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In vitro and in vivo antioxidant properties of ferulic acid: A comparative study with other natural oxidation inhibitors
Food Chemistry 114 (2009) 466–471
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In vitro and in vivo antioxidant properties of ferulic acid: A comparative study with other natural oxidation inhibitors Shirou Itagaki, Toshimitsu Kurokawa, Chie Nakata, Yoshitaka Saito, Setsu Oikawa, Masaki Kobayashi, Takeshi Hirano, Ken Iseki * Laboratory of Clinical Pharmaceutics and Therapeutics, Department of Biopharmaceutical Sciences and Pharmacy, Division of Biopharmaceutical Sciences and Pharmacy, Faculty of Pharmaceutical Sciences, Hokkaido University, Kita 12-jo, Nishi 6-chome, Kita-ku, Sapporo 060-0812, Japan
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Article history: Received 27 June 2008 Received in revised form 12 August 2008 Accepted 24 September 2008
Keywords: Reactive oxygen substances Ferulic acid EGCG Ascorbic acid Chain-breaking
a b s t r a c t Ferulic acid exhibits a wide range of therapeutic effects that are attributed to its potent antioxidant capacity. However, in vitro antioxidant properties of ferulic acid have not been elucidated in detail. Evidence that polyphenols, including ferulic acid, act as antioxidants in vivo is also limited. In order to elucidate in more detail the scientific background of antioxidant activities of ferulic acid, we carried out in vitro and in vivo experiments. We focused on superoxide anion scavenging activity, xanthine oxidase inhibition activity, and chain-breaking activity. The combined antioxidant activity from radical scavenging and xanthine oxidase inhibition of ferulic acid was much weaker than that of ()-epigallocatechin gallate (EGCG) and ascorbic acid. On the other hand, EGCG, ascorbic acid and ferulic acid exhibited chain-breaking activity and prevented ischaemia-reperfusion-associated intestinal injury. Chain-breaking activity may play a contributory role in the protective effect of ferulic acid on oxidative injury in humans and in in vivo studies. Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction Production of reactive oxygen species (ROS) in tissue contributes to the development of various chronic diseases such as cancer, neurodegenerative diseases, and cardiovascular diseases (Benzie, 2000; Stocker, 1999). Administration of antioxidants to patients may therefore help in removing ROS and thus improve the clinical outcome. Dietary antioxidants can enhance cellular defence and help to prevent oxidation damage to cellular components. There has been considerable public and scientific interest in therapeutic use of natural antioxidants. Among the natural antioxidants, polyphenols play a very important role. Dietary polyphenols are thought to be beneficial to human health by exerting various biological effects such as free radical scavenging, metal chelation, modulation of enzymatic activity, and alteration of signal transduction pathways (Singh & Aggarwal, 1995; Stocker, 1999; Yoshioka, Deng, Cavigelli, & Karin, 1995). Epidemiological studies have shown relationships between consumption of polyphenol-rich foods and prevention of diseases such as cancer, coronary heart disease, and osteoporosis, and results of these studies have promoted interest in polyphenols (Adlercreutz & Mazur, 1997; Steinmetz & Potter, 1996). * Corresponding author. Tel./fax: +81 11 706 3770. E-mail address: [email protected] (K. Iseki). 0308-8146/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2008.09.073
Polyphenols are classified into phenolic acids, flavonoids, and less common stilbenes and lignans. Many studies have focused on the antioxidant activities of flavonoids. Although several flavonoids are highly efficacious free radical scavengers in vitro, there is little information on the importance of dietary flavonoids as antioxidants in vivo, or evidence for such activity in vivo. Moreover, there have been few studies on phenolic acids compared to the number of studies on flavonoids, despite the high contents of phenolic acids in fruits, cereals, and some vegetables (Clliford, 1999). Ferulic acid is a phenolic compound that exhibits a wide range of therapeutic effects against various diseases, including cancer, diabetes, and cardiovascular and neurodegenerative diseases. Moreover, ferulic acid has been approved in Japan as a food additive to prevent oxidation. Ferulic acid is a ubiquitous plant constituent that arises from the metabolism of phenylalanine and tyrosine. Ferulic acid is a major constituent of fruits and vegetables such as orange, tomato, carrot, sweet corn, and rice bran (Balasubashini, Rukkumani, & Menon, 2003). Ferulic acid is a phenolic compound and it possesses three distinctive structural motifs that can possibly contribute to its free radical scavenging capability. The presence of electron donating groups on the benzene ring (3 methoxy and more importantly 4-hydroxyl) of ferulic acid gives the additional property of terminating free radical chain reactions. The next functionality – the carboxylic acid group in ferulic acid with an adjacent unsaturated C–C double bond – can provide
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additional attack sites for free radicals and thus prevent them from attacking the membrane. In addition, this carboxylic acid group also acts as an anchor of ferulic acid, by which it binds to the lipid bilayer, providing some protection against lipid peroxidation (Kanaski, Aksenova, Stoyanova, & Butter field, 2002). Ferulic acid is commercially prepared and used as a functional food ingredient. Orally administered compounds are absorbed in the intestine. It is now recognised that several transporters contribute to the absorption of administered compounds from the intestine. Recently, we have reported that intestinal uptake of ferulic acid is associated with an H+-driven transport system, which is identical to the nateglinide/fluorescein transport system (Itagaki et al., 2005). The physiological importance of ferulic acid depends on its availability for absorption and subsequent interaction with target tissues. Since there is limited information on the importance of ferulic acid as an antioxidant in vivo, we focused on the protective effect of ferulic acid on intestinal oxidative injury. The intestinal mucosa is extremely sensitive to ROS (Illyes & Hamar, 1992; Kong, Blennerhassett, Heel, McCauley, & Hall, 1998). It is well known that ROS are responsible for intestinal ischaemia–reperfusion (I/R) injury (Adam et al., 2002; Granger, Rutili & McCord; 1981). Intestinal I/R is a common clinical problem in the settings of severe burns, circulatory shock, and strangulation ileus. Furthermore, intestinal I/R injury is a serious medical problem often necessitating surgical intervention. In this study, we used a rat mesenteric I/R injury model as a model of oxidative injury and investigated the antioxidant activities of ferulic acid in vivo.
