Materials
Aluminium chloride, ethyl acetate, chloroform, ethanol and methanol were purchased from Lobachemie. Butylatedhydroxytoluene (BHT), Trolox, gallic acid and quercetin were purchased from Sigma-Aldrich. 2,2-Diphenyl-1-picrylhydrazyl (DPPH), molybdate reagent, dimethyl sulfoxide (DMSO), Dulbecco’s modified Eagle’s medium (DMEM), actinomycotic 100X solution and ascorbic acid were purchased from Thermo Fisher Scientific.
Collection and authentication of the plant part
The plant C. zeylanica was collected from the rural area in Kolhapur district, Maharashtra, India. The plant was identified and authenticated (MB/RW/02/013) by Dr. M. Y. Bachulkar, Ex-Principal and Taxonomist, Department of Botany, Shri Vijaysinha Yadav Arts and Science College, Kolhapur, Maharashtra. A fresh leaves were collected, washed and dried in shade for 2–3 weeks. Dried leaves were blended to fine powder and stored in airtight container and used for further studies.
Preparation of crude extract
A 50 g of powdered crude drug was subjected to extraction by maceration method using ethyl acetate, chloroform, ethanol, methanol and aqueous solvents for 6–8 days to extract soluble compounds [12, 13]. Filtered the liquid using filter paper and concentrated the extracts. Concentrated extracts were collected and stored at 4 °C until further use.
Phytochemical analysis
The preliminary phytochemical evaluation of the extracts was done using different biochemical tests as per reported method [14]. Further, ethanolic and methanolic extracts of C. zeylanica were selected for further studies based on the qualitative analysis from five extracts.
Estimation of total phenolic content
The total phenolic content of C. zeylanica was estimated by the Folin-Ciocalteu reagent method [15] with slight modification. Briefly, 200 μl of methanolic and ethanolic extracts were added in equal volume of Folin-Ciocalteu reagent (tenfold diluted) and incubated for 10 min. Then, 1.25 mL of aqueous sodium carbonate was added to neutralize the mixture, incubated for 45 min at 37 °C with intermittent shaking to generate colour. Distilled water was used as a blank. The extracts were analysed for total phenolic content by measuring the blue colour at 765 nm using UV-Vis spectrophotometer (UV-2700i/2600i, Shimdzu, Japan) in triplicate. Gallic acid was used as a reference standard for calibrating the method of analysis and to construct the calibration curve. The total phenolic content of the extract was calculated from the linear equation of calibration curve of gallic acid, in terms of milligram per gram of gallic acid equivalent (GAE) of dry extract.
Estimation of total flavonoid content
The total flavonoid content of C. zeylanica was estimated as per the procedure reported [16]. Briefly, 0.5 mL of methanolic and ethanolic extracts were mixed with equal volume of 2% AlCl3 and incubated at room temperature for about 1 h. Evaluation of extract samples was carried out at a final concentration of 1 mg/mL. Quercetin was used to construct the calibration curve, and the total flavonoid content was calculated as milligram per gram quercetin equivalent (QE). The results were carried out in triplicate by measuring absorbance at 420 nm. The procedure was repeated as above for both the extracts.
Determination of total antioxidant capacity (TAC)
The total antioxidant capacity of C. zeylanica leaf extracts was determined by the phosphomolybdenum assay using the method described by an earlier report [17]. Briefly, 1.0 mL of different concentrations of methanolic and ethanolic extracts (100 to 500 μg/mL) was mixed with 3.0 mL phosphomolybdenum reagent containing 28 mM sodium phosphate and 4 mM ammonium molybdate in 0.6 M sulphuric acid in capped test tubes. Distilled water was added to make the final volume to 5 mL. The test tubes containing mixture was incubated for 90 min in a water bath set at 95 °C in dark. After incubation, the mixture was cooled at room temperature and absorbance of resulting solution was measured in triplicate at 695 nm against ethanol and methanol as blank using UV-visible spectrophotometer. The TAC results were expressed as Trolox equivalents (mg TE/g of dry extract). Butylatedhydroxytoluene was used as reference controls.
