Determination of total flavonoid content
The basic principle behind the use of the aluminium chloride colorimetric method is that AlCl3 forms acid-stable complexes with the C4 keto group and either the C3 or C5 hydroxyl group of flavones or flavonols, resulting in a yellow colour which can then be quantified using a spectrophotometer at an absorbance range of 410–437 nm [17].
One millimetre of 2% aluminium chloride was added to 1 mL of varying concentrations of rutin (0.25–1.0 mg/mL)/ plant extracts (5 mg/mL) and incubated at room temperature for 10 min. As a blank solution, 1 mL of 2% aluminium chloride was added to 1 mL of distilled water. The flavonoid content of the plant extract was spectrophotometrically quantified against a rutin standard curve at 430 nm. The result was then expressed as mg RE /g dry weight of the extract [17].
Determination of total polyphenols
The Folin–Ciocalteu assay works on the principle of reducing the Folin–Ciocalteu reagent (FCR) in the presence of phenolics to produce molybdenum–tungsten blue, which can then be measured spectrophotometrically at 740 nm. The colour intensity rises in direct proportion to the amount of phenolics in the reaction medium. An oxidation/reduction reaction is the basic mechanism. Sodium carbonate was used to adjust the pH, and an incubation time of 120 min at room temperature is required for the reaction to be completed [18].
One millilitre of the plant extracts (0.5 mg/mL)/standard (0.25–1 mg/mL) to 5 mL of diluted Folin–Ciocalteu reagent (10:1) was added, and ~ 5 min later 4 mL of sodium carbonate (7.5%) was then added in the mixture. The test tubes were then thoroughly vortexed mixed and incubated at room temperature in the dark for two hours. The blue complex formed was then spectrophotometrically quantified against a gallic acid standard curve at an absorbance of 740 nm [18]. Results were expressed as mg GAE/g dry weight of the extract.
In vitro free radical scavenging assay
DPPH radical scavenging activity
Two millilitres of DPPH solution (0.004% in methanol) was mixed with 1 mL of varying concentrations of plant extract (– 0.1-1 mg/mL)/standard ascorbic acid (0.02 - 0.1 mg/mL). The solution mixture was incubated for 30 min at room temperature and allowed to stand in a dark environment. Two millilitres of DPPH added to 1 mL of methanol was used as a control. The absorbance was spectrophotometrically estimated at 517 nm against a blank according to the method of Brand-Williams [19] with slight modification.
$$\% {\text{Inhibition}} = {\text{Abs}}\left( {{\text{control}}} \right){-}{\text{Abs}}\left( {{\text{Sample}}} \right)/{\text{Abs}}\left( {{\text{control}}} \right) \times 100$$
ABTS radical scavenging activity
Two millilitres of ABTS radical cation working solution was allowed to react with 1 mL of varying concentrations of standard ascorbic (0.02- 0.1 mg/mL) or plant extract (0.1- 0.5 mg/mL) and incubated at room temperature in the dark for 4 min. The control was prepared by adding 2 mL of ABTS radical cation working solution to 1 mL of distilled water. The absorbance was spectrophotometrically quantified at 734 nm against a blank according to the proposed method of Re [20] with slight modification.
$$\% {\text{Inhibition}} = {\text{Abs}}\left( {{\text{control}}} \right){-}{\text{Abs}}\left( {{\text{Sample}}} \right)/{\text{Abs}}\left( {{\text{control}}} \right) \times 100$$
HPTLC profiling analysis
Sample preparation and application
1 mg/mL of standard reference compound and 10 mg/mL of ethanolic crude extract were prepared in methanol of chromatographic grade and then filtered through Whatman filter paper No. 1. Prepared extract/standard was applied on a TLC aluminium sheets silica gel 60 F 254 (Merck). One microliter of standard reference in the order of ascorbic acid, caffeine, gallic acid, pyrogallol, quercetin, rutin, and 10 μL of plant crude extract were applied to a 200 × 100 mm plate, each with a band length of 8.0 mm using a Linomat 5 sample applicator set at a dosage speed of 150 nL/s.
Solvent system development
For the separation of compounds in the extract, a variety of solvent system combinations were tried, but a satisfactory resolution and a maximum number of spots were obtained in the following solvent system comprising of 2-butanol/1-propanol/water in the ratio of 3:1:1.
Development of the chromatogram
The plates were developed in a twin through glass chamber of 20 × 10 cm and saturated with the above solvent system combination for 20 min at room temperature up to a distance of 80 mm. The plate was then allowed to dry at room temperature for 5 min. The Rf values and colour of the resolved bands were noted.
Scanning and detection of spots
The air-dried plates were visualized using CAMAG® TLC Visualizer in the ultraviolet (254 nm and 366 nm) and white light. Spots were visible without derivatization at 254 nm (Deuterium lamp, absorption mode, filter K320) and 366 nm (Mercury lamp, fluorescence mode, filter K320) wavelengths, but the best results were observed when the TLC plates were derivatized. Anisaldehyde sulphuric acid (derivatizing agent) was sprayed, and the plates were heated at 100 °C using CAMAG® TLC Plate Heater 3.
Spectrum scanning of the developed plate was performed on a CAMAG® TLC Scanner 4, using a deuterium lamp at 254 nm wavelength and a spectrum speed of 100 nm/s. Baseline correction of the lowest slope with noise of 0.05, peak detection-Gauss (legacy) with a sensitivity of 0.1, a threshold of 0.1, and separation of 1 was used. The chromatogram peaks with their Rf value were noted.
GCMS profiling analysis
Chemical profiling analysis to find out the bioactive compound present in the freeze-dried ethanolic extract of K. galanga (rhizome) was performed using GCMS analysis. The study was performed on the Clarus 680 Gas chromatography/Clarus 600 Mass Spectrometer, PerkinElmer Turbo Mass Spectrophotometer (USA) model (GC having a Liquid Autosampler). PerkinElmer Elite Elite-35MS capillary column with a length of 60 m and an internal diameter of 0.2 mm was used. An electron impact technique of 5.0 mV was used. A carrier gas of pure helium gas (99.99%) with a flow rate of 1 mL/min was used. At 280 °C and 0 °C, respectively, the temperatures of the injector and detector were preserved. The oven temperature was initially set at 60 °C with an initial holding time of 1.0 min, at an equilibration time of 2.0 min, and then gradually increased to 350 °C (maximum) with ramp rate 1 of 7.0 °C/min to 200 °C and a holding time of 3.0 min, and ramp rate 2 of 10.0 °C/min to 300 °C and a holding time of 5 min. 1.5 μL of the plant extract (diluted in 1% methanol) was injected (normal velocity) and a split injection technique was used (10:1:1 split ratio). The analysis was conducted and permitted to run for 40 min with a sampling rate of 1.5625 pts/s. Data were assessed for compound identification and quantification using a total ion chromatogram (TIC). The identification of the compound was based on the comparison of their retention indexes (RI) and retention time and also verified by comparison with the library spectra of the National Institute of Standard and Technology (NIST) and published literature data.