CN114452313A - Application of fructus gleditsiae in preparing medicine for treating cerebral ischemia reperfusion injury - Google Patents
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Abstract
The invention provides application of fructus gleditsiae volatile oil in preparing a medicine for treating cerebral ischemia reperfusion injury, and the preparation method of the fructus gleditsiae volatile oil comprises the steps of taking a proper amount of fructus gleditsiae decoction pieces, adding water and normal hexane, carrying out steam distillation to obtain volatile oil, collecting the volatile oil, adding a proper amount of anhydrous sodium sulfate to remove water, and standing overnight to obtain the fructus gleditsiae volatile oil. The prepared fructus gleditsiae volatile oil acts on key targets of VEGFA, SRC, MAPK8, PIK3CA and TNF through active ingredients of paeonol, linalool, methyl eugenol, anethole and eugenol, and regulates multiple signal paths of sphingolipid, TNF and VEGF, inhibits oxidative stress injury of cells, and inhibits release of inflammatory factors and nerve function regulation, thereby playing a role in treating cerebral ischemia reperfusion injury, and laying a foundation for further deep research on a therapeutic action mechanism of the fructus gleditsiae for 'opening orifices after passing through the meridians'.
Description
Technical Field
The invention relates to a new application of gleditsia sinensis volatile oil, in particular to an application of the gleditsia sinensis volatile oil in preparation of a medicine for treating cerebral ischemia reperfusion injury.
Background
The brain is the most sensitive organ to hypoxia, and ischemia (ischemia) of the brain tissue causes damage to local brain tissue and functions thereof, the damage degree is related to the ischemia time and residual blood flow, short-term incomplete ischemia only causes reversible damage, and long-term complete ischemia or severe ischemia causes infarction. Histological changes the most obvious histological changes of the brain are cerebral edema and brain cell necrosis. Brain edema occurs as a result of membrane lipid peroxidation which results in structural destruction of the membrane and dysfunction of the sodium pump. The pathophysiological mechanism is as follows: 1 free radical and cerebral ischemia reperfusion injury, 2 calcium overload and cerebral ischemia reperfusion injury, 3 excitatory amino acid and cerebral ischemia reperfusion injury, 4NO and cerebral ischemia reperfusion injury, 5 inflammatory reaction and cerebral ischemia reperfusion injury, and 6 cell apoptosis. Cerebral ischemia reperfusion injury is also called ischemic cerebral apoplexy in traditional Chinese medicine, and can be treated by selecting traditional Chinese medicines for resuscitation and resuscitation.
Fructus Gleditsiae Abnormalis is dry sterile fruit of Gleditsia sinensis (Gleditia sinensis Lam) of Leguminosae. In late autumn, the ripe fruits are picked, the impurities are removed, and the fruits are dried in the sun. The cylindrical shape is slightly flat and curved, and has a length of 5-11 cm and a width of 0.7-1.5 cm. The surface is purple brown or purple brown, and is wiped off by grey white waxy cream to be glossy, and fine warty bulges and linear or reticular cracks are formed. The top end is provided with a beak-shaped style residual, and the base part is provided with a fruit stem residual mark. Hard and brittle, easy to break, brown yellow cross section, loose middle, light green or light brown yellow filament, occasionally having underdeveloped seed. Slight smell, pungent and sweet taste. Pungent, salty and warm in nature; has little toxicity. It enters lung and large intestine meridians. Has the functions of eliminating phlegm, inducing resuscitation, dissipating stagnation and relieving swelling. Can be used for treating apoplexy, trismus, epilepsy, excessive phlegm, obstruction of orifices, sore throat, phlegm stagnation, intractable phlegm, asthma, cough, and constipation; it is indicated for abscess and swelling. Can be used for treating acute schistosomiasis, acute intestinal obstruction, etc. At present, the chemical components and the