Enhanced solvent-free microwave extraction of Foeniculum vulgare Mill. essential oil seeds using double walled reactor

2.1 Plants material

Fennel seeds (Foeniculum vulgare Mill.) were purchased from a local herb market in Gabes (South of Tunisia). The seeds are originally from the province of Gabes (Matmata), harvested in 2013. Seed species was identified by Dr. Ezzeddine Saadaoui, from the herbarium of the regional station of Gabes, National Institute of Research in Rural Engineering, Waters and Forests (INRGREF, Gabes, Tunisia). The particle size chosen was less than 2 mm. The initial moisture content of dried fennel seeds was verified in the laboratory as 6.8 ± 0.08%.

2.2 ESFME apparatus and procedure

The ESFME apparatus consisted of a microwave oven, a double-walled reactor, and a Clevenger apparatus. A schematic diagram of the ESFME apparatus is illustrated in Fig. 1. The microwave oven (MW71C) used in the ESFME has been modified in the laboratory. Power and time can be changed with a regulable transformer. Its maximum output power was 800 W with 2450 MHz of microwave irradiation frequency. The dimensions of the interior cavity of the microwave oven were 31 cm × 21 cm × 32 cm. During experiments, the temperature was monitored by the digital temperature controller (KRK DT96, Turkey) with 3 digits display and memory protection, its measurement temperature range was between -99 °C and 999 °C with a frequency of 60 Hz, and its thermocouple sensor type (J, K) was inserted directly into the sample container. Temperature variations in the reactor were measured continuously. The Wattmeter placed at the microwave generator entrance monitored the input power consumptions. The microwave reactor consisted of the cylindrical double-walled vessel made of Pyrex glass and having a capacity of 500 mL. The neck of the reactor was connected to the Clevenger apparatus through the hole of the microwave cavity. The microwave irradiation energy was transmitted continuously to the surroundings of the microwave reactor during the extraction process. The attractive features of this microwave reactor were evidenced by comparison with the conventional single-walled microwave vessel used in the conventional SFME process, having the same shape and volume.

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Figure 1 Schematic diagram of Enhanced Solvent Free Microwave Extraction (ESFME) process.

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In the ESFME procedure performed at atmospheric pressure, 100 g of fennel seeds was heated using a different microwave power (300, 450, 600, 700 and 800 W) without adding any solvent or water after being soaked in 500 mL of distilled water at room temperature for 20 min. A cooling system placed outside the microwave oven condensed the distillate. The excess of water was returned to the extraction vessel in order to recover the ‘in situ’ water to the plant material. The extraction was continued until no essential oil was obtained. The essential oil was collected, dried under anhydrous sodium sulfate and stored at 4 °C until analyzed. Extraction yield was calculated according to the following equation:

2.3 Conventional SFME apparatus and procedure

To facilitate rigorous comparisons, the same microwave equipment and the same operating conditions have been used for the conventional SFME process (Lucchesi et al., 2004a), but with using the single-walled reactor. The essential oil was collected, dried under anhydrous sodium sulfate and stored at 4 °C until analyzed. The extraction yield was calculated according to Eq. (1).

2.4 Hydrodistillation apparatus and procedure

100 g of dried fennel seeds was submitted to hydrodistillation with a Clevenger-type apparatus according to the European Pharmacopoeia and extracted with 1 L of water during 180 min. This period was sufficient to isolate all quantities of essential oil from the sample. The Wattmeter was placed in the electrical heater entrance monitored the input power consumptions. The thermocouple sensor was inserted directly into the reactor to measure the temperature continuously. The essential oil was collected, dried under anhydrous sodium sulfate and stored at 4 °C until analyzed. The extraction yield was calculated according to Eq. (1).

All extractions experiments were performed in triplicated, and the mean values were given herein.

2.5 GC–FID and GC–MS analyses

The essential oils extracted by ESFME, conventional SFME and hydrodistillation methods were analyzed by a gas chromatography–flame ionization detection (GC–FID) and gas chromatography–mass spectrometry (GC–MS), respectively (El Babili et al., 2011).

