Memory Enhancing Efficacy of an Ayurvedic Polyherbal Formulation on Scopolamine-Induced Memory Deficit Experimental Models

Memory and learning are crucial mental processes occurring in the cerebrum of the brain. Memory is an important factor for all types of learning due to gathering and retrieving information after the learning process.1 Learning and memory processes based on electrochemical signaling within the neuronal networks in the brain.2 Synaptic plasticity is a connection at the synapse of neurons that hypothetically to be modified during learning and memory disturbance.3 It is expected that modification of synapse function may lead to remodeling of neurons signaling as cognitive behavioral therapy.4 Cognitive behavioral therapy (CBT) helps in improving mental health by providing cognitive information and causing training-induced neuroplasticity.5


INTRODUCTION
Memory and learning are crucial mental processes occurring in the cerebrum of the brain. Memory is an important factor for all types of learning due to gathering and retrieving information after the learning process. 1 Learning and memory processes based on electrochemical signaling within the neuronal networks in the brain. 2 Synaptic plasticity is a connection at the synapse of neurons that hypothetically to be modified during learning and memory disturbance. 3 It is expected that modification of synapse function may lead to remodeling of neurons signaling as cognitive behavioral therapy. 4 Cognitive behavioral therapy (CBT) helps in improving mental health by providing cognitive information and causing training-induced neuroplasticity. 5 Medicinal Plants have been used since ancient times in the treatment of several disorders. 6 Natural medicines are now attracting researchers in the development of several formulations due to low side effects and higher efficacy. 7 The traditional medicines are used worldwide in the form of crude herbal extracts or herbal formulation in alleviating and curing nervous disorders. 8 Alpinia galanga (L.) wild belongs to family Zingiberaceae, it is a well-known medicinal plant containing mild spicy fragrance, has been used in the Asian countries for treating various diseases and disorders such as diabetes mellitus, inflammation, oxidative stress, diarrhea, ulcer, stomach ache, spasm, and microbial and insecticidal infection. [9][10][11][12][13] It is used as a nervine tonic in India. The plant of A. galanga is a class of the edible group and its rhizome possesses several active constituents in the form of essential oils such as p-hydroxycinnamaldehyde, galangin, galanganol B, methyl cinnamate, trans-p-coumaric acid, alpinin, kampheride, and cincole. [14] Glycyrrhiza glabra L. (liquorice) belongs to family Leguminosae. 15,16 It is an ayurvedic herb traditionally used in the treatment of insomnia and anxiety. 17 It possesses several biological activities including neuroprotective, sedative-hypnotic, anticancer, immunomodulatory and hepatoprotective activities. [18][19][20] This plant possess bioactive compounds such as glycyrrhizin, glycyrrhetinic acid, octadecane, octanoic acid and paeonol, benzaldehyde and 4-terpineol. 21,22 It is a widely used food industry due to its sweet taste.
Convolvulus pluricaulis Linn. is a native plant of India belongs to family Convolvulaceae, it has been used traditionally as a memory enhancer and neuroprotective herb 23,24 and in the treatment of several disorders such as liver diseases, diabetes, ulcer, anxiety, and oxidative stress. [25][26][27][28] It is used as a tonic of the brain, reduces age-related degradation of neurons and also performs the anti-stress activity. 29 The plant possessess numerous active constituents including shankhapushpine, scopoletin, and 29-oxodotriacontanol. 30 Thus, due to wide utilization of selected folk medicines and phytotherapy as well, the protocol of the study was designed to evaluate the efficacy of these herbs in the form of polyherbal formulation (PHF) as brain tonic against Alzheimer's disease, mental problems and other brain disorders including Schizophrenia, and Autism. Biochemical analysis was performed to determine the level of acetylcholinesterase due to treatment with PHF.

Drugs and reagents
Donepezil, Scopolamine, Propylparaben, Methylparaben, Tween 80 and Sodium carboxymethyl cellulose were procured from Sigma Aldrich, USA. All other solvents were of analytical grade and distilled water was used throughout the study.

Preparation of polyherbal formulation (PHF)
The prepared PHF containing 0.5014 g, 0.3343 g and 0.3343 g of Alpinia galanga (AG), Glycyrrhiza glabra (GG) and Convolvulus pleuricaulis (CP) extracts, respectively; further, the addition of parabens, tween 80 and a little amount of sodium carboxymethyl cellulose then uniformly triturated to form a smooth paste. The paste was rinsed with distilled water (100 ml) and vortexed using a mechanical stirrer (500 rpm) to get a suspension.

