Okra is a multipurpose plant which can be consumed freshly or dried. The pod and vegetable has been used for salads, stew and soup [15]. Kahlon et al. [16] and Varmudy, [17] reported that 100g of okra leaf can contain 0.70mg of iron and 385.00 μg of β-carotene. Okra is also rich in phenolic compounds such as flavonol and some these flavonol derivatives and isoflavonoid have been reported to be rutin and quercetin [15]. Furthermore, Okra has shown promising but not confirmed anti-inflammatory properties [18].
The objective of this study was to determine the effect of Abelmoschus esculentus leaf on haematological parameters, iron profile parameters and inflammatory markers in Sprague Dawley rats. Serum iron, TIBC, transferrin saturation, ferritin, hepcidin and ferroportin, were all significantly high in all groups fed with okra leaf. The result suggests a high iron content in okra leaf used for this study. In developing countries, iron deficiency is usually a consequence of absorption disorders, loss of blood, or the intake of a restricted diet (vegan diet). Foods present different forms of iron that differ in their bioavailability, depending on the source. Hemic iron is present mainly in animal proteins such as hemoglobin and myoglobin, which has a higher bioavailability; these proteins are present in meat, fish, and shellfish [19].
Non-hemic iron is present in different chemical forms, which significantly affects its absorption. This type of iron is present in both organic and inorganic forms. The most common sources of non-hemic iron present in foods are low-molecular-weight compounds such as ferric citrate, phosphate, phytate, oxalate, hydroxide and in high-molecular-weight compounds such as ferritin. The best sources of non-hemic iron are seeds, grains, nuts, and the green leaf of vegetables [19]. Likewise, the absorption of iron depends to a large extent on the concentration of iron present in the body and enhancers such as ascorbic acid and some muscle tissue proteins. Although, Okra (Abelmoschus esculentus) has been reported to contain iron [20], reported that iron-rich vegetable diet did not necessarily translate into high iron absorption rate and stated that other factors such as vitamin C and β –carotene should be investigated. The reported presence of vitamin C and β –carotene in okra leaf [21] may have contributed to high serum iron observed in this study.
One of the objectives of this study was to evaluate some iron associated proteins involved in iron metabolism. Most dietary iron is in the ferric form (Fe2+) which are converted to ferrous form (Fe3+) by duodenal ferric reductase and then transported by divalent metal transporter 1 (DMT-1) [22]. Hepcidin is a hormone secreted by the liver which regulates iron homeostasis. It is the master regulator of systemic iron homeostasis, coordinating the use and storage of iron with iron acquisition [23]. In this study, hepicidin concentration was lower in the groups fed with higher concentrations of okra leaf. This is probably due to high iron levels which induce the synthesis of ferroportin which in turn causes a negative feedback mechanism in hepcidin activity. The hepcidin results from this study suggest that hepcidin regulation is probably a negative feedback mechanism to high serum iron content. Another mechanism could probably be due to the fact that estrogen down regulates hepcidin expression through the estrogen responsive elements [24]. Kuhnle et al. [25] using LC-MS reported that okra contains a high concentration of phytoestrogens and these natural compounds have been shown to elicit estrogen like properties [26]. Young et al. [27] had also previously reported increased serum hepcidin levels in women who had iron supplements from food sources.
