Fennel is a native plant of Mediterranean Flora, well known and used since antiquity. Because of its great industrial and commercial value, it is widely cultivated all over the Balkan area. Fennel is mainly used for its anethole content in local and national spirits beverages, as far as in pharmaceutical, cosmetic products and in the confectionery as well. The essential oils of several fennel commercial samples (Foeniculum vulgare Mill.) from three Balkan countries (Bulgaria, Greece and Turkey) were studied, considering the yield and the quality of the obtained essential oils. The obtained yields of the essential oils ranged among 1.8-5.5%, whereas the anethole percentage varied from 65% up to 83%. The qualitative analysis of the oils was determined by GC/MS.

During the last decades an increasing interest is emerging on the natural products uses in the industry or in therapeutics. Among the aromatic and medicinal plants, fennel possesses a predominant position because of its multiple utilisation.

Fennel (Foeniculum vulgare Mill., fam. Apiaceae) is a native plant of Mediterranean Flora and it is cultivated in Europe, Asia, and many areas of Africa and South America. Fennel's therapeutic properties are known since antiquity; it has many uses as anti-inflammatory, stimulant, carminative, antispasmodic and lactagenic (1). Also it is far utilised in culinary as a condiment, and in the bakery and the confectionery as additive. More recently are recorded its antioxidant properties, due to the vitamin E content (2).

The various parts of fennel plant and mainly its seeds, produce essential oil, which is one of the main sources of anethole; and due to this has great industrial interest. It is widely used in cosmetics, in pharmaceuticals, in the perfumery, and in the food and beverages industry (flavouring sauces, liqueurs and various alcoholic distillates). In Greece the fennel seeds are mostly used, together with other rich in anethole aromatic plants, like anise, for the production of traditional drinks, and particularly the well-known "ouzo".

Numerous articles are the last years referred to the fennel's "aroma" (2,3,4), to the technological parameters influencing its yield and quality (5), and furthermore, studies on fennel's biochemical constituents (6) or its chemotaxonomy (7) were also performed.

Various cultivars of fennel are widely cultivated in Balkans, and the most exploitable part of the plants - the seeds, are exported from country to country. Because of the great trade importance of this crop, our aim in the present work is to study and compare commercial fennel's samples from three different Balkan countries - Bulgaria, Greece and Turkey in regard to their essential oils yield and quality, as it is well known how greatly the synthesis of the volatiles is influenced by the plant origin, the conditions (soil, climate) and the cultivation practices.

Plant material

The plant material from Bulgaria and Turkey were commercial samples of ripe fennel seeds, purchased from the local markets. The Greek seed sample was originated from native fennel cultivations, from districts of North Greece (Chalkidiki peninsula).

Isolation and yield of the essential oils

The percentage oil yield of the three different samples was determined using the European Pharmacopoeia distillation apparatus (Clevenger type) and after 3 replications, in a relative standard error Srel of ±0.1%. The seeds were placed in liquid N₂ for 30 min, and then comminuted in a closed type mill with a degree of comminution less than 1 mm, and then immediately were submitted to hydrodistillation. Samples of 20 g of the grinding plant material were distilled for 2 hours with 340 ml deionised water, at distillation rates of 3-3.5 ml/min (8). The lighter than water, colourless to pale yellow oils were dried over anhydrous Na₂SO₄ and stored within 3 ml of n-pentane in sealed glass vials under refrigeration (-20°C).

Gas liquid chromatography

The essential oil samples were analysed by gas chromatography, using Shimadzu 17A Ver.3 gas chromatograph, equipped with flame ionisation detector and connected to the PC workstation for gas chromatography. The analyses were carried out using a fused silica capillary column Carbowax 20M (30 m, 0.32 mm i.d., and film thickness 0.25 mm). Multi-ramp column temperature program was used [45°-53°C (0.5°/min), 53-150°C (2.5°/min), 150-200°C (4°/min), 200-260°C (10°/min)]. Injector temperature was 260°C, split ratio 1:10, detector temperature 250°C, while carrier gas was helium. The percentage compositions were computed from the GC peak areas without correction factors.

Gas chromatography and mass spectrometry

The analysis was performed on a fused silica column DB-5, 30m x 0.25 mm i.d., film thickness 0.25 mm (J & W Scientific Inc., Rancho Cordova, California, USA) using 17A Ver.3 gas chromatograph interfaced with a mass spectrometer (ion selective mode) Shimadzu QP-5050A supported by the Class 5000 software. Chromatographic conditions were as follows: oven temperature 55-120°C (3°/min), 120-200°C 4°/min, 200-250°C (6°/min), 250°C for 5 min.; carrier gas He; split ratio 1:30. Ionisation energy was 1.30 kV and scan time 0.5 s.

The constituents were identified by comparing the retention times with those of authentic samples, and by mass spectrometry matching the peaks mass spectra to those of NIST 98 mass spectra library, and of the SZTERP Library for GC/MS.

In Table 1 the main constituents of the essential oils of the Bulgarian, Greek and Turkish fennel samples are listed, representing the 99.0%, 99.6% and 99.4 % respectively of the oils, with more than 30 components identified. The percentages given are based on the data after three chromatographic runs (Srel.±0.2 - ±1.2%).

