CN110999860B

CN110999860B - Method for researching insomnia resistance action mechanism of Sugelia based on amino acid metabonomics

Info

Publication number: CN110999860B
Application number: CN201911301769.6A
Authority: CN
Inventors: 田继兰
Original assignee: Individual
Current assignee: Individual
Priority date: 2019-12-17
Filing date: 2019-12-17
Publication date: 2021-12-17
Anticipated expiration: 2039-12-17
Also published as: CN110999860A

Abstract

The method for researching the insomnia resistance action mechanism of the Sugelia based on the amino acid metabonomics is to analyze amino acid metabonomics in each tissue of a PCPA insomnia model rat brain before and after the Sugelia is perfused by a GC-MS method, identify endogenous compounds by a standard map library, analyze the change of metabolite spectrums of each part of the brain by PCA and PLS-DA modes and identify related metabolic markers; carrying out targeted metabonomics research by applying LC-MS, and investigating the content changes of neurotransmitters such as 5-hydroxytryptamine, GABA, Glu and the like and related metabolites in brain tissues; the method comprises the steps of determining an anti-insomnia action mechanism and action sites of the Scorzonera ussuriensis through searching different metabolites and metabolic pathways thereof related to the curative effect of the Scorzonera ussuriensis in a clinical rat brain tissue sample; the invention firstly applies metabonomics research means to preliminarily explore the action mechanism of the SugeMuller for regulating and controlling the sleep-wake rhythm through a neurotransmitter system.

Description

Method for researching insomnia resistance action mechanism of Sugelia based on amino acid metabonomics

Technical Field

The invention belongs to the cross technical field of traditional Chinese medicine technology and metabonomics technology, and particularly relates to a method for researching the insomnia resistance action mechanism of Sugeli based on amino acid metabonomics.

Background

Sleep accounts for about one third of the time of normal people every day, and is an important physiological process for maintaining normal life activities of human bodies. In recent years, with the rapid development of economy, the life rhythm is accelerated, the occupation and environmental pressure is increased, the incidence rate of insomnia is increased, the trend of lightening the year is more and more obvious, and the normal work, life and health of people are seriously influenced. Insomnia is a common disease and frequently encountered disease in an outpatient department of neurology, and is clinically manifested by difficulty in falling asleep, easy waking, dreaminess, early waking, difficulty in falling asleep again after waking, discomfort after waking and the like. Su Ge Muller-3 decoction is a Mongolian medicine prescription, and is recorded in Mongolian medicine classic works, namely, the four medical classics, the Mongolian medicine encyclopedia and the Mongolian medicine prescriptions. According to the research, according to the improved traditional formula of the examined professor of the dried husband, 15g of fried wild jujube kernels are added on the basis of 15g of round cardamom, 10g of chicory and 5g of long pepper. Mainly treats insomnia and is a good medicine for soothing the nerves. In the formula, the spina date seed is sweet in taste, mild in nature, bitter in taste and pungent in flavor of the cardamom, and warm in nature, and is used as a main drug for suppressing heyi; the chicory has sweet flavor and mild nature and is an assistant drug for Zheheyi; the prescription for treating the heyi disease is prepared by adding the voxel regulating medicine Piper longum and combining the four medicines which are coordinated with each other.

Sleep is a very complex process, and abnormal metabolism of central neurotransmitters and corresponding changes in receptor function are one of the current research hotspots for the pathogenesis and treatment of the disease. Monoamine neurotransmitters 5-hydroxytryptamine (5-HT), Norepinephrine (NE), Dopamine (DA) and amino acid neurotransmitters gamma-aminobutyric acid (GABA) and glutamic acid (G1u) are all involved in the regulation of sleep mechanism, and the balance relationship between the neurotransmitters is affected by the change of the content of any transmitter and the imbalance of the proportion of the transmitters, so that insomnia is caused. 5-HT is an important chemical substance causing sleep, and neurons of the chemical substance are mainly located in the midsuture nucleus in the midsuture area of the lower brainstem, and the insomnia patients are generally accompanied with the reduction of 5-HT content. Escimirtazapine as H1/5-HT_2AThe receptor antagonist can remarkably improve the difficulty in falling asleep and prolong the sleep time, the spina date seed decoction can be combined with 5-HT and GABA receptors, the high-dose spina date seed decoction (4g/kg/2ml) can remarkably increase the time of non-oculomotor sleep (NREMS), and the increase of the NREMS time can be eliminated by adding the 5-HT receptor antagonist. DA is a precursor substance of NE, is a key neurotransmitter in hypothalamus and pituitary gland, belongs to excitatory neurotransmitters, and increases the content of the neurotransmitter when stimulated, so that the arousal of animals is increased, the sleep is reduced, and NE has specific effects which are obviously different at different parts. The 5-HT content in hypothalamus and brainstem of the sleep deprivation model is obviously reduced, and the NE and DA content is obviously increased. GABA is the most important inhibitory transmitter in the central nervous system, and approximately 20% to 40% of central synapses in the central nervous system are predominantly GABA as the neurotransmitter. Compared with the blank group, the GABA content of hypothalamus and brainstem in the rat brain is reduced, and the Glu content is increased in the sleep deprivation animal model. The results of several studies also indicate that the medicine can improve sleep by changing the content of neurotransmitter, and the adenosine analog B2 separated from rhizoma Gastrodiae can shorten sleep latency and prolong NREM sleep by increasing GAD enzyme activity and GABA concentration in rat cerebral cortex and hypothalamus. The current common resistanceThe insomnia drugs are benzodiazepine drugs and non-Z anti-insomnia drugs, the acting target of the insomnia drugs is GABA (gamma-aminobutyric acid) receptors, and the insomnia drugs have the characteristics of slow response, large side effect and the like.

