In Silico, in Vitro and in Vivo Memory Enhancing Activity

Home , Afzelechin, Azaleatin, Chrysosplenetin, Ermanin, Eupalitin, Fisetin

IN SILICO, IN VITRO AND IN VIVO MEMORY ENHANCING ACTIVITY OF CERTAIN COMMERCIALLY AVAILABLE FLAVONOIDS IN SCOPOLAMINE AND ALUMINIUM-INDUCED LEARNING IMPAIRMENT IN MICE

Thesis submitted to The Tamil Nadu Dr. M.G.R. Medical University, Chennai

for the award of the degree of DOCTOR OF PHILOSOPHY

in PHARMACY

Submitted by A. MADESWARAN, M. Pharm.,

Under the guidance of Dr. K. ASOK KUMAR, M. Pharm., Ph.D.

College of Pharmacy, Sri Ramakrishna Institute of Paramedical Sciences, Coimbatore – 641 044, Tamil Nadu, India.

JUNE 2017 Certificate

This is to certify that the Ph.D. dissertation entitled “IN SILICO, IN VITRO AND IN VIVO MEMORY ENHANCING ACTIVITY OF CERTAIN COMMERCIALLY AVAILABLE FLAVONOIDS IN SCOPOLAMINE AND ALUMINIUM- INDUCED LEARNING IMPAIRMENT IN MICE” being submitted to The Tamil Nadu Dr. M.G.R. Medical University, Chennai, for the award of degree of DOCTOR OF PHILOSOPHY in the FACULTY OF PHARMACY was carried out by Mr. A. MADESWARAN, in College of Pharmacy, Sri Ramakrishna Institute of Paramedical Sciences, Coimbatore, under my direct supervision and guidance to my fullest satisfaction. The contents of this thesis, in full or in parts, have not been submitted to any other Institute or University for the award of any degree or diploma.

Dr. K. Asok Kumar, M.Pharm., Ph.D. Professor & Head, Department of Pharmacology, College of Pharmacy, Sri Ramakrishna Institute of Paramedical Sciences, Coimbatore, Tamil Nadu - 641 044.

Place: Coimbatore – 44. Date: Certificate

This is to certify that the Ph.D. dissertation entitled “IN SILICO, IN VITRO AND IN VIVO MEMORY ENHANCING ACTIVITY OF CERTAIN COMMERCIALLY AVAILABLE FLAVONOIDS IN SCOPOLAMINE AND ALUMINIUM- INDUCED LEARNING IMPAIRMENT IN MICE” being submitted to The Tamil Nadu Dr. M.G.R. Medical University, Chennai, for the award of degree of DOCTOR OF PHILOSOPHY in the FACULTY OF PHARMACY was carried out by Mr. A. MADESWARAN, in College of Pharmacy, Sri Ramakrishna Institute of Paramedical Sciences, Coimbatore, under my co-guidance to my fullest satisfaction. The contents of this thesis, in full or in parts, have not been submitted to any other Institute or University for the award of any degree or diploma.

Dr. Sonia George, M. Pharm., Ph.D. Assistant Professor, Dept. of Pharmaceutical Chemistry, College of Pharmacy, Sri Ramakrishna Institute of Paramedical Sciences, Coimbatore, Tamil Nadu - 641 044.

Place: Coimbatore - 44 Date: Certificate

This is to certify that the Ph.D. dissertation entitled “IN SILICO, IN VITRO AND IN VIVO MEMORY ENHANCING ACTIVITY OF CERTAIN COMMERCIALLY AVAILABLE FLAVONOIDS IN SCOPOLAMINE AND ALUMINIUM- INDUCED LEARNING IMPAIRMENT IN MICE” being submitted to The Tamil Nadu Dr. M.G.R. Medical University, Chennai, for the award of degree of DOCTOR OF PHILOSOPHY in the FACULTY Of PHARMACY was carried out by Mr. A. MADESWARAN under the direct supervision and guidance of Dr. K. ASOK KUMAR, M. Pharm., Ph.D. and the co-guidance of Dr. SONIA GEORGE, M. Pharm., Ph.D. in College of Pharmacy, Sri Ramakrishna Institute of Paramedical Sciences, Coimbatore.

Dr. T.K. Ravi, M.Pharm., Ph.D., FAGE. Principal, College of Pharmacy, Sri Ramakrishna Institute of Paramedical Sciences, Coimbatore, Tamil Nadu - 641 044.

Place: Coimbatore - 44 Date: Declaration

I hereby declare that the Ph.D. dissertation entitled “IN SILICO, IN VITRO AND IN VIVO MEMORY ENHANCING ACTIVITY OF CERTAIN COMMERCIALLY AVAILABLE FLAVONOIDS IN SCOPOLAMINE AND ALUMINIUM- INDUCED LEARNING IMPAIRMENT IN MICE” being submitted to The Tamil Nadu Dr. M.G.R. Medical University, Chennai, for the award of degree of DOCTOR OF PHILOSOPHY in the FACULTY OF PHARMACY was carried out by me under the direct supervision and guidance of Dr. K. ASOK KUMAR, M. Pharm., Ph.D. and the co- guidance of Dr. SONIA GEORGE, M. Pharm., Ph.D. in College of Pharmacy, Sri Ramakrishna Institute of Paramedical Sciences, Coimbatore. The contents of this thesis, in full or in parts, have not been submitted to any other Institute or University for the award of any degree or diploma.

A. Madeswaran

Place: Coimbatore -44 Date: ACKNOWLEDGEMENTS Apart from my efforts, the success of this thesis depends largely on the encouragement and advice of many others. I take this opportunity to express my gratitude to the people who have been involved in the successful completion of this research work. I wish to express my most sincere gratitude to my guide Dr. K. Asok Kumar M. Pharm., Ph.D. Professor and Head, Department of Pharmacology, College of Pharmacy, SRIPMS, Coimbatore and it is my duty to thank him, for his constant support, help, guidance, encouragement and ultimately inspiration. I take this opportunity to accord my sedulous gratitude to my beloved co-guide Dr. Sonia George, M.Pharm., Ph.D. Assistant Professor, Department of Pharmaceutical Chemistry, for her valuable guidance and moral support throughout the period of my study. I express my heartfelt thanks to Prof. Dr. T.K. Ravi, Principal, College of Pharmacy, Sri Ramakrishna Institute of Paramedical Sciences, Coimbatore for providing me this chance, for his kind and constant support and timely guidance in the completion of this work. I am elated to keep on record my indebtedness and thankfulness to our beloved Managing Trustee Shri. R. Vijayakumhar and the Joint Managing Trustee Shri. D. Lakshminarayanaswamy, former Managing Trustees, Shri. C. Soundararaj and Sevaratna Dr. R. Venkatesalu Naidu and the Management for permitting me and providing all the facilities to carry out this project. I am greatly thankful to Dr. M. Gopal rao, Vice-Principal, College of Pharmacy, SRIPMS for his kind support and timely help in the completion of this work. I am greatly expressing my acknowledgement of gratitude to my well- wisher Dr. M. Uma Maheswari, for her consent, endurance and help in completing this thesis work on time. I also extend my heartfelt thanks to Dr. B. Rajalingam, Dr. P. Bharathi, Dr. A.T. Sivashanmugam, Dr. S. Krishnan, Dr. M. Gandhimathi, Dr. J. Bagyalakshmi and Mr. Sunnapu Prasad for their constant support, endurance and help in completing this thesis work on time. I also extend my heartfelt gratitude to Dr. S. Sriram, Prof. M. Francis Saleshier and Dr. R. Vijayaraj for their valuable guidance for the successful completion of this task on time. I extend my deep sense of gratitude to my colleagues Dr. A.S. Manjuladevi, Dr. Beena S Menon, Dr. R.M. Akhila, Dr. R. Jayaprakasam, Mrs. V. Subhadra Devi, Dr. A. Suganthi, Mrs. R. K. Sangeetha, Dr. S. Lakshmidevi, Mrs. M. Dhanamani, Mr. V. Shivashankar and Mr. P. Jagannath for their help, support and best wishes offered, which helped me a lot in completing this work. I would like to express my thanks to Mrs. R. Vathsala, Store In- charge for helping me in timely acquiring the required chemicals. I extend my gratitude to Mrs. A. Nirmala, Office In-charge. I take this opportunity, with pride and immense pleasure in expressing my heartfelt thanks to Mr. John, Mrs. Dhanalakshmi, Dr. R. Venkataswamy for their skilled technical assistance and for their timely help during this research work and also to the lab attendants Mrs. R. Rajeswari, Mrs. Karpagam, Mrs. Kalaivani, Mr. Murganantham, Mrs. Beula Hepsibah, Mrs. F. Stella and Mrs. Vanitha for their support in the due course of this work. I would like to thank all other teaching and non-teaching faculties of this institution and other friends whose name I might have not spelt it here, for their direct or indirect help in completing this work. I’m acknowledging my family members D. Parameswaran, P. Jamuna, A. Srinivasan, and S. Shanthi for their prayers and also I would like to express my gratitude to P. Dhanush, P. Harish ram, S. Jaisurya and S. Lidhiksha which will be always be remembered. I’m greatly indebted forever to my lovely wife M. Pradeepa for her care, prayers, encouragement and the support rendered throughout my tough times and tremendous patience, understanding and inestimable help which will be always be remembered. Finally, I would be ever indebted to the two persons who had given me life and brought me to this level. First, my beloved Mother and second my beloved Father.

A. MADESWARAN Contents

Page S. No. Chapters No. Abbreviations List of Tables List of Figures 1. Introduction 1 1.1 Neurodegenerative disorders 1 1.1.1 Overview of dementia 1 1.2 Types of dementia 2 1.2.1 Alzheimer’s disease 2 1.2.2 Vascular dementia 2 1.2.3 Dementia with Lewy bodies (DLB) 2 1.2.4 Fronto-temporal lobar degeneration (FTLD) 3 1.2.5 Mixed dementia 3 1.2.6 Creutzfeldt-Jakob disease 3 1.2.7 Parkinson’s disease (PD) dementia 3 1.2.8 Normal pressure hydrocephalus 4 1.3 Alzheimer's disease (AD) 4 1.3.1 History of Alzheimer’s disease 4 1.3.2 Neurotransmitters in AD 5 1.3.3 Signs of AD 5 1.3.4 Clinical facts of AD 6 1.3.5 Etiology of AD 6 1.3.6 Stages of AD 7 1.3.7 Pathophysiology of AD 9 1.3.8 Acetylcholine 13 1.3.9 Synthesis, storage and release of acetylcholine 13 1.3.10 Management of AD 14 1.3.11 Alzheimer’s disease inducing agents 16 1.4 Drug discovery and development 20 1.4.1 Drug design 20 1.4.2 Types of drug design 20 1.4.3 Molecular docking 22 1.4.4 Identification of right target and active site 22 Contents

1.4.5 Lipinski’s rule of Five 23 1.4.6 Types of docking 23 1.4.7 Docking approaches 23 1.4.8 Mechanics of docking 24 1.4.9 Scoring functions 26

1.4.10 Various docking software’s used for molecular 27 docking 1.4.11 Applications of drug design 31 1.5 Flavonoids 31 1.5.1 Structure of flavonoids 31 1.5.2 Classification of flavonoids 32 1.5.3 Pharmacological activity of flavonoids 32 2. Review of Literature 36 2.1 Related to docking studies 36 2.2 Enzyme inhibition assay 39 2.3 Scopolamine-induced amnesia 42 2.4 Aluminium chloride-induced amnesia 45 3. Aim and Objectives 54 4. Plan of Work 55 5. Materials and Methods 58 5.1 Evaluation of drug likeness properties 58 5.2 In silico docking binding interaction studies 59 5.3 Reagents and chemicals 66 5.4 Instruments used 66 5.5 In vitro enzyme inhibition assay 66 5.5.1 In vitro acetylcholinesterase inhibition 66 5.5.2 In vitro butyrylcholinesterase inhibition 67 5.6 Acute toxicity studies 67 5.7 In vivo experimental design 68 5.7.1 Experimental animals 68 5.7.2 In vivo scopolamine-induced amnesia model 69 5.7.3 Aluminium-induced learning impairment 69 5.8 Behavioral assessments 70 5.8.1 Rectangular maze test 70 Contents

5.8.2 Morris water maze test 71 Assessment of gross behavioral activity 5.8.3 72 (Locomotor activity) 5.8.4 Pole climbing test 72 Attention in a 5 choice serial reaction time task (5- 5.8.5 73 CSRT) 5.9 Biochemical estimations 74 5.9.1 Estimation of AChE in mice brain 74 5.9.2 Estimation of total protein content 74 5.9.3 Enzymatic anti-oxidants 75 5.9.3.1 Estimation of catalase 75 5.9.3.2 Estimation of superoxide dismutase (SOD) 75 5.9.3.3 Estimation of glutathione reductase (GSSH) 75 5.9.3.4 Estimation of glutathione peroxidase (GPx) 76 5.9.4 Non-enzymatic antioxidants 76 5.9.4.1 Estimation of reduced glutathione (GSH) 76 5.9.5 Estimation of lipid peroxidation (LPO) 76 5.9.6 (2,2-Diphenyl-1-picrylhydrazyl) (DPPH) assay 77 5.10 Histopathological studies 77 5.11 Statistical analysis 77 6. Results and Analysis 78 6.1 Drug likeness properties of the flavonoids 78 In silico docking studies against AChE and 6.2 78 BuChE 6.3 In vitro AChE and BuChE inhibitory assay 105 6.3.1 Role of flavonoids in AChE inhibitory activity 106 6.3.2 Role of flavonoids in BuChE inhibitory activity 109 Acute toxicity studies and selection of dose for 6.4 109 in vivo studies Neuroprotective effects of baicalein and 6.5 117 scopoletin in scopolamine-induced model Behavioural assessment of baicalein and 6.5.1 117 scopoletin in scopolamine treated mice 6.5.1.1 Effect on transfer latency in the rectangular maze 117 Effect on escape latency time in the Morris water 6.5.1.2 117 maze 6.5.1.3 Locomotor activity in scopolamine treated mice 120 Conditioned avoidance response in scopolamine 6.5.1.4 120 treated mice Contents

Effect of scopoletin and baicalein response time in 6.5.1.5 121 5-choice serial time task method Biochemical estimations in scopolamine induced 6.5.2 121 mice 6.5.2.1 Estimation of AChE activity in the brain 121 6.5.2.2 Determination of total protein content 124 6.5.2.3 Enzymatic antioxidant levels 126 6.5.2.4 Non enzymatic antioxidant activity 129 6.5.2.5 DPPH assay 129 6.5.2.6 Estimation of lipid peroxidation 130 Histopathological studies of the baicalien and 6.5.3 scopoletin in acute scopolamine induced amnesia 131 in mice Neuroprotective effect of baicalein and scopoletin 6.6 131 on behavioural changes in alcl3-induced model Effect of baicalein and scopoletin on behavioural 6.6.1 131 changes in alcl3 treated mice Evaluation of transfer latency in the rectangular 6.6.1.1 131 maze Estimation of escape latency time in the Morris 6.6.1.2 133 water maze

6.6.1.3 Locomotor activity in alcl3 treated mice 133 Conditioned avoidance response in AlCl treated 6.6.1.4 3 137 mice Effect of baicalein and scopoletin on response 6.6.1.5 140 time in 5-choice serial reaction time task method Biochemical evaluations in AlCl -induced 6.6.2 3 140 learning impairment in mice 6.6.2.1 Estimation of ache level in the brain 140 6.6.2.2 Estimation of total protein content 141 6.6.3 Measurement of enzymatic antioxidant levels 141 6.6.4 Non enzymatic antioxidant assay 144 6.6.4.1 Determination of reduced glutathione 144 6.6.5 Estimation of DPPH assay 145 6.6.6 Determination of lipid peroxidation level 145 Histopathological studies of the baicalien and 6.6.7 145 scopoletin against chronic alcl3 treatment in mice 7. Discussion 147 7.1 Evaluation of drug likeness properties 147 7.2 In silico AChE and BuChE inhibition 147 7.3 In vitro AChE and BuChE inhibition 149 Contents

7.4 Acute toxicity studies 150 7.5 Acute scopolamine-induced amnesia 151 Chronic in vivo aluminium chloride-induced 7.6 153 dementia 8. Conclusion 156 9. Impact of the study 160 10 Bibliography Appendices Plagiarism percentage report IAEC certificate copy

Research Publications Oral and Poster Presentations List of Abbreviations

AD Alzheimer's disease PD Parkinson's disease HD Huntington's disease ALS Amyotrophic lateral sclerosis AChE Acetylcholinesterase BuChE Butyrylcholinesterase ACh Acetylcholine CHAT Choline acetyltransferase APP Amyloid Precursor Protein NMDA (N-methyl-D-aspartate) receptor NFT Neurofibrillary tangle ROS Reactive oxygen species NSAID Non-steroidal anti-inflammatory drugs Al Aluminium MC Monte Carlo methods GA Genetic algorithm MD Molecular dynamics GOLD Genetic Optimization for Ligand Docking GLIDE Grid Based Ligand Docking with Energetics FlexX Future Leaders Exchange SLIDE Screening for ligands by induced-fit docking CADD Computer-aided drug design CAMM Computer-aided molecular modeling CAMD Computer-aided molecular design HDAC Histone deacetylase EPM Elevated plus maze MWM Morris water maze

AlCl3 Aluminium chloride CAL Conditioned-avoidance and learning CAT Catalase MDA Malondialdehyde SOD Superoxide dismutase TAC Total antioxidant capacity STZ Streptozotocin ALA Alpha-lipoic acid LGA Lamarckian genetic algorithm DTNB (5,5'-dithiobis-(2-nitrobenzoic acid) Organization for economic co-operation and OECD development List of Abbreviations

IAEC Institutional animal ethical committee Committee for the Purpose of Control and CPCSEA Supervision of Experiments on Animals ELT Escape latency time CAR Conditioned avoidance response

H2O2 Hydrogen peroxide GSSH Glutathione reductase GPx Glutathione peroxidase GSH Reduced glutathione LPO Lipid peroxidation DPPH (2,2-Diphenyl-1-picrylhydrazyl) assay ANOVA Analysis of Variance SEM Standard Error Mean LT Latency time

IC50 Inhibition constant min Minutes UV Ultraviolet

List of Tables

Table Page Details No. No. In silico molecular interactions scores of the flavonoids against 1. 80 AChE and BuChE 2. Observations done for the acute oral toxicity study- Baicalein 111 3. Mortality record for baicalein in acute oral toxicity study 113 4. Observations done for the acute oral toxicity study- Scopoletin 114 5. Mortality record for scopoletin in acute oral toxicity study 116 Effect of baicalein and scopoletin on protein content in scopolamine 6. 126 treated mice Effect of baicalein and scopoletin on enzymatic and non-enzymatic 7. 128 antioxidant level in scopolamine treated mice Effect of baicalein and scopoletin on DPPH and MDA level in 8. 130 scopolamine-induced mice Effect of baicalein and scopoletin on protein content in AlCl 9. 3 141 treated mice Effect of baicalein and scopoletin on enzymatic and non-enzymatic 10. 142 antioxidant level in AlCl3-induced mice Effect of baicalein and scopoletin on DPPH and MDA level in 11. 144 AlCl3 treated mice

List of Figures

Figure Details Page No. No. 1. Brain image showing different stages of AD 7 2. Cascade of pathophysiological events in AD 9 3. Aggregation of tau protein cascade events 10 4. Synthesis, release and binding of ACh to its receptors 11 5. Structure of acetylcholine chloride 13 6. Mechanism of scopolamine induced AD 17

7. Mechanism of AlCl3-induced AD 18 8. Different phases of drug discovery cycle 20 9. Various steps involved in structure based drug design 21 Difference between structure based drug design and ligand based 10. 22 drug design 11. Basic structure of flavonoids 32 12. Classification of flavonoids 32 13. Schematic representation of the plan of work 57 14. Refined crystal structure of mouse acetylcholinesterase (5HCU) 59 15. Human butyrylcholinesterase in complex with tacrine (4BDS) 60 16. Rectangular maze 71 17. Morris water maze 72 18. Actophotometer 72 19. Pole climbing apparatus 73 20. Five choice serial reaction time task apparatus 74 21. Drug likeness properties of the flavonoids 78 22. Binding energy of the flavonoids against AChE 79 23. Molecular interactions of scopoletin and donepezil against AChE 103 24. Binding energy of the flavonoids against BuChE 103 Molecular interactions of scopoletin and donepezil against 25. 104 BuChE 26. Selection of flavonoids for in vitro enzyme inhibition studies 106 27. In vitro AChE enzyme inhibitory assay of the selected flavonoids 107 In vitro BuChE enzyme inhibitory assay of the selected 28. 108 flavonoids Effect of baicalein and scopoletin on latency time using 29. 118 rectangular maze test in scopolamine-induced amnesia Effect of baicalein and scopoletin on escape latency time using 30. 119 Morris water maze test in scopolamine-induced amnesia List of Figures

Effect of baicalein and scopoletin on locomotor activity using 31. 122 actophotometer in scopolamine-induced amnesia Effect of baicalein and scopoletin on percentage of conditioned 32. avoidance response using pole climbing test in scopolamine- 123 induced amnesia Effect of baicalein and scopoletin on response time using 5 33. 124 CSRT model in scopolamine-induced amnesia Percentage of AChE inhibition in the mice brain in scopolamine- 34. 125 induced amnesia The demonstration of the effects of baicalein and scopoletin in 35. 132 scopolamine-induced amnesia Effect of baicalein and scopoletin on latency time using 36. 134 rectangular maze test in AlCl3-induced learning impairment Effect of baicalein and scopoletin on escape latency time using 37. 135 Morris water maze test in AlCl3-induced learning impairment Effect of baicalein and scopoletin on locomotor activity in AlCl - 38. 3 136 induced learning impairment Effect of baicalein and scopoletin on percentage of conditioned 39. 138 avoidance response in AlCl3-induced learning impairment Effect of baicalein and scopoletin on response time using 5- 40. 139 CSRT model in AlCl3-induced learning impairment Percentage of AChE inhibition in the mice brain in AlCl - 41. 3 140 induced learning impairment The demonstration of the effects of baicalein and scopoletin in 42. 146 AlCl3-induced AD

1. INTRODUCTION 1.1 NEURODEGENERATIVE DISORDERS

Neurodegenerative disorders are categorized by progressive and irreversible loss of neurons from particular regions of the brain. Prototypical neurodegenerative disorders include Parkinson's disease (PD) and Huntington's disease (HD), results in deviations in the control of drive due to the loss of neurons from structures of the basal ganglia1, 2. Alzheimer's disease (AD), results in the loss of cortical and hippocampal neurons with impairment of memory and cognitive ability and amyotrophic lateral sclerosis (ALS), producing muscular weakness due to degeneration of bulbar, spinal, and cortical motor neurons3, 4.

Neurodegenerative disorders are comparatively common and represent a substantial societal and medical problem. They are principal disorders of later life, developing in individuals who are neurologically normal, although childhood-onset forms of each of the disorders are known. PD is detected in more than 1% of population above the age of 655, 6, whereas AD affects as many as 10% of the same individuals7. HD, which is a genetically stable autosomal dominant disorder, is less common in the population as a whole but disturbs, on average, 50% of each generation in families booming the gene. ALS also is moderately rare but sometimes leads to disability and death8, 9.

At present, the pharmacological therapy of neurodegenerative disorders is restricted mostly to symptomatic treatments that do not change the course of the underlying disease. Symptomatic management for PD, where the neurochemical deficit created by the disease is well defined. The available treatments for AD, HD, and ALS are much more limited in effectiveness, and there is a need for new strategies10, 11.

1.1.1 Overview of dementia

1 hypopituitarism, pulmonary insufficiency, cardiac dysfunction and hepatolenticular degeneration), deficiency states (vitamin B12, niacin and folate deficiency), collagen (vascular disorders, systemic lupus erythematous, Behcet’s syndrome, temporal arteritis and sarcoidosis), intracranial disorders (tumor, epilepsy, neurosyphilis, encephalitis, multiple sclerosis and cerebrovascular insufficiency) and exogenous intoxication (alcohol, lead, carbon monoxide, organophosphates, mercury, manganese and toluene). It is not a part of normal aging and constantly turns as pathological process. Alzheimer’s is the most common cause of dementia13-15.

1.2 TYPES OF DEMENTIA

1.2.1 Alzheimer’s disease

Alzheimer’s disease is a chronic, progressive and disabling organic brain disorder characterized by disturbances of multiple cortical functions including memory, comprehension, orientation, language and learning capacity16. AD is accompanied by three main structural changes in the brain including neuronal loss, formation and accumulation of hyperphosphorylated tau protein termed neurofibrillary tangles (NFT) and aggregation of β-amyloid (Aβ) peptides termed senile or amyloid plaques17. These changes are most protruding in the cholinergic system, primarily in cortex and hippocampus, which is closely associated with memory loss and cognitive dysfunction in AD18.

1.2.2 Vascular dementia

Earlier known as post stroke or multi-infract dementia, for about 10% of population associated with vascular dementia and it is less common as a cause of dementia than Alzheimer’s disease. In most of the cases, the infarct dementia co-occurs with Alzheimer’s pathology. Vascular dementia occurs most commonly from damage leading to infarcts or blood vessel blockages or bleeding in the brain19, 20.

1.2.3 Dementia with Lewy bodies (DLB)

Lewy bodies are abnormal aggregations of the protein alpha-synuclein that accumulate in neurons and dementia can result when they develop in cortex region of the brain. Aggregation of alpha-synuclein in the brains of people with Parkinson’s disease (PD), in which it is produced by extreme neuronal loss in a part of the brain

2 called the substantia nigra. While people with dementia with Lewy bodies DLB and PD both have Lewy bodies, the onset of the disease is marked by cognitive impairment in DLB and motor impairment in PD20.

1.2.4 Fronto-temporal lobar degeneration (FTLD)

FTLD includes dementias such as behavioural-variant FTLD, primary progressive aphasia, corticobasal degeneration, Pick’s disease and progressive supranuclear palsy. Initial symptoms include marked changes in behaviour and personality and difficulty with comprehending language. Nerve cells in the frontal lobe and temporal lobes of the brain are affected, and these regions become significantly shrunken. In addition, the upper layers of the cortex typically become spongy and soft and have protein insertions (tau protein or the transactive DNA-binding protein) 21.

1.2.5 Mixed dementia

Mixed dementia is characterized by the hallmark abnormalities of more than one cause of dementia most commonly Alzheimer’s combined with vascular dementia, followed by Alzheimer’s with DLB, and Alzheimer’s with vascular dementia and DLB. Vascular dementia with DLB is less common. Recent studies suggest that mixed dementia is more common than previously recognized, with about half of those with dementia having pathologic evidence of more than one cause of dementia 20, 22.

1.2.6 Creutzfeldt-Jakob disease

This is very rare fatal disorder and rapidly impairs memory and coordination and causes behaviour changes, results from a misfolding protein (prion) that causes other proteins throughout the brain to misfold and malfunction. It may be hereditary sporadic or caused by a known infection. A specific form called variant Creutzfeldt- Jakob disease is believed to be caused by consumption of products from cattle affected by mad cow disease23.

1.2.7 Parkinson’s disease (PD) dementia

Problems with movement (slowness, rigidity, tremor and changes in gait) are common symptoms of PD. In PD, alpha-synuclein aggregates appear in an area deep in the brain called the substantia nigra. The aggregates are thought to cause degeneration of the nerve cells that produce dopamine. The incidence of PD is about one-tenth that

3 of Alzheimer’s. As PD progresses, it often results in dementia secondary to the accumulation of Lewy bodies in the cortex (similar to DLB) or the accumulation of beta-amyloid clumps and tau tangles24, 25.

1.2.8 Normal pressure hydrocephalus

Symptoms include difficulty in walking, memory loss and inability to control urination. Accounts for less than 5% of dementia cases and caused by impaired reabsorption of cerebrospinal fluid and the consequent build-up of fluid in the brain, increasing pressure in the brain. People with a history of brain haemorrhage (particularly subarachnoid haemorrhage) and meningitis are at increased risk. It can sometimes be corrected with surgical installation of a shunt in the brain to drain excess fluid26.

1.3 ALZHEIMER'S DISEASE (AD)

AD produces an impairment of cognitive abilities that is gradual in onset but relentless in progression. Impairment of short-term memory usually is the first clinical feature, whereas retrieval of distant memories is preserved relatively well into the course of the disease. As the condition progresses, additional cognitive abilities like, the ability to calculate, exercise visuospatial skills, and use common objects and tools (ideomotor apraxia) are impaired27. The level of arousal or alertness of the patient is not affected until the condition is very advanced, nor is there motor weakness, although muscular contractures are an almost universal feature of advanced stages of the disease. Death, most often from a complication of immobility such as pneumonia or pulmonary embolism, usually ensues within 6 to 12 years of onset. The diagnosis of AD is based on careful clinical assessment of the patient and appropriate laboratory tests to exclude other disorders that may mimic AD; at present, no direct antemortem confirmatory test exists28.

1.3.1 History of Alzheimer’s disease

4 both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) play an important role in Aβ-aggregation during plaque formation31. Many research papers published in the last decade have indicated that the structures of compounds that exhibit cholinesterase inhibitory activity are diverse32, 33.

1.3.2 Neurotransmitters in AD

The brain, in AD, shows the loss of cholinergic neurons in the basal forebrain, decreased acetylcholine (ACh) levels, and a decrease in the acetylcholine synthesizing enzyme choline acetyltransferase (CHAT) in the cerebral cortex. Animal models show that ACh plays a crucial role in information processing and memory34. Although other neurotransmitters (noradrenaline, serotonin, somatostatin and other peptides) are also deficient in AD, the cognitive impairment correlates best with the loss of cholinergic input. AChE inhibitors (tacrine) and ACh receptor agonists, including nicotine, have been used to treat AD. The marginal success of this approach suggests that, in addition to ACh deficiency, there are other profound alterations that contribute to the cognitive dysfunction35.

The relation between accumulation of plaques in brain and dementia was first identified by Dr. Alois Alzheimer in the year 1906. Later Emil Kraepelin, a German psychiatrist coined this disease as Alzheimer’s disease in 1910. Dr. Alois Alzheimer described the case of a 51-year-old woman, Auguste D in the year 1906. The patient was admitted to a hospital for 5 years with a cluster of unusual symptoms with loss of memory, inability to speak, disorientation, behavioral problems, and hallucinations. After the death, Dr. Alzheimer examined her brain tissue and found two hallmarks of Alzheimer’s disease (AD): Beta-amyloid plaques and neuroﬁbrillary tangles. Hence the name of this neurodegenerative disorder delivers his name36, 37.

1.3.3 Signs of AD

The first sign of AD is generally related to memory loss. The person may replicate the things many times, have trouble in naming items, may lose things like their wallet, glasses, keys, or get lost themselves. These changes may become more pronounced with time. Simple tasks can prove difficult for the person with Alzheimer’s, such as dressing, preparing meals, using the phone, or playing a game38.

The Alzheimer's Association has identified ten warning signs concerned someone might have Alzheimer's or dementia:

 Memory loss;

 Problems with abstract thinking;

 Difficulty performing familiar tasks;

 Changes in mood or behavior;

 Disorientation to time and place;

 Misplacing things in unusual places;

 Poor or decreased judgment;

 Changes in personality;

 Problems with language; and,

 Loss of initiative.

1.3.4 Clinical facts of AD

The possibility of AD is 6% to 10% for men and 12% to 19% for women after 65 years of age. About 47% of individuals develop AD beyond the age of 80 years. This disease last for 20 years and the persons would survive around 8 years after initial diagnosis39.

1.3.5 Etiology of AD

1.3.5.1 Genetic etiology of AD

The genetic etiology of AD includes apolipoprotein E gene polymorphism, amyloid-β protein precursor and presenilin I and II40.

1.3.5.2 Non-genetic etiology of AD

Non- genetic etiology of AD includes age, tauopathy and other risk factors like hyperlipidemia, hypertension, diabetes mellitus, stroke, smoking, traumatic head injury and familial history39, 41.

1.3.6 Stages of AD

Different stages of AD are:

1. Mild cognitive decline

2. Moderate cognitive decline

3. Severe cognitive decline

Fig 1: Brain image showing different stages of AD

1.3.6.1 Mild cognitive decline

People suffering from mild Alzheimer’s disease may appear to be healthy, but they actually have trouble making sense of their surroundings. Because mild Alzheimer’s is commonly mistaken for normal aging, it may take time to realize the condition of mild Alzheimer’s disease42.

1.3.6.2 Moderate cognitive decline

In the moderate stages of Alzheimer’s disease, the degeneration of the brain worsens and spreads to other areas that control language, reasoning, sensory processing and thought. The signs of the disease become more pronounced and behavioral problems often occur43.

1.3.6.3 Severe cognitive decline

Damage to the brain’s nerve cells is widespread in severe stages of AD. People may lose motor coordination and the ability to walk, speak, feed themselves, and recognize others (Fig. 1). Typically at this stage, full time care is required20, 43.

Each of the three stages; mild, moderate and severe, can be further broken down into seven levels:

Level 1 is considered to be a normal adult with no decline in function or of their memory.

Level 2 is a slight decline from stage 1. A person in stage 2 will complain of deficits in recalling familiar names and places. This stage is often overlooked because forgetfulness is assumed to be a natural process associated with aging.

Level 3 is characterized by a mild decline defined as early Alzheimer’s disease. In stage 3, anxiety and denial are large features under intensive interviews. Some signs include getting lost when traveling to unknown locations, low performance in the workplace and loss of words or vocabulary.

Level 4 is identified when the person needs assistance in complicated tasks such as handling individual’s finances. In this stage people tend to exhibit problems remembering their past events such as family vacations.

Level 5 brings a severe decline in a person’s ability to do everyday activities. In this stage one will need constant assistance by another individual to do simple tasks of picking out cloths, preparing meals and even starting a car. They will have difficultly recalling current information, but will still remember major information involving themselves and their families.

Level 6 is when there is a moderately severe decline in the disease. By this stage, Alzheimer’s patients forget significant amount of information including personal facts. When this stage approaches, a person needs assistance getting dressed and going to the bathroom, and has disturbed sleep patterns. The individual’s personality change becomes highly apparent and noticeable. Feeling frustrated and acting out with violence may occur because of the person’s inability to remember or process information long enough to react to their own thoughts.

Level 7 is the last stage in Alzheimer’s disease. This is when there is a severe decline in vocabulary, emotions and the connection of the brain to body parts. Patients are now limited to six or seven words at a time. They lose the ability to walk, sit up straight, hold up their head, and even smile. In this stage there is no ability to have a

8 hand-to-mouth motion, to place one foot in front of another, or even to urinate on their own.

1.3.7 Pathophysiology of AD

AD is driven by two processes: extracellular deposition of beta amyloid-Aβ and intracellular accumulation of tau protein. Both these compounds are insoluble. Aβ is the main component of senile plaques and tau is the component of neurofibrillary tangles. Aβ deposition is specific for AD and is thought to be primary. Tau accumulation is also seen in other degenerative diseases and is thought to be secondary44, 45.

1.3.7.1 Beta Amyloid hypothesis

Aβ is a 36 to 43 amino acid peptide, which is part of a larger protein, the Amyloid Precursor Protein (APP). APP is a transmembrane protein, made by neurons and other brain cells. It is also found in extraneural tissues and is especially abundant in platelets. Its function is unknown. The Aβ amyloid residue includes part of the transmembrane domain of APP and is derived from cleavage of APP by the enzymes β-and γ-secretase. Aβ monomers and oligomers are further degraded by other enzymes46. Defective clearance of Aβ from aberrant cleavage of APP and other mechanisms results in its accumulation (Fig. 2). Aβ monomers polymerize initially into soluble oligomers and then into larger insoluble fragments such as Aβ42, which precipitate as amyloid fibrils.

Fig 2: Cascade of pathophysiological events in AD

The beta amyloid hypothesis is the basis of a novel prevention and treatment method for AD that was reported recently in transgenic mice that overexpress a mutant APP and develop AD neuropathology. Active immunization of young animals with Aβ and passive immunization with Aβ antibodies prevented the development of AD; immunization of older animals reduced the extent and severity of AD pathology. Based on these findings, a human vaccine consisting of synthetic Aβ was developed. Phase I trials went uneventfully but phase II studies were terminated because some patients developed an autoimmune meningoencepalitis47, 48.

1.3.7.2 Tau protein aggregation

Neurofibrillary degeneration is characterized by the deposition in the neuronal body and processes of insoluble polymers of over-phosphorylated microtubule associated protein tau. Tau aggregates as pairs of filaments that are twisted around one another (paired helical filaments). These deposits interfere with cellular functions by displacing organelles (Fig. 3).

Fig. 3: Aggregation of tau protein cascade events

By distorting the spacing of microtubules, they impair the axonal transport thus affecting the nutrition of axon terminals and dendrites. No mutations of the tau gene

10 occur in AD. Abnormal tau first appears in the entorhinal cortex, then in the hippocampus, and at later stages in association cortex. Recent observations in transgenic mice suggest that the spread of the pathology to anatomically linked areas occurs by passage of abnormal tau across synapses49, 50.

1.3.7.3 Cholinergic deficiency

Neuronal and pharmacological evidences correlate the severity of memory impairment with cholinergic hypo function. Cholinergic neurotransmitter plays an important role in patients with AD. AChE is an enzyme that hydrolyzes the acetylcholine to acetate and choline at the cholinergic synapses51. Reduction in the level of acetylcholine in the brain produces memory and learning impairment. ACh binds to postsynaptic receptors such as muscarinic and nicotinic receptors (Fig. 4). Presynaptic nicotinic receptors are responsible for the release of neurotransmitters important for memory and mood (acetylcholine, glutamate, serotonin and norepinephrine). Loss of nicotinic receptor subtypes in the hippocampus and cortex has been observed in AD. Muscarinic receptors are not involved in the development of dementia, but blocking of this receptor produces confusion52, 53.

Fig 4: Synthesis, release and binding of ACh to its receptors

11 inhibitors. Drugs that inhibit both enzymes may be preferably used for treating AD. In AD, there is an involvement of pyramidal neurons there by neuronal loss, synapse loss, reduced glutamate concentration and tangle formation leads to the formation of neurofibrillary tangles. Glutamate is the neurotransmitter of pyramidal neurons54-56.

1.3.7.4 Glutamatergic pathway

Glutamate is the primary excitatory neurotransmitter present in the brain. In the central nervous system, it is ubiquitous and involved roughly 66% of all brain synapse. Glutamatergic neurons are critical, because they form projections to other areas of brain including cholinergic neurons and the cholinergic neurons help for cognition and memory. (N-methyl-D-aspartate) (NMDA) receptor undergoes sustained low level activation in AD brains and produce sustained low level neurotransmission56. This dysregulation of NMDA glutamate receptor leads to neuronal damage and the continuous activation of the receptor leads to the chronic calcium influx within the neuron. This interfere the normal signal transduction pathway of glutamate and leads to increased production of amyloid precursor protein (APP). APP increases the plaque development and hyperphosporylation of tau protein (Neurofibrillary tangle (NFT) formation) followed by neuronal toxicity. AD patients appeared to have reduced number of glutamate reuptake receptor sites and abnormal expression of glutamate transporter leads to the formation of NFT and these tangles are pathogenic55.

Other neurotransmitters like serotonin and norepinephrine levels become altered in brain which affect wide spread areas of cerebral cortex and produce behavioral and psychological symptoms of dementia57.

1.3.7.5 Oxidative stress

In AD brains, Aβ induces lipid peroxidation and generate reactive nitrogen and reactive oxygen species. The reactive nitrogen and oxygen molecules contain unpaired electrons in the outer shell. These unpaired electrons react with other molecules to achieve a stable configuration and produce free radicals. These free radicals cause cellular and molecular damage. This reaction is permanent and occurs in all types of neuronal macromolecules such as proteins, lipids, carbohydrates and nucleic acids. Brain is unprotected to damage from oxidative stress, because of high oxygen consumption rate, high lipid content and relative loss (paucity) of anti-oxidant enzymes

12 compared to other organs. Neuronal oxidation can cause numerous problems such as irreparable DNA damage and up-regulation of pro inflammatory cytokines57, 58.

1.3.7.6 Chronic inflammation

Senile plaques, NFT and damaged neurons may stimulate inflammation and leads to cell damage. Microglia is a type brain cells and it includes neurons, astrocytes and oligodendrocytes. These are involved in the infection or injury within the brain. Microglia gets activated and release cytotoxic molecules like pro-inflammatory cytokines, reactive oxygen species (ROS), complement proteins and proteinases during AD pathogenesis. Cytokines stimulate inflammatory process and promote apoptosis of neurons and oligodendrocytes and induce myelin damage55, 59.

1.3.8 Acetylcholine

Acetylcholine (ACh) is a neurotransmitter present in the cholinergic synapses and neuroeffector junctions in the central and peripheral nervous systems. Acetylcholine acts through muscarinic and nicotinic cholinergic receptors. With the help of these receptors, signals will transduce via different mechanisms. Cholinergic agonists are drugs, which mimics the effects of acetylcholine at these receptors60.

CH 3 + H 3 C N O CH 3 CH 3 O . Cl-

Fig 5: Structure of acetylcholine chloride

1.3.9 Synthesis, storage and release of acetylcholine

In the synthesis of acetylcholine, Acetyl CoA (Acetyl co-enzyme A) is derived from pyruvate via multistep pyruvate dehydrogenase reaction. Choline is taken up from the extracellular fluid into the axoplasm by active transport mechanism. In the presence of choline acetyltransferase, choline and acetyl CoA gets converted to acetylcholine. After its synthesis, ACh is taken up by storage vesicles present in the nerve terminals. The vesicles contain both ACh and ATP in the ratio of 10:1 and the

13 release of ACh and other neurotransmitters takes place through calcium-mediated exocytosis. Release of ACh inhibited by botulinum and tetanus toxins from clostridium60, 61.

1.3.10 Management of AD

Several pathogenic pathways are involved in the progression of AD via senile plaques, NFT, oxidative stress, inflammatory cascade, glutamatergic and cholinergic deficiency. Based on the pathological mechanism, several pharmacological treatments are available. Currently available drug therapy for AD consist of cholinesterase inhibitors and NMDA receptor antagonists approved by the U.S. Food and Drug Administration (FDA) and some neuroprotective agents improve the psychological and behavioral symptoms of AD62.

A major approach to the treatment of AD has involved attempts to augment the cholinergic function of the brain63. An early approach was the use of precursors of acetylcholine synthesis, such as choline chloride and phosphatidyl choline (lecithin). Physostigmine, a rapidly acting, reversible AChE inhibitor, produces improved responses in animal models of learning, and some studies have demonstrated mild transitory improvement in memory following physostigmine treatment in patients with AD. The use of physostigmine has been limited because of its short half-life and tendency to produce symptoms of systemic cholinergic excess at therapeutic doses64.

Four inhibitors of AChE currently approved by the FDA for treatment of Alzheimer's disease are tacrine (1,2,3,4-tetrahydro-9-aminoacridine; COGNEX), donepezil (ARICEPT), rivastigmine (EXCELON), and galantamine (RAZADYNE)65.

Tacrine is a potent centrally acting inhibitor of AChE66. Studies of oral tacrine in combination with lecithin have confirmed that there is indeed an effect of tacrine on some measures of memory performance, but the magnitude of improvement observed with the combination of lecithin and tacrine is modest at best67. The side effects of tacrine often are significant and dose-limiting; abdominal cramps, anorexia, nausea, vomiting, and diarrhoea are observed in up to one-third of patients receiving therapeutic doses, and elevations of serum transaminases are observed in up to 50% of those treated. Because of significant side effects, tacrine is not clinically widely used68.

Donepezil is a selective inhibitor of AChE in the CNS with little effect on AChE in peripheral tissues. It produces modest improvement in cognitive scores in Alzheimer's disease patients and has a long half-life, allowing once-daily dosing. Rivastigmine and galantamine are dosed twice daily and produce a similar degree of cognitive improvement. Adverse effects associated with donepezil, rivastigmine, and galantamine are similar in character but generally less frequent and less severe than those observed with tacrine; they include nausea, diarrhea, vomiting, and insomnia. Donepezil, rivastigmine, and galantamine are not associated with the hepatotoxicity that limits the use of tacrine69.

An alternative strategy for the treatment of AD is the use of the NMDA glutamate-receptor antagonist memantine. Memantine produces a use-dependent blockade of NMDA receptors. In patients with moderate to severe AD, use of memantine is associated with a reduced rate of clinical deterioration. Whether this is due to a true disease-modifying effect, possibly reduced excitotoxicity, or is a symptomatic effect of the drug is unclear. Adverse effects of memantine usually are mild and reversible and may include headache or dizziness70.

1.3.10.1 Cholinesterase inhibitor

Donepezil, rivastigmine, galantamine and tacrine are cholinesterase inhibitors. These drugs prevent the breakdown of acetylcholine in the brain by inhibition of acetylcholinesterase enzyme and increase the expression of nicotinic receptors. These drugs help in maintaining the acetylcholine level in brain and compensate the loss of functioning of brain cells. Cholinesterase inhibitors are used for the treatment of mild, moderate and severe AD and the side effects observed are nausea, vomiting, loss of appetite and increased frequency of bowel movement71.

1.3.10.2 NMDA receptor antagonist

Memantine is a NMDA receptor antagonist. It is used for the treatment of moderate to severe AD. Memantine restores the function of damaged nerve cells and reduce abnormal excitatory signals by modulating the NMDA receptor activity. Some compounds are mainly under preclinical and clinical evaluations based on the mechanism of β-amyloid (Aβ) cascade and tau protein hyperphosporylation72.

1.3.10.2.1 Aβ-targeting strategies

 α secretase activators/ modulators

 β secretase inhibitors (GSK188909, PMS777)

 γ secretase inhibitors/ modulators (BMS299897,MRK-560)

 Aβ- aggregation inhibitors

 M1 muscarinic agonists (AF102B, AF267B)

 Immunotherapy

 Apo lipoprotein (ApoE) promotes Aβ clearance

 Aβ-degrading enzyme

 Drugs influencing Aβ blood- brain barrier transport

1.3.10.2.2 Treatment based on tau pathology

 Prevention of phosphorylation of tau

 Prevention of the aggregation of tau

 Tau immunotherapy

 Prevent the misfolding of tau

1.3.10.2.3 Non-steroidal anti-inflammatory drugs (NSAIDs)

 Trials with COX-2 selective inhibitors

1.3.10.2.4 Antioxidants nutrients and agents: Vitamins C and E, oestrogen, statins, omega-3 polyunsaturated fatty acids.

1.3.11 Alzheimer’s disease inducing agents

Alzheimer’s disease or learning impairment was induced by drugs and chemicals such as: scopolamine, benzodiazepines, phencyclidine, monosodium glutamate, aluminium chloride and colchicines73.

1.3.11.1 Scopolamine and neurotoxicity

16 cholinergic dysfunction and impaired cognition in rats74. Recently, it has been reported that Indigofera tinctoria Linn (Fabaceae) attenuates cognitive and behavioral deficits in scopolamine-induced amnesic mice75. Munshi et al., 2016 reported the efficacy of Brahmi ghrita against scopolamine induced amnesia in rats76.

Fig. 6: Mechanism of scopolamine induced AD

The administration of the antimuscarinic agent scopolamine to young human volunteers produces transient memory deficits77. Analogously, scopolamine has been shown to impair memory retention when given to mice shortly before training in a dark avoidance task78-80. The ability of a range of different cholinergic agonist drugs to reverse the amnesic effects of scopolamine is now well documented in animals and human volunteers. However, the neuropathology of dementia of the Alzheimer type is not confined to the cholinergic system81.

Nabeshima et al., studied the antagonism against phencyclidine-induced retrograde amnesia in mice82. Lenegre et al., investigated the effects of piracetam on amnesia induced by scopolamine, diazepam and electroconvulsive shock83. Yamaoto et al., studied the effect of drugs on the impairment of working memory produced by scopolamine, ethylcholine aziridinium (AF64A) or cerebral ischemia using a repeated acquisition procedure in a three-panel runway apparatus84. Braida et al., investigated the short (120 min) and long-lasting (360 min) antagonism of scopolamine- induced amnesia in rats in an eight-arm radial maze by a cholinesterase inhibitor85.

1.3.11.2 Aluminum and neurotoxicity

The hypothesis that there is a link between aluminum and AD was first brought out in the 1960s by Terry and Pena and by Klatzo and collaborators in 196586. Early in 1976, high levels of aluminum have been found in brain lesions, such as plaques and tangles, in patients with Alzheimer’s disease and also in other conditions such as Parkinson’s disease (PD), pre-senile dementia, amyotrophic lateral sclerosis, neuro fibrilar degeneration, dialysis encephalopathy syndrome and nigro striatal syndrome87 (Fig. 7).

Fig. 7: Mechanism of Aluminium chloride induced AD

Extended exposure to 100 mM Al lactate increased striatal levels of the dopamine metabolite in turns, suggests that exposure to Al may cause increased turnover of dopamine. The development of an encephalopathy, characterized by cognitive deficits, in-coordination, tremor and spinocerebellar degeneration, among workers in the aluminum industry also indicates that exposure to the metal can be profoundly deleterious89.

Abnormal neurological symptoms have been observed in several patients receiving intramuscular injections of Al-containing vaccines. There have been many experimental studies on animals and on isolated cells showing that aluminum has toxic effects on the nervous system. In 1991, Guy showed that the uptake of aluminum by human neuroblastoma cells display an epitope associated with Alzheimer's diseases. Chronic exposure of animals to aluminum is associated with behavioral, neuropathological and neurochemical changes90. Among them, deficits of learning and behavioral functions are most evident. Several metals interact with β-amyloid (Aβ) in senile plaques. It is interesting to note that, compared to other Aβ-metal complexes (Aβ-Fe, Aβ-Zn, Aβ-Cu), Aβ-Al is unique in promoting a specific form of Aβ oligomerization that has marked neurotoxic effects91. There are a lot of ways by which Al can damage neural cells:

 Interfering with glucose metabolism, leading to low amounts of ACh precursors.

 Inhibiting the binding of Ca++ ions

 Increasing the production of cAMP

 Changes in the cytoskeleton protein, leading to phosphorylation, proteolysis, transport and synthesis disruption

 Inducing oxidative damage by lipid peroxidation

 Interacting directly to genomic structures

Being involved in the production of reactive oxygen species (ROS), aluminum may cause impairment in mitochondrial bioenergetics and may lead to the generation of oxidative stress which may lead to a gradual accumulation of oxidatively modified cellular proteins, lipids and affects endogenous antioxidant enzyme activity, leading to degeneration of neuronal cells92, 93. In this way, aluminum is a strong candidate for

19 consideration as a subtle promoter of events typically associated with brain aging and neurodegenerative disorders.

1.4 DRUG DISCOVERY AND DEVELOPMENT Drug discovery process was started in the beginning of nineteenth century, by John Langley in the year of 1905. It is considered as the fastest developing directions of the high-tech investigations that depend on the achievements of molecular chemistry, information technologies, quantum physics, biology and bio-informatics94.

Drug discovery process involves the identification of target, target validation, lead identification, lead optimisation, synthesis, screening for its therapeutic efficacy (Fig. 8). Once the testing is completed, drug development process will started prior to the clinical trials95, 96.

Fig 8: Different phases of drug discovery cycle

1.4.1 Drug design

Rational drug design uses different computational methods to identify novel compounds. The compounds having maximum efficacy, safety and selectivity towards the target will be identified. In rational drug design the biological and physical properties of the target was studied97.

1.4.2 Types of drug design

There are two types of drug design:

Structure based drug design Ligand based drug design

1.4.2.1 Structure based drug design Structural based virtual screening is being with the identification of a potential ligand binding site on the target molecule (Fig. 9). Target site possesses a variety of hydrogen bond donors and acceptors, hydrophobic characteristics, and molecular adherence surfaces. The structure of a target protein is determined by NMR, X-ray crystallography and initialization of the target is the key steps in the structure based drug design98, 99. The experimental structures of ligands bound to the enzymes, provide ideas for analog design and intern useful for the docking studies100.

Fig. 9 Various steps involved in structure based drug design

1.4.2.2 Ligand based drug design It is a most effective method used in the absence of the structural information of the target. Ligand based method is used to know about the inhibitors of the target receptor101. The active lead molecule is detected by using structural properties or topological similarity properties. Several criteria for structure similarity comparisons are shape of an individual fragment or electrostatic properties of the molecule. The lead molecules are generated by ranked based on their function score obtained by using different algorithms98.

In this method, the known active molecules (ligands) and the potential hit molecules are compared using pre-defined mathematical expressions (grids) to quantify molecular similarity (Fig. 10). These approaches essentially neglect any information about the target biomolecule as well as the 3D structure of the ligand compounds. Ligand based drug design is efficient method and applied in combination with structure-based approaches to identify potential bioactive leads that can be fed into

21 docking experiments101.

Fig. 10 Difference between structure based drug design and ligand based drug design

1.4.3 Molecular docking

Molecular docking may be defined as an optimization problem, which would describe the “best-fit” orientation of a ligand that binds to a particular protein of interest. The aim of molecular docking is to achieve an optimized conformation for both the protein and ligand and relative orientation between protein and ligand such that the free energy of the overall system is minimized102.

In the field of molecular modeling, docking is a method which predicts the preferred orientation of one molecule to a second when bound to each other to form a stable complex. Knowledge of the preferred orientation may be used to predict the binding affinity between two molecules or strength of association (scoring functions). During the docking process, the ligand and the protein adjust their conformation to achieve an overall “best-fit” and this kind of conformational adjustments results in the overall binding and can be referred to as “induced-fit”103.

1.4.4 Identification of right target and active site

Target is a protein molecule and it is closely related to disease. It plays an important role in signal transduction pathway that often disrupted in the diseased condition. Enzymes are one of the targets as they have binding pockets for inhibition as

22 well as substrates104.

1.4.5 Lipinski’s rule of Five

This rule was postulated by Christopher A. Lipinski in 1997, based on the observation that most of the drugs are relatively small and lipophilic in nature. Lipinski's rule of five is a rule of thumb to evaluate druglikeness. This rule states that, an orally active "drug-like" molecule has:

 Partition coefficient (log P) less than 5  Molecular weight below 500 daltons  Not more than 10 hydrogen bond acceptors (O and Ngroup)  Not more than 5 hydrogen bond donors (OH and NH group)  Number of violations less than 5  All the numbers should be multiples of 5, which is the basis for the rules name.

The rule describes about pharmacokinetic properties of a drug in the human body, including their absorption, distribution, metabolism, and excretion (ADME). But this rule does not predict if a compound is pharmacologically active or not105. This set of rules suggests that the necessary properties for good oral bioavailability and reflects the notion that pharmacokinetics, toxicity and other adverse effects are directly linked to the chemical structure of a drug106.

1.4.6 Types of docking107

1.4.6.1 Rigid docking The rigid docking is suitable position for the ligand in receptor environment obtained while maintaining its rigidity. 1.4.6.2 Flexible docking In this process, receptor-ligand interaction was obtained by changing internal torsions of ligand into the active site while receptor remains fixed.

1.4.7 Docking approaches108

Two approaches mainly popular in molecular docking.

1.4.7.1 Shape complementarity: This technique is used to describe the matching of

23 ligand and protein as complementarity surfaces. 1.4.7.2 Simulation: It is the actual docking process additionally calculating the interaction energies between ligand and protein molecule.

1.4.8 Mechanics of docking

To perform a docking screen, the first requirement is a structure of a protein. Protein structure has been determined by using x-ray crystallography or NMR spectroscopy. The protein structure and database of potential ligands serve as inputs to a docking programme. The success of docking program depends on: search algorithm and scoring function109.

1.4.8.1 Search algorithm A strict search algorithm would completely elucidate all possible binding modes between ligand and receptor. Various searching algorithms have been developed and widely used in molecular docking software. But it would be too expensive to computationally generate all the possible conformations. Some commonly used searching algorithms are: Monte Carlo (MC) methods, fragment based method, distance geometry, matching method, ligand fit method, point complementarity method, blind docking, inverse docking, genetic algorithms and molecular dynamics110.

1.4.8.2 Monte Carlo (MC) method

MC methods are among the most established and widely used stochastic optimization techniques. These methods use sampling technique and are able to generate states of low energy conformations. The system makes random moves and accepts or rejects each conformation based on Boltzmann probability. Simulated annealing is a generalization of a Monte Carlo method for examining the equations of state and frozen states of n-body systems. The initial state of the system has random thermal motion within a specified potential force field. The effective temperature of the system (the degree of random motion) is decreased overtime, until a final stable docked position is obtained. The random motion of the ligand allows for exploration of the local search space, and the decreasing temperature of the system acts to drive it to a minimum energy111. One of the most widely used simulated annealing procedures is the Metropolis Monte Carlo simulated annealing algorithm.

1.4.8.3 Matching method

This method focuses on complementarity. Ligand atom is placed at the ‘best’ position in the site, generating a ligand receptor configuration that may require optimization103.

1.4.8.4 Distance geometry

Many types of structural information can be expressed as intra or intermolecular distances. The distance geometry formalism allows these distance to be assembled and 3 dimensional structures consistent with them to be calculated. The fast sampling of the conformational space do not always results in reliable results. An example of a program using distance geometry in docking problem is Dock It102.

1.4.8.5 Fragment based method

Fragment based methods can be described as dividing the ligand into separate protons or fragments, docking the fragments and finally linking these fragments together with target protein. Some mainly used fragment based methods are FlexX104.

1.4.8.6 Ligand fit method

Ligand fit term provide a rapid accurate protocol for docking small molecules ligand into protein active sites for considering shape complimentarity between ligand and protein active sites103.

1.4.8.7 Inverse docking

This method uses computer for finding toxicity and side effects of protein targets on a small molecule. Knowledge of these targets combined with that of proteomics pharmacokinetic profile can facilitate the assessment of potential toxicities side effect of drug candidate. One of these protocols is selected for docking studies of particular ligand108.

1.4.8.8 Genetic algorithm (GA)

25 mutation and crossover transformations. The algorithm maintains a selective pressure towards an optimal solution, with a randomized information exchange permitting exploration of the search space. A range of programs implements GA for docking110.

1.4.8.9 Molecular dynamics (MD)

Molecular dynamics (MD) is widely used as a powerful simulation method in many fields of molecular modeling. In the context of docking, by moving each atom separately in the field of the rest atoms, MD simulation represents the flexibility of both the ligand and protein more effectively than other algorithms. The disadvantage of MD simulations is that they progress in very small steps and thus have difficulties in stepping over high energy conformational barriers, which may lead to inadequate sampling. MD simulations are often efficient at local optimization. Thus a current strategy is to use random search in order to identify the conformation of the ligand, followed by the further subtle MD simulations106.

1.4.9 Scoring functions

The purpose of the scoring function is to delineate the correct poses from incorrect poses, or binders from inactive compounds in a reasonable computation time. Scoring functions involve estimating, rather than calculating the binding affinity between the protein and ligand and through these functions, adopting various assumptions and simplifications104. Scoring functions can be divided into:

i. Force-field-based scoring functions ii. Knowledge-based scoring functions. iii. Empirical based scoring functions 1.4.9.1 Force-field-based scoring functions

Classical force-field-based scoring functions assess the binding energy by calculating the sum of the non-bonded (electrostatics and van der Waals) interactions. The electrostatic terms are calculated by a Columbic formulation. Force-field-based scoring functions also have the problem of slow computational speed. Thus cut-off distance is used to handle the non-bonded interactions106.

Extensions of force-field-based scoring functions consider the hydrogen bonds, solvation’s and entropy contributions. Software programs, such as DOCK, GOLD and AutoDock, offer users such functions. They have some differences in the treatment of

26 hydrogen bonds, the form of the energy function etc. The results of docking with force- field-based functions can be further refined with other techniques, such as linear interaction energy and free-energy perturbation methods (FEP) to improve the accuracy in predicting binding energies109.

1.4.9.2 Knowledge-based scoring functions

Knowledge based scoring functions use statistical analysis of ligand-protein complexes crystal structures to obtain the inter-atomic contact frequencies and distances between the ligand and protein. The scoring is done by statistically observing the intermolecular relation between the ligand and the biological target protein using “Potential of Mean Force”. The intermolecular interactions are mainly taken into account for the functional group or atoms that occur frequently. The result of this method is evaluated based on the binding interactions. PMF, Drug Score, SMoG and Bleep are examples of knowledge-based functions110.

1.4.9.3 Empirical scoring functions

In empirical scoring functions, binding energy decomposes into several energy components, such as hydrogen bond, ionic interaction, hydrophobic effect and binding entropy. Each component is multiplied by a coefficient and then summed up to give a final score. Coefficients are obtained from regression analysis fitted to a test set of ligand-protein complexes with known binding affinities. Empirical scoring functions may be treated in a different manner by different software, and the numbers of the terms included are also different. LUDI, PLP, Chem Score are examples derived from empirical scoring functions112.

1.4.10 Various docking software’s used for molecular docking

Over 60 docking software systems and more than 30 scoring functions are reported. Molecular docking is implemented as part of software packages for molecule design and simulation. More than one search method and scoring functions are provided in order to increase the accuracy of the simulations113. Only some of the software was made available and a limited number of them are widely used. Commonly used software’s are:

1.4.10.1 DOCK

DOCK is one of the oldest and best known ligand-protein docking programs. It systematically describes the geometries of ligands and binding sites by sets of spheres, attempting to fit each compound from a database into the binding site and the spheres could be overlapped by means of an approximate clique-detection procedure. The initial version used rigid ligands and edibility was later incorporated via incremental construction of the ligand in the binding pocket of the target protein. In the recent version of DOCK, steric matching-scores with electrostatic and molecular mechanics interaction energies are considered for the ligand-receptor complex. In scoring, the docked orientations of atomic hydrophobicity descriptors are being considered114, 115.

1.4.10.2 GOLD (Genetic Optimization for Ligand Docking)

GOLD uses a flexible docking mode for small molecules into protein binding site which utilizes genetic algorithm for the conformational search that forms a powerful tool for screening and identification of novel lead compounds. It provides docking of flexible ligand and protein with flexible hydroxyl groups. Otherwise the protein is considered to be rigid. This makes it a good choice when the binding pocket contains amino acids that form hydrogen bonds with the ligand. Gold is very highly regarded within the molecular modeling community for its accuracy and reliability and its genetic algorithm parameters are optimized for wide range of virtual screening applications. GOLD has one of the most comprehensive validation test sets114, 116.

1.4.10.3 GLIDE (Grid Based Ligand Docking with Energetics)

GLIDE approximates a close and complete systematic search for the conformational, orientation and positional space of the docked ligand. Glide uses a series of hierarchical filters to search for possible locations of the ligand in the active- site region of the receptor. A grid representation for the shape and properties of the receptor is used that progressively scores for ligand posing114.

1.4.10.4 FlexX (Future Leaders Exchange)

28 ligand again, using the other pieces. For a protein with known three dimensional structures and a small ligand molecule, FlexX predicts the geometry of the protein- ligand complex and estimates the binding affinity. It has a bit lower hit rate than DOCK but provides better estimates of Root Mean Square Distance for compounds with correctly predicted binding mode117.

1.4.10.5 SLIDE (Screening for ligands by induced-fit docking)

SLIDE represents a general approach to organic and peptidyl database screening. It can handle large binding-site templates and uses multi-stage indexing to identify feasible subsets of template points for ligand docking. An optimization approach based on mean-field theory is applied to model induced-fit complementarities, balancing flexibility between the ligand and the protein side chains. SLIDE can screen 100,000 compounds within a few days and returns a ranked list of sterically feasible ligand candidates, ranked by complementarities to the protein's binding site114.

1.4.10.6 Hammerhead Hammerhead is suitable for screening large databases of flexible molecules by binding to a protein of known structure. It precisely docks a variety of known flexible ligands, and it spends an average of only a few seconds on each compound during a screen. The approach is completely automated, from the elucidation of protein binding sites, through the docking of molecules, to the final selection of compounds for assay114.

1.4.10.7 FRED FRED takes a multi-conformer library or database of one or more ligands, a target protein structure, a box defining the active site of the protein and several optional parameters. Various options are available for optimization with respect to the built-in scoring functions: optimization of hydroxyl group rotamers, rigid body optimization, torsion optimization, and reduction of the number of poses that are passed on to the next scoring function. The ligand conformers and protein structure are treated as rigid during the majority of the docking process. FRED's docking strategy is to exhaustively score all possible positions of each ligand in the active site. The exhaustive search is based on rigid rotations and translations of each conformer. This novel strategy completely avoids the sampling issues associated with stochastic methods used by most other docking programs (such as Gold, FlexX, Dock and Glide). FRED jobs can also be

29 easily distributed over multiple processors to further reduce docking time115.

1.4.10.8 AutoDock AutoDock is a script driven flexible automated and random search docking technique operated by altering the ligand or a subset of ligand with several rotatable bonds to predict the binding interaction between small molecules to the known receptor three-dimensional structure. The robustness of AutoDock can be attributed to the Monte Carlo simulated annealing, evolutionary, genetic and Lamarckian genetic algorithm methods. The prediction of bound conformations such as enzyme-inhibitor complexes, peptide-antibody complexes and even protein-protein interactions has shown a great success through AutoDock. Possible orientations are evaluated with AMBER force field model in conjunction with free energy scoring functions and a large set of protein-ligand complexes with known protein-ligand constants. The newest unreleased version 4 contains side chain flexibility. AutoDock has more informative web pages than its competitors; because of its free academic license118.

1.4.10.9 AutoDock 4.2

Auto Dock 4.2 uses a semi-empirical free energy force field to evaluate conformations during docking simulations. The force field evaluates binding in two steps. The ligand and protein start in an unbound conformation. In the first step, the intra molecular energies are estimated for the transition from these unbound states to the conformation of the ligand and protein in the bound state. The second step then evaluates the intermolecular energies of combining the ligand and protein in their bound conformations. The parameters are based on the amber force field119.

