Natural Iron Chelator. Chemistry, Biology, and Therapeutic Applications

Inhaltsverzeichnis

I. Introduction

1. Iron disorders in the human body
Metabolism of Iron
Iron blood disorders
Classification and causes of iron overload
Complications of iron overload
Iron overloads medications

2. Natural compounds as alternative chelating agents

3. Agricultural wastes with reported iron chelating activity

4. Chemical and biological activity of Mangifera Indica L. leaves

5. Iron Chelation assays
Determination of iron chelation activity by 2-2` bipyridyl reagent
Materials

Methods

II. Phytochemical screening and iron chelating activity of Mangifera indica L. leaves

III. Bio-guided fractionation of iron chelators from Mangifera indica L. leaves

Identification of the isolated compounds

IV. A comparative study of the iron chelating property of mangiferin Vs. desferal®

V. In-vivo investigation of the total extract (TM) and the ethyl acetate fraction (EM) of Mangifera indica L. leaves in iron overloaded rats

Isolation, characterization, and biological evaluation of iron chelators from some natural sources

Conclusion

References

Summary

The book is divided into five chapters: -

Chapter I: Screening of some agricultural wastes for iron chelating activity

In this chapter, sixteen extract of agricultural wastes were subjected to iron chelation assay, mango leaves extract showed the highest iron chelation activity. Preliminary phytochemical screening showed that mango leavesا has a rich content of tannins and flavonoids.

Chapter II: Phytochemical screening and iron chelating activity of M. indica L. leaves

In this chapter, each of methanolic extract and ethyl acetate fraction of mango leaves were subjected to further biological assays (total flavonoid, total phenolic and ABTS). The ethyl acetate fraction showed the best results.

Chapter III : Bio-guided fractionation of iron chelators from M.indica L. leaves

In this chapter, the phytochemical investigation of the ethyl acetate fraction of mango leaves led to isolate two compounds (iriflophenone-3-C- β -D-glucoside ), mangiferin showed the highest iron chelation activity.

Chapter IV : A comparative study of the iron chelating property of mangiferin Vs. desferal®

In this chapter, the chelation pattern of mangiferin was determined, also the antioxidant activity of mangiferin-iron complex was better than desferal®-iron complex.

Chapter V : In-vivo investigation of the total extract (TM) and the ethyl acetate (EM) fraction of M. indica L. leaves in iron overloaded rats

The ethyl acetate fraction showed significant decreases in iron accumulation within the liver and spleen in iron overloaded rats. Accordingly, it showed the best restored of all biological parameters toward normal value as compared to total extract of mango leaves.

I. Introduction

Iron is an important trace element in the human body (Sarkar et al., 2012). It is essential for oxygen and electrons transport within cells and as an integrated part of several enzyme systems (Gupta, 2014). Iron overload (hemochromatosis) maybe caused by genetic disorder of protein involved in regulation of iron absorption or due to multiple transfusion of iron as in chronic anemia (Queiroz-Andrade et al., 2009). Accumulation of iron in the body results in initiation and propagation of reactive oxygen species, which start to attack the cell vital macromolecules such as proteins, lipids, RNA and DNA causing cell damage, DNA mutation and ultimately cell death. This produced state of oxidative stress is associated with many health problems such as heart failure, liver cirrhosis, fibrosis, gallbladder disorders, diabetes, arthritis, infertility, and cancer (Gupta, 2014).

Iron chelators can remove the accumulated iron from the body before causing irreversible tissue damage by forming soluble, stable complexes that can be excreted in the feces and/or urine. Although available iron chelators can reduce iron-related complications, their severe side effects, poor oral bioavailability or short plasma half-life as well as their expensive cost make them suboptimal such as deferoxamine (Desferal®), deferasirox (Exjade®) and deferiprone (Ferriprox®) (Ebrahimzadeh et al., 2008).

The unmet need for iron chelators, could be satisfied by natural products, providing the advantage of being less toxic, wider in safety margin and more economic. The main interest of our study is agricultural residues, being a potential source for biologically active compounds (Duarte and Rai, 2015, Schmidl et al., 2008) with economic and environmental merits(Jain et al., 2014, Zhang et al., 2011).

Mangifera indica L. is a tropical fruit, belonging to family Anacardiaceae. It is widely spread in India, Central America, Asia, and Africa (Lauricella et al., 2017). The Egyptian Agriculture and Land Reclamation provided a statistical study indicates that, the total production of mango is 0.596 million tons in Egypt and about 151,000 Fadden are dedicated to mango trees (Mansour et al., 2008).

