Iron is the most common cofactor within the oxygen handling biological machinery and, specifically, lipid peroxidation of biological membranes is the main pathogenic mechanism of iron overload induced tissue damage [38
]. Harmful effects of extreme iron deposition in liver are likely during iron overload states (e.g., genetic hemochromatosis, thalassemia major and transfusional siderosis). In such conditions, iron has been associated with the initiation and propagation of ROS induced oxidative damage to all biomacromolecules (proteins, lipids, sugar and DNA) that can lead to a critical failure of biological functions and ultimately cell death [39
]. An effective therapeutic approach can play a double role in reducing the rate of oxidation - one by sequestering and chelating cellular iron stores [40
] and other as radical trap (i.e., antioxidant activity). Since TCME has shown antioxidant and free radical scavenging activity [24
], the present study, primarily incorporates the in vitro
iron chelation potency of TCME, and inhibition of iron mediated DNA breakdown. Consequently, in vivo
ameliorating effect of TCME on iron accumulation and oxidative damage in liver of iron overloaded mice is studied. Intraperitoneal iron-dextran injection resembled the hemochromatosis secondary to iron loaded anemias (anemias treated with repeated transfusions) and high iron oral intake [41
], while avoiding direct interruption of fruit extract on intestinal iron absorption leading to hepatic and serum iron overload.
Intracellular defense mechanism against free radical generation and pathogenesis involves antioxidant enzymes such as SOD, CAT, GST or compounds such as GSH [42
]. Excess iron imbalances their levels with excess ROS production thus resulting oxidative stress, followed by peroxidative decomposition of cellular membrane lipids which is a postulated mechanism of hepatocellular injury in iron overload [43
]. Alongside, the iron overload generated ROS can lead to oxidation of protein backbone resulting in modification of catalytic and structural integrity of various important proteins [44
] contributing to the pathogenesis of liver fibrosis [45
]. In turn, hepatic injury by iron results in the leakage of cellular enzymes into the bloodstream, resulting in augmented levels of serum ALAT, ASAT, ALP and bilirubin [1
The in vitro results from Figure suggest that TCME has iron chelating activity, although not as good as the standard EDTA. The significant dose-dependent reduction in the formation of Fe2+-dependent hydroxyl radical induced nicked DNA and increase in supercoiled DNA in the presence of TCME reveal its excellent iron chelating activity. The in vivo results showed that TCME administration in iron overloaded mice restored the antioxidant enzymes level significantly. Chiefly, the present study demonstrated the lipid peroxidation and protein oxidation inhibiting capability of TCME, which is supposed to be associated with its iron chelating activity. Iron overload causes a significant increase of hydroxyproline, a marker of liver fibrosis. Treatment with TCME significantly reduced hydroxyproline content in iron intoxicated mice, thus demonstrating the hepatic fibrosis inhibitory potency of the fruit extract. Moreover, the direct effect of TCME to reduce hepatic iron content in treated mice supported its iron chelating potency. Above all, TCME reduced the serum enzymes as well as the total Bilirubin levels, indicating its protective effect over liver damage by iron overload and improvement in its functional efficiency.
Ferritin is a ubiquitous intracellular protein that stores iron in a non-toxic ferric form and also helps prevent iron from mediating oxidative damage to cell constituents [46
]. Serum ferritin concentration is the most sensitive indicator of the severity of iron overload and its level usually increases when body’s iron stores increase. In this study, the ferritin level was found enhanced in iron overloaded mice, whereas, the level significantly reduced after the treatment with TCME.
Maximum iron chelators depend on the availability of Fe2+
, which in turn depends on the rate of reductive release of iron from ferritin. Therefore, successive chelation therapy includes the supplementation of ascorbate as reducing agent to increase the availability of storage iron to chelators [47
]. Previously, TCME had shown reductive ability [24
] as well as in the present study; a significant positive correlation between reducing power and iron released from ferritin has been well established. Therefore, TCME can also be used as drug to treat iron overload as the present results show its reductive release activity of ferritin iron dose dependently as well as time dependently.