Tetradenia (T.) riparia (Hochst.) Codd (Lamiaceae), formerly known as Iboza riparia (Hochst.) N.E.Br., is one of the most frequently used medicinal plants in traditional Rwandese medicine. It was used as a remedy against a wide range of diseases including malaria, angina, yaws, dental abscesses, headache, worm infections and several kinds of fevers and aches.
However, the active compounds responsible for anthelmintic activity in Tetradenia riparia have not been identified so far. Recently, we found robust antihelmintic activity in the leaves of T. riparia against Caenorrhabitis elegans (C. elegans). In this study, a bioassay-guided fractionation using a C. elegans testing model led to the isolation of the active compound.
Materials And Methods:
The anthelmintic assay was carried out in a 96-well microplate (flat-bottom, TPP Techno Plastic Products AG, Switzerland). Freshly cultivated young adults were collected in M9 buffer and adjusted approximately to 3000 larvae/mL. Then, 15 μL of this suspension (containing about 45 larvae) was added to each well of a 96-well microplate containing 184 μL of E. coli OP50 culture (O.D. = 0.5). Subsequently, 1 μL of plant extract, fraction or pure compound stock soution was added to each well; the starting concentration in the microplate was 1000 μg/mL for total extracts and 100 μg/mL for fractions, and DMSO (1 μL) was used in a separate well as a solvent control. After proper mixing, the 96-well microplate was placed into a WMicroTracker (Phylumtech, Argentina) apparatus and incubated for 16 hours at 20 °C. The movement of worms in each well was measured every 30 minutes and recorded by the WMicroTracker. The percentage of the average movement over 16 hours (except for the first hour) of test samples with extract, compared to the DMSO control, was used to estimate the relative anthelmintic activity. Levamisole (final concentration 50 μM) was systematically used as a positive control. Data from dose-response experiments were represented as the percentage of inhibition, and analyzed with GraphPad Prism 6 software (San Diego, CA). A log (inhibitor) vs. response non-linear fit was used to estimate the IC50.
The bioassay-guided fractionation showed that only the hexane extract was active, which led after liquid/liquid extraction of the hexane fraction (hexane and dichloromethane) and column chromatography of the dichloromethane fraction over silica gel to the isolation of the anthelmintic active principle, i.e. 8(14),15-sandaracopimaradiene-7α,18-diol (see Figure 1) from the leaves of Tetradenia riparia. The anthelminthic activity of this diterpenediol explains the use of T. riparia against parasitic worms in Rwandese traditional medicine. Its IC50 value was 5.4 ± 0.9 μg/mL (17.8 ± 2.9 μM) in our C. elegans assay. Activity against C. elegans of an ethylacetate extract of Tetradenia riparia has been described before (Okem et al., 2012), with a minimum lethal concentration of 0.004 mg/mL, but the bioactive compounds were not identified. Remarkably, the potency of this extract is comparable to that of our purified compound, suggesting that the reported extract mainly contained the compound that we identified, or that its potency was boosted by the presence of synergistic compounds.
J Ethnopharmacol. 2018 Jan 31;216:229-232. doi: 10.1016/j.jep.2018.01.024.
Van Puyvelde L, Liu M, Veryser C, De Borggraeve WM, Mungarulire J, Mukazayire MJ, Luyten W.