Neuroprotective effect of Bacopa monnieri on beta-amyloid-induced cell death in primary cortical culture

Journal of Ethnopharmacology 120 (2008) 112–117

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Neuroprotective effect of Bacopa monnieri on beta-amyloid-induced cell death in primary cortical culture Nanteetip Limpeanchob a,∗ , Somkiet Jaipan a , Saisunee Rattanakaruna a , Watoo Phrompittayarat b , Kornkanok Ingkaninan b a b

Department of Pharmacy Practice, Faculty of Pharmaceutical Sciences, Naresuan University, Phitsanulok 65000, Thailand Department of Pharmaceutical Chemistry and Pharmacognosy, Faculty of Pharmaceutical Sciences, Naresuan University, Phitsanulok 65000, Thailand

a r t i c l e

i n f o

Article history: Received 18 February 2008 Received in revised form 9 July 2008 Accepted 28 July 2008 Available online 5 August 2008 Keywords: Bacopa monnieri Brahmi Alzheimer’s disease Neuroprotection Amyloid Glutamate

a b s t r a c t Aim of the study: Bacopa monnieri (Brahmi) is extensively used in traditional Indian medicine as a nerve tonic and thought to improve memory. To examine the neuroprotective effects of Brahmi extract, we tested its protection against the beta-amyloid protein (25–35) and glutamate-induced neurotoxicity in primary cortical cultured neurons. Materials and Methods: Neuroprotective effects were determined by measuring neuronal cell viability following beta-amyloid and glutamate treatment with and without Brahmi extract. Mechanisms of neuroprotection were evaluated by monitoring cellular oxidative stress and acetylcholinesterase activity. Results: Our result demonstrated that Brahmi extract protected neurons from beta-amyloid-induced cell death, but not glutamate-induced excitotoxicity. This neuroprotection was possibly due to its ability to suppress cellular acetylcholinesterase activity but not the inhibition of glutamate-mediated toxicity. In addition, culture medium containing Brahmi extract appeared to promote cell survival compared to neuronal cells growing in regular culture medium. Further study showed that Brahmi-treated neurons expressed lower level of reactive oxygen species suggesting that Brahmi restrained intracellular oxidative stress which in turn prolonged the lifespan of the culture neurons. Brahmi extract also exhibited both reducing and lipid peroxidation inhibitory activities. Conclusions: From this study, the mode of action of neuroprotective effects of Brahmi appeared to be the results of its antioxidant to suppress neuronal oxidative stress and the acetylcholinesterase inhibitory activities. Therefore, treating patients with Brahmi extract may be an alternative direction for ameliorating neurodegenerative disorders associated with the overwhelming oxidative stress as well as Alzheimer’s disease. © 2008 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Alzheimer’s disease is one of the most common neurodegenerative disorders affecting many elderly people worldwide. In addition to the neuropathologic hallmarks of this disease namely neurofibrillary tangles and amyloid plaques, it is also characterized by the loss of cholinergic neurons in the basal forebrain (Michaelis, 2003). The 40–42 amino acids beta-amyloid peptide (A␤1–40,42) derived

Abbreviations: A␤25–35, beta-amyloid protein (25–35); A␤1–40,42, 40–42 amino acids beta-amyloid peptide; AChE, acetylcholinesterase; APP, amyloid precursor protein; FRAP, ferric reducing antioxidant power; MTT, [3-(4,5dimethylthiazol-2,5-diphenyltetrazolium bromide]; NMDA, N-methyl-d-aspartate; ROS, reactive oxygen species; TBARs, thiobarbituric acid reactive substances. ∗ Corresponding author. Tel.: +66 81 554 3013; fax: +66 55 261 057. E-mail addresses: [email protected], [email protected] (N. Limpeanchob). 0378-8741/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2008.07.039

