Dimethyl sulfoxide (dmso)

Water was purified using a Milli-Q Plus system from Millipore (Amsterdam, The Netherlands). Acetonitrile (ACN) (ULC/MS grade) and formic acid (FA) were obtained from Biosolve (Valkenswaard, The Netherlands). Tris, ZnCl₂, glycerol, hydrochloric acid, angiotensin-converting enzyme (ACE) from rabbit lung (≥2.0 units/mg protein), and captopril were obtained from Sigma-Aldrich (Zwijndrecht, The Netherlands). o-Aminobenzoylglycyl-p-nitro-l-phenylalanyl-l-proline (Abz-Gly-p-nitroPhe-Pro-OH; M1100) was obtained from Bachem (Bubendorf, Switzerland). DMSO was obtained from Riedel-de Haën (Zwijndrecht, The Netherlands). Most snake venoms were obtained commercially from Kentucky Reptile Zoo (USA), Ventoxin (USA), Biotoxin (USA), Venom Supplies (Tanunda, Australia), and African Reptiles & Venoms (Johannesburg, South Africa). The Boiga irregularis and Gloydius blomhoffii venoms were kind gifts from Prof. Steve Mackessy (University of Northern Colorado, USA) and Prof. Sadaaki Iwanaga (Kyushu University, Japan), respectively. Lyophilized venoms were kept at −20 °C. Prior to analysis, snake venoms were diluted in water to an end concentration of approximately 5.0 ± 0.1 mg/mL. After analysis, the samples were kept at −80 °C for later re-analysis.

The PGEO α-amylase inhibitory activity was estimated according to the method reported by Ali et al. [48 (link)] with minor modifications. The EO was dissolved in DMSO (Riedel-de-Haen, Hamburg, Germany) and then diluted with a buffer (Na₂HPO₄/NaH₂PO₄ (0.02 M), NaCl (0.006 M) at pH 6.9) to a concentration of 1000 µg/mL. Concentration series of 10, 50, 70, 100, and 500 µg/mL were prepared. A total of 0.2 mL of porcine pancreatic α-amylase enzyme solution (Sigma-Aldrich, St. Louis, MO, USA) with a concentration of 2 units/mL was mixed with 0.2 mL of the PGEO and incubated at 30 °C for 10 min. After that, 0.2 mL of freshly prepared starch solution (1%) was added and the mixture was incubated for at least 3 min. The reaction was stopped by the addition of 0.2 mL dinitrosalicylic acid (DNSA) (Alfa-Aesar, Lancashire, UK). The mixture was then diluted with 5 mL of distilled water and heated in a water bath at 90 °C for 10 min. The mixture was left to cool down to room temperature, and the absorbance was measured at 540 nm. A blank was prepared following the same procedure by replacing the PGEO with 0.2 mL of the buffer. Acarbose (Sigma-Aldrich, Burlington, MA, USA) was utilized as a positive control and prepared to adopt the same procedure described above. The α-amylase inhibitory activity was calculated using the following equation: $$\mathsf{\alpha }-\mathrm{Amylase} \%=\frac{\mathrm{ABSblank}-\mathrm{ABStest}}{\mathrm{ABSblank}}\times 100$$

The biofilm metabolic activity was evaluated by the reduction of 3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) as previously described by (Walenka et al. 2005) (link). After the biofilm maturation process (6, 12, and 24 h), the supernatant of the infected cells was discarded, followed by one washing step with PBS. After the washing step, 100 µl of PBS and 100 µl of the MTT solution (SIGMA^®, St. Louis, MO, USA) at 0.3% were added to each well. The cells were incubated for 2 h at 37°C. After the incubation period, the supernatant on each well was discarded, and 150 µl of dimethyl sulfoxide (DMSO) (Riedel-de Haën™, Seelze, Germany) in 25 µl of glycine buffer (0.1 M, pH 10.2) were added to each well followed by an incubation of 15 min at room temperature under light shaking. Finally, the microplates were read at 540 nm using the ELISA microplate reader Multiskan. Three individual experiments for each infection model (monospecies biofilm and mixed biofilm with HLFCs and without HLFCs) were evaluated.

All solvents used were of reagent grade. Tryptone and soy peptone were purchased from Biolife, Milano, Italy. Agar was purchased from Sigma-Aldrich St. Louis, MO, USA product of Spain. Sodium cloride, D(+)–Glucose, and di potassium hydrogen phosphate trihydrate were purchased from Merck, Darmstadt, Germany. DMSO was purchased from Riedel–de Haen (Seelze, Germany). Melting points were measured in open tubes with a Stuart Scientific apparatus and are uncorrected. A UV–1600 PC series spectrophotometer of VWR international GmbH, Darmstadt, Germany was used to obtain electronic absorption spectra. ATR-FTIR spectra in the region of 4000–370 cm^–1 were obtained with a Cary 670 FTIR spectrometer, Agilent Technologies Agilent Technologies. XRF measurement was carried out using an Am–241 radio isotopic source (exciting radiation 59.5 keV).

Trolox (6 hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) was purchased from Sigma-Aldrich, Denmark, 2,2-diphenyl-1-picrylhydrazyl (DPPH) was obtained from Sigma-Aldrich, Germany, and methanol was from Loba Chemie, India, and they were used to evaluate the antioxidant activity of R. chalepensis essential oil.
Nutrient broth was purchased from Himedia, India, and dimethyl sulfoxide (DMSO) was purchased from Riedel-de Haen, Germany, which were used for the screening of the antimicrobial activity of the R. chalepensis essential oils.

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from AppliChem (Darmstadt, Germany), dimethylsulfoxide (DMSO) was ordered from Riedel-de Haën, (Seelze, Germany), bovine serum albumin (BSA), all-trans-retinoic acid and cisplatin were obtained from Sigma-Aldrich (Steinheim, Germany), foetal calf serum, penicillin-streptomycin solution, IMDM^TM Medium, trypsin-EDTA solution were ordered from PAN^TM Biotech (Aidenbach, Germany) and ultrapure water was obtained using a Purelab Plus ^TM system from ELGA Labwater (Celle, Germany).