INTRODUCTION:

Alzheimer's disease (AD), a condition related to age, is caused due to neurodegeneration^1,2. It is associated with memory loss, linguistic impairment, cognitive dysfunction, and behavioural problems^3,4. AD is a multifactorial disorder characterized by inflammation, beta-amyloid plaque build-up, neurofibrillary tangles, decreased central functioning, and brain oxidative stress⁵.

There are currently three types of cholinesterase inhibitors, rivastigmine, donepezil and galantamine prescribed for the treatment of mild to moderate types of AD and memantine, an NMDA receptor antagonist is approved for more severe forms of AD⁶. These drugs only improve cognitive function and fail to treat the underlying biological cause of the disease. Aducanumab and lecanemab, the FDA-approved medications modify the pathological progression of the disease but fail to eliminate the root cause of AD. In individuals with early-stage of Alzheimer's, these drugs effectively remove beta-amyloid from the brain, leading to a delay in cognitive and functional decline^6,9. They are reported not to be suitable for all individuals and are associated with adverse effects such as intracranial bleeding^10.Hence, for the treatment of AD, a drug is needed to target several pathological factors like oxidative stress, cholinergic dysfunction, tau hyperphosphorylation, mitochondrial dysfunction, and neuroinflammation.

A polyherbal formulation may be a ray of light with a multitargeted approach for the treatment of AD. The Siddha system is a traditional drug system in India that provides preventive, promotional, restorative, rejuvenating, and rehabilitative health care using a scientific and holistic approach¹¹. Bhiramiyadhi bhavanai choornam (BBC) is a polyherbal Siddha formulation that contains 15 herbs; roots of Withania somnifera, Picrorhiza scrophulariflora; fruits of Trachyspermum roxburghianum, Cuminum cyminum, Phyllanthus emblica, Piper longum, Piper nigrum, Terminalia chebula, Terminalia bellirica; seeds of Nigella sativa; rhizomes of Zingiber officinale, Acorus calamus and whole plant of Eclipta prostrata, Enicostemma axillare, Centella asiatica and has been reported to be used for enhancing memory, cognition, physical development and also tonic for the brain¹². However, scientifically limited literature is available regarding this formulation, and no pharmacological evidence exists for BBC as a memory enhancer. Hence, the present work aimed to explore the traditional claim of BBC as a memory booster using scopolamine-induced impaired learning process and memory in male Wistar rats.

MATERIALS AND METHODS:

Materials:

1. Procurement and authentication of plant:

The roots of Withania somnifera Dunal (Solanaceae), Picrorhiza scrophulariflora Royal ex Benth (Scropulariaceae), fruits of Trachyspermum roxburghianum (DC) H. Wolff (Apiaceae), Cuminum cyminum Linn. (Apiaceae), Piper nigrum Linn., Piper longum Linn. (Piperaceae), Terminalia chebula Retz (Combretaceae), Terminalia bellirica Roxb (Combretaceae), Phyllanthus emblica Linn. (Euphorbiaceae), seeds of Nigella sativa Linn. (Ranunculaceae), rhizomes of Zingiber officinale Rose (Zingiberaceae), Acorus calamus Linn. (Araceae) and whole plant of Eclipta prostrata, Enicostemma axillare, Centella asiatica were purchased from authentic vendors of Siddha medicinal plants and were authenticated by the National Institute of Siddha, Tamil Nadu, India (certificate number NISMB3852019).

2. Drugs and Chemicals:

Scopolamine butyl bromide, a label stating 20mg/ml (Buscopan, German Remedies Zydus Cadila Health) was procured from a retail pharmacy in Pune, Maharashtra, India. TNF α ELISA kit (Thermo Scientific, USA, Product no: ER3TNFA, Lot. No. LF144120) was purchased and stored at -20ºC. Donepezil hydrochloride tablets (Aricep 10), Eisai Pharmaceuticals, India were used as standard. The honey was purchased from the Central Bee Research Institute located in Pune, Maharashtra, and the cow ghee of Santulan Annayog was used.

