Neuroprotective effects of bergenin in Alzheimer’s disease: Investigation through molecular docking, in vitro and in vivo studies

Accepted Manuscript Title: Neuroprotective effects of bergenin in Alzheimer’s disease: Investigation through molecular docking, in vitro and in vivo studies Authors: Priyal Barai, Nisith Raval, Sanjeev Acharya, Ankit Borisa, Hardik Bhatt, Niyati Acharya PII: DOI: Reference:

S0166-4328(18)30740-X https://doi.org/10.1016/j.bbr.2018.08.010 BBR 11534

To appear in:

Behavioural Brain Research

Received date: Revised date: Accepted date:

25-5-2018 20-7-2018 11-8-2018

Please cite this article as: Barai P, Raval N, Acharya S, Borisa A, Bhatt H, Acharya N, Neuroprotective effects of bergenin in Alzheimer’s disease: Investigation through molecular docking, in vitro and in vivo studies, Behavioural Brain Research (2018), https://doi.org/10.1016/j.bbr.2018.08.010 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.

Neuroprotective effects of bergenin in Alzheimer’s disease: Investigation through molecular docking, in vitro and in vivo studies Priyal Barai1, Nisith Raval1, Sanjeev Acharya2, Ankit Borisa1, Hardik Bhatt1, Niyati Acharya1* Institute of Pharmacy, Nirma University, S. G. Highway, Ahmedabad, Gujarat – 382481, India 2 Principal, SSR College of Pharmacy, Sayli, Silvassa – 306230, U. T. of D&NH, India

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Graphical Abstract

Highlights

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Bergenin reported as neuroprotective, BACE-1 inhibitor, tyrosine hydroxylase inhibitor as well as hPTP1B inhibitor was screened using in silico, in vitro as well as in vivo studies for AD management Docking of bergenin into various targets of AD i.e. AChE, BuChE, BACE-1 and Tau Protein KinaseI (GSK-3β) showed high GOLD Fitness and Internal GOLD score indicating its binding interactions in these site Bergenin was safe upto 50 µM in MTT and Resazurin reduction-based assay and also prevented NMDA induced cytotoxicity in SH-SY5Y cells Bergenin was evaluated in scopolamine induced amnesia and ICV STZ induced model of AD in rats led to reduction mean escape latency (MEL) in MWM and improvement in mean percentage alternations (MPA) in Y maze Bergenin inhibited acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) enzymes and attenuated of oxidative stress through augmentation of decreased GSH levels Bergenin caused restoration of cytoarchitecture of hippocampus damaged by scopolamine as well as STZ ELISA assays showed reduction in Aβ1-42 levels, phosphorylated tau protein as well as GSK-3β levels in brain homogenates of rats

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ABSTRACT: Alzheimer’s disease (AD) is an enervating and chronic progressive neurodegenerative disorder, occurring frequently in the elderly and adversely affecting intellectual capabilities and the cognitive processes. Bergenin possesses efficacious antioxidant, antiulcerogenic, anti-HIV, hepatoprotective, neuroprotective, antiinflammatory and immunomodulatory activity along with antinociceptive effect and wound healing properties.

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Previous studies have shown that bergenin has in vitro bovine adrenal tyrosine hydroxylase inhibitory activity, mushroom tyrosinase inhibitory activities, β-secretase (BACE-1) enzyme inhibitory activity and prevented neuronal death in the primary culture of rat cortical neurons. Protein tyrosine phosphatase-1B (PTP1B) is an intriguing target for anticancer and antidiabetic drugs and has recently been implicated to act as a positive regulator of neuroinflammation. Bergenin is also found to inhibit human protein tyrosine phosphatase-1B (hPTP1B) in vitro. Thus, bergenin was screened by molecular docking study using GOLD suite (version 5.2), CCDC for predicting its activity against targets of AD management like acetylcholinesterase (AChE) (1B41), butyrylcholinesterase (BuChE) (1P0I), Tau protein kinase 1 (GSK-3β) (1J1B), BACE-1 (1FKN) wherein the GOLD score and fitness of bergenin were comparable to those of standard drugs like donepezil, galanthamine, physostigmine, etc. Bergenin demonstrated dose-dependent inhibition of both AChE and BuChE in vitro and found to be safe up to 50 µM when screened in vitro on SH-SY5Y cell lines by cytotoxicity studies using MTT and Alamar blue assays. It also led to dose-dependent prevention of NMDA induced toxicity in these cells. Pretreatment with bergenin (14 days) in rats at three dose levels (20, 40 and 80 mg/kg; p.o.) significantly (p < 0.01) and dose-dependently alleviated amnesia induced by scopolamine (2 mg/kg, i.p.). The therapeutic effect of bergenin supplementation for 28 days, at three dose levels, was also evaluated in streptozotocin (3 mg/kg, ICV, unilateral) induced AD model in Wistar rats using Morris water maze and Y maze on 7 th, 14th, 21st and 28th days. STZ caused significant (p < 0.001) cognitive impairment and cholinergic deficit and increased oxidative stress in rats. Bergenin could significantly ameliorate STZ induced behavioral deficits, inhibit the AChE and BuChE activity in parallel with an increase in the diminished GSH levels in a dose-dependent fashion. The histopathological investigations were also supportive of this datum. The bergenin treatment at 80 mg/kg led to significant (p < 0.05) abatement of the raised Aβ-1-42 levels and alleviated the perturbed p- tau levels leading to significantly low (p < 0.01) levels of p-tau in brain homogenates of rats as compared to ICV STZ injected rats. In conclusion, the observed effects might be attributed to the cholinesterase inhibitory activity of bergenin coupled with its antioxidant effect, anti-inflammatory activity and reduction of Aβ-1-42 and p-tau levels which could have collectively helped in the attenuation of cognitive deficits. The current findings of the study are indicative of the promising preventive and ameliorative potential of bergenin in the management of AD through multiple targets. Keywords: Bergenin; SH-SY5Y; streptozotocin; scopolamine; Alzheimer’s; cholinesterase

1. Introduction

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Alzheimer’s disease (AD) is a debilitating and chronic persistent neurodegenerative disorder, a common cause of dementia frequently occurring in the elderly, and characterized clinically into psychological symptoms which collectively form the intellectual abilities and the cognitive processes [1]. The prevalence of this enervating disorder has increased affecting millions of people worldwide and it stands as a major issue of concern due to the increased average life expectancy. It lays a high socio-economic burden on the medical and healthcare systems of the developed and underdeveloped countries [1,2]. The majority of AD cases are of the sporadic AD (SAD, hereafter referred to as AD), which involves many etiopathogenic mechanisms including environmental, genetic and metabolic factors [3]. AD involves an important role played by levels and turnover regulation of acetylcholine (ACh) present in neuron and synaptic junctions [4]. The AD is accompanied by the destruction of cholinergic neurons, playing a vital role in cognition, leading to scarcity of the neurotransmitter ACh in the synapses and cognitive impairment [5]. The cholinergic hypothesis suggests that the prolongation of the effect of symptomatic ACh by enhancement of its signal transmission and counteracting its deficit by its replenishment can be brought about with the help of cholinesterase inhibitors [4]. The disparity in the homeostasis between the pro-oxidant and antioxidant homeostasis results in oxidative stress which is a major player a major role in AD pathology [6]. The other prominent neuropathological hallmarks of this disease are extracellularly occurring senile neuritic plaques of aggregated amyloid β (Aβ) and intracellularly prevailing neurofibrillary tangles (NFTs) comprised of hyperphosphorylated tau protein interspersed in subcortical nuclei and the cerebral cortex [7,8]. The Aβ peptides act as the primary inducer of the AD as proposed by the amyloid hypothesis, whereas, NFTs are essentially involved in the manifestation of the disease clinically [9]. BACE-1 (β- site APP cleaving enzyme-1) which is a β- secretase plays a key role by cleaving the amyloid precursor protein (APP) into APPβ and C-99 which is a membrane-associated C-terminal fragment cleaved by γ- secretase to form Aβ peptides [10]. These pathological features occur primarily in the cerebral cortex and hippocampal areas and are conjectured to be linked with cognitive impairment through execution of cholinergic hypofunction, reactive oxygen/ nitrogen species (ROS/RNS) generation, inflammation in neurons and eventual forfeiture of neuronal cells [4,11].

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The first line pharmacotherapy being used for the symptomatic treatment of AD and slowing AD progression include the acetylcholinesterase (AChE) inhibitors (tacrine, donepezil, galantamine and rivastigmine) which are capable of effectively elevating the attenuated ACh concentrations in the affected brain by reducing the ACh breakdown. The AChE inhibitors can lengthen the half-life of ACh but lead to the only modest symptomatic relief which is demonstrated by the beneficial effects on cognitive, functional and behavioral symptoms. Moreover, these enzyme inhibitors are associated with various side effects due to the cholinergic stimulus in the brain and peripheral tissues [12]. Many promising agents have been proven unsuccessful in the clinical trials due to tolerability issues and their properties to provide only symptomatic relief of cognitive dysfunction. Thus, such limitations of the current therapeutic and growing interest in preventive approaches, persuade the researchers to search for the agents with properties to prevent cognitive dysfunction accompanied by protective action against neuronal death [12,13]. This builds interest in the phytotherapy which leads to potential improvement in the AD symptoms by targeting the cholinergic pathway along with other related downstream pathways with less number of adverse effects and can be employed as a preventive approach. Many plants have been reported to be used traditionally by the folks for curing various neurodegenerative disorders and bioactives derived from them have been reported to show cognitive improvement. Galanthamine, physostigmine, rivastigmine,

huperzine A, etc. are few naturally or semisynthetically derived AChE inhibitors indicated for AD management [14].

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Bergenin is a colorless crystalline isocoumarin primarily obtained from Bergenia species and is a C-glucoside of 4-O-methyl gallic acid. It has been reported with antioxidant, antiinflammatory, antiarthritic, immunomodulatory, antinarcotic, wound healing, antidiabetic and in vitro neuroprotective activity. Bergenin also demonstrates hepatoprotective, hypolipidemic, antitussive, antiarrhythmic, antimicrobial, antiviral and anticancer effects [15]. Bergenin pretreatment leads to a significant reduction of lipid peroxidation in the liver, brain and red blood cell of rats against 2, 4-dinitrophenyl hydrazine-induced tissue damage [16–18]. Various analogs of bergenin have demonstrated dose-dependent BACE-1, antioxidant activity and in vitro tyrosinase inhibitory activity [19,20]. Bergenin has been proven to inhibit human protein tyrosine phosphatase-1B (hPTP1B) activity in vitro [21]. Few norbergenin derivatives have been reported to show potent neuroprotective activity in the primary cultures of rat cortical neurons [22]. Potential neuroprotective effects of crude extracts of Bergenia ciliata as observed in our preliminary studies in intracerebroventricular streptozotocin (ICV STZ) induced model in rats and multiple reports of bergenin as an antioxidant, anti-inflammatory and antidiabetic raised our interest in exploring its beneficial effects in SAD [23]. To the best of our knowledge, the neuroprotective potential of bergenin in animal models of the AD has not been explored before.

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The in silico molecular docking studies were performed to get a preliminary idea about the binding interactions of the compounds with various targets such as AChE, butyrylcholinesterase (BuChE), BACE-1 and tau protein kinase 1 (Glycogen Synthase Kinase 3 beta, GSK-3β). SH-SY5Y neuroblastoma cells were used to evaluate its cytotoxicity and preventive effects against the N- methyl D- aspartate (NMDA) induced toxicity through MTT and Alamar blue assays. Scopolamine, a potent non-selective muscarinic ACh receptor antagonist, on intraperitoneal injection acts as an inducer of cognitive impairment similar to the AD in experimental animals by inhibiting depolarization of receptors and thereby blocking cholinergic neurotransmission [24,25]. ICV STZ injection generates an “insulin-resistant brain state” and triggers temporal fruition of various characteristics that replicate occurring in the SAD patients in the actual scenario such as the presence of Aβ aggregates in the cerebral capillaries and hyperphosphorylated tau protein in the rodent brains. The ICV STZ has been proven to cause the cholinergic deficit, cerebral hypometabolism and increased oxidativenitrosative stress as an indication of the disturbance of the cell homeostasis [26–29]. Thus, the current study was undertaken with an aim to determine the beneficial effects of the bergenin pre-treatment and post-treatment in vivo at three dose levels i.e. 20 mg/kg, 40 mg/kg and 80 mg/kg on the cognitive impairment induced by scopolamine and STZ respectively and to determine whether the beneficial effects are exerted through cholinergic receptors, mitigation of oxidative stress, amyloid β reduction, inhibition of tau phosphorylation and/or GSK3β. The study was done with an aim to screen the effects of bergenin on multiple targets and the promising results of the study are indicative of its potential use in AD management as a preventive approach or as an adjuvant to the currently used drugs owing to its actions on multiple targets.

