Q exactive orbitrap lc ms ms system

The antioxidant activities ABTS⁺ (2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonate), DPPH (2,2-diphenyl-1-picrylhydrazyl), and FRAP (ferric reduction antioxidant potency) were determined by UV-VIS spectrophotometry (Shimadzu, Japan) according to the protocols described by Formisano et al. (2021) (link). Results were expressed as mmol Trolox equivalents kg^–1 dw. Phenolic Concentration Determination
One hundred milligrams of freeze-dried basil were used to quantify and determine polyphenols using a UHPLC system (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a thermo-stated column (T=25°C, 100 × 2.1 mm, Kinetex 1.7 µm biphenyl, Phenomenex, Torrance, CA, USA) and a quaternary pump (Ultimate 3000, Dionex, Sunnyvale, CA, USA). Mass spectrometry analysis was facilitated by a Q Exactive Orbitrap LC-MS/MS system (Thermo Fisher Scientific, Waltham, MA, USA). Phenolic compound sampling was performed according to the protocol detailed by Pannico et al. (2020) (link). The accuracy and calibration of the instruments used were set and checked using a mixture of reference standards (Thermo Fisher Scientific, Waltham, MA, USA). Data processing and analysis were performed using Xcalibur software, version 3.0.63 (Thermo Fisher Scientific, Waltham, MA, USA), and the results were expressed as µg g^–1 dw.

The optical rotation was measured in MeOH by an Autopol IV polarimeter (Rudolph Research Analytical, Hackettstown, NJ, United States). UV spectra were obtained by a UH5300 UV–VIS double beam spectrophotometer (Hitachi Co., Tokyo, Japan). 1D and 2D NMR spectra were obtained by a Bruker AVANCE IIITM 500 and 600 MHz spectrometers (Bruker, Ettlingen, Germany) in methanol-d₄, DMSO-d₆ using TMS as internal standard. HR-ESI-MS data was obtained on a Thermo Scientific Q Exactive Orbitrap LC-MS/MS System (Thermo Scientific, Waltham, MA, United States). An Ultimate 3000 HPLC system (Dionex Co., Sunnyvale, CA, United States) with an UltiMate 3000 pump and UltiMate 3000 variable wavelength detector was employed to carry out semi-preparative HPLC, with a Nacalai Tesque 5C₁₈-MS-II column (250 × 10 mm, 5 μm). Silica gel (200–300 mesh and 300–400 mesh) for open column chromatography (CC) was purchased from Qingdao Haiyang Chemical Group Co., Ltd. (Qingdao, China). The organic solvents were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Reagents used for α-glucosidase inhibitory assay (α-glucosidase, 4-nitrophenyl-α-D-glucopyranoside, and acarbose) were purchased from Shanghai Yuanye Biology Co., Ltd. (Shanghai, China), and the absorbance was measured by a full-wavelength microplate reader (Thermo Fisher Scientific Shier Technology Co., Ltd.).

Exosome samples (250 μg) were lysed in STD buffer (4% SDS, 100 mM Tris/Hcl, and 1 mM dithiothreitol, pH 7.6) and centrifugated at 1000 g for 10 min to collect the supernatants. Proteins were identified using Q Exactive Orbitrap LC-MS/MS system (ThermoFisher Scientific).