3 Animals:

Male Wistar rats (150-180g) were bought from Agharkar Research Institute, Pune, Maharashtra. They were caged at 25°C with RH 45–55 % and maintained at a 12-hour day-night cycle. The feed pellets (Amrut Laboratory animal feed, Sangli, Maharashtra) and water were provided ad libitum. All experimental methods were approved by the IAEC, Poona College of Pharmacy, Pune (Registration number: 1703/PO/C/13/CPCSEA). The approval code was IAEC/PCP/PCL/06/2019-2020.

Methods:

1. Preparation of Bhiramiyadhi bhavanai choornam:

One part of each of all the ingredients listed above except Centella asiatica was powdered separately and placed in a mortar. After adding the juice of Centella asiatica, the mixture was ground until the juice was completely absorbed by the mixture and was placed in the shade for drying. To increase the potency of the medicine the mixture was scrapped, mixed and shade dried daily for 21 days. Finally, the dried powder was collected in a dry container¹².

2. Organoleptic and Phytochemical Evaluation of BBC:

Organoleptic and phytochemical investigations of BBC were carried out according to procedures mentioned in the Protocol for testing Ayurvedic, Siddha and Unani medicines¹³ and Practical Pharmacognosy techniques and experiments¹⁴. The methanolic extract of BBC was subjected to phytochemical tests.

3. Dose selection:

BBC is clinically administered as a 2-4g daily dose along with honey and ghee in a 1:1 ratio. The human dose of 4g was converted to the animal dose by applying the formula AED (mg/kg) = Human Dose (mg/kg) * Km ratio (6.2)¹⁵. The final dose selected was 215mg/kg, 430mg/kg and 860mg/kg.

4. Experimental design:

The experimental design of Upadhyay et al¹⁶, with slight modification, was used. Rats were separated into 6 groups each containing 8 rats. The vehicle control group (VC) received ghee and honey in a 1:1 ratio p.o., Group 2 (SC) received scopolamine (2mg/kg)¹⁷ i.p., and Group 3 was treated with standard donepezil (1mg/kg) once daily orally. Groups 4, 5 and 6 received BBC (215,430 and 860mg/kg) orally once daily for 14 days. Groups 3 to 6 were administered with scopolamine 2mg/kg i.p. half an hour before the respective treatment from day 1 to 14 of the experiment. Behavioural parameters were evaluated during the study. After the retrieval trial of MWM on day 15, all animals were euthanized. The brains were isolated, rinsed with isotonic ice-cold saline solution, homogenized in a phosphate buffer and stored at -80°C to be further utilized for biochemical estimation.

5. Behavioural Parameters:

a. Spontaneous locomotor activity:

On day 6 of the experiment, spontaneous locomotor activity in rats was evaluated using a digital actophotometer (Orchid Scientific and Innovative, India). After a 10 min actophotometer acclimatization period before treatment, the rats were kept back in their home cages. After half an hour of respective treatment rats were positioned in an actophotometer for 5 min to record locomotor activity. As soon as the animal broke the laser beam, its movement was recorded¹⁸.

b.Y maze:

For evaluation of spontaneous locomotor, the Y maze test was carried out with slight modification in the procedure described by Hritcu L et al¹⁹. On day 7 of the experiment, after half an hour of respective treatments rats were placed at the end of one of the arms and allowed to move freely in the maze for 5 min. When the rat's hind paws were entirely within the arm, it was considered to have entered the arm. Entry of the rat with its choice in all three successive arms was referred to as spontaneous alternation behaviour. The percentage of spontaneous alternation was computed as (actual alternations/maximum alternations)*100. Before the next animal was tested, the maze was cleaned with 0.1% sodium bicarbonate to remove olfactory clues^20,21.

c. Novel Object Recognition Test (NORT):

