title: Identification of Small Molecules in Traditional Chinese Medicine Using Cytological Profiling
Creators: name: Miao, William Gang
Publisher: name: Griffith University
Year: 2020
Subjects: subject: Chinese Medicine
subject: Traditional Chinese Medicine
subject: Parkinson’s disease
subject: Liguisticum chuanxiong Hort
subject: Gastrodia elata Bl

description: Traditional Chinese Medicine (TCM) plays an important role in healthcare in China and increasingly in western countries. However, the functions and mechanisms of TCMs have not been precisely researched which affect their acceptance by the regulatory authorizations in the western world. Parkinson’s disease (PD) is a chronic neurodegenerative disorder with a high prevalence rate worldwide, imposing serious burden to individuals, their families and society. Unfortunately, the molecular mechanisms of PD remain poorly understood, and there are no drugs available to cure the disease. Current medical treatment only provides symptomatic relief but are not able to stop, slow or reverse the progression of neurodegeneration. The objective of the project is to utilize the tremendous knowledge of TCM that have neuroprotective properties, isolate molecules from the TCM and use the molecules as tools to better understand PD biology. More specifically, we chose two TCM materials, Liguisticum chuanxiong Hort and Gastrodia elata Bl, which are traditionally used to treat PD symptoms, and isolated major compounds from the ethanol extracts and evaluated their actions on PD patients derived hONS cells using cytological profiling assay. However, the current project focused on individual compound that was isolated from neuroprotective TCMs, which was considered as a cutting point for in-depth research of TCM. The application of TCM formulation was far more complex, and the bioactivity was the sum of all ingredients. The thesis began with Chapter 1, the introduction of traditional Chinese medicine in drug discovery. The chapter firstly introduced the unique concept of compatibility in establishing TCM formulation, and emphasized the efficacies of TCM, especially for complex and chronic diseases, such as PD. The chapter then introduced cell based cytological profiling assay, a novel, unbiased image-based screening technique. Chapter 2 detailed the isolation of two TCM herbal medicines, Liguisticum chuanxiong Hort and Gastrodia elata Bl. The ethanol extracts of Chuanxiong and Gastrodia were fractionated by using flash column chromatography, and further purified by repeated HPLC under the guidance of 1H-NMR. 53 pure compounds, including 6 new structures, were isolated from Chuanxiong and 32 pure compounds from Gastrodia. The chemical structures of new compounds were elucidated by 1D, 2D NMR and HRESIMS, and chemical structures of known compounds were determined by 1H-NMR, LRESIMS and confirmed by comparison with literature data. Chapter 3 described the cytological profiling of the compounds, fractions and crude extracts using PD patient derived hONs cells. 38 parameters related to the function of nucleu, lysosomes, early endosome, microtubule cytoskeleton, mitochondria and autophagosome were evaluated. The bio-clusters of analogous compounds from both TCM plants showed strong structure-activity relationship. Analogues of phenolic compounds, phthalide lactones in Chuanxiong, analogues of gastrodin and parishins, analogues that contain bis (4-hydroxybenzyl) group from Gastrodia showed high level of similarity in the cytological profiling. Statistics analysis of the cytological profiling data identified 13 molecules that significantly perturbed the functions of lysosomes, mitochondrion, early endosomes, autophagosomes. These 12 molecules could be used as tools to further study the biology of PD. Chapter 4 studied the TCM extraction methods and compared their extraction efficiency with a sequential organic solvent extraction method. In this study, 6 TCM herbal materials were extracted using traditional methods by hot water and ethanol, and sequential organic solvent extraction method by using hexane, dichloromethane and methanol accordingly. Different extracts were analysed by HPLC and 1H-NMR. The physicochemical properties of isolated compounds were calculated against Lipinski’s Ro5 and ChemGPS. The results showed that traditional ethanol extraction, which mimicked TCM wine tincture, was as effective as the sequential organic solvent extraction method and delivered molecules with drug-like physicochemical properties. Chapter 5 provided an overview of major findings and perspectives of this project. The three major findings included (1) 13 hit molecules were identified by cytological profiling assay; (2) structure activity relationship was observed among analogues; (3) ethanol extraction, which mimicked TCM wine tincture, was proved to deliver drug-like molecules. The results of my research highlighted that TCM work on multiple targets, and component based TCM medicine, especially fractionated TCM, could provide higher efficacy, quicker and more precise TCM therapy in the future. Chapter 6 described the materials, equipment and general methods used in this project.