Asparagus adscendens root extract enhances cognition and protects against scopolamine induced amnesia: An in-silico and in-vivo studies

Asparagus adscendens root extract enhances cognition and protects against scopolamine induced amnesia: An in-silico and in-vivo studies

Accepted Manuscript Asparagus adscendens root extract enhances cognition and protects against scopolamine induced amnesia: An in-silico and in-vivo st...

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Accepted Manuscript Asparagus adscendens root extract enhances cognition and protects against scopolamine induced amnesia: An in-silico and in-vivo studies Priyanka Pahwa, Rajesh Kumar Goel PII:

S0009-2797(16)30430-6

DOI:

10.1016/j.cbi.2016.10.007

Reference:

CBI 7828

To appear in:

Chemico-Biological Interactions

Received Date: 7 June 2016 Revised Date:

11 September 2016

Accepted Date: 3 October 2016

Please cite this article as: P. Pahwa, R.K. Goel, Asparagus adscendens root extract enhances cognition and protects against scopolamine induced amnesia: An in-silico and in-vivo studies, Chemico-Biological Interactions (2016), doi: 10.1016/j.cbi.2016.10.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Asparagus adscendens root extract enhances cognition and protects against

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scopolamine induced amnesia: An in-silico and in-vivo studies

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Priyanka Pahwa and Rajesh Kumar Goel*

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Department of Pharmaceutical Sciences and Drug Research,

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Corresponding Author:

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Punjabi University, Patiala, Punjab, India.

Prof. Rajesh Kumar Goel

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Head, Pharmacology Division,

Department of Pharmaceutical Sciences and Drug Research, Punjabi University, Patiala 147002, Punjab, India Email: [email protected] Phone No: +919417881189

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Abstract Asparagus adscendens Roxb. commonly known as safed musli and belonging to the Liliaceae family is cultivated mainly in Asian countries. In traditional medicine, safed musli is

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recommended as nerve tonic and remedy for memory impairment. The present study was aimed to evaluate nootropic and antiamnesic activities of Asparagus adscendens extract (AAE) using in silico and in vivo approach. Phytoconstituents of A. adscendens root reported in literature were subjected to in silico prediction using PASS and Pharmaexpert. The radial

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arm maze and passive shock avoidance paradigm were employed to evaluate nootropic activity. Subsequently, the anti-amnesic activity was evaluated in scopolamine induced amnesia model. To elucidate the mechanism of nootropic activity, the effect of AAE on the

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activities of acetylcholinesterase and antioxidant enzymes in the cortex and hippocampus of mice were also evaluated. In silico activity spectrum for all of A. adscendens phytoconstituents exhibited excellent prediction score for nootropic activity. Pretreatment with AAE (50, 100 & 200 mg/kg, i.p.) for 15 days showed significant decrease in working memory error, reference memory error and retrieval latency in radial arm maze and

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decrease in step down latency in passive shock avoidance paradigm were observed. Further, AAE significantly reduced acetylcholinesterase and oxidative stress parameters in cortex and hippocampus of mice. Thus, in silico and in vivo results suggest that A. adscendens root may exert its nootropic activity through both anti-acetylcholinesterase and

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antioxidant activities.

Keywords: Asparagus adscendens, nootropic, anti-amnesic, in silico, acetylcholinesterase,

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antioxidant Introduction

Alzheimer’s disease (AD) is a neurodegenerative disorder has been characterized by a progressive loss of cognitive abilities, leading to learning and memory dysfunctioning in everyday activities [1, 2]. According to the WHO, it affects nearly 47.5 million people worldwide, which is likely to be increased to 75.6 million by 2030 [3]. Dysfunctioning of cholinergic system and elevated oxidative stress in forebrain and hippocampus appears to play a critical role in the pathogenesis of Alzheimer’s disease [4-6]. Currently,

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acetylcholinesterase inhibitors such as physostigmine, tacrine and donepezil are widely used for the treatment of AD, which are known to increase the availability of acetylcholine in cholinergic synapses [7, 8]. However, the adverse effects such as loss of appetite, vomiting, headache, diarrhea, narrow therapeutic range, hepatotoxicity etc. limit their use

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[9, 10]. Therefore, it is an urgent need to find complementary and alternative medicines (CAM) for the management of AD. Several medicinal plants and their phytoconstituents have shown the neuroprotective and cognition enhancing properties in experimental

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model of AD [11-13].

Asparagus adscendens Roxb. (Liliaceae) commonly known as safed musali or dholi musali, is a climbing herb found mainly in Asian countries. It has been attributed to a

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number of therapeutic uses in traditional medicine system such as diarrhea, dysentery, sexual debilities, nerve tonic and as a remedy in memory impairment [14]. The literature revealed that A. adscendens roots have been investigated for antifilarial [15], Insulin enhancing [16], antistress [17], Chemomodulatory [18], aphrodisiac [19] and antioxidant activities [18, 20-21]. Moreover, the available literature reveals that this plant has not been

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explored with respect to its therapeutic potential as nootropic activity. Also, nowadays the virtual screening of the phytochemical is believed to be a useful tool for drug discovery beyond their ethnic use [22]. Therefore, an effort has been made to explore the pharmacological activities of A. adscendens root using Prediction of Activity Spectra for

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Substances (PASS) and Pharmaexpert. A PASS based analysis (Table 1) revealed the presence of different bioactive phytoconstituents which might be responsible for nootropic potential with the most significant possibility of getting effective in the management of

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Alzheimer’s disease. Hence, based on traditional knowledge and in silico support the present study was envisaged to evaluate nootropic and anti-amnesic activities of Asparagus adscendens extract (AAE). Material and Methods

2.1 Drugs and Chemicals Shatavarin IV was procured from Natural Remedies Private Limited (Bangalore, India), Piracetam was obtained as a gift sample from Micro Labs Ltd. (Bangalore, India) and Griess

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reagent was procured from Sigma-Aldrich, Company (St. Louis, MO, USA). All other chemicals used in the present study were of analytic grade. 2.2. Animals

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Swiss albino mice, weighing 20-30 g were purchased from Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar, Haryana. The animals were housed on a 12h light/dark cycle under controlled temperature (22 ± 2 ºC) and humidity (50 ± 10%). They were allowed to acclimatize for 1 week with free access of food and water ad libitum.

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Animal experiments were performed according to the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of

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Environment and Forest, Government of India and approved by the Institutional Animal Ethical Committee (IAEC) (107/99/CPCSEA -2012-01).

