Therapeutic Potential of Ganoderma Lucidum (Fr.) P. Karst. Against the Declined Antioxidant Status in the Mitochondria of Post-Mitotic Tissues of Aged Mice

Clinical Nutrition 29 (2010) 406–412

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Clinical Nutrition

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Original Article Therapeutic potential of Ganoderma lucidum (Fr.) P. Karst. against the declined antioxidant status in the mitochondria of post-mitotic tissues of aged mice

N.P. Sudheesh a, T.A. Ajith b, V. Ramnath c, K.K. Janardhanan a,* a Department of Microbiology, Amala Cancer Research Centre, Amala Nagar, Thrissur 680 555, Kerala, India b Department of Biochemistry, Amala Institute of Medical Sciences, Amala Nagar, Thrissur 680 555, Kerala, India c Department of Veterinary Physiology, College of Veterinary and Animal Science, Kerala Agricultural University, Mannuthy, Thrissur 680 651, Kerala, India article info summary

Article history: Background & aims: Post-mitotic cells such as brain and heart cells are particularly vulnerable to Received 14 August 2009 oxidative damages during ageing. In this study, we evaluated the effect of Ganoderma lucidum on the Accepted 9 December 2009 antioxidant status in the mitochondria of heart and brain of aged mice. Methods: The effect was evaluated by estimating the activities of manganese-superoxide dismutase (Mn Keywords: SOD), glutathione peroxidase (GPx), glutathione-S-transferase (GST) and catalase (CAT) as well as levels Ganoderma lucidum of reduced glutathione (GSH), lipid peroxidation, advanced oxidation protein products (AOPP) and Reactive oxygen species reactive oxygen species (ROS) in the heart and brain mitochondria of aged mice after oral administration Mitochondrial ageing Antioxidants of ethanolic extract of G. lucidum (50 and 250 mg/kg), once daily for 15 days. The effect was compared Neurodegenerative diseases with that of aged and young control animals. DL-a-lipoic acid (100 mg/kg) was taken as the positive Congestive heart failure control. Results: Administration of G. lucidum extract signiﬁcantly (p < 0.05) elevated the levels of GSH as well as activities of Mn SOD, GPx, and GST and decreased signiﬁcantly (p < 0.05) the levels of lipid peroxidation, AOPP and ROS. Conclusion: G. lucidum administration could improve the age-related decline of antioxidant status which was partly ascribed to free radical scavenging activity. Ó 2009 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

1. Introduction The degree of age-related changes in cardiomyocytes was found to be high.4 Heart is dependent on molecular oxygen and oxidative Ageing is generally accompanied by a gradual decline in phosphorylation (OXPHOS) to provide high-energy compounds biochemical and physiological functions of most organs, ultimately necessary for contraction, but this exposes the myocardium to leading to an increase in the susceptibility to age-associated harmful ROS that are generated continuously as normal by-prod- disorders. According to the free radical and the oxidative stress ucts of the mitochondrial electron transport chain (ETC). The ageing theories of ageing, the disruption of the delicate balance between heart undergoes significant functional and structural alterations generation of reactive oxygen species (ROS) and antioxidant scav- leading to atrophy and a compensatory hypertrophy, followed by enging systems with increasing age could lead to a shift towards myocardial fibrosis.5 Loss of mitochondrial function and an increase oxidative cellular damage.1 During ageing, antioxidant functions in oxidative stress has been proposed to be one of the key factors in decline in almost all mammals with concomitant increase in myocardial ageing.6 In addition, there is an age-related decline in oxidative damage to biomolecules.2 Though ageing affects all types the capacity to withstand stress like ischemia or reperfusion.7 In its of nucleated cells, tissues with few or no cellular division will be most severe form, cardiac decay results in congestive heart failure theoretically more susceptible to accumulative damage induced by which is observed during old age. ROS. Many of the significant age-related changes are exhibited in Cumulative free radical damage also leads to significant changes post-mitotic tissues such as brain, heart and skeletal muscle.3 in brain mitochondrial function with ageing.8 Of particular interest are the changes in the activity of enzyme complexes of the respiratory chain during the ageing process and the role of such changes in the progression of neurodegenerative diseases.9 Any decline in * Corresponding author. Fax: þ91 487 2307020/868. the activity of brain respiratory chain enzyme complexes has E-mail address: [email protected] (K.K. Janardhanan). a significant impact on brain function particularly on the etiology of

