Antioxidant Activity of Phytic Acid Hydrolysis Products on Iron Ion

Downloaded by NANYANG TECHNOLOGICAL UNIV on June 1, 2016 | http://pubs.acs.org Publication Date: March 4, 2002 | doi: 10.1021/bk-2002-0807.ch018

Chapter 18

Antioxidant Activity of Phytic Acid Hydrolysis Products on Iron Ion-Induced Oxidative Damage in Biological System 1

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Sayuri Miyamoto , Kaeko Murota , Goro Kuwataz , Masatake Imai , Akihiko Nagao , and Juliji Terao 2

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Department of Nutrition, School of Medicine, The University of Tokushima, Kuramoto-cho 3, Tokushima 770-8503, Japan Research Institute of Morinaga & Company Ltd., Simosueyoshi 2-1-1 Tsurumi-ku,, Yokohama 230-0012, Japan National Food Research Institute, Ministry of Agriculture, Forestry and Fisheries, Tsukuba 305-0856, Japan 3

Phytic acid (IP ) is capable of chelating iron ion and thereby blocking the generation of reactive oxygen radicals via Fenton reactions. Some evidence suggests that the consumption of diets rich in phytic acid protect intestinal epithelial cells against iron ion-induced oxidative damage. During digestion, phytic acid is dephosphorylated yielding lower phosphorylated forms of inositol phosphate (IP - IP ). In this study, we evaluated the antioxidant properties of these hydrolysis products. Their ability to chelate iron ion decreased with the decrease in the number of phosphate groups on the inositol structure. However, IP showed a unique ability to prevent iron 6

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© 2002 American Chemical Society Morello et al.; Free Radicals in Food ACS Symposium Series; American Chemical Society: Washington, DC, 2002.

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242 ion-induced deoxyribose degradation, and similar to IP , IP showed a strong inhibitory effect against iron ion-induced oxidation of large intestinal mucosa homogenate. Interestingly, addition of vitamin Ε into the liposomal suspension greatly increased the antioxidant activity of IP , suggesting a synergistic antioxidant effect. Moreover, oral administration of IP protected large intestinal mucosa against iron ion-induced lipid peroxidation. These observations indicate an important antioxidant function for phytic acid hydrolysis products and suggest that their synergistic effect with vitamin Ε is an essential factor in the prevention of oxidative damage occurring in large intestinal mucosa. 6

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Dietary iron remains largely unabsorbed in the small intestine, thus it is available to participate in the generation of reactive oxygen radicals in the large intestine (/,2). These radicals are capable of attacking all kind of molecules including protein, DNA, and lipids. In particular, radical attack on lipids constituting cellular membranes promotes lipid peroxidation, and thereby enhances oxidative tissue damage (3) (Figure 1). Recent studies have shown a positive correlation between high iron intake and colon cancer (4-6). On the other hand, epidemiological and animal model studies indicate that diets rich in phytic acid fwyo-inositol hexaphosphate; IP ) (Figure 2) may prevent colon carcinogenesis (7-10). The anticarcinogenic effect of IP is probably related to its ability to chelate iron and block iron ion-catalyzed redox reactions (77). Since IP is partly hydrolyzed during digestion (12), this study aimed to evaluate the antioxidant properties of phytic acid hydrolysis products. In the study, we evaluated the effectiveness of IP - IP against iron ion-induced oxidative reactions. It was demonstrated that IP and IP possess considerable antioxidant functions and oral administration of IP was effective in protecting large intestinal mucosa against iron ion-induced lipid peroxidation. 6

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Effect of Phytic Acid Hydrolysis Products on Deoxyribose Degradation Assay Iron ion-binding ability of phytic acid (IP ) and its hydrolysis products (IP , IP IP and IP ) were assessed by the deoxyribose degradation assay (75). In the assay, the sugar 2-deoxyribose (2.8 mM) was degraded in the presence of Fe(N0 ) (10 μΜ), ascorbic acid (100 μΜ), and H 0 (1 mM), generating malonaldehyde-likefragments,which were determined by the TBA assay. IP 6

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Morello et al.; Free Radicals in Food ACS Symposium Series; American Chemical Society: Washington, DC, 2002.


Figure L Possible mechanism of iron ion-induced oxidative damage in the large intestine.

Bacterial Metabolism

intestinal lumen


244 IP and Desferal (100 μΜ) were added to the reaction mixture and their inhibitory ratio was calculated (Figure 3). The strong iron chelator Desferal, used as a positive control, showed the highest inhibitory effect. IP provided less inhibition than IP and IP and IP showed no inhibitory effect, suggesting that dephosphorylation reduces the ability of IP to inhibit Fenton reaction. However, IP showed a unique, high ability to prevent deoxyribose degradation. This phenomenon can not be explained by its iron chelating activity alone. It has been suggested that deoxyribose degradation involves a series of reactions (14) and IP probably has the ability to inhibit one of these reactions. 2

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Synergistic Effect of IP and Vitamin Ε on Iron Ion-induced Oxidation of Liposomal Membrane 3