pharmacological activity of the fructus gleditsiae are researched, but no relevant data report is found in the research on cerebral ischemia-reperfusion. The research analyzes 64 components of the gleditsia sinensis volatile oil by using a GC-MS technology, and 24 active components are screened by a Swiss ADME platform, wherein the content of paeonol is the highest. According to the volatile oil-component-target point network diagram, the first 5 components with the largest matching target points are paeonol, linalool, methyl eugenol, anethole and eugenol. The research shows that the paeonol given in advance can obviously relieve inflammatory reaction and cerebral ischemia inflammation and improve the nerve defect symptoms of rats, thereby playing the role of resisting cerebral ischemia injury. Linalool has anti-inflammatory and antioxidant properties, has protective effects in vitro models of glutamate-induced oxidative stress and in vitro models of excitotoxicity, and can be used as a potential therapeutic agent against neurodegenerative brain diseases. Methyl eugenol can enter brain tissue through blood brain barrier to exert the function of treating brain diseases. The anethole can reduce the nerve function defect of mice, and reduce the cerebral infarction volume and cerebral edema, thereby having the protective effect on ischemic stroke. Eugenol can increase brain BDNF content through inhalation passage, and regulate brain function, thereby improving injury after ischemia. Based on the research reports and network pharmacological analysis, the gleditsia sinensis volatile oil is presumed to exert the cerebral ischemia-reperfusion treatment effect through the 5 core components.
Disclosure of Invention
In order to solve the technical problems, the invention provides application of fructus gleditsiae in preparation of a medicine for treating cerebral ischemia-reperfusion injury, and solves the problem of how to extract fructus gleditsiae volatile oil for treating cerebral ischemia-reperfusion injury. In order to realize the purpose, the invention is realized by the following technical scheme:
application of fructus Gleditsiae Abnormalis volatile oil in preparing medicine for treating cerebral ischemia reperfusion injury is provided.
A medicine for treating cerebral ischemia reperfusion injury is mainly prepared from fructus Gleditsiae Abnormalis volatile oil.
The medicine for treating cerebral ischemia-reperfusion injury comprises the following active ingredients in percentage by weight: 33.2 to 41.2 percent of paeonol, 3.8 to 11.8 percent of eugenol, 0.7 to 8.7 percent of methyl eugenol, 19.0 to 27.0 percent of linalool and 23.3 to 31.3 percent of anethole.
The medicine for treating cerebral ischemia-reperfusion injury comprises the following active ingredients in percentage by weight: 35.2 to 39.2 percent of paeonol, 5.8 to 9.8 percent of eugenol, 2.7 to 6.7 percent of methyl eugenol, 21.0 to 25.0 percent of linalool and 25.3 to 29.3 percent of anethole.
Specifically, the medicine for treating cerebral ischemia-reperfusion injury comprises the following active ingredients in percentage by weight: 37.2 percent of paeonol, 7.8 percent of eugenol, 4.7 percent of methyl eugenol, 23.0 percent of linalool and 27.3 percent of anethole.
The preparation method of the volatile oil of the fructus gleditsiae comprises the following steps: taking a proper amount of fructus gleditsiae decoction pieces, adding water and n-hexane, performing steam distillation to obtain volatile oil, collecting the volatile oil, adding a proper amount of anhydrous sodium sulfate to remove water, and standing overnight to obtain the fructus gleditsiae volatile oil.
Specifically, the preparation method of the volatile oil of the fructus gleditsiae comprises the following steps: cutting appropriate amount of fructus Gleditsiae Abnormalis decoction pieces, adding 8-12 times of water, collecting 5-15% n-hexane of fructus Gleditsiae Abnormalis decoction pieces according to g/ml, steam distilling for 6-14 hr to obtain volatile oil, collecting volatile oil, adding appropriate amount of anhydrous sodium sulfate to remove water, standing overnight to obtain fructus Gleditsiae Abnormalis volatile oil.