2.3 PASS (Prediction of Activity Spectra for Substances) analysis

In-silico methodology was used as reported by Goel et al., (2015) and Gawande and Goel, (2015). An extensive literature search was carried out using various data bases to collect

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information regarding the phytoconstituents already reported in the roots of A. adscendens. Thereafter, biological activity spectrum of phytoconstituents was obtained by Prediction of Activity Spectra for Substances (version: 2012.10.22). The updated software has the ability to predict 6400 types of biological activity. The predicted activity spectrum in PASS was

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presented with the list of activities with probabilities «to be active» Pa and «to be inactive» Pi. Being probabilities, the Pa and Pi values varied from 0.000 to 1.000. The list of predicted

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activities is arranged in a descending order of Pa-Pi values. The PASS prediction results were interpreted and used in a flexible manner; (i) only activities with Pa0.7, the chance to find the activity experimentally was high; (iii) if 0.5
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2.4 Plant collection and preparation of extract A. adscendens roots were collected from “CSIR- Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh, India. The botanical identification of the plant

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material was done by Prof. V.K. Singla, and authentication was done by CSIR, Palampur (Voucher no: PLP16565). Authenticated roots were washed with water, shade-dried, ground to a moderately coarse powder. The powdered roots were subjected to extraction by percolation method with ethanol: water (50: 50 v/v) at room temperature. The

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resultant extract was evaporated to dryness using rotary evaporator (Buchi Type Rotary Vacuum Evaporator, Axiva, Shanghai, China) followed by lyophilization (Delvac, India) and stored at 4 ºC for further use. The percentage yield of the extract was found to be 44.7%

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w/w.

2.5 Phytochemical analysis and standardization of plant extract using high performance thin layer chromatography

A. adscendens extract was subjected to preliminary phytochemical screening tests to determine the presence of alkaloids, carbohydrates, glycosides, saponins, steroids,

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triterpenoids, flavonoids, tannins, proteins and amino acids [25]. Shatavarin IV being one of the principal phytoconstituent present in A. racemosus root was used as a biomarker for the standardization of the hydroethanolic extract. The presence of Shatavarin IV in the hydroethanolic extract was confirmed by using high

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performance thin layer chromatography (HPTLC) method as reported by Gohel et al., (2015) with slight modifications. Initially, the presence of Shatavarin IV was confirmed by

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thin layer chromatography (TLC) using Ethyl acetate: Methanol: Water (7.5:1.5:0.5, v/v/v) as a mobile phase. The plate was derivatised by spraying with freshly prepared anisaldehyde in sulfuric acid and then dried in an oven at 100 ºC for 10 min. Further, A. racemosus extract was standardized with Shatavarin IV using HPTLC. A stock solution of A. racemosus extract (30 mg/ml) and Shatavarin IV (1 mg/ml) were prepared in methanol. The mobile phase and spraying reagent for developing the chromatogram was same as used in TLC. Detection was done by measurement of absorbance at 426 nm. The study was carried out using pre-coated silica gel aluminum plate 60F254 (20 cm × 20 cm, E. Merck, Germany); Camag-HPTLC instrumentation (Camag, Switzerland) equipped with Camag

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Linomat V sample applicator, Camag TLC scanner IV and CAMAG Win CATS (1.4.8.2031) for data interpretation. The Rf values were recorded and the developed plate was screened at wavelength (λmax) of 426 nm. Standard curve was prepared with Shatavarin IV (Y= 2. 239x +712.7) with regression coefficient (R2) of 0.997 [26].

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2.6 Acute Toxicity test of the extract

Animals were subjected to acute toxicity studies as per guideline (AOT No: 420) suggested by OECD (The Organization of Economic Cooperation Development) (2001). The AAE was

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administered to the animals (n = 6) at different doses, up to ten times of effective dose i.e. 50, 100 and 200 mg/kg; i.p. One group served as vehicle control and received equal volume of vehicle. Percentage mortality and other gross behavioral changes were observed up to

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48 h after treatment [27, 28]. The neurotoxicity was evaluated using rota rod [29]. Locomotor activity was monitored by using actophotometer (Rolex, India), animals were individually placed in activity meter after 30 min of treatment and total activity count was recorded for 5 min. The locomotor activity was expressed in terms of total photobeam

2.7 Experimental Protocol

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interruption counts/5 min [30].

Six groups of animals (n = 6) were investigated. Group I: saline (control) (10 ml/kg/day, i.p.); Group II: Piracetam (standard drug) (200 mg/kg/day, i.p.); Group III: Shatavarin IV

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(1.9 mg/kg) (Based on the Shatavarin IV content present in AAE 200 mg/kg); Group IV, V and VI: A. adscendens extract (50, 100 and 200 mg/kg/day, i.p. respectively); Group VII: Scopolamine (1 mg/kg, i.p.); Group VIII: Piracetam + Scopolamine; Group IX: AAE 200

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mg/kg + Scopolamine. After 30 min of Piracetam and AAE administration in Group VIII and IX respectively, animals were injected with scopolamine (1 mg/kg; i.p.) in order to induce amnesia. Drug treatment and day wise behavioral evaluation of in-vivo experiments of all groups were respectively carried out as depicted in Fig 1.

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Fig 1: Schematic presentation of experimental protocol. RAM: Radial arm maze; WME: Working memory error; RME: Reference memory error 2.8 Cognitive Assessment 2.8.1 Radial Arm Maze

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The radial 8 arm-maze used in the present study consisted of 8 arms, numbered from 1 to 8 (30 cm × 6 cm), extending radially from a central area (16 cm in diameter). The apparatus was placed 50 cm above the floor, and surrounded by various extramaze visual cues placed at the same position during the study. At the end of each arm there was a food cup that had

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a single 50 mg food pellet. The movements of the animals were monitored via a video camera mounted above the maze. Prior to the performance of the maze task, the animals

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were kept on restricted diet and body weight was maintained at 85% of their free-feeding weight over a week period, with water being available ad libitum. Before the actual training began, three or four animals were simultaneously placed in the radial maze and allowed to explore for 5 min and take food freely. The food was initially available throughout the baited arms, but was gradually restricted to the food cup. The animals were trained for 7 days to run to the end of the arms and consume the food. To evaluate basal activity of animals in radial 8 arm-mazes, the animals were given 2 consecutive training trials per day to run to the end of the arms and consume the food. The training trial continued until all the four food cups had been consumed or until 5 min has elapsed. Criterion performance

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was defined as consumption of all four food cups or until 5 min had elapsed. After adaptation, all animals were trained with 1 trial per day. Briefly, 30 min after the treatments, each animal was placed individually in the center of the maze and subjected to working and reference memory tasks. An arm entry was counted when all four limbs of the

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animal were within an arm. Measures were made of the number of working memory errors (entering an arm containing food, but previously entered), reference memory errors (entering an arm that was not baited). The time taken to consume all four baits was also recorded. Reference memory is regarded as a long-term memory for information that

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remains constant over repeated trials (memory for the positions of baited arms), whereas working memory is considered a short time memory in which the information to be

been visited in each trial) [31, 32].

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remembered changes in every trial (memory for the positions of arms that had already

2.8.2. Passive Shock Avoidance Paradigm

For the evaluation of contextual fear memory, modified passive shock avoidance paradigm previously standardized in our laboratory was used [24, 33]. On day 0 animals were

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trained to stay on shock free zone (SFZ) for at least 120s and numbers of trials required were recorded. Further retrieval of learned task was evaluated by recording the changes in the number of mistakes and step down latency on day 1 and 8.

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2.9 Neurochemical estimation

After the behavioral evaluation on day 8, all the animals were scarified by cervical

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dislocation, and their brains were dissected to isolate the different brain regions (cortex and hippocampus). Isolated brain parts were homogenized in an ice-cold solution of 10% w/v (0.05 M, pH 7.4) phosphate buffer, and then centrifuged at 6000 g for 20 min at 4 ºC. The clear supernatant obtained was utilized for estimation of thiobarbituric acid reactive substances, reduced glutathione, catalase, total nitrite levels and acetylcholinesterase activity.

