Bacopa monnieri modulates endogenous cytoplasmic and mitochondrial oxidative markers in prepubertal mice brain.
Bacopa monnieri (BM) an herb, found throughout the Indian subcontinent in wet, damp and marshy areas is used in Ayurvedic system of medicine for improving intellect/memory, treatment of anxiety and neuropharmacological disorders. Although extensively given to children as a memory enhancer, no data exists on its ability to modulate neuronal oxidative stress in prepubertal animal models. Hence in this study, we examined if dietary intake of BM leaf powder has the propensity to modulate endogenous markers of oxidative stress, redox status (reduced GSII, thiol status), response of antioxidant defenses (enzymic), protein oxidation and cholinergic function in various brain regions of prepubertal (PP) mice. PP mice maintained on a BM-enriched died (0.5 and 1%) for 4 weeks showed a significant diminution of basal oxidative markers (malondialdehyde levels, reactive species generation, hydroperoxide levels and protein carbonyls) in both cytoplasm and mitochondria of all brain regions. This was accompanied with enhanced reduced glutathione, thiol levels and elevated activities of antioxidant enzymes (catalase, glutathione peroxidase, superoxide dismutase). Siginificant reduction in the activity of acetyl cholinesterase enzyme in all brain regions suggested the potential of BM leaf powder to modulate cholinergic function. Further evidence that dietary intake of BM leaf powder confers the prepubertal brain with additional capacity to cope up with neurotoxic prooxidants was obtained by exposing cortical/cerebellar synaptosomes of normal and BM fed mice to 3-nitropropionic acid (3-NPA). While synaptosomes obtained from BM fed mice showed only a marginal induction at the highest concentration clearly suggesting their increased resistance to 3-NPA-induced oxidative stress. Collectively these data clearly indicate the potential of Bacopa monnieri to modulate endogenous markers of oxidative stress in brain tissue of PP mice. Based on these results, it is hypothesized that dietary intake of BM leaf powder confers neuroprotective advantage and is likely to be effective as a prophylactic/therapeutic agent for neurodegenerative disorders involving oxidative stress.
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Bacopa monnieri (Brahmi, Family: Scrophulariaceae), a traditional Ayurvedic medicinal plant is extensively used for centuries for treatment of epilepsy, insomnia and anxiety and also as a mild sedative and memory enhancer (Tripathi et al. 1996; Kishore and Singh 2005; Ernst 2006). Besides, B. monnieri (BM) displays antioxidant, antistress, anxiolytic properties in experimental animals (Shanker and Singh 2000; Chowdhuri et al. 2002). Further, it improves the performance of rats in various learning situations such as shock-motivated brightness discrimination reaction, an active conditioned flight reaction, continuous avoidance response (Singh and Dhawan 1982) and attenuates experimentally induced amnesia in experimental animals (Kishore and Singh 2005; Saraf et al. 2008).
Several clinical studies have confirmed the beneficial actions of BM (Russo and Borrelli 2005) and the pharmacological actions are mainly attributed to the saponin compounds present in the alcoholic extract of the plant. The major chemical constituents isolated and characterized from Bacopa are dammarane type of tri terpenoid saponins. Several pharmacological (Singh et al. 2001l; Singh and Dhawan 1997) and clinical studies (Nathan et al. 2001; Stough et al. 2001) on the extracts of BM standardized to the bacosides A and B have been reported. Bacoside A is shown to alleviate the amnesic effects of scopolamine (Russo and Borrelli 2005) and provide protection against phenytoin-induced deficit in cognitive function in mice (Vohora et al. 2000). Earlier studies have reported that BM revitalizes the intellectual functions among children (Sharma et al. 1987). Recently, preclinical studies have demonstrated cognitive enhancing effects with various BM extracts, although the precise mechanism/s of its action is not clear (Stough et al. 2001; Roodenrys et al. 2002; Russo and Borrelli 2005).
The neuroprotective and cognitive enhancing effects of BM extracts are explained to be due to several mechanisms such as chelation of metal ions (Tripathi et al. 1996), scavenging of free radicals (Russo et al. 2003) and enhanced antioxidative defense enzymes (Bhattacharya et al. 1999, 2000; Russo et al. 2003). Further, the antistress activity of BM in experimental animals is attributed to its propensity to modulate Hsp70 expression, cytochrome P450 levels, activity of SOD (Chowdhuri et al. 2002), enhanced kinase activity, neuronal synthesis coupled with restoration of synaptic activity, and nerve impulse transmission (Kishore and Singh 2005). Other biological effects of BM reported in animal model include hepatoprotection against morphine (Sumathy et al. 2001) and antiulcerogenic activity (Sairam et al. 2001). Despite the extensive human usage of BM-derived products, knowledge with regard to its ability to modulate endogenous oxidative markers in different brain regions either in young or adult experimental animals is limited.
3-Nitropropionic acid (3-NPA), a fungal mitochondrial toxin causes selective neuronal degeneration in the striatum and produces anatomical changes similar to that of Huntington's disease in experimental animals (Brouillet et al. 1999). 3-NPA inactivates the mitochondrial enzyme, succinic dehydrogenase, a step in the tricarboxylic acid cycle and oxidative phosphorylation (complex II) reactions under in vivo conditions (Coles et al. 1979). Enhanced reactive species generation (ROS) generation and malondialdehyde (MDA) levels have been amply demonstrated in brain of rats challenged with 3-NPA suggesting the vital role of oxidative stress in the manifestation of neurotoxicity. Following 3-NPA administration, the concentration of free fatty acids are demonstrated to increase in all brain regions which is accompanied with higher generation of free radicals resulting in elevated oxidative stress (Binienda and Kim 1997; Binienda et al. 1998; Kim et al. 2005).