Computer-aided drug design (CADD) is a key component in the process of drug discovery and development, as it offers great promise to drastically reduce cost and time120. It belongs to pre-clinical phase and it plays a key role in identifying new investigational drug-likes and developing new potential inhibitors related to a determinate target. Computational strategy protocol characterized by the use of bioinformatics tools for lead optimization which reduce the cycle time and cost121. AutoDock 4.2 is the most recent version which has been widely used for virtual screening, due to its enhanced docking speed122.

1.4.11 Applications of drug design

Use of computational techniques in drug discovery and development process is rapidly gaining in implementation, popularity, and appreciation. Different terms are being applied to this area, including computer-aided drug design (CADD), computer- aided molecular modeling (CAMM), computational drug design, computer-aided molecular design (CAMD), computer-aided rational drug design, and in silico drug design. Both computational and experimental techniques have important roles in drug discovery and development and represent complementary approaches123, 124. CADD entails:

 Streamline drug discovery and development process  Leverage of chemical and biological information about ligands and/or targets to identify and optimize new drugs  Design of in silico filters to eliminate compounds with undesirable properties (poor activity and/or poor absorption, distribution, metabolism, excretion and toxicity, ADMET) and develop most promising drug candidates.

1.5 FLAVONOIDS Flavonoids are the large group of biologically active water-soluble plant compounds that include pigments present naturally in fruits, vegetables, and herbs. Flavonoids are inherent part of human and animal diet however, these are not synthesized within the body of animals including human125.

Flavonoids are present in different parts of plant materials include leaves, fruits, vegetables, herbs and beverages such as tea and red wine. Flavonoids are associated with a broad spectrum of health promoting effects126-128. They are an indispensable component in a variety of nutraceutical, pharmaceutical, medicinal and cosmetic applications129-130. Most of them are present in our everyday’s life.

1.5.1 Structure of flavonoids

Flavonoids occur in aglycones, glycosides and methylated derivatives131. Glycosylation makes it less active, more water soluble and permits its storage in the cell vacuole. Chemically flavonoids may be defined as a group of polyphenolic compounds consisting of two substituted benzene rings (Ring A and C) connected by the side chain

31 of three carbon atoms and an oxygen bridge (Ring B)132 (Fig. 11).

Fig. 11 Basic structure of flavonoids

All flavonoids share a basic C6-C3-C6 phenyl-benzopyran backbone. Ring A usually shows a phloroglucinol substitution pattern133.

1.5.2 Classification of flavonoids

Over 5000 naturally occurring flavonoids have been characterized from various plants sources134 (Fig. 12).

Fig. 12 Classification of flavonoids

1.5.3 Pharmacological activity of flavonoids

Flavonoids exhibit several biological effects such as:

1.5.3.1 Antimicrobial activity Flavonoids are esters of phenolic acids; it has antibacterial, antifungal and

32 antiviral activities. Naringin, quercetin, hesperetin and catechin possess various spectrum of antiviral activity. It affects the replication and infectivity of certain RNA and DNA viruses. Flavones selectively inhibit HIV-1 and HIV-2 or similar immunodeficiency virus infections. Quercetin and apigenin exhibit antibacterial activity135.

1.5.3.2 Hepatoprotective activity Flavonoids have also been found to possess hepatoprotective activity. In a study carried out to investigate the flavonoid derivatives silymarin, apigenin, quercetin, and naringenin, as putative therapeutic agents against microcrystin LR-induced hepatotoxicity, silymarin was found to be the most effective one. The flavonoid, rutin and venoruton, showed regenerative and hepatoprotective effects in experimental cirrhosis131.

1.5.3.3 Anti-oxidant activity

Flavonoids possess antioxidant potential due to its structural characteristics. They protect cells against the damaging effects of reactive oxygen species. Anti- oxidant activity of flavonoids is due to their ability to reduce free radical formation, scavenge free radicals, and chelate metal ions. Flavonoids require a hydroxyl group at carbon 3 of the C- ring and two hydroxyl groups at carbons 3' and 4' of the B- ring for their anti-oxidant activity. Luteolin, genistein, daidzein, kaempferol, morin, naringenin, quercetin, apigenin, myricetin, robinin and cyanidin are possess antioxidant activity. Catechins are a most powerful flavonoid for protecting the body against reactive oxygen species131, 134.

1.5.3.4 Antidiabetic activity

Flavonoids, especially quercetin, have been reported to possess antidiabetic activity. Quercetin brings about the regeneration of pancreatic islets and probably increases insulin release in streptozotocin-induced diabetic rats. Quercetin stimulates insulin release and enhanced Ca2+ uptake from isolated islets cell which suggest a place for flavonoids in noninsulin-dependent diabetes135.

1.5.3.5 Anti-inflammatory activity

Flavonoids possess the anti-inflammatory activity. Hesperidin possesses significant anti-inflammatory and analgesic effect. Recently apigenin, luteolin and

33 quercetin have been reported to exhibit anti-inflammatory activity. Kaempferol, quercetin, myricetin, fisetin can modulate arachidonic acid metabolism via the inhibition of cyclooxygenase (COX) and lipoxygenase activity (LOX) activities131.

1.5.3.6 Antiulcer activity

Flavonoids possess anti-ulcerogenic activity. Flavonoid glycosides decreased ulcer and inhibited gastric acid and pepsin secretions. Quercetin, rutin, and kaempferolare used for the treatment of peptic ulcer132, 134.

1.5.3.7 Cardioprotective effects

Flavonoids have been stimulated by the potential health benefits arising from the antioxidant activity. It results in the high propensity to transfer electrons, chelate ferrous ions, and scavenge reactive oxygen species. Because of these properties, flavonoids have been considered as potential protectors against chronic cardiotoxicity caused by the cytostatic drug doxorubicin. Doxorubicin is a very effective antitumor agent but its clinical use is limited by the occurrence of a cumulative dose-related cardiotoxicity, resulting in, for example, congestive heart failure (negative inotropic effect) In a recent report, the cardiotoxicity of doxorubicin on the mouse left atrium has been inhibited by flavonoids, 7- monohydroxyethylrutoside and 7’,3’,4’- trihydroxyethylrutoside131.

1.5.3.8 Anticancer effect

Dietary flavonoid reduces the risk of cancer. Flavonoids are dietary antioxidants, which act as nutritional supplements during treatment of cancer or during inflammatory disorders. Flavonoids and their synthetic analogues are used in the treatment of ovarian, breast, cervical, pancreatic, and prostate cancer. Daidzein and genistein inhibit both hormonal and non-hormonal types of cancer. Tangeritin is a citrus flavonoid and inhibit cancer cell proliferation. Flavonoids such as 3-hydroxyflavone, 3',4' dihydroxy flavone, 2',3'-dihydroxy flavone, fisetin, apigenin, and luteolin are potential inhibitors of tumour cell proliferation. Quercetin and apigenin inhibit melanoma growth. Kaempferol, catechin, toxifolin and fisetin also suppressed cell growth136.

1.5.3.9 Immunomodulatory activity

Certain flavonoids act as immune boosters. Flavonoids help in the homeostasis within the immune system and also affect the functions of inflammatory cells. Quercetin helps to inhibit several initial process of inflammation and stimulate immune functions137.

1.5.3.10 Neuroprotective property

Neurodegenerative disorders were developed by the combined effect of oxidative stress, inflammation and transition metal accumulation. Alzheimer’s and dementias are the major disorder of neurodegeneration. Flavonoids have attracted potential interest for their neuroprotective properties, because of their antioxidant, anti- inflammatory and metal chelating properties. Flavones and flavonones play an effective role in the treatment of neurodegenerative diseases138.

2. LITERATURE REVIEW

2.1 RELATED TO DOCKING STUDIES

Hamulakova et al., 2017 synthesized a novel series of acridine-coumarin hybrids and biologically evaluated their inhibitory potential of AChE and BuChE. The newly synthesized derivatives 9a-d have shown higher activity against human AChE compared with 7-MEOTA as the standard drug. Among them derivative 9b exhibited the most potent AChE inhibitory activity, with an IC50 value of 5.85μM compared with

7-MEOTA (IC50=15μM). Molecular modelling studies were performed to predict the binding modes of compounds 9b, 9c and 9f with hAChE/hBuChE139.

Park et al., 2017 studied the AChE and BuChE inhibitory potential of 3,4- dihydroquinazoline derivatives. In particular, compound 8b and 8d were the most active compounds in the series against BuChE with IC50 values of 45nM and 62nM, as well as 146- and 161-fold higher affinity to BuChE, respectively. Molecular docking simulations were performed to get better insights into the mechanism of binding of 3,4- dihydroquinazoline derivatives. As expected, compound 8b and 8d bind to both catalytic anionic site (CAS) and peripheral site (PS) of BuChE with better interaction energy values than AChE. Furthermore, the non-competitive/mixed-type inhibitions of both compounds confirmed their dual binding nature in kinetic studies140.

Sehgal et al., 2016 carried out detailed screening analyses by 2-D similarity search against prescribed antidepressant drugs using their physicochemical properties. Ligand Scout was employed to ascertain novel molecules and pharmacophore properties. In this study, three novel compounds showed maximum binding affinity with HSPB8. Docking analysis elucidated that Met37, Ser57, Ser58, Trp60, Thr63, Thr114, Lys115, Asp116, Gly117, Val152, Val154, Leu186, Asp189, Ser190, Gln191, and Glu192 are their critical residues for ligand-receptor interactions. Site-directed mutagenesis could be significant for further analysis of the binding pocket. The novel findings based on an in silico approach may be momentous for potent drug design against depression and Charcoat Marie Tooth disease141.

Leu-95, Val-98, Ser-100, Glu-112, Tyr-116, Lys-120, Asp-121, and Arg-122 were critical residues for receptor-ligand interaction. The C-terminal of selected isoforms is conserved, and binding was observed on the conserved region of isoforms. Further analysis of this inhibitor through site-directed mutagenesis could be helpful for exploring the details of ligand-binding pockets142.

Ahmad et al., 2014 generated the ligand based pharmacophore model using the Discovery studio 2.0 software. In addition to this a structure based approach has been used to validate the developed pharmacophoric features to gain a deeper insight into its molecular recognition process. This validated pharmacophore and the docking model was then implemented as a query for pharmacophore based virtual screening to prioritize the probable hits for the caspase-3. Two ligands, ZINC12405015 and ZINC12405043 were finally selected on the basis of their fit values and docking scores. This study also reveals the vital amino acids His-121, Ser-205, Arg-207 which were found to be playing crucial role in the binding of the selected compounds within the active site of caspase-3143.

Jalkute et al., 2013 performed molecular dynamics simulation of crystal structure complex of testis truncated version of ACE (tACE) and its inhibitor lisinopril along with Zn2+ to understand the dynamic behavior of active site residues of tACE. Root mean square deviation results revealed the stability of tACE throughout simulation. The residues Ala 354, Glu 376, Asp 377, Glu 384, His 513, Tyr 520 and Tyr 523 of tACE stabilized lisinopril by hydrogen bonding interactions. Using this information, in subsequent part of study, molecular docking of tACE crystal structure with Aβ-peptide was investigated and the interactions of Aβ-peptide with enzyme tACE was explored. The residues Asp 7 and Ser 8 of Aβ-peptide were found in close contact with Glu 384 of tACE along with Zn2+. This study has demonstrated that the residue Glu 384 of tACE might play key role in the degradation of Aβ-peptide by cleaving peptide bond between Asp 7 and Ser 8 residues. Molecular basis generated by this attempt could provide valuable information towards designing of new therapies to control Aβ concentration in Alzheimer’s patient144.

37 strategy with different linker lengths (n = 2–8) joining the well-known NMDA antagonist adamantine and the hAChE inhibitor 7-methoxytacrine (7-MEOTA). Based on in silico studies, these inhibitors proved dual binding site character capable of simultaneous interaction with the peripheral anionic site (PAS) of human AChE and the catalytic active site (CAS). These structural derivatives exhibited very good inhibitory activity towards human BuChE resulting in more selective inhibitors of this enzyme. The most potent cholinesterase inhibitor was found to be thiourea analogue 14 (with an

IC50 value of 0.47 µM for human AChE and an IC50value of 0.11 µM for hBuChE, respectively). Molecule 14 is a suitable novel lead compound for further evaluation proving that the strategy of dual binding site inhibitors might be a promising direction for development of novel AD drugs145.

Grover et al., 2012 elucidated the ChE inhibitory potential of withanolide A and its associated binding mechanism. Binding interactions of the ligand to the receptor were predicted with the help of computational docking studies. Ligand interaction with the human AChE residues was found to be Thr78, Trp81, Ser120 and His442 as active sites, it could be responsible for its inhibitory activity. The study further proved the evidence for consideration of withanolide A as a potential molecule in prevention and management of AD146.

Dayangac-Erden et al., 2009 found that (E)-resveratrol, inhibited histone deacetylase activity in a concentration-dependent manner and half-maximum inhibition was observed at 650 μm. Molecular docking studies showed that (E)-resveratrol had more favorable free energy of binding (−9.09 kcal/mol) and inhibition constant values (0.219 μm) than known inhibitors. To evaluate the effect of (E)-resveratrol on survival motor neuron (SMN2) expression, spinal muscular atrophy type I fibroblast cell lines was treated with (E)-resveratrol. The level of full-length SMN2 mRNA and protein showed 1.2 to 1.3 fold increases after treatment with 100 μm (E)-resveratrol in only one cell line. These results indicated that response to (E)-resveratrol treatment was variable among cell lines. This data demonstrated a novel activity of (E)-resveratrol and that it could be a promising candidate for the treatment of spinal muscular atrophy147.

38 acid HDAC inhibitors. HDAC inhibition activities were investigated in vitro by using HeLa nuclear extract in a fluorimetric assay. Molecular docking was also carried out for the human HDAC8 enzyme in order to predict inhibition activity and the 3D poses of inhibitor–enzyme complexes. Among the compounds tested caffeic acid derivatives chlorogenic acid and curcumin were found to be highly potent compared to sodium butyrate, a well-known HDAC inhibitor148.

2.2 ENZYME INHIBITION ASSAY

Lan et al., 2017 developed novel family of cinnamic acid derivatives and evaluated cholinesterase inhibitory activity by fusing N-benzyl pyridinium moiety and different substituted cinnamic acids. In vitro studies showed that most compounds were endowed with a noteworthy ability to inhibit cholinesterase, self-induced Aβ (1-42) aggregation, and to chelate metal ions. Especially, compound 5l showed potent cholinesterase inhibitory activity (IC50, 12.1 nM for eeAChE, 8.6 nM for human AChE, 2.6 μM for eqBuChE and 4.4 μM for hBuChE) and the highest selectivity toward AChE over BuChE. It also showed good inhibition of Aβ (1-42) aggregation (64.7% at 20 μM) and good neuroprotection on PC12 cells against amyloid-induced cell toxicity. Finally, compound 5l could penetrate the BBB, as forecasted by the PAMPA-BBB assay and proved in OF1 mice by ex vivo experiments. Overall, compound 5l seems to be a promising lead compound for the treatment of Alzheimer's disease149.

Saeedi et al., 2017 evaluated the in vitro ChEI activity of various plants including betel nuts (Areca catechu L.), clove buds (Syzygium aromaticum L.), aerial parts of dodder (Cuscuta chinensis Lam.), common polypody rhizomes (Polypodium vulgare L.) and turpeth roots (Ipomoea turpethum R. Br.) which were recommended for the treatment of AD symptoms in Iranian traditional medicine (ITM). Among them, aqueous extract of A. catechu L. was found to be potent anti-AChE 150 (IC50 = 32.00 μg/mL) and anti-BuChE (IC50 = 48.81 ± 0.1200 μg/mL) agent .

39 inhibition activity was observed with all prepared extracts and however, Ocimum basilicum extract (OBE) showed most marked free radical scavenging, reducing power and AChE inhibition (IC50 0.65±0.15mg/ml) activity. The in-vivo studies showed that OBE pre-treatment (200 and 400 mg/kg, p.o.) reversed the memory deficit induced by scopolamine in mice, evident by significant (p<0.05) decrease in the transfer latency time and increase in step down latency in elevated plus maze and passive shock avoidance task, respectively. These biochemical changes were responsible for the anti- amnesic and neuroprotective activities of Ocimum basilicum which may be attributed to the presence of phenolic and flavonoid compounds and can be developed as an effective anti-amnesic drug151.

Hajlaoui et al., 2016 investigated the chemical composition and evaluated the antioxidant, antimicrobial, cytotoxic and anti-acetylcholinesterase properties of Tunisian Origanum majorana essential oil. The findings showed that the oil exhibited high activity, particularly in terms of reducing power and β-carotene bleaching, inducing higher IC50 values than butylated hydroxyl toluene. The oil showed an important antimicrobial activity against 25 bacterial and fungal strains. In fact, the inhibitory zone, minimum inhibitory concentration and minimum bactericidal concentration values were recorded. A low cytotoxic effect was observed against cancer (Hep-2 and HT29) and continuous cell lineage (Vero), with cytotoxic concentration (CC50) values ranging from 13.73 to 85.63 mg/mL. The oil was also evaluated for anti-AChE effects, which showed that it exhibited 152 significant activity with IC50 values reaching 150.33 ± 2.02 μg/mL .

Formagio et al., 2015 investigated the in vitro antiproliferative and anticholinesterase activities of 11 extracts from 5 Annonaceae species. Antiproliferative activity was assessed using 10 human cancer cell lines. Thin-layer chromatography and a microplate assay were used to screen the extracts for AChE inhibitory activity. The chemical compositions of the active extracts were investigated using high performance liquid chromatography. Eleven extracts obtained from five Annonaceae plant species were active and were particularly effective against the UA251, NCI-470 lung, HT-29, NCI/ADR, and K-562 cell lines with growth inhibition (GI50) values of 0.04-0.06, 0.02-0.50, 0.01-0.12, 0.10-0.27, and 0.02-0.04 mg/mL, respectively. The Annona cacans extract displayed the lowest activity. Based on the microplate assay, the percent AChE inhibition of the extracts ranged from 12 to 52%, and the Annona coriacea seed

40 extract resulted in the greatest inhibition (52%). Caffeic acid, sinapic acid, and rutin were present at higher concentrations in the Annona crassiflora seed samples153.

Ali-Shtayeh et al., 2014 investigated the in vitro AChEI in herbal medicines traditionally used in Palestine to treat cognitive disorders. The effect on AChE activity of 92 extracts belonging to 47 medicinal plants were evaluated using micro-well plate AChE activity (NA-FB) and Ellman’s assays and antioxidant activity using DPPH assay.67.4% and 37% of extracts inhibited AChE by >50% using the NA-FB and Ellman’s assays. Using NA-FB assay, 84 extracts interacted reversibly with the enzyme, of which Mentha spicata (94.8%), Foeniculum vulgare (89.81), and Oxalis pes-caprae (89.21) were most potent, and 8 showed irreversible inhibitions of leaves of Lupinus pilosus (92.02%) were most active. Antioxidant activity was demonstrated by

73 extracts Majorana syriaca (IC50 0.21mg/ml), and Rosmarinus officinalis (0.38) were the most active. Palestinian flora has shown to be a rich source for, new and promising agents (AChEIs) for the treatment of AD154.

Liu et al., 2014 focused on the synthesis and anti-cholinesterase evaluation of new donepezil like analogs. A new series of phthalimide derivatives (compounds 4a-4j) were synthesized via Gabriel protocol and subsequently amidation reaction was performed using various benzoic acid derivatives. Then, the corresponding anti- acetylcholinesterase activity of the prepared derivatives (4a-4j) was assessed by utilization of the Ellman’s test and obtained results were compared to donepezil. Besides, docking study was also carried out to explore the likely in silico binding interactions. According to the obtained results, electron withdrawing groups (Cl, F) at position 3 and an electron donating group (methoxy) at position 4 of the phenyl ring enhanced the AChE inhibitory activity. Compound 4e (m-Fluoro, IC50 = 7.1 nM) and 4i

(p-Methoxy, IC50 = 20.3 nM) were the most active compounds in this series and exerted superior potency than donepezil (410 nM). Moreover, a similar binding mode was observed in silico for all ligands in superimposition state with donepezil into the active site of AChE155.

41 revealed that the selected compounds were inhibitors of AChE dual binding site. A kinetic study was carried out and mixed type of enzyme inhibition was found. The compounds were evaluated for their antioxidant activity and no significant activity was witnessed156.

2.3 SCOPOLAMINE INDUCED AMNESIA

Akinyemi et al., 2017 assessed the combined pretreatment effect of curcumin, the major polyphenolic compound of turmeric (Curcuma longa) rhizomes, with donepezil, a ChE inhibitor, on cognitive function in scopolamine- induced memory impairment in rats. Rats were pretreated with curcumin (50 mg/kg) and/or donepezil (2.5 mg/kg) via oral administration (p.o.) for seven successive days. Dementia was induced at the end of the treatment period by a single i.p. injection of scopolamine (1 mg/kg). Thereafter, the changes in spatial and episodic memory were conducted; then, the estimation of some biochemical parameters associated with cognitive function was determined. Scopolamine-treated rats showed impaired learning and memory and increased activities of AChE and BuChE, adenosine deaminase (ADA), and lipid peroxidation with a concomitant decrease in levels of nitric oxide (NO) and reduced glutathione (GSH), superoxide dismutase (SOD), and catalase when compared with control. Thus, the observed anti-amnestic effect could be linked to their inhibitory effect on key enzyme of cholinergic system associated with memory function157.

Erfanparast et al., 2017 investigated the effects of microinjection of vitamin

B12 into the hippocampus on the orofacial pain and memory impairments induced by scopolamine and orofacial pain. The memory impairment induced by orofacial pain was improved by vitamin B12 and physostigmine used alone. Naloxone prevented, whereas physostigmine enhanced the memory improving effect of vitamin B12 in the pain-induced memory impairment. All the above-mentioned chemicals did not alter locomotor activity. The results of the present study showed that at the level of the dorsal hippocampus, vitamin B12 modulated orofacial pain through a mu-opioid receptor mechanism. In addition, vitamin B12 contributed to hippocampal cholinergic system in processing of memory. Moreover, cholinergic and opioid systems may be 158 involved in improving effect of vitamin B12 on pain-induced memory impairment .

Imam et al., 2017 investigated the neuro-cognitive implications of oral cannabis use in rats. Cannabis significantly reduced rearing frequencies in the OFT and EPM, and increased freezing period in the OFT. It also reduced percentage alternation similar to scopolamine in the Y maze, and these effects were coupled with alterations in the cortico-hippocampal neuronal architectures. These results pointed the detrimental impacts of cannabis on cortico-hippocampal neuronal architecture and morphology, and consequently cognitive deficits159.

Rahmati et al., 2017 studied the effects of L. officinalis extract in scopolamine- induced memory impairment, anxiety and depression-like behaviour. Memory impairment was assessed by Y-maze task, while, elevated plus maze and forced swimming test were used to measure anxiolytic and antidepressive-like activity. Spontaneous alternation percentage in Y maze is reduced by scopolamine (36.42 ± 2.60) (p ≤ 0.001), whereas lavender (200 and 400 mg/kg) enhanced it (83.12 ± 5.20 and 95 ± 11.08, respectively) (p ≤ 0.05). Also, lavender pretreatment in 200 and 400 mg/kg enhanced time spent on the open arms (15.4 ± 3.37 and 32.1 ± 3.46, respectively) (p ≤ 0.001). On the contrary, while immobility time was enhanced by scopolamine (296 ± 4.70), 100, 200 and 400 mg/kg lavender reduced it (193.88 ± 22.42, 73.3 ± 8.25 and 35.2 ± 4.22, respectively) in a dose-dependent manner (p ≤ 0.001). Lavender extracts improved scopolamine-induced memory impairment and also reduced anxiety and depression-like behaviour in a dose-dependent manner160.

Kim et al., 2016 investigated the cognition-enhancing effects of aqueous extract of Indigofera tinctoria Linn. (ITE, Fabaceae) in experimental amnesic mice. The cognitive-enhancing activity of the ITE (5, 10 and 20 μg/mL) was studied by passive avoidance response, elevated plus maze and Y-maze behavioral paradigm in normal and scopolamine-induced amnesic mice. Antioxidant activities were also determined based on the ability of ITE to inhibit lipid peroxide, superoxide and hydroxyl radicals. Scopolamine-induced cognitive deficits were significantly reversed by ITE (p < 0.001 at 20 mg/kg) in a dose-dependent fashion in all the behavioral paradigms tested. Furthermore, ITE dose dependently scavenged lipid peroxide, superoxide and hydroxyl free radicals with 50 % inhibition concentration (IC50) of 7.28 ± 0.37, 5.25 ± 0.28 and 7.62 ± 0.43 μg/mL, respectively. ITE possesses cognitive-enhancing properties in amnesic mice due to its potent antioxidant action75.

AChE and up-regulating BDNF, muscarinic M1 receptor and CREB expression in brain hippocampus confirms the potent neuroprotective role and these results are in corroboration with the earlier in vitro studies. BME administration showed significant protection against scopolamine-induced toxicity by restoring the levels of antioxidant and lipid peroxidation. These results indicate that, cognition-enhancing and neuromodulatory propensity of BME is through modulating the expression of AChE, BDNF, MUS-1, CREB and also by altering the levels of neurotransmitters in hippocampus of rat brain161.

Setorki, 2016, observed the effect of Ziziphus spina-christi extract against anxiety related behavior induced by scopolamine. Experimental groups received Z. spina-christi extract (50, 100 and 200 mg/kg IP) 30 min after scopolamine injection. Anxiety related behaviors were assessed using the elevated plus maze. The rotarod test was used to evaluate motor coordination. Administration of Z. spina-christi extract (200 mg/kg) significantly increased the time spent in the open arm of elevated plus maze. The extract also reduced the percentage of closed arms entries and time spent in the closed arms. Different concentration of Z. spina-christi extract didn’t affect motor coordination and balance. Hydro-alcoholic extract of Z. spina-christi significantly ameliorated scopolamine-induced anxiety162.

He et al., 2015 examined the effect on scopolamine-induced memory impairment mice and APP/PS1 transgenic mice using Morris water maze test. Results showed that harmine (20 mg/kg) administered by oral gavage for 2 weeks could effectively enhance the spatial cognition of C57BL/6 mice impaired by intraperitoneal injection of scopolamine (1 mg/kg). Meanwhile, long-term consumption of harmine (20 mg/kg) for 10 weeks also slightly benefited the impaired memory of APP/PS1 mice. Furthermore, harmine could pass through blood brain barrier, penetrate into the brain parenchyma shortly after oral administration, and modulate the expression of Egr-1, c- Jun and c-Fos. Molecular docking assay disclosed that harmine molecule could directly dock into the catalytic active site of acetylcholinesterase, which was partially confirmed by its in vivo inhibitory activity on AChE. Results suggested that harmine impaired

44 memory by enhancement of cholinergic neurotransmission via inhibiting the activity of AChE, which may contribute to its clinical use in the therapy of neurological diseases characterized with AChE deficiency163.

Lee et al., 2015 evaluated the neuropharmacological effects of 30% ethanolic pine needle extract (PNE) on memory impairment caused by scopolamine injection in mice hippocampus. Mice were orally pretreated with PNE (25, 50, and 100 mg/kg) or tacrine (10 mg/kg) for 7 days, and scopolamine (2 mg/kg) was injected intraperitoneally, 30 min before the Morris water maze task on first day. To evaluate memory function, the Morris water maze task was performed for 5 days consecutively. Scopolamine increased the escape latency and cumulative path-length but decreases the time spent in target quadrant, which were ameliorated by pretreatment with PNE. These findings suggest that PNE could be a potent neuro pharmacological drug against amnesia, and its possible mechanism might be modulating cholinergic activity via CREB-BDNF pathway164.

2.4 ALUMINIUM CHLORIDE INDUCED AMNESIA

Lee et al., 2017 investigated the effect of the ethanol extract of Acanthopanax koreanum (EEAK) on cholinergic blockade- induced memory impairment in mice. The phosphorylation levels of both Akt and CaMKII were significantly increased by approximately two-fold compared with the control group because of the administration of EEAK (100 or 200 mg/kg) (p < 0.05). Moreover, the phosphorylation level of CREB was also significantly increased compared with the control group by the administration of EEAK (200 mg/kg) (p < 0.05). The study suggested that EEAK ameliorates the cognitive dysfunction induced by the cholinergic blockade, in part, via several memory- associated signalling molecules and may hold therapeutic potential against cognitive dysfunction, such as that presented in neurodegenerative diseases165.

45 superoxide dismutase and catalase were significantly increased, whereas the thiobarbituric acid reactive substance was significantly decreased after 8 consecutive days of treatment with M. inermis at the dose of 393 mg/kg. These results suggest that M. inermis leaf extract possess potential anti-amnesic effects166.

Intraperitoneal injection of AlCl3 (100 mg/kg., b.w.) for 60 days significantly enhanced the learning and memory deficits, levels of thiobarbituric acid reactive substances and diminished the levels of reduced glutathione, activities of enzymatic antioxidants and the expression of B-cell lymphoma-2 as compared to control group in the hippocampus, cortex, and cerebellum. Co administration of hesperidin (100 mg/kg., b.w. oral) for 60 days prevented the cognitive deficits, biochemical anomalies and apoptosis induced by

AlCl3 treatment. Results of the study demonstrated that hesperidin could be a potential therapeutic agent in the treatment of oxidative stress and apoptosis associated neurodegenerative diseases including AD18.

Al-Amin et al., 2016 investigated the effect of astaxanthin on AlCl3-exposed behavioral brain function and neuronal oxidative stress. Aluminum exposed group showed a significant reduction in spatial memory performance and anxiety-like behavior. Moreover, aluminium group exhibited a marked deterioration of oxidative markers; lipid peroxidation (MDA), nitric oxide (NO), glutathione (GSH) and advanced oxidation of protein products (AOPP) in the brain. To the contrary, co-administration of astaxanthin and aluminium has shown improved spatial memory, locomotor activity, and reduced OS. These results indicate that astaxanthin improves aluminium-induced impaired memory performances presumably by the reduction of OS in the distinct brain regions167.

46 test (FST), Morris water maze (MWM) task and conditioned-avoidance and learning (CAL) test. Histo pathological changes in the brain and biochemical changes in AChE as well as oxidative parameters; malondialdehyde (MDA), superoxide dismutase (SOD), total antioxidant capacity (TAC) were also evaluated for all groups. Results of the behavioral tests showed that caffeine and nicotine co-administration had more pronounced protecting effect from learning and memory impairment induced by AlCl3 than each one alone. Caffeine and nicotine co-administration also prevented neuronal degeneration in the hippocampus and the eosinophilic plagues in the striatum induced by AlCl3 while nicotine alone showed mild gliosis in striatum. The marked protection of caffeine and nicotine co-administration was also confirmed by the significant increase in TAC and SOD and decrease in MDA and AChE in brain tissue. Co- administration of caffeine and nicotine reduced the risk of neuronal degeneration in the hippocampus and attenuated the impairment of learning and memory associated with AD16.

Jayant et al., 2016 investigated the ameliorative role of selective modulator of cannabinoid receptor type 2 (CB2), 1-phenylisatin in experimental AD condition. Intra cerebro ventricular (i.c.v.) administration of streptozotocin (STZ) and AlCl3+d- galactose were given to animals. Morris water maze (MWM) and attentional set shifting test (ASST) were used to assess learning and memory. Hematoxylin-eosin and Congo red staining were used to examine the structural variation in brain. Brain oxidative stress (thiobarbituric acid reactive substance and glutathione), nitric oxide levels (nitrites/nitrates), AChE activity, myeloperoxidase and calcium levels were also estimated. i.c.v. STZ as well as AlCl3+d-galactose have impaired spatial and reversal learning with executive functioning, increased brain oxidative and nitrosative stress, cholinergic activity, inflammation and calcium levels. Furthermore, these agents have also enhanced the burden of Aβ plaque in the brain. Treatment with 1-phenylisatin and donepezil attenuated i.c.v. STZ as well as AlCl3+d-galactose induced impairment of learning-memory, brain biochemistry and brain damage. Hence, this study concludes that CB2 receptor modulation can be a potential therapeutic target for the management of AD168.

47 segregated into four groups: group 1-control rats, group 2- rats received AlCl3 (300 mg/kg b.w. every day orally) for 60 days, rats in group 3-received CA (500 mg/kg b.w. orally) and group 4 rats were initiated with both AlCl3 and CA treatment.

Administration of AlCl3 developed behavioral deficits, triggered lipid peroxidation (LPO), compromised acetylcholine esterase (AChE) activity, and reduced the levels of superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH) and glutathione-S-transferase (GST) and caused histologic aberrations. AlCl3–induced alterations in the activities of SOD, CAT, GSH and GST, levels of LPO, activity of AChE, behavioral deficits and histologic aberrations were attenuated on treatment with

CA. Results of the study demonstrate neuroprotective potential of CA against AlCl3- induced oxidative damage and cognitive dysfunction169.

de Vargas et al., 2016 evaluated the in vitro antioxidant and anti-peroxidases potential of the ethanol extracts of five plants from the Brazilian Amazon. 2,2- diphenyl-1-picrylhydrazyl (DPPH) assay, 2,2’-azinobis(3-ethylbenzothiazoline-6- sulfonic acid) (ABTS) assay, superoxide anion radical, singlet oxygen and the β- carotene bleaching methods were employed for characterization of free radical scavenging activity and total polyphenols were determined. Antioxidant activities were evaluated using murine fibroblast NIH3T3 cell. The stem bark extracts of Calycophyllum spruceanum and Mytella guyanensis provided the highest free radical scavenging activities. C. spruceanum exhibited IC50 = 7.5 ± 0.9, 5.0 ± 0.1, 18.2 ± 3.0 and 92.4 ± 24.8 μg/mL for DPPH(•), ABTS(+•), O2 (-•) and O2 assays, respectively. Ptychopetalum olacoides and C. spruceanum extracts also inhibited free radicals formation in the cell-based assay. At a concentration of 100 μg/mL, the extracts of C. spruceanum, Byrsonima japurensis inhibited horseradish peroxidase by 62 and 50 %, respectively. C. spruceanum, M. guyanensis, B. japurensis also inhibited myeloperoxidase in 72, 67 and 56 %, respectively170.

48 neurotrophic factor (BDNF) content. Mangiferin (40 mg/kg) prevented the cognitive deficits, hippocampal BDNF depletion, and biochemical anomalies induced by AlCl3- treatment. Mangiferin offered neuroprotection in AlCl3-induced neurotoxicity and reduced oxido-nitrosative stress and inflammation-associated neurotoxicity171.

Li et al., 2016 explored the therapeutic effect of ginseng protein (GP) on Alzheimer's disease and its correlation with the PI3K/Akt signaling pathway to understand the mechanism underlying the neuroprotective effect of ginseng. The expression of mRNAs and proteins of PI3K, Akt, phosphorylated Akt (p-Akt), Bcl-2, and Bax in the hippocampus was detected by real-time quantitative reverse transcription polymerase chain reaction and Western blot assay. GP was found to significantly improve the memory ability of AD rats and prolong the times of crossing the platform and the percentage of residence time in the original platform quadrant of spatial probe test. GP also reduced the content of Aβ1-42 and p-tau and improved the mRNA and protein expression of PI3K, p-Akt/Akt, and Bcl-2/Bax in the hippocampus. GP improved the memory ability and reduced the content of Aβ1-42 and p-tau in AD rats. The anti-AD effects of GP were in part mediated by PI3K/Akt signaling pathway activation172.

Mahboob et al., 2016 evaluated the neuroprotective effect of alpha-lipoic acid

(ALA) in AlCl3-induced neurotoxicity mouse model. Effect of ALA (25mg/kg/day) was evaluated in the AlCl3-induced neurotoxicity (AlCl3 150 mg/kg/day) mouse model on learning and memory using behaviour tests and on the expression of muscarinic receptor genes (using RT-PCR), in hippocampus and amygdala. ALA enhanced fear memory (p<0.01) and social novelty preference (p<0.001) comparative to the AlCl3- treated group. Fear extinction memory was remarkably restored (p<0.001) in ALA- treated group demonstrated by reduced freezing response as compared to the AlCl3- treated group which showed higher freezing. In-silico analysis showed that racemic mixture of ALA has higher binding affinity for M1 and M2 compared to acetylcholine. These novel findings highlight the potential role of ALA in cognitive functions and cholinergic system enhancement thus presenting it an enviable therapeutic candidate for the treatment of neurodegenerative disorders173.

Prema et al., 2016 explored the neuroprotective effect of fenugreek seed powder (FSP) against AlCl3-induced experimental AD model. Administration of

49 germinated FSP (2.5, 5 and 10% mixed with ground standard rat feed) protected AlCl3 induced memory and learning impairments, Al overload, AChE hyperactivity, amyloid β (Aβ) burden and apoptosis via activating Akt/GSK3β pathway. Further these results could lead a possible therapeutics for the management of neurodegenerative diseases including AD in future12.

Lakshmi et al., 2015 evaluated the effect of selenium on AlCl3- induced Alzheimer's disease in rats. The animals showed increase in time to reach platform in Morris water maze and decreased step-down latencies in passive avoidance test indicating learning and memory impairment in AlCl3-treated group, but administration of selenium decreased time to reach platform in Morris water maze, increased step-down latencies, and strengthened its memory action in drug-treated animals. There was decrease in muscle strength measured by rotarod test indicating motor incoordination and decrease in locomotor activity assessed by actophotometer test in AlCl3 control group, whereas in selenium-AlCl3 group, there was improvement in muscle strength and locomotion. Biochemical analysis of the brain revealed that chronic administration of AlCl3 significantly increased lipid peroxidation and decreased levels of AChE, catalase, reduced glutathione and glutathione reductase, an index of oxidative stress process. Administration of selenium attenuated lipid peroxidation and ameliorated the biochemical changes. There were marked changes at subcellular level observed by histopathology studies in AlCl3 group, and better improvement in these changes was observed in selenium + AlCl3 group. Therefore, this study strengthened the hypothesis that selenium helped to combat oxidative stress produced by accumulation 175 of AlCl3 in the brain and helped in prophylaxis of Alzheimer's diseases .

Huma et al., 2013 analyzed thirteen plant-derived alkaloids, namely pleiocarpine, kopsinine, pleiocarpamine, oliveroline, noroliveroline, liridonine, isooncodine, polyfothine, darienine and eburnamine, eburnamonine, eburnamenine and geissoschizol for their anti-cholinergic action through docking with AChE as target. Among the alkaloids, pleiocarpine showed promising anti-cholinergic potential, while its amino derivative showed about six-fold higher anti-cholinergic potential than pleiocarpine. Pleiocarpine and its amino derivative were found to be better inhibitors of AChE, as compared to commonly used drugs tacrine and rivastigmine, suggesting development of these molecules as potential therapeutics in future174.

Thippeswamy et al., 2013 evaluated the synergistic activity of Bacopa monniera with rivastigmine against AlCl3-induced cognitive impairment in rats.

Chronic administration of AlCl3 caused significant memory impairment associated with increased RL in the Morris water maze task and increased TL in the elevated plus maze test. Interestingly, animals treated with oral administration of B. monniera (100 and 200 mg/kg), rivastigmine (5 mg/kg) or a combination of B. monniera (100 mg/kg) with rivastigmine (5 mg/kg) showed significant protection against AlCl3- induced memory impairment compared to animal treated with AlCl3 alone. Additionally, the neuroprotective effect of B. monniera (100 and 200 mg/kg) was significantly improved when supplemented with rivastigmine (5 mg/kg). These findings suggest that treatment with a combination of B. monniera with rivastigmine highly 176 beneficial compared to their AlCl3 alone treated animals .

Thirunavukkarasu et al., 2012 examined the neuroprotective effect of Manasamitra vatakam (MMV) against aluminum (Al)-induced memory impairment and oxidative damage in rats. Neurotoxicity was induced by the administration of Al [100 mg/kg body weight (b.w.) per oral (p.o.)/day] to Wistar albino rats for 90 days. It was observed that the administration of MMV (100 mg/kg b.w./p.o./day) along with AlCl3 improved memory performance and antioxidant activity against Al-induced neurotoxicity in rats. These data suggest that MMV can prevent brain damage from Al- induced neurotoxicity in rats and thus can be used as a neuroprotective agent177.

Kumar et al., 2011 explored the possible role of carvedilol, an adrenergic antagonist against AlCl3-induced neurotoxicity in rats. AlCl3 (100 mg/kg) administered daily for six weeks, significantly increased cognitive dysfunction in the Morris water maze and oxidative damage as indicated by a rise in lipid peroxidation and nitrite concentration and depleted reduced glutathione, superoxide dismutase, catalase and glutathione S-transferase activity compared to sham treatment. Chronic aluminium chloride treatment also significantly increased acetylcholinesterase activity and the aluminium concentration in brain compared to sham. Chronic administration of carvedilol (2.5 and 5 mg/kg, po) daily to rats for a period of 6 weeks significantly improved the memory performance tasks of rats in the Morris water maze test, attenuated oxidative stress (reduced lipid peroxidation, nitrite concentration and restored reduced glutathione, superoxide dismutase, catalase and glutathione S- transferase activity), decreased acetylcholinesterase activity and aluminium

51 concentration in aluminium-treated rats compared to control rats (p < 0.05). Results of this study demonstrated the neuroprotective potential of carvedilol in aluminium chloride-induced cognitive dysfunction and oxidative damage178.

Butler et al., 2010 tested tubastatin A, in primary cortical neuron cultures in which it was found to induce elevated levels of acetylated α-tubulin, but not histone, consistent with its HDAC6 selectivity. Tubastatin A also conferred dose-dependent protection in primary cortical neuron cultures against glutathione depletion-induced oxidative stress. Importantly, when given alone at all concentrations tested, this hydroxamate-containing HDAC6-selective compound displayed no neuronal toxicity, thus, forecasting the potential application of this agent and its analogues to neurodegenerative conditions179.

Bihaqi et al., 2009 investigated neuroprotective effects of aqueous extract from Convolvulus pluricaulis (CP) against AlCl3-induced neurotoxicity in rat cerebral cortex. Daily administration of CP (150 mg/kg) for 3 months along with AlCl3 (50 mg/kg) decreased the elevated enzymatic activity of AChE and also inhibited the decline in Na+/K+ATPase activity which resulted from aluminium intake. Oral administration of CP preserved the mRNA levels of muscarinic receptor 1 (M1 receptor), choline acetyltransferase (ChAT) and Nerve growth factor-tyrosine kinase A receptor (NGF-TrkA). The potential of CPE to inhibit aluminium induced toxicity was compared with rivastigmine tartrate (1 mg/kg), which was taken as standard. The potential of the extract to prevent aluminium-induced neurotoxicity was also reflected at the microscopic level, indicative of its neuroprotective effects. C. pluricaulis possesses neuroprotective potential, thus validating its use in alleviating toxic effects of aluminium180.

52 aluminium chloride administration resulted in poor retention of memory in Morris water maze, elevated plus maze task paradigms and caused marked oxidative damage. It also caused a significant increase in the acetylcholinesterase activity and aluminium concentration in aluminium treated rats. Chronic administration of curcumin significantly improved memory retention in tasks, attenuated oxidative damage, AChE activity and aluminium concentration in aluminium treated rats (P < 0.05). Curcumin has neuroprotective effects against aluminium-induced cognitive dysfunction and oxidative damage181.

3. AIM & OBJECTIVES

In the light of literature review, the study was aimed to evaluate the possible neuroprotective effects of commercially available flavonoids using in silico, in vitro and in vivo approaches.

The objectives of the present study are to

 Evaluate drug likeness properties of the flavonoids

 Carry out in silico docking studies of the selected flavonoids using AutoDock 4.2

 Perform in vitro AChE and BuChE enzyme inhibitory assay for the selected flavonoids

 Carry out the acute toxicity studies for the selected flavonoids

 Assess the selected flavonoids against scopolamine and aluminium chloride induced Alzheimer models

 Carry out various behavioural studies using selected flavonoids using in vivo models

 Explore the biochemical alterations after induction of learning impairment and correction of biochemical parameters after drug treatment.

 Perform histopathological studies to corroborate the results of in vivo studies.

 To correlate the results obtained using statistical tools using GraphPad Prism software.

4. PLAN OF WORK

STEP I

Selection of flavonoids and determination of acetylcholinesterase inhibitory activity.

STEP II

Determination of drug likeness score by using MedChem Designer software.

STEP III

To carry out the molecular docking studies using AutoDock 4.2 against AChE and BuChE based on the bioactive score.

STEP IV

Selection of flavonoids based on their docking score and drug likeness properties.

STEP V

In vitro screening of selected flavonoids by AChE and BuChE inhibitory assay.

STEP VI

Acute toxicity studies following OECD guidelines-420 (fixed dose procedure) for selected flavonoids based on the in vitro results.

STEP VII

In vivo screening of the flavonoids by acute scopolamine-induced learning impairment in mice.

STEP VIII

In vivo screening of the flavonoids by chronic aluminium chloride-induced learning impairment in mice.

STEP IX

Assessment of neuroprotective activity using various behavioral experiments.

STEP X

Dissection and preparation of tissue homogenates using brain for biochemical estimations.

STEP XI

Histopathological studies for both scopolamine and aluminium chloride-induced models.

STEP XII

Tabulation, compilation of results and statistical analysis of data obtained.

Literature review

In silico prediction of neuroprotective potential of certain commercially

available flavonoids • Acetylcholinesterase Butyrylcholinesterase

In vitro enzyme inhibition Assay • Acetylcholinesterase inhibitory activity

• Butyrylcholinesterase inhibitory activity

Acute toxicity studies

Scopolamine-induced amnesia Aluminium Chloride-induced amnesia

Biochemical Estimations . Estimation of cholinesterase activity Behavioural Assessments . Estimation of protein . Enzymatic antioxidants . Rectangular maze test Catalase activity . o Morris water maze test SOD assay . Attention in a 5 choice serial o o Estimation of glutathione reaction time task reductase . Assessment of gross behavioral o Estimation of glutathione activity peroxidase . Pole climbing test . Non-Enzymatic antioxidants . Estimation of reduced glutathione . TBARS assay

Statistical analysis

Tabulation and compilation of results

Fig.13 Schematic representation of the plan of work

5. MATERIALS AND METHODS

5.1 Evaluation of drug likeness properties The pharmacologically active substituents are characterized by calculating steric, hydrophobic, electronic, and hydrogen bonding properties as well as by the drug- likeness score. The theory of drug-likeness score helps to optimize pharmaceutical and pharmacokinetic properties, for example, chemical stability, solubility, distribution profile and bioavailability. The molecular descriptors have developed as rationally predictive and informative, for example, the Lipinski's Rule-of-Five182. The better oral absorption of the ligands and drug likeness scores are constructed by getting information about the solubility, diffusion, log P, molecular weight etc. The theory of drug-likeness score helps to optimize pharmaceutical and pharmacokinetic properties. Lipinski’s rule of five describes the important molecular properties required for the drug’s pharmacokinetics183. Lipinski’s rule states that, the drug to have good pharmacokinetic profile, should not have more than one violation of the following criteria:

 Not more than 5 hydrogen-bond donors

 Not more than 10 hydrogen-bond acceptors

 Molecular weight should be less than 500 daltons

 The partition coefficient (log P) not greater than 5 Two hundred and forty five flavonoids were evaluated for their pharmacokinetic profile based on Lipinski’s rule of five using MedChem Designer software.

5.1.1 Lipinski’s rule of five calculations

 Open the MedChem designer home page.

 Click calculation of molecular properties of drug likeness  Draw the structure of flavonoids in the active window.  Click and calculate properties.  Save the properties.

5.2 In silico docking binding interaction studies 5.2.1 Softwares used

 AutoDock 4.2 which combines

 AutoDock Tools 1.5.4

 Python Molecule Viewer 1.5.4

 Vision 1.5.4

 Python 2.7

 Cygwin

 Accerlys discovery studio viewer

 ChemSketch

 RCSB protein data bank

 Online SMILES translator

5.2.2 In silico docking study on AChE enzyme using AutoDock 4.2

5.2.2.1 Identification of target enzyme and selection of flavonoids and related compounds

The crystal structures of acetylcholinesterase (AChE) (PDB ID: 5HCU) and butyrylcholinesterase (BuChE) (PDB ID: 4BDS) were obtained from the research collaboratory for structural bioinformatics (RCSB) protein data bank and refined proteins were further used for in silico studies.

Fig. 14 Refined crystal structure of mouse acetylcholinesterase (5HCU)

A total of 245 flavonoids were taken for the study and the structures were drawn using ChemSketch and optimized using “Prepare Ligands” in the AutoDock 4.2 for docking studies184.

Fig. 15 Human butyrylcholinesterase in complex with tacrine (4BDS)

5.2.2.2 Docking studies for the selected flavonoids185

Step I - Protein structure refinement

The proteins downloaded from PDB have to be refined for docking. The steps involved in the protein structure refinement are,

 Open Accelrys discovery studio viewer

 Select Target protein PDB file

 Click View, and then click Hierarchy.

 Click water molecules

 Ctrl + shift and click the last water molecule (select all water molecules)

 Give right click and cut

 Select ligand, which is unnecessary, give right click and cut.

 Save the molecule in our desired area.

Step II - Ligand file format conversion

 The ligands which are desired are drawn in Chem Sketch.

 Select tools and click generate, finally click SMILES notation [simplified molecular input line entry system, which is a file format]

 Save the SMILES in a word document

 Open the online smiles translator – cactus.nci.nih.gov/services/translate/

 Upload the SMILES.

 By choosing the required file format we can save the file (pdb file).

Online smiles translator allows the user to convert SMILES format into PDB, MOL, SDF and smile text file format. Thus the selected ligand molecule of canonical smile formats was converted to pdb formats. The protein and ligand files which are prepared by above said procedures are selected for docking.

Step III - Docking with AutoDock 4.2

Docking calculation in AutoDock 4.2 was performed using the refined protein and the desired ligand in pdb format.

Preparing the protein

 At first give the right click on the PMV molecules for to open the file.

 Choose the particular refined enzyme file.

 Click under atom to give color to the atoms.

 The elimination of the water is carried by the following steps.

press select option

click select from string option

then write HOH* in the residue line & * in the atom line

click add and then dismiss.

 After that click edit option and delete and then delete atom set.

 Addition of hydrogens is take placed by,

Press edit option

Click the hydrogens

Then click add

Finally choose hydrogens and no bond order, and finally yes to renumbering.

 Next add the Kollmann charges which is present in the edit option

 For to hide the protein click on the gray under show molecules.

Preparing the ligand

 Confirm that all the hydrogens are added in the ligand.

 Toggle the Autodock tools button

 For to open the ligand file press the Input option and click open and then all files finally choose the suitable ligand file and finally open

 AutoDock tools itself computes Gasteiger charges, and merges nonpolar hydrogens, and finally assigns AutoDock type to each atom.

 The torsions are designed by following steps,

In the ligand option select torsion tree

Select detect root option

Click torsion tree

Then select the choose torsions option

Amide bonds should not be active.

 After that click the torsion tree and select set number of torsions.

 Number of rotatable bonds is choosen.

 Finally save the ligand files by selecting the output option (pdbqt file).

 Hide the ligand.

Conversion of pdb files of protein into pdbqt file

 Select the grid option and open the macromolecule pdb file

 AutoDock adds the charges and itself merges the hydrogens.

 Save the object as pdbqt in desired area.

Running AutoGrid Calculation (glg file)

 Open the grid and click macromolecule option and choose the rigid protein then yes to preserve the existing charges.

 The preparation of grid parameter file is carried out by,

Open grid

Select the set map types

Choose ligand

Accept it.

 Setting of grid properties

Open grid

Select the grid box

Set the proper grid dimensions

Adjust the spacing

Select the file and click close saving current.

 Save the grid settings as gpf file in the output option (gpf file).

 Make sure the AutoGrid is in the same directory as like of the other input files.

 Select the run option and click run AutoGrid for to launch.

 It will be saved as glg file.

Running AutoDock (dlg file)

 The rigid molecule specification is carried out by,

Select the docking option

Click the macromolecule

Set rigid file name.

 The ligand specification is carried out by,

Click the docking option

Select the ligand

And then accept it.

 In the next step, click docking option and select search parameters in that click genetic algorithm and finally accept it.

 In the docking options select docking parameters and choose the defaults.

 After that, in the docking option clicks output and adds Lamarckian genetic algorithm.

 The confirmation is required that whether all the files (macromolecule, ligand, gpf, dpf ) are in the same directory.

 Finally select the run command and click run AutoDock and then launch.

Step IV - Viewing docking results

Reading the docking log file .dlg

 Toggle the AutoDock tools button

 Click analyze and dockings then open it.

 In the next step, click analyze option and conformations then load.

 Double click on the conformation for to view it.

Visualizing docked conformations

 Click analyze and dockings then play.

 Load dlg file

 Choose the suitable conformations

 In the next step, click analyze and docking then show interactions.

Obtaining snap shots of docked pose

 Open the file and read the molecule

 Open analyze option click dockings and open dlg file

 Open analyze option click macromolecule and choose pdbqt file.

 Open analyze option click conformations and load

 Double click the desired conformation

 In the next step, click analyze and docking then show interactions.

Lamarckian genetic algorithm (LGA) for ligand conformational searching is used for the docking, which is a hybrid of genetic algorithm and local search algorithm. This algorithm first builds a population of individuals, each being a different random conformation of the docked molecule. Each individual is then mutated to acquire a slightly different translation and rotation and the local search algorithm then performs energy minimizations on a user-specified proportion of the individuals. The individuals with the low resulting energy are transferred to the next generation and the process is then repeated. The algorithm is called lamarckian genetic algorithm because every new generation of individuals is allowed to inherit the local search adaptations of their parents186.

The preparation of the target protein 5HCU and 4 BDS with the AutoDock tools software involved adding all hydrogen atoms to the macromolecule, which is a step necessary for correct calculation of partial atomic charges. Gasteiger charges are calculated for each atom of the macromolecule in AutoDock 4.2 instead of kollman charges which were used in the previous versions of the program. Three-dimensional affinity grids of size 277 × 277 × 277 Å with 0.6 Å spacing were centered on the geometric center of the target protein and were calculated for each of the following atom types: HD, C, A, N, OA, and SA, representing all possible atom types in a protein. Additionally, an electrostatic map and a desolvation map were also calculated187.

Rapid energy evaluation was achieved by precalculating atomic affinity potentials for each atom in the ligand molecule. In the AutoGrid procedure, the target enzyme was embedded on a three dimensional grid point. The energy of interaction of each atom in the ligand was encountered. The selected important docking parameters for the LGA as follows: population size of 150 individuals, 2.5 million energy evaluations, maximum of 27000 generations, number of top individuals to automatically survive to next generation of 1, mutation rate of 0.02, crossover rate of 0.8, 10 docking runs, and random initial positions and conformations. The probability of performing local search on an individual in the population was set to 0.06. AutoDock was run several times to get various docked conformations, and used to analyze the predicted docking energy. The binding sites for these molecules were selected based on

65 the ligand-binding pocket of the templates. AutoDock Tools provide various methods to analyze the results of docking simulations such as, conformational similarity, visualizing the binding site and its energy and other parameters like intermolecular energy and inhibition constant. For each ligand, ten best poses were generated and scored using AutoDock 4.2 scoring functions188.

5.3 REAGENTS AND CHEMICALS Donepezil, acetylcholinesterase, butyrylcholinesterase, apigenin, baicalein, catechin, chrysin, diosmin, epigallocatechin, hesperetin, hesperidin, kaempferol, morin, myrecetin, naringenin, robinetin, rutin, scopoletin, silibinin, taxifolin, tetra hydroflavone, theaflavin and tricetin were purchased from Sigma Aldrich, St. Louis, MO. All other drugs and chemicals used in the study were obtained commercially and were of analytical grade. Bovine serum albumin (BSA), p-nitro benzaldehyde, p- fluro benzaldehyde, p-chloro benzaldehyde, p-methyl benzaldehyde, p-methoxy benzaldehyde, p-hydroxy benzaldehyde ethanol, potassium phosphate, Folin phenol reagent, 5,5'-dithiobis-2-nitro benzoic acid, acetylthiocholine iodide, carboxy methyl cellulose and tris hydrochloride were purchased from Hi Media Laboratories, Mumbai. Sodium thiosulphate, sodium hydroxide, hydrochloric acid, sodium potassium tartarate, sodium carbonate, copper sulphate, hydrogen peroxide, potassium dichromate, glacial acetic acid and sodium citrate were purchased from SD Fine chem limited, Mumbai. Scopolamine and aluminium chloride were purchased from Sigma Aldrich, USA. All other chemicals and solvents used were of AR grade.

5.4 INSTRUMENTS USED Digital Balance (Sartorius Ltd., USA), Eppendorff minispin, incubator (Technico), Jasco FTIR-420 series, Mass spectrometer, Shimadzu-Jasco V-530 UV/Vis spectrophotometer, ELCO 1/27 pH meter.

5.5 In vitro enzyme inhibition assay

5.5.1 In vitro acetylcholinesterase inhibition

Based on in silico results, twenty compounds were selected for their AChE inhibitory activities189. About 1.3 ml of the Tris-HCl buffer (pH 8.0; 50 mM) was treated with 0.4 ml of different concentrations (2 to 128 µg/ml) of the compounds and 0.1 ml of the AChE (0.28 U/ml) was added. This mixture was incubated for 15 minutes and 0.3 ml of acetylthiocholine iodide (0.023mg/ml) and 1.9 ml (5,5'-dithiobis-(2-

66 nitrobenzoic acid) DTNB (3 mM) solution were added. This final reaction mixture (4.0 ml) was kept for further incubation at room temperature for 30 minutes and the absorbance of the reaction mixture was measured at 405 nm. The assay was done in triplicate and the results were expressed as mean±SEM. Donepezil was used as standard190. The percentage inhibition was calculated for the selected flavonoids using the following formula,

A0= Absorbance of the control

A1= Absorbance of the standard/compounds.

5.5.2 In vitro butyrylcholinesterase inhibition

In our work, 20 flavonoids were examined for their BuChE inhibitory activities at various concentrations (1 to 128 µg/ml) and were dissolved in 1.3 ml of the Tris-HCl buffer (pH 8.0; 50 mM) following the spectrophotometric method189. In this method, 0.1 ml of the BuChE (0.377 U/ml) solution were added to the initial mixture, to start the reaction and then the absorbance was determined. 0.3 ml of s-butyrylthiocholine iodide (1.5 mM) and 1.9 ml DTNB (3 mM) solution and 0.4 ml of drug solution at the different concentrations evaluated, which were mixed and incubated for 15 min at 30ºC. Then, the mixture was monitored spectrophotometrically at 405 nm. The control sample contained all components except the test compounds and rivastigmine was used as standard190. The percentage of BuChE inhibitory activity was calculated by using the following equation:

A0 = Absorbance of the control

A1 = Absorbance of the standard/ compounds. 5.6 ACUTE TOXICITY STUDIES

5.6.1 Experimental animals

67 used for the acute toxicity studies were procured from Kerala Veterinary and Animal Sciences University, Mannuthy, Thrissur, Kerala, India. Animals were 8-12 weeks old and weight was not deviated from ± 20%. Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) approval number for experimental protocols for the toxicological studies was COPSRIPMS/IAEC/PhD/P’CHEMISTRY/ 03/ 2012-2013.

5.6.2 Acute toxicity study procedure

Due to absence of toxicity information for flavonoids, in the sighting study, one animal was administered with 5 mg/kg dose and in the absence of toxicity 50mg/kg dose was given to second animal to check for toxicity. If no mortality was observed for 50mg/kg dose then the next dose of 300 mg/kg was given to third animal and if no mortality was observed then the fourth animal was administered with 2000 mg/kg dose. If no mortality was observed for upper dose limit of 2000 mg/kg, then the specific dose was selected for main study. Keen observations were done to determine the onset of signs, onset of recovery and time to death.

In main study a total of 5 animals of one sex was used. The 5 animals would be made of one animal from the sighting study dosed at the selected dose level together with an additional four animals. Dose of 2000 mg/kg dose of the drug was administered and food was withheld for further 3-4 hours. Animals were observed individually at least once during the first 30 minutes after dosing, periodically during the first 24 hours (with special attention during the first 4 hours) and daily thereafter for a period of 14 days. Once daily cage side observations included changes in skin and fur, eyes and mucous membrane (nasal), and also changes in respiratory rate, circulatory parameters (heart rate and blood pressure), autonomic parameters (salivation, lacrimation, perspiration, piloerection, urinary incontinence and defecation) and CNS parameters (ptosis, drowsiness, gait, tremors and convulsions)192.

5.7 IN VIVO EXPERIMENTAL DESIGN

5.7.1 Experimental animals

Male swiss albino mice (20-25 g) were procured from Kerala Veterinary and Animal Sciences University, Mannuthy, Thrissur, Kerala, India. Animals were fed with commercial pellet diet and drinking water ad libitum and maintained at an ambient temperature of 22 ± 2°C with a 12-h light: dark cycle. The experiments were conducted

68 in accordance with guidelines of Institutional animal ethical committee (IAEC) governed by CPCSEA guidelines, Government of India. All the readings were taken during the same time of the day that is between 10.00 am and 2.00 pm. CPCSEA approval number for experimental protocols for the pharmacological studies was COPSRIPMS/IAEC/PhD/P’CHEMISTRY/ 03/ 2012-2013.

5.7.2 In vivo scopolamine-induced amnesia model

Animals were classified into six groups (n=6, Swiss albino mice). Treatments were given p.o. for seven successive days. Group I served as control received only normal saline (10ml/kg). Animals in group II were treated with baicalein (100 mg/kg/day) and group III mice were treated with baicalein (200 mg/kg/day). Animals in group IV received scopoletin (100 mg/kg/day) and group V mice were administered with scopoletin (200 mg/kg/day). Group VI mice were treated with donepezil (5 mg/kg, p.o.). For all the groups, treatments were given for 7 consecutive days. One hour after the last dose of test agents, all animals were i.p. injected with scopolamine hydrobromide (1 mg/kg) except the control group193. The values for the negative control group were taken from archives163. The neuroprotective effects of the selected flavonoids were studied using various behavioral assessments such as rectangular maze test, Morris water maze, gross behavioral activity, 5 choice serial reaction time task and pole climbing test during the treatment period.

On day 8, after behavioral assessments, animals were sacrificed by excess of anesthesia. The brain was removed and each brain had been separately kept after rinsing with ice-cold isotonic saline. A (10% w/v) homogenate was prepared with 0.1 M phosphate buffer (pH 7.4) and centrifuged at 3000 rpm for 15 minutes and aliquots of supernatant had separated and used for the various biochemical estimations {cholinesterase activity, protein estimation, enzymatic (Catalase activity, 2,2-diphenyl- 1-picrylhydrazyl assay, superoxide dismutase assay, glutathione reductase, glutathione peroxidase) and non-enzymatic antioxidant activities (reduced glutathione) and lipid peroxidation indices with thiobarbituric acid reactive substances assay}.

5.7.3 Aluminium-induced learning impairment

(200 mg/kg/day). In group IV mice received scopoletin (100 mg/kg/day), group V received scopoletin (200 mg/kg/day). Group VI mice were treated with donepezil (5 mg/kg, p.o.) for 3 months. All the groups were treated for a period of 3 months. Groups

III to VII received Aluminum chloride (AlCl3) dissolved in water at a dose of 50 mg/kg/day194, 195, for a period of 3 months. The values for the negative control group were taken from archives. The values for the negative control group were taken from archives18. The effects of the selected flavonoids were studied using various behavioral assessments such as rectangular maze test, Morris water maze, gross behavioral activity, 5 choice serial reaction time task and pole climbing test during the treatment period.

After treatment period, animals were sacrificed by excess of anesthesia. The brain of the each animal was removed and rinsed with ice-cold isotonic saline. A (10% w/v) homogenate was prepared with 0.1 M phosphate buffer (pH 7.4) and centrifuged at 3000 rpm for 15 minutes and aliquots of supernatant separated and used for various biochemical estimations {cholinesterase activity, protein estimation, enzymatic (Catalase activity, 2,2-diphenyl-1-picrylhydrazyl assay, superoxide dismutase assay, glutathione reductase, glutathione peroxidase} and non-enzymatic antioxidant activities (reduced glutathione) and lipid peroxidation by thiobarbituric acid reactive substances assay}.

5.8 BEHAVIORAL ASSESSMENTS

5.8.1 Rectangular maze test

The maze consists of completely enclosed rectangular box with an entry and reward chamber partitioned with wooden slots into blind passages leaving just twisting corridor leading from the entry to the reward chamber (Fig. 16). All the mice were familiarized with the rectangular maze. Well trained animals were taken for the experiment. The time taken for the mouse to reach the reward chamber was taken as the transfer latency. Lower scores of assessment indicate efficient learning while higher scores indicate poor learning in animals. The time taken by the animals to reach the reward chamber from the entry chamber was tabulated196. All the values were calculated in seconds.

Fig. 16 Rectangular maze

5.8.2 Morris water maze test

This test was carried out in circular pool (150 cm in diameter and 60 cm in height) of water with ten centimeter hidden platform (Fig. 17). In the water maze experiments, the day prior to the experiment the animals were trained to swim for 60 s in the absence of the platform. In the following days, the mice were given the trial session each day for four consecutive days. During each trial, the escape latencies of mice were recorded. The point of entry of the mouse into the pool and the location of the platform for escape between trials changed each day thereafter. During acquisition, trial escape latency time (ELT), measure to locate the hidden platform, was noted as an index of acquisition. Escape latency time was calculated in seconds. All mice were tested for spatial memory 30 minutes after the last dose of the treatment197.

Fig. 17 Morris water maze

5.8.3 Assessment of gross behavioral activity (Locomotor activity)

Most of the CNS drugs influence the locomotor activity in man and animals. The locomotor activity of drug was studied using actophotometer which operates on photoelectric cells which are connected in circuit with a counter (Fig. 18). When the beam of light falling on photocell was cut off by the animal, then a count was recorded. Animals were placed individually in the activity cage for 10 min and the activity was monitored. The photo cell count was noted and a decrease or increase in locomotor activity was calculated198.

