Insect rearing
Spodoptera litura larvae used in this study were obtained from a laboratory colony maintained in the Animal Toxicology and Physiology Specialty Research Unit (ATPSRU), Department of Zoology, Faculty of Science, Kasetsart University. The culture was continuously maintained on an artificial diet (mixture of 240 g of green bean, 25 g of agar, 40 ml of mixed vitamin solution, 5 g of ascorbic acid, 40 ml of amoxicillin solution, 3 g of sorbic acid, 5 g of methylparaben, 20 g of yeast, 4 ml of 40% formalin and 1.41 l of water) in the insect-rearing room of the Department of Zoology, Faculty of Science, Kasetsart University, at 26 °C with 75% RH and a 16:8-h L:D photoperiod. Second and third instar larvae were used randomly for the treatment. All experimental procedures in this research were performed with the approval of an appropriate animal Ethics Committee of Kasetsart University, Thailand, under the reference number OACKU01059.
Plant materials and extraction methods
The rhizomes of A. galanga, C. longa, and A. calamus; the leaves and stem of S. trilobata; and the fruits of P. nigrum and P. retrofractum were obtained from Banphoromyen, Amphawa, Samut Songkhram province, Thailand. Each plant was rinsed with water to remove debris and air dried under shade. Dried plants were chopped finely to a powder. One kilogram of each powder sample was soaked in ethanol for 14 days. Each crude extract was filtered using a vacuum pump, dried by a rotary evaporator to obtain the solidified crude extracts and stored at 4 °C in a refrigerator until further processing.
Preliminary test of the contact toxicity bioassay for crude extract
Contact toxicity bioassays were performed with second instar larvae of S. litura. Each ethanolic crude extract was evaluated individually to determine efficacy levels upon topical application to the thorax region with various concentrations of extracts (2–140 µg/larva) using acetone as a carrier. Each second instar larva received 2 µl of extract per treatment for the thoracic region, and acetone alone served as the control. Thirty insects at each concentration were used with five biological replicates. After treatment, larvae were maintained in the insect-rearing room and allowed to feed on an artificial diet. Mortality was recorded every day post-treatment. The median lethal dose (LD50) and sublethal dose (LD10 and LD30) at 24 and 48 h after exposure were calculated by Probit analysis using the Statplus program (version 2017, Analyst company, Canada).
Three extracts that showed the best control efficiency were chosen to make compound mixtures and subsequent analysis of the active ingredient compounds.
Isolation method
Major components of the fruits of P. retrofractum extract were isolated by preparative thin layer chromatography (PTLC) with 30% ethyl acetate (EtOAc) in hexane to yield a major compound identified as piperine (15.6%), whereas (2E,4E,14Z)-N-isobutylicosa-2,4,14-trienamide (6.2%) was obtained by PTLC using 10% EtOAc in hexane followed by 15% EtOAc in hexane. The 1H and 13C NMR spectra were recorded on a Bruker 400 MHz AVANCE III HD spectrometer operating at 400 MHz (1H) and 100 MHz (13C). The high-resolution mass spectra (HRMS) were recorded on a MAXIS (Bruker).
Piperine
Pale yellow solid; 1H NMR (400 MHz, CDCl3): δ 7.39 (ddd, J = 14.7, 8.4, 1.8 Hz, 1 H), 6.96 (d, J = 1.6 Hz, 1 H), 6.87 (dd, J = 8.0, 1.6 Hz, 1 H), 6.78–6.70 (m, 3 H), 6.42 (d, J = 14.7 Hz, 1 H), 5.94 (d, J = 4.7 Hz, 2H), 3.56 (s, 4 H), 1.74–1.46 (m, 6 H). 13C NMR (100 MHz, CDCl3): δ 165.59, 148.34, 148.26, 142.64, 138.38, 131.12, 125.51, 122.65, 120.20, 108.64, 105.82, 101.42, 47.07, 43.38, 26.87, 25.80, 24.81. HRMS (ESI) Calcd for C17H19NNaO3 308.1263 ([M+Na]+), Found 308.1278.
(2E,4E,14Z)-N-isobutylicosa-2,4,14-trienamide
Pale yellow oil; 1H NMR (400 MHz, CDCl3) δ 7.18 (dd, J = 15.0, 9.9 Hz, 1H), 6.16–5.99 (m, 2H), 5.77 (d, J = 15.1 Hz, 1H), 5.72 (s, 1H), 5.36–5.31 (m, 2H), 3.14 (dd, J = 12.3, 5.8 Hz, 2H), 2.12 (dd, J = 13.7, 7.1 Hz, 2H), 2.05–1.97 (m, 4H), 1.85–1.73 (m, 1H), 1.62 (dt, J = 15.2, 7.8 Hz, 1H), 1.43–1.37 (m, 2H), 1.29 (ddd, J = 19.8, 9.3, 4.0 Hz, 16H), 0.93–0.84 (m, 9H). 13C NMR (100 MHz, CDCl3) δ 166.72, 143.43, 141.55, 129.98, 128.32, 121.76, 47.10, 32.08, 31.89, 29.90–29.19, 28.93, 28.72, 27.30, 27.02, 22.45, 20.24, 14.11. HRMS (ESI) Calcd for C24H43NNaO 384.3242 ([M+Na]+), Found 384.3235.
For the ethanolic extract of the rhizomes of A. calamus, the major spot on TLC was isolated by PTLC using 25% EtOAc in hexane to obtain fraction 1 (17.1%). For structural analysis using 1H NMR, this fraction was identified as a mixture of beta-asarone and alpha-asarone (ratio 4.38/1) and confirmed by authentic compounds purchased from Sigma-Aldrich.
