Larvicidal activity of the essential oil of Youngia japonica aerial parts and its constituents against Aedes albopictus

2.1 Plant material and essential oil extract

Fresh aerial parts of Y. japonica at flowering stage (20 kg) were harvested from Lishui City (27.54° N and 119.20° E, Zhejiang Province, China) in August 2013. The herb was identified by Dr. Q.R. Liu (College of Life Sciences, Beijing Normal University, Beijing 100875, China), and a voucher specimen (No. ENTCAU-Compositae-10239) was deposited at the Department of Entomology, China Agricultural University, Beijing, China. The sample was air-dried, ground to powder using a grinding mill (Retsch Mühle, Haan, Germany); subjected to hydrodistillation, using a modified Clevenger-type apparatus, for 6 h, and then extracted with n-hexane. The oil was dried over anhydrous Na₂SO₄ after evaporation of n-hexane using a rotary evaporator and kept in a refrigerator (4 °C) pending subsequent experiments.

2.2 Insect

Mosquito eggs of A. albopictus utilized in bioassays were obtained from a laboratory colony maintained in the Department of Vector Biology and Control, Institute for Infectious Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China. The original eggs of A. albopictus were collected from Nanjing, Jiangsu Province, China, in 1997. Adults were maintained in a cage (60 cm×30 cm×30 cm) at 28 °C–30 °C and 75%–85% relative humidity (RH). The females were fed with blood (Mus musculus, Beijing Laboratory Animal Research Center, Chaoyang District, Beijing, China) every alternate day, whereas the males were fed with 10% glucose solution soaked on cotton pad, which were hung in the middle of the cage. A beaker with strips of moistened filter paper was kept for oviposition. The eggs laid on paper strips were kept wet for 24 h and then dehydrated at room temperature. The dehydrated eggs were placed into a plastic tray containing tap water in our laboratory at 26 °C–28 °C and natural summer photoperiod for hatching and dog food served as food for the emerging larvae. Larvae were controlled daily until they reached the fourth instar (within 12 h), at which point they were utilized for bioassays.

2.3 Gas chromatography-mass spectrometry (GC-MS)

The essential oil of Y. japonica aerial parts was subjected to GC-MS analysis on an Agilent (Santa Clara, CA, USA) system consisting of a model 6890N gas chromatograph, a model 5973N mass selective detector (EIMS; electron energy, 70 eV), and an Agilent ChemStation data system. The GC column was an HP-5ms (Santa Clara, CA, USA) fused silica capillary with a 5% phenyl-methylpolysiloxane stationary phase, film thickness of 0.25 μm, a length of 30 m, and an internal diameter of 0.25 mm. The GC settings were as follows: the initial oven temperature was held at 60 °C for 1 min and ramped at 10 °C/min to 180 °C, held for 1 min, ramped at 20 °C/min to 280 °C, and then held for 15 min. The injector temperature was maintained at 270 °C. The sample (1 μL; 1:100, v/v, in acetone) was injected with a split ratio of 1:10. The carrier gas was helium at a flow rate of 1.0 mL/min. Spectra were scanned from m/z 20–550 at 2 scans/s. Most constituents were identified by gas chromatography by comparison of their retention indices with those found in the literature or with those of authentic compounds available in our laboratories. The retention indices were determined in relation to a homologous series of n-alkanes (C₈–C₂₄) under the same operating conditions. Further identification was made by comparison of their mass spectra with those stored in the NIST 08 and Wiley 275 libraries or with mass spectra in the literature [17]. Component relative percentages were calculated based on the normalization method without using correction factors.

2.4 Purification and characterization of two constituents

The crude essential oil of Y. japonica aerial parts (25 mL) was chromatographed on a silica gel (Merck 9385, 230–400 mesh, 1000 g; Merck Millipore, Darmstadt, Germany) column (85 mm i.d., 850 mm long) by gradient elution with a mixture of solvents [n-hexane:ethyl acetate, from 100:0, 100:1, 100:2, ···, to 0:100 (v/v)]. Fractions of 500 mL each were collected and concentrated at 40 °C; similar fractions according to their TLC profiles were combined to yield 13 fractions. The screening experiments were conducted using 500 ppm solutions (initially dissolved in acetone) of various fractions. The larvicidal activity bioassay was carried out as described below. Fractions 4–6 and 9–10 that possessed larvicidal activity, with similar TLC profiles, were pooled and further purified by preparative silica gel column chromatography (pre-coated GF254 plate; Qingdao Marine Chemical Plant, Qingdao, China) with petroleum ether/acetone (50:1, v/v) to obtain the pure compounds the structures of which were determined as menthol (1) (0.15 g), and α-asarone (2) (0.16 g). The structures of the compounds were elucidated based on nuclear magnetic resonance. Next, ¹H and ¹³C NMR spectra were recorded on Bruker ACF300 [300 MHz (H)] and AMX500 [500 MHz (H)] instruments, using CDCl₃ as the solvent with tetramethylsilane (TMS) as internal standard. Electron impact (EI) mass spectra were determined on a Micromass VG7035 mass spectrometer (VG Micromass, Manchester, UK) at 70 eV (probe).

