Sesquiterpenes

The larval toxicities of CINEX, the isolated sesquiterpenes, and cypermethrin were evaluated using an established assay [46 (link), 47 (link)]. In brief, five 1^st-instar larvae were added to the wells of a 24-well Falcon Multiwell plate (Becton Dickinson Labware, Franklin Lakes, NJ) containing 985 μl of dH₂O and 5 μl of a food solution (13 mg/ml of finely ground fish food flakes in dH₂O; Tetramin, Blacksburg, VA). To each well, 10 μl of CINEX, a sesquiterpene, or cypermethrin (all dissolved in 100% acetone) at various concentrations was added. Control wells received 10 μl of 100% acetone. The plates were held under normal rearing conditions (28°C, 80% relative humidity, 12:12 light:dark) for 24 h before assessment of larvae. The efficacy of a concentration was determined as the percentage of larvae in a well that died within 24 h. Larvae were counted as dead if they did not move after gentle touching with a fine needle or pipette tip.
The adult toxicities of CINEX and the isolated sesquiterpenes were evaluated using an established assay [46 (link), 48 (link)]. In brief, groups of 10 adult female mosquitoes (3–10 days post-emergence) were immobilized on ice and treated with 500 nl (Ae. aegypti, Cx. pipiens) or 200 nl (An. gambiae) of a compound (dissolved in 100% acetone) at various concentrations. The compounds were delivered to the thorax of mosquitoes with a repeating dispenser (PB600-1, Hamilton, Reno, NV). As a control, 100% acetone was used. Immediately after treatment, the mosquitoes were transferred to small cages (32 oz. containers) with access to 10% sucrose and held under normal rearing conditions for 24 h before assessment. The efficacy of a dose was defined as the percentage of incapacitated mosquitoes (i.e., dead or unable to fly) in a cage within 24 h [46 (link), 48 (link)–51 (link)].
The concentration/dose-response curves for CINEX, the sesquiterpenes, and cypermethrin were evaluated with GraphPad Prism (version 6.07) software. In brief, percent efficacies were plotted against the log transformations of the concentrations/doses. The EC₅₀, ED₅₀, and Hill slope values were determined with non-linear regressions using the log(agonist) vs. normalized response function. Statistical comparisons of the best fit values were made with F-tests using GraphPad Prism software.

PTR-MS analysis allows online VOC quantification at high temporal resolution without differentiation of isomeric compounds (Ghirardo et al., 2010 ). To identify the VOCs and separate different monoterpene and sesquiterpene isomers, GC-MS analysis was performed by trapping 4 l of air leaving the cuvettes onto polydimethylsiloxane-foam-adsorbent tubes (Gerstel, Mülheim an der Ruhr, Germany) at flow rates of 100ml min⁻¹. This procedure was optimized for determination of nonpolar compounds; some polar volatiles such as methanol, ethanol, and C6 LOX products were not included in this analysis. Samples were analysed after thermal desorption and cryofocusing by GC-MS, as described by Ghirardo et al. (2012) . VOCs from control experiments were used for background subtraction. For the quantification of VOCs, individual response factors were determined using the total ion count from calibration curves (R²>0.98) of pure standards (α-pinene, sabinene, 3-carene, p-cymene, limonene, linalool, trans-β-caryophyllene, α-farnesene, and nerolidol) at four different concentrations (1–100 pmol (l hexane)^–1). Other monoterpenes not present in the standard were quantified using sabinene; other monoterpene alcohols using linalool; other sesquiterpenes using (−)-β-caryophyllene; and other sesquiterpene alcohols using nerolidol. For the quantification of aliphatic and aromatic compounds, a response factor was calculated for each compound by using the response factor of sabinene (R²>0.99) and was normalized based on molecular weight in order to consider the changes of total ion count responses due to different molecular masses. The same procedure was used for the quantification of the aliphatic and aromatic alcohols, except that the response factor of the linalool standard (R²>0.99) was used as reference.

For the data obtained from the first experiment, analysis of variance (ANOVA) of a completely randomized design (CRD), also known as one-way ANOVA, was conducted to determine the effect of species (five levels: H. hirsutum, H. maculatum, H. perforatum, H. perforatum (Com, USA), and H. perforatum (Com, Bul), on the concentrations of 15 constituents (2-methyloctane, nonane, α-pinene, 3-methylnonane, β-pinene, (Z)-β-ocimene, (E)-β-ocimene, undecane, caryophyllene oxide, (E)-caryophyllene, (E)-β-farnesene, germacrene D, δ-cadinene, α-epi-cadinol, and α-cadinol), and four classes (monoterpenes, sesquiterpenes, alkanes, and other). The analyses were completed using the GLM Procedure of SAS [76 ]. Since the effect of species was significant (p-value < 0.05) on the concentrations of all 19 constituents, further multiple means comparison was completed using Tukey’s multiple range test at 5% level of significance and letter groupings were generated. For each response variable, the validity of the normal distribution of the error terms assumption was verified by generating a normal probability plot of residuals and testing for normality of the error terms using the residuals, and the validity of the constant variance assumption of the error terms was verified by plotting the residuals vs. the fitted values, as described in Montgomery [75 ].
For the data from the second experiment, descriptive statistics (mean and standard deviation) were calculated using the three replicates.

A total of 153 natural compounds in the two types of agarwood were collected from the literature for building a focused compound library. The compound library includes PECs and sesquiterpenoids (SESs), and the details of the compound library are presented in Table S2. SwissADME was used to evaluate and screen the ADME parameters (pharmacokinetic screening under GI absorption condition was high, and at least more than two of 5 different rule-based filters) of the small molecule compounds library. A total of 54 potential targets for treating related diseases with agarwood ethanol extract were initially identified from the literature, DrugBank database, and STITCH database, and the related diseases include diabetes, asthma, depression, Alzheimer’s disease, coronary artery disease, gastric ulcer, myocardial infarction and gastric tumor. The selection and acquisition of the target proteins with a high-resolution crystalline structure was based on the UniProt database and RCSB database, and either ligand molecules contained in the downloaded protein structure models or FDA-approved drugs were selected as positive drugs. The details of the genes and target proteins are presented in Table S3.