Asarone mixture
Colorless oil; 1H NMR (400 MHz, CDCl3): beta-asarone: δ 6.84 (s, 1H), 6.53, (s, 1H), 6.48 (m, 1H), 5.77 (dq, 1H, J = 11.5, 7.0 Hz), 3.90 (s, 3H), 3.84 (s, 3H), 3.81 (s, 3H), 1.84 (dd, 3H, J = 6.6, 1.8 Hz); alpha-asarone: δ 6.94 (s, 1H), 6.65 (dq, 1H, J = 15.8, 1.7 Hz), 6.49 (s, 1H), 6.09 (dq, 1H, J = 16.0, 6.6 Hz), 3.88 (s, 3H), 3.85 (s, 3H), 3.82 (s, 3H), 1.88 (dd, 3H, J = 6.6, 1.8 Hz).
Contact toxicity bioassay for pure compounds
Similar to the crude extract, a contact toxicity bioassay was performed with second instar larvae of S. litura. Each purified compound was evaluated individually to determine their efficacy levels upon 2-µl topical application to the thorax region with various concentrations (2–100 µg/larva) using acetone as a carrier at concentration. Thirty insects at each concentration were used with five biological replicates. After treatment, larvae were maintained in the insect-rearing room and allowed to feed on an artificial diet. Mortality was recorded at 24 h post-treatment. The median lethal dose (LD50) at 24 h after exposure was calculated by Probit analysis using the Statplus program (version 2017, Analyst Company, Canada).
Mixture concentration preparation methods
The compound mixtures of plants were prepared and modified from Hummelbrunner and Isman [13] by choosing the dose at LD30 or LD10 values of S. litura after contact toxicity analysis.
The LD10 and LD30 values of two extracts that showed the best control efficiency were chosen to make compound mixtures. Each crude extract was prepared to a specific concentration by dissolving in acetone. Then, 1 ml of each crude extract was mixed at a ratio of 1:1 v/v. The mixture was then used to analyze the contact toxicity and antifeedant efficiency.
Contact toxicity assay for compound mixtures
As described above, each mixture was treated with S. litura larvae by topical application. A minimum of 30 insects/combination was used for each experiment, and five replicates were performed. After 24 h, mortality was recorded. Actual mortalities were compared with expected mortalities based on the formula described as follows:
$$E = O_{\text{a}} + O_{\text{b}} \left( { 1- O_{\text{a}} } \right),$$
where E is the expected mortality and Oa and Ob are the observed mortalities of extracts at the given concentration. The effects of mixtures were designated antagonistic, additive, or synergistic by analysis using χ2 comparisons from the following formula:
$$\chi^{ 2} = \left( {\left( {O_{\text{m}} - E} \right)^{ 2} /E} \right),$$
where Om is the observed mortality from the binary mixture and E is the expected mortality. In addition, χ2 with df = 1 and α = 0.05 is 3.84. A pair with χ2 values > 3.84 and having higher than expected mortality was considered to be synergistic (negative = antagonist effect), with χ2 values < 3.84 representing additive effects. An observed mortality less than expected suggested an antagonistic effect of the mixtures. The mixtures that showed a synergistic effect were used for antifeedant and enzyme assays.
Antifeedant bioassay for compound mixtures
The no-choice bioassay investigated the antifeedant effect. Each binary mixture was applied to kale leaf discs (4 cm2) using a micropipette with 2 µl on each side [22] and allowed to air dry at room temperature before releasing early third instar larva onto the discs. Each larva that was starved for 4 h was placed in a Petri dish with one treated leaf disc and allowed to feed. Each treatment used 30 larvae with three replicates. The uneaten area of the leaf disc was measured using a digitizing leaf area meter after 3 h of feeding. The percent feeding inhibition was calculated by using the formula from [23], \(\left( {C{-}T} \right)/\left( {C + T} \right) \times 100,\) where C is the consumption of the control leaf and T is the treated leaf cut.
Enzyme assays
Enzyme extraction method
Enzyme assays were performed in an in vivo experiment. The combined mixtures were tested with the second instar of S. litura larvae to optimize its effect on detoxification enzyme activities. Acetone was used as a control group. After 24 h, the surviving larvae were used for enzyme extraction to determine the activities of esterase and glutathione-S-transferase. The extraction method was modified from Feyereisen [24], and surviving larvae were placed in a microtube and kept on ice. Then, larvae were ground with homogenized buffer (0.1 M potassium phosphate buffer mixed with 1 mM EDTA at pH 7.2). Homogenates were centrifuged at 4 °C and 12,000 rpm for 15 min. The supernatants were transferred to new tubes and kept on ice immediately to study the different enzyme activities.
Esterase activity (EST)
The esterase activity was determined by the method of Bullangpoti et al. [25] with modifications. Enzyme solution (40 µl) was mixed with p-nitrophenylacetate (pNPA) (10 mM in DMSO) and potassium phosphate buffer (50 mM, pH 7.4). Enzyme activity was measured at 410 nm and 37 °C for 90 s in a 96-well plate in a microplate reader using the kinetic mode. EST activity was determined using the extinction coefficient of 176.4705 for pNPA.
Glutathione-S-transferase activity (GST)
The glutathione-S-transferase method was modified from Oppenoorth et al. [26]. The mixtures containing 50 mM phosphate buffer (pH 7.2) were mixed with glutathione solution, supernatant, and 1-chloro-2,4′-dinitrobenzene (CDNB). Then, the activity of the mixtures was measured at a wavelength of 340 nm using a microplate reader. The GST activity was determined from the extinction coefficient of 0.000137 for CDNB. Three biological replicates per treatment were estimated.