More specifically, the preparation method of the volatile oil of the fructus gleditsiae comprises the following steps: cutting appropriate amount of fructus Gleditsiae Abnormalis decoction pieces, adding 10 times of water, collecting 10% n-hexane of fructus Gleditsiae Abnormalis decoction pieces, steam distilling for 10 hr to obtain volatile oil, collecting volatile oil, adding appropriate amount of anhydrous sodium sulfate to remove water, and standing overnight to obtain fructus Gleditsiae Abnormalis volatile oil.
Application of fructus Gleditsiae Abnormalis in preparing medicine for treating cerebral ischemia reperfusion injury is provided.
A medicine for treating cerebral ischemia reperfusion injury comprises fructus Gleditsiae Abnormalis.
The invention has the beneficial effects that:
the method optimizes the preparation method of the volatile oil of the fructus gleditsiae on the basis of the traditional method, and the prepared volatile oil of the fructus gleditsiae acts on key targets such as VEGFA, SRC, MAPK8, PIK3CA, TNF and the like through active ingredients such as paeonol, linalool, methyl eugenol, anethole and eugenol, so that multiple signal paths such as sphingolipid, TNF, VEGF and the like are regulated, the oxidative stress injury of cells is inhibited, the release of inflammatory factors and the adjustment of nerve functions are inhibited, and therefore, the effect of treating the cerebral ischemia reperfusion injury is achieved, and the foundation is laid for further deeply researching the 'clearance and resuscitation' treatment action mechanism of the fructus gleditsiae.
Drawings
FIG. 1 EOGSL total ion diagram;
FIG. 2 is a Wein diagram of the intersection gene of EOGSL and CIRI;
FIG. 3 EOGSL-active ingredient-target network;
FIG. 4 EOGSL protein interaction network;
FIG. 5 core target GO enrichment analysis;
FIG. 6 core target KEGG enrichment analysis;
FIG. 7 is a schematic view of molecular docking;
FIG. 8 TTC staining results of rats in each group after operation (note: A. sham operation group; B. model group; C. nimodipine group; D. volatile oil group; E, F, H. effective component composition low, medium and high dose group);
FIG. 9 is a diagram of neuronal cell morphology in cortical regions of the sham operated group, model group and nimodipine group;
FIG. 10 is a diagram of the morphology of neuronal cells in cortical areas of the low dose group of the volatile oil component, the effective formulation, and the medium dose group of the effective formulation;
figure 11 is a graphical representation of neuronal cell morphology in cortical regions of the effective formulation high dose group.
In order that those skilled in the art will better understand the technical solution of the present invention, the present invention will be further described with reference to the following specific examples, which are not intended to limit the present invention.
Detailed Description
Example 1:
the process comprises the following steps:
taking a proper amount of fructus gleditsiae decoction pieces, cutting into pieces, precisely weighing 100g, adding 10 times of water and 10ml of n-hexane, performing steam distillation for 10h to obtain volatile oil, collecting the volatile oil, adding a proper amount of anhydrous sodium sulfate to remove water, and standing overnight to obtain the fructus gleditsiae volatile oil.
Taking 25mg of fructus Gleditsiae Abnormalis volatile oil, adding 3% Tween-80, and making into 10ml normal saline to obtain fructus Gleditsiae Abnormalis volatile oil nasal drop.
The efficacy is as follows: dispel phlegm and induce resuscitation, dissipate stagnation and relieve swelling, and treat cerebral ischemia reperfusion injury (ischemic cerebral apoplexy).
The using method comprises the following steps: the sprayer is sprayed into nasal cavity.
The usage and dosage are as follows: 0.8 ml/day (calculated according to the daily dosage of 1.5g for adult fructus Gleditsiae Abnormalis in the first part of 2020 edition of Chinese pharmacopoeia and the extraction rate of volatile oil of fructus Gleditsiae Abnormalis of 0.132%), and spraying into nasal cavity with sprayer three times a day.
