Catalpol

After subtraction of the extremities, the remaining parts of each leaf sample were crushed with a sterile pestle in liquid nitrogen and the frozen powder was freeze‐dried for 48 h. The extraction was processed using previously described protocol, and ¹H‐NMR spectra of the metabolites were recorded (Supporting Information; Kim et al., 2010). NMR spectra were processed with MNOVA software v.10.0.2 (Mestrelab research S.L., Spain). Model compounds of Aucubin (Sigma‐Aldrich, Germany), Catalpol (Sigma‐Aldrich) and Verbascoside (Extrasynthese, France) were used for signal assignments of P. lanceolata defensive metabolites. Other primary or secondary metabolite shifts and J‐coupling constants obtained from plant material using similar solvents were used as reference (Kim et al., 2010; Lubbe et al., 2011; Yang et al., 2012; Agudelo‐Romero et al., 2014; Gallo et al., 2014). For multivariate analysis, the signal was binned to 0.04 ppm and integrated. The trimethylsilylpropanoic acid (TSP) and methanol signals were removed and the relative intensity of the chemical signals was normalized according to the dry mass of the samples and the TSP intensity.

Ellagic acid, quercitrin hydrate, and catalpol were purchased from Sigma Chemicals (Saint Louis, MO). Purity of standard compounds was guaranteed to be higher than 95% by HPLC. HPLC grade acetonitrile, methanol, and formic acid were purchased from J. T. Baker (Phillipsburg, NJ). catalpol, Ellagic acid, and quercitrin were chosen as Marker compounds to standardize the extract sample. CP001 was dissolved in distilled water at a concentration of 100 mg/mL and the solution was filtered through a 0.45 μm membrane filter. A 10 μL aliquot of the sample solution was injected into a HPLC system (Agilent Technologies, Palo Alto, CA). The sample was analyzed on a Capcell Pak UG120 C₁₈ analytical column (250 × 4.6 mm, 5 μm; Shiseido, Japan).

The following standards for iridoid glycosides and phenylpropanoid glycosides were purchased from APTBio (Shanghai, China): catalpol, rehmannioside A, rehmannioside D, leonuride, aucubin, and verbascoside, n-alkanes (C8–C20; Sigma-Aldrich, USA). The structures of these standards are shown in Figure 1. Methanol, acetonitrile, and formic acid (HPLC grade) were supplied by Fisher Scientific (Fairlawn, NJ, USA). Ethyl acetate and petroleum ether (HPLC grade) were purchased from Shanghai Chemical Reagent Co. (Shanghai, China). Water was purified by a Milli-Q system (Millipore, Milford, MA, USA).

Catalpol was purchased from Sigma-Aldrich (St. Louis, MO, USA). To prepare plates supplemented with Catalpol, the stock solution in dimethyl sulfoxide (DMSO) was inserted into autoclaved NGM plates (at 50°C). A final DMSO concentration of 0.1% (v/v) was maintained under all conditions. Bristol N2 (wild-type) and Escherichia coli OP50 strain were kindly provided by Dr. Myon-Hee Lee (East Carolina University, NC, USA). All other strains were obtained from the Caenorhabditis Genetic Center (CGC; University of Minnesota, Minneapis, MN). The transgenic strain CF1553 (muIs84) was used to visualize SOD-3 expression. Mechanistic study was performed using several null mutant strains including GR1307 (mgDf50), VC199 (ok434), EU1 (zu67), DR1572 (e1368), TJ1052 (hx546), and FK171 (ks54). The worms were grown at 22°C on nematode growth medium (NGM) agar plate with E. coli OP50 as described previously [13 (link)].

Catalpol (molecular weight 362.33) and aconitine (molecular weight 645.74) were purchased from Sigma-Aldrich (Shanghai, China). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay kit (cell proliferation) (ab211091), lactate dehydrogenase (LDH) assay kit (colorimetric) (ab102526), anti-Caspase-3 antibody (ab4051) and anti-caspase-9 antibody (ab25758) were purchased from Abcam (UK).

High purity chemicals: sodium carbonate, sodium acetate trihydrate, and anhydrous aluminum chloride were acquired from Sigma-Aldrich (Germany). Folin-Ciocâlteu reagent was purchased from Merck (Germany). The standard chemicals; chlorogenic acid, p-coumaric acid, caffeic acid, rutin, apigenin, quercetin, isoquercitrin, hyperoside, kaempferol, quercetol, myricetol, fisetin, gallic acid, aucubin, catalpol, and harpagoside were sourced from Sigma-Aldrich (Germany), ferulic acid, sinapic acid, gentisic acid, patuletin, luteolin from Roth (Germany), caftaric acid from Dalton (United States), harpagide, and 8-O-acetyl-harpagide from PhytoLab GmbH & Co. (Germany). HPLC grade solvents (methanol, acetonitrile, ammonium acetate, and silver nitrate) were purchased from Sigma-Aldrich. Distilled, deionised water was produced by a Direct Q-5 Millipore (Millipore SA, Molsheim, France) water system.