Fig. 18 Actophotometer

5.8.4 Pole climbing test

72 chamber that was enclosed in a dim light, sound attenuated box (Fig. 19). Each mouse was allowed to acclimatize for 2 min and then exposed to a buzzer noise. After 10 sec of putting on the buzzer, mild electric shocks were given through the grid floor. The magnitude of the voltage was adequate (30v) to stimulate the mice to escape from the floor and climb the pole. As soon as the mice climbed the pole, both the buzzer and foot-shock were switched off. At least 10 such trails were given to each mouse at an interval of 1 min. per day for 3 days. After training schedule, most of the mice learned to climb the pole within 10 sec of starting the buzzer to avoid the electric foot shocks. On the test days, after 2 min of acclimatization period, each mouse was exposed to the buzzer for 10 sec; ten such trails were given at an interval of 1 min, without giving any shock. Mice, responding by climbing the pole when exposed to the buzzer noise, were considered to have retained the conditional avoidance response198.

Fig. 19 Pole climbing apparatus

5.8.5 Attention in a 5 choice serial reaction time task (5-CSRT)

The 5 choice serial reaction time task (5-CSRT) tests specific types of attention, including sustained, divided, and selected attention. The 5-CSRT was implemented in a specifically designed operant chamber with multiple response locations using light source (Fig. 20). The usual paradigm requires the mice to detect brief flashes of light presented in a pseudorandom order in one of five locations over a large number of trials. The response time of the animals were calculated in seconds199.

Fig. 20 Five choice serial reaction time task apparatus

5.9 BIOCHEMICAL ESTIMATIONS

5.9.1 Estimation of AChE in mice brain

The cholinergic marker, acetylcholinesterase, was estimated in the whole brain using Ellman method. Ellman’s reagent is 5,5’-dithiobis(2-nitrobenzoate) and is also abbreviated as DTNB. Ellman’s reagent is the mixture of 19.8 mg of DTNB in 100 ml of 0.1% sodium citrate solution. About 0.1 ml of brain homogenate was incubated for 5 min with 2.7 ml of phosphate buffer and 0.1 ml of DTNB. Then, 0.1 ml of freshly prepared acetylthiocholine iodide (pH 8) was added and the absorbance was measured at 412 nm200.

5.9.2 Estimation of total protein content

The amount of total protein present in the tissue homogenate was estimated using bovine serum albumin as the standard. To 0.1 ml of tissue homogenate, 4.0 ml of alkaline copper tartarate solution was added and allowed to stand for 10 min. To the mixture 0.4 ml of phenol reagent was added rapidly, mixed quickly and incubated at room temperature for 30 min for colour development. Reading was taken against blank prepared with distilled water at 610 nm in an UV-visible spectrophotometer. The protein content was calculated from the standard curve prepared with bovine serum albumin (BSA) (1 mg/ml) and expressed as mmoles/min/mg brain tissue201.

5.9.3 Enzymatic anti-oxidants

5.9.3.1 Estimation of catalase

Catalase (hydrogen peroxide oxidoreductase) enzyme is involved in the decomposition of hydrogen peroxide into water and oxygen molecules. Catalase activity is measured either by decomposition of H2O or by liberation of O2. The decrease in the absorbance by H2O2 as a function of time is used to follow the hydrogen-peroxide reaction. The spectral region for hydrogen peroxide is 210-240 nm. The difference in absorbance per minute is a measure of catalase activity. Catalase plays a major role in cellular antioxidant defense system by decomposing H2O2, thereby preventing the generation of hydroxyl radical through the Fenton reaction202.

About 0.1 ml of tissue homogenate was added to 1.0 ml of 0.01 M phosphate buffer of pH 7.0 and was incubated at 37C for 15 min. The reaction was started by the addition of 0.4 ml of 2M H2O2. The reaction was then stopped by the addition of 2 ml of dichromate - acetic acid reagent (a mixture of 5% potassium dichromate and glacial acetic acid in a ratio of 1:3). CAT was assayed colorimetrically at 620 nm. Unit of catalase activity is expressed as the amount of enzyme causing the decomposition of 203 µmoles H2O2/mg protein/min .

5.9.3.2 Estimation of superoxide dismutase (SOD)

The first enzyme involved in the antioxidant defense is superoxide dismutase. It is a metalloprotein found in both prokaryotic and eukaryotic cells. The oxygen-derived 2+ free radicals generated by the interaction of Fe and H2O2 are the species responsible for the oxidation of epinephrine and they were strongly inhibited by the enzyme, superoxide dismutase204.

About 0.1 ml of tissue homogenate was mixed with sodium pyrophosphate buffer, and nitroblue tetrazolium (NBT). The reaction was initiated by the addition of NADH. The reaction mixture was incubated at 30°C for 90 seconds and it was stopped by the addition of 1 ml of glacial acetic acid. The absorbance of the chromogen formed was measured at 560nm. SOD activity is expressed as nmoles/min/mg protein205.

5.9.3.3 Estimation of glutathione reductase (GSSH)

The reaction mixture contained 2.1 ml of 0.25 mM potassium phosphate buffer of pH 7.6, 0.1ml of 0.001M NADPH, 0.2 ml of 0.0165 M oxidized glutathione and 0.1

75 ml of bovine serum albumin (10 mg/ml). The reaction was started by the addition of 0.2 ml of tissue homogenate with mixing and the decrease in the absorbance at 340 nm was measured for 3 minutes against the blank. Glutathione reductase activity is expressed as nmoles NADPH oxidized /min/mg protein205.

5.9.3.4 Estimation of glutathione peroxidase (GPx)

Glutathione peroxidase (GPx) has a major role in degrading the levels of H2O2 in cells. Since GPx acts on the hydroperoxides of unsaturated fatty acids, this enzyme plays an important role in protecting membrane lipids, and thus the cell membranes from oxidative disintegration206.

The reaction mixture consisted of 0.2 ml of 0.4 M Tris-HCl buffer, 0.1 ml of sodium azide, 0.1 ml of hydrogen peroxide, 0.2 ml of glutathione and 0.2 ml of supernatant. The mixture was incubated at 37C for 10 min. The reaction was arrested by the addition of 10 % trichloro acetic acid and the absorbance was measured at 340 nm. GPx activity is expressed as nmoles/min/mg protein207.

5.9.4. Non-enzymatic antioxidants

5.9.4.1 Estimation of reduced glutathione (GSH)

The most widely used method for the determination of GSH in biological samples is by Ellman reagent (DTNB), which reacts with sulfydryl compounds to give a relatively stable yellow colour. About 1 ml of supernatant was treated with 0.5 ml of Ellman’s reagent (19.8 mg of 5, 5’-dithiobis-(2-nitro) benzoic acid (DTNB) in 100 ml of 0.1% sodium citrate solution) and 3.0 ml of 0.2 M phosphate buffer of pH 8.0. The absorbance was read at 412 nm using a spectrophotometer. The activity of GSH was expressed as nmoles/min/mg protein204.

5.9.5 Estimation of lipid peroxidation (LPO)

Lipid peroxidation as evidenced by the formation of malondialdehyde was measured by the method of Niehaus and Samuelson, 1986. About 0.1 ml of tissue homogenate was treated with 2 ml of (1:1:1 ratio) TBA-TCA-HCl reagent (0.37% thiobarbituric acid, 15% trichloroacetic acid and 0.25 N hydrochloric acid) placed in a water bath for 15 min, cooled and centrifuged at room temperature for 10 min at 3000 rpm. The absorbance of the clear supernatant was measured against a reference blank at 535 nm. The values are expressed as nmoles of MDA formed /min/mg protein208.

5.9.6 (2,2-Diphenyl-1-picrylhydrazyl) (DPPH) assay

DPPH is a stable, nitrogen-centered free radical which produces violet colour in ethanol solution. It was reduced to a yellow colored product, diphenylpicryl hydrazine, with the addition of the fractions in a concentration-dependent manner. The reduction in the number of DPPH molecules can be correlated with the number of available hydroxyl groups. About 1 ml of tissue homogenate was mixed with 4 ml of 0.004% methanolic solution of DPPH solution. After 30 min incubation, absorbance was read at 517 nm. Percentage of inhibition was calculated 209.

5.10 Histopathological studies

Brain of animals in different groups was perfusion-fixed with 4% paraformaldehyde in 0.1 M phosphate buffer. The brain was removed and post fixed in the same fixative overnight. The brain was embedded in paraffin and stained with hematoxylin-eosin. The hippocampal lesions were assessed microscopically at 40 X magnification210.

5.11 Statistical analysis

Statistical analysis was carried out using GraphPad Prism 4.0 by One-way Analysis of Variance (ANOVA) followed by Dunnett’s test. Results are expressed as mean±SEM from six mice in each group. P values < 0.05 were considered significant.

6. RESULTS AND ANALYSIS

6.1 Drug likeness properties of the flavonoids

In the present study, two hundred and forty five flavonoids were identified and the structures were drawn with the help of MedChem designer. The drug likeness properties of the flavonoids were evaluated with the help of MedChem designer. All the 245 flavonoids showed excellent score (partition coefficient, molecular weight, hydrogen bond donors and acceptors) which indicate that these compounds have no violations or acceptable violations in Lipinski’s rule of 5 (Fig. 21). Based on the drug likeness score, all the 245 flavonoids exhibited considerable pharmacokinetic profile and were selected for in silico biological screening against AChE and BuChE using AutoDock 4.2.

Fig. 21 Drug likeness properties of the flavonoids

6.2 In silico docking studies against AChE and BuChE

Totally 245 flavonoids selected were subjected to in silico docking studies based on drug likeness properties. In the docking studies of the flavonoids, the following docking parameters such as binding energy, inhibition constant and intermolecular energy were evaluated against AChE and BuChE enzymes. The selected flavonoids showed excellent docking parameters and orientations when compared with the standard (donepezil) against AChE and BuChE.

Scopoletin and baicalein showed remarkably excellent binding energy (-6.95 kcal/mol and -6.93 kcal/mol) when compared to other flavonoids (Fig. 22). The minimum binding energy indicated that the selected flavonoids have excellent inhibitory potential toward AChE protein.

Inhibition constant is an important score to consider, by which the effectiveness of the compounds were evaluated through both virtual screening and in vitro pharmacological experimental studies. In silico studies proved that, the flavonoids could exhibit excellent inhibition constant toward the AChE ranging between 8.37 µM to 990.45 µM when compared with standard (994.14 µM). Scopoletin and baicalein showed remarkably excellent inhibition constant (8.37 µM and 8.32 µM) when compared to other flavonoids.

Intermolecular energy is also directly proportional to binding energy. We found a decrease in intermolecular energy of all the flavonoids with a simultaneous decrease in binding energy. The flavonoids showed intermolecular energy towards the AChE ranging from -8.94 kcal/mol to -5.51 kcal/mol when compared with standard (-5.46 kcal/mol). Scopoletin and baicalein showed remarkably excellent intermolecular energy (-8.94 kcal/mol and -8.91 kcal/mol) when compared to other flavonoids. This further proved the inhibitory potential of scopoletin and baicalein against AChE.

The ligand scopoletin interacted with the binding sites of AChE at the amino acid residues Tyr 124, Trp 286, Leu 289, Ser 293, Ile 294, Phe 295, Tyr 341 and it was in correlation with the donepezil Tyr 72, Tyr 124, Trp 286, Phe 295 and Tyr 341 (Fig. 23).

Fig. 22 Binding energy of the flavonoids against AChE

Table 1: In silico molecular interactions scores of the flavonoids against AChE and BuChE (Cont.)

Acetylcholinesterase Butyrylcholinesterase

Flavonoids Binding Inhibition Intermolecular Binding Inhibition Intermolecular Energy constant Energy Energy constant Energy (kcal/mol) (µM) (kcal/mol) (kcal/mol) (µM) (kcal/mol)

Acacatechin -4.84 287.59 -7.37 -2.97 134.85 -4.81

Acacetin -4.54 335.28 -6.92 -2.79 156.98 -4.54

Afzelechin -5.26 136.28 -7.80 -3.20 71.63 -5.09

Angoletin -5.14 162.14 -7.73 -3.12 80.92 -5.06

Annulatin -4.86 282.94 -7.40 -2.98 131.50 -4.85

Apigenin -6.33 39.87 -8.67 -3.88 19.08 -5.68

Apometzgerin -5.31 114.42 -7.87 -3.26 54.77 -5.16

Aromadedrin, Dihydro Kaempferol -5.37 90.21 -7.96 -3.29 43.32 -5.23

Aurantinidin -5.46 84.04 -8.10 -3.35 39.25 -5.25

Aureusidin -5.54 80.12 -8.19 -3.40 37.76 -5.37

Ayanin -5.63 74.78 -8.24 -3.45 35.09 -5.41

Table 1: In silico molecular interactions scores of the flavonoids against AChE and BuChE (Cont.)

Acetylcholinesterase Butyrylcholinesterase

Flavonoids Binding Inhibition Intermolecular Binding Inhibition Intermolecular Energy constant Energy Energy constant Energy (kcal/mol) (µM) (kcal/mol) (kcal/mol) (µM) (kcal/mol)

Azaleatin -4.24 730.98 -6.30 -2.60 342.77 -4.11

Baicalein -6.93 8.32 -8.91 -4.51 2.12 -6.11

Baicalin mono hydrate -3.94 964.84 -5.64 -2.41 459.96 -3.66

Baptigenin -5.34 98.84 -7.94 -3.26 51.69 -5.21

Bavachinin -4.04 914.52 -5.84 -2.47 439.08 -3.77

Biochanin A -4.36 368.66 -6.62 -2.68 170.41 -4.33

Bis Hydroxy Coumarin -5.68 70.32 -8.35 -3.48 33.11 -5.46

Bracteatin -5.46 83.74 -7.99 -3.33 39.69 -5.25

Buceracidin-B -5.54 80.56 -8.18 -3.40 37.96 -5.36

Butein -4.92 276.36 -7.45 -3.02 126.27 -4.87

Calycopterin -4.30 581.36 -6.41 -2.63 320.78 -4.20

Table 1: In silico molecular interactions scores of the flavonoids against AChE and BuChE (Cont.)

Acetylcholinesterase Butyrylcholinesterase

Flavonoids Binding Inhibition Intermolecular Binding Inhibition Intermolecular Energy constant Energy Energy constant Energy (kcal/mol) (µM) (kcal/mol) (kcal/mol) (µM) (kcal/mol)

Calycosin -4.67 321.84 -7.14 -2.85 150.65 -4.66

Candidone -5.05 200.12 -7.62 -3.09 102.49 -4.95

Cannflavin -4.77 298.34 -7.30 -2.92 142.95 -4.77

Capensinidin -4.50 339.68 -6.89 -2.75 160.72 -4.50

Carajuflavone -4.23 729.36 -6.24 -2.60 341.73 -4.07

Carpachromene -3.96 954.36 -5.71 -2.42 451.12 -3.68

Casticin -4.69 315.64 -7.16 -2.87 150.86 -4.69

Catechin -5.34 136.44 -6.99 -3.28 45.30 -5.21

Chrysin -6.30 45.39 -8.64 -3.83 25.37 -5.65

Chrysoeriol -4.59 328.45 -6.97 -2.79 157.16 -4.54

Chrysosplenetin -5.43 84.68 -7.99 -3.33 40.58 -5.23

Table 1: In silico molecular interactions scores of the flavonoids against AChE and BuChE (Cont.)

Acetylcholinesterase Butyrylcholinesterase

Flavonoids Binding Inhibition Intermolecular Binding Inhibition Intermolecular Energy constant Energy Energy constant Energy (kcal/mol) (µM) (kcal/mol) (kcal/mol) (µM) (kcal/mol)

Cirsililol -4.16 832.21 -6.01 -2.52 399.50 -3.91

Cirsilineol -5.63 74.86 -8.24 -3.45 35.76 -5.40

Cirsimaritin -5.72 69.92 -8.38 -3.49 32.93 -5.48

Cladrin -5.80 68.84 -8.44 -3.53 32.46 -5.53

Combretol -4.23 729.64 -6.23 -2.59 339.48 -4.07

Cyanidin -4.50 338.64 -6.87 -2.75 160.37 -4.49

Cyclocommunol -4.77 298.81 -7.29 -2.92 142.72 -4.77

Daidzein -5.04 218.24 -7.58 -3.08 109.86 -4.95

Datin -5.31 118.42 -7.86 -3.26 54.04 -5.15

Datiscetin -4.24 731.26 -6.30 -2.60 343.07 -4.10

Delphinidin -3.94 962.38 -5.61 -2.41 459.45 -3.64

Table 1: In silico molecular interactions scores of the flavonoids against AChE and BuChE (Cont.)

Acetylcholinesterase Butyrylcholinesterase

Flavonoids Binding Inhibition Intermolecular Binding Inhibition Intermolecular Energy constant Energy Energy constant Energy (kcal/mol) (µM) (kcal/mol) (kcal/mol) (µM) (kcal/mol)

Demethylbellidifolin -4.64 321.62 -7.12 -2.83 151.89 -4.59

Desmethoxycentaureidin -5.58 79.05 -8.20 -3.42 37.00 -5.38

Dihydrobaicalein -5.84 67.86 -8.50 -3.57 31.99 -5.55

Dihydroflavonol -4.77 298.72 -7.27 -2.91 143.73 -4.77

Dihydrokaempferide -5.04 218.64 -7.54 -3.08 108.39 -4.94

Dihydromyricetin -5.31 116.84 -7.86 -3.24 57.58 -5.14

Dihydrorobinetin -5.58 78.94 -8.20 -3.42 36.88 -5.38

Dihydrotricetin -4.54 334.98 -6.92 -2.77 157.71 -4.53

Dihydrowogonin -4.34 396.44 -6.55 -2.65 197.60 -4.27

Diosmetin -6.03 60.86 -8.57 -3.66 29.87 -5.61

Diosmin -6.03 36.86 -7.84 -3.69 28.66 -5.61

Table 1: In silico molecular interactions scores of the flavonoids against AChE and BuChE (Cont.)

Acetylcholinesterase Butyrylcholinesterase

Flavonoids Binding Inhibition Intermolecular Binding Inhibition Intermolecular Energy constant Energy Energy constant Energy (kcal/mol) (µM) (kcal/mol) (kcal/mol) (µM) (kcal/mol)

Dorsilurin-F -5.68 70.26 -8.34 -3.52 32.57 -5.51

Epicatechin -5.74 69.64 -8.42 -3.52 32.64 -5.53

Epicatechin Gallate -5.80 68.98 -8.44 -3.17 73.26 -5.09

Epigallocatechin -5.24 124.12 -7.04 -3.24 58.29 -5.13

Epigallocatechin Gallate -5.31 115.28 -7.84 -3.40 37.56 -5.38

Eriocitrin -5.58 78.68 -8.20 -3.57 31.95 -5.55

Eriodictyol -5.84 67.24 -8.48 -2.68 172.81 -4.35

Ermanin -4.37 357.45 -6.67 -2.69 164.74 -4.42

Esculetin -4.38 354.98 -6.75 -3.12 79.86 -5.05

Eupafolin -5.09 168.42 -7.72 -3.39 38.31 -5.34

Eupalitin -5.53 81.12 -8.17 -3.44 36.51 -5.39

Table 1: In silico molecular interactions scores of the flavonoids against AChE and BuChE (Cont.)

Acetylcholinesterase Butyrylcholinesterase

Flavonoids Binding Inhibition Intermolecular Binding Inhibition Intermolecular Energy constant Energy Energy constant Energy (kcal/mol) (µM) (kcal/mol) (kcal/mol) (µM) (kcal/mol)

Eupatilin -5.60 77.94 -8.21 -3.47 33.24 -5.44

Eupatolitin -5.67 70.64 -8.32 -3.52 32.59 -5.52

Eupatorin -5.75 69.24 -8.43 -3.42 36.85 -5.38

Europetin -5.60 77.89 -8.21 -3.38 38.42 -5.32

Europinidin -5.52 81.72 -8.13 -3.30 42.29 -5.23

Farobin-A -5.39 89.12 -7.97 -3.23 63.88 -5.12

Fisetin -5.28 122.84 -7.83 -3.15 76.00 -5.07

Fisetinidol -5.17 156.28 -7.75 -3.09 102.30 -4.98

Flavoxate -5.05 198.89 -7.67 -3.02 126.66 -4.88

Formononetin -4.94 264.69 -7.45 -2.95 136.96 -4.79

Fustin -4.82 290.86 -7.32 -2.87 148.82 -4.69

Table 1: In silico molecular interactions scores of the flavonoids against AChE and BuChE (Cont.)

Acetylcholinesterase Butyrylcholinesterase

Flavonoids Binding Inhibition Intermolecular Binding Inhibition Intermolecular Energy constant Energy Energy constant Energy (kcal/mol) (µM) (kcal/mol) (kcal/mol) (µM) (kcal/mol)

Galangin -4.71 312.28 -7.18 -2.81 155.39 -4.56

Gallocatechin -4.60 326.54 -6.97 -3.36 38.85 -5.32

Gallocatechin Gallate -5.51 81.96 -8.11 -3.44 36.53 -5.39

Ganhuangenin -5.62 76.28 -8.23 -3.51 32.78 -5.50

Gardenin B -5.74 69.47 -8.39 -3.56 32.27 -5.54

Gartanin -5.82 68.24 -8.46 -2.65 197.10 -4.22

Genistein -4.32 421.54 -6.50 -2.50 412.75 -3.87

Genistin -4.08 886.24 -5.94 -2.58 338.97 -4.06

Geraldone -4.22 724.23 -6.20 -2.97 134.81 -4.84

Gericudranin-B -4.86 283.16 -7.40 -2.77 157.50 -4.53

Glabridin -4.54 334.20 -6.92 -2.69 165.22 -4.40

Table 1: In silico molecular interactions scores of the flavonoids against AChE and BuChE (Cont.)

Acetylcholinesterase Butyrylcholinesterase

Flavonoids Binding Inhibition Intermolecular Binding Inhibition Intermolecular Energy constant Energy Energy constant Energy (kcal/mol) (µM) (kcal/mol) (kcal/mol) (µM) (kcal/mol)

Glaziovianin-A -4.38 352.12 -6.74 -2.91 144.53 -4.75

Glycitein -4.76 304.95 -7.27 -2.38 464.27 -3.62

Gnetifolin E -3.92 981.25 -5.58 -2.98 132.73 -4.86

Gomphrenol -4.88 278.98 -7.42 -2.96 136.34 -4.81

Gossypetin -4.84 289.64 -7.34 -2.50 413.84 -3.86

Guibourtinidol -4.08 884.16 -5.93 -2.63 311.37 -4.19

Helmon -4.28 698.28 -6.41 -3.70 27.38 -5.63

Herbacetin -6.08 57.96 -8.58 -3.22 25.63 -5.11

Hesperedin -5.28 148.38 -7.08 -4.15 8.09 -5.72

Hesperetin -6.72 17.02 -8.81 -3.83 24.67 -5.64

Hibiscetin -6.28 48.42 -8.63 -3.56 32.33 -5.54

Table 1: In silico molecular interactions scores of the flavonoids against AChE and BuChE (Cont.)

Acetylcholinesterase Butyrylcholinesterase

Flavonoids Binding Inhibition Intermolecular Binding Inhibition Intermolecular Energy constant Energy Energy constant Energy (kcal/mol) (µM) (kcal/mol) (kcal/mol) (µM) (kcal/mol)

Hirsutidin -5.82 68.16 -8.45 -3.52 32.56 -5.51

Hispudulin -5.74 69.53 -8.41 -3.35 38.98 -5.32

Homoeriodictyol -5.48 82.87 -8.10 -3.26 53.63 -5.17

Homoorientin -5.32 110.28 -7.94 -2.82 153.96 -4.58

Hydrangenol -4.61 324.03 -6.99 -2.73 163.43 -4.48

Hypolaetin -4.46 345.24 -6.84 -2.63 316.01 -4.20

Icaritin -4.30 561.28 -6.41 -2.69 164.18 -4.40

Irigenin -4.38 350.92 -6.74 -2.93 140.03 -4.77

Isalpinin -4.77 298.36 -7.30 -2.97 135.77 -4.82

Isoetin -4.86 284.31 -7.38 -2.50 413.50 -3.88

Isoimperatorin -4.11 852.26 -5.96 -2.58 338.62 -4.06

Table 1: In silico molecular interactions scores of the flavonoids against AChE and BuChE (Cont.)

Acetylcholinesterase Butyrylcholinesterase

Flavonoids Binding Inhibition Intermolecular Binding Inhibition Intermolecular Energy constant Energy Energy constant Energy (kcal/mol) (µM) (kcal/mol) (kcal/mol) (µM) (kcal/mol)

Isokaempferide -4.23 728.14 -6.22 -2.74 161.83 -4.49

Isookanin -4.48 344.23 -6.85 -2.76 158.74 -4.51

Isopongaflavone -4.52 336.45 -6.90 -2.65 196.09 -4.22

Isoquercitrin -4.32 421.28 -6.48 -3.73 27.17 -5.63

Isorhamnetin -6.22 57.28 -8.58 -3.06 114.78 -4.92

Isosakuranetin -5.02 236.24 -7.54 -3.04 119.75 -4.90

Isoscutellarein -4.98 244.87 -7.50 -2.95 136.61 -4.81

Isosilybin -4.84 287.31 -7.34 -3.03 124.07 -4.89

Isothymusin -4.96 252.36 -7.48 -2.65 195.11 -4.21

Isovitexin -4.32 420.84 -6.46 -2.74 161.80 -4.48

Isoxanthohumol -4.48 342.86 -6.85 -2.99 130.77 -4.87

Table 1: In silico molecular interactions scores of the flavonoids against AChE and BuChE (Cont.)

Acetylcholinesterase Butyrylcholinesterase

Flavonoids Binding Inhibition Intermolecular Binding Inhibition Intermolecular Energy constant Energy Energy constant Energy (kcal/mol) (µM) (kcal/mol) (kcal/mol) (µM) (kcal/mol)

Jaceidin -4.92 272.38 -7.44 -3.11 85.53 -5.04

Kaempferide -5.08 172.63 -7.71 -3.85 22.70 -5.68

Kaempferol -6.32 42.90 -8.65 -3.06 112.98 -4.92

Kumatakenin -5.02 234.36 -7.54 -2.68 171.17 -4.33

Ladanein -4.36 364.28 -6.61 -2.71 163.76 -4.43

Laricitrin -4.42 349.84 -6.81 -2.50 412.54 -3.85

Leptosidin -4.08 882.14 -5.91 -2.69 166.40 -4.43

Leucoanthocyanidin -4.42 349.21 -6.79 -3.14 77.49 -5.07

Leucocyanidin -5.16 160.76 -7.74 -2.71 163.99 -4.47

Leucodelphinidin -4.46 345.18 -6.83 -2.69 164.29 -4.40

Leucofisetinidin -4.38 351.44 -6.73 -2.60 341.89 -4.10

Table 1: In silico molecular interactions scores of the flavonoids against AChE and BuChE (Cont.)

Acetylcholinesterase Butyrylcholinesterase

Flavonoids Binding Inhibition Intermolecular Binding Inhibition Intermolecular Energy constant Energy Energy constant Energy (kcal/mol) (µM) (kcal/mol) (kcal/mol) (µM) (kcal/mol)

Leucomalvidin -4.24 731.04 -6.30 -2.90 145.70 -4.75

Leucopelargonidin -4.76 304.46 -7.27 -2.93 139.86 -4.79

Leucopeonidin -4.81 291.44 -7.32 -2.99 130.04 -4.86

Leucorobinetinidin -4.92 270.21 -7.43 -2.83 152.19 -4.58

Leukoefdin -4.64 321.38 -7.10 -2.62 346.13 -4.18

Licoflavonol -4.28 674.16 -6.40 -2.63 327.32 -4.21

Limocitrin -4.31 454.26 -6.42 -2.67 180.12 -4.33

Limocitrol -4.36 362.18 -6.61 -2.69 165.06 -4.42

Liquiritigenin -4.42 349.12 -6.77 -2.71 163.69 -4.46

Luteoforol -4.46 345.02 -6.83 -2.66 188.85 -4.29

Luteolin -4.35 384.26 -6.59 -2.58 338.73 -4.06

Table 1: In silico molecular interactions scores of the flavonoids against AChE and BuChE (Cont.)

Acetylcholinesterase Butyrylcholinesterase

Flavonoids Binding Inhibition Intermolecular Binding Inhibition Intermolecular Energy constant Energy Energy constant Energy (kcal/mol) (µM) (kcal/mol) (kcal/mol) (µM) (kcal/mol)

Luteolinidin -4.23 729.02 -6.20 -2.48 428.68 -3.83

Maesopsin -4.08 880.54 -5.89 -2.50 415.43 -3.90

Malvidin -4.12 842.15 -5.99 -2.60 342.02 -4.09

Maritimetin -4.24 731.24 -6.26 -2.61 343.32 -4.14

Matteucinol -4.26 734.22 -6.34 -2.65 197.27 -4.24

Mearnsitrin -4.33 402.89 -6.53 -2.69 164.10 -4.39

Meciadanol -4.38 352.46 -6.70 -2.58 339.12 -4.06

Melacacidin -4.21 722.38 -6.19 -2.71 163.65 -4.44

Melanoxetin -4.45 348.65 -6.82 -2.68 170.76 -4.34

Mesquitol -4.37 359.12 -6.67 -4.44 3.92 -5.74

Morin -6.92 8.29 -8.90 -3.88 18.69 -5.69

Table 1: In silico molecular interactions scores of the flavonoids against AChE and BuChE (Cont.)

Acetylcholinesterase Butyrylcholinesterase

Flavonoids Binding Inhibition Intermolecular Binding Inhibition Intermolecular Energy constant Energy Energy constant Energy (kcal/mol) (µM) (kcal/mol) (kcal/mol) (µM) (kcal/mol)

Myricetin -6.34 38.96 -8.68 -4.21 6.18 -5.72

Naringenin -6.76 17.26 -8.84 -3.59 31.52 -5.57

Naringin -5.88 65.86 -8.51 -3.45 35.05 -5.41

Natsudaidain -5.64 72.68 -8.28 -3.31 41.78 -5.23

Negletein -5.42 86.56 -7.97 -3.12 78.95 -5.07

Nepetin -5.16 160.32 -7.73 -2.97 134.68 -4.82

Nobiletin -4.86 280.53 -7.38 -2.89 146.38 -4.71

Nodifloretin -4.73 310.83 -7.23 -2.68 167.55 -4.38

Norathyriol -4.38 350.25 -6.70 -2.84 151.38 -4.66

Norwogonin -4.67 321.15 -7.14 -2.93 140.07 -4.79

Okanin -4.80 292.19 -7.30 -2.50 414.45 -3.89

Table 1: In silico molecular interactions scores of the flavonoids against AChE and BuChE (Cont.)

Acetylcholinesterase Butyrylcholinesterase

Flavonoids Binding Inhibition Intermolecular Binding Inhibition Intermolecular Energy constant Energy Energy constant Energy (kcal/mol) (µM) (kcal/mol) (kcal/mol) (µM) (kcal/mol)

Ombuin -4.12 840.14 -5.98 -2.60 341.32 -4.08

Ononin -4.24 731.88 -6.25 -2.48 427.71 -3.82

Orientin -4.08 882.85 -5.88 -2.64 212.93 -4.21

Orobol -4.32 420.48 -6.43 -2.55 390.10 -3.95

Oroxylin-A -4.18 784.46 -6.13 -2.68 168.34 -4.38

Osajin -4.38 350.48 -6.70 -2.87 150.54 -4.69

Pachypodol -4.68 318.65 -7.15 -2.62 346.42 -4.16

Patuletin -4.28 684.33 -6.39 -2.43 447.36 -3.75

Pectolinarigenin -4.04 912.44 -5.82 -2.43 448.38 -3.73

Pedalitin -4.02 934.27 -5.76 -2.64 263.10 -4.21

Pelargonidin -4.32 418.33 -6.43 -2.53 394.76 -3.94

Table 1: In silico molecular interactions scores of the flavonoids against AChE and BuChE (Cont.)

Acetylcholinesterase Butyrylcholinesterase

Flavonoids Binding Inhibition Intermolecular Binding Inhibition Intermolecular Energy constant Energy Energy constant Energy (kcal/mol) (µM) (kcal/mol) (kcal/mol) (µM) (kcal/mol)

Penduletin -4.18 782.15 -6.12 -3.94 17.01 -5.70

Peonidin -6.42 36.28 -8.71 -2.60 342.65 -4.14

Persicarin -4.26 735.08 -6.33 -2.75 161.36 -4.50

Persicogenin -4.52 336.01 -6.90 -2.81 155.24 -4.56

Petunidin -4.61 324.68 -6.98 -2.74 161.73 -4.48

Pinoquercetin -4.48 342.12 -6.84 -2.87 149.37 -4.69

Platanetin -4.71 311.88 -7.19 -2.64 272.51 -4.21

Pomiferin -4.32 416.24 -6.42 -2.53 393.82 -3.92

Pratensein -4.18 780.14 -6.03 -2.65 197.48 -4.25

Pratol -4.34 398.78 -6.55 -2.42 452.27 -3.70

Primetin -4.02 936.71 -5.74 -2.61 344.17 -4.16

Table 1: In silico molecular interactions scores of the flavonoids against AChE and BuChE (Cont.)

Acetylcholinesterase Butyrylcholinesterase

Flavonoids Binding Inhibition Intermolecular Binding Inhibition Intermolecular Energy constant Energy Energy constant Energy (kcal/mol) (µM) (kcal/mol) (kcal/mol) (µM) (kcal/mol)

Propolin B -4.28 664.26 -6.38 -2.21 488.27 -2.92

Prosogerin E -3.92 980.16 -5.55 -2.18 508.27 -2.82

Prunetin -3.90 990.45 -5.51 -2.47 437.94 -3.78

Prunin -4.08 880.08 -5.86 -2.79 157.02 -4.54

Pulchellidin -4.58 331.18 -6.95 -2.82 153.07 -4.58

Quercetagetin -4.63 322.94 -7.10 -2.57 367.72 -4.02

Quercetagetinidin -4.21 722.62 -6.18 -3.26 55.51 -5.16

Ranupetin -5.32 108.12 -7.90 -3.09 102.36 -4.99

Retusin -5.06 182.46 -7.68 -2.93 139.85 -4.79

Rhamnazin -4.81 291.25 -7.31 -3.04 118.74 -4.89

Rhamnetin -4.98 241.02 -7.50 -2.66 185.83 -4.30

Table 1: In silico molecular interactions scores of the flavonoids against AChE and BuChE (Cont.)

Acetylcholinesterase Butyrylcholinesterase

Flavonoids Binding Inhibition Intermolecular Binding Inhibition Intermolecular Energy constant Energy Energy constant Energy (kcal/mol) (µM) (kcal/mol) (kcal/mol) (µM) (kcal/mol)

Rhusflavanone -4.36 363.55 -6.60 -2.76 159.23 -4.52

Rhynchosin -4.54 334.89 -6.91 -1.25 231.18 -2.41

Robinetin -6.45 24.25 -8.73 -3.96 11.37 -5.71

Robinetinidin -5.66 70.96 -8.30 -3.45 34.93 -5.42

Robinetinidol -5.02 231.24 -7.52 -3.04 118.29 -4.91

Rosinidin -5.08 170.15 -7.69 -3.10 93.23 -5.04

Rutin -6.51 23.27 -8.76 -4.03 9.23 -5.72

Sakuranetin -4.96 255.46 -7.47 -3.02 129.54 -4.89

Salvigenin -4.84 287.68 -7.33 -2.95 136.52 -4.80

Santal -5.08 170.36 -7.69 -3.10 93.81 -5.00

Santin -5.28 124.36 -7.82 -3.20 70.46 -5.10

Table 1: In silico molecular interactions scores of the flavonoids against AChE and BuChE (Cont.)

Acetylcholinesterase Butyrylcholinesterase

Flavonoids Binding Inhibition Intermolecular Binding Inhibition Intermolecular Energy constant Energy Energy constant Energy (kcal/mol) (µM) (kcal/mol) (kcal/mol) (µM) (kcal/mol)

Scopoletin -6.95 8.37 -8.94 -4.62 1.29 -6.23

Scutellarein -5.72 69.87 -8.38 -3.49 32.96 -5.48

Scutevulin -5.12 165.31 -7.72 -3.12 79.76 -5.05

Selagin -4.96 253.31 -7.45 -3.02 127.68 -4.88

Silibinin -5.66 70.92 -7.45 -3.46 34.07 -5.44

Silybin -5.74 69.48 -8.40 -3.51 32.75 -5.50

Sinensetin -5.04 218.36 -7.60 -3.08 110.74 -4.95

Skullcapflavone Ii -4.75 306.62 -7.26 -2.90 145.79 -4.73

Sorbifolin -4.37 358.98 -6.64 -2.68 169.77 -4.34

Sternbin -4.26 736.24 -6.32 -2.60 342.78 -4.14

Sterubin -4.42 349.36 -6.75 -2.69 164.49 -4.42

Table 1: In silico molecular interactions scores of the flavonoids against AChE and BuChE (Cont.)

Acetylcholinesterase Butyrylcholinesterase

Flavonoids Binding Inhibition Intermolecular Binding Inhibition Intermolecular Energy constant Energy Energy constant Energy (kcal/mol) (µM) (kcal/mol) (kcal/mol) (µM) (kcal/mol)

Syringetin -4.36 365.16 -6.59 -2.66 186.93 -4.30

Tamarixetin -4.68 317.48 -7.15 -2.85 150.76 -4.67

Tambulin -4.74 308.32 -7.24 -2.89 146.19 -4.72

Tanetin -4.58 331.49 -6.95 -2.79 156.66 -4.54

Tangeritin -6.24 56.62 -8.59 -3.82 26.19 -5.63

Taxifolin -5.22 150.32 -7.01 -3.17 75.36 -5.08

Tectorigenin -4.21 723.14 -6.18 -2.57 366.63 -4.02

Tephrosin -4.92 269.38 -7.42 -2.98 132.63 -4.86

Teracacidin -4.72 311.02 -7.20 -2.88 147.96 -4.70

Ternatin -4.88 277.42 -7.40 -2.98 133.27 -4.85

Tetra Hydroflavone -6.38 37.08 -8.70 -3.89 18.26 -5.70

100

Table 1: In silico molecular interactions scores of the flavonoids against AChE and BuChE (Cont.)

Acetylcholinesterase Butyrylcholinesterase

Flavonoids Binding Inhibition Intermolecular Binding Inhibition Intermolecular Energy constant Energy Energy constant Energy (kcal/mol) (µM) (kcal/mol) (kcal/mol) (µM) (kcal/mol)

Texasin -5.34 96.64 -7.94 -3.26 50.68 -5.19

Theaflavin -5.88 66.25 -8.52 -3.59 31.81 -5.58

Theaflavin-3-Gallate -5.58 78.62 -8.20 -3.42 37.05 -5.38

Thunberginol A -4.27 738.41 -6.36 -2.61 345.11 -4.15

Tomentin -4.21 723.46 -6.18 -2.57 365.69 -3.96

Topazolin -4.38 350.08 -6.68 -2.68 168.27 -4.36

Tricetin -4.27 739.02 -6.06 -2.61 344.57 -4.16

Tricin -5.36 92.42 -7.96 -3.28 46.33 -5.21

Veronicoside -4.25 732.41 -6.31 -2.60 342.68 -4.14

Viscidulin Ii -5.46 83.16 -8.10 -3.35 39.39 -5.32

Vitexin -5.96 63.72 -8.54 -3.61 30.87 -5.59

101

Table 1: In silico molecular interactions scores of the flavonoids against AChE and BuChE (Cont.)

Acetylcholinesterase Butyrylcholinesterase

Flavonoids Binding Inhibition Intermolecular Binding Inhibition Intermolecular Energy constant Energy Energy constant Energy (kcal/mol) (µM) (kcal/mol) (kcal/mol) (µM) (kcal/mol)

Vitexycarpin -6.01 61.15 -8.54 -3.61 31.05 -5.59

Wogonin -6.86 9.48 -8.87 -4.25 4.81 -5.73

Wogonoside -5.54 80.98 -8.17 -3.39 38.03 -5.36

Xanthohumol -5.21 152.82 -7.75 -3.17 75.15 -5.07

Donepezil -3.87 994.14 -5.46 -3.47 33.26 -5.45

102

Fig. 23 Molecular interactions of scopoletin and donepezil against AChE

In silico studies with BuChE revealed that all the flavonoids showed good binding energy ranging between -4.62 to -2.18 kcal/mol. The scopoletin binding energy was found to be -4.62 kcal/mol as compared to donepezil (standard) -1.25 kcal/mol (Fig. 24). In case of BuChE target, inhibition constant of the flavonoids were found to be 1.29 µM to 508.27 µM when compared to the standard (231.18 µM). The results of inhibition constant coincide with the binding energy scores (Table 1).

Fig. 24 Binding energy of the flavonoids against BuChE

The flavonoid molecules having 2D structure were converted to energy minimized 3D structures and were further used for in silico protein–ligand docking. The active pocket of the BuChE for the scopoletin was found to be His 77, Lys 131, Gln 279, Val 204, Ile 272, Ser 287, Thr 346, Pro 449, Arg 515 and for donepezil were His 77, Pro 102, Lys 131, Val 204, Ser 287, Thr 346 (Fig. 25).

103

Fig. 25 Molecular interactions of scopoletin and donepezil against BuChE

These amino acid residues were dynamically involved in the formation of hydrophilic and hydrophobic contacts and they were initiated to play an important role in the binding site of target. This proves that the flavonoids possess potential ChE inhibitory binding sites when compared to the standard. These molecular docking analyses could lead to the further development of potent acetylcholinesterase inhibitors for the treatment of AD.

The in silico ChE (AChE and BuChE) inhibitory activities of the 245 flavonoid compounds decreased in the order of Scopoletin > Baicalein > Morin > Wogonin > Naringenin > Hesperetin > Rutin > Robinetin > Peonidin > Tetra Hydroflavone > Myricetin > Apigenin > Kaempferol > Chrysin > Hibiscetin > Tangeritin > Isorhamnetin > Herbacetin > Diosmin > Diosmetin > Vitexycarpin > Vitexin > Theaflavin > Naringin > Dihydrobaicalein > Eriodictyol > Gartanin > Hirsutidin > Cladrin > Epicatechin Gallate > Eupatorin > Epicatechin > Hispudulin > Silybin > Gardenin B > Scutellarein > Cirsimaritin > Bis Hydroxy Coumarin > Dorsilurin-F > Eupatolitin > Silibinin > Robinetinidin > Natsudaidain > Ayanin > Cirsilineol > Ganhuangenin > Eupatilin > Europetin > Theaflavin-3-Gallate > Desmethoxycentaureidin > Dihydrorobinetin > Eriocitrin > Aureusidin > Buceracidin- B > Wogonoside > Eupalitin > Europinidin > Gallocatechin Gallate > Homoeriodictyol > Viscidulin II > Aurantinidin > Bracteatin > Chrysosplenetin > Negletein > Farobin-A > Aromadedrin > Tricin > Catechin > Baptigenin > Texasin > Homoorientin > Ranupetin > Apometzgerin > Datin > Dihydromyricetin > Epigallocatechin Gallate >

104

Fisetin > Hesperedin > Santin > Afzelechin > Epigallocatechin > Taxifolin > Xanthohumol > Fisetinidol > Leucocyanidin > Nepetin > Angoletin > Scutevulin > Eupafolin > Kaempferide > Rosinidin > Santal > Retusin > Flavoxate > Candidone > Sinensetin > Daidzein > Dihydrokaempferide > Isosakuranetin > Kumatakenin > Robinetinidol > Isoscutellarein > Rhamnetin > Isothymusin > Sakuranetin > Selagin > Formononetin > Butein > Jaceidin > Leucorobinetinidin > Tephrosin > Gomphrenol > Ternatin > Annulatin > Gericudranin-B > Isoetin > Nobiletin > Acacatechin > Gossypetin > Isosilybin > Salvigenin > Fustin > Leucopeonidin > Rhamnazin > Okanin > Isalpinin > Cannflavin > Cyclocommunol > Dihydroflavonol > Glycitein > Leucopelargonidin > Skullcapflavone II > Tambulin > Nodifloretin > Teracacidin > Platanetin > Galangin > Casticin > Pachypodol > Tamarixetin > Calycosin > Norwogonin > Demethylbellidifolin > Leukoefdin > Quercetagetin > Hydrangenol > Petunidin > Gallocatechin > Chrysoeriol > Pulchellidin > Tanetin > Acacetin > Dihydrotricetin > Glabridin > Rhynchosin > Isopongaflavone > Persicogenin > Capensinidin > Cyanidin > Isookanin > Isoxanthohumol > Pinoquercetin > Hypolaetin > Leucodelphinidin > Luteoforol > Melanoxetin > Laricitrin > Leucoanthocyanidin > Liquiritigenin > Sterubin > Esculetin > Glaziovianin-A > Irigenin > Leucofisetinidin > Meciadanol > Norathyriol > Osajin > Topazolin > Ermanin > Mesquitol > Sorbifolin > Biochanin A > Ladanein > Limocitrol > Rhusflavanone > Syringetin > Luteolin > Dihydrowogonin > Pratol > Mearnsitrin > Genistein > Isoquercitrin > Isovitexin > Orobol > Pelargonidin > Pomiferin > Limocitrin > Icaritin > Calycopterin > Helmon > Licoflavonol > Patuletin > Propolin B > Tricetin > Thunberginol A > Matteucinol > Persicarin > Sternbin > Veronicoside > Azaleatin > Datiscetin > Leucomalvidin > Maritimetin > Ononin > Carajuflavone > Combretol > Isokaempferide > Luteolinidin > Geraldone > Melacacidin > Quercetagetinidin > Tectorigenin > Tomentin > Oroxylin-A > Penduletin > Pratensein > Cirsililol > Malvidin > Ombuin > Isoimperatorin > Genistin > Guibourtinidol > Leptosidin > Maesopsin > Orientin > Prunin > Bavachinin > Pectolinarigenin > Pedalitin > Primetin > Carpachromene > Delphinidin > Gnetifolin E > Prosogerin E > Prunetin > Donepezil.

6.3 In vitro AChE and BuChE inhibitory assay

105 inhibitors are commonly employed in the treatment of AD. Inhibition of AChE results in a decreased breakdown of acetylcholine into acetate and choline, when measured spectroscopically. Out of 245 flavonoids taken for in silico docking studies, 20 flavonoids were selected for the in vitro enzyme inhibitory assay. The selection of the flavonoids was based upon the predicted docked score and the compounds were divided conventionally into those of high, moderate and low neuroprotective activity against the targets.

Hence, to prove the reliability of in silico studies the fourteen flavonoids with highest activity (Scopoletin > Baicalein > Morin > Diosmin > Naringenin > Catechin > Hesperetin > Silibinin > Rutin > Robinetin > Taxifolin > Tetra hydroflavone > Hesperidin > Myrecetin), three with moderate (Apigenin > Kaempferol > Tricetin) and three with lowest (Chrysin > Epigallocatechin > Theaflavin) neuroprotective activities were selected for the in vitro enzyme inhibition studies (Fig. 26).

Fig. 26 Selection of flavonoids for in vitro enzyme inhibition studies

The absorbance of all the selected flavonoids decreased upon increasing the concentrations in a dose dependent manner. The percentage inhibition was calculated for all the flavonoids and for standard. A dose dependent increase in percentage of inhibition was observed for standard.

6.3.1 Role of flavonoids in AChE inhibitory activity

Among the different flavonoids tested, scopoletin and baicalein exhibited the highest AChE inhibitory activity. The inhibition constant (IC50) of scopoletin was

106

Values are expressed as mean ± SEM; n = 6.

*P <0.05 considered as significant (One way ANOVA followed by Dunnett’s test).

Fig. 27 In vitro AChE enzyme inhibitory assay of the selected flavonoids

107

Values are expressed as mean ± SEM; n = 6. *P <0.05 considered as significant (One way ANOVA followed by Dunnett’s test). Fig. 28 In vitro BuChE enzyme inhibitory assay of the selected flavonoids

108 found to be 10.18 ± 0.68 µg/ml and for baicalein 10.92 ± 0.38 µg/ml when compared to the standard donepezil showed 22.14 ± 0.56 µg/ml. All the selected flavonoids showed

AChE inhibitory activity at a concentration below 100 µg/ml, with IC50 values ranging from 10.18 to 21.48 µg/ml (Fig. 27).

6.3.2 Role of flavonoids in BuChE inhibitory activity

The inhibitory activity of the flavonoids against BuChE was determined by Ellman’s method with some modifications. This method estimates BuChE using s- butyrylythiocholine iodide (substrate) and DTNB. The enzymatic activity was measured by the yellow color compound produced by thiocholine when it reacts with dithiobisnitro benzoate ion. The inhibitory activity of the flavonoids increased with increasing concentration. All the flavonoids selected showed BuChE inhibitory activity at a concentration below 100 µg/ml, with IC50 values ranging from 28.18 to 6.12 µg/ml.

Noteworthy IC50 values were found for scopoletin and baicalein against BuChE.

Scopoletin showed the IC50 value 28.18 ± 0.92 µg/ml and for baicalein 31.24 ± 1.12 µg/ml against BuChE when compared to that of donepezil was found to be 64.38± 0.42 µg/ml (Fig. 28).

The order of potency of flavonoids to ChE (AChE and BuChE) inhibitory activities of the compounds decreased in the order of Scopoletin > Baicalein > Morin > Diosmin > Naringenin > Catechin > Hesperetin > Silibinin > Rutin > Robinetin > Taxifolin > Tetra hydroflavone > Hesperidin > Myrecetin > Apigenin, Kaempferol > Tricetin > Chrysin > Epigallocatechin > Theaflavin > Donepezil.

Among the selected flavonoids, scopoletin and baicalein showed excellent inhibition constant (IC50) values against AChE and BuChE and thereby proved that these compounds possess potential cholinesterase inhibitory activity. The inhibitory activity of scopoletin and baicalein against cholinesterase enzymes were found to be greater than the standard, donepezil (22±0.56 µg/ml). Hence, the compounds scopoletin and baicalein were further selected for the in vivo studies to prove its neuroprotective activity against scopolamine and aluminium induced learning impairment in mice.

6.4 Acute toxicity studies and selection of dose for in vivo studies

109 for further in vivo studies. Acute toxicity was determined as per OECD guidelines (420) employing fixed dose procedure for selecting the dose for biological activity. For acute toxicity studies 10 female Wistar rats weighing 150-200 g were used and they were fasted overnight before the experimental day.

The animals were observed no toxic effect and after sufficient interval of time (2-3 days) the second animal was administered with 50 mg/kg dose of test flavonoids. Similar observations were made as before and since the dose was non-toxic the third animal was administered with 300 mg/kg. This dose also did not exhibit any toxic effects or morbidity and therefore the last and highest dose in fixed dose procedure of 2000 mg/kg was administered to fourth animal and no toxicity was noticed for highest dose (Table 2, 3).

Signs and symptoms of toxicity and death if any were observed individually for each rat at 0, 0.5, 1, 2, 3 and 4h for first 24 h and thereafter daily for 14 days. Food was given to the animals after 4 h of dosing. The animals were observed twice daily for 14 days and body weight changes, food and water consumption, etc., were noted. In acute toxicity studies, it was found that the animals were safe up to a maximum dose of 2000 mg/kg of body weight. There were no changes in normal behavioural pattern and no signs and symptoms of toxicity and mortality were observed (Table 4, 5).

As per the OECD 420 guidelines, selected flavonoids can be included in the category 5 or unclassified category of globally harmonized classification system (GHS). Hence, based on these results, the flavonoids baicalein and scopoletin were considered non-toxic and 1/10th and 1/20th of the maximum tolerated (2000 mg/kg) dose was used for the biological evaluation (memory enhancing activity) and thus the studies were conducted at dose levels of 100 and 200 mg/kg body weight.

110

Table 2: Observations done for the acute oral toxicity study- Baicalein

Day Day Day Day Day Day Day Parameters observed 0 h 0.5h 1 h 2 h 4 h 2&3 4&5 6&7 8&9 10&11 12&13 14 Dyspnea ------Respiratory Apnea ------Nostril discharges ------Tremor ------Hyper activity ------Hypo activity ------Motor activity Ataxia ------Jumping ------Catalepsy ------Locomotor activity ------Corneal reflex ------Reflexes Pinna reflex ------Righting reflex ------Convulsion Tonic and clonic convulsion ------Hypertonia ------Muscle tone Hypotonia ------Lacrimation ------Miosis ------Ocular sign Mydriasis ------Ptosis ------Edema ------Skin Skin and fur ------Erythema ------Bradycardia ------Cardiovascular signs Tachycardia ------Contraction of erectile tissue of ------Piloerection hair ------Gastro intestinal signs Diarrhoea ------

111

Live phase animals Observations 1. Body weight every day Normal 2. Food consumption daily Normal 3. Water consumption daily Normal 4. Home cage activity Normal

Note: Acute toxicity study of baicalein and scopoletin was performed and it was non-toxic up to 2000 mg/kg dose

112

Table 3: Mortality record for baicalein in acute oral toxicity study Sighting study Main study (2000 mg/kg) Dose 5 mg/kg 50 mg/kg 300 mg/kg 2000 mg/kg No. of animals 1 2 3 4 5 6 7 8 9 Body weight (g) 170 174 168 172 172 196 186 170 184 Sex Female Female Female Female Female Female Female Female Female 30 min Nil Nil Nil Nil Nil Nil Nil Nil Nil 1 h Nil Nil Nil Nil Nil Nil Nil Nil Nil 2 h Nil Nil Nil Nil Nil Nil Nil Nil Nil 3 h Nil Nil Nil Nil Nil Nil Nil Nil Nil 4 h Nil Nil Nil Nil Nil Nil Nil Nil Nil Day 1 Nil Nil Nil Nil Nil Nil Nil Nil Nil Day 2 Nil Nil Nil Nil Nil Nil Nil Nil Nil Day 3 Nil Nil Nil Nil Nil Nil Nil Nil Nil Day 4 Nil Nil Nil Nil Nil Nil Nil Nil Nil Day 5 Nil Nil Nil Nil Nil Nil Nil Nil Nil Day 6 Nil Nil Nil Nil Nil Nil Nil Nil Nil Day 7 Nil Nil Nil Nil Nil Nil Nil Nil Nil Day 8 Nil Nil Nil Nil Nil Nil Nil Nil Nil Day 9 Nil Nil Nil Nil Nil Nil Nil Nil Nil Day 10 Nil Nil Nil Nil Nil Nil Nil Nil Nil Day 11 Nil Nil Nil Nil Nil Nil Nil Nil Nil Day 12 Nil Nil Nil Nil Nil Nil Nil Nil Nil Day 13 Nil Nil Nil Nil Nil Nil Nil Nil Nil Day 14 Nil Nil Nil Nil Nil Nil Nil Nil Nil Mortality 0/1 0/1 0/1 0/1 0/5 Note: Acute toxicity study of baicalein compound was performed and it was non-toxic up to 2000 mg/kg dose

113

Table 4: Observations done for the acute oral toxicity study- Scopoletin

Day Day Day Day Day Day Parameters observed 0 h 0.5h 1 h 2 h 4 h Day 14 2&3 4&5 6&7 8&9 10&11 12&13 Dyspnea ------Respiratory Apnea ------Nostril discharges ------Tremor ------Hyper activity ------Hypo activity ------Motor activity Ataxia ------Jumping ------Catalepsy ------Locomotor activity ------Corneal reflex ------Reflexes Pinna reflex ------Righting reflex ------Convulsion Tonic and clonic convulsion ------Hypertonia ------Muscle tone Hypotonia ------Lacrimation ------Miosis ------Ocular sign Mydriasis ------Ptosis ------Edema ------Skin Skin and fur ------Erythema ------Bradycardia ------Cardiovascular signs Tachycardia ------Contraction of erectile tissue ------Piloerection of hair ------Gastro intestinal signs Diarrhoea ------

114

Live phase animals Observations 1. Body weight every day Normal 2. Food consumption daily Normal 3. Water consumption daily Normal 4. Home cage activity Normal

Note: Acute toxicity study of baicalein and scopoletin was performed and it was non-toxic up to 2000 mg/kg dose

115

Table 5: Mortality record for scopoletin in acute oral toxicity study Sighting study Main study (2000 mg/kg) Dose 5 mg/kg 50 mg/kg 300 mg/kg 2000 mg/kg No. of animals 1 2 3 4 5 6 7 8 9 Body weight (g) 184 166 182 174 196 168 172 196 183 Sex Female Female Female Female Female Female Female Female Female 30 min Nil Nil Nil Nil Nil Nil Nil Nil Nil 1 h Nil Nil Nil Nil Nil Nil Nil Nil Nil 2 h Nil Nil Nil Nil Nil Nil Nil Nil Nil 3 h Nil Nil Nil Nil Nil Nil Nil Nil Nil 4 h Nil Nil Nil Nil Nil Nil Nil Nil Nil Day 1 Nil Nil Nil Nil Nil Nil Nil Nil Nil Day 2 Nil Nil Nil Nil Nil Nil Nil Nil Nil Day 3 Nil Nil Nil Nil Nil Nil Nil Nil Nil Day 4 Nil Nil Nil Nil Nil Nil Nil Nil Nil Day 5 Nil Nil Nil Nil Nil Nil Nil Nil Nil Day 6 Nil Nil Nil Nil Nil Nil Nil Nil Nil Day 7 Nil Nil Nil Nil Nil Nil Nil Nil Nil Day 8 Nil Nil Nil Nil Nil Nil Nil Nil Nil Day 9 Nil Nil Nil Nil Nil Nil Nil Nil Nil Day 10 Nil Nil Nil Nil Nil Nil Nil Nil Nil Day 11 Nil Nil Nil Nil Nil Nil Nil Nil Nil Day 12 Nil Nil Nil Nil Nil Nil Nil Nil Nil Day 13 Nil Nil Nil Nil Nil Nil Nil Nil Nil Day 14 Nil Nil Nil Nil Nil Nil Nil Nil Nil Mortality 0/1 0/1 0/1 0/1 0/5 Note: Acute toxicity study of scopoletin compound was performed and it was non-toxic up to 2000 mg/kg dose

116

6.5 NEUROPROTECTIVE EFFECTS OF BAICALEIN AND SCOPOLETIN IN SCOPOLAMINE-INDUCED MODEL

6.5.1 Behavioural assessment of baicalein and scopoletin in scopolamine treated mice 6.5.1.1 Effect on transfer latency in the rectangular maze

In this test, baicalein and scopoletin treated mice showed significant (P<0.01) decrease in latency time (LT) when compared to scopolamine treated group on the 7th day. Moreover the higher LT induced by scopolamine was significantly reversed by scopoletin and baicalein treated groups than the standard donepezil treated group. Administration of scopolamine resulted in learning impairment as evidenced by a significant (P<0.05) increase in the LT from 124.45±1.15 on the 1st day to 174.88±0.76 on the 7th day in the negative control group when compared to the control group (120.34±1.18 on 1st day and 118.34±0.68 on the 7th day). Treatment with the scopoletin and baicalein for seven days reversed the LT significantly (P<0.01) when compared with negative control group (Fig. 29). The potency of the reduction in LT was produced by the scopoletin 200mg/kg followed by baicalein 200 mg/kg among the flavonoids treated groups (77.92±0.63 sec, 82.35±0.78 sec on the 7th day). Donepezil, at a dose of 10 mg/kg treated for successive 7 days acts as positive control, elicited a significant (P<0.01) reduction in LT from 122.44±1.05 sec to 92.81±0.67 sec compared to the negative control group using Dunnet’s test.

6.5.1.2 Effect on escape latency time in the Morris water maze

The Morris water maze (MWM) plays an important role in the validation of rodent models for neurocognitive disorders such as Alzheimer’s disease. In MWM, escape latency time (ETL) was calculated, i.e., the time taken by an animal to find the platform. The scopolamine administration group showed increased ELT from 64.53±0.94 sec to 86.78±1.14 sec, whereas the control group was found to be 56.35±0.84. This relative increase in ELT when compared to the control group indicated a spatial memory deficit in the scopolamine treated mice. It was found that baicalein and scopoletin treated mice significantly (P<0.01) reduced ELT when compared with the scopolamine treated group.

117

Fig. 29 Effect of baicalein and scopoletin on latency time using rectangular maze test in scopolamine-induced amnesia

118

Fig. 30 Effect of baicalein and scopoletin on escape latency time using Morris water maze test in scopolamine-induced amnesia

119

In the treatment groups, higher dose of scopoletin and baicalein successfully reversed the scopolamine-induced memory deficits compared to other flavonoids treated groups and standard. The significant reduction of ELT was produced by the scopoletin and baicalein (200 mg/kg) among the flavonoids treated groups on the 7th day was found to be 24.18±0.63 sec, 31.26±1.18. Donepezil (10 mg/kg) treated animals, produced a significant (P<0.01) decrease in ELT 36.42±0.84 sec on the 7th day compared to the negative control group (Fig. 30).

6.5.1.3 Locomotor activity in scopolamine treated mice

Reduction of metabolic rate occurs in rodents in response to intoxication with several neurotoxic agents. Scopolamine decreased locomotor activity as compared to control mice indicating locomotor deficits. Scopolamine-treated group showed lower locomotion values on the 7th day 164.85±0.82 sec when compared to control group 280.46±0.64 sec which in turn replicate the learning impairment in negative control group.

The locomotor activity of the flavonoids treated groups significantly (P<0.01) increased when compared to the negative control group. A significant (P<0.01) increase in the values of locomotion 346.17±1.04 sec on the 7th day was observed in the standard group. In the treatment groups, scopoletin (200 mg/kg and 100 mg/kg) treated mice significantly increased the locomotor values (P<0.01), 380.78±0.82 sec and 368.52±1.02 sec respectively and baicalein (200 mg/kg and 100 mg/kg) significantly elevated locomotor values 356.32±0.78 and 352.43±0.62 on the 7th day compared to the scopolamine-treated group (Fig. 31).

6.5.1.4 Conditioned avoidance response in scopolamine treated mice

In pole climbing method, the percentage of conditioned avoidance response (CAR) were calculated based on the number of successful events and number of errors produced by each groups. Scopolamine treated mice showed significantly decreased percentage of CAR activity 48.21±0.51 sec as compared to control group 74.15±0.58 sec on the 7th day and donepezil treated animals exhibited significant (P<0.05) increase in percentage of CAR activity 88.92±0.38. Scopoletin (100 mg/kg and 200 mg/kg) treated animals significantly (P<0.01) increased CAR activity to 92.15±0.26 and 96.84±0.62 and baicalein (100 mg/kg and 200 mg/kg) significantly (P<0.01) elevated

120

CAR values to 88.08±0.64 and 92.62±0.82 compared to negative control group (Fig. 32).

6.5.1.5 Effect of scopoletin and baicalein response time in 5-choice serial time task method

Five-choice serial reaction time task (5-CSRTT) is an operant based task and was used to study attention and impulse control in rodents. The 5-CSRTT can be used to explore aspects of both attentional and inhibitory control mechanisms. The basic test design involves training animals to respond to a brief visual stimulus presented unpredictably in one of five locations. Increasingly, the test is also used to examine aspects of response control. Increasingly this test is being applied in drug discovery efforts, primarily to identify novel drug treatments for conditions associated with attention deficits.

Administration of scopolamine resulted in learning impairment as evidenced by significant delayed response values (68.24±0.84) when compared to control group (36.18±0.44). In treatment groups, scopoletin (100 mg/kg and 200 mg/kg) showed quicker responses (23.12±1.08 and 18.54±0.72) and baicalein (100 mg/kg and 200 mg/kg) treated animals also showed significant (P<0.01) improved responses time (25.74±0.56 and 22.32±0.44). Standard drug, donepezil administered animals exhibited significantly improved responses (19.42±0.63) (Fig. 33).

These observations made the scopolamine-induced mice as a useful learning impairment model for studying neuroprotective effects of the baicalein and scolpoletin. It has been found that higher dose of scopoletin followed by baicalein treated mice effectively reversed the cognitive impairments produced by the scopolamine when compared to the lower dose of flavonoids treated animals. These results further augmented the neuroprotective effect of the scopoletin against the Alzheimer's disease.

6.5.2 Biochemical estimations in scopolamine induced mice

6.5.2.1 Estimation of AChE activity in the brain

AChE is a key enzyme involved in cholinergic neurotransmission and is a marker of extensive loss of cholinergic neurons in the brain. A significant increase (P<0.01) in the level of AChE was observed in the scopolamine treated mice.

121

Fig. 31 Effect of baicalein and scopoletin on locomotor activity using Actophotometer in scopolamine-induced amnesia

122

Fig. 32 Effect of baicalein and scopoletin on percentage of conditioned avoidance response using Pole climbing test in scopolamine- induced amnesia

123

Administration of baicalein and scopoletin significantly (P<0.01) attenuated AChE levels and no change was observed in control group. Scopoletin (100 mg/kg and 200 mg/kg) exhibited the percentage AChE inhibition in the brain homogenate as 70.48% and 85.22%, which were significantly (P<0.01) increased values when compared to scopolamine treated mice (22.14%). Administration of baicalein (100 mg/kg and 200mg/kg) exhibited the percentage AChE inhibition as 57.18% and 72.14%. Donepezil significantly (P<0.05) reduced the level of AChE into 66.72%. Scopoletin (200 mg/kg) effectively inhibited the AChE and thereby it increased the level of acetylcholine in the brain (Fig. 34).

Fig. 34 Percentage of AChE inhibition in the mice brain in scopolamine-induced amnesia

Values are expressed as mean ± SEM; n = 6.

*P <0.05 when compared to vehicle control; **P <0.01 when compared to negative control (One way ANOVA followed by Dunnett’s test).

6.5.2.2 Determination of total protein content

124

Fig. 33 Effect of baicalein and scopoletin on response time using 5 CSRT model in scopolamine-induced amnesia

125

Treatment with donepezil (5 mg/kg, p.o.) resulted in a significant increased protein content level (1.12±0.96 µmoles/min/mg protein) in the brain (Table 6). The total protein content was greatly increased in higher dose of scopoletin (200 mg/kg) treated group when compared to all other groups.

