Chemicals
The analytical grade chemicals were purchased from Hi-Media and Merck, India. Standard drugs were purchased from Sigma-Aldrich, India. 2,4,6-tris(2-pyridyl)-s-triazine TPTZ and MTT reagent were procured from Merck, India. Dulbecco's Modified Eagle's Medium (DMEM), fetal bovine serum (FBS) and penicillin (5000 U/mL), streptomycin (2500 U/mL) from Gibco (USA).
Sample collection
Seeds of A. precatorius were collected from the Khari Baoli, Kucha Challan, Chandni Chowk, Delhi complies with relevant institutional, national, and international guidelines and legislation. Voucher specimens were deposited at Raw Material Herbarium and Museum, Delhi (RHMD), India and were authenticated by Dr. Sunita Garg, (Emeritus Scientist, CSIR-NISCAIR), with Ref No: NISCAIR/RHMD/Consult/2020/3697-98-2.
Genomic DNA isolation and molecular identification
Genomic DNA was isolated from seeds of A. precatorius using Cetyltrimethylammonium bromide (CTAB) method 26. The genomic DNA was amplified using rbcLa-forward primer (ATGTCACCACAAACAGAGACTAAAGC) and rbcLa- reverse primer (GTAAAATCAAGTCCACCRCG). PCR amplification was carried out in Veriti model of Applied Biosystem Thermo cycler 27.
Cell culture
Human cervix carcinoma Hep2C cell line was obtained from National Centre for Cell Sciences (NCCS), Pune, India. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) FBS, 100 U/ml penicillin, and 100 μg/mL streptomycin at 37°C in a humidified atmosphere of 5% CO2.
Preparation of Extracts
Seeds were washed with distilled water to remove dirt and soil particles, followed by drying and grinding to form powder and used throughout the study.
1. Soxhlet Extraction Method
Alcoholic extracts were prepared by soxhlet method using ethyl acetate and 70% ethanol as solvent. 10g of seed powder was placed inside a thimble made from thick filter paper and loaded into the soxhlet extractor. The Soxhlet extractor was placed onto the flask containing the solvent (500 mL) equipped with a condenser. The extractor was then allowed to heat to reflux for 16 h at 70 °C. Extracts were filtered twice through a Whatman No.1 paper filter and concentrated to the dry mass with the aid of rotary evaporator 28.
2. Maceration Extraction Method:
10g of seed powder was dissolved in 100ml of solvent (Ethyl acetate and 70% Ethanol) and then kept in the dark for 7 days at room temperature. The conical flasks of the extract were covered with cotton plugs to avoid the evaporation. After 7 days of incubation, they were filtered with muslin cloth followed by Whatman No.1 filter paper and concentrated to the dry mass with the aid of rotary evaporator 29.
The dried extracts were dissolved in absolute dimethyl sulfoxide (DMSO) as 50 mg/mL and diluted with phosphate-buffered saline (PBS, pH 7.4) to give final concentrations.
Quantification of Phytochemical Constituents
Total flavonoid content was measured by Aluminium Chloride Spectrophotometric method. Absorbance was measured against the prepared blank at 510 nm and results were represented as quercetin equivalents (mg QE)/g of extract. Similarly, total phenolic content and total tannins content was quantified by Folin-Ciocalteau method. Absorbance of mixture was measured at 725 nm. Final results were represented as quercetin equivalents (mg QE)/g of extract and tannic Acid equivalents (mg QE)/g of extract, respectively 28,30,31. All the concentrations were calculated using a standard calibration plot.
Antioxidant Assay
Antioxidant potential in the seed extracts was determined by electron transfer assay i.e. (2,2-Diphenyl-1-picrylhydrazyl) Radical scavenging assay (DPPH) and Ferric reducing antioxidant power (FRAP) assay. DPPH free radical scavenging assay was performed to measure the hydrogen donating or radical scavenging ability. Briefly, 0.04mM DPPH radical solution was prepared in methanol and then 900 μL of this solution was mixed with 100 μL of extract solution containing different concentrations of seed extracts. The absorbance was measured at 517 nm after 30 min of incubation. Methanol (95%), DPPH solution and were used as blank, and control respectively. Quercetin was used as the standard.50% inhibitory concentrations (IC50 values) of the extracts were calculated from graph as concentration versus percentage inhibition. Radical scavenging activity was expressed as percentage of inhibition. Measurements were taken in triplicate. The IC50 of the extract and standards were determined graphically 32.