Amino acids are the basic unit of protein, and are also the essential precursor molecules of important neurotransmitters, and are involved in signal transduction, immune regulation, gene expression processes, and the like. In addition, there are studies showing that rapid depletion of circulating tryptophan or phenylalanine can induce transient depressive-like behavior in healthy people with a family history of depression, as well as in depressed patients in remission. The antidepressant drug is supplemented with specific amino acid (such as methionine) as adjuvant for antidepressant therapy, and has more obvious therapeutic effect than single drug of antidepressant drug. The amino acid metabolism detection and analysis can help us to further understand the pathophysiology process and pathogenesis of mental diseases such as insomnia, depression and the like from the metabolic level. Metabonomics (Metabolomics) is another representative research technology in system biology research and is an emerging discipline taking the change characteristics of small molecule metabolites in organisms as important research contents. These small molecule metabolites can be broadly classified into amino acids, organic acids, fatty acids, saccharides, and the like. The expression level of the small molecule metabolite, which is the final product of the metabolic pathway, is closely related to the activity of the corresponding enzymes in the metabolic pathway and the effectors of the enzymes, and is closely related to the pathophysiological state of the human body. Therefore, metabonomics is an emerging technical method for exploring pathophysiological mechanisms and discovering disease biomarkers. The gas chromatography-mass spectrometry (GC-MS) is one of the analysis technologies, has the characteristics of high flux, high sensitivity, high separation and analysis capability and self-contained structure identification spectrum library, and is widely applied to the field of metabonomics research. Metabolites in the body, such as amino acids, fatty acids, amines, sugars and nucleotides, have the common characteristics of strong polarity and low volatility, are easily adsorbed by a chromatographic column, and are not suitable for direct GC-MS analysis. The methods mainly comprise an oximation-silanization derivation method and a chloroformate derivation method. Compared with silanization derivatization, the chloroformate derivatization method has higher derivatization efficiency and better derivatization stability for metabolites such as amino acid, organic acid and the like. Plasma metabolites were detected using GC-MS metabolomics approach, and static (CUMS model replication endpoint) and dynamic (CUMS model replication whole process) metabolomic data were analyzed using S-Plot and variance-synchronized principal components, respectively. The results show that static metabonomic analysis finds 3 different metabolites of propionic acid, D-allose and linolenic acid; dynamic metabonomics analysis obtains 7 different metabolites such as propionic acid, D-allose, inositol, butyric acid and the like. The differential analysis of urine metabolites of hepatitis B patients accompanied with depression is carried out by adopting GC-MS-based metabonomics research, and the 6 compounds are found to have significant difference with a control group, so that the method can be used for screening clinical markers of hepatitis B patients suffering from depression.

In conclusion, insomnia is a common disease and frequently-occurring disease in an outpatient department of neurology, and the clinical curative effect and mouse pharmacodynamic research prove that the anti-insomnia activity of the Sugelheir-4 is proved. However, the pathogenesis and the treatment mechanism of insomnia are not determined and elucidated, so that the influence of SugeMuller-4 on the metabolic pathway related to neurotransmitter in the brain of a rat is researched by adopting non-targeted metabonomics based on GC-MS and targeted metabonomics based on LC-MS, and the action mechanism and the target point of the disease are preliminarily proved.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provides a method for researching the insomnia resistance action mechanism of Sugeluer based on amino acid metabonomics, which can determine and clarify pathogenesis and treatment mechanism of insomnia on the basis that clinical curative effect and mouse pharmacodynamics research confirm the insomnia resistance activity of the Sugeluer-4;

the method for researching the insomnia resistance action mechanism of the Sugelia based on the amino acid metabonomics is to analyze amino acid metabolome in each tissue of a PCPA insomnia model rat brain before and after the gastric lavage of the Sugelia by applying a derivatization GC-MS method, identify endogenous compounds by a standard map library, analyze the change of metabolite spectrums of each part of the brain by adopting PCA and PLS-DA modes and identify related metabolic markers; carrying out targeted metabonomics research by applying LC-MS, and investigating the content changes of neurotransmitters such as 5-hydroxytryptamine, GABA, Glu and the like and related metabolites in brain tissues; the invention finds the differential metabolite and the metabolic pathway thereof related to the curative effect of the Sukiusjohne-4 in the brain tissue sample of the clinical rat, and discusses the mechanism and the site of action of the Sukiusjohne-4 in resisting insomnia.

In order to achieve the purpose of the invention, the following technical scheme is adopted for implementation.

The method for researching the insomnia resistance action mechanism of Sugeluer based on amino acid metabonomics is characterized in that: the method comprises the following steps:

firstly, constructing a PCPA insomnia rat model;

secondly, deriving the rat brain tissue metabolites by adopting a chloroformate derivation method, and optimizing the determination conditions;

thirdly, performing peak identification by GC-MS analysis and NIST database retrieval, and performing area normalization treatment;

fourthly, performing data analysis by using Principal Component Analysis (PCA) and partial least squares discriminant analysis (PLS-DA);

fifthly, simulating and analyzing a metabolic pathway by using Metabionalayst3.0 and a KEGG database, and determining potential biomarkers and related metabolic pathways of insomnia;

sixthly, determining the content of 5-hydroxytryptamine, 5-hydroxyindoleacetic acid, dopamine, norepinephrine, taurine, gamma-aminobutyric acid, glutamic acid neurotransmitter and related precursors and metabolites in the cortex, brainstem, hypothalamus and hippocampus tissues of the rat after administration by adopting an LC-MS/MS multi-response monitoring mode, and determining the action site of the Suogluca-4 on the synthesis and metabolism of the neurotransmitter in the brain.