Table 6: Effect of baicalein and scopoletin on protein content in scopolamine treated mice

Protein Content Groups (mmoles/min/mg)

Control 1.08±1.26 Negative Control (Scopolamine) 0.34±1.32*

Baicalein (100 mg/kg) 1.16±0.84**

Baicalein (200 mg/kg) 1.25±1.23** Scopoletin (100 mg/kg) 1.22±1.08** Scopoletin (200 mg/kg) 1.31±0.82** Standard (Donepezil) 1.12±0.96**

Values are expressed as mean ± SEM; n = 6.

*P <0.05 when compared to vehicle control; **P <0.01 when compared to negative control (One way ANOVA followed by Dunnett’s test).

6.5.2.3 Enzymatic antioxidant levels

Administration of scopolamine caused marked oxidative stress, which was evident by the decrease in the antioxidant enzyme levels marking the occurrence of AD. The role of baicalien and scopoletin in the antioxidant level (CAT, SOD, GSSH, GPx and GSH) in the brain homogenate were estimated.

6.5.2.3.1 Estimation of catalase

A significant (P<0.05) reduction in the catalase level was found in scopolamine treated mice (1.24±0.48 µmoles/min/mg protein) compared to the control group (3.18±0.62 µmoles/min/mg protein). Scopoletin (100 mg/kg and 200 mg/kg, p.o.) treated mice resulted in significant (P<0.01) increase in the level of catalase (3.34±1.08 and 3.46±0.38 µmoles/min/mg protein) and administration of baicalein (100 mg/kg and 200 mg/kg, p.o.) significantly (P<0.01) increased the catalase level to 3.26±0.72 and

126

3.38±0.64 µmoles/min/mg protein when compared to negative control. Treatment with donepezil (5 mg/kg) showed significant (P<0.05) increased in the level of catalase to 3.30±0.52 µmoles/min/mg protein in the brain (Table 7). The catalase level was greatly increased in higher dose of scopoletin treated group animals compared to other groups used in the study.

6.5.2.3.2 Estimation of superoxide dismutase

Superoxide dismutase is an enzymatic antioxidant which scavenges the superoxide anion to hydrogen peroxide, hence diminishing the toxic effect caused by this radical. The SOD level was significantly (P<0.01) reduced to 0.98±1.08 units/mg tissue protein in negative control when compared to control (2.96±0.58 units/mg tissue protein). Scopoletin (100 mg/kg and 200 mg/kg, p.o.) treated group significantly (P<0.01) increased the SOD level to 3.28±0.82 and 3.42±0.74 µmoles/min/mg protein and treatment with baicalein (100 mg/kg and 200 mg/kg, p.o.) significantly (P<0.01) elevated the SOD level to 3.08±0.64 and 3.36±1.12 µmoles/min/mg protein when compared to negative control. Donepezil treated mice (5 mg/kg, p.o.) resulted in a significant (P<0.05) elevation of SOD level (3.28±0.88 µmoles/min/mg protein) in the brain (Table 7). The SOD level was greatly increased in higher dose of scopoletin group treated animals compared to all other groups.

6.5.2.3.3 Estimation of glutathione peroxidase

The GPx level was significantly decreased (P<0.05) in negative control group to 0.42±0.48 when compared to the control group (1.48±0.36) and enzymatic activity of GPx in donepezil treated group showed elevation of GPx level (1.62±0.34). It reveals that in baicalein treated mice (100 and 200 mg/kg) restoration of the activity of the glutathione peroxidase enzyme was noticed to 1.52±0.28 and 1.66±0.32 and scopoletin (100 and 200 mg/kg) treatment elevated GPx values significantly to 1.58±0.40 and 1.76±0.36 respectively and possibly this could be due to reduced generation of free radicals and subsequent neuronal damage (Table 7). This suggests that the higher dose of scopoletin has greater capability to restore level of GPx and reduced formation of free radicals and associated oxidative stress.

127

Table 7: Effect of baicalein and scopoletin on enzymatic and non-enzymatic antioxidant level in scopolamine treated mice

Groups CAT SOD GSSH GPx GSH

Control 3.18±0.62 2.96±0.58 22.45±1.32 1.48±0.36 3.88±0.46

Negative Control 1.24±0.48* 0.98±1.08* 11.31±1.43* 0.42±0.48* 1.28±0.28* (Scopolamine)

Baicalein 3.26±0.72** 3.08±0.64** 21.08±1.61** 1.52±0.28** 3.74±0.24** (100 mg/kg)

Baicalein (200 3.38±0.64** 3.36±1.12** 24.82±1.17** 1.66±0.32** 3.98±0.48** mg/kg)

Scopoletin 3.34±1.08** 3.28±0.82** 25.03±1.72** 1.58±0.40** 4.14±0.54** (100 mg/kg)

Scopoletin 3.46±0.38** 3.42±0.74** 27.21±1.54** 1.76±0.36** 4.32±1.08** (200 mg/kg)

Standard 3.30±0.52** 3.28±0.88** 23.13±1.38** 1.62±0.34** 3.96±0.32** (Donepezil)

Values are expressed as mean ± SEM; n = 6.

*P <0.05 when compared to vehicle control; **P <0.01 when compared to negative control (One way ANOVA followed by Dunnett’s test).

6.5.2.3.4 Estimation of glutathione reductase (GSSH)

The glutathione reductase level was significantly reduced (P<0.05) in negative control group to 11.31±1.43 when compared to control group (22.45±1.32) and enzymatic activity in donepezil resulted in an increased level of glutathione reductase (23.13±1.38). Administration of baicalein (100 and 200 mg/kg) could reverse the level of the glutathione reductase to 21.08±1.61 and 24.82±1.17 and scopoletin (100 and 200 mg/kg) increased GR significantly (P<0.01) to 25.03±1.72 and 27.21±1.54 respectively (Table 7). This suggests that the higher dose of scopoletin has the capability to reverse the level of glutathione reductase.

128

6.5.2.4 Non enzymatic antioxidant activity

6.5.2.4.1 Estimation of reduced glutathione

Glutathione is an important tripeptide which can remove the free radical species · such as H2O2, O2 , alkoxy radical and maintain the membrane protein thiols and acts as a substrate for glutathione peroxidase and glutathione transferase. The level of reduced glutathione in the brain was reduced in the negative control group (1.28±0.28 nmoles/min/mg tissue protein) when compared to the control (1.28±0.28 nmoles/min/mg tissue protein). Treatment with baicalein (100 and 200 mg/kg) resulted in a significant (P<0.01) elevation of reduced glutathione level to 3.74±0.24 and 3.98±0.48 and administration of scopoletin (100 and 200 mg/kg) significantly (P<0.01) elevated the reduced glutathione levels to 4.14±0.54 and 4.32±1.08 respectively. Donepezil (5mg/kg, p.o.) significantly (P<0.01) increased the level (3.96±0.32 nmoles/min/mg tissue protein) of reduced glutathione in the brain (Table 7). The present research depicts that scopoletin (100 mg and 200 mg/kg) reduced oxidative stress by replenishing the levels of catalase, SOD, GPx, and GSH in mice.

6.5.2.5 DPPH assay

DPPH is a stable, nitrogen-centered free radical which produces violet colour. It was reduced to a yellow colored product, diphenylpicryl hydrazine, with the addition of the fractions in a concentration-dependent manner. The reduction in the number of DPPH molecules can be correlated with the number of available hydroxyl groups. The DPPH scavenging activity was significantly decreased (P<0.05) in negative control group to 7.24±1.89 when compared to the control group (12.18±1.58). The highest DPPH radical scavenging activity was detected in the higher concentration of scopoletin (200 mg/kg) 15.48±1.15, followed by the scopoletin 100 mg/kg, baicalein (200 mg/kg, 100mg/kg) showed the values 14.12±0.84, 13.98±1.06 and 12.48±0.81 respectively. These activities were less than that of the standard, donepezil (13.08±0.98) (Table 8). Scopoletin and baicalein treated mice showed significantly (P<0.01) higher DPPH scavenging values compared to negative control group.

129

Table 8: Effect of baicalein and scopoletin on DPPH and MDA level in scopolamine-induced mice

DPPH MDA Groups (nmoles/min/mg) (nmoles/min/mg)

Control 12.18±1.58 5.86±0.24

Negative Control (Scopolamine) 7.24±1.89* 13.88±0.64*

Baicalein (100 mg/kg) 12.48±0.81** 5.63±0.34**

Baicalein (200 mg/kg) 13.98±1.06** 5.48±0.28**

Scopoletin (100 mg/kg) 14.12±0.84** 4.52±0.46**

Scopoletin (200 mg/kg) 15.48±1.15** 4.03±0.38**

Standard (Donepezil) 13.08±0.98** 6.26±0.73**

Values are expressed as mean ± SEM; n = 6.

*P <0.05 when compared to vehicle control; **P <0.01 when compared to negative control (One way ANOVA followed by Dunnett’s test).

6.5.2.6 Estimation of lipid peroxidation

Lipid peroxidation (LPO) is one of the main manifestations of oxidative damage, which implicates free radical oxidative cell injury in mediating the toxicity of scopolamine. Effect of baicalein and scopoletin on the levels of malondialdehyde (MDA) during scopolamine-induced neurotoxicity was observed. Increase in the level of lipid peroxides in the brain reflects the neuronal damage. Elevated level of MDA was observed in scopolamine-treated mice (13.88±0.64 nmoles/min/mg tissue protein), whereas it was significantly (P<0.01) reversed in flavonoids-treated groups (scopoletin and baicalein).

Administration of baicalein (100 and 200 mg/kg) significantly (P<0.01) decreased (5.63±0.34 and 5.48±0.28 nmoles/min/mg tissue protein) MDA level in brain and scopoletin (100 and 200 mg/kg) treated group also reduced the level of MDA significantly to 4.52±0.46 and 4.03±0.38 when compared to the negative control group. Donepezil (5 mg/kg, p.o) significantly (P<0.01) decreased the level (6.26±0.73

130 nmoles/min/mg tissue protein) of MDA in the brain (Table 8). The results revealed that scopoletin and baicalein possess antioxidant property with concentration gradient and thereby it reduces the oxidative stress induced membrane lipid peroxidation.

6.5.3 Histopathological studies of the baicalien and scopoletin in acute scopolamine induced amnesia in mice

The results for the histopathological studies in the brains (H&E x 400) of control group showed normal histological architecture without any degenerative changes and scopolmine induced mice indicated severe neurodegenerative changes and also resulted in the formation of neurofibrillary tangles. Administration of baicalein (100 mg/kg) with scopolamine showed faintly appearing neurofibrillary tangles suggesting moderate therapeutic efficacy of the flavonoids and baicalein (200 mg/kg) resulted in mild infiltration of inflammatory cells indicating the repair mechanism. Treatment with scopoletin (100 mg/kg) showed moderately appearing neurofibrillary tangles representing the minimal efficacy and scopoletin (200 mg/kg) resulted in normal histological architecture with mild infiltration of inflammatory cells which indicates the effective repair mechanism at the high dose of scopoletin. Administration of standard donepezil exhibited minimal neuro degenerative changes indicating neuroprotective effect of the standard drug (Fig. 35).

6.6 NEUROPROTECTIVE EFFECT OF BAICALEIN AND

SCOPOLETIN ON BEHAVIOURAL CHANGES IN AlCl3-INDUCED MODEL

6.6.1 Effect of baicalein and scopoletin on behavioural changes in AlCl3 treated mice

6.6.1.1 Evaluation of transfer latency in the rectangular maze

In this test, latency time (LT) of the animals was calculated for all (Group I to

VII) the groups. Administration of AlCl3 to mice for 3 months resulted in learning impairment by a significant (P<0.05) increase in the LT (175.32±0.98) in the negative control group when compared to the control group (112.21±0.54). Baicalein and scopoletin treated mice resulted in reversal of LT significantly (P<0.01) when compared with negative control.

131

Fig. 35 The demonstration of the effects of baicalein and scopoletin in scopolamine-induced amnesia (SCP-scopolamine)(A-Normal histological architecture without any degenerative changes; B-Severe neurodegenerative changes; C-Formation of neurofibrillary tangles; D- Faintly appearing neurofibrillary tangles; E-Mild infiltration of inflammatory cells indicating the repair mechanism; F-Moderately appearing neurofibrillary tangles; G-normal histological architecture with mild infiltration of inflammatory cells; H-minimal neuro degenerative changes)

132

The LT was effectively decreased by scopoletin (200 mg/kg, 100 mg/kg) treated mice among the all other groups tested showing the values of 46.72±0.38 and 54.48 ±0.82 respectively. Donepezil resulted in significant (P<0.01) decrease in LT 72.54±0.64 compared to the negative control group using Dunnet’s test (Fig. 36).

6.6.1.2 Estimation of escape latency time in the Morris water maze

In MWM, escape latency time (ETL) was calculated, which was the time taken by an animal to find the platform. AlCl3 treated animals showed significantly (P<0.01) increased ETL values on 90th day (76.38±1.18) as compared to control group (49.48±0.72) and it indicates the impairment in learning and memory. Baicalein (100 mg/kg and 200 mg/kg) treated mice significantly (P<0.01) decreased ETL, 31.54±0.84 and 27.06±0.78 and scopoletin (100 mg/kg and 200 mg/kg) significantly (P<0.01) decreased ETL, 30.82±1.02 and 21.42±0.62 when compared to negative control. Administration of donepezil significantly (P<0.01) decreased ETL values (32.18±0.48). Among the treatment groups, scopoletin (200 mg/kg) treated mice effectively reduced ETL values when compared to all other groups (Fig. 37).

6.6.1.3 Locomotor activity in AlCl3 treated mice

AlCl3 treated mice exhibited significantly (P<0.05) decreased locomotor activity on 90th day (176.46±1.38) as compared to control group (286.53±0.82) and it indicates the learning and memory impairment in mice. Baicalein (100 mg/kg and 200 mg/kg) treated mice significantly (P<0.01) elevated locomotor values 386.76±0.46 and 448.54±0.68 and scopoletin (100 mg/kg and 200 mg/kg) significantly (P<0.01) increased the locomotion to 426.34±0.92 and 492.54±1.08 when compared to negative control. Donepezil significantly (P<0.01) elevated the locomotor values (394.38±0.62) on the 90th day. Higher dose of scopoletin resulted in effective increase in locomotor activity which in turns results in its neuroprotective activity (Fig. 38).

133

Fig. 36 Effect of baicalein and scopoletin on latency time using rectangular maze test in AlCl3-induced learning impairment

134

Fig. 37 Effect of baicalein and scopoletin on escape latency time using Morris water maze test in AlCl3-induced learning impairment

135

Fig. 38 Effect of baicalein and scopoletin on locomotor activity in AlCl3-induced learning impairment

136

6.6.1.4 Conditioned avoidance response in AlCl3 treated mice

In pole climbing method, the percentage of conditioned avoidance response (CAR) was calculated based on the number of successful events and number of errors produced by the animals. AlCl3 alone treated mice showed significantly (P<0.05) decreased percentage of CAR activity on 90th day (32.82±1.18) as compared to control group (66.28±0.76). Administration of baicalein (100 mg/kg and 200 mg/kg) resulted in significant (P<0.01) elevation of CAR values 89.52±0.74 and 93.43±0.46 and scopoletin (100 mg/kg and 200 mg/kg) significantly (P<0.01) increased the values to 91.08±0.52 and 96.62±0.78 when compared to negative control. Donepezil treated mice significantly (P<0.01) increased percentage of CAR activity (84.45±0.82) compared to negative control group (Fig. 39).

6.6.1.5 Effect of baicalein and scopoletin on response time in 5-choice serial reaction time task method

5-CSRT is an operant based task used to study attention and impulse control in rodents. AlCl3 treated mice showed significantly (P<0.05) quicker response time (72.86±0.98) on the 91st day when compared to control group (35.24±0.68). Treatment with baicalein (100 mg/kg and 200 mg/kg) significantly (P<0.01) faster response time 22.92±0.58 and 18.26±0.46 and scopoletin (100 mg/kg and 200 mg/kg) significantly (P<0.01) decreased the learning impairment values to 21.38±0.68 and 15.72±0.74 when compared to negative control. Donepezil treated mice significantly (P<0.01) decreased memory impairment to 19.56±0.76.

From the behavioural assessments it has been evidenced that, the chronic administration of AlCl3 (3 months) resulted in the learning impairment in mice and the flavonoids (baicalein and scopoletin) treated mice significantly (P<0.01) reversed the learning impairment produced by AlCl3 (Fig. 40). Scopoletin (200 mg/kg) treated mice, effectively attenuated the learning impairment produced by AlCl3 when compared to all other groups and this further emphasized the neuroprotective role of scopoletin.

137

Fig. 39 Effect of baicalein and scopoletin on percentage of conditioned avoidance response in AlCl3-induced learning impairment

138

Fig. 40 Effect of baicalein and scopoletin on response time using 5-CSRT model in AlCl3-induced learning impairment

139

6.6.2 Biochemical evaluations in AlCl3-induced learning impairment in mice

6.6.2.1 Estimation of AChE level in the brain

AChE is an essential enzyme involved in cholinergic neurotransmission. Acetylcholine is broken into acetate and choline in the presence of AChE and thereby it act as a marker of extensive loss of cholinergic neurons in the brain. In the present study, a significant increase in AChE level was observed in the AlCl3 treated mice (Negative control). The percentage of the AChE inhibition in the brain homogenate was calculated for all the groups.

The percentage of the AChE inhibition of the scopoletin (100 mg/kg and 200 mg/kg) was found to be 68.25% and 78.41% and the values compared with AlCl3 alone treated group was found to be only 18.08% on the 91st day. The baicalein (100 mg/kg and 200 mg/kg) administered mice resulted in elevation of percentage of AChE inhibition 53.21% and 68.48%. Donepezil (5 mg/kg, p.o.) increased the percentage of AChE inhibition to 58.38% (Fig. 41). These results clearly demonstrated that, scopoletin (200 mg/kg) effectively inhibited the AChE level and thereby it prevents the breakdown of acetylcholine.

Fig. 41 Percentage of AChE inhibition in the mice brain in AlCl3-induced learning impairment

Values are expressed as mean ± SEM; n = 6.

*P <0.05 when compared to vehicle control; **P <0.01 when compared to negative control (One way ANOVA followed by Dunnett’s test).

140

6.6.2.2 Estimation of total protein content

The total protein content was significantly (P<0.05) reduced to 0.28±1.24 units/mg tissue protein in negative control group compared to control (1.24±0.81 units/mg tissue protein). Administration of baicalein (100 mg/kg and 200 mg/kg) significantly (P<0.01) reversed the protein content values (1.21±1.12 and 1.25±0.84 µmoles/min/mg protein) when compared to negative control and treatment with scopoletin (100 mg/kg and 200 mg/kg) significantly (P<0.01) increased the values (1.28±0.94 and 1.34±1.07 µmoles/min/mg protein) when compared to negative control. Donepezil (5 mg/kg, p.o.) increased the total protein level (1.26±1.04 µmoles/min/mg protein) (Table 9). The total protein content was greatly increased in higher dose of both scopoletin and baicalein (200 mg/kg) group compared to all other groups.

Table 9: Effect of baicalein and scopoletin on protein content in AlCl3 treated mice

Protein Content Groups (mmoles/min/mg) Control 1.24±0.81

Negative Control (AlCl3 50 mg/kg) 0.28±1.24* Baicalein (100 mg/kg) 1.21±1.12** Baicalein (200 mg/kg) 1.25±0.84** Scopoletin (100 mg/kg) 1.28±0.94** Scopoletin (200 mg/kg) 1.34±1.07** Standard (Donepezil) 1.26±1.04**

Values are expressed as mean ± SEM; n = 6.

*P <0.05 when compared to vehicle control; **P <0.01 when compared to negative control (One way ANOVA followed by Dunnett’s test).

6.6.3 Measurement of enzymatic antioxidant levels

6.6.3.1 Estimation of catalase level

Chronic administration of AlCl3 caused marked oxidative stress, which led to decrease in the antioxidant enzyme levels. In the present study, a significant (P<0.05) decrease in the catalase level was found in AlCl3 treated group (0.98±0.34 µmoles/min/mg protein) compared to the control (3.58±0.46 µmoles/min/mg protein).

141

Treatment with baicalein (100 mg/kg and 200 mg/kg) resulted in significant (P<0.01) increase in the catalase level (3.66±0.38 and 3.78±0.42 µmoles/min/mg protein) and scopoletin (100 mg/kg and 200 mg/kg) significantly (P<0.01) reversed the catalase level (3.76±0.66 and 3.88±0.42 µmoles/min/mg protein). Donepezil (5 mg/kg) increased the catalase level (3.72±0.44 µmoles/min/mg protein) compared to the negative control group (Table 10).

6.6.3.2 Evaluation of superoxide dismutase

Table 10: Effect of baicalein and scopoletin on enzymatic and non-

enzymatic antioxidant level in AlCl3-induced mice

Groups CAT SOD GPx GSSH GSH

Control 3.58±0.46 2.74±0.38 1.82±0.42 25.38±0.94 3.24±0.36

Negative Control 0.98±0.34* 0.62±0.74* 0.58±0.24* 16.25±1.31* 0.94±0.48*

(AlCl3)

Baicalein 3.66±0.38** 2.78±0.34** 1.90±0.48** 24.16±0.88** 3.34±0.44** (100 mg/kg)

Baicalein 3.78±0.42** 2.94±0.64** 2.08±0.28** 26.58±0.97** 3.48±0.38** (200 mg/kg)

Scopoletin 3.76±0.66** 2.92±1.02** 2.02±0.36** 26.48±1.08** 3.40±0.42** (100 mg/kg)

Scopoletin 3.88±0.42** 3.12±0.48** 2.28±0.62** 28.35±0.94** 3.64±0.72** (200 mg/kg)

Standard 3.72±0.44** 2.88±0.68** 1.96±0.52** 26.18±1.24** 3.42±0.38** (Donepezil)

Values are expressed as mean ± SEM; n = 6.

*P <0.05 when compared to vehicle control; **P <0.01 when compared to negative control (One way ANOVA followed by Dunnett’s test).

142

Superoxide dismutase scavenges the superoxide anion to hydrogen peroxide, hence diminishing the toxic effect caused by the radicals. The SOD level was significantly (P<0.05) decreased to 0.62±0.74 units/mg tissue protein in negative control group compared to control group (2.74±0.38 units/mg tissue protein). Donepezil (5 mg/kg, p.o.) resulted in a elevated SOD level (2.88±0.68 µmoles/min/mg protein) in the brain compared to negative control. In the baicalein (100 mg/kg and 200 mg/kg) treated mice, there was a significant (P<0.01) reversal in the level of SOD to 2.78±0.34 and 2.94±0.64 µmoles/min/mg protein and scopoletin (100 mg/kg and 200 mg/kg) significantly (P<0.01) increased the SOD level (2.92±1.02 and 3.12±0.48 µmoles/min/mg protein) emphasising the antioxidant potential of baicalein and scopoletin (Table 10).

6.6.3.3 Determination of glutathione peroxidase level

AlCl3 treated mice significantly (P<0.05) decreased the GPx level to 0.58±0.24 when compared to the control group (1.82±0.42) and enzymatic activity of GPx in donepezil treated group was found to be 1.96±0.52. Administration with baicalein (100 and 200 mg/kg) significantly (P<0.01) increased the level of GPx (1.90±0.48 and 2.08±0.28) and scopoletin (100 mg/kg and 200 mg/kg) treated animals showed elevated levels of GPx to 2.02±0.36 and 2.28±0.62 when compared to negative control (Table 10). Effective restoration of GPx level and the generation of free radicals and neuronal damage was reduced in the scopoletin (200 mg/kg) treated group.

6.6.3.4 Estimation of glutathione reductase level

The glutathione reductase level was significantly (P<0.01) decreased in negative control group to 16.25±1.31 when compared to the control group (25.38±0.94) and enzymatic activity in donepezil treated group was found to be 26.158±1.24. Baicalein treated (100 and 200 mg/kg) animals significantly (P<0.01) increased the level of glutathione reductase to 1.52±0.28 and 1.66±0.32 and treatment with scopoletin (100 and 200 mg/kg) significantly (P<0.01) elevated the glutathione reductase level (1.58±0.40 and 1.76±0.36) (Table 10). Flavonoids treated animals showed increased level of antioxidant enzyme which in turn results in the improvement of neuroprotective potential of scopoletin and baicalein.

143

6.6.4 Non enzymatic antioxidant assay

6.6.4.1 Determination of reduced glutathione

Glutathione is a tripeptide which can remove the free radical species and maintain the membrane protein thiols and act as a substrate for GPx and glutathione transferase. Reduced glutathione level in brain of the negative control (AlCl3) group was significantly (P<0.05) reduced to 0.94±0.48 nmoles/min/mg tissue protein when compared to the control group. Donepezil (5mg/kg) treated animals significantly increased the reduced glutathione level 3.42±0.38 nmoles/min/mg tissue protein in the brain. Administration with baicalein (100 and 200 mg/kg) showed significantly (P<0.01) increased values 3.34±0.44 and 3.48±0.38 and scopoletin (100 and 200 mg/kg) increased significantly (P<0.01) the levels of reduced glutathione to 3.40±0.42 and 3.64±0.72 (Table 10).

6.6.5 Estimation of DPPH assay

Table 11: Effect of baicalein and scopoletin on DPPH and MDA level in AlCl3 treated mice

Groups MDA (nmoles/min/mg) DPPH (nmoles/min/mg)

Control 0.98±0.36 16.22±0.86

Negative Control (AlCl3) 4.12±0.48* 8.16±1.24*

Baicalein (100 mg/kg) 0.72±0.54** 16.18±1.18**

Baicalein (200 mg/kg) 0.60±1.08** 17.52±0.96**

Scopoletin (100 mg/kg) 0.62±0.36** 18.08±1.08**

Scopoletin (200 mg/kg) 0.54±0.28** 19.24±0.95**

Standard (Donepezil) 0.66±0.62** 17.12±1.28**

Values are expressed as mean ± SEM; n = 6.

*P <0.05 when compared to vehicle control; **P <0.01 when compared to negative control (One way ANOVA followed by Dunnett’s test).

The DPPH scavenging activity was significantly decreased (P<0.05) in negative control group to 8.16±1.24 when compared to the control group (16.22±0.86).

144

Baicalein and scopoletin demonstrated remarkable H-donor activity. The highest DPPH radical scavenging activity was detected in the higher concentration of scopoletin (200 mg/kg) 19.24±0.95, followed by the scopoletin 100 mg/kg, baicalein (200 mg/kg, 100 mg/kg) exhibited lower DPPH scavenging activity compared to sscopoletin treated mice with the values of 18.08±1.08, 17.52±0.96 and 16.18±1.18 respectively. These activities were less than that of the standard, donepezil (17.12±1.28) (Table 11).

6.6.6 Determination of lipid peroxidation level

Increase in the level of lipid peroxides in the brain reflects the neuronal damage and thus leads to AD. Elevated level of MDA was observed in AlCl3-treated mice (4.12±0.48 nmoles/min/mg tissue protein), whereas it was significantly (P<0.05) reversed in flavonoids-treated groups. Donepezil (5 mg/kg, p.o) decreased the MDA level to 0.66±0.62 nmoles/min/mg tissue protein. Baicalein (100 and 200 mg/kg) treated mice showed significantly (P<0.05) decreased MDA level in brain to 0.72±0.54 and 0.60±1.08 nmoles/min/mg tissue protein as compared to the negative control group and scopoletin (100 and 200 mg/kg) significantly (P<0.01) reduced the level of MDA (0.62±0.36 and 0.54±0.28) when compared to negative control group. The results suggested that scopoletin possibly has potent antioxidant property to reduce oxidative stress induced membrane lipid peroxidation (Table 11).

6.6.7 Histopathological studies of the baicalien and scopoletin against chronic

AlCl3 treatment in mice

AlCl3 treated mice brain demonstrated severe degenerative changes and cytoplasmic vacuolation which in turn results in the cognitive impairment when compared to control group with normal histological architecture without any degenerative changes. Administration of donepezil exhibited mild congestion and less degenerative changes indicating the therapeutic efficacy. Mice treated with baicalein at a dose of 100 mg/kg presented moderate degenerative changes with cytoplasmic vacuolation and baicalein at higher dose of 200 mg/kg resulted in the intact histological structure with stray vacuolation and congested vessels. Treatment with scopoletin (100 mg/kg) showed moderate neurodegenerative changes with vacuolation indicating the moderate therapeutic efficacy and high dose of scopoletin (200 mg/kg) resulted in normal histological features with appearance of normal glial cell population which further emphasize the therapeutic efficacy of the scopoletin (Fig. 42).

145

Fig. 42 The demonstration of the effects of baicalein and scopoletin in AlCl3induced AD (A- Normal histological architecture without any degenerative changes; B-Severe neurodegenerative changes; C- Cytoplasmic vacuolation; D- Moderate degenerative changes; E- Cytoplasmic vacuolation; F- Stray vacuolation and congested vessels; G- Moderate neurodegenerative changes with vacuolation; H- Normal glial cell population; I-Mild congestion and less degenerative changes)

146

7. DISCUSSION Drug design is an important tool in the field of medicinal chemistry where new compounds are synthesized by molecular or chemical manipulation of the lead moiety in order to produce highly active compounds with minimum steric effect211. Docking of small molecules in the receptor binding site and estimation of binding affinity of the complex is a vital part of structure based drug design212, 213. A huge breakthrough in the process of drug design was the development of in silico method to predict the therapeutic efficacy of the molecule214. 7.1 Evaluation of drug likeness properties

The pharmacologically active substituents were characterized by calculating steric, hydrophobic, electronic, and hydrogen bonding properties as well as by the drug- likeness score. The theory of drug-likeness score helps to optimize pharmaceutical and pharmacokinetic properties, for example, chemical stability, solubility, distribution profile and bioavailability. The molecular descriptors have developed as rationally predictive and informative, for example, the Lipinski's Rule-of-Five119. The better oral absorption of the ligands and drug likeness scores are constructed by the solubility, diffusion, Log P, molecular weight etc. Before the docking studies, the ligands were evaluated for its drug likeness properties. MedChem Designer software was used to evaluate the Lipinski’s rule of five for the flavonoids215.

In the present study, lead optimization of the selected flavonoids was done by computation of druglikeness properties. Two hundred and forty five flavonoids were evaluated for its drug likeness properties with the help of MedChem Deisgner. All the 245 flavonoids showed excellent drug likeness score which indicate that these compounds have no violations or acceptable violations in Lipinski’s Rule of 5. All the selected flavonoids were exhibiting considerable pharmacokinetic profile and were selected for in silico biological screening against AChE and BuChE using AutoDock 4.2.

7.2 In silico AChE and BuChE inhibition

147 libraries of small molecules to known target structures is an increasingly important component in the drug discovery process217, 218. There is a wide range of software packages available for the conduct of molecular docking simulations like, AutoDock, GOLD, FlexX etc.122, 219. Among these, AutoDock 4.2 is the most recent version which has been widely employed for virtual screening, due to its enhanced docking speed220. Its default search function is based on Lamarckian Genetic Algorithm (LGA), a hybrid genetic algorithm with local optimization that uses a parameterized free-energy scoring function to estimate the binding energy221.

AChE is a key enzyme in the central nervous system and inhibition of it leads to increase of neural acetylcholine levels which is one of the therapies for symptomatic relief of mild to moderate AD 222. Hence the inhibition of AChE is one of the central focuses for drug development to combat AD223.

In the present study, 245 flavonoids were selected for the in silico docking studies. The docking studies were performed by the use of AutoDock4.2. Its principle is based on Monte carlo and Simulated annealing methods in combination with genetic algorithm which is used for the global optimization. In the docking studies, if a compound shows lesser binding energy compared to the standard it proves that the compound has higher activity. The inhibition constant was correlated to half-maximal inhibitory concentration (IC50) at which 50% of the target was inhibited. This quantitative measure indicates the amount of a particular drug needed to inhibit a biological process.

Based upon the chemical structure classification of our selected flavonoids, the AChE inhibitory activity was found to be, Flavones > Coumarin derivatives > Flavonones > Iso flavones > Flavonols. The minimum binding energy indicated that AChE and BuChE were successfully docked with selected flavonoids. These flavonoids showed an excellent binding mode when compared with the standard donepezil against AChE and BuChE.

148 bonds formed by quercetin involve the same amino acid residues Tyr 124, Ser 293 and Tyr 341 as for conventional drugs. Moreover, different atoms of other amino acid residues such as Phe 295, Arg 296, Tyr 337 and Tyr 341 participate to form the hydrogen bonds225.

While binding at the active site, a number of hydrogen bonds between different atoms of AChE and the inhibitors participate to interact with each other to make the binding affinity much stronger. In the present study, the flavonoids showed the similar amino acid residues Tyr 124, Trp 286, Phe 295 and Tyr 341 against AChE as for the standard donepezil. Similarly, in case of BuChE flavonoids showed the binding interactive residues such as His 77, Lys 131, Val 204, Ser 287, Thr 346. This proves that the selected flavonoids possess potential AChE and BuChE inhibitory binding sites when compared to the standard.

The presence of a benzopyran ring in their basic nucleus of the flavonoids would have contributed to its cholinesterase inhibitory activity. From these results, it was proved that all the selected flavonoids showed higher activity than the standard donepezil. This molecular docking analysis may further lead to the development of potent ChE inhibitors for the prevention and treatment of AD.

7.3 In vitro AChE and BuChE inhibition

Alzheimer’s disease (AD), a common type of progressive neurodegenerative disease, is characterized by low level of neurotransmitter (acetylcholine), oxidative stress and neuro-inflammation in brain stream226. Effective treatment strategies rely mostly on either enhancing the cholinergic function of the brain by stimulating the cholinergic receptors, improve the level of acetylcholine from being a breakdown by cholinesterase enzymes or induce antioxidant therapy and anti-inflammatory agents227.

Acetylcholine (ACh) is the main neurotransmitter that is depleted in AD. This ACh plays an important role in learning things and in memory function. ACh is generally stored and released from the nerve terminals. In AD patients’ large amount of AChE and BuChE released in the system that makes the working half-life of ACh is too short. AChE enzyme hydrolyses ester bond of the ACh molecule to make it choline and ester. BuChE enzyme is prominent in glial cells, which have almost same activity on ACh228.

149

Till date the most promising target for the symptomatic treatment and slowing of AD progression is cholinesterase inhibitors. In AD the destruction of cholinergic neurones causes a depletion of ACh229. By inhibiting AChE, the enzyme which catalyses break down of ACh, levels of this neurotransmitter can be elevated and function improved. This approach has a particular success as these cholinergic neurones are found mainly in regions associated with learning and memory - spreading from the basal forebrain230 and hippocampus up to the cerebral cortex231, 232. Therefore, inhibition of AChE and BuChE level is an important strategy to treat AD. The enzyme inhibitory activity of the flavonoids was determined by Ellman’s method. This method based on the reaction between acetylthiocholine iodide or butyrylthiocholine iodide, DTNB, flavonoids and the enzyme solution. The enzymatic activity was measured spectrophotometrically, by the formation of a yellow coloured complex produced by reaction with DTNB ion.

The selected flavonoids exhibited an excellent AChE and BuChE inhibitory activity than the standard. The results indicated that the flavonoids may be a potential source of cholinergic inhibition. In the current study, out of 245 flavonoids taken for in silico docking studies, 20 flavonoids were selected for the in vitro enzyme inhibitory assay. The selection of the flavonoids was based on high, moderate and low neuroprotective activity against the targets. The absorbance of all the selected flavonoids decreased upon increasing concentrations in a dose dependent manner. The in vitro enzymatic activity of the flavonoids exhibited the potential AChE and BuChE inhibitory activity than the standard donepezil. The results validated the in silico docking studies and it further confirms that the selected flavonoids could be a promising remedy for the AD.

7.4 Acute toxicity studies The acute toxicity studies were performed as per the OECD guidelines. Based on the acute toxicity studies, we had selected the two different doses of the selected flavonoids baicalein and scopoletin. The results of acute toxicity studies have revealed that, these compounds were considered non-toxic up to the dose of 2000 mg/kg and therefore 1/10th and 1/20th dose was used for the biological evaluation and the in vivo scopolamine and aluminium induced learning impairment studies were conducted at dose levels of 100 and 200 mg/kg body weight.

150

7.5 Acute scopolamine-induced amnesia Scopolamine is widely utilized as an experimental tool to study the anti-amnesic potential of new drugs233. Scopolamine hydrobromide is an anticholinergic drug, which produces dementia by reducing the levels of acetylcholine, which is considered to be an important neurotransmitter for learning and memory234. From the behavioral tests like, rectangular maze test and Morris water maze test, it is clearly seen that there was a significant decrease in the transfer latency in all the flavonoids treated groups compared to the scopolamine-treated group. The memory loss effect of scopolamine is more prominent compared to the control group. In comparison with Donepezil, the higher dose (200 mg/kg) of scopoletin and baicalein treated groups had excellent performance which indicates neuroprotective effect against scopolamine. Administration of scopoletin and baicalein at a dose of 200 mg/kg/p.o. for 7 days to animals successfully reversed the learning impairment produced by the scopolamine when compared to the lower doses of the selected flavonoids. There was a gradual increase in the locomotor activity and percentage of conditioned avoidance response in the flavonoids treated animals when compared to the standard. Thus, the potential neuroprotective behavioural changes in the flavonoids treated animals further augmented their role in the management of AD. The effect of the selected flavonoids in scopolamine-induced amnesia in mice was compared with donepezil, a known nootropic drug. It was observed that both the flavonoids showed significant improvement in conditional avoidance response (CAR) indicating protective effect of these flavonoids against scopolamine induced damage of memory. Along with behavioural parameters, the effect of flavonoids on the AChE activity, protein estimation enzymatic and non-enzymatic anti-oxidants were also evaluated. The selected flavonoids showed a reduction of the AChE level that was increased after administration of scopolamine. It was reported that a decrease in AChE activity can lead to improvement in learning and memory235. The non-enzymatic production of lipid peroxidation (LPO) is associated mostly with cellular damage as a result of oxidative stress. In this process, malanoldehyde is generated as secondary products. The increased generation of reactive oxygen species (ROS) is implicated in the pathogenesis of many diseases and in the toxicity of a wide range of compounds. Brain tissue is rich in polyunsaturated lipids, has high iron content, and critically dependent on aerobic metabolism and thus highly vulnerable to ROS mediated

151 oxidative damage. There are reports suggesting accumulation of LPO products in the brain of patients with Alzheimer's disease236. Our results also further emphasized the memory protective effect of the flavonoids. Scopoletin and baicalein significantly reduced brain AChE level, indicating the remarkable elevation of ACh level in the brain and thereby the flavonoids might possess anti-ChE activity. The selected flavonoids significantly improved antioxidant levels when compared to the scopolamine treated group. These results further augmented the potential effect of the selected flavonoids against the Alzheimer's disease. Our study revealed that scopoletin and baicalein enhanced the oxygen species markers in most of the brain regions by improving antioxidant enzymes. Administration of scopoletin (200 mg/kg) exhibited the highest activity followed by the scopoletin (100 mg/kg) and baicalein treated groups. It is well known that cholinergic neuronal systems play an important role in the cognitive deficits associated with AD, ageing and neurodegenerative diseases. Flavonoids treated animals showed potential in inhibition of oxidative stress-induced neuronal damage in the neurodegeneration and also in antioxidant assays and thus represents promising neuroprotective activities. Superoxide anion plays an important role in the formation of other reactive oxygen species such as hydrogen peroxide, hydroxyl radical and singlet oxygen in living systems. SOD is considered to be a stress protein which is synthesized in response to oxidative stress237. Glutathione peroxidase catalyses the oxidation of reduced glutathione (GSH) to oxidized glutathione (GSSG) at the expense of hydrogen 238 peroxide . Catalase primarily causes the decomposition of H2O2 to water at a much faster rate, in association with glutathione peroxidase. Thus, a decrease in the activities of both catalase and glutathione peroxidase leads to an accumulation of H2O2. A decrease in the activity of glutathione reductase is due to the depletion of thiol which leads to related pathologies239.

Treatment with scopoletin and baicalein significantly (P<0.01) enhanced the total protein and enzymatic antioxidant levels in the brain. In scopolamine treated mice, significant (P<0.05) reduction in antioxidant enzyme level was observed in the mice brain. Higher dose (200 mg/kg) of scopoletin and baicalein treated mice significantly (P<0.05) increased the level of the enzymatic antioxidant enzymes such as superoxide dismutase, catalase, peroxidase and glutathione reductase, thus showing the neuroprotective beneficial effect against scopolamine treatment.

152

Glutathione is a major non-protein thiol in living organisms and protects the cellular defense system against the toxic effects of lipid peroxidation. Due to oxidative stress, the brain is unable to maintain the GSH redox state and oxidized glutathione (GSSG) is concentrated in some regions of the brain 240. The cellular levels of the active forms of vitamin C and α-tocopherol are also maintained by GSH by neutralizing the free radicals. A decrease in GSH level leads to a decrease in cellular levels of α- tocopherol241. In the present study, a significant decrease in the levels of non-enzymatic antioxidants like GSH was observed in scopoletin and baicalein treated mice. This could be due to an increased utilization of GSH for scavenging the toxic products. Mice treated with higher dose of scopoletin and baicalein significantly restored the levels of GSH in the brain suggesting the involvement of GSH-dependent detoxification of free radical. These observations reveal that scopoletin and baicalein treated groups has enhanced the antioxidant potential.

Increased lipid peroxidation in ethanol treated rats showed in a depletion of the cellular antioxidant defense system. Inhibition of SOD results in the generation of partially reduced oxygen species. When SOD activity is decreased, neurons are more vulnerable to oxyradical injury242. There was a significant elevation in MDA and a decrease in total protein and antioxidant enzymes in scopolamine treated mice (negative control) compared to normal control. These results suggest that scopoletin and baicalein can be used for the treatment of AD attributing to their antioxidant and neuroprotective activities. In the histopathological studies, scopolmine treated mice indicated severe neurodegenerative changes and also resulted in the formation of neurofibrillary tangles and scopoletin (200 mg/kg) administered animals resulted in normal histological architecture with mild infiltration of inflammatory cells which indicates the effective repair mechanism of the high dose of scopoletin.

7.6 Chronic in vivo aluminium chloride-induced dementia

153 neuroprotective effect against AlCl3-induced cell damages in various experimental models181. Our current study explores the role of the selected flavonoids during chronic administration of AlCl3-induced neurotoxicity.

AlCl3 is cholinotoxin, which produces functional alterations in the cholinergic and noradrenergic neurotransmission. AChE is a specific cholinergic indicator has received wide attention in the study of AlCl3 neurotoxicity. AChE is more accountable for acetylcholine metabolism whose modifications caused neurobehavioral fluctuations particularly in memory and cognitive function. AChE catalyzes the hydrolysis of acetylcholine into inactive metabolites, choline and acetate. Numerous literatures have shown that augmented AChE level is a prelude to oxidative stress244. Consistent with the previous discoveries, a substantial elevation in AChE activity was observed in 245 AlCl3 exposure rats, which signifies impaired cholinergic function . Scopoletin and baicalein at a dose of 200 mg/kg/p.o. for 90 days reversed the learning impairment produced by the AlCl3 in mice. Flavonoids treated animals significantly decreased the transfer latency time in rectangular maze test and escape latency time in Morris water maze test which results in improvement of the learning and memory. There was a substantial elevation of locomotor function and percentage of conditioned avoidance response in pole climbing method in scopoletin and baicaelin treated mice. These remarkable behavioural changes in the animals further highlight the role of scopoletin and baicalein in the management of AD. Catalase plays a major role in cellular antioxidant defense system by decomposing H2O2, thereby preventing the generation of hydroxyl radical through the Fenton reaction246. Accumulating evidence suggests that aluminium decreased the level of enzymatic antioxidants in the brain, such as catalase (CAT), superoxide dismutase (SOD) and glutathione (GSH). The effect of Al contact is consistent with the previous studies that result in the decreased levels of CAT and SOD in the brain247. Scopoletin and baicalein treated mice significantly elevated the level of total protein and enzymatic and non-enzymatic antioxidant levels and thus lead to a reduction of neural stress which enhances the synaptic communication. These results further augmented the long term potential effect of the selected flavonoids against the Alzheimer's disease. Our study demonstrated that, the selected flavonoids enhanced the oxygen species markers in most of the brain regions by improving the levels of antioxidant enzymes. Thus result implied that, because of elevated the level of antioxidant enzyme

154 by the selected flavonoids shows the reduction of neural stress, and enhances the synaptic communication. Our results also further emphasize the memory enhancing effect of the flavonoids against chronic administration of aluminium chloride. The selected flavonoids significantly improved antioxidant levels. These results further augmented the long term potential effect of the selected flavonoids against the Alzheimer's disease. In the histopathological studies, negative control group showed severe degenerative changes and cytoplasmic vacuolation results in cognitive impairment. Scopoletin (200 mg/kg) treated group resulted in normal histological features with appearance of normal glial cell population. The short and long-term administration of the scopoletin 200 mg/kg and baicalein 200 mg/kg exhibited prominent effect in the reversal of the both scopolamine and aluminium-induced learning impairment in both behavioural and biochemical estimations.

The long term chronic neuroprotective effect of the scopoletin and baicalein was investigated against aluminium-induced learning impairment in mice. The long term administration of flavonoids (90 days) further improved the neuroprotective activities which further correlates with the results of acute scopolamine model.

155

8. CONCLUSION

Alzheimer’s disease is a neurodegenerative disorder associated with a decline in cognitive abilities. The deficiency of acetylcholine is one of the characteristics of AD and responsible for most of its symptoms, such as a decline in memory and cognition. As a consequence of its role as a target for AD therapy, AChE is one of the most studied target in the Alzheimer’s field and the vast majority of reports in the field relate with treatment strategies associated with the use of AChE inhibitors. Despite the severity and high prevalence of this disease, the allopathic system of medicine is yet to provide a satisfactory drug candidate. Hence, the present study was focused on exploration of the memory enhancing activity of the flavonoids in scopolamine and aluminium-induced learning impairment in mice.

The current study suggests that the flavonoids, baicalein and scopoletin possessed excellent binding interactions and docking score with AChE and BuChE enzymes than the standard (donepezil) in in silico studies. The in silico studies addressed the flavonoids stability against AChE and BuChE (protein-ligand complex) by molecular dynamic simulation. The in silico studies is actually an added advantage to screen the ChE enzyme inhibition. Two hundred and forty five compounds were selected and screened in the computational studies and among these compounds 20 flavonoids have been selected for the in vitro enzyme inhibition studies based on their docking results.

In vitro enzyme inhibition studies also proved that selected 20 flavonoids were effective in inhibiting ChE enzymes, which is considered to be related to the mechanism of memory dysfunction. The results of the in vitro studies were in correlation with the in silico predictions. Among the 20 selected flavonoids, scopoletin and baicalein demonstrated excellent inhibition constant (IC50) values in both computational and experimental studies towards AChE and BuChE and thereby these compounds possess potential ChE inhibitory activity. In the light of these findings, it can be concluded that scopoletin and baicalein showed less IC50 values against both enzyme targets (AChE and BuChE) and could be considered worthwhile to check its in vivo memory enhancing activity to be used in treatment of AD.

156

The results of acute toxicity studies have revealed that, baicalein and scopoletin were considered non-toxic up to the dose of 2000 mg/kg and therefore 1/10th and 1/20th dose was used for the biological evaluation and the in vivo scopolamine and aluminium induced learning impairment studies were conducted at dose levels of 100 and 200 mg/kg body weight.

The findings of acute model with scopolamine induced amnesia model suggested that the higher dose of scopoletin and baicalein (200 mg/kg) treated animals showed excellent neuroprotective activity in all the behavioural assessment studies performed. Administration of scopoletin and baicalein (200 mg/kg/p.o. for 7 days) to animals reversed the learning impairment produced by the scopolamine. Scopoletin and baicalein treated animals effectively decreased the transfer latency time in rectangular maze test, escape latency time in morris water maze test which emphasize the improvement of the learning and memory skills of the animals. There is a gradual increase in the locomotor function in actophotometer and percentage of conditioned avoidance response in pole climbing method was observed in the flavonoids treated animals. These potential neuroprotective behavioural changes in the flavonoids treated animals further proved the role of scopoletin and baicalein in the management of AD.

It is well known that cholinergic neuronal systems play an important role in the cognitive deficits associated with AD, ageing and neurodegenerative diseases. In our study, scopoletin and baicalein significantly reduced brain AChE activity, indicating the stimulating actions of these drugs on the cholinergic system. Hence, the memory- enhancing effect of the scopoletin and baicalein can be attributed to its anti-ChE activity. Flavonoids treated animals showed potential inhibition of oxidative stress- induced neuronal damage in the neurodegeneration and also in antioxidant assays and represent promising neuroprotective activities.

157 as superoxide dismutase, catalase, peroxidase and glutathione reductase. Glutathione which is widely distributed in cells is an intracelluar reductant and plays a major role in catalysis and metabolism. The selected flavonoids significantly (P<0.01) increased the level of non-enzymatic antioxidant GSH whereas in the negative control, the levels were decreased.

In the histopathological studies, the brains of negative control group (scopolamine treated animals) showed severe neurodegenerative changes resulting in formation of neurofibrillary tangles which was reversed in the flavonoid treated groups. Scopoletin (200 mg/kg) treated group resulted in normal histological architecture with mild infiltration of inflammatory cells which indicates the effective repair mechanism at a high dose of scopoletin.

The findings of chronic AlCl3 model suggested that the higher dose (200 mg/kg) of scopoletin and baicalein treated animals showed excellent neuroprotective activity in all the behavioural assessment studies performed. Scopoletin and baicalein (200 mg/kg/p.o. for 90 days) treated animals reversed the learning impairment produced by the AlCl3. Scopoletin and baicalein treated animals significantly decreased the transfer latency time in rectangular maze test and escape latency time in morris water maze test which results in improvement of the learning and memory. There is a substantial elevation of locomotor function and percentage of conditioned avoidance response in pole climbing method was shown in the scopoletin and baicaelin treated mice. These remarkable behavioural changes in the animals further highlight the role of scopoletin and baicalein in the management of AD.

Scopoletin and baicalein significantly reduced brain AChE level, indicating the remarkable elevation of ACh level in the brain and thereby the flavonoids possess anti- ChE activity. Our study revealed that, high dose of scopoletin and baicalein improved antioxidant enzymes in most of the brain regions. Administration of scopoletin (200 mg/kg) exhibited the highest activity followed by the scopoletin (100 mg/kg) and baicalein treated groups.

158 glutathione reductase. Scopoletin and baicalein treated mice significantly elevated the level of total protein and enzymatic and non-enzymatic antioxidant levels and thus lead to reduction of neural stress which enhances the synaptic communication. These results further augmented the long term potential effect of the selected flavonoids against the Alzheimer's disease.

In the histopathological analysis, the brains of negative control group showed severe degenerative changes and cytoplasmic vacuolation which in turn results in the cognitive impairment which was recovered by the flavonoid treated groups so effectively than the standard drug. Scopoletin (200 mg/kg) treated group resulted in normal histological features with appearance of normal glial cell population. The short term (7 days) and long-term (90 days) administration of the scopoletin 200 mg/kg exhibited pronounced effect in the reversal of the both scopolamine and aluminium- induced learning impairment in both behavioural and biochemical estimations.

In conclusion, it has been clearly demonstrated that the approach utilized in this study is successful in finding novel anti-alzhiemer’s drugs. In particular, higher dose of scopoletin and baicalein, showed excellent neuroprotective effects against in silico, in vitro enzyme inhibitory and in vivo acute scopolamine and chronic AlCl3 induced learning impairment models. Hence, scopoletin and baicalein could be considered as a therapeutic agent to prevent or slow down the development of neurodegenerative diseases such as AD. Further research is required to explore the detailed mechanism of action of the scopoletin and baicalein specifically targeting the pathogenetic mechanisms like amyloid deposition and tau hyperphosphorylation which might provide a definite therapeutic edge. Further extensive studies are needed to establish scopoletin and baicalein as potential drug candidates for clinical use in the prevention and treatment of Alzhiemer’s disease.

159

9. IMPACT OF THE STUDY

Alzhiemer’s disease is a progressive neurodegenerative disorder characterized by a gradual decline in memory associated with shrinkage of brain tissue, with localized loss of neurons mainly in the hippocampus and basal forebrain, with diminished level of acetylcholine. It has also reported to be associated with accumulation of beta-amyloid and neurofibrillary tangles in the brain. WHO reports (2015) confirms that 47 million people are suffering from dementia globally and 4.1 million people are living with dementia in India. Based on this alarming situation, the search for the identification of novel drug candidate with lesser toxicity and increased efficacy is essential. Clinical trials allow the health sector people to learn the symptoms and causes of AD and also to test new management strategies. Treatment with AChE inhibitors is one of the important treatment strategies for AD patients. In the management of AD, initiation of an AChE inhibitor is suggested as early as possible. Despite the issues of cost, it is important that AChE inhibitor therapy is deliberated for patients with mild to moderate AD.

The prevalence of AD and dementia has increased significantly nowadays. As none of the available medications appears to be able to reduce the disease progression, there is enormous medical need for the development of novel therapeutic strategies that target the underlying pathogenic mechanisms in AD.

The present study revealed that scopoletin and baicalein potentiates the neuroprotective activity in in silico, in vitro studies and in both acute scopolamine and chronic AlCl3-induced AD in mice. Scopoletin and baicalein possess significant advantages over existing drug candidates and these compounds may possess industrial acceptability. The observed neuroprotective and enhanced cognitive functions could be due to reducing the AChE level and antioxidant activities of the flavonoids in mice brain. Hence, this could be an effective drug of choice in the management of AD. Keeping in mind that scopoletin and baicalein has broad neuroprotective properties as observed in the current study. Its use might slow the progression of the multifactorial Alzhiemer’s disease.

160

Bibliography

1. Colton CA. Heterogeneity of microglial activation in the innate immune response in the brain. J Neuroimmune Pharmacol 2009; 4: 399-418. 2. Garden GA, La Spada AR. Molecular pathogenesis and cellular pathology of spinocerebellar ataxia type 7 neurodegeneration. Cerebellum 2008; 7: 138-49. 3. Rangasamy SB, Soderstrom K, Bakay RA, Kordower JH. Neurotrophic factor therapy for Parkinson’s disease. Prog Brain Res 2006; 184: 237-64. 4. Small GW, Rabins PV, Berry PP. Diagnosis and treatment of Alzheimer's disease and related disorders: consensus statement of the American Association for Geriatric Psychiatry, the Alzheimer's Association, and the American Geriatrics Society. J Am Med Assoc 1997; 278: 1363-71. 5. Gilley DW, Bienias JL, Wilson RS. Influence of behavioural symptoms on rates on institutionalization for person’s with Alzheimer’s disease. Psychol Med 2004; 34: 1129 -35. 6. Urdinguio RG, Sanchez-Mut JV, Esteller M. Epigenetic mechanisms in neurological diseases: genes, syndromes, and therapies. Lancet Neurol 2009; 8(11): 1056-72. 7. Chahrour M, Sung YJ, Shaw C. MeCP2, a key contributor to neurological disease, activates and represses transcription. Science 2008; 320(5880): 1224-9. 8. Mehler MF. Epigenetic principles and mechanisms underlying nervous system functions in health and disease. Prog Neurobiol 2008; 86(4): 305-41. 9. Kurtzke JF. Epidemiology of amyotrophic lateral sclerosis. Adv Neurol 1982; 36: 281– 302. 10. Ross CA, Akimov SS. Human-induced pluripotent stem cells: potential for neurodegenerative diseases. Hum Mol Gen 2014; 23(1): R17–R26. 11. Pirooznia SK, Elefant F. Targeting specific HATs for neurodegenerative disease treatment: translating basic biology to therapeutic possibilities. Front Cell Neurosci 2013; 7(30): 1-10. 12. Prema A, Thenmozhi AJ, Manivasagam T, Essa MM. Fenugreek seed powder nullified aluminium chloride induced memory loss, biochemical changes, Aβ burden and apoptosis via regulating Akt/GSK3β signaling pathway. PLoS ONE 2016; 11(11): e0165955-62. Bibliography 13. Perry NS, Bollen C, Perry EK, Ballard C. Salvia for dementia therapy: review of pharmacological activity and pilot tolerability clinical trial. Pharmacol Biochem Behav 2003; 75(3): 651-9. 14. Nivsarkar M, Banerjee A, Deval S, Jaimic T, Manoj P, Bapu C, et al. Reduction in aluminium induced oxidative stress by meloxicam in rats brain. Iran Biomed J 2006; 10(3): 151-5. 15. Thompson SB. Alzheimers disease: comprehensive review of aetiology, diagnosis, assessment recommendations and treatment. Webmed Central Review Article 2011; 81: 1-42. 16. Azza AA, Ahmed HI, El-Samea HAA, El-Demerdash E. The potential effect of caffeine and nicotine co-administration against aluminum induced Alzheimer’s disease in rats. J Alzheimers Dis Parkinsonism 2016; 6: 236-40. 17. Humpel C. Chronic mild cerebrovascular dysfunction as a cause for Alzheimer's disease? Exp Gerontol 2011; 46: 225-32. 18. Thenmozhi AJ, Raja TRW, Manivasagam T, Janakiraman U. Hesperidin ameliorates cognitive dysfunction, oxidative stress and apoptosis against aluminium chloride induced rat model of Alzheimer's disease. Nutr Neurosci 2016; Article in Press. 19. Alzheimer’s association (2007). Understanding the stages of Alzheimer’s disease: 1-4. 20. Alzheimer’s association (2015). Understanding the stages of Alzheimer’s disease: 1-10. 21. Harrison IF, Dexter DT. Epigenetic targeting of histone deacetylase: therapeutic potential in Parkinson's disease? Pharmacol. Ther 2013; 140(1): 34-52. 22. Kanthasamy A, Jin H, Anantharam V. Emerging neurotoxic mechanisms in environmental factors-induced neurodegeneration. NeuroToxicol 2012; 33(4): 833–7. 23. Jia H, Morris CD, Williams RM, Loring JF, Thomas EA. HDAC inhibition imparts beneficial transgenerational effects in Huntington's disease mice via altered DNA and histone methylation Proc Natl Acad Sci U.S.A. 2015; 112(1): E56–E64. 24. Marchetto MCN, Winner B, Gage FH. Pluripotent stem cells in neurodegenerative and neurodevelopmental diseases. Hum Mol Gen 2010; 19(1): R71–R76. 25. Alexander H, Rolf H. Modern methods of drug discovery. Spinger (India) private limited: New Delhi: 2009; 207-18. 26. Kiefer M, Unterberg A. The Differential Diagnosis and Treatment of Normal-Pressure Hydrocephalus. Dtsch Arztebl Int 2012; 109(1-2): 15-26. Bibliography 27. Williams B, Eriksdotter-Jonhagen M, Granholm A. Nerve growth factor in treatment and pathogenesis of Alzheimer’s disease. Prog Neurobiol 2006; 80: 114-28. 28. Clinebell K, Azzam PN, Gopalan P, Haskett R. Guidelines for preventing common medical complications of catatonia: case report and literature review. J Clin Psychiatry 2014; 75(6): 644-51. 29. Ghosh A, Brindisi M, Tang J. Developing β-secretase inhibitors for treatment of Alzheimer’s disease. J Neurochem 2012; 120: 71–83. 30. Hansen R, Gartlehner G, Webb A, Morgan L, Moore C, Jonas D. Efficacy and safety of donepezil, galantamine, and rivastigmine for the treatment of Alzheimer’s disease: a systematic review and meta-analysis. Clin Interv Aging 2008; 3: 211–25. 31. Galimberti D, Scarpini E. Disease-modifying treatments for Alzheimer’s disease. Ther Adv Neurol Disord 2011; 4: 203-16 32. Imbimbo B, Giardina G. γ-Secretase inhibitors and modulators for the treatment of Alzheimer’s disease: disappointments and hopes. Curr Top Med Chem 2011; 11: 1555– 70. 33. Wisniewski T, Konietzko U. Amyloid-β immunization for Alzheimer’s disease. Lancet Neurol 2008; 7: 805–11. 34. Francis PT. The interplay of neurotransmitters in Alzheimer's disease. CNS Spectr 2005; 10: 6-9. 35. Calabrese EJ. Alzheimer's disease drugs: an application of the hormetic dose-response model. Crit Rev Toxicol 2008; 38(5): 419-51. 36. Korolev IO. Alzheimer’s disease: A Clinical and Basic Science Review. Am Med Stud Res J 2014; 4: 24-33. 37. Lipton SA. The molecular basis of memantine action in Alzheimer's disease and other neurologic disorders: low-affinity, uncompetitive antagonism. Curr Alzheimer Res 2005; 2(2): 155-65. 38. de Kort GJ, Steggerda S, Shigemoto R, Ribeiro-Da-Silva A, Cuello AC. Structural involvement of the glutamatergic presynaptic boutons in a transgenic mouse model expressing early onset amyloid pathology. Neurosci Lett 2003; 353: 143–7. 39. Aliev G, Mark EO, Prakash R ,Justin CS , Paula IM ,Akihiko N et al. Antioxidant therapy in Alzheimer’s disease: theory and practice. National Institute of Health Science 2008; 8(13):1395-406. Bibliography 40. Kolarova M, Garcia-Sierra F, Bartos A, Ricny J, Ripova D. Structure and pathology of tau protein in Alzheimer disease. Int J Alzheimers Dis 2012; 2012: 731526-38. 41. Goate A, Chartier-Harlin MC, Mullan M, Brown J, Crawford F, Fidani L. Segregation of a missense mutation in the amyloid precursor protein gene with familial Alzheimer’s disease. Nature 1991; 349(6311): 704-6. 42. Hanger DP, Anderton BH, Noble W. Tau phosphorylation: the therapeutic challenge for neurodegenerative disease. Trends Mol Med 2009; 15: 112-9.