The aim of this work:

- To explore agriculture residues rich in polyphenolic compounds for iron chelating activity.
- To correlate the iron chelating activity with the total flavonoid and phenolic content as well as the antioxidant activity.
- To isolate the natural iron chelators from agricultural wastes using bio-guided-fractionation protocol.
- To evaluate the isolated natural iron chelators Vs. commercially available drug.
- To evaluate the effect of the most potent fraction and/or pure compound in iron overloaded rats (in-vivo study).

This chapter deals with five main sections:

1. Iron disorders in the human body.
2. Natural compounds as alternative chelating agents.
3. Agricultural wastes with reported iron chelating activity.
4. Chemical and biological activity of Mangifera i ndica L. leaves.
5. Iron chelation assays.

1. Iron disorders in the human body

- Iron functions in the human Body:

Iron is an essential trace element in the human body{Sarkar, 2012 #15;Gupta, 2014 #16}. About, 60- 70% of the body iron is present in the hemoglobin in the red blood cells and about, 10% is in the muscle myoglobin. Iron is essential in hemoglobin for oxygen transport from the lungs to tissues and in myoglobin for regulation of oxygen storage and transport. It is needed for oxygen and electrons transport within cells and as an integrated part of important enzyme systems in various tissues (Gupta, 2014). Only about, 1% of the body iron is implicated as an integrated part in a variety of important iron-containing enzymes such as cytochrome P450 and less than 0.2% is incorporated in the plasma transport where most of it is bound to transferrin. The remaining body iron, about, 20–30 %, is stored in the liver and macrophages as ferritin and haemosiderin, which is a degraded form of ferritin according to (Jackson, 2010) as shown in (Figure 1). Body iron content is approximately 4.0 and 3.5 g in men and women, respectively (Jackson, 2010).

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Figure (1): Illustration of vital roles of iron in the human body, (Author’s own work).

Metabolism of Iron

A. Absorption:

Iron absorption takes place in the enterocytes of the small intestinal predominantly in the duodenum and upper jejunum by a transport protein called divalent metal transporter 1, (Abbaspour et al., 2014). After reduction by duodenal cytochrome b of Fe (III) to Fe (II), within enterocytes, the iron transferred to the vascular site of the cell from the luminal, then released to the circulation by ferroportin (metal transporter). Thereafter, excreted Fe (II) is oxidized to Fe (III) by hephaestin, and by serum transferrin. The resulting ferric iron will be carried out into the cells or the bone marrow for erythropoiesis process as shown in (Figure 2), (Kohgo et al, 2008). .

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Figure (2): Absorption process of iron in small intestine, ( Kumar et al., 2017 ) .

B. Regulation of iron homeostasis:

Iron homeostasis is regulated by circulating peptide hormone secreted via the liver called hepcidin, through binding with ferroportin, leading to degradation of ferroportin from the cell surface. Consequently, this will lead to decreased iron entry into plasma and low transferrin saturation, accordingly low iron is trasported to the developing erythroblast. In contrary, decreased hepcidin expression lead to increased ferroportin on the cell surface and enhance iron absorption as shown in (Figure 3), (Abbaspour et al., 2014).

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Figure (3): Iron homeostasis mediated by Hepcidin, ( Abbaspour et al., 2014 ) .

C. Storage:

Liver is a major organ for storage of excess iron in the body in the form of ferritin and hemosiderin. Free iron fractions is present within cells as labile iron pool (LIP) which is biologically active in intracellular metabolism, but if present in excess levels it will be an hazard on the human health (Abbaspour et al., 2014).

D. Excretion:

Body losses iron through bleeding, menstruation or pregnancy. In addition, some obligatory loss as the physiological exfoliation from epithelial surfaces of cell, such as the skin, gastrointestinal tract and genitourinary tract. Nevertheless, these losses are very limited about, ≈1 mg/day, (Abbaspour et al., 2014).

