Determination of phenolic content
Total phenolic content was determined using Folin-Ciocalteu’s reagent method. 1 mL of individual extract solution, at an appropriate dilution level, was mixed with 500 µL of Folin-Ciocalteu’s reagent and 7.5 mL of double-deionized water. To this, mL of 5% Na2CO3 (W/V) solution is further added whose resulting mixture was set to incubation at room temperature for a 90-minute period.
The absorbance is measured, and the total phenolic contents was given as μg gallic acid equivalents (GAEs) per mg of extract.
Total flavonoid content
Aluminum chloride method is used to determine total flavonoid content. Extract solutions, at 0.5 mL, is mixed with 1.5 mL of 95% ethanol (V/V), 0.1 mL of 10% aluminum chloride (m/V), 0.1 mL of 1 mol L–1 potassium acetate and 2.8 mL of water. The resulting mixture, like before, is set to incubation at room temperature for a 30-minute period. Absorbance is measured using a spectrophotometer and the total flavonoid content is given as µg quercetin equivalents (QEs) per mg of plant extract.
HPLC analysis of phenolic compounds
The hydrolysis technique for A. rutifolia leaf extracts follows the methods of this study with Mengkudu leaf extracts.
50g of the test sample extracts are dissolved in 24 mL of methanol to allow for homogenization. 16 mL of distilled water and 10 mL of 6M HCl are added. This followed thermostatic process for 2 hours at 95°C. Later, the solution is filtered and subsequently goes through high-performance liquid chromatography (HPLC) analysis.
The separation of plant samples on the gradient HPLC is done. Phenolic acid and flavonoid identities are established, as well as the limit of detection (LOD) and
limit of quantification (LOQ) are calculated.
Free radical scavenging activity (DPPH assay)
This is carried about as per the methods applied in the study exploring the antioxidant properties of flowers and roots of Pyrostegia venusta (Ker Gawl) Miers.
Extract solution is mixed with equal volumes of 100 µM DPPH solution in methanol whereby its resulting mixture is incubated for 15 minutes at room temperature. The absorbance is recording by using a UV–visible spectrophotometer at 517nm.
Ferric reducing–antioxidant power (FRAP) assay
Reducing power was determined following ferric reducing–antioxidant power (FRAP) assay. Detailed information on it by reading the antioxidant and antibacterial activity of leaves of Etlingera species (Zingiberaceae).
The absorbance of reaction mixtures, whose details and steps are better explained in the report, is measured via UV–vis spectrophotometer set to 700nm. Total oxidant activity is recorded as well as documenting total oxidant content as mg gallic acid equivalent/g of plant extract.
Antimicrobial activity
In this step, in order to be able to calculate the antimicrobial activity of A. rutifolia leaf extracts, the researchers used agar disc diffusion assay Inoculation of nutrient agar and potato dextrose took place. Afterwards, these were placed into sterilized petri plates. Dilutions of the extracts were made with biological-grade dimethyl sulfoxide (DMSO). Sterile filter discs impregnated with 50 µL of diluted plant extract solution are then placed into the aforementioned petri plates. These places undergo incubation at 37°C for 24-hour and at 27°C for 48-hour time period. Later, Antibacterial and antifungal activities are determined by using a zone reader.
Estimation of minimum inhibitory concentration (MIC) values
The calculation of the minimum inhibitory concentration (MIC) was carried out by following the methods laid out in the
broth microdilution method. After experimentation, the plates are incubated at 37°C for 24 hours for bacteria and 27°C for 48 hour for fungi. It is noted that the reason for the color change of indicator, resazurin, varies as per microbial growth.