43. Limon A, Reyes-Ruiz JM, Miledi R. Loss of functional GABAA receptors in the Alzheimer diseased brain. Proc Natl Acad Sci U S A 2012; 109: 10071-6. 44. Mitew S, Kirkcaldie MT, Dickson TC, Vickers JC. Altered synapses and gliotransmission in Alzheimer’s disease and AD model mice. Neurobiol Aging 2013; 34: 2341-51. 45. Iqbal K, Alonso AAC, Chen S, Chohan MO, El-AkkadE, Gong CX. Tau pathology in Alzheimer disease and other tauopathies. Biochim Biophys Acta 2005; 1739: 198-210. 46. Bell KF, Ducatenzeiler A, Ribeiro-Da-Silva A, Duff K, Bennett DA, Cuello AC. The amyloid pathology progresses in a neurotransmitter-specific manner. Neurobiol Aging 2006; 27: 1644-57. 47. Braak H, Braak E. Neuropathological stageing of Alzheimer-related changes. Acta Neuropathol 1991; 82: 239-59. 48. Adlard P, Bush A. Metals and Alzheimer’s disease. J Alzheimer’s Dis 2006; 10: 145- 63. 49. Zec R, Burkett N. Non-pharmacological and pharmacological treatment of the cognitive and behavioral symptoms of Alzheimer disease. Neuro Rehabilitation 2008; 23: 425-38. 50. Vassar R. BACE1: the beta-secretase enzyme in Alzheimer’s disease. J Mol Neurosci 2004; 23: 105-14. 51. Martinez A, Perez D. GSK-3 inhibitors: a ray of hope for the treatment of Alzheimer’s disease? J Alzheimers Dis 2008; 15: 181-91. 52. Herrmann N, Chau S, Kircanski I, Lanctot K. Current and emerging drug treatment options for Alzheimer’s disease: a systematic review. Drugs 2011; 71: 2031-65. 53. Cummings J, Back C. The cholinergic hypothesis of neuropsychiatric symptoms in Alzheimer’s disease. Am J Geriatr Psychiatry 1998; 6: 64-78. Bibliography 54. Palmer T. Neurotransmission in the autonomic and somatic motor nervous system. Goodman & Gilman’s - The pharmacological basis of therapeutics, 11th Edition, McGraw-Hill medical publishing division, New York: 2002; 125-9. 55. Morrison AS, Lyketsos C. The pathophysiology of Alzheimer’s disease and directions in treatment. J Adv Nurs 2005; 3(8): 256-70. 56. Jackson, Siegal J. Our current understanding of pathophysiology of Alzheimer’s disease. Adv\ Stud Pharm 2005; 2(4): 126-35. 57. Butterfield DA, Pocernich CB. The glutamatergic system and Alzheimer’s disease. Adis data information BV 2003; 17(9): 641-52. 58. Christen Y. Oxidative stress and Alzheimer disease. Am J Clin Nutr 2000; 7: 621-629. 59. Francis PT, Alan M, Michael S, Wilcock KG. The cholinergic hypothesis of Alzheimer’s disease: a review of progress. J Neurol Neurosurg Psychiatry 1999; 16: 137-47. 60. Brunton LL, Chabner BA, Knollmann BC. Goodman & Gillman's the pharmacological basis of therapeutics. 12th edition. The McGraw-Hill companies: United States. 2011; 549-560. 61. Rang HP, Dale MM, Ritter JM, Moore PK. Pharmacology. Elsevier. 5th edition; 2003; 518-35. 62. Rossor MN, Garrett NJ, Johnson AL, Mountjoy CQ, Roth M, Iversen LL. A post- mortem study of the cholinergic and GABA systems in senile dementia. Brain 1982; 105: 313-30. 63. Selkoe DJ. The cell biology of β-amyloid precursor protein and presenilin in Alzheimer’s disease. Trends Cell Biol 1998; 8: 447-53. 64. Farlow M. A clinical overview of cholinesterase inhibitors in Alzheimer’s disease. Int Psychogeriatr 2002; 14(Suppl. 1): 93-126. 65. Knopman D, Donohue J, Gutterman E. Patterns of care in the early stages of Alzheimer's disease: impediments to timely diagnosis. J Am Geriatr Soc 2000; 48: 300- 4. 66. Lim A, Tsuang D, Kukull W. Clinico-neuropathological correlation of Alzheimer's disease in a community-based case series. J Am Geriatr Soc 1999; 47: 564-9. 67. Lopez O, Becker J, Klunk W. Research evaluation and diagnosis of probable Alzheimer's disease over the last two decades. Neurology 2000; 55: 1854-62. Bibliography 68. Birks J. Cholinesterase inhibitors for Alzheimer’s disease. Cochrane Database Syst Rev 2006; (1): CD005593-8. 69. Hachinski V, Lassen N, Marshall J. Multi-infarct dementia: a cause of mental deterioration in the elderly. Lancet 1974; 2: 207-10. 70. Sunderland T, Hill J, Mellow A. Clock drawing in Alzheimer's disease: a novel measure of dementia severity. J Am Geriatr Soc 1989; 37: 725-9. 71. Alva G, Cummings J. Relative tolerability of Alzheimer’s disease treatments. Psychiatry (Edgmont) 2008; 5: 27-36. 72. Maidment I, Fox C, Boustani M, Rodriguez J, Brown R, Katona C. Efficacy of memantine on behavioral and psychological symptoms related to dementia: a systematic meta-analysis. Ann Pharmacother 2008; 42: 32-8. 73. Vogel HG, Schlkens BA, Sandow J. Drug effects on learning and memory in Drug Discovery and Evaluation: Pharmacological Assays. Springer, Berlin, Germany, 2nd edition, 2002; 595-643. 74. Oh JH, Choi BJ, Chang MS, Park SK. Nelumbo nucifera semen extract improves memory in rats with scopolamine-induced amnesia through the induction of choline acetyltransferase expression. Neurosci Lett 2009; 461(1): 41-4. 75. Kim J, Kopalli RS, Koppula S. Indigofera tinctoria Linn (Fabaceae) attenuates cognitive and behavioral deficits in scopolamine-induced amnesic mice. Trop J Pharm Res 2016; 15(4): 773-9. 76. Munshi R, Bhalerao S, Nesari T. Evaluation of the efficacy of Brahmi ghrita in scopolamine induced amnesia in rats using Cook’s pole apparatus. Int J Basic Clin Pharmacol 2016; 5(3): 829-33. 77. Bejar C, Wang RH, Weinstock M. Effect of Rivastigmine on scopolamine-induced memory impairment in rats. Eur J Pharmacol 1999; 383: 231-40. 78. Gene VW, David RV. Intrahippocampal scopolamine impairs both acquisition and consolidation of contextual fear conditioning. Neurobiol Learn Mem 2001; 75: 245–52. 79. Blokland A. Scopolamine-induced deficits in cognitive performance: A review of animal studies. Scopolamine Rev 2005; 1-76. 80. Abel T, Lattal KM. Molecular mechanisms of memory acquisition, consolidation and retrieval. Curr Opin Neurobiol 2001; 11: 180–7. Bibliography 81. Das A, Kapoor K, Sayeepriyadarshini AT, Dikshit M, Palit G, Nath C. Immobilization stress-induced changes in brain acetylcholinesterase activity and cognitive function in mice. Pharmacol Res 2000; 42: 213–7. 82. Nabeshima T, Noda Y, Kamei H. Anti-dementia drugs for Alzheimer disease in present and future. Nippon Yakurigaku Zasshi 2002; 120: 24-9. 83. Roger D. Porso R, Lenegre A, Avril I, Doumont G. Antagonism by exifone, a new cognitive enhancing agent, of the amnesias induced by four benzodiazepines in mice. Psychopharmacology 1988; 95: 291-7. 84. Yamamoto Y, Nakano S. Kawashima S, Nakamura S, Urakami K, Katot T, et al. Plasma and serum G4 isoenzyme of acetylcholinesterase in patients with Alzheimer- type dementia and vascular dementia. Ann Clin Biochem 1990; 27: 321-6. 85. Green RC. Risk assessment for Alzheimer's disease with genetic susceptibility testing: has the moment arrived? Alzheimer's Care Q. 2002; 208-14. 86. Rabins P, Mace N, Lusas M. The impact of dementia on the family. J Am Med Assoc 1982; 248: 333-5. 87. Yuan CY, Lee YJ, Hsu GS. Aluminum overload increases oxidative stress in four functional brain areas of neonatal rats. J Biomed Sci 2012; 19: 51-8. 88. Nayak P, Sharma SB, Chowdary NV. Augmentation of aluminum-induced oxidative stress in rat cerebrum by presence of pro-oxidant (graded doses of ethanol) exposure. Neurochem Res 2010; 35: 1681-90. 89. Flora SJ, Mehta A, Satsangi K, Kannan GM, Gupta M. Aluminum-induced oxidative stress in rat brain: response to combined administration of citric acid and HEDTA. Comp Biochem Physiol C Toxicol Pharmacol 2003; 134: 319-28. 90. Provan SD, Yokel RA. Aluminum inhibits glutamate release from transverse rat hippocampal slices: role of G proteins. Ca channels and protein kinase C. Neurotoxicol 1992; 13: 413-20. 91. Prakash D, Gopinath K, Sudhandiran G. Fistein enhances behavioral performances and attenuates reactive gliosis and inflammation during aluminum chloride-induced neurotoxicity. Neuromol Med 2013; 15: 192–208. 92. Abd-Elhady RM, Elsheikh AM, Khalifa AE. Anti-amnestic properties of Ginkgo biloba extract on impaired memory function induced by aluminum in rats. Int J Dev Neurosci 2013; 31(7): 598-607. Bibliography 93. Al-Kahtani MA. Renal damage mediated by oxidative stress in mice treated with aluminium chloride: protective effect of taurine. J Biol Sci 2010; 10: 584-95. 94. Cushnie TP, Lamb AJ. Recent advances in understanding the antibacterial properties of flavonoids. Int J Antimicrob Agents 2011; 38(2): 99-107. 95. Carrier L. Donepezil and paroxetine: possible drug interaction. J Am Geriatr Soc 1999; 47: 1037-42. 96. Khedkar V, Verma J, Coutiho E. Small molecule screening techniques in drug discovery. Biobytes 2009; 4: 7-4. 97. Soma M, Moudgil M, Mandal SK. Rational drug design. Eur J Pharmacol 2009; 625: 90-100. 98. Hachinski V, Lassen N, Marshall J. Multi-infarct dementia: a cause of mental deterioration in the elderly. Lancet 1974; 2: 207-10. 99. Lavecchia A, Giovanni CD. Virtual screening strategies in drug discovery: a critical review. Curr Med Chem 2013; 20(1): 1-22. 100. Chakraborty C, Hsu CH, Wen ZH, Lin CS, Agoramoorthy G. Zebrafish: a complete animal model for in vivo drug discovery and development. Curr Drug Metab 2009; 10(2):116-24. 101. Solt I, Anna T, Krisztian N. New approaches to virtual screening. Drug Discovery and Development 2014; 4-7. 102. Durdagi S, Supuran CT, Strom TA, Doostdar N, Kumar MK, Barron AR, et al. In silico drug screening approach for the design of magic bullets: a successful example with anti-HIV fullerene derivatized amino acids. J Chem Inf Model 2009; 49: 1139- 43. 103. Rangaraju A, Veerabhadra AR. A review on molecular docking – Novel tool in drug design and analysis. J Har Res Pharm 2013; 2(4): 215-21. 104. Ashokraj. A recent technology in drug discovery and development. International Journal of Innovative Drug Discovery 2012; 2(1): 22-39. 105. Alexander H, Rolf H. Modern methods of drug discovery. Spinger (India) private limited: New Delhi: 2009; 207-26. 106. Sliwoski G, Sandeepkumar K, Meiler J, Edward W. Application of computational methods for the design of BACE-1 inhibitors: validation of in silico modelling. Computational Methods in Drug Discovery 2013; 66(1): 334-95. Bibliography 107. Hirashima A, Huang H. Homology modeling, agonist binding site identiﬁcation, and docking in octopamine receptor of Periplaneta americana. Comput Biol Chem 2008; 32: 185-90. 108. Mukesh B, Rakesh K. Molecular docking a review. Int J Res Ayurveda Pharm 2011; 2(6): 1746-51. 109. Breda A, Basso LA, Santos DS, Walter F. Virtual screening of drugs: score functions, docking, and drug design. Curr Comp Aided Drug Des 2008; 4: 265-72. 110. Zhang HX, Xuan-Yu M, Mihaly M, Meng C. Molecular docking: a powerful approach for structure-based drug discovery. NIH Public Access 2011; 7(2): 146-57. 111. Venkatachalam CM, Jiang X, Oldfield T and Waldman M. LigandFit: A novel method for the shape-directed rapid docking of ligands to protein active sites. J Mol Graph Model 2003; 21: 289-307. 112. Sherman DH, Li S, Yermaliskaya VL, Kim Y, Smith AJ, Waterman RM, et al. The structural basis for substrate anchoring, active site selectivity, and product formation by P450 PikC from Streptomyces venezuelae. J Biol Chem 2006; 281: 26289-97. 113. Yasutake Y, Imoto N, Fujii T, Arisawa A, Tamura T. Crystal structure of cytochrome P450 MoxA from Nonomuraea recticatena (CYP105). Biochem Biophys Res Commun 2007; 361: 876–82. 114. Reddy S, Priyadarshini PS, Praveen KP, Pradeep HN, Narahari SG. Virtual screening in drug discovery – A computational perspective. Current Protein and Peptide Science 2007; 8: 329-351. 115. Gregory AP, Dagmar R. X-ray crystallography in the service of structure-based drug design. Drug design-structure and ligand-based approaches. 2nd ed. 2010; 17-29. 116. Joshi UJ, Amol SG, Priscilla DM, Ragini S, Sudha S, Girjesh G. Anti-inflammatory, antioxidant and anticancer activity of quercetin and its analogues. Int J pharm biomed res 2011; 2(4): 1756-66. 117. Jones G, Willett P, Glen RC, Leach AR, Taylor R. Development and validation of a genetic algorithm for flexible docking. J Mol Biol 1997; 2: 727-48. 118. Morris GM, Goodsell DS, Halliday RS. Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function. J Comput Chem 1998; 19(14): 1639-62. Bibliography 119. Morris GM, Huey R, Lindstrom W, Sanner MF. AutoDock 4 and AutoDock Tools 4: automated docking with selective receptor flexibility. J Comput Chem 2009; 19(14): 2785-91. 120. Gao LB, Yu XF, Chen Q, Zhou D. Alzheimer’s Disease therapeutics: current and future therapies. Minerva Med 2016; 107: 108-13. 121. Sunseri J, Koes DR. Pharmit: interactive exploration of chemical space. Nucleic Acids Res 2016; 44: W442-W448.

122. Collignon B, Schulz R, Smith JC. Task-parallel message passing interface implementation of Autodock4 for docking of very large databases of compounds using high-performance super-computers. J Comput Chem 2011; 32: 1202-12. 123. Apostolakis J, Pluckthun A, Caflisch A. Molecular docking: A problem with thousands of degrees of freedom. J Comput Chem 1998; 19: 21-37. 124. Borges F, Feranandes E, Roleira F. Progress towards the discovery of xanthine oxidase inhibitors. Curr Med Chem 2002; 9: 195-217. 125. Kumar A, Nisha CM, Silakari C, Sharma I, Anusha K, Gupta N, et al. Current and novel therapeutic molecules and targets in Alzheimer's disease. Journal of the Formosan Medical Association 2016; 115: 3-10. 126. Krishnendu G, Pratibha A, Greg H. Alzheimer's disease - Not an exaggeration of healthy aging. Indian J Psychol Med. 2011; 33(2):106-14. 127. Agrawal AD. Pharmacological activity of flavonoids: A review. Int J Pharm Sci Ntech 2011; 4(2): 1394-8. 128. Srivasthava N, Rao S, Thosar A. Flavonoids: health booster. Int Res J Pharm 2012; 5:142-6. 129. Meda NTR, Lamien MA, Kiendrebeogo M, Lamien CE, Coulibaly AY, Mjllogo RJ, et al. In vitro antioxidant, xanthine oxidase and acetylcholinesterase inhibitory activities of Balanites aegyptiaca (L.) Del. (Balanitaceae). Pak J Biol Sci 2010; 13: 362-8. 130. Manach C, ScalbertA, Morand C, Remesy C, Jimenez L. Polyphenols: food sources and bioavailability. Am J Clin Nutr 2004; 79(5): 727-47. 131. Tapas AR, Sakarkar DM, Kakde RB. Flavonoids as nutraceuticals: a review. Trop J Pharm Res 2008; 7(3): 1089-99. Bibliography 132. Krishna DR, Sripal RM, Chaluvadi MR. Bioflavonoids classification, pharmacological, biochemical effects and therapeutic potential. Indian J Pharmacol 2001; 13: 2-16. 133. Swaminathan M, Chin CF, Chin SP, Buckle MJC, Rahman NA, Doughty SW, et al.

Flavonoids with M1 muscarinic acetylcholine receptor binding activity. Molecules 2014; 19(7): 8933-48. 134. Janicijevic J, Tosic S, Mitrovic T. Flavonoids in plants. Proceeding of the 9th Symposium of flora of Southeastern Serbia and Neighbouring Regions, Nis 2007; 1(3): 153-6. 135. Raina GS. Significant emergent role of flavonoids as valuable nutraceuticals: systemic review. Int J Pharmacol 2013; 3(2): 44-51. 136. Agarwal A, Aponte-Mellado A, Premkumar BJ, Shaman A, Gupta S. The effects of oxidative stress on female reproduction: a review. Reprod Biol Endocrinol 2012; 10: 49-58. 137. Karrasch T, Kim JS, Jang IB and Jobin C. The flavonoid luteolin worsens chemical- induced colitis in NF-κBEGFP transgenic mice through blockade of NF-κB-dependent protective molecules. PLoS ONE 2007; 2: 596-8. 138. Malmstrom RE, Godman BB, Diogene E, Baumgartel C, Bennie M, Bishop I, et al. Dabigatran – a case history demonstrating the need for comprehensive approaches to optimize the use of new drugs. Frontiers in Pharmacology 2013; 4(39): 1-31. 139. Hamulakova S, Janovec L, Soukup O, Jun D, Kuca K. Synthesis, in vitro acetylcholinesterase inhibitory activity and molecular docking of new acridine- coumarin hybrids. Int J Biol Macromol 2017; 104: 333-8. 140. Park B, Nam JH, Kim JH, Kim HJ, Onnis V, Balboni G, et al. 3, 4- Dihydroquinazoline derivatives inhibit the activities of cholinesterase enzymes. Bioorg Med Chem Lett 2017; 27(5): 1179-85. 141. Sehgal AS, Mannan S, Ali S. Pharmacoinformatic and molecular docking studies reveal potential novel antidepressants against neurodegenerative disorders by targeting HSPB8. Drug Des Devel Ther 2016; 10: 1605-18. 142. Sehgal AS, Mannan S, Kanwal S, Naveed I, Mir A. Adaptive evolution and elucidating the potential inhibitor against schizophrenia to target DAOA (G72) isoforms. Drug Des Devel Ther 2015; 9: 3471-80. Bibliography 143. Ahmad K, Balaramnavar MV, Baig HM, Srivastava KA, Khan S, Kamal AM. Identification of Potent Caspase-3 Inhibitors for Treatment of Multi- Neurodegenerative Diseases Using Pharmacophore Modeling and Docking Approaches. CNS Neurol Disord Drug Targets 2014; 13(8): 1346-53. 144. Jalkute BC, Barage HS, Dhanavade JM, Sonawane DK. Molecular Dynamics Simulation and Molecular Docking Studies of Angiotensin Converting Enzyme with Inhibitor Lisinopril and Amyloid Beta Peptide. Protein J 2013; 32(5): 356-64. 145. Spilovska K, Korabecny J, Kral J, Horova A, Musilek K, Soukup O, et al. 7- Methoxytacrine-adamantylamine heterodimers as cholinesterase inhibitors in Alzheimer's disease treatment-synthesis, biological evaluation and molecular modeling studies. Molecules 2013; 18(2): 2397-418. 146. Grover A, Shandilya A, Agrawal V, Bisaria VS, Sundar D. Computational evidence to inhibition of human acetyl cholinesterase by withanolide a for Alzheimer treatment. J Biomol Struct Dyn 2012; 29(4): 651-62. 147. Dayangac-Erden D, Bora G, Ayhan P, Kocaefe C, Dalkara S, Yelekçi K, et al. Histone deacetylase inhibition activity and molecular docking of (e)-resveratrol: its therapeutic potential in spinal muscular atrophy. Chem Biol Drug Des 2009; 73(3): 355-64. 148. Bora-Tatar G, Dayangac-Erden D, Demir AS, Dalkara S, Yelekçi K, Erdem-Yurter H. Molecular modifications on carboxylic acid derivatives as potent histone deacetylase inhibitors: Activity and docking studies. Bioorg Med Chem 2009; 17(14): 5219-28. 149. Lan JS, Hou JW Liu Y, Ding Y, Zhang Y, Li L, et al. Design, synthesis and evaluation of novel cinnamic acid derivatives bearing N-benzyl pyridinium moiety as multifunctional cholinesterase inhibitors for Alzheimer's disease. J Enzyme Inhib Med Chem 2017; 32(1): 776-88. 150. Saeedi M, Babaie K, Karimpour-Razkenari E, Vazirian M, Akbarzadeh T, Khanavi M, et al. In vitro cholinesterase inhibitory activity of some plants used in Iranian traditional medicine. Nat Prod Res 2017; 6: 1-5. 151. Singh V, Kahol A, Singh IP, Saraf I, Shri R. Evaluation of anti-amnesic effect of extracts of selected Ocimum species using in-vitro and in-vivo models. J Ethnopharmacol 2016; 193: 490-9. Bibliography 152. Hajlaoui H, Mighri H, Aouni M, Gharsallah N, Kadri A. Chemical composition and in vitro evaluation of antioxidant, antimicrobial, cytotoxicity and anti- acetylcholinesterase properties of Tunisian Origanum majorana L. essential oil. Microb Pathogenesis 2016; 95: 86-94. 153. Formagio AS, Vieira MC, Volobuff CR, Silva MS, Matos AI, Cardoso CA, et al. In vitro biological screening of the anticholinesterase and antiproliferative activities of medicinal plants belonging to Annonaceae. Braz J Med Biol Res 2015; 48(4): 308-15.

154. Ali-shtayeh MS, Rana MJ, Salam YAZ, Iman BQ. In-vitro screening of acetylcholinesterase inhibitory activity of extracts from Palestinian indigenous flora in relation to the treatment of Alzheimer’s disease. Functional Foods in Health and Disease 2014; 4(9): 381-400. 155. Liu H, Huang X, Lou D, Liu X, Liu W, Wang Q. Synthesis and acetylcholinesterase inhibitory activity of Mannich base derivatives flavokawain B. Bioorg Med Chem Lett 2014; 24(19): 4749-53. 156. Alipour M, Khoobi M, Nadri H, Sakhteman A, Moradi A, Ghandi N, et al. Synthesis of some new 3-coumaranone and coumarin derivatives as dual inhibitors of acetyl and butyrylcholinesterase. Archiv der Pharmazie: Chem Life Sci 2013; 346: 1-16. 157. Akinyemi AJ, Oboh G, Oyeleye SI, Ogunsuyi O. Anti-amnestic Effect of Curcumin in combination with Donepezil, an anticholinesterase drug: Involvement of cholinergic system. Neurotox Res 2017; 31(4): 560-9. 158. Erfanparast A, Tamaddonfard E, Nemati S. Effects of intra-hippocampal microinjection of vitamin B12 on the orofacial pain and memory impairments induced by scopolamine and orofacial pain in rats. Physiol Behav 2016; 170: 68-77. 159. Imam A, Ajao MS, Akinola OB, Ajibola MI, Ibrahim A, Amin A, et al. Repeated acute oral exposure to Cannabis sativa impaired neurocognitive behaviours and cortico-hippocampal architectonics in Wistar rats. Niger J Physiol Sci 2017; 31(2): 153-9. 160. Rahmati B, Kiasalari Z, Roghani M, Khalili M, Ansari F. Antidepressant and anxiolytic activity of Lavandula officinalis aerial parts hydroalcoholic extract in scopolamine-treated rats. Pharm Biol 2017; 55(1): 958-65. Bibliography 161. Pandareesh MD, Anand T, Khanum F. Cognition enhancing and neuromodulatory propensity of Bacopa monniera extract against scopolamine induced cognitive impairments in rat hippocampus. Neurochem Res 2016; 41(5): 985-99. 162. Setorki M. Effect of hydro-alcoholic extract of Ziziphus spina-christi against scopolamine-induced anxiety in rats. Bangladesh J Pharmacol 2016; 11(2): 1-8. 163. He D, Wu H, Wei Y, Liu W, Huang F, Shi H, et al. Effects of harmine, an acetylcholinesterase inhibitor, on spatial learning and memory of APP/PS1 transgenic mice and scopolamine-induced memory impairment mice. Eur J Pharmacol 2015; 768: 96-107. 164. Lee J, Kim H, Lee H, Han J, Lee S, Kim D, et al. Hippocampal memory enhancing activity of pine needle extract against scopolamine-induced amnesia in a mouse model. Sci Rep 2015; 5: 9651-8. 165. Lee S, Park HJ, Jeon SJ, Kim E, Lee HE, Kim H, et al. Cognitive ameliorating effect of Acanthopanax koreanum against scopolamine-induced memory impairment in mice. Phytother Res 2017; 31(3): 425-32. 166. Pahaye DB, Bum EN, Taiwe GS, Ngoupaye GT, Sidiki N, Moto FCO, et al. Neuroprotective and antiamnesic effects of Mitragyna inermis Willd (Rubiaceae) on scopolamine-induced memory impairment in mice. Behav Neurol 2017; 2017: 1-11. 167. Al-Amin MM, Reza MH, Saadi MH, Mahmuda W. Astaxanthin ameliorates aluminum chloride-induced spatial memory impairment and neuronal oxidative stress in mice. Eur J Pharmacol 2016; 777: 60-9. 168. Kaushik A, Jayant RD, Tiwari S, Vashist A, Nair M. Nano-biosensors to detect beta- amyloid for Alzheimer's disease management. Biosensors and Bioelectronic 2016; 1- 15. 169. Liu L, Chan C. The role of inflammasome in Alzheimer’s disease. Ageing Res Rev 2014; 0: 6-15. 170. de Vargas FS, Almeida PD, de Boleti AP, Pereira MM, de Souza TP, de Vasconcellos MC, et al. Antioxidant activity and peroxidase inhibition of Amazonian plants extracts traditionally used as anti-inflammatory. BMC Complement Altern Med 2016; 16: 83-8. 171. Kasbe P, Jangra A, Lahkar M. Mangiferin ameliorates aluminium chloride-induced cognitive dysfunction via alleviation of hippocampal oxido-nitrosative stress, Bibliography proinflammatory cytokines and acetylcholinesterase level. J Trace Elem Med Biol 2015; 31: 107-12. 172. Li H, Kang T, Qi B, Kong L, Jiao Y, Cao Y, et al. Neuroprotective effects of ginseng protein on PI3K/Akt signaling pathway in the hippocampus of D-galactose/AlCl3 inducing rats model of Alzheimer's disease. J Ethnopharmacol 2016; 179: 162-9. 173. Mahboob A, Farhat SM, Iqbal G, Babar MM, Zaidi NU, Nabavi SM, et al. Alpha- lipoic acid-mediated activation of muscarinic receptors improves hippocampus- and amygdala-dependent memory. Brain Res Bull 2016; 122: 19-28. 174. Naaz H, Singh S, Pandey VP, Dwivedi U. Anti-cholinergic alkaloids as potential therapeutic agents for Alzheimer’s disease: An in Silico approach. Indian J Biochem Biophys 2013; 50(2): 120-5. 175. Lakshmi BV, Sudhakar M, Prakash KS. Protective effect of selenium against aluminum chloride-induced Alzheimer's disease: Behavioral and biochemical alterations in rats. Biol Trace Elem Res 2015; 165(1): 67-74. 176. Thippeswamy AH, Rafiq M, Viswantha GL, Kavya KJ, Anturlikar SD, Patki PS. Evaluation of Bacopa monniera for its synergistic activity with rivastigmine in reversing aluminum-induced memory loss and learning deficit in rats. J Acupunct Meridian Stud 2013; 6(4): 208-13. 177. Thirunavukkarasu SV, Venkataraman S, Raja S, Upadhyay L. Neuroprotective effect of Manasamitra vatakam against aluminium induced cognitive impairment and oxidative damage in the cortex and hippocampus of rat brain. Drug Chem Toxicol 2012; 35(1): 104-15. 178. Kumar A, Prakash A, Dogra S. Neuroprotective effect of carvedilol against aluminium induced toxicity: Possible behavioral and biochemical alterations in rats. Pharmacol Rep 2011; PR63 (4): 915-23. 179. Butler KV, Kalin J, Brochier C, Vistoli G, Langley B, Kozikowski AP. Rational design and simple chemistry yield a superior, neuroprotective HDAC6 inhibitor, tubastatin A. J Am Chem Soc 2010; 132(31): 10842-6. 180. Bihaqi SW, Singh AP, Tiwari M. Supplementation of Convolvulus pluricaulis attenuates scopolamine-induced increased tau and amyloid precursor protein (AβPP) expression in rat brain. Indian J Pharmacol 2012; 44(5): 593-8. Bibliography 181. Kumar AI, Dogra S, Prakash A. Protective effect of curcumin (Curcuma longa), against aluminium toxicity: Possible behavioral and biochemical alterations in rats. Behav Brain Res 2009; 205: 384-390. 182. Giulio PD, Toscani F, Villani D, Brunelli C, Gentile S, Spadin P. Dying with advanced dementia in long‐term care geriatric institutions: a retrospective study. J Palliat Med 2008; 11(7): 1023-8. 183. Gschwend DA, Good AC, Kuntz ID. Molecular docking towards drug discovery. J Mol Recog 1996; 9: 175-86. 184. Asokkumar K, Madeswaran A, George S. Combining in silico and in vitro approaches to evaluate the acetylcholinesterase inhibitory profile of some commercially available flavonoids in the management of Alzheimer’s disease. Int J Biol Macromolec 2017; 95: 199-203. 185. Goodsell DS, Morris GM, Olson AJ. Automated docking of flexible ligands: Applications of Autodock. J Mol Recogn 1996; 9: 1-5. 186. Sander T, Liljefors T, Balle T. Prediction of the receptor conformation for iGluR2 agonist binding: QM/MM docking to an extensive conformational ensemble generated using normal mode analysis. J Mol Graph Model 2008; 26: 1259-68. 187. Zhong S, Zhang Y, Xiu Z. Rescoring ligand docking poses. Curr Opin Drug Discov Devel 2010; 13: 376-34. 188. Ellman GL, Courtney DK, Andres V, Featherstone RM. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 1959; 7: 88-95. 189. Sacan O, Yanardag R. Antioxidant and antiacetylcholinesterase activities of chard (Beta vulgaris L. Var. Cicla). Food Chem Toxicol 2010; 48: 1275-80. 190. Organisation for Economic Cooperation and Development, Test No.420: Acute oral toxicity – fixed dose procedure, OECD Guidelines for the testing of chemicals, Section 4: Health effects. OECD publishing, Paris: 2002; doi: 10.1787/9789264070943-en. 191. Mo S, Zhou F, Lv Y, Hu Q, Zhang D, Kong LD. Hypouricemic action of selected flavonoids in mice: structure-activity relationships. Biol Pharm Bull 2007; 30: 1551-6. 192. Sujith K, Darwin CR, Sathish, Suba V. Memory-enhancing activity of Anacyclus pyrethrum in albino Wistar rats. Asian Pac J Trop Biomed 2012; 2(4): 307-11. Bibliography 193. Rebai O, Djebli NE. Chronic Exposure to Aluminum Chloride in Mice: Exploratory Behaviors and Spatial Learning. Adv Biol Res 2008; 2(1-2): 26-33. 194. Hu B, Liao C, Millson SH, Mollapour M. Qri2/Nse4, a component of the essential Smc5/6 DNA repair complex. Mol Microbiol 2005; 55(6): 1735-50. 195. Saraf MK, Prabhakar S, Khanduja KL, Anand A. Bacopa monniera attenuates scopolamine-induced impairment of spatial memory in mice. Evid Based Complement Alternat Med 2011; 2011: 1-10. 196. Rani A, Prasad S. A special extract of Bacopa monnieri (CDRI-08)-restored memory in CoCl2-hypoxia mimetic mice is associated with upregulation of Fmr-1 gene expression in hippocampus. Evid Based Complement Alternat Med 2015; 2015: 1-12. 197. Vogel HG, Schlkens BA, Sandow J. Drug effects on learning and memory in Drug Discovery and Evaluation: Pharmacological Assays. Springer, Berlin, Germany, 2nd edition, 2002; 595–643. 198. Robbins TW. The 5 choice serial reaction time task: behavioral pharmacology and functional neurochemistry. J Psychopharmacol 2002; 163(3-4): 362-80. 199. Kumar AI, Dogra S, Prakash A. Protective effect of curcumin (Curcuma longa), against aluminium toxicity: Possible behavioral and biochemical alterations in rats. Behav Brain Res 2009; 205: 384-90. 200. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with protein phenol reagent. J Biol Chem 1951; 193: 265-75. 201. Halliwell B, Gutteridge JMC. Free radicals in Biology and Medicine, 3rd ed. Oxford University Press, Oxford, 1989. 202. Sinha KA. Colorimetric assay of catalase. Anal Biochem 1972; 47:389-94. 203. Misra HP, Fridovich I. The role of superoxide anion in the autooxidation of epinephrine and a simple assay for superoxide dismutase. J Biol Chem 1972; 247:3170-5. 204. Taju G, Jayanthi M, Basha N, Nambi KSN. Hepatoprotective effect of Indian medicinal plant Psidium Guajava Linn. Leaf extract on paracetamol induced liver toxicity in albino rats. J Pharm Res 2010; 3:1759-63. 205. Racker E. Glutathione reductase from bakers’ yeast and beef liver. J Biol Chem 1955; 217:855-66. Bibliography 206. Paglia DE, Valentine WN. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med 1967; 70:158-69. 207. Niehaus WG, Samuelson B. Formation of MDA from phospholipids arachidonate during microsomal lipid peroxidation. Eur J Biochem 1986; 6:126-30. 208. Kaur IP, Geetha T. Screening methods for antioxidants-A review. Mini Rev Med Chem 2006; 6(3):305-312. 209. Yu ZF, Cheng GJ, Hu BR. Mechanism of colchicine impairment of learning and memory, and protective effect of CGP36742 in mice. Brain res 1997; 750 (1-2):53-8. 210. Seeliger D, Groot BL. Ligand docking and binding site analysis with PYMOL and Autodock/Vina. J Comput Aided Mol Des 2010; 24:417-24. 211. Cavasotto CN, Abagyan RA. Protein flexibility in ligand docking and virtual screening to protein kinases. J Mol Biol 2004; 12:209-25. 212. Sharma MC, Kohli DV, Sharma S, Sharma AD. Design, synthesis and biological activity of some benzimidazoles derivatives 3-chloro-4-(substituted--phenyl-1-(4-{1- [2'-(2H-tetrazol-5-yl)-biphenyl-4-ylmethyl]-1H-benzimidazol-2yl}-phenyl)-azetidin- 2-ones. D Chem Sin 2010; 1:92-105. 213. Liu J, Mori A. Anti-oxidant and free radical scavenging activities of Gastrodia elata and Uncaria Rhynchophylla (MiQ) Jacks. Neuropharmacol 1992; 31:1287-98. 214. Saleshier FM, Suresh S, Anitha N, Karim J, Divakar MC. Design, docking and synthesis of some 6-benzimidazoyl pyrans and screening of their anti-tubercular activity. Eur J Exp Biol 2011; 1:150-9. 215. Madeswaran A, Umamaheswari M, Asokkumar K, Sivashanmugam A, Subhadradevi V, Jagannath P. Computational drug discovery of potential TAU protein kinase I inhibitors using in silico docking studies. Bangladesh J Pharmacol 2013; 8:131-5. 216. Morgan D, Immunotherapy for Alzheimer’s disease. J Intern Med 2011; 269(1):54- 63. 217. Shoichet BK. Virtual screening of chemical libraries. Nature 2004; 43:862-8. 218. Koppen H. Virtual screening – what does it give us? Current opinion in drug discovery & development 2009; 12:397-402. 219. Dawane BS, Chobe SS, Mandaward GG, Shaikh BM, Konda SG, Patil SD. Design, synthesis of some new pyrazolo [3, 4-c] pyrazol thiazolone and evaluation of their antimicrobial activity. D Pharm Sin 2010; 1:140-8. Bibliography 220. Schames JR, Henchman RH, Seigel JS, Sotriffer CA, Ni H, McCammon A. Discovery of a Novel Binding Trench in HIV Integrase. J Med Chem 2004; 47:1879-81. 221. Cosconati S, Forli S, Perryman AL, Harris R, Goodsell DS, Olson AJ. Virtual screening with AutoDock: Theory and practice. Expert Opin Drug Discov 2010; 5:597-607. 222. Perry EK, Perry RH, Blessed G, Tomlinson BE. Changes in brain cholinesterases in senile dementia of Alzheimer type. Neuropathol Appl Neurobiol 1978; 4(4):273-7. 223. Panche AN, Diwan AD, Chandra SR. Flavonoids: an overview. Nutr Sci 2016; 5:e47- e54. 224. Zarotsky V, Sramek JJ, Cutler NR. Galanthamine hydrobromide: an agent for Alzheimer’s disease. Am J Health Syst Pharm 2003; 60:446-52. 225. Reiman EM. A 100-Year Update on Alzheimer's disease and related disorders. J Clin Psychiatry 2006; 67:1784-1800. 226. Wenk GL. Neuropathologic changes in Alzheimer's disease: potential targets for treatment. J Clin Psychiatry 2006; 67(3):3-7. 227. Bierer LM, Haroutunian V, Gabriel S, Knott PJ, Carlin LS, Purohit DP, et al. Neurochemical correlates of dementia severity in Alzheimer's disease: Relative importance of the cholinergic deficits. J Neurochem 1995; 64:749-60. 228. Wu CK, Thal L, Pizzo D, Hansen L, Masliah E, Geula C. Apoptotic signals within the basal forebrain cholinergic neurons in Alzheimer's disease. Exp Neurol 2005; 195:484-96. 229. Gron G, Brandenburg I, Wunderlich AP, Riepe MW. Inhibition of hippocampal function in mild cognitive impairment: targeting the cholinergic hypothesis. Neurobiol Aging 2006; 27:78-87. 230. Descarries L, Aznavour N, Hamel E. The acetylcholine innervation of cerebral cortex: new data on its normal development and its fate in the hAPP SW, IND mouse model of Alzheimer's disease. J Neural Transm 2005; 112:149-62. 231. Milind P, Dev C. Anti-Alzheimer potential of organic juice. Int Res J Pharm 2012; 3(9): 312-16. 232. Goverdhan P, Sravanthi A, Mamatha T. Neuroprotective effects of meloxicam and selegiline in scopolamine-induced cognitive impairment and oxidative stress. Int J Alzheimers Dis 2012; 1-6. Bibliography 233. Sudha S, Lakshmana MK, Pradhan N. Changes in learning and memory, acetylcholinesterase activity and monoamines in brain after chronic carbamazepine administration in rats. Epilepsia 1995; 36(4):416-22. 234. Zeng X, Zhang S, Zhang L, Zhang K, Zheng X. A study of the neuroprotective effect of the phenolic glucoside gastrodin during cerebral ischemia in vivo and in vitro. Planta Med 2006; 72: 1359-65. 235. Ray G, Husain SA. Oxidants, antioxidants and carcinogenesis. Indian J Exp Biol 2002; 40: 1213-32. 236. Zhu JH, Zhang X, McClung JP, Lei XG. Impact of Cu, Zn-superoxide dismutase and Se-dependent glutathione peroxidase-1 knockouts on acetaminophen-induced cell death and related signaling in murine liver. Exp Biol Med 2006; 231: 1726-32. 237. Scapangnini G, Ravagna A, Bella R, Colombrita C, Pennisi G, Calvani M, et al. Long- term ethanol administration enhances age-dependent modulation of redox state in brain and peripheral organs of rat: protection by acetylcarnitine. Int J Tissue React 2002; 24: 89-96. 238. El-Sokkary GH, Reiter JR, Tan D, Kim SJ, Cabrera J. Inhibitory effect of melatonin on products of lipid peroxidation resulting from chronic ethanol administration. Alcohol Alcoholism 1999; 34:842-850. 239. Winkler BS. Unequivocal evidence in support of the non-enzymatic redox coupling between glutathione/glutathione disulphide and ascorbic acid/dehydroascorbic acid. Biochimica et Biophysica Acta 1992; 1117:287-90. 240. Jaya DS, Augstin J, Venugopal PM. Role of lipid peroxides, glutathione and antiperoxidative enzymes in alcohol and drug toxicity. Indian J Exp Biol 1993; 31:453-9. 241. Guochuan ET, Ragan P, Chang BSR, Chen BSS, Markku V, Linnoila I, et al. Increase glutaminergic neurotransmission and oxidative stress after alcohol withdrawal. Am J Psychiatry 1998; 155:726-32. 242. Savory J, Herman MM, Ghribi O. Mechanisms of aluminum-induced neurodegeneration in animals: Implications for Alzheimer's disease. J Alzheimers Dis 2006; 10(2-3):135-44. Bibliography 243. Zatta P, Ibn-Lkhayat-Idrissi M, Zambenedetti P, Kilyen M, Kiss T. In vivo and in vitro effects of aluminum on the activity of mouse brain acetylcholinesterase. Brain Res Bull 2002; 59(1):41-5. 244. da Silva GFZ, Ming L. Metallo-ROS in Alzheimer's disease: Oxidation of neurotransmitters by CuII-β-amyloid and neuropathology of the disease. Angew Chem 2007; 46(18): 3337-41. 245. Halliwell B. Oxidants and the central nervous system: some fundamental questions is oxidant damage relevant to Parkinson’s disease, Alzheimer’s disease, traumatic injury or stroke? Acta Neurol Scand 1989; 126:23-33. 246. Halliwell, B, Gutteridge, JMC. Free radicals in Biology and Medicine, 3rd ed. Oxford University Press, Oxford, 1989. 247. Jung JW, Yoon BH, Oh HR, Ahn JH, Kim SY, Park SY, et al. Anxiolytic-like effects of Gastrodia elata and its phenolic constituents in mice. Biol Pharm Bull 2006; 29:261–5.

Appendixes U R K U N D

Urkund Analysis Result

Analysed Document: Madeswaran Final Thesis.doc (D29512176) Submitted: 2017-06-28 09:13:00 Submitted By: [email protected] Significance: 3 % Sources included in the report:

Dinesh_Thakkar_Pharmacy_Thesis.pdf (D24003545) Lubhan Singh.doc (D21327176) Lubhan Singh With references.doc (D21785446) thesis plagiarism check.docx (D27235467) D Sheeja Malar Ph.D Thesis 501 2012-13.docx (D27246331) Lubhan Singh without references.doc (D21785482) 16_chapter10.pdf (D21209962) Thesis Final Work 27062017.docx (D29511320) 107cd289-b95b-422a-b194-c980f3b9a6c7 Instances where selected sources appear:

59 U R K U N D Madeswaran Final Thesis.doc (D29512176)

IN SILICO, IN VITRO AND IN VIVO MEMORY ENHANCING ACTIVITY OF CERTAIN COMMERCIALLY AVAILABLE FLAVONOIDS IN SCOPOLAMINE AND ALUMINIUM-INDUCED LEARNING IMPAIRMENT IN MICE

Thesis submitted to

The Tamil Nadu Dr. M.G.R. Medical University, Chennai

0: Dinesh_Thakkar_Pharmacy_Thesis.pdf 34%

for the award of the degree of

DOCTOR OF PHILOSOPHY

PHARMACY

Submitted by

A. MADESWARAN, M. Pharm.,

Under the guidance of

Dr. K. ASOK KUMAR, M. Pharm., Ph.D.

College of Pharmacy,

Sri Ramakrishna Institute of

Paramedical Sciences,

Coimbatore – 641 044,

Tamil Nadu, India.

JUNE 2017

1. INTRODUCTION

1.1

0: Lubhan Singh.doc 71%

1: Lubhan Singh With references.doc 71%

2: Lubhan Singh without references.doc 71%

NEURODEGENERATIVE DISORDERS

2 U R K U N D Madeswaran Final Thesis.doc (D29512176)

drive due to the

0: Lubhan Singh.doc 58%

1: Lubhan Singh With references.doc 58%

2: Lubhan Singh without references.doc 58%

loss of neurons from structures of the basal ganglia1, 2. Alzheimer's disease (AD), results in the loss of cortical and hippocampal neurons with impairment of memory and cognitive ability and amyotrophic lateral sclerosis (ALS), producing muscular weakness

due to

0: Lubhan Singh.doc 54%

1: Lubhan Singh With references.doc 54%

2: Lubhan Singh without references.doc 54%

degeneration of bulbar, spinal, and cortical motor neurons3, 4.

Neurodegenerative disorders are comparatively common and represent a substantial societal and medical problem. They are

principal disorders

0: Lubhan Singh.doc 72%

1: Lubhan Singh With references.doc 72%

2: Lubhan Singh without references.doc 72%

of later life, developing in individuals who are neurologically normal, although childhood-onset forms of each of the disorders are known. PD is detected in more than 1% of population above the age of 655, 6, whereas AD affects as many as 10% of the same individuals7. HD, which is a genetically stable autosomal dominant disorder, is less common in the population as a whole but disturbs, on average, 50% of each generation in families booming the gene. ALS also is moderately rare but sometimes leads to disability and death8, 9.

0: Lubhan Singh.doc 83%

1: Lubhan Singh With references.doc 83%

3 U R K U N D Madeswaran Final Thesis.doc (D29512176)

2: Lubhan Singh without references.doc 83%

The available treatments for AD, HD, and ALS are much more limited in effectiveness, and

there is a need for new strategies10, 11.

1.1.1 Overview of dementia

Dementia is a depleting disorder, which gradually declines the cognitive functions including memory, language, speech, orientation, judgement and learning capacity. More than 47 million people are currently living with dementia worldwide and about 75% of this population that is, 27 million people, are estimated to be affected by AD12. It may be due to therapeutic drug use (atropine and phenytoin), metabolic disorders (hypoglycemia, acid base disorders, hematological disorders, hyperglycemia, hypopituitarism, pulmonary insufficiency, cardiac dysfunction and hepatolenticular degeneration), deficiency states (vitamin B12, niacin and folate deficiency), collagen (vascular disorders, systemic lupus erythematous, Behcet’s syndrome, temporal arteritis and sarcoidosis), intracranial disorders (tumor, epilepsy, neurosyphilis, encephalitis, multiple sclerosis and cerebrovascular insufficiency) and exogenous intoxication (alcohol, lead, carbon monoxide, organophosphates, mercury, manganese and toluene). It is not a part of normal aging and constantly turns as pathological process. Alzheimer’s is the most common cause of dementia13-15.

1.2 TYPES OF DEMENTIA

1.2.1 Alzheimer’s disease

1.2.2 Vascular dementia

1.2.3 Dementia with Lewy bodies (DLB)

Lewy bodies are abnormal aggregations of the protein alpha-synuclein that accumulate in neurons and dementia can result

0: 107cd289-b95b-422a-b194-c980f3b9a6c7 52%

when they develop in cortex region of the brain. Aggregation of alpha-synuclein in the brains of people with

Parkinson’s disease (PD), in which it is produced by extreme neuronal loss in a part of the brain called the substantia nigra. While people with dementia with Lewy bodies DLB and PD both have

4 U R K U N D Madeswaran Final Thesis.doc (D29512176)

Lewy bodies, the onset of the disease is marked by cognitive impairment in DLB and motor impairment in PD20.

1.2.4 Fronto-temporal lobar degeneration (FTLD)

0: 107cd289-b95b-422a-b194-c980f3b9a6c7 67%

FTLD includes dementias such as behavioural-variant FTLD,

0: thesis plagiarism check.docx 47%

1: D Sheeja Malar Ph.D Thesis 501 2012-13.docx 47%

primary progressive aphasia, corticobasal degeneration, Pick’s disease and progressive supranuclear palsy. Initial symptoms include marked changes in behaviour and personality and difficulty with

comprehending language. Nerve cells in the frontal lobe and temporal lobes of the brain are affected,

and these regions become significantly shrunken.

0: thesis plagiarism check.docx 78%

1: D Sheeja Malar Ph.D Thesis 501 2012-13.docx 78%

In addition, the upper layers of the cortex typically become spongy and soft and have protein insertions (tau protein or the transactive DNA-binding protein) 21.

1.2.5

Mixed dementia

Mixed dementia is

0: thesis plagiarism check.docx 79%

1: D Sheeja Malar Ph.D Thesis 501 2012-13.docx 79%

characterized by the hallmark abnormalities of more than one cause of dementia most commonly Alzheimer’s combined with vascular dementia, followed by Alzheimer’s with DLB, and Alzheimer’s with vascular dementia and DLB. Vascular dementia with

DLB is less common.

0: 107cd289-b95b-422a-b194-c980f3b9a6c7 100%

Recent studies suggest that mixed dementia is more common than previously

5 U R K U N D Madeswaran Final Thesis.doc (D29512176)

recognized, with about half of those with dementia having pathologic evidence of more than one cause of dementia 20, 22.

1.2.6 Creutzfeldt-Jakob disease

This is very rare fatal disorder and rapidly impairs memory and coordination and causes behaviour changes, results from a misfolding

0: 107cd289-b95b-422a-b194-c980f3b9a6c7 96%

protein (prion) that causes other proteins throughout the brain to misfold and malfunction.

It may be hereditary sporadic or caused by a known infection. A specific form called

0: thesis plagiarism check.docx 100%

1: D Sheeja Malar Ph.D Thesis 501 2012-13.docx 100%

2: 107cd289-b95b-422a-b194-c980f3b9a6c7 100%

variant Creutzfeldt-Jakob disease is believed to be caused by consumption of products from cattle affected by mad cow disease23.

1.2.7

Parkinson’s disease (PD) dementia

Problems with movement (slowness, rigidity, tremor and changes in gait) are common symptoms of PD. In PD,

0: 107cd289-b95b-422a-b194-c980f3b9a6c7 92%

alpha-synuclein aggregates appear in an area deep in the brain called

0: thesis plagiarism check.docx 100%

1: D Sheeja Malar Ph.D Thesis 501 2012-13.docx 100%

the substantia nigra. The aggregates are thought to cause degeneration of the nerve cells that produce dopamine.

The incidence of PD is about one-tenth that of

Alzheimer’s. As PD progresses, it often results in dementia secondary to the accumulation of Lewy bodies in the cortex (similar to DLB) or the accumulation of beta-amyloid clumps and tau tangles24, 25.

1.2.8 Normal pressure hydrocephalus

0: thesis plagiarism check.docx 100%

6 U R K U N D Madeswaran Final Thesis.doc (D29512176)

1: D Sheeja Malar Ph.D Thesis 501 2012-13.docx 100%

Symptoms include difficulty in walking, memory loss and inability to control urination.

Accounts for less than 5% of dementia cases and caused by impaired reabsorption of cerebrospinal fluid and the consequent build-up of fluid in the brain, increasing pressure in the brain. People with a history of brain haemorrhage (particularly subarachnoid haemorrhage) and meningitis are at increased risk. It

0: 107cd289-b95b-422a-b194-c980f3b9a6c7 95%

1: thesis plagiarism check.docx 94%

2: D Sheeja Malar Ph.D Thesis 501 2012-13.docx 94%

can sometimes be corrected with surgical installation of a shunt in the brain to drain excess fluid26.

1.3 ALZHEIMER'S

DISEASE (

AD)

1.3.1 History of Alzheimer’s disease

AD, the most common form of dementia, is an increasing medical, social and public health concern29. AD is characterized by deficits in the cholinergic system and the deposition of β-amyloid (Aβ) protein30. The cholinergic system has been targeted for the design of anti-Alzheimer’s drugs. Cholinesterase inhibitors increase both the level and duration of action of acetylcholine. In addition, it has been demonstrated that both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) play an important role in Aβ-aggregation during plaque formation31. Many research papers published in the last decade have indicated that the structures of compounds that exhibit cholinesterase inhibitory activity are diverse32, 33.

1.3.2 Neurotransmitters in AD

7 U R K U N D Madeswaran Final Thesis.doc (D29512176)

information processing and memory34. Although other neurotransmitters (noradrenaline, serotonin, somatostatin and other peptides) are also deficient in AD, the cognitive impairment correlates best with the loss of cholinergic input. AChE inhibitors (tacrine) and ACh receptor agonists, including nicotine, have been used to treat AD. The marginal success of this approach suggests that, in addition to ACh deficiency, there are other profound alterations that contribute to the cognitive dysfunction35.

1.3.3 Signs of AD

➢ Memory loss;

➢ Problems with abstract thinking;

➢ Difficulty performing familiar tasks;

➢ Changes in mood or behavior;

➢ Disorientation to time and place;

➢ Misplacing things in unusual places;

➢ Poor or decreased judgment;

➢ Changes in personality;

➢ Problems with language; and,

➢ Loss of initiative.

1.3.4 Clinical facts of AD

1.3.5 Etiology of AD

1.3.5.1 Genetic etiology of AD

8 U R K U N D Madeswaran Final Thesis.doc (D29512176)

The genetic etiology of AD includes apolipoprotein E gene polymorphism, amyloid-β protein precursor and presenilin I and II40.

1.3.5.2 Non-genetic etiology of AD

Non- genetic etiology of AD includes age, tauopathy and other risk factors like hyperlipidemia, hypertension, diabetes mellitus, stroke, smoking, traumatic head injury and familial history39, 41.

1.3.6 Stages of AD

Different stages of AD are:

1. Mild cognitive decline

2. Moderate cognitive decline

3. Severe cognitive decline

Fig 1: Brain image showing different stages of AD

1.3.6.1 Mild cognitive decline

1.3.6.2 Moderate cognitive decline

1.3.6.3 Severe cognitive decline

Each of the three stages; mild, moderate and severe, can be further broken down into seven levels:

Level 1 is considered to be a normal adult with no decline in function or of their memory.

9 U R K U N D Madeswaran Final Thesis.doc (D29512176)

meals and even starting a car. They will have difficultly recalling current information, but will still remember major information involving themselves and their families.

1.3.7 Pathophysiology of AD

1.3.7.1 Beta Amyloid hypothesis

Fig 2: Cascade of pathophysiological events in AD

1.3.7.2 Tau protein aggregation

Fig. 3: Aggregation of tau protein cascade events

By distorting the spacing of microtubules, they impair the axonal transport thus affecting the nutrition of axon terminals and dendrites. No mutations of the tau gene occur in AD. Abnormal tau first 10 U R K U N D Madeswaran Final Thesis.doc (D29512176)

appears in the entorhinal cortex, then in the hippocampus, and at later stages in association cortex. Recent observations in transgenic mice suggest that the spread of the pathology to anatomically linked areas occurs by passage of abnormal tau across synapses49, 50.

1.3.7.3 Cholinergic deficiency

Fig 4: Synthesis, release and binding of ACh to its receptors

The function of BuChE is less clearly defined, and it can hydrolyze acetylcholine as well as other esters. Membrane bound form of AChE decreases the acetylcholine level in the brain of patients with AD whereas the BuChE remains unchanged or even increased. But ACh level can be maintained by selective BuChE inhibitors. Drugs that inhibit both enzymes may be preferably used for treating AD. In AD, there is an involvement of pyramidal neurons there by neuronal loss, synapse loss, reduced glutamate concentration and tangle formation leads to the formation of neurofibrillary tangles. Glutamate is the neurotransmitter of pyramidal neurons54-56.

1.3.7.4 Glutamatergic pathway

1.3.7.5 Oxidative stress

11 U R K U N D Madeswaran Final Thesis.doc (D29512176)

consumption rate, high lipid content and relative loss (paucity) of anti-oxidant enzymes compared to other organs. Neuronal oxidation can cause numerous problems such as irreparable DNA damage and up-regulation of pro inflammatory cytokines57, 58.

1.3.7.6 Chronic inflammation

1.3.8 Acetylcholine

. Cl-

Fig 5: Structure of acetylcholine chloride

1.3.9 Synthesis, storage and release of acetylcholine

1.3.10 Management of AD

12 U R K U N D Madeswaran Final Thesis.doc (D29512176)

1.3.10.1 Cholinesterase inhibitor

1.3.10.2 NMDA receptor antagonist

1.3.10.2.1 Aβ-targeting strategies

➢ α secretase activators/ modulators

➢ β secretase inhibitors (GSK188909, PMS777)

➢ γ secretase inhibitors/ modulators (BMS299897,MRK-560)

➢ Aβ- aggregation inhibitors

➢ M1 muscarinic agonists (AF102B, AF267B)

➢ Immunotherapy

➢ Apo lipoprotein (ApoE) promotes Aβ clearance

13 U R K U N D Madeswaran Final Thesis.doc (D29512176)

➢ Aβ-degrading enzyme

➢ Drugs influencing Aβ blood- brain barrier transport

1.3.10.2.2 Treatment based on tau pathology

➢ Prevention of phosphorylation of tau

➢ Prevention of the aggregation of tau

➢ Tau immunotherapy

➢ Prevent the misfolding of tau

1.3.10.2.3 Non-steroidal anti-inflammatory drugs (NSAIDs)

➢ Trials with COX-2 selective inhibitors

1.3.10.2.4 Antioxidants nutrients and agents: Vitamins C and E, oestrogen, statins, omega-3 polyunsaturated fatty acids.

1.3.11 Alzheimer’s disease inducing agents

Alzheimer’s disease or learning impairment was induced by drugs and chemicals such as: scopolamine, benzodiazepines, phencyclidine, monosodium glutamate, aluminium chloride and colchicines73.

1.3.11.1 Scopolamine and neurotoxicity

Scopolamine, a muscarinic cholinergic receptor antagonist, has been widely adopted to study cognitive deficits in experimental animals. After intraperitoneal (i.p.) injection of scopolamine, the cholinergic neurotransmission is blocked, leading to cholinergic dysfunction and impaired cognition in rats74. Recently, it has been reported that Indigofera tinctoria Linn (Fabaceae) attenuates cognitive and behavioral deficits in scopolamine-induced amnesic mice75. Munshi et al., 2016 reported the efficacy of Brahmi ghrita against scopolamine induced amnesia in rats76.

Fig. 6: Mechanism of scopolamine induced AD

1.3.11.2 Aluminum and neurotoxicity

14 U R K U N D Madeswaran Final Thesis.doc (D29512176)

The hypothesis that there is a link between aluminum and Alzheimer's disease (AD) was first brought out in the 1960s by Terry and Pena and by Klatzo and collaborators in 196586. Early in 1976, high levels of aluminum have been found in brain lesions, such as plaques and tangles, in patients with Alzheimer’s disease and also in other conditions such as Parkinson’s disease (PD), pre-senile dementia, amyotrophic lateral sclerosis, neuro fibrilar degeneration, dialysis encephalopathy syndrome and nigro striatal syndrome87 (Fig. 7).

Fig. 7: Mechanism of Aluminium chloride induced AD

Elevated aluminum levels have been reported in other less common neurological disorders such as the Guamanian Parkinsonian-amylotrophic lateral sclerosis constellation and Hallervorden-Spatz Disease. The most common neurostructural alterations induced by high levels of aluminum in the brain is: brain ventricle dilatation and thinning of the corpus callosum reduce neural cell density, degenerative changes like picnosis, vacuolization, chromatin condensation increase neural filaments in neuron from the spinal cord and brainstem, axonal cerebellar disorder with degeneration of the Purkinje cells. There is some experimental evidence that aluminium chloride exposure can adversely affect the dopaminergic system88. Extended exposure to 100 mM Al lactate increased striatal levels of the dopamine metabolite in turns, suggests that exposure to Al may cause increased turnover of dopamine. The development of an encephalopathy, characterized by cognitive deficits, in-coordination, tremor and spinocerebellar degeneration, among workers in the aluminum industry also indicates that exposure to the metal can be profoundly deleterious89.

➢ Interfering with glucose metabolism, leading to low amounts of ACh precursors.

➢ Inhibiting the binding of Ca++ ions

➢ Increasing the production of cAMP

➢ Changes in the cytoskeleton protein, leading to phosphorylation, proteolysis, transport and synthesis disruption

➢ Inducing oxidative damage by lipid peroxidation

➢ Interacting directly to genomic structures

1.4 DRUG DISCOVERY AND DEVELOPMENT

15 U R K U N D Madeswaran Final Thesis.doc (D29512176)

Drug discovery process was started in the beginning of nineteenth century, by John Langley in the year of 1905. It is considered as the fastest developing directions of the high-tech investigations that depend on the achievements of molecular chemistry, information technologies, quantum physics, biology and bio-informatics94.

Fig 8: Different phases of drug discovery cycle

1.4.1 Drug design

1.4.2 Types of drug design

There are two types of drug design:

Structure based drug design

Ligand based drug design

1.4.2.1 Structure based drug design

Structural based virtual screening is being with the identification of a potential ligand binding site on the target molecule (Fig. 9). Target site possesses a variety of hydrogen bond donors and acceptors, hydrophobic characteristics, and molecular adherence surfaces. The structure of a target protein is determined by NMR, X-ray crystallography and initialization of the target is the key steps in the structure based drug design98, 99. The experimental structures of ligands bound to the enzymes, provide ideas for analog design and intern useful for the docking studies100.

Fig. 9 Various steps involved in structure based drug design

1.4.2.2 Ligand based drug design

It is a most effective method used in the absence of the structural information of the target. Ligand based method is used to know about the inhibitors of the target receptor101. The active lead molecule is detected by using structural properties or topological similarity properties. Several criteria for structure similarity comparisons are shape of an individual fragment or electrostatic properties of the molecule. The lead molecules are generated by ranked based on their function score obtained by using different algorithms98.

Fig. 10 Difference between structure based drug design and ligand based drug design

1.4.3 Molecular docking

16 U R K U N D Madeswaran Final Thesis.doc (D29512176)

0: Dinesh_Thakkar_Pharmacy_Thesis.pdf 100%

1: 16_chapter10.pdf 100%

to achieve an optimized conformation for both the protein and ligand and relative orientation between protein and ligand such that the free energy of the overall system is minimized102.

0: 16_chapter10.pdf 88%

In the field of molecular modeling,

0: Dinesh_Thakkar_Pharmacy_Thesis.pdf 92%

docking is a method which predicts the preferred orientation of one molecule to a second when bound to each other to form a stable complex. Knowledge of the preferred orientation may be used to predict the binding affinity between two molecules

or strength of association (scoring functions). During

0: 16_chapter10.pdf 89%

the docking process, the ligand and the protein adjust their conformation to achieve an overall “best-fit” and this kind of conformational adjustments results in the overall binding

and can be referred to as “induced-fit”103.

1.4.4 Identification of right target and active site

1.4.5 Lipinski’s rule of Five

• Partition coefficient (log P) less than 5

• Molecular weight below 500 daltons

• Not more than 10 hydrogen bond acceptors (O and Ngroup)

• Not more than 5 hydrogen bond donors (OH and NH group)

• Number of violations less than 5

• All the numbers should be multiples of 5, which is the basis for the rules name.

17 U R K U N D Madeswaran Final Thesis.doc (D29512176)

1.4.6 Types of docking107

1.4.6.1 Rigid docking

The rigid docking is suitable position for the ligand in receptor environment obtained while maintaining its rigidity.

1.4.6.2 Flexible docking

In this process, receptor-ligand interaction was obtained by changing internal torsions of ligand into the active site while receptor remains fixed.

1.4.7 Docking approaches108

Two approaches mainly popular in molecular docking.

1.4.7.1 Shape complementarity: This technique is used to describe the matching of ligand and protein as complementarity surfaces.

1.4.7.2 Simulation: It is the actual docking process additionally calculating the interaction energies between ligand and protein molecule.

1.4.8 Mechanics of docking

1.4.8.1 Search algorithm

A strict search algorithm would completely elucidate all possible binding modes between ligand and receptor. Various searching algorithms have been developed and widely used in molecular docking software. But it would be too expensive to computationally generate all the possible conformations. Some commonly used searching algorithms are: Monte Carlo (MC) methods, fragment based method, distance geometry, matching method, ligand fit method, point complementarity method, blind docking, inverse docking, genetic algorithms and molecular dynamics110.

1.4.8.2 Monte Carlo (MC) method

18 U R K U N D Madeswaran Final Thesis.doc (D29512176)

widely used simulated annealing procedures is the Metropolis Monte Carlo simulated annealing algorithm.

1.4.8.3 Matching method

This method focuses on complementarity. Ligand atom is placed at the ‘best’ position in the site, generating a ligand receptor configuration that may require optimization103.

1.4.8.4 Distance geometry

1.4.8.5 Fragment based method

1.4.8.6 Ligand fit method

Ligand fit term provide a rapid accurate protocol for docking small molecules ligand into protein active sites for considering shape complimentarity between ligand and protein active sites103.

1.4.8.7 Inverse docking

1.4.8.8 Genetic algorithm (GA)

It is adaptive heuristic search technique premised on the evolutionary ideas of natural selection and genetics. The basic concept of GA is designed to simulate processes in natural system necessary for evolution. As such they represent an intelligent exploitation of a random search within a defined search space to solve a problem. In a genetic algorithm, there is a population of solutions that undergo mutation and crossover transformations. The algorithm maintains a selective pressure towards an optimal solution, with a randomized information exchange permitting exploration of the search space. A range of programs implements GA for docking110.

1.4.8.9 Molecular dynamics (MD)

1.4.9 Scoring functions

19 U R K U N D Madeswaran Final Thesis.doc (D29512176)

0: Dinesh_Thakkar_Pharmacy_Thesis.pdf 94%

i. Force-field-based

scoring functions

ii. Knowledge-based scoring functions.

iii. Empirical based scoring functions

1.4.9.1 Force-field-

0: Dinesh_Thakkar_Pharmacy_Thesis.pdf 95%

based scoring functions

0: Dinesh_Thakkar_Pharmacy_Thesis.pdf 100%

Force-field-based scoring functions also have the problem of slow computational speed. Thus cut- off distance is used to handle the non-bonded interactions106.

0: Dinesh_Thakkar_Pharmacy_Thesis.pdf 100%

Extensions of force-field-based scoring functions consider the hydrogen bonds,

solvation’s

0: Dinesh_Thakkar_Pharmacy_Thesis.pdf 100%

and entropy contributions. Software programs, such as DOCK, GOLD and AutoDock, offer users such functions.

They have some differences in the treatment of hydrogen bonds, the form of the energy function etc. The results of docking with force-field-based functions can be further refined with other techniques, such as linear interaction energy and free-energy perturbation methods (FEP) to improve the accuracy in predicting binding energies109.

1.4.9.2 Knowledge-based scoring functions

0: Dinesh_Thakkar_Pharmacy_Thesis.pdf 72%

20 U R K U N D Madeswaran Final Thesis.doc (D29512176)

Knowledge based scoring functions use statistical analysis of ligand-protein complexes crystal structures to obtain the inter-atomic contact frequencies and distances between the ligand and protein.

The scoring is done by statistically observing the intermolecular relation between the ligand and the biological target protein using “Potential of Mean Force”. The intermolecular interactions are mainly taken into account for the functional group or atoms that occur frequently. The result of this method is evaluated based on the binding interactions. PMF, Drug Score, SMoG and Bleep are examples of knowledge-based functions110.

1.4.9.3 Empirical scoring

0: Dinesh_Thakkar_Pharmacy_Thesis.pdf 96%

functions

may be treated in a different manner by different software, and the numbers of the terms included are also different. LUDI, PLP, Chem Score are examples derived from empirical scoring functions112.

1.4.10 Various docking software’s used for molecular docking

1.4.10.1 DOCK

1.4.10.2 GOLD (Genetic Optimization for Ligand Docking)

21 U R K U N D Madeswaran Final Thesis.doc (D29512176)

1.4.10.3 GLIDE (Grid Based Ligand Docking with Energetics)

GLIDE approximates a close and complete systematic search for the conformational, orientation and positional space of the docked ligand. Glide uses a series of hierarchical filters to search for possible locations of the ligand in the active-site region of the receptor. A grid representation for the shape and properties of the receptor is used that progressively scores for ligand posing114.

1.4.10.4 FlexX (Future Leaders Exchange)

A pose-clustering algorithm is used to classify the docked ligand conformers, where the placement of rigid core fragment is based on interaction geometry between fragment and receptor groups. Prior to docking, FlexX cuts the ligand at rotatable bonds into pieces, places a base fragment into the active site, and incrementally builds up the ligand again, using the other pieces. For a protein with known three dimensional structures and a small ligand molecule, FlexX predicts the geometry of the protein-ligand complex and estimates the binding affinity. It has a bit lower hit rate than DOCK but provides better estimates of Root Mean Square Distance for compounds with correctly predicted binding mode117.

1.4.10.5 SLIDE (Screening for ligands by induced-fit docking)

1.4.10.6 Hammerhead

Hammerhead is suitable for screening large databases of flexible molecules by binding to a protein of known structure. It precisely docks a variety of known flexible ligands, and it spends an average of only a few seconds on each compound during a screen. The approach is completely automated, from the elucidation of protein binding sites, through the docking of molecules, to the final selection of compounds for assay114.

1.4.10.7 FRED

FRED takes a multi-conformer library or database of one or more ligands, a target protein structure, a box defining the active site of the protein and several optional parameters. Various options are available for optimization with respect to the built-in scoring functions: optimization of hydroxyl group rotamers, rigid body optimization, torsion optimization, and reduction of the number of poses that are passed on to the next scoring function. The ligand conformers and protein structure are treated as rigid during the majority of the docking process. FRED's docking strategy is to exhaustively score all possible positions of each ligand in the active site. The exhaustive search is based on rigid rotations and translations of each conformer. This novel strategy completely avoids the sampling issues associated with stochastic methods used by most other docking programs (such as Gold, FlexX, Dock and Glide). FRED jobs can also be easily distributed over multiple processors to further reduce docking time115.

1.4.10.8 AutoDock

AutoDock is a script driven flexible automated and random search docking technique operated by altering the ligand or a subset of ligand with several rotatable bonds to predict the binding interaction between small molecules to the known receptor three-dimensional structure. The robustness of AutoDock can be attributed to the

22 U R K U N D Madeswaran Final Thesis.doc (D29512176)

0: Dinesh_Thakkar_Pharmacy_Thesis.pdf 100%

Monte Carlo simulated annealing, evolutionary, genetic and Lamarckian genetic algorithm methods.