Iron blood disorders

A. Iron blood deficiency.
B. Iron overload.

A. Iron blood deficiency:

Iron blood deficiency can be defined as decreased total iron body content. Reduction of iron stores occurs when iron absorption is not enough for iron metabolic requirements to maintain growth and to compensate iron loss. The main causes of iron deficiency include decreased intake of bioavailable iron, increased iron demands such as pregnancy, rapid growth, menstruation, and pathologic infections by hook worm and whipworm leading to gastrointestinal blood loss. Iron deficiency has damaging effects on intellectual capacity, nervous system, immune response and physical performance (Al-Momen et al., 1996). Also iron deficiency causes anemia due to the insufficient iron for hemoglobin synthesis and erythropoiesis resulting in increased morbidity rate (Abbaspour et al., 2014).

- Types of anemia:

-Sickle cell anemia: Red blood cells become sickle in the shape ("C"-shaped) and it contains an abnormal haemoglobin. The sickle-shape of RBCs does not allow them to move easily in blood vessels and block blood flow (Soundarya and Suganthi, 2017).

-Thalassemia: It is considered as genetic blood disorder, in which the RBCs production is partially healthy with less haemoglobin. The body produces fewer red blood cells with less haemoglobin. Alpha- and beta thalassaemia are the major type of thalassemia (Soundarya and Suganthi, 2017).

-Plastic anemia: In this type of anemia the bone marrow is unable to produce new blood cells, due to many of health problems such as heart failure and damage in stem cells bone marrow (Soundarya and Suganthi, 2017).

-Diamond Blackfan anemia (DBA): It is a genetic blood disorder type, as a result of malfunction of bone marrow where the RBCS production is not enough. Patients with DBA are more susceptible to develop bone marrow cancer (Vlachos et al., 2008).

- Treatment of iron deficiency:

Iron deficiency could be treated by iron supplementation (tablets, elixirs), ferrous iron salts (ferrous sulphate, ferrous gluconate and ferrous fumarate) are preferred because of their low cost and high bioavailability (Alleyne et al., 2008, Abbaspour et al., 2014). Parenteral iron preparation are used in cases where rapid repair of hemoglobin is required or with patients who suffer from intestinal malabsorption (Clarke and Dodds, 2014). Proper nutrition includes iron rich food (flesh foods) besides fruits and vegetable rich in ascorbic acid to enhance iron absorption (Abbaspour et al., 2014).

B. Iron blood-overload (hemochromatosis):

Iron overload is defined as iron accumulation in the body above than the normal level which is 4.0 and 3.5 g in men and women, respectively. The excessive content of iron can be investigated by elevation of serum ferritin concentration or iron stores expansion demonstrated histologically through liver biopsy or bone marrow (Bentley, 2014).

Classification and causes of iron overload

Iron overload can be classified as primary or secondary hemochromatosis:

A. Primary hemochromatosis (hereditary)
B. Secondary hemochromatosis (acquired)

A. Primary hemochromatosis (hereditary): It is a recessive autosomal genetic disorder arises from mutation in the protein included in the regulation of iron absorption

B. Secondary hemochromatosis (acquired): It is mainly caused by chronic iron transfusions used in treatment of iron deficiency disorders and bone-marrow failures (Kohgo et al., 2008). Also patients suffering from chronic kidney disease are more affected by iron overload through hemodialysis processes, as well as patients with hepatic dysfunction (e.g. liver cirrhosis) (Puntarulo, 2005).

Complications of iron overload

Iron is potentially toxic, since it can accept or donate electrons (Bystrom et al., 2014). Accumulation of iron in tissues, especially in form of labile iron pool, leads to initiation and propagation of highly reactive oxygen species (ROS) including superoxide anion (O2-), hydrogen peroxide (H2O2) and the hydroxyl radical (HO•) through Fenton reaction; where iron act as a catalyst.

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Fenton reaction

ROS starts to attack the cell vital macromolecules such as proteins, lipids, RNA and DNA causing cell damage, DNA mutation and ultimately cell death. This produced state of oxidative stress is associated with many health problems such as heart failure, liver cirrhosis, fibrosis, gallbladder disorders, diabetes, arthritis, infertility, and cancer (Gupta, 2014). other pathological conditions that are associated with iron overload include: infection, noplasia, cardiomyopathy and atherosclerosis diseases (Puntarulo, 2005, Kohgo et al., 2008).

- Treatment of iron overload:

Phlebotomy (Bloodletting) is the common therapy for hemochromatosis (Pietrangelo, 2010). Also by using synthetic chelators (drugs) which can bind with iron, forming a stable soluble complex that can be excreted through faeces and/or urine.