0261-5614/$ – see front matter Ó 2009 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved. doi:10.1016/j.clnu.2009.12.003 N.P. Sudheesh et al. / Clinical Nutrition 29 (2010) 406–412 407 neurodegenerative disorders.10,11 Parkinson’s disease, Alzheimer’s of the extract was 5% (w/w). The extract was suspended in distilled disease (AD), Huntington’s disease and Friedreich’s ataxia have water and employed for the experiment. been associated with several mitochondrial alterations including impaired OXPHOS.10 2.4. Effect of G. lucidum on the antioxidant status of heart and brain Several attempts have been made to slow down the age-asso- mitochondria ciated mitochondrial decay by means of dietary interventions with antioxidants and some have succeeded in partially restoring Animals were divided into 5 groups of 6 animals each. Group 1: mitochondrial structure and function by reducing the oxidative aged control administered with distilled water, Group 2: young damage to lipids, proteins and nucleic acids.12,13 Oriental herbal control administered with distilled water, Group 3 and 4: aged mice medicines have been widely investigated for drug development administered with G. lucidum extract 50 and 250 mg/kg body probably due to fewer side effects. Ganoderma lucidum popularly weight respectively, Group 5: aged mice administered with DL a- known as Reshi or Lingzhi has been traditionally used as a popular Lipoic acid (100 mg/kg b.w.). The DL a-lipoic acid was dissolved in folk medicine for the promotion of health in the Orient. G. lucidum alkaline saline (0.5% NaOH, w/v). The DL a-lipoic acid, G. lucidum has been shown to be safe and prevents many chronic diseases in extract and distilled water (to controls) were administered orally clinical practice.14–16 Previous investigations in our laboratory once daily for 15 days. Twenty-four hours after the completion of showed that G. lucidum occurring in South India possessed signif- drug administration, the animals were sacrificed by cervical icant antioxidant, antitumor, anti-inflammatory, antimutagenic, decapitation. The heart and brain were excised immediately and and radioprotective properties.17–19 G. lucidum has also been kept in 70 C for the determination of enzymatic and non-enzy- reported to be effective against the AD by attenuating the b- matic mitochondrial antioxidant status. amyloid (Ab).20 Recently, we have reported that G. lucidum enhanced the activities of respiratory chain complexes and mito- 2.4.1. Isolation of mitochondria and determination of enzymatic chondrial dehydrogenases in the brain and heart of aged rats.21,22 antioxidant activity This prompted us to evaluate the effect of G. lucidum on the age- Mitochondria were isolated from the heart and brain homoge- related decline of enzymatic and non-enzymatic antioxidant status nates by differential centrifugation according to the method in the heart and brain mitochondria of aged mice. described by Ajith et al.21 Mitochondrial fraction was frozen and thawed 3–5 times to release the enzymes. The supernatant 2. Materials and methods (approximately 3 mg/ml protein) was used for the determination of activities of manganese-superoxide dismutase (Mn SOD), catalase 2.1. Chemicals (CAT), glutathione peroxidase (GPx), glutathione-S-transferase (GST) using a double beam spectrophotometer (2202-Systronics Glutathione (GSH), 5,50-dithio-dinitro bisbenzoic acid (DTNB), India Ltd, Hyderabad, India). 