Chemical composition and larvicidal activity of Algerian Foeniculum vulgare seed essential oil

2.1 Plant material

F. vulgare seeds (5 kg) were collected from cultivated plants in Sétif region (Eastern Algeria, 1096 m above sea level) during May 2007. Only full green seeds are harvested.

2.2 Essential oil isolation

Dried F. vulgare seed were subjected to hydrodistillation in Clevenger-type apparatus. The resulting essential oil was dried over anhydrous sodium sulfate and stored at +4 °C until use. The effect of three extraction parameters on the yield and chemical composition of the extracted oil was studied. The considered parameters are: soaking time (0, 6, 12, 18 and 12 h), extraction time (2, 3, 4, 6 and 8 h) and the ratio of solid to liquid (50, 100, 150, 200, 250 and 300 g/L). On the basis of single-factor test, the optimum ratio of solid to liquid after the optimum soaking time was placed in a 4 L round bottomed flask. The flask was connected to a hydrodistillation apparatus (Clevenger-type apparatus) and the water was boiled for the optimum extraction time. The yield of essential oil was calculated by three extractions time.

2.3 GC–FID and GC–MS analysis

Analytical gas chromatography was carried out on a Hewlett–Packard 6890 gas chromatograph equipped with a flame ionization detector. An apolar HP-5 column (30 m × 0.32 mm, 0.25 μm film thickness) was used. The flow of the carrier gas (Helium) was 1 mL/min. The split ratio was 50:1. The analysis was performed using the following temperature program: oven isotherm at 40 °C for 8 min, from 40 to 250 °C at the rate of 2 °C/min and isotherm at 250 °C for 5 min. Injector and detector temperatures were held at 250 °C. The injection volume was 0.5 μL. GC–MS analysis was performed on a gas chromatograph HP 6890 coupled with an HP 5973 mass spectrometer with electron impact ionization (70 eV). An HP-5MS capillary column (30 m × 0.25 mm, 0.25 μm film thickness) was used. GC conditions were the same as described above.

The identification of the essential oil constituents was made based on matching retention time and mass spectrum of unknown components with the identified standards. The retention indices were calculated, for all volatile constituents using a homologous series of n-alkanes C₇–C₂₇. Essential oil components are reported as a relative percent of the total oil by peak area.

2.4 Larvicidal investigations

The larvicidal activity assays were developed using a known methodology (WHO, 2005). Concentrations ranging from 1 to 250 mg/L of the dissolved emulsified oil in water were prepared and three replicates were run for each concentration. Control tests were carried out in parallel, using the emulsifier solution without oil. Number of dead larvae was counted after 0.5, 1, 2, 4 and 24 h of exposure and the percentage mortality was reported from the average of three replicates.

3.1 Chemical composition of the essential oils

The qualitative and semi-quantitative essential oil compositions for the three hydrodistillation conditions are presented in Table 1. Compounds are listed in order of their elution on the HP-5MS column. Results have shown that soaking time had no significant influence on F. vulgare essential oil yield. We calculated a yield of 0.93 ± 0.07%, this is still true for the considered extraction time and ration solid to liquid, 2 h and 100 g/L, respectively. Through Fig. 1a we notice that soaking time has no influence on the chemical composition. For the studied F. vulgare samples we calculate a variation of ±1.6% for trans-anethol, ±0.8% for fenchone, ±0.5% for limonene, ±0.3% for estragol, and ±0.05% for α-pinene. For the second studied parameter we perceive that F. vulgare essential oil yield increases parallely with the extraction time. We calculate a yield of 0.70% after 2 h versus 1.20% after 6 h extraction time. The four major compound relative chemical variations were presented in Fig. 1b. For the present study, 6 h extraction time was fit to achieve the effective yield. The ration solid to liquid study allowed us to say that there is no significant yield variation for this parameter, and the observed yield fluctuations might be the result of the non-uniform grain shape distribution. Therefore, an extraction time of 6 h and a ration solid to liquid of 300 g/L were the conditions to get 72.86 ± 2.00% of trans-anethol toward fenchone and limonene without soaking the seeds.

Close

Figure 1

Soaking (a) and extraction (b) times effects on majors Foeniculum vulgare seed essential oil components.

Export to PPT

In addition, we summarize in Table 2 previous investigations of authors on the analysis of the volatile oils from fennel. The chemical composition of the examined Algerian F. vulgare seed oil was different from that observed with Turkish (Akgül and Bayrak, 1988; Telci et al., 2009), Serbian (Damjanovic et al., 2005), Indian (Singh et al., 2006), and Chinese (Fang et al., 2006) materials. The similar oil constituents are α-pinene (3.18%, Akgül and Bayrak, 1988), β-myrcene (1.68%, Damjanovic et al., 2005), limonene (6.37%, our work), fenchone (20.30%, Damjanovic et al., 2005), estragol (5.95%, Fang et al., 2006) and trans-anethol (87.85%, Telci et al., 2009). However, in the studied Algerian F. vulgare seed oil, β-pinene (1.17%, Akgül and Bayrak, 1988), 1,8-cineol (1.20%, Damjanovic et al., 2005), β-ocimene (0.83%, Telci et al., 2009), γ-terpinene (1.05%, Damjanovic et al., 2005), linalool (1.2%, Singh et al., 2006), anisketone (1.12%, Akgül and Bayrak, 1988), and 4-methoxy-benzaldehyde (1.99%, Fang et al., 2006) are not detected.

3.2 Mosquito larvicidal effect of F. vulgare seed essential oil

The essential oil was subjected to laboratory bioassay studies on second, fourth instars and pupae. Results of C. pipiens mortalities (Fig. 2) show that dose and time increases amplify the observed mortalities percentages. As a general observation, we perceive that for a dosage of 50 mg/L the mortality value doubles when we pass from 2 to 4 h of exposure for the two larvae considered stages.

Close

Figure 2

Mortality of Culex pipiens resulting from 0.5, 1, 2, 4 and 24 h after treatment with different dosages of Foeniculum vulgare seed essential oil. Means (n = 3) using 50 larvae per replicate.

Export to PPT

Fig. 2a shows that the second instars are very sensitive and the use of the concentration at 250 mg/L ensure 90% mortality after only 30 min treatment time. Moreover, we notice that we reach the 100% mortality for all the studied dosage after 4 h treatment time. The total elimination of the second instars is also insured after 2 h for the concentration at 150 mg/L. The fourth instars are less sensitive (Fig. 2b). To register a 100% mortality using the concentration at 150 mg/L, 4 h treatment time is needed. Besides, to record 100% mortality after 2 h of exposure the use of the concentration at 250 mg/L is required. Pupae are more resistant. Mortalities still less than 23% after 2 h of exposure (Fig. 2c). The use of the concentration at 200 mg/L guarantees 40% mortality after 4 h of exposure. Results became promising after 24 h of exposure. Fig. 3 shows lethal concentrations that insure 50% and 90% mortalities. These results could be useful in search of newer, safer and more effective natural compounds as larvicides.

Close

Figure 3

Lethal dosages of Foeniculum vulgare seed essential oil for 0.5, 1, 2, 4 and 24 h exposure time to record 50% and 90% mortality of Culex pipiens second, four instars and pupae.

Export to PPT