Iron overloads medications

- Deferoxamine (Desferal®): DFO binds with iron in ferritin and hemosiderin as well as non-transferin bound iron. It forms a ferrioxamine molecule which is eliminated by kidneys. Ferrioxamine makes iron unavailable for chemical reactions therefore preventing ROS formation. DFO also induces autophagy in lysosomes hence promotes degradation of ferritin (Temraz et al., 2014).

- Limitations: In addition to being very expensive drug, it is also painful since its administration only via subcutaneous and intravenous routes or by infusion which is time-consuming and may cause induration or redness at the site of infusion. Moreover, chronic treatment by DFO leads to auditory and visual neurotoxicity and other acute effects including hypotension, bone abnormalities, abdominal pain, diarrhoea, vomiting and nausea. It is also associated with increased blood pressure in lungs, enhancing the risk of infection of vibrio, mucormycosis and Yersinia (Poggiali et al., 2012).

-Deferiprone (Ferriprox®): DFP has the ability to chelate cytosolic iron and extract iron from ferritin before degradation of ferritin via proteasomes. It is also connected with increased hepcidin levels resulting in removing ferroportin from the enterocyte membrane and its later degradation via lysosomes (Temraz et al., 2014).

- Limitations: The side effects of DFP therapy are increased liver enzymes, gastrointestinal disorders, arthralgia, neutropenia and agranulocytosis . Also in a previous study, it is reported that DFP is not successful enough with thalassemia patients for controlling iron overload (Poggiali et al., 2012).

- Deferasirox (Exjade®): It has a similar mechanism of action of DFB, where it binds to cytosolic iron and extracts iron from ferritin before degradation of ferritin via proteasomes (Temraz et al., 2014).

- Limitations: It is a very expensive drug and it shows many complications such as nausea, abdominal pain, vomiting, skin rashes, diarrhea, ophthalmic complication, kidney dysfunction, drastic level of metabolic acidosis, hypophosphatemia and hypokalemia {Poggiali, 2012 #91}(Poggiali et al., 2012). The available pharmaceutical iron chelators in the Egyptian market are recorded in (Table 1)

Table (1): Some pharmaceutical iron chelators in the Egyptian market:

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2. Natural compounds as alternative chelating agents

Although available iron chelators alleviate iron overload complications and therefore, enhance life quality and overall survival, but their severe side effects, poor oral bioavailability and short plasma half-life make them suboptimal (Ebrahimzadeh et al., 2008). Accordingly, researchers have been interested in finding an alternative and effective source with lower side effect and higher safety margin. In this context, natural products can serve as a vast platform for development of new drugs with wider safety margin and more favorable properties.

Examples of natural compounds with reported iron chelation activity:

- Curcumin:

A natural phenol isolated from curcuma longa L. root (Hewlings and Kalman, 2017) . Many studies have proven that curcumin has a potential iron chelating activity and potent antioxidant capacity (Fibach and Rachmilewitz, 2010). A previous study in iron-overloaded rats showed that curcumin significantly inhibited lipid peroxidation and decreased accumulated iron in the liver and spleen (Badria et al., 2015).

- Quercetin and Rutin:

Quercetin and rutin are major flavonoids in various plant species. Both are previously reported to have the ability to prevent lipid peroxidation of lecithin liposomes even at high iron ion concentrations (1 mM). They can chelate ferrous iron into a stable complex that is unable to initiate lipid peroxidation. Iron complexes of flavonoids retain their free radical scavenging activities, in the same time they make iron unable to catalyse generation of the active oxygen species (Afanas' ev et al., 1989).

- Catechins:

They are tea flavonoids derived from Camellia sinensis L. they are reported as strong chelating agents with potential antioxidant activity. In a previous study, catechins showed the ability to penetrate blood brain barrier and chelate with free iron. Hence it prevented iron-induced oxidative stress and aggregation of alpha-synuclein and beta-amyloid peptides. Consequently, they could be useful in attenuation of Parkinson's and Alzheimer's diseases (Mandel et al., 2006).

- Kolaviron:

A natural biflavonoid isolated from Garsinia kola L. seed. It prevented lipoprotein oxidation in iron overload rats through chelating the excess iron and showed a protective effect against atherosclerosis (Hatcher et al., 2009).

- Floranol:

A new flavonoid isolated from Dioclea grandiflora L. roots. Through a previous in-vivo study, it showed the ability to protect low density lipoprotein against oxidation by chelating iron and preventing free radical formation (Hatcher et al., 2009).