NORT was conducted using the methodology outlined by Ennaceur and Delacour²² with some changes. On day 8 of treatment before the conduction of NORT, rats were acclimatized to the testing arena for 10 min and then were placed back in their home cage. The first trial (T1) of NORT was conducted on the 9^th day of the experiment 30 minutes after the respective treatment. Five minutes were given to the rats to investigate the two similar objects in the arena. Two hours after the first trial, a second trial (T2) was carried out where one of the familiar objects was substituted with a new one. Time to explore familiar object (TF) and novel object (TN) was noted. 0.1% sodium bicarbonate was used to clean the open arena and the objects after every animal trial to erase the existence of olfactory clues^23,24. The discrimination index (DI) and % novelty preference (NP)²¹ were then calculated by using the formulas, DI = (TN- TF) / TE, and NP = (TN/ TE) *100 respectively, where TE is the total exploration time (TN + TF) in T2

d. Morris Water Maze (MWM):

MWM is a circular pool (1.5m in diameter and 0.45m in height) divided into four quadrants with four visual clues of varying colours and dimensions in each quadrant. Throughout the experiment, these hints were kept constant, and the maze's water temperature (24 ± 2°C) and the water level (30cm from the bottom) were also maintained. In one of the four quadrants, a circular platform was submerged (15cm in diameter and 2cm below the water's surface). The rats were trained for 5 days at regular intervals by conducting 4 trials per day, i.e., from day 10 to day 14 of the experiment to swim towards the visible platform with respective clues. After introducing rats into the pool 120 s were allotted to them for locating the platform. The animals who could not locate the platform were taken towards it and were given a 15 s window of time to observe their surroundings. On day 15, the trial was conducted with the hidden platform for evaluation of retention memory. To conceal the platform, the water level was raised by 1 cm above the platform and nontoxic fabric white colour paint was added to the water. The total time spent by each rat in the target quadrant (TSTQ), distance (path length) and the speed with which the rat reached the hidden platform and the duration taken to reach the hidden platform (escape latency) were recorded on the day 15 of the study^25,26.

6. Biochemical estimation in brain tissue homogenate:

a. Estimation of Total protein:

Lowry’s method was used to determine total protein by measuring absorbance at 640nm absorbance against distilled water as a blank²⁷.

b. Estimation of MDA:

An equal volume of rat brain homogenate and 10% trichloroacetic acid were mixed for 15 min and then kept in an ice bath to estimate malondialdehyde (MDA), a lipid peroxidation marker. The mixture was centrifuged to collect the supernatant. To the supernatant, an equal volume of TBA was added, which was then heated for 10 min and then cooled on an ice bath for 5 min. Against the reagent blank at 532nm absorbance was measured after the development of colour²⁸.

c. Estimation of SOD, GSH and catalase enzymes:

To estimate superoxide dismutase (SOD) activity 100µl of brain homogenate was mixed with 800µl of ethanol and 100µl of cold chloroform and centrifuged. Then to resultant supernatant 500µl of EDTA (0.6mM) and 1000µl of bicarbonate buffer (0.1 M) were added. After that, 500µl of 1.8mM epinephrine was added and the absorbance was measured every 30 sec for two to three min at 480nm²⁹.

To estimate reduced glutathione (GSH) in brain homogenate TCA (20%) and brain tissue homogenate were combined in equal parts and centrifuged. Then supernatant was collected and DTNB reagent was added. Phosphate buffer was used to make the final amount. Against a reagent blank, the colour produced was read at 412nm³⁰.

According to Sinha, 1972 technique,³¹ catalase (CAT) activity was evaluated using a theory based on its capacity to cause the disappearance of hydrogen peroxide. The quantity of enzyme required to cause the breakdown of 1 mmol of peroxide per minute equals one unit of CAT. A mixture of 400µl of 0.1M phosphate buffer, 200µl of 2 M hydrogen peroxide and 40µl sample were mixed and then after 1min 800µl of dichromate acetic acid was added to stop the reaction. After heating the mixture for 10 minutes in a water bath at 90°C, 160µl of water was added and measured at ƛ max 583 nm.

d. Estimation of nitric oxide (NO):

The Griess^32,33technique was used to determine the amount of NO in brain tissue homogenate.

e. Estimation of AChE activity by Ellman’s method:

Fresh acetylthiocholine iodide (pH 8.0) was added to the brain tissue homogenate to which Ellman reagent and phosphate buffer were added. Then at 412nm absorbance was measured³⁴.

f. Estimation of TNF α by ELISA technique:

TNF α level was measured as stated in the manufacturer’s instructions in the rat TNF alpha kit (Thermo Scientific, USA, Product No.: ER3TNFA, Lot. No. LF144120) and expressed as pg/ml.