1-chloro-2,4-dinitrobenzene (CDNB), nitroblue tetrazolium (NBT), Mn SOD activity was estimated according to the method of 23 thiobarbituric acid (TBA), riboflavin and sodium azide (NaN3) were McCord and Fridovich. Briefly, 0.01 ml of mitochondrial sample obtained from SRL, Mumbai, India. Hydrogen peroxide (H2O2), was mixed with 7 mmol/l EDTA (containing 0.0015% NaCN), ethylene diamine tetraacetic acid (EDTA), n-butanol, trichloroacetic 0.05 mmol/l NBT and phosphate buffer (67 mmol/l, pH 7.8) in a net acid (TCA) and pyridine were from Merck India Ltd. Mumbai, India. volume of 1.3 ml. Absorbance of the solution was measured at DL-a-Lipoic acid was a gift from Garnett McKeen Laboratory, Inc., 560 nm after the addition of 0.05 ml of riboflavin and again after USA. Protein assay kit was purchased from Genei Pvt. Ltd., Banga- uniform photo illumination of solution under an incandescent lamp lore, India. All other chemicals and reagents used were of analytical for 15 min. The volume of the mitochondrial sample required to reagent grade. scavenge 50% of the generated superoxide anion was considered as 1 unit of Mn SOD activity and is expressed in U/mg protein. 2.2. Animals CAT activity was estimated by the method of Beers and Sizer.24 Briefly, mitochondrial sample containing 0.1 mg of protein was Male BALB/c mice strain weighing approximately 40 5 g (age mixed with phosphate buffer (0.5 mol/l, pH 7) in a net volume of 19–21 months) and 15 5 g (age 2–4 months) were considered as 1 ml. The decrease in extinction was measured at 240 nm, 1 min aged and young mice respectively. The animals were purchased interval for 3 min, immediately after the addition of 3.6 mmol/l from Small Animal Breeding Centre, Kerala Agricultural University, H2O2 against the tissue control. Activity of the CAT was calculated 1 1 Mannuthy, Thrissur, Kerala, India. The animals were maintained for using the molar extinction coefficient of H2O2 (43.6 M cm ) and a week under standardized environmental conditions (22–28 C, expressed in U/mg protein. 60–70% relative humidity, 12 h dark/light cycle) with free access to Activity of GPx was determined by the method of Hafemann standard food (Sai Durga Feeds and Foods, Bangalore, India) and et al.25 Briefly, 0.025 ml of the mitochondrial sample was incu- water ad libitum. The experiment was carried out with the approval bated with 8 mmol/l GSH, 0.1 mmol/H2O2, 2 mmol/l NaN3 and of Institutional Animal Ethics Committee, which follows the phosphate buffer (1 mol/l, pH 7) in a net volume of 1.25 ml at 37 C guidelines of Committee for the Purpose of Control and Supervision for 10 min. After the reaction was stopped with 1 ml of 1.65% of Experiments on Animals (CPCSEA), Govt. of India. metaphosphoric acid, 1 ml of the supernatant was mixed with 1 ml of 0.4 mol/l disodium hydrogen phosphate and 1 ml of 1 mmol/l 2.3. Preparation of the extract DTNB. The absorbance of the solution was measured at 412 nm. Reaction mixture without mitochondrial sample was processed in Fruiting bodies of G. lucidum growing on the Caesalpinia coriaria the same way and kept as non-enzymatic reaction. One unit of GPx wild trees in the local area were collected. The type specimen was activity was defined as decrease in log GSH by 0.001/min after deposited in the herbarium of Centre for Advanced Studies in subtracting from the non-enzymatic reaction and is expressed in Botany, University of Madras, Chennai, India (Herbarium of Madras U/mg protein. University Botany Laboratory number, HERB. MUBL-3175). Seventy GST activity was determined by the method of Habig et al.26 percent ethanol extract of G. lucidum was prepared as described from the rate of increase in conjugate formation between reduced previously.22 The ethanol extract was finally lyophilized. The yield glutathione and CDNB. Briefly, the reaction mixture contained 408 N.P. Sudheesh et al. / Clinical Nutrition 29 (2010) 406–412