BM plant, plant extracts and isolated bacosides have been extensively investigated for their biological activities. BM leaves as such are often consumed in a variety of ways in this part of the world. Hence, we have addressed two questions: (a) whether short-term dietary intake of BM leaf powder could significantly diminish the levels of endogenous oxidative markers in different brain regions of prepubertal (PP) mice and (b) if BM supplements confer any significant advantage against exogenous exposure to a neurotoxicant. These questions were addressed in an experimental design which involved feeding of PP (4-week old) male mice with BM leaf powder enriched diet (0.5 and 1.0%) for four weeks. Biochemical investigations comprised of assessment of both cytoplasmic and mitochondrial oxidative markers in different brain regions (cortex, cerebellum, hippocampus and striatum), antioxidant defenses (enzymic/non-enzymic), protein carbonyl levels and the activity of acetylcholinesterase in brain regions. Further, employing an ex vivo approach, we compared the 3-NPA induced 'oxidative response' in synaptosomes (isolated from cortex and cerebellar brain regions) of normal and BM fed mice.
Materials and methods
Animals and care
Prepubertal male mice (CFT-Swiss, 4-week old) were drawn from the stock colony of the 'institute animal house facility'. They were housed in rectangular polypropylene cages (three per cage) kept on racks built of slotted angles and the cages were provided with dust free paddy husk as a bedding material. The animals were housed in a controlled atmosphere with a 12 h light/dark cycle. They were acclimatized for 1 week prior to the start of the experiment and were maintained on a commercial pellet diet which was coarsely powdered (M/s Saidurga feeds, Mumbai, India) and tap water ad libitum. All procedures with animals were conducted strictly in accordance with approved guidelines by the local Institute Animal Ethical Committee (IAEC) regulated by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), constituted by the Ministry of Social justice and Empowerment, Government of India, India (Registration number 49/1999/CPCSEA).
Bacopa monnieri (BM) leaf powder
Bacopa monnieri plant was collected during early summer from the state of Kerala and authenticated by Prof. CM. Joy, Department of Botany, Sacred Heart College, Thevara, Mahatma Gandhi University (MGU), Kerala, India. The voucher specimen was kept at the departmental Herbarium, MGU, and Kerala. Fresh leaves of BM were shade dried and powdered in a mill without producing much heat. Whole leaf powder was employed as such for incorporation in to the commercial powdered diet. The powder was subjected to physical and chemical analysis employing standard procedures and the salient results are presented in Table 1. The total triterpenoid saponins were quantified in the leaf powder by HPLC method (Houghton and Raman 1998). The HPLC-finger print analysis of the sample was carried out as per the conditions and procedures described previously (Deepak et al. 2005). A typical HPLC chromatogram is presented in Fig. 1. The quantities of major saponins expressed as % w/w are: luteolin (1: 0.29%); Apigenin (2: 0.12%); Bacopaside-I (3: 1.43%); Bacoside-A3 (4: 1.60%); Bacopaside-II (5: 2.74%); Jujobogenin isomer of Bacopasaponin C (6: 1.96%); Bacopasaponin-C (7: 1.70%); Bacoside-A (8: 7.96%) and Bacosine (9: 0.61%).
[FIGURE 1 OMITTED]
Table 1 Quality control specifications of the Bacopa monnieri leaf powder. Physiochemical Loss on drying (moisture) NMT 5% (w/w) analysis Ash content NMT 10% (w/w) Heavy metals NMT 20 ppm Microbiological Total viable aerobic count <100 cfu/g analysis Total fungal count (yeast and moulds) <100 cfu/g E. coli Absent Salmonella typhimuirum Absent S. aureus Absent Phytochemical Bacosides 40% (w/w) analysis NMT: not more than and cfu: colony forming units.
Extraction of Bacopa leaf powder
A known (500 mg) amount of Bacopa monnieri extract was dissolved in methanol (50 ml), sonicated for 3 min, boiled on steam water bath for 5 min, cooled, made up to 100 ml with methanol and filtered through 0.45 [micro]m membrane filter paper.
Estimation of bacosides by HPLC analysis
The separation was performed using a Shimadzu High Performance Liquid Chromatographic system equipped with LC10A pump with SPD-M 10Avp Photo diode Array Detector or UV detector in combination with Class-VP software or LC 2010 A and LC 2010HT integrated system equipped with Quaternary gradient, auto injector in combination with Lab solution software. The mobile phase consisted of 0.2% orthophosphoric acid and acetonitrile. The total run time was 45 min. All peaks were integrated at the wavelength of 205 nm. The flow rate was 1.5 ml/min. The coulmn used was Pinnacle DB C18 column, 250 mm, 4.6 mm/5 [micro]m obtained from Restek, Part No. 9414575. Calibration curves of five saponin glycosides, bacoside A3, bacopasiside II, bacopasaponin C isomer, bacopasaponin C, bacopaside I were prepared based on peak areas of reference standards.