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the multilabel counter Wallac 1420 ARVOse (Perkin Elmer, Wellesley, MA). 2.4. Measurement of radical chain-breaking activity The assay was carried out as described in a previous report with some modification (Beretta et al., 2006). Stock solutions of the fluorescent lipophilic probe BODIPY were prepared in dimethyl sulfoxide and stored under nitrogen at 70 °C. For BODIPY incorporation, 25 ll of the BODIPY stock solution (2 mM) was diluted 100-fold with PBS. Then aliquots of 50 ll were added to 50 ll of rat plasma, diluted to 350 ll of PBS with or without tested compounds (final concentrations of 0.001, 0.01, 0.1, 1, and 10 mM, respectively), vortexed for 20 s at the lowest speed, and incubated under aerobic conditions for 10 min at 37 °C. The final volume was adjusted to 480 ll with PBS, yielding BODIPY at a final concentration of 2 lM. MeO-AMVN was dissolved in CH3CN immediately before use, and 20 ll of the solution was added to the sample at a final concentration of 2 mM. Then aliquots of 200 ll were transferred to a 96-microwell plate, and the lipid oxidation kinetics was monitored by measuring the green fluorescence (kex = 485, kem = 535 nm; cycle time of 10 min for 18 cycles) of the oxidation product of BODIPY. Light emission was measured with the multilabel counter Wallac 1420 ARVOse. The results are expressed as total antioxidant performance (TAP) values, representing the percentage of inhibition of BODIPY oxidation in human plasma with respect to that occurring in a control sample: TAP = [(AUCcontrolAUCplasma)/AUCcontrol] 100, where AUCcontrol and AUCsample represent the area under the curve (AUC) of BODIPY oxidation kinetics in the control and plasma samples, respectively. Control samples were prepared using PBS.
2. Materials and methods 2.5. Intestinal I/R model 2.1. Chemicals
a-Lipoic acid, ferulic acid, and hypoxanthine were purchased from Sigma (St Louis, MO). b-Carotene, ()-epigallocatechin gallate (EGCG) and, 2,20 -Azobis(4-methoxy-2,4-dimethylvaleronitrile) (MeO-AMVN) were purchased from Wako (Osaka, Japan). Xanthine oxidase was purchased from Nacalai Tesque (Kyoto, Japan). 4,4-Difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a,-diaza-s-indacene-3propionic acid saccinimidyl ester (BODIPY) was purchased from Invitrogen (Carlsbad, CA). All other reagents were of the highest grade available and used without further purification. 2.2. Animals Male Wistar rats, aged six weeks (180–200 g in weight), were obtained from Japan Laboratory Animals (Tokyo, Japan). The housing conditions were the same as those described previously (Ochiai et al., 2007). The rats were housed at least one week at 23 ± 3 °C and 50 ± 10% relative humidity and were maintained on a 12 h light/dark cycle. During the acclimatisation the rats were allowed freely to access food and water. The experimental protocols were reviewed and approved by the Hokkaido University Animal Care Committee in accordance with the ‘‘Guide for the Care and Use of Laboratory Animals”.
Surgical procedures were carried out as described in a previous report with some modification (Ogura, Kobayashi, Itagaki, Hirano, & Iseki, 2008). Wistar rats were anesthetized with sodium pentobarbital (40 mg/kg weight, i.p.). The rats were fixed after the operation. A small midline incision was made in the abdomen. A 5-cm-long loop of the jejunum was identified and ligated at both ends. Through a midline laparotomy, the superior mesenteric artery (SMA) was isolated and a bulldog arterial clamp was applied at the aortic origin. The abdomen was then covered with a sterile plastic wrap. After 30 min of intestinal ischaemia, the arterial clamp was removed. Five hundred microlitres of tested compounds (1 mM) was administered directly into the loops 1 h before the induction of ischaemia. 2.6. Tissue sampling The 5-cm-long intestine was excised, the contents were removed, and the intestine was cleansed in ice-cold saline. The intestine was then homogenised in 2.5 ml saline using a glass Teflon homogenizer with 20 strokes. Protein was measured by the method of Lowry, Rosebrough, Farr, and Randall (1951) with bovine serum albumin as a standard. 2.7. TBA analysis
2.3. Measurement of superoxide anion scavenging activity Superoxide anion scavenging activity was measured by the chemiluminescent superoxide anion probe method, using a superoxide anion-2-methyl-6-methoxyphenylethynylimidazopyrazynone (MPEC) reaction kit (ATTO Corp., Osaka, Japan) according to the manufacturer’s instructions. Superoxide anions were generated by xanthine/xanthine oxidase. Light emission was measured with
The amount of lipid peroxide in the intestine was determined as that of malondialdehyde (MDA) by the method of Ohkawa, Ohishi, and Yagi (1979) with some modification (Kurokawa et al., 2006). Thiobarbituric acid (TBA) solution was composed of 2.6 mM TBA, 918 mM trichloroacetic acid, 0.3 mM HCl, and 1.8 mM 2,6-ditert-butyl-4-metylphenol (BHT) in 22% ethanol. The reaction mixture contained 0.2 ml of tissue homogenate, 0.2 ml of 8.1% sodium
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dodecyl sulphate (SDS), 1.5 ml of 20% acetic acid solution (pH 3.5), and 1.5 ml of 0.8% aqueous solution of TBA. The mixture was heated at 95 °C for 60 min. After cooling with tap water, 1.0 ml of distilled water and 5.0 ml of n-butanol were added, and the mixture was shaken vigorously. After centrifugation at 3000x g for 10 min, the absorbance of the organic layer (upper layer) was measured at 535 nm with 1,1,3,3-tetraethoxypropane as a standard. 2.8. Evaluation of changes in vascular permeability Extravasation of Evans blue dye into tissue was used as an index of increased vascular permeability (Matos, Souza, Seabra, FreireMaia, & Teixeira, 1999). Evans blue dye (20 mg/kg) was injected (1 ml/kg) through the jugular vein 5 min prior to reperfusion of the ischaemic artery. Sixty min after reperfusion, fragments of the loop were cut and allowed to dry in a petri dish for 24 h at 37 °C. The dry weight of the tissue was calculated and Evans blue dye was extracted using 4 ml of formamide (24 h at 37 °C). The dye concentration of the samples was measured by spectrophotometry at 620 nm using JASCO V-630 BIO (Tokyo, Japan). Results are presented as the amount of Evans blue in lg per 100 mg of tissue. 2.9. Data analysis Non-linear regression analysis was performed by using OriginÒ (version 6.1J). Student’s t-test was used to determine the significance of differences between two group means. Statistical significance among means of more than two groups was determined by one-way analysis of variance (ANOVA). A value of p < 0.05 was considered significant.