1 mmol/l GSH, 1 mmol/l CDNB, 0.01 ml of mitochondrial sample using a Nanodrop ﬂuorimeter (ND 3300, Wilmington, USA) (lex and phosphate buffer (0.1 mol/l, pH 6.5) in a net volume of 3 ml. 488 nm, lem 525 nm). The reaction was started by the addition of sample and the absor- Protein content was determined by the method of Bradford31 bance measured at 340 nm, 1 min interval for 3 min, against the using the diagnostic kit of Genei Pvt. Ltd., Bangalore, India. Bovine control. The activity was calculated using the extinction coefﬁcient serum albumin (BSA) was used as standard. of CDNB (9.6 mM1 cm1) and expressed as U/mg protein (nmol of CDNB-GSH conjugate formed/min/mg protein). 2.5. Statistical analysis

2.4.2. Determination of reduced glutathione All data were represented as mean SD. The mean values were Reduced GSH, non-enzymatic antioxidant, level was determined statistically analyzed using one-way analysis of variance (ANOVA) according to the method of Moron et al.,27 and based on the (using the Graph Pad Instat software package). The significant formation of a yellow colored complex with Ellman’s reagent. differences between the groups were further analyzed by Bonfer- Briefly, 0.1 ml of mitochondrial sample was mixed with 0.063 ml of roni’s t-test. p values less than 0.05 were considered as significant. 25% TCA. After incubation in ice for 5–10 min, 0.3 ml of 5% TCA was added. The protein-free supernatant (0.15 ml) was mixed with 3. Results 0.4 mmol/l of DTNB and sodium phosphate (0.2 mol/l, pH 8) in net volume of 1.5 ml. The absorbance was measured at 412 nm against The experimental results showed that the antioxidant status the reagent blank. The concentration was calculated from the declined with age. The antioxidant status in the heart and brain standard graph of GSH and is expressed as U/mg protein (nmol/mg mitochondria of young mice was significantly (p < 0.05) higher protein). than that of aged mice. Treatment with G. lucidum extract effec- tively ameliorated the oxidative stress in aged mice as evident from 2.4.3. Determination of lipid peroxidation and advanced oxidation the enhanced antioxidant status in the brain and heart tissues. protein product levels The level of lipid peroxidation was measured as thiobarbituric 3.1. Effect of G. lucidum in the heart mitochondrial acid reacting substance (TBARS) by the method of Ohkawa et al.28 antioxidant status 0.2 ml of mitochondrial sample was mixed with 0.4% SDS, 0.3% TBA and 7.5% acetic acid (pH 3.5) and distilled water in a net volume of The activities of the antioxidant enzymes such as Mn SOD, CAT, 2 ml. The reaction mixture was incubated in boiling water bath for GPx and GST were lowered in the heart mitochondria of aged mice 1 h, followed by extraction of the organic layer with n-buta- (Table 1). There were approximately 1.75, 0.91, 1.83 and 1.82 fold nol:pyridine mixture (15:1, v/v). The absorbance of the supernatant decrease in the activities of Mn SOD, CAT, GPx and GST respectively was measured at 532 nm and expressed as equivalents of malon- in aged mice with respect to young mice. The G. lucidum treatment dialdehyde (MDA)/mg protein, using 10103030-tetramethoxypropane at 50 and 250 mg/kg for 15 days improved the activities of anti- as standard. oxidant enzymes in the heart of aged mice. The fold increase in the Advanced oxidation protein products’ (AOPP) level was deter- activity of Mn SOD, GPx and GST were approximately 1.72, 1.73 and mined according to the method described in Kayali et al.29 Briefly, 1.63 fold in the case of 250 mg/kg and 1.37 and 1.25 fold in the case 0.2 ml of mitochondrial sample was mixed with 0.09 mol/l potas- of 50 mg/kg treated group for Mn SOD and GST respectively. sium iodide and phosphate buffered saline (pH 7.4) in net volume of The levels of GSH in the heart mitochondria were found to be 1.3 ml. Two minutes later, 0.2 ml of glacial acetic acid was added decreased during ageing (Fig. 1). The level of GSH had an approxi- and the absorbance was immediately measured at 340 against the mate of 1.09-fold decrease in the heart mitochondria of aged mice reagent blank. The concentration of AOPP was calculated using the than that of young mice. G. lucidum extract improved the levels of extinction coefficient 26 mM1 cm1 and expressed as nmol/mg GSH in the heart mitochondria of aged mice. There was approxi- protein. mately 1.25-fold increase in 250 mg/kg G. lucidum treated group compared to that of aged control. 2.4.4. Determination of reactive oxygen species level in the heart The levels of lipid peroxidation and AOPP were significantly and brain mitochondria (p < 0.05) higher in the heart mitochondria of aged mice than that Mitochondrial ROS level was assessed as described previously of young mice (Figs. 2 and 3). The fold increases in the levels of lipid by Wasilewski and Wojtczak30 with modifications. Briefly, mito- peroxidation and AOPP in the heart mitochondria of aged mice chondria (0.05 mg) were added to 0.1 M phosphate buffer con- were 1.13 and 1.63 respectively. The treatment of G. lucidum had taining 100 mM of dichlorodihydrofluorescein diacetate (H2DCFDA). significantly (p < 0.05) lowered the levels of lipid peroxidation and Fluorescence was quantified after 20 min of incubation at 37 C AOPP in the aged mice. There was approximately 0.98 and 1.36-fold

Table 1 Effect of G. lucidum on the activities of Mn SOD, CAT, GPx and GST in the heart and brain mitochondria of aged BALB/c mice.

