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Year : 2016  |  Volume : 12  |  Issue : 47  |  Page : 482-487  

Elucidation of molecular mechanism(s) of cognition enhancing activity of Bacomind® : A standardized extract of bacopa monnieri

Natural Remedies R and D Centre No. 5B, Veerasandra, Bangalore, India

Date of Submission10-Nov-2015
Date of Decision10-Feb-2016
Date of Web Publication30-Sep-2016

Correspondence Address:
Shekhar Dethe
Natural Remedies R and D Centre No. 5B, Veerasandra Industrial Area, 19th km, Hosur Road, Electronic City, Bangalore
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0973-1296.191464

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Background: Bacopa monnieri (L.) Wettst., commonly known as Brahmi, is renowned in Indian traditional system for its potent memory enhancing activity, which has been validated by various scientific studies. Objective: The objective of this study was to understand the molecular mechanism of memory enhancing activity of BacoMind® (BM), a standardized extract of B. monnieri. Materials and Methods: BM was screened in vitro in a panel of cell-free and receptor-transfected cell assays. The purified enzymes/membrane homogenates/cells were incubated with substrate/standard ligand in the absence or presence of the test compound. The IC 50 values and EC 50 values were determined by nonlinear regression analysis of the concentration-response curves generated with mean replicate values using Hill equation curve fitting. Results: BM was found to inhibit three enzymes; Catechol-O-methyl transferase (COMT), Prolyl endopeptidase (PEP), and Poly (ADP-ribose) polymerase (PARP). It also had an antagonistic effect on serotonin 6 and 2A (5-HT 6 and 5-HT 2A ) receptors , known to influence the different neurological pathways, associated with memory and learning disorders, age-associated memory impairment. Conclusion: BM was found to inhibit three enzymes namely, Catechol-O-methyl transferase (COMT), Prolyl endopeptidase (PEP), and Poly (ADP-ribose) polymerase (PARP). It also exhibited an antagonistic effect on 5-HT 6 and 5-HT 2A receptors.
Abbreviations used: HTRF: Homogenous time resolved fluorescence, cAMP: Cyclic adenosine monophosphate, CHO : Chinese hamster ovary, RFU : Relative fluorescence unit, pNP: Para nitro phenol, AMC: 7-amino-4-methylcoumarin, ELISA: Enzyme linked immunosorbent assay, Z-Pro-Pro-CHO: Z-prolyl-prolinal, HEK: Human embryonic kidney, TE: Trolox equivalent.

Keywords: Bacopa monnieri, Bacomind, cognition, enzyme inhibition, learning and memory, mechanism of action, serotonin receptors

How to cite this article:
Dethe S, Deepak M, Agarwal A. Elucidation of molecular mechanism(s) of cognition enhancing activity of Bacomind® : A standardized extract of bacopa monnieri. Phcog Mag 2016;12, Suppl S4:482-7

How to cite this URL:
Dethe S, Deepak M, Agarwal A. Elucidation of molecular mechanism(s) of cognition enhancing activity of Bacomind® : A standardized extract of bacopa monnieri. Phcog Mag [serial online] 2016 [cited 2022 Aug 14];12, Suppl S4:482-7. Available from: http://www.phcog.com/text.asp?2016/12/47/482/191464


This study was conducted to understand the molecular mechanism of memory enhancing activity of a standardized extract of B. monnieri by was screening it in vitro in a panel of cell-free and receptor-transfected cell assays. The purified enzymes/membrane homogenates/cells were incubated with substrate/standard ligand in the absence or presence of the test compound. BM was found to inhibit three enzymes; Catechol-O-methyl transferase (COMT), Prolyl endopeptidase (PEP), and Poly (ADP-ribose) polymerase (PARP). It also had an antagonistic effect on serotonin 6 and 2A (5-HT6 and 5-HT2A) receptors, known to influence the different neurological pathways, associated with memory and learning disorders, age-associated memory impairment.

