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

#### **5.1. Administration in thalassemia patients**

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.

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

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

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

7) Xanthine oxidase inhibitors (e.g. oxypurinol and allopurinol) improve endothelial function in hypercholesterolemic subjects, type II diabetic mellitus (DM) patients, congestive heart

**Apocynin S17834 Simvastatin** 

**Enalapril Losartan Amlodipine** 

 **Estrogen M40401 Allopurinol** *N***-Acetylcysteine** 


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

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

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

**4.1 Administration in thalassemia patients**

16

 **Pharmacology and Nutritional Intervention in the Treatment of Disease** 

expression and synthesis.

130 Pharmacology and Nutritional Intervention in the Treatment of Disease

superoxide anion [108].

failure (CHF) and cigarette smokers.

endothelin.

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‐ teracted the lipid peroxidation processes [114].

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 of RBC to undergo hemolysis and phagocytosis [118]. Thalassemic lymphocytes exhibited approximately a two-fold increase in the sensitivity to treatment of food mutagen/carcinogen, 3-amino-1-methyl-5H-pyrido(4,3-b)indole (or Trp-P-2) *in vitro*. However, treatment with flavonoids (quercetin and kaempferol) reduced the responses to Trp-P-2 to untreated control levels, significantly [119]. Transfusion-dependent thalassaemia patients were vitamin E deficient (0.45±0.21 mg/dl in all patients, 0.43±0.19 mg/dl in splenectomized patients), when compared to healthy subjects (0.76±0.22 mg/dl). Replacement therapy with vitamin E is necessary to correct its low serum levels easily (0.36±0.13 mg/dl before therapy and 1.19±0.35 mg/dl after therapy) [120]. Paraoxonase (PON) and arylesterase activities were significantly lower in β-thalassemia major patients than in the control healthy subjects, whereas total oxidant status, total peroxide concentration levels, and the oxidative stress index were significantly higher in the patients than in the controls in reference [121]. In addition, the activity of prolidase, a hydrolytic enzyme involved in collagen degradation was significantly increased in β-thalassemia major patients (53.7±8.7 U/l) compared to the control group (49.2±7.2 U/l). Total oxidant status was significantly increased in the patients (5.31±3.14 mmol H2O2 equivalent/l) compared to the controls (3.49±2.98 µmol H2O2 equivalent/l). Oxidative stress index was significantly increased in the patients (3.86±3.28 arbitrary unit) compared to the controls (2.53±2.70 arbitrary unit), while antioxidant capacity expressed as TEAC (1.61±0.30 µM) in the patients' plasma and this was not significantly different from that (1.64±0.33 µM) of the controls' plasma [122]. Administration of lipophilic antioxidant vitamin E (10 mg/kg/day for 4 weeks) is beneficial in the management of transfusion-dependant β-thalas‐ semia HbE patients [123]. A current study has demonstrated that levels of iron parameters, such as serum ferritin, NTBI and transferrin receptors, were significantly increased in βthalassemia major and thalassemia intermedia patients, compared to the controls and in severe cases when compared to the mild cases. Levels of serum ferritin, MDA, NTBI and GSSG/GSH were significantly increased in thalassemia intermedia patients; activities of serum glutathione reductase (GR), GPx and SOD were significantly reduced in these patients; while serum ascorbic acid concentrations were mildly reduced in the patients [124]. Interestingly, DFP has been reported to be a potent pharmaceutical antioxidant [125].

**5.2. Intervention of antioxidants in β-knockout (BKO) thalassemia mice**

wild type (WT) C57BL/6, β-knock (BKO) thalassemia (Hbβth-3/Hbβ<sup>+</sup>

(DH) and rescued β-thalassemia Hb E (LCRε<sup>G</sup>γ<sup>A</sup>γδβE) Hbβ<sup>0</sup>

/Hbβ<sup>+</sup>

(LCRε<sup>G</sup>γ<sup>A</sup>γδβE) Hbβ<sup>+</sup>

**WT liver iron**

0

2

4

6

8

10

12

**Iron accumulation (mg/g dry weight)**

**TG liver iron**

(0.28+0.03) (0.35+0.08)

**BKO liver iron**

**DH liver iron**

mia and double heterozygous (DH) thalassemia mice (S. Srichairatanakool, unpublished data)

(1.29+0.46)

**WT splenic iron**

**Figure 6.** Iron deposition in the livers and spleens of the wild type (WT), transgenic (TG), β-knockout (BKO) thalasse‐

(0.56+0.34)

**TG splenic iron**

(1.77+0.62)

**BKO splenic iron**

(1.50+0.31)

**DH splenic iron**

Nowadays, all clinical trials in humans and animals need to be approved by highly experienced ethical committees. Accurate animal models that recapitulate the phenotype and genotype of patients with β-thalassemia would enable researchers to develop possible therapeutic ap‐ proaches. In this case thalassemic mice have been developed by groups of researchers [132-135]. The Thalassemia Research Center at Mahidol University, Salaya Campus in collaboration with the Murdoch Children Research Institute Melbourne, Australia have inbred

order to investigate the properties of the potential antioxidants and iron-chelating agents.

) (HT HbE), double heterozygous (LCRε<sup>G</sup>γ<sup>A</sup>γδβE) Hbβ<sup>+</sup>

/Hbβ<sup>0</sup>

(7.18+1.80)

Antioxidants as Complementary Medication in Thalassemia

), β-thalasssemia/HbE

) (rescued β/HbE) mice in

http://dx.doi.org/10.5772/57372

(3.73+1.94)

/Hbβth-3)

133

Patients with Hb H disease, β-thalassemia/Hb E and β-thalassemia major had vitamin E deficiency; however, supplements of vitamin E and selenium to the patients prevented RBC oxidation and increased RBC resistance to oxidative damage [126, 127]. There were no significant differences in the mean serum vitamin A, E concentrations and the vitamin E/ cholesterol ratio between pregnant women with normal hemoglobin and hemoglobinopathies (Hb E and thalassemia) [128]. Patients with β<sup>0</sup> -thalassemia/Hb E disease with lower blood Hb concentration had significantly higher erythrocyte SOD activity and the Hb concentrations were inversely correlated with the SOD activities (*p* <0.001) [129]. Supplementation of vitamin C plus vitamin E has greater benefits than vitamin E alone in promoting antioxidant status [130]. Administration of CoQ10 (100 mg/day) for six months markedly increased activities of plasma SOD, CAT and GPx, and decreased plasma MDA concentration of β-thalassemia HbE patients [131]. Increased oxidative stress in β-thalassemia/Hb E patients relates to higher levels of MDA, SOD and GPx in RBC, serum NTBI, and lower level of RBC GSH.

#### **5.2. Intervention of antioxidants in β-knockout (BKO) thalassemia mice**

of RBC to undergo hemolysis and phagocytosis [118]. Thalassemic lymphocytes exhibited approximately a two-fold increase in the sensitivity to treatment of food mutagen/carcinogen, 3-amino-1-methyl-5H-pyrido(4,3-b)indole (or Trp-P-2) *in vitro*. However, treatment with flavonoids (quercetin and kaempferol) reduced the responses to Trp-P-2 to untreated control levels, significantly [119]. Transfusion-dependent thalassaemia patients were vitamin E deficient (0.45±0.21 mg/dl in all patients, 0.43±0.19 mg/dl in splenectomized patients), when compared to healthy subjects (0.76±0.22 mg/dl). Replacement therapy with vitamin E is necessary to correct its low serum levels easily (0.36±0.13 mg/dl before therapy and 1.19±0.35 mg/dl after therapy) [120]. Paraoxonase (PON) and arylesterase activities were significantly lower in β-thalassemia major patients than in the control healthy subjects, whereas total oxidant status, total peroxide concentration levels, and the oxidative stress index were significantly higher in the patients than in the controls in reference [121]. In addition, the activity of prolidase, a hydrolytic enzyme involved in collagen degradation was significantly increased in β-thalassemia major patients (53.7±8.7 U/l) compared to the control group (49.2±7.2 U/l). Total oxidant status was significantly increased in the patients (5.31±3.14 mmol H2O2 equivalent/l) compared to the controls (3.49±2.98 µmol H2O2 equivalent/l). Oxidative stress index was significantly increased in the patients (3.86±3.28 arbitrary unit) compared to the controls (2.53±2.70 arbitrary unit), while antioxidant capacity expressed as TEAC (1.61±0.30 µM) in the patients' plasma and this was not significantly different from that (1.64±0.33 µM) of the controls' plasma [122]. Administration of lipophilic antioxidant vitamin E (10 mg/kg/day for 4 weeks) is beneficial in the management of transfusion-dependant β-thalas‐ semia HbE patients [123]. A current study has demonstrated that levels of iron parameters, such as serum ferritin, NTBI and transferrin receptors, were significantly increased in βthalassemia major and thalassemia intermedia patients, compared to the controls and in severe cases when compared to the mild cases. Levels of serum ferritin, MDA, NTBI and GSSG/GSH were significantly increased in thalassemia intermedia patients; activities of serum glutathione reductase (GR), GPx and SOD were significantly reduced in these patients; while serum ascorbic acid concentrations were mildly reduced in the patients [124]. Interestingly, DFP has

132 Pharmacology and Nutritional Intervention in the Treatment of Disease

been reported to be a potent pharmaceutical antioxidant [125].