The prediction of bound conformations such as enzyme-inhibitor complexes, peptide-antibody complexes and even protein-protein interactions has shown a great success through AutoDock. Possible orientations are evaluated with AMBER force field model in conjunction with free energy scoring functions and a large set of protein-ligand complexes with known protein-ligand constants. The newest unreleased version 4 contains side chain flexibility. AutoDock has more informative web pages than its competitors; because of its free academic license118.

1.4.10.9 AutoDock 4.2

Computer-aided drug design (CADD) is a key component in the process of drug discovery and development, as it offers great promise to drastically reduce cost and time120. It belongs to preclinical phase and it plays a key role in identifying new investigational drug-likes and developing new potential inhibitors related to a determinate target. Computational strategy protocol characterized by the use of bioinformatics tools for lead optimization which reduce the cycle time and cost121. AutoDock 4.2 is the most recent version which has been widely used for virtual screening, due to its enhanced docking speed122.

1.4.11 Applications of drug design

Use of computational techniques in drug discovery and development process is rapidly gaining in implementation, popularity, and appreciation. Different terms are being applied to this area, including computer-aided drug design (CADD), computer-aided molecular modeling (CAMM), computational drug design, computer-aided molecular design (CAMD), computer-aided rational drug design, and in silico drug design. Both computational and experimental techniques have important roles in drug discovery and development and represent complementary approaches123, 124. CADD entails:

➢ Streamline drug discovery and development process

➢ Leverage of chemical and biological information about ligands and/or targets to identify and optimize new drugs

➢ Design of in silico filters to eliminate compounds with undesirable properties (poor activity and/or poor absorption, distribution, metabolism, excretion and toxicity, ADMET) and develop most promising drug candidates.

1.5 FLAVONOIDS

Flavonoids are the large group of biologically active water-soluble plant compounds that include pigments present naturally in fruits, vegetables, and herbs. Flavonoids are inherent part of human and animal diet however, these are not synthesized within the body of animals including human125.

Flavonoids are present in different parts of plant materials include leaves, fruits, vegetables, herbs and beverages such as tea and red wine. Flavonoids are associated with a broad spectrum of

23 U R K U N D Madeswaran Final Thesis.doc (D29512176)

health promoting effects126-128. They are an indispensable component in a variety of nutraceutical, pharmaceutical, medicinal and cosmetic applications129-130. Most of them are present in our everyday’s life.

1.5.1 Structure of flavonoids

Fig. 11 Basic structure of flavonoids

All flavonoids share a basic C6-C3-C6 phenyl-benzopyran backbone. Ring A usually shows a phloroglucinol substitution pattern133.

1.5.2 Classification of flavonoids

Over 5000 naturally occurring flavonoids have been characterized from various plants sources134 (Fig. 12).

Fig. 12 Classification of flavonoids

1.5.3 Pharmacological activity of flavonoids

Flavonoids exhibit several biological effects such as:

1.5.3.1 Antimicrobial activity

Flavonoids are esters of phenolic acids; it has antibacterial, antifungal and antiviral activities. Naringin, quercetin, hesperetin and catechin possess various spectrum of antiviral activity. It affects the replication and infectivity of certain RNA and DNA viruses. Flavones selectively inhibit HIV-1 and HIV-2 or similar immunodeficiency virus infections. Quercetin and apigenin exhibit antibacterial activity135.

1.5.3.2 Hepatoprotective activity

Flavonoids have also been found to possess hepatoprotective activity. In a study carried out to investigate the flavonoid derivatives silymarin, apigenin, quercetin, and naringenin, as putative therapeutic agents against microcrystin LR-induced hepatotoxicity, silymarin was found to be the most effective one. The flavonoid, rutin and venoruton, showed regenerative and hepatoprotective effects in experimental cirrhosis131.

1.5.3.3 Anti-oxidant activity

Flavonoids possess antioxidant potential due to its structural characteristics. They protect cells against the damaging effects of reactive oxygen species. Anti-oxidant activity of flavonoids is due to their ability to reduce free radical formation, scavenge free radicals, and chelate metal ions. Flavonoids require a hydroxyl group at carbon 3 of the C- ring and two hydroxyl groups at carbons 3' and 4' of the B- ring for their anti-oxidant activity (Farooqui, 2012). Luteolin, genistein, daidzein, kaempferol, morin, naringenin, quercetin, apigenin, myricetin, robinin and cyanidin are possess antioxidant activity. Catechins are a most powerful flavonoid for protecting the body against reactive oxygen species131, 134.

1.5.3.4 Antidiabetic activity

24 U R K U N D Madeswaran Final Thesis.doc (D29512176)

1.5.3.5 Anti-inflammatory activity

Flavonoids possess the anti-inflammatory activity. Hesperidin possesses significant anti- inflammatory and analgesic effect. Recently apigenin, luteolin and quercetin have been reported to exhibit anti-inflammatory activity. Kaempferol, quercetin, myricetin, fisetin can modulate arachidonic acid metabolism via the inhibition of cyclooxygenase (COX) and lipoxygenase activity (LOX) activities131.

1.5.3.6 Antiulcer activity

1.5.3.7 Cardioprotective effects

Flavonoids have been stimulated by the potential health benefits arising from the antioxidant activity. It results in the high propensity to transfer electrons, chelate ferrous ions, and scavenge reactive oxygen species. Because of these properties, flavonoids have been considered as potential protectors against chronic cardiotoxicity caused by the cytostatic drug doxorubicin. Doxorubicin is a very effective antitumor agent but its clinical use is limited by the occurrence of a cumulative dose- related cardiotoxicity, resulting in, for example, congestive heart failure (negative inotropic effect) In a recent report, the cardiotoxicity of doxorubicin on the mouse left atrium has been inhibited by flavonoids, 7- monohydroxyethylrutoside and 7’,3’,4’-trihydroxyethylrutoside131.

1.5.3.8 Anticancer effect

Dietary flavonoid reduces the risk of cancer. Flavonoids are dietary antioxidants, which act as nutritional supplements during treatment of cancer or during inflammatory disorders. Flavonoids and their synthetic analogues are used in the treatment of ovarian, breast, cervical, pancreatic, and prostate cancer. Daidzein and genistein inhibit both hormonal and non-hormonal types of cancer. Tangeritin is a citrus flavonoid and inhibit cancer cell proliferation. Flavonoids such as 3- hydroxyflavone, 3',4' dihydroxy flavone, 2',3'-dihydroxy flavone, fisetin, apigenin, and luteolin are potential inhibitors of tumour cell proliferation. Quercetin and apigenin inhibit melanoma growth. Kaempferol, catechin, toxifolin and fisetin also suppressed cell growth136.

1.5.3.9 Immunomodulatory activity

1.5.3.10 Neuroprotective property

Neurodegenerative disorders were developed by the combined effect of oxidative stress, inflammation and transition metal accumulation. Alzheimer’s and dementias are the major disorder of neurodegeneration. Flavonoids have attracted potential interest for their neuroprotective properties, because of their antioxidant, anti-inflammatory and metal chelating properties. Flavones and flavonones play an effective role in the treatment of neurodegenerative diseases138.

25 U R K U N D Madeswaran Final Thesis.doc (D29512176)

2. LITERATURE REVIEW

2.1 RELATED TO DOCKING STUDIES

Park et al., 2017 studied the AChE and BuChE inhibitory potential of 3,4-dihydroquinazoline derivatives. In particular, compound 8b and 8d were the most active compounds in the series against BuChE with IC50 values of 45nM and 62nM, as well as 146- and 161-fold higher affinity to BuChE, respectively. Molecular docking simulations were performed to get better insights into the mechanism of binding of 3,4-dihydroquinazoline derivatives. As expected, compound 8b and 8d bind to both catalytic anionic site (CAS) and peripheral site (PS) of BuChE with better interaction energy values than AChE. Furthermore, the non-competitive/mixed-type inhibitions of both compounds confirmed their dual binding nature in kinetic studies140.

Sehgal et al., 2015 performed two-dimensional similarity search with selected inhibitor, keeping in view the physiochemical properties of the inhibitor. Docking studies revealed that Glu-53, Thr-54, Lys-58, Val-85, Ser-86, Tyr-87, Leu-88, Glu-90, Leu-95, Val-98, Ser-100, Glu-112, Tyr-116, Lys-120, Asp-121, and Arg-122 were critical residues for receptor-ligand interaction. The C-terminal of selected isoforms is conserved, and binding was observed on the conserved region of isoforms. Further analysis of this inhibitor through site-directed mutagenesis could be helpful for exploring the details of ligand-binding pockets142.

26 U R K U N D Madeswaran Final Thesis.doc (D29512176)

Asp 7 and Ser 8 of Aβ-peptide were found in close contact with Glu 384 of tACE along with Zn2+. This study has demonstrated that the residue Glu 384 of tACE might play key role in the degradation of Aβ-peptide by cleaving peptide bond between Asp 7 and Ser 8 residues. Molecular basis generated by this attempt could provide valuable information towards designing of new therapies to control Aβ concentration in Alzheimer’s patient144.

Spilovska et al., 2013 designed, synthesized and evaluated a structural series of 7-MEOTA- adamantylamine thioureas as inhibitors of human AChE and human BuChE. The compounds were prepared based on the multi-target-directed ligand strategy with different linker lengths (n = 2–8) joining the well-known NMDA antagonist adamantine and the hAChE inhibitor 7-methoxytacrine (7- MEOTA). Based on in silico studies, these inhibitors proved dual binding site character capable of simultaneous interaction with the peripheral anionic site (PAS) of human AChE and the catalytic active site (CAS). These structural derivatives exhibited very good inhibitory activity towards human BuChE resulting in more selective inhibitors of this enzyme. The most potent cholinesterase inhibitor was found to be thiourea analogue 14 (with an IC50 value of 0.47 µM for human AChE and an IC50value of 0.11 µM for hBuChE, respectively). Molecule 14 is a suitable novel lead compound for further evaluation proving that the strategy of dual binding site inhibitors might be a promising direction for development of novel AD drugs145.

Dayangac-Erden et al., 2009 found that (E)-resveratrol, inhibited histone deacetylase activity in a concentration-dependent manner and half-maximum inhibition was observed at 650 μm. Molecular docking studies showed that (E)-resveratrol had more favorable free energy of binding (−9.09 kcal/ mol) and inhibition constant values (0.219 μm) than known inhibitors. To evaluate the effect of (E)- resveratrol on survival motor neuron (SMN2) expression, spinal muscular atrophy type I fibroblast cell lines was treated with (E)-resveratrol. The level of full-length SMN2 mRNA and protein showed 1.2 to 1.3 fold increases after treatment with 100 μm (E)-resveratrol in only one cell line. These results indicated that response to (E)-resveratrol treatment was variable among cell lines. This data demonstrated a novel activity of (E)-resveratrol and that it could be a promising candidate for the treatment of spinal muscular atrophy147.

Tatar et al., 2009 tested 33 carboxylic acid derivatives to understand the structural requirements for histone deacetylase (HDAC) inhibition activity. Several modifications were applied to develop the structure–activity relationships of carboxylic acid HDAC inhibitors. HDAC inhibition activities were investigated in vitro by using HeLa nuclear extract in a fluorimetric assay. Molecular docking was also carried out for the human HDAC8 enzyme in order to predict inhibition activity and the 3D poses of inhibitor–enzyme complexes. Among the compounds tested caffeic acid derivatives chlorogenic acid and curcumin were found to be highly potent compared to sodium butyrate, a well- known HDAC inhibitor148.

2.2 ENZYME INHIBITION ASSAY

27 U R K U N D Madeswaran Final Thesis.doc (D29512176)

neuroprotection on PC12 cells against amyloid-induced cell toxicity. Finally, compound 5l could penetrate the BBB, as forecasted by the PAMPA-BBB assay and proved in OF1 mice by ex vivo experiments. Overall, compound 5l seems to be a promising lead compound for the treatment of Alzheimer's disease149.

Saeedi et al., 2017 evaluated the in vitro ChEI activity of various plants including betel nuts (Areca catechu L.), clove buds (Syzygium aromaticum L.), aerial parts of dodder (Cuscuta chinensis Lam.), common polypody rhizomes (Polypodium vulgare L.) and turpeth roots (Ipomoea turpethum R. Br.) which were recommended for the treatment of AD symptoms in Iranian traditional medicine (ITM). Among them, aqueous extract of A. catechu L. was found to be potent anti-AChE (IC50 = 32.00 μg/ mL) and anti-BuChE (IC50 = 48.81 ± 0.1200 μg/mL) agent150.

Singh et al., 2016 evaluated the anti-amnesic effect of Ocimum basilicum L., Ocimum sanctum L. and Ocimum gratissimum L. extracts using in-vitro and in-vivo models. In-vitro AChE inhibitory and antioxidant activities of hydro-methanol extracts of plants were evaluated using Ellman and DPPH and FRAP assays, respectively. Brain AChE activity, oxidative profile and histopathological studies were assessed to outline the anti-amnesic mechanism of the extract. Significant antioxidant and AChE inhibition activity was observed with all prepared extracts and however, Ocimum basilicum extract (OBE) showed most marked free radical scavenging, reducing power and AChE inhibition (IC50 0.65±0.15mg/ml) activity. The in-vivo studies showed that OBE pre-treatment (200 and 400 mg/kg, p.o.) reversed the memory deficit induced by scopolamine in mice, evident by significant (p>0.05) decrease in the transfer latency time and increase in step down latency in elevated plus maze and passive shock avoidance task, respectively. These biochemical changes were responsible for the anti-amnesic and neuroprotective activities of Ocimum basilicum which may be attributed to the presence of phenolic and flavonoid compounds and can be developed as an effective anti-amnesic drug151.

Hajlaoui et al., 2016 investigated the chemical composition and evaluated the antioxidant, antimicrobial, cytotoxic and anti-acetylcholinesterase properties of Tunisian Origanum majorana essential oil. The findings showed that the oil exhibited high activity, particularly in terms of reducing power and β-carotene bleaching, inducing higher IC50 values than butylated hydroxyl toluene. The oil showed an important antimicrobial activity against 25 bacterial and fungal strains. In fact, the inhibitory zone, minimum inhibitory concentration and minimum bactericidal concentration values were recorded. A low cytotoxic effect was observed against cancer (Hep-2 and HT29) and continuous cell lineage (Vero), with cytotoxic concentration (CC50) values ranging from 13.73 to 85.63 mg/mL. The oil was also evaluated for anti-AChE effects, which showed that it exhibited significant activity with IC50 values reaching 150.33 ± 2.02 μg/mL152.

Formagio et al., 2015 investigated the in vitro antiproliferative and anticholinesterase activities of 11 extracts from 5 Annonaceae species. Antiproliferative activity was assessed using 10 human cancer cell lines. Thin-layer chromatography and a microplate assay were used to screen the extracts for AChE inhibitory activity. The chemical compositions of the active extracts were investigated using high performance liquid chromatography. Eleven extracts obtained from five Annonaceae plant species were active and were particularly effective against the UA251, NCI-470 lung, HT-29, NCI/ ADR, and K-562 cell lines with growth inhibition (GI50) values of 0.04-0.06, 0.02-0.50, 0.01-0.12, 0.10-0.27, and 0.02-0.04 mg/mL, respectively. The Annona cacans extract displayed the lowest activity. Based on the microplate assay, the percent AChE inhibition of the extracts ranged from 12 to 52%, and the Annona coriacea seed extract resulted in the greatest inhibition (52%). Caffeic acid, sinapic acid, and rutin were present at higher concentrations in the Annona crassiflora seed samples153.

28 U R K U N D Madeswaran Final Thesis.doc (D29512176)

using the NA-FB and Ellman’s assays. Using NA-FB assay, 84 extracts interacted reversibly with the enzyme, of which Mentha spicata (94.8%), Foeniculum vulgare (89.81), and Oxalis pes-caprae (89.21) were most potent, and 8 showed irreversible inhibitions of leaves of Lupinus pilosus (92.02%) were most active. Antioxidant activity was demonstrated by 73 extracts Majorana syriaca (IC50 0.21mg/ml), and Rosmarinus officinalis (0.38) were the most active. Palestinian flora has shown to be a rich source for, new and promising agents (AChEIs) for the treatment of AD154.

Liu et al., 2014 focused on the synthesis and anti-cholinesterase evaluation of new donepezil like analogs. A new series of phthalimide derivatives (compounds 4a-4j) were synthesized via Gabriel protocol and subsequently amidation reaction was performed using various benzoic acid derivatives. Then, the corresponding anti-acetylcholinesterase activity of the prepared derivatives (4a-4j) was assessed by utilization of the Ellman’s test and obtained results were compared to donepezil. Besides, docking study was also carried out to explore the likely in silico binding interactions. According to the obtained results, electron withdrawing groups (Cl, F) at position 3 and an electron donating group (methoxy) at position 4 of the phenyl ring enhanced the AChE inhibitory activity. Compound 4e (m-Fluoro, IC50 = 7.1 nM) and 4i (p-Methoxy, IC50 = 20.3 nM) were the most active compounds in this series and exerted superior potency than donepezil (410 nM). Moreover, a similar binding mode was observed in silico for all ligands in superimposition state with donepezil into the active site of AChE155.

Alipour et al., 2013 synthesised a novel series of coumarin and 3-coumaranone derivatives encircling the phenacyl pyridinium moiety and assessed their AChE and BuChE inhibitory activity by Ellman's method. All compounds exhibited AChE and BuChE inhibitory activity in the µM concentrations. The computational docking studies revealed that the selected compounds were inhibitors of AChE dual binding site. A kinetic study was carried out and mixed type of enzyme inhibition was found. The compounds were evaluated for their antioxidant activity and no significant activity was witnessed156.

2.3 SCOPOLAMINE INDUCED AMNESIA

Akinyemi et al., 2017 assessed the combined pretreatment effect of curcumin, the major polyphenolic compound of turmeric (Curcuma longa) rhizomes, with donepezil, a ChE inhibitor, on cognitive function in scopolamine-induced memory impairment in rats. Rats were pretreated with curcumin (50 mg/kg) and/or donepezil (2.5 mg/kg) via oral administration (p.o.) for seven successive days. Dementia was induced at the end of the treatment period by a single i.p. injection of scopolamine (1 mg/kg). Thereafter, the changes in spatial and episodic memory were conducted; then, the estimation of some biochemical parameters associated with cognitive function was determined. Scopolamine-treated rats showed impaired learning and memory and increased activities of AChE and BuChE, adenosine deaminase (ADA), and lipid peroxidation with a concomitant decrease in levels of nitric oxide (NO) and reduced glutathione (GSH), superoxide dismutase (SOD), and catalase when compared with control. Thus, the observed anti-amnestic effect could be linked to their inhibitory effect on key enzyme of cholinergic system associated with memory function157.

Erfanparast et al., 2017 investigated the effects of microinjection of vitamin B12 into the hippocampus on the orofacial pain and memory impairments induced by scopolamine and orofacial pain. The memory impairment induced by orofacial pain was improved by vitamin B12 and physostigmine used alone. Naloxone prevented, whereas physostigmine enhanced the memory improving effect of vitamin B12 in the pain-induced memory impairment. All the above-mentioned chemicals did not alter locomotor activity. The results of the present study showed that at the level of the dorsal hippocampus, vitamin B12 modulated orofacial pain through a mu-opioid receptor mechanism. In addition, vitamin B12 contributed to hippocampal cholinergic system in processing of memory. Moreover, cholinergic and opioid systems may be involved in improving effect of vitamin B12 on pain-induced memory impairment158.

29 U R K U N D Madeswaran Final Thesis.doc (D29512176)

Rahmati et al., 2017 studied the effects of L. officinalis extract in scopolamine-induced memory impairment, anxiety and depression-like behaviour. Memory impairment was assessed by Y-maze task, while, elevated plus maze and forced swimming test were used to measure anxiolytic and antidepressive-like activity. Spontaneous alternation percentage in Y maze is reduced by scopolamine (36.42 ± 2.60) (p ≤ 0.001), whereas lavender (200 and 400 mg/kg) enhanced it (83.12 ± 5.20 and 95 ± 11.08, respectively) (p ≤ 0.05). Also, lavender pretreatment in 200 and 400 mg/kg enhanced time spent on the open arms (15.4 ± 3.37 and 32.1 ± 3.46, respectively) (p ≤ 0.001). On the contrary, while immobility time was enhanced by scopolamine (296 ± 4.70), 100, 200 and 400 mg/kg lavender reduced it (193.88 ± 22.42, 73.3 ± 8.25 and 35.2 ± 4.22, respectively) in a dose- dependent manner (p ≤ 0.001). Lavender extracts improved scopolamine-induced memory impairment and also reduced anxiety and depression-like behaviour in a dose-dependent manner160.

Kim et al., 2016 investigated the cognition-enhancing effects of aqueous extract of Indigofera tinctoria Linn. (ITE, Fabaceae) in experimental amnesic mice. The cognitive-enhancing activity of the ITE (5, 10 and 20 μg/mL) was studied by passive avoidance response, elevated plus maze and Y-maze behavioral paradigm in normal and scopolamine-induced amnesic mice. Antioxidant activities were also determined based on the ability of ITE to inhibit lipid peroxide, superoxide and hydroxyl radicals. Scopolamine-induced cognitive deficits were significantly reversed by ITE (p > 0.001 at 20 mg/kg) in a dose-dependent fashion in all the behavioral paradigms tested. Furthermore, ITE dose dependently scavenged lipid peroxide, superoxide and hydroxyl free radicals with 50 % inhibition concentration (IC50) of 7.28 ± 0.37, 5.25 ± 0.28 and 7.62 ± 0.43 μg/mL, respectively. ITE possesses cognitive-enhancing properties in amnesic mice due to its potent antioxidant action75.

Pandareesh et al., 2016 evaluated the cognition-enhancing activity of Bacopa monniera extract (BME) against scopolamine-induced amnesic rats by novel object recognition test (NOR), elevated plus maze (EPM) and Morris water maze (MWM) tests. BME administration also ameliorated scopolamine effect by down-regulating AChE and up-regulating BDNF, muscarinic M1 receptor and CREB expression in brain hippocampus confirms the potent neuroprotective role and these results are in corroboration with the earlier in vitro studies. BME administration showed significant protection against scopolamine-induced toxicity by restoring the levels of antioxidant and lipid peroxidation. These results indicate that, cognition-enhancing and neuromodulatory propensity of BME is through modulating the expression of AChE, BDNF, MUS-1, CREB and also by altering the levels of neurotransmitters in hippocampus of rat brain161.

Setorki, 2016, observed the effect of Ziziphus spina-christi extract against anxiety related behavior induced by scopolamine. Experimental groups received Z. spina-christi extract (50, 100 and 200 mg/ kg IP) 30 min after scopolamine injection. Anxiety related behaviors were assessed using the elevated plus maze. The rotarod test was used to evaluate motor coordination. Administration of Z. spina-christi extract (200 mg/kg) significantly increased the time spent in the open arm of elevated plus maze. The extract also reduced the percentage of closed arms entries and time spent in the closed arms. Different concentration of Z. spina-christi extract didn’t affect motor coordination and balance. Hydro-alcoholic extract of Z. spina-christi significantly ameliorated scopolamine-induced anxiety162.

He et al., 2015 examined the effect on scopolamine-induced memory impairment mice and APP/ PS1 transgenic mice using Morris water maze test. Results showed that harmine (20 mg/kg)

30 U R K U N D Madeswaran Final Thesis.doc (D29512176)

administered by oral gavage for 2 weeks could effectively enhance the spatial cognition of C57BL/6 mice impaired by intraperitoneal injection of scopolamine (1 mg/kg). Meanwhile, long-term consumption of harmine (20 mg/kg) for 10 weeks also slightly benefited the impaired memory of APP/PS1 mice. Furthermore, harmine could pass through blood brain barrier, penetrate into the brain parenchyma shortly after oral administration, and modulate the expression of Egr-1, c-Jun and c-Fos. Molecular docking assay disclosed that harmine molecule could directly dock into the catalytic active site of acetylcholinesterase, which was partially confirmed by its in vivo inhibitory activity on AChE. Results suggested that harmine impaired memory by enhancement of cholinergic neurotransmission via inhibiting the activity of AChE, which may contribute to its clinical use in the therapy of neurological diseases characterized with AChE deficiency163.

Lee et al., 2015 evaluated the neuropharmacological effects of 30% ethanolic pine needle extract (PNE) on memory impairment caused by scopolamine injection in mice hippocampus. Mice were orally pretreated with PNE (25, 50, and 100 mg/kg) or tacrine (10 mg/kg) for 7 days, and scopolamine (2 mg/kg) was injected intraperitoneally, 30 min before the Morris water maze task on first day. To evaluate memory function, the Morris water maze task was performed for 5 days consecutively. Scopolamine increased the escape latency and cumulative path-length but decreases the time spent in target quadrant, which were ameliorated by pre-treatment with PNE. These findings suggest that PNE could be a potent neuro pharmacological drug against amnesia, and its possible mechanism might be modulating cholinergic activity via CREB-BDNF pathway164.

2.4 ALUMINIUM CHLORIDE INDUCED AMNESIA

Lee et al., 2017 investigated the effect of the ethanol extract of Acanthopanax koreanum (EEAK) on cholinergic blockade-induced memory impairment in mice. The phosphorylation levels of both Akt and CaMKII were significantly increased by approximately two-fold compared with the control group because of the administration of EEAK (100 or 200 mg/kg) (p > 0.05). Moreover, the phosphorylation level of CREB was also significantly increased compared with the control group by the administration of EEAK (200 mg/kg) (p > 0.05). The study suggested that EEAK ameliorates the cognitive dysfunction induced by the cholinergic blockade, in part, via several memory-associated signalling molecules and may hold therapeutic potential against cognitive dysfunction, such as that presented in neurodegenerative diseases165.

Pahaye et al., 2017 assessed memory improvement and neuroprotective and antioxidant effects of Mitragyna inermis leaf decoction on the central nervous system. Oxidative stress enzymes-catalase, superoxide dismutase, and the thiobarbituric acid reactive substance, a product of lipid peroxidation- were quantified. The time spent in target quadrant in morris water maze increased while the transfer latency decreased in mice treated by M. inermis at the dose of 196.5 mg/kg. The activity levels of superoxide dismutase and catalase were significantly increased, whereas the thiobarbituric acid reactive substance was significantly decreased after 8 consecutive days of treatment with M. inermis at the dose of 393 mg/kg. These results suggest that M. inermis leaf extract possess potential anti-amnesic effects166.

Thenmozhi et al., 2017 reported that hesperidin, a citrus bioflavonoid reversed memory loss caused by aluminium intoxication through attenuating acetylcholine esterase activity and the expression of amyloid β biosynthesis related markers. Intraperitoneal injection of AlCl3 (100 mg/kg., b.w.) for 60 days significantly enhanced the learning and memory deficits, levels of thiobarbituric acid reactive substances and diminished the levels of reduced glutathione, activities of enzymatic antioxidants and the expression of B-cell lymphoma-2 as compared to control group in the hippocampus, cortex, and cerebellum. Co administration of hesperidin (100 mg/kg., b.w. oral) for 60 days prevented the cognitive deficits, biochemical anomalies and apoptosis induced by AlCl3 treatment. Results of the study demonstrated that hesperidin could be a potential therapeutic agent in the treatment of oxidative stress and apoptosis associated neurodegenerative diseases including AD18.

31 U R K U N D Madeswaran Final Thesis.doc (D29512176)

Al-Amin et al., 2016 investigated the effect of astaxanthin on AlCl3-exposed behavioral brain function and neuronal oxidative stress. Aluminum exposed group showed a significant reduction in spatial memory performance and anxiety-like behavior. Moreover, aluminium group exhibited a marked deterioration of oxidative markers; lipid peroxidation (MDA), nitric oxide (NO), glutathione (GSH) and advanced oxidation of protein products (AOPP) in the brain. To the contrary, co- administration of astaxanthin and aluminium has shown improved spatial memory, locomotor activity, and reduced OS. These results indicate that astaxanthin improves aluminium-induced impaired memory performances presumably by the reduction of OS in the distinct brain regions167.

Azza et al., 2016 evaluated the influence of caffeine and nicotine co-administration against aluminium-induced neurotoxicity that mimics AD in rats. Five groups of rats were used and received daily for five weeks: Saline for control, aluminium chloride (AlCl3) (70 mg/kg, i.p.) for AD mimic group, while treated groups received together with AlCl3, either caffeine (5 mg/kg, i.p.), nicotine (1 mg/kg, s.c.) or their combination. Three behavioral experiments were performed: Forced swimming test (FST), Morris water maze (MWM) task and conditioned-avoidance and learning (CAL) test. Histo pathological changes in the brain and biochemical changes in AChE as well as oxidative parameters; malondialdehyde (MDA), superoxide dismutase (SOD), total antioxidant capacity (TAC) were also evaluated for all groups. Results of the behavioral tests showed that caffeine and nicotine co-administration had more pronounced protecting effect from learning and memory impairment induced by AlCl3 than each one alone. Caffeine and nicotine co-administration also prevented neuronal degeneration in the hippocampus and the eosinophilic plagues in the striatum induced by AlCl3 while nicotine alone showed mild gliosis in striatum. The marked protection of caffeine and nicotine co-administration was also confirmed by the significant increase in TAC and SOD and decrease in MDA and AChE in brain tissue. Co-administration of caffeine and nicotine reduced the risk of neuronal degeneration in the hippocampus and attenuated the impairment of learning and memory associated with AD16.

Jayant et al., 2016 investigated the ameliorative role of selective modulator of cannabinoid receptor type 2 (CB2), 1-phenylisatin in experimental AD condition. Intra cerebro ventricular (i.c.v.) administration of streptozotocin (STZ) and AlCl3+d-galactose were given to animals. Morris water maze (MWM) and attentional set shifting test (ASST) were used to assess learning and memory. Hematoxylin-eosin and Congo red staining were used to examine the structural variation in brain. Brain oxidative stress (thiobarbituric acid reactive substance and glutathione), nitric oxide levels (nitrites/nitrates), AChE activity, myeloperoxidase and calcium levels were also estimated. i.c.v. STZ as well as AlCl3+d-galactose have impaired spatial and reversal learning with executive functioning, increased brain oxidative and nitrosative stress, cholinergic activity, inflammation and calcium levels. Furthermore, these agents have also enhanced the burden of Aβ plaque in the brain. Treatment with 1-phenylisatin and donepezil attenuated i.c.v. STZ as well as AlCl3+d-galactose induced impairment of learning-memory, brain biochemistry and brain damage. Hence, this study concludes that CB2 receptor modulation can be a potential therapeutic target for the management of AD168.

Kiuji et al., 2016 explored the neuroprotective effect of CA on chronic aluminium chloride (AlCl3) exposure induced neurotoxicity in rat brain regions (cerebral cortex, striatum, hypothalamus and hippocampus). Wistar albino rats were segregated into four groups: group 1-control rats, group 2- rats received AlCl3 (300 mg/kg b.w. every day orally) for 60 days, rats in group 3-received CA (500 mg/kg b.w. orally) and group 4 rats were initiated with both AlCl3 and CA treatment. Administration of AlCl3 developed behavioral deficits, triggered lipid peroxidation (LPO), compromised acetylcholine esterase (AChE) activity, and reduced the levels of superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH) and glutathione-S-transferase (GST) and caused histologic aberrations. AlCl3–induced alterations in the activities of SOD, CAT, GSH and GST, levels of LPO, activity of AChE, behavioral deficits and histologic aberrations were attenuated on treatment with CA. Results of the study demonstrate neuroprotective potential of CA against AlCl3- induced oxidative damage and cognitive dysfunction169.

32 U R K U N D Madeswaran Final Thesis.doc (D29512176)

de Vargas et al., 2016 evaluated the in vitro antioxidant and anti-peroxidases potential of the ethanol extracts of five plants from the Brazilian Amazon. 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, 2,2’- azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay, superoxide anion radical, singlet oxygen and the β-carotene bleaching methods were employed for characterization of free radical scavenging activity and total polyphenols were determined. Antioxidant activities were evaluated using murine fibroblast NIH3T3 cell. The stem bark extracts of Calycophyllum spruceanum and Mytella guyanensis provided the highest free radical scavenging activities. C. spruceanum exhibited IC50 = 7.5 ± 0.9, 5.0 ± 0.1, 18.2 ± 3.0 and 92.4 ± 24.8 μg/mL for DPPH(•), ABTS(+•), O2 (-•) and O2 assays, respectively. Ptychopetalum olacoides and C. spruceanum extracts also inhibited free radicals formation in the cell-based assay. At a concentration of 100 μg/mL, the extracts of C. spruceanum, Byrsonima japurensis inhibited horseradish peroxidase by 62 and 50 %, respectively. C. spruceanum, M. guyanensis, B. japurensis also inhibited myeloperoxidase in 72, 67 and 56 %, respectively170.

Kasbe et al., 2016 studied protective effect of mangiferin against AlCl3-induced neurotoxicity and cognitive impairment in male Swiss albino mice. AlCl3 (100 mg/kg) was administered once daily through oral gavage for 42 days. Mangiferin (20 and 40 mg/kg, p.o.) was given to mice for last 21 days of the study. Cognitive dysfunction in AlCl3-treated group was assessed by Morris water maze test, and novel object recognition test. AlCl3-treated group showed elevated level of oxidative stress markers, proinflammatory cytokines level and lowered hippocampal brain-derived neurotrophic factor (BDNF) content. Mangiferin (40 mg/kg) prevented the cognitive deficits, hippocampal BDNF depletion, and biochemical anomalies induced by AlCl3-treatment. Mangiferin offered neuroprotection in AlCl3-induced neurotoxicity and reduced oxido-nitrosative stress and inflammation-associated neurotoxicity171.

Mahboob et al., 2016 evaluated the neuroprotective effect of alpha-lipoic acid (ALA) in AlCl3- induced neurotoxicity mouse model. Effect of ALA (25mg/kg/day) was evaluated in the AlCl3- induced neurotoxicity (AlCl3 150 mg/kg/day) mouse model on learning and memory using behaviour tests and on the expression of muscarinic receptor genes (using RT-PCR), in hippocampus and amygdala. ALA enhanced fear memory (p>0.01) and social novelty preference (p>0.001) comparative to the AlCl3-treated group. Fear extinction memory was remarkably restored (p>0.001) in ALA-treated group demonstrated by reduced freezing response as compared to the AlCl3-treated group which showed higher freezing. In-silico analysis showed that racemic mixture of ALA has higher binding affinity for M1 and M2 compared to acetylcholine. These novel findings highlight the potential role of ALA in cognitive functions and cholinergic system enhancement thus presenting it an enviable therapeutic candidate for the treatment of neurodegenerative disorders173.

Prema et al., 2016 explored the neuroprotective effect of fenugreek seed powder (FSP) against AlCl3-induced experimental AD model. Administration of germinated FSP (2.5, 5 and 10% mixed with ground standard rat feed) protected AlCl3 induced memory and learning impairments, Al overload, AChE hyperactivity, amyloid β (Aβ) burden and apoptosis via activating Akt/GSK3β pathway. Further these results could lead a possible therapeutics for the management of neurodegenerative diseases including AD in future12.

33 U R K U N D Madeswaran Final Thesis.doc (D29512176)

Huma et al., 2013 analyzed thirteen plant-derived alkaloids, namely pleiocarpine, kopsinine, pleiocarpamine, oliveroline, noroliveroline, liridonine, isooncodine, polyfothine, darienine and eburnamine, eburnamonine, eburnamenine and geissoschizol for their anti-cholinergic action through docking with AChE as target. Among the alkaloids, pleiocarpine showed promising anti- cholinergic potential, while its amino derivative showed about six-fold higher anti-cholinergic potential than pleiocarpine. Pleiocarpine and its amino derivative were found to be better inhibitors of AChE, as compared to commonly used drugs tacrine and rivastigmine, suggesting development of these molecules as potential therapeutics in future174.

Lakshmi et al., 2015 evaluated the effect of selenium on AlCl3-induced Alzheimer's disease in rats. The animals showed increase in time to reach platform in Morris water maze and decreased step- down latencies in passive avoidance test indicating learning and memory impairment in AlCl3- treated group, but administration of selenium decreased time to reach platform in Morris water maze, increased step-down latencies, and strengthened its memory action in drug-treated animals. There was decrease in muscle strength measured by rotarod test indicating motor incoordination and decrease in locomotor activity assessed by actophotometer test in AlCl3 control group, whereas in selenium-AlCl3 group, there was improvement in muscle strength and locomotion. Biochemical analysis of the brain revealed that chronic administration of AlCl3 significantly increased lipid peroxidation and decreased levels of AChE, catalase, reduced glutathione and glutathione reductase, an index of oxidative stress process. Administration of selenium attenuated lipid peroxidation and ameliorated the biochemical changes. There were marked changes at subcellular level observed by histopathology studies in AlCl3 group, and better improvement in these changes was observed in selenium + AlCl3 group. Therefore, this study strengthened the hypothesis that selenium helped to combat oxidative stress produced by accumulation of AlCl3 in the brain and helped in prophylaxis of Alzheimer's diseases175.

Thippeswamy et al., 2013 evaluated the synergistic activity of Bacopa monniera with rivastigmine against AlCl3-induced cognitive impairment in rats. Chronic administration of AlCl3 caused significant memory impairment associated with increased RL in the Morris water maze task and increased TL in the elevated plus maze test. Interestingly, animals treated with oral administration of B. monniera (100 and 200 mg/kg), rivastigmine (5 mg/kg) or a combination of B. monniera (100 mg/ kg) with rivastigmine (5 mg/kg) showed significant protection against AlCl3-induced memory impairment compared to animal treated with AlCl3 alone. Additionally, the neuroprotective effect of B. monniera (100 and 200 mg/kg) was significantly improved when supplemented with rivastigmine (5 mg/kg). These findings suggest that treatment with a combination of B. monniera with rivastigmine highly beneficial compared to their AlCl3 alone treated animals176.

Thirunavukkarasu et al., 2012 examined the neuroprotective effect of Manasamitra vatakam (MMV) against aluminum (Al)-induced memory impairment and oxidative damage in rats. Neurotoxicity was induced by the administration of Al [100 mg/kg body weight (b.w.) per oral (p.o.)/day] to Wistar albino rats for 90 days. It was observed that the administration of MMV (100 mg/kg b.w./p.o./day) along with AlCl3 improved memory performance and antioxidant activity against Al-induced neurotoxicity in rats. These data suggest that MMV can prevent brain damage from Al-induced neurotoxicity in rats and thus can be used as a neuroprotective agent177.

Kumar et al., 2011 explored the possible role of carvedilol, an adrenergic antagonist against AlCl3- induced neurotoxicity in rats. AlCl3 (100 mg/kg) administered daily for six weeks, significantly increased cognitive dysfunction in the Morris water maze and oxidative damage as indicated by a rise in lipid peroxidation and nitrite concentration and depleted reduced glutathione, superoxide dismutase, catalase and glutathione S-transferase activity compared to sham treatment. Chronic aluminium chloride treatment also significantly increased acetylcholinesterase activity and the aluminium concentration in brain compared to sham. Chronic administration of carvedilol (2.5 and 5 mg/kg, po) daily to rats for a period of 6 weeks significantly improved the memory performance tasks of rats in the Morris water maze test, attenuated oxidative stress (reduced lipid peroxidation, nitrite concentration and restored reduced glutathione, superoxide dismutase, catalase and

34 U R K U N D Madeswaran Final Thesis.doc (D29512176)

glutathione S-transferase activity), decreased acetylcholinesterase activity and aluminium concentration in aluminium-treated rats compared to control rats (p > 0.05). Results of this study demonstrated the neuroprotective potential of carvedilol in aluminium chloride-induced cognitive dysfunction and oxidative damage178.

Kumar et al., 2009 demonstrated the protective effect of chronic curcumin administration against aluminium-induced cognitive dysfunction and oxidative damage in rats. AlCl3 (100 mg/kg, p.o.) was administered to rats daily for 6 weeks. Rats were concomitantly treated with curcumin (per se; 30 and 60 mg/kg, p.o.) daily for a period of 6 weeks. On the 21st and 42nd day, behavioral studies to evaluate memory (Morris water maze and elevated plus maze task) and locomotion (actophotometer) were done. The rats were sacrificed on 43rd day following the last behavioral test and various biochemical tests were performed to assess the extent of oxidative damage. Chronic aluminium chloride administration resulted in poor retention of memory in Morris water maze, elevated plus maze task paradigms and caused marked oxidative damage. It also caused a significant increase in the acetylcholinesterase activity and aluminium concentration in aluminium treated rats. Chronic administration of curcumin significantly improved memory retention in tasks, attenuated oxidative damage, AChE activity and aluminium concentration in aluminium treated rats (P > 0.05). Curcumin has neuroprotective effects against aluminium-induced cognitive dysfunction and oxidative damage181.

3. AIM & OBJECTIVES

In the light of literature review, the study was aimed to evaluate the possible neuroprotective effects of commercially available flavonoids using in silico, in vitro and in vivo approaches.

The objectives of the present study are to

• Evaluate drug likeness properties of the flavonoids

• Carry out in silico docking studies of the selected flavonoids using AutoDock 4.2

• Perform in vitro AChE and BuChE enzyme inhibitory assay for the selected flavonoids

• Carry out the acute toxicity studies for the selected flavonoids

35 U R K U N D Madeswaran Final Thesis.doc (D29512176)

• Assess the selected flavonoids against scopolamine and aluminium chloride induced Alzheimer models

• Carry out various behavioural studies using selected flavonoids using in vivo models

• Explore the biochemical alterations after induction of learning impairment and correction of biochemical parameters after drug treatment.

• Perform histopathological studies to corroborate the results of in vivo studies.

• To correlate the results obtained using statistical tools using GraphPad Prism software.

4. PLAN OF WORK

STEP I

Selection of flavonoids and determination of acetylcholinesterase inhibitory activity.

STEP II

Determination of drug likeness score by using MedChem Designer software.

STEP III

To carry out the molecular docking studies using AutoDock 4.2 against AChE and BuChE based on the bioactive score.

STEP IV

Selection of flavonoids based on their docking score and drug likeness properties.

STEP V

In vitro screening of selected flavonoids by AChE and BuChE inhibitory assay.

STEP VI

Acute toxicity studies following OECD guidelines-420 (fixed dose procedure) for selected flavonoids based on the in vitro results.

STEP VII

In vivo screening of the flavonoids by acute scopolamine-induced learning impairment in mice.

STEP VIII

In vivo screening of the flavonoids by chronic aluminium chloride-induced learning impairment in mice.

STEP IX

Assessment of neuroprotective activity using various behavioral experiments.

STEP X

Dissection and preparation of tissue homogenates using brain for biochemical estimations.

36 U R K U N D Madeswaran Final Thesis.doc (D29512176)

STEP XI

Histopathological studies for both scopolamine and aluminium chloride-induced models.

STEP XII

Tabulation, compilation of results and statistical analysis of data obtained.

Scopolamine-induced amnesia Aluminium Chloride-induced amnesia

Fig.13 Schematic representation of the plan of work

5. MATERIALS AND METHODS

5.1 Evaluation of drug likeness properties

The pharmacologically active substituents are characterized by calculating steric, hydrophobic, electronic, and hydrogen bonding properties as well as by the drug-likeness score. The theory of drug-likeness score helps to optimize pharmaceutical and pharmacokinetic properties, for example, chemical stability, solubility, distribution profile and bioavailability. The molecular descriptors have developed as rationally predictive and informative, for example, the Lipinski's Rule-of-Five182.

The better oral absorption of the ligands and drug likeness scores are constructed by getting information about the solubility, diffusion, log P, molecular weight etc. The theory of drug-likeness score helps to optimize pharmaceutical and pharmacokinetic properties. Lipinski’s rule of five describes the important molecular properties required for the drug’s pharmacokinetics183. Lipinski’s rule states that, the drug to have good pharmacokinetic profile, should not have more than one violation of the following criteria:

• Not more than 5 hydrogen-bond donors

• Not more than 10 hydrogen-bond acceptors

• Molecular weight should be less than 500 daltons

• The partition coefficient (log P) not greater than 5

Two hundred and forty five flavonoids were evaluated for their pharmacokinetic profile based on Lipinski’s rule of five using MedChem Designer software.

5.1.1 Lipinski’s rule of five calculations

• Open the MedChem designer home page.

• Click calculation of molecular properties of drug likeness

• Draw the structure of flavonoids in the active window.

• Click and calculate properties.

• Save the properties.

5.2 In silico docking binding interaction studies

5.2.1 Softwares used

• AutoDock 4.2 which combines

37 U R K U N D Madeswaran Final Thesis.doc (D29512176)

➢ AutoDock Tools 1.5.4

➢ Python Molecule Viewer 1.5.4

➢ Vision 1.5.4

• Python 2.7

• Cygwin

• Accerlys discovery studio viewer

• ChemSketch

• RCSB protein data bank

• Online SMILES translator

5.2.2 In silico docking study on AChE enzyme using AutoDock 4.2

5.2.2.1 Identification of target enzyme and selection of flavonoids and related compounds

Fig. 14 Refined crystal structure of mouse acetylcholinesterase (5HCU)

A total of 245 flavonoids were taken for the study and the structures were drawn using ChemSketch and optimized using “Prepare Ligands” in the AutoDock 4.2 for docking studies184.

Fig. 15 Human butyrylcholinesterase in complex with tacrine (4BDS)

5.2.2.2 Docking studies for the selected flavonoids185

Step I - Protein structure refinement

The proteins downloaded from PDB have to be refined for docking. The steps involved in the protein structure refinement are,

• Open Accelrys discovery studio viewer

• Select Target protein PDB file

• Click View, and then click Hierarchy.

• Click water molecules

• Ctrl + shift and click the last water molecule (select all water molecules)

• Give right click and cut

• Select ligand, which is unnecessary, give right click and cut.

• Save the molecule in our desired area.

Step II - Ligand file format conversion

38 U R K U N D Madeswaran Final Thesis.doc (D29512176)

• The ligands which are desired are drawn in Chem Sketch.

• Select tools and click generate, finally click SMILES notation [simplified molecular input line entry system, which is a file format]

• Save the SMILES in a word document

• Open the online smiles translator – cactus.nci.nih.gov/services/translate/

• Upload the SMILES.

• By choosing the required file format we can save the file (pdb file).

Step III - Docking with AutoDock 4.2

Docking calculation in AutoDock 4.2 was performed using the refined protein and the desired ligand in pdb format.

Preparing the protein

• At first give the right click on the PMV molecules for to open the file.

• Choose the particular refined enzyme file.

• Click under atom to give color to the atoms.

• The elimination of the water is carried by the following steps.

• press select option

• click select from string option

• then write HOH* in the residue line & * in the atom line

• click add and then dismiss.

• After that click edit option and delete and then delete atom set.

• Addition of hydrogens is take placed by,

• Press edit option

• Click the hydrogens

• Then click add

• Finally choose hydrogens and no bond order, and finally yes to renumbering.

• Next add the Kollmann charges which is present in the edit option

• For to hide the protein click on the gray under show molecules.

39 U R K U N D Madeswaran Final Thesis.doc (D29512176)

Preparing the ligand

• Confirm that all the hydrogens are added in the ligand.

• Toggle the Autodock tools button

• For to open the ligand file press the Input option and click open and then all files finally choose the suitable ligand file and finally open

• AutoDock tools itself computes Gasteiger charges, and merges nonpolar hydrogens, and finally assigns AutoDock type to each atom.

• The torsions are designed by following steps,

• In the ligand option select torsion tree

• Select detect root option

• Click torsion tree

• Then select the choose torsions option

• Amide bonds should not be active.

• After that click the torsion tree and select set number of torsions.

• Number of rotatable bonds is choosen.

• Finally save the ligand files by selecting the output option (pdbqt file).

• Hide the ligand.

Conversion of pdb files of protein into pdbqt file

• Select the grid option and open the macromolecule pdb file

• AutoDock adds the charges and itself merges the hydrogens.

• Save the object as pdbqt in desired area.

Running AutoGrid Calculation (glg file)

• Open the grid and click macromolecule option and choose the rigid protein then yes to preserve the existing charges.

• The preparation of grid parameter file is carried out by,

• Open grid

• Select the set map types

• Choose ligand

• Accept it.

• Setting of grid properties

40 U R K U N D Madeswaran Final Thesis.doc (D29512176)

• Open grid

• Select the grid box

• Set the proper grid dimensions

• Adjust the spacing

• Select the file and click close saving current.

• Save the grid settings as gpf file in the output option (gpf file).

• Make sure the AutoGrid is in the same directory as like of the other input files.

• Select the run option and click run AutoGrid for to launch.

• It will be saved as glg file.

Running AutoDock (dlg file)

• The rigid molecule specification is carried out by,

• Select the docking option

• Click the macromolecule

• Set rigid file name.

• The ligand specification is carried out by,

• Click the docking option

• Select the ligand

• And then accept it.

• In the next step, click docking option and select search parameters in that click genetic algorithm and finally accept it.

• In the docking options select docking parameters and choose the defaults.

• After that, in the docking option clicks output and adds Lamarckian genetic algorithm.

• The confirmation is required that whether all the files (macromolecule, ligand, gpf, dpf ) are in the same directory.

• Finally select the run command and click run AutoDock and then launch.

Step IV - Viewing docking results

Reading the docking log file .dlg

• Toggle the AutoDock tools button

• Click analyze and dockings then open it.

• In the next step, click analyze option and conformations then load.

41 U R K U N D Madeswaran Final Thesis.doc (D29512176)

• Double click on the conformation for to view it.

Visualizing docked conformations

• Click analyze and dockings then play.

• Load dlg file

• Choose the suitable conformations

• In the next step, click analyze and docking then show interactions.

Obtaining snap shots of docked pose

• Open the file and read the molecule

• Open analyze option click dockings and open dlg file

• Open analyze option click macromolecule and choose pdbqt file.

• Open analyze option click conformations and load

• Double click the desired conformation

• In the next step, click analyze and docking then show interactions.

The preparation of the target protein 5HCU and 4 BDS with the AutoDock tools software involved adding all hydrogen atoms to the macromolecule, which is a step necessary for correct calculation of partial atomic charges (Calderone et al. 2000). Gasteiger charges are calculated for each atom of the macromolecule in AutoDock 4.2 instead of kollman charges which were used in the previous versions of the program. Three-dimensional affinity grids of size 277 × 277 × 277 Å with 0.6 Å spacing were centered on the geometric center of the target protein and were calculated for each of the following atom types: HD, C, A, N, OA, and SA, representing all possible atom types in a protein. Additionally, an electrostatic map and a desolvation map were also calculated187.

42 U R K U N D Madeswaran Final Thesis.doc (D29512176)

and inhibition constant. For each ligand, ten best poses were generated and scored using AutoDock 4.2 scoring functions188.

5.3 REAGENTS AND CHEMICALS

Donepezil, acetylcholinesterase, butyrylcholinesterase, apigenin, baicalein, catechin, chrysin, diosmin, epigallocatechin, hesperetin, hesperidin, kaempferol, morin, myrecetin, naringenin, robinetin, rutin, scopoletin, silibinin, taxifolin, tetra hydroflavone, theaflavin and tricetin were purchased from Sigma Aldrich, St. Louis, MO. All other drugs and chemicals used in the study were obtained commercially and were of analytical grade. Bovine serum albumin (BSA), p-nitro benzaldehyde, p- fluro benzaldehyde, p-chloro benzaldehyde, p-methyl benzaldehyde, p-methoxy benzaldehyde, p-hydroxy benzaldehyde ethanol, potassium phosphate, Folin phenol reagent, 5,5'- dithiobis-2-nitro benzoic acid, acetylthiocholine iodide, carboxy methyl cellulose and tris hydrochloride were purchased from Hi Media Laboratories, Mumbai. Sodium thiosulphate, sodium hydroxide, hydrochloric acid, sodium potassium tartarate, sodium carbonate, copper sulphate, hydrogen peroxide, potassium dichromate, glacial acetic acid and sodium citrate were purchased from SD Fine chem limited, Mumbai. Scopolamine and aluminium chloride were purchased from Sigma Aldrich, USA. All other chemicals and solvents used were of AR grade.

5.4 INSTRUMENTS USED

Digital Balance (Sartorius Ltd., USA), Eppendorff minispin, incubator (Technico), Jasco FTIR-420 series, Mass spectrometer, Shimadzu-Jasco V-530 UV/Vis spectrophotometer, ELCO 1/27 pH meter.

5.5 In vitro enzyme inhibition assay

5.5.1 In vitro acetylcholinesterase inhibition

0: Lubhan Singh.doc 71%

1: Lubhan Singh With references.doc 71%

2: Lubhan Singh without references.doc 71%

at room temperature for 30 minutes and the absorbance of the reaction mixture was measured at 405 nm.

The assay was done in triplicate and the results were expressed as mean±SEM. Donepezil was used as standard190. The percentage inhibition was calculated for the selected flavonoids using the following formula,

A0= Absorbance of the control

A1= Absorbance of the standard/compounds.

43 U R K U N D Madeswaran Final Thesis.doc (D29512176)

5.5.2 In vitro butyrylcholinesterase inhibition

A0 = Absorbance of the control

A1 = Absorbance of the standard/ compounds.

5.6 ACUTE TOXICITY STUDIES

5.6.1 Experimental animals

Acute toxicity studies were performed for the baicalein and scopoletin according to organization for economic co-operation and development (OECD) guidelines 420 (fixed dose procedure) 191. Healthy young adult female rats (200-250 g) used for the acute toxicity studies were procured from Kerala Veterinary and Animal Sciences University, Mannuthy, Thrissur, Kerala, India. Animals were 8-12 weeks old and weight was not deviated from ± 20%. CPCSEA approval number for experimental protocols for the toxicological studies was COPSRIPMS/IAEC/PhD/P’CHEMISTRY/ 03/ 2012-2013.

5.6.2 Acute toxicity study procedure

Due to absence of toxicity information for flavonoids, in the sighting study, one animal was administered with 5 mg/kg dose and in the absence of toxicity 50mg/kg dose was given to second animal to check for toxicity. If no mortality was observed for 50mg/kg dose then the next dose of 300 mg/kg was given to third animal and if no mortality was observed then the fourth animal was administered with 2000 mg/kg dose. If no mortality was observed for upper dose limit of 2000 mg/ kg, then the specific dose was selected for main study. Keen observations were done to determine the onset of signs, onset of recovery and time to death.

0: Thesis Final Work 27062017.docx 71%

Animals were observed individually at least once during the first 30 minutes after dosing, periodically during the first 24 hours (with special attention during the first 4 hours) and daily thereafter for a

period of 14 days. Once daily cage side observations included changes in skin and fur, eyes and mucous membrane (nasal), and also changes in respiratory rate, circulatory parameters (heart rate and blood pressure), autonomic parameters (salivation, lacrimation, perspiration, piloerection, urinary incontinence and defecation) and CNS parameters (ptosis, drowsiness, gait, tremors and convulsions)192.

44 U R K U N D Madeswaran Final Thesis.doc (D29512176)

5.7 IN VIVO EXPERIMENTAL DESIGN

5.7.1 Experimental animals

0: Thesis Final Work 27062017.docx 100%

by Committee for the Purpose of Control and Supervision of Experiments on Animals (

CPCSEA) guidelines, Government of India. All the readings were taken during the same time of the day that is between 10.00 am and 2.00 pm. CPCSEA approval number for experimental protocols for the pharmacological studies was COPSRIPMS/IAEC/PhD/P’CHEMISTRY/ 03/ 2012-2013.

5.7.2 In vivo scopolamine-induced amnesia model

On day 8, after behavioral assessments, animals were sacrificed by excess of anesthesia. The brain was removed and each brain had been separately kept after rinsing with ice-cold isotonic saline. A (10% w/v) homogenate was prepared with 0.1 M phosphate buffer (pH 7.4) and centrifuged at 3000 rpm for 15 minutes and aliquots of supernatant had separated and used for the various biochemical estimations {cholinesterase activity, protein estimation, enzymatic (Catalase activity, 2,2-diphenyl-1-picrylhydrazyl assay, superoxide dismutase assay, glutathione reductase, glutathione peroxidase) and non-enzymatic antioxidant activities (reduced glutathione) and lipid peroxidation indices with thiobarbituric acid reactive substances assay}.

5.7.3 Aluminium-induced learning impairment

Swiss albino mice were divided into 6 different groups, each comprising of six animals. Group I served as control which received normal saline. In group II mice were treated with baicalein (100 mg/kg/day) and group III were administered with baicalein (200 mg/kg/day). In group IV mice received scopoletin (100 mg/kg/day), group V received scopoletin (200 mg/kg/day). Group VI mice were treated with donepezil (5 mg/kg, p.o.) for 3 months. All the groups were treated for a period of 3 months. Groups III to VII received Aluminum chloride (AlCl3) dissolved in water at a dose of 50 mg/kg/day194, 195, for a period of 3 months. The values for the negative control group were taken from archives. The values for the negative control group were taken from archives18. The effects of the selected flavonoids were studied using various behavioral assessments such as rectangular maze test, Morris water maze, gross behavioral activity, 5 choice serial reaction time task and pole climbing test during the treatment period.

45 U R K U N D Madeswaran Final Thesis.doc (D29512176)

5.8 BEHAVIORAL ASSESSMENTS

5.8.1 Rectangular maze test

Fig. 16 Rectangular maze

5.8.2 Morris water maze test

Fig. 17 Morris water maze

5.8.3 Assessment of gross behavioral activity (Locomotor activity)

Fig. 18 Actophotometer

5.8.4 Pole climbing test

This test was used to study the neuroprotective and nootropic effect of test drugs. The mice were trained for conditioned avoidance response (CAR) by using pole climbing apparatus. Prior to the experiment, animals were trained in a 25 × 25 × 40 cm chamber that was enclosed in a dim light, sound attenuated box (Fig. 19). Each mouse was allowed to acclimatize for 2 min and then exposed to a buzzer noise. After 10 sec of putting on the buzzer, mild electric shocks were given through the 46 U R K U N D Madeswaran Final Thesis.doc (D29512176)

grid floor. The magnitude of the voltage was adequate (30v) to stimulate the mice to escape from the floor and climb the pole. As soon as the mice climbed the pole, both the buzzer and foot-shock were switched off. At least 10 such trails were given to each mouse at an interval of 1 min. per day for 3 days. After training schedule, most of the mice learned to climb the pole within 10 sec of starting the buzzer to avoid the electric foot shocks. On the test days, after 2 min of acclimatization period, each mouse was exposed to the buzzer for 10 sec; ten such trails were given at an interval of 1 min, without giving any shock. Mice, responding by climbing the pole when exposed to the buzzer noise, were considered to have retained the conditional avoidance response198.

Fig. 19 Pole climbing apparatus

5.8.5 Attention in a 5 choice serial reaction time task (5-CSRT)

Fig. 20 Five choice serial reaction time task apparatus

5.9 BIOCHEMICAL ESTIMATIONS

5.9.1 Estimation of AChE in mice brain

5.9.2 Estimation of total protein content

5.9.3 Enzymatic anti-oxidants

5.9.3.1 Estimation of catalase

About 0.1 ml of tissue homogenate was added to 1.0 ml of 0.01 M phosphate buffer of pH 7.0 and was incubated at 37C for 15 min. The reaction was started by the addition of 0.4 ml of 2M H2O2. 47 U R K U N D Madeswaran Final Thesis.doc (D29512176)

The reaction was then stopped by the addition of 2 ml of dichromate - acetic acid reagent (a mixture of 5% potassium dichromate and glacial acetic acid in a ratio of 1:3). CAT was assayed colorimetrically at 620 nm. Unit of catalase activity is expressed as the amount of enzyme causing the decomposition of µmoles H2O2/mg protein/min203.

5.9.3.2 Estimation of superoxide dismutase (SOD)

The first enzyme involved in the antioxidant defense is superoxide dismutase. It is a metalloprotein found in both prokaryotic and eukaryotic cells. The oxygen-derived free radicals generated by the interaction of Fe2+ and H2O2 are the species responsible for the oxidation of epinephrine and they were strongly inhibited by the enzyme, superoxide dismutase204.

About 0.1 ml of tissue homogenate was mixed with sodium pyrophosphate buffer, and nitroblue tetrazolium (NBT).

0: Lubhan Singh.doc 83%

1: Lubhan Singh With references.doc 83%

2: Lubhan Singh without references.doc 83%

The reaction was initiated by the addition of NADH. The reaction mixture

was incubated at 30°C for 90 seconds and it was stopped by the addition of 1 ml of glacial acetic acid. The absorbance of the chromogen formed was measured at 560nm. SOD activity is expressed as nmoles/min/mg protein205.

5.9.3.3 Estimation of glutathione reductase (GSSH)

The reaction mixture contained 2.1 ml of 0.25 mM potassium phosphate buffer of pH 7.6, 0.1ml of 0.001M NADPH, 0.2 ml of 0.0165 M oxidized glutathione and 0.1 ml of bovine serum albumin (10 mg/ml). The reaction was started by the addition of 0.2 ml of tissue homogenate with mixing and the decrease in the absorbance at 340 nm was measured for 3 minutes against the blank. Glutathione reductase activity is expressed as nmoles NADPH oxidized /min/mg protein205.

5.9.3.4 Estimation of glutathione peroxidase (GPx)

The reaction mixture consisted of 0.2 ml of 0.4 M Tris-HCl buffer, 0.1 ml of sodium azide, 0.1 ml of hydrogen peroxide, 0.2 ml of glutathione and 0.2 ml of supernatant. The mixture was incubated at 37C for 10 min. The reaction was arrested by the addition of 10 % trichloro acetic acid and the absorbance was measured at 340 nm. GPx activity is expressed as nmoles/min/mg protein207.

5.9.4. Non-enzymatic antioxidants

5.9.4.1 Estimation of reduced glutathione (GSH)

48 U R K U N D Madeswaran Final Thesis.doc (D29512176)

nitro) benzoic acid (DTNB) in 100 ml of 0.1% sodium citrate solution) and 3.0 ml of 0.2 M phosphate buffer of pH 8.0. The absorbance was read at 412 nm using a spectrophotometer. The activity of GSH was expressed as nmoles/min/mg protein204.

5.9.5 Estimation of lipid peroxidation (LPO)

5.9.6 (2,2-Diphenyl-1-picrylhydrazyl) (DPPH) assay

5.10 Histopathological studies

5.11 Statistical analysis

Statistical analysis was carried out using GraphPad Prism 4.0 by

0: Thesis Final Work 27062017.docx 90%

One-way Analysis of Variance (ANOVA) followed by Dunnett’s test.

Results are expressed as mean±SEM from six mice in each group. P values > 0.05 were considered significant.

6. RESULTS

6.1 Drug likeness properties of the flavonoids

Fig. 21 Drug likeness properties of the flavonoids

6.2 In silico docking studies against AChE and BuChE

49 U R K U N D Madeswaran Final Thesis.doc (D29512176)

All the selected flavonoids when docked with AChE target protein showed binding energy ranging between -6.95 kcal/mol to -3.90 kcal/mol as compared to donepezil (standard) which showed binding energy of -3.87 kcal/mol (Table 1). Scopoletin and baicalein showed remarkably excellent binding energy (-6.95 kcal/mol and -6.93 kcal/mol) when compared to other flavonoids (Fig. 22). The minimum binding energy indicated that the selected flavonoids have excellent inhibitory potential toward AChE protein.