3. Results 3.1. Inhibition of xanthine oxidase-induced light emission The superoxide radical anion appears to play a central role since other ROS are formed in reaction sequences starting with superoxide radical anion. Xanthine/xanthine oxidase is also a main source of ROS (Chambers et al., 1985). Moreover, intestinal mucosa is one of the richest sources of xanthine oxidase. In this study, we therefore used a xanthine/xanthine oxidase system to generate superoxide anions. A mixture of xanthine and xanthine oxidase generated superoxide anions, which were reacted with MEPC to give light emission by chemiluminescence. Since light emission induced by xanthine oxidase is inhibited in the presence of a radical scavenger or xanthine oxidase inhibitor, the inhibitory effect of a compound on the increase in chemiluminescence intensity indicates the combined antioxidant activity from both superoxide anion scavenging and xanthine oxidase inhibition. In the first part of this study, the antioxidant activities of various compounds were evaluated using the xanthine/xanthine oxidase system and MPEC. As shown in Fig. 1, four antioxidants, EGCG, ascorbic acid, b-carotene and a-lipoic acid, inhibited the light emission induced by xanthine oxidase in a concentration-dependent manner. IC50 values of these compounds for light emission induced by xanthine oxidase are listed in Table 1. We then investigated the effects of ferulic acid on xanthine oxidase-induced light emission. The IC50 value of ferulic acid for light emission induced by xanthine oxidase was different from the values of EGCG, ascorbic acid, b-carotene and a-lipoic acid (Fig. 1, Table 1).
Fig. 1. Dose–response relationship for the inhibition of xanthine oxidase-induced light emission by various compounds. Each point represents the mean with SD of three independent experiments. Abbreviations: EGCG, ()-epigallocatechin gallate.
Table 1 IC50 values of various compounds on xanthine oxidase-induced light emission Compounds
IC50 (lM)
EGCG Ascorbic acid b-Carotene a-Lipoic acid Ferulic acid
5.34 22.5 89.1 479 6614
Abbreviations: EGCG, ()-epigallocatechin gallate.
of ferulic acid were quite different from those of EGCG and ascorbic acid. Since the influence of free radical-mediated oxidation is amplified because it proceeds by a chain mechanism, the role of chain-breaking activity is important as well as radical scavenger activity (Riley, 1994). Recently, Beretta et al. (2006) reported a method that enables specific measurements of chain-breaking activities of tested compounds using BODIPY and showed that EGCG and ascorbic acid act as chain-breaking antioxidants as well as radical scavengers. We therefore investigated the chain-breaking activity of ferulic acid using BODIPY. Fig. 2 shows the oxidation kinetics of BODIPY in the presence of ascorbic acid. Ascorbic acid inhibited BODIPY oxidation, and the effect was concentrationdependent. Thus, the TAP value of ascorbic acid also increased in a concentration-dependent manner (Fig. 3). We then examined the effects of EGCG and ferulic acid on BODIPY oxidation. Similar
3.2. Inhibitory effects of EGCG, ascorbic acid and ferulic acid on BODIPY oxidation Results of the MPEC assay showed that combined antioxidant activities from radical scavenging and xanthine oxidase inhibition
Fig. 2. Dose–response relationship for the inhibition of BODIPY oxidation by ascorbic acids. Abbreviations: BODIPY, 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4bora-3a,4a,-diaza-s-indacene-3-propionic acid saccinimidyl ester.
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Fig. 3. Antioxidant activity of EGCG, ascorbic acid and ferulic acid as determined by the TAP assay. Each point represents the mean with SD of three independent experiments. Abbreviations: EGCG, ()-epigallocatechin gallate; TAP, total antioxidant performance.
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Fig. 5. Effects of EGCG, ascorbic acid and ferulic acid (1 mM) on the changes in vascular permeability in the small intestine. Data are given as mean with SD of four rats (I/R+EGCG, I/R+Ascorbic acid, I/R+ferulic acid) or five rats (sham, I/R). *P < 0.05, significantly different from non ischaemia control animals. P < 0.05, significantly different from animals not treated with compounds (I/R animals). Abbreviations: I/ R, ischaemia–reperfusion; EGCG, ()-epigallocatechin gallate.
duction of lipid peroxide contributes to small intestinal injury. The increase in the amount of lipid peroxide after I/R was significantly inhibited by treatment with EGCG, ascorbic acid, and ferulic acid (Fig. 4). Since vascular permeability has been shown to be significantly higher in rats with I/R than in sham-operated rats, elevated vascular permeability is also one of the indicators of I/R injury (Pompermayer et al., 2007). We therefore investigated the effects of these compounds on the increased vascular permeability, as assessed by reduction in the level of Evans blue dye. Elevation of vascular permeability by intestinal I/R was attenuated by treatment with EGCG and ascorbic acid (Fig. 5). Ferulic acid also prevented the elevation of vascular permeability following I/R injury in the intestine (Fig. 5). Fig. 4. Effects of EGCG, ascorbic acid and ferulic acid (1 mM) on the amount of lipid peroxide in the small intestine after I/R. Data are given as mean with SD of four rats. * P < 0.05, significantly different from non ischaemia control animals. P < 0.05, significantly different from animals not treated with compounds (I/R animals). Abbreviations: MDA, malondialdehyde; I/R, ischaemia–reperfusion; EGCG, ()epigallocatechin gallate.
