**4. Potential antioxidants for use in thalassemias**

Oxidative stress is not only a simple imbalance between the production and scavenging of ROS, but also a dysfunction of the enzymes involved in ROS production. Dysfunction of NOX, uncoupling endothelial nitric oxide synthase (eNOS), activation of xanthine oxidase (XO) and dysfunction of the mitochondria are underlying signs of oxidative stress; therefore, NOX is one of important therapeutic targets [99]. Pharmacological agents and approaches, as shown in Figure 5, can be implemented to relieve oxidative stress by the following mechanisms.

1) Inhibition of NAD(P)H oxidase activity:

evidence and caution should be imposed in their use. Most importantly, some natural

Advantages and disadvantages of the antioxidants are mentioned in Table 1. Most natural antioxidants are obtained in the diet from natural sources, especially from food of plant origin. Vegetables, fruits, and other foodstuffs are the best sources of these natural antioxidants. These antioxidants include vitamin C, vitamin E, carotenoids and polyphenol compounds. Antiox‐ idants that are derived from natural sources are preferred by consumers. This is due to concerns over the toxic and carcinogenic effects of the synthetic antioxidants. Phenolic compounds include a large class of phytochemicals with interesting biological properties. Recently, the roles of these natural compounds in counteracting the negative effects of ROS/RNS and maintaining the redox homeostasis of biological fluids have been reported. Commonly, antioxidants can neutralize potentially harmful ROS in the cells before they induce lipid and proteins oxidation. Antioxidants from plants are believed to be useful in preventing aging, atherosclerosis, cancer, peptic ulcer, liver diseases and other degenerative pathologies,

Antioxidants are any substances that significantly delay or inhibit oxidation of the oxidized substrate. These can greatly reduce the adverse damage to oxidants by crumbling or scaveng‐ ing free radicals before they react with biological targets such as biomolecules, subcellular organelles, cells and tissues. The defense systems against the damage induced by ROS/reactive nitrogen species (RNS) fall into three categories: 1) preventive antioxidants that suppress free radical formation, 2) radical-scavenging antioxidants that inhibit initiation of chain reactions and intercept chain propagation and repair processes, and 3) adaptation to generate and transfer appropriate antioxidant enzymes. The hydrophilic antioxidants include vitamin C, uric acid, bilirubin, albumin, and the lipophilic antioxidants include vitamin E, ubiquinol and carotenoids. The radicals-scavenging antioxidant is described as a primary antioxidant, such as flavonoids and vitamin E (α-tocopherol), and the others that do not involve direct scavenge

antioxidants are more potent, efficiency and safer than synthetic antioxidants.

**Synthetic antioxidant Natural antioxidant**

Widely applied Use restricted to some products Medium to high antioxidant activity Wide-ranging antioxidant activity Increasing safety concerns Perceived as innocuous substances

Low water solubility Broad range of solubility Decreasing interest Increasing interest

**Table 1.** Advantage and disadvantage of natural and synthetic antioxidants.

such as cancer, diabetes, Alzheimer's and Parkinson's diseases.

radical are secondary antioxidants.

Use banned in some cases Increasing use and expanding application

Inexpensive Expensive

128 Pharmacology and Nutritional Intervention in the Treatment of Disease

1.1) Apocynin (methoxy-substituted catechol) is a natural compound that has a structure related to vanillin and exhibits anti-inflammatory activity, blocks an assembly of p47*phox* onto membrane complex, decreases production of superoxide, enhances production of nitric oxide and disrupts active NOX complex [100].

1.2) Chimeric peptides, such as polyethylene glycol-superoxide and NOX inhibitory peptide (gp91dstat), can inhibit association of p47*phox* with gp91*phox* [101] and synthesis of Angiotensin converting enzyme II (ACE-II) a dipeptidyl carboxy-peptidase that generates the vasocon‐ stricting peptide angiotensin II [102].

1.3) Compound S17834 (e.g. benzo(b)pyran-4-one) inhibits NOX activity and superoxide radical production and attenuates atherosclerotic lesions in apolipoprotein-E-deficient mice [103].

1.4) Statins (e.g. simvastatin) are hydroxymethylglutaryl CoA (HMG CoA) reductase inhibitor hypocholesterolemic drugs that can prevent production of hydrogen peroxide and superoxide radicals, interfere with the renin-angiotensin-aldosterone system, and inhibit NOX activation and expression [99]. Currently, Zacharski and colleagues have demonstrat‐ ed that statins increased HDL/LDL ratios and reduced serum ferritin levels in subjects with advanced peripheral arterial disease. The results imply that statins may improve cardiovas‐ cular disease (CVD) outcomes, possibly by countering the pro-inflammatory effects of excess iron stores [104].

1.5) ACE-II inhibitors (e.g. captopril, enalapril and quinapril) may act as antioxidants and down-regulate the vascular NOX system. Angiotensin II, through Angiotensin I (AT1) receptor activation, can induce vasoconstriction, cell growth, actions of pro-inflammatory cytokines and profibrogenic agents, and the production of vascular ROS, such as superoxide radicals [105]. Hence, the ACE-II inhibitors would lower ROS production and prevent vascular and renal changes.

1.6) AT1 receptor antagonists (e.g. candesartan, losartan and irbesartan) reduce the expression of NOX enzyme components, resulting in lower superoxide levels and increased nitric oxide bioavailability in rat blood vessels [106].