Preferably, in the first step, the PCPA insomnia rat model is constructed by randomly dividing rats into a normal control group, a model control group, a Mongolian medicine group and a nerve-soothing and brain-nourishing oral liquid group according to weight, wherein each group comprises 10 rats; injecting PCPA suspension 1ml/100g into abdominal cavity of rats of other 3 groups except the normal control group at 8:00 am every day for 1 time every day, and injecting continuously for 2 days, wherein the normal control group is injected with alkalescent normal saline with the same volume into abdominal cavity; after 24-28h of the first abdominal cavity injection, the circadian rhythm of the animal disappears, and the activity is ceaseless in the daytime, which indicates that the model is successfully copied; injecting PCPA into abdominal cavity of rat for two days, after model replication is successful, beginning intragastric administration at 8:00 of morning every day on the third day, and once a day; the Mongolian medicine is administrated to the stomach in high, medium and low dose groups; the model control group is administrated with the oral liquid for soothing the nerves and nourishing the brain by intragastric administration with the same volume; the normal control group and the model control group are administered with distilled water with the same volume through intragastric administration; the administration is carried out continuously for 7 days by intragastric administration, and cervical vertebra removing treatment is carried out on the rat after the last administration for 60 min.

Preferably, in the second step, the derivation comprises the following steps:

firstly, cutting off the head of a rat, taking the brain, separating cortex, hippocampus, brainstem and hypothalamus on an ice tray, cutting each tissue along the central axis, and respectively storing the left side and the right side of each tissue in a refrigerator at the temperature of-80 ℃;

secondly, taking out the tissue from a refrigerator at the temperature of-80 ℃, placing the tissue in a homogenizer, adding 4 times of water, homogenizing for 30s until the tissue is free from fragments, taking 200 mu L of homogenate liquid, placing the 200 mu L of homogenate liquid in an EP tube, adding 125 mu L of chloroform and 375 mu L of methanol, carrying out ice bath ultrasound for 10min, taking 300 mu L of supernatant liquid, placing the supernatant liquid in a sample injection vial, and drying by nitrogen;

thirdly, 30. mu.L (15 mg. mL) of the residue was added^-1) Reacting with methoxylamine hydrochloride pyridine solution at 70 ℃ for 1h, adding 50 mu L of MSTFA (containing 1% TMCS), and reacting at 40 ℃ for 90 min;

fourthly, 500. mu.L of n-heptane (containing 0.1 mg. mL) is added finally^-1Tetracosane), vortexed for 1min, and then subjected to GC-MS analysis.

Preferably, in the second step, the determination conditions comprise chromatography and mass spectrometry detection conditions, the chromatography detection conditions comprise a chromatographic column, namely a DB-5MS capillary column, the sample injection amount is 1 muL, the sample injection port temperature is 260 ℃, the ion source temperature is 200 ℃, the temperature programming starting temperature is 60 ℃, the temperature is kept for 2min, and the temperature is controlled at 7 ℃ min^-1Heating to 140 deg.C, and cooling at 5 deg.C/min^-1Heating to 180 deg.C, maintaining for 66min, and heating to 5 deg.C/min^-1Raising to 280 ℃, keeping for 2min, carrier gas: helium, carrier gas flow rate: 1mL min-1;

mass spectrum detection conditions: ionization mode EI, electron energy 70eV, mass spectrometry scan range: full scan mode, m/z50 ~ 650.

Preferably, in step three, the GC-MS analysis comprises the following steps:

converting the RAW format into a netCDF format by GC-MS data through GC-MS instrument workstation software Xcalibur;

secondly, importing the txt file generated by the software into Excel software for surface normalization processing;

thirdly, carrying out Principal Component Analysis (PCA) and least square discriminant analysis (PLS-DA) by adopting a metabonomic software SIMCA P11.0 software package, and searching for biomarkers through VIP values (the contribution of each principal component variable to each separated group) in a loading map (loading map) on the premise of separating the score map;

and fourthly, searching a matched binding standard control through a standard mass spectrum database (NIST05) and identifying partial metabolites.

Preferably, the load map in step three is a loading map.

Preferably, the VIP value is the magnitude of each principal variable's contribution to separating groups.

Preferably, in the sixth step, the neurotransmitter and related metabolites content in the brain tissue is determined by cutting the head of a rat to take out the brain, separating the cortex, hippocampus, brainstem and hypothalamus from the ice tray, cutting each tissue along the central axis, and storing each tissue on the left and right sides in a-80 ℃ refrigerator; weighing 100mg of brain tissues of different parts in a freezing environment respectively, preparing brain tissue homogenate on ice by using 0.1mol/L phosphate buffer solution, removing protein by using 0.4mol/L perchloric acid, centrifuging and precipitating, and measuring the supernatant; the determination conditions include: chromatographic conditions are as follows: c18 chromatographic column with flow rate of 0.2ml/min and mobile phase of 0.1% formic acid solution (A) and acetonitrile (B) for gradient elution, wherein the flow rate is 0.2ml/min, the mobile phase is 0.1% formic acid solution (A) and acetonitrile (B), the gradient elution is carried out for 0-2min, 20% B, 2-4min, 20% B, 40-5 min, the gradient elution is carried out for 40% B, 5-7min, 20% B and the column temperature is 40 ℃; the ionization being ESI⁺The atomization gas is nitrogen, the collision gas is argon, the temperature of the DL tube is 250 ℃, the flow rate of the heating gas is 10L/min, the flow rate of the atomization gas is 3L/min, the temperature of the heating module is 400 ℃, and the temperature of the ion source is 300 ℃; the detection mode is MRM, and the quantification is carried out by an external standard method.

Preferably, after the MRM is optimized by a preliminary experiment, the quantitative parameters of the LC-MS/MS-determination neurotransmitter MRM are determined as follows:

advantageous effects

The invention firstly applies a metabonomics research means, systematically researches the influence of the Suaeda glauca-4 on the synthesis, metabolism, distribution and sum of neurotransmitters in the brain, explores an action mechanism of the Mongolian medicine, namely the Suaeda glauca-4, for regulating the content of the neurotransmitters at different parts in the brain, determines an action target point of the anti-insomnia active component of the Suaeda glauca-4, and preliminarily discovers the action mechanism of the Suaeda glauca-4 for regulating sleep-wake rhythm through the neurotransmitter system.