to ascorbic acid, EGCG, and ferulic acid also inhibited BODIPY oxidation, and the TAP values of these compounds increased in a concentration-dependent manner (Fig. 3). 3.3. Protective effects of EGCG, ascorbic acid and ferulic acid on intestinal I/R injury We confirmed in vivo antioxidant activities of EGCG, ascorbic acid, and ferulic acid using a rat I/R injury model. Although 1 mM EGCG and ascorbic acid completely inhibited the light emission induced by xanthine oxidase, 1 mM ferulic acid did not inhibit the light emission (Fig. 1). On the other hand, 1 mM ferulic acid significantly inhibited BODIPY oxidation (Fig. 3). We therefore used that concentration (1 mM) to evaluate the protective effects of these compounds on intestinal I/R injury in rats. Lipid peroxidation is an integral process in the oxidation of unsaturated fatty acids via a radical chain reaction and overpro-
4. Discussion ROS are continuously generated by metabolism in the body and exert physiological actions (Gate, Paul, Ba, Tew, & Tapiero, 1999; Hensley & Floyd, 2002). Although the action of ROS is normally limited by the antioxidant defence system of the body, an excess of ROS induces oxidative damage in vulnerable targets such as membrane unsaturated fatty acids, protein thiols, and DNA bases (Ceconi, Boraso, Cargnoni, & Ferrari, 2003). Since oxidative stress is an imbalance between ROS production and antioxidant defence, the antioxidant capacity in patients is likely to be compromised. Much interest has been shown in the discovery of new antioxidants in recent years due to their potential applications in the treatment of ROS-induced diseases. For the prevention of these diseases, an approach focusing on the health aspects of functional foods should be beneficial (Swinbanks & O’Brien, 1993). However, the assessment of functional foods for approval for human consumption is not as severe as that for pharmaceutical drugs. Thus, there is not so much available experimental evidence of their physiological functions. In order to develop new functional foods, it is important to select a suitable ingredient that has a scientific background. In Japan, ferulic acid has been approved as a food additive to prevent oxidation. Moreover, ferulic acid is used as a functional food ingredient. However,
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antioxidant properties of ferulic acid have not been elucidated in detail. In this study, we carried out in vitro and in vivo experiments to elucidate in more detail the scientific background of antioxidant activities of ferulic acid. It is difficult to quantitate superoxide anion because of its reactive nature and short lives, however, MPEC-enhanced chemiluminescence is a simple and reproducible tool indicating the involvement of superoxide anion. Thus, the MPEC assay is suitable for measuring the superoxide anion scavenging activities of various compounds. Since the intestinal mucosa is one of the richest sources of xanthine oxidase, xanthine oxidase inhibition activity also contributes to the protective effect on oxidation damage in the rat small intestine. In the MPEC assay, superoxide anions were generated by the xanthine/xanthine oxidase system. Thus, an inhibitory effect on xanthine oxidase itself also leads to a decrease in light emission. We can evaluate combined antioxidant activity from radical scavenging and xanthine oxidase inhibition using MPEC. In this study, we found that EGCG and ascorbic acid had much higher levels of antioxidant activity and almost completely inhibited the light emission at 50 lM. In addition to EGCG and ascorbic acid, b-carotene and a-lipoic acid also exhibited significant antioxidant activity from both superoxide anion scavenging and xanthine oxidase inhibition. On the other hand, ferulic acid had only weak superoxide anion scavenging and xanthine oxidase inhibition activities. Highly reactive free radicals, such as peroxyl radicals involved in auto-oxidation of lipoproteins and of biological membranes, are responsible for microvascular damage. Since antioxidant evaluation is currently conducted in many cases merely by means of radical scavenging tests, it is important to investigate the chainbreaking ability of antioxidants. Recently, a simple method that enables specific measurements of chain-breaking activities of tested compounds using BODIPY has been developed. We investigated the chain-breaking effects of EGCG, ascorbic acid, and ferulic acid by this method. All of the tested compounds, including ferulic acid, exhibited chain-breaking activity. Evidence supporting an antioxidant function for polyphenols is usually derived from in vitro assays. However, evidence that polyphenols, including ferulic acid, act directly or indirectly as antioxidants in vivo is limited. Antioxidant studies in laboratory animals should be carried out in order to obtain a mechanistic basis and safety profiles before they can be applied to humans for intervention trials. It has been reported that ferulic acid is absorbed by passive diffusion or by a facilitated transporter that appears not to be saturated even at a luminal concentration of 50 lmol/l (Chan, Zhang, Fung, Guo, & Tam, 1999). Under the conditions used in that study, 56.1% of perfused ferulic acid enters enterocytes. We therefore focused on the small intestine in order to investigate the in vivo antioxidant activities of ferulic acid. It is well known that ROS are responsible for I/R injury and that the intestine is highly sensitive to I/R injury (Adam et al., 2002; Granger, et al., 1981; Illyes & Hamar, 1992; Kong et al., 1998). In this study, we investigated the antioxidant activities of ferulic acid in in vivo studies using an intestinal I/R model. Reperfusion of the ischaemic intestine results in an increase in lipid peroxide and in vascular permeability to Evans blue dye. The production of lipid peroxide and the increase in vascular permeability were attenuated by pretreatment with EGCG and ascorbic acid, which have superoxide anion scavenging activity, xanthine oxidase inhibition activity and chain-breaking activity. Ferulic acid, which has only chain-breaking activity, also showed a protective effect on I/R injury in the rat small intestine. The results suggest that chain-breaking activity, not superoxide anion scavenging activity, xanthine oxidase inhibition activity and NADPH oxidase inhibition activity, plays a contributory role in the protective effect of ferulic acid on intestinal I/R injury. I/R in-
jury occurs clinically during abdominal aortic aneurysm surgery, small bowel transplantation, cardiopulmonary bypass, strangulated hernias, and neonatal necrotizing enterocolitis. Moreover, reperfusion of ischaemic vascular beds may lead to recruitment and activation of leukocytes, release of mediators of the inflammatory process and further injury to the affected vascular bed and remote sites (Lefer & Lefer, 1996). Thus, strategies that limit the damage induced by the reperfusion process may be useful in the treatment of ischaemic disorders in various organs (Willerson, 1997). The protective effect of ferulic acid on intestinal I/R injury may be relevant to human health. As stated above, I/R injury is considered to be a major clinical problem and occurs in many kinds of tissues, including the stomach, pancreas, and cardiac and skeletal muscle (Harris, Leiderer, Peer, & Messmer, 1996; Reiter & Tan, 2003). In addition to I/R injury, ROS are thought to play an important role in many diseases (Benzie, 2000; Gate et al., 1999; Stocker, 1999). Although there have been few studies on the tissue distribution of ferulic acid after oral administration, ferulic acid stays longer in blood than do other antioxidants such as ascorbic acid. Ferulic acid would therefore be useful for the prevention of oxidation damage in various tissues. Further studies are needed to clarify the pharmacological effects of ferulic acid in other tissues. In summary, we have found that EGCG and ascorbic acid, which have combined antioxidant activity from radical scavenging, xanthine oxidase inhibition and chain-breaking effects, have protective effects on I/R injury in the rat small intestine. Although combined antioxidant activity from radical scavenging and xanthine oxidase inhibition of ferulic acid was much weaker than the combined antioxidant activities of EGCG and ascorbic acid, treatment with ferulic acid also prevented the increase in vascular permeability caused by intestinal I/R. It is possible that chainbreaking activity plays a contributory role in the protective effect of ferulic acid on oxidative injury in humans and in in vivo studies. Furthermore, the results of this study should provide a scientific background of the usefulness of ferulic acid as a functional food ingredient.
