Preparation of extract
The aqueous extract was prepared from the heartwood powder of P. Santalinus as described previously (Bulle et al. 2013a). Before use, the extract was dissolved in water.
LC-MS analysis
Standardization of Pterocarpus santalinus extract was performed using LC/MS, Waters model -3100 coupled with LC-29960 system. The chromatographic separation was performed using Hypersil GOLD, THERMO C-18, 3µm, 4.6 X 50 mm. 0.1% formic Acid (1ml of formic acid in 1000 mL of HPLC grade water) and acetonitrile were used as mobile phase A and B respectively and used with a flow rate of 0.8 mL/min. The gradient elution started with 95% A/5% B 0-10 min. Photodiode array detector was set at 285 nm for acquiring chromatograms. Approximately 2mg of compound is weighed and dissolved in water for injections. The injection volume was 5 μL and peaks were monitored at 250 nm. A mass spectrum was recorded in positive ionization mode between m/z 100 to 800 as per the standard operating procedure and Electroscopy ionization (ESI) technique was used for the ionization of the sample.
Animals
Two-month-old male albino wistar rats (weight 120-140 g) were maintained on a regular rodent diet and water ad libitum at Sri Krishnadevaraya University vivarium. After adaptation for seven days, animals were divided into four groups (n=8).
1. Group I- Controls
2. Group II- 20% alcohol-treated
3. Group III - PSE-treated and
4. Group IV - 20% alcohol and PSE-treated.
Alcohol (20%) was administered at a dose of 5g/kg body weight per day, and PSE was administered at a dose of 250 mg/kg body weight per day. Iso-caloric glucose was administered to control rats. Alcohol and PSE were administered using an oral gavage feeding needle for 60 days. All animal studies were conducted following Institutional regulations with approved protocol from Sri Krishnadevaraya University institutional animal ethics committee (471/06/c/CPCSEA, dt.20th Sep 2016). After two months, the animals were fasted overnight and euthanized by cervical dislocation to collect blood (cardiac puncture) and tissues including the brain. Cortex was separated from brain and used immediately for analysis.
2.4.Measurement of TBARS, protein carbonyls, and NADPH oxidase activity:
Cortex was minced in tris-buffer (0.1M, pH 7.4, 10% w/v), centrifuged for twenty minutes (10,000xg for at 4ºC). The supernatant was separated and used for all the biochemical parameters. Thiobarbituric acid reactive substances (TBARS) levels were determined by measuring the formation of malondialdehyde as published earlier (Padmavathi et al. 2018). Protein carbonyls content was determined using a 2,4-dinitrophenylhydrazine (DNPH) assay (Reddy et al. 2011) NADPH oxidase activity levels were determined using cytochrome-c reduction assay (Yuan et al. 2013). The reaction mix was made in modified HEPES buffer (25mM, pH 7) and consisted of NADPH (100 mM), cytochome-C (150 mM) and membrane protein (100mg) with/without superoxide dismutase (200 units/mL). The activity (reduction of cytochome-c) was measured using a microplate reader (550nm). The activity levels of NADPH oxidase was determined with extinction coefficient of 21 mmol/ (L-cm) and was presented in nmol/min/mg protein.
Measurement of CYP-450 activity
CYP-450 enzyme activity was determined as described earlier (Reddy et al. 2014). The CYP-450 content was measured using the extinction coefficient difference (∆E450–490nm of 91 cm−1mM−1).
Measurement of T3, T4, TSH, Cortisol, Testosterone, and Estradiol Levels:
Concentrations of thyroid hormones (T3, T4, and TSH), testosterone, estradiol, and cortisol in plasma samples of alcohol administered rats and control rats were performed using chemiluminescence based immunoassay system (Advia Centaur TM, Bayer Health Care Diagnostika, Vienna, Austria) (Padmavathi et al. 2015).
Histopathology:
Dissected brain tissue was fixed immediately in 10% neutral buffered formalin solution followed by embedding in paraffin. Five µm thickness sections were cut and conducted histopathological studies following standard methods. Hematoxilin and eosin staining were done to observe pathological changes.
Assay of non-enzymic and enzymic antioxidant status:
Total reduced glutathione content was determined using Ellman’s reagent. Enzyme activities of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione-s-transferase (GST) and glutathione peroxidase (GPX) were measured as described before (Bulle et al. 2016a). Protein concentration was estimated by the method of (Lowry et. a 1951).
Measurement of mitochondrial complex activities
Complex-I (NADH-dehydrogenase), complex-II (succinate-dehydrogenase), complex-III (Cytochrome-bc1-complex/ubiquinol-ferricytochrome-c-oxidoreductase) and complex-IV (cytochrome-c-oxidase) activities were determined following published protocols (Remsberg et al. 2008, Fressco et al.2006, Faustino et al. 2019, Tiwari et al.2013).
Immunoblotting analysis
Brain tissue samples were cleaned, washed and grinded followed by sonication in RIPA lysis buffer with phosphatase inhibitors and protease inhibitors. The lysates were centrifuged at 14,000 rpm for 15min at 4oC to collect the supernatant. Laemmli buffer was added to the lysates and electrophoresis (8%). The resolved proteins/lysates were PVDF membranes. Blocking of the membranes was done using 3% BSA followed by incubating with primary antibody (1:1000 dilution, overnight). Anti-total protein kinase C (Cat # 2056), anti-phospho PLCγ1(Tyr783) (Cat # 2821), anti-total PLCγ1 (Cat # 2822) and anti-pan PKCγThr514 (Cat # 9379) were purchased from Cell signalling Technology, USA. Anti-NOX 2 antibody (Cat # sc-74514, Santa Cruz Biotechnology, USA) and anti-α-tubulin (Cat # T-5168) were also used. Later blots were incubated with HRP conjugated secondary antibodies (1:10,000 dilution) and developed using an ECL solution. The images were captured using Alpha Innotech Imager.
Statistical analysis:
Statistical analysis was conducted using Student "t" test and one-way ANOVA. A p value, <0.01 and <0.05 were considered as significant.