(Hb E and thalassemia) [128]. Patients with β<sup>0</sup>

Patients with Hb H disease, β-thalassemia/Hb E and β-thalassemia major had vitamin E deficiency; however, supplements of vitamin E and selenium to the patients prevented RBC oxidation and increased RBC resistance to oxidative damage [126, 127]. There were no significant differences in the mean serum vitamin A, E concentrations and the vitamin E/ cholesterol ratio between pregnant women with normal hemoglobin and hemoglobinopathies

concentration had significantly higher erythrocyte SOD activity and the Hb concentrations were inversely correlated with the SOD activities (*p* <0.001) [129]. Supplementation of vitamin C plus vitamin E has greater benefits than vitamin E alone in promoting antioxidant status [130]. Administration of CoQ10 (100 mg/day) for six months markedly increased activities of plasma SOD, CAT and GPx, and decreased plasma MDA concentration of β-thalassemia HbE patients [131]. Increased oxidative stress in β-thalassemia/Hb E patients relates to higher levels

of MDA, SOD and GPx in RBC, serum NTBI, and lower level of RBC GSH.


Nowadays, all clinical trials in humans and animals need to be approved by highly experienced ethical committees. Accurate animal models that recapitulate the phenotype and genotype of patients with β-thalassemia would enable researchers to develop possible therapeutic ap‐ proaches. In this case thalassemic mice have been developed by groups of researchers [132-135]. The Thalassemia Research Center at Mahidol University, Salaya Campus in collaboration with the Murdoch Children Research Institute Melbourne, Australia have inbred wild type (WT) C57BL/6, β-knock (BKO) thalassemia (Hbβth-3/Hbβ<sup>+</sup> ), β-thalasssemia/HbE (LCRε<sup>G</sup>γ<sup>A</sup>γδβE) Hbβ<sup>+</sup> /Hbβ<sup>+</sup> ) (HT HbE), double heterozygous (LCRε<sup>G</sup>γ<sup>A</sup>γδβE) Hbβ<sup>+</sup> /Hbβth-3) (DH) and rescued β-thalassemia Hb E (LCRε<sup>G</sup>γ<sup>A</sup>γδβE) Hbβ<sup>0</sup> /Hbβ<sup>0</sup> ) (rescued β/HbE) mice in order to investigate the properties of the potential antioxidants and iron-chelating agents.

**Figure 6.** Iron deposition in the livers and spleens of the wild type (WT), transgenic (TG), β-knockout (BKO) thalasse‐ mia and double heterozygous (DH) thalassemia mice (S. Srichairatanakool, unpublished data)

Having measured the amounts of tissue iron of the WT and thalassemia mice, the BKO mice had the highest iron content accumulated in the livers and spleens when compared to the TG mice and WT mice, which the splenic iron content was far higher than the liver iron content (Figure 7). The results indicated that anemic conditions persisting in the BKO mice were similar to β-thalassemia intermedia and enhanced an increase of duodenal iron absorption for accelerating erythropoiesis. In our experimentations, feeding the BKO mice with a high iron diet (such as iron ferrocene) can load iron into their blood and tissue compartments, leading to iron overload and oxidative tissue damage. Their hematological parameters were deter‐ mined and are illustrated in Table 2 [136]. Most importantly, the BKO thalassemia mice were found to be the most anemic when compared to the other types of thalassemia mice and their phenotype mimiced the human thalassemia intermedia patients. The mice heterozygous for deletion of the β-globin gene appear normal, but their hematologic indices show the charac‐ teristics that were typical of thalassemia intermedia. These include dramatically decreased hematocrit (Hct), hemoglobin (Hb) and red blood cell counts [136]. Bone deformities and splenic enlargement due to increased hematopoiesis [137], and iron overload in the vital organs (e.g. spleen, liver and kidneys) were also found in the heterozygous β-thalassemic mice, in reference [138].

 **Pharmacology and Nutritional Intervention in the Treatment of Disease** 

 **Curcumin Demethoxycurcumin** *Bis***-demethoxycurcumin Figure 6** Chemical structures of curcumin, demethoxycurcumin and *bis*-demethoxycurcumin found in turmeric *Curcuma longa* Linn (Redrawn from http://www.molecular-cancer.com/18

**Figure 7.** Chemical structures of curcumin, demethoxycurcumin and *bis*-demethoxycurcumin found in turmeric Curcu‐

Interestingly, curcumin can bind ferric and ferrous ions, in a concentration-dependent manner and with a molar ratio of 1:1, to form a red colored complex with a predominant peak at 500 nm. The curcumin itself chelates biological iron (e.g. plasma NTBI) and can work with DFP in lowering plasma NTBI levels more efficiently *in vitro* [139]. Curcuminoids effectively reduced levels of plasma NTBI, and liver weight index, non-heme iron, plasma and liver MDA concentrations, but increased the plasma GSH concentrations of iron-loaded BKO thalassemic mice [138]. Our results of the atomic absorption spectrometric and Perl's staining examinations demonstrated that curcumin was able to decrease the accumulation of heart iron and to depress heart rate variability of BKO mice with iron overload, suggesting the cardioprotective effect of curcumin [140]. Treatment of β-thalassemia HbE patients with curcuminoids (Thailand GPO product, 500 mg/day) for three months increased erythrocyte SOD as well as the GPx activities and GSH concentrations, and lowered levels of plasma NTBI and platelet factor-3 like activity [141, 142]. The same researcher group has been using a cocktail containing DFP, vitamin E, NAC and the curcuminoids for treatment of the thalassemia patients and expected that the treatment would improve their iron overload and oxidative stress more effectively (Kalpra‐ vidh and coworkers, personal communication). A recent study has reported that iron content of HDL-2 and HDL-3 from β-thalassemia/HbE patients was higher while the cholesterol content was lower than those levels found in the healthy subjects. Thalassemic HDL-2 and HDL-3 had increased levels of conjugated diene, lipid hydroperoxide and TBARS. The thalassemic HDL had lower peroxidase activity than the control HDL and could not protect

Curcuminoids derived from *Curcuma longa* Linn. (turmeric) contained curcumin (diferuloylmethane), demethoxycurcumin (*p*,*p*'-dihydroxyldicinnamoylmethane), as well as *bis*-demethoxycurcumin (*p*-hydroxylcinnamoylmethane), which curcumin was found to be the most abundant and major active compound (**Figure 6**). Phenolic, methylene and diketo groups in the curcumin molecule participated in antioxidant, iron-chelating, free

Antioxidants as Complementary Medication in Thalassemia

http://dx.doi.org/10.5772/57372

135

Interestingly, curcumin can bind ferric and ferrous ions, in a concentrationdependent manner and with a molar ratio of 1:1, to form a red colored complex with a predominant peak at 500 nm. The curcumin itself chelates biological iron (e.g. plasma NTBI) and can work with DFP in lowering plasma NTBI levels more efficiently *in vitro* [139]. Curcuminoids effectively reduced levels of plasma NTBI, and liver weight index, non-heme iron, plasma and liver MDA concentrations, but increased the plasma GSH concentrations of iron-loaded BKO thalassemic mice [138]. Our results of the atomic absorption spectrometric and Perl's staining examinations demonstrated that curcumin was able to decrease the accumulation of heart iron and to depress heart rate variability of BKO mice with iron overload, suggesting the cardioprotective effect of curcumin [140]. Treatment of thalassemia HbE patients with curcuminoids (Thailand GPO product, 500 mg/day) for three months increased erythrocyte SOD as well as the GPx activities and GSH concentrations, and lowered levels of plasma NTBI and platelet factor-3 like activity [141, 142]. The same researcher group has been using a cocktail containing DFP, vitamin E, NAC and the curcuminoids for treatment of the thalassemia patients and expected that the treatment would improve their iron overload and oxidative stress more effectively (Kalpravidh and coworkers, personal communication). A recent study has reported that iron content of HDL-2 and HDL-3 from -thalassemia/HbE patients was higher while the cholesterol content was lower than those levels found in the healthy subjects. Thalassemic HDL-2 and HDL-3 had increased levels of conjugated diene, lipid hydroperoxide and TBARS. The thalassemic HDL had lower peroxidase activity than the control HDL and could not protect against CuSO4-

*Curcuminoids* 

September 2013)

induced oxidation of LDL [143].

against CuSO4-induced oxidation of LDL [143].

*Tea*

*5.2.2. Tea*

radical-scavenging and anti-lipid peroxidation activities.