Fig. 22 Binding energy of the flavonoids against AChE

Table 1: In silico molecular interactions scores of the flavonoids against AChE and BuChE Flavonoids Acetylcholinesterase Butyrylcholinesterase Binding Energy (kcal/mol) Inhibition constant (µM) Intermolecular Energy (kcal/mol) Binding Energy (kcal/mol) Inhibition constant (µM) Intermolecular Energy (kcal/mol) Acacatechin -4.84 287.59 -7.37 -2.97 134.85 -4.81 Acacetin -4.54 335.28 -6.92 -2.79 156.98 -4.54 Afzelechin -5.26 136.28 -7.80 -3.20 71.63 -5.09 Angoletin -5.14 162.14 -7.73 -3.12 80.92 -5.06 Annulatin -4.86 282.94 -7.40 -2.98 131.50 -4.85 Apigenin -6.33 39.87 -8.67 -3.88 19.08 -5.68 Apometzgerin -5.31 114.42 -7.87 -3.26 54.77 -5.16 Aromadedrin, Dihydro Kaempferol -5.37 90.21 -7.96 -3.29 43.32 -5.23 Aurantinidin -5.46 84.04 -8.10 -3.35 39.25 -5.25 Aureusidin -5.54 80.12 -8.19 -3.40 37.76 -5.37 Ayanin -5.63 74.78 -8.24 -3.45 35.09 -5.41 Azaleatin -4.24 730.98 -6.30 -2.60 342.77 -4.11 Baicalein -6.93 8.32 -8.91 -4.51 2.12 -6.11 Baicalin mono hydrate -3.94 964.84 -5.64 -2.41 459.96 -3.66 Baptigenin -5.34 98.84 -7.94 -3.26 51.69 -5.21 Bavachinin -4.04 914.52 -5.84 -2.47 439.08 -3.77 Biochanin A -4.36 368.66 -6.62 -2.68 170.41 -4.33 Bis Hydroxy Coumarin -5.68 70.32 -8.35 -3.48 33.11 -5.46 Bracteatin -5.46 83.74 -7.99 -3.33 39.69 -5.25 Buceracidin-B -5.54 80.56 -8.18 -3.40 37.96 -5.36 Butein -4.92 276.36 -7.45 -3.02 126.27 -4.87 Calycopterin -4.30 581.36 -6.41 -2.63 320.78 -4.20 Calycosin -4.67 321.84 -7.14 -2.85 150.65 -4.66 Candidone -5.05 200.12 -7.62 -3.09 102.49 -4.95 Cannflavin -4.77 298.34 -7.30 -2.92 142.95 -4.77 Capensinidin -4.50 339.68 -6.89 -2.75 160.72 -4.50 Carajuflavone -4.23 729.36 -6.24 -2.60 341.73 -4.07 Carpachromene -3.96 954.36 -5.71 -2.42 451.12 -3.68 Casticin -4.69 315.64 -7.16 -2.87 150.86 -4.69 Catechin -5.34 136.44 -6.99 -3.28 45.30 -5.21 Chrysin -6.30 45.39 -8.64 -3.83 25.37 -5.65 Chrysoeriol -4.59 328.45 -6.97 -2.79 157.16 -4.54 Chrysosplenetin -5.43 84.68 -7.99 -3.33 40.58 -5.23 Cirsililol -4.16 832.21 -6.01 -2.52 399.50 -3.91 Cirsilineol -5.63 74.86 -8.24 -3.45

50 U R K U N D Madeswaran Final Thesis.doc (D29512176)

35.76 -5.40 Cirsimaritin -5.72 69.92 -8.38 -3.49 32.93 -5.48 Cladrin -5.80 68.84 -8.44 -3.53 32.46 -5.53 Combretol -4.23 729.64 -6.23 -2.59 339.48 -4.07 Cyanidin -4.50 338.64 -6.87 -2.75 160.37 -4.49 Cyclocommunol -4.77 298.81 -7.29 -2.92 142.72 -4.77 Daidzein -5.04 218.24 -7.58 -3.08 109.86 -4.95 Datin -5.31 118.42 -7.86 -3.26 54.04 -5.15 Datiscetin -4.24 731.26 -6.30 -2.60 343.07 -4.10 Delphinidin -3.94 962.38 -5.61 -2.41 459.45 -3.64 Demethylbellidifolin -4.64 321.62 -7.12 -2.83 151.89 -4.59 Desmethoxycentaureidin -5.58 79.05 -8.20 -3.42 37.00 -5.38 Dihydrobaicalein -5.84 67.86 -8.50 -3.57 31.99 -5.55 Dihydroflavonol -4.77 298.72 -7.27 -2.91 143.73 -4.77 Dihydrokaempferide -5.04 218.64 -7.54 -3.08 108.39 -4.94 Dihydromyricetin -5.31 116.84 -7.86 -3.24 57.58 -5.14 Dihydrorobinetin -5.58 78.94 -8.20 -3.42 36.88 -5.38 Dihydrotricetin -4.54 334.98 -6.92 -2.77 157.71 -4.53 Dihydrowogonin -4.34 396.44 -6.55 -2.65 197.60 -4.27 Diosmetin -6.03 60.86 -8.57 -3.66 29.87 -5.61 Diosmin -6.03 36.86 -7.84 -3.69 28.66 -5.61 Dorsilurin-F -5.68 70.26 -8.34 -3.52 32.57 -5.51 Epicatechin -5.74 69.64 -8.42 -3.52 32.64 -5.53 Epicatechin Gallate -5.80 68.98 -8.44 -3.17 73.26 -5.09 Epigallocatechin -5.24 124.12 -7.04 -3.24 58.29 -5.13 Epigallocatechin Gallate -5.31 115.28 -7.84 -3.40 37.56 -5.38 Eriocitrin -5.58 78.68 -8.20 -3.57 31.95 -5.55 Eriodictyol -5.84 67.24 -8.48 -2.68 172.81 -4.35 Ermanin -4.37 357.45 -6.67 -2.69 164.74 -4.42 Esculetin -4.38 354.98 -6.75 -3.12 79.86 -5.05 Eupafolin -5.09 168.42 -7.72 -3.39 38.31 -5.34 Eupalitin -5.53 81.12 -8.17 -3.44 36.51 -5.39 Eupatilin -5.60 77.94 -8.21 -3.47 33.24 -5.44 Eupatolitin -5.67 70.64 -8.32 -3.52 32.59 -5.52 Eupatorin -5.75 69.24 -8.43 -3.42 36.85 -5.38 Europetin -5.60 77.89 -8.21 -3.38 38.42 -5.32 Europinidin -5.52 81.72 -8.13 -3.30 42.29 -5.23 Farobin-A -5.39 89.12 -7.97 -3.23 63.88 -5.12 Fisetin -5.28 122.84 -7.83 -3.15 76.00 -5.07 Fisetinidol -5.17 156.28 -7.75 -3.09 102.30 -4.98 Flavoxate -5.05 198.89 -7.67 -3.02 126.66 -4.88 Formononetin -4.94 264.69 -7.45 -2.95 136.96 -4.79 Fustin -4.82 290.86 -7.32 -2.87 148.82 -4.69 Galangin -4.71 312.28 -7.18 -2.81 155.39 -4.56 Gallocatechin -4.60 326.54 -6.97 -3.36 38.85 -5.32 Gallocatechin Gallate -5.51 81.96 -8.11 -3.44 36.53 -5.39 Ganhuangenin -5.62 76.28 -8.23 -3.51 32.78 -5.50 Gardenin B -5.74 69.47 -8.39 -3.56 32.27 -5.54 Gartanin -5.82 68.24 -8.46 -2.65 197.10 -4.22 Genistein -4.32 421.54 -6.50 -2.50 412.75 -3.87 Genistin -4.08 886.24 -5.94 -2.58 338.97 -4.06 Geraldone -4.22 724.23 -6.20 -2.97 134.81 -4.84 Gericudranin-B -4.86 283.16 -7.40 -2.77 157.50 -4.53 Glabridin -4.54 334.20 -6.92 -2.69 165.22 -4.40 Glaziovianin-A -4.38 352.12 -6.74 -2.91 144.53 -4.75 Glycitein -4.76 304.95 -7.27 -2.38 464.27 -3.62 Gnetifolin E -3.92 981.25 -5.58 -2.98 132.73 -4.86 Gomphrenol -4.88 278.98 -7.42 -2.96 136.34 -4.81 Gossypetin -4.84 289.64 -7.34 -2.50 413.84 -3.86 Guibourtinidol -4.08 884.16 -5.93 -2.63 311.37 -4.19 Helmon -4.28 698.28 -6.41 -3.70 27.38 -5.63 Herbacetin -6.08 57.96 -8.58 -3.22 25.63 -5.11 Hesperedin -5.28 148.38 -7.08 -4.15 8.09 -5.72 Hesperetin -6.72 17.02 -8.81 -3.83 24.67 -5.64 Hibiscetin -6.28 48.42 -8.63 -3.56 32.33 -5.54 Hirsutidin -5.82 68.16 -8.45 -3.52 32.56 -5.51 Hispudulin -5.74 69.53 -8.41 -3.35 38.98 -5.32 Homoeriodictyol -5.48 82.87 -8.10 -3.26 53.63 -5.17 Homoorientin -5.32 110.28 -7.94 -2.82 153.96 -4.58 Hydrangenol -4.61 324.03 -6.99 -2.73 163.43 -4.48 Hypolaetin -4.46 345.24 -6.84 -2.63 316.01 -4.20 Icaritin -4.30 561.28 -6.41 -2.69 164.18 -4.40 Irigenin -4.38 350.92 -6.74 -2.93 140.03 -4.77 Isalpinin -4.77 298.36 -7.30 -2.97 135.77 -4.82 Isoetin -4.86 284.31 -7.38 -2.50 413.50 -3.88 Isoimperatorin -4.11 852.26 -5.96 -2.58 338.62 -4.06 Isokaempferide -4.23 728.14 -6.22 -2.74 161.83 -4.49 Isookanin -4.48 344.23 -6.85 -2.76 158.74 -4.51 Isopongaflavone -4.52 336.45 -6.90 -2.65 196.09 -4.22 Isoquercitrin -4.32 421.28 -6.48 -3.73 27.17 -5.63 Isorhamnetin -6.22 57.28 -8.58 -3.06 114.78 -4.92 Isosakuranetin -5.02 236.24 -7.54 -3.04 119.75 -4.90 Isoscutellarein -4.98 244.87 -7.50 -2.95 136.61 -4.81 Isosilybin -4.84 287.31 -7.34 -3.03 124.07 -4.89 Isothymusin -4.96 252.36 -7.48 -2.65 195.11 -4.21 Isovitexin -4.32 420.84 -6.46 -2.74 161.80 -4.48 Isoxanthohumol -4.48 342.86 -6.85 -2.99 130.77 -4.87 Jaceidin -4.92 272.38 -7.44 -3.11 85.53 -5.04 Kaempferide -5.08 172.63 -7.71 -3.85 22.70 -5.68 Kaempferol -6.32 42.90 -8.65 -3.06 112.98 -4.92 Kumatakenin -5.02 234.36 -7.54 -2.68 171.17 -4.33 Ladanein -4.36 364.28 -6.61 -2.71 163.76 -4.43 Laricitrin -4.42 349.84 -6.81 -2.50 412.54 -3.85 Leptosidin -4.08 882.14 -5.91 -2.69 166.40 -4.43 Leucoanthocyanidin -4.42 349.21 -6.79 -3.14 77.49 -5.07 Leucocyanidin -5.16 160.76 -7.74 -2.71 163.99 -4.47 Leucodelphinidin -4.46 345.18 -6.83 -2.69 164.29 -4.40 Leucofisetinidin -4.38 351.44 -6.73 -2.60 341.89 -4.10 Leucomalvidin -4.24 731.04 -6.30 -2.90 145.70 -4.75 Leucopelargonidin -4.76 304.46 -7.27 -2.93 139.86 -4.79 Leucopeonidin -4.81 291.44 -7.32 -2.99 130.04 -4.86 Leucorobinetinidin -4.92 270.21 -7.43 -2.83 152.19 -4.58 Leukoefdin -4.64 321.38 -7.10 -2.62 346.13 -4.18 Licoflavonol -4.28 674.16 -6.40 -2.63 327.32 -4.21 Limocitrin -4.31 454.26

51 U R K U N D Madeswaran Final Thesis.doc (D29512176)

-6.42 -2.67 180.12 -4.33 Limocitrol -4.36 362.18 -6.61 -2.69 165.06 -4.42 Liquiritigenin -4.42 349.12 -6.77 -2.71 163.69 -4.46 Luteoforol -4.46 345.02 -6.83 -2.66 188.85 -4.29 Luteolin -4.35 384.26 -6.59 -2.58 338.73 -4.06 Luteolinidin -4.23 729.02 -6.20 -2.48 428.68 -3.83 Maesopsin -4.08 880.54 -5.89 -2.50 415.43 -3.90 Malvidin -4.12 842.15 -5.99 -2.60 342.02 -4.09 Maritimetin -4.24 731.24 -6.26 -2.61 343.32 -4.14 Matteucinol -4.26 734.22 -6.34 -2.65 197.27 -4.24 Mearnsitrin -4.33 402.89 -6.53 -2.69 164.10 -4.39 Meciadanol -4.38 352.46 -6.70 -2.58 339.12 -4.06 Melacacidin -4.21 722.38 -6.19 -2.71 163.65 -4.44 Melanoxetin -4.45 348.65 -6.82 -2.68 170.76 -4.34 Mesquitol -4.37 359.12 -6.67 -4.44 3.92 -5.74 Morin -6.92 8.29 -8.90 -3.88 18.69 -5.69 Myricetin -6.34 38.96 -8.68 -4.21 6.18 -5.72 Naringenin -6.76 17.26 -8.84 -3.59 31.52 -5.57 Naringin -5.88 65.86 -8.51 -3.45 35.05 -5.41 Natsudaidain -5.64 72.68 -8.28 -3.31 41.78 -5.23 Negletein -5.42 86.56 -7.97 -3.12 78.95 -5.07 Nepetin -5.16 160.32 -7.73 -2.97 134.68 -4.82 Nobiletin -4.86 280.53 -7.38 -2.89 146.38 -4.71 Nodifloretin -4.73 310.83 -7.23 -2.68 167.55 -4.38 Norathyriol -4.38 350.25 -6.70 -2.84 151.38 -4.66 Norwogonin -4.67 321.15 -7.14 -2.93 140.07 -4.79 Okanin -4.80 292.19 -7.30 -2.50 414.45 -3.89 Ombuin -4.12 840.14 -5.98 -2.60 341.32 -4.08 Ononin -4.24 731.88 -6.25 -2.48 427.71 -3.82 Orientin -4.08 882.85 -5.88 -2.64 212.93 -4.21 Orobol -4.32 420.48 -6.43 -2.55 390.10 -3.95 Oroxylin-A -4.18 784.46 -6.13 -2.68 168.34 -4.38 Osajin -4.38 350.48 -6.70 -2.87 150.54 -4.69 Pachypodol -4.68 318.65 -7.15 -2.62 346.42 -4.16 Patuletin -4.28 684.33 -6.39 -2.43 447.36 -3.75 Pectolinarigenin -4.04 912.44 -5.82 -2.43 448.38 -3.73 Pedalitin -4.02 934.27 -5.76 -2.64 263.10 -4.21 Pelargonidin -4.32 418.33 -6.43 -2.53 394.76 -3.94 Penduletin -4.18 782.15 -6.12 -3.94 17.01 -5.70 Peonidin -6.42 36.28 -8.71 -2.60 342.65 -4.14 Persicarin -4.26 735.08 -6.33 -2.75 161.36 -4.50 Persicogenin -4.52 336.01 -6.90 -2.81 155.24 -4.56 Petunidin -4.61 324.68 -6.98 -2.74 161.73 -4.48 Pinoquercetin -4.48 342.12 -6.84 -2.87 149.37 -4.69 Platanetin -4.71 311.88 -7.19 -2.64 272.51 -4.21 Pomiferin -4.32 416.24 -6.42 -2.53 393.82 -3.92 Pratensein -4.18 780.14 -6.03 -2.65 197.48 -4.25 Pratol -4.34 398.78 -6.55 -2.42 452.27 -3.70 Primetin -4.02 936.71 -5.74 -2.61 344.17 -4.16 Propolin B -4.28 664.26 -6.38 -2.21 488.27 -2.92 Prosogerin E -3.92 980.16 -5.55 -2.18 508.27 -2.82 Prunetin -3.90 990.45 -5.51 -2.47 437.94 -3.78 Prunin -4.08 880.08 -5.86 -2.79 157.02 -4.54 Pulchellidin -4.58 331.18 -6.95 -2.82 153.07 -4.58 Quercetagetin -4.63 322.94 -7.10 -2.57 367.72 -4.02 Quercetagetinidin -4.21 722.62 -6.18 -3.26 55.51 -5.16 Ranupetin -5.32 108.12 -7.90 -3.09 102.36 -4.99 Retusin -5.06 182.46 -7.68 -2.93 139.85 -4.79 Rhamnazin -4.81 291.25 -7.31 -3.04 118.74 -4.89 Rhamnetin -4.98 241.02 -7.50 -2.66 185.83 -4.30 Rhusflavanone -4.36 363.55 -6.60 -2.76 159.23 -4.52 Rhynchosin -4.54 334.89 -6.91 -1.25 231.18 -2.41 Robinetin -6.45 24.25 -8.73 -3.96 11.37 -5.71 Robinetinidin -5.66 70.96 -8.30 -3.45 34.93 -5.42 Robinetinidol -5.02 231.24 -7.52 -3.04 118.29 -4.91 Rosinidin -5.08 170.15 -7.69 -3.10 93.23 -5.04 Rutin -6.51 23.27 -8.76 -4.03 9.23 -5.72 Sakuranetin -4.96 255.46 -7.47 -3.02 129.54 -4.89 Salvigenin -4.84 287.68 -7.33 -2.95 136.52 -4.80 Santal -5.08 170.36 -7.69 -3.10 93.81 -5.00 Santin -5.28 124.36 -7.82 -3.20 70.46 -5.10 Scopoletin -6.95 8.37 -8.94 -4.62 1.29 -6.23 Scutellarein -5.72 69.87 -8.38 -3.49 32.96 -5.48 Scutevulin -5.12 165.31 -7.72 -3.12 79.76 -5.05 Selagin -4.96 253.31 -7.45 -3.02 127.68 -4.88 Silibinin -5.66 70.92 -7.45 -3.46 34.07 -5.44 Silybin -5.74 69.48 -8.40 -3.51 32.75 -5.50 Sinensetin -5.04 218.36 -7.60 -3.08 110.74 -4.95 Skullcapflavone Ii -4.75 306.62 -7.26 -2.90 145.79 -4.73 Sorbifolin -4.37 358.98 -6.64 -2.68 169.77 -4.34 Sternbin -4.26 736.24 -6.32 -2.60 342.78 -4.14 Sterubin -4.42 349.36 -6.75 -2.69 164.49 -4.42 Syringetin -4.36 365.16 -6.59 -2.66 186.93 -4.30 Tamarixetin -4.68 317.48 -7.15 -2.85 150.76 -4.67 Tambulin -4.74 308.32 -7.24 -2.89 146.19 -4.72 Tanetin -4.58 331.49 -6.95 -2.79 156.66 -4.54 Tangeritin -6.24 56.62 -8.59 -3.82 26.19 -5.63 Taxifolin -5.22 150.32 -7.01 -3.17 75.36 -5.08 Tectorigenin -4.21 723.14 -6.18 -2.57 366.63 -4.02 Tephrosin -4.92 269.38 -7.42 -2.98 132.63 -4.86 Teracacidin -4.72 311.02 -7.20 -2.88 147.96 -4.70 Ternatin -4.88 277.42 -7.40 -2.98 133.27 -4.85 Tetra Hydroflavone -6.38 37.08 -8.70 -3.89 18.26 -5.70 Texasin -5.34 96.64 -7.94 -3.26 50.68 -5.19 Theaflavin -5.88 66.25 -8.52 -3.59 31.81 -5.58 Theaflavin-3-Gallate -5.58 78.62 -8.20 -3.42 37.05 -5.38 Thunberginol A -4.27 738.41 -6.36 -2.61 345.11 -4.15 Tomentin -4.21 723.46 -6.18 -2.57 365.69 -3.96 Topazolin -4.38 350.08 -6.68 -2.68 168.27 -4.36 Tricetin -4.27 739.02 -6.06 -2.61 344.57 -4.16 Tricin -5.36 92.42 -7.96 -3.28 46.33 -5.21 Veronicoside -4.25 732.41 -6.31 -2.60 342.68 -4.14 Viscidulin Ii -5.46 83.16 -8.10 -3.35 39.39 -5.32 Vitexin -5.96 63.72 -8.54 -3.61 30.87 -5.59 Vitexycarpin -6.01 61.15 -8.54 -3.61 31.05 -5.59 Wogonin -6.86 9.48 -8.87 -4.25 4.81 -5.73 Wogonoside -5.54 80.98 -8.17 -3.39 38.03 -5.36 Xanthohumol -5.21 152.82 -7.75 -3.17 75.15 -5.07 Donepezil -3.87 994.14 -5.46 -3.47 33.26 -5.45

52 U R K U N D Madeswaran Final Thesis.doc (D29512176)

Fig. 23 Molecular interactions of scopoletin and donepezil against AChE

Fig. 24 Binding energy of the flavonoids against BuChE

Fig. 25 Molecular interactions of scopoletin and donepezil against BuChE

The in silico ChE (AChE and BuChE) inhibitory activities of the 245 flavonoid compounds decreased in the order of Scopoletin < Baicalein < Morin < Wogonin < Naringenin < Hesperetin < Rutin < Robinetin < Peonidin < Tetra Hydroflavone < Myricetin < Apigenin < Kaempferol < Chrysin < Hibiscetin < Tangeritin < Isorhamnetin < Herbacetin < Diosmin < Diosmetin < Vitexycarpin < Vitexin < Theaflavin < Naringin < Dihydrobaicalein < Eriodictyol < Gartanin < Hirsutidin < Cladrin < Epicatechin Gallate < Eupatorin < Epicatechin < Hispudulin < Silybin < Gardenin B < Scutellarein < Cirsimaritin < Bis Hydroxy Coumarin < Dorsilurin-F < Eupatolitin < Silibinin < Robinetinidin < Natsudaidain < Ayanin < Cirsilineol < Ganhuangenin < Eupatilin < Europetin < Theaflavin-3-Gallate < Desmethoxycentaureidin < Dihydrorobinetin < Eriocitrin < Aureusidin < Buceracidin-B < Wogonoside < Eupalitin < Europinidin < Gallocatechin Gallate < Homoeriodictyol < Viscidulin II < Aurantinidin < Bracteatin < Chrysosplenetin < Negletein < Farobin-A < Aromadedrin < Tricin < Catechin < Baptigenin < Texasin < Homoorientin < Ranupetin < Apometzgerin < Datin < Dihydromyricetin < Epigallocatechin Gallate < Fisetin < Hesperedin < Santin < Afzelechin < Epigallocatechin < Taxifolin < Xanthohumol < Fisetinidol < Leucocyanidin < Nepetin < Angoletin < Scutevulin < Eupafolin < Kaempferide < Rosinidin < Santal < Retusin < Flavoxate < Candidone < Sinensetin < Daidzein < Dihydrokaempferide < Isosakuranetin < Kumatakenin < Robinetinidol < Isoscutellarein < Rhamnetin < Isothymusin < Sakuranetin < Selagin < Formononetin < Butein < Jaceidin < Leucorobinetinidin < Tephrosin < Gomphrenol < Ternatin < Annulatin < Gericudranin-B < Isoetin < Nobiletin < Acacatechin < Gossypetin < Isosilybin < Salvigenin < Fustin < Leucopeonidin < Rhamnazin < Okanin < Isalpinin < Cannflavin < Cyclocommunol < Dihydroflavonol < Glycitein < Leucopelargonidin < Skullcapflavone II < Tambulin < Nodifloretin < Teracacidin < Platanetin < Galangin < Casticin < Pachypodol < Tamarixetin < Calycosin < Norwogonin < Demethylbellidifolin < Leukoefdin < Quercetagetin < Hydrangenol < Petunidin < Gallocatechin < Chrysoeriol < Pulchellidin < Tanetin < Acacetin < Dihydrotricetin < Glabridin < Rhynchosin < Isopongaflavone < Persicogenin < Capensinidin < Cyanidin < Isookanin < Isoxanthohumol < Pinoquercetin < Hypolaetin < Leucodelphinidin < Luteoforol < Melanoxetin < Laricitrin < Leucoanthocyanidin < Liquiritigenin < Sterubin < Esculetin < Glaziovianin-A < Irigenin < Leucofisetinidin < Meciadanol < Norathyriol < Osajin < Topazolin < Ermanin < Mesquitol < Sorbifolin < Biochanin A < Ladanein < Limocitrol < Rhusflavanone < Syringetin < Luteolin < Dihydrowogonin < Pratol < Mearnsitrin < Genistein < Isoquercitrin < Isovitexin < Orobol < Pelargonidin < Pomiferin < Limocitrin < Icaritin < Calycopterin < Helmon < Licoflavonol < Patuletin < Propolin B < Tricetin < Thunberginol A < Matteucinol < Persicarin < Sternbin < Veronicoside < Azaleatin < Datiscetin < Leucomalvidin < Maritimetin <

53 U R K U N D Madeswaran Final Thesis.doc (D29512176)

Ononin < Carajuflavone < Combretol < Isokaempferide < Luteolinidin < Geraldone < Melacacidin < Quercetagetinidin < Tectorigenin < Tomentin < Oroxylin-A < Penduletin < Pratensein < Cirsililol < Malvidin < Ombuin < Isoimperatorin < Genistin < Guibourtinidol < Leptosidin < Maesopsin < Orientin < Prunin < Bavachinin < Pectolinarigenin < Pedalitin < Primetin < Carpachromene < Delphinidin < Gnetifolin E < Prosogerin E < Prunetin < Donepezil.

6.3 In vitro AChE and BuChE inhibitory assay

An increase in AChE leads to an increase in breakdown of ACh to acetate and choline, leading to the development of AD. Recent findings suggest that the occurrence of AD is increasing worldwide, possibly due to life style modifications. AChE inhibitors are commonly employed in the treatment of AD. Inhibition of AChE results in a decreased breakdown of acetylcholine into acetate and choline, when measured spectroscopically. Out of 245 flavonoids taken for in silico docking studies, 20 flavonoids were selected for the in vitro enzyme inhibitory assay. The selection of the flavonoids was based upon the predicted docked score and the compounds were divided conventionally into those of high, moderate and low neuroprotective activity against the targets.

Hence, to prove the reliability of in silico studies the fourteen flavonoids with highest activity (Scopoletin < Baicalein < Morin < Diosmin < Naringenin < Catechin < Hesperetin < Silibinin < Rutin < Robinetin < Taxifolin < Tetra hydroflavone < Hesperidin < Myrecetin), three with moderate (Apigenin < Kaempferol < Tricetin) and three with lowest (Chrysin < Epigallocatechin < Theaflavin) neuroprotective activities were selected for the in vitro enzyme inhibition studies (Fig. 26).

Fig. 26 Selection of flavonoids for in vitro enzyme inhibition studies

6.3.1 Role of flavonoids in AChE inhibitory activity

Among the different flavonoids tested, scopoletin and baicalein exhibited the highest AChE inhibitory activity. The inhibition constant (IC50) of scopoletin was

found to be 10.18 ± 0.68 µg/ml and for baicalein 10.92 ± 0.38 µg/ml when compared to the standard donepezil showed 22.14 ± 0.56 µg/ml. All the selected flavonoids showed AChE inhibitory activity at a concentration below 100 µg/ml, with IC50 values ranging from 10.18 to 21.48 µg/ml (Fig. 27).

Values are expressed as mean ± SEM; n = 6.

*P >0.05 considered as significant (One way ANOVA followed by Dunnett’s test).

Fig. 27 In vitro AChE enzyme inhibitory assay of the selected flavonoids

Values are expressed as mean ± SEM; n = 6.

*P >0.05 considered as significant (One way ANOVA followed by Dunnett’s test).

Fig. 28 In vitro BuChE enzyme inhibitory assay of the selected flavonoids

6.3.2 Role of flavonoids in BuChE inhibitory activity

The inhibitory activity of the flavonoids against BuChE was determined by Ellman’s method with some modifications. This method estimates BuChE using s-butyrylythiocholine iodide (substrate)

54 U R K U N D Madeswaran Final Thesis.doc (D29512176)

and DTNB. The enzymatic activity was measured by the yellow color compound produced by thiocholine when it reacts with dithiobisnitro benzoate ion. The inhibitory activity of the flavonoids increased with increasing concentration. All the flavonoids selected showed BuChE inhibitory activity at a concentration below 100 µg/ml, with IC50 values ranging from 28.18 to 6.12 µg/ml. Noteworthy IC50 values were found for scopoletin and baicalein against BuChE. Scopoletin showed the IC50 value 28.18 ± 0.92 µg/ml and for baicalein 31.24 ± 1.12 µg/ml against BuChE when compared to that of donepezil was found to be 64.38± 0.42 µg/ml (Fig. 28).

The order of potency of flavonoids to ChE (AChE and BuChE) inhibitory activities of the compounds decreased in the order of Scopoletin < Baicalein < Morin < Diosmin < Naringenin < Catechin < Hesperetin < Silibinin < Rutin < Robinetin < Taxifolin < Tetra hydroflavone < Hesperidin < Myrecetin < Apigenin, Kaempferol < Tricetin < Chrysin < Epigallocatechin < Theaflavin < Donepezil.

6.4 Acute toxicity studies and selection of dose for in vivo studies

Among the twenty compounds selected for in vitro AChE inhibitory assay two flavonoids (scopoletin and baicalein) were found to possess excellent activity in inhibiting the AChE and BuChE enzymes and these compounds were selected and used for further in vivo studies. Acute toxicity was determined as per OECD guidelines (420) employing fixed dose procedure for selecting the dose for biological activity. For acute toxicity studies 10 female Wistar rats weighing 150-200 g were used and they were fasted overnight before the experimental day.

Table 2: Observations done for the acute oral toxicity study- Baicalein

Parameters observed 0 h 0.5h 1 h 2 h 4 h Day 2&3 Day 4&5 Day 6&7 Day 8&9 Day 10&11 Day 12&13 Day 14 Respiratory Dyspnea ------Apnea ------Nostril discharges ------Motor activity Tremor ------Hyper activity ------Hypo activity ------Ataxia ------Jumping ------Catalepsy ------Locomotor activity ------Reflexes Corneal reflex ------Pinna reflex ------Righting reflex ------Convulsion Tonic and clonic convulsion ------Muscle tone Hypertonia ------Hypotonia ------Ocular sign Lacrimation ------Miosis ------Mydriasis ------Ptosis ------Skin Edema ------Skin and fur ------Erythema ------Cardiovascular signs Bradycardia ------Tachycardia ------Piloerection Contraction of erectile tissue of hair ------Gastro intestinal signs Diarrhoea ------

Live phase animals Observations Body weight every day Normal Food consumption daily Normal Water consumption daily Normal Home cage activity Normal

Note: Acute toxicity study of baicalein and scopoletin was performed and it was non-toxic up to 2000 mg/kg dose

Table 3: Mortality record for baicalein in acute oral toxicity study

Dose Sighting study Main study (2000 mg/kg) 5 mg/kg 50 mg/kg 300 mg/kg 2000 mg/kg No. of animals 1 2 3 4 5 6 7 8 9 Body weight (g) 170 174 168 172 172 196 186 170 184 Sex Female Female Female Female Female Female Female Female Female 30 min Nil Nil Nil Nil Nil Nil Nil Nil

55 U R K U N D Madeswaran Final Thesis.doc (D29512176)

Nil 1 h Nil Nil Nil Nil Nil Nil Nil Nil Nil 2 h Nil Nil Nil Nil Nil Nil Nil Nil Nil 3 h Nil Nil Nil Nil Nil Nil Nil Nil Nil 4 h Nil Nil Nil Nil Nil Nil Nil Nil Nil Day 1 Nil Nil Nil Nil Nil Nil Nil Nil Nil Day 2 Nil Nil Nil Nil Nil Nil Nil Nil Nil Day 3 Nil Nil Nil Nil Nil Nil Nil Nil Nil Day 4 Nil Nil Nil Nil Nil Nil Nil Nil Nil Day 5 Nil Nil Nil Nil Nil Nil Nil Nil Nil Day 6 Nil Nil Nil Nil Nil Nil Nil Nil Nil Day 7 Nil Nil Nil Nil Nil Nil Nil Nil Nil Day 8 Nil Nil Nil Nil Nil Nil Nil Nil Nil Day 9 Nil Nil Nil Nil Nil Nil Nil Nil Nil Day 10 Nil Nil Nil Nil Nil Nil Nil Nil Nil Day 11 Nil Nil Nil Nil Nil Nil Nil Nil Nil Day 12 Nil Nil Nil Nil Nil Nil Nil Nil Nil Day 13 Nil Nil Nil Nil Nil Nil Nil Nil Nil Day 14 Nil Nil Nil Nil Nil Nil Nil Nil Nil Mortality 0/1 0/1 0/1 0/1 0/5 Note: Acute toxicity study of baicalein compound was performed and it was non-toxic up to 2000 mg/kg dose

Table 4: Observations done for the acute oral toxicity study- Scopoletin

Live phase animals Observations Body weight every day Normal Food consumption daily Normal Water consumption daily Normal Home cage activity Normal

Note: Acute toxicity study of baicalein and scopoletin was performed and it was non-toxic up to 2000 mg/kg dose

Table 5: Mortality record for scopoletin in acute oral toxicity study

Dose Sighting study Main study (2000 mg/kg) 5 mg/kg 50 mg/kg 300 mg/kg 2000 mg/kg No. of animals 1 2 3 4 5 6 7 8 9 Body weight (g) 184 166 182 174 196 168 172 196 183 Sex Female Female Female Female Female Female Female Female Female 30 min Nil Nil Nil Nil Nil Nil Nil Nil Nil 1 h Nil Nil Nil Nil Nil Nil Nil Nil Nil 2 h Nil Nil Nil Nil Nil Nil Nil Nil Nil 3 h Nil Nil Nil Nil Nil Nil Nil Nil Nil 4 h Nil Nil Nil Nil Nil Nil Nil Nil Nil Day 1 Nil Nil Nil Nil Nil Nil Nil Nil Nil Day 2 Nil Nil Nil Nil Nil Nil Nil Nil Nil Day 3 Nil Nil Nil Nil Nil Nil Nil Nil Nil Day 4 Nil Nil Nil Nil Nil Nil Nil Nil Nil Day 5 Nil Nil Nil Nil Nil Nil Nil Nil Nil Day 6 Nil Nil Nil Nil Nil Nil Nil Nil Nil Day 7 Nil Nil Nil Nil Nil Nil Nil Nil Nil Day 8 Nil Nil Nil Nil Nil Nil Nil Nil Nil Day 9 Nil Nil Nil Nil Nil Nil Nil Nil Nil Day 10 Nil Nil Nil Nil Nil Nil Nil Nil Nil Day 11 Nil Nil Nil Nil Nil Nil Nil Nil Nil Day 12 Nil Nil Nil Nil Nil Nil Nil Nil Nil Day 13 Nil Nil Nil Nil Nil Nil Nil Nil Nil Day 14 Nil Nil Nil Nil Nil Nil Nil Nil Nil Mortality 0/1 0/1 0/1 0/1 0/5 Note: Acute toxicity study of scopoletin compound was performed and it was non-toxic up to 2000 mg/kg dose

56 U R K U N D Madeswaran Final Thesis.doc (D29512176)

safe up to a maximum dose of 2000 mg/kg of body weight. There were no changes in normal behavioural pattern and no signs and symptoms of toxicity and mortality were observed (Table 4, 5).

6.5 NEUROPROTECTIVE EFFECTS OF BAICALEIN AND SCOPOLETIN IN SCOPOLAMINE- INDUCED MODEL

6.5.1 Behavioural assessment of baicalein and scopoletin in scopolamine treated mice 6.5.1.1 Effect on transfer latency in the rectangular maze

In this test, baicalein and scopoletin treated mice showed significant (P>0.01) decrease in latency time (LT) when compared to scopolamine treated group on the 7th day. Moreover the higher LT induced by scopolamine was significantly reversed by scopoletin and baicalein treated groups than the standard donepezil treated group. Administration of scopolamine resulted in learning impairment as evidenced by a significant (P>0.05) increase in the LT from 124.45±1.15 on the 1st day to 174.88 ±0.76 on the 7th day in the negative control group when compared to the control group

(120.34±1.18 on 1st day and 118.34±0.68 on the 7th day). Treatment with the scopoletin and baicalein for seven days reversed the LT significantly (P>0.01) when compared with negative control group (Fig. 29). The potency of the reduction in LT was produced by the scopoletin 200mg/kg followed by baicalein 200 mg/kg among the flavonoids treated groups (77.92±0.63 sec, 82.35±0.78 sec on the 7th day). Donepezil, at a dose of 10 mg/kg treated for successive 7 days acts as positive control, elicited a significant (P>0.01) reduction in LT from 122.44±1.05 sec to 92.81±0.67 sec compared to the negative control group using Dunnet’s test.

Fig. 29 Effect of baicalein and scopoletin on latency time using rectangular maze test in scopolamine-induced amnesia

Fig. 30 Effect of baicalein and scopoletin on escape latency time using Morris water maze test in scopolamine-induced amnesia

6.5.1.2 Effect on escape latency time in the Morris water maze

The Morris water maze (MWM) plays an important role in the validation of rodent models for neurocognitive disorders such as Alzheimer’s disease. In MWM, escape latency time (ETL) was calculated, i.e., the time taken by an animal to find the platform. The scopolamine administration group showed increased ELT from 64.53±0.94 sec to 86.78±1.14 sec, whereas the control group was found to be 56.35±0.84. This relative increase in ELT when compared to the control group indicated a spatial memory deficit in the scopolamine treated mice. It was found that baicalein and scopoletin treated mice significantly (P>0.01) reduced ELT when compared with the scopolamine treated group.

In the treatment groups, higher dose of scopoletin and baicalein successfully reversed the scopolamine-induced memory deficits compared to other flavonoids treated groups and standard. The significant reduction of ELT was produced by the scopoletin and baicalein (200 mg/kg) among the flavonoids treated groups on the 7th day was found to be 24.18±0.63 sec, 31.26±1.18. Donepezil (10 mg/kg) treated animals, produced a significant (P>0.01) decrease in ELT 36.42±0.84 sec on the 7th day compared to the negative control group (Fig. 30).

57 U R K U N D Madeswaran Final Thesis.doc (D29512176)

6.5.1.3 Locomotor activity in scopolamine treated mice

The locomotor activity of the flavonoids treated groups significantly (P>0.01) increased when compared to the negative control group. A significant (P>0.01) increase in the values of locomotion 346.17±1.04 sec on the 7th day was observed in the standard group. In the treatment groups, scopoletin (200 mg/kg and 100 mg/kg) treated mice significantly increased the locomotor values (P>0.01), 380.78±0.82 sec and 368.52±1.02 sec respectively and baicalein (200 mg/kg and 100 mg/ kg) significantly elevated locomotor values 356.32±0.78 and 352.43±0.62 on the 7th day compared to the scopolamine-treated group (Fig. 31).

Fig. 31 Effect of baicalein and scopoletin on locomotor activity using Actophotometer in scopolamine-induced amnesia

Fig. 32 Effect of baicalein and scopoletin on percentage of conditioned avoidance response using Pole climbing test in scopolamine-induced amnesia

6.5.1.4 Conditioned avoidance response in scopolamine treated mice

In pole climbing method, the percentage of conditioned avoidance response (CAR) were calculated based on the number of successful events and number of errors produced by each groups. Scopolamine treated mice showed significantly decreased percentage of CAR activity 48.21±0.51 sec as compared to control group 74.15±0.58 sec on the 7th day and donepezil treated animals exhibited significant (P>0.05) increase in percentage of CAR activity 88.92±0.38. Scopoletin (100 mg/kg and 200 mg/kg) treated animals significantly (P>0.01) increased CAR activity to 92.15±0.26 and 96.84±0.62 and baicalein (100 mg/kg and 200 mg/kg) significantly (P>0.01) elevated CAR values to 88.08±0.64 and 92.62±0.82 compared to negative control group (Fig. 32).

6.5.1.5 Effect of scopoletin and baicalein response time in 5-choice serial time task method

Administration of scopolamine resulted in learning impairment as evidenced by significant delayed response values (68.24±0.84) when compared to control group (36.18±0.44). In treatment groups, scopoletin (100 mg/kg and 200 mg/kg) showed quicker responses (23.12±1.08 and 18.54±0.72) and baicalein (100 mg/kg and 200 mg/kg) treated animals also showed significant (P>0.01) improved responses time (25.74±0.56 and 22.32±0.44). Standard drug, donepezil administered animals exhibited significantly improved responses (19.42±0.63) (Fig. 33).

58 U R K U N D Madeswaran Final Thesis.doc (D29512176)

Fig. 33 Effect of baicalein and scopoletin on response time using 5 CSRT model in scopolamine- induced amnesia

compared to the lower dose of flavonoids treated animals. These results further augmented the neuroprotective effect of the scopoletin against the Alzheimer's disease.

6.5.2 Biochemical estimations in scopolamine induced mice

6.5.2.1 Estimation of AChE activity in the brain

Fig. 34 Percentage of AChE inhibition in the mice brain in scopolamine-induced amnesia

Values are expressed as mean ± SEM; n = 6.

*P >0.05 when compared to vehicle control; **P >0.01 when compared to negative control (One way ANOVA followed by Dunnett’s test).

AChE is a key enzyme involved in cholinergic neurotransmission and is a marker of extensive loss of cholinergic neurons in the brain. A significant increase (P>0.01) in the level of AChE was observed in the scopolamine treated mice. Administration of baicalein and scopoletin significantly (P>0.01) attenuated AChE levels and no change was observed in control group.

Scopoletin (100 mg/kg and 200 mg/kg) exhibited the percentage AChE inhibition in the brain homogenate as 70.48% and 85.22%, which were significantly (P>0.01) increased values when compared to scopolamine treated mice (22.14%). Administration of baicalein (100 mg/kg and 200mg/kg) exhibited the percentage AChE inhibition as 57.18% and 72.14%. Donepezil significantly (P>0.05) reduced the level of AChE into 66.72%. Scopoletin (200 mg/kg) effectively inhibited the AChE and thereby it increased the level of acetylcholine in the brain (Fig. 34).

6.5.2.2 Determination of total protein content

The total protein content was significantly reduced to 0.34±1.32 units/mg tissue protein in negative control group compared to control (1.08±1.26 units/mg tissue protein). Scopoletin (100 mg/kg and 200 mg/kg, p.o.) treated mice significantly reversed the protein content values (1.22±1.08 and 1.31 ±0.82 µmoles/min/mg protein) and administration of baicalein (100 mg/kg and 200 mg/kg, p.o.) significantly increased the values (1.16±0.84 and 1.25±1.23 µmoles/min/mg protein) when compared to negative control. Treatment with donepezil (5 mg/kg, p.o.) resulted in a significant increased protein content level (1.12±0.96 µmoles/min/mg protein) in the brain (Table 6). The total protein content was greatly increased in higher dose of scopoletin (200 mg/kg) treated group when compared to all other groups.

Table 6: Effect of baicalein and scopoletin on protein content in scopolamine treated mice

Groups Protein Content (mmoles/min/mg) Control 1.08±1.26 Negative Control (Scopolamine) 0.34 ±1.32* Baicalein (100 mg/kg) 1.16±0.84** Baicalein (200 mg/kg) 1.25±1.23** Scopoletin (100 mg/kg) 1.22±1.08** Scopoletin (200 mg/kg) 1.31±0.82** Standard (Donepezil) 1.12±0.96**

Values are expressed as mean ± SEM; n = 6.

*P >0.05 when compared to vehicle control; **P >0.01 when compared to negative control (One way ANOVA followed by Dunnett’s test).

6.5.2.3 Enzymatic antioxidant levels

59 U R K U N D Madeswaran Final Thesis.doc (D29512176)

6.5.2.3.1 Estimation of catalase

A significant (P>0.05) reduction in the catalase level was found in scopolamine treated mice (1.24 ±0.48 µmoles/min/mg protein) compared to the control group (3.18±0.62 µmoles/min/mg protein). Scopoletin (100 mg/kg and 200 mg/kg, p.o.) treated mice resulted in significant (P>0.01) increase in the level of catalase (3.34±1.08 and 3.46±0.38 µmoles/min/mg protein) and administration of baicalein (100 mg/kg and 200 mg/kg, p.o.) significantly (P>0.01) increased the catalase level to 3.26 ±0.72 and 3.38±0.64 µmoles/min/mg protein when compared to negative control. Treatment with donepezil (5 mg/kg) showed significant (P>0.05) increased in the level of catalase to 3.30±0.52 µmoles/min/mg protein in the brain (Table 7). The catalase level was greatly increased in higher dose of scopoletin treated group animals compared to other groups used in the study.

6.5.2.3.2 Estimation of superoxide dismutase

Superoxide dismutase is an enzymatic antioxidant which scavenges the superoxide anion to hydrogen peroxide, hence diminishing the toxic effect caused by this radical. The SOD level was significantly (P>0.01) reduced to 0.98±1.08 units/mg tissue protein in negative control when compared to control (2.96±0.58 units/mg tissue protein). Scopoletin (100 mg/kg and 200 mg/kg, p.o.) treated group significantly (P>0.01) increased the SOD level to 3.28±0.82 and 3.42±0.74 µmoles/min/mg protein and treatment with baicalein (100 mg/kg and 200 mg/kg, p.o.) significantly (P>0.01) elevated the SOD level to 3.08±0.64 and 3.36±1.12 µmoles/min/mg protein when compared to negative control. Donepezil treated mice (5 mg/kg, p.o.) resulted in a significant (P>0.05) elevation of SOD level (3.28±0.88 µmoles/min/mg protein) in the brain (Table 7). The SOD level was greatly increased in higher dose of scopoletin group treated animals compared to all other groups.

6.5.2.3.3 Estimation of glutathione peroxidase

The GPx level was significantly decreased (P>0.05) in negative control group to 0.42±0.48 when compared to the control group (1.48±0.36) and enzymatic activity of GPx in donepezil treated group showed elevation of GPx level (1.62±0.34). It reveals that in baicalein treated mice (100 and 200 mg/kg) restoration of the activity of the glutathione peroxidase enzyme was noticed to 1.52±0.28 and 1.66±0.32 and scopoletin (100 and 200 mg/kg) treatment elevated GPx values significantly to 1.58±0.40 and 1.76±0.36 respectively and possibly this could be due to reduced generation of free radicals and subsequent neuronal damage (Table 7). This suggests that the higher dose of scopoletin has greater capability to restore level of GPx and reduced formation of free radicals and associated oxidative stress.

Table 7: Effect of baicalein and scopoletin on enzymatic and non-enzymatic antioxidant level in scopolamine treated mice

Groups CAT SOD GSSH GPx GSH Control 3.18±0.62 2.96±0.58 22.45±1.32 1.48±0.36 3.88±0.46 Negative Control (Scopolamine) 1.24±0.48* 0.98±1.08* 11.31±1.43* 0.42±0.48* 1.28±0.28* Baicalein (100 mg/kg) 3.26±0.72** 3.08±0.64** 21.08±1.61** 1.52±0.28** 3.74±0.24** Baicalein (200 mg/kg) 3.38±0.64** 3.36±1.12** 24.82±1.17** 1.66±0.32** 3.98±0.48** Scopoletin (100 mg/kg) 3.34 ±1.08** 3.28±0.82** 25.03±1.72** 1.58±0.40** 4.14±0.54** Scopoletin (200 mg/kg) 3.46±0.38** 3.42 ±0.74** 27.21±1.54** 1.76±0.36** 4.32±1.08** Standard (Donepezil) 3.30±0.52** 3.28±0.88** 23.13 ±1.38** 1.62±0.34** 3.96±0.32**

Values are expressed as mean ± SEM; n = 6.

60 U R K U N D Madeswaran Final Thesis.doc (D29512176)

*P >0.05 when compared to vehicle control; **P >0.01 when compared to negative control (One way ANOVA followed by Dunnett’s test).

6.5.2.3.4 Estimation of glutathione reductase (GSSH)

The glutathione reductase level was significantly reduced (P>0.05) in negative control group to 11.31±1.43 when compared to control group (22.45±1.32) and enzymatic activity in donepezil resulted in an increased level of glutathione reductase (23.13±1.38). Administration of baicalein (100 and 200 mg/kg) could reverse the level of the glutathione reductase to 21.08±1.61 and 24.82±1.17 and scopoletin (100 and 200 mg/kg) increased GR significantly (P>0.01) to 25.03±1.72 and 27.21 ±1.54 respectively (Table 7). This suggests that the higher dose of scopoletin has the capability to reverse the level of glutathione reductase.

6.5.2.4 Non enzymatic antioxidant activity

6.5.2.4.1 Estimation of reduced glutathione

Glutathione is an important tripeptide which can remove the free radical species such as H2O2, O2·, alkoxy radical and maintain the membrane protein thiols and acts as a substrate for glutathione peroxidase and glutathione transferase. The level of reduced glutathione in the brain was reduced in the negative control group (1.28±0.28 nmoles/min/mg tissue protein) when compared to the control (1.28±0.28 nmoles/min/mg tissue protein). Treatment with baicalein (100 and 200 mg/kg) resulted in a significant (P>0.01) elevation of reduced glutathione level to 3.74±0.24 and 3.98±0.48 and administration of scopoletin (100 and 200 mg/kg) significantly (P>0.01) elevated the reduced glutathione levels to 4.14±0.54 and 4.32±1.08 respectively. Donepezil (5mg/kg, p.o.) significantly (P>0.01) increased the level (3.96±0.32 nmoles/min/mg tissue protein) of reduced glutathione in the brain (Table 7). The present research depicts that scopoletin (100 mg and 200 mg/kg) reduced oxidative stress by replenishing the levels of catalase, SOD, GPx, and GSH in mice.

6.5.2.5 DPPH assay

DPPH is a stable, nitrogen-centered free radical which produces violet colour. It was reduced to a yellow colored product, diphenylpicryl hydrazine, with the addition of the fractions in a concentration- dependent manner. The reduction in the number of DPPH molecules can be correlated with the number of available hydroxyl groups. The DPPH scavenging activity was significantly decreased (P>0.05) in negative control group to 7.24±1.89 when compared to the control group (12.18±1.58). The highest DPPH radical scavenging activity was detected in the higher concentration of scopoletin (200 mg/kg) 15.48±1.15, followed by the scopoletin 100 mg/kg, baicalein (200 mg/kg, 100mg/kg) showed the values 14.12±0.84, 13.98±1.06 and 12.48±0.81 respectively. These activities were less than that of the standard, donepezil (13.08±0.98) (Table 8). Scopoletin and baicalein treated mice showed significantly (P>0.01) higher DPPH scavenging values compared to negative control group.

Table 8: Effect of baicalein and scopoletin on DPPH and MDA level in scopolamine treated mice

Groups DPPH (nmoles/min/mg) MDA (nmoles/min/mg) Control 12.18±1.58 5.86±0.24 Negative Control (Scopolamine) 7.24±1.89* 13.88±0.64* Baicalein (100 mg/kg) 12.48±0.81** 5.63±0.34** Baicalein (200 mg/kg) 13.98±1.06** 5.48±0.28** Scopoletin (100 mg/kg) 14.12±0.84** 4.52±0.46** Scopoletin (200 mg/kg) 15.48±1.15** 4.03±0.38** Standard (Donepezil) 13.08±0.98** 6.26±0.73**

Values are expressed as mean ± SEM; n = 6.

*P >0.05 when compared to vehicle control; **P >0.01 when compared to negative control (One way ANOVA followed by Dunnett’s test).

6.5.2.6 Estimation of lipid peroxidation

61 U R K U N D Madeswaran Final Thesis.doc (D29512176)

Lipid peroxidation (LPO) is one of the main manifestations of oxidative damage, which implicates free radical oxidative cell injury in mediating the toxicity of scopolamine. Effect of baicalein and scopoletin on the levels of malondialdehyde (MDA) during scopolamine-induced neurotoxicity was observed. Increase in the level of lipid peroxides in the brain reflects the neuronal damage. Elevated level of MDA was observed in scopolamine-treated mice (13.88±0.64 nmoles/min/mg tissue protein), whereas it was significantly (P>0.01) reversed in flavonoids-treated groups (scopoletin and baicalein).

Administration of baicalein (100 and 200 mg/kg) significantly (P>0.01) decreased (5.63±0.34 and 5.48±0.28 nmoles/min/mg tissue protein) MDA level in brain and scopoletin (100 and 200 mg/kg) treated group also reduced the level of MDA significantly to 4.52±0.46 and 4.03±0.38 when compared to the negative control group. Donepezil (5 mg/kg, p.o) significantly (P>0.01) decreased the level (6.26±0.73 nmoles/min/mg tissue protein) of MDA in the brain (Table 8). The results revealed that scopoletin and baicalein possess antioxidant property with concentration gradient and thereby it reduces the oxidative stress induced membrane lipid peroxidation.

6.5.3 Histopathological studies of the baicalien and scopoletin in acute scopolamine induced amnesia in mice

Fig. 35 The demonstration of the effects of baicalein and scopoletin in scopolamine-induced amnesia (SCP-scopolamine)(A-Normal histological architecture without any degenerative changes; B-Severe neurodegenerative changes; C-Formation of neurofibrillary tangles; D-Faintly appearing neurofibrillary tangles; E-Mild infiltration of inflammatory cells indicating the repair mechanism; F- Moderately appearing neurofibrillary tangles; G-normal histological architecture with mild infiltration of inflammatory cells; H-minimal neuro degenerative changes)

6.6 NEUROPROTECTIVE EFFECT OF BAICALEIN AND SCOPOLETIN ON BEHAVIOURAL CHANGES IN AlCl3-INDUCED MODEL

6.6.1 Effect of baicalein and scopoletin on behavioural changes in AlCl3 treated mice

6.6.1.1 Evaluation of transfer latency in the rectangular maze

In this test, latency time (LT) of the animals was calculated for all (Group I to VII) the groups. Administration of AlCl3 to mice for 3 months resulted in learning impairment by a significant (P>0.05) increase in the LT (175.32±0.98) in the negative control group when compared to the control group (112.21±0.54). Baicalein and scopoletin treated mice resulted in reversal of LT significantly (P>0.01) when compared with negative control.

The LT was effectively decreased by scopoletin (200 mg/kg, 100 mg/kg) treated mice among the all other groups tested showing the values of 46.72±0.38 and 54.48 ±0.82 respectively. Donepezil resulted in significant (P>0.01) decrease in LT 72.54±0.64 compared to the negative control group using Dunnet’s test (Fig. 36).

62 U R K U N D Madeswaran Final Thesis.doc (D29512176)

6.6.1.2 Estimation of escape latency time in the Morris water maze

In MWM, escape latency time (ETL) was calculated, which was the time taken by an animal to find the platform. AlCl3 treated animals showed significantly (P>0.01) increased ETL values on 90th day (76.38±1.18) as compared to control group (49.48±0.72) and it indicates the impairment in learning and memory. Baicalein (100 mg/kg and 200 mg/kg) treated mice significantly (P>0.01) decreased ETL, 31.54±0.84 and 27.06±0.78 and scopoletin (100 mg/kg and 200 mg/kg) significantly (P>0.01) decreased ETL, 30.82±1.02 and 21.42±0.62 when compared to negative control. Administration of donepezil significantly (P>0.01) decreased ETL values (32.18±0.48). Among the treatment groups, scopoletin (200 mg/kg) treated mice effectively reduced ETL values when compared to all other groups (Fig. 37).

Fig. 36 Effect of baicalein and scopoletin on latency time using rectangular maze test in AlCl3- induced learning impairment

Fig. 37 Effect of baicalein and scopoletin on escape latency time using Morris water maze test in AlCl3-induced learning impairment

Fig. 38 Effect of baicalein and scopoletin on locomotor activity in AlCl3-induced learning impairment

6.6.1.3 Locomotor activity in AlCl3 treated mice

AlCl3 treated mice exhibited significantly (P>0.05) decreased locomotor activity on 90th day (176.46 ±1.38) as compared to control group (286.53±0.82) and it indicates the learning and memory impairment in mice. Baicalein (100 mg/kg and 200 mg/kg) treated mice significantly (P>0.01) elevated locomotor values 386.76±0.46 and 448.54±0.68 and scopoletin (100 mg/kg and 200 mg/ kg) significantly (P>0.01) increased the locomotion to 426.34±0.92 and 492.54±1.08 when compared to negative control. Donepezil significantly (P>0.01) elevated the locomotor values (394.38±0.62) on the 90th day. Higher dose of scopoletin resulted in effective increase in locomotor activity which in turns results in its neuroprotective activity (Fig. 38).

6.6.1.4 Conditioned avoidance response in AlCl3 treated mice

In pole climbing method, the percentage of conditioned avoidance response (CAR) was calculated based on the number of successful events and number of errors produced by the animals. AlCl3 alone treated mice showed significantly (P>0.05) decreased percentage of CAR activity on 90th day (32.82±1.18) as compared to control group (66.28±0.76). Administration of baicalein (100 mg/kg and 200 mg/kg) resulted in significant (P>0.01) elevation of CAR values 89.52±0.74 and 93.43±0.46 and scopoletin (100 mg/kg and 200 mg/kg) significantly (P>0.01) increased the values to 91.08±0.52 and 96.62±0.78 when compared to negative control. Donepezil treated mice significantly (P>0.01) increased percentage of CAR activity (84.45±0.82) compared to negative control group (Fig. 39).

Fig. 39 Effect of baicalein and scopoletin on percentage of conditioned avoidance response in AlCl3-induced learning impairment

Fig. 40 Effect of baicalein and scopoletin on response time using 5-CSRT model in AlCl3-induced learning impairment

6.6.1.5 Effect of baicalein and scopoletin on response time in 5-choice serial reaction time task method

5-CSRT is an operant based task used to study attention and impulse control in rodents. AlCl3 treated mice showed significantly (P>0.05) quicker response time (72.86±0.98) on the 91st day when compared to control group (35.24±0.68). Treatment with baicalein (100 mg/kg and 200 mg/kg) significantly (P>0.01) faster response time 22.92±0.58 and 18.26±0.46 and scopoletin (100 mg/kg and 200 mg/kg) significantly (P>0.01) decreased the learning impairment values to 21.38±0.68 and 63 U R K U N D Madeswaran Final Thesis.doc (D29512176)

15.72±0.74 when compared to negative control. Donepezil treated mice significantly (P>0.01) decreased memory impairment to 19.56±0.76.

From the behavioural assessments it has been evidenced that, the chronic administration of AlCl3 (3 months) resulted in the learning impairment in mice and the flavonoids (baicalein and scopoletin) treated mice significantly (P>0.01) reversed the learning impairment produced by AlCl3 (Fig. 40). Scopoletin (200 mg/kg) treated mice, effectively attenuated the learning impairment produced by AlCl3 when compared to all other groups and this further emphasized the neuroprotective role of scopoletin.

6.6.2 Biochemical evaluations in AlCl3-induced learning impairment in mice

6.6.2.1 Estimation of AChE level in the brain

Fig. 41 Percentage of AChE inhibition in the mice brain in AlCl3-induced learning impairment

Values are expressed as mean ± SEM; n = 6.

*P >0.05 when compared to vehicle control; **P >0.01 when compared to negative control (One way ANOVA followed by Dunnett’s test).

6.6.2.2 Estimation of total protein content

Table 9: Effect of baicalein and scopoletin on protein content in AlCl3 treated mice

Groups Protein Content (mmoles/min/mg) Control 1.24±0.81 Negative Control (AlCl3 50 mg/kg) 0.28±1.24* Baicalein (100 mg/kg) 1.21±1.12** Baicalein (200 mg/kg) 1.25±0.84** Scopoletin (100 mg/kg) 1.28±0.94** Scopoletin (200 mg/kg) 1.34±1.07** Standard (Donepezil) 1.26±1.04**

Values are expressed as mean ± SEM; n = 6.

*P >0.05 when compared to vehicle control; **P >0.01 when compared to negative control (One way ANOVA followed by Dunnett’s test).

The total protein content was significantly (P>0.05) reduced to 0.28±1.24 units/mg tissue protein in negative control group compared to control (1.24±0.81 units/mg tissue protein). Administration of baicalein (100 mg/kg and 200 mg/kg) significantly (P>0.01) reversed the protein content values (1.21±1.12 and 1.25±0.84 µmoles/min/mg protein) when compared to negative control and treatment with scopoletin (100 mg/kg and 200 mg/kg) significantly (P>0.01) increased the values (1.28±0.94 and 1.34±1.07 µmoles/min/mg protein) when compared to negative control. Donepezil (5 mg/kg, p.o.) increased the total protein level (1.26±1.04 µmoles/min/mg protein) (Table 9). The total

64 U R K U N D Madeswaran Final Thesis.doc (D29512176)

protein content was greatly increased in higher dose of both scopoletin and baicalein (200 mg/kg) group compared to all other groups.

6.6.3 Measurement of enzymatic antioxidant levels

6.6.3.1 Estimation of catalase level

Chronic administration of AlCl3 caused marked oxidative stress, which led to decrease in the antioxidant enzyme levels. In the present study, a significant (P>0.05) decrease in the catalase level was found in AlCl3 treated group (0.98±0.34 µmoles/min/mg protein) compared to the control (3.58 ±0.46 µmoles/min/mg protein). Treatment with baicalein (100 mg/kg and 200 mg/kg) resulted in significant (P>0.01) increase in the catalase level (3.66±0.38 and 3.78±0.42 µmoles/min/mg protein) and scopoletin (100 mg/kg and 200 mg/kg) significantly (P>0.01) reversed the catalase level (3.76 ±0.66 and 3.88±0.42 µmoles/min/mg protein). Donepezil (5 mg/kg) increased the catalase level (3.72±0.44 µmoles/min/mg protein) compared to the negative control group (Table 10).

6.6.3.2 Evaluation of superoxide dismutase

Superoxide dismutase scavenges the superoxide anion to hydrogen peroxide, hence diminishing the toxic effect caused by the radicals. The SOD level was significantly (P>0.05) decreased to 0.62 ±0.74 units/mg tissue protein in negative control group compared to control group (2.74±0.38 units/ mg tissue protein). Donepezil (5 mg/kg, p.o.) resulted in a elevated SOD level (2.88±0.68 µmoles/ min/mg protein) in the brain compared to negative control. In the baicalein (100 mg/kg and 200 mg/ kg) treated mice, there was a significant (P>0.01) reversal in the level of SOD to 2.78±0.34 and 2.94 ±0.64 µmoles/min/mg protein and scopoletin (100 mg/kg and 200 mg/kg) significantly (P>0.01) increased the SOD level (2.92±1.02 and 3.12±0.48 µmoles/min/mg protein) emphasising the antioxidant potential of baicalein and Scopoletin (Table 10).

Table 10: Effect of baicalein and scopoletin on enzymatic and non-enzymatic antioxidant level in AlCl3 treated mice

Groups CAT SOD GPx GSSH GSH Control 3.58±0.46 2.74±0.38 1.82±0.42 25.38±0.94 3.24±0.36 Negative Control (AlCl3) 0.98±0.34* 0.62±0.74* 0.58±0.24* 16.25±1.31* 0.94±0.48* Baicalein (100 mg/kg) 3.66±0.38** 2.78±0.34** 1.90±0.48** 24.16±0.88** 3.34±0.44** Baicalein (200 mg/kg) 3.78 ±0.42** 2.94±0.64** 2.08±0.28** 26.58±0.97** 3.48±0.38** Scopoletin (100 mg/kg) 3.76±0.66** 2.92 ±1.02** 2.02±0.36** 26.48±1.08** 3.40±0.42** Scopoletin (200 mg/kg) 3.88±0.42** 3.12±0.48** 2.28 ±0.62** 28.35±0.94** 3.64±0.72** Standard (Donepezil) 3.72±0.44** 2.88±0.68** 1.96±0.52** 26.18 ±1.24** 3.42±0.38**

Values are expressed as mean ± SEM; n = 6.

*P >0.05 when compared to vehicle control; **P >0.01 when compared to negative control (One way ANOVA followed by Dunnett’s test).

6.6.3.3 Determination of glutathione peroxidase level

AlCl3 treated mice significantly (P>0.05) decreased the GPx level to 0.58±0.24 when compared to the control group (1.82±0.42) and enzymatic activity of GPx in donepezil treated group was found to be 1.96±0.52. Administration with baicalein (100 and 200 mg/kg) significantly (P>0.01) increased the level of GPx (1.90±0.48 and 2.08±0.28) and scopoletin (100 mg/kg and 200 mg/kg) treated animals showed elevated levels of GPx to 2.02±0.36 and 2.28±0.62 when compared to negative control (Table 10). Effective restoration of GPx level and the generation of free radicals and neuronal damage was reduced in the scopoletin (200 mg/kg) treated group.

6.6.3.4 Estimation of glutathione reductase level

65 U R K U N D Madeswaran Final Thesis.doc (D29512176)

The glutathione reductase level was significantly (P>0.01) decreased in negative control group to 16.25±1.31 when compared to the control group (25.38±0.94) and enzymatic activity in donepezil treated group was found to be 26.158±1.24. Baicalein treated (100 and 200 mg/kg) animals significantly (P>0.01) increased the level of glutathione reductase to 1.52±0.28 and 1.66±0.32 and treatment with scopoletin (100 and 200 mg/kg) significantly (P>0.01) elevated the glutathione reductase level (1.58±0.40 and 1.76±0.36) (Table 10). Flavonoids treated animals showed increased level of antioxidant enzyme which in turn results in the improvement of neuroprotective potential of scopoletin and baicalein.

6.6.4 Non enzymatic antioxidant assay

6.6.4.1 Determination of reduced glutathione

Glutathione is a tripeptide which can remove the free radical species and maintain the membrane protein thiols and act as a substrate for GPx and glutathione transferase. Reduced glutathione level in brain of the negative control (AlCl3) group was significantly (P>0.05) reduced to 0.94±0.48 nmoles/min/mg tissue protein when compared to the control group. Donepezil (5mg/kg) treated animals significantly increased the reduced glutathione level 3.42±0.38 nmoles/min/mg tissue protein in the brain. Administration with baicalein (100 and 200 mg/kg) showed significantly (P>0.01) increased values 3.34±0.44 and 3.48±0.38 and scopoletin (100 and 200 mg/kg) increased significantly (P>0.01) the levels of reduced glutathione to 3.40±0.42 and 3.64±0.72 (Table 10).

6.6.5 Estimation of DPPH assay

The DPPH scavenging activity was significantly decreased (P>0.05) in negative control group to 8.16±1.24 when compared to the control group (16.22±0.86). Baicalein and scopoletin demonstrated remarkable H-donor activity. The highest DPPH radical scavenging activity was detected in the higher concentration of scopoletin (200 mg/kg) 19.24±0.95, followed by the scopoletin 100 mg/kg, baicalein (200 mg/kg, 100 mg/kg) exhibited lower DPPH scavenging activity compared to sscopoletin treated mice with the values of 18.08±1.08, 17.52±0.96 and 16.18±1.18 respectively. These activities were less than that of the standard, donepezil (17.12±1.28) (Table 11).

Table 11: Effect of baicalein and scopoletin on DPPH and MDA level in AlCl3 treated mice

Groups MDA (nmoles/min/mg) DPPH (nmoles/min/mg) Control 0.98±0.36 16.22±0.86 Negative Control (AlCl3) 4.12±0.48* 8.16±1.24* Baicalein (100 mg/kg) 0.72±0.54** 16.18±1.18** Baicalein (200 mg/kg) 0.60±1.08** 17.52±0.96** Scopoletin (100 mg/kg) 0.62±0.36** 18.08±1.08** Scopoletin (200 mg/kg) 0.54±0.28** 19.24±0.95** Standard (Donepezil) 0.66±0.62** 17.12±1.28**

Values are expressed as mean ± SEM; n = 6.

*P >0.05 when compared to vehicle control; **P >0.01 when compared to negative control (One way ANOVA followed by Dunnett’s test).

6.6.6 Determination of lipid peroxidation level

Increase in the level of lipid peroxides in the brain reflects the neuronal damage and thus leads to AD. Elevated level of MDA was observed in AlCl3-treated mice (4.12±0.48 nmoles/min/mg tissue protein), whereas it was significantly (P>0.05) reversed in flavonoids-treated groups. Donepezil (5 mg/kg, p.o) decreased the MDA level to 0.66±0.62 nmoles/min/mg tissue protein. Baicalein (100 and 200 mg/kg) treated mice showed significantly (P>0.05) decreased MDA level in brain to 0.72 ±0.54 and 0.60±1.08 nmoles/min/mg tissue protein as compared to the negative control group and scopoletin (100 and 200 mg/kg) significantly (P>0.01) reduced the level of MDA (0.62±0.36 and 0.54 ±0.28) when compared to negative control group. The results suggested that scopoletin possibly

66 U R K U N D Madeswaran Final Thesis.doc (D29512176)

has potent antioxidant property to reduce oxidative stress induced membrane lipid peroxidation (Table 11).