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2.9.1 Estimation of thiobarbituric acid reactive substance (TBARS) The quantitative measurement of TBARS, an index of lipid peroxidation was performed using the method described by Oakes and Van der Kraak, (2003). The absorbance was

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measured at 532 nm using microplate reader (BIORAD-Microplate Reader, Logotech, ISE Group, Germany) [34]. A standard calibration curve was prepared using 1–10 nM of 1,1,3,3-

nanomoles per g of wet tissue. 2.9.2 Estimation of reduced glutathione (GSH)

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tetra methoxy propane (Y = 0.0105x + 0.0562; R2 = 0.992). TBARS value was expressed as

The reduced glutathione content in tissues was estimated by using the method described

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by Beutler et al. (1963). Absorbance was measured using microplate reader (BIORADMicroplate Reader, Logotech, ISE Group, Germany) at 412 nm [35]. Reduced GSH standard curve was plotted using a 10-100 µM of reduced glutathione. The results were calculated using equation (Y = 0.001x + 0.085; R2 = 0.994). All the values were expressed as micromoles per mg protein.

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2.9.3 Estimation of catalase

CAT activity was determined by using the modified method of Chance et al., (1955). Catalase activity was calculated by using the molar extinction coefficient of H2O2 (0.071mM

[36].

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cm-1) and was expressed as micromoles of H2O2 oxidised per minute per milligram protein

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2.9.4. Estimation of total nitrite levels The total nitrite level was estimated employing the method described by Choudhary et al (2013). Absorbance was noted using microplate reader (BIORAD-Microplate Reader, Logotech, ISE Group, Germany) at 540 nm [37]. The standard curve was plotted using 10 100 mM of sodium nitrite. The results were calculated using equation (y = 0.0472x + 0.1034; R2 = 0.9982). All the values were expressed as ng per mg protein.

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2.9.5 Estimation of acetylcholinesterase activity Acetylcholinesterase (AChE) activity was assayed according to the Ellman's method with slight modification [38, 39]. Briefly, 40 μL of filtered brain homogenate (source of AChE)

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was mixed with 80 μL of Ellman's reagent and 200 μL phosphate buffer (pH = 8) in a 96 well-plate and shaken properly. The absorbance of the reaction mixture was recorded prior to the addition of the substrate at 412 nm using microplate reader (BIORAD-Microplate Reader, Logotech, ISE Group, Germany). The reaction was initiated by adding 10 μL of the

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enzyme substrate (10 mM acetylthiocholine chloride) to each well and was allowed to incubate for 15 min. A yellow color developed, and the absorption of the solution was measured at 412 nm. A molar extinction coefficient of 14,150M-1 cm-1 was used to calculate

hydrolyzed per mg protein. 2.9.6. Estimation of total brain protein

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enzyme activity. Enzyme activity was expressed as micro-moles of acetylcholine

The brain total protein was determined by Lowry’s method with slight modifications [40]

India) 2.10 Statistical analysis

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using total protein modified biuret, end point assay test kit (Span diagnostics Ltd., Surat,

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The statistical analysis was performed by using Graphpad prism® version 5 (Graph-Pad Software Inc., San Diego, CA, USA). Statistical significance was calculated using one-way ANOVA followed by Student-Newman-Keuls test. Each value was expressed as Mean ± SEM,

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and statistical significance was considered at p < 0.05. 3 Results

3.1 In silico screening of A. adscendens root phytoconstituents So far, 23 phytoconstituents have been reported as per literature, 3-heptadecanone, 8hexadecenoic acid, methyl pentacosanoate, tetratriacontane, tritriacontane, methyl palmitate, tetracosyl tetracosanoate, palmitic acid, stearic acid, asparanin C, asparanin D, asparoside C, asparoside D, 3-β-O-{β-D-2-tetracosylxylopyranosyl}-stigmasterol, 3-β-O-{β-

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D-glucopyranosyl (1-2)-α-L-arabinopyranosyl}-stigmasterol, β-sitosterol-β-D-glucoside, Shatavarin IV, stigmasterol, sarsasapogenin, xanthophyll, ascorbic acid, α-tocopherol and βcarotene. PASS (in silico) screening of phytoconstituents has shown good score for treating dementia and may have nootropic effect. Moreover, these observed predicted effects have

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also been supported by the several mechanisms as analyzed by Pharmaexpert (Table 1). Table 1: PASS predicted effects and mechanisms for nootropic activity of Asparagus

Phytoconstituent

Predicted effects

Shatavarin IV

0.907 0.000 Vascular dementia

0.375 0.015 Antioxidant 0.280 0.109 Calcium regulator

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treatment

Predicted relevant mechanisms

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adscendens root phytoconstituents.

0.902 0.000 Dementia treatment

0.244 0.077 Lipid peroxidase

0.381 0.246 Neuroprotector

inhibitor

0.185 0.077 Neurodegenerative diseases treatment

0.489 0.143 Nootropic

0.943 0.002 Acetylesterase

0.447 0.178 Neuroprotector

inhibitor

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3-heptadecanone

0.600 0.029 Calcium channel

0.312 0.038 Vascular dementia

(voltage-sensitive) activator

treatment

0.325 0.030 Lipid peroxidase

0.302 0.030 Amyotrophic lateral

inhibitor

sclerosis treatment

0.288 0.100 Calcium regulator

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0.424 0.049 Dementia treatment

0.271 0.091 Calcium channel activator 0.268 0.044 Free radical scavenger 0.177 0.071 Antioxidant 0.142 0.015 Acetylcholine release stimulant

8-hexadecenoic acid

0.741 0.027 Neuroprotector

0.873 0.004 Acetylesterase

0.404 0.061 Dementia treatment

inhibitor

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0.521 0.074 Calcium channel

0.310 0.039 Vascular dementia

(voltage-sensitive) activator

treatment

0.415 0.029 Calcium regulator

0.264 0.065 Amyotrophic lateral

0.294 0.040 Lipid peroxidase

sclerosis treatment

inhibitor

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0.361 0.255 Nootropic

0.267 0.032 Antioxidant

0.198 0.182 Calcium channel activator 0.682 0.045 Neuroprotector 0.478 0.154 Nootropic

0.832 0.006 Acetylesterase

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Methyl pentacosanoate

0.642 0.015 Calcium channel

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0.437 0.043 Dementia treatment

inhibitor

0.320 0.034 Vascular dementia

(voltage-sensitive) activator

treatment

0.376 0.045 Calcium regulator

0.266 0.062 Amyotrophic lateral

0.335 0.044 Calcium channel

sclerosis treatment

activator 0.170 0.005 Acetylcholine

0.665 0.051 Neuroprotector

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Tetratriacontane

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release stimulant 0.194 0.059 Antioxidant 0.166 0.136 Lipid peroxidase inhibitor 0.942 0.002 Acetylesterase inhibitor

0.365 0.089 Dementia treatment

0.681 0.008 Calcium channel

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0.513 0.121 Nootropic

0.323 0.019 Amyotrophic lateral

(voltage-sensitive) activator

sclerosis treatment

0.394 0.037 Calcium regulator

0.278 0.059 Vascular dementia

0.377 0.027 Calcium channel

treatment

activator 0.329 0.030 Lipid peroxidase inhibitor 0.190 0.005 Acetylcholine release stimulant

