Methanol p a

To screen for alterations in the metabolite profile of ∆hda1A compared to the WT, metabolites were extracted from the supernatants of untreated (PDB; control) and sorbitol treated liquid cultures and subjected to HPTLC. An optimized extraction method of our previously described protocol (69 (link)) was applied for metabolite extraction from liquid cultures. Liquid cultures of the WT and the ∆hda1A mutant were obtained like described above. Six milliliters of the culture supernatants were aliquoted and mixed with 1.1 mL of acetone p.a. (CarlRoth GmbH + Co KG, Karlsruhe, Germany) each. The mixture was incubated for 15 min at room temperature in the ultrasonic bath. After addition of 4.5-mL ethyl acetate p.a. (EtOAc; CarlRoth GmbH + Co KG, Karlsruhe, Germany), the samples were mixed well. Phase separation was obtained by centrifugation at 3,000 × g for 1 min, and the upper phase was transferred to a broad glass vial. The EtOAc extraction step was repeated a second time, and the EtOAc extracts were evaporated overnight. The next day, evaporated extracts were re-collected in 140 µL of methanol p.a. (Merck KGaA, Darmstadt, Germany). Chromatographic separation was done on silica gel plates (HPTLC silica gel 60 F254S, Merck KGaA, Darmstadt, Germany) via HPTLC as previously described (69 (link)).

This study used the rhizome of K. parviflora, 96% ethanol (Merck, Rahway, NJ, USA), 96% methanol PA (Merck), distilled water (Onelab, PT. Jayamas Medica Industri, Indonesia), RPMI Medium (Gibco, Billings, MT, USA), DMEM High Glucose (Gibco), MTT Reagent (Merck), Trypsin (Gibco), Fetal Bovine Serum (Gibco), sodium bicarbonate, Hepes, penicillin–streptomycin (Thermo Fischer, Waltham, MA, USA), 96-well plate (Biologic, Seyssinet-Pariset, France), micropipette (Dragonlab), rotary evaporator (RV 10), vortex (B-One, Cedar Knolls, NJ, USA), Elisa Reader (Epoch, Fremont, CA, USA), glassware (Iwaki, Tokyo, Japan), and freeze dryer.

Ethanol (HPLC degree) and aseptic acid (HPLC degree) were obtained from Merck Co. (Darmstadt, Germany) and methanol (PA) from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). A cellulose ester membrane filter of 0.45 μm (SLCR025NS, Millipore® Co., Bedford, Massachusetts, USA) was used. The carbon dioxide (CO₂) used in the extraction had 99.9% purity (White Martins Gases Industrials–São Paulo, Brazil). The standard 3,5-diprenil-4-hidroxicinamic (Artepillin C–cas number 72944-19-5) was acquired from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) and the Acid 4-hidroxicinamic (p-coumaric acid–cas number 501-98-4), 2,2-diphenyl-1-picrylhydrazyl (DPPH), Acid Gallic (cas number 149-91-7), Quercetin (cas number 117-39-5), 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) (cas number 30931-67-0) and (±)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) (cas number 53188-07-1) were acquired from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA).

Ethanol (HPLC degree) and acetic acid (HPLC degree) were obtained from Merck Co. (Darmstadt, Germany) and methanol (PA) from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). A cellulose ester membrane filter of 0.45 μm (SLCR025NS, Millipore Co., Bedford, Massachusetts, USA) was used. The carbon dioxide (CO₂) used in the extraction had 99.9% purity (White Martins Industrials Gases–São Paulo, Brazil). The standard 3,5-diprenyl-4-hydroxicinnamic acid (Artepillin C–cas number 72944-19-5) was acquired from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) and the acid 4-hydroxycinnamic (p-coumaric acid–cas number 501-98-4), 1,1-diphenil-2-picrilhidrazil (DPPH), Acid Gallic (cas number 149-91-7) and Quercetin (cas number 117-39-5) were acquired from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA).

Drops from total hemolymph (plasma and hemocytes) of untreated ticks (negative control group), ticks inoculated with 0.1% Tween^®80 and ticks infected with 10⁷ conidia mL⁻¹ were collected 24 h after infection and placed directly onto glass slides, dried at room temperature for 20–30 min, and fixed in methanol PA (Sigma-Aldrich) for 3 min. Slides were then stained with Giemsa (Sigma-Aldrich) [diluted 1:9 in buffered distilled water Brayner et al. (2005) (link)] for 30 min, rapidly washed with buffered distilled water, and observed by light microscope. Five stained slides were performed per group. One hundred cells per slide were count for differential hemocytes counts. Differential hemocytes counts were expressed based on the amount of each hemocyte type in the total cells counted.

DPPH free scavenging activity was assessed by a modified microplate assay method previously described by Li et al. (20 (link)). Initially, 200 μM of DPPH stock solution (Sigma Aldrich, St. Louis, MO, USA) was prepared in methanol p.a. (Synth, Diadema, SP, Brazil) 10–15 min before experimentation, stored in a sealed bottle, and kept away from light. For this assay, stock drug solutions were prepared, using methanol, at concentrations ranging from 3.13 to 800 μM/well. HU, as well as the antioxidant external controls BHT and L-ascorbate, were incubated for 30 or 60 min at a volume of 0.1 mL on 96-well flat-bottom microtiter plates (Greiner Bio-one, Monroe, North Carolina, USA) at a ratio of 1:1 (v/v), with the addition of DPPH (100 μM/well). All plates were covered and kept in the dark to minimize evaporation and to avoid the photosensitization of DPPH radicals. Finally, the plated solutions were homogenized for 5 sec, and absorbance was measured on a microplate reader (SpectraMax 190, Molecular Devices Corporation, Sunnyvale, CA) using Softmax software v. 5.0 (Molecular Devices, Sunnyvale, CA, USA) at a wavelength of 517 nm. DPPH radical scavenging activity was determined using the following equation: Scavenging activity of DPPH (%) = [(Abs_dpph-Abs_drug) × 100]/Abs_dpph.