7. Statistical Analysis:

The values of 8 animals were represented as mean ± SEM. The statistical evaluation involved one-way ANOVA and a post hoc Dunnett test with the help of Graph pad Prism 5.

RESULTS:

1. Evaluation of Organoleptic Characteristics of BBC:

BBC appeared to be a black solid smooth fine powder that passed through mesh with 180 µm mesh aperture size with characteristic odour and taste.

2. Phytochemical Evaluation of BBC:

The methanolic extract of BBC yield was 9.48% w/w and showed the presence of steroids, phenols, flavonoids, terpenoids, tannins, quinones, alkaloids, glycosides and carbohydrates.

3 Evaluation of BBC for Behavioural Assessment:

a. Effect of BBC on locomotor activity in scopolamine-induced impaired learning and memory in rats:

The locomotor activity of rats was evaluated in an actophotometer after half an hour of respective treatments. Non-significant change in locomotor activity of donepezil (1mg/kg) and BBC-treated groups (215,430,860 mg/kg) was observed as compared to the VC and SC groups (Figure 1).

Figure 1: Effect of BBC on locomotor activity in scopolamine-induced impaired learning and memory in rats. The values are of 8 animals represented as mean ± SEM. The statistical evaluation involved a one-way analysis of variance and a post hoc Dunnett’s test;(VC: vehicle control group, SC: scopolamine control group, BBC: Bhiramiyadhi bhavanai choornam).

b. Effects of BBC on exploratory behaviour in scopolamine-induced impaired learning and memory in rats:

In the Y-maze test, the SC group decreased significantly (p < 0.001) the percentage of spontaneous alteration as compared to the VC group. Oral treatment of donepezil (1mg/kg) and BBC (215,430,860 mg/kg) significantly (p < 0.01, p < 0.001) enhanced spontaneous alternation percentage compared to the SC group indicating enhanced exploratory behaviour which was altered due to scopolamine injection (Figure 2).

Figure 2: Effect of BBC on spontaneous alteration in scopolamine-induced impaired learning and memory in rats. The values are of 8 animals represented as mean ± SEM. The statistical evaluation involved a one-way analysis of variance and a post hoc Dunnett’s test. Significance is denoted by ^###p < 0.001 when compared to the VC group and **p < 0.01, ***p < 0.001 when compared to the SC group;(VC: vehicle control group, SC: scopolamine control group, BBC: Bhiramiyadhi bhavanai choornam).

c. Effects of BBC discrimination index and percentage novelty preference in scopolamine-induced impaired learning and memory in rats:

Rats injected with scopolamine exhibited a significant decrease (p < 0.001) in DI and percentage novelty preference indicating the rat's inability to differentiate between familiar and novel objects in the retention phase. A significant increase in DI (p < 0.01, p < 0.001) (Figure 3(a)) and percentage novelty preference (p < 0.01, p < 0.001) (Figure 3(b)) was observed in rats treated with donepezil (1mg/kg) and BBC (215, 430, and 860 mg/kg) indicating that animals were able to distinguish familiar and novel objects.

