Espécies lenhosas do gênero Croton L. (Euphorbiaceae) no Estado do Acre. Revista Brasileira de Biociências, 5(2), 177–179. DOI: http://www.ufrgs.br/seerbio/ojs/index.php/rbb/article/view/261/179
Downloads
Published
How to Cite
Issue
Section
License
Copyright (c) 2022 Karen Almeida da Silva; Ana Lívia do Nascimento Silva; Yara Raphaela Maia dos Santos Gomes; Maria Karoline Sales de Sá; Taiane Nunes Magalhães; Nayara Nágila Neves Alves; Rebeka Alves Ramos; Almeida Andrade Casseb; Júlio Sancho Linhares Teixeira Militão; Elieth Afonso de Mesquita
This work is licensed under a Creative Commons Attribution 4.0 International License.
Authors who publish with this journal agree to the following terms:
1) Authors retain copyright and grant the journal right of first publication with the work simultaneously licensed under a Creative Commons Attribution License that allows others to share the work with an acknowledgement of the work's authorship and initial publication in this journal.
2) Authors are able to enter into separate, additional contractual arrangements for the non-exclusive distribution of the journal's published version of the work (e.g., post it to an institutional repository or publish it in a book), with an acknowledgement of its initial publication in this journal.
3) Authors are permitted and encouraged to post their work online (e.g., in institutional repositories or on their website) prior to and during the submission process, as it can lead to productive exchanges, as well as earlier and greater citation of published work.