Enzyme activity Organ Aged control Young control G. lucidum G. lucidum DL-a-Lipoic acid 50 mg/kg b.w. 250 mg/kg b.w. (100 mg/kg) Mn SOD (U/mg protein) Heart 27.13 7.27a 81.68 21.4 52.96 5.88* 71.23 11.94* 53.67 10.73* Brain 6.19 1.7a 21.44 6.43 18.62 5.45* 19.05 4.55* 16.23 6.35* CAT (U/mg protein) Heart 1.98 0.58a 4.40 2.06 2.13 1.06ns 2.40 0.56ns 2.89 1.08ns Brain 2.83 0.86a 7.29 1.01 6.10 1.65* 7.33 1.83* 5.99 1.55* GPx (U/mg protein) Heart 23.86 5.24a 71.38 18.78 43.18 14.45ns 59.40 9.55* 63.41 26.58* Brain 74.96 19.38a 116.33 18.43 95.89 15.87ns 120.86 35.80* 67.98 27.94ns GST (U/mg protein) Heart 157.43 42.27a 482.23 117.36 289.60 40.56* 366.80 41* 397.33 59.12* Brain 33.73 13.71a 102.20 15.42 59.13 7.05ns 88.37 21.94* 48.88 18.99ns

Values are the mean SD; n ¼ 6. ap < 0.05 significantly different from young control (Bonferroni test). *p < 0.05 significantly and nsp > 0.05 non-significantly different from aged control (Bonferroni test). N.P. Sudheesh et al. / Clinical Nutrition 29 (2010) 406–412 409

80 35 * a 70 ns * 30 )niet )nietorp gm/lom n( HSG n( gm/lom )nietorp 60 25 * o rp gm/lom gm/lom rp 50 a * * 20 * * 40 * 15

a n

30 ( P

PO 10 20

A a 10 5 * * *

0 0 Aged Control Young G. lucidum G. lucidum Lipoic acid Aged Young G. lucidum G. lucidum Lipoic acid control 50 mg/Kg 250 mg/Kg (100mg/Kg) Control control 50 mg/Kg 250 mg/Kg (100mg/Kg)

Heart Brain Heart Brain

Fig. 1. Effect of G. lucidum on the levels of GSH in the heart and brain mitochondria of Fig. 3. Effect of G. lucidum on the levels of advanced oxidation protein products (AOPP) aged BALB/c mice treated with G. lucidum for 15 days. Values are the mean SD; n ¼ 6. in the heart and brain mitochondria of aged BALB/c mice treated with G. lucidum for 15 a ap < 0.05 significantly different from young control (Bonferroni test). *p < 0.05 signif- days. Values are the mean SD; n ¼ 6. p < 0.05 significantly different from young * icantly and nsp > 0.05 non-significantly different from aged control (Bonferroni test). control (Bonferroni test). p < 0.05 significantly different from aged control (Bonferroni test). decrease in the lipid peroxidation in 50 and 250 mg/kg G. lucidum treated groups respectively, where as the fold decrease of AOPP mitochondria of aged control group when compared to young were respectively 1.36 and 1.3 in 50 and 250 mg/kg treated groups control group (Figs. 2 and 3). The treatment of G. lucidum had with respect to the aged control. significantly (p < 0.05) lowered the levels of lipid peroxidation and AOPP in the aged mice. The fold decrease in the brain lipid 3.2. Effect of G. lucidum in the brain mitochondrial antioxidant peroxidation was approximately 1.21 and 1.39 for 50 and status 250 mg/kg treated groups. Similarly, the AOPP level was decreased 1.21 and 1.19-fold for 50 and 250 mg/kg treated group There were approximately 1.74,1.70,1.04 and 1.83 fold decreases respectively. in the activities of Mn SOD, CAT, GPx and GST in the brain mitochondria of aged mice than that of young control (Table 1). The G. lucidum treatment at 50 and 250 mg/kg for 15 days improved the 3.3. Effect of G. lucidum in the heart and brain mitochondrial antioxidant status in the brain. The fold increases in the activity of ROS level brain Mn SOD, CAT, GPx and GST were respectively 1.72, 1.49, 0.90 and 1.63 in the case of 250 mg/kg treated group and 1.53, 2.08 and The study clearly showed that during ageing, the level of ROS is 1.25 for Mn SOD, CAT and GST in the 50 mg/kg treated group. increasing. There was approximately 14.9 and 1.94-fold increase in The level of GSH had an approximate of 2.05-fold increase in the the level of ROS in the heart and brain mitochondria during ageing. brain mitochondria of young mice than that of aged mice. G. luci- The G. lucidum treatment at 50 and 250 mg/kg for 15 days had dum at 50 and 250 mg/kg treated group showed 1.96-fold and 1.50- decreased the ROS levels in heart and brain mitochondria. There fold increase of GSH in brain mitochondria when compared to that was approximately 1.02 and 0.93-fold decrease in the ROS level in of aged control (Fig. 1). the heart mitochondria and 1.0 and 1.09-fold decrease in the case of There was approximately 1.46 and 1.79-fold increases in the brain mitochondria for 50 and 250 mg/kg G. lucidum treated groups levels of lipid peroxidation and AOPP respectively for brain respectively (Fig. 4). ) n ietor 16 70000 p gm 14 a a

