Phytochemical profiling followed by antimicrobial and anthelmintic activity evaluation of the Australian plant Geijera parviflora, known for its customary use in Indigenous Australian ceremonies and bush medicine, was performed. In the present study, seven previously reported compounds were isolated. Subsequently, a subset of these compounds as well as crude extracts from the plant were evaluated for their antimicrobial and anthelmintic activities. Anthelmintic activity assays showed that two of the isolated compounds, auraptene and flindersine, as well as the dichloromethane and methanol crude extracts of G. parviflora, displayed significant activity against a parasitic nematode (Haemonchus contortus). This is the first report of the anthelmintic activity associated with these compounds and indicates the importance of such fundamental explorations for the discovery of bioactive phytochemicals for therapeutic application(s).
Methods
Anthelmintic Activity Assessment
The G. parviflora dichloromethane(DCM) and methanol (MeOH) crude extracts and the purified compounds 1, 2, 3, 5, and 9 were evaluated for activity against exsheathed third-stage larvae (xL3s) of the H. contortus (Haecon-5 strain) nematode worm, to evaluate their effects on larval motility and/or development using an established protocol. The assessment of anthelmintic activity was carried out in a screening (extracts) and a dose-response assay (compounds) using for exsheathed third-stage larvae (xL3s) of Haemonchus contortus (Haecon-5 strain).
G. parviflora extracts were prepared at a concentration of 1 mg/mL (in 50 μL of LB supplemented*; final assay concentration of 0.5 mg/mL) and compounds were prepared in two-fold serial dilution, starting at a concentration of 100 μM (18-points; in 50 μL of LB*; final assay concentrations of 50 μM to 0.76 nM), in 96-well plates with larvae dispensed in 50 μL at a density of 300. LB*+ 0.5% DMSO serving as negative control and two commercial anthelmintic compounds, monepantel and moxidectin, were prepared as positive controls and applied to the 96-well microtiter plates. Following a 168-h incubation at 38 ◦C, worm activity was captured using a WmicroTracker ONE. Over a period of 15 min, interference of an infrared beam in individual wells was recorded as a worm ‘activity count’. Activity counts were then normalised to the positive and negative controls. To observe compound effects, the half-maximal inhibitory concentrations (IC50 values) were estimated. Worms were fixed with 40 μL of Lugol’s solution, and assessed microscopically via the development of a mouth. Individual crude extracts were evaluated at a single concentration of 0.5 mg/mL (vehicle: DMSO), and the activity of the purified compounds was assessed in dose-response assays (50 μM to 0.2 μM; vehicle: DMSO). The compounds assessed were selected based on availability and chemical stability.
Results
G. parviflora extracts and compounds were evaluated against xL3s of H. contortus to establish whether they inhibited larval motility and/or development, and/or induced a non-wildtype morphology . The crude extracts were assayed at a single concentration (0.5 mg/mL) and compounds were assessed in a dose-response assay (50 μM to 0.2 μM). The effect of the crude extracts and the purified compounds on the motility, development, and phenotype of xL3s at 168 h is summarised in Tables 4 and 5. All the DCM crude extracts were active (≥70% motility reduction) and the resinous components of the MeOH extracts also displayed anthelmintic activity (100% motility reduction) (Table 4). The DCM crude extracts of leaves and flowers and the resinous component of the leaf MeOH extract induced a skinny (Skn) phenotype in affected larvae. The solid component of the leaf MeOH extract also displayed some anthelmintic activity, but the MeOH extract of the flowers did not. As a result, the DCM crude extracts of the leaves and bark were prioritised and subjected to further fractionation and compound isolation.
Although the compounds from the bark were not obtained in a sufficient quantity for anthelmintic activity assessment, five purified compounds obtained from the leaves were evaluated (Table 5). Of these, auraptene and flindersine 1 exhibited significant activity against H. contortus (see Table 5 and Figure 4). Flindersine 1 inhibited motility (IC50 3.7 μM) and auraptene 9 inhibited development (100% at 25 μM) of the xL3 stage of H. contortus. In addition, geiparvarin 3 induced a Skn phenotype (100% at 21.7 μM) in the affected larvae.
This study demonstrates the importance of exploring, in detail, the constituents of individual plant parts and in evaluating the bioactivity of pure compounds in a variety of biological assays. This approach has the potential to lead to the discovery of new and yet undescribed phytochemical compounds with therapeutic activities.
Metabolites 2024, 14, 259.https://www.mdpi.com/2218-1989/14/5/259
Deepika Dugan, Rachael J. Bell, Robert Brkljaca, Colin Rix, Aya C. Taki, Robin B. Gasser and Sylvia Urban