2. Materials and methods 2.1 Drugs and chemicals

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(-) Scopolamine hydrobromide trihydrate (hereafter referred to as scopolamine) was procured from Sigma Aldrich, Steinheim, Germany and was dissolved in physiological saline. Streptozotocin (STZ) (MP Biomedicals, California, USA) was solubilized in ACSF (artificial cerebrospinal fluid) (CMA). Bergenin (> 98 % purity) was procured from Adooq Bioscience, Irvine, CA, USA through Biogenuix Medsystem Pvt. Ltd, New Delhi, India. Memantine and donepezil HCl (DPZ) were received as a gift sample from Sun Pharmaceutical Industries Ltd., Vadodara, Gujarat, India and Torrent Pharmaceuticals Limited, Ahmedabad, Gujarat, India respectively. DPZ was administered orally by dissolving in deionized water. Bergenin was administered orally in the form of a suspension in 0.3% sodium carboxymethylcellulose (NaCMC). 2,2-Diphenyl-2-picrylhydrazyl (DPPH), acetylcholinesterase (AChE) from electric eel, butyrylcholinesterase (BuChE) from equine serum, butyrylthiocholine iodide (BuTCI) and Resazurin sodium (Alamar blue) were procured from Sigma Aldrich, Steinheim, Germany. AcTCI (acetylthiocholine iodide), DTNB [5,5’-dithiobis (2-nitrobenzoic acid)] and NMDA were purchased from Tokyo Chemical Industry, Tokyo, Japan whereas thiobarbituric acid and reduced glutathione (GSH) were procured from Sisco Research Laboratories Pvt Ltd., Mumbai, Maharashtra, India. Various chemicals and reagents employed for the experimentation were of analytical grade and all the solutions were always prepared freshly before use. SH-SY-5Y cell lines were procured from National Center for Cell Science (NCCS), Pune, Maharashtra, India. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) reagent (MTT), the Nutrient mixture F-12 Ham (Kaighn’s modification), trypsin, antibiotic-antimycotic mixture and Fetal Calf Serum (FCS) were procured from HiMedia Laboratories Pvt. Ltd., Mumbai, India. The ELISA kits for Aβ-1-42 (KMB3441), rat phosphor tau protein (E-EL-R1090) and GSK3β protein (SED317Ra) estimation were procured from Invitrogen™ Corporation, Frederick, MD, USA, Elabscience, Houston, TX, USA and Cloud-Clone Corp., Katy, TX, USA respectively.

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2.2 In silico molecular docking study using Genetic Optimization Ligand Docking GOLD (version 5.2)

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The molecular docking studies were carried out the binding interactions of bergenin as compared to the using GOLD (version 5.2), CCDC. Bergenin along with various reported drugs used for AD management was docked into AChE (1B41; human acetylcholinesterase complexed with fasciculin-II, glycosylated protein), BuChE (1P0I; human butyrylcholinesterase), Tau protein kinase 1 (GSK-3β) (1J1B; binary complex structure of human tau protein kinase 1 with AMPPNP) and BACE-1 (1FKN; structure of beta secretase complexed with inhibitor) with an aim to check preliminarily, the probable binding interactions and orientations with these targets using GOLD (version 5.2). The docking procedure involved retrieval of structures of various targets from PDB after which the ligand binding site of the enzyme/ protein was analyzed. This was followed by the elimination of ligand from the cocrystallized proteins and docked on the PDB using reference binding site and pose. The binding pose was then validated by using actual ligand binding followed by which the test compounds were docked to obtain the GOLD scores, GOLD fitness and interactive amino acids. Comparative GOLD scores and fitness of bergenin were compared to those of the standard reference drugs such as donepezil, physostigmine, and galanthamine which are few of the currently approved drugs used for AD management. 2.3 In vitro evaluation of bergenin for beneficial effects in AD management

2.3.1 Cell culture, treatments and cytotoxicity evaluation by cell viability and cell proliferation assays

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2.3.1.1 Cell culture conditions SH-SY5Y cells were maintained in a monolayer under in vitro conditions in tissue culture flasks in a CO2 incubator maintained at 37ºC which provided an ambiance of humidified atmosphere along with 5% CO2 and 95% air to the cells. The cells were cultured in Nutrient Mixture Ham’s F12 medium supplemented with 10 % FCS and antibiotic-antimycotic solution. For plating into 96 well plates, the counting was done using trypan blue exclusion assay using 0.4% trypan blue solution. All the experiments were conducted in cells of passage between 16 and 24 [30,31].

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2.3.1.2 Measurement of cell proliferation by MTT assay and Resazurin reduction-based cell viability assay MTT assay and resazurin reduction based assays were explored to check for the effects of bergenin on the viability of SH-SY5Y. Briefly, the cells were seeded in aliquots of 100 µL into 96-well plates at a density of 1 × 105 cells per well [32] for MTT assay and 5 × 104 cells/well [33] for the resazurin assay and were incubated in the ambient conditions in a CO2 incubator where they lied for 12 h. The cells were then treated with 100 µL solution of bergenin solubilized in a minimum amount of DMSO (final concentration < 0.01%) and suspended in serum-free culture medium (insult medium) at the effective concentrations 50 nm, 0.5 µM and 50 µM and incubated for 48 h in the incubator at 37 ºC with humidified environment and 5% CO2. Each test was performed in triplicates for statistical significance. Thereafter, 0.02 mL of MTT solution [5 mg/mL in sterile PBS (0.01 M; pH 7.4)] or 0.01 mL resazurin sodium solution (0.4 mg/mL in sterile PBS) and filtered through 0.2-µ filter, were added aseptically in dark conditions to the wells and kept in dark for a span of 4 h in humidified surroundings containing 5% CO2 at 37 ºC in the incubator [34,35]. For MTT assay, the medium was aspirated, and cells were resuspended with 0.1 mL DMSO with gentle shaking for a period of 10 min to homogenize the formazan crystals and ensure its complete solubilization [36] and the absorbance was recorded at 578 nm using ELISA plate reader. For resazurin assay, the O.D. was measured at room temperature [37] using primary wavelength 578 nm and secondary wavelength 630 nm using a microplate reader [38,39]. The results were normalized with respect to the control well absorbance which consisted of cells in media only indicating 100 % cell viability and with DMSO i.e. solvent control to account for the cell viability in absence of compound and in presence of the minimum amount of DMSO (<0.1%). Blank well consisted of wells which were not seeded with cells but were filled with cell culture medium only and wells not seeded with cells but added with bergenin.

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2.3.1.3 Evaluation of the preventive effect of bergenin on NMDA induced cytotoxicity in SHSY5Y cells In the preliminary pilot trial, the concentration at which around 50 % cell death was observed in SH-SY5Y cells i.e. 2.5 mM effective concentration was selected to observe the preventive effects of bergenin on NMDA induced toxicity in cells [40,41]. SH-SY5Y cells (1 × 105 cells per well; 100 µL) were plated in 96 well plates and incubated for 12 h in the incubator and on the next day, the drugs were treated with 50 µL volumes of bergenin solubilized in insult medium at effective concentration 5-50,000 nM for 24 h. Thereafter, the cells were treated with 50 µL NMDA dissolved in sterile PBS to induce excitotoxicity induced cell death. Then, 20 μL MTT solution (5 mg/mL) was added after 24 h of incubation and the remaining procedures followed as mentioned previously [30,31]. Suitable solvent controls, cell controls, blank, NMDA controls were run alongside, and the percent cell viability was expressed as mean ±

SEM with respect to controls after deducting the O.D. values from the corresponding blank readings. 2.3.2 In vitro acetylcholinesterase and butyrylcholinesterase inhibition assay

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The assay was accomplished by slightly modifying the generic assay procedure reported by Ellman et al., 1961 [42]in 96 well plates. The reaction mixture (200 µL) consisted of 160 µL phosphate buffer (0.1 M, pH 8.0), 5 μL of 5 mM DTNB, 20 μL of enzyme (0.5 units/mL), 5 μL of 10 mM AcTI and test compounds (10 μL). The rise in absorbance was measured at intervals of 2.5 min up to 30 min at 405 nm using a microplate reader and the plates were shaken for a span of 5 sec prior to each recording of absorbance. The BuChE activity was determined similarly except that the concentration of enzyme employed was 0.25 units/mL and BuTCI was used as the substrate at the similar concentrations. Each of the samples was evaluated in triplicates against appropriate blanks and percentage inhibition was calculated in terms of the difference in absorbance per min (∆ O.D.) with respect to that of the enzyme control. 2.3.3 DPPH free radical scavenging assay

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The percentage antioxidant capacity of bergenin was determined by DPPH free-radical scavenging assay in 96 well plates. The detailed procedure has been mentioned in the Supplementary information.

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2.4 In vivo evaluation of bergenin using scopolamine-induced amnesia model and ICV STZinduced AD model in rats

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2.4.1 Animals Healthy male albino Wistar rats weighing 250– 300 g and aged 8–9 weeks were housed in the departmental vivarium (22 ± 1 ºC and relative humidity 55 to 75 %). The rats were maintained in a colony in a density of three per polyacrylic cage with 12h/12h light: dark cycles and were provided the rodent chow and water ad libitum. The rats were fasted overnight prior to the day of icv STZ injection. The behavioral observations were performed between 10:00 a.m. and 20:00 p.m. in a research laboratory with quietude and diffused lights to eradicate the interference caused due to the reflections but at the same time to help the location of animal motion. Every day, the rats were transported to this laboratory, half an hour before the commencement of behavioral studies for the purpose of acclimatization. The handling of animals was done gently to evade the effects of stress on behavioral assessment [13,23,43]. The experimental procedures followed were in agreement with the CPCSEA guidelines and National Institute of Health Guide for the Care and Use of Laboratory Animals and the ethical approval was obtained from the Institutional Animal Ethics Committee (IAEC) prior to the experiments with IAEC protocol numbers IP/PCOG/PHD/19/018 and IP/PCOG/FAC/20/021 [44,45].

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2.4.2 Experimental protocol and group assignment for scopolamine-induced amnesia in rats The male Wistar rats were randomly alienated into six groups (n=6 per group) [25]. Normal control (NC) and Disease control (DC) i.e. scopolamine treatment groups were administered with vehicle (0.3% sodium CMC suspension) orally while DPZ (5 mg/kg, b. wt., p.o.) acted as reference drug group. Bergenin was dosed orally at 20, 40 and 80 mg/kg b. wt., p.o., daily for 14 consecutive days. The rats were imparted five-day training of Morris Water Maze (MWM) prior to the initiation of the protocol [46]. The behavioral assessment was carried out once on the 7th-day post initiation of dosing protocol to ensure that rats remember the behavioral task. Terminally, on the 14th day, after 60 min of oral administration [47], all the rats except those

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belonging to NC group were injected with a single dose of scopolamine in physiological saline (2 mg/kg b.wt., i.p.) to induce amnesia. The dose of scopolamine was selected based on a preliminary pilot study carried out in the laboratory and on the basis of previous reports of the anti-amnesic action of scopolamine. Thirty min after the scopolamine injection, the behavioral assessment was undertaken [48]. The experimental timelines and behavioral assessment schedules have been depicted in Fig. 1.

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Fig. 1. Experimental schedule, dosage regimen and behavioral assessment time frame for the scopolamineinduced AD model in rats.