*ma longa* Linn (Redrawn from http://www.molecular-cancer.com/18 September 2013)

21

Recently, the demand for green tea has increased due to current trends in human health concerns and preference. The main components found in green tea are polysaccharides, flavonoids, vitamins B, C, E, γ-aminobutyric acid, catechin compounds and fluoride. Among them, catechin compounds have been of focus for their strong antioxidant capacity. The pharmaceutical activities of the components have been studied. Tea (*Camellia sinensis*) is an excellent source of polyphenols, namely catechins, including (-)-epicatechin (EC), (-)-epicate‐ chin 3-gallate (ECG), (-)-epigallocatechin (EGC), (-)-epigallocatechin 3-gallate (EGCG), (+) catechin (C) and (-)-gallocatechin (GC). Among them, EGCG exerted the strongest antioxidant capacity and was found to be the most abundant, as well. It has been reported that catechins possess free radical scavenging abilities and iron chelating properties [144]. Green tea also

showed a protective effect under various oxidative-related pathologic conditions.

Recently, the demand for green tea has increased due to current trends in human health concerns and preference. The main components found in green tea are polysaccharides, flavonoids, vitamins B, C, E, -aminobutyric acid, catechin compounds and fluoride. Among them, catechin compounds have been of focus for their strong antioxidant capacity. The pharmaceutical activities of the components have been studied. Tea (*Camellia* 


Abbreviations: Hb = hemoglobin, Hct = hematocrit, MCV = mean corpuscular volume, MCH = mean corpuscular hemoglobin, RDW = RBC distribution width, ROS = reactive oxygen species, PS = phosphatidylserine.

**Table 2.** Hematological parameters (mean±SD) of erythrocyte WT and β-thalassemia mice (Redrawn from Wannasuphaphol et al. [136]

#### *5.2.1. Curcuminoids*

Curcuminoids derived from *Curcuma longa* Linn. (turmeric) contained curcumin (diferuloyl‐ methane), demethoxycurcumin (*p*,*p*'-dihydroxyldicinnamoylmethane), as well as *bis*-deme‐ thoxycurcumin (*p*-hydroxylcinnamoylmethane), which curcumin was found to be the most abundant and major active compound (Figure 7). Phenolic, methylene and β-diketo groups in the curcumin molecule participated in antioxidant, iron-chelating, free radical-scavenging and anti-lipid peroxidation activities.

(diferuloylmethane), demethoxycurcumin (*p*,*p*'-dihydroxyldicinnamoylmethane), as well as *bis*-demethoxycurcumin (*p*-hydroxylcinnamoylmethane), which curcumin was found to be the most abundant and major active compound (**Figure 6**). Phenolic, methylene and - Antioxidants as Complementary Medication in Thalassemia http://dx.doi.org/10.5772/57372 135

diketo groups in the curcumin molecule participated in antioxidant, iron-chelating, free

radical-scavenging and anti-lipid peroxidation activities.

 **Pharmacology and Nutritional Intervention in the Treatment of Disease** 

Curcuminoids derived from *Curcuma longa* Linn. (turmeric) contained curcumin

*Curcuminoids* 

**Figure 6** Chemical structures of curcumin, demethoxycurcumin and *bis*-demethoxycurcumin found in turmeric *Curcuma longa* Linn (Redrawn from http://www.molecular-cancer.com/18 September 2013) **Figure 7.** Chemical structures of curcumin, demethoxycurcumin and *bis*-demethoxycurcumin found in turmeric Curcu‐ *ma longa* Linn (Redrawn from http://www.molecular-cancer.com/18 September 2013)

Interestingly, curcumin can bind ferric and ferrous ions, in a concentrationdependent manner and with a molar ratio of 1:1, to form a red colored complex with a predominant peak at 500 nm. The curcumin itself chelates biological iron (e.g. plasma NTBI) and can work with DFP in lowering plasma NTBI levels more efficiently *in vitro* [139]. Curcuminoids effectively reduced levels of plasma NTBI, and liver weight index, non-heme iron, plasma and liver MDA concentrations, but increased the plasma GSH concentrations of iron-loaded BKO thalassemic mice [138]. Our results of the atomic absorption spectrometric and Perl's staining examinations demonstrated that curcumin was able to decrease the accumulation of heart iron and to depress heart rate variability of BKO mice with iron overload, suggesting the cardioprotective effect of curcumin [140]. Treatment of thalassemia HbE patients with curcuminoids (Thailand GPO product, 500 mg/day) for three months increased erythrocyte SOD as well as the GPx activities and GSH concentrations, and lowered levels of plasma NTBI and platelet factor-3 like activity [141, 142]. The same researcher group has been using a cocktail containing DFP, vitamin E, NAC and the curcuminoids for treatment of the thalassemia patients and expected that the treatment would improve their iron overload and oxidative stress more effectively (Kalpravidh and coworkers, personal communication). A recent study has reported that iron content of HDL-2 and HDL-3 from -thalassemia/HbE patients was higher while the cholesterol content was lower than those levels found in the healthy subjects. Thalassemic HDL-2 and HDL-3 had increased levels of conjugated diene, lipid hydroperoxide and TBARS. The thalassemic HDL had lower peroxidase activity than the control HDL and could not protect against CuSO4 induced oxidation of LDL [143]. *Tea* Recently, the demand for green tea has increased due to current trends in human health concerns and preference. The main components found in green tea are polysaccharides, flavonoids, vitamins B, C, E, -aminobutyric acid, catechin compounds and fluoride. Among them, catechin compounds have been of focus for their strong antioxidant capacity. The pharmaceutical activities of the components have been studied. Tea (*Camellia*  Interestingly, curcumin can bind ferric and ferrous ions, in a concentration-dependent manner and with a molar ratio of 1:1, to form a red colored complex with a predominant peak at 500 nm. The curcumin itself chelates biological iron (e.g. plasma NTBI) and can work with DFP in lowering plasma NTBI levels more efficiently *in vitro* [139]. Curcuminoids effectively reduced levels of plasma NTBI, and liver weight index, non-heme iron, plasma and liver MDA concentrations, but increased the plasma GSH concentrations of iron-loaded BKO thalassemic mice [138]. Our results of the atomic absorption spectrometric and Perl's staining examinations demonstrated that curcumin was able to decrease the accumulation of heart iron and to depress heart rate variability of BKO mice with iron overload, suggesting the cardioprotective effect of curcumin [140]. Treatment of β-thalassemia HbE patients with curcuminoids (Thailand GPO product, 500 mg/day) for three months increased erythrocyte SOD as well as the GPx activities and GSH concentrations, and lowered levels of plasma NTBI and platelet factor-3 like activity [141, 142]. The same researcher group has been using a cocktail containing DFP, vitamin E, NAC and the curcuminoids for treatment of the thalassemia patients and expected that the treatment would improve their iron overload and oxidative stress more effectively (Kalpra‐ vidh and coworkers, personal communication). A recent study has reported that iron content of HDL-2 and HDL-3 from β-thalassemia/HbE patients was higher while the cholesterol content was lower than those levels found in the healthy subjects. Thalassemic HDL-2 and HDL-3 had increased levels of conjugated diene, lipid hydroperoxide and TBARS. The thalassemic HDL had lower peroxidase activity than the control HDL and could not protect against CuSO4-induced oxidation of LDL [143].

#### *5.2.2. Tea*

Having measured the amounts of tissue iron of the WT and thalassemia mice, the BKO mice had the highest iron content accumulated in the livers and spleens when compared to the TG mice and WT mice, which the splenic iron content was far higher than the liver iron content (Figure 7). The results indicated that anemic conditions persisting in the BKO mice were similar to β-thalassemia intermedia and enhanced an increase of duodenal iron absorption for accelerating erythropoiesis. In our experimentations, feeding the BKO mice with a high iron diet (such as iron ferrocene) can load iron into their blood and tissue compartments, leading to iron overload and oxidative tissue damage. Their hematological parameters were deter‐ mined and are illustrated in Table 2 [136]. Most importantly, the BKO thalassemia mice were found to be the most anemic when compared to the other types of thalassemia mice and their phenotype mimiced the human thalassemia intermedia patients. The mice heterozygous for deletion of the β-globin gene appear normal, but their hematologic indices show the charac‐ teristics that were typical of thalassemia intermedia. These include dramatically decreased hematocrit (Hct), hemoglobin (Hb) and red blood cell counts [136]. Bone deformities and splenic enlargement due to increased hematopoiesis [137], and iron overload in the vital organs (e.g. spleen, liver and kidneys) were also found in the heterozygous β-thalassemic mice, in

reference [138].

RBC (x106 cells/μl)

Hb (g/dl)

134 Pharmacology and Nutritional Intervention in the Treatment of Disease

Hct (%)

RDW = RBC distribution width, ROS = reactive oxygen species, PS = phosphatidylserine.

