Waters 717 plus

The concentration of ICA in EE was determined by HPLC–PDA method modified from Pharmacopoeia of The People's Republic of China⁶⁴ . Briefly, an HPLC system consisted of Waters 717plus autosampler (Waters, Milford, MA, USA), Waters 2996 photodiode array detector, Waters 600 Gradient delivery system, and Sugai U-620 Column heater (Sugai Chemie Inc.,Wakayama, Japan). Chromatographic separation of compounds was performed using a Cosmosil 5C18-MS-II (5 µm, 4.6 mm × 250 mm; Nacalai tesque, Kyoto, Japan) maintaining at 35℃. Elution was performed at a flow rate of 1 ml/min in a gradient. The mobile phase consisted of potassium dihydrogen phosphate aqueous solution, acetonitrile, and water. The UV detection wavelength was set at 270 nm. The peak of icariin eluted at approximately 44.6 min. Sample preparation was performed by adding 0.5 g of EE powder into 20 ml of methanol/water (70/30, v/v) for 35 min, then centrifuged at 3000 rpm for 10 min. The supernatant solution was collected and injected to HPLC for assay after filtered by a 0.45 µm PVDF filter. The results from HPLC chromatogram showed that each gram of dried EE powder contained 5.8 mg ICA (supplementary Fig. S4A,B).

To determine the potential components, the XZP was prepared and analyzed by high performance liquid chromatography (HPLC) using >98% purity of the standards of tetrahydropalmatine, imperatorin, isoimperatorin, coptisine, and palmatine chloride (in methanol) that were purchased from the National Institute for Food and Drug Control (Beijing, China). Methanol and acetonitrile (HPLC grade, Fisher Scientific, New Jersey, USA) and pure water (Wahaha Group, China) were used for HPLC analysis. All other chemicals were of analytical grade. The solutions were filtered through 0.45 μm membranes and subjected to analysis at 280 nm using an Agilent Zorbax Eclips XDB C18 column (250 × 4.6 mm, 4.5 μm) on a Waters HPLC system (Waters Corporation, USA) equipped with 2487 pump, an UV detector, an Empower 2 system controller, and a waters 717 plus autosampler at 30°C. Samples at 10 μL were injected and acetonitrile-methanol-water-triethylamine (24 : 32 : 44 : 0.5) at a flow-rate of 1.0 mL per min was used at the mobile phase. According to the chromatographic characteristics of these standard compounds, the peak areas of individual compounds in the samples were used for determining the contents of these compounds and expressed as the percent.

The left anterior striata were dissected (approximately 1.54 mm to 0.62 mm from bregma), homogenized in 250 μL of 0.1 N HClO₄ at 4 °C and then centrifuged at 10,000× g for 30 min (4 °C) to precipitate proteins. The striatal contents of DA and its metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC), 3-methoxytyramine (3-MT), and homovanillic acid (HVA), as well as serotonin (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA), were measured by high-performance liquid chromatography (HPLC) with electrochemical detection. Supernatants of striatal tissue were directly injected into the chromatograph consisting of a Waters 717 plus autosampler automatic injector, a Waters 515 pump equipped with a C-18 column (Waters Nova-Pak C18, 3 μm, 3.9 mm × 150 cm, Waters Corp., Milford, MA, USA), a BAS LC-4C electrochemical detector, and a glassy carbon electrode. The mobile phase consisted of 0.025 M citric acid, 1.7 mM 1-heptane-sulfonic acid, and 10% methanol, in filtered distilled water, delivered at a flow rate of 0.8 mL/min. The final pH of 3.98 was obtained by addition of NaOH. The electrochemical potential was set at 0.8 V with respect to an Ag/AgCl reference electrode. Proteins were assayed with a Micro BCA protein assay kit (Thermo Scientific, Waltham, MA, USA) and results were expressed in nanograms of amine per milligram of protein.

The solvents hexane, dichloromethane, ethanol, ethyl acetate and butanol were purchased from Trinity College Dublin HMF facilities. HPLC grade acetonitrile and chloroform were purchased from Sigma Aldrich. HPLC grade water was obtained from a deionized water treatment system from PureLab Option. The HPLC system (Waters) was used in conjunction with Waters 1525 binary HPLC pump, Waters 2487 dual λ absorbance detector, Waters 717 plus auto sampler and breeze software program. A reverse phase Thermo C18 column (250 x 4.6mm, 5μm) was used.

Mice were sacrificed, their brains removed and placed on ice. Dorsal striatum and nucleus accumbens were dissected and weighed, then flash-frozen and stored at −80 °C. Tissue samples were ultrasonicated in 0.1 M perchloric acid and stored at −80 °C until extraction. Upon thawing, the samples were homogenized in 0.1 M perchloric acid and centrifuged at 25,000 g for 12 min. Dopamine levels were measured by HPLC with electrochemical detection. The analytical column was a SunFire C18 5 lm (4.6–150.0 mm) from Waters (Milford, MA, USA). The mobile phase was 0.01 M sodium dihydrogen phosphate, 0.01 M citric acid, 1.2 mM sodium EDTA, 1.2 mM sodium 1- heptane sulfonic acid, 10% methanol, pH 3.5; the flow rate was 1.0 mL/min and the column temperature was 34 °C. The installation consisted of a Waters 717 Plus automated injection system, a Waters 1525 Binary pump, and an ESA Coulochem III detector (Dionex, Sunnyvale, CA, USA). Waters Breeze system was used for analysis.