2.4.3 Surgical procedure and experimental timelines for generation of ICV STZ-induced AD model animals

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The rats were first injected intraperitoneally with sodium thiopental (30 mg/kg) for anesthesia and placed onto a heating pad maintained at 36-38 ˚C. The head was shaved, the scalp was disinfected, and the skull was exposed with a midline sagittal incision. The rat was then placed on the stereotaxic frame (Stoelting, USA). The coordinates were selected as 0.8 mm AP, 1.8 mm ML and 3.6 mm DV. A burr hole was created at the juncture of AP and ML co-ordinates using a micromotor drill (diameter: 0.027-inch, Micro-drill system, 230V AC, Circuitmedic, Haverhill, MA, USA) and the DV co-ordinate was determined. Then, with the help of a 25 µL Hamilton microsyringe (Hamilton®, Switzerland), STZ (3 mg/kg b. wt.) was slowly injected into the right cerebral ventricle of the rat. The incision made was then sutured. The rats injected with an equivalent volume of ACSF served as the vehicle control. The site of injection being cerebral ventricles was confirmed by the microscopic examination after Hematoxylin-Eosin (HE) staining of the brain sections of few of the rats killed suddenly after being injected with a yellow non-toxic dye and macroscopic examination of the sections [13,23,49]. In the postoperative period, excessive care was taken until the rats gained consciousness and recuperated their normal activity to avoid any post-surgical physical trauma. Tramadol (12.5 mg/kg, i.p.) was injected as an analgesic for two post-surgical days to evade pain and gentamycin (5 mg/kg, i.p.) was injected as an antibiotic to avert infection. The normal motor function, body condition, body weight and dehydration were regularly monitored for entire week post-surgery [13]. Six days after surgery, the rats were selected based on general health and normal behavior in the tail suspension test [23,50]. One day prior to the initiation of dosing on the seventh post-surgical day, rats were randomly estranged into seven groups (n = 6 per group) and the therapeutic regimen for the experiment included [49]:  The normal control group (NC): Rats dosed orally with 0.3 % NaCMC suspended in PBS for 28 days. No surgery was performed on these rats.  The sham control group (SC): Rats injected ICV with 10 µL of ACSF and orally dosed with a NaCMC suspension prepared in PBS for 28 days  Disease control group (DC): Rats injected ICV with STZ and fed orally with the NaCMC suspension for 28 days  BERG (20 mg/kg), BERG (40 mg/kg) and BERG (80 mg/kg) groups: Rats ICV injected with STZ that received oral doses of bergenin at 20, 40 and 80 mg/kg b.w., in form of NaCMC suspension for 28 days

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The standard control group (DPZ 5 mg/kg): ICV STZ injected rats receiving DPZ 5 mg/kg b.w., p.o. for 28 days A pilot study (data not revealed) was also undertaken to observe any toxicity at the selected dose levels and nonappearance of any adverse effects or toxicity was confirmed prior to the initiation of the protocol. The behavioral manipulations were performed on 7th, 14th, 21st and 28th days ensuing the day from which the treatment began [13]. The experimental timelines are depicted in Fig. 2 in form of a symbolic diagram.

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Fig. 2. Experimental schedule, dosage regimen and behavioral assessment time frame for the streptozotocininduced AD model in rats.

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2.4.4 Morris Water Maze (MWM)

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A round tank (1.40 m diameter and 0.60 m height) was filled with water till 0.35 m height, rendered opaque with a non-toxic yellow colored herbal dye and sustained at 25-29ºC to avert the effects of hypothermia on the task performance of rats. The maze was virtually alienated into four equal quadrants (NE, NW, SE, SW) as depicted in Fig. 3(A). The polyacrylic escape platform (100 mm long x 8 mm wide) was placed amidst the SW quadrant. N, E, NW and SE positions were chosen as the release positions of the rats, being equidistant from the platform [51]. The behavioral prescreening, training and evaluation were performed using the methods mentioned in our earlier studies [23] and the detailed procedures have also been included in the supplementary data. The average time taken by the rats to reach the platform i.e. mean escape latency (MEL) were calculated for all the groups and compared to know the learning and memory deficits caused to the rats. The reduction in MEL and mean path lengths (MPL) were indicative of the reference memory task [13].

(A) (B) Fig. 3: (A) Division of four zones and position of the platform in a water maze and (B) Division of various zones in Y-maze.

2.4.5 Spatial Alternation Behavior (SAB) using Y maze

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The Y maze consisted of three wooden rectangular arms in shape of alphabet Y, two of which termed as B and C were equal in dimensions (length 0.50 m x height 0.30 m x width 0.15 m) and the third arm (length 0.70 m x height 0.30 m x width 0.15 m) was slightly longer than the other two. The software was used to divide the maze into various zones; viz A, B, C, and D (Fig. 3 (B)). The behavioral prescreening and evaluation were performed using the methods mentioned in our earlier studies [23] and the detailed procedures have also been included in the supplementary data. The SAB, observed as an indication of spatial memory, was assessed manually. The mean percentage alternation (MPA) was obtained as the average of the percentage of actual alternations out of the total alternations possible for the rats belonging to various test groups [52,53].

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2.4.6 Biochemical and Histopathological parameters Terminally, the rats were anesthetized by injecting sodium thiopental (30 mg/kg b. wt., i.p.. [49] to withdraw blood from the retroorbital cavity and separate serum immediately after behavioral analysis on the terminal day. Soon after, rats were injected with higher dose of sodium thiopental and whole brain samples were removed within 30 s after decapitation from the skull, washed with ice‑ cold isotonic saline and half of the samples were kept for neurochemical assays whereas the other half were stored in 4% formaldehyde at 4ºC for the histopathological examination [54,55]. For the preparation of homogenates, the isolated brains were, weighed and homogenized with ice-cold PBS (pH 7.4, 0.1M) to form 20% w/v homogenates which were subjected to centrifugation at 10,000g at 4ºC for a period of 15 min. The serum samples and supernatant obtained from homogenates was aliquoted into separate vials and stored at -80 ºC until use to avoid multiple freeze-thaw cycles [56]. 2.4.6.1 Preparation of paraffin-embedded tissues and HE-staining For H&E staining the tissue processing was performed in agreement with standard procedures of fixation, dehydration, impregnation, embedding and sectioning followed by staining with hematoxylin and eosin [57]. The micrographs of these sections (5-μm thickness) were captured with aid of a camera attached to an inverted light microscope (Olympus CKX41, Japan) (400X).

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2.4.6.2 Protein determination For the determination of protein concentration in the serum and brain homogenates of rats the method described by Lowry et al was employed using Bovine Serum Albumin (1 mg/mL) as standard [58].

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2.4.6.3 AChE and BuChE inhibitory activity The assay was performed in microplates by slightly modifying of the method of [42] by employing 5 µL of serum sample or 10 µL supernatant obtained from the brain homogenate in a final assay volume of 200 µL for determination of serum and brain AChE activity respectively. Microplate reader was used to monitor the rise in absorbance at 405 nm at intervals of 2.5 min for 30 min. Cholinesterase activity was determined according to the previously reported methods [23]. 20 µL of serum samples and 40 µL of supernatant from brain homogenates were employed for serum and brain BuChE activity determination and the substrate employed was BuTCI for BuChE activity determination. 2.4.6.4 Reduced glutathione (GSH) The reaction mixture (0.2 mL) consisted of 0.01 mL serum sample or 0.03 mL brain homogenates, 0.02 mL DTNB solution and the remaining volume was made up with 0.1 M phosphate buffer (pH 8). The absorbance was measured spectrophotometrically at 405 nm with the assistance of a microplate reader and GSH levels were calculated [59]

2.4.6.5 Determination of Aβ1-42 protein, phospo-tau (p-tau) and GSK3β from brain homogenates of ICV-STZ rats The determination of Aβ1-42, p-tau and GSK3β levels were performed in accordance with the user guide supplied with the respective kits using sandwich ELISA assays. Levels of Aβ42 and p-tau were expressed in terms of pg/mg protein whereas those of GSK3β were expressed in terms of ng/mg protein. 2.5 Statistical analysis

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For all the behavioural, biochemical and histopathological studies, values have been expressed as Mean ± SEM, where a, b and c indicate values that differ significantly from those of DC group at p < 0.05, 0.01 and 0.001 respectively, whereas a’, b’ and c’ represent values differing significantly from those of NC at p < 0.05, 0.01 and 0.001 respectively using one-way ANOVA followed by Bonferroni’s post-hoc test. The statistical data were scrutinized using GraphPad Prism® software (version 5.01, California, USA). 3. Results

3.1 In silico molecular docking study using GOLD (version 5.2)

Bergenin

57.9546

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Donepezil

67.7309

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Galanthamine

61.9206

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Physostigmine

56.9314

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47.5438

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57.3035 55.9427

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Interactive Amino acids

1B41 (Acetylcholinesterase) ARG 24, LYS 32, LEU 339, VAL 340, GLY 342, ALA -14.9545 343, PHE 346 TYR 61, ARG 24, VAL 340, GLY 342, ALA 343, PHE -8.1400 346 LYS 32, VAL 340, TYR 341, GLY 342, ALA 343, PHE -5.3697 346, SER 347 -9.7334 VAL 340, GLY 342, ALA 343, PHE 346 1P0I (Butyrylcholinesterase) ASN 245, LYS 248, PHE 278, VAL 279, VAL 280, PRO -14.9669 281 -9.2707 ASN 245, PHE 278, VAL 280, PRO 281 -5.3967 ARG 242, ASN 245, PHE 278, VAL 280

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GOLD Fitness

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51.1272

-9.7363

ASN 245, PHE 278, VAL 280, PRO 281

1J1B (Tau protein kinase 1 (GSK-3β)) 60.5180 -14.8216 LYS 85, ASP 133, TYR 134, VAL 135, ASP 200 ILE 62, GLY 68, LYS 85, LEU 132, ASP 133, VAL 135, 77.3513 -8.1485 ASP 200 68.8832 -5.3816 LYS 85, ASP 133, TYR 134, ARG 141, ASP 200 64.3742 -9.7218 ASP 200 1FKN (β- Secretase) ASP 32, GLY 34, TYR 71, THR 72, ASP 228, GLY 230, 61.0480 -15.0337 THR 231, ARG 235 ASP 32, GLY 34, PRO 70, TYR 71, THR 72, ASP 228, 78.6108 -8.1399 GLY 230, THR 231 65.9790 -5.3910 ASP 32, GLN 73, ASP 228, THR 231, ARG 235 58.1433 -9.7182 GLN 73, ASP 228, THR 231

Bergenin was docked into various identified targets for AD management AChE, BuChE, Tau protein kinase 1 (GSK-3β) and β- amyloid converting enzyme i.e. BACE-1. Bergenin showed high GOLD score and fitness values against all the targets which were comparable to those of

the well-known standard drugs like donepezil, galanthamine, and physostigmine and indicated strong binding interactions of the bergenin in the respective binding pockets. In case of molecular docking into 1B41, the most common amino acids which are responsible for interaction with standard compounds are ARG 24, LYS 32, VAL 340, GLY 342, ALA 343 and PHE 346. Bergenin interacted with all of them in various docking poses. The oxygen atoms of bergenin interact with VAL 340, GLY 342, PHE 346 forming hydrogen bonds at distances of 2.217 A˚, 2.790 A˚, and 2.976 A˚ respectively. While molecular docking into 1P0I, the amino acids interacting with standard compounds were found to be ASN 245, PHE 278, VAL 280 and

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PRO 281 respectively. The hydrogen bond interactions of bergenin were found to be with all of them forming hydrogen bonds with ASN 245, PHE 278 at distances 2.373 A˚ and 2.410 A˚ respectively.

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During molecular docking into 1J1B, the major interacting amino acids were found to be ILE 62, GLY 68, LYS 85, LEU 132, ASP 133, TYR 134, VAL 135, ARG 141 and ASP 200 out of which bergenin interacts with most of them forming hydrogen bonds. The hydrogen bonds formed by bergenin with LYS 85 and ASP 200 were at distances of 2.964 A˚ and 2.959 A˚ respectively. When compounds were docked into 1FKN, the major interacting amino acids were found to be ASP 32, GLY 34, PRO 70, TYR 71, THR 72, GLN 73, ASP 228, GLY 230, THR 231, ARG 235. Bergenin interacts with the maximum number of amino acids forming hydrogen bonds with ASP 32, GLY 34, ASP 228, THR 231 and ARG 235 at distances 2.792 A˚, 2.864 A˚, 2.969 A˚, 2.405 A˚ and 2.953 A˚ respectively. In many cases, the oxygen atom of bergenin formed multiple hydrogen bonds with same or different amino acids. These interactions owing to the presence of multiple oxygen atoms might be responsible for the strong binding resulting in good GOLD scores and fitness values. The GOLD score values, GOLD fitness and the interactive amino acids of various docking studies have been briefly summarized in table 1 whereas the docking interactions are given in Fig. 4.

