Plant materials and essential oil extraction method
Dried barks of C. verum, C. cassia, and C. loureiroi, purchased from Chao Krompoe pharmacy, Chakkrawat, Bangkok 10100, Thailand, were extracted of their essential oils. Images of the three plant species and the chemical structure of their major constituent are shown in Fig.1. Specimens of all Cinnamomum spp. were positively identified by a botanist from the botanical center, King Mongkut’s Institute of Technology Ladkrabang (KMITL), Bangkok, Thailand. All specimens were cleaned, crushed, and extracted of essential oils (EOs) by a hydro-distillation method. After 6-7 h, the process was completed. Each EO was collected from the separating funnel, removed of water with anhydrous sodium sulfate (Na2SO4), preserved in airtight vials, and kept at 4°C for further chemical composition analysis and bioassays (Table 2). All EOs and their combinations were diluted with ethyl alcohol into several formulations shown in Table 1.
Chemical composition analysis of the three Cinnamomum spp. EOs
Chemical compositions of EOs from C. verum, C. cassia, and C. loureiroi were analyzed at the Center Laboratory of King Mongkut’s Institute of Technology Ladkrabang (KMITL), Bangkok, Thailand by Gas chromatography-mass spectrometry (GC-MS). The GC-MS analysis was performed with an Agilent Technology (USA) GC-MS system. All chemical constituents were identified with Agilent software (version G1701DA D.00.00) in combination with a mass spectral library from the National Institute of Standard and Technology (NIST; Wiley 7n.1). The GC-MS identified constituents were confirmed of their identity by comparing their retention indices to those of reference compounds reported in the literature. In this composition analysis, the RI of each chemical constituent was determined and calculated with respect to a homologous series of n-alkanes (C7- C30). Then, it was compared to the RI of a corresponding reference chemical reported in the literature49-50.
Chemicals
Cinnamaldehyde, the major constituent of C. verum, C. cassia, and C. loureiroi EOs was purchased from Sigma-Aldrich Co., LTD., 3050 Spruce Street, Saint Louis, MO 63103, USA. The positive control was 1% w/v cypermethrin (Kumakai 10®), manufactured by MD Industry Co. LTD., 22 Phahonyothin Rd., Wang-Noi district, Phranakhonsri Ayutthaya province, Thailand. The negative control was 70% v/v ethyl alcohol, manufactured by Hong Huat Co. LTD., 77/82-87 Krugthonburi Rd, Klongsarn, Bangkok 10600, Thailand.
Ae. aegypti and Ae. albopictus rearing
Colonies of Ae. aegypti and Ae. albopictus were maintained at the entomological laboratory, Faculty of Agricultural Technology, KMITL, Bangkok, Thailand. The conditions in the laboratory were a temperature of 26±2°C and a 75±5% RH with a photoperiod cycle of 12.5-h light: 11.5-h dark. The eggs of Ae. aegypti and Ae. albopictus were obtained from the Mosquito Laboratory, KMITL. Eggs were hatched and reared for 1-2 days in a white plastic tray (the size of 23.0 cm wide x 32.0 cm long x 6.5 cm high) containing 2,000 ml of clean water until the larvae emerged. A total of 200 larvae were reared in the white plastic tray and fed with fish food pellets one time per day for 12-14 days until they pupated. A total of 100 pupae were collected in a 250 ml beaker containing 200 ml of clean water and then transferred into an entomological cage (the size of 30x30x30 cm3). After 3-5 days, the pupae developed into adults that were reared in an entomological cage. Adults of both sexes were fed with 5% glucose solution + 5% multivitamin syrup solution. Two-day-old female adults of each mosquito species were used in an adulticidal bioassay.
Adulticidal bioassay
The toxicity of each EO, each formulation of combined EOs, and the major constituent of these EOs against female adults of Ae. aegypti and Ae. albopictus were determined by a standard WHO susceptibility assay 51. A WHO susceptibility assay kit was purchased from the WHO Vector Control Unit in Penang, Malaysia. Following the WHO susceptibility assay guide lines51, 25 females of each mosquito species were exposed to 2 ml of each EO formulation (shown in Table 1). Namely, two millimeters of each formulation were dropped onto a filter paper (the size of 12x15 cm2) in the exposure tube (red spot tube, 4.4 cm in diameter and 12.5 cm in length). The mosquitoes were exposed to each formulation for 1 h and then transferred to the holding tube (green spot tube). The knockdown rate of each formulation against the two mosquito species was observed and recorded at 1, 5, 10, 30, and 60 min after exposure, while the mortality rate was observed and recorded at 24 h after exposure. The knockdown and mortality criterion were no movement of head, antenna, leg, wing, or other body parts26. Each treatment was performed in five replicates with positive (1% w/v cypermethrin) and negative (70% v/v ethyl alcohol) controls. The knockdown rate (K) and Mortality rate (M) were calculated by the following formula:
Knockdown rate (%K) = [(K/T) × 100],
Mortality rate (%M) = [(M/T) × 100],
where K was the mean number of knocked-down adults; M was the mean number of dead adults; and T was the mean number of treated adults.
Statistical Analysis
The means and percentages of knockdown and mortality results were statistically analyzed by one-way analysis of variance (ANOVA). The means were compared by Duncan’s Multiple Range Test (DMRT) at P<0.05. At the same P<0.05, 50% Knockdown Time (KT50) was determined by a standard probit regression analysis (SPSS, Version 19).
- The increasing knockdown value (%IKV) was calculated by the following formula26:
%IKV = [(%K of EOs combination - %K of Individual EO) / % K of EOs combination] × 100
- The increasing mortality value (%IMV) was calculated by the following formula26:
%IMV = [(%M of EOs combination - %M of Individual EO) / % M of EOs combination] × 100
- The synergistic value (SV) of each formulation was calculated by the following
formula36:
SV = [KT50 of individual EO / KT50 of combined EOs].
SV >1 indicated that the combined EOs were synergistic; SV <1 indicated that the combined EOs were antagonistic; and SV= 1 indicated that the combined EOs did not show any synergistic or antagonistic effect.
- The effective knockdown index (EKI) was calculated by the following formula:
EKI = [KT50 of individual EO or combined EOs / KT50 of 1% w/v cypermethrin].
EKI <1 indicated that the individual EO or combined EOs was more toxic than 1% w/v cypermethrin; EKI >1 indicated that the individual EO or combined EOs was less toxic than 1% w/v cypermethrin; and EKI =1 indicated that the individual EO or combined EOs was as toxic as 1% w/v cypermethrin.
- The effective mortality index (EMI) was calculated by the following formula:
EMI = [%M of individual EO or combined EOs / %M of 1% w/v cypermethrin].
EMI = 0 or >1 indicated that the individual EO or combined EOs was more toxic than 1%w/v cypermethrin, and EMI <1 indicated that the individual EO or combined EOs was less toxic than 1% w/v cypermethrin.