Chemicals: All the chemicals excluding CuO(1-x)ZnOx were purchased from commercial sources and used without further purification. (±)-α-Bisabolol (TCI), PEG-400 (TCI), PEG-4000 (TCI), capric triglyceride (Spectrum Chemical), polysorbate 80 (Spectrum chemical). Synthesis of CuO(1-x)ZnOx was described in a previous publication.13
Preparation of ointment: Ointment was produced in two steps. In the first step, 1% CuO(1-x)ZnOx suspension was prepared in PEG-400 using a homogenizer. In the second step, PEG-400 (572 g) and PEG-4000 (200 g) were mixed at 70 °C and to this added, capric triglyceride (8g), Tween 80 (8g), and (±)-α-bisabolol (10g) while continuously mixing at 70 °C for 30 min. Finally, added 1% CuO(1-x)ZnOx PEG-400 (200g) suspension and mixed for additional 60 min. Brown semi-solid formulation was obtained after slowly cooling the above mixture.
Antimicrobial activity: Antimicrobial test was based on ASTM E2315-16. Inoculum was grown from -80oC in 40 mL TSB (Tryptic Soy Broth, Himedia, Mumbai, India) in a 50mL conical tube for 24h @36oC, 100 RPM in a shaker-incubator (ES-60, MIULAB). Cells were washed twice (3000g, 5 min) in saline and concentration estimated using a nephelometer (PhoenixSpec, BECTON DICKINSON, Israel). Bacterial suspension and inoculum adjusted in saline (0.86% NaCl) containing 5% FBS (Fetal Bovine Serum, Biological Industries, Beit Haemek, Israel) to 2x108 cfu/mL.
9.5g ointment was mixed manually to homogenization with 0.5mL FBS to simulate a protein-rich wound exudate environment. After 2h at room temperature the ointment/5% FBS mixture was inoculated with 0.5mL inoculum and vortexed to homogenization. At T0 and after 2h incubation @30oC, samples of 1g were weighted in a 15mL conical tube and added with 9mL saline. Homogenization was reached after ~30sec vortex, and this “-1” dilution was further serially diluted to “-5” dilution. Using the pour plate method, each dilution was plated in duplicate in molten TSA (Tryptic Soy Agar, HiMedia, Mumbai, India) @45±2oC. Plates were incubated @36oC for 48h (bacteria) of 72h (C. albicans).
When running the test for C. albicans the addition of FBS was omitted, both in the inoculum (saline only) and in the test article (10g ointment, w/o FBS).
For calculation of log10 reduction, inoculum count was performed by plating dilutions of the inoculum in TSA as described above. Average duplicate plate counts of the inoculum and counts (T0/T2) were multiplied by the corresponding dilution factor and converted to log10 scale. Log10 reduction was calculated by subtraction of log10(Tx) from log10(inoculum).