Significant differences were observed within the samples, concerning the percentage of the most constituents. trans-Anethole was the most abundant component in the samples, varying through the samples from 65.83% in the sample from Bulgaria, and 80.38% in the sample from Turkey to 82.73% in the Greek one. Estragole, fenchone and limonene, typical components of the fennel essential oil were present in all the studied samples, but with great variations in their proportions. The amount of estragole varied also within the samples; from 2.85% in the Bulgarian fennel to the intermediate 4.19% of the Turkish sample, up to the highest 5.75% of the Greek one. One of the main constituents of the Bulgarian fennel sample fenchone (20.76%), was identified only in small quantities in the Turkish and Greek samples (1.98 and 0.61% respectively). Noteworthy differences were also recorded in the percentage yield of limonene (7.70% in the fennel sample from Turkey, 5.64% in the Greek one and 2.85% in the Bulgarian sample).

Concerning the amounts of several monoterpene hydrocarbons such as a-pinene, camphene, b-pinene, myrcene and a-phellandrene they occurred in much greater proportion in the Bulgarian sample than in the other two ones. Particularly, a-pinene was the most abundant monoterpene hydrocarbon (2.96%) in the fennel oil from Bulgaria, whereas it represented only the 0.63% and 0.52% in the samples from Turkey and Greece respectively.

Carvone, of the group of the oxygenated monoterpenes, was identified in the Turkish sample (1.06%), in minor quantities in the Greek one (0.10%), and only in traces in that from Bulgaria. The constituent p-anisaldehyde, an autoxidation product of trans-anethole, ranged among the samples from 0.03% to 1.46% and 1.51% in the oils originated from Bulgaria, Greece and Turkey respectively. Totally, the percentage yield of the oxygenated monoterpenes represents the 89.6, 91.18 and 89.37% in the samples from Bulgaria, Greece and Turkey.

Germacrene-D resulted as the main sesquiterpene compound in the samples, identified among 0.04 and 0.09%, whereas other sesquiterpenes were detected just only in traces.

The fennel seeds samples were also examined regarding their essential oil content. The three samples differ greatly to the yields (vol/wt) obtained, with the Bulgarian one being the richest in essential oil (5.5%), followed by the Greek (2.4%) and the Turkish one (1.8%).

The essential oils of fennel seeds originated from the three Balkan countries were definitely of anethole type, because of the predominance of this component against estragole, the second representative constituent of fennel essential oils. Especially the anethole yields of the Greek and Turkish samples 82.73% and 80.38% respectively, are classified among the higher scattered in the literature. Previously, Toth (9), studying numerous fennel samples from different geographic origin, recorded the maximum yields of anethole; 88% in fennel var. dulcis samples, and 81.7% in samples of Italian origin. The percentage yield of anethole was determined to 84.4% in fennel samples from Israel (10), whereas Lawrence (11) noticed yields 71.5% in oils exported from Romania. The findings of the Greek and Turkish oils, concerning the anethole content, are also in accordance with previous studies on Greek fennel; Katsiotis (8) applying different processing methods on Greek sweet fennel seeds, recorded anethole values from 83% up to 95%.

More recently, Muckensturm et al. (7), studying different cultivars of Foeniculum vulgare Mill. subs. vulgare, determined the maximum anethole content 60% in the varieties dulce and bronze. The estragole / anethole proportion varied also within the samples. This ratio was determined in the Bulgarian sample to 4.34% (parts of estragole/100 parts of anethole) and this is also in agreement with analogous findings from a previous study on Bulgarian fennel as well (12). This ratio was higher in the Greek sample 6.95%, while in the Turkish one it was estimated somehow lower (5.21%).
 

Considerable differences were noticed also among the oils concerning the amounts of fenchone, limonene and a-pinene (Figure 1). These constituent yields in the Greek sample matched more to those originated from Turkey, and are more resembled to those of sweet fennel (9). On the contrary, the particularly high content of fenchone in the Bulgarian fennel is rather indicative of the var. vulgare.

The undesirable component of fennel essential oils, cis-anethole, was determined only in small quantities in the samples. While the presence of p-anisaldehyde in the Greek and Turkish oils is an evidence of a probable partial transformation of anethole. p-Anisaldehyde and p-anisylketone as well, are considered as autoxidation products of trans-anethole, formatted especially in commercial samples during the storage of fresh fennel (11). This demonstrates the extremely importance of the treatment (drying) and the storage conditions on the quality of the commercial fennel samples.

The analyses of the commercial fennel samples essential oils showed that they are of superior quality; particularly the Greek and Turkish fennel samples are more enriched in anethole, the most valuable constituent of this oil. Moreover, the fennel seeds from Bulgaria obtain one of the highest, so far known essential oil yield. Taking into consideration the necessity for appropriate conditions of treatment and storage, the cultivation of fennel undoubtedly could be proved as extremely profitable for the growers of these Balkan countries, due to the high commercial significance of this crop.

The authors are grateful to Prof. S.T. Katsiotis, of the School of Pharmacy of the Aristotle University of Thessaloniki, for the comparison of some spectral data.

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