Experimental protocol - in vivo study
BM leaf powder was mixed with powdered diet at two concentrations viz., 0.5 and 1.0%. These dietary concentrations were selected based on a preliminary study in which the palatability of enriched diet by PP mice was ascertained. Male mice (n = 6) were fed with either powdered commercial diet or B. monneri incorporated diet for a period of 4 weeks. Mice fed with only commercial diet served as the normal controls. BM incorporated diet was prepared on every alternate days. During the experimental period, known amount of diet was provided and the residual diet weighed in order to obtain the exact diet consumption by each group of mice. Body weights were recorded once a week. An interim sampling (after 2 weeks of feeding) was also carried out. Mice from control and BM groups were subjected to mild ether anesthesia and autopsied at the end of 4 weeks. Whole brain was excised and frozen immediately. The brain regions, cerebral cortex, cerebellum, hippocampus and striatum were subsequently dissected over ice. From each brain region cytosol and mitochondrial fractions were prepared and subjected to quantification of various biochemical parameters.
Ex vivo study--exposure of synaptosomes to 3-NPA
Freshly isolated cortex and cerebellum were used to prepare synaptosomes for the ex vivo study. Synaptosomes prepared from both control and BM fed mice were exposed to 3-NPA in vitro (as described below) and 'lipid peroxidation induction response' was determined by quantification of both malondialdehyde (MDA) levels and ROS generation.
Preparation of homogenates and mitochondria
Different brain regions were separately processed for isolation of cytosol and mitochondria by differential centrifugation method as described earlier (Moreadith and Fiskum 1984) with minor modifications. Briefly, 10% homogenates of the brain regions were prepared in ice-cold Tris-sucrose buffer (0.25 M, pH 7.4) using a glass-teflon grinder at 4[degrees]C. The homogenates were centrifuged at 1000 x g for 10 min at 4[degrees]C to obtain the nuclear pellet. Mitochondria were obtained by centrifuging the post-nuclear supernatant at 10,000 x g for 20 min at 4[degrees]C. The pellet was washed three times in Mannitol-Sucrose-HEPES buffer and resuspended in the same buffer. The resuspended mitochondria were stored at 4[degrees]C until further use. The post-mitochondrial supernatent was further subjected to ultracentrifugation to sediment the microsomal fraction to obtain the cytosol fraction.
Preparation of synaptosomes
Synaptosomes were prepared as per the method described earlier (Gil et al. 2001). Cortex and cerebellum were separately homogenized in 40 volume (w/v) phosphate buffer, pH 7.4 supplemented with 0.32 M sucrose. The homogenization was performed with twelve strokes (900 RPM) of a glass-teflon grinder at 4[degrees]C. The homogenate is centrifuged at 1000 x g for 10 min at 4[degrees]C. The supernatant obtained was subjected to further centrifugation at 12,000 x g for 20 min. The crude synaptosome was resuspended in 10 ml of HEPES--sodium buffer (NaCl: 140 mM; KCl: 5 mM; [NaHCO.sub.3]: 5 mM; [MgCl.sub.2]: 1 mM; HEPES/NaOH: 20 mM and glucose: 10 mM, pH 7.4).
Ex vivo exposure of synaptosomes to 3-NPA
The induction response of cortex/cerebellar synaptosomes of control and of BM fed mice was determined in vitro as follows. An aliquot of synaptosomes (200 [micro]g protein) was added to Locke's buffer and challenged with varying concentrations (0.5-2.0 mM) of 3-NPA and incubated for 1 h. The reaction was stopped by addition of 200 [micro]l of SDS (8%) followed by 1.5 ml acetic acid (20%, pH 3.5) and 1.5 ml TBA (0.8%), vortexed and kept in boiling water bath for 45 min. The pink colored complex was extracted into 1 -butanol and read at 532 nm and expressed as [eta] moles MDA/mg protein. In a separate experiment ROS generation was determined as follows. Briefly an aliquot of synaptosomes (200 [micro]g protein) was challenged with 3-NPA (0.5-2 mM) followed by incubation at 37[degrees]C in a water bath for 1 h. After 30 min of further incubation, the conversions of DCFH-DA to the fluorescent product DCF was measured in a spectrofluorimeter with excitation at 485 nm and emission at 530 nm. Appropriate blanks with and with out homogenates/synaptosomes were included to remove interferences with results. ROS formation was quantified from a DCF-standard curve and results were expressed as pmol DCF formed/min/mg protein.
Status of oxidative markers
Lipid peroxidation (LPO): Induction of oxidative damage was ascertained by measuring the extent of LPO in cytosol and mitochondrial fractions of different brain regions (cortex, cerebellum, hippocampus and striatum) LPO was quantified by measuring the formation of thiobarbituric acid reactive substances (TBARS). Briefly, the reaction mixture contained 0.2 ml of brain region cytosol or mitochondria (1 mg protein), 1.5 ml of acetic acid (pH 3.5, 20%), 1.5 ml of 0.8% thiobarbituric acid (0.8%, w/v) and 0.2 ml SDS (8%, w/v). The mixture was heated to boiling for 45 min and TBARS adducts were extracted into 3 ml of 1-butanol and was measured in a UV--vis spectrophotometer at 532 nm and quantified as malondialdehyde (MDA) equivalents using 1,1,3.3-tetramethoxypropane as the standard (Ohkawa et al. 1979).
ROS generation: ROS generation was assayed using dihydro dichloro-fluorescein diacetate ([H.sub.2] DCFH-DA), a non-polar compound, after conversion to a polar derivative by intracellular esterases, can rapidly react with ROS to form the highly fluorescent compound dichloro-fluorescein (Shinomol and Muralidhara 2007). The reaction mixture (1 ml) containing Locke's buffer (pH 7.4), 0.2 ml cytosol or mitochondria (0.5 mg protein) and 10 [micro]l of DCFH-DA (5 [micro]M) was incubated for 15 min at room temperature to allow the DCFH-DA to be incorporated into any membrane-bound vesicles and the diacetate group cleaved by esterases. After 30 min of further incubation, the conversion of DCFH-DA to the fluorescent product DCF was measured with excitation at 485 nm and emission at 530 nm. Background fluorescence (conversion of DCFH-DA in the absence of homogenate) was corrected by the inclusion of parallel blanks. ROS formation was quantified from a DCF-standard curve and data are expressed as pmol DCF formed/min/mg protein.
