Perchloric Acid

The total phenolic content was determined by employing the methods given in the literature (Slinkard and Singleton, 1977 (link)) with some modification. Sample solution (1 mg/mL; 0.25 mL) was mixed with diluted Folin–Ciocalteu reagent (1 mL, 1:9, v/v) and shaken vigorously. After 3 min, Na₂CO₃ solution (0.75 mL, 1%) was added and the sample absorbance was read at 760 nm after a 2 h incubation at room temperature. The total phenolic content was expressed as milligrams of gallic acid equivalents (mg GAE/g extract) (Vlase et al., 2014 ).
The total flavonoids content was determined using AlCl₃ method (Zengin et al., 2014 (link)). Briefly, sample solution (1 mg/mL; 1 mL) was mixed with the same volume of aluminum trichloride (2%) in methanol. Similarly, a blank was prepared by adding sample solution (1 mL) to methanol (1 mL) without AlCl₃. The sample and blank absorbances were read at 415 nm after a 10 min incubation at room temperature. The absorbance of the blank was subtracted from that of the sample. Rutin was used as a reference standard and the total flavonoid content was expressed as milligrams of rutin equivalents (mg RE/g extract) (Mocan et al., 2015 (link)).
The total saponins content of the extract was determined by the vanillin-sulfuric acid method (Aktumsek et al., 2013 (link)). Sample solution (1 mg/mL; 0.25 mL) was mixed with vanillin (0.25 mL, 8%) and sulfuric acid (2 mL, 72%). The mixture was incubated for 10 min at 60°C. Then the mixture was cooled for another 15 min, followed by the sample absorbance measurement at 538 nm. The total saponin content was expressed as milligrams of quillaja equivalents (mg QAE/g extract).
The total triterpenoids content of the extracts was determined according to Zhang et al. (2010) (link) method with some modifications. Briefly, sample solution (1 mg/mL; 500 μL) was mixed with the vanillin–glacial acetic acid (5%, w/v, 0.5 mL) and 1 mL of perchloric acid. The mixture was incubated at 60°C for 10 min, cooled in an ice water bath for 15 min and then 5 mL glacial acetic acid was added and mixed well. After 6 min, the absorbance was read at 538 nm. Oleanolic acid was used as a reference standard and the content of total triterpenoids was expressed as oleanolic acid equivalents (mg OAE/g extract) through a calibration curve with oleanolic acid.
HPLC-PDA analyses were performed on a Waters liquid chromatograph equipped with a model 600 solvent pump and a 2996 photodiode array detector, and Empower v.2 Software (Waters Spa, Milford, MA, United States) was used for acquisition of data. A C18 reversed-phase packing column (Prodigy ODS (3), 4.6 × 150 mm, 5 μm; Phemomenex, Torrance, CA, United States) was used for the separation and the column was thermostated at 30 ± 1°C using a Jetstream2 Plus column oven. The injection volume was 20 μL. The mobile phase was directly on-line degassed by using Biotech DEGASi, mod. Compact (LabService, Anzola dell’Emilia, Italy). Gradient elution was performed using the mobile phase water-acetonitrile (93:7, v/v, 3% acetic acid) (Zengin et al., 2016 (link)). The UV/Vis acquisition wavelength was set in the range of 200–500 nm. The quantitative analyses were achieved at maximum wavelength for each compound.

A single colony of strain S288C was inoculated into 10 ml of YPD (1%(w/v) yeast extract/2%(w/v) Bacto peptone/2%(w/v) glucose) and grown with shaking at 30°C for overnight. To prepare cells under a rich condition, they were resuspended in 100 ml of YPD at an OD₆₀₀ of 0.1 and grown at 30°C for 6 hrs. To prepare cells under a poor condition, the cells grown overnight in YPD media were washed with ddH₂O, resuspended in 100 ml of SD (0.67%(w/v) yeast nitrogen base without amino acids/2%(w/v) glucose) at an OD₆₀₀ of 0.5, and grown at 30°C for 6 hrs. The cells were collected by centrifugation, resuspended in ddH₂O, aliquoted in microtubes (400 μl), frozen in liquid nitrogen, and stored at -80°C until use. The number of cells was directly counted using a hematocytometer.
Total RNA was extracted using a hot-phenol method [12 (link)] with some modifications. To the 400-μl cell suspension described above, 100 μl of 5× lysis buffer (50 mM Tris-HCl, pH 7.5/50 mM EDTA/2.5%(w/v) SDS) and 500 μl of water-saturated phenol were added and mixed well on a shaker at 65°C for 1 hr. The tubes were chilled on ice for 5 min and centrifuged for phase separation. While the aqueous phase was saved in another tube, the phenol phase was mixed with 500 μl of 1× lysis buffer and shaked at 65°C for 1 hr. The second aqueous phase was combined with the first one and extracted once with water-saturated phenol and once with chloroform. The RNAs was precipitated by adding isopropanol to the aqueous phase, rinsed with 75%(v/v) ethanol, and dissolved in ddH₂O. To remove contaminating genomic DNA, the RNA was treated with RNase-free DNase I (Promega) and purified with TRIzol reagent (Invitrogen) according to the manufacturer's instruction. The concentration of RNA was determined by measuring OD₂₆₀ on spectrophotometer based on an assumption that one OD₂₆₀ unit corresponds to 40 ng/μl of RNA.
Total amount of cellular RNAs was also determined using selective extraction of ribonucleotides by NaOH [13 (link)] with some modifications. To the 400-μl cell suspension, 100 μl of 1.2 N perchloric acid (PCA) was added and the obtained mixture was placed in ice-cold water for 1 hr. Following centrifugation, the supernatant was removed and the cell pellet was washed again with 500 μl of 0.25 N PCA. Following careful removal of residual PCA solution, the cell pellet was resuspended in 300 μl of 0.3 N NaOH and incubated at 37°C for 1 hr. After neutralization with adding 150 μl of 1.2 N PCA, the concentration of RNA was determined from OD₂₆₀ and a standard curve obtained from the measurement of various known amounts of purified yeast RNA subjected to the same NaOH/PCA treatment.

