Kinetic studies of the spasmogenic effects of serotonin and isolates from Byrsonima crassifolia leaves on rat fundus

Leaf and bark extracts of a Mexican medicinal plant, Byrsonima crassifolia (Malpighiaceae), exhibited spasmogenic effects on isolated rat fundus and biphasic effects on jejunum and ileum. Preliminary evaluations using rat fundus in Krebs solution indicated that the activity of a 2% acetic acid extract of eaves (HOAcE) could be split into two types: (i) high-efficacy, low-potency, n-butanol-extracted, pargyline- and 1-(1-naphtylpiperazine) (1-NP)-sensitive, atropine-insensitive activity, and (ii) low-efficacy, high-potency, ethyl acetate-extracted, pargyline-insensitive, atropine- and 1-NP-sensitive activity. HOAcE lacked muscarinic and nicotinic effects on rat jejunum and frog rectus abdominis. Serotonin (5-HT) and HOAcE curves in fundus were parallel and 5-HT potency was 6,037 times that of HOAcE (95% confidence limits: 4,624-7,852). The pD2 (affinity constant) for 5-HT was 7.96 (7.92-8.00) with pargyline added to the medium. 5-HT receptor-interaction kinetics using cholinergics and 5-HT agonists and antagonists was carried out. 1-NP competitively antagonized 5-HT. The 5-HT, antagonist s-(-)propranolol did not significantly antagonize 5-HT. The 5-HT2 blocker ketanserin noncompetitively antagonized 5-HT and α-Me-5-HT (pD\u272 = 5.6 and 6.7, respectively). The 5-HT3 antagonist MDL-72222 inactivated only a small proportion of receptors (pD\u272 = 6.46). Atropine did not significantly modify the curve of 5-HT while fluoxetine noncompetitively antagonized 5-HT (pD\u272 = 5.8). 5-HT and α-Me-5-HT curves were biphasic indicating two receptor interactions (high and low affinity). High-affinity pD2 values for six different 5-HT agonists and 1-NP on rat fundus correlate well with reported rat brain (radioligand binding) pKd values at the 5-HT1C receptor (r = 0.94). Large scale extraction and fractionation of a methanol extract of leaves yielded two peaks of activity (Peak 1, lipophilic; Peak 2, polar). Peak 1 contained Compounds 1 to 7 (C1-C7); Peak 2 included C8-C15. Compound 1, C2, C3, C4, C10 and C11 were inactive while C8, C12 and C13 showed equivocal effects. Compound 5, C6, C7, C9, C14, C15, quercetin and gallic acid were active. Potencies were: C5 \u3e C6 \u3e C7 = quercetin \u3e C9 \u3e gallic acid. Efficacy (IA) was: C15 ≥ C14 \u3e gallic acid \u3e C9 \u3e C5 \u3e C7 \u3e quercetin \u3e C6. Compound 9 and its aglycone quercetin were partial agonists (C9 IA = 70%, pD2 = 6.35; quercetin IA = 60%, pD2 = 6.58). Compound 9 noncompetitively antagonized 5-HT (pD\u272 = 6.10), while quercetin did not. Compound 14 and C15, the most active compounds, had similar response curves but these curves were not parallel to 5-HT. Spasmogenic ED50 values for C14 = 0.76 (0.38-1.54) μg/mL and C15 = 0.76 (0.41-1.42) μg/mL. Gram for gram 5-HT was 181 x C14 and 182 x C15