60000 /demrof AD M fo sel fo M AD /demrof 12 50000 * * 10 * a 8 40000 * UFR a 6 * * * 30000 o

m n m ** 4 * * ( 20000 * noi 2 t adixore 10000 0 Aged Young G. lucidum G. lucidum Lipoic acid

p 0

d Control control 50 mg/Kg 250 mg/Kg (100mg/Kg) ipi Aged Control Young control G. lucidum 50 G. lucidum 250 Lipoic acid mg/Kg mg/Kg (100mg/Kg) L Heart Brain Heart Brain Fig. 2. Effect of G. lucidum on the levels of lipid peroxidation (as equivalent of MDA) in the heart and brain mitochondria of aged BALB/c mice treated with G. lucidum for 15 Fig. 4. Effect of G. lucidum on the levels of ROS in the heart and brain mitochondria of days. Values are the mean SD; n ¼ 6. ap < 0.05 significantly different from young aged BALB/c mice treated with G. lucidum for 15 days. Values are the mean SD; n ¼ 6. control (Bonferroni test). *p < 0.05 significantly different from aged control (Bonferroni ap < 0.05 significantly different from young control (Bonferroni test). *p < 0.05 signif- test). icantly different from aged control (Bonferroni test). 410 N.P. Sudheesh et al. / Clinical Nutrition 29 (2010) 406–412

