PhD: Study of chemical compositon of Foeniculum vulgare from Macedonia using chromatographic methods

Mence Najdoska-Bogdanov
Institute of Chemistry, Faculty of Natural Sciences and Mathematics, Ss. Cyril and Methodius University, Skopje, Macedonia
April, 2016


Fennel (Foeniculum vulgare Mill.) is commercially valuable plant from the Apiaceae family. It originates from the Mediterranean basin, but has spread to several areas in the world. In this work, more than forty samples of fennel (wild and cultivated) have been collected from different locations in Macedonia, and were analyzed in order to determine the diversity between species from both, morphological and chemical point of view, as well as determination of the quality in terms of their use in medicinal purposes and as food additive. The samples were analyzed for volatile secondary metabolites, polyphenolic compounds and fatty acid composition. The cultivated varieties have the highest content of trans-anethole (above 75%) and low content of fenchone (< 2%). IR spectroscopy (ATR-FTIR) was found as valuable for initial qualitative assessment of the essential oils.
The chemical composition was determined using gas chromatography with flame-ionization detection (GC-FID, column SP2560, 100 m, 0.25 mm i.d., 0.20 μm) and with mass spectrometric detection (GC-MS, column HP-5MS, 30 m, 0.25 mm i.d., 0.25 μm) using authentic standards. The main components of the essential oil of sweet fennel were identified as: trans-anethole (>80%), estragole (<6%), limonene (<6%), anisaldehyde (<1%) and fenchone (<1%). The analyzed samples of wild fennel had a variable composition, but in general had a higher content of fenchone (>8%). An alternative method for extraction and analysis of both volatile and non-volatile nonpolar fractions was developed. The extraction was carried out using 3:1 (V/V) n-hexane and dichloromethane followed by transesterification of the extract without prior evaporation of the solvents. The extracts obtained with this procedure, containing the components of the essential oil and fatty acids methyl esters were characterized using GC-FID and GC-MS. The dominant fatty acids were found to be C18:1 positional isomers (petroselinic and oleic acid), and their content in the analyzed samples was in range from 75.0-82.8%. The differences in chemical composition of volatile fraction after extraction and transesterification were very similar to the ones obtained for the pure essential oils implying that the volatile components did not undergo chemical transformation during extraction and derivatization and the developed sample preparation method can be used for simultaneous determination of volatile components and fatty acids in fennel.
For analysis and comparison of fennel volatile fraction, a static headspace gas-chromatographic method (SHS-GC-FID, HP-5 column 30 m, 0.25 mm i.d., 0.25 μm) was developed and compared with the “standard” hydrodistillation/GC method (HD/GC). Аs the fennel fruit ages, new products such as limonene oxides, carvone and carveole were detectable and other biosynthesized products were also present: threo-anethole β-hydroxy methylether, erythro-anethole β-hydroxy methylether and anethole glycol. The relative abundance of the major components is quite comparable between these two methods for fennel fruits up to 3 years from the harvest (HD/GC, SHS-GC-FID) and they can be used interchangeably depending on the purpose and amount of material. In order of further testing of the stability of the volatiles, photochemical behavior of pure trans-anethole was compared to that of sweet fennel essential oil (85% trans-anethole content) by exposing each to sunlight for different time intervals and then analyzing them by GC-MS. In the case of pure trans-anethole the dominant process was found to be dimerization, whereas for the essential oil the dominant process was isomerization to cis-anethole.
In the last stage of the study, fennel samples were extracted with 70% methanol and analyzed using HPLC-DAD-MSn to determine the polyphenolic profile. Hydroxybenzoic acid together with derivatives of hydroxycinnnamic acids such as isomers of caffeoylquinic and dicaffeoylquinic acid were determined, as well as their malonyl derivatives. The main flavonoid component that was detected in all analyzed samples was quercetin-3-O-glucuronide.