Here, we focused in the antioxidant properties of IP and tried to evaluate whether the addition of vitamin Ε has any effect on its antioxidant activity. Large unilamellar vesicle liposomes composed of phosphatidylcholine were prepared by the method previously described (15). The liposomal suspension was incubated with IP (100 μΜ) with or without the addition of α-tocopherol (1 μΜ), and oxidation was initiated by addition of Fe(N0 ) (10 μΜ) and ascorbic acid (100 μΜ). The reaction mixture was incubated at 37 °C, and accumulation of phosphatidylcholine hydroperoxides (PC-OOH) and the disappearance of atocopherol were determined by HPLC analysis (Figure 4). IP slightly retarded the accumulation of PC-OOH, whereas α-tocopherol inhibited PC-OOH accumulation during the first 5 hours. However, the combination of IP and atocopherol was much more effective in preventing the oxidation. Together they inhibited oxidation during approximately 15 hours. The disappearance of atocopherol was also retarded during this period. It is therefore likely that IP and α-tocopherol have a synergistic antioxidant effect, and its combination may play an important role in the prevention of iron ion-induced oxidative damage. 3

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Antioxidant Activity of Phytic Acid Hydrolysis Products in the Rat Large Intestine: in vitro and ex vivo Study We examined the antioxidant activity of phytic acid hydrolysis products using rat large intestinal mucosa homogenate. Wistar male rats (6 weeks, 160180 g) were used for the test. In the ex vivo study, 0.5 ml of an aqueous solution of phytic acid (100 mg) was administered intragastrically to the rats. The large intestinal mucosa was removed by scraping and then homogenized in Tris-HCl buffer (0.1 M , pH 7.4; containing 0.135 M KC1). The in vitro study was carried



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Figure 3. Effect ofphytic acid hydrolysis products on iron ion-induced deoxyribose degradation.


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Figure 4. Effect of IP $ and α-tocopherol on iron ion-induced LUV-liposome lipid peroxidation. (A) PC-OOH accumulation. (B) a-tocopherol disappearance. (Εφ Control, (Jk)IP (Φ) α-tocopherol, (O) a-tocopherol plus IP 3t

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247 out by the addition of IP , IP , or Desferal (1 mM) into the homogenate obtained from untreated animals. In both cases mucosal homogenate was oxidized by the addition of Fe(N0 ) (10 μΜ) and ascorbic acid (100 μΜ). The incubation was carried out at 37 °C for 4 hours, and the oxidation level was analyzed by TBA assay (Table I). Similar to IP and Desferal, IP strongly inhibited formation of TBARS, suggesting that IP is effective in preventing iron ion-induced lipid peroxidation in the large intestine. It is therefore expected that IP can protect large intestinal mucosa against iron ion-induced lipid peroxidation even after digestion. Indeed in the ex vivo study, oral administration of IP significantly protected large intestinal mucosa against iron ion-induced lipid peroxidation. Large intestinal mucosa from treated animals showed a significantly lower level of TBARS compared to results for untreated animals (Table I). Although the level of hydrolysis was not determined, phytic acid digestion products are suggested to exert an important antioxidantfonctionin the large intestine. Further work is in progress to examine the ex vivo effects of IP and IP . 6

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Summary Phytic acid is hydrolysed by the action of phytase during the digestive process (Figure 5). Dephosphorylation apparently decreases the ability of phytic acid hydrolysis products to inhibit hydroxyl radical generation via Fenton reaction. However, our results demonstrate that IP and IP still have important antioxidant functions. Indeed, administration of IP protected large intestinal mucosa against iron ion-induced lipid peroxidation. Furthermore, the possible synergistic antioxidant effect of IP with vitamin Ε implies that the combined effect of antioxidants exerts an essential role in the prevention of iron ioninduced oxidative damage. 2

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References 1. 2. 3. 4. 5. 6. 7. 8.

Babbs, C.F. Free Radic. Biol. Med. 1990, 8, 191-200. Lund E.K., Wharf S.G., Fairweather-Tait S.J., Johnson I.T. Am. J. Clin.Nutr. 1999, 69, 250-5. Halliwell, B.; Gutteridge, J.M.C. Methods Enzymol. 1990, 186, 1-85. Nelson, R.L. Free Rad. Biol. Med. 1992, 12, 161-168. Weinberg, E.D. Biometals 1994, 7, 211-216. Lund E.K., Wharf S.G., Fairweather-Tait S.J., Johnson IT. J. Nutr. 1998, 128. 175-179. Graf, E.; Eaton J.W. Cancer 1985, 56, 717-718. Graf E.; Eaton J.W. Nutr. Cancer 1993, 19, 11-19.



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8.89 ± 2.07

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9.28 ± 1.42

Control a

6.06 ± 1.59

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1.25 ± 0.31

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IP

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2.15 ± 0.77

IPs

TBARS (nmol/mg protein)

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0.84 ± 0.27

Desferal b

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IP aqueous solution (100 mg) was administered orally to rats.

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IP , IP and Desferal (1 mM) were added to large intestinal mucosa homogenate.

n.d.= not determined

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Large intestinal mucosa homogenate was oxidized by the addition of Fe(N0 ) and ascorbic acid solution

(100 μΜ and 1 mM).

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(p

Antioxidant Activity of Phytic Acid Hydrolysis Products on Iron Ion

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