Figure 3: Effect of BBC on discrimination index (Figure 3 (a)) and novelty preference (Figure 3(b))in scopolamine-induced impaired learning and memory in rats. The values are of 8 animals represented as mean ± SEM. The statistical evaluation involved a one-way analysis of variance and a post hoc Dunnett’s test. Significance is denoted by ###p < 0.001 in comparison with the VC group and **p < 0.01, ***p < 0.001 in comparison with the SC group;(VC: vehicle control group, SC: scopolamine control group, BBC: Bhiramiyadhi bhavanai choornam)

d. Effects of BBC on spatial memory in scopolamine-induced impaired learning and memory in rats:

The group which received scopolamine only exhibited a significant increase (p < 0.001) in escape latency and path length while a decrease (p < 0.001) in TSTQ on day 15^th of the experiment. Donepezil (1mg/kg) and BBC (215, 430, and 860mg/kg) treated group significantly decreased (p < 0.001, p < 0.01) escape latency and path length while significantly improved TSTQ (p < 0.05, p < 0.01). The SC group, donepezil and BBC treatment groups did not show any significant difference in average speed measured in the MWM test when compared with the VC group (Figure 4).

Figure 4: Effect of BBC on Escape latency 4 (a), TSTQ 4(b), Path length 4(c) and Speed 4(d), in scopolamine-induced impaired learning and memory in rats. The values are of 8 animals represented as mean ± SEM. The statistical evaluation involved a one-way analysis of variance and a post hoc Dunnett’s test. Significance is denoted by ###p < 0.001 in comparison with VC group and *p<0.05, **p < 0.01, ***p < 0.001 in comparison with SC group;(VC: vehicle control group, SC: scopolamine control group, BBC: Bhiramiyadhi bhavanai choornam).

4. Effect on Biochemical Parameters:

a. Effect of BBC on Oxidative markers in the brain in scopolamine-induced impaired learning and memory in rats:

MDA and NO levels were found to be significantly raised (p < 0.001) while antioxidant enzymes SOD, GSH and catalase were significantly decreased (p < 0.001) in the SC group compared to the VC group. Treatment with donepezil (1mg/kg) and BCC (215,430 and 860mg/kg) significantly (p < 0.001) decreased MDA and NO levels and significantly (p < 0.001) improved levels of SOD, GSH and CAT in comparison with the SC group (Table No-1).

b. Effect of BBC on the activity of AChE in scopolamine-induced impaired learning and memory in rats:

Animals which received scopolamine only significantly (p < 0.001) increased brain AChE levels in comparison to the VC group. Treatment with BBC (215, 430 and 860 mg/kg) and donepezil (1mg/kg) significantly (p < 0.01, p < 0.001) reduced AChE activity in rat brains. Treatment with various doses of BCC appears to improve memory by preventing an increase in acetylcholinesterase activity compared to the group that received only scopolamine (2mg/kg) (Figure 5).

Figure 5: Effect of BBC on AChE activity in scopolamine-induced impaired learning and memory in rats. The values are of 8 animals represented as mean ± SEM. The statistical evaluation involved a one-way analysis of variance and a post hoc Dunnett’s test. Significance is denoted by ###p < 0.001 when compared to the VC group and **p < 0.01, ***p < 0.001 when compared to the SC group;(VC: vehicle control group, SC: scopolamine control group, BBC: Bhiramiyadhi bhavanai choornam).

c. Effect of BBC on TNF-α in scopolamine-induced impaired learning and memory in rats:

An approximately 2-fold surge (p < 0.001) in TNF-α level was observed in the SC group after comparing with the VC group. Treatment with donepezil (1mg/kg) and BBC (215, 430 and 860mg/kg) decreased significantly (p < 0.01, p < 0.001) the amount of TNF-α levels in the brain (Figure 6).

Figure 6: Effect of BBC on brain TNF α in scopolamine-induced impaired learning and memory in rats. The values are of 8 animals represented as mean ± SEM. The statistical evaluation involved a one-way analysis of variance and a post hoc Dunnett’s test. Significance is denoted as ###p < 0.001 in comparison to the VC group, and **p < 0.01, ***p < 0.001 in comparison to the SC group;(VC: vehicle control group, SC: scopolamine control group, BBC: Bhiramiyadhi bhavanai choornam).