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(A) 1B41

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(C) 1J1B (D) 1FKN Fig. 4. Docking images of Bergenin into various targets of AD (A) AChE (PDB ID: 1B41), (B) BuChE (PDB ID: 1P0I), (C) Tau protein kinase 1 (GSK-3β) (PDB ID: 1J1B) and (D) BACE-1 (PDB ID: 1FKN) depicting the binding interactions with various amino acids in the binding pocket.

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3.2 In vitro evaluation of bergenin for beneficial effects in AD management

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(C) Fig. 5. (A) The MTT assay in undifferentiated SH-SY5Y cells exposed to bergenin at different concentrations (5 nM – 50 µM) for 48 h. (B) Resazurin metabolism-based cell viability assay in undifferentiated SH-SY5Y cells exposed to bergenin at different concentrations (5 nM – 50 µM) for 48 h and (C) Preventive effects of bergenin of NMDA induced toxicity in SH-SY5Y cells.

Bergenin treatment at all tested doses led to non- significant reduction in cell viability when tested using both MTT and resazurin-based assay indicating its safety at all tested concentrations. The NMDA group showed significant (p < 0.001) reduction in the cell viability as compared to both Normal control (NC) and DMSO control groups. Bergenin at all tested doses led to significant (p < 0.001) prevention of the NMDA induced cell death with 500 nM concentration leading to highest cell viability as all other test groups except BERG 500 nM depicted significantly (p < 0.001) low cell viability compared to NC and DMSO control groups Values are expressed as Mean ± SEM, where #- p < 0.001 as compared to NC; * p < 0.001 as compared to NMDA control; @ p < 0.05 as compared to DMSO control; @@ p< 0.01 as compared to normal control; $- p < 0.001 as compared to BERG 500 nM; when compared using one-way ANOVA followed by Bonferroni’s posthoc test (n = 3)

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3.2.1 Cytotoxicity assays (MTT assay and resazurin reduction-based cell viability assay) The in vitro cytotoxicity evaluation using MTT assay and resazurin metabolism-based cell viability assay were performed to ascertain the consequence of exposure of bergenin at different concentrations (5 nM – 50 µM) for 48 h on the viability of undifferentiated SH-SY5Y cells. It was observed that the DMSO concentration (0.01 %) did not adversely influence the viability of SH-SY5Y cells (Data not shown). The results of the MTT assay revealed that at all the tested concentrations, bergenin did not manifest any marked cytotoxic activity on the SHSY5Y cells (Fig. 5 (A)). This was further confirmed by the resazurin reduction-based cell viability assay showed that the cells were metabolically active in the presence of bergenin as compared to control (Fig. 5 (B)). Both the assays revealed that the two lower concentrations tested corresponding to the therapeutic concentrations (50 nM-500 nM) showed around 80-97 % viability whereas the higher concentrations corresponding to the hyper-therapeutic concentration (50 µM) showed around 71-79% cell viability. Thus, bergenin did not show any adverse effects on cell proliferation.

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3.2.2. Evaluation of the preventive effect of bergenin on NMDA induced cytotoxicity in SHSY5Y cells The results of cytotoxicity assays indicated that bergenin is non-toxic to neuronal cells and hence it was further tested to check whether it could reverse the cytotoxic effects of NMDA challenge on SH-SY5Y cells. The NMDA treatment at concentration of 2.5 mM for 24 h led to significant (p<0.001) reduction in cell viability (49.33 ± 5.01% cell viability) indicating its toxic effect on the SH-SY5Y cells. This concentration of NMDA was selected for induction of cytotoxicity in SH-SY5Y cells. The effect of pre-treatment of bergenin at different concentrations (5 nM, 50 nM, 500 nM, 5,000 nM, 50,000 nM) on NMDA (2.5 mM) induced cytotoxicity was evaluated using MTT assay. The results of the study revealed that bergenin pre-treatment led to significant (p < 0.001) concentration-dependent reversal of NMDA induced cytotoxicity in the concentration range 5-500 nM. Bergenin at 500 nM led to the highest increase in cell survival (% cell survival: 81.75 ± 5.91; 32.4 % increase in cell survival) compared to all other groups tested against NMDA induced toxicity. The protective action of bergenin at this concentration was significantly (p < 0.001) higher when compared to that rendered by memantine (NMDA receptor antagonist) and DPZ standard. Bergenin at 5,000 and 50,000 nM could also reverse the NMDA toxicity significantly (p<0.001) but the cell viability was slightly below that obtained using 500 nM bergenin. Donepezil at 5 nM (67.34 ± 5.75 % cell survival, 18.01 %: increase in cell survival) and Memantine at 5 nM (71.85 ± 0.62 % cell survival; 22.52 %: increase in cell survival) and 50,00 nM (71.63 ± 4.98, 22.30 %: increase in cell survival) also led to significant (p<0.001) increase in cell survival but no specific dosedependent effect was observed in case of prevention of cytotoxicity. Thus, 500 nM bergenin afforded the most effective prevention against NMDA induced cytotoxicity (Fig. 5 (C)). 3.2.3 In vitro AChE inhibition assay

It was found that bergenin led to an increase in the % inhibition with an increase in concentration from 0.03 mM, 1.25 mM, 2.5 mM, 5 mM and 10 mM. The higher concentration showed inhibition which was comparable to that of 0.03 µM DPZ. The 0.03 mM led to an initial delay in the increase in absorbance, but the final absorbance value was similar to that of the enzyme control. Thus, higher concentration ranges of 1.25 mM to 10 mM were tried which led to a dose-dependent increase in percentage inhibition of the AChE enzyme activity (Fig. 6 (A)). 3.2.4 In vitro BuChE inhibition assay

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It was found that bergenin led to an increase in the % inhibition with an increase in concentration from 0.03 mM, 1.25 mM, 2.5 mM, 5 mM and 10 mM. The higher concentrations showed inhibition which was comparable to that of 3 µM DPZ. It was observed that all the tested concentrations led to mild and concentration-dependent inhibition of BuChE enzyme activity. The inhibition was mild as observed by the moderate reduction in the absorbance (Fig. 6 (B)). 3.2.5 In vitro DPPH inhibition assay

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The IC50 value for bergenin screened for in-vitro DPPH inhibition assay was found to be 330.28 µg/mL.

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Fig. 6. (A) In -vitro AChE inhibition assay and (B) In- vitro BuChE inhibition assay. BERG 1, BERG 2, BERG 3, BERG 4 and BERG 5 indicate 10 mM, 5 mM, 2.5 mM, 1.25 mM and 0.03 mM final concentration of bergenin in wells respectively; E: Enzyme only; M: Enzyme in presence of methanol at maximum concentration employed; DPZ- 0.03 µM and 3 µM final concentration of DPZ in well employed in AChE and BuChE assays respectively The absorbance versus time curves indicates that bergenin in a concentration-dependent manner led to decrease in the total absorbance of the test mixture with the highest dose leading to a maximum reduction in the absorbance and hence indicating highest degree of inhibition of AChE and BuChE. The effect of bergenin on inhibition of BuChE was less as compared to that of AChE Values are expressed as Mean ± SEM (n = 3)

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3.3 Evaluation of the prophylactic effect of bergenin treatment on the reversal of scopolamineinduced amnesia in rats 3.3.1 Effect of bergenin pretreatment on the performance of rats in MWM

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The MEL of the rats was significantly (around 1.6-fold, p < 0.001) increased along with the MPL compared to NC group post scopolamine injection on the 14th-day post oral dosing regimen (Fig. 7 (A) and 8). The bergenin pretreatment at 40 mg/kg and 80 mg/kg significantly (p < 0.05 and 0.01 respectively) attenuated the increase in MEL of the scopolamine injected rats. Bergenin 20 mg/kg pretreatment was ineffective in preventing the cognitive deficits incurred due to scopolamine injection. DPZ (5 mg/kg) led to the most significant (p<0.001) reduction in the MEL in scopolamine treated rats.

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3.3.2 Effect of bergenin pretreatment on Spontaneous Alternation Behaviour (SAB) of rats in Y maze task The MPA of the rats was significantly (around 1.97-fold, p < 0.001) decreased subsequent to administration of scopolamine on the 14th day compared to NC group. The bergenin pretreatment at 80 mg/kg led to significant (p < 0.001) improvement of the significantly abridged SAB deliberated owing to scopolamine. The advantageous effects of bergenin on SAB augmentation were analogous to those of DPZ (5 mg/kg) which also led to significant (p < 0.001) increase in the MPA (Fig. 7 (B)).

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(A) (B) Fig. 7. (A) Mean Escape Latencies (MELs) and (B) Mean Percentage Alternations (MPA) manifested by test groups on 14th day thirty min post scopolamine injection. Rats belonging to the DC group presented significantly (p < 0.001) higher MELs and significantly (p < 0.001) lower MPAs when compared to the NC group rats. Bergenin treatment in a dose-dependent manner led to amelioration of the behavioural deficits caused by dose-dependent decrease in MELs and dose-dependent increase in MPAs with 80 mg/kg treatment affording maximum benefit with significant (p < 0.01) reduction of MEL and significant (p < 0.001) increase in the MPA as compared to the DC group and the results were comparable to those of the DPZ group (n=6)

DPZ (5 mg/kg)

Fig. 8. The illustration of the path voyaged by the various test groups in MWM attained using SMART 3.0 Video Tracking Software of 14th day, 30 min post scopolamine injection. The path traveled by the DC group rats was noticeably longer than that traveled by the NC group rats to find the hidden platform and bergenin treatment could dose-dependently reduce the time taken and hence the path covered by the rats to discover the hidden platform which was analogous to that obtained using DPZ as reference drug

3.3.3 Effect of bergenin pretreatment on biochemical parameters in serum and brain homogenates of scopolamine injected rats

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3.3.3.1 Effect of bergenin pretreatment on the serum and brain AChE activity After scopolamine injection on 14th day a significant (p < 0.001) surge of the AChE activity was experiential in the serum samples and brain homogenates (2-fold) of DC group rats. The bergenin treatment at 80 mg/kg could significantly diminish the escalation in the AChE activity in serum (p < 0.01) and brain homogenates (1.5-fold, p < 0.001) of the pretreated rats comparable to that observed in DPZ (p < 0.001) (1.7-fold in the brain) treated rats. The low dose counterparts of bergenin pretreatment depicted no significant effects on the diminution of AChE activity in both serum and brain homogenates of rats (Fig. 9 (A and B)).

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3.3.3.2 Effect of bergenin pretreatment on serum and brain BuChE activity The BuChE activity evaluation in the serum and brain homogenates (1.38-fold) of the rats revealed significant outpouring in the BuChE activity (p < 0.05) after the scopolamine injection compared to the untreated rats. In case of brain homogenates, both DPZ (1.4-fold) and bergenin at 80 mg/kg (1.4-fold) portrayed significant (p < 0.05) dwindling of BuChE activity as compared to the DC group rats (Fig. 9 (C and D)). The 80 mg/kg bergenin treatment led to a lowering of serum BuChE activity, but the effect was non-significant as compared to the DC group. DPZ (5 mg/kg) led to a significant reduction (p<0.01) of the levels of serum BuChE activity as compared to the DC group.

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3.3.3.3 Effect of bergenin pretreatment on serum and brain GSH activity The assessment of the GSH levels in the serum and brain homogenates of the rats after behavioral analyses showed that scopolamine led to significant attenuation of the GSH levels in the plasma (p < 0.001) and brain homogenates (around 39 %, p < 0.01) of rats. It was perceived from the results that bergenin treatment led to dose-dependent improvement in the reduced GSH levels. Bergenin (80 mg/kg) lead to maximum escalation in the compromised GSH levels (p < 0.001) followed by that at 40 mg/kg (p < 0.01) and 20 mg/kg (p < 0.05). This replenishment of GSH levels was comparable to that revealed by DPZ (p < 0.01). Identical reversal of the raised brain GSH level was accomplished by bergenin treatment which led to significant improvement (p < 0.01) at all the tried dose echelons while DPZ was incapable of causing a significant rise in the reduced levels. The results of the study are in harmony with the previous reports of bergenin leading to an increase in GSH levels in various other animal models (Fig. 9 (E and F)) [60,61].