6.6.7 Histopathological studies of the baicalien and scopoletin against chronic AlCl3 treatment in mice

7. DISCUSSION

Drug design is an important tool in the field of medicinal chemistry where new compounds are synthesized by molecular or chemical manipulation of the lead moiety in order to produce highly active compounds with minimum steric effect211. Docking of small molecules in the receptor binding site and estimation of binding affinity of the complex is a vital part of structure based drug design212, 213. A huge breakthrough in the process of drug design was the development of in silico method to predict the therapeutic efficacy of the molecule214.

7.1 Evaluation of drug likeness properties

The pharmacologically active substituents were characterized by calculating steric, hydrophobic, electronic, and hydrogen bonding properties as well as by the drug-likeness score. The theory of drug-likeness score helps to optimize pharmaceutical and pharmacokinetic properties, for example, chemical stability, solubility, distribution profile and bioavailability. The molecular descriptors have developed as rationally predictive and informative, for example, the Lipinski's Rule-of-Five119. The better oral absorption of the ligands and drug likeness scores are constructed by the solubility, diffusion, Log P, molecular weight etc. Before the docking studies, the ligands were evaluated for its drug likeness properties. MedChem Designer software was used to evaluate the Lipinski’s rule of five for the flavonoids215.

7.2 In silico AChE and BuChE inhibition

67 U R K U N D Madeswaran Final Thesis.doc (D29512176)

Molecular docking is a frequently used tool in computer-aided structure-based rational drug design. It evaluates how small molecules called ligands (flavonoids) and the target macromolecule fit together216. The use of computers to predict the binding of libraries of small molecules to known target structures is an increasingly important component in the drug discovery process217, 218. There is a wide range of software packages available for the conduct of molecular docking simulations like, AutoDock, GOLD, FlexX etc.122, 219. Among these, AutoDock 4.2 is the most recent version which has been widely employed for virtual screening, due to its enhanced docking speed220. Its default search function is based on Lamarckian Genetic Algorithm (LGA), a hybrid genetic algorithm with local optimization that uses a parameterized free-energy scoring function to estimate the binding energy221.

Based upon the chemical structure classification of our selected flavonoids, the AChE inhibitory activity was found to be, Flavones < Coumarin derivatives < Flavonones < Iso flavones < Flavonols. The minimum binding energy indicated that AChE and BuChE were successfully docked with selected flavonoids. These flavonoids showed an excellent binding mode when compared with the standard donepezil against AChE and BuChE.

The amino acid residues are dynamically involved in the formation of hydrophilic and hydrophobic contacts and there were initiated to play important role in the binding site of target. Islam et al., studied the in silico QSAR analysis of quercetin and found that quercetin has higher binding score than the conventional drugs when allowed to bind at the same pocket of the AChE as conventional drugs224. The hydrogen bonds formed by quercetin involve the same amino acid residues Tyr 124, Ser 293 and Tyr 341 as for conventional drugs. Moreover, different atoms of other amino acid residues such as Phe 295, Arg 296, Tyr 337 and Tyr 341 participate to form the hydrogen bonds225.

7.3 In vitro AChE and BuChE inhibition

68 U R K U N D Madeswaran Final Thesis.doc (D29512176)

cholinergic function of the brain by stimulating the cholinergic receptors, improve the level of acetylcholine from being a breakdown by cholinesterase enzymes or induce antioxidant therapy and anti-inflammatory agents227.

7.4 Acute toxicity studies

The acute toxicity studies were performed as per the OECD guidelines. Based on the acute toxicity studies, we had selected the two different doses of the selected flavonoids baicalein and scopoletin. The results of acute toxicity studies have revealed that, these compounds were considered non- toxic up to the dose of 2000 mg/kg and therefore 1/10th and 1/20th dose was used for the biological evaluation and the in vivo scopolamine and aluminium induced learning impairment studies were conducted at dose levels of 100 and 200 mg/kg body weight.

7.5 Acute scopolamine-induced amnesia

Scopolamine is widely utilized as an experimental tool to study the anti-amnesic potential of new drugs233. Scopolamine hydrobromide is an anticholinergic drug, which produces dementia by reducing the levels of acetylcholine, which is considered to be an important neurotransmitter for learning and memory234.

From the behavioral tests like, rectangular maze test and Morris water maze test, it is clearly seen that there was a significant decrease in the transfer latency in all the flavonoids treated groups compared to the scopolamine-treated group. The memory loss effect of scopolamine is more prominent compared to the control group. In comparison with Donepezil, the higher dose (200 mg/ kg) of scopoletin and baicalein treated groups had excellent performance which indicates neuroprotective effect against scopolamine. Administration of scopoletin and baicalein at a dose of

69 U R K U N D Madeswaran Final Thesis.doc (D29512176)

200 mg/kg/p.o. for 7 days to animals successfully reversed the learning impairment produced by the scopolamine when compared to the lower doses of the selected flavonoids.

There was a gradual increase in the locomotor activity and percentage of conditioned avoidance response in the flavonoids treated animals when compared to the standard. Thus, the potential neuroprotective behavioural changes in the flavonoids treated animals further augmented their role in the management of AD.

The effect of the selected flavonoids in scopolamine-induced amnesia in mice was compared with donepezil, a known nootropic drug. It was observed that both the flavonoids showed significant improvement in conditional avoidance response (CAR) indicating protective effect of these flavonoids against scopolamine induced damage of memory. Along with behavioural parameters, the effect of flavonoids on the AChE activity, protein estimation enzymatic and non-enzymatic antioxidants were also evaluated.

The selected flavonoids showed a reduction of the AChE level that was increased after administration of scopolamine. It was reported that a decrease in AChE activity can lead to improvement in learning and memory235. The non-enzymatic production of lipid peroxidation (LPO) is associated mostly with cellular damage as a result of oxidative stress. In this process, malanoldehyde is generated as secondary products. The increased generation of reactive oxygen species (ROS) is implicated in the pathogenesis of many diseases and in the toxicity of a wide range of compounds. Brain tissue is rich in polyunsaturated lipids, has high iron content, and critically dependent on aerobic metabolism and thus highly vulnerable to ROS mediated oxidative damage. There are reports suggesting accumulation of LPO products in the brain of patients with Alzheimer's disease236.

Our results also further emphasized the memory protective effect of the flavonoids. Scopoletin and baicalein significantly reduced brain AChE level, indicating the remarkable elevation of ACh level in the brain and thereby the flavonoids might possess anti-ChE activity. The selected flavonoids significantly improved antioxidant levels when compared to the scopolamine treated group. These results further augmented the potential effect of the selected flavonoids against the Alzheimer's disease. Our study revealed that scopoletin and baicalein enhanced the oxygen species markers in most of the brain regions by improving antioxidant enzymes. Administration of scopoletin (200 mg/ kg) exhibited the highest activity followed by the scopoletin (100 mg/kg) and baicalein treated groups. It is well known that cholinergic neuronal systems play an important role in the cognitive deficits associated with AD, ageing and neurodegenerative diseases. Flavonoids treated animals showed potential in inhibition of oxidative stress-induced neuronal damage in the neurodegeneration and also in antioxidant assays and thus represents promising neuroprotective activities.

Superoxide anion plays an important role in the formation of other reactive oxygen species such as hydrogen peroxide, hydroxyl radical and singlet oxygen in living systems. SOD is considered to be a stress protein which is synthesized in response to oxidative stress237. Glutathione peroxidase catalyses the oxidation of reduced glutathione (GSH) to oxidized glutathione (GSSG) at the expense of hydrogen peroxide238. Catalase primarily causes the decomposition of H2O2 to water at a much faster rate, in association with glutathione peroxidase. Thus, a decrease in the activities of both catalase and glutathione peroxidase leads to an accumulation of H2O2. A decrease in the activity of glutathione reductase is due to the depletion of thiol which leads to related pathologies239.

Treatment with scopoletin and baicalein significantly (P>0.01) enhanced the total protein and enzymatic antioxidant levels in the brain. In scopolamine treated mice, significant (P>0.05) reduction in antioxidant enzyme level was observed in the mice brain. Higher dose (200 mg/kg) of scopoletin and baicalein treated mice significantly (P>0.05) increased the level of the enzymatic antioxidant enzymes such as superoxide dismutase, catalase, peroxidase and glutathione reductase, thus showing the neuroprotective beneficial effect against scopolamine treatment.

70 U R K U N D Madeswaran Final Thesis.doc (D29512176)

Glutathione is a major non-protein thiol in living organisms and protects the cellular defense system against the toxic effects of lipid peroxidation. Due to oxidative stress, the brain is unable to maintain the GSH redox state and oxidized glutathione (GSSG) is concentrated in some regions of the brain 240. The cellular levels of the active forms of vitamin C and α-tocopherol are also maintained by GSH by neutralizing the free radicals. A decrease in GSH level leads to a decrease in cellular levels of α-tocopherol241. In the present study, a significant decrease in the levels of non-enzymatic antioxidants like GSH was observed in scopoletin and baicalein treated mice. This could be due to an increased utilization of GSH for scavenging the toxic products. Mice treated with higher dose of scopoletin and baicalein significantly restored the levels of GSH in the brain suggesting the involvement of GSH-dependent detoxification of free radical. These observations reveal that scopoletin and baicalein treated groups has enhanced the antioxidant potential.

7.6 Chronic in vivo aluminium chloride-induced dementia

Based on the in vivo acute scopolamine studies, the flavonoids were selected for the chronic in vivo aluminium chloride induced dementia. Aluminium is a ubiquitous metal and has been associated in the etiology of Alzheimer’s disease where it intensifies brain oxidative damage. Based on the literatures AlCl3 is considered as a recognized etiological factor in a series of neurodegenerative disorders for many decades243. Several natural compounds such as curcumin and naringin exhibit neuroprotective effect against AlCl3-induced cell damages in various experimental models181. Our current study explores the role of the selected flavonoids during chronic administration of AlCl3- induced neurotoxicity.

Scopoletin and baicalein at a dose of 200 mg/kg/p.o. for 90 days reversed the learning impairment produced by the AlCl3 in mice. Flavonoids treated animals significantly decreased the transfer latency time in rectangular maze test and escape latency time in Morris water maze test which results in improvement of the learning and memory. There was a substantial elevation of locomotor function and percentage of conditioned avoidance response in pole climbing method in scopoletin and baicaelin treated mice. These remarkable behavioural changes in the animals further highlight the role of scopoletin and baicalein in the management of AD.

Catalase plays a major role in cellular antioxidant defense system by decomposing H2O2, thereby preventing the generation of hydroxyl radical through the Fenton reaction246. Accumulating evidence suggests that aluminium decreased the level of enzymatic antioxidants in the brain, such

71 U R K U N D Madeswaran Final Thesis.doc (D29512176)

as catalase (CAT), superoxide dismutase (SOD) and glutathione (GSH). The effect of Al contact is consistent with the previous studies that result in the decreased levels of CAT and SOD in the brain247.

Scopoletin and baicalein treated mice significantly elevated the level of total protein and enzymatic and non-enzymatic antioxidant levels and thus lead to a reduction of neural stress which enhances the synaptic communication. These results further augmented the long term potential effect of the selected flavonoids against the Alzheimer's disease.

Our study demonstrated that, the selected flavonoids enhanced the oxygen species markers in most of the brain regions by improving the levels of antioxidant enzymes. Thus result implied that, because of elevated the level of antioxidant enzyme by the selected flavonoids shows the reduction of neural stress, and enhances the synaptic communication. Our results also further emphasize the memory enhancing effect of the flavonoids against chronic administration of aluminium chloride. The selected flavonoids significantly improved antioxidant levels. These results further augmented the long term potential effect of the selected flavonoids against the Alzheimer's disease.

In the histopathological studies, negative control group showed severe degenerative changes and cytoplasmic vacuolation results in cognitive impairment. Scopoletin (200 mg/kg) treated group resulted in normal histological features with appearance of normal glial cell population. The short and long-term administration of the scopoletin 200 mg/kg and baicalein 200 mg/kg exhibited prominent effect in the reversal of the both scopolamine and aluminium-induced learning impairment in both behavioural and biochemical estimations.

8. CONCLUSION

72 U R K U N D Madeswaran Final Thesis.doc (D29512176)

possess potential ChE inhibitory activity. In the light of these findings, it can be concluded that scopoletin and baicalein showed less IC50 values against both enzyme targets (AChE and BuChE) and could be considered worthwhile to check its in vivo memory enhancing activity to be used in treatment of AD.

It is well known that cholinergic neuronal systems play an important role in the cognitive deficits associated with AD, ageing and neurodegenerative diseases. In our study, scopoletin and baicalein significantly reduced brain AChE activity, indicating the stimulating actions of these drugs on the cholinergic system. Hence, the memory-enhancing effect of the scopoletin and baicalein can be attributed to its anti-ChE activity. Flavonoids treated animals showed potential inhibition of oxidative stress-induced neuronal damage in the neurodegeneration and also in antioxidant assays and represent promising neuroprotective activities.

The end products of lipid peroxidation viz. malondialdehyde and lipid hydroperoxides and the levels of tissue protein, enzymatic and non-enzymatic antioxidants in the brain were estimated. Treatment with the scopoletin and baicalein significantly enhanced the total protein and enzymatic and non- enzymatic antioxidant levels in the brain. In scopolamine treated mice, significant (P>0.05) diminution was observed in brain antioxidant enzyme activity. Scopoletin and baicalein (200 mg/kg) treated mice significantly (P>0.05) increased the level of the antioxidant enzymes such as superoxide dismutase, catalase, peroxidase and glutathione reductase. Glutathione which is widely distributed in cells is an intracelluar reductant and plays a major role in catalysis and metabolism. The selected flavonoids significantly (P>0.01) increased the level of non-enzymatic antioxidant GSH whereas in the negative control, the levels were decreased.

73 U R K U N D Madeswaran Final Thesis.doc (D29512176)

remarkable behavioural changes in the animals further highlight the role of scopoletin and baicalein in the management of AD.

Scopoletin and baicalein significantly reduced brain AChE level, indicating the remarkable elevation of ACh level in the brain and thereby the flavonoids possess anti-ChE activity. Our study revealed that, high dose of scopoletin and baicalein improved antioxidant enzymes in most of the brain regions. Administration of scopoletin (200 mg/kg) exhibited the highest activity followed by the scopoletin (100 mg/kg) and baicalein treated groups.

AlCl3 treated mice showed significant reduction in total protein and enzymatic and non-enzymatic antioxidant levels which in turn results in cognitive impairment. Scopoletin and baicalein (200 mg/kg) treated mice significantly (P>0.05) increased the level of the antioxidant enzymes such as superoxide dismutase, catalase, peroxidase and glutathione reductase. Scopoletin and baicalein treated mice significantly elevated the level of total protein and enzymatic and non-enzymatic antioxidant levels and thus lead to reduction of neural stress which enhances the synaptic communication. These results further augmented the long term potential effect of the selected flavonoids against the Alzheimer's disease.

In the histopathological analysis, the brains of negative control group showed severe degenerative changes and cytoplasmic vacuolation which in turn results in the cognitive impairment which was recovered by the flavonoid treated groups so effectively than the standard drug. Scopoletin (200 mg/ kg) treated group resulted in normal histological features with appearance of normal glial cell population. The short term (7 days) and long-term (90 days) administration of the scopoletin 200 mg/kg exhibited pronounced effect in the reversal of the both scopolamine and aluminium-induced learning impairment in both behavioural and biochemical estimations.

In conclusion, it has been clearly demonstrated that the approach utilized in this study is successful in finding novel anti-alzhiemer’s drugs. In particular, higher dose of scopoletin and baicalein, showed excellent neuroprotective effects against in silico, in vitro enzyme inhibitory and in vivo acute scopolamine and chronic AlCl3 induced learning impairment models. Hence, scopoletin and baicalein could be considered as a therapeutic agent to prevent or slow down the development of neurodegenerative diseases such as AD. Further research is required to explore the detailed mechanism of action of the scopoletin and baicalein specifically targeting the pathogenetic mechanisms like amyloid deposition and tau hyperphosphorylation might provide a definite therapeutic edge and thereby extensive clinical trials are needed to identify a potential chemical entity for clinical use in the prevention and treatment of alzhiemer’s disease.

9. IMPACT OF THE STUDY

The prevalence of AD and dementia has increased significantly nowadays. As none of the available medications appears to be able to reduce the disease progression, there is enormous medical need

74 U R K U N D Madeswaran Final Thesis.doc (D29512176)

for the development of novel therapeutic strategies that target the underlying pathogenic mechanisms in AD.

In silico prediction of neuroprotective potential of certain commercially

available flavonoids

• Acetylcholinesterase Butyrylcholinesterase

Literature review

In vitro enzyme inhibition Assay

• Acetylcholinesterase inhibitory activity

• Butyrylcholinesterase inhibitory activity

Acute toxicity studies

Tabulation and compilation of results

Statistical analysis

Behavioural Assessments

▪ Rectangular maze test

▪ Morris water maze test

▪ Attention in a 5 choice serial reaction time task

▪ Assessment of gross behavioral activity

▪ Pole climbing test

Biochemical Estimations

▪ Estimation of cholinesterase activity

▪ Estimation of protein

▪ Enzymatic antioxidants

□ Catalase activity

□ SOD assay

75 U R K U N D Madeswaran Final Thesis.doc (D29512176)

□ Estimation of glutathione reductase

□ Estimation of glutathione peroxidase

▪ Non-Enzymatic antioxidants

▪ Estimation of reduced glutathione

▪ TBARS assay

▪ DPPH assay

Histopathological studies

76 U R K U N D Madeswaran Final Thesis.doc (D29512176)

Hit and source - focused comparison, Side by Side:

Left side: As student entered the text in the submitted document. Right side: As the text appears in the source.

Instances from: Dinesh_Thakkar_Pharmacy_Thesis.pdf

0: Dinesh_Thakkar_Pharmacy_Thesis.pdf 34% 0: Dinesh_Thakkar_Pharmacy_Thesis.pdf 34%

for the award of the degree of The source document can not be shown. The most likely reason is that the submitter has opted to exempt the document as a source in DOCTOR OF PHILOSOPHY Urkund's Archive.

PHARMACY

Submitted by

A. MADESWARAN, M. Pharm.,

Under the guidance of

Dr. K. ASOK KUMAR, M. Pharm., Ph.D.

College of Pharmacy,

Sri Ramakrishna Institute of

37: Dinesh_Thakkar_Pharmacy_Thesis.pdf 100% 37: Dinesh_Thakkar_Pharmacy_Thesis.pdf 100%

77 U R K U N D Madeswaran Final Thesis.doc (D29512176)

to achieve an optimized conformation for both the protein and ligand The source document can not be shown. The most likely reason is and relative orientation between protein and ligand such that the free that the submitter has opted to exempt the document as a source in energy of the overall system is minimized102. Urkund's Archive.

39: Dinesh_Thakkar_Pharmacy_Thesis.pdf 92% 39: Dinesh_Thakkar_Pharmacy_Thesis.pdf 92%

docking is a method which predicts the preferred orientation of one The source document can not be shown. The most likely reason is molecule to a second when bound to each other to form a stable that the submitter has opted to exempt the document as a source in complex. Knowledge of the preferred orientation may be used to Urkund's Archive. predict the binding affinity between two molecules

42: Dinesh_Thakkar_Pharmacy_Thesis.pdf 94% 42: Dinesh_Thakkar_Pharmacy_Thesis.pdf 94%

The purpose of the scoring function is to delineate the correct poses The source document can not be shown. The most likely reason is from incorrect poses, or binders from inactive compounds in a that the submitter has opted to exempt the document as a source in reasonable computation time. Scoring functions involve estimating, Urkund's Archive. rather than calculating the binding affinity between the protein and ligand and through these functions, adopting various assumptions and simplifications104. Scoring functions can be divided into:

i. Force-field-based

43: Dinesh_Thakkar_Pharmacy_Thesis.pdf 95% 43: Dinesh_Thakkar_Pharmacy_Thesis.pdf 95%

based scoring functions The source document can not be shown. The most likely reason is that the submitter has opted to exempt the document as a source in Classical force-field-based scoring functions assess the binding Urkund's Archive. energy by calculating the sum of the non-bonded (electrostatics and van der Waals) interactions. The electrostatic terms are calculated by a Columbic formulation.

78 U R K U N D Madeswaran Final Thesis.doc (D29512176)

44: Dinesh_Thakkar_Pharmacy_Thesis.pdf 100% 44: Dinesh_Thakkar_Pharmacy_Thesis.pdf 100%

Force-field-based scoring functions also have the problem of slow The source document can not be shown. The most likely reason is computational speed. Thus cut-off distance is used to handle the that the submitter has opted to exempt the document as a source in non-bonded interactions106. Urkund's Archive.

45: Dinesh_Thakkar_Pharmacy_Thesis.pdf 100% 45: Dinesh_Thakkar_Pharmacy_Thesis.pdf 100%

Extensions of force-field-based scoring functions consider the The source document can not be shown. The most likely reason is hydrogen bonds, that the submitter has opted to exempt the document as a source in Urkund's Archive.

46: Dinesh_Thakkar_Pharmacy_Thesis.pdf 100% 46: Dinesh_Thakkar_Pharmacy_Thesis.pdf 100%

and entropy contributions. Software programs, such as DOCK, The source document can not be shown. The most likely reason is GOLD and AutoDock, offer users such functions. that the submitter has opted to exempt the document as a source in Urkund's Archive.

47: Dinesh_Thakkar_Pharmacy_Thesis.pdf 72% 47: Dinesh_Thakkar_Pharmacy_Thesis.pdf 72%

Knowledge based scoring functions use statistical analysis of ligand- The source document can not be shown. The most likely reason is protein complexes crystal structures to obtain the inter-atomic that the submitter has opted to exempt the document as a source in contact frequencies and distances between the ligand and protein. Urkund's Archive.

48: Dinesh_Thakkar_Pharmacy_Thesis.pdf 96% 48: Dinesh_Thakkar_Pharmacy_Thesis.pdf 96%

79 U R K U N D Madeswaran Final Thesis.doc (D29512176)

functions The source document can not be shown. The most likely reason is that the submitter has opted to exempt the document as a source in In empirical scoring functions, binding energy decomposes into Urkund's Archive. several energy components, such as hydrogen bond, ionic interaction, hydrophobic effect and binding entropy. Each component is multiplied by a coefficient and then summed up to give a final score. Coefficients are obtained from regression analysis fitted to a test set of ligand-protein complexes with known binding affinities. Empirical scoring functions

49: Dinesh_Thakkar_Pharmacy_Thesis.pdf 100% 49: Dinesh_Thakkar_Pharmacy_Thesis.pdf 100%

Monte Carlo simulated annealing, evolutionary, genetic and The source document can not be shown. The most likely reason is Lamarckian genetic algorithm methods. that the submitter has opted to exempt the document as a source in Urkund's Archive.

80 U R K U N D Madeswaran Final Thesis.doc (D29512176)

Instances from: Lubhan Singh.doc

1: Lubhan Singh.doc 71% 1: Lubhan Singh.doc 71%

NEURODEGENERATIVE DISORDERS The source document can not be shown. The most likely reason is that the submitter has opted to exempt the document as a source in Neurodegenerative disorders are categorized by progressive and Urkund's Archive. irreversible loss of neurons from particular regions of the brain. Prototypical neurodegenerative disorders include Parkinson's disease (PD) and Huntington's disease (HD), results in deviations in the control of

4: Lubhan Singh.doc 58% 4: Lubhan Singh.doc 58%

loss of neurons from structures of the basal ganglia1, 2. Alzheimer's The source document can not be shown. The most likely reason is disease (AD), results in the loss of cortical and hippocampal neurons that the submitter has opted to exempt the document as a source in with impairment of memory and cognitive ability and amyotrophic Urkund's Archive. lateral sclerosis (ALS), producing muscular weakness

7: Lubhan Singh.doc 54% 7: Lubhan Singh.doc 54%

degeneration of bulbar, spinal, and cortical motor neurons3, 4. The source document can not be shown. The most likely reason is that the submitter has opted to exempt the document as a source in Neurodegenerative disorders are comparatively common and Urkund's Archive. represent a substantial societal and medical problem. They are

10: Lubhan Singh.doc 72% 10: Lubhan Singh.doc 72%

of later life, developing in individuals who are neurologically normal, The source document can not be shown. The most likely reason is although childhood-onset forms of each of the disorders are known. that the submitter has opted to exempt the document as a source in PD is detected in more than 1% of population above the age of 655, Urkund's Archive.

81 U R K U N D Madeswaran Final Thesis.doc (D29512176)

6, whereas AD affects as many as 10% of the same individuals7. HD, which is a genetically stable autosomal dominant disorder, is less common in the population as a whole but disturbs, on average, 50% of each generation in families booming the gene. ALS also is moderately rare but sometimes leads to disability and death8, 9.

13: Lubhan Singh.doc 83% 13: Lubhan Singh.doc 83%

The available treatments for AD, HD, and ALS are much more The source document can not be shown. The most likely reason is limited in effectiveness, and that the submitter has opted to exempt the document as a source in Urkund's Archive.

50: Lubhan Singh.doc 71% 50: Lubhan Singh.doc 71%

at room temperature for 30 minutes and the absorbance of the The source document can not be shown. The most likely reason is reaction mixture was measured at 405 nm. that the submitter has opted to exempt the document as a source in Urkund's Archive.

55: Lubhan Singh.doc 83% 55: Lubhan Singh.doc 83%

The reaction was initiated by the addition of NADH. The reaction The source document can not be shown. The most likely reason is mixture that the submitter has opted to exempt the document as a source in Urkund's Archive.

82 U R K U N D Madeswaran Final Thesis.doc (D29512176)

Instances from: Lubhan Singh With references.doc

2: Lubhan Singh With references.doc 71% 2: Lubhan Singh With references.doc 71%

5: Lubhan Singh With references.doc 58% 5: Lubhan Singh With references.doc 58%

8: Lubhan Singh With references.doc 54% 8: Lubhan Singh With references.doc 54%

11: Lubhan Singh With references.doc 72% 11: Lubhan Singh With references.doc 72%

83 U R K U N D Madeswaran Final Thesis.doc (D29512176)

14: Lubhan Singh With references.doc 83% 14: Lubhan Singh With references.doc 83%

51: Lubhan Singh With references.doc 71% 51: Lubhan Singh With references.doc 71%

56: Lubhan Singh With references.doc 83% 56: Lubhan Singh With references.doc 83%

84 U R K U N D Madeswaran Final Thesis.doc (D29512176)

Instances from: thesis plagiarism check.docx

18: thesis plagiarism check.docx 47% 18: thesis plagiarism check.docx 47%

primary progressive aphasia, corticobasal degeneration, Pick’s The source document can not be shown. The most likely reason is disease and progressive supranuclear palsy. Initial symptoms that the submitter has opted to exempt the document as a source in include marked changes in behaviour and personality and difficulty Urkund's Archive. with

comprehending language. Nerve cells in the frontal lobe and temporal lobes of the brain are affected,

20: thesis plagiarism check.docx 78% 20: thesis plagiarism check.docx 78%

In addition, the upper layers of the cortex typically become spongy The source document can not be shown. The most likely reason is and soft and have protein insertions (tau protein or the transactive that the submitter has opted to exempt the document as a source in DNA-binding protein) 21. Urkund's Archive.

1.2.5

22: thesis plagiarism check.docx 79% 22: thesis plagiarism check.docx 79%

characterized by the hallmark abnormalities of more than one cause The source document can not be shown. The most likely reason is of dementia most commonly Alzheimer’s combined with vascular that the submitter has opted to exempt the document as a source in dementia, followed by Alzheimer’s with DLB, and Alzheimer’s with Urkund's Archive. vascular dementia and DLB. Vascular dementia with

26: thesis plagiarism check.docx 100% 26: thesis plagiarism check.docx 100%

85 U R K U N D Madeswaran Final Thesis.doc (D29512176)

variant Creutzfeldt-Jakob disease is believed to be caused by The source document can not be shown. The most likely reason is consumption of products from cattle affected by mad cow disease23. that the submitter has opted to exempt the document as a source in Urkund's Archive. 1.2.7

29: thesis plagiarism check.docx 100% 29: thesis plagiarism check.docx 100%

the substantia nigra. The aggregates are thought to cause The source document can not be shown. The most likely reason is degeneration of the nerve cells that produce dopamine. that the submitter has opted to exempt the document as a source in Urkund's Archive.

32: thesis plagiarism check.docx 100% 32: thesis plagiarism check.docx 100%

Symptoms include difficulty in walking, memory loss and inability to The source document can not be shown. The most likely reason is control urination. that the submitter has opted to exempt the document as a source in Urkund's Archive.

34: thesis plagiarism check.docx 94% 34: thesis plagiarism check.docx 94%

can sometimes be corrected with surgical installation of a shunt in The source document can not be shown. The most likely reason is the brain to drain excess fluid26. that the submitter has opted to exempt the document as a source in Urkund's Archive. 1.3 ALZHEIMER'S

86 U R K U N D Madeswaran Final Thesis.doc (D29512176)

Instances from: D Sheeja Malar Ph.D Thesis 501 2012-13.docx

19: D Sheeja Malar Ph.D Thesis 501 2012-13.docx 47% 19: D Sheeja Malar Ph.D Thesis 501 2012-13.docx 47%

comprehending language. Nerve cells in the frontal lobe and temporal lobes of the brain are affected,

21: D Sheeja Malar Ph.D Thesis 501 2012-13.docx 78% 21: D Sheeja Malar Ph.D Thesis 501 2012-13.docx 78%

1.2.5

23: D Sheeja Malar Ph.D Thesis 501 2012-13.docx 79% 23: D Sheeja Malar Ph.D Thesis 501 2012-13.docx 79%

27: D Sheeja Malar Ph.D Thesis 501 2012-13.docx 100% 27: D Sheeja Malar Ph.D Thesis 501 2012-13.docx 100%

87 U R K U N D Madeswaran Final Thesis.doc (D29512176)

30: D Sheeja Malar Ph.D Thesis 501 2012-13.docx 100% 30: D Sheeja Malar Ph.D Thesis 501 2012-13.docx 100%

33: D Sheeja Malar Ph.D Thesis 501 2012-13.docx 100% 33: D Sheeja Malar Ph.D Thesis 501 2012-13.docx 100%

35: D Sheeja Malar Ph.D Thesis 501 2012-13.docx 94% 35: D Sheeja Malar Ph.D Thesis 501 2012-13.docx 94%

88 U R K U N D Madeswaran Final Thesis.doc (D29512176)

Instances from: Lubhan Singh without references.doc

3: Lubhan Singh without references.doc 71% 3: Lubhan Singh without references.doc 71%

6: Lubhan Singh without references.doc 58% 6: Lubhan Singh without references.doc 58%

9: Lubhan Singh without references.doc 54% 9: Lubhan Singh without references.doc 54%

12: Lubhan Singh without references.doc 72% 12: Lubhan Singh without references.doc 72%

89 U R K U N D Madeswaran Final Thesis.doc (D29512176)

15: Lubhan Singh without references.doc 83% 15: Lubhan Singh without references.doc 83%

52: Lubhan Singh without references.doc 71% 52: Lubhan Singh without references.doc 71%

57: Lubhan Singh without references.doc 83% 57: Lubhan Singh without references.doc 83%

90 U R K U N D Madeswaran Final Thesis.doc (D29512176)

Instances from: 16_chapter10.pdf

38: 16_chapter10.pdf 100% 38: 16_chapter10.pdf 100%

40: 16_chapter10.pdf 88% 40: 16_chapter10.pdf 88%

In the field of molecular modeling, The source document can not be shown. The most likely reason is that the submitter has opted to exempt the document as a source in docking is a method which predicts the preferred orientation of one Urkund's Archive. molecule to a second when bound to each other to form a stable complex. Knowledge of the preferred orientation may be used to predict the binding affinity between two molecules

41: 16_chapter10.pdf 89% 41: 16_chapter10.pdf 89%

the docking process, the ligand and the protein adjust their The source document can not be shown. The most likely reason is conformation to achieve an overall “best-fit” and this kind of that the submitter has opted to exempt the document as a source in conformational adjustments results in the overall binding Urkund's Archive.

91 U R K U N D Madeswaran Final Thesis.doc (D29512176)

Instances from: Thesis Final Work 27062017.docx

53: Thesis Final Work 27062017.docx 71% 53: Thesis Final Work 27062017.docx 71%

Animals were observed individually at least once during the first 30 animals were observed individually after dosing atleast once during minutes after dosing, periodically during the first 24 hours (with the first 30 minutes periodically and during the first 24 hours with special attention during the first 4 hours) and daily thereafter for a special attention given during the first 4 hours and daily thereafter for a

54: Thesis Final Work 27062017.docx 100% 54: Thesis Final Work 27062017.docx 100%

by Committee for the Purpose of Control and Supervision of by committee for the purpose of control and Supervision of Experiments on Animals ( Experiments on Animals (

58: Thesis Final Work 27062017.docx 90% 58: Thesis Final Work 27062017.docx 90%

One-way Analysis of Variance (ANOVA) followed by Dunnett’s test. one-way analysis of variance (ANOVA), which was followed by Dunnett’s test.

92 U R K U N D Madeswaran Final Thesis.doc (D29512176)

Instances from: 107cd289-b95b-422a-b194-c980f3b9a6c7

16: 107cd289-b95b-422a-b194-c980f3b9a6c7 52% 16: 107cd289-b95b-422a-b194-c980f3b9a6c7 52%

when they develop in cortex region of the brain. Aggregation of When they develop in a part of the brain called the cortex, dementia alpha-synuclein in the brains of people with can result. Alpha-synuclein also aggregates in the brains of people with

17: 107cd289-b95b-422a-b194-c980f3b9a6c7 67% 17: 107cd289-b95b-422a-b194-c980f3b9a6c7 67%

FTLD includes dementias such as behavioural-variant FTLD, FTLD Includes dementias such as behavioral-variant FTLD, primary progressive aphasia, Pick’s disease, and progressive supranuclear primary progressive aphasia, corticobasal degeneration, Pick’s palsy. disease and progressive supranuclear palsy. Initial symptoms include marked changes in behaviour and personality and difficulty Typical symptoms include changes in personality and behavior, and with difficulty with

24: 107cd289-b95b-422a-b194-c980f3b9a6c7 100% 24: 107cd289-b95b-422a-b194-c980f3b9a6c7 100%

Recent studies suggest that mixed dementia is more common than Recent studies suggest that mixed dementia is more common than previously previously

25: 107cd289-b95b-422a-b194-c980f3b9a6c7 96% 25: 107cd289-b95b-422a-b194-c980f3b9a6c7 96%

protein (prion) that causes other proteins throughout the brain to protein (prion) that causes other proteins throughout the brain to misfold and malfunction. misfold and thus malfunction.

28: 107cd289-b95b-422a-b194-c980f3b9a6c7 100% 28: 107cd289-b95b-422a-b194-c980f3b9a6c7 100%

93 U R K U N D Madeswaran Final Thesis.doc (D29512176)

variant Creutzfeldt-Jakob disease is believed to be caused by Variant Creutzfeldt-Jakob disease is believed to be caused by consumption of products from cattle affected by mad cow disease23. consumption of products from cattle affected by mad cow disease.

1.2.7

31: 107cd289-b95b-422a-b194-c980f3b9a6c7 92% 31: 107cd289-b95b-422a-b194-c980f3b9a6c7 92%

alpha-synuclein aggregates appear in an area deep in the brain Alpha-synuclein aggregates are likely to begin in an area deep in the called brain called the substantia nigra. The aggregates are thought to cause degeneration of the nerve cells that produce dopamine. the substantia nigra. The aggregates are thought to cause degeneration of the nerve cells that produce dopamine. The incidence of PD is about one-tenth that of

The incidence of PD is about one-tenth that of

36: 107cd289-b95b-422a-b194-c980f3b9a6c7 95% 36: 107cd289-b95b-422a-b194-c980f3b9a6c7 95%

can sometimes be corrected with surgical installation of a shunt in Can sometimes be corrected with surgical installation of a shunt in the brain to drain excess fluid26. the brain to drain excess fluid. Abbreviations: AD, Alzheimer’s disease; 1.3 ALZHEIMER'S

DISEASE (

International Journal of Biological Macromolecules 95 (2017) 199–203

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

Combining in silico and in vitro approaches to evaluate the

acetylcholinesterase inhibitory proﬁle of some commercially available

ﬂavonoids in the management of Alzheimer’s disease

a a,∗ b

Asokkumar Kuppusamy , Madeswaran Arumugam , Sonia George

Department of Pharmacology, College of Pharmacy, Sri Ramakrishna Institute of Paramedical Sciences, Coimbatore, 641 044, Tamil Nadu, India

Department of Pharmaceutical Chemistry, College of Pharmacy, Sri Ramakrishna Institute of Paramedical Sciences, Coimbatore, 641 044, Tamil Nadu, India

a r t i c l e i n f o a b s t r a c t

Article history: The current objective of the study is to identify inhibitory afﬁnity potential of the certain commer-

Received 24 August 2016

cially available ﬂavonoids, against crystal structure of acetylcholinesterase (AChE) enzyme using in silico

Received in revised form

and in vitro studies. The inhibitory proﬁles of the compounds have been compared with standard AChE

16 November 2016

inhibitor donepezil. In the docking studies, conformational site analysis and docking parameters like

Accepted 17 November 2016

binding energy, inhibition constant and intermolecular energy were determined using AutoDock 4.2.

Available online 18 November 2016

Docking studies conducted with diosmin, silibinin, scopoletin, taxifolin and tricetin exhibited tight bind-

ing forces prevailing with the enzyme than between donepezil. Based on the in silico studies, compounds

Keywords:

were selected for the in vitro AChE inhibitory assay. In vitro results showed that all the selected ﬂavonoids

Alzheimer’s disease

displayed excellent concentration-dependant inhibition of AChE. Scopoletin was found to be the most

Binding energy

Inhibition constant potent and speciﬁc inhibitor of the enzyme with IC50 values of 10.18 ± 0.68 ␮M. Scopoletin showed sev-

Amino acid residues eral strong hydrogen bonds to several important amino acid residues against target enzyme. A number of

Molecular interactions hydrophobic interactions could also explain the potency of the compounds to inhibit AChE. These molec-

ular docking and in vitro analyses could lead to the further development of potent acetylcholinesterase

inhibitors for the treatment of Alzheimer’s disease.

1. Introduction (ACh), preventing breakdown by cholinesterase enzymes and anti-

inﬂammatory agents [5].

Drug design is an important tool in the ﬁeld of medicinal ACh is the major neurotransmitter located at all autonomic

chemistry where new compounds are synthesized by molecular ganglia, at various autonomically innervated organs, at the neuro-

or chemical manipulation of the lead moiety in order to produce muscular junction, and also found in various synapses in central

highly active compounds with minimum steric effect [1]. Docking nervous system. In central nervous system, ACh is found pre-

of small molecules in the receptor binding site and estimation of dominantly in interneurons, and a few vital long-axon cholinergic

binding afﬁnity of the complex is a vital part of structure based drug pathways have also been recognized. Degeneration of this pathway

design [2,3]. is one of the pathologies associated with AD [6]. During neuro-

Alzheimer’s disease (AD), a common type of progressive neu- transmission, ACh is released from the nerve into the synaptic

rodegenerative disease, is characterized by low level of neurotrans- cleft and binds to ACh receptors (nicotinic and muscarinic) on

mitter (acetylcholine), oxidative stress and neuro-inﬂammation in the post-synaptic membrane, relaying the signal from the nerve.

brain stream [4]. Effective treatment methodologies rely mostly on Acetylcholinesterase (AChE), also located on the post-synaptic

either increasing the cholinergic function of the brain by stimulat- membrane, terminates the signal transmission by hydrolyzing ACh

ing the cholinergic receptors, improving the level of acetylcholine [7].

AChE is found in several types of conducting tissue such as nerve

and muscle, motor and sensory ﬁbers, central and peripheral tis-

sues, and cholinergic and noncholinergic ﬁbers. The activity of AChE

∗ is higher in motor neurons than in sensory neurons [8–10]. Pseu-

Corresponding author at: Department of Pharmacology, College of Pharmacy, Sri

Ramakrishna Institute of Paramedical Sciences, Coimbatore, 641 044, Tamil Nadu, docholinesterase (BuChE), also known as plasma cholinesterase

India. or butyrylcholinesterase, is primarily found in the liver. Different

E-mail address: [email protected] (M. Arumugam).

http://dx.doi.org/10.1016/j.ijbiomac.2016.11.062

200 A. Kuppusamy et al. / International Journal of Biological Macromolecules 95 (2017) 199–203

from AChE, BuChE hydrolyzes butyrylcholine faster than ACh [11].

AChE enzyme hydrolyzes ester bond of the ACh to breakdown into

choline and ester. BuChE enzyme is rich in glial cells, which have

similar action on ACh. In AD enhanced concentration of AChE and

BuChE results into the decreased half-life of ACh [12].

Currently the most favourable target for the symptomatic treat-

ment and slowing of AD development is cholinesterase inhibitors

[13]. Cholinergic neurones are located mainly in regions associated

with learning and memory – spreading from the basal forebrain

[14] and hippocampus [15]. By inhibiting acetylcholinesterase

(AChE), the enzyme which catalyses break down of ACh, levels of

this neurotransmitter can be elevated and thus improve the learn-

ing and memory function.

Flavonoids and their related compounds are a group of

natural products which exhibits various biological and phar-

macological activities like antibacterial, antiviral, antioxidant,

anti-inﬂammatory, anti-allergic, hepatoprotective, antithrombotic,

antiviral and antimutagenic effects and inhibition of several

Fig. 1. Reﬁned crystal structure of mouse acetylcholinesterase (5HCU).

enzymes [16,17]. Flavonoids can be a promising remedy to treat

AD by inhibiting AChE and thereby it can increase the ACh level.

Hence, the objective of the current study is to identify the AChE

2.5. In silico acetylcholinesterase inhibition

inhibitory afﬁnity potential of the certain commercially available

ﬂavonoids using in silico and in vitro studies.

Lamarckian genetic algorithm (LGA) was used for ligand confor-

mational searching, which is a hybrid of a genetic algorithm and a

2. Materials and methods

local search algorithm. This algorithm ﬁrst builds a population of

individuals (genes), each being a different random conformation of

2.1. Software used

the docked molecule. Each individual is then mutated to acquire

a slightly different translation and rotation and the local search

Python 2.7 – language was downloaded from www.python.

algorithm then performs energy minimizations on a user-speciﬁed

com [18], Cygwin (a data storage) c:\program and Python 2.5

proportion of the population of individuals. The individuals with

were simultaneously downloaded from www.cygwin.com [19],

the low resulting energy are transferred to the next generation and

Molecular graphics laboratory (MGL) tools and AutoDock 4.2

the process is then repeated. The algorithm is called Lamarckian

was downloaded from www.scripps.edu [20], Discovery stu-

because every new generation of individuals is allowed to inherit

dio visualizer 2.5.5 was downloaded from www.accelerys.com

the local search adaptations of their parents. Gasteiger charges are

[21], Chemsketch was downloaded from www.acdlabs.com [22].

calculated for each atom of the macromolecule in AutoDock 4.2

Online smiles translatory notation was carried out using cac-

instead of Kollman charges which were used in the previous ver-

tus.nci.nih.gov/translate/ [23].

sions of this program [26].

Docking calculation in AutoDock 4.2 was performed using the

2.2. Chemicals used

reﬁned protein and the desired ligand in pdb format. Running Auto-

Grid calculation is carried out by the .glg ﬁle. Running AutoDock

Donepezil, acetylcholinesterase, diosmin, silibinin, scopoletin,

is carried out by the .dlg ﬁle. Finally 10 different docked con-

and taxifolin were purchased from Sigma Aldrich, St. Louis, MO.

formations were studied for a single compound. The parameters

All other drugs and chemicals used in the study were obtained

like binding energy, intermolecular energy and inhibition constant

commercially and were of analytical grade.

were studied for all the selected ﬂavonoids [27]. For each lig-

and, 10 best docking simulations were obtained against the target

2.3. Preparation of target enzyme

molecule. AutoDock 4.2 was run numerous times to get different

docked conformational poses [28]. Based upon the docking param-

The crystal structure of mouse acetylcholinesterase (5HCU) pro-

eters obtained using AutoDock 4.2, the ﬂavonoids was selected for

tein database was downloaded from the research collaboratory

further in vitro studies.

for structural bioinformatics (RCSB) protein data bank (Fig. 1).

The preparation of the target protein 5HCU with the AutoDock

Tools involved adding all hydrogen atoms to the macromolecule,

2.6. In vitro acetylcholinesterase inhibition

which is a step necessary for correct calculation of partial atomic

charges. Three-dimensional afﬁnity grids of size 277 × 277 × 277 Å

The assay for the measurement of the enzyme inhibition was

with 0.6 Å spacing were centered on the geometric center of the

carried out based on Ellman method [29]. About 1.3 ml of the Tris-

target protein and were calculated for each of the following atom

HCl buffer (pH 8.0; 50 mM) was treated with 0.4 ml of different

types: HD, C, A, N, OA, and SA, representing all possible atom types

concentrations of the drug solution and to it 0.1 ml of the AChE

in a protein [24].

(0.28 U/ml) was added. This mixture was incubated for 15 min and

to it 0.3 ml of Acetylthiocholine iodide (0.023 mg/ml) and 1.9 ml

′

2.4. Drug likeness properties of the ligands (5,5 -dithiobis-(2-nitrobenzoic acid) [DTNB] (3 mM) solution were

added. This ﬁnal reaction mixture was kept for further incubation

The ﬂavonoid ligands like diosmin, silibinin, scopoletin, taxi- at room temperature for 30 min and the absorbance of the reaction

folin, tricetin, and donepezil were built using ChemSketch (Fig. 2) mixtures was taken at 405 nm. The assay was done in triplicate

and optimized using “Prepare Ligands” in the AutoDock 4.2 for and the results were expressed as mean ± SEM. The percentage

docking studies [25]. inhibition was calculated for the selected ﬂavonoids [30].

A. Kuppusamy et al. / International Journal of Biological Macromolecules 95 (2017) 199–203 201

Fig. 2. The optimized ligand molecules (A–Diosmin, B–Silibinin, C–Scopoletin, D–Taxifolin, E–Tricetin, and F–Donepezil).

2.7. Statistical analysis

Statistical analysis of the results was carried out using Graph-

Pad by one way analysis of variance followed by Dunnett’s test. All

results are expressed as mean ± standard deviation (SD) and values

average from 3 independent experiments.

3. Results

3.1. In silico acetylcholinesterase inhibitory proﬁling

Fig. 3. Docked pose of AChE with scopoletin and donepezil.

In silico AChE inhibitory proﬁling were evaluated based on the

binding energy, inhibition constant, intermolecular energy and root 286, Leu 289, Ser 293, Ile 294, Phe 295, Tyr 341 and the binding

mean square deviation (RMSD) of the starting and the ﬁnal docked site of the Donepezil (Fig. 3) was found to possess, Tyr 72, Tyr

conformation against AChE enzyme. All the selected ﬂavonoids 124, Trp 286, Phe 295 and Tyr 341 amino acid residues. This result

showed the excellent docking parameters when compared with proves that the effective binding orientations were present in the

the standard against AChE. Diosmin when compared with the standard Donepezil. Based on

As shown in Table 1, the selected compounds had lesser bind- the most stable conformations, we predict that the interactions

ing energy when compared to the standard (−3.87 kcal/mol). observed with molecular docking calculations of the ﬂavonoids lead

The binding energy of ﬂavonoids ranged from −6.93 kcal/mol to to reversible AChE inhibitors.

−4.27 kcal/mol. Thus proving those ﬂavonoids has potential AChE Hydrogen bonding between a protein and its ligands delivers

inhibitory binding sites when compared to donepezil. a directionality and speciﬁcity of interaction that is a fundamen-

All the selected compounds had lesser inhibition constant when tal part of molecular recognition. The energetics and kinetics of

compared to the standard (529.73 ␮M). Inhibition constant is hydrogen bonding therefore require to allow the rapid sampling

directly proportional to binding energy. We found a decrease in and kinetics of folding, conferring stability to the target structure

inhibition constant of all the selected compounds with a simultane- and providing the speciﬁcity needed for selective macromolecular

ous decrease in the binding energy. Flavonoids showed inhibition interactions.

constant ranging from 8.37 ␮M to 739.02 ␮M. The selected com- Hydrophobic interactions make the major contribution to the

pounds had lesser intermolecular energy when compared to the stability of 5HCU (AChE) and the major contributors are: Ile 294

standard (994.14 ␮M). (4.5), Leu 289 (3.8), and Phe 295 (2.8) (Table 2). The globular con-

We found a decrease in intermolecular energy of all the selected formation of proteins is stabilized predominately by hydrophobic

compounds with a progressive decrease in the binding energy. interactions.

Flavonoids showed intermolecular energy ranging from −8.72 to

−6.06. Root mean square deviation is indirectly proportional to

3.3. In vitro acetylcholinesterase inhibitory assay

binding energy. We found an increase in RMSD of all the compounds

with an increase in the binding energy. Flavonoids showed RMSD

From the in silico docking studies the ﬂavonoids were selected

ranging from 26.46 to 35.19. The selected compounds had excel-

for the in vitro cholinesterase inhibitory studies based on the dock-

lent RMSD when compared to the standard (42.19). The in silico

ing parameters. The in vitro enzymatic activity of the ﬂavonoids

AChE inhibitory activities of the compounds were in the order of

exhibited the potential AChE inhibitory activity than the standard

scopoletin > diosmin > silibinin > taxifolin > tricetin > donepezil.

donepezil (Fig. 4). The results further validated the in silico dock-

ing studies and it further conﬁrms that the ﬂavonoids could be a

3.2. Amino acid interactions in the molecular docking studies promising remedy for the AD.

An increase in AChE leads to an increase in breakdown of ACh

The ﬂavonoids showed the excellent binding sites when com- to acetate and choline, leading to the development of AD. Recent

pared with the standard against AChE. These amino acid residues ﬁndings suggest that the occurrence of AD is increasing world-

play an important role in the binding site of target enzyme. This wide, possibly due to life style modiﬁcations. AChE inhibitors are

proves that the ﬂavonoids possess potential AChE inhibitory bind- commonly employed in the treatment of AD.

ing sites when compared to the standard. Inhibition of AChE resulted in a decreased breakdown of

The aminoacid residues responsible for the binding interactions acetylcholine into acetate and choline, when measured spectro-

of the Scopoletin (Fig. 3) with the enzyme AChE were Tyr 124, Trp scopically. Among the selected ﬂavonoids, scopoletin was found

202 A. Kuppusamy et al. / International Journal of Biological Macromolecules 95 (2017) 199–203

Table 1

Molecular docking scores of the ﬂavonoids against AChE.

Name of the Compound Binding Energy(Kcal/mol) Inhibition Constant (kI) (␮M) Intermolecular Energy (Kcal/mol) RMSD

Diosmin −6.03 36.86 −7.84 27.00

Silibinin −5.66 70.92 −7.45 22.59

Scopoletin −6.95 8.37 −8.72 26.46

Taxifolin −5.22 150.32 −7.01 25.01

Tricetin −4.27 739.02 −6.06 35.19

Donepezil −3.87 994.14 −5.46 42.19

The root mean square deviation of the starting and the ﬁnal docked conformation.

Table 2

Hydrophobic interactions and interacting residues of the AChE with ﬂavonoids.

Amino acid Residue Name of the amino acid Hydrophobicity Average Isotropic Displacement

TYR72 Tyrosine −1.3 60.989

TYR124 Tyrosine −1.3 49.835

TRP286 Tryptophan −0.9 64.389

LEU289 Leucine 3.8 101.06

SER293 Serine −0.8 136.315

ILE294 Isoleucine 4.5 112.474

PHE295 Phenylalanine 2.8 91.122

TYR341 Tyrosine −1.3 87.133

the AChE enzyme and the essential active site residues involved in

the intermolecular interactions [34].

AChE that has been covalently inhibited by organophosphate

compounds (OPCs), such as nerve agents and pesticides, has tra-

ditionally been reactivated by using nucleophilic oximes. The

recognized need for new classes of compounds with the ability

to reactivate inhibited AChE with improved in vivo efﬁcacy. Katz

et al., 2015 described the discovery of new functional groups-

Mannich phenols and general bases-that are capable of reactivating

OPC-inhibited AChE more efﬁciently than standard oximes and the

cooperative mechanism by which these functionalities are deliv-

ered to the active site [35].

Ali et al. [36] evaluated the tea polyphenols of green tea and

reported its AChE and BChE inhibitory potential and its use in

the management of AD. The molecular docking analysis revealed

that polyphenols exhibited binding interactions with AChE and

Fig. 4. in vitro AChE inhibitory activity of the ﬂavonoids (Values are Mean ± SEM of

three parallel measurements). BChE. The amount of energy to bind with AChE and BChE required

by Epigallocatechin-3-gallate was lowest at about −14.45 and

−13.30 kcal/mol, respectively.

to possess highest activity (IC50 10.18 ± 0.68 ␮g/ml). The activ- Phyllanthus acidus is used in traditional medicine to treat sev-

ity of scopoletin was found to be greater than the standard, eral inﬂammatory, pain and oxidative stress related diseases such

donepezil (22 ± 0.56 ␮g/ml). All the compounds demonstrated as rheumatism, respiratory disorder, and its potential to pro-

AChE inhibitory activity at a concentration below 100 ␮g/ml, mote intellect and memory enhancement, thus supporting its

with IC50 values ranging from 10.18 to 18.28 ␮g/ml. The AChE anti-Alzheimer’s properties. The methanolic extract of P. acidus

inhibitory activities of the compounds were in the order of scopo- exhibited inhibition of rat brain AChE and human blood BuChE in

letin > Diosmin > silibinin > Taxifolin > Tricetin > Donepezil. a dose dependent manner and the inhibition constant value was

found to be 1009.87 ␮g/ml and 449.51 ␮g/ml respectively [37].

In the present study, we had investigated the AChE-drug com-

4. Discussion

plexes, ligand-binding sites calculation within the active site of the

enzyme, pharmacophore perception of ﬂavonoids and in vitro AChE

AD is a complex neurodegenerative disorder of the central ner-

inhibitory activity of the ﬂavonoids. The ﬂavonoids showed the

vous system, characterized by amyloid-␤ deposits, tau-protein

better binding modes when compared with the standard against

aggregation, oxidative stress and reduced levels of acetylcholine in

AChE. These amino acid residues are dynamically involved in the

the brain [31]. AD is a progressive neurodegenerative disorder clin-

formation of hydrophilic and hydrophobic contacts and there were

ically characterized by loss of memory and cognition. Cholinergic

initiated to play important role in the binding site of target. This

deﬁcit and oxidative stress have been implicated in the patho-

proves that the ﬂavonoids possess potential AChE inhibitory bind-

genesis of AD. Therefore, inhibition of acetylcholinesterase and

ing sites when compared to the standard. All the selected ﬂavonoids

oxidation are the two promising strategies in the development of

contributed AChE inhibitory activity because of its structural prop-

drug for AD [32,33].

erties.

The functionalized piperidine were evaluated in vitro for their

In in vitro study, a dose dependent AChE inhibitory activity was

AChE inhibitory activity, and in silico studies for all the target com-

observed for all the selected ﬂavonoids. These molecular dock-

pounds were carried out using pharmacophore mapping, molecular

ing and in vitro analyses could lead to the further development of

docking and quantitative structure-activity relationship analysis in

potent acetylcholinesterase inhibitors for the treatment of AD. The

order to know the structural features needed for interaction with

A. Kuppusamy et al. / International Journal of Biological Macromolecules 95 (2017) 199–203 203

in vitro results were in corroboration with the in silico studies as [14] C.K. Wu, L. Thal, D. Pizzo, L. Hansen, E. Masliah, C. Geula, Apoptotic signals

within the basal forebrain cholinergic neurons in Alzheimer’s disease, Exp.

evidenced by the docking orientations and the parameters. Based

Neurol. 195 (2005) 484–496.

on the in silico and in vitro AChE inhibitory studies, scopoletin was

[15] G. Gron, I. Brandenburg, A.P. Wunderlich, M.W. Riepe, Inhibition of

found to be potential lead in the management of AD. hippocampal function in mild cognitive impairment: targeting the cholinergic

hypothesis, Neurobiol. Aging 27 (2006) 78–87.

[16] T.P. Cushnie, A.J. Lamb, Recent advances in understanding the antibacterial

5. Conclusions

properties of ﬂavonoids, Int. J. Antimicrob. Agents 38 (2011) 99–107.

[17] S. Nishiumi, S. Miyamoto, Dietary ﬂavonoids as cancer-preventive and

therapeutic biofactors, Front. Biosci. 3 (2011) 1332–1362.

AChE inhibitors provide symptomatic beneﬁt in AD across key

[18] Python Software Foundation, Available from: www.python.com, Python

symptom domains. From the in silico docking studies it is concluded

2.7–language, downloaded on 24 July 2013.

that ﬂavonoids possess the potential binding sites and the dock- [19] Redhat, Available from: www.cygwin.com, Cygwin and Python 2.5,

downloaded on 24 July 2013.

ing parameters against the AChE. This in silico studies is actually an

[20] Molecular Graphics Laboratory, The Scripps research Institute Available from:

added advantage to screen the AChE inhibition. The results from the

www.scripps.edu, graphics laboratory (MGL) tools and AutoDock4.2,

in vitro study emphasise the ﬂavonoids inhibitory potential against downloaded on 25 July 2013.

AChE. The stronger effect of scopoletin than the other ﬂavonoids [21] Accelrys, Available from: www.accelerys.com, Discovery studio visualizer

2.5.5 downloaded on 22 July 2013.

and donepezil, is interesting and merits further characterization.

[22] Advanced Chemistry Development Available from: www.acdlabs.com,

Scopoletin showed a particularly favourable AChE inhibitory pro-

ChemSketch, ACD/Labs downloaded on 29 July 2013.

ﬁle and thus could be a great potential candidate for AD treatment. [23] NCI CADD Group, Available from: cactus.nci.nih.gov/translate/, National

cancer Institute, downloaded on 22 July 2013.

Further investigations on the above compound and in vivo stud-

[24] J. Konc, J.T. Konc, M. Penca, M. Janezic, Binding-sites prediction assisting

ies are necessary to develop clinical useful AChE inhibitor in the

protein–protein docking, Acta Chim. Solv. 58 (2011) 396–401.

prevention and management of AD. [25] Z. Bikadi, E. Hazai, Application of the PM6 semi-empirical method to

modeling proteins enhances docking accuracy of AutoDock, J. Cheminform. 1

(2011) 15–17.

Conﬂict of interest

[26] A. Madeswaran, M. Umamaheswari, K. Asokkumar, A. Sivashanmugam, V.

Subhadradevi, P. Jagannath, Computational drug discovery of potential TAU

protein kinase I inhibitors using in silico docking studies, Bangladesh J.

The authors declare that there are no conﬂicts of interest for this

Pharmacol. 8 (2013) 131–135.

work.

[27] G.M. Morris, R. Huey, W. Lindstrom, M.F. Sanner, R.K. Belew, D.S. Goodsell, A.J.

Olson, AutoDock 4 and AutoDock Tools 4: automated docking with selective

References receptor ﬂexibility, J. Comput. Chem. 19 (2009) 2785–2791.

[28] D.S. Goodsell, G.M. Morris, A.J. Olson, Automated docking of ﬂexible ligands:

applications of Autodock, J. Mol. Recognit. 9 (1996) 1–5.

[1] C.N. Cavasotto, R.A. Abagyan, Protein ﬂexibility in ligand docking and virtual

[29] G.L. Ellman, D.K. Courtney, V. Andres, R.M. Featherstone, A new and rapid

screening to protein kinases, J. Mol. Biol. 12 (2004) 209–225.

colorimetric determination of acetylcholinesterase activity, Biochem.

[2] D. Seeliger, B.L. Groot, Ligand docking and binding site analysis with PyMOL

Pharmacol. 7 (1961) 88–95.

and Autodock/Vina, J. Comput. Aided Mol. Des. 24 (2010) 417–424.

[30] O. Sacan, R. Yanardag, Antioxidant and antiacetylcholinesterase activities of

[3] M.C. Sharma, D.V. Kohli, S. Sharma, A.D. Sharma, Design, synthesis and

chard (Beta vulgaris L. Var. Cicla), Food Chem. Toxicol. 48 (2010) 1275–1280.

biological activity of some benzimidazoles derivatives

[31] V.B. da Silva, P. de Andrade, D.F. Kawano, P.A. Morais, J.R. de Almeida, I.

3-chloro-4-(substituted–phenyl-1-(4-{1-[2’-(2H-tetrazol-5-yl)-biphenyl-4-

Carvalho, C.A. Taft, C.H. da Silva, In silico design and search for

ylmethyl]-1H-benzimidazol-2yl}-phenyl)-azetidin-2-ones, Der Chem. Sin. 1

acetylcholinesterase inhibitors in Alzheimer’s disease with a suitable

(2010) 92–105.

pharmacokinetic proﬁle and low toxicity, Future Med. Chem. 3 (2011)

[4] V. Zarotsky, J.J. Sramek, N.R. Cutler, Galanthamine hydrobromide: an agent for 947–960.

Alzheimer’s disease, Am. J. Health Syst. Pharm. 60 (2003) 446–452.

[32] P.T. Francis, A.M. Palmer, M. Snape, G.K. Wilcock, The cholinergic hypothesis

[5] E.M. Reiman, A 100-Year Update on Alzheimer’s disease and related

of Alzheimer’s disease: a review of progress, J. Neurol. Neurosurg. Psychiatry

disorders, J. Clin. Psychiatry 67 (2006) 1784–1800.

66 (1999) 137–147.

[6] E. Perry, M. Walker, J. Grace, R. Perry, Acetylcholine in mind a

[33] M. Asaduzzaman, M.J. Uddin, M.A. Kader, A.H. Alam, A.A. Rahman, M. Rashid,

neurotransmitter correlate of consciousness? Trends Neurosci. 22 (1999)

K. Kato, T. Tanaka, M. Takeda, G. Sadik, In vitro acetylcholinesterase inhibitory

273–280.

activity and the antioxidant properties of Aegle marmelos leaf extract:

[7] R. Wang, X.C. Tang, Neuroprotective effects of huperzine A, Neurosignals 14

implications for the treatment of Alzheimer’s disease, Psychogeriatrics 14

(2005) 71–82.

(2014) 1–10.

[8] J. Massoulie, L. Pezzementi, S. Bon, E. Krejci, F.M. Vallette, Molecular and

[34] G. Brahmachari, C. Choo, P. Ambure, K. Roy, In vitro evaluation and in silico

cellular biology of cholinesterases, Prog. Neurobiol. 41 (1993) 31–91.

screening of synthetic acetylcholinesterase inhibitors bearing functionalized

[9] L.W. Chacho, J.A. Cerf, Histochemical localization of cholinesterase in the

piperidine pharmacophores, Bioorg. Med. Chem. 23 (2015) 4567–4575.

amphibian spinal cord and alterations following ventral root section, J. Anat.

[35] F.S. Katz, S. Pecic, T.H. Tran, I. Trakht, L. Schneider, Z. Zhu, L. Ton-That, M.

94 (1960) 74–81.

Luzac, V. Zlatanic, S. Damera, J. Macdonald, D.W. Landry, L. Tong, M.N.

[10] G.B. Koelle, The histochemical localization of cholinesterases in the central

Stojanovic, Discovery of new classes of compounds that reactivate

nervous system of the rat, J. Comp. Anat. 100 (1954) 211–235.

acetylcholinesterase inhibited by organophosphates, Chembiochem 16 (2015)

[11] Y.J. Huang, Y. Huang, H. Baldassarre, B. Wang, A. Lazaris, M. Leduc, A.S. 2205–2215.

Bilodeau, A. Bellemare, M. Cote, P. Herskovits, M. Touati, C. Turcotte, L.

[36] B. Ali, Q.M. Jamal, S. Shams, N.A. Al-Wabel, M.U. Siddiqui, M.A. Alzohairy, M.A.

Valeanu, N. Lemee, H. Wilgus, I. Begin, B. Bhatia, K. Rao, N. Neveu, E. Brochu, J.

Al-Karaawi, K.K. Kesari, G. Mushtaq, M.A. Kamal, In silico analysis of green tea

Pierson, D.K. Hockley, D.M. Cerasoli, D.E. Lenz, C.N. Karatzas, S. Langermann,

polyphenols as inhibitors of AChE and BChE enzymes in Alzheimer’s disease

Recombinant human butyrylcholinesterase from milk of transgenic animals

treatment, CNS Neurol. Disord. Drug Targets 15 (2016) 624–628.

to protect against organophosphate poisoning, Proc. Natl. Acad. Sci. U. S. A.

[37] M. Moniruzzaman, M. Asaduzzaman, M.S. Hossain, J. Sarker, S.M.A. Rahman,

104 (2007) 13603–13608.

M. Rashid, M.M. Rahman, In vitro antioxidant and cholinesterase inhibitory

[12] G.L. Wenk, Neuropathologic changes in Alzheimer’s disease: potential targets

activities of methanolic fruit extract of Phyllanthus acidus, BMC Complement.

for treatment, J. Clin. Psychiatry 67 (2006) 3–7.

Altern. Med. 15 (2015) 403–413.

[13] L.M. Bierer, V. Haroutunian, S. Gabriel, P.J. Knott, L.S. Carlin, D.P. Purohit, D.P.

Perl, J. Schmeidler, P. Kanof, K.C. Davis, Neurochemical correlates of dementia

severity in Alzheimer’s disease: relative importance of the cholinergic

deﬁcits, J. Neurochem. 64 (1995) 749–760.