from proteolysis of amyloid precursor protein (APP) is the major component of the senile plaque found in Alzheimer’s disease brains (Glenner and Wong, 1984). Currently, there is no drug therapy that provides definite solution for curing Alzheimer’s disease. The pharmacological treatment conventionally used to maintain cognitive functions of patients consists of two classes of drugs, the acetylcholinesterase inhibitors (AChEI) and the glutamate modulators (Knopman, 2006). In addition, several alternative approaches for controlling the symptoms of this disease have been displayed such as anti-inflammatory drugs, antioxidants, and recently reported beta-amyloid based immunotherapy (Liu et al., 2007; Solomon, 2007; Weggen et al., 2007). Although those drugs are available for Alzheimer’s disease, the outcomes are often unsatisfactory, and hence there is a place for alternative herbal medicines. Many herbal treatments have been tested and demonstrated beneficial effects in different Alzheimer’s disease-related models as well as in clinical trials (Anekonda and

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Reddy, 2005; Dos Santos-Neto et al., 2006). This study, therefore, is focused on the effect of the extract of a perennial creeping plant named Bacopa monnieri. Bacopa monnieri (Brahmi) is a traditional Ayurvedic medicinal plant and is used in India as a nerve tonic. It has been reported to have several pharmacological effects acting as anti-inflammation, antimicrobial and anti-depressant (Sairam et al., 2002; Chaudhuri et al., 2004; Channa et al., 2006). The active constituents in Brahmi was found to be a mixture of saponins, for example, bacoside A, bacopasides I and II, and bacopasaponin C (Hou et al., 2002; Deepak et al., 2005; Phrompittayarat et al., 2007). During the last decade, a lot of research has been focused on cognitive and memory improvement of Brahmi. A memory enhancing effect of Brahmi was established in animal experiments as well as in healthy volunteers (Stough et al., 2001; Das et al., 2002; Roodenrys et al., 2002; Kishore and Singh, 2005). Therefore, this plant might have potential to ameliorate memory loss in Alzheimer’s disease patients. A previous study reported that Brahmi extract administration reduced the beta-amyloid levels in the brain of an Alzheimer’s disease transgenic mouse model (PSAPP mice), expressing the “Swedish” amyloid precursor protein and M146L presenilin-1 mutations with spontaneous amyloid plaque formation (Holcomb et al., 2006). Currently, the mechanism of action of Brahmi regarding its neuroprotective effect are still not completely clear. One mode of action was thought to be involved with Brahmi’s antioxidant properties including metal ions reduction, free radical scavenging, and lipid peroxidation inhibitory activities as well as enhancement of antioxidant enzymes (Bhattacharya et al., 2000; Dhanasekaran et al., 2007). Currently, there are still only limited numbers of studies on Brahmi’s neuroprotective effect both in in vitro and in vivo Alzheimer’s disease models. The present study, therefore, aims to investigate the neuroprotection of Brahmi extract in cultured primary cortical neurons against amyloid peptide and glutamateinduced toxicity. 2. Material and methods 2.1. Materials [3-(4,5-Dimethylthiazol-2,5-diphenyltetrazolium bromide] (MTT), 2,4,6-tripyridyl-s-trizine (TPTZ), malondialdehyde (MDA), 5,5 -dithio(bis)nitrobenzoic acid (DTNB), aprotinin, leupeptin, pepstatin A, phenylmethylsulphonyl fluoride (PMSF), acetylthiocholine iodide, beta-amyloid25–35 (A␤25–35), trichloroacetic acid, thiobarbituric acid, 2 ,7 -dichlorofluorescein diacetate (DCFHDA), cytosine arabinoside and all the materials for cell culture were purchased from Sigma (St. Louis, MO). Fetal bovine serum was purchased from Gibco. Bacoside A3 , Bacopaside II, 3-O-(-lara-(1→2)-[(-d-glc-(1→3)]-(-ara-jujubogenin, and Bacopasaponin C were kindly provided by Professor I. Khan, The National Center for Natural Products Research, University of Mississippy, USA. Bacopaside l was purchased from Chromadex, USA. 2.2. Preparation of Brahmi extract Brahmi was collected from Phetburi province, Thailand. It was identified by Assoc. Prof. Dr. Wongsatit Chuakul, Faculty of Pharmacy, Mahidol University, Thailand. The voucher specimen (Phrompittayarat001) was kept at the PBM Herbarium, Mahidol University, Thailand. The aerial part was collected, cut into small pieces and dried in a hot-air oven at 50 ◦ C for 12 h. The dried plant material was coarsely powdered. The dried plant material (18 kg) was soaked in water. After 24 h, the water was mechanically