MCV (fl)

MCH (pg)

8.87+0.87 11.08±1.17 38.02±3.54 42.93±2.06 12.52±0.51 20.03±1.43 15.12±3.33 117.4±67.2 1.24±0.72

**WT** 8.37±0.54 13.35±0.85 39.32±3.22 46.95±1.41 15.96±0.48 12.64±1.25 3.88±0.74 7.51±3.85 1.06±0.56 **HT Hb E** 7.95±0.36 12.24±0.56 35.69±1.58 44.93±0.84 15.40±0.35 14.47±0.79 4.65±0.86 3.28±0.43 0.87±0.29 **BKO** 5.34±0.63 7.06±0.61 22.86±2.05 43.05±2.95 13.32±1.09 23.17±2.53 29.01±5.76 170.1±24.6 3.35±0.75 **DH** 8.32±0.51 12.81±1.09 38.55±3.15 46.34±2.33 15.41±0.96 14.15±1.16 3.64±1.70 13.86±2.98 1.07±0.35

Abbreviations: Hb = hemoglobin, Hct = hematocrit, MCV = mean corpuscular volume, MCH = mean corpuscular hemoglobin,

Curcuminoids derived from *Curcuma longa* Linn. (turmeric) contained curcumin (diferuloyl‐ methane), demethoxycurcumin (*p*,*p*'-dihydroxyldicinnamoylmethane), as well as *bis*-deme‐ thoxycurcumin (*p*-hydroxylcinnamoylmethane), which curcumin was found to be the most abundant and major active compound (Figure 7). Phenolic, methylene and β-diketo groups in the curcumin molecule participated in antioxidant, iron-chelating, free radical-scavenging and

**Table 2.** Hematological parameters (mean±SD) of erythrocyte WT and β-thalassemia mice (Redrawn from

RDW (%)

Reticulocyte (%)

ROS (Mean FI) PS cell (%)

**Mice**

**Rescued β/ Hb E**

Wannasuphaphol et al. [136]

anti-lipid peroxidation activities.

*5.2.1. Curcuminoids*

Recently, the demand for green tea has increased due to current trends in human health concerns and preference. The main components found in green tea are polysaccharides, flavonoids, vitamins B, C, E, γ-aminobutyric acid, catechin compounds and fluoride. Among them, catechin compounds have been of focus for their strong antioxidant capacity. The pharmaceutical activities of the components have been studied. Tea (*Camellia sinensis*) is an excellent source of polyphenols, namely catechins, including (-)-epicatechin (EC), (-)-epicate‐ chin 3-gallate (ECG), (-)-epigallocatechin (EGC), (-)-epigallocatechin 3-gallate (EGCG), (+) catechin (C) and (-)-gallocatechin (GC). Among them, EGCG exerted the strongest antioxidant capacity and was found to be the most abundant, as well. It has been reported that catechins possess free radical scavenging abilities and iron chelating properties [144]. Green tea also showed a protective effect under various oxidative-related pathologic conditions.

21

pathologic conditions.

Drinking tea produced a 41 – 95% inhibition of dietary iron absorption in five -

 **Pharmacology and Nutritional Intervention in the Treatment of Disease** 

*sinensis*) is an excellent source of polyphenols, namely catechins, including (-)-epicatechin (EC), (-)-epicatechin 3-gallate (ECG), (-)-epigallocatechin (EGC), (-)-epigallocatechin 3-

been reported that catechins possess free radical scavenging abilities and iron chelating properties [144]. Green tea also showed a protective effect under various oxidative-related

> U/ml of SOD. This also revealed the pharmacologic effects modulating gene expression that were related to the inflammatory response [152]. Mangiferin xanthone modulates the expres‐ sion of many genes critical for apoptosis regulation, viral replication, tumorigenesis, inflam‐ mation and autoimmune diseases, suggesting its possible value in the treatment of inflammatory diseases and/or cancer [153]. Vimang mango peel extract with a high mangiferin content acted as an antioxidant and complexed with Fe3+ efficiently, leading to protection of iron-induced oxidative liver damage and DNA fragmentation [154, 155]. Hydrolysable gallotannin present in mango kernels showed the inhibitory effects of bacterial growth, which

Mangoes can be considered a good source of dietary antioxidants, such as ascorbic acid, carotenoids and phenolic compounds. *β*-carotene was found to be the most abundant carotenoid in several cultivars. The nutritional value of mango is that it is a source of vitamin C and provitamin A. Mangiferin (1,3,6,7-tetrahydroxyxanthone-2-glucopyranoside) (**Figure 8**) can interact with iron and other cations and also shows antioxidant activity by eliminating the superoxide radical *in vitro*, in which 100 μM of mangiferin was equivalent to the activity of 1 U/ml of SOD. This also revealed the pharmacologic effects modulating gene expression that were related to the inflammatory response [152]. Mangiferin xanthone modulates the expression of many genes critical for apoptosis regulation, viral replication, tumorigenesis, inflammation and autoimmune diseases, suggesting its possible value in the treatment of inflammatory diseases and/or cancer [153]. Vimang mango peel extract with a high mangiferin content acted as an antioxidant and complexed with Fe3+ efficiently, leading to protection of iron-induced oxidative liver damage and DNA fragmentation [154, 155]. Hydrolysable gallotannin present in mango kernels showed the inhibitory effects of bacterial

O OH

O

OH

Antioxidants as Complementary Medication in Thalassemia

http://dx.doi.org/10.5772/57372

137

Glc

HO

OH

Green peel Kaew (OD 534 nm) Ripe peel Kaew (OD 572 nm)

> 6.25 12.5 25 50 100 200 Mango extract (g/ml)

**Iron-chelating activity**

6.25 12.5 25 50 100 200 Mango extract (g/ml)

23

**Absorbance unit**

0.000 .025 .050 .075 .100 .125 .150 .175 .200

As shown in **Table 2**, the degree of antioxidant activity of the mango peel extracts

**Figure 9** Iron-chelating activity of aqueous extract of Kaew mango peel (Srichairatanakool,

**Figure 10.** Iron-chelating activity of aqueous extract of Kaew mango peel (Srichairatanakool, S. unpublished data)

were found to be Mahajanaka (ripe > green) > Chok-anan (green > ripe) > Namdocmai (green > ripe) > Kaew (ripe green). The antioxidant activities were well correlated with their total phenolic contents and vitamin C concentrations. Incredibly, amounts of vitamin C in the extracts were lower than those of the total phenolic compounds. Mangiferin contents in the

6.25 12.5 25 50 100 200 Mango extract (g/ml)

6.25 12.5 25 50 100 200 Mango extract (g/ml)

+ 200 M ferrous citrate + 200 M ferrous citrate + 200 M ferric NTA + 200 M ferric NTA

24

Ripe and green Mahajanaka mango mangiferin

Kaew mango peel extract can chelate both Fe3+ and Fe2+ to form the products with different predominant wavelengths, of which the binding was found to be dose-dependent and affinitydifferent. The green peel extract tended to exhibit stronger iron-binding abilities than the ripe peel extract and it is likely that the green peel might contain different kinds and amounts of

 **Pharmacology and Nutritional Intervention in the Treatment of Disease** 

Kaew mango peel extract can chelate both Fe3+ and Fe2+ to form the products with different predominant wavelengths, of which the binding was found to be dose-dependent and affinity-different. The green peel extract tended to exhibit stronger iron-binding abilities than the ripe peel extract and it is likely that the green peel might contain different kinds and

**Figure 8** Fresh mango and chemical structure of mangiferin (Redrawn from [157])

 **Pharmacology and Nutritional Intervention in the Treatment of Disease** 

was probably due to their iron-complexing properties [156].

amounts of phytochemical ingredients (**Figure 9**).

**Iron-chelating activity**

**Figure 9.** Fresh mango and chemical structure of mangiferin (Redrawn from [157])

1.0 mg/ml Green peel Kaew (OD 534 nm) 1.0 mg/ml Ripe peel Kaew (OD 572 nm)

> 6.25 12.5 25 50 100 200 Ferric NTA (M)

growth, which was probably due to their iron-complexing properties [156].

**Mango (***Mangifera indica* **L.)** 

(www.nanagarden.com)

phytochemical ingredients (Figure 10).

(www.nanagarden.com)

**Absorbance unit**

0.0

6.25 12.5 25 50 100 200 Ferrous citrate (M)

S. unpublished data)

peel extracts were very low.

6.25 12.5 25 50 100 200 Ferrous citrate (M)

.1

.2

.3

.4

.5

**Figure 7** Chemical structures of catechins in tea (*Camellia sinensis*) (Redrawn from [145]) **Figure 8.** Chemical structures of catechins in tea (*Camellia sinensis*) (Redrawn from [145])

