Isolation and Chelation Activity of Plant Phenolic Root Exudates As Potential Clinical Iron Chelators

Introduction:Hemoglobinopathies such as β-Thalassemia (Thal) and other genetic disorders such as neurodegeneration with brain iron accumulation have complications of iron overload, which can impair vital processes such as cardiac and neurological function. In Thal, ineffective erythropoiesis leads to elevated gastrointestinal iron absorption and in severe cases may necessitate blood transfusions exacerbating the condition. Iron chelating agents such as deferoxamine (DFO), deferiprone, and deferasirox must be used, but can have unwanted side effects. While effective in treating iron overload, the increased risk of side effects at higher doses highlights the importance of finding new potent iron-chelating agents, either as replacements or as adjuvants. Plant based treatments may be better tolerated by patients, offering an alternative therapeutic approach. Plant secondary metabolites secreted from roots under iron limiting conditions may provide a novel source for this bioactivity. Plants rich in phenolic compounds and those tolerating alkaline soils, which necessitate efficient mobilization of soil-precipitated iron, are our focus here. The aim of this project is to functionally and analytically characterize naturally occurring plant root exudates as possible clinical iron chelators.

Methods : We cultivated Populus trichocarpa ( Poplar), Thuja plicata (Western red-cedar), and Lavendula X intermedia (Lavender) cuttings under iron normal and iron limiting conditions. From their roots, exudates were isolated to characterize and determine their effectiveness as iron chelators. Total phenolic concentrations of exudates were evaluated using Folin-Ciocalteu reagent. The ferrozine assay was used as a competition assay to characterize total iron binding ability of root exudates and standard chelators, which was sensitive enough to differentiate chelation effectiveness between DFO, EDTA, and isolated root exudates at 800 nM. Furthermore, known plant iron chelators chlorogenic acid (CA), a phenolic, and the more lipophilic β-thujaplicin (hinokitiol) were compared to DFO, then used tomodel root exudates in a cell culture bioassay. The bioassay was developed using monocytic THP-1 cells (as an RE system model) to quantify intra- and extracellular iron, comparing iron uptake in cells exposed to varying concentrations of iron chelators as a way of evaluating chelation strengths of crude root exudates, β-thujaplicin, and CA in comparison to DFO.

Results:Total phenolic production in P. trichocarpa root exudates increased significantly (p < 0.05) in response to iron depletion after 12 hrs. Iron binding ability increased almost four-fold upon iron depletion. In the ferrozine competition assay, iron depleted root exudates inhibited iron binding by 15% whereas iron normal exudates inhibited by only 4%. Iron binding of root exudates was similar to EDTA (13% inhibition) and about half that of DFO (30% inhibition). In the THP-1 cell bioassay, DFO and CA showed comparable trends of chelation activity. Cells exposed to either 50 or 100 μM CA or DFO reduced intracellular iron by at least 47% or 89%, respectively. However, at 50 and 100 µM β-thujaplicin increased iron uptake by 82 to 86% respectively. Further testing of plant root exudates is ongoing.

Conclusions: Exposing P. trichocarpa to an iron deficient environment resulted in plants that secreted root exudates with significantly higher level of phenolics and increased iron chelation ability. This was shown in the ferrozine competition assay where iron depleted root exudates showed higher binding affinity for iron when compared to the iron normal exudates resulting in an iron chelation ability similar to the known iron chelator EDTA. In the bioassay using THP-1 monocytic cells, testing the plant phenolic CA, there was reduction of intracellular iron comparable to DFO, whereas the lipophilic chelator β-thujaplicin increased cellular iron. Consequently, chelators such as CA exclude iron from being taken up by the cells, while more lipophilic chelators, like β-thujaplicin enable transport through membranes, and thus have the capacity to transport iron both into and out of cells. Thus, we have shown that plant root exudates can yield potential iron chelators and that in general plant phenolics have iron chelation capacity comparable to the classic iron chelator DFO.