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squeezed out of the plant material. The plant material was percolated with circulating 95% ethanol for 8 h. The residue was extracted again twice using the same procedure. The combined extract was filtrated and dried under reduced pressure. 2.3. Chemical constituent analysis by HPLC The separation was performed using a Shimadzu HPLC system equipped with a SPD-M10AVP photodiode array detector (PDA). The mobile phase consisted of 0.2% phosphoric acid and acetonitrile (65:35 v/v). The pH of the mobile phase was adjusted to 3.0 with 5 M NaOH. The flow rate was 1.0 ml/min. The total run time was 20 min. All peaks were integrated at the wavelength of 205 nm. They were initially assigned by comparing retention times with the standards and confirmed with characteristic spectra obtained from the PDA. The purity of the peak was also confirmed by the PDA. Calibration curves of five saponin glycosides, bacoside A3 , bacopaside II, bacopasaponin C isomer, bacopasaponin C, bacopaside I were prepared based on peak areas of seven concentrations. Linearity was obtained in the concentration range of 500–7.8 ␮g/ml. All data were processed using Class-VP software (Shimadzu, Japan). 2.4. Preparation of primary cortical cell cultures Primary cortical cell cultures were prepared from 18-day old Sprague-Dawley rat fetuses. The cerebral cortex was dissected under a stereomicroscope. The isolated tissue was cut into small pieces and suspended in 25 ml of 0.25% trypsin for 15 min and then mechanically dissociated with a pasteur pipette. Neuronal cells were collected by centrifugation 1000 × g for 5 min and resuspended in the defined medium DMEM/F12 supplemented with 10% FBS. Cells were resuspended in defined medium and plated in polyd-lysine coated 96-well plates at 5 × 104 cells/well. At 20–24 h after plating the medium was replaced by 2.5% serum-DMEM/F12. To minimize glial proliferation, the cultures were treated with 4 ␮M cytosine arabinoside. Experiments were performed after 10–14 days of seeding the culture. Animal experiments were approved by the Animal Ethical Committee of Naresuan University. 2.5. Preparation of aggregated B-amyloid A␤25–35 was reconstituted in sterile water at a concentration of 1 mM. Aliquots were incubated at 37 ◦ C for 72 h to form aggregated amyloid. During the experiment, A␤25–35 was directly added to cultured medium to achieve a final concentration of 50 ␮M. 2.6. Cell viability test Neuronal survival was quantified by MTT assay. MTT assay is based on the ability of a mitochondria dehydrogenase enzyme from viable cells to cleave the tetrazolium rings of the pale yellow MTT and form dark blue formazan crystals. Two hours before the end of cell treatments, 20 ␮l of MTT (5 mg/ml in phosphate buffer saline, PBS) were added to each well. At the end of treatment, the medium was remove, 200 ␮l of DMSO:ethanol (1:1) were added to each well to dissolve crystals. The color was quantified using ELISA reader at 550 nm. 2.7. Lipid peroxidation determination To initiate lipid peroxidation, rat brain homogenates were incubated with 400 ␮M FeCl2 and 200 ␮M ascorbic acid at 37 ◦ C for 1 h. Brahmi extract was added to the brain homogenate before lipid peroxidation induction. At the end of the incubation period, TBAR solution (10% trichloroacetic acid, 7% thiobarbituric acid, and 4%

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HCl final) was added to the samples. The mixtures were heated at 95 ◦ C for 1 h, cooled to room temperature, and spun at 3000 × g for 5 min to pellet precipitated protein. The clarified supernatant was read on a plate reader at 532 nm. An malondialdehyde (MDA) standard curve was established from 1,1,3,3 tetramethoxypropane. Lipid peroxidation was calculated and converted to percentage of untreated controls.