The role of hepcidin in the regulation of the bioavailability of dietary iron is associated with its interaction with transmembrane protein, ferroportin (Fpn) [28]. In this study, ferroportin levels were higher in the treatment groups which is indicative of iron been pooled into circulation from the labile pool. Ferroportin is both the hepcidin receptor and the only known cellular iron exporter in vertebrates [29]. Ferroportin is expressed on duodenal enterocytes absorbing dietary iron, macrophages in liver and spleen recycling old erythrocytes, hepatocytes storing iron and placental trophoblasts transferring iron to the fetus during pregnancy [30]. The biological role of ferroportin is the transportation of iron from cells to blood vessels. Hepcidin regulates the expression of ferroportin on a post-translational level [31]. It can directly bind to ferroportin molecules, with subsequent internalization of the resultant hepcidin-ferroportin complex. Ferroportin undergoes lysosomal degradation inside the endosome. Its loss from the cellular surface induces a secondary decrease in the release of iron from the cell [29, 31]. Consequently, the interaction between hepcidin and ferroportin inhibits the efflux of iron ions from enterocytes to blood vessels [32]. Whenever the systemic deposits of iron are either sufficient or too high, the liver synthesis of hepcidin is enhanced; the hormone is released into circulation, reaches the enterocytes, and binds to ferroportin, thereby inducing its endocytosis and degradation [33]. Ferroportin is also expressed in erythroid precursor cells, and it has been proposed that its presence enhances the sensitivity of precursors to systemic iron levels and helps determine their commitment to expansion and differentiation [34]. Furthermore, enhanced hepcidin expression will exert a negative feedback regulation on iron absorption which will culminate in the degradation of ferroportin over time. The results from this study is suggestive that lower concentration of hepcidin correlates with high concentration of ferroportin, which resulted in the high serum iron observed in this study.
Serum ferritin which is the main storage protein for iron was significantly high in all test groups compared to controls. Similar result was reported by Nyakundi et al. [35], where they stated that serum ferritin levels directly correlates with consumption of iron and ascorbate-rich foods. Although, high serum ferritin level does not necessary connote high iron level because it can also be a marker for an inflammatory process [36], the result from this study with the high serum iron and TIBC suggest that the increase in serum ferritin is probably due to increase iron levels. In this study, there was also a significant increase in transferrin saturation in groups fed with okra leaf. This suggest over saturation of iron and ferritin within the entire metabolic process thereby increasing the capacity for the body to transport more iron. Intracellular iron levels regulate ferritin biosynthesis and transferrin expression affinity [37]. This regulation is mediated by the interaction of iron responsive elements (IREs) with cytosol RNA-binding proteins called iron regulatory proteins (IRPs). A careful balance between the IREs and IRPs, swings the pendulum of iron transportation (transferrin levels) and iron storage (increased ferritin levels) [37]. The binding of IRPs to IREs lead to changes in expression of iron-regulated genes such as hepcidin, with subsequent alterations in intracellular iron uptake, use and storage. When iron levels are high, the IREs do not bind to IRPs and the translation of ferritin mRNA occurs normally.
The study showed a significant increase in RBC count, haemoglobin concentration and haematocrit in the experimental groups when compared to control. This indicates that okra leaf can be used to help boost red cells and haemoglobin concentration. Okra has been previously reported to be rich in iron and folate [10, 38] which may also contribute to the increase in red cell parameters observed in this study.
The rapid evolution of molecular information about iron transport and homeostasis has uncovered a comprehensive understanding of the complex mechanisms involved in this process. It has long been recognized that iron levels must be tightly regulated to provide an essential nutrient that is involved in oxygen delivery, metabolism and redox regulation while guarding against excessive levels of a primary toxicant that can generate reactive oxygen species (ROS) to produce cellular damage and death. Unlike other essential minerals, the delicate balance between iron nutrition and toxicity is maintained by systemic control mechanisms that drive iron conservation and limit uptake until needs are presented [39]. Excessive inflammation and oxidative stress is closely linked to the development of many human chronic illnesses. This study also investigated the effects of okra leaf on some anti-inflammatory and pro-inflammatory cytokines. The results shows that IL- 6 and IL- 2 which are pro-inflammatory cytokines were all lower in groups fed with okra leaf. Although, the levels of IL-6 was not statistically significant in male rats but in female rats 40% okra leaf fed group had lower levels when compared to 10% okra leaf fed group. Inflammatory cytokines, in particular IL-6 have been reported to regulate hepcidin transcription via the JAK-STAT3 pathway [40]. In addition, data for MIP - 1β (anti-inflammatory cytokine) showed a significant increase in the groups fed with okra leaf. This result indicates the anti-inflammatory effect of okra. This results corroborate the reports by other researchers, where they reported a decrease in IL- 6 [18, 41, 42] in animals fed with okra. Some of the most abundant phytochemicals in okra leaf extracts have been identified as quercetin, quercitrin, rutin and kaempferol [43]. Some of these derivatives, in fact have been previously studied for their anti-inflammatory activities [44, 45].