Anti-inflammatory properties of Nauclea latifolia extracts and isolation of strictosamide by a high-content screening approach

Abstract

Crude extracts of Nauclea latifolia (Rubiaceae), commonly known as pin cushion tree, are employed in traditional or folk medicine and are used in the treatment of several diseases, such as malaria, gastrointestinal tract disorders, and hypertension. We were able to demonstrate that treatment with the root bark ethanolic extract of N. latifolia inhibited the formation of edema in a rat inflammation model. Carragenan-induced inflammation was inhibited by 62% after 1h treatment (i.p.) with the crude extract at 1 g/kg. In addition, administration (i.v.) of the ethanolic extract decreased the blood pressure in rats that were treated with arachidonic acid. Following bioactivity-guided screening we aimed at characterizing single plant metabolites that are responsible for the activity observed with crude extracts. In a high-content screening approach using the stable CHO/NFκBp65-GFP cell line we found that the crude extract of N. latifolia (4h treatment, 20 µg/ml) completely inhibited the IL-1β induced translocation of the transcription factor NFκB from cytoplasm to nuclei. Further, we found this effect to be induced by certain HPLC fractions of the chloroform extract of N. latifolia. We were then able to identify strictosamide as the active ingredient and could validate our results in reporter gene assays. HEK293T/17 cells were transiently transfected with a reporter construct that contains five copies of an NFκB response element that drives transcription of a luciferase transporter gene. At a concentration of 50 µg/ml strictosamide inhibited the IL-1β induced transcription by approximately 70%. Previously, it was reported that strictosamide may account for the folk use of plant extracts on hypertension. However, the compounds mechanism-of-action remains to be elucidated. We assume that strictosamide, at least to a certain extent, accounts for the anti-inflammatory properties of the crude N. latifolia extracts observed in rat models.