Table 1 The amount of saponin glycosides in Brahmi extract Saponins

Mean ± S.D. (%w/w)

1. BacosideA3 2. Bacopaside II 3. Bacopasaponin C isomer 4. Bacopasaponin C 5. Bacopaside I

0.588 1.228 0.717 0.630 1.882

2.8. Ferric reducing antioxidant power (FRAP assay)

Total

5.045 ± 0.400

The FRAP assay was used to determine the reducing ability of Brahmi extract. The method was previously described (Benzie and Strain, 1996). Briefly, FRAP reagent was freshly prepared by mixing 3 mM acetate buffer, pH 3.6, 10 mM TPTZ in 40 mM HCl, 20 mM FeCl3 (10:1:1). Brahmi extract (50 ␮l) was added to 150 ␮l of FRAP reagent. The absorbance was read at 593 nM. The results were expressed in ␮M of Fe2+ ions.

most enriched constituent in the extract was bacopaside I which was present at almost 2% (w/w) of the total extract.

2.9. Reactive oxygen species (ROS) determination ROS determination was performed by using a fluorescent probe DCFH-DA. 10 ␮M of DCFH-DA in methanol were added to medium and incubated at 37 ◦ C for 30 min. ROS in the cells causes oxidation of DCFH-DA, yielding the fluorescent product 2 ,7 dichlorofluorescein (DCF). Cells were washed twice with PBS to remove excess of DCFH-DA. The DCF fluorescence was measured after lysing the cells with lysis buffer (25 mM Tris phosphate, pH 7.8, 2 mM dithiothreitol, 2 mM 1,2-diaminocyclohexane-N,N,N ,N tetraacetic acid, 10% glycerol, 1% Triton X-100). The fluorescence was monitored by spectrofluorometer at excitation 485 nm and emission 530 nM.

± ± ± ± ±

0.007 0.036 0.033 0.026 0.311

3.2. Evaluation of the effect of Brahmi extract on cultured neurons Prior to testing the neuroprotective effect of Brahmi extract, its direct effect on cell viability of primary cortical cultures was evaluated. Cell viability was determined following incubating cells with various concentrations (1–1000 ␮g/ml) of Brahmi extract in cultured medium for 24 h. The results as shown in Fig. 1 demonstrated that cell survival was decreased in the presence of high concentrations of Brahmi extracts (data at 1000 ␮g/ml not shown). The 50% inhibitory concentration (IC50) was 242.8 ± 1.5 ␮g/ml. The concentration at 100 ␮g/ml did not affect cell viability and was used for later experiments. Interestingly, we found that when cultured cortical cells were treated with 100 ␮g/ml Brahmi extract for 48 and 72 h, cell viability was increased 37% and 20%, respectively (Fig. 2). After 48 h of incubation with Brahmi extract, the increment of cell viability was statistically significant compared to control cells. This observation suggests that certain compounds present in Brahmi extract were likely to promote cell survival or delay the natural death of neurons in culture medium.

2.10. Acetylcholinesterase activity assay 3.3. In vitro antioxidant activities Cells were washed and lysed in lysis buffer (15 mM Tris–HCl, pH 7.4) containing protease inhibitors (2 ␮g/ml aprotinin, 2 ␮g/ml leupeptin, 2 ␮g/ml pepstatin A, and 1 mM PMSF). AChE activity was measured by using acetylthiocholine iodide as a substrate in a concentration of 0.5 mM. 0.3 mM of DTNB was used to inhibit nonspecific esterase. The hydrolysis rate of acetylthiocholine iodide was measured spectrophotometrically at 414 nm, and used to calculate the AChE activity.