Menthol (1) (Figure 1): Crystalline solid, C₁₀H₂₀O. – ¹H NMR (CDCl₃, 500 MHz): δ=3.41 (1H, m, J=10.5, 4.3 Hz, H-6), 2.78 (2H, m, J=2.8 Hz, H-7), 1.96 (H, m, J=4.3, 2.1 Hz, H-5a), 1.66 (1H, m, H-3a), 1.62 (1H, m, H-2a), 1.42 (1H, m, H-4), 1.11 (1H, m, H-1), 0.94 (1H, m, H-2b), 0.93 (3H, s, H-9), 0.92 (1H, m, H-5b), 0.91 (3H, s, H-10), 0.83 (1H, m, H-3b), 0.81 (3H, s, H-8). – ¹³C NMR (CDCl₃, 125 MHz): δ=72.5 (C-6), 50.2 (C-1), 45.1 (C-5), 34.6 (C-3), 31.7 (C-4), 25.9 (C-7), 23.3 (C-2), 22.2 (C-10), 21.0 (C-9), 16.2 (C-8). – EI-MS: m/z (%)=138 (27.6), 123 (31.3), 96 (29.0), 95 (70.2), 81 (76.7), 71 (100), 67 (25.7), 55 (40.1), 41 (40.9), 27 (15.3). The spectral data matched those of a previous report [18].

Figure 1:

Mosquito larvicidal constituents isolated from the essential oil of Y. japonica

α-Asarone (2) (Figure 1). Crystalline solid, C₁₂H₁₆O₃. – ¹H NMR (500 MHz, CDCl₃): δ=6.95 (1H, s, H-2), 6.66 (1H, dd, J=16, 1.6 Hz, H-7), 6.48 (1H, s, H-5), 6.09 (1H, dq, J=16, 7.2 Hz, H-8), 3.90 (3H, s, 10-OCH₃), 3.84 (3H, s, 11-OCH₃), 3.81 (3H, s, 12-OCH₃), 1.89 (3H, dd, J=16, 7.2 Hz, H-10). – ¹³C NMR (125 MHz, CDCl₃): δ=159.9 (C-4), 148.9 (C-6), 143.5 (C-1), 125.2 (C-7), 124.5 (C-8), 119.2 (C-3), 109.9 (C-2), 98.1 (C-5), 56.8 (C-10), 56.6 (C-12), 56.2 (C-11), 18.9 (C-9). – EI-MS: m/z (%)=208 (100), 165 (38), 137 (21), 105 (13), 91 (19), 69 (25), 41 (11). The spectral data matched those of a previous report [19].

2.5 Bioassay

Range-finding tests were conducted to determine the appropriate testing concentrations. The essential oil or constituents were first diluted to 10% (v/v), using acetone as a solvent, after which the final concentrations of 160, 80, 40, 20, and 10 μg/mL of the essential oil or constituents were tested. The larval mortality bioassays were carried out according to the test method of larval susceptibility as recommended by WHO [20]. A total of 20 larvae were placed in a glass beaker with 250 mL of aqueous solutions of test material at various concentrations, to which the emulsifier dimethylsulfoxide (DMSO; final content 0.05%, v/v) was added in the final test solution. Five replicates were run simultaneously per concentration. With each experiment, a set of controls made up of 0.05% DMSO and acetone and untreated sets of larvae in tap water were also run for comparison. Mortality was recorded after 24 h of exposure.

2.6 Data analysis

The observed mortality data were corrected for control mortality using Abbott’s formula. The mortality of larval mosquitoes in the control was <5%. Regression analysis was used to examine the linear relationship between concentrations and the mean value of larval mortalities [21]. When the goodness-of-fit test was significant (p<0.15) (Table 2), a heterogeneity factor was used in the calculation of confidence limits. The results from all replicates of the larvicidal bioassay were subjected to Probit analysis using PriProbit Program V1.6.3 to determine LC₅₀ values and their 95% confidence intervals [21]. Significant differences in LC₅₀ were based on non-overlap of the 95% confidence intervals.