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0.192 0.060 Antioxidant 0.117 0.044 Acetylcholine agonist 0.539 0.011 Dementia Treatment

0.942 0.002 Acetylesterase

0.517 0.117 Nootropic

inhibitor

0.323 0.019 Amyotrophic lateral

0.681 0.008 Calcium channel

sclerosis treatment

(voltage-sensitive) activator

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Tritriacontane

0.394 0.037 Calcium regulator

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0.377 0.027 Calcium channel activator

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0.329 0.030 Lipid peroxidase inhibitor 0.117 0.044 Acetylcholine agonist

0.682 0.045 Neuroprotector

0.170 0.005 Acetylcholine

0.437 0.043 Dementia treatment

release stimulant

0.478 0.154 Nootropic

0.194 0.059 Antioxidant

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Methyl palmitate

0.166 0.136 Lipid peroxidase

treatment

inhibitor

0.266 0.062 Amyotrophic lateral

0.832 0.006 Acetylesterase

sclerosis treatment

inhibitor

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0.320 0.034 Vascular dementia

0.642 0.015 Calcium channel (voltage-sensitive) activator 0.335 0.044 Calcium channel activator 0.376 0.045 Calcium regulator

Tetracosyl

0.665 0.051 Neuroprotector

0.865 0.004 Acetylesterase

tetracosanoate

0.513 0.121 Nootropic

inhibitor

0.365 0.089 Dementia treatment

0.551 0.054 Calcium channel

0.278 0.059 Vascular dementia

(voltage-sensitive) activator

treatment

0.378 0.045 Calcium regulator

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0.273 0.054 Amyotrophic lateral

0.290 0.074 Calcium channel

sclerosis treatment

activator 0.216 0.082 Lipid peroxidase inhibitor

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0.192 0.060 Antioxidant

0.190 0.005 Acetylcholine release stimulant

0.916 0.003 Acetylesterase

0.484 0.149 Nootropic

inhibitor

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0.696 0.041 Neuroprotector

0.413 0.056 Dementia treatment

0.591 0.033 Calcium channel

0.314 0.037 Vascular dementia

(voltage-sensitive) activator

treatment

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Palmitic acid

0.400 0.034 Calcium regulator

0.275 0.052 Amyotrophic lateral

0.263 0.101 Calcium channel

sclerosis treatment

activator 0.250 0.062 Lipid peroxidase inhibitor

0.112 0.055 Acetylcholine release stimulant

0.696 0.041 Neuroprotector

0.916 0.003 Acetylesterase

0.413 0.056 Dementia treatment

inhibitor

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Stearic acid

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0.212 0.050 Antioxidant

0.591 0.033 Calcium channel

0.314 0.037 Vascular dementia

(voltage-sensitive) activator

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0.484 0.149 Nootropic

treatment

0.400 0.034 Calcium regulator

0.275 0.052 Amyotrophic lateral

0.263 0.101 Calcium channel

sclerosis treatment

activator 0.250 0.062 Lipid peroxidase inhibitor 0.212 0.050 Antioxidant 0.112 0.055 Acetylcholine release stimulant

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Asparanin C

0.787 0.001 Dementia treatment

0.323 0.021 Antioxidant

0.763 0.001 Vascular dementia

0.297 0.091 Calcium regulator

treatment

0.253 0.060 Lipid peroxidase inhibitor

0.827 0.001 Dementia treatment

0.395 0.014 Antioxidant

0.819 0.001 Vascular dementia

0.291 0.041 Lipid peroxidase

treatment

inhibitor

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Asparanin D

0.246 0.155 Calcium regulator 0.836 0.001 Dementia treatment

0.546 0.006 Antioxidant

0.812 0.001 Vascular dementia

0.334 0.029 Lipid peroxidase

Asparoside D

inhibitor

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treatment

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Asparoside C

0.882 0.000 Vascular dementia

0.619 0.005 Antioxidant

treatment

0.339 0.028 Lipid peroxidase

0.880 0.000 Dementia treatment

inhibitor

0.384 0.242 Neuroprotector 3-β-O-{β-D-2-

0.246 0.245 Dementia treatment

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tetracosylxylopyranosyl 0.206 0.144 Vascular dementia }-stigmasterol

treatment

3-β-O-{β-D-

0.423 0.050 Dementia treatment

0.540 0.008 Calcium regulator 0.286 0.028 Antioxidant 0.252 0.060 Lipid peroxidase inhibitor 0.429 0.025 Calcium regulator 0.388 0.014 Antioxidant

L-arabinopyranosyl}-

0.315 0.033 Lipid peroxidase

treatment

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stigmasterol

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glucopyranosyl (1-2)-α- 0.358 0.020 Vascular dementia

inhibitor

β-sitosterol-β-D-

0.604 0.005 Dementia treatment

0.506 0.012 Calcium regulator

glucoside

0.531 0.003 Vascular dementia

0.405 0.013 Antioxidant

treatment

0.330 0.029 Lipid peroxidase

0.350 0.280 Neuroprotector

inhibitor

0.518 0.117 Nootropic

0.685 0.004 Calcium regulator

0.432 0.191 Neuroprotector

0.330 0.029 Lipid peroxidase

0.389 0.070 Dementia treatment

inhibitor

0.315 0.037 Vascular dementia

0.195 0.058 Antioxidant

Stigmasterol

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treatment Sarsasapogenin

0.886 0.005 Nootropic

0.398 0.035 Calcium regulator

0.701 0.002 Dementia treatment

0.175 0.073 Antioxidant

treatment 0.320 0.317 Neuroprotector 0.363 0.091 Dementia treatment

0.777 0.004 Antioxidant

0.243 0.091 Vascular dementia

0.471 0.017 Calcium regulator

treatment

0.183 0.111 Lipid peroxidase

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Xanthophyll

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0.625 0.002 Vascular dementia

inhibitor

0.887 0.008 Neuroprotector

0.941 0.003 Antioxidant

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Ascorbic acid

0.479 0.153 Nootropic

0.893 0.002 Lipid peroxidase

0.317 0.137 Dementia treatment

inhibitor

0.242 0.093 Vascular dementia

0.545 0.057 Calcium channel

treatment

(voltage-sensitive) activator 0.447 0.073 Acetylesterase

0.709 0.094 Antioxidant

0.337 0.073 Vascular dementia

0.406 0.002 Calcium regulator

treatment

0.432 0.151 Calcium channel (voltage-sensitive) activator

0.456 0.034 Dementia treatment

0.794 0.004 Antioxidant

0.313 0.037 Vascular dementia

0.506 0.012 Calcium regulator

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β-carotene

0.581 0.004 Dementia treatment

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α-tocopherol

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inhibitor

treatment

0.369 0.221 Calcium channel (voltage-sensitive) activator 0.353 0.144 Acetylesterase inhibitor 0.245 0.064 Lipid peroxidase inhibitor 0.205 0.173 Calcium channel activator

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Pa: Prediction activity; Pi: Prediction inactivity 3.2 Phytochemical analysis and standardization of plant extract Preliminary phytochemical screening confirmed the presence of carbohydrates, glycosides,

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flavanoid, saponins, steroids, triterpenoids and amino acids in the hydroethanolic extract. The HPTLC analysis depicted well-resolved peaks of A. adscendens showing the presence of Shatavarin IV. The spots of the entire chromatogram were visualized at 426 nm and the quantity of Shatavarin IV was found to be 9.4 mg/kg of the hydroethanolic extract (Figure

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1A and B in supplementary file).

