Bioassay Guided Isolation Of Human Acetylcholinesterase Inhibitory Trans Tephrostachin Nanogel Mediated Blood Brain Barrier Permeability And Crispr Cas9 Based Sorl1 Knock Out Development In Zebrafish Model For Alzheimers Disease

Abstract

More than 450 million people around the world are believed to be suffering from some form of mental illness but the hunt for new medications to treat psychiatric and central nervous system disorders is in crisis. Novel drugs, drug delivery system and disease model are urgently required to treat and understand neurological disorder mainly for Alzheimer s Disease (AD). This study was aimed to identify neuroactive small molecules from the medicinal herb Tephrosia purpurea, developing the smart nanomaterials for delivering the drugs to the brain and to develop the neurospecific zebrafish knock out (KO) model using the CRISPR/Cas9 technology. newlineThe neuroactive compound was isolated from Tephrosia purpurea by bioassay guided fractionation using zebrafish as model. The active molecule trans-tephrostachin was isolated and structurally characterized by RP-HPLC, LC-HRMS/MS and NMR. The enzyme inhibition and molecular simulation studies against the acetylcholinesterase (AChE) had confirmed the molecule possessed mixed type of inhibition. The active molecule and four derivatives were chemically synthesized and evaluated in vitro and in silico analysis against AChE. Most of the drugs are not able to reach the brain because the presence of blood brain barrier (BBB). To overcome this problem, PNIPAM nano hydrogel carrier was developed. The fabricated nanogel potentially crossed the zebrafish BBB by apoe receptor mediated endocytic pathway and delivered trans- tephrostachin to the brain. newline newline newline newlineIn order to develop a specific model to study the progress of AD, a sorl1 gene knock out was developed by CRISPR/Cas9 in zebrafish model. Various reports showed that SORL1 protein is a receptor for APOE and its predominant expression is observed in neurons of rat, human and zebrafish brains, hence it was selected to understand the sporadic form of AD. The sorl1 KO zebrafish model was successfully generated and confirmed through genotyping. The behavior studies revealed that the KO larvae was found to be hyperactive than the c