22 thalassemia major and one -thalassemia intermedia cases, which the iron absorption increased strikingly in the -thalassemia intermedia cases, in references [146, 147]. Interestingly, Thai researchers have elucidated that green tea extract (GTE) and EGCG fraction were able to decrease iron (as NTBI) in plasma, eliminate plasma lipid-peroxidation product (as TBARS) and destroy the formation of erythrocyte ROS *in vitro* [144, 148] and in iron-loaded rats [149]. In addition, the GTE inhibited or delayed the deposition of hepatic iron in regularly iron-loaded BKO thalassemic mice effectively. This implies a prevention of ironinduced ROS generation and consequently liver damage and fibrosis by green tea consumption [150]. Our group has found that elevated levels of plasma NTBI and lipid peroxidation tended to be normalized in the BKO mice in response to oral therapy with GTE, while their plasma GSH concentrations were also increased by up to 2-times. The mice exhibited a decrease of the lipid peroxidation product and an improvement in the oxidant– antioxidant balance in erythrocytes. Importantly, GTE was effective in inhibiting hemolysis and thereby prolonged RBC half-life in the BKO mice (Sakaewan Ounjaijean PhD thesis. Chiang Mai University; 2011). Our current study has shown that the treatment of iron-loaded mouse hepatocytes and human hepatoma (HepG2) cells with GTE (0 – 100 mg/dl) and EGCG (0 – 200 M) removed intracellular LIP and ROS efficiently, and relieved the mitochondrial membrane collapse, implying a hepatoprotective effect of green tea catechins in the hepatocytes with iron overload [151]. Drinking tea produced a 41 – 95% inhibition of dietary iron absorption in five β-thalassemia major and one β-thalassemia intermedia cases, which the iron absorption increased strikingly in the β-thalassemia intermedia cases, in references [146, 147]. Interestingly, Thai researchers have elucidated that green tea extract (GTE) and EGCG fraction were able to decrease iron (as NTBI) in plasma, eliminate plasma lipid-peroxidation product (as TBARS) and destroy the formation of erythrocyte ROS *in vitro* [144, 148] and in iron-loaded rats [149]. In addition, the GTE inhibited or delayed the deposition of hepatic iron in regularly iron-loaded BKO thalas‐ semic mice effectively. This implies a prevention of iron-induced ROS generation and conse‐ quently liver damage and fibrosis by green tea consumption [150]. Our group has found that elevated levels of plasma NTBI and lipid peroxidation tended to be normalized in the BKO mice in response to oral therapy with GTE, while their plasma GSH concentrations were also increased by up to 2-times. The mice exhibited a decrease of the lipid peroxidation product and an improvement in the oxidant–antioxidant balance in erythrocytes. Importantly, GTE was effective in inhibiting hemolysis and thereby prolonged RBC half-life in the BKO mice (Sakaewan Ounjaijean PhD thesis. Chiang Mai University; 2011). Our current study has shown that the treatment of iron-loaded mouse hepatocytes and human hepatoma (HepG2) cells with GTE (0 – 100 mg/dl) and EGCG (0 – 200 µM) removed intracellular LIP and ROS efficiently, and relieved the mitochondrial membrane collapse, implying a hepatoprotective effect of green tea catechins in the hepatocytes with iron overload [151].

#### *5.2.3. Mango (Mangifera indica L.)*

Mangoes can be considered a good source of dietary antioxidants, such as ascorbic acid, carotenoids and phenolic compounds. *β*-carotene was found to be the most abundant carote‐ noid in several cultivars. The nutritional value of mango is that it is a source of vitamin C and provitamin A. Mangiferin (1,3,6,7-tetrahydroxyxanthone-2-glucopyranoside) (Figure 9) can interact with iron and other cations and also shows antioxidant activity by eliminating the superoxide radical *in vitro*, in which 100 µM of mangiferin was equivalent to the activity of 1 U/ml of SOD. This also revealed the pharmacologic effects modulating gene expression that were related to the inflammatory response [152]. Mangiferin xanthone modulates the expres‐ sion of many genes critical for apoptosis regulation, viral replication, tumorigenesis, inflam‐ mation and autoimmune diseases, suggesting its possible value in the treatment of inflammatory diseases and/or cancer [153]. Vimang mango peel extract with a high mangiferin content acted as an antioxidant and complexed with Fe3+ efficiently, leading to protection of iron-induced oxidative liver damage and DNA fragmentation [154, 155]. Hydrolysable gallotannin present in mango kernels showed the inhibitory effects of bacterial growth, which was probably due to their iron-complexing properties [156]. Mangoes can be considered a good source of dietary antioxidants, such as ascorbic acid, carotenoids and phenolic compounds. *β*-carotene was found to be the most abundant carotenoid in several cultivars. The nutritional value of mango is that it is a source of vitamin C and provitamin A. Mangiferin (1,3,6,7-tetrahydroxyxanthone-2-glucopyranoside) (**Figure 8**) can interact with iron and other cations and also shows antioxidant activity by eliminating the superoxide radical *in vitro*, in which 100 μM of mangiferin was equivalent to the activity of 1 U/ml of SOD. This also revealed the pharmacologic effects modulating gene expression that were related to the inflammatory response [152]. Mangiferin xanthone modulates the expression of many genes critical for apoptosis regulation, viral replication, tumorigenesis, inflammation and autoimmune diseases, suggesting its possible value in the treatment of inflammatory diseases and/or cancer [153]. Vimang mango peel extract with a high mangiferin content acted as an antioxidant and complexed with Fe3+ efficiently, leading to protection of iron-induced oxidative liver damage and DNA fragmentation [154, 155].

Hydrolysable gallotannin present in mango kernels showed the inhibitory effects of bacterial

growth, which was probably due to their iron-complexing properties [156].

Ripe and green Mahajanaka mango mangiferin

**Mango (***Mangifera indica* **L.)** 

(www.nanagarden.com) (www.nanagarden.com)

S. unpublished data)

peel extracts were very low.

 **Pharmacology and Nutritional Intervention in the Treatment of Disease** 

*sinensis*) is an excellent source of polyphenols, namely catechins, including (-)-epicatechin (EC), (-)-epicatechin 3-gallate (ECG), (-)-epigallocatechin (EGC), (-)-epigallocatechin 3 gallate (EGCG), (+)-catechin (C) and (-)-gallocatechin (GC). Among them, EGCG exerted the strongest antioxidant capacity and was found to be the most abundant, as well. It has been reported that catechins possess free radical scavenging abilities and iron chelating properties [144]. Green tea also showed a protective effect under various oxidative-related

**(-) Epicatechin (EC) (-) Epicatechin 3-gallate (ECG) (-) Epigallocatechin (EGC)** 

**(-) Epigallocatechin 3-gallate (EGCG) (+) Catechin (C) (-) Gallocatechin(GC) Figure 7** Chemical structures of catechins in tea (*Camellia sinensis*) (Redrawn from [145])

Drinking tea produced a 41 – 95% inhibition of dietary iron absorption in five β-thalassemia major and one β-thalassemia intermedia cases, which the iron absorption increased strikingly in the β-thalassemia intermedia cases, in references [146, 147]. Interestingly, Thai researchers have elucidated that green tea extract (GTE) and EGCG fraction were able to decrease iron (as NTBI) in plasma, eliminate plasma lipid-peroxidation product (as TBARS) and destroy the formation of erythrocyte ROS *in vitro* [144, 148] and in iron-loaded rats [149]. In addition, the GTE inhibited or delayed the deposition of hepatic iron in regularly iron-loaded BKO thalas‐ semic mice effectively. This implies a prevention of iron-induced ROS generation and conse‐ quently liver damage and fibrosis by green tea consumption [150]. Our group has found that elevated levels of plasma NTBI and lipid peroxidation tended to be normalized in the BKO mice in response to oral therapy with GTE, while their plasma GSH concentrations were also increased by up to 2-times. The mice exhibited a decrease of the lipid peroxidation product and an improvement in the oxidant–antioxidant balance in erythrocytes. Importantly, GTE was effective in inhibiting hemolysis and thereby prolonged RBC half-life in the BKO mice (Sakaewan Ounjaijean PhD thesis. Chiang Mai University; 2011). Our current study has shown that the treatment of iron-loaded mouse hepatocytes and human hepatoma (HepG2) cells with GTE (0 – 100 mg/dl) and EGCG (0 – 200 µM) removed intracellular LIP and ROS efficiently, and relieved the mitochondrial membrane collapse, implying a hepatoprotective effect of

Drinking tea produced a 41 – 95% inhibition of dietary iron absorption in five thalassemia major and one -thalassemia intermedia cases, which the iron absorption increased strikingly in the -thalassemia intermedia cases, in references [146, 147]. Interestingly, Thai researchers have elucidated that green tea extract (GTE) and EGCG fraction were able to decrease iron (as NTBI) in plasma, eliminate plasma lipid-peroxidation product (as TBARS) and destroy the formation of erythrocyte ROS *in vitro* [144, 148] and in iron-loaded rats [149]. In addition, the GTE inhibited or delayed the deposition of hepatic iron in regularly iron-loaded BKO thalassemic mice effectively. This implies a prevention of ironinduced ROS generation and consequently liver damage and fibrosis by green tea consumption [150]. Our group has found that elevated levels of plasma NTBI and lipid peroxidation tended to be normalized in the BKO mice in response to oral therapy with GTE, while their plasma GSH concentrations were also increased by up to 2-times. The mice exhibited a decrease of the lipid peroxidation product and an improvement in the oxidant– antioxidant balance in erythrocytes. Importantly, GTE was effective in inhibiting hemolysis and thereby prolonged RBC half-life in the BKO mice (Sakaewan Ounjaijean PhD thesis. Chiang Mai University; 2011). Our current study has shown that the treatment of iron-loaded mouse hepatocytes and human hepatoma (HepG2) cells with GTE (0 – 100 mg/dl) and EGCG (0 – 200 M) removed intracellular LIP and ROS efficiently, and relieved the mitochondrial membrane collapse, implying a hepatoprotective effect of green tea catechins

O

OH

HO

OH

OH OH

OH

pathologic conditions.

