Natural products are compounds or extracts that originate from natural sources and usually exhibit biological activity. They are raw materials for natural medicines. Pharmacognosy encompasses their selection, characterization, identification and use. Bioassay-guided fractionation of plant extracts linked to chromatographic separation techniques leads to the isolation of biologically active compounds whose chemical structures can readily be determined by modern spectroscopic and spectrometric methods, followed by testing of their pharmacological activity.Buckwheat contains many healthy nutrients, and its consumption is therefore increasing. The plant contains phototoxic compounds called fagopyrins that can act as photosensitisers upon excitation with visible light causing fagopyrism, but these compounds have received little research attention. To assure adequate safety of buckwheat and its products, fagopyrins should be characterized from a nutritional and pharmaceutical point of view. Nevertheless, fagopyrins are not only health-threatening compounds but also potent photodynamic agents that deserve further research.
In this work, fagopyrins extraction procedure was improved employing a mixture of acetone and water. Flowers were recognized as the best material for the extraction. A chromatographic analysis of fagopyrins was performed by developing a simple, sensitive HPLC analytical method of high-resolution employing fluorescence detection. The developed method is a good starting point for a preparative method that would enable the isolation of pure compounds. At least eight fagopyrin derivatives were observed upon isolation and were characterized via UV-Vis absorption, NMR spectroscopy and mass spectrometry. The structures of two new fagopyrins were elucidated. Fagopyrin F is a previously known compound that consists of naphthodianthrone scaffold and two piperidine moieties. Fagopyrin A differentiates by two pyrrolidine moieties instead of piperidine moieties, and fagopyrin E consists of one pyrrolidine and one piperidine moiety. The existence of protofagopyrins that originate in buckwheat and can transform into fagopyrins upon light exposure was confirmed. Information on solubility, fluorescence and UV-Vis absorbance in different solvents were obtained. The need for reference compounds of different fagopyrins that would allow proper quantitative evaluation of buckwheat samples still remains. Researchers use hypericin for that purpose, but we recognize its use as inadequate due to the different structure and consequently uncomparable absorbance in spectrophotometric detection. Our methods complement the existing knowledge regarding fagopyrins and will facilitate their further isolation and analysis of their biological activity. In the second part of our work, silver fir trunk extracts were investigated. Extracts from the bark of different conifer species are known to contain various polyphenols that possess interesting pharmacological activities. So far the most extensive research was done on the antioxidative extract of the maritime pine (Pinus maritima) bark Pycnogenol®, which is widely used in food supplements and cosmetic products. We have shown that antioxidant activity of silver fir (Abies alba) extract is higher comparing to the maritime pine bark extract in vitro and in cultured cells. Components of the extract were separated with normal phase flash chromatography and reversed phase HPLC. Structures of individual compounds were elucidated by mass spectrometry, UV-Vis absorption spectroscopy and comparison to reference compounds. Six phenolic acids were identified (gallic, homovanillic, protocatehuic, p-hydroxybenzoic, vanillic and p-coumaric), three flavonoids (catechin, epicatechin and catechin tetramethyl eter) and four lignans (taxiresinol, 7-(2-methyl-3,4-dihydroxytetrahydropyran-5-yloxy)-taxiresinol, secoisolariciresinol and laricinresinol).The research was continued with pharmacological testing. It was found that the extract exerts strong antioxidative and protective effects against atherogenic diet-induced arterial wall damage. Protective effects of the silver fir extract and its compounds as well as of two phenolic acids (p-coumaric and protocatechuic acids) were also evauated against ischemia-reperfusion-induced damage in isolated rat hearts. The silver fir extract significantly reduced tissue damage (measured as the lactate dehydrogenase release rate) and shortened the duration of arrhythmias, which was not observed in the case of phenolic acids. It was concluded that the two acids are only partially responsible for the observed effects and synergism of more compounds could be present. Silver fir extract, solubilized in polyethylene glycol was developed as an appropriate raw material for research and use. A need for water determination in such samples emerged. The most accessible technique for water content determination is Karl-Fischer titration with visual detection of a titration endpoint, which was unfortunately not suitable for the analysis of the silver fir extract due to its strong colour. Therefore an appropriate method for moisture determination in coloured samples was developed employing simple equipment and spectrophotometric detection of a titration endpoint. Silver fir extract, characterized in such manner is a suitable ingredient for development of food supplements, cosmetic products and medicines.
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