Chemicals

All the chemicals used in this research were of analytical grade and including 2,2-diphenyl-1-picrylhydrazyl (DPPH), butylated hydroxytoluene (BHT), butylated hydroxy anisole (BHA), ABTS, sodium nitroprusside, sodium carbonate, FRAP (ferric reducing antioxidant power), NBT (nitro blue tetrazolium), TBA (thiobarbituric acid assay) chloroform and ethanol were purchased from sigma (Sigma Aldrich GmbH, Steinheim, Germany) while Merck supplied the penicillin G (Quality level 200), streptomycin (Quality level 100) and doxorubicin.

Cell culture technique

The Human tumour cell line HePG2 was purchased from the National Centre for Cell Sciences (NCCS), Pune, India. Cells were maintained in DMEM supplemented with 2 mM l-glutamine and balanced salt solution (BSS) adjusted to contain 1.5 g/l Na2CO3, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 2 mM l-glutamine, 1.5 g/l glucose, 10 mM HEPES (4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid) and 10% fatal bovine serum (GIBCO, USA). Penicillin and streptomycin (100 IU/100 µg) were adjusted to 1 mL/l. The cells were maintained at 37 °C with 5% CO2 in a humidified CO2 incubator.

Microscopic studies

For microscopic examination, several parts of Oxalis corniculata were examined under magnifying lens by Labomed Lx-300 trinocular halogen microscope in KLE College of Pharmacy, Belagavi.

Physicochemical evaluation

Physicochemical parameters like percentage of loss on drying (LOD), total ash, acid insoluble ash and water-soluble ash were examined as per the guidance of Indian Pharmacopeia.

Fluorescence study

On a grease-free, clean microscopic slide, a totally powdered plant material was placed and 1–2 drops of reagent solutions (ethanol, 1 N HCL, 1 N H2SO4, 1 N NaOH, iodine and FeCl3) were added. After mixing well for 1–2 min, the slide was seen in the UV chamber using long (365 nm) and short (254 nm) ultraviolet radiations. The colours observed by application of different reagents in different radiations were reported [15].

Preparation of extract

The plant of Oxalis corniculata was collected from Kurani village of Hukkeri Tq (Dist. Belgaum, Karnataka). Authentication of plant is done from Shri B.M.K. Ayurveda Mahavidyalaya, Central Research Facility, (Shahpur, Belgaum). The samples were collected, cleaned, drained, cut and dried before being used. A crude extract was prepared by adding 750 g of powder in 2.5 l mixture of ethanol and water (70:30) and leaving the mixture for 7 days while stirring often. The extract was then filtered, added to a soxhlet apparatus with the same solvent system and left for 3 days for the extraction of a lipid from a solid material and extraction of compound has a limited solubility in solvent.

Preliminary phytochemical analysis

Preliminary phytochemical screening of the Oxalis corniculata plant extract for the determination of the presence of secondary metabolites like Flavonoids, Alkaloids, Saponins, Triterpenoids, Steroids, Tannins, Glycosides and Phenolics by standard methods [16].

Preparation of flavonoid fraction

Ethyl acetate was used for sequential liquid–liquid extraction on the hydroalcoholic extracts. The ethyl acetate fraction (Flavonoid Fraction/EAF) was then obtained by collecting and drying each fraction under reduced pressure.

In-silico anticancer test

The protein data bank-obtained molecule combines the native ligand and the binding pocket molecule. Native ligand molecule 4-anilinoquinazoline inhibitor and binding pocket molecule epidermal growth factor receptor tyrosine kinase domain with protein code 1M17. The coordinates for the epidermal growth factor receptor tyrosine kinase were downloaded from the RCSB protein data bank (PDB ID:1M17) [17, 18]. The protein preparation wizard (PPW) was used to process the protein structure, and the prime module of the Schrödinger suite was used to verify and alter its integrity by adding the missing residues. The protein was reduced by using the OPLS 2005 force field, and the pH range was set to 7.0. Finally, restricted minimization was followed until the non-hydrogen atoms average root mean square deviation (RMSD) converged to 0.30 Å [19].

A total of 23 molecules were created using Schrödinger's maestro molecule builder and then optimized using the OPLS 2005 force field in the LigPrep module at a pH of 7.4, ionizer was used to identify all the protomers and ionisation states for ligands.

The GLIDE (Grid-based Ligand Docking with Energetics) docking module of the Schrödinger suite was used to carry out molecular docking studies. The prepared ligands were docked into the generated receptor grid using glide XP docking precision. It utilizes an energy-based grid-based ligand docking approach to investigate for advantageous interactions between a ligand and a protein-like receptor molecule. Using the Schrödinger suite, the interactions of each complex were analyzed, and the 3D poses indicative of the molecular recognition interactions were obtained.

The pharmacokinetic properties like absorption, distribution, metabolism and excretion of Oxalis corniculata play an important role in drug development process. So, we have used physico-chemical and ADME properties, which were estimated using the Qikprop module of Schrödinger, assist in predicting both physico-chemical significant descriptors and pharmacokinetically important properties of the molecules.

In- vitro determination of antioxidant activity and free radical scavenging activities

The radical scavenging activity of the extracts was studied using DPPH free radical assay, ferrous ion chelating, nitric oxide scavenging activity, ABTS radical scavenging activity, and ferric‑reducing antioxidant power (FRAP). Deoxyribose assay was carried out according to the method proposed by Halliwell et al. [20]. Superoxide radical scavenging activity of the extracts (NBT method) was determined and Antilipid peroxidation activity (thiobarbituric acid [TBA] method) was carried out as described by Gupta et al. [21].

In-vitro determination of anticancer activityEvaluation of cytotoxicity

Determination of the inhibitory concentration (IC50) value using an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay. Cultured cells (1 × 105) were seeded in a 96-well plate and incubated for 48 h at 37 °C at 5% CO2 incubator. After 48 h, the monolayer was washed with medium and 100 µL of different test concentrations (10, 20, 30, 40 and 50 µg/ml) of samples were added on to the monolayer and the cells were further incubated at the same conditions. Removed the cultured medium and 100 µl of the MTT solution was added to each well and incubated at 37 °C for 4 h. After the supernatant has been removed, 100 µL of DMSO was added to each of the wells and incubated for 10 min to dissolve the crystals of formazan Measurement of the optical density at 590 nm. The percentage growth inhibition was calculated and results were expressed as IC50 values using dose response curve. [22].

Statistical analysis

All the estimation was carried out in triplicates. The data obtained in this study were analyzed using relative standard deviation method and the results were expressed as mean ± SD.