Hydroperoxide levels: An aliquot of cytosol or mitochondria (100 [micro]g protein) was added to 1 ml FOX reagent (100 [micro]M xylenol orange; 250 [micro]M ammonium ferrous sulphate; 100 [micro]M sorbitol; 25 mM [H.sub.2] [SO.sub.4])and incubated for 30 min at room temperature. The mixture was centrifuged at 600 x g and the supernatant was read at 560 nm. Results were expressed as [mu]mole hydroperoxide/mg protein (Wolff 1994).
Activity of antioxidant enzymes
The activities of enzymes viz., catalase, glutathione-S-transferase, and glutathione peroxidase and superoxide dismutase were measured by following the original methods with minor modifications. Catalase activity was assayed by the method of Aebi (1984) and the enzyme activity was expressed as (mole [H.sub.2] [O.sub.2] consumed/min/mg protein (e = 43.6m[M.sup.-1] [cm.sup.-1). The activity of glutathione peroxidase was determined using t-butyl hydroperoxide as the substrate according to the method of Flohe and Gunzler (1984) and the activity was expressed as [eta] moles of NADPH oxidized/min/mg protein ([e.sub.340] = 6.22 m[M.sup.-1] [cm.sup.-1]). Glutathione-S-transferase was assayed by measuring the rate of enzyme catalyzed conjugation of GSH with 1-chloro-2,4-dinitro benzene (CDNB) according to the method of Guthenberg et al. (1985) and the enzyme activity was expressed as [eta] moles of S-2,4-dinitrophenyl glutathione formed/min/mg protein (M EC -9.6 m[M.sup.-1] [cm.sup.-1]). Superoxide dismutase (SOD) activity was measured by monitoring the inhibition of ferricytochrome-c reduction using xanthine-xanthine oxidase as the source of [O.sub.2]. One unit of SOD is calculated as the amount of protein required to inhibit 50% of the SOD independent cytochrome 'c' reduction (McCord and Fridovich 1969).
Determination of reduced glutathione (GSH)
GSH was measured according to the fluorimetric method of Mokrasch and Teschke (1984). Briefly 100 [micro]l of cytosol/mitochondria was added to 2 ml formic acid (0.1 M) and centrifuged at 10,000 x g for 20 min. 100 [micro]l of the supernatant was used for the assay. Concentration of GSH was calculated from the standard curve and the values were expressed as [mu]g GSH/mg protein.
Determination of protein carbonyls
Protein carbonyl content was determined in supernatants obtained after centrifugation of cytosol/mitochondria at 10,000 x g for 15 min by measuring the hydrazone derivatives between 360 and 390 nm according to the method of Levine et al. (1990).
Total thiols and non-protein thiols
Estimation of total thiols and non-protein thiols was done according to the method of Ellman (1959). To estimate total thiols, 150 [micro]g of protein (cytosol and mitochondria) was added to tubes containing 375 [micro]l of 0.2 M Tris buffer (pH.8.2), 25 [micro]l of DTNB (10 mM in absolute methanol) and 1.975 ml of methanol in a final volume of 25 ml. Allowed to stand for 30 min at room temperature with occasional shaking. The tubes were centrifuged at 3000 x g for 15 min and the absorbance of the supernatant was read at 412 nm against distilled water as blank. For non-protein thiols 150 [micro]g (in 125 [micro]l) of cytosolic/mitochondrial protein was added to 1 ml of distilled water and 250 [micro]l 5% TCA and centrifuged at 3000 x g for 15 min. An aliquot (0.5 ml) of supernatant was added to 1 ml of 0.4M Tris buffer PH.8.9 and 25 [micro]l of DTNB (10 mM in absolute methanol), kept at room temperature for 15 min and centrifuged at 3000 x g (5 min). Supernatant was taken and read at 412 nm against a distilled water blank and calculated using MEC - 13.6 [mM.sup.-1] [cm.sup.-1] and expressed as [eta] moles oxidized DTNB formed/mg protein.
Acetylcholinesterase (AChE) activity
AChE activity was determined according to the method of Ellmann et al. (1961). To the reaction mixture containing 2.85 ml phosphate buffer (0.1 M, pH 8.0), 50(1 of DTNB (10 mM), 50(1 sample (cytosol) and 20(1acetylthiocholine iodide (78 mM) were added and the change in absorbance was monitored at 412 nm for 5 min in a spectrophotometer. The enzyme activity was expressed as [eta] moles of substrate hydrolyzed/min/mg protein.
Determination of protein
Protein concentrations in the tissue homogenates, cytosol, synaptosomes and mitochondria were determined by the method of Lowry et al. (1951), using bovine serum albumin as the standard.
Data expressed as mean[+ or -]SD were analyzed by one-way analysis of variance (ANOVA) followed by Duncan's Multiple Range Test (DMRT) to compare the control and treatment groups: p values less than 0.05 were considered as statistically significant. All statistical analysis were performed using the SPSS statistical software package version 13.0
Food intake and growth
No significant alterations were observed in daily food intake among BM fed mice. Further, no significant alterations in body weights and liver weights were noticeable among mice fed BM-enriched diet (data not shown).
