harmacologyOnLine April 30, 2013 Archives • 2013 • vol.1 • 113 - 127 Preliminary evaluation of the acute toxicity related to Abarema auriculata to mice and investigation of cytotoxicity of isolated flavonones Daniela Fernandes Gusmão1, Dirce Mimoto Estork1, Mateus Luís Barradas Paciencia2, Ingrit Elida Collantes Díaz2, Sergio Alexandre Frana2, Paula Andreotti Rodrigues1, Ivana Barbosa Suffredini1,2,*, Antonio Drauzio Varella2, Riad Naim Younes2,3, Luiz Fernando Lima Reis3, Edna Frasson de Souza Montero4, Maria Martha Bernardi1 1 Graduate Program in Veterinary, Graduate and Research Vice-Dean Office, Paulista University, São Paulo, Brazil 2 Center for Research in Biodiversity, Botany Laboratory and Herbarium UNIP and Extraction Laboratory, Paulista University, São Paulo, Brazil 3Education and Research Center, Sírio Libanês Hospital, São Paulo, SP, Brazil 4Medicine College, São Paulo University, LIM-62, SP, São Paulo, SP, Brazil. *Prof. Dr. Ivana Barbosa Suffredini. Núcleo de Pesquisas em Biodiversidade, Laboratório de Extração, Universidade Paulista, Av. Paulista, 900, 1º andar - Bela Vista - São Paulo – SP. CEP 01310-100, São Paulo, Brazil E-mail: ibsuff[email protected] - Phone: +55 11 3170-3776 Fax: +55 11 3170-3978 Summary Abarema auriculata crude extract EB689 showed cytotoxic activity against prostate, central nervous system and head-and-neck cancers, and may be useful in veterinary and human care in the future. For the first time, the presence of steroids and phenolic compounds were related to A. auriculata. Spinasterol, a bioactive steroid, was isolated from the non polar fractions of EB689, and the phenolic compounds neoastil- bin, astilbin, isoastilbin, neoisoastilbin and engeletin were isolated from polar fractions of EB689. Preliminary toxicological results showed that EB689 exhibited a non-lethal dose of 4.90 mg/kg and a LD50 of 15.0 mg/kg. Higher doses of EB689 IP administered influenced general activity of male mice, provoking significant altera- tions in sensory system functions, including corneal reflex and tail squeeze and touch responses. Effects were also observed in psychomotor system function, particularly in hindquarter fall, surface-righting reflex, grasp reflex and body tone and in the autonomic nervous system, where piloerection, defecation, hypother- mia, cyanosis and breathing were influenced. Significant alterations were observed in general activity, cyano- sis and breath after the administration of the non-lethal dose (4.90 mg/kg), corroborating the initial observa- tions. Diminishment of general activity and breathing and appearance of cyanosis are possibly related to intestine hemorrhage observed in necropsy, which led to animal death. The occurrence of the isolated compounds in EB 689 is unlikely related to animal death and it is the first time they are isolated from Abarema auriculata. Key words: Toxicity, Abarema auriculata (Fabaceae), steroid, flavanones, Amazon plant extract http://pharmacologyonline.silae.it ISSN: 1827-8620 PhOL 114 (113 - 127) Introduction of animals (8). In addition, testosterone concentra- tion in serum was evaluated in order to overview Plants have played a significant role in the treat- the possible alterations caused by the administra- ment of cancer and infectious diseases for the last tion of EB689 to male mice. four decades (1,2). Natural products have been rediscovered as important tools for drug develop- The present study aims to evaluate the general ment, as they play an important role in combinato- activity and acute toxicity after the administration rial chemistry approaches based on natural pro- of different doses of the extract EB689 obtained ducts scaffolds that may resemble drug-like com- from A. auriculata species, assessing the general pounds. signs of toxicity, including the influence on sex hormone levels in an animal model, and to isolate The Brazilian flora, the most diverse in the world, and identify compounds from the active extract. has become an interesting source for the prospec- ting of new chemical leads due to its species diver- sity and associated chemical richness. Screening Methods programs have been established in Brazil (3,4) as a strategy to identify potentially active substances Plant collection and extract preparation (5). The extract obtained from the stem of Abarema Plant material was collected in the Brazilian auriculata (Benth.) Barneby & J.W.Grimes (designa- Amazon rain forest, under Brazilian Government ted as EB689), a plant found in the Amazon rain licenses (license CGen/MMA#12A/2008) for collec- forest, has shown significant antitumor activity in in ting and bioprospecting genetic resources in protec- vitro assays against the PC3 prostate cancer cell line ted areas of Brazilian forests. The collection was showing