The percentage of inhibition was calculated by using the formula:
Percentage of inhibition = [(absorbance of control- absorbance of reaction mixture)/absorbance of control] ×100
Further for the FRAP assay, FRAP reagent solution was prepared with 300 mM sodium acetate buffer (pH 3.6), 10 mM 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ) in 40 mM HCl and 20 mM FeCl3·6H2O (10:1:1 v/v/v). The absorbance was measured at 593 nm after a 30 min incubation at room temperature against 50% ethanol as blank. A calibration curve was prepared using FeSO4x 7H2O. FRAP activity was expressed as mM Fe (II)/ g dry weight of extract 28,33,34.
High Pressure Liquid Chromatography
The presence of phenolic compounds and alkaloids in the prepared extracts was screened against standards rutin, tannic acid and piperine by HPLC. The analysis was performed using a C-18 reversed phase column (Phenomenex, Gemini 5 μ, 250 mm length × 4.6 mm internal diameter). The mobile phase consisted of methanol: 0.1% orthophosphoric acid (77:23) for quercetin, methanol: water (50:50) for tannic acid and 1% acetic Acid: acetonitrile (52:48) for piperine were chosen for the separation at a constant flow rate of 1 mL/min. The column temperature was set to 38°C and the injection volume was 20 μL. The wavelengths were set to 370 nm for the detection of rutin, 280 nm for tannic acid, and 343nm for piperine. Retention time was observed by using HPLC 28,35.
Antiproliferative activity
Cytotoxic activity of A. precatorius seed extracts on Hep2C cells were determined by using a modified 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium (MTT) assay. Briefly, Hep2c cells were seeded (~1×104 cells/well) onto flat-bottomed 96-well culture plates. Further, different concentrations of seed extracts and doxorubicin (standard)were added to each well. After 48 h of incubation 10 μl of MTT reagent (5 mg/ml) were added and mixtures were re-incubated for 4 h. The resulting formazan was solubilized with DMSO (100 μl). Finally, the absorbance of formazan was measured at 570 nm using an automated microplate reader (Bio-Rad, Illinois, USA). Experiments were carried out in triplicates. The cytotoxicity was recorded as concentration causing 50% growth inhibition (IC50) for cell line 36.
The percent inhibition was calculated by using the following formula:
%Inhibition = 100 – (mean OD of test compound−mean OD of negative control) / (mean OD of positive control − mean OD of negative control) ×100
Antioxidant Enzyme Activity Assay on Hep2C cells
To estimate the effect of A. precatorius seed extracts on Hep2C cells, the enzymatic activity [superoxide dismutase (SOD), catalase (CAT), Glutathione-S-Transferase (GST)] and non-enzyme content [Glutathione content (GSH) and lipid peroxidation (Malondialdehyde (MDA) content)] were evaluated. A total of 1.51 × 105 cells/well were seeded in 24 well plates and incubated for 24 h in a CO2 incubator at 37 °C followed by a 48 h treatment with the obtained IC50 specific values of each of these extracts. The cells were harvested by washing with PBS followed by trypsinization. Further, the samples were centrifuged at 10,000 rpm for 20 min at 4°C to obtain the cell lysate which was further used for estimation of enzyme and non-enzyme content in accordance with standardized procedures 37.
Molecular Docking and MM-GBSA refinement
The molecular docking was performed on cervical cancer targets using molecules found in HPLC. The X-ray crystallographic structures of targets were obtained from Protein Data Bank (https://www.rcsb.org/search) using PDB ID:3LMY (HEXB), 5MY6(4LSJ), and HER2 (5MY6). Molecular docking was performed using Schrödinger Maestro Suite 2020-3 (Schrödinger, LLC, New-York, NY) 38,39. All the structures were optimized prior to docking, any structural inconsistencies were rectified using Schrodinger’s in-built protein preparation wizard module. HEX-B and GCR both have co-crystallized ligands which were chosen to act as the center of the receptor grid. In case of HER2 active site prediction was made using SiteMap module, active sites with a score <0.9 were rejected. The internal grid size x*y*z was fixed 15*15*15 Å. Further, MM-GBSA refinements were carried out on the docked poses of each isoform with a flexible residue distance of 5.0 Å.
ADMET Analysis and Drug Score
The computational ADME/TOX analysis for identified compounds for 23 molecular descriptors were computed using ADMETLAB (http://admet.scbdd.com/) 40. Additional Descriptors like mutagenic and drug score were computed using Osiris Property Explorer 41.
Statistical analysis
The statistical data were represented as mean ± standard deviation. Statistical analysis was performed using one-way analysis of variance (ANOVA). A probability value of ≤ 0.05 was considered as statistically significant. All analyses were performed using IBM-SPSS statistical software (version 13.0).