Absolute ethanol

Cetyltrimethylammonium bromide (CTAB), tetraethyl orthosilicate (TEOS, 99.98%), (3-aminopropyl) triethoxysilane (APTES), and eugenol (4-allyl-2-methoxyphenol) were purchased from Sigma-Aldrich. Casein, trypsin solution, absolute ethanol (EtOH), succinic anhydride, 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysulfosuccinimide sodium salt (sulfo-NHS), N,N-dimethylformamide (DMF), 2-(N-morpholino)ethanesulfonic acid (MES) buffer, and glacial acetic acid were purchased from ThermoFisher Scientific, USA. Chemical reagents were of analytical grade and were used as received.

Leaves were collected and dried, then 10 g were ground in a mortar and pestle. The essential oil was extracted employing the Soxhlet method, using absolute ethanol (Thermo Fisher Scientific, Loughborough, UK) as a solvent [28 (link)]. Through this method, 10 g of dry lemon balm was extracted for 4 h. Once the extraction was complete, essential oil was separated from the solvent, using a BÜCHI R-124 Rotary Evaporator System (BUCHI UK Ltd., Suffolk, UK). The essential oils were then collected in a vial. The vial was weighed before and after the extraction to calculate the quantity of the essential oil obtained.

Alexa Fluor 546 (AF546) rat anti–mouse Ly6G mAb (clone 1A8) and Violet 450 (V450) rat anti–mouse CD49b mAb (clone DX5) were purchased from BioLegend. FITC dextran (MW 70 KDa) was purchased from Thermo Fisher Scientific. RLT+ lysis buffer (catalog 1030963) and RNeasy Plus Micro Kit (catalog 74034) were purchased from Qiagen. High-capacity cDNA reverse transcription kit (catalog 4368814) and PowerUp SYBR Green Master Mix (catalog A25742) were purchased from Thermo Fisher Scientific. Ambion nuclease-free water (catalog AM9906) was purchased from Invitrogen. Quantitative PCR (qPCR) primers were purchased from Integrated DNA Technologies. Sterile saline solution (catalog 65207-807-60) was purchased from Nova-Tech. Gibco PBS without Ca²⁺ and Mg²⁺ (catalog 10010072), buffered 10% formalin (catalog SF100-4), and absolute ethanol (catalog BP2818100) were purchased from Thermo Fisher Scientific. AnaSed Xylazine (catalog 59399-111-50) was purchased from Akorn Pharmaceuticals. Ketamine was purchased from Covetrus.

Tin(II) 2-ethylhexanoate (stannous octoate), ε-caprolactone (CL), methoxy poly(ethylene glycol) (mPEG) molecular weight (MW) 5000, Span 80, and thymoquinone (TQ) were obtained from Sigma Aldrich (St. Louis, MO, USA). Castor oil was provided by Philadelphia Pharmaceuticals (Amman, Jordan). Polysorbate 80 (Tween 80,) was purchased from RFCL Ltd. (New Delhi, India). Absolute ethanol, acetone, methanol (HPLC grade), isopropanol, formic acid, water (HPLC grade), and dichloromethane (DCM) were obtained from Fisher Chemical (Thermo Fisher Scientific, Waltham, MA, USA). Diethyl ether and dimethyl sulfoxide were provided by Tedia (Fairfield, OH, USA). Phosphate buffered saline (PBS) 10×, pH 7.4, was obtained from Biowest (Nuaillé, France). Ultrapure water (~18.2 MΩ·cm) was prepared using a Millipore Direct-Q 5UV system (EMD Millipore, Billerica, MA, USA).

The chemicals used in this study are of analytical grades and had been prepared immediately before use. The following drugs were used: ranitidine (Sigma Aldrich, USA), absolute ethanol (Fischer Scientific, USA), N-ethylmaleimide (NEM) (Sigma-Aldrich, USA), N^G-nitro-l-arginine methyl esters (L-NAME) (Sigma-Aldrich, USA), carbenoxolone (CBX) (Sigma-Aldrich, USA) and diethyl ether (Fischer Scientific, USA).

D-glucose (Gibco, Waltham, MA, USA), insulin (Sigma Aldrich, Saint Louis, MI, USA), recombinant TNF-α (Abcam, Cambridge, UK), recombinant IL-6 (Abcam, Cambridge, UK), PA (Sigma Aldrich, Saint Louis, MI, USA), LA (Sigma Aldrich, Saint Louis, MI, USA), and Chol (Sigma Aldrich, Saint Louis, MI, USA) were used to establish the obesity-like models. D-glucose, TNF-α, and IL-6 were diluted using sterilized 1X phosphate-buffered saline (PBS). insulin was diluted using sterilized acidic distilled water, and the pH was adjusted to be between 2.0 and 3.0 with diluted HCl. Chol was diluted using absolute ethanol (Thermo Fisher Scientific, Waltham, MA, USA). PA and LA were conjugated with bovine serum albumin (BSA; GenDEPOT, Barker, TX, USA). For BSA conjugation, PA and LA were diluted using absolute ethanol, then boiled at 40 °C for at least two hours while vortexing. The solution was filtrated using a syringe filter (0.2 μm; Millipore, Saint Louis, MI, USA) and mixed with a 10% BSA solution at a 1:100 ratio. The cells were treated with D-glucose (4.5 g/L [91 (link)]), insulin (100 nM [91 (link)]), TNF-α (25 ng/mL [92 (link)]), IL-6 (25 ng/mL [93 (link)]), BSA-conjugated PA (50 μM [94 (link)]), BSA-conjugated LA (50 μM [95 (link)]), and Chol (50 μM [96 (link)]) for 48 h.