Effect of BM on oxidative markers in brain--interim sampling
The brain regions of prepubertal mice fed BM diet for 15 days showed significant decrease in oxidative markers. Major changes consisted of reduced MDA (15-21%), ROS levels (18-25%) and protein carbonyls content (17-24%) in all brain regions of BM fed mice. These were accompanied with significant increase in reduced glutathione (14-27%) and elevated activities of antioxidant enzymes (CAT: 26%; GPx: 22%; SOD: 21%) in all brain regions (Data not shown).
Modulatory effect on oxidative stress markers in brain regions
Effect on oxidative stress markers
In general there was a decrease in the basal levels of oxidative markers in both cytosol (Table 2) and mitochondria (Fig. 2) of various brain regions of mice fed BM. The MDA levels were significantly diminished in both cytosol (27-30%) and mitochondria (22-25%). Likewise the ROS generation were reduced in all brain regions both in cytosol (15-30%) and mitochondria (10-29%), while decrease in hydroperoxide levels were more robust at the higher dose (cytosol: 34-40%: mitochondria: Ct: 35-40%).
[FIGURE 2 OMITTED]
Table 2 Oxidative stress markers in cytosol of brain regions of prepubertal male mice fed with Bacopa monnieri (BM)-enriched diet for 30 days. BM (%) Brain regions Cortex Cerebellum Hippocampus Striatum ROS (a) 0 9.96 [+ 10.9 [+ or 10.56 [+ or 11.57 [+ or or -] -] 0.75a -] 0.60a -] 0.70a 0.65a 0.5 8.26 [+ 9.06 [+ or 8.99 [+ or -] 9.91 [+ or -] or -] -] 0.70b 0.70b 0.60b 0.50b 1.0 7.45 [+ 7.45 [+ or 7.42 [+ or -] 8.43 [+ or -] or -] -] 0.60c 0.50c 0.65c 0.55c Malondialdehyde (b) 0 9.54 [+ 10.11 [+ or 9.96 [+ or -] 9.50 [+ or -] or -] -] 0.80a 0.58a 0.60a 0.89a 0.5 8.07 [+ 8.85 [+ or 8.59 [+ or -] 8.59 [+ or -] or -] -] 0.56b 0.50b 0.65b 0.78b 1.0 6.84 [+ 7.07 [+ or 7.01 [+ or -] 6.95 [+ or -] or -] -] 0.64c 0.45c 0.66c 0.61c Hydroperoxides (c) 0 2.33 [+ 2.35 [+ or 2.43 [+ or -] 2.55 [+ or -] or -] -] 0.13a 0.1a 0.09a 0.09a 0.5 2.04 [+ 2.11 [+ or 2.05 [+ or -] 1.99 [+ or -] or -] -] 0.10b 0.09b 0.07b 0.08b 1.0 1.53 [+ 1.34 [+ or 1.45 [+ or -] 1.42 [+ or -] or -] -] 0.09c 0.08c 0.07c 0.07c Values are mean [+ or -] SD (n = 6); data analyzed by one-way ANOVA (p<0.05) appropriate to completely randomized design with replicates. Means followed by different letters differ significantly according to Duncan's Multiple Range Test (DMRT). (a) [rho] moles DCF/min/mg protein. (b) [eta] moles MDA/mg protein. (c) [mu]mole [H.sub.2][O.sub.2]/mg protein.
The protein carbonyl levels were significantly reduced in both cytosol (Ct: 14-34%: Cb: 16-31%; Hc: 18-34%; St: 15-30%) and mitochondrial fractions in all brain regions (Ct: 19-41%; Cb: 18-44%; Hc: 20-35%; St: 22-33%) of mice fed BM-enriched diet (Fig. 3).
[FIGURE 3 OMITTED]
Status of glutathione (GSH) and thiol levels
Reduced glutathione and thiol levels were significantly enhanced in both cytosol (Table 3) and mitochondria (Fig. 4) of various brain regions of mice fed BM. In cytosol, the elevations in GSH levels were 10-23% and increase in total thiols were 11 -29%. Significant increases were also observed in reduced glutathione (10-32%), total thiols (16-35%) and non-protein thiols (10-31%) in mitochondria of brain regions among mice fed BM-enriched diet.
[FIGURE 4 OMITTED]
Table 3 Levels of reduced glutathione, total thiols and non-protein thiols in cytosol of brain regions of prepubertal male mice fed with B. monnieri (BM)-enriched diet for 30 days. BM (%) Brain regions Cortex Cerebellum Hippocampus Striatum Reduced GSH (a) 0 8.72 [+ 8.69 [+ or 8.58 [+ or -] 8.77 [+ or -] or -] -] 0.78a 0.59a 0.65a 0.58a 0.5 9.66 [+ 9.61 [+ or 9.77 [+ or -] 9.68 [+ or -] or -] -] 0.60b 0.76b 0.59b 0.55b 1.0 10.72 [+ 10.62 [+ or 10.56 [+ or 10.60 [+ or -] or -] -] 0.63c -] 0.77c 0.63c 0.57c Total thiols (b) 0 10.20 [+ 10.32 [+ or 9.99 [+ or -] 10.01 [+ or -] or -] -] 0.49a 0.65a 0.35a 0.50a 0.5 11.31 [+ 11.47 [+ or 11.50 [+ or 11.40 [+ or -] or -] -] 0.45b -] 0.60b 0.62b 0.51b 1.0 12.34 [+ 12.49 [+ or 12.40 [+ or 12.91 [+ or -] or -] -] 0.50c -] 0.48c 0.45c 0.45c Non-protein thiols (c) 0 0.107 [+ 0.112 [+ or 0.105 [+ or 0.107 [+ or -] or -] -] 0.004a -] 0.003a 0.006a 0.008a 0.5 0.119 [+ 0.116 [+ or 0.116 [+ or 0.116 [+ or -] or -] -] 0.004b -] 0.005b 0.007b 0.008b 1.0 0.127 [+ 0.125 [+ or 0.126 [+ or 0.126 [+ or -] or -] -] 0.003c -] 0.004c 0.005c 0.007c Values are mean [+ or -] SD (n = 6) data analyzed by one-way ANOVA (p <0.05) appropriate to completely randomized design with replicates. Means followed by different letters differ significantly according to DMRT. (a) [mu]g GSH/mg protein. (b) [eta] moles DTNB oxidized/mg protein. (c) [eta] moles DTNB oxidized/mg protein.