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Fig. 9. Alterations in biochemical parameters in serum and brain homogenates scopolamine injected rats (A) Serum AChE activity, (B) Brain AChE activity, (C) Serum BuChE activity (D) Brain BuChE activity, (E) Serum GSH levels (F) Brain GSH levels. The intraperitoneal scopolamine administration led to the significant elevation of AChE (p < 0.001) and BuChE (p < 0.05) activity in the serum and brain homogenates of the DC group rats as compared to that of the NC group rats. A significant decrease in the GSH levels in the serum (p < 0.001) and brain homogenates (p < 0.01) of DC group rats was observed. Bergenin treatment at 80 mg/kg led to most significant reduction of the elevated AChE (p < 0.01) and BuChE (p < 0.05) activity along with the refurbishment of the diminished GSH levels (p < 0.01) in the serum and brain homogenates of the rats when compared to the DC group rats and the results were analogous to those attained employing DPZ as standard (n=3)

3.3.4 Histopathological observations made in the brain post hematoxylin and eosin staining in scopolamine injected rats The histopathological observations of the hippocampal regions of rats i.e. CA1, CA2, CA3 and dentate gyrus of all the rats were compared for all the groups to observe the deleterious effects of scopolamine and preventive effects of bergenin treatment. The scopolamine injected rats demonstrated shriveled neuronal cell bodies, activated glia, increased numbers of vacuoles,

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indistinct membranes and became hyperchromatic. The hippocampus of the NC group, on the contrary, was unaffected and had well-outlined soma and with no or minimum vacuoles. The bergenin treatment exerted dose-dependent preventive effects on the hippocampal cytology by reducing the vacuolation and contraction of the perikaryal. The cells could retain the intact membranes and become normochromic (Fig. 10). The neuronal count performed using ImageJ software (1.51p, Wayne Rasband National Institute of Health, USA) revealed that the scopolamine injected DC group rats depicted significantly (p < 0.05) a low number of neurons in the hippocampal area when compared to that of the NC group rats. The bergenin (80 mg/kg) treated groups showed most significant (p < 0.05) improvement in the average neuronal count was comparable to that achieved by DPZ. This was followed by the 40 mg/kg dose which led to non-significant improvement and the 20 mg/kg dose which was ineffective in improving the neuronal count (Fig. 11).

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Fig. 10. The HE-stained histopathological images of various regions of hippocampus i.e. CA1, CA2, CA3 and dentate gyrus of rats belonging to various test groups in scopolamine-induced amnesia model observed under 400X magnification (scale bar-50 μm). The neurons in CA1, CA2, CA3 and dentate gyrus of hippocampi of NC group rats were healthy and compactly arranged with less or no intercellular spaces (designated with black arrows) but those belonging to DC group had numerous intercellular spaces (designated with black arrows) accompanied by noticeable loss of neurons in these regions. Bergenin pre-treatment could prevent this loss in a dose-dependent manner which was comparable to that attained by DPZ treatment (n=3).

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Fig. 11. Average neuron count per unit area in the hippocampal region of rats in scopolamine-induced amnesia model. The scopolamine injected DC group rats depicted significantly (p < 0.05) low number of neurons in the hippocampal area when compared to that of the NC group rats. The bergenin (80 mg/kg) treated groups showed most significant (p < 0.05) improvement in the average neuronal count was comparable to that achieved by DPZ. The neuronal count was performed by an experimenter blind to the groups with the aid of ImageJ software (n=3)

3.4 Evaluation of the ameliorative effect of bergenin treatment on ICV STZ induced AD model in rats

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3.4.1 Effect of bergenin post-treatment on the reversal of cognitive deficits in MWM

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The progressive decline in memory performance through ICV STZ was evinced through the 2.4-fold increase (p < 0.001) in the MEL at the end of the first week which was aggravated to around 7-fold (p < 0.001) increase by the end of the fourth week as compared to the NC group on corresponding evaluation days. Bergenin treatment could mitigate the significant increase (p < 0.001) in MEL fostered by STZ intervention (Fig. 12). The observed effect increased with the dose administered as the bergenin administration at 40 mg/kg and 80 mg/kg led to significant reversal (p < 0.001) of the deficits in spatial learning persuaded by STZ on all the evaluation days. The BM intervention at 20 mg/kg, during the 7th day of assessment, led to a moderate but non-significant reduction of the MEL but on chronic treatment, it could cause significant curtailment (p < 0.001) in the MEL indicating its late onset of action. The effect was also perceived from the reduction in MPL with DC group depicting the highest path length corresponding to the longer time taken to find the hidden platform (Fig. 13).

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(C) (D) Fig. 12. Mean Escape Latencies (MELs) of the test groups of ICV STZ induced model on (A) 7th day, (B) 14th day, (C) 21st day and (D) 28th day. The ICV STZ administration caused significant (p < 0.001) rise in the MELs of the DC group rats when compared to that of NC and SC group rats. The bergenin treatment could significantly (p < 0.001) and dosedependently decreased the raised MELs as compared to the DC group rats and the effect was found to be improving with time. The effect shown by the higher dose levels i.e. 40 and 80 mg/kg were equivalent to that of DPZ standard group (n=6).

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Fig. 13. The illustration of path trekked by the test groups in ICV STZ induced model MWM attained using SMART 3.0 Video Tracking Software. The path traveled by the DC group rats was markedly longer than that traveled by the NC and SC group rats to discover the hidden platform because of ICV STZ administration which also corresponded to longer MELSs. Bergenin treatment led to the dose-dependent reduction in the path covered by the rats

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3.4.2 Effect of bergenin post-treatment on the reversal of cognitive deficits in Y maze

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The deleterious effects of STZ were observed through 53.34 % - 71.875% reduction (p < 0.001) in MPA as compared to NC group when evaluated using the Y maze task over 28 days. Bergenin treatment could also mitigate the dwindling of MPA fostered by STZ intervention significantly (p < 0.001) (Fig. 14). The effect was dose-dependent with 80 mg/kg which was evident through the significantly higher (p < 0.001) MPA depicted by rats as compared to those injected with STZ. Bergenin treatment at 40 mg/kg exhibited gradual improvement in the mean percentage alternations with time showing that chronic treatment is required to completely exterminate the effects of STZ on SAB. The 20 mg/kg dose was not as effective as the higher dose analogs as it led to slight improvement in the SAB but could not completely bring the SAB back to normal levels. The effects of higher dose levels on augmentation of MPA were identical to those of DPZ treated group.

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Fig. 14. MPAs of the test groups of ICV STZ induced model on (A) 7th day, (B) 14th day, (C) 21st day and (D) 28th day. ICV STZ injection led to a significant decrease in the MPAs of the DC group rats when compared to NC and SC group rats. Bergenin treatment at 80 mg/kg led to highest MPAs in rats which were significantly (p < 0.001) higher than that of the DC group and the results were comparable to those of DPZ taken as reference drug. The 40 mg/kg and 20 mg/kg dose were less effective in improving the cognitive deficits during initial weeks but on longterm intervention, showed significant improvement (p < 0.001) (n=6).

3.4.3 Effect of bergenin pretreatment on biochemical parameters in serum and brain homogenates of ICV STZ injected rats

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3.4.3.1 Effect of bergenin treatment on the STZ induced serum and brain AChE activity STZ intrusion led to significant elevation (p < 0.001) in the serum (1.3-fold) and brain (1.9fold) AChE activity which was relegated back to normal levels with bergenin oral gavage at 40 mg/kg and 80 mg/kg (p < 0.001). The results of the study were comparable to the reduction caused by the DPZ standard. The lower dose at 20 mg/kg even on chronic intervention led to the significant reduction (p < 0.05) in the AChE activity but could not bring down the levels back to normal (Fig. 15 (A and B)).

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3.4.3.2 Effect of bergenin treatment on the STZ induced serum and brain BuChE activity The BuChE activity was significantly elevated (p < 0.001) in both serum (2.4-fold) and brain homogenates (1.8-fold) of rats injected ICV with STZ. The evaluation of BuChE activity in the serum samples revealed that the bergenin treatment at 40 and 80 mg/kg led to a significant reduction (p < 0.01) while that with 20 mg/kg led to moderately significant (p < 0.05) reduction of the BuChE activity. The DPZ treatment was able to completely reverse the augmented BuChE activity to normal levels. In brain homogenates, it was evidenced that higher dose analogs of 40 mg/kg and 80 mg/kg were capable of significantly reducing (p < 0.001) the amplified BuChE activity comparable to that achieved by DPZ treatment. Bergenin 20 mg/kg treatment, on the contrary, did not affect the raised BuChE levels, even on chronic administration, indicating that higher doses are mandatory for the desired effect (Fig. 15 (C and D)).

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3.4.3.3 Effect of bergenin treatment on the STZ induced serum and brain GSH activity The detrimental effect of STZ led to a significant reduction (p < 0.001) of GSH levels in serum (32 % reduction) and brain homogenates (42 % reduction) of rats as an indication of increased oxidative stress. This was alleviated with bergenin treatment in a dose-dependent manner wherein the highest dose levelled to the most significant (p < 0.001) elevation of the decreased GSH levels in the serum and brain samples. The 40 mg/kg treatment led to significant increase in GSH levels in serum (p < 0.05) and brain homogenates (p < 0.001) followed by 20 mg/kg which was only moderately effective in elevating the serum GSH levels and significantly (p < 0.01) effective in elevating the brain GSH levels (Fig. 15 (E and F)).

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Fig. 15. Alterations in biochemical parameters in plasma and brain homogenates ICV-STZ injected rats (A) Serum AChE activity, (B) Brain AChE activity, (C) Serum BuChE activity (D) Brain BuChE activity, (E) Serum GSH levels (F) Brain GSH levels. The ICV STZ administration led to significant (p < 0.001) elevation of AChE and BuChE activity coupled with a significant decrease (p < 0.001) in the GSH levels in the serum and brain homogenates of the rats belonging to DC group compared to that of the NC group rats. Bergenin treatment at 80 mg/kg and 40 mg/kg led to significant reduction of the elevated AChE (p < 0.001) and BuChE (p < 0.01) activity along with the refurbishment of the diminished GSH levels (p < 0.001 and 0.05 respectively) in the serum and brain homogenates of the rats when compared to the DC group rats and the results for the cholinesterase inhibitory activity were comparable to those achieved employing DPZ as standard (n=3)

3.4.4 The histopathological investigations of the HE-stained slides to evaluate the effect of bergenin supplementation on ICV STZ injection The histopathological examinations of the HE-stained slides of the sections of rat brains belonging to various groups indicated that the STZ intervention led to the prominent deterioration of the hippocampal cytoarchitecture. The density of the neurons in the hippocampal areas of DC group rats was markedly lower with many neurons depicting emaciated cell bodies. This led to the appearance of more number of clear spaces surrounding

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the cell bodies. Many neuronal cells lost their original form and clearly distinct cell membranes. The nuclei turned out to be hyperchromatic with few pyknotic nuclei present in the sections. All these were indicative of neuroinflammation. There were few dystrophic neurites observed after HE stain. The rat brains of NC group showed distinct and healthy neurons with distinct cell membranes and absence of any emaciation. The bergenin treatment led to the restoration of the detrimental effects caused by STZ injection. The cells could retain original shape and bergenin could also prevent the reduction in the density of the neurons. The number of clear spaces was reduced noticeably along with the number of pyknotic and hyperchromic nuclei. The effect was dose-dependent with highest dose level i.e. 80 mg/kg showing highest neuronal density and lowest number of shrunken soma, which was comparable to that achieved using DPZ. The results of the histopathological examination clearly indicate the neuroprotective potential of bergenin in AD management (Fig. 16).

