Kaurenoic Acid from Sphagneticola trilobata Inhibits Inflammatory Pain: Effect on Cytokine Production and Activation of the NO-Cyclic GMP-Protein Kinase G-ATP-Sensitive Potassium Channel Signaling Pathway Sandra S. Mizokami, † Nilton S. Arakawa, ‡ Sergio R. Ambrosio, § Ana C. Zarpelon, † Rubia Casagrande, ‡ Thiago M. Cunha, ⊥ Sergio H. Ferreira, ⊥ Fernando Q. Cunha, ⊥ and Waldiceu A. Verri, Jr.* ,† † Departamento de Ciê ncias Patoló gicas-Centro de Ciê ncias Bioló gicas, Universidade Estadual de Londrina, 86051990 Londrina, Brazil ‡ Departamento de Ciê ncias Farmacê uticas-Centro de Ciê ncias de Saú de, Universidade Estadual de Londrina, 86039440 Londrina, Brazil § Nú cleo de Pesquisa em Ciê ncias Exatas e Tecnoló gicas, Universidade de Franca, Franca 14404600, Brazil ⊥ Department of Pharmacology, Faculty of Medicine of Ribeirã o Preto, University of Sã o Paulo, Ribeirã o Preto 14049900, Brazil ABSTRACT: Kaurenoic acid [ent-kaur-16-en-19-oic acid (1)] is a diterpene present in several plants including Sphagneticola trilobata. The only documented evidence for its antinociceptive effect is that it inhibits the writhing response induced by acetic acid in mice. Therefore, the analgesic effect of 1 in different models of pain and its mechanisms in mice were investigated further. Intraperitoneal and oral treatment with 1 dose- dependently inhibited inflammatory nociception induced by acetic acid. Oral treatment with 1 also inhibited overt nociception-like behavior induced by phenyl-p-benzoquinone, complete Freund’s adjuvant (CFA), and both phases of the formalin test. Compound 1 also inhibited acute carrageenin- and PGE 2 -induced and chronic CFA-induced inflammatory mechan- ical hyperalgesia. Mechanistically, 1 inhibited the production of the hyperalgesic cytokines TNF-α and IL-1β. Furthermore, the analgesic effect of 1 was inhibited by L-NAME, ODQ, KT5823, and glybenclamide treatment, demonstrating that such activity also depends on activation of the NO-cyclic GMP-protein kinase G-ATP-sensitive potassium channel signaling pathway, respectively. These results demonstrate that 1 exhibits an analgesic effect in a consistent manner and that its mechanisms involve the inhibition of cytokine production and activation of the NO-cyclic GMP-protein kinase G-ATP-sensitive potassium channel signaling pathway. K aurenoic acid [ent-kaur-16-en-19-oic acid (1)] is a diterpene obtained from a number of plants and is a major compound in Sphagneticola trilobata (L.) Pruski (syn. Wedelia paludosa, Acmella brasiliensis; Asteraceae), 1 which is known popularly in Brazil as “arnica-do-mato”, “pseudo-arnica”, “picã o-da-praia”, and “vede ́ lia”. There is evidence that 1 exhibits its biological effects by inhibiting the inflammatory process such as in the carrageenin-induced paw edema 2,3 and TPA-induced ear edema models. 4 Furthermore, in a model of asthma in guinea pigs, 1 inhibited ovalbumin challenge-induced airway resistance in immunized animals as well as the production of histamine and activity of phospholipase A 2 . 5 In vitro experi- ments also demonstrated that 1 inhibits LPS-induced production of nitric oxide 2,3 and prostaglandin E 2 (PGE 2 ) as well as the expression of cyclooxygenase-2 and inducible NO synthase (iNOS) in RAW 264.7 macrophages. 3 This inhibition of cyclooxygenase-2 and iNOS expression is probably related to the inhibition of NFκB activation. 3 On the other hand, there is also evidence that 1 does not inhibit LPS-induced NFκB activation, nitrite production, or mRNA expression of pro- inflammatory cytokines such as TNF-α and IL-1β, and cyclooxygenase-2. 6 Thus, the possible effects of 1 on these inflammatory pathways is still controversial and has been addressed only in vitro. Additional relevant biological effects of 1 are the vasorelaxant effect via NO/cyclic guanosine monophosphate (cGMP)/ protein kinase G (PKG)/ATP-sensitive potassium and calcium channels, depending on the experimental model, 7 and antimicrobial effects against Bacillus cereus and Mycobacterium tuberculosis. 8,9 Received: December 21, 2011 Published: May 10, 2012 Article pubs.acs.org/jnp © 2012 American Chemical Society and American Society of Pharmacognosy 896 dx.doi.org/10.1021/np200989t | J. Nat. Prod. 2012, 75, 896-904