34,36 3.4. Effect of DL-a-lipoic acid in the heart and brain mitochondrial reported data. The possible reason for the declined mitochon- antioxidant status drial antioxidant enzyme activity can be explained with the excess generation of ROS during ageing. Mn SOD and CAT are prone to age- DL-a-Lipoic acid (100 mg/kg b.w.) treatment improved the heart associated oxidative damage due to hydroxyl radicals ( OH) mitochondrial antioxidant status in the aged mice. There was generated from Fenton’s reaction.37 ROS-induced oxidative modi- approximately 1.24, 1.28 and 1.69-fold increases in the activities of fication of many enzyme proteins results in structural alteration 38 Mn SOD, GPx and GST in the DL-a-lipoic acid treated group than that and their functional inactivation. The decreased GPx activity can of aged control. There was approximately 1.09-fold increase in the be attributed to the decreased GSH level. Increased production of level of GSH in the DL a-lipoic acid than that of aged control. ROS with concomitant decreases in antioxidant status, DNA Similarly, the level of lipid peroxidation and AOPP were decreased modifications and a progressive decline of overall protein synthesis 39,40 significantly (p < 0.05) in the DL-a-lipoic acid treated group than has been reported to accompany ageing. that of aged control. The fold decreases were approximately 1.39 GSH, a water-soluble tripeptide, is the most abundant non- and 1.15 than that of aged control group. protein thiol molecule in tissues with predominant defense against 41 Similarly, the DL-a-lipoic acid (100 mg/kg b.w.) treatment ROS mainly in brain. GSH reacts directly with ROS and electro- improved the brain mitochondrial antioxidant status in the aged philic metabolites, protects essential thiol groups from oxidation, mice. There was approximately 1.15 and 1.20-fold increase for Mn promotes the regeneration of a-tocopherol, and serves as 42 SOD and CAT in the DL-a-lipoic acid treated group than that of aged a substrate for GSH-related enzymes, such as GPx and GST. In this control. There was approximately 1.50-fold increase in the level of study, we found that the level of mitochondrial GSH (mGSH) in the GSH in the DL-a-lipoic acid than that of aged control. Similarly, the heart as well as brain was lowered significantly in the aged mice levels of lipid peroxidation and AOPP were decreased significantly with respect to that of young. The declined heart and brain mGSH 34,36 (p < 0.05) in the DL-a-lipoic acid treated group than that of aged level in ageing was reported previously. The reduction in the control. The fold decreases were approximately 1.43 and 1.04 than level of GSH was due to increased ROS-induced degradation, or that of aged control group. direct reaction of GSH with the increased lipid peroxidation products such as MDA and 4-hydroxynonenal or due its decreased 3.5. Effect of DL-a-lipoic acid in the heart and brain mitochondrial synthesis. Treatment with G. lucidum for 15 days significantly ROS level elevated the level of GSH in mitochondria of both heart and brain. The excessive generation of free radicals leads to peroxidative DL-a-Lipoic acid (100 mg/kg b.w.) treatment decreased the heart changes that ultimately result in enhanced lipid peroxidation and brain mitochondrial ROS level significantly (p < 0.05) in the (LPO).43 In our experiments, we found a marked increase in the aged mice. There was approximately 1.2 and 1.04-fold decrease in levels of lipid peroxidation in heart and brain mitochondria of the the ROS level in the heart and brain mitochondria for DL-a-lipoic aged mice. Age-related increase in LPO might be a reflection of treated groups respectively than that of aged control. decrease in enzymatic and non-enzymatic antioxidant protection.44 Therefore, the increased LPO observed in the aged group 4. Discussion resulted from the declined activities of Mn SOD, CAT, and GPx as well as decreased level of GSH. By enhancing the antioxidant level Living tissues are endowed with innate antioxidant defense G. lucidum, is able to protect the LPO significantly. mechanisms, including the antioxidant enzymes SOD, CAT, and The G. lucidum treated group also showed improved activity of GPx. Antioxidant enzymes are considered to be the primary mitochondrial GST, when compared to that of aged control mice. defense that prevents biological macromolecules from oxidative GST catalyzes the addition of glutathione moiety to a great variety damage. Attention has been focused on mitochondria in ageing of acceptor molecules including organic hydroperoxides, and lipid biology due to the central role of mitochondria in producing peroxides. Therefore, the declined LPO level observed in the G. chemical energy (ATP), the decline of basal metabolic rate, and of lucidum treated groups might be correlated to the enhanced GST physiological performances which are the characteristics of aged activity. AOPP, dityrosine containing cross-linked protein products, mammals. In the present study, the activities of the antioxidant have been considered as a reliable marker to estimate the degree of enzymes such as CAT, GPx, and Mn SOD were declined significantly oxidant mediated protein damage.45 Major mechanism leading to in the heart and brain mitochondria of the aged mice when oxidation of proteins is initiated from the iron, in the mitochondrial compared to that of young mice. Treatment with G. lucidum membrane, mediated generation of OH via Fenton reaction. significantly maintained the enzymatic antioxidant activity as Therefore, greater levels of O2 and H2O2 in the heart and brain evident from the improved activities of Mn SOD and GPx with mitochondria of aged animals can accelerate the generation of respect to that of aged control. The effect was found to be non- oxidative protein damage. A number of studies have shown significantly different