Materials and reagents
Hawthorn (Batch number: 170801, Guangzhou Zhixin Chinese Medicine Pieces Co., Ltd., Origin: Shandong); Domperidone tablets (Zhejiang Deend Pharmaceutical Co., Ltd.); Atropine sulfate injection (Tianjin Jinyao Pharmaceutical Co., Ltd.); Chloral hydrate (Macleans, AR > 99.0%); Petroleum ether (Tianjin Baishi Chemical Co., Ltd., analytical grade); Ethyl acetate (Tianjin Baishi Chemical Co., Ltd., analytical grade); N-butanol (Tianjin Zhiyuan Chemical Reagent Co., Ltd., analytical grade); Formic acid (Aladdin Reagent Shanghai Co., Ltd., chromatographic grade); Methanol (Germany Merck shares two companies, chromatographic grade); Acetonitrile (Germany Merck shares two companies, chromatographic grade).
Preparation of the extracts
Hawthorn was pulverized by a high speed automatic Chinese medicine grinder. Then, 500 g of the coarse powder (No. 4 sieve) of hawthorn was extracted three times with 6000 mL of water, 100 minutes each . Three water extraction solutions of hawthorn were mixed and concentrated under reduced pressure at 50 ℃. The water concentrated solution of hawthorn was extracted by three different solvents (petroleum ether, ethyl acetate, and n-butanol), respectively for 3 times, and then concentrated, freeze dried into powder to obtain four different polarity parts, including water part (T1), n-butanol part (T2), ethyl acetate part (T3) and petroleum ether part (T4) .
Drug administration and model preparation
Equal numbers of male and female SD rats (body mass: 200 ± 20 g) were provided by the Guangdong Provincial Laboratory Animal Center, China. They were housed at a temperature of 24 ± 2 ℃, a relative humidity of 50 to 60%, and a 12 h light-dark cycle was set in the SPF animal laboratory. Standard diet and water was provided. The animal protocol was approved by The Animal Ethics Committee of Guangdong Pharmaceutical University.
All the rats were acclimatized for 3 days and then randomly divided into 7 groups (each with 8 rats): a normal control group (NC group), a model group (MOD group), a domperidone, group (DOMP group), and 4 sample groups of hawthorn extracts (T1-T4 groups). Rats in the NC and Mod groups were administrated with 0.5% CMC-Na solution, and those in the DOMP group were administrated with domperidone (2.8 mg·kg− 1). Rats in the 4 sample groups of hawthorn were administrated with T1-T4 samples (2100 mg·kg− 1), respectively. All the groups received their administrations once a day for 5 days.
After 4 days of treatment, all eating was stopped except for water for 18 hours. After 20 min of administration on the 5th day, each group was subcutaneously injected with atropine sulfate at a dose of 1.4 mg·kg− 1, except for the NC group that received subcutaneous normal saline injections. After 20 min, the rats in each group were intragastrically administered the semi-solid black nutrient food. The nutrient food containing 10 g of carboxymethylcellulose sodium, 16 g of milk powder, 8 g of starch, 8 g of sugar, and 2 g of activated carbon powder was added in 250 mL of distilled water. The mixture was uniformly stirred to prepare 300 g of a semi-solid black nutrient paste, prepared for animal treatments.
Plasma sample collection and preparation
After the semi-solid black nutrient food administration for 20 minutes, blood was withdrawn from the abdominal aorta using a sodium heparinized blood collection vacuum tube, and centrifuged at 4000 rpm for 10 min to obtain supernatant, which was stored at − 80 ℃.
The plasma samples were thawed at 4 ℃, and then 100 µL of plasma was transferred to a new tube and diluted with 200 µL of cold acetonitrile (4 ℃). After vortex for 1 min, the diluted plasma solution was centrifuged at 4 ℃ at 13000 g for 15 min to remove proteins. The supernatant was transferred to a new tube after passing through a 0.22 µL microporous filter membrane, and then the filtrate was transferred to vials for UPLC-MS/MS analysis.