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In vitro and in vivo antioxidant properties of ferulic acid: A comparative study with other natural oxidation inhibitors Shirou Itagaki, Toshimitsu Kurokawa, Chie Nakata, Yoshitaka Saito, Setsu Oikawa, Masaki Kobayashi, Takeshi Hirano, Ken Iseki * Laboratory of Clinical Pharmaceutics and Therapeutics, Department of Biopharmaceutical Sciences and Pharmacy, Division of Biopharmaceutical Sciences and Pharmacy, Faculty of Pharmaceutical Sciences, Hokkaido University, Kita 12-jo, Nishi 6-chome, Kita-ku, Sapporo 060-0812, Japan
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Article history: Received 27 June 2008 Received in revised form 12 August 2008 Accepted 24 September 2008
Keywords: Reactive oxygen substances Ferulic acid EGCG Ascorbic acid Chain-breaking
a b s t r a c t Ferulic acid exhibits a wide range of therapeutic effects that are attributed to its potent antioxidant capacity. However, in vitro antioxidant properties of ferulic acid have not been elucidated in detail. Evidence that polyphenols, including ferulic acid, act as antioxidants in vivo is also limited. In order to elucidate in more detail the scientific background of antioxidant activities of ferulic acid, we carried out in vitro and in vivo experiments. We focused on superoxide anion scavenging activity, xanthine oxidase inhibition activity, and chain-breaking activity. The combined antioxidant activity from radical scavenging and xanthine oxidase inhibition of ferulic acid was much weaker than that of ()-epigallocatechin gallate (EGCG) and ascorbic acid. On the other hand, EGCG, ascorbic acid and ferulic acid exhibited chain-breaking activity and prevented ischaemia-reperfusion-associated intestinal injury. Chain-breaking activity may play a contributory role in the protective effect of ferulic acid on oxidative injury in humans and in in vivo studies. Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction Production of reactive oxygen species (ROS) in tissue contributes to the development of various chronic diseases such as cancer, neurodegenerative diseases, and cardiovascular diseases (Benzie, 2000; Stocker, 1999). Administration of antioxidants to patients may therefore help in removing ROS and thus improve the clinical outcome. Dietary antioxidants can enhance cellular defence and help to prevent oxidation damage to cellular components. There has been considerable public and scientific interest in therapeutic use of natural antioxidants. Among the natural antioxidants, polyphenols play a very important role. Dietary polyphenols are thought to be beneficial to human health by exerting various biological effects such as free radical scavenging, metal chelation, modulation of enzymatic activity, and alteration of signal transduction pathways (Singh & Aggarwal, 1995; Stocker, 1999; Yoshioka, Deng, Cavigelli, & Karin, 1995). Epidemiological studies have shown relationships between consumption of polyphenol-rich foods and prevention of diseases such as cancer, coronary heart disease, and osteoporosis, and results of these studies have promoted interest in polyphenols (Adlercreutz & Mazur, 1997; Steinmetz & Potter, 1996). * Corresponding author. Tel./fax: +81 11 706 3770. E-mail address: [email protected] (K. Iseki). 0308-8146/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2008.09.073
Polyphenols are classified into phenolic acids, flavonoids, and less common stilbenes and lignans. Many studies have focused on the antioxidant activities of flavonoids. Although several flavonoids are highly efficacious free radical scavengers in vitro, there is little information on the importance of dietary flavonoids as antioxidants in vivo, or evidence for such activity in vivo. Moreover, there have been few studies on phenolic acids compared to the number of studies on flavonoids, despite the high contents of phenolic acids in fruits, cereals, and some vegetables (Clliford, 1999). Ferulic acid is a phenolic compound that exhibits a wide range of therapeutic effects against various diseases, including cancer, diabetes, and cardiovascular and neurodegenerative diseases. Moreover, ferulic acid has been approved in Japan as a food additive to prevent oxidation. Ferulic acid is a ubiquitous plant constituent that arises from the metabolism of phenylalanine and tyrosine. Ferulic acid is a major constituent of fruits and vegetables such as orange, tomato, carrot, sweet corn, and rice bran (Balasubashini, Rukkumani, & Menon, 2003). Ferulic acid is a phenolic compound and it possesses three distinctive structural motifs that can possibly contribute to its free radical scavenging capability. The presence of electron donating groups on the benzene ring (3 methoxy and more importantly 4-hydroxyl) of ferulic acid gives the additional property of terminating free radical chain reactions. The next functionality – the carboxylic acid group in ferulic acid with an adjacent unsaturated C–C double bond – can provide
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additional attack sites for free radicals and thus prevent them from attacking the membrane. In addition, this carboxylic acid group also acts as an anchor of ferulic acid, by which it binds to the lipid bilayer, providing some protection against lipid peroxidation (Kanaski, Aksenova, Stoyanova, & Butter field, 2002). Ferulic acid is commercially prepared and used as a functional food ingredient. Orally administered compounds are absorbed in the intestine. It is now recognised that several transporters contribute to the absorption of administered compounds from the intestine. Recently, we have reported that intestinal uptake of ferulic acid is associated with an H+-driven transport system, which is identical to the nateglinide/fluorescein transport system (Itagaki et al., 2005). The physiological importance of ferulic acid depends on its availability for absorption and subsequent interaction with target tissues. Since there is limited information on the importance of ferulic acid as an antioxidant in vivo, we focused on the protective effect of ferulic acid on intestinal oxidative injury. The intestinal mucosa is extremely sensitive to ROS (Illyes & Hamar, 1992; Kong, Blennerhassett, Heel, McCauley, & Hall, 1998). It is well known that ROS are responsible for intestinal ischaemia–reperfusion (I/R) injury (Adam et al., 2002; Granger, Rutili & McCord; 1981). Intestinal I/R is a common clinical problem in the settings of severe burns, circulatory shock, and strangulation ileus. Furthermore, intestinal I/R injury is a serious medical problem often necessitating surgical intervention. In this study, we used a rat mesenteric I/R injury model as a model of oxidative injury and investigated the antioxidant activities of ferulic acid in vivo.