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3.3 Acute toxicity studies

Animals administered with AAE were found to be non-toxic up to 2000 mg/kg (ten times of highest effective dose) during the 48 h of study. The animals did not show any typical symptoms associated with toxicity, such as ataxia, convulsions etc. Most of the CNS depressants are suspected to alter motor coordination but the extract was found to have no neurotoxic effects with respect to disturbances in motor coordination up to highest dose.

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3.4 In-vivo studies

3.4.1 Effect on spontaneous locomotor activity

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No significant difference in spontaneous locomotor activity was observed on first intraperitoneal injection of respective treatment (- 7th day) as well as on last

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intraperitoneal injection of respective treatment (8th day) (Table 2). Table 2: Effect of Asparagus adscendens extract on locomotor count on day -7th and on day 8

Treatment Groups Control Pira SHA-IV AAE 50 AAE 100 AAE 200

Day (-7) 113 ± 2.39 108 ± 2.98 112.3 ± 1.17 119.33 ± 4.40 120.66 ± 2.4 119 ± 0.73

Day 8 119 ± 2.9 116.33 ± 2.34 115.3 ± 3.65 112.33 ± 2.01 116.33 ± 1.83 115.33 ± 1.47

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Values are presented as mean ± SEM (n=6). Pira: Piracetam (200 mg/kg); SHA-IV: Shatavarin IV (1.9 mg/kg); AAE 50, 100 and 200: Asparagus adscendens extract 50, 100 and 200 mg/kg respectively.

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3.4.2 Effect on RME, WME and Retrieval latency in radial arm maze In radial arm maze task, three parameters were observed which include Reference memory error, working memory error and retrieval latency. These above parameters represent the

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memory capacity and learning ability of the animals.

Long term memory assessment was determined by the number of reference memory errors (RME). The scopolamine treated animals showed significant (p < 0.05)

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increase in RME on all the days as compared to control animals. However, the piracetam and AAE treated animals significantly (p < 0.05) reduced the RME on day 6 onwards as compared to control animals. Similarly, the piracetam and AAE treated animals significantly (p < 0.05) reduced the RME on all the days as compared to scopolamine treated animals. Also, the Shatavarin-IV treated animals significantly (p < 0.05) reduced the RME on day 4 onwards as compared to control animals. The AAE 200 mg/kg treated

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animals significantly (p < 0.05) reverses the scopolamine induced amnesia as evidenced by reduced the RME on all the days as compared to scopolamine treated animals. Moreover, the piracetam treated animals also significantly (p < 0.05) reverses the scopolamine

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induced amnesia (Table 3).

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Table 3: Effect of Asparagus adscendens on the number of reference memory errors during eight days of memory assessment

Control

Piracetam #

SHA-IV

AAE-50 #

4.3 ± 0.2

3.7 ± 0.2 #

4.6 ± 0.2 #

4 ± 0.4 #

3.2 ± 0.16 #

4.3 ± 0.2 #

4.3 ± 0.2 #

4.33±0.2 3.7 ± 0.2

2

4±0

3

4.3 ± 0.2 3.7 ± 0.2 #

4

4 ± 0.36

3.3 ± 0.2 #

2.8 ± 0.16 *#

4 ± 0.37 #

3.67 ± 0.21 #

5

3.67±0.2 2.7±0.42 #

2.5 ± 0.22 *#

4.3 ± 0.2

6

3.7 ± 0.2 2.3± 0.2 *#

2.2 ± 0.4 *#

7

4 ± 0.37

8

4.3 ± 0.2 2 ± 0 *#

AAE-200

3.6 ± 0.2

#

Scopolamine

6.3 ± 0.2

*

Scop + Pira

Scop + AAE 200

4.7 ± 0.2

#

4.3 ± 0.21 #

3.3 ± 0.2 #

5.7 ± 0.2 *

4.6 ± 0.2 #

4±0#

3.7 ± 0.2 #

5.6 ± 0.2 *

4.3 ± 0.2 #

4.3 ± 0.2 #

3.33±0.21 #

5.3 ± 0.21 *

4.7 ± 0.2 #

3.67 ± 0.2 #

3 ± 0.36 #

2.7 ± 0.2 #

5.3 ± 0.2 *

4.3 ± 0.2

3.67 ± 0.2 #

3.7 ± 0.2 #

3.3 ± 0.2 #

2.7 ± 0.2 *#

4.7 ± 0.2 *

3.6 ± 0.2 #

3.3 ± 0.2 #

2.16±0.16 *#

3.7 ± 0.2 #

2.6 ± 0.2 *#

2 ± 0 *#

5±0*

3.3 ± 0.2 #

2.7 ± 0.2 *#

2.2 ± 0.2 *#

3.3 ± 0.2 *#

3 ± 0 *#

2 ± 0 *#

5.3 ± 0.2 *

3.3 ± 0.2 *#

2.7 ± 0.2 *#

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2 ± 0 *#

4.7 ± 0.2

#

1

3.3 ± 0.4 #

3.6 ± 0.2

*#

AAE-100

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Days

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in radial arm maze task

Values were expressed in mean ± SEM (n=6). * compared to control animals; # compared to scopolamine induced animals;

mg/kg respectively.

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Pira: Piracetam; SHA-IV: Shatavarin IV (1.9 mg/kg); AAE 50, 100 and 200: Asparagus adscendens extract 50, 100 and 200

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The scopolamine induced animals showed elevation in working memory error (p < 0.05) as compared to control group, which indicates memory impairment. However, the piracetam and AAE treated animals significantly (p < 0.05) reduced the WME on day 3 onwards as compared to control animals. Similarly, the piracetam and AAE treated animals significantly (p < 0.05) reduced the WME on all the days as compared to scopolamine treated animals. Also, the Shatavarin-IV treated animals significantly (p < 0.05) reduced the WME on day 3 onwards as compared to control animals. The AAE 200 mg/kg treated animals significantly (p < 0.05) reverses the scopolamine induced amnesia as evidenced by reduced the WME on all the days

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as compared to scopolamine treated animals. Moreover, the piracetam treated animals also significantly (p < 0.05) reverses

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the scopolamine induced amnesia (Table 4). Table 4: Effect of Asparagus adscendens on the number of working memory errors during eight days of memory assessment in