Drawings

FIG. 1 is a technical roadmap for the present invention;

FIG. 2 is a GC-MS analysis spectrum of Scotland Muller-4 essential oil;

FIG. 3 is an LC-MS/MS separation profile (TIC and MRM) of 9 neurotransmitters.

Detailed Description

The invention is further described with reference to the accompanying drawings and examples.

In one example, as shown in figure 1, the method for studying the mechanism of action of scotch on insomnia based on amino acid metabolomics is as follows:

firstly, making PCPA-insomnia rat model

Randomly dividing rats into a normal control group, a model control group, a Mongolian medicine group and a nerve-soothing and brain-nourishing oral liquid group according to weight, wherein each group comprises 10 rats; injecting PCPA suspension l ml/100g into abdominal cavity of rats of other 3 groups except the normal control group at 8:00 am every day for 1 time every day for 2 days continuously, and injecting alkalescent normal saline with the same volume into abdominal cavity of the normal control group; after 24-28h of the first abdominal cavity injection, the circadian rhythm of the animal disappears, and the activity is ceaseless in the daytime, which indicates that the model is successfully copied; injecting PCPA into abdominal cavity of rat for two days, after model replication is successful, beginning intragastric administration at 8:00 of morning every day on the third day, and once a day; the Mongolian medicine is administrated to the stomach in high, medium and low dose groups; the positive control group is administrated with the oral liquid for soothing the nerves and nourishing the brain by intragastric administration with the same volume; the blank control group and the model control group are subjected to intragastric administration with distilled water of the same volume; the administration is carried out continuously for 7 days by intragastric administration, and cervical vertebra removing treatment is carried out on the rat after the last administration for 60 min.

Secondly, deriving the rat brain tissue metabolites by adopting a chloroformate derivation method, and optimizing the determination conditions.

Cutting rat head, collecting brain, separating cortex, Hippocampus, brainstem and hypothalamus on ice tray, cutting each tissue along central axis, and storing each tissue in refrigerator at-80 deg.C;

taking out the tissue from a refrigerator at minus 80 ℃, placing the tissue in a homogenizer, adding 4 times of water, homogenizing for 30s until no tissue fragments, taking 200 mu L of homogenate liquid, placing in an EP tube, adding 125 mu L of chloroform and 375 mu L of methanol, carrying out ice bath ultrasonic treatment for 10min, taking 300 mu L of supernatant liquid, placing in a sample injection vial, and drying by nitrogen;

to the residue was added 30. mu.L (15 mg. mL)^-1) Methoxamidine hydrochloride solution was reacted at 70 ℃ for 1h, then 50. mu.L MSTFA (containing 1% TMCS) was added and reacted at 40 ℃ for 90 min. Finally, 500. mu.L of n-heptane (containing 0.1 mg. multidot.mL) was added^-1Tetracosane), vortexed for 1min, and subjected to GC-MS analysis.

Drawing chromatographic and mass spectrum conditions, and chromatographic detection conditions: the chromatographic column comprises DB-5MS capillary column, with sample volume of 1 μ L, sample inlet temperature of 260 deg.C, ion source temperature of 200 deg.C, programmed temperature start temperature of 60 deg.C, and holding time of 7 deg.C/min^-1Heating to 140 deg.C, and cooling at 5 deg.C/min^-1Heating to 180 deg.C, maintaining for 66min, and heating to 5 deg.C/min^-1Raising to 280 ℃, keeping for 2min, carrier gas: helium, carrier gas flow rate: 1 mL. min^－1。

Thirdly, performing peak identification and area normalization processing by adopting GC-MS analysis and NIST database retrieval, and comprising the following steps of:

converting the RAW format into a netCDF format by GC-MS instrument workstation software Xcalibur;

then importing the txt file generated by the software into Excel software for surface normalization processing;

and fourthly, performing data analysis by using Principal Component Analysis (PCA) and partial least squares discriminant analysis (PLS-DA).

Adopting a metabonomics software SIMCA P11.0 software package to carry out Principal Component Analysis (PCA) and least square discriminant analysis (PLS-DA), and searching for biomarkers through VIP values (the size of contribution of each principal component variable to each separated group) in a loading map (loading map) on the premise of separating the score map;

finally, the matching and the standard substance control are retrieved through a standard mass spectrum database (NIST05) to identify partial metabolites.

And fifthly, analyzing the metabolic pathways by using Metabionalayst3.0 and KEGG databases, and exploring potential biomarkers and related metabolic pathways of insomnia.

Sixth, determination of neurotransmitter and related metabolite content in brain tissue

100mg of brain tissue of different parts are weighed respectively in a freezing environment, brain tissue homogenate is prepared on ice by using 0.1mol/L phosphate buffer solution, protein is removed by using 0.4mol/L perchloric acid, centrifugal precipitation is carried out, and the supernatant is used for determination.

Establishing a measuring method, wherein the detection mode to be adopted is MRM, and quantifying by an external standard method.

The measurement conditions include: chromatographic determination conditions: c18 chromatographic column (Waters BEH, 2.0 x 100mm, 1.7 μm), flow rate of 0.2ml/min, mobile phase of 0.1% formic acid solution (A) and acetonitrile (B) gradient elution, 0-2min, 20% B, 2-4min, 20% B-40% B, 4-5min, keeping 40% B, 5-7min, 20% B, column temperature of 40 deg.C;

mass spectrometry conditions: the ionization being ESI⁺The atomization gas is nitrogen, the collision gas is argon, the temperature of the DL tube is 250 ℃, the flow rate of the heating gas is 10L/min, the flow rate of the atomization gas is 3L/min, the temperature of the heating module is 400 ℃, and the temperature of the ion source is 300 ℃.