Our results demonstrated the beneficial effect of Brahmi extract on the cortical growth in culture media. This cell survival promotion could be the result of oxidative stress suppression as Brahmi has been previously shown to have antioxidant properties (Jyoti and Sharma, 2006; Vijayan and Helen, 2007). In the present study, the antioxidant activities of the tested Brahmi extract were determined by two conventional in vitro assays, TBARs and FRAP assays to evaluate the lipid peroxidation inhibition and metal ion reduc-

2.11. Statistical analysis Results are expressed as the mean ± S.E.M. of n experiments. The data were analyzed by repeated measurements of one-way analysis of variance (ANOVA). Differences were considered to be significant when p < 0.05. 50% inhibitory concentration (IC50) values were calculated using the Prism program (GraphPad Software Inc). 3. Results 3.1. Determination of the chemical components from Brahmi extract As the active components of Brahmi were thought to be compounds from the group of saponins, we determined the amount of such compounds as ethanolic extracts by HPLC. The results demonstrated that ethanolic extraction of the aerial part of Brahmi plants contained 5.04 ± 0.40% (w/w) of mixtures comprising of at least five saponin glycosides including bacoside A3 , bacopaside II, bacopasaponin C isomer, bacopasaponin C and bacopaside l (Table 1). The

Fig. 1. Effect of Brahmi extract on primary cortical cell growth in culture. Cortical cells were treated with various concentration of Brahmi extract (1–1000 ␮g/ml) in DMSO. Final concentration of DMSO is 0.5% which was not toxic to the cells. MTT assay was determined at 24 h incubation period to assess cell viability.

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Fig. 2. Brahmi extract promotes cortical cell viability in culture media. Cortical cells were treated with 100 ␮g/ml of Brahmi extract for 24, 48 and 72 h. Cell viability was determined by MTT assay. The results are mean ± S.E.M. from more than four experiments. (* p-value < 0.05).

ing activity, respectively. Our results showed that Brahmi extract inhibited the lipid peroxidation reaction of brain homogenate in a dose-dependent manner (Fig. 3). The generation of lipid peroxide products was almost completely blocked (80%) at concentrations higher than 150 ␮g/ml of the extract. The IC50 of lipid peroxidation inhibitory activity of Brahmi extract was 82.97 ± 1.24 ␮g/ml. Furthermore, Brahmi exhibited dose-dependent activity for reducing ferric to ferrous ions (Fig. 4). The reducing activity of Brahmi at 400 ␮g/ml is almost equivalent to 100 ␮M of standard antioxidant agents used in this experiment including ascorbic acid and trolox (water soluble vitamin E analog). 3.4. Brahmi reduced intracellular ROS of cultured neurons To investigate whether antioxidant activities of Brahmi extract play a role in cortical cell viability in culture media, we tested the level of intracellular ROS of Brahmi-treated cells. Compared to untreated cells, cells treated with 100 ␮g/ml Brahmi for 48 h showed a decrease in the amount of intracellular ROS which was

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Fig. 4. Metal ion reducing activity of Brahmi extract. The amount of Fe2+ ions reduced from Fe3+ ions were measured by FRAP assay. Brahmi extracts at concentration 50–400 ␮g/ml were tested comparing to 50 and 100 ␮M of both ascorbic acid and trolox.

