Iron-Chelating Potential of Novel Phytochemicals in Poplar and Cedar Trees

Introduction: Iron overload is not only a consequence of diseases such as thalassemia and hereditary hemochromatosis (HFE), but also of neurodegeneration with brain iron accumulation (NBIA). In HFE, iron increases the risk of developing hepatocellular and colorectal cancers. Excess iron resulting from disruptions in normal iron homeostasis can accumulate in major organs including liver, heart and brain, and has devastating effects if left untreated. Currently, treatment includes using iron chelators, which at higher concentrations can have significant adverse effects and require constant medical supervision. Therefore, the search for alternative unique or adjuvant iron chelators that have reduced toxicity could be of significant benefit. Plants that grow in alkaline soils may be a good source of chelators for this purpose. Since iron is generally unavailable in such soils, plant roots have evolved mechanisms to solubilize iron for uptake, such as soil acidification, but need additional strategies to overcome high alkalinity. This may include producing secondary metabolites that are exuded into the soil and can chelate iron directly, including phenolic acids that may chelate iron at physiological pH in humans. This project is focused on finding, isolating, and testing bioactivity of compounds from western red cedar (alkaline tolerant) and poplar (rich in phenolics).

Methods: Plants are grown in iron-normal and -deficient conditions in an innovative aeroponic system to stimulate the production of secondary metabolites related to Fe deficiency. Plant tissue extracts and root washings are collected and concentrated with solid phase extraction chromatography to form plant-derived concentrates (PDC) that are analyzed by UPLC-MS and colourimetric assays to isolate, identify, and characterize compounds induced by iron-deficiency. For bio-activity testing, PDCs are introduced to cultures of THP-1 cells, a model human monocytic cell line, to study their effect on Fe homeostasis. Prior to treatment with chelators, cells are cultured under normal (Con) and Fe-overload (CrFe) conditions (produced by treatment with 10 and 20 µM Fe-citrate) for one week to model human chronic iron overload. Deferoxamine (DFO), a well-known clinical iron chelator, model phenolics like caffeic (CafA) and chlorogenic acid (CGA), and PDCs have been applied to cultures as potential chelators.

Results: Leaf compared to root tissues from poplar vary greatly in their CGA and phenolic content. Leaf extracts contained 5 times more phenolics than root extracts, and root extracts from iron-deficient plants produced 66% more phenolic compounds than those from iron-normal plants. Compared to leaf extracts, root extracts showed a 4-fold increase in iron-binding activity in vitro. PDCs including these extracts were found to contain compounds responsive to iron deficiency, which are semi-polar and low in molecular weight (140 - 340 m/z). Distinct iron-responsive compounds were also identified from cedar. Following acute dosage with Fe-citrate, THP-1 cells showed a moderate reduction in iron content after treatment with CafA, CGA, and PDCs from roots, with no influence on cell viability. On-going work includes dose-dependency with CafA and PDCs and co-operative effects of PDCs with DFO. Iron-loading in THP-1 cells is time sensitive, with maximum iron uptake measured at 8 hours following delivery of 20 µM Fe-citrate. Detailed kinetics of cellular iron-loading in the presence of iron-chelators is currently being investigated.

Conclusions: We found that low-molecular weight and water-soluble PDCs from iron-deficient plants had excellent iron-binding capacity in vitro, and inhibited iron uptake in THP-1 cells. Effects of chelators on cellular iron uptake is both dose and time dependent. Screening plants for novel chelators provides an abundance of opportunity to search for new chelators for human medicinal use.