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The counting of neurons was obtained using ImageJ software indicated that the DC group rats depicted significantly (p < 0.001) low number of neurons in the hippocampal area when compared to that of the NC group rats. The bergenin treated groups showed improvement in the average neuronal count in a dose-dependent manner with 80 mg/kg dose leading to most significant improvement (p < 0.001) which was analogous to that achieved by DPZ. This was followed by the 40 mg/kg (p < 0.001) and 20 mg/kg (p < 0.05) treatment with bergenin which also led to significant improvements in the average neuronal count as an indication of improvement in hippocampal cytoarchitecture damaged by ICV STZ administration (Fig. 17) CA3

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Fig. 16. The HE-stained histopathological imaginings of various regions of hippocampus i.e. CA1, CA2, CA3 and dentate gyrus of rats that belong to various test groups in ICV-STZ injected model observed under 400X magnification (scale bar-50 μm). The neurons belonging to the CA1, CA2, CA3 and dentate gyrus areas of hippocampi of the NC and SC group rats were densely packed, had distinct cell membrane and had very few intercellular spaces (designated with black arrows) whereas those of DC group had markedly lower density with many neurons depicting emaciated cell bodies. This led to the appearance of more number of clear spaces surrounding the cell bodies (designated with black arrows). Many neuronal cells lost their original form and clearly distinct cell membranes. Bergenin led to the dose-dependent improvement of these incongruities on chronic administration which was comparable to that obtained using DPZ (n=3).

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Fig. 17. Average neuron count per unit area in the hippocampal region of ICV – STZ injected rats. The DC group rats depicted significantly (p < 0.001) low number of neurons in the hippocampal area when compared to that of the NC group rats. The bergenin treated groups showed dose-dependent improvement in the average neuronal count with 80 mg/kg dose leading to most significant improvement (p < 0.001) which was analogous to that achieved by DPZ. The neuronal count was performed with the aid of ImageJ software (n=3)

3.4.5 The effect of bergenin supplementation on the Aβ-1-42 levels in brain homogenates of rats

The results of the sandwich ELISA assay for Aβ-1-42 levels in brain homogenates of the rats indicated a significant surge (p < 0.01) in the Aβ-1-42 levels in the DC groups injected ICV with STZ only as compared to that of the control rats. This indicates an enhanced immunoreactivity to Aβ-1-42. The bergenin treatment at 80 mg/kg led to most significant (p < 0.05) abatement of the Aβ-1-42 levels detected in the brain homogenates comparable to that of DPZ and was followed by that of the 40 mg/kg which led to non-significant reduction and the 20 mg/kg treatment which was incapable of reducing the Aβ-1-42 levels in the brain homogenates of the ICV STZ injected rats as compared to that in DC group rats (Fig. 18 (A)).

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3.4.6 The effect of bergenin supplementation on the p-tau (phosphorylated tau) in brain homogenates of rats

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The results of the sandwich ELISA assay for the detection of the p-tau protein in the rat brain homogenates were suggestive of a significant rise (p < 0.001) in the p-tau levels after STZ administration compared to that of the control rats. The perturbation caused by STZ was significantly alleviated by the bergenin with 80 mg/kg and 40 mg/kg treatment leading to significantly low (p < 0.01) levels of p-tau which was analogous to that achieved by DPZ treatment followed by that with 20 mg/kg bergenin administration which also reduced the ptau levels significantly (Fig. 18 (B)).

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3.4.7 The effect of bergenin administration on the GSK3β in brain homogenates of rats

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The immunoassay for GSK3β performed using sandwich ELISA assay led to the conclusion that the STZ induction led to slightly significant (p < 0.05) rise in the levels of GSK3β in the brain homogenates of the rats belonging to the DC groups induced with STZ. This rise in GSK3β was reduced by bergenin administration in a dose-dependent manner but the reduction was found to be non-significant (Fig. 18 (C)).

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(C) Fig. 18. The effect of bergenin supplementation on (A) Aβ-1-42 levels (B) p-tau (phosphorylated tau) levels and (C) GSK-3β levels in brain homogenates of ICV-STZ injected rats. The ICV STZ administration led to moderately significant (p < 0.01) rise Aβ-1-42 levels, highly significant (p < 0.001) rise in the p-tau levels and slightly significant (p < 0.01) rise in the GSK-3β levels in brain homogenates of rats. Bergenin treatment at 80 mg/kg could significantly curb the rise in Aβ-1-42 levels (p < 0.05) and p-tau levels (p < 0.001) and reduced the raised GSK-3β levels but the reduction was found to be non-significant. The effect of bergenin was dose-dependent and the results were comparable to that observed using DPZ standard (n=3)

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4. Discussion

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The present study was undertaken with an aim to screen bergenin using in silico techniques, in vitro assays and in vivo models for its beneficial effects in the AD management. The molecular docking studies suggested strong binding interactions of bergenin with AChE, BuChE, BACE1 and tau protein kinase-1 with good gold scores and fitness values comparable to the reference drugs. Bergenin was found to interact with more number of amino acids compared to the reference drugs. Bergenin has five hydroxy groups and a lactone ring in its structure due to which it forms numerous hydrogen bond interactions and are responsible for the high binding interactions and scores. The promising results of this preliminary study led us to further in vitro and in vivo evaluation of bergenin for its activity on multiple targets involved in the AD.

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SH-SY5Y neuroblastoma cells derived from bone marrow were selected being most widely explored to test the cytotoxic and neuroprotective effects of various compounds [32]. MTT assay and resazurin assays were employed with the cell concentrations of 1 lakh cells and 50,000 cells per well respectively, being the maximum concentrations up to which cells give linear correlations in respective assays [62]. The major players in the reduction of MTT are mitochondrial and microsomal enzyme whereas, for reduction of resazurin, mitochondrial and cytosolic enzymes are more active [33,63]. Thus, both the assays were employed to afford independent and overlapping ways to determine the cytotoxicity of bergenin [35]. The broad concentration range of the compounds was selected to render clinical significance to the in vitro study by the inclusion of probable therapeutic, hyper-therapeutic, and overdose concentrations. The time for study was fixed to 48 h after drug exposure to eradicate the effects of massive cell proliferation on the results [35]. The results of our study demonstrated that bergenin at concentrations 50 nM-500 nM showed high cell viability (80-97 %) and the higher concentrations also depicted high cell viability indicating that the compound is non-toxic to the SH-SY5Y cells.

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NMDA has been shown to elicit time and concentration-dependent cell death in SH-SY5Y cells as per Corasaniti et al., 2007 and Hu et al., 2015 who employed 1 mM and 2 mM NMDA to check the neuroprotective effects of test compounds on SH-SY5Y cells against NMDA challenge [41,64]. Eugenin et al., 2003 showed maximum cell death after NMDA insult was observed in the primary neuronal cultures during 24 - 72 h but insignificant 12 h post NMDA treatment. Thus, the exposure duration of NMDA was set to 24 h and concentration to 2.5 mM NMDA [65]. NMDARs have been implicated in brain development, brain function, learning, and memory by playing an integral role in synaptogenesis, synaptic remodeling etc. via Ca2+ influx through glutamate-gated ion channels. Excitotoxicity due to sustained stimulation of NMDARs leads to enormous Ca+2 and Na+ accumulation intracellularly, upregulation of numerous detrimental signaling pathways and oxido- nitrosative leading to terminal cell death [41,66,67]. NMDA has been depicted to rapidly activate protease calpain I and deactivate Akt kinase which triggers downstream activation of GSK-3β which is detrimental to cells [41]. In our study, bergenin treatment at 500 nM led to around 32.4 % increase in cell survival of NMDA treated SH-SY5Y cells as compared to those treated with NMDA alone. The protective effects of bergenin were found to be better as compared to memantine and donepezil but it could not reestablish cell viability up to 100 %. The plausible explanation for this might be the action of bergenin on only some of these pathways to prevent the NMDA toxicity and bergenin does not have proliferative activity as observed from the results of MTT and resazurin-based assay. Thus, bergenin might not able to completely combat the toxic effects of NMDA though the in-depth investigation is still required to completely determine its mechanism. The protective effects exerted by bergenin could be attributed to its antioxidant properties leading to the prevention of cell death elicited by NMDA.

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Based on the promising results of the docking analysis and in vitro cell line studies, bergenin was evaluated for the in vitro AChE and BuChE inhibitory activities. AChE hydrolyzes ACh into choline and acetate and regulates levels of ACh in the synaptic cleft and cognitive function of learning and memory [45]. AChE along with BuChE regulates non-cholinergic functions neurogenesis, cellular proliferation and neurite growth [68–70]. Raised levels of AChE and BuChE in the AD are mostly associated with plaques and tangles [71]. The AChE and BuChE inhibitors have been proven effective in significantly ameliorating scopolamine-induced reduction in neuroblast differentiation, cell proliferation, survival and neuron integration in the dentate gyrus of rats without significantly affecting mature neurons [72–74]. Bergenin could inhibit both the enzymes in a concentration-dependent fashion with higher inhibitory effects on AChE as compared to BuChE in a manner similar to DPZ. The gold fitness values of bergenin were higher for AChE (57.9546) as compared to its fitness for BuChE (47.5438) which was supported by the observations of the in vitro studies. The effects comparable to those of DPZ were observed at quite high concentrations but the lower concentrations also led to a minimum to moderate inhibition of the enzymes.

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Bergenin was further screened in vivo in the scopolamine-induced amnesia model and ICV STZ induced AD model in rats. Scopolamine penetrates the blood-brain barrier and leads to a significant increase in the AChE levels, elevation of oxidative stress, neuroinflammation and mitochondrial dysfunction in rodent brain simulating cognitive decline in the AD which has been proven through impaired spatial working memory in various behavioral studies [25,48,75]. Thus, it was selected being a quick and apt for evaluating the potential therapeutic application of new drugs in attenuating cognitive decline and neuronal cell death in dementia in general and AD in particular [25]. The ICV STZ injection inhibits insulin receptors resulting in dysregulation of glucose energy metabolism leading to decreased hippocampal choline acetyltransferase (ChAT) activity and increased cholinesterase activity in the cerebral cortex and hippocampus [13,76]. The consequential diminution of cholinergic activity is hypothesized

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to play a pivotal role in the memory impairment in rats. The exact mechanism of action of STZ has not yet been derived but is supposed to be a consequence of overexpression of AChE encoding genes or because of β-amyloid deposit in the neurons and astrocytes [77]. The upregulation of AChE activity leads to accelerated ACh hydrolysis, neurodegeneration and cell demise [45,78]. The incidence of neurobehavioral and neuropathological changes aroused by central ICV injection of STZ observed through spatial memory deficits in behavioral tests, cholinergic deficit, oxidative stress, and degenerative change in the histoarchitecture obtained in present study were similar to those in SAD and resembled the results of various other studies employing ICV STZ injection [56,79]. To evaluate the cognitive behavior of rats, MWM, a well-recognized hippocampus-dependent spatial reference memory task, which is expedient especially for spatial learning and memory was utilized [80]. The SAB was assessed using Y maze a measure of spatial working memory task pertaining to the hippocampal area which helps to avoid the effects of traumatic circumstances like swimming, electric shock, fasting overnight etc. on the assessment paradigm [52,53].

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Preventive effects of bergenin were evaluated on the scopolamine-induced amnesia in rats and ameliorative effects on ICV-STZ induced model in rats. This impairment of memory and learning caused by intraperitoneal scopolamine injection and ICV STZ administration evident through significantly (p < 0.001) high MEL and low MPA are in line with the previous reports [25,56,79,81]. This impairment in the spatial learning and memory as observed from the battery of behavioral experiments was a clear indication of hippocampal damage in the rats belonging to the DC group. This agrees to the fact that the spatial learning is an outcome of the synchronized action of hippocampus and cortex. in the brain which collectively forms a functionally unified neural network [56]. Bergenin pretreatment at 80 mg/kg was found most effective in bringing back the MEL and MPA to normal levels in both the models which were similar to that observed after DPZ treatment, though the DPZ treatment proved more effective owing to its potent cholinesterase inhibition. The dose-dependent improvement arbitrated via bergenin oral dosing in ICV STZ model was easily demonstrated through the declining MEL and the shortening of path lengths in MWM task and increasing mean MPA when evaluated over definite time intervals in a concentration reliant manner. The nonappearance of any significant differences in the locomotor activity amongst various test groups, as observed from the average swim speeds, omitted the possibility of these differences to skew the results. These results are indicative of the improvement in acquisition and consolidation of short-term and long-term spatial working memory by bergenin administration at 80 mg/kg [25,51].