1.7) Other blockers including calcium channel blocker (e.g. amlodipine, nifedipine and dihydropyridine), β-blocker (e.g. metoprolol, propranolol and nebivolol) and α-receptor blocker (e.g. doxazosin) can inhibit or interfere with functions of NOX activity [106, 107].

2) Vitamins and dietary antioxidants (e.g. vitamin C, vitamin E, β-carotene, polyphenols, flavonoids, olive oil and nuts) can decrease the oxidation of LDL (bad cholesterol), improve vascular endothelial functions, enhance NOS activity and attenuate NOX activity in rat aorta.

**5. Benefits of antioxidants in thalassemia patients**

β-Thalassemia major patients who suffered from leg ulcers and were treated with ascorbic acid (3 g/day) for eight weeks showed a high rate of either complete or partial healing [109]. Improvement of ulcer healing in β-thalassemia major patients was observed when they were orally administered with pentoxifylline (1.2 g/day) which is xanthine derivative and functions as a competitive non-selective phosphodiesterase inhibitor [110]. Plasma antioxidant enzyme activity and selenium concentration increased in subjects with the Hb Lepore trait and were found to be significantly low in those patients with the α-thalassemia trait, while erythrocyte Se content was significantly increased in α-thalassemia patients [111]. *N*-allylsecoboldine may act as an effective antioxidant and protect cells against oxidative damage in β-thalassemic red blood cells [112]. Plasma vitamin E levels were lower in β-thalassemia intermedia patients compared to the controls and these levels were positively correlated with vitamin E in the LDL [113]. Treatment of the patients with vitamin E (600 mg/day, orally) for nine months improved the antioxidant/oxidant balance in the plasma, LDL particles and red blood cells, and coun‐

Antioxidants as Complementary Medication in Thalassemia

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Significant decrease of GSH and proliferation in peripheral blood mononuclear cells (PBMC) were found in β-thalassemia major patients, probably due to the abnormality of cell mediated immunity (CMI) under iron overload conditions. Treatment of the patients with silymarin, which is an extract of milk thistle seed containing anti-hepatotoxic silibinin, led to a restoration of the GSH levels and PBMC proliferation, suggesting antioxidant and immunostimulatory activities and the benefits of using flavonoids to normalize immune dysfunction in β-thalas‐ semia major patients [115]. *In vitro* treatment of blood cells from β-thalassemia patients with *N*-acetylcysteine amide (AD4), NAC and vitamin C increased the reduced GSH content of RBC, platelets and PMN leukocytes, and reduced their ROS. Intra-peritoneal injection of AD4 to βthalassemic mice (150 mg/kg) reduced the parameters of oxidative stress (*p* <0.001). These may imply the superiority of AD4, compared to NAC, in reducing oxidative stress markers in thalassemic cells, both *in vitro* and *in vivo*, and also providing a significant reduced sensitivity of thalassemic RBC to hemolysis and phagocytosis by macrophages [116]. *In vitro* treatment of blood cells from β-thalassemia patients with fermented papaya preparation (FPP) increased the GSH content in the RBC, platelets and PMN leukocytes, and reduced their ROS, membrane lipid peroxidation and phosphatidylserine (PS) externalization. Importantly, oral administra‐ tion of FPP to β-thalassemia mice (50 mg/mouse/day for 3 months) and to thalassemia patients (3 g x 3 times/day for 3 months) reduced the levels of the oxidative stress parameters signifi‐ cantly. Suggestively, the FFP would be beneficial in reducing thalassemic RBC sensitivity to hemolysis and phagocytosis by macrophages, improving PMN ability to generate the oxida‐

tive burst and to reduce the platelet tendency to undergo activation [117].

Treatment of thalassemic RBC with erythropoietin (Epo) increased their GSH content and reduced their ROS, membrane lipid hydroperoxides, and PS exposure. Injection of Epo into heterozygous β-thalassemia mice reduced the oxidative markers. Probably, Epo might likely be an antioxidant that can alleviate symptoms of hemolytic anemia and reduced susceptibility

**5.1. Administration in thalassemia patients**

teracted the lipid peroxidation processes [114].

3) L-Arginine is a nitric oxide generator that can improve vascular function and regulate NOS expression and synthesis.

4) Thiol-containing compounds (e.g. α-lipoic acid and NAC) potentially inhibit LDL oxidation, decrease oxidative stress, and attenuate hypertension, insulin resistance and oxidative stress.

5) Estrogen and hormone replacement therapy can reduce the morbidity and mortality associated with CVD, lower production of superoxide radicals, up-regulate NOS gene expression, activate cyclo-oxygenase (COX) gene and reduce the production of vasoconstrictor endothelin.

6) Cu/Zn SOD (MW = 31 kD) mimetics are selective synthetic compounds; for example, M40401 (MW = 483), M40403 (MW = 501), SC-55858, that either inhibit their formation or remove superoxide anion [108].

7) Xanthine oxidase inhibitors (e.g. oxypurinol and allopurinol) improve endothelial function in hypercholesterolemic subjects, type II diabetic mellitus (DM) patients, congestive heart failure (CHF) and cigarette smokers.  **Pharmacology and Nutritional Intervention in the Treatment of Disease** 

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**Figure 5** Drugs used for therapeutic purposes exhibits antioxidant activity **Figure 5.** Drugs used for therapeutic purposes exhibits antioxidant activity

**4.1 Administration in thalassemia patients**

**4. Benefits of antioxidants in thalassemia patients**