Experimental design and drug administrations
The effect of Alpinia galanga (AG), Glycyrrhiza glabra (GG) and Convolvulus pleuricaulis (CP) extracts and their PHF was evaluated inmemory impairment models induced by scopolamine.

Social recognition test (SRT)
Male adult rats weighing 225-250 g were used to evaluate the efficacy of PHF Scopolamine induced social recognition memory impairment. Scopolamine testing was performed in the cages on the 5 th day of drug administration in scopolamine (1.25 mg/kg) 31 models. The social stimuli were generated using small rats weighing 50-60 g. The time interval of 5 min was recorded between adult rats and juvenile rats as social interaction between them (T1). The juvenile rats were removed from cages and re-introduced after 2 h to again record the social interaction time (T2).

Morris water maze (MWM) test
In this and afterward animal models, the highest dose of individual plant extracts and PHF i.e. 200 mg/kg was selected based on its efficacy in SRT. Male adult rats (225-250 g) were subjected to the Morris water maze (MWM) test from the 5 th day in the scopolamine model (1 mg/ kg). 32 The water pool of 45 cm diameter and 26 cm height with four different starting points-N-E-SE-NW was used in this model. Animals were placed from anyone starting point of pool and any animal failed to escape from the pool within 120 s were placed at the side and allowed to stay for 30 s. The index of learning was determined by escape latency time in the water maze.

Pole climbing test (PCT)
Pole climbing test was performed to evaluate the efficacy of PHF, it is an apparatus enclosed with a chamber (25 cm × 25 cm × 40 cm) with light and sound arrangement. This chamber containing a grid floor to provide electricity. The rat jump to the pole to avoid foot shock while delivering an electrical stimulus, it was recorded as escape and electricity were terminated, however, another response 'avoidance' was noted when jumping occurs due to the sound of the buzzer. The experiment was terminated after 10 trials with intervals of 30 s. The animals were given the respective treatment and again subjected to the test procedure for 5 consecutive days after the completion of training. Reduction in escape latency time was considered as successful retention of avoidance memory. 33

Elevated plus maze (EPM) test
Efficacy of PHF was studied on scopolamine-induced memory impairment using the EPM test. The apparatus containing open and closed arms in all four directions with a central platform. The height of the maze is approximately 25 cm from the floor. Animals were placed on the open arm and latency time was noted when the animal moves to a closed arm. latency time was assigned 90 s if one of the two arms was pushed gently. Examination of memory retention was done after a 24 h post-first-day trial. 34

Biochemical analysis
Evaluation of MDA level in scopolamine-induced amnesic rat brain Malondialdehyde (MDA) level was measured using a thiobarbituric acid reaction (TBAR) colorimetric assay. Briefly, TBA reagent was mixed with brain homogenate and were mixed in a test tube followed by incubation at 90°C for 60 min. The test tube was then placed on ice cubes for cooling, it was centrifuged at 1000 RPM for 10 min. The supernatant was measured at 534 nm in a microplate reader. 35

Evaluation of GSH level in scopolamine-induced amnesic rat brain
In this method, GSH was measured by preparing brain homogenate, it was precipitated in trichloroacetic acid in the concentration of 10% followed by centrifugation at 12,000 rpm for 5 min. The supernatant was incubated and absorbance was measured at 425 nm. 36

Evaluation of AChE level in scopolamine-induced amnesic rat brain
The acetylcholinesterase (AChE) level was measured as Ellman reagent (100 μL) was filled in an individual well of 96 well plates. Brain homogenate (10 μL) was added in each well containing Ellman reagent and the absorbance was measured at 456 nm in microplate reader followed by determination of protein content in brain homogenate. 35

Effect of PHF in SRT behavioral model
Results exhibited that SIT2 was significantly decreased compared to SIT1. It indicated successful learning due to the administration of PHF. However, the scopolamine-induced group without treatment with test drug showed no change in the time during trial 2 comparing to trial 1. A significant reduction in SIT2 was noted in the piracetam (200 mg/ kg) administered group. Treatment with AG, CP and GG extracts at 200 mg/kg each have shown a significant reduction in SIT2 comparing to SIT1. It indicated the prevention of Scopolamine induced memory impairment (Figure 1). PHF was more effective than individual plant extracts in preventing scopolamine-induced memory impairment. Further, there was a significant difference in the recognition index of control, vehicle and Scopolamine group, confirming impairment of memory ( Figure 2). The recognition index was significantly lower in piracetam, individual herbal extracts (200 mg/kg) and PHF (200 mg/ kg) treated groups.