The determination conditions of MRM are optimized through preliminary experiments, and the determined MRM conditions are detailed in the following table:

LC-MS/MS determination of quantitative parameters of neurotransmitter MRM

In one embodiment, the pharmacodynamics of Sugelle-4 against insomnia

1 materials and methods

1.1 animals

100 ICR mice, male, with a body mass of 18-22 g, were provided by Schbefu (Beijing) Biotechnology Inc., and the animal certification number SCXK (Beijing) 2016-.

1.2 instruments

Toy activity recorder, model: YLS-1C, Jinan Yiyan science and technology development Limited; electronic balance, model: AL204-IC, mettler-toledo; vacuum drying oven, model: DZF-2B, Yongguanming medical instruments, Inc., Beijing; rotary evaporator, type: r-215, Buchi; a bench high speed refrigerated centrifuge, model TGL-20M.

1.3 herbs

The fructus Amomi rotundus is fruit of Amomum cardamomum and Amomum javanicum of Zingiberaceae, and has batch number: 20160801, respectively; the chicory peptide is the seeds of the lettuce of the compositae, and has the batch number: 20160401; the fructus Piperis Longi is dry mature or mature fruit cluster of fructus Piperis Longi of Piper of Piperaceae, and has a lot number: 20170101, respectively; the spina date seed is dry mature seed of Rhamnaceae plant ziziphus jujuba, and has the following batch number: 20161201. the medicinal materials are all purchased from Anhui and Jitang Chinese medicinal decoction pieces Limited. Pentobarbital sodium was purchased from Shanghai preparation II.

1.4 preparation of the medicament

The traditional Chinese medicine composition is prepared from 15g of amomum cardamomum, 10g of chicory, 5g of long pepper and 15g of fried spina date seed, 10 parts of the 4 medicinal materials are weighed, the total amount is 450g, 10 times of water is added, the medicinal materials are soaked for 1 hour and then decocted, the medicinal materials are filtered after being boiled for 30min, the medicinal residues are added with 5 times of water and then decocted, the medicinal residues are filtered after being boiled for 20min, the filtrates are combined, and a rotary evaporator is used for reducing pressure and drying the mixture to obtain extract, so that 58g of extract is obtained. It is diluted with distilled water to obtain the corresponding crude drug concentration.

1.5 mouse autonomic Activity assay

100 ICR male mice are screened after adaptive feeding for 7 days and before experiments. The screening method comprises the following steps: 100 mice are respectively placed in a mouse spontaneous activity instrument, 8 mice are placed each time, after the mice are adaptive to the environment for 5min, the activity times within 5min are recorded, and 80 mice with the same activity number are selected for experiments. The experiment was carried out in a barrier environment with a light rhythm of 12L: 12D (9:00-21:00), quiet, ventilated, and room temperature of about 22 ℃. The screened mice were randomly divided into 5 groups by body weight: normal control group, positive control group of tranquilizing and brain tonifying solution, and high, medium, and low dosage groups of Sugeli-4 decoction. The mice are sequentially placed in a mouse spontaneous activity instrument, and after the mice are adapted to the environment for 5min, the number of autonomous activities for 5min is recorded. Thereafter, each group of mice was gavaged with a volume of 0.2ml/10 g. The control group of the tranquilizing and brain-nourishing liquid is administered by intragastric administration at a concentration of 0.13ml/10g, and the high, medium and low dosage groups of the Sugeli-4 decoction are administered by intragastric administration at a concentration of 52mg/10g, 26mg/10g and 13mg/10g, respectively. Before gavage, the mice are fasted for 12 hours without water prohibition, the mice are continuously gavaged for 7 days after 1 time of gavage every day, and the number of the autonomic activities of the mice is recorded by the same method after the last gavage administration for 60 min.

1.6 pentobarbital sodium hypnotic effect

1.6.1 Experimental methods

Each group of mice was administered 1 time daily by gavage for 7 days continuously, and a threshold dose of sodium pentobarbital was injected intraperitoneally 1h after the last administration (39 mg kg as determined by preliminary experiments)^-1)。

1.6.2 judgment index

The righting reflection disappears: the mice do not turn over within 1min after lying on the back, which indicates that the turning-over positive reflection disappearance stage is started; (2) righting reflection recovery: the mouse can automatically turn over from the supine position and does not turn over within 1min, indicating that the righting reflex is recovered; (3) latency, the time from the start of injection to the disappearance of the righting reflex; (4) sleep time: the time taken from disappearance of the righting reflection to restoration of the righting reflection.

1.7 experiment of subthreshold hypnotic effect of sodium pentobarbital in mice

1.7.1 Experimental methods

Each group of mice was administered 1 time daily by gavage for 7 days continuously, and the abdominal cavity was injected with a threshold dose of sodium pentobarbital 1h after the last administration (28 mg. kg as determined by preliminary experiments)^-1)。

1.7.2 judgment index

And (3) after the mice are injected with the pentobarbital sodium, the inversion and positive reflection disappear within 15min, and the mice are positive if the inversion and positive reflection disappear for more than 1min, and the sleep latency period and the inversion and positive reflection disappear duration time of the mice are recorded.

1.8 serum Biochemical index detection

The mice were anesthetized 1 hour after the completion of gavage on the seventh day and then the eyeballs were removed to obtain blood. The blood was centrifuged in a 1.5ml EP tube in a high speed centrifuge and the supernatant was taken for biochemical index detection.