136 Pharmacology and Nutritional Intervention in the Treatment of Disease

**Figure 8.** Chemical structures of catechins in tea (*Camellia sinensis*) (Redrawn from [145])

in the hepatocytes with iron overload [151].

*5.2.3. Mango (Mangifera indica L.)*

green tea catechins in the hepatocytes with iron overload [151].

22

Mangoes can be considered a good source of dietary antioxidants, such as ascorbic acid, carotenoids and phenolic compounds. *β*-carotene was found to be the most abundant carote‐ noid in several cultivars. The nutritional value of mango is that it is a source of vitamin C and provitamin A. Mangiferin (1,3,6,7-tetrahydroxyxanthone-2-glucopyranoside) (Figure 9) can interact with iron and other cations and also shows antioxidant activity by eliminating the superoxide radical *in vitro*, in which 100 µM of mangiferin was equivalent to the activity of 1

and affinity-different. The green peel extract tended to exhibit stronger iron-binding abilities than the ripe peel extract and it is likely that the green peel might contain different kinds and amounts of phytochemical ingredients (**Figure 9**). Kaew mango peel extract can chelate both Fe3+ and Fe2+ to form the products with different predominant wavelengths, of which the binding was found to be dose-dependent and affinitydifferent. The green peel extract tended to exhibit stronger iron-binding abilities than the ripe peel extract and it is likely that the green peel might contain different kinds and amounts of phytochemical ingredients (Figure 10). **Pharmacology and Nutritional Intervention in the Treatment of Disease** 

Kaew mango peel extract can chelate both Fe3+ and Fe2+ to form the products with different predominant wavelengths, of which the binding was found to be dose-dependent

**Figure 9** Iron-chelating activity of aqueous extract of Kaew mango peel (Srichairatanakool, **Figure 10.** Iron-chelating activity of aqueous extract of Kaew mango peel (Srichairatanakool, S. unpublished data)

As shown in **Table 2**, the degree of antioxidant activity of the mango peel extracts

were found to be Mahajanaka (ripe > green) > Chok-anan (green > ripe) > Namdocmai (green > ripe) > Kaew (ripe green). The antioxidant activities were well correlated with their total phenolic contents and vitamin C concentrations. Incredibly, amounts of vitamin C in the extracts were lower than those of the total phenolic compounds. Mangiferin contents in the

24

**Figure 8** Fresh mango and chemical structure of mangiferin (Redrawn from [157]) **Figure 9.** Fresh mango and chemical structure of mangiferin (Redrawn from [157])

As shown in Table 2, the degree of antioxidant activity of the mango peel extracts were found to be Mahajanaka (ripe > green) > Chok-anan (green > ripe) > Namdocmai (green > ripe) > Kaew (ripe ~ green). The antioxidant activities were well correlated with their total phenolic contents and vitamin C concentrations. Incredibly, amounts of vitamin C in the extracts were lower than those of the total phenolic compounds. Mangiferin contents in the peel extracts were very low.


and nicotinamine, and sequesters iron from the ground for their growth and development [162]. Interestingly, wheat grass (WG) juice which has been used as a general-purpose health tonic in Indian medicine and has shown at least a 25% reduction in the number of blood transfusions needed in β-thalassemia major patients [163] and also increased their blood hemoglobin levels [164]. Nonetheless, a contradictory report has shown that the oral admin‐ istration of WG juice tablets for one year was not effective in reducing blood transfusions in

 Spinach pheophytin a (max 409 nm) and pheophytin b (max 435 nm) can chelate ferric ion and produce the Fe-pheophytin a complex (max 393 nm) and Fe-pheophytin b complex (max 421 nm) [161]. Germinating rice grain synthesizes iron-chelating compounds, deoxymugineic acid and nicotinamine, and sequesters iron from the ground for their growth and development [162]. Interestingly, wheat grass (WG) juice which has been used as a general-purpose health tonic in Indian medicine and has shown at least a 25% reduction in the number of blood transfusions needed in -thalassemia major patients [163] and also increased their blood hemoglobin levels [164]. Nonetheless, a contradictory report has shown that the oral administration of WG juice tablets for one year was not effective in reducing blood transfusions in transfusion-dependent -thalassemia major patients [165].

**Figure 10** Chemical structures of chlorophyll, pheophytin, iron-pheophytin complex and

**Chlorophyll Pheophytin Fe-pheophytin complex Heme** 

**Figure 11.** Chemical structures of chlorophyll, pheophytin, iron-pheophytin complex and heme [161]

http://dx.doi.org/10.5772/57372

139

Antioxidants as Complementary Medication in Thalassemia

 **Pharmacology and Nutritional Intervention in the Treatment of Disease** 

Rice is an economic cereal plant that is grown in many countries in Asian and Africa. Varieties of Asian rice include Thai rice (*O. sativa* cv. indica), Indonesian rice (*O. sativa* cv. javanica) and Japanese rice (*O. sativa* cv. japonica). Additionally, we have listed African rice (*O. glaberrima*). Regarding its nutritional values, rice grain contains carbohydrates (e.g. amylose and amylopectin) and rice bran is abundant in inositol, inositol hexaphosphate (or phytate), oil, ferulic acid, -oryzanol, phytosterol and tocotrienol. The ingredients in rice bran likely prevent carcinogenesis, hyperlipidemia, fatty liver, gallstone disease and heart diseases [158]. Red and purple rice bran possesses several fold higher hydrophilic and lipophilic anti-oxidative phenolics and flavonoids (predominantly cyanidine-3 glycoside) levels than freeze-dried blueberry and broccoli. Rice grass contains chlorophyll as a major ingredient and pheophytin (or pheo) as the second most abundant component (**Figure 10**). Pheophytin is synthesized in rice grass, spinach (*Spinacia oleracea*) leaves and *Michelia alba* leaves, but is not a degradation product of chlorophyll [159], and the compound reveals a level of antioxidant activity that is similar to that of ubiquinone [160].

Pimpilai and colleagues from Maejo University, Chiang Mai investigated the antioxidant activity of the Thai black rice grass (TBRG), Thai red rice grass (TRRG) and Thai fragrant rice grass (TFRG) extracts by using the TEAC colorimetric method and found that they exhibited antioxidant properties. The extracts dose dependently bound Fe3+ (ferric NTA) and Fe2+ (ferrous citrate) and formed the complex(s) with maximal absorption values at 393, 378 and 580 nm, respectively (personal communication). HPLC analysis showed different amounts of catechin derivatives as follows: 3.27 mg C, 4.51 mg EC and 5.19 mg EGCG in 1 g TBRG extract; 4.48 mg C, 6.22 mg EC and 5.23 mg EGCG in 1 g TRRG extract; and 5.89 mg C, 4.38 mg EC and 5.26 mg EGCG in 1 g TFRG extract. Currently, Pimpilai and Srichairatanakool have found that feeding rice grass (Sukhothai 1) extract (100 mg/kg body weight) along with a high iron diet to β-thalassemia mice for three months slightly increased their blood hemoglobin concentra‐ tions from 10.04±0.51 to 11.04±1.14 g/dl (unpublished data), while treatment with the extract (0, 50 and 100 µg/ml) decreased levels of liver MDA (153.6±44.7, 102.9±22.4 and 102.4±58.0 µg/ mg tissue protein, respectively) and levels of labile iron (100±13, 120±1 and 125±5% fluorescent intensity, respectively) in iron-loaded HepG2 cell cultures. Therefore, the rice grass extracts would contain antioxidant compounds including chlorophyll, pheophytin and catechin derivatives, of which the two latter may play important roles in iron chelation and anti-lipid peroxidation to ameliorate oxidative tissue damage in the thalassemia cases with iron over‐ load. Consequently, rice grass extracts need to be clinically studied in thalassemia patients in

Pimpilai and colleagues from Maejo University, Chiang Mai investigated the antioxidant activity of the Thai black rice grass (TBRG), Thai red rice grass (TRRG) and Thai fragrant rice grass (TFRG) extracts by using the TEAC colorimetric method and found that they exhibited antioxidant properties. The extracts dose dependently bound Fe3+ (ferric NTA) and Fe2+ (ferrous citrate) and formed the complex(s) with maximal absorption values at 393, 378 and 580 nm, respectively (personal communication). HPLC analysis showed different amounts of catechin derivatives as follows: 3.27 mg C, 4.51 mg EC and 5.19 mg EGCG in 1 g TBRG extract; 4.48 mg C, 6.22 mg EC and 5.23 mg EGCG in 1 g TRRG extract; and 5.89

26

**6. Evaluation of oxidative stress and antioxidant status in thalassemias**

Electron paramagnetic resonance (EPR) 'radical probe' was used to determine the total oxidative status in patients affected by thalassemia, and to evaluate new strategies of chelation,

transfusion-dependent β-thalassemia major patients [165].

heme [161]

**Rice (***Oryza sativa* **and** *Oryza glaberrima***)**

the near future.

ND = not determined.

