Acetylthiocholine iodide atchi

AChE activity was determined in accordance with the method of Chen et al. (2018) [27 (link)]. A spectrophotometric assay was used in a multi-detection microplate reader (Infinite M200 PRO, Tecan, Switzerland) at 412 nm. The activity was assessed in vivo, and the reaction mixture included 25 uL of enzyme extract, 50 uL of 0.4 mM 5,5′-Dithiobis (2-nitrobenzoic acid) (DTNB) (Sigma-Aldrich, Shanghai, China), 50 uL of 0.4 mM acetylthiocholine iodide (ATChI) (Sigma-Aldrich, Shanghai, China) and 50 uL of sodium phosphate buffer (0.05 M, pH 7.4) (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China). The reading was taken after incubation for 10 min at 37 °C. Each treatment was replicated three times.

Recombinant human membrane preparations for M₁ (expressed in CHO cells; Lot no. 1706299), 5-HT_1A (HEK293 cells; no. 1812363), 5-HT_2A (CHO cells; no. 1813092), D₁ (L cells; no. 1840059), and D_2S (CHO cells; no. 1820984) receptors and MicroScint-O scintillation cocktail were procured from Perkin Elmer (Waltham, MA, USA). [³H] N-methylscopolamine (NMS), [³H] 8-OH-DPAT, [³H] ketanserin, [³H] SCH 23390 and [³H] methylspiperone were purchased from American Radiolabeled Chemicals (St. Louis, MO, USA). Atropine, 5-carboxamidotryptamine (5-CT), ketanserin, SCH 23390 hydrochloride, haloperidol, serotonin hydrochloride, mianserin, human recombinant acetylcholinesterase expressed in HEK 293 cells, acetylthiocholine iodide (ATChI), 5, 5’-dithiobis [2-nitrobenzoic acid] (DTNB) and donepezil hydrochloride were obtained from Sigma-Aldrich (St. Louis, MO, USA). Bacoside A, bacoside A3, bacopaside II, bacopaside X and bacopasaponin C were purchased from Chromadex Inc. (Irvine, CA, USA). Jujubogenin and ebelin lactone were purchased from Shanghai IS Chemical Technology Ltd. (Jinshan, Shanghai, China). Unless stated otherwise, all other reagents of analytical grade were obtained through standard commercial sources.