1.9 statistics of data

The experimental data measurement data are expressed by means of +/-standard deviation of mean values, the experimental results are processed by adopting One-Way ANOVA (One-Way ANOVA) for multi-group comparison, Leven's variance homogeneity test is firstly carried out, multiple comparison is carried out by adopting an LSD (least squares difference) method when variance homogeneity and the overall comparison groups have significant differences, and multiple comparison is not carried out when variance analysis is not significant. Variance was checked by non-parametric Kruskal-Wallis test. The counting data are expressed as percentages and checked by chi-square. The statistical analysis of the normality test and the homogeneity test of variance takes P & gt 0.1 as the boundary of significance of difference, and all the other statistical analyses take P & lt 0.05 as the boundary of significance of difference.

2 results

2.1 Effect on the number of autonomous mouse Activities

Multiple comparisons were further performed using analysis of variance, alignment of variances, statistical significance for the overall comparison (F3.442, P0.012 < 0.05). The scotch-4 high dose group was statistically significant compared to the normal group (P ═ 0.004); the dose group in scotch-4 was statistically significant compared to the normal group (P ═ 0.037); the low dose group of the Sugeli-4 has statistical significance (P is 0.01) compared with the normal group, the oral liquid for soothing the nerves and nourishing the brain has no statistical significance (P is 0.07) compared with the normal group, and the oral liquid for soothing the nerves and nourishing the brain has no statistical significance (P is more than 0.05) compared with each dose group of the Sugeli-4. The comparison of each dose group of Sugeli-4 and the group of the oral liquid for soothing the nerves and tonifying the brain before and after the administration has statistical significance (P is less than 0.05). The results are shown in Table 1.

TABLE 1 comparison of the number of autonomic activity in mice in each group

2.2 Effect on the hypnotic Effect of sodium pentobarbital in mice

The sleep latency is statistically significant by anova, which is uniform in variance and overall comparison (F2.879, P0.032 < 0.05). Compared with a blank control group, the scotch-4 high dose group (P ═ 0.016) and the mind-tranquilizing and brain-nourishing oral liquid group (P ═ 0.003) can effectively shorten the sleep latency of mice, and the effects of the medium dose group (P ═ 0.055) and the low dose group (P ═ 0.164) are not obvious. The results are shown in Table 2.

TABLE 2 comparison of the Effect of groups of mice on sleep latency of sodium pentobarbital

The sleep time does not conform to the normal distribution, so the overall comparison is meaningful by a nonparametric test (P is 0.02< 0.05). Each dose group of Sugeli-4 and the oral liquid group for soothing the nerves and tonifying the brain can effectively prolong the sleeping time of mice caused by suprathreshold dose of sodium pentobarbital and enhance the sleep-promoting effect of the sodium pentobarbital, and the results are shown in Table 3.

TABLE 3 comparison of the Effect of groups of mice on sleep time of sodium pentobarbital

2.3 Effect on subthreshold hypnotic Effect of sodium pentobarbital in mice

Compared with a blank control group, each dosage group of the Sukimueller-4 and the nerve-soothing and brain-nourishing oral liquid group can improve the sleep rate of mice, and the high dosage effect of the Sukimueller-4 is sequentially higher than that of a medium dosage low dosage group and a nerve-soothing and brain-nourishing oral liquid group. The results are shown in Table 3.

TABLE 4 comparison of Effect of groups of mice on subthreshold hypnotic dose of sodium pentobarbital

2.4 Biochemical index detection results

The biochemical test results of each dose group of Sugemuer-4 and a distilled water group are expressed by mean plus or minus standard deviation, the test results are processed by adopting One-Way analysis of variance (One-Way ANOVA) for comparison among multiple groups, Leven analysis of variance homogeneity is firstly carried out, multiple comparison is carried out by adopting an LSD (least squares differential) method when the analysis of variance is not obvious and the multiple comparison is not carried out when the analysis of variance is not obvious. Variance was checked by non-parametric Kruskal-Wallis test. The results after data processing showed that the administration groups of ALT, AST/ALT, GLU, GGT, UA-G, CRE-G, TBIL, DBIL, IDBIL indices had no statistical significance (P > 0.05) compared with the blank group. The CHOL index makes sense to compare the overall results (P0.014 <0.05), two-by-two comparisons show that the low dose group makes sense to the blank dose group (P0.049), the low dose group makes sense to the medium dose group (P0.02), and the rest are meaningless. The TG index has significance in comparison with the overall result, but the analysis of variance is not obvious, and multiple comparison is not carried out. The results are shown in Table 5.

TABLE 5 Biochemical index test results of mice in each group

3 conclusion

In the experiment, the representative index of the sedative effect is the number of the independent activities of the mouse, and the representative index of the hypnotic effect is the sleep-in latent period and the disappearance duration of the positive turning reflex of the mouse after the pentobarbital sodium is injected. The results show that:

(1) the times of autonomous activity of mice in each dose group of Sugeli-4 and the group of the oral liquid for soothing nerves and nourishing brain are reduced compared with the normal control group and the groups before and after administration. However, the Sugeller-4 dose groups have statistical significance compared with the normal group, and show more obvious sedation. The oral liquid for soothing the nerves and nourishing the brain has no statistical significance compared with the normal group, and the oral liquid for soothing the nerves and nourishing the brain has no statistical significance compared with the Sugeli-4 dosage groups. The comparison between each dose group of Sugeli-4 and the oral liquid group for soothing the nerves and tonifying the brain before and after the administration has statistical significance.

(2) Compared with a blank control group, the high-dose group and the nerve-soothing and brain-nourishing oral liquid group of the Sugeli-4 can effectively shorten the sleep latency of mice, but the effects of the medium-dose group and the low-dose group are not obvious. The suger Muller-4 is suggested to have a correlation between the sedative-hypnotic effect on mice and the dose thereof, and the larger the dose is, the stronger the sedative-hypnotic effect on mice is.