2.6 Scanning electron micrographs

Before and after the extraction methods, the microstructure of the samples was performed by scanning electron microscope (SEM) (JEOL-JSM-6700F, UMS 3623 Centre de microcaracterisation Raimond Castaing Toulouse). Single Fennel seeds were fixed on aluminum support and sputter coated with platinum. Structural analyses were examined with the SEM, under standard vacuum conditions and secondary electron detector.

2.7 Statistical analysis

All extractions with ESFME, SFME, and hydrodistillation methods were expressed as mean ± SD of triplicate measurements. SDs did not exceed 5% for the majority of the values obtained. The differences were considered statistically significant at p < 0.05 level.

3.1 Determination of optimal extraction time and power

In order to evaluate the optimal extraction time (it was until no more essential oil was obtained), different microwave irradiation powers of 300, 450, 600, 700 and 800 W were examined. Data presented in Fig. 2a show the evolutions of the extraction yield obtained via different microwave irradiation powers as a function of the extraction time. Fig. 2b shows the evolutions of microwave irradiations powers as a function of the total extraction time in the ESFME process, respectively. Results indicated that almost similar essential oil yields were found in different levels. At 600, 700 and 800 W power settings allow shorter extraction time of 30 min, but the power should not be too high to avoid the loss of volatile compounds. So, the microwave irradiation power of 600 W for 100 g of fennel seeds was chosen as an optimum power for later experiments, because this power gave a complete extraction of fennel essential oil in only 30 min.

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Figure 2 (a) Yield profile of essential oil from fennel seeds obtained by ESFME as a function of the microwave power. (b) Microwave power profile as a function of total extraction time with ESFME. Data were mean values ± SD; n = 3.

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3.2 Comparison of extraction kinetics

As shown in Fig. 3 we have the evolution of the extraction yield, as a function of the extraction time. The experiments were performed at atmospheric pressure and optimal operating conditions (600 W). An extraction time of 30 min with ESFME provided yield comparable to those obtained after 37 min with conventional SFME and 180 min with hydrodistillation. Consequently, the main advantage of the ESFME process was its rapidity. The overall yield of essential oils obtained from fennel seeds was 0.48% by the ESFME and 0.46% by both conventional SFME and hydrodistillation methods. These yields were high in comparison with the yield of fennel essential oil obtained by Jigar Patel et al. (2012) (0.4%) by improved microwave assisted extraction.

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Figure 3 Yield profile of fennel essential oils obtained by ESFME (600 W) (♦), SFME (600 W) (■), and HD (▴) as function of the extraction time (mean values ± SD; n = 3).

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From Fig. 3, three distinctive phases can be observed in the kinetic extractions for both ESFME and SFME methods.

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  The first part (AB or A′B′) was represented by an increasing line, due to the facility of extraction of the first quantities, located at the surface of the plant cells, representing approximately 88% of the total yield obtained by ESFME and 83% of the total yield obtained by SFME method.

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  The second part (BC or B′C′) was followed by a second increasing line, and the oil amounts extracted represented approximately 12% for ESFME and 17% for SFME of the global yield. It is important to underline that this phase was very short in ESFME compared to SFME method. In fact, in the ESFME, the internal diffusion of the essential oil was involved by the thermal conduction of the water located into the interlayer of the reactor (as heat-transfer fluid). Therefore, the internal heating of the ‘in situ’ water was increased very quickly to extract the essential oil from fennel seeds.

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  The third part (CD or C’D’) was represented by the horizontal line which marked the end of the extraction.

The profile of the conventional hydrodistillation method (Fig. 3) represented also three distinctive phases, which were not similar to those obtained with the ESFME and SFME. The first step represented 83% of the yield obtained for 150 min. The end of the extraction reached after 180 min. Therefore, the processing time of hydrodistillation was longer than the ESFME and the conventional SFME methods, because the mass transfer was in the opposite direction with the heat transfer, unlike the ESFME and SFME methods, the process acceleration and high extraction yield due to the synergistic combination of both transfer phenomena: mass and heat gradients working in the same direction (Berka-Zougali et al., 2012).

The yield profile described here was similar to that found by Bousbia et al. (2009), using Microwave hydrodiffusion and gravity for extraction of essential oil from Citrus peels.

