Structure Determination of Bioactive Metabolites from Marine Ascidians and Simultaneous Analysis of Herbal Medicines by HPLC-DAD-MS

Abstract

Part A. Structure Determination of Bioactive Metabolites from Marine Ascidians Marine natural products have been proven to be a prolific source of novel therapeutic agents that have demonstrated significant activities in various pathological conditions including cancer, viral infection, inflammation, analgesia, and immune modulation. Marine ascidians belong to the phylum Chordata, which is one of the highly evolved animals among the groups of marine organisms. Ascidians have been widely recognized as a prolific sources of structurally unique and biologically active secondary metabolites. Many studies have been reported that bioactive compounds derived from ascidians show diverse biological activities such as cytotoxic, antiviral, antimicrobial, and enzyme inhibitory for therapeutic uses. The purpose of this study is to investigate new marine natural products from ascidians and determine their biological activities. Benthic colonial invertebrates were collected from various locations of southern coast of Korea. During the course of searching for bioactive metabolites from Korean marine inverterbrate, three ascidians, Synoicum sp., Pseudodistoma sp., and Aplidium sp., were selected for chemical investigation based on chemical analysis and bioassay screening of the crude extracts as well as the partitioned fractions. Secondary metabolites from the selected organisms were isolated and their congeners were synthesized through various laboratory techniques and chromatographic methods. As a result, 28 novel compounds and 7 known compounds were structurally determined using integrated spectroscopic analysis and chemical approaches. These compounds belonged to various structural classes with diverse biogenetic origins: beta-carboline alkaloids, brominated aromatic furanones, amino alcohols, and iodobenzene containing dipeptides. The biological activities of the isolated compounds were determined using various bioactivity tests: cytotoxicity, antimicrobial activities, and inhibition against the enzymes isocitrate lyase (ICL), sortase A (SrtA) and Na+/K+-ATPase. Several compounds displayed significant antimicrobial and/or enzyme-inhibitory activities. Structure-activity relationships were also deduced.

Part B. Simultaneous Analysis of Herbal Medicines by HPLC-DAD-MS Traditional herbal medicines have been practiced to maintain health and treat diseases especially in the Asia communities for more than 2,000 years. Increment of worldwide attention and concomitant pharmaceutical research has made it essential to carry out the quality control measurement for the herbal medicines. However, serious hindrance has been attributed to the lack of recognition and regulation of professions, qualified practitioners, quality-controlled herbal medicines, and evidence-based clinical studies. Thus it is urgently needed to establish a comprehensive qualified evaluation method that could accurately reflect the quality of herbal medicines. The purpose of this study is developing simultaneous analysis methods of bioactive compounds in herbal medicines. Four widely used herbal medicines, which are Rehmanniae radix preparata, Rehmanniae radix, Forsythia suspensa, and Forsythia viridissima were subjected to the current study. In herbal medicines, it is hard to consider that single or a few compounds would determine the overall pharmacological activities. Rather, it is more likely that multiple compounds act simultaneously and attribute to the therapeutic function of the herbal medicine. For this reason, every bioactive metabolites in the selected herbal medicines were analyzed and determined. In particular, four HMF derivatives and five representative metabolites of the iridoid and phenylethanoid glycoside classes were selected for the analysis of Rehmanniae Radix preparata and Rehmannia glutinosa, respectively. Moreover, phenylethanoid glycoside and four lignans from Forsythia suspensa and three lignans from Forsythia viridissma were also analyzed. The methods based on HPLC with diode array detector (DAD) and electrospray ionization mass spectrometry (ESI-MS) was established for the quantitative and qualitative analysis of a selected folk medicines. Various validation parameters, such as linearity, limit of detection, limit of quantification, recovery, accuracy, and precision, were successfully obtained. In addition, the efficiencies of diverse extraction methods were compared for the development of a standard analytic method. The verified methods were successfully applied to the quantitative determination of representative metabolites in commercial samples from Korea and China.