calculated as the ratio of ROS/cell viability (Table 2). This result suggests that Brahmi is able to reduce the intracellular oxidative stress of cultured cortical neurons which might lead to a prolongation of neuronal life-span in culture media. 3.5. Neuronal protection of Brahmi extract against Aˇ25–35 but not against glutamate-induced cell death The neuroprotective activity of Brahmi extract was evaluated by assessing the viability of cultured cortical cells injured with A␤25–35 and glutamate in the presence and absence of Brahmi extract. Prior to cell treatment, A␤25–35 solution was incubated for 48 h at 37 ◦ C to generate its aggregated form. Neuronal cells which survived from these insults were assessed by the MTT assay. Incubation of cortical cells with A␤25–35 (50 ␮M) for 48 h caused 21.4 ± 2.4% of cell death (Fig. 5). Addition of Brahmi extract (100 ␮g/ml) to the culture medium prior to A␤25–35 led to the increase in the number of survival cells, suggesting that Brahmi extract prevented cell damage mediated by A␤25–35. Similar to the A␤25–35 treatment, neuronal cell viability was reduced after incubating cells with 4 mM glutamate in culture medium for 48 h. Glutamate treatment induced approximately 30% of cell death (Fig. 5). On the contrary to A␤25–35-treated neurons, Brahmi extract did not show the protective effect against glutamate-induced toxicity (Fig. 5). These results demonstrated that certain compounds in Brahmi extract were likely to protect cultured neurons from A␤25–35- but not glutamate-mediated neuronal damage. 3.6. Brahmi reduced intracellular AChE activity in Aˇ25–35-treated cells There are evidences showing that amyloid peptides could increase AChE in cultured cells (Sberna et al., 1997; Fodero et al., 2004), which could be part of its neurotoxic properties. We, Table 2 Oxidative stress evaluation in primary cortical cultures

Fig. 3. Lipid peroxidation inhibitory activity of Brahmi extract. Lipid peroxide products were measured by the TBARs assay. Lipid peroxidation reactions were generated in the presence of different concentration of Brahmi extracts. Degrees of inhibitory activity were calculated as % of control in which lipid peroxidation was conducted in the absence of Brahmi extract.

Treatment

Cell viability (%control)

ROS (%control)

Ratio (ROS/cell viability)

Untreated cells Brahmi (100 ␮g/ml)

100 137.07 ± 18.28*

100 92.11 ± 7.65

1 0.67

Values are means ± S.E.M. of four to six separate experiments. * p < 0.05 when compared to untreated group.

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Fig. 5. Neuroprotective effect of Brahmi extract on A␤25–35- and glutamateinduced neurotoxicity. Primary cortical cells were treated with 50 ␮M aggregated A␤25–35 or 4 mM glutamate (Glu) in the absence and presence of 100 ␮g/ml Brahmi extract for 48 h. Cell viability was determined by MTT assay.

Fig. 6. Effect of Brahmi extract on AChE activity in A␤25–35-treated cells. Primary cortical cells were treated with 50 ␮M aggregated A␤25–35 in the absence and presence of 100 ␮g/ml Brahmi extract for 48 h. After treatments, cell viability and intracellular AChE activity were determined from survival cells. The ratio of AChE activity/cell viability was calculated and referred as AChE activity of individual cells.

therefore, tested the effect of A␤25–35 on AChE by determining the intracellular AChE activity following treatment of cortical cells with A␤25–35 in the presence and absence of Brahmi extract (100 ␮g/ml). The result showed that neurons treated with A␤25–35 exhibited the elevation of intracellular AChE activity almost twice as much as untreated neurons presenting as the ratio of AChE activity/cell viability (Fig. 6). Cells treated with A␤25–35 in the presence of Brahmi extract exhibited lower AChE activity than did the A␤25–35-treated cells. Brahmi alone did not have a significant effect on the AChE activity (as indicated by the ratio closed to 1). This result suggests that A␤25–35 increases the intracellular AChE activity in cultured cortical cells and this increase was significantly counteracted by Brahmi extract. 4. Discussions In the in vitro model of Alzheimer’s disease, beta-amyloid peptides have been used to initiate neurotoxicity in various types of cultured cells (Boyd-Kimball et al., 2004; Martin et al., 2004; Puttfarcken et al., 1996). In addition to A␤1–42, numerous stud-

