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Journal of Ethnopharmacology Antiinflammatory And Journal of Ethnopharmacology 125 (2009) 102–107 Contents lists available at ScienceDirect Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm Antiinflammatory and antinociceptive activities of Blechnum occidentale L. extract Fabiana Regina Nonato a, Tais Adelita Almeida Barros a, Angélica Maria Lucchese b, Carlos Eduardo Cordeiro Oliveira b, Ricardo Ribeiro dos Santos a,c, Milena Botelho Pereira Soares a,c, Cristiane Flora Villarreal a,d,∗ a Centro de Pesquisas Gonc¸ alo Moniz, Fundac¸ ão Oswaldo Cruz, Rua Waldemar Falcão 121, CEP 40296-710 Salvador, Bahia, Brazil b Laboratório de Química de Produtos Naturais e Bioativos, Departamento de Ciências Exatas, Universidade Estadual de Feira de Santana, Avenida Transnordestina s/n, CEP 44036-900 Feira de Santana, Bahia, Brazil c Hospital São Rafael, Av. São Rafael 2152, CEP 41253-190 Salvador, Bahia, Brazil d Faculdade de Farmácia, Universidade Federal da Bahia, Rua Barão de Geremoabo s/n, CEP 40170-290 Salvador, Bahia, Brazil article info abstract Article history: Aim of study: Blechnum occidentale L. is a terrestrial fern that ranges from the United States to South Amer- Received 5 March 2009 ica, and is employed in Brazilian folk medicine. In the present study we investigated the antinociceptive Received in revised form 29 May 2009 and antiinflammatory activities of the methanolic extract of Blechnum occidentale L. (MEB) in animal mod- Accepted 5 June 2009 els of pain and inflammation to support its medicinal use in treatment of inflammatory and pulmonary Available online 12 June 2009 diseases, urinary infections and liver diseases. Materials and methods: The antinociceptive activity of MEB was evaluated using the writhing, formalin, Keywords: and tail flick tests. The antiinflammatory activity of MEB was evaluated in carrageenan-induced paw Blechnum Pteridophyte oedema and neutrophil migration. In order to discard possible non-specific muscle relaxant or sedative Antinociception effects of MEB, mice motor performance was evaluated in the rota rod test and its toxicity evaluated over Antiinflammatory 14 days. Biological activity Results: Intraperitoneal (IP) administration of MEB (0.01–100 mg/kg) produced a dose-related antinoci- ception on acetic acid-induced writhing in mice. Oral administration of MEB, at a different range of doses (100–400 mg/kg), also produced significant antinociceptive effect on the writhing test. Further- more, treatment with MEB (100 and 200 mg/kg IP) inhibited significantly both the early and late phases of formalin-induced hypernociception in rats. In contrast, treatment with MEB (100 and 200 mg/kg IP) did not prevent the thermal nociception in the tail flick test. The IP administration of MEB (100 and 300 mg/kg) significantly reduced the paw oedema induced by carrageenan. Moreover, systemic treatment with MEB (11–300 mg/kg) reduced the neutrophil migration in the carrageenan-induced migration to the peritoneal cavity. In the rota rod test, MEB-treated mice did not show any significant motor performance alterations with the dose of 300 mg/kg. In addition, over the study duration of 14 days, there were no deaths or toxic signs recorded in the mice given 100 or 1000 mg/kg of MEB. Conclusion: The results described here are the first report of pharmacological studies of Blechnum occi- dentale L. and indicate that this plant has antinociceptive and antiinflammatory activities which support its folk medicine use. © 2009 Elsevier Ireland Ltd. All rights reserved. 1. Introduction 2007). Nonsteroidal antiinflammatory drugs (NSAID) reduce the pain and inflammation by blocking the metabolism of arachi- The clinical treatment of inflammatory diseases is dependent donic acid by cyclooxygenase enzyme (COX), and thereby the on nonsteroidal or steroidal chemical therapeutics (Rainsford, production of prostaglandin (Vane, 1971). Since prostaglandins are cytoprotective, long-term administration of NSAID may induce gastro-intestinal ulcers, bleeding, and renal disorders due to their non-selective inhibition of both isoforms of the COX enzyme, ∗ Corresponding author at: Laboratório de Engenharia Tecidual e Imunofarma- the constitutive (COX-1) and the inducible (COX-2) isoforms cologia, Centro de Pesquisa Gonc¸ alo Moniz, Fundac¸ ão Oswaldo Cruz, Waldemar (Robert, 1976; Peskar, 1977; Tapiero et al., 2002). On the other Falcão 121 Candeal, CEP 40296-710 Salvador, BA, Brazil. Tel.: +55 71 31762260; fax: +55 71 31762327. hand, fully selective and reversible COX-2 inhibitors with reduced E-mail address: [email protected] (C.F. Villarreal). gastro-intestinal toxicity have been associated with adverse car- 0378-8741/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2009.06.005 F.R. Nonato et al. / Journal of Ethnopharmacology 125 (2009) 102–107 103 diovascular effects (Dogné et al., 2005). Furthermore, the use All efforts were made to minimize the number of animals used of steroidal drugs as antiinflammatory agents is also becoming and any discomfort. All behavioral testing was performed