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the multilabel counter Wallac 1420 ARVOse (Perkin Elmer, Wellesley, MA). 2.4. Measurement of radical chain-breaking activity The assay was carried out as described in a previous report with some modification (Beretta et al., 2006). Stock solutions of the fluorescent lipophilic probe BODIPY were prepared in dimethyl sulfoxide and stored under nitrogen at 70 °C. For BODIPY incorporation, 25 ll of the BODIPY stock solution (2 mM) was diluted 100-fold with PBS. Then aliquots of 50 ll were added to 50 ll of rat plasma, diluted to 350 ll of PBS with or without tested compounds (final concentrations of 0.001, 0.01, 0.1, 1, and 10 mM, respectively), vortexed for 20 s at the lowest speed, and incubated under aerobic conditions for 10 min at 37 °C. The final volume was adjusted to 480 ll with PBS, yielding BODIPY at a final concentration of 2 lM. MeO-AMVN was dissolved in CH3CN immediately before use, and 20 ll of the solution was added to the sample at a final concentration of 2 mM. Then aliquots of 200 ll were transferred to a 96-microwell plate, and the lipid oxidation kinetics was monitored by measuring the green fluorescence (kex = 485, kem = 535 nm; cycle time of 10 min for 18 cycles) of the oxidation product of BODIPY. Light emission was measured with the multilabel counter Wallac 1420 ARVOse. The results are expressed as total antioxidant performance (TAP) values, representing the percentage of inhibition of BODIPY oxidation in human plasma with respect to that occurring in a control sample: TAP = [(AUCcontrolAUCplasma)/AUCcontrol] 100, where AUCcontrol and AUCsample represent the area under the curve (AUC) of BODIPY oxidation kinetics in the control and plasma samples, respectively. Control samples were prepared using PBS.
2. Materials and methods 2.5. Intestinal I/R model 2.1. Chemicals
a-Lipoic acid, ferulic acid, and hypoxanthine were purchased from Sigma (St Louis, MO). b-Carotene, ()-epigallocatechin gallate (EGCG) and, 2,20 -Azobis(4-methoxy-2,4-dimethylvaleronitrile) (MeO-AMVN) were purchased from Wako (Osaka, Japan). Xanthine oxidase was purchased from Nacalai Tesque (Kyoto, Japan). 4,4-Difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a,-diaza-s-indacene-3propionic acid saccinimidyl ester (BODIPY) was purchased from Invitrogen (Carlsbad, CA). All other reagents were of the highest grade available and used without further purification. 2.2. Animals Male Wistar rats, aged six weeks (180–200 g in weight), were obtained from Japan Laboratory Animals (Tokyo, Japan). The housing conditions were the same as those described previously (Ochiai et al., 2007). The rats were housed at least one week at 23 ± 3 °C and 50 ± 10% relative humidity and were maintained on a 12 h light/dark cycle. During the acclimatisation the rats were allowed freely to access food and water. The experimental protocols were reviewed and approved by the Hokkaido University Animal Care Committee in accordance with the ‘‘Guide for the Care and Use of Laboratory Animals”.
Surgical procedures were carried out as described in a previous report with some modification (Ogura, Kobayashi, Itagaki, Hirano, & Iseki, 2008). Wistar rats were anesthetized with sodium pentobarbital (40 mg/kg weight, i.p.). The rats were fixed after the operation. A small midline incision was made in the abdomen. A 5-cm-long loop of the jejunum was identified and ligated at both ends. Through a midline laparotomy, the superior mesenteric artery (SMA) was isolated and a bulldog arterial clamp was applied at the aortic origin. The abdomen was then covered with a sterile plastic wrap. After 30 min of intestinal ischaemia, the arterial clamp was removed. Five hundred microlitres of tested compounds (1 mM) was administered directly into the loops 1 h before the induction of ischaemia. 2.6. Tissue sampling The 5-cm-long intestine was excised, the contents were removed, and the intestine was cleansed in ice-cold saline. The intestine was then homogenised in 2.5 ml saline using a glass Teflon homogenizer with 20 strokes. Protein was measured by the method of Lowry, Rosebrough, Farr, and Randall (1951) with bovine serum albumin as a standard. 2.7. TBA analysis
2.3. Measurement of superoxide anion scavenging activity Superoxide anion scavenging activity was measured by the chemiluminescent superoxide anion probe method, using a superoxide anion-2-methyl-6-methoxyphenylethynylimidazopyrazynone (MPEC) reaction kit (ATTO Corp., Osaka, Japan) according to the manufacturer’s instructions. Superoxide anions were generated by xanthine/xanthine oxidase. Light emission was measured with
The amount of lipid peroxide in the intestine was determined as that of malondialdehyde (MDA) by the method of Ohkawa, Ohishi, and Yagi (1979) with some modification (Kurokawa et al., 2006). Thiobarbituric acid (TBA) solution was composed of 2.6 mM TBA, 918 mM trichloroacetic acid, 0.3 mM HCl, and 1.8 mM 2,6-ditert-butyl-4-metylphenol (BHT) in 22% ethanol. The reaction mixture contained 0.2 ml of tissue homogenate, 0.2 ml of 8.1% sodium
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dodecyl sulphate (SDS), 1.5 ml of 20% acetic acid solution (pH 3.5), and 1.5 ml of 0.8% aqueous solution of TBA. The mixture was heated at 95 °C for 60 min. After cooling with tap water, 1.0 ml of distilled water and 5.0 ml of n-butanol were added, and the mixture was shaken vigorously. After centrifugation at 3000x g for 10 min, the absorbance of the organic layer (upper layer) was measured at 535 nm with 1,1,3,3-tetraethoxypropane as a standard. 2.8. Evaluation of changes in vascular permeability Extravasation of Evans blue dye into tissue was used as an index of increased vascular permeability (Matos, Souza, Seabra, FreireMaia, & Teixeira, 1999). Evans blue dye (20 mg/kg) was injected (1 ml/kg) through the jugular vein 5 min prior to reperfusion of the ischaemic artery. Sixty min after reperfusion, fragments of the loop were cut and allowed to dry in a petri dish for 24 h at 37 °C. The dry weight of the tissue was calculated and Evans blue dye was extracted using 4 ml of formamide (24 h at 37 °C). The dye concentration of the samples was measured by spectrophotometry at 620 nm using JASCO V-630 BIO (Tokyo, Japan). Results are presented as the amount of Evans blue in lg per 100 mg of tissue. 2.9. Data analysis Non-linear regression analysis was performed by using OriginÒ (version 6.1J). Student’s t-test was used to determine the significance of differences between two group means. Statistical significance among means of more than two groups was determined by one-way analysis of variance (ANOVA). A value of p < 0.05 was considered significant.
