Leucine enkephalin

LC-MS-grade acetonitrile, water, and methanol were purchased from Merck Millipore (Warsaw, Poland). L-Arginine, SDMA, ADMA, L-citrulline, sodium tetraborate, benzoyl chloride (BCl), and HPLC-grade formic acid (FA) were obtained from Sigma-Aldrich (Poznan, Poland). Isotope-labeled asymmetric dimethylarginine (2,3,3,4,4,5,5-D7-ADMA, 98%) and L-Arginine:HCl (D7-arginine, 98%) were acquired from Cambridge Isotope Laboratories (MA, USA). Leucine-enkephalin was purchased from Waters (Warsaw, Poland).

UPLC-grade acetonitrile was purchased from Merck (Darmstadt, Germany). Formic acid was purchased from Sigma-Aldrich (Mo, USA). Leucine enkephalin was obtained from Waters Corporation (Milford, MA, USA). Distilled water was obtained from Watson's Food & Beverage (Guangzhou, China).
The PC12 cells were purchased from Jiangsu KeyGEN BioTECH Corp., Ltd (Nanjing, China). Ginsenoside Rb1, ginsenoside Rb2, ginsenoside Rb3, ginsenoside Rc, ginsenoside Rd, ginsenoside Re, ginsenoside Rf, ginsenoside Rg1, notoginsenoside R1, 20(S)-notoginsenoside R2, notoginsenoside Fa, gypenosides A, and gypenosides XLIX were purchased from Chengdu Must Bio-Technology Co., Ltd. (Chengdu, China). The 20(S)-ginsenoside Rb2, galantamine HBr, and berberine were purchased from the National Institutes for Food and Drug Control (Beijing, China). The structure of these compounds is shown in Figure 2. Dulbecco's modified Eagle medium (DMEM) and phosphate buffer saline (PBS) were purchased from Jiangsu KeyGEN BioTECH Corp., Ltd. (Nanjing, China). Fetal bovine serum (FBS), trypsin, and dimethyl sulfoxide (DMSO) were purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Amyloid β-protein fragment 25–35 was purchased from Sigma-Aldrich (St. Louis, MO, USA). YZG was produced by Guangxi Wanshoutang Pharmaceutical Co., Ltd., and formulated by water into suspension.

LC–MS grade acetonitrile, methanol, and acetic acid, purchased from Fisher Chemical (Raleigh, NC, USA) and ultrapure water with 18.2 MΩ·cm resistivity (Thermo Scientific Barnstead MicroPure UV ultrapure water system, Sunnyvale, CA, USA) were used to prepare solutions and chromatographic mobile phases. Leucine enkephalin was purchased from Waters Corp. (Milford, MA, USA). L-Leucine-1-13C (purity = 99%) was purchased from Eurisotop, Saint-Aubin, France). Lysophosphatidylcholine USP standard (approximate content of LysoPC 0:0/16:0 = 65%, approximate content of LysoPC 0:0/18:0 = 25%), L-phenylalanyl-L-phenylalanine and L-tryptophan (≥98%), were purchased from Sigma-Aldrich (St. Louis, MO, USA). Alogliptin was purchased from DC Chemicals (Shanghai, China).

Liquid chromatography–mass spectrometry (LC–MS/MS) analyses were performed using liquid chromatography coupled to a Xevo G2-S Q-Tof mass spectrometer (Waters Corp., Milford, MA, USA) equipped with an electrospray source. The samples were analyzed in a BEH C18 column (Waters Corp.; 50 mm; 2.1 mm; 1.7 μm particle size) using ultra-pure water (solvent A) and acetonitrile (solvent B) as mobile phases, both acidified with 0.1% formic acid. The column temperature was set at 40 °C and a flow rate of 0.3 mL/min. A total of 2 μL of extract (1 mg.mL⁻¹) was analyzed in the UPLC system, using gradient elution from 5% to 95% solvent B, for 11 min. Leucine-enkephalin (Waters Corp., Milford, MA, USA) was used as a reference mass. The source temperature was set up to 120 °C with a cone gas flow of 30 L/h. The desolvation gas flow was set up to 600 L/h at a temperature of 250 °C. The capillary sampling was set up to 3.5 kV with the cone voltage of 40 V. A DDA method data was used in positive mode (top5 most intense ions), mass range from m/z 50 to 1200 Da (MS¹ and MS²) with a scan time of 0.1 s. A profile of normalized collision energy (10–40 eV) was applied and MassLynx software (Waters Corp., Milford, MA, USA) was used to control the system and data acquisition.

Leucine enkephalin was purchased from Waters Corporation (Milford, MA, USA). Chromatography-grade methanol, mass-spectrometry-grade acetonitrile, and formic acid were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Folin & Ciocalteu phenol reagent was bought from Sigma-Aldrich, Co., Ltd. (St. Louis, MO, USA). Ferrous sulfate, ferric chloride, and analytical-grade ethyl alcohol were procured from Tianjin Xinbo Chemical Co., Ltd. (Tianjin, China). Analytical-grade anhydrous sodium carbonate (Na₂CO₃), aluminum chloride (AlCl₃), potassium acetate, and gallic acid monohydrate (purity 99.0%) were obtained from Tianjin Yongsheng Fine Chemical Co., Ltd. (Tianjin, China). The phenolic standards such as rutin, quercetin, ferulic acid, caffeic acid, isorhamnetin, kaempferol 3-O-beta-rutinoside, p-coumaric acid, chlorogenic acid, and isoquercetin were purchased from Anpel Laboratory Technologies Inc. (Shanghai, China). Hydrochloric acid was purchased from Xi’an Chemical Reagent Factory (Xi’an, China). The 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,4,6-tris (2-pyridyl)-s-triazine (TPTZ), and sodium acetate were provided by Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Ultrapure water was purchased from Watsons Group Ltd. (Hong Kong, China).

Optima-grade formic acid, acetonitrile, methanol, acetone, and food-grade hydrochloric acid (HCl) were purchased from Fisher Scientific (Waltham, MA, USA). Quinic acid, methylparaben, deuterated methanol, and deuterated water were purchased from Millipore Sigma (Burlington, MA, USA). 3-Caffeoylquinic acid (3-CQA), 4-caffeoylquinic acid (4-CQA), and 5-caffeoylquinic acid (5-CQA) were purchased from BOC Sciences (Shirley, NY, USA). Nanopure water was purified through Barnstead Nanopure Diamond Water Purification System (Thermo Fisher, Dubuque, IA, USA). Leucine enkephalin was purchased from Waters Co. (Milford, MA, USA). Organic green Arabica and Robusta coffee beans were purchased from local suppliers (Columbus, OH, USA).