Example 2:
the process comprises the following steps:
taking a proper amount of fructus gleditsiae decoction pieces, cutting into pieces, precisely weighing 100g, adding 8 times of water and 5ml of n-hexane, performing steam distillation for 6h to obtain volatile oil, collecting the volatile oil, adding a proper amount of anhydrous sodium sulfate to remove water, and standing overnight to obtain the fructus gleditsiae volatile oil.
Taking 25mg of fructus Gleditsiae Abnormalis volatile oil, adding 3% Tween-80, and making into 10ml normal saline to obtain fructus Gleditsiae Abnormalis volatile oil nasal drop.
The efficacy is as follows: dispel phlegm and induce resuscitation, dissipate stagnation and relieve swelling, and treat cerebral ischemia reperfusion injury (ischemic cerebral apoplexy).
The using method comprises the following steps: the sprayer is sprayed into nasal cavity.
The usage and dosage are as follows: 0.8 ml/day (calculated according to the daily dosage of 1.5g for adult fructus Gleditsiae Abnormalis in the first part of 2020 edition of Chinese pharmacopoeia and the extraction rate of volatile oil of fructus Gleditsiae Abnormalis of 0.132%), and spraying into nasal cavity with sprayer three times a day.
Example 3:
the process comprises the following steps:
taking a proper amount of fructus gleditsiae decoction pieces, cutting into pieces, precisely weighing 100g, adding 12 times of water and 15ml of n-hexane, performing steam distillation for 14h to obtain volatile oil, collecting the volatile oil, adding a proper amount of anhydrous sodium sulfate to remove water, and standing overnight to obtain the fructus gleditsiae volatile oil.
Taking 25mg of fructus Gleditsiae Abnormalis volatile oil, adding 3% Tween-80, and making into 10ml normal saline to obtain fructus Gleditsiae Abnormalis volatile oil nasal drop.
The efficacy is as follows: dispel phlegm and induce resuscitation, dissipate stagnation and relieve swelling, and treat cerebral ischemia reperfusion injury (ischemic cerebral apoplexy).
The using method comprises the following steps: the sprayer is sprayed into nasal cavity.
The usage and dosage are as follows: 0.8 ml/day (calculated according to the daily dosage of 1.5g for adult fructus Gleditsiae Abnormalis in the first part of 2020 edition of Chinese pharmacopoeia and the extraction rate of volatile oil of fructus Gleditsiae Abnormalis of 0.132%), and spraying into nasal cavity with sprayer three times a day.
Example 4:
the process comprises the following steps:
taking 3mg of the active ingredients of the volatile oil of the fructus gleditsiae (calculated according to the fact that the active ingredients of the volatile oil of the fructus gleditsiae account for 11.86% of the total components of the volatile oil of the fructus gleditsiae), wherein 37.2% of paeonol, 7.8% of eugenol, 4.7% of methyl eugenol, 23.0% of linalool and 27.3% of anethole, adding 3% of tween-80, and preparing into 10ml of normal saline to obtain the nasal drop of the volatile oil of the fructus gleditsiae.
The efficacy is as follows: dispel phlegm and induce resuscitation, dissipate stagnation and relieve swelling, and treat cerebral ischemia reperfusion injury (ischemic cerebral apoplexy).
The using method comprises the following steps: the sprayer is sprayed into nasal cavity.
The usage and dosage are as follows: 0.8 ml/day, sprayed into nasal cavity by sprayer three times a day.
Example 5:
the process comprises the following steps:
taking 6mg of the active ingredients of the volatile oil of the fructus gleditsiae, wherein the active ingredients of the volatile oil of the fructus gleditsiae comprise 37.2% of paeonol, 7.8% of eugenol, 4.7% of methyl eugenol, 23.0% of linalool and 27.3% of anethole, adding 3% of tween-80, and preparing into 10ml of normal saline to obtain the nasal drop of the volatile oil of the fructus gleditsiae.