between highly controversial due to their multiple side effects (Schäcke et 8:00 a.m. and 4:00 p.m. al., 2002; Reinke et al., 2002). Therefore, developing new agents with more powerful analgesic and antiinflammatory activities 2.4. Nociceptive tests and with lesser side effects is, at present, of great inter- est. In the present study we used the term hypernociception rather Since ancient times, pteridophytes have been considered an than hyperalgesia or allodynia to define the decrease in the noci- excellent source of medicines, as Dioscorides and Galeno stated in ceptive withdrawal threshold, since the pain perception in animals their manuscripts (Barros and Andrade, 1997), and have been used is not obvious. to treat different types of pain (Cambie and Ash, 1994; Christensen, 1997; Gogoi, 2002; Bresciani et al., 2003). Some species of Blechnum, 2.4.1. Writhing test an example of pteridophyte genus, are employed in folk medicine The intraperitoneal and oral antinociceptive doses of MEB were worldwide. Blechnum occidentale L. is a terrestrial and/or rupes- determined in mice using the writhing test. Acetic acid (0.8%, v/v, trial fern that ranges from the United States to South America, 10 ml/kg) was injected into the peritoneal cavities of mice, which including Colombia, Venezuela, Guayanas, Ecuador, Peru, Bolivia, were placed in a large glass cylinder and the intensity of nociceptive Brazil, Paraguay, Chile, and Argentina (Moran, 1995). In Brazil this behavior was quantified by counting the total number of writhes fern has been used to treat inflammatory and pulmonary dis- occurring between 0 and 30 min after the stimulus injection (Collier eases, urinary infections and liver diseases (Barros and Andrade, et al., 1968). The writhing response consists of a contraction of the 1997). However, few works about pteridophyte medicinal prop- abdominal muscle together with a stretching of the hind limbs. The erties have been reported, especially in oriental countries where antinociceptive activity was expressed as the writhing scores over traditional medicine employs many species of herbs and is largely 30 min. used by a large number of people. In fact, to the best of our knowledge, this is the first report of pharmacological studies of 2.4.2. Formalin test Blechnum occidentale L. As part of our continuous interest in Brazil- Rats were placed in an open Plexiglas observation chamber for ian native plants and in order to support the use of this fern in 30 min to accommodate to their surroundings, and then removed folk medicine, the present study was undertaken to establish the for formalin administration. Rats were gently restrained while the antiinflammatory and antinociceptive properties of the methanolic dorsum of the hind paw was subcutaneously administered with extract of Blechnum occidentale L. (MEB). In addition, we evaluated 50 ␮l of formalin 1% (1:100 dilution of stock formalin solution, 37% acute toxicity and motor performance alterations associated with formaldehyde in 0.9% saline) using a 30 gauge needle. Following MEB. injection, the rat was returned to the observation chamber for a 60 min observation period. A mirror was placed behind the cham- 2. Materials and methods ber to enable unhindered observation of the formalin-injected paw. Rats were observed from 0 to 10 min (early phase) and from 15 to 2.1. Plant material 60 min (late phase) and a nociception score was determined for each period by counting the number of flinches of the injected limb Plant specimens were collected in the Atlantic Forest region at during the observation time (Dubuisson and Dennis, 1977). Flinches Salvador, Bahia State, Brazil, in August, 2006, in authorized areas were discrete and easily quantifiable. by IBAMA (Brazilian Institute for the Environment and Natural Resources) and received botanic identification by Dr. Fabiana R. 2.4.3. Tail flick test Nonato. A voucher specimen has been deposited at the Herbarium The tail flick test (Analgesiometer, Insight, Brazil) in rats was of Universidade Estadual de Feira de Santana, Bahia, Brazil (HUEFS conducted as described elsewhere (D’Amour and Smith, 1941), with 142950). minor modifications. Each animal was placed in a ventilated tube with the tail laid across a wire coil which was at room temperature (23 ± 2 ◦C). The coil temperature was then raised by the passage 2.2. Preparation of the methanolic extract of electric current and the latency for the tail withdrawal reflex was measured. The heating was applied to a portion of the ventral The air-dried and powdered blades of Blechnum occidentale surface of the tail 4 cm from the tip. Each trial was terminated after (37.6 g) were exhaustively extracted with methanol. The obtained 6 s to minimize the probability of skin damage. Tail flick latency was extracts were filtered and evaporated under reduced pressure, on measured before and 40 min after the MEB or saline administration. a rotary evaporator at 40–45 ◦C, to yield 5.6 g (15%, w/w) of crude methanolic extract. 2.5. Paw oedema induced by carrageenan 2.3.
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