ies have used the smaller fragment, A␤25–35, as an alternative in Alzheimer’s disease investigation. The toxic effects of A␤25–35 were demonstrated by many laboratories (Behl et al., 1992; Michaelis et al., 1998; Brera et al., 2000; Xu et al., 2001). On this basis, this amino-acid fragment seems to be appropriate as an in vitro model for Alzheimer’s disease. Brahmi has received much attention from many laboratories as it is likely to have an ability to enhance memory function of the brain (Jyoti and Sharma, 2006; Vijayan and Helen, 2007). It, therefore, was thought to have some potential in delaying the progression of memory loss in Alzheimer’s disease. Our present study demonstrates that Brahmi extract can prevent cultured cortical neurons from A␤25–35-induced cell death. These data support previous studies showing neuroprotection of Brahmi extract by reducing beta-amyloid deposition in the brain of an Alzheimer’s disease mouse model (Holcomb et al., 2006). These combined evidences support the hypothesis of a beneficial effect of Brahmi in Alzheimer’s patients. Alzheimer’s diseased brain is also associated with glutamate receptor overstimulation, since the moderate affinity N-methyl-d-aspartate (NMDA) receptor antagonist memantine shows therapeutic efficacy in Alzheimer’s disease. Recent investigation using cultured cortical neurons demonstrated that A␤25–35 induced cell damage by increasing glutamate release (Ban et al., 2007). We therefore tested whether Brahmi extract could prevent neuronal damage mediated by glutamate exposure. Unexpectedly, our observation showed that Brahmi extract had no protective effect against glutamate toxicity. Brahmi-diminished A␤25–35induced cell death was likely to be the result of others mechanisms rather than blocking glutamate excitotoxicity. Brahmi treatment was reported to significantly reverse or up-regulate the expression of NMDA receptor 1 (NMDA R1) and metabotropic glutamate-8 receptor (mGluR8) to near control level in epileptic rats (Paulose et al., 2008; Khan et al., 2008). The neuroprotective role of Brahmi is probably associated with the regulation of neuronal protein transcription rather than direct inhibition at glutamate receptor. We further investigated the mechanism underlying the protection of Brahmi against A␤25–35-mediated neurotoxicity. This present study demonstrates that A␤25–35 induced the elevation of intracellular AChE activity which is corresponding to a previous report (Hu et al., 2003). The increment of AChE activity herein was diminished by co-treatment of cortical cells with Brahmi extract. AChE was suggested to be neurotoxic both in vitro and in vivo models (Small et al., 1996; Yang et al., 2002). This observation suggests the neuroprotection of Brahmi through its inhibitory effect on amyloid peptide-activated intracellular AChE activity. Although Brahmi extract previously displayed direct inhibitory activity against AChE testing in in vitro enzymatic assays, its activity was not so strong as ginkgo extract since it showed no more than 50% inhibition of all tested concentrations (Das et al., 2002). In the cellular level, Brahmi itself did not suppress the activity of AChE within control cells suggesting the inability to directly inhibit the intracellular AChE. In animal models, Brahmi extract significantly decreased AChE activity in scopolamine-induced dementic mice which consequently led to improved acetylcholine level in the brains (Das et al., 2002; Kishore and Singh, 2005). The mechanism of anticholinesterase activity of Brahmi is appeared to be complicated and further investigation is required. In addition to the prevention of A␤25–35-induced neuronal damage, Brahmi appeared to increase cell viability of cultured cortical neurons. It is plausible that substances in Brahmi extract are able to prolong the lifespan of neurons in culture media somehow by promoting cell survival and/or delaying cell death. The elevation of cell viability could be the result of the ability of some components in Brahmi to repress cellular oxidative stress as we found that the