**Table 3.** Antioxidant activity, total phenolics, vitamin C and mangiferin contents (mean±SD) in aqueous extracts from ripe and green mango peels (Sricharatanakool, S. unpublished data)

#### *5.2.4. Rice (Oryza sativa and Oryza glaberrima)*

Rice is an economic cereal plant ±that is grown in many countries in Asian and Africa. Varieties of Asian rice include Thai rice (*O. sativa* cv. indica), Indonesian rice (*O. sativa* cv. javanica) and Japanese rice (*O. sativa* cv. japonica). Additionally, we have listed African rice (*O. glaberrima*). Regarding its nutritional values, rice grain contains carbohydrates (e.g. amylose and amylo‐ pectin) and rice bran is abundant in inositol, inositol hexaphosphate (or phytate), oil, ferulic acid, γ-oryzanol, phytosterol and tocotrienol. The ingredients in rice bran likely prevent carcinogenesis, hyperlipidemia, fatty liver, gallstone disease and heart diseases [158]. Red and purple rice bran possesses several fold higher hydrophilic and lipophilic anti-oxidative phenolics and flavonoids (predominantly cyanidine-3-glycoside) levels than freeze-dried blueberry and broccoli. Rice grass contains chlorophyll as a major ingredient and pheophytin (or pheo) as the second most abundant component (Figure 11). Pheophytin is synthesized in rice grass, spinach (*Spinacia oleracea*) leaves and *Michelia alba* leaves, but is not a degradation product of chlorophyll [159], and the compound reveals a level of antioxidant activity that is similar to that of ubiquinone [160].

Spinach pheophytin a (λmax 409 nm) and pheophytin b (λmax 435 nm) can chelate ferric ion and produce the Fe-pheophytin a complex (λmax 393 nm) and Fe-pheophytin b complex (λmax 421 nm) [161]. Germinating rice grain synthesizes iron-chelating compounds, deoxymugineic acid

compound reveals a level of antioxidant activity that is similar to that of ubiquinone [160].

 **Pharmacology and Nutritional Intervention in the Treatment of Disease** 

Rice is an economic cereal plant that is grown in many countries in Asian and Africa. Varieties of Asian rice include Thai rice (*O. sativa* cv. indica), Indonesian rice (*O. sativa* cv. javanica) and Japanese rice (*O. sativa* cv. japonica). Additionally, we have listed African rice (*O. glaberrima*). Regarding its nutritional values, rice grain contains carbohydrates (e.g. amylose and amylopectin) and rice bran is abundant in inositol, inositol hexaphosphate (or phytate), oil, ferulic acid, -oryzanol, phytosterol and tocotrienol. The ingredients in rice bran likely prevent carcinogenesis, hyperlipidemia, fatty liver, gallstone disease and heart diseases [158]. Red and purple rice bran possesses several fold higher hydrophilic and lipophilic anti-oxidative phenolics and flavonoids (predominantly cyanidine-3 glycoside) levels than freeze-dried blueberry and broccoli. Rice grass contains chlorophyll

**Rice (***Oryza sativa* **and** *Oryza glaberrima***)**

**Figure 10** Chemical structures of chlorophyll, pheophytin, iron-pheophytin complex and heme [161] **Figure 11.** Chemical structures of chlorophyll, pheophytin, iron-pheophytin complex and heme [161]

As shown in Table 2, the degree of antioxidant activity of the mango peel extracts were found to be Mahajanaka (ripe > green) > Chok-anan (green > ripe) > Namdocmai (green > ripe) > Kaew (ripe ~ green). The antioxidant activities were well correlated with their total phenolic contents and vitamin C concentrations. Incredibly, amounts of vitamin C in the extracts were lower than those of the total phenolic compounds. Mangiferin contents in the peel extracts were very low.

> **Total phenolics (mg GAE/g extract)**

**Table 3.** Antioxidant activity, total phenolics, vitamin C and mangiferin contents (mean±SD) in aqueous extracts from

Rice is an economic cereal plant ±that is grown in many countries in Asian and Africa. Varieties of Asian rice include Thai rice (*O. sativa* cv. indica), Indonesian rice (*O. sativa* cv. javanica) and Japanese rice (*O. sativa* cv. japonica). Additionally, we have listed African rice (*O. glaberrima*). Regarding its nutritional values, rice grain contains carbohydrates (e.g. amylose and amylo‐ pectin) and rice bran is abundant in inositol, inositol hexaphosphate (or phytate), oil, ferulic acid, γ-oryzanol, phytosterol and tocotrienol. The ingredients in rice bran likely prevent carcinogenesis, hyperlipidemia, fatty liver, gallstone disease and heart diseases [158]. Red and purple rice bran possesses several fold higher hydrophilic and lipophilic anti-oxidative phenolics and flavonoids (predominantly cyanidine-3-glycoside) levels than freeze-dried blueberry and broccoli. Rice grass contains chlorophyll as a major ingredient and pheophytin (or pheo) as the second most abundant component (Figure 11). Pheophytin is synthesized in rice grass, spinach (*Spinacia oleracea*) leaves and *Michelia alba* leaves, but is not a degradation product of chlorophyll [159], and the compound reveals a level of antioxidant activity that is

Spinach pheophytin a (λmax 409 nm) and pheophytin b (λmax 435 nm) can chelate ferric ion and produce the Fe-pheophytin a complex (λmax 393 nm) and Fe-pheophytin b complex (λmax 421 nm) [161]. Germinating rice grain synthesizes iron-chelating compounds, deoxymugineic acid

Mahajanaka/Ripe 225.6±4.4 107.6±9.6 5.49±0.23 1.49±0.25 Mahajanaka/Green 114.3±7.4 44.8±5.9 0.96±0.07 0.57±0.10 Chok-Anan/Ripe 120.1±5.4 46.0±7.0 0.75±0.08 4.62±0.37 Chok-Anan/Green 192.4±4.5 85.7±7.7 4.22±0.22 6.80±0.06 Namdocmai/Ripe 102.9±4.9 49.7±8.5 0.69±0.03 0.25±0.01 Namdocmai/Green 33.1±2.6 5.3±0.8 0.27±0.091 0.25±0.01 Kaew/Ripe 63.2±0.2 34.8±0.7 ND 0.02 Kaew/Green 65.3±0.1 55.9±0.3 ND 0.43

**Vitamin C (mg/g extract)**

**Mangiferin (mg/g extract)**

**Mango/Status Antioxidant activity**

ND = not determined.

**(mg TEAC/g extract)**

138 Pharmacology and Nutritional Intervention in the Treatment of Disease

ripe and green mango peels (Sricharatanakool, S. unpublished data)

*5.2.4. Rice (Oryza sativa and Oryza glaberrima)*

similar to that of ubiquinone [160].

and nicotinamine, and sequesters iron from the ground for their growth and development [162]. Interestingly, wheat grass (WG) juice which has been used as a general-purpose health tonic in Indian medicine and has shown at least a 25% reduction in the number of blood transfusions needed in β-thalassemia major patients [163] and also increased their blood hemoglobin levels [164]. Nonetheless, a contradictory report has shown that the oral admin‐ istration of WG juice tablets for one year was not effective in reducing blood transfusions in transfusion-dependent β-thalassemia major patients [165]. Spinach pheophytin a (max 409 nm) and pheophytin b (max 435 nm) can chelate ferric ion and produce the Fe-pheophytin a complex (max 393 nm) and Fe-pheophytin b complex (max 421 nm) [161]. Germinating rice grain synthesizes iron-chelating compounds, deoxymugineic acid and nicotinamine, and sequesters iron from the ground for their growth and development [162]. Interestingly, wheat grass (WG) juice which has been used as a general-purpose health tonic in Indian medicine and has shown at least a 25% reduction in the number of blood transfusions needed in -thalassemia major patients [163] and also increased their blood hemoglobin levels [164]. Nonetheless, a contradictory report has shown that the oral administration of WG juice tablets for one year was not effective in reducing blood transfusions in transfusion-dependent -thalassemia major patients [165].