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The persistent cholinergic deficit and frequent degeneration of cholinergic neurons play a vital role in the pathogenesis and etiology of the AD. Thus, inhibition of AChE and BuChE could be a beneficial strategy for AD management. The activities of AChE and BuChE were found to be increased significantly in brain homogenates and serum samples of DC group rats compared to NC group and are in harmony with the results of many of the studies carried out hitherto [82,83]. The results of our study indicated dose-dependent AChE and BuChE inhibitory activity of bergenin in brain homogenates of scopolamine and ICV STZ injected rats are in correlation with the results of the in vitro cholinesterase inhibition assays and the molecular docking studies. The previous findings on some of the natural bioactive such as quercetin, taurine, resveratrol, etc. with AChE and BuChE inhibitory activity and thereby augmenting the cognitive effects in various animal models of the AD are in the agreement of the fact of the involvement of cholinergic function in cognition [56,82,84–86]. These results corroborate with the premise that the ACh in the cerebral cortex is responsible for attention and recognition whereas that in the hippocampus allows novel information procurement [45,87]. The hippocampus and frontal cortex, are conjectured to play a central role in declarative memory which is the daily memory of the facts and events. Hippocampus helps in the

instantaneous formation of new memories and their consolidation. There is accruing evidence which is suggestive of the involvement of the cholinergic neurons from these areas in the cognition associated processes like learning and memory and their degeneration is involved in the AD and similar memory disorders [55,88]. The invigorating effects of bergenin on the learning and memory paradigm owing to rescue of the synaptic plasticity and the spatial memory and its parallelism with the AChE activity and improvement of cholinergic function via cholinesterase inhibition levels support this datum.

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The GSH levels were found to be raised significantly (p < 0.001) in the brain homogenates and serum samples of rats injected with scopolamine and STZ. Bergenin treatment could recuperate the GSH levels back to normal in a dose-dependent manner and effects were slightly better than those achieved using DPZ as a standard drug which led to non- significant convalescence of the reduced GSH levels at some instances. The main cause of the augmented pathogenesis in most of the neurological diseases and disorders along with memory impairment is the enduring discrepancy amongst the endogenous antioxidant defense mechanism of the body [6,82]. The BMR in the brain tissues is quite high and hence the corresponding oxygen intake also increases. But, the brain tissue has a weak endogenous antioxidant capacity which makes it more prone to the oxidative stress. GSH forms the antioxidant defense mechanism of the body by scavenging free radicals in the brain tissues and its levels are found to be significantly reduced in the AD [13,57,89]. Cholinergic transmission seems to majorly occur in the cortex, hippocampus and amygdala regions of brains and these are the neurons which are highly susceptible to the oxidative damage [13,56,88]. Various studies have uncovered that the antioxidant-rich diet and chemicals impart neuroprotective effects in animal models and humans and they own the potential to slow down the pace of advancement of the cognitive deterioration in AD [79,82,85,86] The antioxidant activity of bergenin manifested through a reduction in GSH levels and resultant oxidative load further consolidates its beneficial effect in both the models. Our findings are supported with the reports of antioxidant activity of bergenin through replenishment of glutathione content, elevation of glutathione S-transferase and glutathione reductase activities in hepatocytes along with the inhibition of lipid peroxidation in the brain tissues in various other in vitro and in vivo models [17,18,60,61]. Thus, it is expected that the advantageous effects of bergenin on the neurocognitive consequences might be exerted not only through the cholinesterase inhibition but also through antioxidant properties of bergenin.

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The histopathological observations of scopolamine and STZ injected rats which depicted noticeably reduced neuron density and pyramidal cells in the hippocampal area coupled with the deterioration of hippocampal architecture as compared to the NC and SC groups as signs of neuroinflammation [57,82]. Bergenin led to dose-dependent prevention of neuronal density and cytoarchitecture of hippocampal neurons from the scopolamine and STZ induced injury, comparable to DPZ used as a standard. These beneficial effects are consistent with the corresponding enrichment in the cognitive function, cholinesterase inhibition and the antioxidant effects of the compound. The cognitive status is highly interlinked with the number of cholinergic neurons in the hippocampus and basal forebrain, loss of which is an early indication AD occurrence. The results of the study corroborate the proposition that the neurodegeneration or the reduced density of the neurons in the hippocampal region correlate with the cognitive impairments [88]. The management of AD advocates the use of antiinflammatory agents due to the occurrence of neuroinflammation and as the STZ also exacerbates its detrimental effects through inflammatory stimuli [79]. There are reports wherein the anti-inflammatory agents have improved the persistence of the neurons in the hippocampus and subsequent improvements in learning and memory [79]. These studies suggest that bergenin supplementation might also be beneficial in the management of

neuroinflammation related to the AD. The reports on inhibition of carrageenan-induced edema in rats, hPTP1B inhibitory activity and reduction of various inflammatory and proinflammatory mediators like IL-1β, TNF-α, IFN γ, COX-1, COX-2, phospholipase A2, etc. indicate that anti-inflammatory activity of bergenin might have also contributed its part for the improvement of the morphology of neurons by reducing neuroinflammation [15,90,91].

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The significant rise in the Aβ1-42 (p < 0.01) and p-tau (p < 0.001) levels in the brain homogenates of STZ injected rats as a precursor of extracellular plaque and NFT formation were observed. The results of the study are in line with the results of the previous studies indicating increased Aβ1-42 and p-tau immunoreactivity in ELISA assay of hippocampal homogenates of rats one month post 3 mg/kg STZ injection owing to acute compensatory effect to oxidative damage induced mitochondrial incongruities which gradually diffuse to form mature plaques, PHFs and NFTs [92,93]. The levels of Aβ1-42 and p-tau were found reduced significantly and dose-dependently on bergenin supplementation. The previous reports of BACE-1 inhibitory activity of bergenin, ROS scavenging activity and anti-inflammatory activity of bergenin are supportive of these results [19]. The results of molecular docking studies indicating binding interactions with BACE-1 and tau protein kinase-1 are also supportive of these results as this activity might have contributed its role in the reduction of raised Aβ1-42 and p-tau levels. The studies indicating a correlation of this increased immunoreactivity to cognitive deterioration also partly suffice the correlation between improvement of cognitive parameters by bergenin treatment and reduced Aβ1-42 and p-tau immunoreactivity [92,93]. GSK-3β has a vital role in AD development and progression as it partakes in the synthesis and the toxicity of amyloid β and tau hyperphosphorylation [82,94,95]. GSK-3β plays an intrinsic role in the memory and its enhanced expression is indicated to attenuate spatial learning. The results of the ELISA depicted that ICV STZ injection led to an only slightly significant rise in the GSK-3β levels in the brain homogenates of rats whereas there were no significant changes midst, various other groups. The binding interactions with bergenin in molecular docking studies support the prevention the rise in GSK3β afforded by bergenin. Since the phosphorylation of GSK-3β at Serine 9 amino acid by insulin constrains its effect on tau, the ratio of p-Ser-GSK-3β to GSK-3β can be evaluated in future to gain a better idea about the activity of GSK-3β [82].

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The neuroprotective effects of bergenin are evident from successful attenuation of the cognitive decline, neurodegeneration and improvement of cholinergic activity and behavioral abnormalities which were consequences of the STZ injection.

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5. Conclusion

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The study demonstrates multiple benefits of bergenin in the in vitro assays of the present study and in vivo evaluation in scopolamine, and STZ induced models of the AD. It exhibited neuromodulatory activity, cholinesterase inhibition, antioxidant activity, anti-dementia effects, as a supportive of the overall neuroprotective action. From the results of the present study and the supporting literature thereof, it can be proposed that bergenin is capable of restoring the cholinergic function back to normal levels, thereby improving the learning and memory function. In case of short-term pre-treatment, bergenin oral dosing at 80 mg/kg daily proved beneficial in preventing the scopolamine-induced amnesia. Bergenin at 40 mg/kg and 80 mg/kg was found almost similarly effective in invigorating the STZ induced damage except that the onset of duration was a bit delayed in the lower dose. These studies complement previous observations by providing evidence for a subset of the neuroprotective effects of bergenin in a dose-dependent manner through various in silico, in vitro and in vivo studies could be attributed to its anticholinesterase, antioxidant and anti-inflammatory properties along with the reduction

of the Aβ1-42 immunoreactivity and tau phosphorylation. Thus, the prophylactic or therapeutic use of bergenin as an adjuvant might be further explored for prevention and prophylaxis of AD and associated neurodegenerative disorders. Our study provides a foundation for the further illumination on the molecular mechanisms and an insight towards the role of bergenin in signaling pathways for proposed neuroprotective action. Author Contributions

Compliance with ethical standards Authors declare that they do not have any conflict of interest.

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Financial disclosures and acknowledgments

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Designing of experiments, data acquisition and analysis part of this work along with manuscript writing is done by Ms. Priyal Barai as a part of her Ph.D. work under the able supervision of Dr. Niyati Acharya. Mr. Nisith Raval helped in the designing of experiments, data acquisition, and analysis. Dr. Sanjeev Acharya helped by providing critical feedbacks while designing and implementing the work plan, analyzing the data and the preparation of the manuscript. Dr. Hardik Bhatt and Mr. Ankit Borisa helped with the molecular docking studies. All authors provided their feedbacks which helped shape the work along with the final draft of the manuscript.

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The authors thank the Department of Science and Technology (DST) for the provision of the INSPIRE fellowship (IF140234). They also express a sense of gratitude to Department of Biotechnology (DBT) (Grant No. BT/PR6664/NNT/28/628/2012) and Gujarat Council on Science and Technology (GUJCOST) (Grant No. GUJCOST/MRP/15-16/1107) for extending financial support. The authors are also grateful to the Institute of Pharmacy, Nirma University for the provision of various technical and infrastructural facilities for conducting the research work. This research is a portion of the Ph.D. research work of Priyal Barai which will be submitted to Institute of Pharmacy, Nirma University, Ahmedabad, India. References

