Dragon's blood Croton palanostigma induces genotoxic effects in mice Edson Luis Maistro a,n , Giulia Ganthous a , Marina da Silva Machado b , Tailyn Zermiani b , Sérgio Faloni de Andrade b , Paulo Cesar Pires Rosa c , Fabio Ferreira Perazzo c a Universidade Estadual Paulista—UNESP, Faculdade de Filosofia e Ciências, Departamento de Fonoaudiologia. Marília, SP 17525-900, Brazil b Núcleo de Investigações Químico-Farmacêuticas (NIQFAR), Universidade do Vale do Itajaí—UNIVALI, Itajaí, Santa Catarina, Brazil c Universidade Federal de São Paulo—UNIFESP, Departamento de Ciências Exatas e da Terra, Diadema, SP, Brazil article info Article history: Received 21 December 2012 Received in revised form 30 January 2013 Accepted 10 March 2013 Keywords: Croton palanostigma Dragon's blood Sangre de grado Euphorbiaceae Comet assay Micronucleus test abstract Ethnopharmacological relevance: Dragon's blood is a dark-red sap produced by species from the genus Croton (Euphorbiaceae), which has been used as a famous traditional medicine since ancient times in many countries, with scarce data about its safe use in humans. In this research, we studied genotoxicity and clastogenicity of Croton palanostigma sap using the comet assay and micronucleus test in cells of mice submitted to acute treatment. Material and methods: HPLC analysis was performed to identify the main components of the sap. The sap was administered by oral gavage at doses of 300 mg/kg, 1000 mg/kg and 2000 mg/kg. For the analysis, the comet assay was performed on the leukocytes and liver cells collected 24 h after treatment, and the micronucleus test (MN) on bone marrow cells. Cytotoxicity was assessed by scoring 200 consecutive polychromatic (PCE) and normochromatic (NCE) erythrocytes (PCE/NCE ratio). Results and conclusion: The alkaloid taspine was the main compound indentified in the crude sap of Croton palanostigma. The results of the genotoxicity assessment show that all sap doses tested produced genotoxic effects in leukocytes and liver cells and also produced clastogenic/aneugenic effects in bone marrow cells of mice at the two higher doses tested. The PCE/NCE ratio indicated no cytotoxicity. The data obtained suggest caution in the use of Croton palanostigma sap by humans considering its risk of carcinogenesis. & 2013 Elsevier Ireland Ltd. All rights reserved. 1. Introduction An assessment of the cytotoxic and genotoxic potential of medicinal plants is necessary because plants in general can synthesize toxic substances to protect themselves against infec- tions, insects and herbivores in nature, but also may often affect the organisms that feed of them. Croton palanostigma is a plant that belongs to the Euphorbia- ceae family. This traditional medicine is derived from a fast growing tree that is known by different names in various coun- tries: in Peru it is called “sangre de grado”, in Ecuador, “sangre de drago”, and in several other countries “dragon's blood”. Species from this genus contain a red viscous sap that can be obtained from making cuts in the bark (Duke and Vasquez, 1994). This bark sap is used for wound healing (Porras-Reyes et al., 1993), anti- microbial (Ubillas, 1994), controlling diarrhea (Gabriel et al., 1999; Holodniy et al., 1999), gastric ulcer healing, as a treatment for intestinal inflammation that are common in the rainforest regions (Miller et al., 2000), as an anti-cancer agent (Sandoval et al., 2002), as an anti-emetic and anti-itch (Miller et al., 2008), among others (see Gupta et al., 2008 for a review). Despite of some reports that several compounds can be found in the dragon's blood sap (Cai et al., 1993a,b), it has been reported that the bark sap contains fundamentally the alkaloid, Taspine (Bettolo and Scarpati, 1979), and the lignin 3′4′-O-dimethylcedru- sin, which has been claimed to be the active principles responsible for the anti-inflammatory and wound healing activities (Perdue et al., 1979; Vaisberg et al., 1989; Pieters et al., 1993). The complete chemical constituents present in dragon's blood from several sources can be found in a review of Gupta et al. (2008). In our continuing efforts to evaluate the genotoxic potential of medicinal plants found in Brazil, and due to the fact that have been no studies on the likely genotoxic effects of the Croton palanos- tigma sap, the present study evaluated the in vivo genotoxicity and clastogenicity/aneugenicity potential of this sap in different cells of mice using the comet and micronucleus assays. 2. Materials and methods 2.1. Chemicals The alkylating agent cyclophosphamide (CPA, CAS No. C 0768, Sigma) was used as the DNA damaging agent in comet and micronucleus assays. The other main chemicals were obtained Contents lists available at SciVerse ScienceDirect journal homepage: www.elsevier.com/locate/jep Journal of Ethnopharmacology 0378-8741/$ - see front matter & 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jep.2013.03.026 n Corresponding author. Tel.: þ551434021324; fax: þ551434021302. E-mail address: edson.maistro@marilia.unesp.br (E.L. Maistro). Please cite this article as: Maistro, E.L., et al., (2013), http://dx.doi.org/10.1016/j.jep.2013.03.026i Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