Modulatory effect on the activities of antioxidant enzymes
In general the activity of antioxidant enzymes in brain regions of mice fed BM were enhanced (Table 4). A dose dependent increase in catalase (17-47%), glutathione peroxidase (12-37%) and super oxide dismutase (10-38%) was observed in cytosol of brain regions. A similar trend of increase was also observed in mitochondria. While the SOD activity was significantly increased (10-23%), the increase was less robust in case of GPx activity (10-26%). However, no significant alterations were observed in GST activity (data not shown).
Table 4 Activities of antioxidant enzymes in cytosol and mitochondria of brain regions of prepubertal male mice fed Bacopa monneri (BM) for 30 days in diet. BM (%) Brain regions Cortex Cerebellum Hippocampus Striatum Cytosol CAT (a) 0 2.0 [+ or 2.0 [+ or -] 2.0 [+ or -] 2.0 [+ or -] -] 0.08a 0.07a 0.07a 0.04a 0.5 2.3 [+ or 2.5 [+ or -] 2.43 [+ or -] 2.3 [+ or -] -]0.1 lb 0.09b 0.08b 0.05b 1.0 2.9 [+ or 2.9 [+ or -] 2.95 [+ or -] 2.7 [+ or -] -] 0.10c 0.06c 0.07c 0.07c GPx (b) 0 20.6 [+ or 20.8 [+ or 19.11 [+ or 20.1 [+ or -] -] 0.89a -] 0.90a -] 0.38a 0.60a 0.5 23.4 [+ or 23.3 [+ or 23.25 [+ or 23.0 [+ or -] -] 0.78b -] 0.56b -] 0.50b 0.45b 1.0 26.2 [+ or 26.4 [+ or 26.09 [+ or 26.8 [+ or -] -] 1.21c -] 0.24c -] 0.45c 0.56c SOD (c) 0 34.9 [+ or 36.0 [+ or 36.17 [+ or 36.0 [+ or -] -] 1.12a -] 1.50a -] 1.10a 1.23a 0.5 38.8 [+ or 38.4 [+ or 39.77 [+ or 39.1 [+ or -] -] 1.11b -] 1.66b -] 1.34b 1.23b 1.0 47.2 [+ or 48.4 [+ or 49.09 [+ or 49.7 [+ or -] -] 2.34c -] 2.10c -] 1.01c 1.01c Mitochondria GPx (b) 0 27.0 [+ or 29.1 [+ or 29.7 [+ or -] 28.3 [+ or -] -] 0.98a -] 0.65a 0.75a 0.80a 0.5 30.5 [+ or 31.1 [+ or 32.0 [+ or -] 31.7 [+ or -] -] 1.12b -] 1.21b 1.22b 1.05b 1.0 34.0 [+ or 33.0 [+ or 32.7 [+ or -] 32.6 [+ or -] -] 1.0c -] 1.15c 1.21c 1.07c SOD (c) 0 49.6 [+ or 50.5 [+ or 50.0 [+ or -] 49.1 [+ or -] -] 1.23a -] 1.34a 1.67a 1.80a 0.5 55.1 [+ or 54.8 [+ or 54.9 [+ or -] 55.2 [+ or -] -] 2.31b -] 1.13b 1.23b 1.22b 1.0 58.9 [+ or 59.1 [+ or 61.4 [+ or -] 60.5 [+ or -] -] 2.14c -] 2.03c 2.15c 2.09c Values are mean [+ or -] SD(n = 6); data analyzed by one-way ANOVA (p <0.05) appropriate to completely randomized design with replicates. Means followed by different letters differ significantly according to DMRT. CAT: catalase; GPx: glutathione peroxidase; SOD: superoxide dismutase. (a) [mu] mole [H.sub.2][O.sub.2] degraded/min/mg protein. (b) [eta] moles NADPH/min/mg protein. (c) Units/mg protein.
Effect on cholinergic enzymes
Terminally, a significant dose dependent decrease in AChE activity was evident in all brain regions of mice fed BM (Fig. 5). Consistent reduction in enzyme activity was observed in cortex (16-31%), cerebellum (14-27%), hippocampus (15-31%) and striatum (17-32%). Interim analysis also showed diminished activity of AChE enzyme in all brain regions.
[FIGURE 5 OMITTED]
Ex vivo response of brain regions to 3-NPA exposure
Synaptosomes prepared from untreated control mice, 3-NPA exposure caused a concentration dependent increase in ROS levels in both cortex (12-54%) and cerebellum (11-36%). In contrast, synaptosomes obtained from BM treated mice on exposure to 3- NPA showed no induction of ROS at the two lower concentrations, while only a marginal induction ensued at the highest concentration (Fig. 6A). Likewise, 3-NPA exposure induced a concentration dependent increase in MDA levels in both cortex (29-95%) and cerebellar (27-79%) synaptosomes of untreated control mice. On the other hand, synaptosomes of BM treated mice showed no induction of lipid peroxidation at the two lower concentrations, while only a marginal induction ensued even at the highest concentration (Fig. 6B).
[FIGURE 6 OMITTED]
Usage of Bacopa in Ayurvedic system of medicine is on the increase for the treatment of anxiety, to improve intellect and memory among both children and adults (Kapur 1990; Singh and Dhawan 1997). Further it is also employed to treat various neuropharmacological (Russo and Borrelli 2005) disorders. Owing to the large scale applications, several forms of Bacopa formulations are available over the top of counter with specific claims of memory enhancement for all ages. Thus extensive use of BM entails a thorough understanding of its possible mechanisms of action in vivo.
The alcoholic extract of B. monnieri is reported to contain various types of saponin including bacopa saponin A, B, C, D, pseudojujubogenin, bacopaside I, II, II, IV and V. Besides the substance mentioned above, the extract also contained other ingredients such as brahmine, herpestine and monnierin (Deepak and Amit 2004; Kapoor et al. 2009). It is reported to possess anti-inflammatory, analgesic, antipyretic, sedative, free radical scavenging and anti-lipid per- oxidative activities (Anbarasi et al. 2005). However, experimental evidences demonstrating its efficacy to modulate oxidative stress in vivo in brain are limited. More importantly studies on its potency to attenuate endogenous markers of oxidative stress in brain regions are totally lacking. The compelling reasons for employing prepubertal mice for the study were (a) the brain in 4-week-old mice is still in the process of development of new interneuronal connections and will continue during the postnatal development till the adult architecture is established by about 6 weeks (Rao et al. 2005). (b) The prepubertal brain may be more responsive and (c) the enhancement of memory and increased antioxidant levels are necessary in the growing stage. Accordingly, we examined the propensity of BM to modulate the endogenous markers oxidative stress, response of antioxidant enzymes, and redox status in different brain regions of prepubertal mice. Further, its ability to confer neuroprotective advantage against a neurotoxicant (3-NPA) induced oxidative dysfunctions in synaptosomal fractions was also assessed using an ex vivo approach.
Accumulation of neurotoxic free radicals and consequent neurodegeneration in specific brain areas has been proposed as the causal factor in Alzheimer's disease (AD), Parkinson's disease (PD) and other neurodegenerative diseases as well as aging (Glover and Sandler 1993). This accumulation of oxidative free radicals is due to defective antioxidant defense mechanisms resulting from decreased function of the free radical scavenging enzymes. Hence a potential therapy which augments antioxidative defense system may prove useful under various neurodegenerative diseases (NDD). Logically an effective antioxidant agent should be capable augmenting intracellular concentrations of antioxidant enzymes and effectively reduce lipid peroxidation (Halliwell and Gutteridge 1989; Maxwell 1995; Halliwel 2006). Further, evidences suggest that mitochondria have a central role in development of several NDD (Petrozzi et al. 2007). In the present study, BM significantly reduced the levels of various endogenous markers of oxidative stress in different brain regions in both cytosol and mitochondria. This major finding clearly suggests the neuroprotective ability of BM among prepubertal mice. These data are consistent with our recent findings in which BM supplements significantly diminished the basal levels of oxidative markers in Drosophila system (Ravikumar and Muralidhara 2009).
Protein carbonyls, whose formation is considered a detectable marker of protein oxidation, are increased in AD and the resulting chemical modifications appear to be involved in cellular metabolism. Oxidative damage can lead to loss in specific protein function and oxidized proteins are more prone to degradation by proteases (Stadtman 1990). Hence a decrease in basal levels of protein oxidation proves to be a significant characteristic feature of BM which can be exploited further in therapeutics. Further the oxidative damage to proteins is reflected by a decrease in the levels of protein thiols (P-SH) and free radicals are known to cause oxidation of P-SH groups (Takenaka et al. 1991). It is reported that a decrease in GSH concentrations precede impairments of oxidative phosphorylation in PD (Dexter et al. 1994). Interestingly, depletion of mitochondrial GSH, but not cytosolic GSH in PC-12 cells resulted in the generation of ROS and inhibition of oxidative phosphorylation (Seyfried et al. 1999). In the present study, significantincrease in the levels of GSH, total thiols and non-protein thiols in both cytosol and mitochondria of different brain regions of mice fed BM clearly suggests its potential to enhance thiol antioxidants in brain.
BM has been used safely in Ayurvedic medicine for several hundred years and the therapealutic doses are not associated with any known side effects (Allan et al. 2007). While several safety studies have been conducted with BM extracts, to the best of our knowledge, there are no reports which describe the effect of BM on antioxidant defenses in brain regions of prepubertal rodents. In the present study, BM was found to enhance the activities of SOD, CAT and GPX in all the brain regions. This data corroborates with a report in which subchronic administration of BM to adult rats enhanced the oxidative free radical scavenging enzymes in brain regions of adult rats suggesting a possible antioxidant effect (Bhattacharya et al. 2000). The natural cellular antioxidant enzymes include SOD, which removes superoxide radicals by speeding their dismutation, CAT, a haeme enzyme which removes hydrogen peroxide, and GPX, a selenium-containing enzyme which removes hydrogen peroxide and other peroxides (Halliwell and Gutteridge 1989). The radical scavenging activity of SOD is effective only when it is followed by catalytic actions of CAT and GPX, since SOD generates hydrogen peroxide as a metabolite, which is more toxic than oxygen radicals and requires to be scavenged by CAT and/or GPX. Interestingly, dietary intake of BM also resulted in an increase in the activity of mitochondrial enzymes viz., malate dehydrogenase and thioredoxin reductase which is indicative of the redox modulation and increase of mitochondrial functions. Hence the enhanced activity of antioxidant enzymes, mitochondrial enzymes coupled with the decrease in oxidative markers due to BM consumption certainly provides a useful paradigm to mitigate oxidative stress-mediated disease conditions in vivo. This thinking is consistent with recently demonstrated efficacy of BM to protect brain against aluminiuminduced oxidative damage and neurotoxicity (Jyoti and Sharma 2006; Jyoti et al. 2007) and its ability to modulate the antioxidant defenses in brain and kidney of diabetic rats (Kapoor et al. 2009).
The central cholinergic system is considered the most important part involved in the regulation of cognitive functions. It is well-established that the cholinergic neurons are involved in several neuropsychic functions such as learning, memory, sleep and acetylcholine (ACh) may play a vast role in modulating these functions (Mohapel et al. 2005). The central cholinergic deficit is strongly associated with some neurodegenerative diseases such as AD and PD (Perry et al. 1985; Oda 1999). Choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) are specific cholinergic marker proteins for the functional state of cholinergic neurons, both of which can play a key role in the maintenance of ACh levels at the cholinergic neurons (Eckenstein and Sofroniew 1983). Treatment of AD patients with cholinesterase inhibitors causes symptomatic benefit and seems to delay disease progression for 6-12 months (Aarsiand et al. 2004). In a recent report the potency of BM alcoholic extract to enhance cognitive function was demonstrated in an animal model of AD (Uabundita et al. 2010). Subchronic administration of standardized bacosides rich extract of BM reversed the cognitive deficits induced by colchicine and ibotenic acid and also reversed colchicine-induced reduction in frontal cortex and hippocampal acetylcholine concentration, choline acetyltransferase activity and muscarinic cholinergic receptors binding (Bhattacharya et al. 1999). In the present study, we observed a significant reduction in AChE activity in all brain regions suggesting the specific effect of BM on cholinergic systems which may partly explain the cholinoproective action observed in AD patients as weli as its neuroprotective effects in animal models of AD.
Whole brain synaptosomes have been earlier employed as in vitro models to understand the modulatory effect of dietary compounds against 3-NPA induced oxidative dysfunctions and mitochondrial damage. Hence we tested whether BM prophylaxis to prepubertal mice provides any significant neuroprotective advantage against 3-NPA induced oxidative damage in cortex and cerebellar synaptosomes. 3-NPA induced lipid peroxidation induction response in synaptosomes of untreated mice was more robust and occurred at lower concentrations compared to synaptosomes obtained from mice given BM prophylaxis. These data unequivocally illustrate the neuroprotective advantage conferred by BM prophylaxis although the underlying mechanisms cannot be explained at this point. Further studies in cell models such as PC 12 and N 27 cell lines is likely to provide a better understanding of the specific mechanisms by which BM confers neuroprotective resistance. Interestingly, we have also obtained comprehensive data which suggest prophylaxis with an BM provides significant neuroprotection against various neurotoxicants such as rotenone and paraquat in Drosophila (Ravikumar and Muralidhara 2009, 2010) and 3-NPA and rotenone in prepubertal mice models (Shinomol and Muralidhara 2006; Shinomol 2008).
Collectively, our findings in prepubertal mice clearly indicate that dietary intake of BM leaf powder significantly diminished the basal levels of several oxidative markers, enhanced thiol related antioxidant molecules and activities of antioxidant enzymes suggesting its antioxidant potential in vivo. Further, our findings may at least in part, explain the mechanism of nootropic action of BM demonstrated earlier both under experimental and clinical situations. Although speculative, lowering of endogenous oxidative stress may be partly responsible for the memory enhancement property of BM reported among children and aged. Further our results from ex vivo study suggest that dietary BM supplements to young mice impart significant resistance against neurotox-icant induced oxidative damage in brain which suggests its prophylactic therapeutic potential for the treatment of oxidative stress-mediated neuronal dysfunctions.
The authors wish to thank the Director, CFTRI for his keen interest in this study. The first author thanks the Council of Scientific and Industrial research (CSIR), New Delhi for the award of a Junior and Senior Research Fellowship. We also thank M/s Natural remedies, Bengaluru, India for carrying out the HPLC-finger print analysis of our Bacopa monerri extract.
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Abbreviations: LPO, lipid peroxidation; ROS, reactive oxygen species; TBARS, thiobarbituric acid reactive substances; MDA, malondialdehyde; DCF, 2', 7'-dichlorofluorescein; DCF-DA, 2',7'-dichloro-fluorescein diacetate; BM, Bacopa monnieri.
* Corresponding author. Tel.: +91 821 2514876; fax; +91 821 2517233.
E-mail address: mura16@Yahoo.com (Muralidhara).
0944-7113/$ - see front matter[c] 2010 Elsevier GmbH. All rights reserved.
George K. Shinomol, Muralidhara *
Deportment of Biochemistry and Nutrition, Central Food Technological Research Institute (Council of Scientific and Industrial Research), Mysore 570020, Karnataka, India