Extract and Active Principal of the Neotropical Vine Souroubea sympetala Gilg. Block Fear Memory Reconsolidation

Abstract

Background: Souroubea sympetala Gilg. is a neotropical vine native to Central America, investigated as part of a targeted study of the plant family Marcgraviaceae. Our previous research showed that extract of S. sympetala leaf and small branch extract had anxiolytic effects in animals and acts as an agonist for the GABA_A receptor at the benzodiazepine binding site. To date, the potential effects of S. sympetala and its constituents on reconsolidation have not been assessed. Reconsolidation, the process by which formed memories are rendered labile and susceptible to change, may offer a window of opportunity for pharmacological manipulation of learned fear. Here, we assessed the effects of S. sympetala crude extract and isolated phytochemicals (orally administered) on the reconsolidation of conditioned fear. In addition, we explored whether betulin (BE), a closely related molecule to betulinic acid (BA, an active principal component of S. sympetala), has effects on reconsolidation of learned fear and whether BE may synergize with BA to enhance attenuation of learned fear. Method: Male Sprague-Dawley rats received six 1.0-mA continuous foot shocks (contextual training). Twenty-four hours later, rats were re-exposed to the context (but in the absence of foot shocks). Immediately following memory retrieval (recall), rats received oral administration of S. sympetala extract at various doses (8-75 mg/kg) or diazepam (1 mg/kg). In separate experiments, we compared the effects of BA (2 mg/kg), BE (2 mg/kg), and BA + BE (2 mg/kg BA + 2 mg/kg BE). The freezing response was assessed either 24 h later (day 3) or 5 days later (day 7). Effects of phytochemicals on fear expression were also explored using the elevated plus maze paradigm. Results: S. sympetala leaf extract significantly attenuated the reconsolidation of contextual fear at the 25- and 75-mg/kg doses, but not at the 8-mg/kg dose. Furthermore, BA + BE, but not BA or BE alone, attenuated the reconsolidation of learned fear and exerted an anxiolytic-like effect on fear expression.

Keywords: Sin Susto; Souroubea sympetala; betulin; betulinic acid; fear memory; reconsolidation.

Figure 1

Chemical structure of (1) betulinic acid and (2) betulin.

Figure 2

S. sympetala extracts attenuated the freezing response. 25 mg/kg SIN (approximate BA content 0.37 μmol/kg) and 75 mg/kg SIN (approximate BA content 1.12 μmol/kg), but not 8 mg/kg SIN (approximate BA content 0.12 μmol/kg), attenuated freezing on day 3 when administered immediately post-recall. * indicates p < 0.05.

Figure 3

S. sympetala extract administered immediately following recall did not attenuate freezing on day 7.

Figure 4

Isolated phytochemicals betulinic Acid (BA) 2 mg/kg (4.38 μmol/kg) and betulin (BE) 2 mg/kg (4.52 μmol/kg) attenuated reconsolidation of conditioned fear when co-administered, but not alone. The combined dose of BA + BE more closely resembles S. sympetala extract. * indicates p < 0.05.

Figure 5

Isolated phytochemicals 2 mg/kg (4.52 μmol/kg) BE and 2 mg/kg (4.38 μmol/kg) BA did not attenuate freezing on day 7.

Figure 6

SIN 75 mg/kg (approximate BA content 1.12 μmol/kg) and a combined dose of 2 mg/kg (4.52 μmol/kg) BE + 2mg/kg (4.38 μmol/kg) BA did not produce attenuation of the learned fear response when administered in the absence of re-exposure to the fearful stimulus.

Figure 7

Rodents treated with 2 mg/kg (4.52 μmol/kg) BE + 2 mg/kg (4.38 μmol/kg) BA, as well as 1 mg/kg (3.51 μmol/kg) diazepam, exhibited significantly more time in the open arms of the maze than rodents treated with vehicle. * indicates p < 0.05.

Figure 8

Rodents treated with 2 mg/kg (4.52 μmol/kg) BE + 2 mg/kg (4.38 μmol/kg) BA, as well as 1 mg/kg (3.51 μmol/kg) diazepam, exhibited significantly more unprotected head dips than rodents treated with vehicle. * indicates p < 0.05.