- Apocynin:

Apocynin is derived from Picrorbiza korroa L. rhizome. Similar to catechins, it has the ability to cross blood brain barrier and can exert antioxidant and chelating activity in the brain (Hatcher et al., 2009)

- Mangiferin:

It is a xanthone derivative and a major constituent in Mangifera indica L. mangiferin showed a strong chelating activity and a potential antioxidant effect in many studies. (Andreu et al., 2005, Singh et al., 2009). . A previous study conducted on isolated rat liver mitochondria treated with ferrous citrate, showed that mangiferin prevented lipid peroxidation though inhibiting the formation of thiobarbituric acid reactive substances. It also acted as an antioxidant by protecting 2-deoxyribose degradation by ROS, which is mainly due to its chelating activity (Pardo‐Andreu et al., 2006).

3. Agricultural wastes with reported iron chelating activity

Agricultural wastes are an economic source for biologically active compounds (Santana-Méridas et al., 2012). Harvesting natural products from agricultural wastes has both economic and environmental appeal. In contrast, the atmospheric pollution resulting from disposal of agricultural residues by burning, has a serious impact on human health, in particularly the increased risk of respiratory and heart diseases (Ghorani-Azam et al., 2016). Many agricultural wastes are reported to have potential iron chelating activity; we reported some examples in (Table 2).

Table (2): Some agricultural wastes with reported iron chelating activity:

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Previous studies proved that the antioxidant activity and iron chelating ability of some plants may be correlated to their polyphenol contents (Brglez Mojzer et al., 2016). Consequently, in our study we focused on the agricultural wastes rich in polyphenols as mentioned in (Table 3). Particularly, the leaf parts were collected since they represent one of the most accumulated agricultural wastes in Egypt. According to the Egyptian environmental ministry, about 1.68 tons of agricultural wastes are accumulated as a result of trees pruning process per year. Moreover leaves are generally discarded, easily collected, rapidly removed, conveniently transported and easily preserved. In (Table 3), we reported phenolic compounds previously isolated from the agricultural residues used in this study.

Table (3): A gricultural wastes used in the study and their phenolic constituents:

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Among the selected agricultural wastes the leaves of mangifera indica L. leaves showed the highest iron chelation property. Subsequently, we will talk in details about mangifera indica L.

4. Chemical and biological activity of Mangifera Indica L. leaves.

Mangifera indica L. (Anacardiaceae) is classified under kingdom: Plantae; order: Sapindales, family: Anacardiaceae genus: Mangifera and species: Indica (Shah et al., 2010). It grows naturally as well as being cultivated in the tropical or subtropical regions as an important source for foods and medicine. The Egyptian Agriculture and Land Reclamation provided a previous statistics study indicated that the total production of mango is about 0.596 million tons in Egypt and that about 151,000 Fadden are dedicated to mango trees (Mansour et al., 2008). The leaf parts of mango fruit are agricultural waste that is rich in antioxidant polyphenols, including flavonoids, phenolic acids and benzophenones. The main active constituent of mango is mangiferin which is responsible for its iron chelating activity (Shah et al., 2010). Herein we report the previous studies regarding the chemistry and biological activity of M. indica leaves

A. Chemistry:

Tables from 4 to 11 represent the different categories of compounds isolated from Mangifera indica leaves.

- Flavonoids:

A. Flavones:

Table (4): Flavones isolated from Mangifera indica leaves:

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B. Flavonols:

Table (5): Flavonols isolated from Mangifera indica leaves:

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C. Catechins:

Table (6): Catechins isolated from Mangifera indica leaves:

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D. Aurone:

Table (7): Aurone isolated from Mangifera indica leaves:

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E. Flavanonols:

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Table (8): Flavanonols isolated from Mangifera indica leaves:

F. Isoflavone:

Table (9): Isoflavone isolated from Mangifera indica leaves:

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- Phenolic compounds:

Table (10): Phenolic compounds isolated from Mangifera indica leaves:

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- Xanthones

Table (11): Xanthones isolated from Mangifera indica leaves:

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B. Reported biological activities of Mangifera indica:

Mangifera indica is a rich source of bioactive compounds including: phenols, flavonoids, tannins, carbohydrates, protein, minerals and vitamins. Therefore, it shows a variety of medicinal properties which have been exploited in folk medicine as well as pharmacological uses.

