Synthesis and bioaccessibility of Fe-pheophytin derivatives from crude spinach extract

Heme iron is recognized as a highly bioavailable source of iron suitable for treatment of iron deficiency anemia. However, the animal origin of purified heme limits its broad applicability due to religious, personal, and food safety issues. Development of chlorophyll-derived heme mimetics offers opportunities to expand current iron fortification strategies. The objective of this study was the synthesis of Fe-pheophytin (FePhe) derivatives from natural chlorophyll and subsequent evaluation of their digestive behavior and bioaccessibility in vitro. FePhe a and a' were synthesized from crude spinach extracts by treatment with 1.3 M iron(II)chloride and 0.25 M Na-acetate dissolved in glacial acetic acid at 80 degrees C for 30 min. FePhe-rich extracts (approximately 1 mM) were formulated into corn starch based test meals (7.5% lipid) and subjected to a 2-step in vitro digestion designed to simulate in vivo gastric and small intestinal conditions. Recovery of FePhe following digestion and transfer of FePhe and pheophytins (Phe) from test meal matrix to mixed micelles was assessed by RP C18-HPLC to determine the digestive stability and micellarization efficiency (bioaccessibility). FePhe a and a' derivatives were moderately stable to digestive conditions with recoveries of 52.3% and 58.7%, respectively. Residual Phe a was stable to digestion. Micellarization efficiency of FePhe a (4%) and a' (3.4%) was significantly (P < 0.05) lower than Phe a (25.8%) from test meals. While digestive stability and micellarization efficiency are limiting, the presence of lipophilic FePhe derivatives in mixed micelles suggests that these compounds would be available for subsequent absorption in the intestinal tract.