| Traditional Chinese medicines (TCM) are Chinese national treasures. These medicines have unique advantages and can achieve impressive outcomes, and independent intellectual property rights for TCM can be obtained. Ketone, including flavonoid and furantone, are a broad categories of active ingredient in TCM. Among them, flavonoid compounds are a major active ingredient widely distributed in TCM and are demonstrated to have many beneficial pharmaceutical effects. Chromatography is an important modern analytical technology and is indispensable to both qualitative and quantitative analyses of the chemical composition of TCM. There are still many analytical challenges due to the complexity of and diversity of TCM compositions, the instability of active TCM components, and the various concentrations of components. Therefore, development of a new method for coupled chromatographic analysis with high sensitivity and high efficiency has profound significance and broad application prospects for the analysis of active TCM ingredients. This work is aimed at establishing a new type of coupled chromatographic method based on evaluating the effects of TCM. Two monomers were separated from Phellinus igniarius as the index ingredients using High Speed Counter Current Chromatography (HSCCC). Furthermore, a simultaneous quantitative method, Ultra Performance Liquid Chromatography-Photodiode Array Detection-Electrospray Ionization Tandem Mass Spectrometry (UPLC-PAD-MS), was established and applied to distinguish the real samples. A functionalized pipette tip (PT) was designed; next, an analytical method coupled with PT Solid Phase Extraction and High Performance Liquid Chromatography (PT-SPE-HPLC) was established for the simultaneous determination of four Epimedium flavone glycosides in biological samples. The research is divided into the following six chapters.Chapter One:Provides a summary describing the effects of flavonoids, their distribution among TCM, and the analytical methods used for determining flavonoids. The value of TCM containing flavonoids was analyzed based on a summary of the biological activity of flavonoids. The application of chromatographic methods for the analysis of TCM, including sample preparation and pharmaceutical analysis in vivo is introduced.Chapter Two:Antioxidant activity of Phellinus igniarius in vitro was evaluated using chemical methods. The results illustrated that Phellinus igniarius has hydroxyl radical scavenging capacity, high oxygen anion free radical scavenging ability and reducing power. Twenty compositions were identified from the alcohol extraction of Phellinus igniarius by UPLC-PAD-MS. High concentrations of Inoscavin A and B Hypholomine were identified in Phellinus igniarius, and Hypholomine B was detected in blood samples after oral administration.An animal experiment was performed to evaluate the effect of Epimedium on the treatment of chronic osteomyelitis in model animals. The results show that the total flavone of Epimedium is mainly composed of four kinds of flavonoid glycosides. Oral administration with the dose of 242.70 mg kg-1 per day for eight weeks had an obvious bone healing effect in the treatment group with a significant difference between the model group and vancomycin group. Therefore, it is necessary to establish quantitative methods for both in vivo and in vitro sample determination.Chapter 3:First, because of a lack of standards, purification of substances was required. Separation and purification were conducted using HSCCC. The purified Inoscavin A and Hypholomine B were carefully evaluated using Nuclear Magnetic Resonance Spectroscopy (NMR), Mass Spectrometry (MS), and Ultra Violet Spectrometry (UVS) such that it could be used as an analytical standard later in the study. Furthermore, a UPLC-PAD-MS method was developed to separate and quantify target compounds found in Phellinus igniarius. Finally, real samples were used to verify the developed method and demonstrate that the UPLC-PAD-MS method might be a useful alternative asses the quality of Phellinus igniarius. Compared with the traditional HPLC methods, the analysis time was reduced from 50 min to 10 min. Limits of quantitation (LOQ) of Inoscavin A and Hypholomine B were 0.39 mg L-1 and 0.28 mg L-1, respectively. Recoveries of Inoscavin A and Hypholomine B were between 103.8% and 105.3% and between 104.4% and 105.0%, respectively. RSD values were less than 3%.Chapter Four:In view of the low concentration of Epimedium flavonoids in vivo, sample pretreatment was a challenging problem, demanding the design of PT with the function of solid phase extraction. C18 packing was chosen by comparing different absorbent packing. The procedure of serum pretreatment on the designed PT was optimized to discover the optimal method as follows:A 100?L sample was withdrawn manually into the tip at rate of approximately 5 ?L s-1. After a dwell time of 1.0 min, the withdrawn sample was released from the tip back into the sample vial at the same speed.The solid phase was subsequently washed with water (500 ?L) to remove the protein and other interferences. Next,100 ?L of acetonitrile was withdrawn into the tip. After a dwell time of 5 min, the solvent was released from the tip into a sample vial.20 ?L aliquots of the elutant were injected into the HPLC. This method could simplify operation steps, reduce the error in the process, improve the efficiency of enrichment, and be suitable for the determination of trace flavonoids in biological samples. At the same time, the PT can be applied to other active ingredients of TCM by replacing adsorption packing and optimizing the extraction process.Chapter 5:A PT-SPE-HPLC method was established for the simultaneous determination of four Epimedium flavonoids in serum. The results show that four target compositions can be well-separated from the other components in the serum. There were good linear relationships within 0.05 to 10.00 mg· L-1 for epimedin A, B, C and icariin, and the average recoveries were 98.82%,97.12%,97.43% and 98.82%, for epimedin A, B, C and icariin, respectively. LODs were 0.042,0.045,0.040 and 0.023 mg L-1 for epimedin A, B, C and icariin, respectively. Using the established method, Epimedium flavonoids in rabbits with chronic osteomyelitis has been determined to clarify concentration changes in vivo, and the pharmacokinetic parameters of Epimedium flavonoids in rats were researched. This method has the advantage of high sensitivity and simple operation and can be used in pharmacokinetics and for drug concentration monitoring of TCM.Chapter 6:The application of chromatography and new coupled chromatographic methods in TCM are summarized and evaluated. It is necessary to develop a convenient and efficient method for biological sample pretreatment to establish high sensitivity, and accurate and rapid analysis of TCM. In the field of TCM research, such a method will greatly promote the modernization of TCM. |
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