The efficacy is as follows: dispel phlegm and induce resuscitation, dissipate stagnation and relieve swelling, and treat cerebral ischemia reperfusion injury (ischemic cerebral apoplexy).
The using method comprises the following steps: spraying into nasal cavity with sprayer
The usage and dosage are as follows: 0.8 ml/day, sprayed into nasal cavity by sprayer three times a day.
Example 6:
the process comprises the following steps:
taking 12mg of the active ingredients of the volatile oil of the fructus gleditsiae, wherein the active ingredients of the volatile oil of the fructus gleditsiae contain 37.2 percent of paeonol, 7.8 percent of eugenol, 4.7 percent of methyl eugenol, 23.0 percent of linalool and 27.3 percent of anethole, adding 3 percent of tween-80, and preparing into 10ml of normal saline to obtain the nasal drop of the volatile oil of the fructus gleditsiae.
The using method comprises the following steps: the sprayer is sprayed into nasal cavity.
The usage and dosage are as follows: 0.8 ml/day, sprayed into nasal cavity by sprayer three times a day.
The inventors carried out a number of experiments and the following were studies of the extraction method of the present invention:
extraction of volatile oil of fructus gleditsiae
1 medicinal materials and reagents
The fructus gleditsiae is purchased from Beijing Tongrentang: lot number 20180408; n-hexane (Rianlong Bohua pharmaceutical chemistry, Ltd., lot No. 20180110); an electric temperature-adjusting electric heating jacket (Tester instruments Co., Ltd., Tianjin, model: 98-1-B); electronic balance (SHIMADZU AUW20D)
2 method
Single factor experiment
Because various factors for extracting the volatile oil of the fructus gleditsiae by adopting a steam distillation method have no interactive influence, a single-factor investigation method is adopted to investigate the extraction process of the volatile oil of the fructus gleditsiae, the water addition amount, the extraction time and the n-hexane addition amount are taken as three factors, the extraction rate of the volatile oil of the fructus gleditsiae is taken as an investigation index, and the optimal extraction process of the volatile oil of the fructus gleditsiae is preferably obtained. The extraction rate is 100% of the volatile oil extraction amount/medicinal material amount.
2.1 investigation of time
Weighing 100g of fructus gleditsiae, 3 parts in total, adding 10 times of water and 10mL of n-hexane, and extracting by adopting a steam distillation method for 6, 8, 10 and 12 hours respectively; and collecting the n-hexane part, drying, weighing and calculating the extraction rate.
2.2 examination of the amount of n-hexane added
Weighing 100g of fructus gleditsiae, 3 parts in total, 10 times of water and 5mL, 10mL and 15mL of n-hexane respectively, and extracting for 10h by adopting a steam distillation method. And collecting the n-hexane part, drying, weighing and calculating the extraction rate.
2.3 examination of the amount of Water added
Weighing 100g of fructus gleditsiae, 3 parts in total, adding 10mL of n-hexane, wherein the water addition amount is respectively 8, 10 and 12 times of the amount of the medicinal materials, and extracting for 10 hours by adopting a steam distillation method. And collecting the n-hexane part, drying, weighing and calculating the extraction rate.
3 results
3.1 investigation of time
As can be seen from Table 1-1, the extraction rate was the best when the extraction time was 10 hours.
TABLE 1-1 Effect of time on extraction yield
3.2 examination of the amount of n-hexane added
As seen from Table 1-2, the extraction yield was the best when the amount of n-hexane was 10 mL.
TABLE 1-2 Effect of n-Hexane on extraction yield
3.3 examination of the amount of Water added
As can be seen from tables 1-3, the extraction yield was the best when the amount of water added was 10 times the amount of the medicinal material.