Days

Control

Piracetam

SHA-IV

AAE-50

AAE-100

AAE-200

3.7 ± 0.2 #

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radial arm maze task

3±0#

3.3 ± 0.2 #

2.7 ± 0.2 #

3.7 ± 0.4 #

Scopolamine Scop

+ Scop + AAE

Pira

200

5.7 ± 0.2 *

4.6 ± 0.2 *#

4.3 ± 0.2 #

6±0*

4.3 ± 0.2 *#

4 ± 0.37 #

2.3± 0.2 *# 5.3 ± 0.2 *

3.7 ± 0.2 #

3.7 ± 0.2 #

2.3 ± 0.2 *#

2.7 ± 0.2 #

3 ± 0.36 #

3 ± 0.4 #

1.7 ± 0.2 *#

1.6± 0.2 *# 4.3 ± 0.2 *

2.6 ± 0.2 #

2.3 ± 0.2 *#

3.7 ± 0.2

4±0#

3.3 ± 0.2 #

4.3 ± 0.2 #

2

3.3 ± 0.2

2.7 ± 0.2 #

3±0#

4±0#

3

4±0

2.3 ± 0.2 *#

2.7 ± 0.2 *#

3.6 ± 0.2 #

4

3.7 ± 0.4

1.7 ± 0.2 *#

2.7 ± 0.2 #

3.6 ± 0.2 #

5

3.3 ± 0.2

1 ± 0 *#

2.3 ± 0.2 *#

3.3 ± 0.2 #

6

3 ± 0.4

0.7 ± 0.4 *#

1.3 ± 0.2 *#

2.7 ± 0.2 #

1.67 ± 0.2 *#

0.6± 0.4 *# 4.3 ± 0.2 *

2.3 ± 0.2 #

2±0#

7

2.7 ± 0.2

0.67 ± 0.2 *#

1 ± 0.4 *#

2.3 ± 0.2 #

1.3 ± 0.2 *#

0.3± 0.2 *# 3.7 ± 0.2 *

1.7 ± 0.6 #

1.3 ± 0.2 *#

8

2.3 ± 0.2

0.33 ± 0.2 *#

0.7 ± 0.2 *#

2.3 ± 0.2 #

1 ± 0 *#

0.3± 0.2 *# 3.3 ± 0.2 *

1.7 ± 0.2 #

1.3 ± 0.2 *#

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1

4.6 ± 0.2

Values were expressed in mean ± SEM (n=6). * compared to control animals; # compared to scopolamine induced animals;

mg/kg respectively.

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Pira: Piracetam; SHA-IV: Shatavarin IV (1.9 mg/kg); AAE 50, 100 and 200: Asparagus adscendens extract 50, 100 and 200

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On last day of behavioral assessment (day 8), the scopolamine induced animals showed significant (p < 0.05) increase in time taken to consume all the baits indicates memory impairment as compared control animals. However, the AAE significantly reduced the retrieval latency only at 50 and 200 mg/kg as compared to control animals. Similarly,

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the Piracetam and AAE 200 mg/kg significantly reverses the scopolamine induced amnesia

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as compared to control and scopolamine induced animals (Fig 2).

Fig 2: Effect of Asparagus adscendens extract on retrieval latency in radial arm maze. Values were expressed in mean ± SEM (n=6). * compared to control animals; # compared to

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scopolamine induced animals; Pira: Piracetam; SHA-IV: Shatavarin IV (1.9 mg/kg); AAE 50, 100 and 200: Asparagus adscendens extract 50, 100 and 200 mg/kg respectively.

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3.4.3 Effect on step down latency in passive shock avoidance test The scopolamine induced animals significantly impaired the memory on all the days as evidenced by increased the number of mistakes as compared to control animals. However, the piracetam and AAE (50, 100 and 200 mg/kg) significantly reduced the number of mistakes as compared to control animals. Moreover, the piracetam and AAE 200 mg/kg reverses the scopolamine induced amnesia as evidenced by reduced number of mistakes as compared to scopolamine induced animals on all the days (Table 5).

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Scopolamine induced animals significantly impaired the memory on all the days as evidenced by decreased the step down latency as compared to control animals. However, the piracetam and AAE (50, 100 and 200 mg/kg) significantly increased the step down latency as compared to control animals. Moreover, the piracetam and AAE 200 mg/kg

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reverses the scopolamine induced amnesia as evidenced by improved step down latency as compared to scopolamine induced animals on all the days (Table 5).

Table 5: Effect of Asparagus adscendens extract on number of mistakes and step down

Number of Mistakes Day 0

Day 1

Control

10.3 ± 0.5

2.3 ± 0.5

Pira

3.3 ± 0.2 *#

SHA-IV

Step Down Latency (sec)

Day 8

Day 0

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Treatments

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latency in passive shock avoidance test

Day 1

Day 8

5.7 ± 1.1

43.7 ± 2.37

45 ± 2.89

0.7 ± 0.18 *#

0.3 ± 0.2 *#

3.7 ± 0.9

89.3 ± 9.8 *#

106 ± 8.8 *#

3 ± 0 *#

0.3 ± 0.1 *#

0.3 ± 0.2 *#

4.3 ± 0.5

109.3±6.7 *#

117 ± 1.89 *#

AAE 50

3.7 ± 0.2 *#

1.6 ± 0.36 #

1.3 ± 0.2 *#

7.3 ± 0.8 #

57.3 ± 7.8 *#

70.6 ± 5.68 *#

AAE 100

2.3 ± 0.2 *#

0.3 ± 0.18 *#

0.3 ± 0.2 *#

2.3 ± 0.2 *

99 ± 13.3 *#

108.6±7.16 *#

AAE 200

2 ± 0.3 *#

0 ± 0 *#

0 ± 0 *#

1.7 ± 0.2*

120 ± 0 *#

120 ± 0 *#

Scol

16 ± 1.4 *

10.7 ± 0.2 *

12.3 ± 0.2 *

3.7 ± 0.2

4.7 ± 0.2 *

6 ± 0.36 *

Scol + Pira

12.33± 0.7 #

3.3 ± 0.2 #

2.3 ± 0.2 *#

4 ± 0.6

34.7 ± 4.3 #

61.67± 2.43 #

Scol + AAE 200

10.3 ± 0.6 #

2.7 ± 0.2 #

1.7 ± 0.2 *#

5.3 ± 0.2

44.3± 2.07 #

70 ± 2.63 *#

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3.3 ± 0.2

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Values were expressed in mean ± SEM (n=6). * compared to control animals; # compared to scopolamine induced animals; Pira: Piracetam; SHA-IV: Shatavarin IV (1.9 mg/kg); AAE

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50, 100 and 200: Asparagus adscendens extract 50, 100 and 200 mg/kg respectively. 3.5 Changes in oxidative stress markers One-way ANOVA showed that lipid peroxidation level was significant different in both cortex [F

(8, 54)

= 178.9, p < 0.0001] and hippocampus [F

(8, 54)

= 262.2, p < 0.0001] in

between the groups. The post hoc analysis revealed a significant elevation in lipid peroxidation level was recorded in cortical and hippocampal regions of brain with scopolamine induced amnesia as compared to control animals. However, piracetam and AAE (50, 100 and 200 mg/kg) significantly (p < 0.05) reduced the lipid peroxidation level

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in cortical and hippocampal regions of brain as compared to control animals. Similarly, the piracetam and AAE 200 mg/kg reverses the scopolamine induced amnesia as evidenced by reduced lipid peroxidation level as compared to scopolamine induced animals (Table 6).