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intracellular ROS level was reduced following Brahmi extract treatment. The antioxidant activity was thought to be one mode of action of Brahmi regarding its neuroprotective effect. Brahmi’s antioxidant properties including metal ions reducing, free radical scavenging, and lipid peroxidation inhibitory activities as well as enhancement of antioxidant enzymes were already reported in literatures (Bhattacharya et al., 2000; Dhanasekaran et al., 2007). Similar to previous studies, our data here demonstrate the antioxidant activities of Brahmi through its reducing ability and lipid peroxidation inhibitory activities. According to the cultured cell-based observation, Brahmi extract may be able to suppress oxidative stress of neuronal cells within the brain possibly indicating its neurotonic as well as memory enhancing effects. Taken all these evidences together, Brahmi seems to have a therapeutic potency possibly as alternative therapy for preventing or delaying progression of Alzheimer’s disease. 5. Conclusions Brahmi has been used as traditional medicine due to its neurotonic and memory enhancing property. This study demonstrates that Brahmi extract diminishes neuronal death induced by amyloid peptide partly through the suppression of AChE activity. Brahmi extract also exhibited antioxidant properties both in vitro and cell-based assays. Overall results from the present study support the potential of Brahmi extract as a remedy to prevent memory loss in natural aging as well as an alternative remedy for neurodegenerative disorders associated with oxidative stress and amyloid-induced memory loss. Acknowledgements The authors would like to thank Prof. Hans E. Junginger and Asst. Prof. Arom Jedsadayanmata for their suggestions in preparing the manuscript. This study was financial support by Naresuan University and the National Research Council of Thailand (NRCT). References Anekonda, T.S., Reddy, P.H., 2005. Can herbs provide a new generation of drugs for treating Alzheimer’s disease? Brain Research Reviews 50, 361–376. Ban, J.Y., Cho, S.O., Jeon, S.Y., Bae, K., Song, K.S., Seong, Y.H., 2007. 3,4Dihydroxybenzoic acid from Smilacis chinae rhizome protects amyloid beta protein (25-35)-induced neurotoxicity in cultured rat cortical neurons. Neuroscience Letters 420, 184–188. Behl, C., Davis, J., Cole, G.M., Schubert, D., 1992. Vitamin E protects nerve cells from amyloid ␤ protein toxicity. Biochemical and Biophysical Research Communications 186, 944–950. Benzie, I.F., Strain, J.J., 1996. The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: the FRAP assay. Analytical Biochemistry 239, 70–76. Bhattacharya, S.K., Bhattacharya, A., Kumar, A., Ghosal, S., 2000. Antioxidant activity of Bacopa monniera in rat frontal cortex, striatum and hippocampus. Phytotherapy Research 14, 174–179. Boyd-Kimball, D., Mohmmad Abdul, H., Reed, T., Sultana, R., Butterfield, D.A., 2004. Role of phenylalanine 20 in Alzheimer’s amyloid beta-peptide (1-42)induced oxidative stress and neurotoxicity. Chemical Research in Toxicology 17, 1743–1749. Brera, B., Serrano, A., de Ceballos, M.L., 2000. ␤-Amyloid peptides are cytotoxic to astrocytes in culture: a role for oxidative stress. Neurobiology of Disease 7, 395–405. Channa, S., Dar, A., Anjum, S., Yaqoob, M., Atta-Ur-Rahman, 2006. Anti-inflammatory activity of Bacopa monniera in rodents. Journal of Ethnopharmacology 104, 286–289. Chaudhuri, P.K., Srivastava, R., Kumar, S., Kumar, S., 2004. Phytotoxic and antimicrobial constituents of Bacopa monnieri and Holmskioldia sanguinea. Phytotherapy Research 18, 114–117. Das, A., Shanker, G., Nath, C., Pal, R., Singh, S., Singh, H., 2002. A comparative study in rodents of standardized extracts of Bacopa monniera and Ginkgo biloba: anticholinesterase and cognitive enhancing activities. Pharmacology, Biochemistry and Behavior 73, 893–900. Deepak, M., Sangli, G.K., Arun, P.C., Amit, A., 2005. Quantitative determination of the major saponin mixture bacoside A in Bacopa monnieri by HPLC. Phytochemical Analysis 16, 24–29.

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