A new local anesthetic isolated from Bufoparacnemis venom (Amphibia, Anura)

Patrocínio, M.C.A., Cortez, J.J.C., Almeida, M.M., Evangelista, I.L., Oliveira, R.G.S., Cardi, B.A., Carvalho, K.M.

Laboratório de Neurofarmacologia, Universidade Estadual do Ceará, Fortaleza, CE, Brazil.

Objectives: The anuran skin secretions contain several pharmacologically active compounds, such as biogenic amines, toxins, peptides, enzymes and alkaloids, protecting anurans from predators and microorganisms. In this study, we demonstrated the isolation, the local anesthetic effects and the structure of a bufodienolide from Bufo paracnemis skin secretions.

Methods and Results:The venom was dissolved in ethanol (1:3, w,v), centrifuged at 5000g/ 20 min and the supernatant was submitted to HPLC using a C₁₈ column (25 x 250 mm)(5 ml/min) eluted with  acetonitrile(0-40%)(30 min). The purified fraction (Bpa) with local anesthetic activity was pooled and lyophilized. High resolution NMR analysis of the purified factor by application of modern pulse sequences such as 1H, 1H-COSY and NOESY, carbon-detected and hydrogen-detected (inverse) hetero-nuclear correlation of directly attached carbon-hydrogen (HETCOR and HMQC, respectively), as well as the long-range equivalent sequences, COLOC and HMBC, allowed its identification as a bufodienolide-type steroid, known in the literature as telecinobufagin. Local anesthetic activity was assayed by infiltration anesthesia in guinea pigs (n=6), mouse tail flick test (n=6) and cornea test in rabbits (n=6). The anesthetic time of the drugs used were: a) infiltration anesthesia: Bpa (0.5%), 181±15 min, bupivacaine(0.5%), 110±10 min, and lidocaine (2%), 49±5 min, b) mouse tail flick test: Bpa (0.5%), 152±12 min, bupivacaine (0.5%), 52±8 min, and lidocaine (2%), 18.4±4 min, c) cornea test: Bpa (0.5%), 49.4±6 min, bupivacaine (0.5%),  46.8±5 min, and lidocaine (2%), 11.5±3min.

Conclusions: A bufodienolide isolated from Bufo paracnemis skin secretions, known as telecinobufagin, induced a strong local anesthesia, more sustained (p<0.05) than those caused by lidocaine and bupivacaine.

CORRESPONDENCE TO:

Krishnamurtide Morais Carvalho, Laboratório de Neurofarmacologia, Universidade Estadual do Ceará, Fortaleza, CE, Brazil. CEP: 60.000-000, Email: carvalhokris@hotmail.com