Effect of PHF in EPM behavioral model
The effects of plant extracts and PHF in the EPM test were evaluated on days 5 th and 6 th . As shown in Figure 5, the transfer latency time in the retention trial was significantly lower than the acquisition trial in control and vehicle groups. However, the scopolamine group showed no significant change in retention latencies in comparison to the acquisition trial. Piracetam at 200 mg/kg significantly reduced latency time during retention trial in comparison to acquisition trial indicating the prevention of memory impairment. Treatment with AG, CP, and GG extracts caused amelioration of scopolamine-induced memory impairment as indicated by significantly lower latency time in retention trial ( Figure 5). The PHF also significantly reduced latency time during the retention test in scopolamine-induced rats.

Effect of PHF on MDA level in scopolamine-induced amnesic rat brain
The MDA level was raised significantly in the cortex and hippocampus of scopolamine-treated rats in comparison to the control and vehicle groups. Preventive treatment with piracetam significantly reduced MDA levels in both brain regions. Administration of 200 mg/kg of plant extracts significantly decreased MDA levels in the cortex and hippocampus of scopolamine-treated rats. PHF treatment also caused a significant decrease in MDA level in brain regions ( Figure 6).

Effect of PHF on GSH level in scopolamine-induced amnesic rat brain
GSH level was observed using the calibration curve obtained with different concentrations of glutathione. Scopolamine caused a significant reduction in GSH levels in brain regions in comparison to control and vehicle groups. As shown in Figure 7, piracetam significantly prevented the scopolamine-induced reduction in GSH levels in the cortex and hippocampus. The administration of plant extract prevented the scopolamine-induced reduction in GSH levels. AG, CC, and GG   extract raised GSH levels in brain regions. PHF at 200 mg/kg also raised the level of GSH in brain regions (Figure 7).

Effect of PHF on AChE level in scopolamine-induced amnesic rat brain
AChE activity was raised significantly in brain regions in Scopolamine induced memory deficit rats. Preventive treatment with piracetam significantly inhibited AChE activity in brain regions (Figure 8). The administration of plant extract prevented scopolamine-induced elevation of AChE activity. AG, CC, and GG extract significantly decreased AChE activity in animals. PHF at 200 mg/kg also reduced AChE activity in the brain regions ( Figure 8).

DISCUSSION
Plants have been used in the development of the drug as an effective source of novel phytoconstituents in the treatment of several diseases. The phytotherapeutic approach has offered several opportunities to produce new drugs for memory disorders. 37 In this study a polyherbal formulation (PHF) of three brain tonic herbs i.e. Alpinia galanga; Glycerrhiza glabra and Convolulus pluricaulis was evaluated against scopolamine-induced memory loss experimental models. In this cognitive-behavioral study, we found that this formulation was effective    in the learning and memory process of different animal models. These medicinal herbs were selected due to their past India history of traditional uses as a memory enhancer and nervine tonic properties.
It has reported that Convolvulus pluricaulis has ameliorated neurotoxicity in the Drosophila model and depicted the neuroprotective herb. [30] In our study, PHF containing CP as one of the herbal drugs attenuated scopolamine-induced amnesia, it is supported by the work of Malik et al. 38 As scopolamine impairs both short-and long-term memory processes in rodents and humans. 39 Researchers also found that CP is an effective neuroprotective herb against the tumor, depression, epilepsy, anxiety and ulcer. 7,23,27,40,41 Several behavioral studies have been used neuroprotective drugs against oxidative stress in the brain because the generation of free radicals indicates the progression of cognitive decline. 42 In this study, PHF reduced AChE activity in both regions of the brain in a dose-dependent manner. Hippocampus consolidates information from short-term to long-term memory and plays a role in forming, organizing and storing memories. 43

CONCLUSION
It can be concluded from the study that PHF reduced scopolamineinduced amnesia via cholinergic function improvement, prevention of oxidative damage and behavior enhancement in the brain. Thus, PHF may be a potential candidate to prevent memory deficit in some neurodegenerative diseases.

DECLARATION
The author declares no conflict of interest.