Measurement of gastric emptying rate and small intestine propulsion rate
After blood collection, gastric emptying rate and small intestine propulsion rate were examined by the following procedure. Briefly, after ligating the gastric cardia and pylorus, the operator took the stomach, wiped it with filter paper and weighed it (labeled “G”). Then the operator cut the stomach along the greater curvature, washed its contents, and again wiped it with filter paper and weighed it (labeled “g”). The “G” and “g” values were used to calculate the gastric emptying rate (Formula 1). The total length of the small intestine was also measured (from the pylorus to the ileocecal area, labeled “H”) along with the distance from the pylorus to the black semi-solid paste front (labeled “h”). The small intestine propulsion rate was calculated based on the values of “H” and “h” (Formula 2).
Gastric emptying rate (%) = 1-(G-g)/2*100% (Formula 1)
Small intestine propulsion rate (%) = h/H*100% (Formula 2)
Chromatography was performed on a UPLC system (Thermo Corp., USA) with an autosampler at 4 ℃. The separation was carried out on a Hypersil GOLD™ C18 column (2.1 mm × 100 mm, 1.9 µm, Thermo Corporation, USA). The column temperature was maintained at 40 ℃. The analysis was performed with gradient elution using (A) water with 0.1% formic acid, and (B) acetonitrile as the mobile phase. Gradient elution was modifed as follows: 0–4 min, 5–30% B; 4–6 min, 40% B; 6–7 min, 60% B; 7–9 min, 95% B; and 9–12 min, 95% B. The flow rate was set to 0.3 mL/min. The sample injection volume was 2 µL. Throughout the analysis, all samples were kept at 4 ℃.
A Quadrupole-Exactive Orbitrap Mass Spectrometer (Thermo Fisher Scientific Corp., USA) was connected to a UPLC system via an electrospray ionization (ESI) interface. The MS data were collected in a centroid mode from 100 to 1000 Da. The temperature of the desolvation was 320 ℃. The collision energy was 15, 35, and 55 eV. The sheath gas flow rate was 45 arb and the auxiliary gas flow rate was 10 arb.
Aliquots of 3 µL of all the plasma samples were mixed together as the quality control (QC) sample. A QC sample was run prior to the test samples for six times to condition or equilibrate the system . The replicability of the method was evaluated by analyzing 6 replicates of QC samples in one day. The post-preparative stability of the samples was tested by running 6 prepared QC samples kept in an autosampler (maintained at 4 ℃) for 12 h and the freshly prepared QC samples (n = 6) continuously in a single batch. In addition, QC samples were measured every 10 samples during the testing process in order to investigate the stability of analytical method by determining relative standard deviations (RSD) of intensity and retention time of 10 randomly selected characteristic ion peaks of QC samples.
The obtained index data were analyzed using GraphPad Prism 6.0. To evaluate the statistically signifcant differences among multiple treatments for given parameters, one-way analysis of variance with Dunnett’s multiple comparison test was used for comparison among various groups. Diferences with P values < 0.05 were considered statistically signifcant.
The raw MS data files were processed using Compound Discoverer 4.0 software (Thermo Scientific Corp., USA) for peak detection, matching, and alignment. All data including detection of mass, retention time, and intensity of the peaks eluted in each chromatogram were normalized to the sum total ion intensity per chromatogram to obtain the relative intensities of the metabolites.
Potential biomarkers had been initially identified and analyzed using the MetaboAnalyst (https://www.metaboanalyst.ca) database for related metabolic pathways. After normalization, principal component analysis(PCA) and orthogonal partial least squares discriminate analysis(OPLS-DA) analysis were performed using a SIMCA-P 14.1 software. The OPLS-DA model was evaluated according to the R2Y and Q2 parameters; the potential biomarkers were screened by selecting VIP > 1 and P < 0.05.