## 2 Materials and Methods
### Xanthine Oxidase Inhibitory Activity
#### 2.12.5 Calculation of MIC and MBC  
To determine the minimum bactericidal concentration (MBC) and minimum inhibitory concentration (MIC) of _C. axillaris_ extracts against the eight pathogenic bacteria, a widely accepted two-fold serial broth microdilution method was selected. For each extract, a total of eleven small-sized test tubes were sterilized and clearly labeled. Out of these, nine tubes were designated for diluted extract solutions, one for the blank control, and one for the negative control. Using a micropipette, 1.5 mL of sterile MHA (Mueller-Hinton Agar) broth media was transferred into each tube. To create nine different diluted concentrations of the extract solution, nine sterile test tubes were appropriately labeled. A stock solution was prepared in the first test tube, consisting of 2 mL of the extract (200 mg extract per mL) mixed with DMSO. Subsequently, serial half dilutions were performed using a diluent composed of an equal ratio mixture of DMSO and water. This process resulted in the generation of nine different dilutions, ranging from 200 mg/mL to 0.78125 mg/mL. Next, 500 \(\upmu\)L of the plant extract solution was meticulously added to the respective broth media tubes, resulting in the creation of nine distinct plant extract-broth media solutions with concentrations ranging from 50 mg/mL to 0.1953125 mg/mL (four-fold dilution). Additionally, each vial was inoculated with a bacterial inoculum containing approximately 1 X 105 CFU/mL. Prior to transfer into the test tubes, the turbidity of each bacterial inoculum was adjusted to match the 0.5 McFarland standards. The tenth tube received 500 \(\upmu\)L of diluent and served as a blank control to verify the sterility of the media. Likewise, the eleventh tube was solely inoculated with the bacterial inoculum and used as a negative control to ensure the suitability of the broth media for bacterial growth. Subsequently, all the samples were placed in an incubator set at 37 \({}^{\circ}\)C for a duration of 24 h. After this incubation period, the minimum inhibitory concentration (MIC) value of both extracts against the eight different bacteria was determined. The MIC was identified as the lowest concentration of the plant extract solution that completely inhibited bacterial growth in the respective test tube. To assess the viability of bacterial cells, 3(4,5 dimethylthiazol-2-yl)-2-5-diphenyl tetrazolium bromide (MTT)was utilized. The samples were incubated for an additional 2 h at 37 \({}^{\circ}\)C, allowing the MTT to react with viable cells. Formazan formation, indicating the metabolic activity of the bacteria, was visually examined [50; 51].  
To determine the minimum bactericidal concentration (MBC), preserved MHA Petri dishes without any bacteria were activated by incubating them for 45 min at a temperature of 37 \({}^{\circ}\)C. Subsequently, the activated Petri dishes were transferred to a sterile room equipped with a continuous laminar airflow system. Following that, samples from each diluted test tube, prepared in a similar manner as in the MIC evaluation, were sub-cultured onto the activated MHA plates. These sub-cultured media were then once again transferred to an incubator and kept at a constant temperature of 37 \({}^{\circ}\)C for one day. Finally, the MBC was determined as the lowest concentration of the plant extract sample that completely inhibited bacterial growth on the surface of the media [50].