Hexane

Solvents for different steps in analyses were partially denatured ethanol (AustrAlco, Spillern, Austria) for extraction, hexane, ethyl acetate (both Carl Roth, Karlsruhe, Baden-Württemberg, Germany) and methanol (VWR, Radnor, PA, USA) for open column chromatography and TLC, chloroform-d₁ (VWR) and pyridine-d₅ (Armar, Döttingen, Aargau, Switzerland) for NMR.
HPLC mobile phases consisted of ultrapure water prepared from deionized water with a Barnstead MicroPure system (Thermo Fisher Scientific, Waltham, MA, USA), HPLC-grade acetonitrile (VWR) and formic acid for LC-MS (Honeywell, Charlotte, NC, USA).
Reference substances were abietic acid contributed by the Department of Pharmaceutical Chemistry, University of Graz, Austria, apigenin (Sigma-Aldrich, St. Louis, MO, USA), chrysin, pinocembrin-7-methyl ether (pinostrobin), linoleic acid (all Carl Roth) and oleic acid (Fluka, Buchs, St. Gallen, Switzerland).

Monobutyl phthalate (MBP, 99% purity), di-n-butyl phthalate (DBP, 99%), diethylhexyl phthalate (DEHP, 99.5%), diethyl phthalate (DEP, 99%), dimethyl phthalate (DMP, 99%), phthalic acid (PA, > 99.5%), 2-ethyl-1-hexanol (99.6%), terephthalic acid (98%), and isophthalic acid (99%) were purchased from Sigma Aldrich Chemie GmbH (Hamburg, Germany). Acetonitrile, ethyl acetate, hexane, and formic acid were of HPLC grade and purchased from Carl Roth (Karlsruhe, Germany).

Ethanolic extracts were prepared from the roots and rhizomes of the plant. Sliced dried plant material was ground and about 50 g was used for the extraction. The lipophilic compounds were removed by pre-extraction with hexane (Carl Roth, Karlsruhe, Germany). Ethanolic extraction with 96% ethanol (Carl Roth, Karlsruhe, Germany) took place in an ultrasonic bath (Elma, Singen, Germany) for 10 min at room temperature, followed by centrifugation at 4000 RPM for 10 min. Ethanolic extraction was repeated three times, the supernatants were collected, and the solvent was evaporated using a rotary evaporator (Heidolph, Schwabach, Germany), followed by freeze drying using a VirTis Sentury freeze dryer (SP Scientific, Buena, CA, USA) resulting in 28.52% (“Mattmark”) and 32.47% (“Rosavine”) extraction yields. The ethanolic extracts, designated as M/R 96% EtOH, were stored in dark glass flasks in a fridge at 4 °C.

Living mealworms were purchased from ENTAVA (Roggentin, Germany), frozen in liquid nitrogen, and subsequently lyophilized. Mealworm proteins were extracted according to Klost et al. [19 (link)]. The protein content was 65.4% (derived from nitrogen content according to Dumas (DUMATHERM DT, C. Gerhardt, Königswinter, Germany), protein factor 5.60 [37 (link)]). Hexane, HCl, NaOH, ethanol, and ZnSO₄ were purchased from Carl Roth (Karlsruhe, Germany) or VWR (Darmstadt, Germany) and were of analytical grade. Denatured ethanol and CO₂ for drying were obtained from Carl Roth (Karlsruhe, Germany) and Nippon Gases Deutschland GmbH (Hürth, Germany), respectively.

Brain slices, fixed, permeabilized, and labeled for GFAP, were cryoprotected stepwise in 0.1 M sodium phosphate buffer pH 7.4 (PB) supplemented with increasing concentrations of glycerol [10–20–30% (v/v)] and left overnight in 30% glycerol in PBS at 4 °C. The tissue was frozen by plunging into hexane (Carl Roth) at −70 °C. Samples were transferred into cold methanol (−90 °C) in a freeze-substitution chamber (Leica EM AFS). Methanol was replaced three times before the specimens were immersed overnight in anhydrous methanol at −90 °C, containing 2% (w/v) uranyl acetate. After rinsing several times with methanol, the temperature was gradually raised to −50 °C and left overnight at −50 °C. Tissue was then infiltrated with a mix of Lowicryl HM20 resin (Polysciences) and methanol (1:2; 1:1; 2:1, 1 h each) and left in pure resin overnight at −50 °C. Samples were transferred to flat embedding molds containing freshly prepared resin at −50 °C. UV polymerization was started at −50 °C (overnight) and then continued for 4 d at temperatures gradually increasing from −50 °C to −20 °C (24 h) and finally to +20 °C (24 h). Ultrathin sections (70 nm) were mounted on 200-mesh formvar-coated nickel grids (Plano). Images were acquired using a Zeiss EM 900 equipped with a digital camera (Proscan 1 K Slow-Scan CCD Camera).