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A

M

Figr-29

A ED

PT

CC E

IP T

SC R

U

N

A

M

Figr-30

A ED

PT

CC E

IP T

SC R

U

N

A

M

Figr-31

A ED

PT

CC E

IP T

SC R

U

N

A

M

Figr-32

A ED

PT

CC E

IP T

SC R

U

N

A

M

Figr-33

A ED

PT

CC E

IP T

SC R

U

N

A

M

Figr-34

A ED

PT

CC E

IP T

SC R

U

N

A

M

Figr-35

A ED

PT

CC E

IP T

SC R

U

N

A

M

Figr-36

A ED

PT

CC E

IP T

SC R

U

N

A

M

Figr-37

A ED

PT

CC E

IP T

SC R

U

N

A

M

Figr-38

A ED

PT

CC E

IP T

SC R

U

N

A

M

Figr-39

A ED

PT

CC E

IP T

SC R

U

N

A

M

Figr-40

A ED

PT

CC E

IP T

SC R

U

N

A

M

Figr-41

A ED

PT

CC E

IP T

SC R

U

N

A

M

Figr-42

A ED

PT

CC E

IP T

SC R

U

N

A

M

Figr-43

A ED

PT

CC E

IP T

SC R

U

N

A

M

Figr-44

A ED

PT

CC E

IP T

SC R

U

N

A

M

Figr-45

A ED

PT

CC E

IP T

SC R

U

N

A

M

Figr-46

A ED

PT

CC E

IP T

SC R

U

N

A

M

Figr-47

A ED

PT

CC E

IP T

SC R

U

N

A

M

Figr-48

A ED

PT

CC E

IP T

SC R

U

N

A

M

Figr-49

A ED

PT

CC E

IP T

SC R

U

N

A

M

Figr-50

A ED

PT

CC E

IP T

SC R

U

N

A

M

Figr-51

A ED

PT

CC E

IP T

SC R

U

N

A

M

Figr-52

A ED

PT

CC E

IP T

SC R

U

N

A

M

Figr-53

A ED

PT

CC E

IP T

SC R

U

N

A

M

Figr-54

A ED

PT

CC E

IP T

SC R

U

N

A

M

Figr-55

Figr-56

A

CC E

PT

ED

M

A

N

U

SC R

IP T

Fig. 1. Experimental schedule, dosage regimen and behavioral assessment time frame for the scopolamineinduced AD model in rats. Fig. 2. Experimental schedule, dosage regimen and behavioral assessment time frame for the streptozotocininduced AD model in rats. Fig. 3: (A) Division of four zones and position of the platform in a water maze and (B) Division of various zones in Y-maze. Fig. 4. Docking images of Bergenin into various targets of AD (A) AChE (PDB ID: 1B41), (B) BuChE (PDB ID: 1P0I), (C) Tau protein kinase 1 (GSK-3β) (PDB ID: 1J1B) and (D) BACE-1 (PDB ID: 1FKN) depicting the binding interactions with various amino acids in the binding pocket. Fig. 5. (A) The MTT assay in undifferentiated SH-SY5Y cells exposed to bergenin at different concentrations (5 nM – 50 µM) for 48 h. (B) Resazurin metabolism-based cell viability assay in undifferentiated SH-SY5Y cells exposed to bergenin at different concentrations (5 nM – 50 µM) for 48 h and (C) Preventive effects of bergenin of NMDA induced toxicity in SH-SY5Y cells. Bergenin treatment at all tested doses led to non- significant reduction in cell viability when tested using both MTT and resazurin-based assay indicating its safety at all tested concentrations. The NMDA group showed significant (p < 0.001) reduction in the cell viability as compared to both Normal control (NC) and DMSO control groups. Bergenin at all tested doses led to significant (p < 0.001) prevention of the NMDA induced cell death with 500 nM concentration leading to highest cell viability as all other test groups except BERG 500 nM depicted significantly (p < 0.001) low cell viability compared to NC and DMSO control groups Values are expressed as Mean ± SEM, where #- p < 0.001 as compared to NC; * p < 0.001 as compared to NMDA control; @ p < 0.05 as compared to DMSO control; @@ p< 0.01 as compared to normal control; $- p < 0.001 as compared to BERG 500 nM; when compared using one-way ANOVA followed by Bonferroni’s post hoc test (n = 3) Fig. 6. (A) In -vitro AChE inhibition assay and (B) In- vitro BuChE inhibition assay. BERG 1, BERG 2, BERG 3, BERG 4 and BERG 5 indicate 10 mM, 5 mM, 2.5 mM, 1.25 mM and 0.03 mM final concentration of bergenin in wells respectively; E: Enzyme only; M: Enzyme in presence of methanol at maximum concentration employed; DPZ- 0.03 µM and 3 µM final concentration of DPZ in well employed in AChE and BuChE assays respectively The absorbance versus time curves indicates that bergenin in a concentration-dependent manner led to decrease in the total absorbance of the test mixture with the highest dose leading to a maximum reduction in the absorbance and hence indicating highest degree of inhibition of AChE and BuChE. The effect of bergenin on inhibition of BuChE was less as compared to that of AChE. Values are expressed as Mean ± SEM (n = 3) Fig. 7. (A) Mean Escape Latencies (MELs) and (B) Mean Percentage Alternations (MPA) manifested by test groups on 14th day thirty min post scopolamine injection. Rats belonging to the DC group presented significantly (p < 0.001) higher MELs and significantly (p < 0.001) lower MPAs when compared to the NC group rats. Bergenin treatment in a dose-dependent manner led to amelioration of the behavioural deficits caused by dose-dependent decrease in MELs and dose-dependent increase in MPAs with 80 mg/kg treatment affording maximum benefit with significant (p < 0.01) reduction of MEL and significant (p < 0.001) increase in the MPA as compared to the DC group and the results were comparable to those of the DPZ group (n=6) Fig. 8. The illustration of the path voyaged by the various test groups in MWM attained using SMART 3.0 Video Tracking Software of 14th day, 30 min post scopolamine injection. The path traveled by the DC group rats was noticeably longer than that traveled by the NC group rats to find the hidden platform and bergenin treatment could dose-dependently reduce the time taken and hence the path covered by the rats to discover the hidden platform which was analogous to that obtained using DPZ as reference drug Fig. 9. Alterations in biochemical parameters in serum and brain homogenates scopolamine injected rats (A) Serum AChE activity, (B) Brain AChE activity, (C) Serum BuChE activity (D) Brain BuChE activity, (E) Serum GSH levels (F) Brain GSH levels. The intraperitoneal scopolamine administration led to the significant elevation of AChE (p < 0.001) and BuChE (p < 0.05) activity in the serum and brain homogenates of the DC group rats as compared to that of the NC group rats. A significant decrease in the GSH levels in the serum (p < 0.001) and brain homogenates (p < 0.01) of DC group rats was observed. Bergenin treatment at 80 mg/kg led to most significant reduction of the elevated AChE (p < 0.01) and BuChE (p < 0.05) activity along with the refurbishment of the diminished GSH levels (p < 0.01) in the serum and brain homogenates of the rats when compared to the DC group rats and the results were analogous to those attained employing DPZ as standard (n=3) Fig. 10. The HE-stained histopathological images of various regions of hippocampus i.e. CA1, CA2, CA3 and dentate gyrus of rats belonging to various test groups in scopolamine-induced amnesia model observed under 400X magnification (scale bar-50 μm).

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M

A

N

U

SC R

IP T

The neurons in CA1, CA2, CA3 and dentate gyrus of hippocampi of NC group rats were healthy and compactly arranged with less or no intercellular spaces (designated with black arrows) but those belonging to DC group had numerous intercellular spaces (designated with black arrows) accompanied by noticeable loss of neurons in these regions. Bergenin pre-treatment could prevent this loss in a dose-dependent manner which was comparable to that attained by DPZ treatment (n=3). Fig. 11. Average neuron count per unit area in the hippocampal region of rats in scopolamine-induced amnesia model. The scopolamine injected DC group rats depicted significantly (p < 0.05) low number of neurons in the hippocampal area when compared to that of the NC group rats. The bergenin (80 mg/kg) treated groups showed most significant (p < 0.05) improvement in the average neuronal count was comparable to that achieved by DPZ. The neuronal count was performed by an experimenter blind to the groups with the aid of ImageJ software (n=3) Fig. 12. Mean Escape Latencies (MELs) of the test groups of ICV STZ induced model on (A) 7th day, (B) 14th day, (C) 21st day and (D) 28th day. The ICV STZ administration caused significant (p < 0.001) rise in the MELs of the DC group rats when compared to that of NC and SC group rats. The bergenin treatment could significantly (p < 0.001) and dose-dependently decreased the raised MELs as compared to the DC group rats and the effect was found to be improving with time. The effect shown by the higher dose levels i.e. 40 and 80 mg/kg were equivalent to that of DPZ standard group (n=6). Fig. 13. The illustration of path trekked by the test groups in ICV STZ induced model MWM attained using SMART 3.0 Video Tracking Software. The path traveled by the DC group rats was markedly longer than that traveled by the NC and SC group rats to discover the hidden platform because of ICV STZ administration which also corresponded to longer MELSs. Bergenin treatment led to the dose-dependent reduction in the path covered by the rats Fig. 14. MPAs of the test groups of ICV STZ induced model on (A) 7th day, (B) 14th day, (C) 21st day and (D) 28th day. ICV STZ injection led to a significant decrease in the MPAs of the DC group rats when compared to NC and SC group rats. Bergenin treatment at 80 mg/kg led to highest MPAs in rats which were significantly (p < 0.001) higher than that of the DC group and the results were comparable to those of DPZ taken as reference drug. The 40 mg/kg and 20 mg/kg dose were less effective in improving the cognitive deficits during initial weeks but on long-term intervention, showed significant improvement (p < 0.001) (n=6). Fig. 15. Alterations in biochemical parameters in plasma and brain homogenates ICV-STZ injected rats (A) Serum AChE activity, (B) Brain AChE activity, (C) Serum BuChE activity (D) Brain BuChE activity, (E) Serum GSH levels (F) Brain GSH levels. The ICV STZ administration led to significant (p < 0.001) elevation of AChE and BuChE activity coupled with a significant decrease (p < 0.001) in the GSH levels in the serum and brain homogenates of the rats belonging to DC group compared to that of the NC group rats. Bergenin treatment at 80 mg/kg and 40 mg/kg led to significant reduction of the elevated AChE (p < 0.001) and BuChE (p < 0.01) activity along with the refurbishment of the diminished GSH levels (p < 0.001 and 0.05 respectively) in the serum and brain homogenates of the rats when compared to the DC group rats and the results for the cholinesterase inhibitory activity were comparable to those achieved employing DPZ as standard (n=3) Fig. 16. The HE-stained histopathological imaginings of various regions of hippocampus i.e. CA1, CA2, CA3 and dentate gyrus of rats that belong to various test groups in ICV-STZ injected model observed under 400X magnification (scale bar-50 μm). The neurons belonging to the CA1, CA2, CA3 and dentate gyrus areas of hippocampi of the NC and SC group rats were densely packed, had distinct cell membrane and had very few intercellular spaces (designated with black arrows) whereas those of DC group had markedly lower density with many neurons depicting emaciated cell bodies. This led to the appearance of more number of clear spaces surrounding the cell bodies (designated with black arrows). Many neuronal cells lost their original form and clearly distinct cell membranes. Bergenin led to the dose-dependent improvement of these incongruities on chronic administration which was comparable to that obtained using DPZ (n=3). Fig. 17. Average neuron count per unit area in the hippocampal region of ICV – STZ injected rats. The DC group rats depicted significantly (p < 0.001) low number of neurons in the hippocampal area when compared to that of the NC group rats. The bergenin treated groups showed dose-dependent improvement in the average neuronal count with 80 mg/kg dose leading to most significant improvement (p < 0.001) which was analogous to that achieved by DPZ. The neuronal count was performed with the aid of ImageJ software (n=3) Fig. 18. The effect of bergenin supplementation on (A) Aβ-1-42 levels (B) p-tau (phosphorylated tau) levels and (C) GSK-3β levels in brain homogenates of ICV-STZ injected rats.

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The ICV STZ administration led to moderately significant (p < 0.01) rise Aβ-1-42 levels, highly significant (p < 0.001) rise in the p-tau levels and slightly significant (p < 0.01) rise in the GSK-3β levels in brain homogenates of rats. Bergenin treatment at 80 mg/kg could significantly curb the rise in Aβ-1-42 levels (p < 0.05) and p-tau levels (p < 0.001) and reduced the raised GSK-3β levels but the reduction was found to be non-significant. The effect of bergenin was dose-dependent and the results were comparable to that observed using DPZ standard (n=3)

Table 1: Comparative GOLD scores, fitness values and interactive amino acids of bergenin and standard drugs against various targets of AD GOLD Fitness

1

Bergenin

57.9546

2

Donepezil

67.7309

3

Galanthamine

61.9206

4

Physostigmine

56.9314

1

Bergenin

47.5438

2 3

Donepezil Galanthamine

57.3035 55.9427

4

Physostigmine

51.1272

1

Bergenin

2

Donepezil

3 4

Galanthamine Physostigmine

1

Bergenin

2

Donepezil

3 4

Galanthamine Physostigmine

Interactive Amino acids

1B41 (Acetylcholinesterase) ARG 24, LYS 32, LEU 339, VAL 340, GLY 342, ALA -14.9545 343, PHE 346 TYR 61, ARG 24, VAL 340, GLY 342, ALA 343, PHE -8.1400 346 LYS 32, VAL 340, TYR 341, GLY 342, ALA 343, PHE -5.3697 346, SER 347 -9.7334 VAL 340, GLY 342, ALA 343, PHE 346 1P0I (Butyrylcholinesterase) ASN 245, LYS 248, PHE 278, VAL 279, VAL 280, PRO -14.9669 281 -9.2707 ASN 245, PHE 278, VAL 280, PRO 281 -5.3967 ARG 242, ASN 245, PHE 278, VAL 280 -9.7363

ASN 245, PHE 278, VAL 280, PRO 281

ED

M

A

N

U

1J1B (Tau protein kinase 1 (GSK-3β)) 60.5180 -14.8216 LYS 85, ASP 133, TYR 134, VAL 135, ASP 200 ILE 62, GLY 68, LYS 85, LEU 132, ASP 133, VAL 135, 77.3513 -8.1485 ASP 200 68.8832 -5.3816 LYS 85, ASP 133, TYR 134, ARG 141, ASP 200 64.3742 -9.7218 ASP 200 1FKN (β- Secretase) ASP 32, GLY 34, TYR 71, THR 72, ASP 228, GLY 230, 61.0480 -15.0337 THR 231, ARG 235 ASP 32, GLY 34, PRO 70, TYR 71, THR 72, ASP 228, 78.6108 -8.1399 GLY 230, THR 231 65.9790 -5.3910 ASP 32, GLN 73, ASP 228, THR 231, ARG 235 58.1433 -9.7182 GLN 73, ASP 228, THR 231

PT CC E A

GOLD score

IP T

Compound

SC R

Sr. No.