between the doses. Therefore, 50 mg/kg may elevated concentrations of oxidized proteins especially in the post- be the preferable dose for the beneficial effect. The selection of mitotic tissue as a function of age.46–48 Increased AOPP level in the lipoic acid as reference standard and its dose was based on the heart of aged rat has been recently reported.48,49 Treatment of aged results of the recent study which reported that lipoic acid (100 mg/ mice with G. lucidum had significantly decreased the levels of AOPP kg, i.p.) daily for 14 days could improve the mitochondrial bioen- in aged mice indicating the beneficial effect of G. lucidum extract. ergetics and the antioxidant status.32 The doses of G. lucidum were We found a positive correlation between levels of AOPP and lipid selected based on the previous study.21,22 peroxidation in the mitochondria of heart and brain, which is an The antioxidant enzymes, Mn SOD and GPx are recognized as indicator of a close relation between protein oxidation and lipid primary defense against superoxide anion (O2 ) and H2O2 in peroxidation during ageing. eukaryotic cell mitochondria. However, presence of a heme-con- The DCFH-DA assay in the present study has been adopted to taining CAT in the rat heart mitochondrial matrix was demon- measure most of the intracellular oxidants. There are reports of strated by Radi et al.33 The CAT activity in the mitochondria of rat increased levels of ROS in the post-mitotic tissues such as heart and liver, brain, and skeletal muscle has been previously reported.34–36 brain during ageing.48,50–52 In our study, the estimation of ROS level Our findings as regards the declined Mn SOD, CAT, and GPx activ- in the heart and brain mitochondria revealed that during ageing the ities in heart and brain mitochondria also agree with the previously level of ROS had increased significantly (p < 0.05) and the N.P. Sudheesh et al. / Clinical Nutrition 29 (2010) 406–412 411 treatment with G. lucidum had significantly reduced the level of cellular compartments or have complementary activity. Clinical ROS in the aged rats which clearly shows the significant effect of trials and epidemiological studies have established an increased G. lucidum in scavenging the ROS during ageing. correlation between the intakes of vegetables in the occurrence of Mitochondrial DNA (mtDNA) mutations occur with strikingly diseases including cardiovascular diseases. Though we have characteristic tissue specificity, in post-mitotic cells of ageing previously reported the Krebs cycle and ETC enzyme activity humans.53 Mitochondria in heart and brain exhibit more than 100- enhancing effect of G. lucidum, the current study could partially fold increase in mutation frequency with age.54 mtDNA mutations explain the possible mechanism of attenuating the mitochondrial can be ascribed to the close proximity of the DNA to the free radical oxidative stress in aged mice that could improve the OXPHOS in generated from the OXPHOS in ETC particularly in complex I and III, heart and brain cells. However, direct evidence of improved mito- which are the primary sites of ROS production.55,56 Approximately chondrial function is yet to be studied in detail. 1–2% of total oxygen consumption gives rise to potentially cytotoxic The results of the study concluded that G. lucidum administra- ROS such as O2 and H2O2. Defect or inhibition of mitochondrial tion could improve the age-related decline of antioxidant status in OXPHOS has been shown to result in increased mitochondrial ROS the heart and brain mitochondria, suggesting the potential thera- production.57 The combination of impairment of mitochondrial peutic use of this mushroom in ageing associated ailments such as Complex I and deficiency of O2 removal can also result in massive cardiovascular diseases and neurodegenerative diseases. However, cell death.58 Brain is particularly susceptible to free radical attack further detailed evaluation is necessary to establish the clinical than any other organ due to higher rate of oxidative metabolism, application of G. lucidum. very low respiratory control ratio, lower activity of antioxidant enzymes, reduced content of non-enzymatic antioxidants and Conflict of Interest higher peroxidation potential due to the high content of PUFA.59 The authors declare that there are no conflicts of interest. Cells of the substantia nigra contain the highest level of mtDNA mutation in the brain60,61 and these cells die earlier in Parkinsonian individuals than in normally ageing humans. Similarly, heart is Acknowledgements vulnerable to damage induced by free radicals because of lower levels of CAT. The myocardial cell mitochondria contribute a greater The specific contribution of each author to the work: 1. N.P. Sudheesh, M.Phil – experimental work and technical assistance, 2. H2O2 production owing to high tissue mitochondrial density and exclusively aerobic metabolism of myocardium than other T.A. Ajith, Ph.D. – experimental design, preparation of manuscript, 33 data analysis, 3. V. Ramnath, M.VSc., Ph.D., spectroflurometric tissues. Therefore, the role of CAT to remove H2O2 especially analysis, 4. K.K. Janardhanan, Ph.D., FNABS – technical guidance, under extensive GSH depletion and high H2O2 concentration is non-significant. It can be concluded that mitochondria of cardiac critical review of manuscript. All of the authors have no conflicts of myocytes and brain cells become less efficient with increasing age, interest or contractual agreements that might cause conflicts of resulting in greater damage to DNA and proteins. interest. The results of the study reveal that G. lucidum is capable of preventing age-related decline of antioxidant status in the heart References and brain mitochondria suggesting the potential role of this mushroom in anti-ageing. 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