DPPH scavenging assay
The free radical scavenging activity of C. zeylanica leaf extract was estimated by the DPPH as per earlier reports [18], with slight modifications. One milliliter of different concentrations of methanolic and ethanolic extracts ranging from 100 to 500 μg/mL was added in each test tube containing 2 mL 1.0 mmol/L DPPH solution. Ascorbic acid solution was prepared separately with the same procedure as a standard. All test tubes were incubated at room temperature for 30 min in dark. The absorbance was measured at 517 nm. The lower the absorbance of reaction mixture, the higher is the free radical scavenging activity. The percentage free radical scavenging activity was calculated using the following equation:
$$ \mathrm{DPPH}\ \mathrm{scavenging}\ \mathrm{activity}\ \left(\%\right)=\frac{\ {A}_c-{A}_t}{\ {A}_c}\times 100 $$
(1)
where Ac is the absorbance of the control (1 mL of ethanol with 1 mL of DPPH solution) and At is the absorbance of the test sample. The results were analysed in triplicate.
Cytotoxicity study
Human breast cancer cell line, MCF-7, and human breast cell line, MCF10A, were procured from the National Centre for Cell Science, Pune, India. The cells were subcultured in Dulbecco’s modified Eagle medium (DMEM) with low glucose having phenol red supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin solution to minimize the microbial contamination. The cells were seeded at a density of 5 × 103 cells/well in a 96-well flat bottom microplate and maintained at 37 °C in 5% CO2 incubator with 95% humidity overnight prior to the experiment. The cells were treated with different concentrations (500, 250, 125, 62.5, 32.75 μg/mL) of methanolic and ethanolic extracts by serial dilution method. Initially, cells were diluted with serum-free medium to achieve twice the desired maximum test concentration. The cells were incubated for another 48 h, and cytotoxicity was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay [19, 20]. MTT assay was initiated with 5 × 103 cells/well, 20 μL of the MTT staining solution (5 mg/mL) in phosphate buffer solution was added to each well and plate was incubated at 37 °C for 4 h. After incubation, the medium was removed and subsequently added with 100 μL DMSO in each well to dissolve the formed formazan crystals. The absorbance was measured at 570 nm using micro plate reader (BioRad, India) in triplicate [21, 22]. The percentage cell inhibition was calculated using equation as.
$$ \mathrm{Surviving}\ \mathrm{cells}\ \left(\%\right)=\frac{\mathrm{Mean}\ \mathrm{OD}\ \mathrm{of}\ \mathrm{the}\ \mathrm{test}\ \mathrm{extract}}{\mathrm{Mean}\ \mathrm{OD}\ \mathrm{of}\ \mathrm{negative}\ \mathrm{control}}\times 100 $$
(2)
$$ \mathrm{Inhibiting}\ \mathrm{cells}\ \left(\%\right)=100-\mathrm{Surviving}\ \mathrm{cells} $$
(3)
The inhibitory concentration (IC50) value for the extracts was determined based on the concentration of extract inhibiting the cells by 50% of its initial cell population.
Molecular docking studies
The molecular docking studies were performed to determine the chemical interactions of the phytoconstituents of plant C. zeylanica Linn. with targeted protein responsible for breast cancer. Molecular docking was initiated by drawing 3D structures of phytoconstituents, considered as ligands and prepared, energy minimized. The target protein HER2 was downloaded from RCSB (PDB id: 1N8Z). The protein was prepared and refined using MMCF-2 forcefield. All the heteroatoms were removed from crystal structure of protein to make all the complex receptors free of ligand before docking using PyMOL-2.3.4 software. AutoDock Tools (version-4.2.6) software was used for the preparation and optimization of protein and ligand molecules. Water molecules were removed, and polar hydrogens and Kollman charges were added in the protein molecule. Since ligands were small molecules, Gasteiger charge was added. The binding site of the protein was selected by selecting a grid box with dimensions size (X = 8.820, Y = 91.432, Z = 127.425) and docked using AutoDock vina (version 1.1.2) [23]. All the AutoDock vina docking runs were performed in Intel Centrino Core2Duo CPU @ 2.20 GHz of IBM system origin, with 4 GB DDR2 RAM. AutoDock vina was compiled and run under Microsoft Windows 10 operating system. The results were analysed on the basis of binding affinity of ligand with the protein and refined mean standard deviation. Images of protein-ligand binding confirmations were visualized and processed using Discovery studio visualizer 2020 software. 2D structures of constituents were drawn using Chemdraw Professional 16.0.
Statistical analysis
The data were expressed as mean ± standard deviation of results obtained from three independent experiments. P ≤ 0.05 was considered significant for all the represented results. All the graphs were plotted using OriginPro 9.1 software.