   Introduction Top

Bacopa monnieri (L.) Wettst., (family: Plantaginaceae) also called Brahmi, is found throughout India. Many Indian traditional literatures like Athar-Ved, Charak Samhita and Susrutu Samhita have detailed the medicinal importance of B. monnieri. The traditional knowledge is aptly supported by modern scientific literature, which emphasizes the medicinal importance of B. monnieri. In addition to cognitive properties, B. monnieri has been reported to possess anti-ulcerogenic, anti-oxidant, adaptogenic, anti-anxiety, anti-depressant and hepatoprotective activities. [1] It contains alkaloids (nicotine and herpestine), flavonoids (luteolin and apigenin) and saponins (bacoside A3, bacopaside I, bacopaside II, jujubogenin isomer of bacopa saponin C, bacopa saponin C). Various preclinical and clinical studies have reported B. monnieri to be effective in improving memory and cognition. It has shown significant reduction in forgetting the acquired information and improvement in memory acquisition and retention in healthy older individuals. [2] The capacity of B. monnieri to improve complications related to neurodegenerative disorders has also been studied and it was found to reduce the deposition of β-amyloid protein in animal model of Alzheimer's. [3] The potential of B. monnieri has also been studied as an anti-parkinsonian agent using C. elegans model, [4] which signifies its importance in neurodegenerative disorders. Even though the role of B. monnieri as a memory and cognition enhancer has been accepted traditionally and proved in scientific literature with profuse evidences, the ambiguity about its mechanism of action still remains to be resolved. We studied the effect of a methanolic extract of B. monnieri standardized to 40% bacosides, BacoMind TM (BM), on the different molecular targets associated with memory and cognition in order to understand its mechanism of memory enhancing action. Memory is not a unitary function as different memory and learning forms are sub served by different neurological pathways, which are closely interweaved with each other. [5] The major neurological pathways that are involved in the memory and cognition include cholinergic, dopaminergic, serotoninergic pathway, and neuroprotective or antioxidant pathway [6] . Among the serotoninergic pathway targets, down regulation of 5HT 2a , 5HT 3 [7] and 5HT 6 receptors [8] have been reported to improve memory. The inhibition of enzymes in the cholinergic pathway like acetyl cholinesterase (AChE) and butryl cholinesterase (BuChE), prevents the degradation of cholinergic neurotransmitters and improves the cholinergic transmission in the brain [9] helping to improve long-term memory processes. In the dopaminergic pathway, the inhibition of monoamine oxidase B (MAO-B), which breaks down monoamines like dopamine, has been found to be beneficial for memory and learning. [10] Oxidative stress in the brain can impair memory and learning. Brain is susceptible to oxidative stress as it is a region with high metabolic activity and there are high levels of unsaturated lipids and pro-oxidant iron. [11] Even though the cognition enhancing properties of BM has been demonstrated in preclinical and clinical studies, [12],[13] its mechanism of action has not been elucidated. The objective of our study was to understand the effect of BM on some of the targets that are associated with central nervous system disorders in order to elucidate its mechanism of memory and cognition enhancing activity.

   Materials and Methods Top

The effect of BM on various receptor and enzymes was assessed by using different validated in vitro assays.

Receptor-based assays

BM was tested in receptor binding assays for Muscarinic 1 (M 1), Serotonin 3 (5-HT 3), Gamma amino butyric acid (GABA), Adrenoreceptor alpha 2A (α 2A), N-methyl-D-aspartate (NMDA) and Glycine site (strychnine insensitive) receptors and in agonist/antagonist functional assays for Canabinoid 1 (CB 1) Dopamine 1 (D 1), 5-HT 2A and 5-HT 6 receptors to determine its interaction with these receptors.

For radio ligand binding assay the cell membrane homogenates were incubated for 90 min at 22°C with labeled standard ligand in the absence or presence BM in a buffer containing 20 mM Tris-HCl (pH 7.4). Following incubation, the samples were filtered rapidly under vacuum through glass fiber filters presoaked with 0.3% polyethyleneimine and rinsed several times with ice-cold 50 mM Tris-HCl. The filters were dried and then radioactivity was counted in a scintillation counter (Topcount, Packard).

BM was tested at a concentration of 5 μg/mL and 25 μg/mL for GABA and α- 2A binding assays. For all other binding assays a concentration of 10 μg/mL and 100 μg/mL was used for testing BM.

For functional assay, respective cells were suspended in DMEM buffer and were distributed in microplates. The fluorescent probe was then added into each well and equilibrated with the cells for 60 min at 37°C then 15 min at 22°C.

Thereafter, the test compound, reference antagonist or HBSS buffer was added and then 5 min later 3 nM of reference agonist or HBSS buffer (basal control), and the changes in fluorescence intensity was measured. The results were expressed as percent inhibition of the control response to 3 nM reference agonist [Table 1].
Table 1: Details of different receptor based assays

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BM was evaluated at 5 μg/mL and 25 μg/mL in various functional assays to check its agonist/antagonist effect on the selected receptors. The agonist effect was determined by studying the binding of BM to different receptors as compared to control (stimulated with agonist).