Catalase (µ moles of H2O2 oxidized/ mg of protein) Cortex

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Table 6: Effect of AAE treatment on oxidative parameters in cortex and hippocampus Nitrite Level (ng/ mg protein)

AChE activity (µ moles of acetylcholine hydrolyzed per mg protein)

TBARS (nmol/ g of wet tissue

Reduced GSH (µmol/ mg protein)

Control

10.07 ± 0.49

42.26 ± 0.58

2.75 ± 0.04

235.08 ± 1.53

Pira

10.82 ± 0.43 #

35.18 ± 0.97 *#

2.61 ± 0.01 *#

634.45±2.25 *# 0.40 ± 0.01 *#

Sha-IV

16 ± 0.55 *#

26.23 ± 0.35 *#

2.13 ± 0.01 *#

614.37±6.62 *# 0.35 ± 0.01 *#

AAE 50

20.72 ± 0.83 *#

20.77 ± 0.56 *#

1.51 ± 0.01 *#

818.7±2.42 *#

AAE 100

15.64 ± 0.52 *#

26.66 ± 0.52 *#

2.16 ± 0.02 *#

662.08±2.42 *# 0.36 ± 0.01 *#

AAE 200

9.54 ± 0.31 #

36.42 ± 0.73 *#

2.66 ± 0.02 #

628.5±1.68 *#

0.34 ± 0.03 *#

Scol

31.72 ± 0.28*

15.94 ± 0.42 *

1.54 ± 0.01 *

699.75± 1.37 *

0.65 ± 0.01 *

Scol + Pira

16.01 ± 0.31 *#

23.97± 0.50 *#

1.99 ± 0.04 *#

538.48±1.49 *# 0.38 ± 0.01 *#

30.31± 1.09 *#

2.12 ± 0.01 *#

609.76±1.85 *# 0.38 ± 0.01 *#

200

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Scol + AAE 13.09 ± 0.65 *#

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Treatments

0.165 ± 0.01

0.56 ± 0.01 *#

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Hippocampus

Control

9.46 ± 0.38

40.82 ± 0.63

*#

*#

2.95 ± 0.02

0.15 ± 0.01 0.47 ± 0.01 *#

33.45± 0.62

Sha-IV

16.12 ± 0.37*#

26.63 ± 0.19 *#

1.92 ± 0.02 * #

578.28±8.18 *# 0.34 ± 0.01 *#

20.11 ± 0.68*#

22.65 ± 1.01 *#

1.67 ± 0.02 *

835.03±1.87 *# 0.54 ± 0.02 *

18.57 ± 0.47*#

26.82± 0.94 *#

1.98 ± 0.04 *#

668.84±1.53 *# 0.36 ± 0.01 *#

9.62 ± 0.33 #

40.36 ± 1.07 *#

2.43 ± 0.01 *#

625.08±1.19 *# 0.30 ± 0.01 *#

Scol

32 ± 0.4 *

17.28 ± 0.35 *

1.69 ± 0.03 *

702.67± 1.22 *

Scol + Pira

16.17 ± 0.53 *#

22.95 ± 0.49 *#

1.88 ± 0.04 *#

520.30±1.34 *# 0.42 ± 0.02 *#

Scol + AAE 11.52 ± 0.26 *#

31.57 ± 0.65 *#

2.08 ± 0.02 *#

625.32±1.47 *# 0.35 ± 0.01 *#

AAE 200

200

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11.92 ± 0.35

AAE 100

604.49±2.28

*#

Pira

AAE 50

2.35 ± 0.02

267.02 ± 1.67 *#

0.56 ± 0.02 *

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Values were expressed in mean ± SEM (n=6). * compared to control animals; # compared to scopolamine induced animals; Pira: Piracetam; SHA-IV: Shatavarin IV (1.9 mg/kg); AAE 50, 100 and 200: Asparagus adscendens extract 50, 100 and 200 mg/kg respectively.

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There was a statistically significant difference in reduced glutathione level in cortex [F (8, 54) = 147.6, p < 0.0001] and hippocampus [F (8, 54) = 122.4, p < 0.0001] in between the groups. The post hoc test (Student-Newman-Keuls test) suggested a significant decrease in reduced glutathione level was recorded in cortical and hippocampal regions of brain with

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scopolamine induced amnesia as compared to control animals. However, piracetam and AAE (50, 100 and 200 mg/kg) significantly (p < 0.05) increased the reduced glutathione level in cortical and hippocampal regions of brain as compared to control animals.

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Similarly, the piracetam and AAE 200 mg/kg reverses the scopolamine induced amnesia as evidenced by increased reduced glutathione level as compared to scopolamine induced animals (Table 6).

One-way ANOVA showed that catalase level was significant different in both cortex [F

(8, 54)

= 326.3, p < 0.0001] and hippocampus [F

(8, 54)

= 212, p < 0.0001] in between the

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groups. The post hoc analysis revealed a significant elevation in catalase level was recorded in cortical and hippocampal regions of brain with scopolamine induced amnesia as compared to control animals. However, piracetam and AAE (50, 100 and 200 mg/kg) significantly (p < 0.05) reduced the catalase level in cortical and hippocampal regions of

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brain as compared to control animals. Similarly, the piracetam and AAE 200 mg/kg reverses the scopolamine induced amnesia as evidenced by reduced catalase level as

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compared to scopolamine induced animals (Table 6). 3.6 Changes in total nitrite level There was a statistically significant difference in nitrite level in cortex [F (8, 54) = 3116, p < 0.0001] and hippocampus [F (8, 54) = 2429, p < 0.0001] in between the groups. The post hoc test (Student-Newman-Keuls test) suggested a significant increase in nitrite level was recorded in cortical and hippocampal regions of brain with scopolamine induced amnesia as compared to control animals. However, piracetam and AAE (50, 100 and 200 mg/kg) significantly (p < 0.05) reduced the total nitrite level in cortical and hippocampal regions of

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brain as compared to control animals. Moreover, the piracetam and AAE 200 mg/kg reverses the scopolamine induced amnesia as evidenced by improved total nitrite level as compared to scopolamine induced animals (Table 6).

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3.7 Changes in acetylcholinestrase activity One-way ANOVA showed that acetylcholinesterase activity was significant different in both cortex [F

(8, 54)

= 185, p < 0.0001] and hippocampus [F

(8, 54)

= 83.58, p < 0.0001] in

between the groups. The post hoc analysis revealed a significant increase in

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acetylcholinesterase activity was recorded in cortical and hippocampal regions of brain with scopolamine induced amnesia as compared to control animals. However, piracetam

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and AAE (50, 100 and 200 mg/kg) significantly (p < 0.05) reduced the acetylcholinesterase activity in cortical and hippocampal regions of brain as compared to control animals. Moreover, the piracetam and AAE 200 mg/kg reverses the scopolamine induced amnesia as evidenced by reduced acetylcholinesterase activity as compared to scopolamine induced animals (Table 6).

