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Natural environment and especially plants continue to provide a dynamic research area in the field of drug discovery, since phytochemicals can be possible starting materials of novel bioactive compounds. In the present drug discovery programmes, natural products or compounds derived from natural products account for > 40% of the new registered drugs. In order to discover new bioactive compounds, extracts from plants or other natural sources should be submitted at the same time to chemical screening and various biological or pharmacological targets. Due to the complexity of the plant extracts, it is rather difficult to characterize them chemically in one step. Usually, their study includes fractionation of the complex mixture, separation and isolation of the individual components with liquid chromatography, and structure elucidation using various spectroscopic methods (UV, IR, MS, NMR). Our experience in studying antioxidants that are present in plant extracts revealed that 1D and 2D NM ...
Natural environment and especially plants continue to provide a dynamic research area in the field of drug discovery, since phytochemicals can be possible starting materials of novel bioactive compounds. In the present drug discovery programmes, natural products or compounds derived from natural products account for > 40% of the new registered drugs. In order to discover new bioactive compounds, extracts from plants or other natural sources should be submitted at the same time to chemical screening and various biological or pharmacological targets. Due to the complexity of the plant extracts, it is rather difficult to characterize them chemically in one step. Usually, their study includes fractionation of the complex mixture, separation and isolation of the individual components with liquid chromatography, and structure elucidation using various spectroscopic methods (UV, IR, MS, NMR). Our experience in studying antioxidants that are present in plant extracts revealed that 1D and 2D NMR spectroscopy can be used as an alternative analytical technique to provide insight into mixtures without any previous separation of the individual components. The aim of this thesis was to use different 1D and 2D NMR methodologies for the characterization of different classes of secondary metabolites in plant extracts without any previous separation and isolation of the individual components. The crude plant extracts, belonging in the Lamiaceae, Oleaceae and Apocynaceae families, were dissolved in deuterated solvents (CD3CN and pyridine-d5) and an attempt was made for the identification, and in some cases, the quantification of their main components, belonging to the classes of flavonoids and phenolic acids, diterpenes and triterpenoids. Ground leaves of Rosmarinus officinalis, Salvia officinalis, Thymus vulgaris, Origanum majorana, Olea europaea and Nerium oleander were subsequently extracted with n-hexane and ethyl acetate. The extracts were concentrated in rotary evaporator and kept into sealed flasks.
As a first step, extracts were investigated for their radical scavenging activity using DPPH∙ assay and the content of total phenolic compounds and flavonoids was measured in plant extracts. Because of their strong radical scavenging activity (of most of the extracts) and the high amounts of total phenolic compounds and flavonoids of the ethyl acetate extracts, their composition was further investigated. All crude extracts were dissolved in CD3CN and the 1H- NMR, 1Η- 1Η COSY, 1H-13C HSQC and HMBC spectra were acquired. In the 1H- NMR spectrum of the extracts a very characteristic region between 11 to 13 ppm, which is of high diagnostic value for the presence of flavonoids, was observed. The highly deshielded signals in this region are attributed to the hydroxyl protons OH(5), which participate in a strong intramolecular hydrogen bond, formed between the hydroxyl and the carbonyl group at CO(4). As a rule the 1H- NMR resonances of the –OH groups appear at room temperature as broad signals especially in protic solvents, owing to the mobility of the hydrogen and its fast exchange, on the NMR time scale, with the protons of the solvent. However, by decreasing the temperature, the proton exchange rate is reduced and the –OH peaks are revealed as sharp peaks. With the use of proper temperature and by spiking with standards, certain flavones, flavonols and flavanones were detected in ethyl acetate extracts. Also in the 1H- NMR spectrum of the ethyl acetate extracts of the Lamiaceae family two doublets resonating at ~ 6.2 and ~ 7.5 ppm respectively (J =15.8 Hz), were observed. Their coupling constant indicates the presence of the trans-isomer of the hydroxycinammic acid derivative. In 1Η- 1Η COSY spectrum the strongest cross-peaks (Η3a, H2a), (Η3a, H3β) και (Η2, Η3α,β) are diagnostic of the caffeic acid moiety. Moreover in the 1H-13C HSQC spectrum certain very strong cross-peaks indicate a coupling network that can be attributed to rosmarinic acid. Both proton and carbon-13 chemical shifts of rosmarinic and caffeic acid are almost the same, but there is a significant difference in the 13C shielding and the number of cross-peaks of the –COOR and –COOH carbons, in the 1H-13C HMBC spectrum, which offers the solution to the problem of the unequivocal assignment of them. The high resolution of the 2D 1H-13C HMBC spectrum allowed us to determine unambiguously the individual cross-peaks of the spin system of rosmarinic acid r(H3a, C1a), r(H2a, C1a), even at room temperature. In addition, in the hexane and ethyl acetate extracts of sage and rosemary, using a combination of 1H-13C HSQC and 1H-13C HMBC NMR spectroscopy, the diterpenes carnosic acid and carnosol were identified. In order to confirm the results from the identification of compounds with 1D and 2D NMR spectroscopy and to ascertain the complexity or not of the composition of crude plant extracts, they were dissolved in methanol and analyzed with LC-ESI-MS. Various compounds were tentatively identified and for some of them using standards were identified, belonging to the classes of flavonoids and phenolic acid (rosmarinic acid), diterpenes, simple phenols and triterpenic acids. The presence of compounds identified using 1D and 2D NMR spectroscopy was confirmed with the LC-ESI-MS analysis. The quantification of the standard compounds that were commercially available was achieved using seven point calibration curves. The levels of rosmarinic acid in the extracts were also examined by the use of 1H- NMR spectroscopy and were in good agreement with those obtained by HPLC.
Finally, a novel strategy for NMR analysis of mixtures of oleanolic (OA) and ursolic acids (UA) that occur in natural products was described. The combination of 1H-13C HSQC and 1H-13C HMBC techniques allows the connection of the proton and carbon-13 spins across the molecular backbone resulting in the identification and, thus, discrimination of OA and UA without resorting to physicochemical separation of the components. The methodology was extended and to other triterpenoids (betulinic acid, betulin, erythrodiol and uvaol) that were identified in some of the above extracts. The quantitative results provided by 2D 1H-13C HSQC NMR data were obtained within a short period of time (~ 14 min) and were in excellent agreement with those obtained by HPLC (using modified cyclodextrins in the mobile phase), which supported the efficiency of the suggested methodology.
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