Tables 1-3 Effect of water addition on extraction yield
4 summary of the invention
Experimental results show that the optimal extraction process for extracting the volatile oil of the fructus gleditsiae by the steam distillation method is that 100g of medicinal materials are added with 10 times of medicinal material water and 10mL of normal hexane, and the steam distillation is carried out for 10 hours.
Second, predicting potential active ingredients of the gleditschia horrida volatile oil for improving cerebral ischemia reperfusion injury by network pharmacology
1. Database and software
A database: online human mendelian genetic database (OMIM, http:// www.omim.org /), Pubchem database (https:// Pubchem. ncbi. nlm. nih. gov /), Swiss ADME (http:// www.swissadme.ch /), Swiss Target Prediction (http:// www.swisstargetprediction.ch /), human gene annotation database (GeneCards, https:// www.genecards.org /), DAVID bioinformation resource website (https:// DAVID. nciff. gov /), veny 2.1.0(http:// bioinfogp. cnb. csnc. ic/tools/Venny/index. html.), STRING platform (https:// str. org /), online mapping website (www.bioinformatics.com.cn), protein structure database (https:// www.rcsb.org).
Software: the system comprises network topology data analysis software Cytospace 3.7.2, file format conversion software Open Babe 2.4.1, protein molecule docking software Auto Dock 4.2.6 and molecule simulation mapping software Py MOL 2.5.
2 method
2.1 determination of active ingredients of volatile oils
Taking a proper amount of fructus gleditsiae decoction pieces, cutting into pieces, precisely weighing 100g, adding 10 times of water and 10ml of n-hexane, performing steam distillation for 10h to obtain volatile oil, collecting the volatile oil, adding a proper amount of anhydrous sodium sulfate to remove water, and standing overnight to obtain the fructus gleditsiae decoction pieces. Weighing a proper amount of sample, placing the sample in a headspace bottle, sealing, and placing the headspace bottle in a water bath at 80 ℃ for balancing for 30 min. Keeping the temperature, extracting for 30min with solid phase micro-extraction needle, analyzing the extraction needle at the injection port for 5min after extraction is finished, searching the mass spectrogram obtained by GC-MS analysis with NIST spectral library, identifying the components of the sample, and calculating the relative content of each component.
Gas chromatography conditions: agilent HP-5MS column (0.25mm × 30m × 0.25 μm); the flow dividing ratio is that the flow is not divided; the temperature of a sample inlet is 270 ℃; temperature programming, wherein the temperature raising process comprises the following steps: the initial temperature is 70 deg.C, holding for 2min, and heating to 280 deg.C at 5 deg.C/min, and holding for 10 min. The carrier gas is high-purity helium, and the flow rate is 1.0 mL/min. Mass spectrum conditions: the ion source temperature is 230 ℃; the scanning mode is as follows: full Scan; the temperature of the quadrupole rods is 150 ℃;
2.2 network pharmacological analysis
2.2.1 screening of the active ingredients of Gleditsia sinensis volatile oil
Performing primary screening according to the matching degree of more than 90% in a NIST17 database, confirming the chemical structural formula of the screened chemical components by using a PubChem database, and screening the active components of the fructus gleditsiae by using two 'yes' in the similarity of the medicaments as screening conditions through a Swiss ADME network platform and gastrointestinal tract absorption 'high'.
2.2.2 query and determination of target
Submitting the 2D structural formula of the active ingredient of the gleditsia sinensis volatile oil obtained by screening to a Swiss Target Prediction platform, predicting the potential Target point of the active ingredient, and setting Probasic > 0.1; using "Cerebral ischemia reperfusion in" as key word, searching in Gene Cards database and OMIM database, setting Relevance score to be more than 10 to obtain Gene combination, and removing repeat value to obtain disease Gene target. And drawing a Wein diagram on the acquired volatile oil active component target spot and the cerebral ischemia reperfusion target spot Venny platform.
