Collection of Zingiber officinale:
The rhizome of Zingiber officinale was procured in the month of March to April 2022, from the local market of Tiruvallur district, Tamil Nadu, India. The ginger was washed under running tap water and it was cut into small pieces and used for extraction process.
Soxhlet extraction and simple distillation:
In the order to get the crude extract of Zingiber officinale, the sample was subjected to solvent extraction in the Soxhlet apparatus using the ethanol as a solvent. Exactly 20 g Fresh sample of ginger was subjected to ethanol extraction using 250 ml of ethanol 85% as solvent at 78°C for 3 hours. Extract obtained after Soxhlet extraction was stored in airtight container in refrigerator at 4°C.After the time that is mentioned above, the crude extract was collected from round bottom flask and non-soluble macro particles were discarded, which remains in the thimble. The extract obtained at the end of Soxhlet extraction process was subjected to the simple distillation process in order to obtain peculiarly the phenolic compounds present in it. The temperature was set up to 85°C which the approx. boiling point of ethanol. Through this process, the ethanol present in the sample was collected into the separate flask. Then the bioactive compounds of the sample got settled at the bottom of the flask. This was stored for the phytochemical analysis process.
Phytochemical analysis of Zingiber officinale
Phytochemical analysis of ethanol extract of ginger was performed by following standard protocols which is mentioned below, to confirm the presence of phytochemical constituents such as phenols, alkaloids, flavonoids, glycosides etc using the standard qualitative analysis [9].
Test for alkaloids- Hager’s test: A few ml of ethanolic extract was treated with Hager’s reagent (saturated picric solution). A yellow-colored precipitate indicates the presence of alkaloids.
Test for flavanoids-Shinoda’s test: A 0.5 ml of ethanolic extract was treated with 5-10 drops of dil. hydrochloric acid followed by a small piece of magnesium. A pink, reddish pink or brown colour indicated the presence of Flavonoids.
Test for steroids-Salkowski test: A few ml of extract was taken in a test tube, three to four drops of concentrated sulphuric acid was added and mixed well. A red color Precipitate in the bottom indicated the presence of Steroids.
Test for phenols-Ferric chloride test: A few ml of extract was taken in a test tube, three to four drops of ferric chloride solution. Formation of bluish black colour indicated the presence of phenols.
Test for saponins- Foam test: About 5 ml of the extract was taken and diluted with 10ml of distilled water. The test tube was shaken for 15 minutes. Formation of foam on top layer showed the presence of saponins.
Test for glycosides-Borntrager’s test: For the detection of glycosides, about 50mg of extract was hydrolyzed with concentrated hydrochloric acid for 2 hours in a Water bath, filtered. About 2 ml of the filtrate was taken and about 3ml of the chloroform was added and after the vigorous shaking, chloroform layer was separated and to this 10% of ammonium solution was added. Appearance of white precipitate confirmed the presence of the glycosides.
Test for tannins- Lead acetate test: A few ml of extract was dissolved in distilled water and 3 ml of 10% of lead acetate solution was added. Appearance of white precipitate confirmed the presence of the tannins.
Gas Chromatography-Mass Spectrometry analysis
Volatile constituents of ethanolic extract of Zingiber officinale was analyzed in GC-MS in a Shimdzu GC-2010 plus gas chromatograph [10] was equipped with a straight deactivated 2mm direct injector liner and a 15m Alltech EC-5 column (250µ I.D., 0.25µ film thickness). A split injection was used for sample introduction and the split ratio was set to 10:1. The oven temperature program was programmed to start at 35℃, hold for 2 minutes, then ramp at 20℃ per minute to 450℃ and hold for 5 minutes. The helium carrier gas was set to 2 ml/minute flow rate (constant flow mode) respectively. A Direct connection with capillary column metal quadruple mass filter mass spectrometer operating in electron ionization (EI) mode with software GC-MS solution ver. 2.6 was used for all analyses. Low-resolution mass spectra were acquired at a resolving power of 1000 (20% height definition) and scanning from m/z 25 to m/z 1000 at 0.3 seconds per scan with a 0.2 second inter-scan delay. 26 on the High-resolution mass spectra were acquired at a resolving power of 5000 (20% height definition) and scanning the magnet from m/z 65 to m/z 1000 at 1 second per scan. Compounds are identified based on the retention time and highest peaks in the chromatogram.
Anticancer activity assay
Preparation of cell suspension:
A subculture of A431 cells in Dulbecco’s Modified Eagle’s Medium (DMEM) was trypsinized separately, after discarding the culture medium. To the disaggregated cells in the flask 25 mL of DMEM with 10% FCS (Fetal calf serum) was added. The cells suspended in the medium by gentle passage and the cells were homogenized.
Seeding of cells:
One mL of the homogenized cell suspension was added to each well of a 24 well culture plate along with different concentration of samples (0 to 100 g/ml) and incubated the cells were observed under an inverted tissue culture microscope. With 80% confluence of cells cytotoxicity assay was carried out.
Cytotoxicity assay:
The assay was carried out using (3-(4,5-dimethyl thiazol-2yl)-2,5- diphenyl tetrazolium bromide (MTT). MTT is cleaved by mitochondrial Succinate dehydrogenase and reductase of viable cells, yielding a measurable purple product formazan. This formazan production is directly proportional to the viable cell number and inversely proportional to the degree of cytotoxicity. After 48 h incubation the wells were added with MTT and left for 3h in room temperature. All wells were removed the content using pipette and 100µl SDS in DMSO were added to dissolve the formazan crystals, absorbance’s were read in Read Well touch microplate reader at 570 nm.
In silico studies:
Target selection:
Target selection involves identification of molecular protein target that are involved in disease progression. The receptor molecule DDX3X of Homo sapiens is selected as the target protein and the 3D structure was retrieved from PDB database [11]. The PDB id of the target protein structure is 4PXA.
Retrieval of lead structure:
The structure of [6]-gingerol compound identified from the GC-MS was retrieved from PUBCHEM database [12]. Also 21 other structural analogs were selected which resembles the [6]-gingerol structure from the PUBCHEM database as lead to check the molecular interactions with the target DDX3X of Homo sapiens.
Docking:
AUTODOCK Vina [13] is an automated tool used to study the interactions of protein binding receptor with the lead. The docking has been performed between the DDX3X target and thoroughly 22 compounds together with [6]-Gingerol and its similar compounds obtained from PUBCHEM database.
ADME profiling:
ADME (Absorption, Distribution, Metabolism and Excretion) analysis designed to check the drug likeliness property of the compounds based on Lipinski’s drug filter. ADMET profiling of compounds were determined using SWISS ADMET web server [14].