Pimpilai and colleagues from Maejo University, Chiang Mai investigated the antioxidant activity of the Thai black rice grass (TBRG), Thai red rice grass (TRRG) and Thai fragrant rice grass (TFRG) extracts by using the TEAC colorimetric method and found that they exhibited antioxidant properties. The extracts dose dependently bound Fe3+ (ferric NTA) and Fe2+ (ferrous citrate) and formed the complex(s) with maximal absorption values at 393, 378 and 580 nm, respectively (personal communication). HPLC analysis showed different amounts of catechin derivatives as follows: 3.27 mg C, 4.51 mg EC and 5.19 mg EGCG in 1 g TBRG extract; 4.48 mg C, 6.22 mg EC and 5.23 mg EGCG in 1 g TRRG extract; and 5.89 mg C, 4.38 mg EC and 5.26 mg EGCG in 1 g TFRG extract. Currently, Pimpilai and Srichairatanakool have found that feeding rice grass (Sukhothai 1) extract (100 mg/kg body weight) along with a high iron diet to β-thalassemia mice for three months slightly increased their blood hemoglobin concentra‐ tions from 10.04±0.51 to 11.04±1.14 g/dl (unpublished data), while treatment with the extract (0, 50 and 100 µg/ml) decreased levels of liver MDA (153.6±44.7, 102.9±22.4 and 102.4±58.0 µg/ mg tissue protein, respectively) and levels of labile iron (100±13, 120±1 and 125±5% fluorescent intensity, respectively) in iron-loaded HepG2 cell cultures. Therefore, the rice grass extracts would contain antioxidant compounds including chlorophyll, pheophytin and catechin derivatives, of which the two latter may play important roles in iron chelation and anti-lipid peroxidation to ameliorate oxidative tissue damage in the thalassemia cases with iron over‐ load. Consequently, rice grass extracts need to be clinically studied in thalassemia patients in the near future. 26 Pimpilai and colleagues from Maejo University, Chiang Mai investigated the antioxidant activity of the Thai black rice grass (TBRG), Thai red rice grass (TRRG) and Thai fragrant rice grass (TFRG) extracts by using the TEAC colorimetric method and found that they exhibited antioxidant properties. The extracts dose dependently bound Fe3+ (ferric NTA) and Fe2+ (ferrous citrate) and formed the complex(s) with maximal absorption values at 393, 378 and 580 nm, respectively (personal communication). HPLC analysis showed different amounts of catechin derivatives as follows: 3.27 mg C, 4.51 mg EC and 5.19 mg EGCG in 1 g TBRG extract; 4.48 mg C, 6.22 mg EC and 5.23 mg EGCG in 1 g TRRG extract; and 5.89

### **6. Evaluation of oxidative stress and antioxidant status in thalassemias**

Electron paramagnetic resonance (EPR) 'radical probe' was used to determine the total oxidative status in patients affected by thalassemia, and to evaluate new strategies of chelation, new chelators, or the efficacy of antioxidant formulas [166, 167]. Raman spectroscopic techni‐ que has been developed for the monitoring of Raman hemoglobin bands to evaluate oxygen‐ ation capability, oxidative stress and deformities of thalassemic erythrocytes and to assess the responses to drug therapies [168]. Consistent with the study, in reference [169], serum PON activity and total antioxidant capacity were significantly lower in patients with the β-thalas‐ semia trait patients, MDA and carotid artery intima-media thickness were significantly higher in β-thalassemia trait. The total antioxidant capacity, MDA, and CIMT levels were correlated with serum PON1 (r = 0.945, -0.900, 0.940 and -0.922 respectively). Additionally, serum total antioxidant capacity and MDA levels were well correlated (r = -0.979) [170].

oxidative damage in plasma samples of β-thalassemia patients [180]. Pumala et al. have found that PON1 activity was significantly reduced in association with oxidative stress in the patients with β-thalassemia hemoglobin E, and significant correlations were observed between HDL-PON1 activity and oxidative stress markers (including plasma α-tocopherol and the ratio of cholesteryl linoleate to cholesteryl oleate in HDL, and a marked increased platelet-activating factor/acetylhydrolase (PAF-AH) activity [181]. The GC/MS-based assay showed that the level of urinary and plasma lipid peroxidation poroduct, F(2)-isoprostane in the thalassemic group was significantly increased [182]. An average antioxidant level in Thai thalassemia patients with the HbE trait (3.276±0.209 mM TEAC) was significantly decreased (*p* = 0.008) when compared to the healthy subjects (3.439±0.220 mM TEAC) [183]. It has been reported that Thai thalassemia major patients are associated with an alteration of CYP2E1 and CYP3A4 activities [184], but not CYP1A2 [185]. Anemia was not pronounced in the rescued mice (C57BL/6) with the Hb E transgene mimicing the human β-thalassemia/HbE phenotype; nonetheless, other hematologic parameters in their RBC include highly oxidative stress, no marked changes in PS and vesicles, and a shortened life span, which were abnormally similar to the BKO

Antioxidants as Complementary Medication in Thalassemia

http://dx.doi.org/10.5772/57372

141

Under iron overload, oxidative stress plays a major role in the pathophysiologic complications of thalassemia patients. Free extracellular toxic iron (e.g. NTBI and LPI) and intracellular redox iron (e.g. LIP and plasma membrane nonheme iron) that have been identified in thalassemic blood and tissues are responsible for the generation of oxidative stress by catalyzing a formation of oxygen radicals over the antioxidant capacity of the cells. Consequently, there is a rationale to support iron chelation therapy for the elimination of the free-iron species and to promote the free-radical scavenging activity of the antioxidants. Not only synthetic (vitamin C, vitamin E and NAC) but natural (e.g. polyphenolics, flavonoids and fish oils) antioxidants are also capable of ameliorating such increased levels of oxidative stress. Taken together with an effective iron chelator, antioxidants may provide a substantial improvement in hemolytic anemia cases, and particularly in thalassemia patients. Most importantly, natural antioxidants are ubiquitous and very cheap whereas antioxidant supplements are free from the side effects

commonly encountered in iron chelation and chemotherapeutic treatments.

thalassemic RBC group [136].

**7. Conclusions**

**Abbreviations**

ACE-II = angiotensin-converting enzyme II

AD4 = *N*-acetylcysteine amide δ-ALA = delta-aminolevulinic acid ALP = alkaline phosphatase ALT = alanine aminotransferase

An earlier study in 1986 showed that patients with Hb H diseases, including α-thalassemia 1 or α-thalassemia 2 and 21 with α-thalassemia 1/Hb Constant Spring, had increased activities of erythrocyte SOD, GPx, and CAT when compared with those of the controls. The α-thalas‐ semia 1/Hb CS patients had higher SOD and GPx activities, but lower CAT activity than the patients with α-thalassemia 1/2 [171, 172]. One year later, a study of oxidative stress and the antioxidants in β-thalassemia/hemoglobin E patients in Thailand was conducted [173]. Significantly higher levels of urine N-acetyl-β-D-glycosaminidase, MDA and β2-microglobulin along with aminoaciduria and proximal tubular abnormalities were found in pediatric αthalassemia patients (Hb H disease and HbS/CS), and this was possibly due to increased oxidative stress [174]. A one-year treatment with DFP significantly decreased serum ferritin, NTBI, and MDA (*p* <0.05) of transfusion-independent β-thalassemia/HbE patients. Mean pulmonary arterial pressure and pulmonary vascular resistance were diminished significantly (*p* <0.05). All those parameters were still improved after subgroup analysis was done for the high ferritin group (>2500 ng/ml). The results imply that DFP therapy alone improved iron overload and oxidative stress and the compliance was positive [175]. Oxidative stress was increased in Thai HbE/β-thalassemia patients, as the blood GSH decreased, GSH/GSSG ratio reduced markedly, superoxide anion released from blood cells elevated highly, and γglutamylcysteine ligase activity was increased. Additionally, basal forearm blood flow was significantly increased whereas forearm vasodilatory response to reactive hyperemia was depressed [176].

When using ESR spectroscopic quantification of hemin, the serum hemin readily catalyzed free radical reactions and it would be a major pro-oxidant in the blood circulation of βthalassemia Hb E patients [177]. A previous study showed a precipitous decrease in αtocopherol and increased TBARS concentrations in both plasma and lipoproteins obtained by Thai β-thalassemia Hb E patients. Cholesteryl linoleate showed a reduction of 70% in LDL, while other cholesterol ester levels showed a lower reduction. A good correlation of NTBI and TBARS (*p* <0.01) in LDL strongly supported the contention that iron overload is responsible for initiating the lipid peroxidation in thalassemia patients [178]. The ESR results demonstrated a magnitude of increased lipid fluidity in thalassemic lipoproteins. Lipid fluidity at the LDL and HDL cores showed a good correlation with the oxidative stress markers and the αtocopherol level, suggesting that the hydrophobic region of the thalassemic lipoprotein would be a target site for oxidative damage [179]. Gas chromatography/mass spectrometric (GS/MS) technique has been validated in quantifying ortho- and meta-tyrosine as biomarkers of protein oxidative damage in plasma samples of β-thalassemia patients [180]. Pumala et al. have found that PON1 activity was significantly reduced in association with oxidative stress in the patients with β-thalassemia hemoglobin E, and significant correlations were observed between HDL-PON1 activity and oxidative stress markers (including plasma α-tocopherol and the ratio of cholesteryl linoleate to cholesteryl oleate in HDL, and a marked increased platelet-activating factor/acetylhydrolase (PAF-AH) activity [181]. The GC/MS-based assay showed that the level of urinary and plasma lipid peroxidation poroduct, F(2)-isoprostane in the thalassemic group was significantly increased [182]. An average antioxidant level in Thai thalassemia patients with the HbE trait (3.276±0.209 mM TEAC) was significantly decreased (*p* = 0.008) when compared to the healthy subjects (3.439±0.220 mM TEAC) [183]. It has been reported that Thai thalassemia major patients are associated with an alteration of CYP2E1 and CYP3A4 activities [184], but not CYP1A2 [185]. Anemia was not pronounced in the rescued mice (C57BL/6) with the Hb E transgene mimicing the human β-thalassemia/HbE phenotype; nonetheless, other hematologic parameters in their RBC include highly oxidative stress, no marked changes in PS and vesicles, and a shortened life span, which were abnormally similar to the BKO thalassemic RBC group [136].
