Safrole

Norfloxacin and ciprofloxacin were used in the tests with the 1199B and K2068 strains, respectively. These strains carry the NorA and MepA efflux proteins, respectively, which confer resistance to the corresponding antibiotic. ethidium bromide was used as an efflux pump inhibitor control. Both antibiotics were dissolved in DMSO and diluted in water, while ethidium bromide was dissolved in water. All drugs were prepared at initial concentrations of 1024 µg/mL and serially diluted in test tubes. Both antibiotics, ethidium bromide and safrole were purchased from SIGMA Chemical Co. (St. Louis, MO, USA).

The essential oil, namely nutmeg oil (Myristica fragrans) was purchased from Imeltech Sdn. Bhd (Kuala Lumpur, Malaysia) with in-house code no. MF-004 (Batch no. NMO7956FT) and stored at the Herbarium Unit, Integrative Medicine Cluster, Advanced Medical and Dental Institute (IPPT). α-pinene (Batch no. SHBB7538V, Sigma-Aldrich, St Louis, United States), safrole (Batch no. BCBH2795V, Sigma-Aldrich, Shanghai, China), and terpinen-4-ol (Batch no. BCBF8537V, Sigma-Aldrich, Madrid, Spain) were used as standards for GCMS analysis.

Standard pure compounds for co-injection in GC/MS, α-pinene, β-phellandrene, safrole, isosafrole, and piperonal were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). The separation and identification of the synthesized compounds was carried out by previously performed methods [24 (link),25 (link)].

All reactions were carried out under air in oven-dried glassware with magnetic stirring at room temperature. Safrole was purchased from Millipore Sigma (St. Louis, MO, USA) and used as received. All reagents and solvents were purchased from Fisher (Hampton, NH, USA) and used as received. Purification of reaction products was carried out via flash column chromatography using silica gel 60 (230–400 mesh). TLC visualization was accompanied by UV light. Concentration in vacuo refers to the removal of volatile solvent using a rotary evaporator attached to a dry diaphragm pump (10–15 mm Hg) followed by pumping to a constant weight with an oil pump (<300 mTorr).
¹H NMR spectra were recorded at 400 MHZ (Jeol, Akishima, Tokyio, Japan), and are reported relative to CDCl₃ (δ = 7.26). ¹H NMR coupling constants (J) are reported in Hertz (Hz) and multiplicities are indicated as follows: s (singlet), d (doublet), t (triplet), m (multiplet). Proton-decoupled ¹³C NMR spectra were recorded at 100 MHz and are reported relative to CDCl₃ (δ = 77). IR experiments were recorded with neat samples on a Jasco FT/IR-4700 (Easton, MD, USA) fitted with diamond ATR sample plate. GCMS data were recorded on a Shimadzu GC-2010 plus (Kyoto, Kyoto, Japan) system (GCMS-QP2010 SE). Elemental Analysis measurement was recorded on a FlashSmart elemental analyzer (Thermo Fisher Scientific, Waltham, MA, USA).

All reactions were carried out under air in oven-dried glassware with magnetic stirring at room temperature. Safrole was purchased from Millipore Sigma (St. Louis, MO, USA) and used as received. All reagents and solvents were purchased from Fisher (Hampton, NH, USA) and used as received. Purification of reaction products was carried out via flash column chromatography using silica gel 60 (230–400 mesh). TLC visualization was accompanied by UV light. Concentration in vacuo refers to the removal of volatile solvent using a rotary evaporator attached to a dry diaphragm pump (10–15 mm Hg) followed by pumping to a constant weight with an oil pump (<300 mTorr).
¹H NMR spectra were recorded at 400 MHZ (Jeol, Akishima, Tokyio, Japan), and are reported relative to CDCl₃ (δ = 7.26). ¹H NMR coupling constants (J) are reported in Hertz (Hz) and multiplicities are indicated as follows: s (singlet), d (doublet), t (triplet), m (multiplet). Proton-decoupled ¹³C NMR spectra were recorded at 100 MHz and are reported relative to CDCl₃ (δ = 77). IR experiments were recorded with neat samples on a Jasco FT/IR-4700 (Easton, MD, USA) fitted with diamond ATR sample plate. GCMS data were recorded on a Shimadzu GC-2010 plus (Kyoto, Kyoto, Japan) system (GCMS-QP2010 SE). Elemental Analysis measurement was recorded on a FlashSmart elemental analyzer (Thermo Fisher Scientific, Waltham, MA, USA).