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Neuroprotective role of Bacopa monniera extract against aluminium-induced oxidative stress in the hippocampus of rat brain
NeuroToxicology 27 (2006) 451–457
Neuroprotective role of Bacopa monniera extract against aluminium-induced oxidative stress in the hippocampus of rat brain Amar Jyoti, Deepak Sharma * Neurobiology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India Received 23 December 2004; accepted 21 December 2005 Available online 24 February 2006
Abstract Bacopa monniera is a nerve tonic used extensively in traditional Indian medicinal system ‘‘Ayurveda’’. Reports regarding its various antioxidative, adaptogenic and memory enhancing roles have already appeared in the last few decades. In the present study, aluminium chloride (AlCl3) was used to generate neurotoxicity. We have investigated the neuroprotective effect of Bacopa extract against aluminium-induced changes in peroxidative products, such as thio-barbituric acid-reactive substance (TBA-RS) and protein carbonyl contents and superoxide dismutase (SOD) activity. Effect on lipofuscin (age pigments) accumulation and ultrastructural changes were also studied. Bacopa effects were compared with those of L-deprenyl. Co-administration of Bacopa extract during aluminium treatment significantly prevented the aluminium-induced decrease in SOD activity as well as the increased oxidative damage to lipids and proteins. Protective effect was also observed at microscopic level. Fluorescence and electron microscopic studies revealed considerable inhibition of intraneuronal lipofuscin accumulation and necrotic alteration in the CA1 region of the hippocampus. Observations showed that Bacopa’s neuroprotective effects were comparable to those of L-deprenyl at both biochemical and microscopic levels. # 2006 Elsevier Inc. All rights reserved. Keywords: Aluminium chloride (AlCl3); Thio-barbituric acid-reactive substances (TBA-RS); Protein oxidation (PO); Lipid peroxidation (LP); Superoxide dismutase (SOD)
1. Introduction Bacopa monniera (called Brahmi in Sanskrit) is a creeping herb extensively investigated for its pharmacological and therapeutic effects. Its ethanolic extract contains a mixture of triterpenoid saponins designated as bacosides A and B (Chatterjee et al., 1963, 1965). Bacoside A comprises a mixture of three saponins, viz. bacogenin A1, A2 and A3, with A3 being the major constituent (Kulshreshtha and Rastogi, 1973). Several saponins designated as bacopsides I and II (Chakravarty et al., 2001), III, IVand V have also been isolated from B. monniera and their structures were studied by 2D NMR and spectral analysis (Chakravarty et al., 2003). As an ayurvedic medicine it is used as a nerve tonic. It is thought to improve intelligence, memory and functioning of sense organs, and it has also been used to treat epilepsy, insomnia and asthma (Das et al., 2002). The ethanolic extract of Bacopa has been found to increase the activity of
* Corresponding author. Tel.: +91 26704508. E-mail address: [email protected] (D. Sharma). 0161-813X/$ – see front matter # 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.neuro.2005.12.007
antioxidative enzymes (e.g. superoxide dismutase (SOD), glutathione peroxidase and catalase) in the frontal cortex, striatum and hippocampus of rats (Bhattacharya et al., 2000). In several behavioral studies, bacoside A has been reported to significantly improve acquisition, consolidation and retention of memory (Singh and Dhawan, 1982). It also attenuated retrograde amnesia induced by immobilization stress and scopolamine (Singh and Dhawan, 1997). Clinical studies suggest that Bacopa extract can significantly improve the speed of visual processing, learning rate and memory consolidation (Stough et al., 2001). Recently, Russo et al. (2003) in their studies on the effects of Bacopa extract on human non-immobilized fibroblasts have reported antioxidative, anticytotoxic and DNA protecting capability of Bacopa. It is evident from the literature that the effects of Bacopa extract on certain markers of oxidative stress, as peroxidative products (like TBA-RS), carbonyl compounds and intraneuronal pigment (lipofuscin) accumulation, need to be investigated. Aluminum metal is abundantly present in the earth’s crust. From the environment it gets access to the human body via the gastrointestinal and the respiratory tracts. Aluminium is a
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constituent of cooking utensils and medicines such as antacids, deodorants and food additives (Yokel, 2000) and this has allowed its easy access into the body. Aluminium has been implicated in aging-related changes (Xu et al., 1992; Deloncle et al., 2001) and neurodegenerative diseases (Flaten, 2001). Aluminium promotes the formation of amyloid-b protein plaques (Deloncle and Guillard, 1990; Kawahara et al., 1994) by aggregating tau proteins in Alzheimer’s disease (Savory et al., 1998). Aluminium also disrupts calcium homeostasis (Julka and Gill, 1996) by interacting with calcium binding sites, which play crucial role in neurodegeneration. The mechanism of aluminium-induced toxicity is not clearly known but it is reported that aluminium potentiates the activity of Fe2+ and Fe3+ ions to cause oxidative damage (Gutteridge et al., 1985; Oteiza, 1993; Good et al., 1992; Xie and Yokel, 1996). Aluminium salts cause oxidative stress-related changes such as increased lipid peroxidation, rised 4-HNE formations and augmented lipofuscin accumulation (Kaur et al., 2003b). Aluminium glutamate administration was shown to cause attenuation of myelin sheath, swollen mitochondria, extensive cellular vacuolation and age pigment accumulation in the hippocampus (Deloncle et al., 2001). Aluminium toxicity was found to be associated with reduced axonal length and dendritic branches in hippocampus (Sreekumaran et al., 2003). It is evident that aluminium neurotoxicity is associated with significant oxidative stress, and even the aluminium concentration that may build up in vivo in humans in normal course would appear to be a risk factor for neurodegenerative disorders and aging-related changes. In the present study, we aimed to investigate the neuroprotective effect of Bacopa against aluminium-induced oxidative stress changes in the rat hippocampus. Furthermore, we also measured the neuroprotective effects of L-deprenyl against aluminium neurotoxicity to see the similarity between the antioxidative effects of the two.
lectone and thus contained a conjugated triene system. The estimation was performed by using a UV-spectrophotometer at 278 nm (Pal and Sarin, 1992). 2.3. Treatment Forty male wistar rats of 6 months of age, weighing 350– 400 g were used for this study. Animals, obtained from the central animal facility of the Jawaharlal Nehru University, New Delhi, were housed in polypropylene cages under hygienic conditions. They were fed standard pellet diet (Hindustan Lever Limited, India). Rats were kept on a 12-h light:12-h dark cycle and checked for health status frequently. Animals were divided into four groups of eight animals each. (a) Group I—control: water only, (b) Group II—AlCl3 treated control: AlCl3 at an oral dose of 50 mg/kg/day in drinking water for 5 weeks, (c) Group III—AlCl3 + Bacopa extract treated group: AlCl3 at an oral dose of 50 mg/kg/day and Bacopa extract in 10% gum acacia at a dose of 40 mg/kg/day for 5 weeks and (d) Group IV—AlCl3 + L-deprenyl treated group: L-deprenyl administered simultaneously with AlCl3. L-Deprenyl, dissolved in saline solution, was administered by intraperitoneal injection at a dose of 1 mg/kg/day (Kaur et al., 2003a). L-Deprenyl given by intraperitoneal injections has been found to be more effective than oral administration (Tatton and Chalmers-Redman, 1996). 2.4. Preparation of tissue homogenates
L-Deprenyl, di-nitrophenylhydrazine (DNPH), guanidine HCl and BSA were purchased from the Sigma Chemical Co., USA. Other chemicals were obtained from Merck, Qualigen and SD Fine Chemical Company. The ethanolic extract of Bacopa was obtained as a kind gift from Dr. H.K. Singh of the Central Drug Research Institute (CDRI), Lucknow, India.
Six rats of each group were killed by cervical dislocation after the treatment period. Brains were quickly taken out and cooled in a deep freezer. Hippocampi were rapidly dissected out on ice plate, according to the stereotaxic atlas of Paxinos and Watson (1982). The left and right hippocampi of the brain of one rat were pooled to make one sample of the tissue. Biochemical assays were performed separately in six animals of each group. Tissue samples were homogenized in 50 mM Tris (pH 7.4) with a PotterElevehijam type homogenizer fitted with Teflon plunger. The homogenate was diluted 1:10 (with Tris, pH 7.4, buffer) and centrifuged at 6000 rpm for 5 min in a refrigerated centrifuge (Sorvall RCS or RC5C). The resulting pellet (P1), consisting of nuclear and cellular material, was discarded. The supernatant (S1), containing mitochondria, synaptosomes, microsomes and cytosol, was further ultracentrifuged at 25,000 rpm for 25 min to form mitochondrial pellet (P2). The resulting supernatant (S2) was used as such as cytosolic fraction.
2.2. Bacopa extract preparation
2.5. Biochemical assays
The whole plant (B. monniera) was dried in shade and then powdered. The powder was extracted with distilled water. The aqueous extract was discarded and the residual plant material was extracted thrice with 90% ethanol. The residue obtained after removing the solvent was dried in vacuo and macerated with acetone to give a free flowing powder. The Bacopa extract so prepared contained 55–60% bacosides estimated as bacoside A. The estimation method involved acid hydrolysis of bacoside, which quantitatively yielded a transformed aglycine ebelin
Aliquots of cytosolic fraction were used for biochemical studies. TBA-RS levels were measured (for estimating lipid peroxidation) spectrophotometrically at 532 nm according to the method of Ohkawa et al. (1979). Lipid peroxidation levels were expressed as micromoles of TBA-RS formed per milligram protein. Protein oxidation was measured by estimating the protein carbonyl levels by the method of Reznick and Packer (1994) and Liu et al. (2003). Protein carbonyl content was determined
2. Materials and methods 2.1. Materials
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in the samples by measuring the DNPH adducts at 375 nm by using a Shimadzu UV-160A spectrophotometer. Carbonyl contents were calculated by using a molar extinction coefficient (e) of 22,000 M 1 cm 1. Data were expressed as nanomoles carbonyl per milligram of soluble extracted protein. Superoxide dismutase activity was measured by the method of Marklund and Marklund (1974) with slight modifications. The SOD activity was assayed by following the auto-oxidation of pyrogallol at 420 nm using a Shimadzu UV-260A spectrophotometer. The activity was expressed as units/milligram protein, where a unit is equivalent to the amount of SOD required to inhibit the 50% of pyrogallol auto-oxidation. Protein estimation was performed by Bradford method (1976). 2.6. Microscopic studies Animals were anesthetized and transcardially perfused first with physiological saline and then with a fixative solution containing 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate buffer. 2.6.1. Fluorescent microscopy After perfusion, the brain was excised and put in 10% formalin solution. Paraffin sections were prepared for microscopic study. Deparaffinilised sections were mounted in DPX and observed under Zeiss orthomat microscope equipped with fluorescence attachments Ploemopak Epi illuminator, H2 tube (wide band) excitation filter 390–490 nm for studying the localization of intraneuronal lipofuscin pigments. 2.6.2. Transmission electron microscopy (TEM) For TEM studies, thin sections (1 cm2) of the hippocampal CA1 subfield were fixed in Kanovsky’s fixative (2% paraformaldehyde + 2.5% glutaraldehyde in PBS) at 4 8C for 18 h. Tissues were post-fixed in 1% osmium tetraoxide (OsO4) at 4 8C for 2 h and then dehydrated in acetone. After clearing the tissue with xylene, infiltration was carried out using resins and the component araldite + hardener (10 ml) + accelerator (0.4 ml). Ultrathin sections were double stained using uranyl acetate and lead citrate. These sections were observed under Fei-Philips Morgagni 268D (100 kV) TEM. 2.7. Statistical analysis Data were expressed as mean S.D. Statistical comparison were performed by one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test. The level of significance was accepted at p < 0.05. 3. Results Data obtained in the present experiments would show that lipid peroxidation and protein oxidation significantly increased in the hippocampus of aluminium treated rats (Fig. 1A and B, respectively) in comparison with controls, whereas the superoxide dismutase activity, significantly decreased (Fig. 1C).
Fig. 1. (A–C) Effect of Bacopa extract and L-deprenyl treatment along with AlCl3 in inhibition of biochemical alterations that results from AlCl3 treatment alone. Different biochemical parameters were assayed as described in Section 2. Results were expressed as percentage of untreated control value (mean value; n = 6 rats). (*) Level of significance ( p < 0.05) in comparison to Group I (untreated control rats). (*) Level of significance ( p < 0.05) in comparison to Group II (AlCl3 treated rats).
3.1. Effect of Bacopa treatment In animals co-administered with Bacopa extract during AlCl3 treatment, the lipid peroxides and the protein oxidation
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Fig. 2. (A–D) Photographs representing the yellow fluorescent lipofuscin granules in the hippocampal cells of rats under different treatment conditions. (For interpretation of the references to colour in this figure caption, the reader is referred to the web version of the article.).
levels significantly decreased whereas SOD activity elevated in comparison to aluminium treated rats.
was significantly inhibited in groups co-treated with Bacopa and L-deprenyl (Fig. 2C and D).
3.2. Effect of L-deprenyl treatment
3.4. Morphological changes in the hippocampus cells
In animals co-administered with L-deprenyl during AlCl3 treatment, the lipid peroxidation and the protein oxidation levels significantly decreased whereas the SOD activity increased in comparison with aluminium treated controls. The present results showed that aluminium treatment results in the elevation of lipid peroxides and protein oxidation, and depression of SOD activity in the hippocampus. These aluminium-induced neurotoxic effects are prevented by Bacopa extract as well as by L-deprenyl. A one-way ANOVA for lipid peroxidation, protein oxidation and superoxide dismutase activity among the aluminium treated group animals co-administered with L-deprenyl showed significant differences in all the measures among the treated groups. (F(3, 20) = 379.5 and p < 0.0001), (F(3, 20) = 25.15 and p < 0.0001) and (F(3, 20) = 358.2 and p < 0.0001), respectively.
AlCl3 administration was found to cause several necrosis like changes in hippocampal CA1 cells such as coarse and clumpy chromatin, multi-vesicular bodies, age pigment accumulation within neurons (Fig. 3D–F). Accumulation of synaptic vesicles in the swollen presynaptic terminals was also observed. In sections from animals co-administered with Bacopa extract or L-deprenyl these changes were found to be absent (Fig. 3G–L).
3.3. Effects of treatment on lipofuscin accumulation Observation of lipofuscin localization by fluorescence microscope showed highly increased accumulation of the pigment in the AlCl3 treated rats when compared to the control animals (Fig. 2A and B). Moreover, accumulation of lipofuscin
4. Discussion The present study was carried out mainly to investigate the protective effects of B. monniera on aluminium-induced oxidative stress in the hippocampus. Hippocampus was chosen for the present study for a number of reasons. First, aluminium affects more severely the hippocampus and neocortex regions than any other area of the central nervous system (Deloncle and Guillard, 1990; Urano et al., 1997). Second, this brain region is known to be particularly susceptible in Alzheimer’s disease, and has an important role in learning and memory functions. The hippocampal CA1 field was selected for microscopic studies as its pyramidal neurons are potentially more vulnerable to aluminium-induced neurotoxicity (Sreekumaran et al., 2003)
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Fig. 3. Electron micrographs showing changes in different cellular structure of hippocampal cells of CA1 region in different groups. (A–C) Control group, (D–F) AlCl3 treated group, (G–I) AlCl3 + Bacopa treated group and (J–L) AlCl3 + L-deprenyl treated group. Lipofuscin pigments (L), synaptic vesicles (Sv), condensed chromatin (C), mitochondria (M) and synapse (S).
and hypoxia (Kawasaki et al., 1990) as compared to those of CA3 and CA2 areas. In the aluminium treated hippocampus, the lipid peroxidation and protein oxidation levels were found to be elevated but the superoxide dismutase activity was reduced. Besides these, increased intraneuronal lipofuscin accumulation and necrotic ultrastructural changes were also observed. Aluminium-induced ultrastructural alterations showed coarse and clumpy chromatin, vacuolation (multivesicular bodies), pigment accumulation, etc. These observa-
tions are similar to the data reported by Deloncle et al. (2001). These data are indicative of aluminium-induced oxidative stress, and are consistent with many earlier studies that have indicated that aluminium intake produces oxidative stressrelated changes that contributes to its neurotoxicity. In addition, it was interesting to note that hippocampus exhibits abnormal accumulation of synaptic vesicles in the swollen nerve terminals. Similar synaptic alterations have also been reported earlier in hypoxia, and suggested to be associated with
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abnormal acetylcholine release (Urano et al., 1997). This however, will be consistent with the aluminium-induced dementia reported in behavioral studies (Miu et al., 2003). Our data clearly showed that co-administration of ethanolic extract of Bacopa during treatment with aluminium prevented the latter’s oxidative stress effects. Aluminium reduced the SOD activity significantly, Bacopa prevented this reduction and restored the SOD activity to near normal. Similarly Bacopa prevented the aluminium-induced increase in TBARS and carbonyls. The protective role of Bacopa extract was also supported by our microscopic observations which showed that Bacopa prevented the aluminium-induced lipofuscin accumulation and ultrastructural changes. These effects of Bacopa against aluminium-induced changes are indicative of the antioxidative potential of Bacopa as well as its ability to counter aluminium neurotoxicity. In order to further confirm the antioxidative potential of Bacopa, we also measured the effects of L-deprenyl on aluminium neurotoxicity to see the similarity of its effects with those of Bacopa. L-Deprenyl (selegiline) is a MAO-B inhibitor and neuroprotectant, as it protects against the effects of neurotoxins and excitatory amino acids (Ebadi et al., 2002). It is a therapeutic agent for Parkinson’s disease (neurodegenerative disorder) involving oxidative stress. L-Deprenyl prevents the apoptosis of dopaminergic neurons associated with Parkinson’s disease by altering the expression of a number of genes such as SOD, Bcl-2, Bcl-XL, NOS, cJUN (Ebadi and Sharma, 2003). L-Deprenyl is also a very well known antioxidant (Wu et al., 1993) and is thought to have antiaging effects. It is known to extend life expectancy (Bickford et al., 1997). Our results clearly showed that L-deprenyl when co-administered with aluminium in rats inhibited TBA-RS formation, protein carbonyl formation and lipofuscin accumulation and elevated the SOD activity. It was also found to counter the aluminium-induced ultrastructural changes. Thus, Bacopa’s antioxidative effects appear to be similar to those of L-deprenyl. Oxidative stress (lipid peroxidation, lipofuscin accumulation, etc.) is thought to contribute to the aging process (Kaur et al., 2003a). Bacopa for its antilipidperoxidative and antilipofuscinogenistic effects could thus be considered as a potential antiaging substance. Furthermore, the similarity of Bacopa’s effects with those of L-deprenyl, which is a candidate aging drug, would also be indicative of its antiaging potential. The alcoholic extract of Bacopa contains several saponins (Chatterjee et al., 1963, 1965; Chakravarty et al., 2001, 2003), flavonoids and alkaloids (Schulte et al., 1972). The triterpenoid saponins (mainly bacosides A and B) have been reported to enhance memory and learning (Singh and Dhawan, 1997) and antioxidative activities (Bhattacharya et al., 2000). The ethanolic extract used in the present study contained 55– 60% bacosides (Pal and Sarin, 1992). The neuroprotective effects of the Bacopa extract observed in this study may have been due to the bacosides. In summary, the present data show Bacopa inhibits lipid peroxidation, protein oxidation and lipofuscin accumulation. It also reverses aluminium-induced oxidative stress and ultrastructural alterations. Antiaging potential of Bacopa also merit consideration.
Acknowledgements One author (A.J.) is thankful to the Council of Scientific and Industrial Research (India) for providing a Junior Research Fellowship (JRF). We express our sincere thanks to Dr. H.K. Singh of the Central Drug Research Institute, Lucknow, for providing us the ethanolic Bacopa extract and its details. References Bhattacharya SK, Bhattacharya A, Kumar A, Ghosal S. Antioxidant activity of Bacopa monniera in rat frontal cortex, striatum and Hippocampus. Phyto Res 2000;14:174–9. Bickford PC, Adams CE, Boyson SJ, Curella P, Gerhardt GA, Heron C, et al. Long term treatment of male F344 rats with deprenyl: assessment of effects on longevity, behavior, and brain function. Neurobiol Aging 1997;18: 309–18. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of dye binding. Anal Biochem 1976;72:248–54. Chakravarty AK, Sarkar T, Masuda K, Shiojima K, Nakane T, Kawahara N. Bacopasides I and II: two psedojujubogenin glycosides from Bacopa monniera. Phytochemistry 2001;58(4):553–6. Chakravarty AK, Garai S, Masuda K, Nakane T, Kawahara N. Bacopasides III– V: three new triterpenoid glycosides from Bacopa monniera. Chem Pharmacol Bull 2003;51:215–7. Chatterjee N, Rastogi RP, Dhar ML. Chemical examination of Bacopa monniera Wettst: Part I. Isolation of chemical constituents. Indian J Chem 1963;1:212–5. Chatterjee N, Rastogi RP, Dhar ML. Chemical examination of Bacopa monniera Wettst: Part II. The constitution of bacoside A. Indian J Chem 1965;3:24–9. Das A, Shanker G, Nath C, Pal R, Singh S, Singh HK. A comparative study in rodents of standardized extracts of Bacopa monniera and Ginkgo biloba anticholinesterase and cognitive enhancing abilities. Pharmacol Biochem Behav 2002;73:893–900. Deloncle R, Guillard O. Mechanism of Alzheimer’s disease: arguments for a neurotransmitter aluminium complex implication. Neurochem Res 1990;15:1239–45. Deloncle R, Huguet E, Fernandez B, Quellard N, Babin PH, Guillard O. Ultrastructural study of rat hippocampus after chronic administration of aluminium L-glutamate: an acceleration of aging process. Exp Gerontol 2001;36:234–44. Ebadi M, Sharma S, Shavali S, El-Refaey H. Neuroprotective action of selegiline. J Neurosci Res 2002;285–9. Ebadi M, Sharma SK. Peroxinitite and mitochondrial dysfunction in the pathogenesis of Parkinson’s disease. Antioxid Redox Signal 2003;5: 319–35. Flaten TP. Aluminium as a risk factor for Alzheimer’s disease with emphasis on drinking water. Brain Res Bull 2001;55:187–96. Good PF, Olanow CW, Perl DP. Neuromelanin-containing neurons of the substantia nigra accumulate iron and aluminum in Parkinson’s disease: a LAMMA study. Brain Res 1992;593:343–6. Gutteridge JM, Quinlan GJ, Clark L, Halliwelli B. Aluminum salts accelerate peroxidation of membrane lipids stimulated by iron salts. Biochem Biophys Acta 1985;835:441–7. Julka D, Gill KD. Altered calcium homeostasis: a possible mechanism of aluminum-induced neurotoxicity. Biochem Biophys Acta 1996;135:47–54. Kawahara M, Muamoto K, Kobayashi K, Mori H, Kuroda Y. Aluminium promotes the aggregation of Alzheimer’s amyloid-b protein in vitro. Biochem Biophys Res Commun 1994;198:531–5. Kawasaki K, Traynelis SF, Dingledine R. Different responses of CA1 and CA3 regions to hypoxia in rat hippocampal slice. J Neurophysiol 1990;63:385– 94. Kaur J, Singh S, Sharma D, Singh R. Neurostimulatory and antioxidative effects of L-deprenyl in aged rat brain regions. Biogerontology 2003a;4:105–11.
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Neuroprotective role of Bacopa monniera extract against aluminium-induced oxidative stress in the hippocampus of rat brain Amar Jyoti, Deepak Sharma * Neurobiology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India Received 23 December 2004; accepted 21 December 2005 Available online 24 February 2006
Abstract Bacopa monniera is a nerve tonic used extensively in traditional Indian medicinal system ‘‘Ayurveda’’. Reports regarding its various antioxidative, adaptogenic and memory enhancing roles have already appeared in the last few decades. In the present study, aluminium chloride (AlCl3) was used to generate neurotoxicity. We have investigated the neuroprotective effect of Bacopa extract against aluminium-induced changes in peroxidative products, such as thio-barbituric acid-reactive substance (TBA-RS) and protein carbonyl contents and superoxide dismutase (SOD) activity. Effect on lipofuscin (age pigments) accumulation and ultrastructural changes were also studied. Bacopa effects were compared with those of L-deprenyl. Co-administration of Bacopa extract during aluminium treatment significantly prevented the aluminium-induced decrease in SOD activity as well as the increased oxidative damage to lipids and proteins. Protective effect was also observed at microscopic level. Fluorescence and electron microscopic studies revealed considerable inhibition of intraneuronal lipofuscin accumulation and necrotic alteration in the CA1 region of the hippocampus. Observations showed that Bacopa’s neuroprotective effects were comparable to those of L-deprenyl at both biochemical and microscopic levels. # 2006 Elsevier Inc. All rights reserved. Keywords: Aluminium chloride (AlCl3); Thio-barbituric acid-reactive substances (TBA-RS); Protein oxidation (PO); Lipid peroxidation (LP); Superoxide dismutase (SOD)
1. Introduction Bacopa monniera (called Brahmi in Sanskrit) is a creeping herb extensively investigated for its pharmacological and therapeutic effects. Its ethanolic extract contains a mixture of triterpenoid saponins designated as bacosides A and B (Chatterjee et al., 1963, 1965). Bacoside A comprises a mixture of three saponins, viz. bacogenin A1, A2 and A3, with A3 being the major constituent (Kulshreshtha and Rastogi, 1973). Several saponins designated as bacopsides I and II (Chakravarty et al., 2001), III, IVand V have also been isolated from B. monniera and their structures were studied by 2D NMR and spectral analysis (Chakravarty et al., 2003). As an ayurvedic medicine it is used as a nerve tonic. It is thought to improve intelligence, memory and functioning of sense organs, and it has also been used to treat epilepsy, insomnia and asthma (Das et al., 2002). The ethanolic extract of Bacopa has been found to increase the activity of
* Corresponding author. Tel.: +91 26704508. E-mail address: [email protected] (D. Sharma). 0161-813X/$ – see front matter # 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.neuro.2005.12.007
antioxidative enzymes (e.g. superoxide dismutase (SOD), glutathione peroxidase and catalase) in the frontal cortex, striatum and hippocampus of rats (Bhattacharya et al., 2000). In several behavioral studies, bacoside A has been reported to significantly improve acquisition, consolidation and retention of memory (Singh and Dhawan, 1982). It also attenuated retrograde amnesia induced by immobilization stress and scopolamine (Singh and Dhawan, 1997). Clinical studies suggest that Bacopa extract can significantly improve the speed of visual processing, learning rate and memory consolidation (Stough et al., 2001). Recently, Russo et al. (2003) in their studies on the effects of Bacopa extract on human non-immobilized fibroblasts have reported antioxidative, anticytotoxic and DNA protecting capability of Bacopa. It is evident from the literature that the effects of Bacopa extract on certain markers of oxidative stress, as peroxidative products (like TBA-RS), carbonyl compounds and intraneuronal pigment (lipofuscin) accumulation, need to be investigated. Aluminum metal is abundantly present in the earth’s crust. From the environment it gets access to the human body via the gastrointestinal and the respiratory tracts. Aluminium is a
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constituent of cooking utensils and medicines such as antacids, deodorants and food additives (Yokel, 2000) and this has allowed its easy access into the body. Aluminium has been implicated in aging-related changes (Xu et al., 1992; Deloncle et al., 2001) and neurodegenerative diseases (Flaten, 2001). Aluminium promotes the formation of amyloid-b protein plaques (Deloncle and Guillard, 1990; Kawahara et al., 1994) by aggregating tau proteins in Alzheimer’s disease (Savory et al., 1998). Aluminium also disrupts calcium homeostasis (Julka and Gill, 1996) by interacting with calcium binding sites, which play crucial role in neurodegeneration. The mechanism of aluminium-induced toxicity is not clearly known but it is reported that aluminium potentiates the activity of Fe2+ and Fe3+ ions to cause oxidative damage (Gutteridge et al., 1985; Oteiza, 1993; Good et al., 1992; Xie and Yokel, 1996). Aluminium salts cause oxidative stress-related changes such as increased lipid peroxidation, rised 4-HNE formations and augmented lipofuscin accumulation (Kaur et al., 2003b). Aluminium glutamate administration was shown to cause attenuation of myelin sheath, swollen mitochondria, extensive cellular vacuolation and age pigment accumulation in the hippocampus (Deloncle et al., 2001). Aluminium toxicity was found to be associated with reduced axonal length and dendritic branches in hippocampus (Sreekumaran et al., 2003). It is evident that aluminium neurotoxicity is associated with significant oxidative stress, and even the aluminium concentration that may build up in vivo in humans in normal course would appear to be a risk factor for neurodegenerative disorders and aging-related changes. In the present study, we aimed to investigate the neuroprotective effect of Bacopa against aluminium-induced oxidative stress changes in the rat hippocampus. Furthermore, we also measured the neuroprotective effects of L-deprenyl against aluminium neurotoxicity to see the similarity between the antioxidative effects of the two.
lectone and thus contained a conjugated triene system. The estimation was performed by using a UV-spectrophotometer at 278 nm (Pal and Sarin, 1992). 2.3. Treatment Forty male wistar rats of 6 months of age, weighing 350– 400 g were used for this study. Animals, obtained from the central animal facility of the Jawaharlal Nehru University, New Delhi, were housed in polypropylene cages under hygienic conditions. They were fed standard pellet diet (Hindustan Lever Limited, India). Rats were kept on a 12-h light:12-h dark cycle and checked for health status frequently. Animals were divided into four groups of eight animals each. (a) Group I—control: water only, (b) Group II—AlCl3 treated control: AlCl3 at an oral dose of 50 mg/kg/day in drinking water for 5 weeks, (c) Group III—AlCl3 + Bacopa extract treated group: AlCl3 at an oral dose of 50 mg/kg/day and Bacopa extract in 10% gum acacia at a dose of 40 mg/kg/day for 5 weeks and (d) Group IV—AlCl3 + L-deprenyl treated group: L-deprenyl administered simultaneously with AlCl3. L-Deprenyl, dissolved in saline solution, was administered by intraperitoneal injection at a dose of 1 mg/kg/day (Kaur et al., 2003a). L-Deprenyl given by intraperitoneal injections has been found to be more effective than oral administration (Tatton and Chalmers-Redman, 1996). 2.4. Preparation of tissue homogenates
L-Deprenyl, di-nitrophenylhydrazine (DNPH), guanidine HCl and BSA were purchased from the Sigma Chemical Co., USA. Other chemicals were obtained from Merck, Qualigen and SD Fine Chemical Company. The ethanolic extract of Bacopa was obtained as a kind gift from Dr. H.K. Singh of the Central Drug Research Institute (CDRI), Lucknow, India.
Six rats of each group were killed by cervical dislocation after the treatment period. Brains were quickly taken out and cooled in a deep freezer. Hippocampi were rapidly dissected out on ice plate, according to the stereotaxic atlas of Paxinos and Watson (1982). The left and right hippocampi of the brain of one rat were pooled to make one sample of the tissue. Biochemical assays were performed separately in six animals of each group. Tissue samples were homogenized in 50 mM Tris (pH 7.4) with a PotterElevehijam type homogenizer fitted with Teflon plunger. The homogenate was diluted 1:10 (with Tris, pH 7.4, buffer) and centrifuged at 6000 rpm for 5 min in a refrigerated centrifuge (Sorvall RCS or RC5C). The resulting pellet (P1), consisting of nuclear and cellular material, was discarded. The supernatant (S1), containing mitochondria, synaptosomes, microsomes and cytosol, was further ultracentrifuged at 25,000 rpm for 25 min to form mitochondrial pellet (P2). The resulting supernatant (S2) was used as such as cytosolic fraction.
2.2. Bacopa extract preparation
2.5. Biochemical assays
The whole plant (B. monniera) was dried in shade and then powdered. The powder was extracted with distilled water. The aqueous extract was discarded and the residual plant material was extracted thrice with 90% ethanol. The residue obtained after removing the solvent was dried in vacuo and macerated with acetone to give a free flowing powder. The Bacopa extract so prepared contained 55–60% bacosides estimated as bacoside A. The estimation method involved acid hydrolysis of bacoside, which quantitatively yielded a transformed aglycine ebelin
Aliquots of cytosolic fraction were used for biochemical studies. TBA-RS levels were measured (for estimating lipid peroxidation) spectrophotometrically at 532 nm according to the method of Ohkawa et al. (1979). Lipid peroxidation levels were expressed as micromoles of TBA-RS formed per milligram protein. Protein oxidation was measured by estimating the protein carbonyl levels by the method of Reznick and Packer (1994) and Liu et al. (2003). Protein carbonyl content was determined
2. Materials and methods 2.1. Materials
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in the samples by measuring the DNPH adducts at 375 nm by using a Shimadzu UV-160A spectrophotometer. Carbonyl contents were calculated by using a molar extinction coefficient (e) of 22,000 M 1 cm 1. Data were expressed as nanomoles carbonyl per milligram of soluble extracted protein. Superoxide dismutase activity was measured by the method of Marklund and Marklund (1974) with slight modifications. The SOD activity was assayed by following the auto-oxidation of pyrogallol at 420 nm using a Shimadzu UV-260A spectrophotometer. The activity was expressed as units/milligram protein, where a unit is equivalent to the amount of SOD required to inhibit the 50% of pyrogallol auto-oxidation. Protein estimation was performed by Bradford method (1976). 2.6. Microscopic studies Animals were anesthetized and transcardially perfused first with physiological saline and then with a fixative solution containing 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate buffer. 2.6.1. Fluorescent microscopy After perfusion, the brain was excised and put in 10% formalin solution. Paraffin sections were prepared for microscopic study. Deparaffinilised sections were mounted in DPX and observed under Zeiss orthomat microscope equipped with fluorescence attachments Ploemopak Epi illuminator, H2 tube (wide band) excitation filter 390–490 nm for studying the localization of intraneuronal lipofuscin pigments. 2.6.2. Transmission electron microscopy (TEM) For TEM studies, thin sections (1 cm2) of the hippocampal CA1 subfield were fixed in Kanovsky’s fixative (2% paraformaldehyde + 2.5% glutaraldehyde in PBS) at 4 8C for 18 h. Tissues were post-fixed in 1% osmium tetraoxide (OsO4) at 4 8C for 2 h and then dehydrated in acetone. After clearing the tissue with xylene, infiltration was carried out using resins and the component araldite + hardener (10 ml) + accelerator (0.4 ml). Ultrathin sections were double stained using uranyl acetate and lead citrate. These sections were observed under Fei-Philips Morgagni 268D (100 kV) TEM. 2.7. Statistical analysis Data were expressed as mean S.D. Statistical comparison were performed by one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test. The level of significance was accepted at p < 0.05. 3. Results Data obtained in the present experiments would show that lipid peroxidation and protein oxidation significantly increased in the hippocampus of aluminium treated rats (Fig. 1A and B, respectively) in comparison with controls, whereas the superoxide dismutase activity, significantly decreased (Fig. 1C).
Fig. 1. (A–C) Effect of Bacopa extract and L-deprenyl treatment along with AlCl3 in inhibition of biochemical alterations that results from AlCl3 treatment alone. Different biochemical parameters were assayed as described in Section 2. Results were expressed as percentage of untreated control value (mean value; n = 6 rats). (*) Level of significance ( p < 0.05) in comparison to Group I (untreated control rats). (*) Level of significance ( p < 0.05) in comparison to Group II (AlCl3 treated rats).
3.1. Effect of Bacopa treatment In animals co-administered with Bacopa extract during AlCl3 treatment, the lipid peroxides and the protein oxidation
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Fig. 2. (A–D) Photographs representing the yellow fluorescent lipofuscin granules in the hippocampal cells of rats under different treatment conditions. (For interpretation of the references to colour in this figure caption, the reader is referred to the web version of the article.).
levels significantly decreased whereas SOD activity elevated in comparison to aluminium treated rats.
was significantly inhibited in groups co-treated with Bacopa and L-deprenyl (Fig. 2C and D).
3.2. Effect of L-deprenyl treatment
3.4. Morphological changes in the hippocampus cells
In animals co-administered with L-deprenyl during AlCl3 treatment, the lipid peroxidation and the protein oxidation levels significantly decreased whereas the SOD activity increased in comparison with aluminium treated controls. The present results showed that aluminium treatment results in the elevation of lipid peroxides and protein oxidation, and depression of SOD activity in the hippocampus. These aluminium-induced neurotoxic effects are prevented by Bacopa extract as well as by L-deprenyl. A one-way ANOVA for lipid peroxidation, protein oxidation and superoxide dismutase activity among the aluminium treated group animals co-administered with L-deprenyl showed significant differences in all the measures among the treated groups. (F(3, 20) = 379.5 and p < 0.0001), (F(3, 20) = 25.15 and p < 0.0001) and (F(3, 20) = 358.2 and p < 0.0001), respectively.
AlCl3 administration was found to cause several necrosis like changes in hippocampal CA1 cells such as coarse and clumpy chromatin, multi-vesicular bodies, age pigment accumulation within neurons (Fig. 3D–F). Accumulation of synaptic vesicles in the swollen presynaptic terminals was also observed. In sections from animals co-administered with Bacopa extract or L-deprenyl these changes were found to be absent (Fig. 3G–L).
3.3. Effects of treatment on lipofuscin accumulation Observation of lipofuscin localization by fluorescence microscope showed highly increased accumulation of the pigment in the AlCl3 treated rats when compared to the control animals (Fig. 2A and B). Moreover, accumulation of lipofuscin
4. Discussion The present study was carried out mainly to investigate the protective effects of B. monniera on aluminium-induced oxidative stress in the hippocampus. Hippocampus was chosen for the present study for a number of reasons. First, aluminium affects more severely the hippocampus and neocortex regions than any other area of the central nervous system (Deloncle and Guillard, 1990; Urano et al., 1997). Second, this brain region is known to be particularly susceptible in Alzheimer’s disease, and has an important role in learning and memory functions. The hippocampal CA1 field was selected for microscopic studies as its pyramidal neurons are potentially more vulnerable to aluminium-induced neurotoxicity (Sreekumaran et al., 2003)
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Fig. 3. Electron micrographs showing changes in different cellular structure of hippocampal cells of CA1 region in different groups. (A–C) Control group, (D–F) AlCl3 treated group, (G–I) AlCl3 + Bacopa treated group and (J–L) AlCl3 + L-deprenyl treated group. Lipofuscin pigments (L), synaptic vesicles (Sv), condensed chromatin (C), mitochondria (M) and synapse (S).
and hypoxia (Kawasaki et al., 1990) as compared to those of CA3 and CA2 areas. In the aluminium treated hippocampus, the lipid peroxidation and protein oxidation levels were found to be elevated but the superoxide dismutase activity was reduced. Besides these, increased intraneuronal lipofuscin accumulation and necrotic ultrastructural changes were also observed. Aluminium-induced ultrastructural alterations showed coarse and clumpy chromatin, vacuolation (multivesicular bodies), pigment accumulation, etc. These observa-
tions are similar to the data reported by Deloncle et al. (2001). These data are indicative of aluminium-induced oxidative stress, and are consistent with many earlier studies that have indicated that aluminium intake produces oxidative stressrelated changes that contributes to its neurotoxicity. In addition, it was interesting to note that hippocampus exhibits abnormal accumulation of synaptic vesicles in the swollen nerve terminals. Similar synaptic alterations have also been reported earlier in hypoxia, and suggested to be associated with
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abnormal acetylcholine release (Urano et al., 1997). This however, will be consistent with the aluminium-induced dementia reported in behavioral studies (Miu et al., 2003). Our data clearly showed that co-administration of ethanolic extract of Bacopa during treatment with aluminium prevented the latter’s oxidative stress effects. Aluminium reduced the SOD activity significantly, Bacopa prevented this reduction and restored the SOD activity to near normal. Similarly Bacopa prevented the aluminium-induced increase in TBARS and carbonyls. The protective role of Bacopa extract was also supported by our microscopic observations which showed that Bacopa prevented the aluminium-induced lipofuscin accumulation and ultrastructural changes. These effects of Bacopa against aluminium-induced changes are indicative of the antioxidative potential of Bacopa as well as its ability to counter aluminium neurotoxicity. In order to further confirm the antioxidative potential of Bacopa, we also measured the effects of L-deprenyl on aluminium neurotoxicity to see the similarity of its effects with those of Bacopa. L-Deprenyl (selegiline) is a MAO-B inhibitor and neuroprotectant, as it protects against the effects of neurotoxins and excitatory amino acids (Ebadi et al., 2002). It is a therapeutic agent for Parkinson’s disease (neurodegenerative disorder) involving oxidative stress. L-Deprenyl prevents the apoptosis of dopaminergic neurons associated with Parkinson’s disease by altering the expression of a number of genes such as SOD, Bcl-2, Bcl-XL, NOS, cJUN (Ebadi and Sharma, 2003). L-Deprenyl is also a very well known antioxidant (Wu et al., 1993) and is thought to have antiaging effects. It is known to extend life expectancy (Bickford et al., 1997). Our results clearly showed that L-deprenyl when co-administered with aluminium in rats inhibited TBA-RS formation, protein carbonyl formation and lipofuscin accumulation and elevated the SOD activity. It was also found to counter the aluminium-induced ultrastructural changes. Thus, Bacopa’s antioxidative effects appear to be similar to those of L-deprenyl. Oxidative stress (lipid peroxidation, lipofuscin accumulation, etc.) is thought to contribute to the aging process (Kaur et al., 2003a). Bacopa for its antilipidperoxidative and antilipofuscinogenistic effects could thus be considered as a potential antiaging substance. Furthermore, the similarity of Bacopa’s effects with those of L-deprenyl, which is a candidate aging drug, would also be indicative of its antiaging potential. The alcoholic extract of Bacopa contains several saponins (Chatterjee et al., 1963, 1965; Chakravarty et al., 2001, 2003), flavonoids and alkaloids (Schulte et al., 1972). The triterpenoid saponins (mainly bacosides A and B) have been reported to enhance memory and learning (Singh and Dhawan, 1997) and antioxidative activities (Bhattacharya et al., 2000). The ethanolic extract used in the present study contained 55– 60% bacosides (Pal and Sarin, 1992). The neuroprotective effects of the Bacopa extract observed in this study may have been due to the bacosides. In summary, the present data show Bacopa inhibits lipid peroxidation, protein oxidation and lipofuscin accumulation. It also reverses aluminium-induced oxidative stress and ultrastructural alterations. Antiaging potential of Bacopa also merit consideration.
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