growth inhibition of 32.66 % of an untrea- made in the surrounds of Manaus city, state of ted control, and against SF268 central nervous Amazonas, in a seasonally flooded forest from Rio system cancer cell line, showing growth inhibition Negro Basin (Igapó forest). The voucher is deposi- of 24.69 % (6). EB 689 has also been cytotoxic to ted at UNIP Herbarium [A.A.Oliveira, 3353 (UNIP)] squamous cell carcinoma, in vitro (7)[Ozi et al., and was identified by Mateus L. B. Paciencia, 2011]. Although in vitro results showed significant curator from Herbarium UNIP. The stem of A. activity against tumor cell lines, further studies are auriculata was dried in an air circulating oven needed to assess pharmacological, toxicological (Fanem) at 40°C and was ground in a hammer-mill and chemical properties of A. auriculata. (Holmes). Next, 545 g of the dried plant ground Before pharmacological assays can be developed, material was placed in a glass percolator (Kontes) a wide array of the general signs of toxicity was and macerated for 24h with 1.80 L of dichlorome- assessed in the present study. The establishment of thane and methanol (1:1) (5)[Younes et al., 2007] LD50 values is not well accepted today due to the (Merck), Solvents were evaporated under vacuum misuse of a large number of animals frequently (Büchii) resulting in 26.56 g of crude extract, which submitted to severe assays, and for that reason, was maintained in a freezer (Revco) until use. analysis of toxicology is being conducted according to recent guidelines (8). Preparation of extract for administration to mice These guidelines describe methods that have been developed to minimize animal use and animal Extract EB689 was suspended in almond oil and suffering and were introduced as a tool to assess the following doses were administered IP: 5,000, the toxicological index of an unknown compound 2,500, 1,250, 625, 312.5, 156.3, 78.1, 39.1, 19.5, 9.7, 4.9 (9). In order to evaluate toxicity after the admini- mg/kg. Almond oil was used in the extract formula- stration of plant extracts, a previously described tion because of its non polar origin and non-toxic method (10,11) was performed using a small number profile, and for that reason, it is compatible as a http://pharmacologyonline.silae.it ISSN: 1827-8620 PhOL 115 (113 - 127) vehicle for the administration of drugs to mammals. tremor, irritability, auricular reflex, corneal reflex, To avoid alterations of extract composition, the tail squeeze, response to touch), psychomotor extract suspensions were not sterilized or filtered. system (contortion, hindquarter fall, surface - The intraperitoneal route was chosen due to the righting reflex, body tone and grasp reflex), central absence of bioavailability loss. nervous system (convulsions, ataxia, anesthesia, hypnosis, straub tail, tremor, stimulation and sedation) and to autonomic nervous system (lacri- Animals mation, breathing, ptosis, piloerection, micturition, Male Balb-C mice (Mus musculus) weighing 25-30 defecation, hypothermia and cyanosis) were asses- g, 6 to 9 weeks old, were obtained from São Paulo sed and a score from 0 to 4 was given for each University Animals Facilities After arrival in the parameter, considering that 0 is the absence of the laboratory, animals were randomly selected, indivi- effect and 4 is the complete manifestation of the dually marked, and housed in groups of five in parameter, except to micturition and defecation, isolated units (polypropylene cages 38 x 32 x 16 cm) which numbers of urination and boli were recorded, with controlled temperature (22 ± 2ºC) and humidity respectively. Body weight for each animal was (65–70%). Artificial lighting was provided (12 h measured immediately before administration of light/12 h dark cycle, lights on at 8:00 a.m.), as well extract and at least twice during the observation as free access to Nuvilab® rodent chow (Nuvital period. Company, São Paulo, Brazil) and an unlimited supply of filtered water. Toxicology Experimental design The experiments began one week after the mice A two-stage experiment was designed in order to arrived, allowing for adaptation to the new labora- probe the lethality, general activity, and the toxicity tory environment and conditions. Animals were of EB689. In the first stage, the LD50 was determi- fasted for a period of one hour before receiving ned utilizing three mice per dose and observations treatments. Animals were observed for toxic of general activity and toxicity were assessed. The responses, and if lethality occurred during a period second stage of the experiment was conducted of observation, a necropsy was performed. On the using the non-lethal dose against groups of ten other hand, if animals survived until the end of the animals each, in which the accuracy