3.3 Comparison of temperature profile of the extraction methods

Fig. 4 shows the temperature profiles during ESME, SFME, and hydrodistillation methods. Two phases were observed in ESFME, SFME, and hydrodistillation methods. The first phase was represented by an increasing line until the temperature reached 100 °C, and thus achieved the evaporation of the first essential oil droplets. In the second phase, the extraction temperature was equal to the water boiling temperature (100 °C) at atmospheric pressure until the end of the extractions.

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Figure 4 Temperature profiles as a function of time during ESFME (600 W) (♢), SFME (600 W) ( ), and HD (Δ) as extractions of essential oil from fennel seeds.

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3.4 Composition of essential oil

The molecular formula, retention indice, and relative percentage in fennel essential oils obtained by the ESFME, conventional SFME and hydrodistillation methods at the optimal conditions, were summarized in Table 1, respectively. A total of 7 volatile components were identified in SFME extracts, 9 components in ESFME extracts and 10 components in hydrodistillation extracts, representing 99.99% of the total amount of essential oil extracted by both hydrodistillation and ESFME and 100% of the total amount of essential oil extracted by SFME method. The main volatile active compounds present in fennel essential oil were cis-anethole and α-terpinolene followed by eucalyptol and γ-terpinene, but in different relative amounts according to the extraction methods used. The quantity of cis-anethole was increased in SFME (83.63%) and ESFME (83.07%) compared to hydrodistillation (77.05%), unlike the quantities of α-terpinolene and eucalyptol were decreased respectively in SFME (11.42%, 2.12%) and ESFME (10.90%, 2.95%) compared to hydrodistillation (14.75%, 4.32%). The remainder consisted of other minor and trace substances in the oil. The chemical composition of the examined Tunisian Foeniculum vulgare Mill. essential oil was different from that observed with Algerian (Zoubiri et al., 2014), Turkish (Telci et al., 2009) and Indian (Singh et al., 2006) fennel essential oil.

Higher amounts of oxygenated monoterpene compounds (86.45% and 86.24% versus 81.8%) and conversely, lower amounts of monoterpene hydrocarbons (13.54% and 13.76% versus 18.19%) were present in the fennel essential oils extracted by ESFME and SFME compared to hydrodistillation. Indeed, the monoterpene hydrocarbons were less valuable than the oxygenated monoterpene compounds as they contributed to the fragrance of the essential oil to a slight extent. Contrariwise, oxygenated monoterpene compounds were greatly odoriferous than the monoterpene hydrocarbons and consequently were the most valuable and desirable. In addition, the oxygenated monoterpenes presented in the essential oil interacted more vigorously with the microwave heating, because they have a higher dipolar moment; hence, they can be extracted more easily than monoterpene hydrocarbons that have lower dipolar moments (Jiao et al., 2013).

The higher amount of oxygenated monoterpene compounds obtained by ESFME and SFME was probably due to the presence of water as an excellent absorber of microwave irradiations and their absence as solvent to limit or eliminate the hydrolyzation and oxidation effects, and other undesirable chemical reactions compared to the hydrodistillation that used a large quantity of water which was responsible for all problems cited previously (Lucchesi et al., 2004a, 2004b).

Therefore, ESFME and SFME produced the better essential oils, concentrated in oxygenated monoterpenes from aromatic plant compared to the hydrodistillation method.

3.5 Microstructural changes after extractions

Scanning electron micrograph was employed in this work to study the physical changes of seeds induced by hydrodistillation, SFME and ESFME methods compared to the micrograph of the untreated fennel seed (Fig. 5a).

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Figure 5 Scanning electron micrographs of the glands from fennel seeds: (a) untreated, (b) after HD for 3 h, (c) after SFME (37 min, 600 W) and (d) after ESFME (30 min, 600 W).

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As shown in Fig. 5a, the external surface of the untreated seed was smooth. After extraction by hydrodistillation (3 h), the seed became atrophic and appeared wrinkled with only a few ruptures (Fig. 5b), whereas after extraction by SFME (37 min) and ESFME (30 min only) (Fig. 5c and d), the huge perforation on the seed external surface was clearly observed, and appeared totally disrupted and empty from EO. The heat transfer in the hydrodistillation method, was mainly implemented by conduction and convection only; in contrast, in SFME and ESFME, a rapid decompression and the violent vaporization of the in situ water within the glands could have exceeded their capacity for expansion and caused their breakdown of the cell wall more speedily than in hydrodistillation method.

