Reagents

Livogrit Vital (Internal batch # PRFT/CHIN/0622/0736) was sourced from Divya Pharmacy, India. Dulbecco’s Modified Eagle Medium (DMEM) (Cat# D2902), Silymarin (Cat# S0292), and Isoniazid (Cat# I3377) were obtained from Sigma-Aldrich, USA. Antibiotic-antimycotic solution (Cat# A002A), FBS (Cat# RM9955), and Alamar blue (Cat# TC235) were purchased from HiMedia, India. CellROX Green Reagent (Cat# C10444) was obtained from Invitrogen, USA. Reagent Z-DEVD-AMC (Cat# 13421) was purchased from AAT Bioquest, USA. Human ALT ELISA kit (Cat# ITLK02461) was obtained from G-Biosciences, USA. GGT detection kit (Cat# 121019) was obtained from Transasia bio-medicals ltd., India.

HPLC based phytometabolite characterization of Livogrit Vital

Quantification was performed by HPLC (Prominence-i LC-2030c 3D Plus, Shimadzu, Japan). Separation of phytometabolites was achieved using a Shimadzu shim pack (3 μm, 3.0 × 100 mm) column subjected to binary gradient elution. Two solvents were used for the analysis consisted of water containing 0.1% orthophosphoric acid in water adjusted to pH 2.5 by dimethylamine (solvent A) and acetonitrile (solvent B). Column temperature was kept 35 °C and flow was set at 0.5 ml/min during the analysis. Gradient programming of the solvent system for mobile phase B was set as 2% for 0 to 4 min, 2 to 10% from 4 to 8 min, 10 to 15% from 8 to 15 min, 15% from 15 to 20 min, 15 to 20% from 20 to 25 min, 20 to 35% from 25 to 35 min, 35 to 95% from 35 to 45 min, 95 to 2% from 45 to 46 min and 2% from 46 to 50 min with a flow rate of 0.5 ml/min. 5 µl of standard and test solution were injected and column temperature was maintain at 35 °C. Wavelengths were set 270 nm (for Gallic acid, Methyl gallate, Corilagin, Ellagic acid, Cinnamic acid and Boeravinone B), 325 nm (for Chlorogenic acid and Rutin). In case of vitamins the separation was achieved using a Shodex C18-4E (5 μm, 4.6 × 250 mm) column subjected to binary gradient elution. Two solvents were used for the analysis consisted of water adjusted to pH 2.5 by ortho-phosphoric acid (solvent A) and methanol (solvent B). Column temperature was kept 35 °C and flow was set at 1 ml/min during the analysis. Gradient programming of the solvent system for mobile phase B was set as 2% for 0 to 10 min, 2 to 20% from 10 to 20 min, 20 to 50% from 20 to 30 min, 50 to 2% from 30 to 31 min, and 2% from 31 to 35 min with a flow rate of 1 ml/min. 10 µl of test and standard solutions were injected. Wavelengths were set at 242 nm (for vitamin C), 283 nm (for vitamin B9) and 365 nm (for vitamin B12).

Cell culture and treatments

HepG2 cells were procured from the National Centre for Cell Science, India (local depository of ATCC). Cells were grown in DMEM supplemented with 10% FBS and 1% antibiotic and antimycotic solution and maintained at 37 °C and 5% CO2. All experiments were conducted within 4 passages post 70–80% confluency. Unless specified otherwise, all cells were pre-treated with LVV for 24 h and then co-treated with isoniazid and LVV for 24 h. The normal control group was denoted as untreated control (UC). Silymarin (10 µM) was used as a positive control [3].

Cell viability

HepG2 cells were seeded at a density of 10,000 cells/well in a 96-well plate. The cytosafety of LVV (10–100 µg/ml) and Silymarin (10 µM) was evaluated post 48 h of incubation by Alamar blue dye. Cytosafety of Isoniazid (20–100 mM) was assessed post 24 h of incubation. The recovery of cell viability in response to LVV (10–100 µg/ml) or Silymarin (10 µM) in isoniazid (60 mM) induced HepG2 cells was also detected. The IC50 shift assay was performed by induction with isoniazid (20, 40, 60, 80, and 100 mM) in presence of LVV (10–100 µg/ml) or Silymarin (10 µM). After incubation with Alamar blue the fluorescence read at Ex. 560/Em. 590 nm on infinite 200Pro (Tecan, Switzerland) plate reader. Isoniazid (60 mM) was used for further analysis in presence of LVV (10–100 µg/ml).

Evaluation of oxidative stress parameters

The intracellular ROS levels were detected using the fluorescent probe, CellROX Green Reagent (5 µM) as per the manufacturer’s instructions. GSH was measured by using, o-phthalaldehyde (OPT) as a fluorescent reagent. Twenty microliter samples and GSH standards were taken in flat black 96 well plate, followed by the addition of 180 µl phosphate buffer [100 mM; pH 8]. After that, 10 µl OPT (1 mg/ml) prepared in methanol was added to each well, and incubated in dark for 20 min at room temperature. The fluorescence was evaluated at Ex. 350/Em. 420 nm. Concentration of GSH was expressed as µM GSH/mg protein. Malondialdehyde (MDA) levels were assessed by Thiobarbituric Acid Reactive Substance (TBARS) method. Samples and MDA standards 50 µl were taken into 1.5 ml centrifuge tubes, and 500 µl TBA-TCA reagent containing 0.5% TBA and 20% TCA was added into it. The mixture was incubated at water bath for 1 h at 95 °C. Samples were cooled at room temperature and absorbance was measured at 560 nm. Concentration of MDA was expressed as µM MDA/mg protein.

