5 hiaa

Mice were immediately sacrificed after captopril administration for 21 days followed by series of behavioral test. Brain tissue blocks were rapidly dissected in ice and homogenized with ice-cold 0.4M perchloric acid, then incubated on ice for 1 h. After centrifugation at 21,130 g for 30 min at 4°C, the supernatant was filtered using an appropriate column (#Sc1000-1Kt, SigmaPrep spin column). Filtered supernatants were directly injected onto the Nova-Pak C18 reversed-phase column. The mobile phase consisted of 0.1M sodium phosphate monobasic, 0.1mM EDTA, 1mM sodium octyl sulfate, 0.003% trimethylamine, and 10% methanol at pH 3.7. The external standard solutions for each analyte were 5-hydroxytryptamine (5-HT; sc-298707, Santa Cruz) and 5-hydroxyindole-3-acetic acid (5-HIAA; #H8876, Sigma-Aldrich) solutions in HPLC grade water with 0.4M perchloric acid. The flow rate was kept constant at 1 ml/min. Chromatographic peak analysis was accompanied by identification of unknown peaks in a sample matched according to retention times.

Dichroa Alkali Salt (DAS) was isolated from D. febrifuga Lour (purity ≥99%), and dissolved in physiological saline. Cisplatin (DDP, Sigma-Aldrich, Beijing, China), metoclopramide hydrochloride (Met, Suicheng Pharmaceutical Co. Ltd. Xinzheng, China) and ondansetron hydrochloride (Ond, Qilu Pharmaceutical Co. Ltd.) were dissolved in physiological saline. Aprepitant (Apr), purchased from Merck Sharp and Dohme Ltd. (Hertfordshire, United Kingdom), was suspended in an aqueous suspension of 0.5% CMC (Sigma-Aldrich Japan). All drugs were prepared immediately before administration. 5-HT, 5-HIAA, gum arabic and kaolin were obtained from Sigma-Aldrich China. The SP ELISA kit was purchased from Cusabio Biotech Co. Ltd (Wuhan, Hubei, China). The anti-5-HT₃ receptor antibody was obtained from Proteintech, Chicago, IL (cat. 10443-1-AP). The anti-NK₁ receptor antibody was obtained from Santa Cruz Biotechnology, Inc (code sc-365091). Goat anti-rabbit IgG was obtained from the Proteintech Group (Chicago, IL, United States). Methanol (MeOH) and acetonitrile (ACN) were purchased from Fisher Chemicals (Hampton, NH, United States). All other substances (analytical grade), such as EDTA disodium, sodium 1-octanesulfonate, were purchased from the Beijing Chemical Factory (Beijing, China). The water for preparing the mobile phase was obtained using a Milli-Q system (Millipore, Madrid, Spain).

All the chemicals and reagents used were commercially available and were of the highest grade purity or analytical purity. Indoxyl sulfate potassium salt, the monoamines (NE, E, DA, 5HT) and their metabolites (DOPAC, 5HIAA), sucrose, sodium acetate, EDTA-2NA were purchased from Sigma-Aldrich, USA. Acetic acid, acetonitrile, citric acid, HClO₄, 1-octanesulfonic acid sodium were purchased from Merck, Germany.

SH-SY5Y cells were harvested and washed with PBS after 0, 30, 60 and 90 min of treatment with 30 μM 5-HIAA (Sigma-Aldrich). The cells were then sonicated in iced Tris buffer (50 mM Tris-HCl, pH 7.4) and store at − 80 °C.
Measurement of neprilysin activity was performed according to the technical details given by the manufacturer for the fluorescent SensoLyte 520 kit (AnaSpec Inc., Fremont, CA, US). Briefly, 100 μg of total protein were placed in the wells of a non-binding 96-well plate (Corning) before adding the substrate working solution. The reagents were mixed by shaking the plate gently for 30 s and the fluorescence signal was immediately measured at Ex/Em = 490 nm/520 nm continuously and the data were recorded every 5 min for 1 h.
The initial reaction velocity was determined by the slope of the linear portion of the data plot and the results were expressed in percentage by reference to control condition.

Reference drugs in this study including MOH, INDO and DZP were donated by the Aromatic Plant Research Center. A23187, GBM, CZP and UFP were from Macklin Biochemical (Shanghai, China). NXH, FM, SCH, WAY, 5-HT, 5-HIAA, glutamate and GABA were from Sigma-Aldrich (St. Louis, MO, USA). D101 macroporous resin (particle size 16–60 mesh, moisture content 65–75%, surface area 500–550 m²/g) was from Huiying Instrument Business Department (Hangzhou, China).
Male ICR mice (25–30 g) were purchased from the SLAC Laboratory Animal (Shanghai, China) and kept in Laboratory Animal Center of Shanghai Jiao Tong University under a standard experimental environment. Animal assays were performed according to the regulation of Institutional Animal Care and Use Committee (IACUC) of Shanghai Jiao Tong University with a code (No. A2020010).

The analysis procedure
followed that described in the literature.^{19 (link)} In brief, the hippocampus tissue samples were homogenized using
deionized water. We then transferred the supernatant to the following
analysis. The Agilent 6470 triple quadrupole liquid chromatography–mass
spectrometer/mass spectrometer system was used. The chromatographic
separation was achieved on an ACQUITY UPLC HSS T3 Column, 100 Å,
1.8 μm, 2.1 × 100 mm (Waters Co., Milford, MA, US) at 40
°C. The mobile phase consisted of (A) water with 0.1% formic
acid (FA) and (B) methanol with 0.1% FA at a flow rate of 0.4 mL/min.
The gradient elution was programmed as follows: 0–8 min, 50%
A; 8–8.1 min, 50–0% A; 8.1–10 min, 0% A; 10–10.1
min, 0–100% A; 10.1–12 min, 100% A.
The mass spectrometric
detection was performed using multiple reaction monitoring with an
electrospray ionization source in positive mode. Ion source parameters
were optimized with the isocratic mobile phase composition without
column separation. The conditions were as follows: ion spray voltage,
5000 V; temperature, 300 °C; Sheath gas flow, 11 psi; nebulizer
gas, 45 psi; and heater gas, 40 psi. Data acquisition and processing
were performed with the Agilent MassHunter Workstation Software. The
standard solutions of 5-HIAA, tryptophan, kynurenine, kynurenic acid
(KA), and 3-hydroxykynurenine (3-HK) were purchased from Sigma-Aldrich
(Burlington, MA, US).