Purification Of Alkaloids From Rauvolfia Vomitoria And Application Of Surface Plasmon Resonance On Mistletoe Lectins

The dissertation is composed of two parts of separated research work. Part I is about purification of alkaloids from Rauvolfia vomitora and Part II is about application of surface plasmon resonance on mistletoe lectins.Part I:Purification of alkaloids from Rauvolfia vomitora.Rauvolfia vomitora, as a natural medicine herb, was introduced from Ghana in Africa to Yunnan and Guangxi provinces of China in1960s. Modern drug analysis indicated that it contains a number of bioactive chemicals in its root, including reserpine, yohimbine and other indole alkaloids. It has become an important raw material used in the production of antihypertension drugs and in the treatment of cardiovascular diseases. The research work designed and assembled a simple preparative reversed-phase chromatography device, by which a new integrated and efficient purification method of alkaloids from Rauvolfia vomitora was developed. This part is composed of three chapters.Chapter1describes the overview of the genus Rauwolfia, main medicinal alkaloids in it, and current status of the mainly used extraction processes, and introduces the scientific significance of this research work.Chapter2establishes a new efficient purification method of alkaloids from Rauvolfia vomitora, which is combined with acidic ethanol extraction, macroporous resin adsorption, macroporous weak-acidic cation exchange resin extraction and C18reversed-phase chromatography in a laboratory scale.Chapter3determines the chemical structure of ten types of alkaloids from Rauvolfia vomitora by UPLC-MS and NMR methods and investigates the pharmacological activity of main alkaloids purified from Rauvolfia vomitora via renovascular hypertensive SD rat models and their acute toxicity in ICR mice.There are three innovations in part Ⅰ as follows:(1) A new preparative reversed-phase chromatography device designed and assembled by our group has more sample capacity, higher recovery yield and more convenient operation compared with commercial devices. It shows a wide range of applicability in separation and purification of natural products with its simple structure and convenient operation.(2) A set of purification technology was established, by which a variety of alkaloids could be obtained simultaneously in same process with high purity and yield.(3) The new extraction process is environmentally benign and energy-saving compared with existing methods. Most of the organic solvent used in the extraction process can be recycled to achieve clean production,Part Ⅱ:Application of surface plasmon resonance on mistletoe lectins.Mistletoe lectins are of growing interest to anti-cancer therapy and pharmaceutical research because of their immunomodulatory effects. SPR is a new biosensory technology with high sensitivity and high accuracy, but it has rarely been applied in research of lectins. This part of research work, based on the Plasmonic(?) SPR sensor platform, explores different types of sensor chip preparation methods, establishes a simple, reagent-saving and rapid detection method of mistletoe lectin Ⅰ(ML-Ⅰ) and observes the cytotoxicity effects of lectins on two cell lines in vitro by SPR sensor. This part is composed of three chapters.Chapter4introduces the principle of SPR technology, structure and operation of Plasmonic(?) SPR sensor device. It overviews current research status of mistletoe lectins and states the scientific significance of this research work.Chapter5establishes a sandwich-mode ML-I detection method based on the choices of sensor chips and antibody response systems. The preparation methods of different types of biologically sensitive film on chip surface were firstly explored. Then the sandwich-mode ML-I detection method was applied in buffer system, serum samples and commercially available injections.Chapter6investigates the interaction of ML-I with two cell lines A549and Calu-3in vitro by SPR sensor and the cytotoxic effect by atomic force microscope (AFM) observation.There are two innovations in part Ⅱ as follows:(1) Some preparation methods of different types of sensor chips were designed and improved, which are easy to operate and time-saving.(2) The SPR sensor was applied to the determination of ML-I content in buffer, serum and injections with less reagents and time.