Inhibition of the Lethal and Myotoxic Activities of Crotalus durissus terrificus Venom by Tabernaemontana catharinensis: Identification of One of the Active Components

Abstract

In Brazilian folk medicine, victims of bites by poisonous animals are usually treated with plant extracts derived from the diverse national flora. The chemical and pharmacological properties of most extracts were yet not investigated. In the rural community of Assis-SP, the root bark of Tabernaemontana catharinensis (“leiteiro”, “cow milk”) is applied to the site of the snake bite and believed to neutralize the effect of the venom. We report here the ability of the lyophilized aqueous extract (AE) and of a pure compound obtained from the ethanolic extract of T. catharinensis to inhibit the lethal and myotoxic activities of C. d. terrificus (South American rattlesnake) venom. Doses of 10 mg AE/100 g, injected (i.m., rat) 20 s after injecting (i.m.) the venom and that of 2.5 mg AE/100 g, incubated for 1 h at 25 °C with the venom before injection (i.m.) were able to neutralize the lethal activity of 2LD₅₀. These data indicate that T. catharinensis could be used as a source of a model molecule able to neutralize the lethality and myotoxicity induced by C. d. terrificus venom. Its ethanolic extract was then fractionated on a silica gel 60 chromatography column affording fractions A to F. Fraction A consisted basically of non-polar compounds, terpenes and sterols. Fraction D showed a pronounced antiophidian activity which was later correlated with the presence of the quaternary alkaloid 12-methoxy-4-methylvoachalotine in this fraction. This alkaloid was isolated and inhibited 100 % lethality when injected 20 s after 2 LD₅₀ at 1.7 mg/100 g.

Key words

Tabernaemontana catharinensis - Apocynaceae - Crotalus durissus terrificus venom - antiophidian activity - myotoxicity - alkaloid

References

• 1 Nakagawa  M,, Nakanishi  K.. Structures of cabenegrins A-I and A-II, potent anti-snake venoms.  Tetrahedron Lett.. 1982;;  23 3855-8
• 2 Rizzini  C T,, Mors  W B,, Pereira  N A.. Plantas Brasileiras tidas como Ativas Contra Peçonhas Animais, Especialmente Venenos de Cobras.  Rev. Bras. Farm.. 1988;;  69 82-6
• 3 Nielsen  H B,, Hazell  A,, Hazell  R,, Ghia  F,, Torssell  K BG.. Indole alkaloids and terpenoids from Tabernaemontana markgrafiana.  Phytochemistry. 1994;;  37 1729-35
• 4 Achenbach  H,, Waibel  R,, Zwanzger  M.. Indole alkaloids from Tabernaemontana glandulosa.   Phytochemistry. 1994;;  37 1737-43
• 5 Van Beek  T A,, Verpoorte  R,, Svendsen  A B,, Leuwenberg  A JM,, Besset  N G.. Tabernaemontana L. (Apocynaceae): A review of its taxonomy, phytochemistry, ethnobotany and pharmacology.  J. Ethnopharmacol.. 1984;;  10 1-156
• 6 Wagner  H,, Bladt  S,, Zgainki  E M.. Plant Drug Analysis. New York,; Springer-Verlag 1984: 300
• 7 Litchfield  J T,, Wilcoxon  FA.. Simplified method of evaluating dose-effect experiments.  J. Pharm. Exp. Ther.. 1949;;  96 99-113
• 8 Itzhaki  R F,, Gill  D M.. A micro-biuret method for estimating proteins.   Anal. Biochem.. 1964;;  9 401-10
• 9 Mors  W B,, Nascimento  M C,, Parente  J P,, Silva  M H,, Melo  P A,, Suarez-Kurtz  G.. Neutralization of lethal and myotoxic activities of South American rattlesnake venom by extracts and constituents of plant Eclipta prostata (Asteraceae).  Toxicon. 1989;;  27 1003-9
• 10 Siegel  S.. Nonparametric Statistics for the Behavioral Sciences. International Student Edition. Japan:; MacGraw-Hill Kogakusha 1959: 312 - 5
• 11 Pereira  P S,, Sampaio  S V,, França  S C,, Dias  D A.. Indole alkaloids from Tabernaemontana catharinensis A. DC.  Acta Horticulturae. 1999;;  501 171-6
• 12 Braga  R M,, Reis  A M.. Quaternary alkaloids from Peschieira fuchsiaefolia.  Phytochemistry. 1987;;  26 833-6
• 13 Kochva  E.. The origin of snakes and evolution of the venom apparatus.  Toxicon. 1987;;  25 65-106
• 14 Hawgood  B J,, Smith  J W.. The mode of action at the mouse neuromuscular junction of the phospholipase A-crotapotin complex-isolated from the South American rattlesnake.  Br. J. Pharmacol.. 1977;;  61 597-606
• 15 Giglio  J R.. Analytical studies on crotamine hydrochloride.  Anal. Chem.. 1975;;  69 207-21
• 16 Vital-Brazil  O,, Prado-Franceschi  J,, Laure  C J.. Repetitive muscle response induced by crotamine.  Toxicon. 1979;;  17 61-7
• 17 Batina  M  de FC,, Giglio  J R,, Sampaio  S V.. Methodological care in the evaluation of the LD₅₀ and neutralization of the lethal effect of Crotalus durissus terrificus venom by plant Peschieira fuschiaefolia (Apocynaceae).  J. Venom. An. Tox.. 1997;;  3 23-31
• 18 Melo  P A,, Nascimento  M C,, Mors  W B,, Suarez-Kurtz  G.. Inhibition of the myotoxic and hemorragic activities of crotalid venoms by Eclipta prostrata (Asteraceae) extracts and constituents.  Toxicon. 1994;;  32 595-603