Opuntia vulgaris, a species native to Tunisia, is widely distributed on the center and the south of Tunisia, and its fruits are an excellent source of antioxidants. The ameliorative effects of O. vulgaris fruit extract (OE) was evaluated against methanol-induced haematological and biochemical toxicity in rats. The methanol induced haematological and biochemical perturbation which significantly decreased the levels of red blood cell (RBC), haemoglobin (Hb) and hematocrit (Ht), and increased glucose, cholesterol and triglycerids, and decreased total protein in serum. Treatment of rats with methanol decreased the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) and significantly increased lipid peroxidation (LPO) level in erythrocytes. OE treatment could increase significantly the levels of RBC, Hb, Ht and total protein, and decrease glucose, cholesterol and triglycerids in serum, and increased the activities of SOD, CAT and GPx in erythrocytes when compared with methanol-treated group. Spleen histopathology showed that OE could significantly reduce the incidence of spleen lesion induced by methanol. These results suggested that the OE exhibit a potential source of natural antioxidants against methanol-induced haematological and biochemical disruption in rats, and the protective effects of OE may be due to the modulation of antioxidant enzymes activities and inhibition of lipid peroxidation.
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Key-words: antioxidative enzymes, haematological profile, Opuntia vulgaris, lipid peroxidation, methanol, modulatory role.
Methanol is used as a solvent in many products such as antifreeze, pesticides, varnish and gasoline. Methanol, first in the alcohol series, is normally used as an industrial solvent and cleanser. People handling products that contain methanol may inhale the toxic vapor during its evaporation from the product surface. Accidental or intentional exposure to this alcohol can yield mild to severe health problems and, in extreme cases, coma and death (Sullivan-Mee and Solis, 1998; Palziac et al., 2003). The neurotoxicity of methanol (through acute, subacute or chronic poisoning) is attributed to its metabolite, the formic acid or formate, which inhibits the cytochrome oxidase system, necessary for ATP production (Eells et al., 1996; McKellar et al., 1997). Formic acid is the toxic metabolite responsible for the metabolic acidosis observed in methanol-intoxicated humans (Jacobsen et al., 1986; Seme et al., 1999) and nonhuman primates (Medinsky et al., 1997). Formic acid is also believed to be the metabolite responsible for the ocular toxicity seen in methanol-poisoned humans (Anderson et al., 1989), nonhuman primates, and rats (Eells et al., 1996). Humans are particularly sensitive to methanol because they have a limited capacity to rapidly oxidize and therefore eliminate the formic acid (Snyder and Andrews, 1996).
Ingestion of methanol can cause severe metabolic acidosis and clinical disturbances such as blindness, serious neurological sequelae and death (Kuteifan et al., 1998). Now methanol is an ever more recognized as a substance that damages the liver cells where it is oxidized to formaldehyde and later to formate (Kurcer et al., 2007). These processes are accompanied by elevation of NADH level and the formation of superoxide anion that may be involved in lipid peroxidation (LPO) (Poli, 1993). This fact can be associated with mitochondrial injury, especially with regard to the cell respiratory chain; partial inhibition of this chain may cause increased auto-oxidation of a redox carrier, resulting in increased production of oxygen radicals (Skrzydlewska & Farbiszewski, 1997). These factors together with the excess of formaldehyde formed during acute methanol intoxication significantly increases the LPO, which is an amplifier for initially formed reactive aldehydes generated during LPO (Gutterridge, 1995). Products of LPO are very harmful to cells causing finally their death and can act as second toxic messengers of the complex chain reaction (Parthasarathy et al., 2006). In addition, immune cells are sensitive to changes in the antioxidant status, as they carry out important functions through the generation of a high number of oxygen free radicals (Pavlovic et al., 2007). Erythrocytes are constantly being subjected to various types of oxidative stress, ingested chemicals and accidentally methanol. Although rat erythrocytes contain an abundance of catalase they are incapable of oxidizing chemicals (Tsakiris et al., 2005). Methanol toxicity, either acute or chronic, is characterized by a severe dearrangement of subcellular metabolism and structural alteration of different cells. The distribution of methanol by the blood to all organs and tissues is proportionate to their water content (Liesivuori & Savolainen, 1991). Exposures of tissue to free radicals in a variety of experimental systems have recognized the ability of free radicals in a variety of experimental systems have documented the ability of free radicals to produce damage. Systematically, methanol is moderately toxic to the liver and produces haematological effects. The mechanism by which methanol produces these effects is unknown. Previous data (Narayanaperumal et al., 2006) demonstrated that methanol induced free radicals and an imbalanced antioxidant system may damage the liver and the kidney functions and probably contributed a disruption of some metabolic and biochemical parameters. It is thought that this toxic effect of methanol can be related to decrease of cellular detoxification capacity or increase of generation of reactive intermediates. Red blood cell membrane is rich in polyunsaturated fatty acids which are very susceptible to free radicals mediated peroxidation. Eventually, haemolysis is induced by membrane lipid peroxidation (Lanping et al., 2000). Lipid peroxidation is associated with a wide variety of toxicological effects, including decreased membrane fluidity and function.
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Opuntia vulgaris, a native species to Tunisia, is widely distributed on the center and the south of Tunisia, and its fruits known as prickly pears or cactus pears are an excellent source of betalain natural colorants and functional compounds. Opuntia spp. fruits represents lower risk for microbiological contamination, have no nitrate content, are highly flavored, show adequate nutritional properties (e.g. high levels of calcium, magnesium and vitamin C), and contains interesting functional compounds like quercetin (Butera et al., 2002; Piga, 2004; Stintzing et al., 2001, 2005). The juice of the plant is used in the treatment of syphilis in Ayurveda (Mhaskar et al, 2001). The aqueous extract of O. vulgaris on preliminary chemical analysis is found to contain saponin and alkaloid (Jiang et al, 2003; Nadkarni, 2000). On the other hand, Opuntia spp. extracts have shown analgesic, anti-inflammatory, hypoglycemic, physiological antioxidant, cancer chemoprevention, and neuroprotective effects (Kim et al. 2006; Tesoriere et al., 2004; Zou et al., 2005). Recent studies have shown that some phenolic compounds can prevent some chemical solvents-induced oxidative damage, and the ability of phenolic compounds might be related to their antioxidant properties. Thus, the objective of this study has performed to evaluate the potential protective effects of aqueous extract of Opuntia vulgaris fruit in methanol-induced haematological and biochemical toxicity in male Wistar rats.
Material and Methods
Preparation of Opuntia vulgaris extracts (OE)
The fruits of Opuntia vulgaris were collected from a culture area located in Kasserine region, Tunisia. Fruit samples were ground, put in water and shake (10g/l, v/w) for 15-20 min, and then filtered using Whatman filter paper. The aqueous extract was given as beverage instead of tap water.
Adult male albino Wistar rats weighing 180 to 200g were obtained from Central Pharmacy of Tunisia (SIPHAT, Tunisia). Animals were quarantined and allowed to acclimate for a week prior to experimentation. The animals were handled under standard laboratory conditions of a 12-h light/dark cycle in a temperature- and humidity-controlled room. Food and water were available ad libitum. Our Institutional Animal Care and Use Committee approved the protocols for the animal study, and the animals were cared for in accordance with the institutional ethical guidelines.
After acclimatization, the rats were divided into two batches: 16 control rats (C) drinking tap water and 16 treated-rats drinking O. vulgaris extract (OE) for six weeks. Then, each group was divided into two subgroups and one of them was injected daily (IP), for four weeks, with methanol (2.37 g/kg b.wt.) accordingly to Parthasarathy et al. (2006). After treatment, 8 rats of each group were sacrificed under anaesthesia by i.p injection of chloral hydrate. All animal procedures were conducted in strict conformation with the local Institute Ethical Committee Guidelines for the care and use of laboratory animals of our Institution.
Determination of haematological parameters
After animal anaesthesia with chloral hydrate by intra-abdominal way, blood samples were collected with heparin by heart puncture to determine blood cell parameters (red blood cell number (RBC), haemoglobin concentration (Hb), haematocrit value (Ht), MCV (mean corpuscular volume), MCH (mean corpuscular haemoglobin) and MCHC (mean corpuscular haemoglobin concentration)). A hematology analyzer Coulter MAXM (Beckman Coulter, Inc. Fullerton, USA) was used to determine these parameters.
Serum samples were obtained by the centrifugation of blood at 4000 rpm for 15 min at 4Â°C, and were then divided into eppendorf tubes. Isolated sera were stored at - 20Â°C until they were used for the analyses. The levels of serum glucose, total protein, cholesterol and triglycerids were measured using commercial kits according to the manufacturer's directions.
Determination of antioxidant enzymes activities and lipid peroxidation
Other blood samples were collected without anticoagulant. The blood was centrifuged at 4000 rpm for 15 min (4 Â°C) and erythrocyte was carefully sampled. They were then rinsed with ice-cold saline and homogenized (Ultra Turrax T25, Germany) (1:2, w/v) in 50 mmol l-1 phosphate buffer (pH 7.4). The homogenate and supernatant were frozen at -30 Â°C in aliquots until used for antioxidant enzymes and lipid peroxidation. The protein content of the supernatant was determined using the method of Lowry et al. (1951).
Measurement of lipid peroxidation levels
The thiobarbaturic acid (TBA) method of Buege and Aust (1972) was used to determine the lipid peroxidation by determining the amount of TBA reactive substances present in the erythrocyte homogenates obtained from rats. The principle of the method is spectrophotometric measurement of the color produced during the reaction to TBA with MDA. The absorbance of the solution was measured at 530 nm. The concentration of MDA was calculated by the extinction coefficient of MDA-TBA complex (1.56 Ã- 105 cmâˆ’1 molâˆ’1 L) and expressed in nanomoles per milligram of protein.
Determination of superoxide dismutase (SOD) activity
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SOD activity was assayed by measuring its ability to inhibit the photoreduction of nitroblue tetrazolium 'NBT' (Beyer and Fridovich, 1987), 50 Âµl of supernatant combined with 50 mM of phosphate buffer (pH 7.8), 39 mM of methionine, 2.6mM of NBT and 2.7mM of EDTA-Riboflavin, was added last to obtain a concentration of 0.26 mM, and switching on the light started the reaction; changes in absorbance at 560 nm were recorded after 20 min. In this assay, the activity was expressed in relative units per milligram of protein [U mg-1]. One unit of SOD activity is defined as the amount of protein that inhibits the rate of NBT reduction by 50%.
Determination of catalase (CAT) activity
CAT activity was measured by the UV colorimetric method of Aebi (1974 ) using H2O2 as substrate.
Determination of glutathione peroxidase (GSH-Px) activity
Glutathione peroxidase activity was performed by estimating the content of oxidized glutathione formed by the action of glutathione peroxidase as described by Flohe and Gunzler (1984), but modified using the H2O2 as substrate with the presence of DTNB. One unit of activity catalyzes the oxidation by H2O2 of 1.0 Âµmol of reduced glutathione to oxidized glutathione per minute at pH 7.0 at 25 â-¦C. The specific enzyme activity was expressed in units per gram of protein [Ugâˆ’1].
For histological studies, the spleen tissues were fixed in bouin solution, dehydrated in graded (50-100%) alcohol and embedded in paraffin. Thin sections (4 - 5 Âµm) were cut and stained with routine hematoxylin-eosin (H&E) (Gabe 1968).
All values are expressed as mean Â± S.E.M. The results were analyzed by one-way analysis of variance (ANOVA) followed by Tukey test for multiple comparisons using SPSS forWindows (version 11). Differences were considered significant at p < 0.05.
The effects of methanol, Opuntia vulgaris fruit extract, and their combination on some haematological parameters in the rats are given in Table 1. Results showed that methanol alone caused a significant decrease (p < 0.01 vs. controls) in RBC count, haemoglobin level and hematocrit percentage. After treatment of rats with methanol plus Opuntia vulgaris fruit extract, RBC count, haemoglobin level and hematocrit percentage were normalized to their control values. However, no statistically significant change was observed in the other haematological parameters (MCV, MCH and MCHC levels) in methanol-treated group compared to control group. The present study shows that administration of Opuntia vulgaris fruit extract alone does not cause any significant alteration on the haematological indices.
The effects of methanol, Opuntia vulgaris fruit extract, and their combination on some biochemical parameters in the rats are given in Table 2. Results indicated that methanol caused a significant increase in glucose, cholesterol and triglycerids levels as compared to the control group. Results revealed that treatment with methanol caused a significant decrease (p < 0.01 vs. controls) in the total protein level. At the same time, the decrease in total protein level induced by methanol alone was increased in the presence of Opuntia vulgaris fruit extract with methanol.
Effect on lipid peroxidation
To explore the oxidative consequences of methanol treatment in erythrocytes of rats and to determine the possible protective effects of OE, we analyzed the lipid peroxidation as a marker for membrane damage. Exposure to methanol increased significantly the LPO levels in the erythrocyte tissues samples as compared to control group (figâ€¦). OE treatment prevented the LPO production induced by methanol. OE alone did not change the degree of LPO formation compared to controls.
Effect on antioxidant enzymes
SOD activity in the erythrocyte tissues of experimental animals have been shown in figâ€¦ SOD levels were significantly reduced (p < 0.01) in the erythrocyte tissues of methanol-treated rats compared to control. Whereas, no significant change was observed in OE-treated rats, as well as in OE+M rats showing the protective effects of Opuntia vulgaris fruit extract against changes induced by methanol treatment. The CAT activities in the erythrocyte tissues of all experimental animals have been shown in figâ€¦ Methanol intoxication significantly decreased the CAT activities and treatment with OE revert the enzyme activity near to normal status. A significant decrease (p < 0.01) in GPx activities has been observed in erythrocyte tissues of methanol treated animals (fig..;).
Histopathological studies of the spleen showed that methanol induced oedema and congestion of the pulp and severe depopulation of the spleen follicle in treated rats compared controls. A massive proliferation and hypertrophy of reticulo-endothelial cells were observed in methanol treated rats. The histopathological disorders were in agreement with biochemical parameters. There were no histological alterations in the spleen of Opuntia vulgaris fruit extract group when compared to control.
The results of this study showed that methanol caused a significant decrease in some haematological parameters of the rats such as RBC counts, haemoglobin level and hematocrit percentage. The present investigation demonstrated that the exposure of rats to methanol caused changes in some haematological parameters. Haematological characteristics have been widely used in the diagnosis of variety of diseases and pathologies induced by industrial compounds, drugs, dyes, heavy metals, pesticides and several others (Mansour and Mossa, 2005; Kalender et al., 2006; Eraslan et al., 2009).
The reduction in RBC count is considered due to the direct injurious action of the methanol on the animals. The erythropenia along with decreased haemoglobin concentration is an indication of a decrease in oxygen carrying capacity in the animals, resulting in insufficient supply of oxygen to the tissues causing adverse effects on animal health. The present results showed that the administration of methanol caused highly decrease in RBC count, haemoglobin concentration and hematocrit percentage. The reduction in haemoglobin level may be due to increased rate of breakdown of red cells and/or reduction in the rate of RBC formation. Armutcu et al. (2005) suggested that the decrease in RBC count is either indicative of excessive damage to erythrocytes or inhibition of erythrocyte formation. Moreover, the hematotoxicity induced by methanol might be explained by the inhibition of erythropoiesis and hemosynthesis and to an increase in the rate of erythrocyte destruction in hemopoietic organs (Ivanov, 2001). The results of this study indicated that Opuntia vulgaris fruit extract given orally for six weeks attenuated the extensive changes in haematological parameters in methanol-treated rats. These disorders in haematological parameters induced by methanol did not appear in OE rats drinking Opuntia vulgaris fruit extract. However, the exact mechanisms by which Opuntia vulgaris fruit extract exert their protective effects against methanol-induced toxicity are not yet known. It has also been explained by the potential source of antioxidants in OE (with direct or indirect actions) able to counteract or to minimize the undesirable effects induced by methanol. Previous studies (Tesoriere et al., 2005) demonstrated that cactus pear (Opuntia ssp.) yield high values of important nutrients and exhibit antioxidant functions. Cactus extracts exhibit anti-tumoral (Zou et al., 2005), anti-inflammatory (Ahmad et al., 1996) and antioxidant effects (Gentile et al., 2004). In the Mediterranean countries, cladodes are not a usual nutritional source for humans, but the fruit are largely consumed (Butera et al., 2002; Lee et al., 2002). The fruits are important sources of vitamins for local people at the natural growth sites of the plant. Both nopal and fruit are consumed as fresh vegetables, cooked, or used in salads, syrups and juices (Lee et al., 2002). Besides its traditional use in human nutrition, the plant is mainly used for cattle feed and for the production of carminic acid (Stintzing et al., 2001).
Also, liver, muscle, and brain are organs involved in glycogenesis, glycogenolysis, gluconeogenesis, and glycolysis. In addition, pancreas keeps hormonal control of glucose by secretion of glucagon and insulin. The present study showed that the increase observed in glucose level in rats treated with methanol might be due to the effect of methanol on pathways involved in glucose homeostasis in these organs. Our results are in agreement with studies of Atrens et al. (1989) which demonstrated that ethanol and tertiary butanol produced hyperglycaemic and hypothermic effects in treated rats. These data suggest that the hyperglycaemic and hypothermic effects of ethanol represent a primary physico-chemical or nerve cell membranes and are not secondary to its energy content or metabolites.
Similarly, Simin et al. (2008) reported that fenitrothion, a pesticide, caused an increase in glucose levels in adult wistar rats. In the present study, we observed a significant increase in serum cholesterol and triglycerids recorded in the methanol administered group which can be an indicator of biochemical and metabolic disruption. It seemed that high triglycerids and total cholesterol concentrations were linked with a greater risk for development of coronary artery disease and other organ complication (Al-Maskari et al., 2007). The increase in serum cholesterol and triglycerids levels indicated liver disorders and cholestasis (Shivanandappa and Krishnakumari, 1981; Zarn et al., 2003). In this work, we observed a significant decrease in total protein level in rats treated with methanol alone when compared to those of the control group. This decrease in total protein level might be explained to protein synthesis deficiency as a result of liver dysfunction induced by the existence of methanol. Similarly, the study of Al-Hashem (2009) demonstrated that aluminium chloride produced a reduction in total protein level.
Our study indicated that treatment of rats with methanol plus Opuntia vulgaris fruit extract decreased serum glucose, triglycerides and cholesterol levels, and increased total protein levels compared to the rats treated with methanol. Although various soluble fibres in medicinal plants, including pectin, have been shown to decrease plasma cholesterol levels, no such effects was observed in rats given Opuntia vulgaris fruit extract for six weeks. In our experimental conditions, OE orally given to rats was found to inhibit the effects of methanol poisoning on metabolism of lipids. Previous data of wolfram et al. (2002) ascribed the cactus anti-hyperlipidemic effects to the pulp pectin, which both reduced lipid absorption and increased faecal sterol excretion. Nevertheless, Fernandez et al. (1992; 1994) claimed that the hypocholesterolemic effect of prickly pear pectin did not result from the reduction of cholesterol absorption but rather from an increase in apolipoprotein B/E receptor expression and changes in hepatic cholesterol homeostasis.
However, aerobic organisms generate superoxide anion radicals, hydrogen peroxide (H2O2) and hydroxyl radicals as a result of oxidative metabolism. Damage at the cell level by oxidants is attenuated by antioxidant enzymes such as SOD, CAT and GPx. Oxidative stress, generated by xenobiotics, induces disturbances in antioxidant enzyme systems (Wills and Asha, 2006). Xenobiotics are oxidized to free radicals within RBCs and induce haemolysis of RBC membrane. As a consequence, haemoglobin is released, which induces a multiple of toxic effects, recently summarized by Everse and Hsia (1997). The present study has demonstrated an increase of MDA in erythrocytes of rats treated with methanol alone demonstrates lipid peroxidation to have developed. The occurrence of a significant increase in erythrocytes LPO levels when compared to the control group is also indicative of damage to have been caused in the tissues examined, as a result of free radicals generated by methanol. On the other hand, for the demonstration of oxidative stress, the changes determined either in the form of the inhibition or stimulation of the activity of antioxidant enzymes points out to the generation of a high level of free radicals in tissues and organs, and also proves that antioxidant enzymes play an active role in the conversion of these harmful and very effective compounds into less harmful or harmless metabolites. Methanol is oxidized via three main oxidative pathways among which the alcohol dehydrogenase (folate dependent) and catalase peroxidative system have been extensively studied (Paula and Namasivayam, 2003). In rats, the oxidation of methanol is performed primarily by catalase. This enzyme forms the catalase-hydrogen peroxide system in the presence of H2O2, which intermediates the oxidizing of various alcohol in to corresponding aldehydes. Lipid peroxidation has been implicated in a number of deleterious effects such as increased membrane rigidity, osmotic fragility, decreased cellular deformation, reduced erythrocyte survival and membrane fluidity (Thampi et al., 1991). Increase in the levels of MDA indicate enhanced lipid peroxidation leading to tissue injury and failure of the antioxidant defense mechanisms to prevent the formation of excess free radicals (Comporti, 1985). In our study, significantly elevated levels of LPO were observed in erythrocytes of rats treated with methanol. These results are in agreement with the observations of previous researchers (Skrzydlewska et al., 1998). The increase in LPO levels observed in this study was an index of oxidative stress. The present study also shows that the changes in LPO are accompanied by the concomitant decrease in the activities of antioxidant enzymes such as SOD, CAT and GPx in methanol exposed rats. Similar results previously reported by Dhabhar and McEwen (1997) reported that methanol increased lipid peroxidation by a direct effect or by decreasing the glutathione content. The reduced activity of CAT and SOD in the presence of methanol may cause the accumulation of O2âˆ’, H2O2 or the products of its decomposition. Loss of CAT and SOD activity results in oxygen intolerance and triggers a number of deleterious reactions. It has been proposed that the contribution of CAT might be enhanced if significant amounts of H2O2 become available through Î²-oxidation of fatty acids in peroxisomes (Decremer et al., 1991). Our results indicated that treatment of rats with methanol plus Opuntia vulgaris fruit extract increased antioxidant enzymes (SOD, CAT and GPx), and decreased lipid peroxidation evaluated in erythrocytes, compared to the rats treated with methanol. This suggest that's OE can modulate the balance of antioxidants and pro-oxidants.
The use of Opuntia is recommended for their beneficial and therapeutic properties (Galati et al., 2002). Previous studies reported that this plant exhibited diverse pharmacological actions, including emollient effect, hypocholesterolemic effect, hypoglycaemic effect, inhibition of stomach ulceration, neuroprotective effects through antioxidant actions and anti-inflammatory effect (Galati et al., 2001; Dok-Go et al., 2003). It has been used traditionally as a herbal medicine for treating diabetes, burns, bronchial asthma and indigestion in many countries over the world (Kim et al., 2006; Park & Chun, 2001). Based on the detailed experimental evidence, subjective methanol intoxication appears to play a major risk factor for neuroimmune dysfunction due to chronic consumption of methanol by both direct and indirect means.
This study has clearly shown that, in addition to causing haematological and biochemical perturbation, and erythrocyte damage, the methanol cause damage to spleen tissues in treated rats compared to those of control. It has been clearly reported that spleen is implicated in haematopoiesis and erythrocyte mechanism. The histopathologic evaluation of spleen showed that treatment group of methanol defects in morphology. Despite the hematotoxicity caused by methanol treatment a significant enhancement in the hemolytical parameters evaluated and a responsive recovery of spleen histomorphology were observed six weeks after administration, suggesting a positive recovery from the hemolytic damage caused by the single intraperitoneal administration of methanol. As a consequence of the hemolysis caused by methanol, an extensive filtration of damaged RBC by the reticuloendothelial system (RES) of the spleen as well as an effective haematopoietic process was observed. The major role and functions of the spleen is to remove damaged erythrocytes, and since such xenobiotics proved to damage erythrocytes by altering its antioxidant status (Hernandez et al., 2005), it is expected that injured erythrocytes will be ultimately scavenged by the spleen generating ROS and subsequent tissue injury.
In summary, this study demonstrates that the administration of methanol at a dose of 2.37 g/kg b.wt/day for a period of six weeks caused significant changes in some haematological parameters (RBC, Hb, and Ht), biochemical (glucose, cholesterol, triglyceride and total protein levels), erythrocyte oxidative damages (LPO level, SOD, CAT and GPx activities) and histopathological changes in spleen of treated group compared to control. The use of Opuntia vulgaris fruit extract was ascertained to alleviate the harmful effects of methanol in the mentioned parameters. This study shows the potential value of Opuntia vulgaris fruit as a good source of natural antioxidants and that consumption of fruit or its products may contribute substantial amounts of antioxidants to the diet.