3.6 Proposed ESFME mechanism

Fig. 6 shows the mechanism of ESFME compared to SFME and hydrodistillation methods. In conventional hydrodistillation, the temperature increased often slowly, depended on the thermal conductivity and on convection currents, where the heat transfer was occurring from the outside to the inside while mass transfer was occurring from the inside to the outside of the plant material.

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Figure 6 Mechanism of enhanced solvent free microwave extraction (ESFME).

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Whereas, in both SFME and ESFME, the cell was subjected to severe thermal stress, the temperature increased much faster than the conventional heating, depending on the effects of microwave irradiations and the internal dielectric heating of the plant material with the action of the “in situ” water, where both the heat and mass transfers were in the same direction from the inside to the outside of the gland. In addition to the internal heat of the “in situ” water under the microwave heating and particularly during ESFME, the external heating was favored by the thermal conduction of the distilled water as the heat-transfer fluid contained into the interlayer of the double walled reactor, as additional ways of heating resulted in a rapid increase in temperature. As a result of internal superheating which leaded to the severe vaporization of the “in situ” water and localized a high pressure gradient inside the gland, a dramatic expansion and a rapid rupture of the cell walls were occurring. Finally, the essential oils were migrated quickly from the inside of the plant to the surrounding.

In fact, this external heat transfer occurred by the following: the rotation of dipole and the ionic conduction phenomena (Thuéry, 1992) through reversals of dipoles and rotation of charged ions due to the alignment on the electric field of the water molecule (Kaufmann and Christen, 2002) as heat-transfer fluid located in the double walled reactor possessing a dipole moment. In the normal state, the molecules of the water were oriented randomly; however, when the microwave irradiation was applied, the orientation of dipole varied with the alternative electromagnetic field. Thus, the heat was produced very quickly with the rotation of the water molecule under the microwave irradiation, which was distributed in all liquid by convection, and consequently, the plant material was heated by conduction effect of the water located in the interlayer of the double wall microwave reactor.

According to the study by Sólyom et al. (2011), the energy emitted by the microwave oven (E_emitted [kJ/ml]) was the sum of energy absorbed and energy losses. The absorbed energy was significantly different in ESFME and SFME processes. Indeed, it was higher in the case of ESFME than SFME, which was considered as a positive point for ESFME than SFME, because the microwave irradiations (or energy emitted) were absorbed by the water and by the plant internal water in the double wall reactor, whereas in the conventional reactor (SFME) the microwave irradiations (or energy emitted) were adsorbed only by the internal water of the plant. In addition, the efficiency η_mw was calculated for both processes as the ratio of absorbed energy to emitted one by the microwave oven as the following equation:

Thus, the efficiency η_mw was higher in ESFME than SFME. So, the excess of consumed energy (absorbed energy) stored it as heat reduced the energy losses and increased both the heat and mass transfers, which justified the reduction of the extraction time in ESFME than in SFME (30 min versus 37 min).

To the best of our knowledge, this is the first time that the mechanism of ESFME using the double wall microwave reactor has been proposed.

3.7 Cost, energy and environmental ecology

The reduced cost of extraction for the ESFME was clearly beneficial in terms of time, energy and environmental impacts. The ESFME procedure required an extraction time of 30 min versus 37 min for SFME and 180 min for hydrodistillation. The energy required to perform all extraction methods (energy emitted from the microwave oven and electrical heater) was, respectively, 0.56 kW h for ESFME, 0.59 kW h for SFME, and 3 kW h for hydrodistillation. The Wattmeter placed at the microwave generator entrance and the electrical heater power supply determined their power consumptions. Regarding environmental impact, the calculated quantity of carbon dioxide emitted into the atmosphere was lower in the case of the ESFME (448 g CO₂ per gram of essential oil) than for the SFME (472 g CO₂ per gram of essential oil) and hydrodistillation (2400 g CO₂ per gram of essential oil). These calculations have been made according to the literature, to obtain 1 kW h from fuel; 800 g of CO₂ will be emitted into the atmosphere (Chemat and Cravotto, 2013). ESFME was proposed as a quick and a clean process for the extraction of volatile compounds from medicinal and aromatic plant.