For the binding and agonist assays, results were expressed as percent activity of the control value in the presence of BM, calculated using the formula:

For antagonistic assays, the results were expressed as % inhibition of control agonist response calculated using the formula:

The EC 50 values (concentration producing a half-maximal response) and IC 50 values (concentration causing a half-maximal inhibition of the control agonist response) were determined by non-linear regression analysis of the concentration-response curves generated with mean replicate values using Hill equation curve fitting.

Enzyme-inhibition assays

The effect of BM on 16 enzymes listed in [Table 2] was evaluated using different assay formats. Purified enzymes were incubated with their respective substrates in presence and absence of BM and the colored/fluorescent product formed was measured using microplate reader [Table 2].
Table 2: Details of different enzyme based assays

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Percentage inhibition was calculated using the formula:

IC 50 , the concentration of inhibitor required to inhibit the activity of enzyme by 50%, was calculated by log-probit analysis, using Graphpad Prism ® version 5.01 software. All the assays were conducted in triplicate and the results were expressed as Mean ± S.D.

   Results Top

A 43.3 ± 9.54% inhibition of control specific binding by BM for M 1 receptor was the highest inhibition observed among all the selected receptors at the tested concentration. [Table 1] represents the percent inhibition and percent of control specific binding by BM in different receptor binding assays. Since, the results of binding of BM to the selected receptors were not significant, further studies using functional assays for evaluating the agonist/antagonistic effects for these receptors were not performed.

BM exhibited agonist effect on Canabinoid 1 (CB 1 ) receptors and the percent binding activity at 25 μg/mL with reference to control for 5-HT 2A and CB 1 receptors was found to be 33.3 ± 1.41% and 105.4 ± 0.42%, respectively [Figure 1].
Figure 1: Agonist effect of BM on different receptors

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BM displayed considerable potency in displacement of respective agonist ligands from D 1 , 5-HT 2A and 5-HT 6 receptors [Figure 2]. A very insignificant antagonist response was exhibited by BM in 5-HT 4e antagonist assay.
Figure 2: Antagonist effect of BM on different receptors

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The notable antagonist effect exhibited by BM at the 5-HT 6 receptors was further confirmed by determining its IC 50 in 5-HT 6 functional assays. BM was tested in 5-HT 6 functional assays at concentrations ranging from 4.29 to 50 μg/mL and its IC 50 in 5-HT 6 antagonistic assay was found to be 52 ± 1.2 μg/mL.

Out of the 16 enzyme evaluated, BM showed inhibitory activity on three enzymes. The IC 50 value of BM in PEP, COMT and PARP inhibition assay was found to be 25.5 ± 7.32 μg/mL, 18.4 ± 1.28 μg/mL and 27.8 ± 0.73 μg/mL, respectively. The response of BM on other enzyme was not significant. The anti-oxidant activity of BM in ORAC assay was found to be 1698 μ moles TE/g. The inhibitory activity of BM in various assays is indicated in [Table 3] and [Table 4].
Table 3: Median inhibitory concentration of BM in different enzymes inhibition assays

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Table 4: Effect of BM on different enzymes targets

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   Discussion Top

In this study, we investigated the effect of BM on various moleculartargets that are part of various neurological pathways like cholinergic,dopaminergic, cannabinoidergic, GABAergic and glutaminergic pathways, in order to elucidate its probable mechanisms of cognition enhancing activity. BM was found to inhibit COMT, an enzyme which controls dopamine metabolism by methylation and thereby modulates memory functions. COMT inhibitors like entacapone, are used as adjuncts to levodopa in the treatment of Parkinson's disease. [14] Since, COMT inhibition has been associated with prefrontal cortex dopamine signaling, this enzyme forms an important component of dopaminergic signaling pathway. [15] Dopamine, which produces a stimulatory effect by acting on D1 receptors, stimulates the cAMP signaling pathway. BM could also be modulating the dopamine signaling pathway by inhibiting the activity of COMT enzyme. Bacosides from B. monnieri are reported to significantly increase the concentration of dopamine and serotonin in aged rat brains. [16] Thus, the potent inhibitory effect of BM on COMT enzyme could be corroborating its effect on memory and cognition via dopamine pathway. Studies have indicated that B. monnieri may prevent degeneration of dopaminergic neurons and increase the level of dopamine in cortex region of rat brain. [17] Even though BM does not directly affect the other molecular targets like D1 receptor itself or the MAO-B, PP1 enzymes of the dopamine signaling pathway, our studies indicate that it could be exerting a protective effect and enhancing the dopaminergic system by increasing the concentration of dopamine, a catecholamine required for long-term memory, by inhibiting the COMT enzyme.