DISCUSSION:

An antimuscarinic drug scopolamine is widely used to induce dementia in rodent models and also to investigate the influence of muscarinic receptors on learning and memory^35,36. Numerous cellular changes brought on by scopolamine include weak antioxidant defense, increased oxidative stress, mitochondrial dysfunction, apoptosis, and neuroinflammation. These pathological alterations resemble those seen in AD and other AD models. The protein alterations induced by scopolamine are indicative of the pathological processes observed in AD and other forms of dementia. Hence, scopolamine-induced dementia is a pharmacological model to investigate alterations associated with AD development at cellular and molecular levels^37,38. This research aims to examine the effect of BBC on the multifaceted effects of scopolamine on memory, cognitive function, and neuroinflammation and explore it as a valuable tool for studying dementia-related disorders.

The current study employed Y Maze, NORT and MWM to study the effect of BBC on behavioural parameters in learning and memory-impaired rats. Locomotor activity was evaluated using an actophotometer before behavioural parameters to ensure that locomotion did not interfere with the behavioural parameters of dementia³⁹. No significant change in locomotor activity of the scopolamine group and BBC-treated was observed in comparison with the VC group.

A Y maze test was conducted on the 7^th day of the treatment to evaluate exploratory behaviour and short-term spatial memory^20,40. BBC reversed scopolamine-induced memory impairment, as evidenced by the Y maze test results, which showed dose-dependent improvement in percentage alteration.

The NORT was carried out on the 9^th day of treatment to measure non-spatial visual recognition working memory. The discrimination index aids in differentiating between familiar and novel objects while novelty preference facilitates understanding of preference shown by animals for novel object. The negative DI indicates the failure of the animals to recognize novel object and a higher percentage of novelty preference better is the recognition memory^41-43. The group which received scopolamine only significantly decreased the discrimination index and novelty preference indicating impairment in non-spatial visual recognition working memory. However, treatment with BBC reversed the scopolamine-induced impairment and conserved visual recognition memory which is evidenced by a positive discrimination index and increased % novelty preference.

MWM test was used to measure the escape latency, path length, TSTQ and speed of the rats in a maze to assess long-term spatial memory⁴⁴. The scopolamine-treated group exhibited a significant increase in path length and escape latency and a decrease in TSTQ indicating memory impairment. However, the groups which received BBC significantly decreased path length and escape latency and increased TSTQ indicating improved spatial learning and memory. There was no significant change observed in speed on rats in MWM. The results of locomotor activity measured using an actophotometer and the speed of the rats in the MWM test suggest that there was no alteration in locomotion nor interference of locomotor activity in MWM parameters, indicating that the observed effects of BBC were solely due to enhanced memory. These results illustrate the pivotal role of BBC in preserving neurons involved in long-term and spatial memory formation.

Memory impairment brought on by the induction of scopolamine is linked to increased brain oxidative stress, characterised by elevated brain MDA and NO levels. Since lipids make up the majority of the brain, the action of ROS causing lipid peroxidation may have an immediate impact on the neurons. The results of the current study demonstrated a breakdown in the brain's antioxidant defence system, which was characterised by greater MDA and NO levels and reduced SOD, GSH, and catalase activity in the SC group compared to VC as earlier suggested by Lee et al^45-47. In this study also SOD, GSH and Catalase activity significantly increased after treatment with BCC, while MDA and NO levels decreased significantly. Many studies have shown that polyphenols neutralize free radicals and shield the brain and nervous system from damage after crossing the blood-brain barrier⁴⁸. The presence of quinones such as thymoquinone⁵² or polyphenolics such as rutin, quercetin, gallic acid, ellagic acid, and tannic acid^49–51 may be responsible for BBC's antioxidant impact.

In the brains of AD patients, neuroinflammation is frequently observed^53,54. A proinflammatory mediator, nitric oxide when released in excess, causes oxidation, apoptosis, and neurodegeneration^55,56.The brain's microglia emit TNF- α, an inflammatory cytokine connected to the development of AD⁵⁷. TNF α levels decreased significantly in BBC-treated animals in the current study indicating the anti-inflammatory action of BBC, which can prevent neuroinflammation and neurodegeneration in scopolamine-induced memory impairment.