3.1 Essential oil analysis

The yield of the essential oil of Y. japonica aerial parts was 0.33% (v/w based on dry weight), and its density was determined to be 0.86 g/mL. A total of 31 compounds were identified from the essential oil of Y. japonica, accounting for 96.53% of the total oil. The principal constituents of Y. japonica essential oil were menthol (23.53%), α-asarone (21.54%), 1,8-cineole (5.36%), and caryophyllene (4.45%) (Table 1). Monoterpenoids represented 17 of the 31 constituents, corresponding to 44.50% of the oil. Sesquiterpenoids represented 12 of the 31 compounds, corresponding to 29.73% of the whole essential oil, and only two phenylpropanoids were identified, corresponding to 22.30% of the oil. The results were quite different from those reported previously [22], in which the essential oil of Y. japonica aerial parts harvested from Hunan province was found to mainly contain isophytol (29.85%), n-heneicosane (10.69%), dibutyl phthalate (8.81%), and farnesyl acetate (7.24%). The huge difference in the composition of the essential oils of Y. japonica aerial parts may be attributed to several factors, such as geographical variation, environmental conditions, and harvesting times as well as population variation.

3.2 Larvicidal activity

A positive correlation was observed between the essential oil/isolated compounds concentration and the larvicidal activity (p<0.15), with the rate of mortality being directly proportional to the concentration, indicating a dose-dependent effect on mortality (Table 2). The essential oil of Y. japonica aerial parts exhibited larvicidal activity against the fourth instar larvae of A. albopictus with an LC₅₀ value of 32.45 μg/mL (Table 2). The two isolated constituents, menthol and α-asarone, had LC₅₀ values of 77.97 μg/mL and 24.56 μg/mL, respectively (Table 2). The commercial insecticide, chlorpyrifos, showed larvicidal activity against the mosquitoes with an LC₅₀ value of 1.86 μg/mL [6]. Thus, the essential oil of Y. japonica aerial parts was 17 times less toxic to A. albopictus larvae compared with chlorpyrifos. However, compared with other essential oils in the literature, which were obtained using the same bioassay, the essential oil of Y. japonica exhibited stronger larvicidal activity against A. albopictus larvae, e.g., essential oils of Allium macrostemon bulbs (LC₅₀=72.86 μg/mL) [23], Eucalyptus urophylla (LC₅₀=95.5 μg/mL) and E. camaldulensis (LC₅₀=31.0 μg/mL) [24], Toddalia asiatica (LC₅₀=69.09 μg/mL) [6], Clinopodium gracile (LC₅₀=42.56 μg/mL) [4], Zanthoxylum avicennae leaves (LC₅₀=48.79 μg/mL) [25], and Foeniculum vulgare (LC₅₀=142.9 μg/mL) [26]. Among the two isolated compounds, α-asarone exhibited stronger larvicidal activity than the crude oil against A. albopictus larvae, while menthol was less toxic (based on non-overlap of the 95% confidence intervals). Thus, it is suggested that α-asarone is a major contributor to the larvicidal activity of the essential oil of Y. japonica. Moreover, compared with chlorpyrifos, α-asarone was 12 times less toxic to A. albopictus larvae (Table 2). In previous reports, α-asarone exhibited larvicidal activity against the third instar larvae of three mosquitoes, Culex pipiens pallens, A. aegyptiand Ochlerotatus togoi, with LC₅₀ values of 22.38 ppm, 26.99 ppm and 26.38 ppm, respectively [27, 28]. Menthol also possessed larvicidal activity against the fourth instar larvae of A. aegypti with a 24-h LC₅₀ value of 365.8 ppm [29, 30]. Moreover, some studies reported that α-asarone possesses insecticidal activity against several grain storage insects [19, 31, 32]. Menthol also possessed insecticidal activity against several insects and mites [33–36]. The abovementioned findings suggest that the essential oil of Y. japonica and the isolated constituents, especially α-asarone, show potential for development as possible natural larvicides for the control of mosquitoes.

Youngia japonica is quite safe for humans, because it has been used in traditional medicine for the treatment of inflammatory diseases, such as angina, leucorrhea, mastitis, conjunctivitis, and rheumatoid arthritis [10]. However, a literature survey revealed that there is no experimental data on the toxicity of the essential oil of Y. japonica to humans. Thus, to develop a practical application for the essential oil and its constituents as novel insecticides, further research into the safety of the essential oil/compounds to humans is needed. Additional studies on the development of formulations are also necessary to improve efficacy and stability as well as to reduce costs. Moreover, field evaluation and further investigations on the effects of the essential oil and its constituents on non-target organisms are necessary.