(3) Each dose group of Sugeli-4 and the nerve-soothing and brain-nourishing oral liquid group can prolong the sleeping time of mice caused by suprathreshold dose of sodium pentobarbital and enhance the hypnotic effect of the sodium pentobarbital.

(4) Compared with a blank control group, each dose group of the Sukimueller-4 and the nerve-soothing and brain-nourishing oral liquid group can improve the sleep rate of mice, and the high dose effect of the Sukimueller-4 is sequentially higher than that of a medium dose low dose group and a nerve-soothing and brain-nourishing oral liquid group. Suggesting that the sleep-promoting effect of the Sugelia-4 on the mice is related to the dosage of the Sugelia-4, and the higher the dosage is, the stronger the sleep-promoting effect on the mice is.

(5) Biochemical detection of mice shows that no obvious difference is found in liver and kidney functions of experimental mice and normal mice.

The experimental result shows that the Sukimueller-4 has obvious sedative hypnotic effect, and the high-dose effect is particularly obvious.

In one example, as shown in figure 2, the chemical composition of the essential oil of sujoller-4 was identified.

Extracting Suaeda salsa-4 essential oil components by adopting a simultaneous distillation extraction method, and performing preliminary qualitative analysis on the essential oil by using GC-MS (gas chromatography-mass spectrometry), wherein the Suaeda salsa-4 extracting solution contains sesquiterpene, organic acid, lipid, alkane, piperine and saponin components, and the currently characterized compounds comprise 65 compounds, namely eucalyptol, alpha-terpineol, beta-elemene, allocarpene, rhynchophyllene, cedrene, copaene, alpha-purple scholarynene, alpha-piperonylene, pentadecane, beta-bisabolene, alpha-caryophyllene, nerolidol, caryophyllene oxide, feulenol, feulegol, nonadecane, cyclooctasiloxane, beta-bisabolol, 3-heptadecene, 8-heptadecene, heptadecane, tetradecanal, ethyl octadecanoate, ethyl octadecanoic acid, ethyl gallate, and the rest of the plant, 5-butylnonane, hexyloctyl sulfite, tetradecyl oxirane, 2- (2-biethylamino) ethanol, 3,7,11, 15-tetramethyl-2-hexadecenol, Z-5-nonadecene, 9-nonadecene, heneicosane, -3, 7,11, 15-tetramethyl-1, 3,6,10, 14-hexadecapentaene, methyl hexadecanoate, dimethyldecalin, hexadecanoic acid, ethyl hexadecanoate, methyl 9, 12-octadecanoate, eicosane, linseed oil, oleic acid, ethyl linoleate, ethyl oleate, ethyl stearate, diglycerol stearate, heneicosane, linoleoyl chloride, trans-9-hexadecenal, isopropyl 9, 10-epoxy-octadecanoate, ethyl 9-hexadecanoate, Eicosanoic acid ethyl ester/arachidic acid, docosane, beta-monoalkene, oleoyl chloride, octadecenoic acid, 1,2, 3-propanetriacetate, 2-methylhexadecanol, fumaric acid, 6, 9-pentadecadienol, 9, 12-octadecadienoic acid (Z, Z) -, 2, 3-dihydroxypropyl ester, 4- (3, 4-methylenedioxybenzyloxy) -furan-3-amine, linoleic acid, gamma-tocopherol, tetracosane, piperine, squalene, heptacosanol, (R) - (-) -14-methyl-8-hexadecen-1-ol, stigmasterol, gamma-sitosterol, stigmast-4-en-3-one. The research result is the initial exploration of the weak polar compound in the Scorzonera-4, and provides reference for the subsequent structural identification of partial compounds of petroleum ether. The Scotusjordii-4 essential oil GC-MS analysis spectrum is shown in figure 2.

In one embodiment, a neurotransmitter LC-MS/MS detection method is established, as shown in FIG. 3.

Ultra-high performance liquid chromatography mass spectrometer (Shima) using triple quadrupoledzu LC-MS/MS 8050) established a qualitative and quantitative detection method for neurotransmitters. The experimental conditions were as follows: chromatographic conditions: c18 chromatographic column (Watts BEH, 2.0 x 100mm, 1.7 μm), mobile phase 0.1% formic acid solution (A) and acetonitrile (B) flow rate 0.2ml/min, gradient elution, 0-2min, keeping 20% B, 2-4min, B from 20% to 40%, 4-5min, keeping 40% B, 5-7min, 20% B, column temperature 40 ℃. The ionization being ESI⁺The atomization gas is nitrogen, the collision gas is argon, the temperature of the DL tube is 250 ℃, the flow rate of the heating gas is 10L/min, the flow rate of the atomization gas is 3L/min, the temperature of the heating module is 400 ℃, and the temperature of the ion source is 300 ℃. The detection mode is MRM (MRM parameters are shown in an attached table in an experimental means in detail), the acquisition time is 5min, the external standard method is used for quantification, and the determined LC-MS/MS determination neurotransmitter MRM quantitative parameters are as follows:

the neurotransmitter standard isolation profile is shown in FIG. 3.

Claims

1. The method for researching the insomnia resistance action mechanism of the Sugelia based on the amino acid metabonomics is characterized by comprising the following steps: the method comprises the following steps:

firstly, constructing a PCPA insomnia rat model;

secondly, injecting PCPA into the abdominal cavity of the rat for two days, and after the model is successfully copied, beginning intragastric administration at 8:00 of the morning every day on the third day, and taking the administration once a day; the Mongolian medicine is administrated to the intragastric administration in high, medium and low dose groups; the positive control group is administrated with the oral liquid for soothing the nerves and nourishing the brain by intragastric administration with the same volume; the blank control group and the model control group are subjected to intragastric administration with distilled water of the same volume; continuously performing intragastric administration for 7 days, and performing cervical vertebra removal treatment on the rat after the last administration for 60 min; adopting a chloroformate derivation method to derive the metabolites of the brain tissues of the rats, and optimizing the determination conditions;

thirdly, performing peak identification and area normalization processing by adopting GC-MS analysis and NIST database retrieval;

sixthly, determining the content of 5-hydroxytryptamine, 5-hydroxyindoleacetic acid, dopamine, norepinephrine, taurine, gamma-aminobutyric acid, glutamic acid neurotransmitter and related precursors and metabolites in tissues of a rat brain cortex, a brain stem, a hypothalamus and a hippocampus after administration by adopting an LC-MS/MS multi-reaction monitoring mode, determining the action site of the Suoglauca-4 on the anabolism of the neurotransmitter in the brain, and clarifying the mechanism of the Suoglauca-4 for resisting insomnia.