The synthesis and release of neuropeptides arginine, vasopressin, substance-P, oxytocin and angiotensin II are known to strongly influence the learning and memory process. [18] BM, when tested for its inhibitory activity against two neuropeptidases; prolyl endopeptidase (PEP) and insulin-regulated aminopeptidase (IRAP), was found to inhibit the activity of PEP enzyme which is known to cleave short peptides with internal proline residues. [19] PEP degrades the neuropeptides arginine-vasopressin, oxytocin, neurotensin and substance-P that play a key role in positive reinforcement, social interactions, emotions and stress responsivity. [20] PEP has been acknowledged for its role in cognitive impairment. [21] Reduced PEP activity amplifies substance P-mediated stimulation of Ins (1, 4, 5)P 3 that stimulates the release of intracellular calcium, known to cause neurotransmitter release. [22] The effect of reduced PEP activity on calcium concentration is a novel intracellular function of this peptidase, that has an impact on the cognitive enhancement. [23]

BM exerted significant antagonist effect on 5-HT 6 receptors, which is an important component of serotonergic pathway. A growing body of evidence suggests the use of 5-HT 6 antagonists for treating cognitive dysfunctions [24] and BM exhibited significant antagonism at these receptors with an IC 50 value of 52 ± 1.2 μg/mL. Also, 5-HT 6 receptor blockade has been reported to enhance cholinergic and glutaminergic neurotransmission [25] and increase dopamine level. [26] Thus, BM with an antagonistic effect on 5-HT 6 receptor could be affecting memory and cognition in more than one way. BM also had significant antagonistic effect on 5HT receptor subtype 2A. 5HT 2A receptor antagonists have been suggested to be beneficial for memory and cognition and have been implicated in insomnia [27] and also reported to increase sleep intensity. Morairty et al.[28] have presented the effect of sleep promotion via 5-HT 6 receptor blockade. So, the change in sleep intensity beacuse of 5HT2A antagonism may improve cognition and vigilance during wakefulness. Oxidative stress has been implicated in age associated memory disorders. Secondary injuries resulting from oxidative stress which produce DNA strand breaks that leads to cellular and neuronal death. [29] The ORAC value of BM was found to be 1698 μmoles TE/g suggesting its potential to scavenge the peroxyl radicals. It also inhibits the activity of PARP enzyme, which suggests that apart from reducing the free radicals generated, it can also reduce the secondary damage that has occurs due to free radical generation, adducing its potential as a good neuroprotective agent. The high ORAC value of BM is one of the reasons for its neuro-protection against oxidative stress.

The neurological pathways for memory have been reported to be overlapping. [30] The neural peptidergic pathway which includes arginine-vassopressin is augmented by the inhibition of enzymes in the dopaminergic pathway. [31] This indicates that inhibition of IRAP might be indirectly augmenting dopamine levels in the brain. The other pathways may also be linked through overlapping targets and thus modulation of one target may similarly affect other targets. Moreover, the metabolites of various Bacopa constituents could also be modulating these molecular targets for its memory enhancing activity as reported recently. [32]

   Conclusions Top

The results of the present study demonstrate that BM exerts its effects on cholinergic, dopaminergic, and serotonergic pathways by affecting at least five different molecular targets of memory. The memory enhancing effects of BM can be linked to its cumulative effects on these enzyme and receptor based targets. Further, the reported clinical efficacy could also be on account of the pharmacological effect of metabolites generated as a result of intestinal and liver metabolism of the various phyto-constituents, on multiple targets implicated in memory and cognition, which warrants further studies to understand the overall mechanism of action of this herb.


We authors are thankful to Ms. Jaya B. and Mr. Anirban Bhaskar for assistance with in vitro assay.

Financial support and sponsorship

The authors declare that there is no conflict of interests regarding the publication of this paper.

Conflicts of interest

There are no conflicts of interest.

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  [Table 1], [Table 2], [Table 3], [Table 4]

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