The central cholinergic system can influence the memory process and lead to memory defects. Deficits in cognitive function are caused by the dysfunction of cholinergic neurons in the elderly⁵⁸. According to Chen et al⁵⁹, scopolamine exacerbates neurodegeneration in the brain by causing severe deficits in the cholinergic system and increases acetylcholinesterase activity in the hippocampus. Although the exact mechanism underlying the rise in AChE in a scopolamine-induced model remains unclear, several studies have documented elevated brain AChE activity in rodents^16,60. The current data support these findings where an increase in AChE activity in the SC group compared to VC was observed. Treatment with BBC significantly decreased AChE activity. Our findings imply that the effects of BBC on memory can be due to the inhibition of acetylcholinesterase activity, increased synaptic acetylcholine release, and enhanced acetylcholine fixation on postsynaptic receptors.

Numerous medicinal herbs and formulations containing these herbs are used to prevent and cure learning and memory impairment, and neurodegenerative diseases like AD and its associated symptoms. In this context, BBC contains 15 ingredients which are reported for free radical scavenging activity, the anti-inflammatory and neuroprotective rodent models^61-79. Moreover, flavonoids, tannin, and phenolic contents were shown to be present by the phytochemical study of the BBC's methanolic extract. These constituents function as antioxidants, scavenge free radicals and reduce the activity of acetylcholinesterase. As a result, secondary plant metabolites may be able to guard against AD caused by oxidative stress.^80,81

The results obtained from the current study and earlier reports on the neuroprotective effects of plants used to formulate BBC imply that BBC has nootropic action and prevents learning and memory impairment by modulating cholinergic and inflammatory pathways and improving oxidative defense mechanisms. The neuroprotective effect may be due to different phytoconstituents like piperine, ascorbic acid, embelin, chebulinic acid, 6-shogaol, β-asarone, gallic acid, asiaticoside, quercetin, rutin, tannic acid, ellagic acid, ellagitannins, withanolides, withanoside, thymoquinone, swertiamarin, stigmasterol, β-sitosterol, widalactone and luteolin present in BBC.

CONCLUSION:

In conclusion, the Siddha formulation Bhiramiyadhi bhavanai choornam is effective as a memory enhancer and is evidenced by the in vivo and biochemical studies. The present study has shown that BBC ameliorates scopolamine-induced impairment of memory in rats using Y maze, NORT and MWM tasks. By reducing lipid peroxidation in the brain and raising antioxidant enzyme concentrations—a crucial component in the prevention of AD, BBC enhanced the body's defence mechanism against free radical damage. AChE inhibition and a decrease in the production of NO and TNF α make BBC a choice of formulation in treating diseases related to memory impairment.

CONFLICT OF INTEREST:

The authors do not have any competing interests.

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Received on 04.07.2023 Modified on 18.10.2023

Research J. Pharm. and Tech 2024; 17(2):553-562.

DOI: 10.52711/0974-360X.2024.00086

Groups	MDA (nmol/mg protein)	SOD (U/mg protein)	GSH (U/mg protein)	CAT (U/mg protein)	NO (µmoles/mg protein)
VC	4.70 ± 0.02	15.30 ± 1.28	39.51 ± 3.08	5.86 ± 0.30	20.46 ± 1.073
SC (2mg/kg)	8.30 ± 0.15^###	4.70 ± 0.73^###	19.70 ± 1.18 ^###	2.94 ± 0.25 ^###	58.64 ± 2.61 ^###
Donepezil (1mg/kg)	4.50 ± 0.08***	14.60±.49***	38.90 ± 2.13***	5.64 ± 0.339***	31.37 ± 2.06***
BBC (215 mg/kg)	6.11 ± 0.15***	11.66 ± 1.05**	30.08 ± 1.63**	4.21 ± 0.200 **	45.71 ± 2.27***
BBC (430 mg/kg)	5.97 ± 0.084***	13.80 ± 1.87***	34.18 ± 0.30***	4.76 ±0.139***	41.29 ± 1.05***
BBC (860 mg/kg)	5.76 ± 0.21***	14.44 ±1.12 ***	38.42 ± 1.63***	5.18 ± 0.277 ***	35.61 ± 3.13***