- Iron-chelating activity and antioxidant ability of Mangifera indica:

A previous in-vitro study on the stem bark extract (Vimang®) showed powerful hydroxyl radicals scavenging and potential iron chelation activity through inhibited phospholipid peroxidation. The study was conducted in rat brain incubated in presence of excess of iron salts (Martínez et al., 2000).

In another in-vivo study in mice, Vimang® prevented oxidative stress of the brain and liver tissue via inhibition of ROS generation. It also significantly alleviated the oxidative stress in serum (Sánchez et al., 2000).

A study on the seed kernel also showed iron chelation capacity using ferrozine assay and antioxidant properties using ABTS and DPPH assay (Pitchaon, 2011).

Few research studies were done on the leaves as a potent antioxidant source. The first study on the leaves as an antioxidant source was conducted by (Ling et al., 2009) through some in-vitro assays. In another comparable antioxidant study the leaves were the most active among the tested parts of the M. indica (Ndoye et al., 2018). It is worth to mention that, no further investigation was achieved on the leaves extract concerning their chelating activity.

In general, previous studies cited that the antioxidant activity and iron chelating ability of plant extracts may be correlated to its polyphenolic content, (Brglez Mojzer et al., 2016). In particular, the best chelation ability was found to be correlated to ortho -dihydroxy polyphenols (Symonowicz and Kolanek, 2012). Two mechanisms may explain the antioxidant capacity, which are metal chelation and free radical scavenging. Antioxidant activity depends on the position and the number of hydroxyl groups at the aromatic ring (Andjelković et al., 2006) Mangiferin is the main polyphenolic compound in M. Indica. (Shah et al., 2010). Iron chelating activity is the primary mechanism of mangiferin to act as antioxidant agent according to (Masibo and He, 2008). As shown in (Figure 4). Through formation of a stable mangiferin–iron complex, mangiferin makes iron unavailable to generate free radicals, consequently it prevents oxidative stress (Andreu et al., 2005). Moreover, mangiferin is reported to decrease concentration of the localized O2. It also can scavenge lipid peroxy/alkoxy radical through formation of mangiferin phenoxy radicals. Therefore, it can stop continued abstraction of hydrogen from cellular lipids and maintain the balance of cellular oxidant–antioxidant and regulate polymer chain initiation by interaction with the resulting (ROS). Furthermore the produced caged oxygen radical (mangiferin –Fe[3]+-O-) reacts with vinylic monomer methylmethacrylate (MMA) forming mangiferin–Fe–PMMA, hence it can inhibit the polymerization of MMA (Masibo and He, 2008). These results were of significant pharmacological relevance, suggesting that M. indica is an antioxidant agent and potential candidate for chelation therapy in diseases related to abnormal intracellular iron distribution or iron overload (Shah et al., 2010).

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Figure (4): Mechanism of action of mangiferin as iron chelator, ( Masibo and He, 2008 ) .

- Other reported biological activities

Other reported biological activities include anti-microbial, anti-bacterial, anti-fungal, antiviral, anti-parasitic, anti-inflammatory, anti-diarrheal, anti-spasmodic- anti-pyretic, anthelmintic, anti-allergenic, anti-diabetic, anti-tumor, anti-HIV immunomodulatory, hepatoprotective and gastroprotective activity (Shah et al., 2010). Furthermore, cardioprotective, laxative, bronchodilator and recognition of memory were also attributed to mangiferin (Parvez, 2016).

5. Iron Chelation assays

Determination of iron chelation activity by 2-2` bipyridyl reagent

-Principle: In a slightly acid conditions, iron (II) binds to three molecules of 2-2` bipyridyl, forming a red-pink complex in which nitrogen atoms occupy coordination positions of iron (Moss and Mellon, 1942). The produced complex is stable and resistant to oxidation. As only iron (II) participates in complex formation, it is important to reduce iron (III) via using hydroxyl amine agent (Jankiewicz et al., 2002). The produced color is measured calorimetrically at wavelength 522 nm, according to (Mallikarjuna et al., 2014). As shown in (Equation 1).

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Equation (1): Structure of 2, 2` bipyridyl and its complex with ferrous iron, (Author’s own work).

- Determination of iron chelation activity by ferrozine reagent:

-Principle

Iron chelators can displace ferrous form the red colored iron-ferrozine complex. Disruption of this complex results in decrease in the red color, which could be quantitatively measured spectrophotometrically at 560 nm (Adjimani and Asare, 2015). (Figure 5).

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Figure (5): Illustration of iron chelating activity via Ferrozine reagent, (Author’s own work).

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