Hippocampus, striatum and cerebellum were dissected from one hemisphere under microscope between 2 and 4 h after the last cycle of alcohol exposure, weighed and snap frozen at −80°C. Hippocampus and striatum samples were each homogenized in 250 μL 0.1 M perchloric acid using an immersion hand disperser (Polytron PT 1200 E, Kinematica Inc., Keyland Court Bohemia, NY, USA). Cerebellum samples were homogenized with the same method in 500 μL milliQ water. Samples were then centrifuged at 14000 rpm at 4°C for 20 min and then supernatant collected using a 0.22 μm filter (Advantec, Sierra Court, CA, USA, 13CP020AS).
The concentration of the cations sodium (Na⁺), magnesium (Mg⁺⁺), potassium (K⁺), and calcium (Ca⁺⁺) in the cerebellum were determined by ion chromatography (IC, Dionex Aquion 1100, Thermo Fisher Scientific, Waltham, MA, USA). The cation chromatography consisted of a CS12A 4 mm analytical and a Dionex IonPac CG12A 4 mm guard column set. 20 mmol/L methanesulfonic acid was the eluent and was sonicated for 20 min followed by degassing with nitrogen for an additional 10 min prior to IC. 10 μl of sample were injected, was eluted for 20 min with isocratic 20 mmol/L methanesulfonic acid. Chromeleon Chromatography Data System software (Thermo Fisher Scientific, Waltham, MA, USA) was used to analyze and calculate the peaks.
The concentrations of norepinephrine (NE), 3,4-dihydroxy- phenylacetic acid (DOPAC), dopamine (DA), 5-hydroxy- indoleacetic acid (5-HIAA), homovanillic Acid (HVA) and serotonin (5-HT) in the striatum and hippocampus were determined by HPLC with electrochemical detection. The monoamines were separated by reverse-phase liquid chromatography with a Prodigy C18 column (DA 2 × 100 mm, particle size 3 μm, Phenomenex, YMC Europe, Schermbeck, Germany). The mobile phase (55 mM sodium acetate, 1 mM octanesulfonic acid, 0.1 mM Na2EDTA and 8% acetonitrile, adjusted to pH 3.7 with 0.1 M acetic acid) was de-gassed with an online de-gasser. 10 μl of sample were injected with a flow rate of 0.15 mL/min. Electrochemical detection was accomplished using an amperometric detector Antec Decade (Antec Scientific, Alphen aan den Rijn, Netherlands) with a glassy carbon electrode set at 0.8 V and an Ag/AgCl reference electrode. The output was recorded, and peak areas were calculated by LC solution software (Shimadzu, Kyoto, Japan).

Leaf relative water content (RWC) was calculated as RWC (%) = (fresh weight – dry weight)/(turgid weight – dry weight). Briefly, leaves were weighed (fresh weight), placed in 1.5 mL microtubes filled with water, and set overnight in the dark at 4°C. Then, leaves were weighed to obtain the turgid weight, and after drying for 7 days at 40°C, the dry weight was obtained.
Total soluble sugars (TSS) were determined according to Irigoyen et al. (1992) (link) with some modifications. First, leaf discs were homogenized with ethanol at 80% (v/v) and placed in a bath at 80°C for one hour. Then, after centrifugation (5 000g for 10 min at 4°C), 30 µl of the supernatant was incubated for 10 min at 100°C with an anthrone solution that contained 40 mg of anthrone, 1 mL of dH₂O and 20 mL of H₂SO₄. After cooling and centrifugation (as described previously), the absorbance of the supernatant was read at 625 nm using a Jenway 7305 spectrophotometer. TSS content was calculated using a glucose standard curve (y=7.197x + 0.07, R² = 0.985).
For starch determination, leaf discs were homogenized with perchloric acid (30%, v/v) and incubated at 60°C for one hour (Osaki et al., 1991 (link)). After centrifugation (10 000g for 10 min at 4°C), the supernatant was incubated with an anthrone solution (as described for TSS) at 100°C for 10 min. Then, the samples were centrifuged (5 000g, 10 min, 4°C), and the absorbance was read at 625 nm using a Jenway 7305 spectrophotometer. Starch content was calculated using a glucose standard curve (y=3.84x + 0.03, R² = 0.992).

A mixture of trypsin (2 ml, 6%), tris-HCl buffer (1 ml, pH 7.4, 20 Mm), extract (1 ml, 50, 100, 200, and 400 µg/ml), or diclofenac (1 ml; 50, 100, 200, and 400 µg/ml) were incubated (5 minutes, 37°C), then casein (1 ml, 0.8%) was added followed by a new incubation (20 minutes, 37°C); subsequently perchloric acid (70%) was introduced into the mixture and the optical density was read at 120 nm against the blank and the percentage inhibition of proteinase activity was calculated [40 ].