Isolation of opioid-active compounds from Tabernaemontana pachysiphon leaves

A procedure for prefractionation of crude plant extracts by centrifugal par tition chromatography (CPC) has been developed to enable rapid identificati on of known-positive compounds or false-positive compounds and to increase the chance of identifying minor unknown-active compounds. The study explore d the use of CPC as a tool in the prefractionation step before investigatio n of bioactivity. Fractions obtained by CPC from an ethanolic extract of Tabernaemontana pach ysiphon Stapf (Apocynaceae) were screened by means of an opiate-receptor-bi nding assay and an adenosine Al-receptor-binding assay. Fractions containin g fatty acids, which had false-positive effects on the assay, were identifi ed, as were unknown-positive fractions from which two opioid-active compoun ds, tubotaiwine and apparicine, were subsequently isolated. The affinities (K-i) of tubotaiwine and apparicine at the opiate receptor were 1.65 +/- 0. 81 and 2.65 +/- 1.56 mu mol, respectively. Both alkaloids had analgesic act ivity in the abdominal constriction test in mice. CPC prefractionation led to the rapid isolation of two opioid-active compou nds, tubotaiwine and apparicine, from the unknown-positive fraction; false- positive fractions were rapidly identified. Both tubotaiwine and apparicine had affinity for adenosine receptors in the micromolar range and also had in-vivo analgesic activity in mice.