| ObjectiveIn order to explore the Sedative and hypnotic effect mechanism of Jiao-Tai-Wan. This study is designed to investigate the change in monoamine neurotransmitters and their metabolites in normal and insomnia rat hippocampus microdialysates after Jiao-Tai-Wan was administered. In addition, the intervention effect of Jiao-Tai-Wan to insomnia rat was investigated by using target metabonomics approach.Methods1. Simultaneous determination of a series of monoamine neurotransmitters in rat hippocampus microdialysates by UPLC-MS/MS combined with dansyl chloride derivatizationA UPLC-MS/MS method to determine dopamine (DA), norepinephrine (NE),3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA),3-methoxy-4-hydroxyphenylglycol (MHPG),5-hydroxytryptamine (5-HT), and 5-hydroxyindoleacetic acid (5-HIAA) in rat hippocampus microdialysates was developed. Dansyl chloride (DNS) was used as a pre-column derivatization reagent. The derivatization conditions, chromatographic conditions and mass spectrometric conditions were optimized. Then, the selectivity, linearity and sensitivity, precision and accuracy and stability were investigated.2. The change in monoamine neurotransmitters and their metabolites in normal and insomnia rat hippocampus microdialysates after Jiao-Tai-Wan administrationThe rats were anesthetized with chloral hydrate and mounted in a stereotaxic apparatus (RWD Life Science, Shenzhen, China). A microdialysis guide cannula (Brainlink B.V., Netherlands) was stereotaxically implanted unilaterally aimed at the hippocampus (Anterior Posterior-5.2 mm, Medial Lateral+4.5 mm, Dorsal Ventral-3.8 mm). The coordinates were taken according to Paxinos and Watson with reference points taken from the bregma and vertically from the skull. The rats were given 7 days to recover from surgery prior to starting the microdialysis experiments.After seven days of recovery, a probe with 4 mm membranes (Brainlink B.V., Netherlands) was implanted through the guide cannula into the hippocampus. A stabilization period of 2 h was maintained, and then, four basal samples were collected at 15 min intervals. Jiao-Tai-Wan extract (300 mg/kg RC extract and 4.7 mg/kg Cinnamon Cassia oil) was administered, and the dialysates were collected at 15 min intervals for an additional 3 h period. The collected samples were maintained at -80℃ and analyzed within 7 days. The samples were determined using the method described above.3. Simultaneous determination of amino acids, monoamine neurotransmitters, purines and pyrimidines in rat serum and brain by HILIC-MS/MSA novel quantitative method utilizing hydrophilic interaction ultra-high-performance liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) that is highly selective and high-throughput with better chromatographic behavior was developed. The method enable a rapid quantification of 37 amino acids, neurotransmitters, purines and pyrimidines within 6.5 min.Calibration curves were prepared in acetonitrile. The analytical response differences between plasma/brain homogenate and acetonitrile were evaluated by a recovery assessment to ensure that the calibration curve built in acetonitrile could be applied to quantify biological samples. The slope coefficient (a) of 6-point concentration curves for all 37 analytes spiked in serum/brain homogenate from 6 different healthy rats were compared with their respective curves spiked in acetonitrile solution. The linearity and sensitivity, precision and accuracy were investigated.4. The target metabolomics investigation of insomnia rat after Jiao-Tai-Wan administrationEighteen rats were randomly divided into three groups (normal, PCPA and treatment group).The animals (PCPA and treatment group) were given i.p. PCPA for a total of 500 mg/kg, according to the schedule:300 mg/kg on day 1,100 mg/kg on days 2 and 3. Jiao-Tai-Wan extract (300 mg/kg RC extract and 4.7 mg/kg Cinnamon Cassia oil) was administered for 7 days to rats of treatment group.0.5 mL blood was obtained in day 7. The brain tissue and 0.5 mL blood were obtained in day 10. All the samples were stored at-80℃. The samples were determined using the method described above. Both univariate and multivariate data analysis were performed with SPSS software (V17.0, Chicago, USA) and SIMCA-P+ package (V12.0, Umetrics, Sweden), respectively. The metabolic pathways were analyzed by MetaboAnalyst 3.0. Results1. A UPLC-MS/MS method to determine dopamine (DA), norepinephrine (NE),3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA),3-methoxy-4-hydroxyphenylglycol (MHPG),5-hydroxytryptamine (5-HT), and 5-hydroxyindoleacetic acid (5-HIAA) in rat hippocampus microdialysates was developed. The selectivity, linearity and sensitivity, precision and accuracy and stability of the method were satisfactory. The method is sensitive and high throughput.2. It was the first to investigate the change in monoamine neurotransmitters and their metabolites in rat hippocampus microdialysates after Jiao-Tai-Wan was administered. Jiao-Tai-Wan can increase the monoamine neurotransmitters and their metabolites in rat hippocampus.3. A hydrophilic interaction ultra-high-performance liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) method to determine amino acids, monoamine neurotransmitters, purines and pyrimidines in rat serum and brain was developed. The sample preparation was simple. The linearity and sensitivity, precision and accuracy were satisfactory. The method was fit to target metabonomics investigation.4. The 5-HT concentration in serum of PCPA and treatment group was lower than that of control group. Jiao-Tai-Wan can adjust the abnormal metabolism of insomnia rats. The effect was related to phenylalanine, tyrosine and tryptophan metabolism, valine, leucine and isoleucine metabolism and glutamic acid and glutamine metabolism. In addition, it may be related to glycine, serine and threonine metabolism, arginine and histidine metabolism and proline metabolism.ConelusionMicrodialysis and metabonomics can be used to pharmacological investigations of Jiao-Tai-Wan. Single administration of Jiao-Tai-Wan can quickly increase the monoamine neurotransmitters and their metabolites in rat hippocampus. But the effect maintains less than 6 hours. Repeated administration ofJiao-Tai-Wan can adjust the abnormal metabolism of insomnia rats. The effect was related to phenylalanine, tyrosine and tryptophan metabolism, valine, leucine and isoleucine metabolism and glutamic acid and glutamine metabolism. |
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