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4. Discussion

The PASS prediction spectra of phytoconstituents present in Asparagus adscendens roots revealed the excellent score for nootropic potential. Also, the above predicted nootropic potential was further supported by relevant mechanisms as analyzed by

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Pharmaexpert. The predictive values of these in silico tools have been validated in our laboratory as well as by other laboratories too [24, 41-43]. Therefore, based on the

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excellent score in PASS prediction, the nootropic and anti-amnesic effect of A. adscendens root was assessed using different behavioral models, namely radial arm maze (RAM), passive shock avoidance paradigm and scopolamine induced amnesic model. Furthermore, the acetylcholinesterase and antioxidant activities were evaluated to elucidate the nootropic mechanism of A. adscendens root. The radial arm maze model has been extensively used to investigate the learning and spatial memory performance in rodents [44-46] and has also been validated in our laboratory too [23]. Decrease in reference memory error, working memory error and

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retrieval latency in the RAM model is taken as an index of enhanced spatial memory [47]. Our results showed that pretreatment with AAE and Shatavarin IV significantly enhanced the memory as evidenced by reduced reference memory error, working memory error and retrieval latency on the eighth day of assessment as compared to control animals. Similarly,

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the piracetam also significantly improved the spatial memory as expected [48, 49]. Moreover, the memory enhancing potential was best at AAE 200 mg/kg and comparable with the Shatavarin IV and standard drug piracetam. Further, AAE also showed the beneficial effect on fear associated memory as evaluated in passive shock avoidance test.

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The pretreatment with AAE and Shatavarin IV significantly decrease the number of mistakes and increase the step down latency as compared to control animals. The AAE 200

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mg/kg dose showed significantly enhanced memory as indicated by no mistake along with increased step down latency on day 8. As AAE significantly improved the memory in the absence of any cognitive deficit, it can be suggested to possess nootropic potential, which is in line with the PASS prediction which revealed that maximum number of phytoconstituents possess nootropic activity.

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After validating the memory enhancing potential of AAE, we then evaluated the AAE in scopolamine-induced amnesia model to find out whether it would be effective in reversing learning and memory deficits due to cholinergic perturbations. Cholinergic neurotransmission plays a critical role in the process of learning and memory [50, 51].

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Many medicinal plants and their phytoconstituents were known to possess memory enhancing potential through favorably modulating the cholinergic system [52, 53]. The cholinergic system is also associated with the pathophysiology of Alzheimer’s disease and

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other neurodegenerative disorders. Scopolamine, a muscarinic cholinergic receptor antagonist, impairs cholinergic signaling and produces a memory deficit [54, 55]. Thus, the scopolamine-induced model has been extensively used as an experimental model to assess medicinal plants and their phytoconstituents [56, 57]. Scopolamine in the current study caused amnesia as indicated by increased working memory error, reference memory error, retrieval latency and increased number of mistakes along with decreased step down latency in radial arm maze and passive shock avoidance test respectively. Pretreatment with AAE 200 mg/kg significantly reversed the scopolamine induced amnesia as observed

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in RAM and PSA compared to scopolamine control animals. Although, the pretreated AAE 200 mg/kg animals significantly reduced the reference memory error, but not the working memory error in scopolamine induced animals, indicating AAE might be helpful in maintaining long term memory as working memory represents the short term memory

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while the reference memory represents long term memory [11, 58]. The AAE 200 mg/kg dose was significantly more effective than piracetam in reversing the memory deficits induced by scopolamine, making it more promising drug for the treatment of cognitive

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disorders.

In context to the behavioral results on scopolamine induced amnesia, we can expect that the mechanism responsible for memory improvement induced by AAE may depend on

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the inhibition of AChE activity. Many studies suggest that scopolamine induced memory impairment can be antagonized by the cholinergic agonist drugs such as physostigmine, tacrine which increases the level of acetylcholine in brain [59, 60]. Taking together, the AAE might have cholinomimetic effect in reversing the scopolamine induced amnesia. To assess whether AAE has any cholinomimetic effect, the acetylcholinesterase activity was

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evaluated in the cortical and hippocampal regions, which are primarily involved in the process of memory [61, 62]. In the present study, scopolamine significantly increased the acetylcholinesterase activity, as speculated [63, 64]. On the other hand, AAE pretreatment significantly inhibited the acetylcholinesterase activity in cortical and hippocampal regions

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as compared to scopolamine control animals. These results suggest that AAE significantly enhances the cholinergic neurotransmission thus enhances the learning and memory process. Moreover, the cholinomimetic effect of AAE by virtue of its acetylcholinesterase

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activity also supported by the PASS prediction suggesting the maximum number of phytoconstituents in AAE act as an acetylesterase inhibitor and acetylcholine release stimulant.

Many clinical and preclinical studies have suggested the strong evidences that

oxidative stress is involved in the pathogenesis of neurodegenerative disorders such as Alzheimer’s disease [65, 66]. Recently, El-Khadragy et al., (2014) reported that oxidative stress induced by scopolamine in rats is associated with the altered level of TBARS, GSH and NO in the discrete parts of the brain. The enhanced brain oxidative status of

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scopolamine induced amnesic animals resembled the clinical pathology observed in Alzheimer’s disease patients [67]. In the present study, scopolamine resulted in a significant increase in TBARS and total nitrite levels and decreased GSH levels in both cortical and hippocampal regions. The pretreatment with AAE 200 mg/kg significantly

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preserved the activity of both TBARS and GSH in the cortex and hippocampus to a similar level observed in control animals. Although the increased total nitrite level induced by scopolamine significantly reduced but not as that of control animals. The above results were in line with PASS prediction suggesting the maximum number of phytoconstituents

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possess antioxidant activity. Moreover, the literature also revealed that the AAE has an antioxidant potential [20, 21], which might be responsible for the decreased oxidative

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stress.

Pretreatment with AAE significantly augment the noradrenergic, dopaminergic and serotonergic neurotransmission (Unpublished data) in both cortical and hippocampal regions. In line with, the role of noradrenergic [68, 69], dopaminergic [70-72] and serotonergic [73, 74] in learning and memory process is well documented. Hence, the

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nootropic potential may also involve monoaminergic neurotransmission apart from the cholinergic system. However, the role of monoaminergic neurotransmission needs to be explored before coming to any conclusions on its precise mechanism of action. In all cognitive tests, AAE at highest dose exhibited almost comparable response as

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that of standard nootropic drug piracetam. Although Shatavarin IV treatment showed the nootropic effect in all the cognitive tests, but the effect was less significant than AAE. This

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might be due to the presence of other phytoconstituents in the plant extract. Albeit steroidal saponins and other phytoconstituents present in the A. adscendens roots which have been reported to cross the blood brain barrier (BBB) [75-77] but the better ability of phytoconstituents in extract form in comparison to isolated pure form to get through BBB may be the another reason for more significant effect of AAE as compared to Shatavarin IV. In conclusion, AAE showed potent memory enhancing activity by the inhibition of acetylcholinesterase activity and by the regulation of oxidative stress. Therefore, AAE might be beneficial in treating and preventing the cognitive deficit disorders.

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Acknowledgement The authors are deeply grateful to the Council of Scientific and Industrial Research (CSIR), Pusa, New Delhi, India for providing financial assistance [Vide F.No. 38(1339)/12/EMR-II]

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for the project, and project fellowship to Miss. Priyanka Pahwa. We are also thankful to Dr. Bikram Singh, CSIR- Institute of Himalayan Bioresource Technology, Palampur, H.P. for the authentication of plant material.

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Conflict of Interest The authors declare that there are no conflicts of interest.

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Research Highlights

Phytoconstituents of AAE (Asparagus adscendens extract) showed excellent score

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for nootropic and antioxidant activities. AAE administration showed nootropic activity in behavioral studies (radial arm maze and passive shock avoidance paradigm)

Pretreatment with AAE significantly reduced the acetylcholinesterase activity.

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Pretreatment with AAE significantly reduced the oxidative stress.