3. Results 3.1. Inhibition of xanthine oxidase-induced light emission The superoxide radical anion appears to play a central role since other ROS are formed in reaction sequences starting with superoxide radical anion. Xanthine/xanthine oxidase is also a main source of ROS (Chambers et al., 1985). Moreover, intestinal mucosa is one of the richest sources of xanthine oxidase. In this study, we therefore used a xanthine/xanthine oxidase system to generate superoxide anions. A mixture of xanthine and xanthine oxidase generated superoxide anions, which were reacted with MEPC to give light emission by chemiluminescence. Since light emission induced by xanthine oxidase is inhibited in the presence of a radical scavenger or xanthine oxidase inhibitor, the inhibitory effect of a compound on the increase in chemiluminescence intensity indicates the combined antioxidant activity from both superoxide anion scavenging and xanthine oxidase inhibition. In the first part of this study, the antioxidant activities of various compounds were evaluated using the xanthine/xanthine oxidase system and MPEC. As shown in Fig. 1, four antioxidants, EGCG, ascorbic acid, b-carotene and a-lipoic acid, inhibited the light emission induced by xanthine oxidase in a concentration-dependent manner. IC50 values of these compounds for light emission induced by xanthine oxidase are listed in Table 1. We then investigated the effects of ferulic acid on xanthine oxidase-induced light emission. The IC50 value of ferulic acid for light emission induced by xanthine oxidase was different from the values of EGCG, ascorbic acid, b-carotene and a-lipoic acid (Fig. 1, Table 1).
Fig. 1. Dose–response relationship for the inhibition of xanthine oxidase-induced light emission by various compounds. Each point represents the mean with SD of three independent experiments. Abbreviations: EGCG, ()-epigallocatechin gallate.
Table 1 IC50 values of various compounds on xanthine oxidase-induced light emission Compounds
IC50 (lM)
EGCG Ascorbic acid b-Carotene a-Lipoic acid Ferulic acid
5.34 22.5 89.1 479 6614
Abbreviations: EGCG, ()-epigallocatechin gallate.
of ferulic acid were quite different from those of EGCG and ascorbic acid. Since the influence of free radical-mediated oxidation is amplified because it proceeds by a chain mechanism, the role of chain-breaking activity is important as well as radical scavenger activity (Riley, 1994). Recently, Beretta et al. (2006) reported a method that enables specific measurements of chain-breaking activities of tested compounds using BODIPY and showed that EGCG and ascorbic acid act as chain-breaking antioxidants as well as radical scavengers. We therefore investigated the chain-breaking activity of ferulic acid using BODIPY. Fig. 2 shows the oxidation kinetics of BODIPY in the presence of ascorbic acid. Ascorbic acid inhibited BODIPY oxidation, and the effect was concentrationdependent. Thus, the TAP value of ascorbic acid also increased in a concentration-dependent manner (Fig. 3). We then examined the effects of EGCG and ferulic acid on BODIPY oxidation. Similar
3.2. Inhibitory effects of EGCG, ascorbic acid and ferulic acid on BODIPY oxidation Results of the MPEC assay showed that combined antioxidant activities from radical scavenging and xanthine oxidase inhibition
Fig. 2. Dose–response relationship for the inhibition of BODIPY oxidation by ascorbic acids. Abbreviations: BODIPY, 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4bora-3a,4a,-diaza-s-indacene-3-propionic acid saccinimidyl ester.
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Fig. 3. Antioxidant activity of EGCG, ascorbic acid and ferulic acid as determined by the TAP assay. Each point represents the mean with SD of three independent experiments. Abbreviations: EGCG, ()-epigallocatechin gallate; TAP, total antioxidant performance.
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Fig. 5. Effects of EGCG, ascorbic acid and ferulic acid (1 mM) on the changes in vascular permeability in the small intestine. Data are given as mean with SD of four rats (I/R+EGCG, I/R+Ascorbic acid, I/R+ferulic acid) or five rats (sham, I/R). *P < 0.05, significantly different from non ischaemia control animals. P < 0.05, significantly different from animals not treated with compounds (I/R animals). Abbreviations: I/ R, ischaemia–reperfusion; EGCG, ()-epigallocatechin gallate.
duction of lipid peroxide contributes to small intestinal injury. The increase in the amount of lipid peroxide after I/R was significantly inhibited by treatment with EGCG, ascorbic acid, and ferulic acid (Fig. 4). Since vascular permeability has been shown to be significantly higher in rats with I/R than in sham-operated rats, elevated vascular permeability is also one of the indicators of I/R injury (Pompermayer et al., 2007). We therefore investigated the effects of these compounds on the increased vascular permeability, as assessed by reduction in the level of Evans blue dye. Elevation of vascular permeability by intestinal I/R was attenuated by treatment with EGCG and ascorbic acid (Fig. 5). Ferulic acid also prevented the elevation of vascular permeability following I/R injury in the intestine (Fig. 5). Fig. 4. Effects of EGCG, ascorbic acid and ferulic acid (1 mM) on the amount of lipid peroxide in the small intestine after I/R. Data are given as mean with SD of four rats. * P < 0.05, significantly different from non ischaemia control animals. P < 0.05, significantly different from animals not treated with compounds (I/R animals). Abbreviations: MDA, malondialdehyde; I/R, ischaemia–reperfusion; EGCG, ()epigallocatechin gallate.
to ascorbic acid, EGCG, and ferulic acid also inhibited BODIPY oxidation, and the TAP values of these compounds increased in a concentration-dependent manner (Fig. 3). 3.3. Protective effects of EGCG, ascorbic acid and ferulic acid on intestinal I/R injury We confirmed in vivo antioxidant activities of EGCG, ascorbic acid, and ferulic acid using a rat I/R injury model. Although 1 mM EGCG and ascorbic acid completely inhibited the light emission induced by xanthine oxidase, 1 mM ferulic acid did not inhibit the light emission (Fig. 1). On the other hand, 1 mM ferulic acid significantly inhibited BODIPY oxidation (Fig. 3). We therefore used that concentration (1 mM) to evaluate the protective effects of these compounds on intestinal I/R injury in rats. Lipid peroxidation is an integral process in the oxidation of unsaturated fatty acids via a radical chain reaction and overpro-
4. Discussion ROS are continuously generated by metabolism in the body and exert physiological actions (Gate, Paul, Ba, Tew, & Tapiero, 1999; Hensley & Floyd, 2002). Although the action of ROS is normally limited by the antioxidant defence system of the body, an excess of ROS induces oxidative damage in vulnerable targets such as membrane unsaturated fatty acids, protein thiols, and DNA bases (Ceconi, Boraso, Cargnoni, & Ferrari, 2003). Since oxidative stress is an imbalance between ROS production and antioxidant defence, the antioxidant capacity in patients is likely to be compromised. Much interest has been shown in the discovery of new antioxidants in recent years due to their potential applications in the treatment of ROS-induced diseases. For the prevention of these diseases, an approach focusing on the health aspects of functional foods should be beneficial (Swinbanks & O’Brien, 1993). However, the assessment of functional foods for approval for human consumption is not as severe as that for pharmaceutical drugs. Thus, there is not so much available experimental evidence of their physiological functions. In order to develop new functional foods, it is important to select a suitable ingredient that has a scientific background. In Japan, ferulic acid has been approved as a food additive to prevent oxidation. Moreover, ferulic acid is used as a functional food ingredient. However,
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antioxidant properties of ferulic acid have not been elucidated in detail. In this study, we carried out in vitro and in vivo experiments to elucidate in more detail the scientific background of antioxidant activities of ferulic acid. It is difficult to quantitate superoxide anion because of its reactive nature and short lives, however, MPEC-enhanced chemiluminescence is a simple and reproducible tool indicating the involvement of superoxide anion. Thus, the MPEC assay is suitable for measuring the superoxide anion scavenging activities of various compounds. Since the intestinal mucosa is one of the richest sources of xanthine oxidase, xanthine oxidase inhibition activity also contributes to the protective effect on oxidation damage in the rat small intestine. In the MPEC assay, superoxide anions were generated by the xanthine/xanthine oxidase system. Thus, an inhibitory effect on xanthine oxidase itself also leads to a decrease in light emission. We can evaluate combined antioxidant activity from radical scavenging and xanthine oxidase inhibition using MPEC. In this study, we found that EGCG and ascorbic acid had much higher levels of antioxidant activity and almost completely inhibited the light emission at 50 lM. In addition to EGCG and ascorbic acid, b-carotene and a-lipoic acid also exhibited significant antioxidant activity from both superoxide anion scavenging and xanthine oxidase inhibition. On the other hand, ferulic acid had only weak superoxide anion scavenging and xanthine oxidase inhibition activities. Highly reactive free radicals, such as peroxyl radicals involved in auto-oxidation of lipoproteins and of biological membranes, are responsible for microvascular damage. Since antioxidant evaluation is currently conducted in many cases merely by means of radical scavenging tests, it is important to investigate the chainbreaking ability of antioxidants. Recently, a simple method that enables specific measurements of chain-breaking activities of tested compounds using BODIPY has been developed. We investigated the chain-breaking effects of EGCG, ascorbic acid, and ferulic acid by this method. All of the tested compounds, including ferulic acid, exhibited chain-breaking activity. Evidence supporting an antioxidant function for polyphenols is usually derived from in vitro assays. However, evidence that polyphenols, including ferulic acid, act directly or indirectly as antioxidants in vivo is limited. Antioxidant studies in laboratory animals should be carried out in order to obtain a mechanistic basis and safety profiles before they can be applied to humans for intervention trials. It has been reported that ferulic acid is absorbed by passive diffusion or by a facilitated transporter that appears not to be saturated even at a luminal concentration of 50 lmol/l (Chan, Zhang, Fung, Guo, & Tam, 1999). Under the conditions used in that study, 56.1% of perfused ferulic acid enters enterocytes. We therefore focused on the small intestine in order to investigate the in vivo antioxidant activities of ferulic acid. It is well known that ROS are responsible for I/R injury and that the intestine is highly sensitive to I/R injury (Adam et al., 2002; Granger, et al., 1981; Illyes & Hamar, 1992; Kong et al., 1998). In this study, we investigated the antioxidant activities of ferulic acid in in vivo studies using an intestinal I/R model. Reperfusion of the ischaemic intestine results in an increase in lipid peroxide and in vascular permeability to Evans blue dye. The production of lipid peroxide and the increase in vascular permeability were attenuated by pretreatment with EGCG and ascorbic acid, which have superoxide anion scavenging activity, xanthine oxidase inhibition activity and chain-breaking activity. Ferulic acid, which has only chain-breaking activity, also showed a protective effect on I/R injury in the rat small intestine. The results suggest that chain-breaking activity, not superoxide anion scavenging activity, xanthine oxidase inhibition activity and NADPH oxidase inhibition activity, plays a contributory role in the protective effect of ferulic acid on intestinal I/R injury. I/R in-
jury occurs clinically during abdominal aortic aneurysm surgery, small bowel transplantation, cardiopulmonary bypass, strangulated hernias, and neonatal necrotizing enterocolitis. Moreover, reperfusion of ischaemic vascular beds may lead to recruitment and activation of leukocytes, release of mediators of the inflammatory process and further injury to the affected vascular bed and remote sites (Lefer & Lefer, 1996). Thus, strategies that limit the damage induced by the reperfusion process may be useful in the treatment of ischaemic disorders in various organs (Willerson, 1997). The protective effect of ferulic acid on intestinal I/R injury may be relevant to human health. As stated above, I/R injury is considered to be a major clinical problem and occurs in many kinds of tissues, including the stomach, pancreas, and cardiac and skeletal muscle (Harris, Leiderer, Peer, & Messmer, 1996; Reiter & Tan, 2003). In addition to I/R injury, ROS are thought to play an important role in many diseases (Benzie, 2000; Gate et al., 1999; Stocker, 1999). Although there have been few studies on the tissue distribution of ferulic acid after oral administration, ferulic acid stays longer in blood than do other antioxidants such as ascorbic acid. Ferulic acid would therefore be useful for the prevention of oxidation damage in various tissues. Further studies are needed to clarify the pharmacological effects of ferulic acid in other tissues. In summary, we have found that EGCG and ascorbic acid, which have combined antioxidant activity from radical scavenging, xanthine oxidase inhibition and chain-breaking effects, have protective effects on I/R injury in the rat small intestine. Although combined antioxidant activity from radical scavenging and xanthine oxidase inhibition of ferulic acid was much weaker than the combined antioxidant activities of EGCG and ascorbic acid, treatment with ferulic acid also prevented the increase in vascular permeability caused by intestinal I/R. It is possible that chainbreaking activity plays a contributory role in the protective effect of ferulic acid on oxidative injury in humans and in in vivo studies. Furthermore, the results of this study should provide a scientific background of the usefulness of ferulic acid as a functional food ingredient.
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