Gene expression analysis

The mRNA expression analysis was performed as previously described [22]. Briefly, the total RNA was extracted using RNeasy mini kit (Cat# 74104, Qiagen, Germany) and the cDNA synthesis was done using Verso cDNA synthesis kit (Cat# AB1453A, Thermo Scientific, USA). The cDNA samples were mixed with PowerUp SYBR Green Master Mix (Cat# A25742, Applied Biosystems, USA) and RT-PCR was performed using qTOWER3 G (Analytik-Jena, Germany). Data were analysed by using fold changes in relative mRNA expression (2−ΔΔCt) as compared to control using housekeeping gene β-actin. Description of primers used is mentioned in Supplementary material 1.

Assessment of caspase-3 activity

Z-DEVD-AMC, a fluorogenic substrate for caspase-3 was used for estimation of caspase-3 activity. A 2× caspase substrate assay solution (50 µM) was prepared as per manufacturer’s protocol and mixed with sample in equal amount into 96 black well plate. Post 2 h incubation fluorescence was taken at Ex. 341/Em. 441 nm by Tecan infinite 200Pro (Tecan, Switzerland) plate reader and the values were normalized with respective protein concentration.

Protein quantification by western blot

After treatment cells were lysed with cold RIPA buffer (100 mM Tris, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA 0.5% sodium deoxycholate, 1% Triton X-100) supplemented with protease inhibitor (Cat# A32963, Thermo Fisher Scientific). The lysates containing 20–30 µg proteins were resolved in 10% SDS-PAGE followed by transfer to a PVDF membrane (Cat# 1620177, Bio-Rad, USA). The primary antibodies used for the analysis were cleaved PARP1 (Cat# 44-698G, Invitrogen, USA), β-actin (Cat# MA5-11869, Invitrogen, USA). The incubation time, antibody concentration, and temperature conditions, were followed according to the manufacturer’s protocol. Protein bands were developed using Enhanced chemiluminescent HRP substrate (Millipore, USA) in an ImageQuant LAS 500 (GE Healthcare, USA) instrument, and blots were further processed and quantified using Image Quant TL version 8.2 software (GE Healthcare, USA) and calculated according to reference bands of β-actin.

Evaluation of isoniazid and hydrazine levels in HepG2 by HPLC

HepG2 cells (1 × 106/well) were pre-treated with LVV (30 and 100 µg/ml) for 24 h and then co-treated with isoniazid (60 mM) and LVV (30 and 100 µg/ml) for 24 h. The quantification of isoniazid and hydrazine in the lysates of treated cells was performed by HPLC (Prominence-i LC-2030c 3D Plus, Shimadzu, Japan). For isoniazid quantification separation was achieved using a Shodex C18-4E (5 μm, 4.6 × 250 mm) column subjected to gradient elution with a flow rate of 1.0 ml/min. The mobile phase solution A was prepared using 3.4 gm of sodium tri acetate dissolved into 900 ml of water and pH 6 was adjusted by orthophosphoric acid, volume make up to 1000 ml was done with water. Methanol was used as solution B. The column temperature was kept 30 °C. 50 µl of standard and test solution were injected and wavelength was set at 262 nm. The hydrazine quantification was performed using a Shodex C18-4E (5 μm, 4.6 × 250 mm) column subjected to gradient elution with a flow rate of 1 ml/min isocratic elution of mobile phase 0.1% orthophosphoric acid in water; adjusted to pH 2.5 by diethylamine and acetonitrile (15:85). Column temperature was kept 40 °C during the analysis. 50 µl of standard and test solution were injected during the analysis and chromatograph was recorded at 308 nm wavelength.

Assessment of ALT and GGT

HepG2 cells were pre-treated with LVV (30 and 100 µg/ml) for 24 h followed by 24 h co-treatment with isoniazid (60 mM) and LVV (30 and 100 µg/ml). Post treatment, cell supernatant was used for detection of the released ALT and GGT levels. ALT quantification was performed using Human ALT ELISA kit as per the manufacturer’s instructions. GGT levels were analysed in Erba 200 (Erba Mannheim, Germany) biochemical analyser using the GGT detection kit.

Data analysis

GraphPad Prism 8 (GraphPad software, USA) was utilized for statistical analysis. Data were presented as mean ± S.E.M. All the experiments were performed in triplicate. Significance of the difference between different treatment groups was determined by one-way ANOVA followed by Dunnett’s post-hoc analysis. Statistical significance was considered at values of p < 0.05. The statistical comparison between UC and isoniazid (60 mM) induced groups was represented as ## (p < 0.01) and ### (p < 0.001). Statistical comparison between isoniazid (60 mM) and LVV or Silymarin treatment was represented as * (p < 0.05), * *(p < 0.01), and *** (p < 0.001).