An Integrative Computational and Experimental Study of Multi-Target-Directed Ligands from spices with anti-AChE, anti-Aβ, and Antioxidant Activities in a Neuronal Cell Model

다국어 초록 (Multilingual Abstract)

The present research focuses on the identification of small-molecule compounds from dietary sources (spices) that possess neuroprotective activity. Six spices (clove, black pepper, carom, dill, cardamom, and fennel) were selected for the study. To understand the antioxidant capacity of the extracts, TPC and TFC were calculated. The main bioactive compounds were identified using the GC-MS method in hexane and ethyl acetate extracts. The identified compounds were analyzed for their pharmacokinetic properties and toxicity profile (ADME/T). The neuroprotective activity was analyzed in H2O2-induced stress in the SH-SY5Y neuroblastoma cell line. In silico docking studies were performed to investigate the protein-compound interactions using Aβ (1IYT and 2BEG) and AChE (4EY7) receptors. To provide experimental verification for the docking results, certain in vitro assays were performed. The spice extracts and their bioactive compounds displayed acetylcholinesterase inhibition (IC50 values), with eugenol displaying the best anti-AChE activity. The inhibition mechanism identified competitive and mixed types of inhibition exhibited by extracts and their compounds. The anti-Aβ activity was analyzed using anti-fibrilization (ThT) and anti-oligomerization (MDS) assays. Best anti-ThT activity was observed by the clove extracts, carom extracts, cardamom extracts, fennel extracts, with inhibition above 50% and only eugenol was able to inhibit ⁓50% compared to other bioactive compounds. However, the results from the MDS assay revealed only TMA and anethol to show ⁓50% inhibition. Piperidine was excluded from the study since it did not show any neuroprotective activity. β-caryophyllene was able to display neuroprotective activity as well as anti-AChE activity; however, it was predicted to be unable to cross the BBB and displayed CYP450 enzyme inhibition. Anethol displayed the best anti-MDS activity; however, it was unable to display any neuroprotection in the SH-SY5Y cell model. More mechanistic studies and optimizations are required to further their development into neuroprotective agents for clinical use.

목차 (Table of Contents)

• CHAPTER 1: GENERAL INTRODUCTION 1
• CHAPTER 2: MATERIALS & METHODS 6
• 2.1. Chemicals and Reagents 7
• 2.2. Spice Extraction Process 8
• 2.3. Gas Chromatography-Mass Spectrometry (GC-MS) Method 8
• 2.4. Total Phenolic Content and Total Flavonoid Content 9
• 2.5. In silico assays 10
• 2.5.1. Ligand and protein structure selection 10
• 2.5.2. Physicochemical and Pharmacokinetic parameters 11
• 2.5.3. Molecular Docking 11
• 2.6. Neuroprotective capability analysis on the cellular level 11
• 2.6.1. Cell Culture 12
• 2.6.2. Cell Viability Assay 12
• 2.6.3. Neuroprotection Assay 12
• 2.6.4. Determination of Reactive Oxygen Species (ROS Assay) 13
• 2.6.5. Mitochondrial Membrane Potential Assay 13
• 2.6.6. Antioxidant parameters estimation 14
• a) Estimation of Glutathione 15
• b) Estimation of Malondialdehyde 15
• 2.7. Anti-Acetylcholinesterase Activity 15
• 2.8. Anti-Aβ activity 16
• 2.8.1. Anti-Fibrilization Activity (ThT assay) 16
• 2.8.2. Anti-Aβ1-42 Oligomerization Activity (MDS assay) 16
• CHAPTER 3: RESULTS 18
• 3.1. GC-MS Analysis 19
• 3.2. TPC/TFC 20
• 3.3. ADME/T properties of the bioactive compounds 20
• 3.3.1. Drug-Likeness and ADME properties 20
• 3.3.2. Toxicity Predictions 25
• 3.4. Docking of bioactive compounds with AChE receptor protein (4EY7) 26
• 3.5. Docking of bioactive compounds with Aβ1-42 receptor protein (1IYT and 2BEG) 30
• 3.6. Non-cytotoxic and neuroprotective effects of extracts and their bioactive compounds 35
• 3.7. Alleviation of ROS generation by extracts and their bioactive compounds 43
• 3.8. Restoration of Mitochondrial Membrane Potential (ΔΨm) by extracts and their bioactive compounds 46
• 3.9. Antioxidant markers restoration 51
• 3.9.1. Restoration of Glutathione levels (GSH/GSSG ratio) 51
• 3.9.2. Attenuation of MDA levels 52
• 3.10. Therapeutic potential of natural compounds against acetylcholinesterase in AD treatment 55
• 3.11. Anti-fibrilization and Anti-oligomerization activity 59
• CHAPTER 4: DISCUSSION 66
• CHAPTER 5: CONCLUSION/SUMMARY 84
• References 87
• Appended Figures 96