2. The method for studying the mechanism of action of scotch on insomnia based on amino acid metabonomics according to claim 1, wherein: in the first step, the construction method of the PCPA insomnia rat model is that rats are randomly divided into a normal control group, a model control group, a Mongolian medicine group and a nerve-soothing and brain-nourishing oral liquid group according to weight, and each group comprises 10 rats; injecting PCPA suspension lml/100g into the abdominal cavity of rats in other 3 groups except the normal control group at 8:00 a.m. every day for 1 time every day and continuously injecting for 2 days, and injecting alkalescent normal saline with the same volume into the abdominal cavity of the normal control group; after the first intraperitoneal injection is carried out for 24-28h, the circadian rhythm of the animal disappears, and the activity is ceaseless in the daytime, which indicates that the model is copied successfully.

3. The method for studying the mechanism of action of scotch on insomnia based on amino acid metabonomics according to claim 1, wherein: in the second step, the derivation comprises the following steps:

thirdly, adding 15 mg/mL-1 of 30 mu L of methoxylamine hydrochloride pyridine solution into the residue, reacting for 1h at 70 ℃, adding 50 mu L of MSTFA containing 1% TMCS, and reacting for 90min at 40 ℃;

fourthly, finally adding 500 mu L of n-heptane containing 0.1 mg/mL-1 tetracosane, vortexing for 1min, and then carrying out GC-MS analysis.

4. The method for studying the mechanism of action of scotch on insomnia based on amino acid metabonomics according to claim 1, wherein: in the second step, the determination conditions comprise chromatographic and mass spectrometric detection conditions, the chromatographic detection conditions comprise a chromatographic column DB-5MS capillary column, the sample introduction amount is 1 mu L, the injection port temperature is 260 ℃, the ion source temperature is 200 ℃, the programmed temperature rise starting temperature is 60 ℃, the temperature is kept for 2min, the temperature is increased to 140 ℃ at 7 ℃ min < -1 >, the temperature is increased to 180 ℃ at 5 ℃ min < -1 >, the temperature is kept for 66min, the temperature is increased to 280 ℃ at 5 ℃ min < -1 >, the temperature is kept for 2min, and the carrier gas: helium, carrier gas flow rate: 1mL min-1; mass spectrum detection conditions: ionization mode EI, electron energy 70eV, mass spectrometry scan range: full scan mode, m/z50 ~ 650.

5. The method for studying the mechanism of action of scotch on insomnia based on amino acid metabonomics according to claim 1, wherein: in step three, the GC-MS analysis method comprises the following steps:

converting RAW format into netCDF format by GC-MS data through GC-MS instrument workstation software Xcalibur;

thirdly, carrying out Principal Component Analysis (PCA) and least square discriminant analysis (PLS-DA) by adopting a metabonomic software SIMCA P11.0 software package, and searching for the biomarker through a VIP value in a load map on the premise of separating a score map;

and fourthly, retrieving a matched combination standard product control through a standard mass spectrum database (NIST05) to identify partial metabolites.

6. The method for studying the mechanism of action of scotch on the basis of amino acid metabonomics according to claim 5, which is characterized in that: the load graph in the third step is a loading graph; the VIP value is the magnitude of each principal variable's contribution to separating groups.

7. The method for studying the mechanism of action of scotch on insomnia based on amino acid metabonomics according to claim 1, wherein: in the sixth step, the determination of the content of the neurotransmitter and the related metabolites in the brain tissue is that the rat is cut off the head to take the brain, the cortex, the hippocampus, the brain stem and the hypothalamus are separated on an ice tray, each tissue is cut along the central axis, and the left side and the right side of each tissue are respectively preserved in a refrigerator at the temperature of minus 80 ℃; weighing 100mg of brain tissues of different parts in a freezing environment respectively, preparing brain tissue homogenate on ice by using 0.1mol/L phosphate buffer solution, removing protein by using 0.4mol/L perchloric acid, centrifuging and precipitating, and using supernatant for determination; the determination conditions include: chromatographic conditions are as follows: c18 chromatographic column with flow rate of 0.2ml/min and mobile phase of 0.1% formic acid solution (A) and acetonitrile (B) for gradient elution, wherein the flow rate is 0.2ml/min, the mobile phase is 0.1% formic acid solution (A) and acetonitrile (B), the gradient elution is carried out for 0-2min, 20% B, 2-4min, 20% B, 40-5 min, the gradient elution is carried out for 40% B, 5-7min, 20% B and the column temperature is 40 ℃; the ionization mode is ESI +, the atomizing gas is nitrogen, the collision gas is argon, the temperature of the DL tube is 250 ℃, the heating gas flow is 10L/min, the atomizing gas flow is 3L/min, the temperature of the heating module is 400 ℃, and the temperature of the ion source is 300 ℃; the detection mode is MRM, and the quantification is carried out by an external standard method.

8. The method for studying the mechanism of action of scotch on insomnia based on amino acid metabonomics according to claim 1, wherein: after the MRM is optimized by a pre-experiment, the determined LC-MS/MS determination neurotransmitter MRM quantitative parameters are as follows: