Pleurotus ostreatus, an edible mushroom, enhances glucose 6-phosphate dehydrogenase, ascorbate peroxidase and reduces xanthine dehydrogenase in major organs of aged rats

Abstract

Context: Aging is now considered to be associated with an elevation in oxidative damage to macromolecules and enhanced levels of inflammation. Therefore, inhibition of age-related oxidative stress by natural supplement is an important study.

Objectives: To investigate whether the treatment with Pleurotus ostreatus (Jacq.: Fr) Kumm, (Pleurotaceae) can ameliorate oxidative damage in aged rats.

Materials and methods: Male Wistar rats were divided into three groups of six each: group 1, normal young rats; group 2, normal aged untreated rats; group 3, normal aged rats treated with P. ostreatus (200 mg/kg body wt administered intraperitoneally for 21 days). On the 22nd day, rats were sacrificed by decapitation; the liver, kidneys, heart and brain were removed from each rat for the biochemical and isozyme analyses of the antioxidant enzymes glucose 6-phosphate dehydrogenase (G6PDH), ascorbate peroxidase (Apx) and xanthine dehydrogenase (XDH).

Results: An elevated activity of XDH was observed in the liver (G2:13.72 ± 4.1 versus G1: 7.57 ± 1.15; p < 0.05), kidneys (G2:101.48 ± 12.3 versus G1: 31.15 ± 1.71; p < 0.01), heart (G2: 63.21 ± 3.96 versus G1: 37.3 ± 2.70; p < 0.01) and brain (G2: 39.02 ± 3.96 versus G1: 19.84 ± 1.22; p < 0.001). The activities of G6PDH and Apx were lowered in major organs of aged untreated rats. However, treatment of P. ostreatus to aged rats resulted in decreased XDH and increased G6PDH and Apx activities in liver, kidneys, heart and brain. Interestingly, analyses of isozyme pattern of these enzymes are support the results obtained from the spectrophotometric determinations.

Conclusion: These results suggest that an extract of P. ostreatus can protect the age-related oxidative damage in major organs of Wistar rats by enhancing the antioxidant enzymes G6PDH and Apx and by reducing XDH.

• Ageing
• antioxidant enzymes
• isozymes
• oxidative stress

Introduction

Ageing is now considered to be associated with an increase in oxidative damage to biomolecules (Goto et al., 2001) and enhanced levels of inflammation (Cheng et al., 2002). Reactive oxygen species (ROS) are believed to be generated in aerobic cells; aerobic organisms are provided with antioxidant defense systems (ADS) that could avert damage due to oxidative stress (Sies, 1985). The primary antioxidant enzymes include superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (Gpx) are highly associated in direct elimination of ROS. Secondary enzymes namely glutathione reductase (GR), glutathione S-transferase (GST), glucose 6-phosphate dehydrogenase (G6PDH) and ascorbate peroxidase (Apx) play a major role in the detoxification of ROS by decreasing peroxide levels or by maintaining a steady supply of metabolic intermediates (e.g., glutathione, NADPH) necessary for optimal functioning of the primary antioxidant enzymes (Singh et al., 2003).

G6PDH is the most important antioxidant enzyme, which catalyzes the initial step of the pentose phosphate pathway whose most important function is the reduction of nicotinamide adenine dinucleotide phosphate (NADP⁺) to NADPH which is used for the reduction of oxidized glutathione (GSSG) to reduced state (GSH) for the reduction of mixed disulfides of glutathione and cellular proteins (Lee et al., 1993). Apx, the main enzyme of the ascorbic acid (ASC)-glutathione (GSH) cycle, has multiple locations and is among the most important key enzymes that scavenge potentially harmful H₂O₂ (Diaz-Vivancos et al., 2006). Several studies are available in the literature investigating decreased antioxidant enzymes in liver, kidneys, heart and brain (Ramesh et al., 2012) in aged rats. Our previous studies have documented that the activities of SOD, CAT, Gpx, GR and GST are reduced in major organs of aged (Jayakumar et al., 2007) and carbon tetrachloride (CCl₄)-induced (Jayakumar et al., 2006, 2008) rats. We also found a decreased glutathione redox system (GSH and GR) in aged rats (Jayakumar et al., 2010). However, there are no reports concerning age-related changes of G6PDH and Apx activities in rat tissues.

The increased oxidative damage observed during ageing might be due to the insufficiency of antioxidants (Reiter, 1995). Supplementation of antioxidants could conceivably protect the human body from free radicals and ROS effects and retard the progress of many chronic diseases (Gulcin et al., 2003). Pleurotus ostreatus (Jacq.: Fr) Kumm, (Pleurotaceae) is reported to contain higher concentrations of cystine, methionine and aspartic acid than other edible mushrooms, such as Agaricus bisporus/brown (Lange) Singh., (Agaricaceae), A. bisporus/white (Lange) Singh., (Agaricaceae) and Lentines edodes (Berk.,), (Marasmiaceae) (Mattila et al., 2002). A recent study has found the anti-allergic effect of pleuran (β-glucan from P. ostreatus) in children with recurrent respiratory tract infections (Jesenak et al., 2013). The gastroprotective activity of a polysaccharide from the fruiting bodies of P. ostreatus in rats was also reported by Yang et al. (2012). Ravi et al. (2013) conducted a study to demonstrate the antidiabetic potential of P. ostreatus in alloxan-induced diabetic mice. The antioxidant activity of the exopolysaccharides (EPS) and intracellular polysaccharides (IPS) obtained from the mycelia of two edible P. ostreatus strains, PQMZ91109 and PSI101109, in a batch bioreactor was demonstrated by Vamanu et al. (2013). Our previous studies have demonstrated that treatment of an extract of P. ostreatus enhances the activities of CAT, SOD, Gpx, GR and GST in the liver, kidney heart and brain of aged (Jayakumar et al., 2007) and CCl₄-induced rats (Jayakumar et al., 2006, 2008). The present study investigates whether treatment with P. ostreatus can ameliorate oxidative damage in liver, kidneys, heart and brain of aged rats via improving of G6PDH, Apx and decreasing of XDH activities.

Materials and methods

Preparation of the mushroom extract

The mushroom P. ostreatus was cultivated adopting the “layer spawning” method. Freshly harvested (December, 2012) whole mushrooms were shade-dried and then finely powdered. The powder (5 g) was extracted with 100 ml of 95% ethanol using a Soxhlet apparatus (Bio Technics India, Maharashtra, India). The material thus obtained was filtered, and the resulting filtrate was concentrated to a dry mass (5.6% yield) by vacuum distillation; this was used as mushroom extract.

Animal experiment

Male albino Wistar rats weighing approximately 80–110 g (4 months old) and 300–375 g (24 months old), procured from the Central Animal House, Rajah Muthiah Medical College, Annamalai Nagar, India, was used for the experiments. The study was approved by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) and conducted according to the regulations of the Institutional Animal Ethics Committee. The animals were acclimated for 20 days prior to dosing, with access to food and water ad libitum. Eighteen acclimated rats were divided into three groups of six each: group I, normal young (4 months old) rats; group II, normal aged (24 months old) untreated rats; group III, normal aged rats treated with the extract of the mushroom P. ostreatus (200 mg/kg body wt administered intraperitoneally) for 21 days. On the 22nd day, rats were sacrificed by decapitation; the liver, kidneys, heart and brain were removed from each rat and stored at −80 °C until further analysis.

Preparation of tissue homogenates

Samples of liver, kidneys, heart and brain (100 mg/ml buffer) were homogenized in 50 mM phosphate buffer (pH 7.0). They were centrifuged at 10 000 rpm for 15 min at 4 °C, the supernatant thus obtained was used for biochemical analysis. The protein concentration in each fraction was determined by the method of Lowry et al. (1951), using crystalline bovine serum albumin as standard.

Estimation of XDH

The activity of XDH was determined according to the method of Schrader et al. (1999). A mixture containing 5 mM xanthine and 0.5 mM NADP in 50 mM phosphate buffer (pH 7.8) was prepared. All solutions were prepared in the absence of oxygen. The reaction was started by the addition of 100 μl of one tissue homogenate. The XDH activity was monitored by recording the increase in absorbance at 365 nm using extinction co-efficient of 3.5 mM⁻¹cm⁻¹. One unit of specific activity was defined as 1 μmol of NADP reduced/min/mg of protein at 37 °C.

Assay of G6PDH

The activity of G6PDH was assayed by the method of Ellis and Kirkman (1961). For each tissue homogenate (0.2 ml) tested, an assay mixture was prepared that contained 1.0 ml of Tris–HCl, 0.5 ml of 0.005% phenozone methosulphate, 0.4 ml of 0.01% 2,6-dichloro phenol indophenol solution, 0.1 ml of 1.0 M magnesium chloride and 0.1 ml of 1 M NADP. The mixture was allowed to stand at room temperature for 10 min to permit the oxidation of endogenous materials. The reaction was then initiated by the addition of 0.5 ml of 0.02 M glucose 6-phosphate. The change in optical density at 640 nm was monitored for 3 min at intervals of 30 s. The activity of G6PDH was expressed as units/min/mg protein.

Assay of Apx

Apx (EC 1.11.1.11) is involved in the detoxification of peroxides such as hydrogen peroxide using ascorbate as a substrate. The activity of Apx was determined essentially as described by Nakano and Asada (1987). Briefly, for each tissue homogenate tested (0.2 ml), a reaction mixture containing 50 mM potassium phosphate buffer (pH 7.0), 0.5 mM ASC and 0.1 mM hydrogen peroxide in a total volume of 1.0 ml was prepared. The hydrogen peroxide-dependent oxidation of ASC was followed by monitoring the decrease in absorbance at 290 nm assuming an absorption coefficient of 2.8 mM⁻¹cm⁻¹. The activity of Apx was expressed as 1 μmole of ASC oxidized/min/mg protein.

Detection of isozymes by electrophoresis

Non-denaturing polyacrylamide gel electrophoresis (native-PAGE) was performed on samples of liver, kidneys, heart and brain essentially as described by Laemmli (1970), except that SDS was omitted from all buffers and the samples were not boiled before electrophoresis. The enzymes were run on the basis of equal amounts of protein (70 µg) in a 10% gel for Apx and 7.5% gel for G6PDH and XDH. Electrophoretic separation was performed at 4 °C with a constant power supply of 50 V for stacking gel and 100 V for separating gel. Staining for the activity of each enzyme was performed separately as follows.

Staining of XDH activity

The isozyme of XDH was detected by the method of Irie and Kato (1984). The principle of this method being that bands of enzyme formed on gels are stained by tetrazolium dye reduction. To detect this activity, the gel was soaked in 50 ml of 0.1 M potassium phosphate buffer (pH 7.5) containing 2.5 mM xanthine, 7 mM NAD⁺, 2 mg phenazine methosulfate and 10 mg nitroblue tetrazolium salt. The reaction was made to proceed in the dark at room temperature for 5–30 min. The XDH activity was observed as a stained band on a white background.

Staining of G6PDH activity

G6PDH activity was detected by the method of Wendel and Weeden (1989). To detect the staining activity of this enzyme, the gel was submerged in 50 ml of 50 mM Tris–HCl buffer (pH 8.0) containing 5 mg NADP, 50 mg MgCl₂, 50 mg glucose 6-phosphate, 10 mg NBT and 2 mg PMS. Incubation was performed at room temperature until a stained band appeared.

Staining of Apx activity

Apx isozyme was identified by the method of Mittler and Zilinskas (1993). Subsequent to electrophoretic separation, the gel was equilibrated with 50 mM sodium phosphate buffer (pH 7.0) and 2 mM ascorbate for a total of 30 min; the equilibration buffer was changed every 10 min. The gel was then incubated with 50 mM sodium phosphate buffer (pH 7.0) containing 4 mM ascorbate and 2 mM H₂O₂ for 20 min. The gel was subsequently washed with sodium phosphate buffer (pH 7.0) for 1 min and submerged in a solution of 50 mM sodium phosphate buffer (pH 7.8), 28 mM TEMED and 2.45 mM NBT with gentle agitation. Apx activity was observed as an unstained band on a purple-blue background. The reaction was allowed to continue approximately 10 more min and stopped by a brief wash with deionized water.

Quantification of the isozyme bands for each enzyme studied was performed by a densitometer (Gs 300 transmittance/reflectance scanning densitometer, Hoefer Scientific Instruments, San Francisco, CA).

Statistical analysis

The results obtained for each group of rats tested were expressed as the means ± SD of six values. Statistical analysis of the data was performed by Student’s t-test.

Results

Effects of mushroom P. ostreatus on the activities of XDH in aged rats

A significant increase in the activities of XDH was observed in the liver (p < 0.05), kidneys, heart (p < 0.01) and brain (p < 0.001) tissues of aged rats, when compared to the activity of XDH in the same tissues of young rats (Table 1). In aged rats that had been treated with the extract of P. ostreatus, the activity of this enzyme was found to be significantly lower (p < 0.05) in the kidneys, heart and brain than in the corresponding organs of aged-untreated rats; however, no significant decrease was observed in the liver tissues (Table 1).

Table 1. Effect of extract of mushroom (Pleurotus ostreatus) on xanthine dehydrogenase (XDH) in liver, kidney, heart and brain tissues of aged rats.

	Animal groups
Tissues	Group I (young rats)	Group II (aged rats)	Group III (aged rats treated with mushroom extract)
Liver	7.57 ± 1.15	13.72 ± 4.1*	9.94 ± 0.15^NS
Kidney	31.15 ± 1.71	101.48 ± 12.3**	89.41 ± 0.83*
Heart	37.3 ± 2.70	63.21 ± 3.96**	51.41 ± 2.13*
Brain	19.84 ± 1.22	39.02 ± 3.96***	26.3 ± 1.85*

Values are expressed as mean ± SD of six rats in each group.

XDH, μ mole of NADP reduced/min/mg protein.

*p < 0.05, **p < 0.01, ***p < 0.001. NS, not significant.

Effects of mushroom P. ostreatus on the activities of G6PDH and Apx in aged rats

Table 2 shows the activity of G6PDH in liver, kidney, heart and brain of young, aged and aged rats that had been treated with the extract of P. ostreatus. The activities of G6PDH enzyme were significantly lower in the liver (p < 0.01), kidneys (p < 0.01), heart (p < 0.01) and brain (p < 0.01) of aged untreated (group II) rats when compared to the values in young (group I) rats (Table 2). However, the activity of this enzyme was increased significantly, while aged rats administered with the extract of P. ostreatus (group III).

Table 2. Effect of extract of mushroom (Pleurotus ostreatus) on glucose 6-phosphate dehydrogenase (G6PDH) in liver, kidney, heart and brain tissues of aged rats.

	Animal groups
Tissues	Group I (young rats)	Group II (aged rats)	Group III (aged rats treated with mushroom extract)
Liver	2.41 ± 0.02	1.89 ± 0.04**	2.38 ± 0.06*
Kidney	1.34 ± 0.01	1.07 ± 0.05**	1.29 ± 0.01*
Heart	1.24 ± 0.05	1.04 ± 0.06**	1.12 ± 0.09*
Brain	1.10 ± 0.01	0.99 ± 0.07**	1.031 ± 0.03^NS

Values are expressed as mean ± SD of six rats in each group.

G6PDH, unit/min/mg protein.

*p < 0.05, **p < 0.01. NS, not significant.

The activity of liver, kidney, heart and brain Apx in young, aged and aged rats that had been treated with the extract of P. ostreatus is presented in Table 3 and it was found to be significantly lower (p < 0.01) in major tissues of aged rats. Treatment with P. ostreatus significantly (p < 0.05) increased Apx activities in liver, kidney, heart and brain of aged rats.

Table 3. Effect of extract of mushroom (Pleurotus ostreatus) on ascorbate peroxidase (Apx) in liver, kidney, heart and brain tissues of aged rats.

	Animal groups
Tissues	Group I (young rats)	Group II (aged rats)	Group III (aged rats treated with mushroom extract)
Liver	3.08 ± 0.01	2.57 ± 0.03**	2.91 ± 0.02*
Kidney	2.75 ± 0.03	1.45 ± 0.04**	2.51 ± 0.01*
Heart	2.41 ± 0.05	1.17 ± 0.02**	2.08 ± 0.01*
Brain	1.59 ± 0.01	0.96 ± 0.02**	1.56 ± 0.03*

Values are expressed as mean ± SD of six rats in each group.

Apx, unit/mg protein.

*p < 0.05, **p < 0.01.

Effects of mushroom P. ostreatus on the isozyme pattern of XDH in major organs of aged rats

The XDH enzyme exhibited a single isozyme in the liver, but four isozymes in the kidney, heart and brain tissues of all three groups of rats (Figure 1A). (a) The liver of group I and group III rats exhibited a single band of low intensity (band areas 195.21 and 199.41, respectively) compared with that in group II (band area 204.82) rats [Figure 1A(a)]. (b) Four isozymes of XDH were detected in the kidneys in all three groups of rats [Figure 1A(b)]. In rats of groups I, II and III, the staining intensity of the XDH3 (band areas 104.89, 114.60 and 109.08, respectively) and XDH4 (band areas 125.21, 127.49 and 126.91, respectively) isozymes was essentially similar. However, the staining intensity of the XDH1 and 2 isozymes was greater (band areas 219. 37 and 98.81, respectively) in group II rats compared with that in group I (band areas 133.49 and 89.76, respectively) and that in group III (band areas 196.48 and 92.75, respectively) rats. (c) In the heart tissue of rats of groups I, II and III, the staining intensity of the XDH2 (band areas 88.12, 90.68 and 89.62, respectively), XDH3 (band areas 130.90, 134.69 and 131.01, respectively) and XDH4 (band areas 136.72, 139.87 and 138.45, respectively) isozymes were essentially similar [Figure 1A(c)]. However, the staining intensity of the XDH1 (band area 98.81) isozyme was greater in group II rats than that in group I (band area 89.76) and that in group III (band areas 92.75) rats. (d) Four isozymes of XDH were noted in the brain tissue of all three groups of rats [Figure 1A(d)]. In rats of groups I, II and III, the staining intensity of XDH1 (band areas 79.58, 82.67 and 80.17, respectively) and XDH2 (band areas 89.09, 90.41 and 89.98, respectively) isozymes was essentially similar. However, the staining intensity of the XDH3 and 4 isozymes was less in group I (band areas 45.71 and 99.90, respectively) and in group III (band areas 50.76 and 103.85, respectively) rats compared to that in group II (band areas 52.63 and 108.47, respectively) rats (Figure 1B).

Figure 1. (A) Effect of extract of mushroom (Pleurotus ostreatus) on xanthine dehydrogenase (XDH) isozymes in (a) liver, (b) kidney, (c) heart and (d) brain tissues of aged rats. (B) Densitometric pattern of XDH isozymes (X1-XDH1, X2-XDH2, X3-XDH3 and X4-XDH4).

Effects of mushroom P. ostreatus on the isozyme pattern of G6PDH in major organs of aged rats

The G6PDH enzyme exhibited a single isozyme in the liver and three isozymes in the kidney, heart and brain tissues of all three groups of rats [Figure 2A(a–d)]. (a) The liver of groups I and III rats exhibited a single band of high intensity staining (band areas 219.10 and 211.85, respectively) compared with that in group II (band area 183.93) rats [Figure 2A(a)]. (b) Three isozymes of G6PDH were noted in the kidneys in all three groups of rats [Figure 2A(b)]. In rats of group II, the staining intensity of the G6PDH1, 2 and 3 isozymes (band areas 41.55, 72.17 and 209.89, respectively) was found to be lower than that in group I (band areas 54.48, 83.68 and 217.89, respectively) and that in group III (band areas 49.22, 78.16 and 212.34, respectively) rats. In the heart and brain tissues, three isozymes of the G6PDH enzyme were observed in all three groups of rats. (c) In the heart tissue of rats of group II, the staining intensity of G6PDH 1, 2 and 3 isozymes was less (band areas 182.58, 214.01 and 207.65, respectively) than that in group I (band areas 205.79, 220.31 and 214.07, respectively) and that in group III (band areas 185.17, 219.96 and 211.85, respectively) rats [Figure 2A(c)]. (d) Three isozymes of G6PDH were detected in the brain tissue in all three groups of rats [Figure 2A(d)]. In rats of group II, the staining intensity of G6PDH1, 2 and 3 isozymes was less (band areas 143.21, 181.63 and 225.46, respectively) than that in group I (band areas 203.84, 216.15 and 229.97, respectively) and that in group III (band areas 197.41, 213.74 and 227.83, respectively) rats (Figure 2B).

Figure 2. (A) Effect of extract of mushroom (Pleurotus ostreatus) on glucose 6-phosphate dehydrogenase (G6PDH) isozymes in (a) liver, (b) kidney, (c) heart and (d) brain tissues of aged rats. (B) Densitometric pattern of G6PDH isozymes.

Effects of mushroom P. ostreatus on the isozyme pattern of Apx in major organs of aged rats

The Apx enzyme exhibited four isozymes in the liver and heart tissues and three isozymes in the kidney and brain tissues of all three groups of rats [Figure 3A(a–d)]. (a) In liver, four isozymes of Apx enzyme were detected in all three groups of rats (Figure 3a). In rats of groups I, II and III, the staining intensity of the Apx1 (band areas 117.87, 114.98 and 115.99, respectively), Apx2 (band areas 74.13, 71.09 and 72.68, respectively) and Apx3 (band areas 96.85, 93.17 and 95.98, respectively) isozymes was essentially similar. However, the staining intensity of the Apx4 isozyme was less (band area 29.86) in group II rats compared with that in group I (band area 37.65) and that in group III (band area 34.17) rats. (b) Three isozymes of the Apx enzyme were observed in the kidneys in all three groups of rats [Figure 3A(b)]. In rats of groups I, II and III, the staining intensity of the Apx1 (band areas 101.71, 99.89 and 100.72, respectively), Apx2 (band areas 53.34, 51.20 and 52.39, respectively) and Apx3 (band areas 79.65, 77.75 and 78.17, respectively) isozymes was essentially similar. (c) Four isozymes of the Apx enzyme were detected in the heart tissue in all three groups of rats [Figure 3A(c)]. In rats of groups I, II and III, the staining intensity of Apx3 (band areas 96.63, 96.55 and 95.68, respectively) and Apx4 (band areas 21.55, 19.99 and 20.97, respectively) isozymes was almost similar. However, the staining intensity of the Apx1 and 2 isozymes was less (band areas 30.98 and 41.74, respectively) in group II rats than that in group I (band areas 49.03 and 50.93, respectively) and that in group III (band areas 45.45 and 48.32, respectively) rats. (d) In the case of brain tissue, the staining intensity of Apx1 (band areas 96.04, 94.39 and 95.12) isozyme was essentially similar in rats of groups I, II and III, respectively [Figure 3A(d)]. However, the staining intensity of the Apx2 and 3 isozymes was less (band areas 27.45 and 50.90, respectively) in group II rats compared with that in group I (band areas 46.24. and 59. 54, respectively) and that in group III (band areas 36.81 and 58.17, respectively) rats (Figure 3B).

Figure 3. (A) Effect of extract of mushroom (Pleurotus ostreatus) on ascorbate peroxidase (Apx) in isozymes in (a) liver, (b) kidney, (c) heart and (d) brain tissues of aged rats. (B) Densitometric pattern of Apx isozymes.

Discussion

Supplementation of the diet with fruits and vegetables is reported to be beneficial in reversing the deleterious effects of ageing. In the present study, administration of mushroom P. ostreatus significantly enhanced the activities of antioxidant enzymes G6PDH and Apx and decreases the activity of XDH in the major organs of aged rats. The activities of these antioxidant enzymes have also been reported to increase in major organs of aged rats treated with grape seed extract (Balu et al., 2005). Due to the fact that isoforms catalyse similar reactions but differ from physicochemical properties, the activity of enzymes in examined major organs can be detected regardless of isoforms. Several spectrophotometric methods were developed for the determination of total G6PDH, Apx and XDH activity. Particular isoform, however, can be detected by the use of non-denaturing electrophoresis with particular substrates. In the current study, the extract of mushroom P. ostreatus increases the intensity of the studied enzymes in the major organs of aged rats.

Administration of the antioxidant component dl-α-lipoic acid to aged rats was found to lead to an elevation in the activity of G6PDH (Arivazhagan et al., 2002). Kumaran et al. (2004) reported a significant increase in the activity of G6PDH in the skeletal muscle and heart of aged rats following treatment with dl-α-lipoic acid and l-carnitine. So also, in the present investigation, administration of P. ostreatus extract appears to have brought about a remarkable improvement in the activity of G6PDH and Apx enzymes in aged rats. The increase in G6PDH activity may be a physiological response to compensate the decrease in the GSH level with age. Interestingly, the G6PDH activity was the highest in liver compared to the other tissues. It is possible that either the requirement of GSH in tissues other than liver is low or that these tissues may use alternate mechanisms such as isocitrate dehydrogenase, malic enzyme and/or mitochondrial energy-linked nicotinamide nucleotide transhydrogenase (Enander & Rydstrom, 1982) for generating NADPH which is ultimately used towards regeneration of GSH. However, these possibilities need to be explored experimentally.

It was found that ageing was characterized by a decrease in both ascorbate level and Apx activity and that conditions which induced ascorbate biosynthesis and accumulation resulted in decreased oxidative stress. Oxidative stress and , ROS, can also originate from extra-mitochondrial systems such as microsomal electron transport chain and other enzymes such as xanthine oxidase, aldehyde oxidase, dihydroorotic dehydrogenase and a group of flavoprotein dehydrogenases (Satav et al., 2000). The ascorbate–glutathione cycle, also known as the Halliwell–Asada or water–water cycle, utilizes serial enzymes, Apx, dehydroascorbate reductase, GR and monodehydroascorbate reductase to scavenge superoxide radicals and H₂O₂ in chloroplasts (Asada, 1999). Inhibition of GR, Apx, CAT and SOD activities have also been shown by Yan et al. (1996) in corn leaves under prolonged flooding. Enzymatic analysis showed that four important scavenger enzymes were found in Funalia trogii (Berk.), (Polyporaceae) extract: SOD, Apx, CAT and GR (Unyayar et al., 2006). From this study, for the first time that we discovered the increased activity of Apx in liver, kidney, heart and brain tissues of aged rat that had been treated with the extract of P. ostreatus.

Xanthine dehydrogenase (XDH) and xanthine oxidase (XOD) are single-gene products that exist in separate but interconvertible forms. XOD utilizes hypoxanthine or xanthine as a substrate and O₂ as a cofactor to produce superoxide () and uric acid. XDH acts on these same substrates but utilizes NAD as a cofactor to produce NADH instead of and uric acid. In the present study, an increased activity of XDH was observed in aged rats suggesting the presence of superoxide radicals, and/or Fenton-type reactions (McCord, 1985). Recently, a lot of research has been conducted in order to discover new, natural and specific XO inhibitors. Various plants extract (Dew et al., 2005) and polyphenolic compounds, especially flavonoids have been previously examined for their inhibitory properties against XO activity (Nagao et al., 1999). Terminalia chebula (Retz.), (Combretaceae), used in Indian system of medicines such as Ayurveda and Siddha, is reported to be reduced the activity of xanthine oxidase in both the liver and kidney of aged rats, while in young rats only the liver showed a decrease in mitochondrial xanthine oxidase (Mahesh et al., 2009). Cos et al. (1998) reported that flavonoids inhibit xanthine oxidase activity. However, there are no reports on regulation of XDH in rat tissues prior supplementation of antioxidant components during ageing. Therefore, for the first time that we demonstrated here the activity of XDH could conceivably increase in major organs of aged rats, whereas its activity significantly decreased upon P. ostreatus treatment.

The application of various isoforms of enzymes is assumed to be one of the primary control mechanisms that regulate cellular metabolism (Sang et al., 2005). We have reported our previous studies that the electrophoretic patterns of antioxidant enzymes CAT, SOD and Gpx in liver, kidney, heart and brain revealed characteristic isoform patterns in aged rats (Jayakumar et al., 2006). However, there are no data on the regulation of expression of the isoforms of antioxidant enzymes G6PDH and Apx during age-related oxidative stress, an aspect that deserves study. Therefore, in this study, an attempt was made to evaluate the pattern of isozymes of G6PDH and Apx during ageing and following supplementation with mushroom extract. In general, the number of bands and the staining intensity of isozymes were less in aged untreated (group II) rats than in the two other groups; tissue-specific variations were also noted. Such a pattern of differential expression of enzyme isoforms has been noted in barley shoot and root exposed to saline stress (Sang et al., 2005). The modification in the pattern of enzyme isoforms during stress has been attributed to some shift in gene expression (El-baky et al., 2003). Remarkably, in the present study, a markedly reduced numbers of Apx isozyme found in the brain tissues of aged untreated rats suggests that the brain tissue may be particularly susceptible to oxidative stress. A possible reason for this could be that since the brain is rich in non-heme iron, it could be catalytically involved in the production of oxygen free-radicals (Subbarao & Richardson, 1990). Another possible explanation is that the presence of unsaturated fatty acids in high concentrations makes the brain tissue good substrates for the occurrence of peroxidation reactions (Ogawa, 1994). In the present investigation, the observed variation in the antioxidant enzyme isoform profile during ageing may possibly be due to an alteration in gene expression. As direct genomic action of melatonin has been well-documented (Steinhiller et al., 1995); supplementation with melatonin during ageing has been proposed as a means of increasing the activity of ADS gene with a view to promoting the synthesis of antioxidant enzymes (Srinivasan, 1999). In the present study, a similar phenomenon possibly occurred, resulting in the increased intensity of staining of antioxidant enzyme isoforms following supplementation with mushroom extract.

Conclusion

The results of this study show that treatment of mushroom P. ostreatus protects the major organs of aged Wistar rats against oxidative stress by reducing the activity of XDH and by enhancing the activities of G6PDH and Apx. Moreover, the isozyme analysis of these enzymes are supported these results. Supplementation of P. ostreatus may offer therapeutic benefit, by assisting the liver, kidney, heart and brain in the management of secondary antioxidant enzymes G6PDH and Apx and oxidative marker XDH, therein augmenting the body’s defenses against age-related oxidative stress.

Declaration of interest

The authors declare they have no conflicts of interest. The authors thank the University Grants Commission, Government of India, for the financial assistance provided. Instrumentation facility provided by DST-FIST is also acknowledged.