2.2.3 construction of Gleditsia sinensis volatile oil-active ingredient-target
And (3) introducing the active ingredients of the medicines and target points of intersection of the medicines and diseases into Cytoscape software, carrying out network visualization, and carrying out data analysis.
2.2.4 construction of protein-protein interaction network
To evaluate and integrate the protein-protein interaction relationship, a protein-protein interaction network, i.e., a PPI network, was constructed. Introducing a common target point of the gleditsia sinensis and cerebral ischemia-reperfusion into a String database, selecting an analyzed organism as a human (Homo Sapiens), with High confidence (High confidence) of more than 0.7, hiding 15 free proteins, obtaining PPI data among the proteins of intersection, storing the PPI data as a 'tsv' file, and introducing the file into Cytoscape3.7.2 software to construct a protein-protein interaction network. And analyzing the target value by using a Network analyzer function, and taking the target with the value more than or equal to 1.5 times of the average value as a core target.
2.2.5 enrichment analysis of GO and KEGG
And recording the core target into a DAVID database, selecting an analyzed organism as a human (Homo Sapiens), performing GO function enrichment and KEGG pathway enrichment analysis, and taking error occurrence rate (FDR) <0.05 as significant enrichment, wherein the smaller the FDR value is, the higher the significance degree is. And sequencing the FDR values from small to large, and drawing a GO function enrichment histogram (namely a secondary classification histogram comprising cell components, molecular functions and biological processes) and a KEGG passage enrichment bubble diagram by using a microbial message online tool from the first 10 positions.
2.2.6 molecular docking
Downloading the 3D structure of the target protein through a PDB database, storing the 3D structure in a PDB format, and introducing into pymol software to remove water molecules and ligands; and downloading a 3D structure of the components through a Pubchem database, storing the 3D structure as an SDF format, and converting the SDF format into a mol2 or PDB format by using Open babel software. And (3) introducing the processed molecular structures of the target protein and the compound into Autodock, carrying out conventional pretreatment on the molecular structures, then selecting a Docking module for molecular Docking, and analyzing the binding activity of the target protein and the compound. And finally, carrying out visual treatment on the docking result through pymol.
3 results
3.1 screening of the active ingredient of Gleditsia sinensis
The total ion flow diagram of the volatile oil of the fructus gleditsiae is shown in figure 1 by GC-MS analysis. 64 components are identified after NIST17 database retrieval, matching and artificial analysis, see Table 2-1, the obtained components are screened by 2.2.1 items, and 24 components are obtained in total, mainly comprising aromatic aldehydes, unsaturated alcohols, esters and terpene compounds.
TABLE 2-1 effective ingredients of EOGSL
Table2-1 Chemical components in EOGSL
3.2 results of network pharmacology
3.2.1 screening of targets
Target Prediction is carried out on 24 active ingredients of the gleditsia sinensis volatile oil in a Swiss Target Prediction database, and 311 potential targets are predicted after repeated items are deleted. Through Gene Cards, OMIM disease database, respectively matched to 62 and 817 CIRI target genes, to obtain 822 duplication. The obtained active ingredients and the disease intersection target point are uploaded to Venny software to construct a Wien diagram, and the result is shown in figure 2, and 95 target points of the drug combined action are obtained.
3.2.2 network construction of Gleditsia sinensis volatile oil-active ingredient-target and core ingredient screening
The network visualization processing diagram of the volatile oil-active ingredients-target point of the fructus gleditsiae is shown in figure 3, wherein the diagram has 122 nodes and 181 edges. And (3) calculating a Degree analysis by using a Network analyzer in software, wherein the more edges connected with the nodes, the higher the value of the nodes. The volatile oil of fructus Gleditsiae Abnormalis is represented by yellow hexagon, the components are represented by blue diamond, the related target point is represented by red circle, and the higher the value, the larger the shape and the darker the color. Wherein the paeonol has 33 interaction targets, and the components with higher degree value in the network diagram also comprise linalool, methyl eugenol, eugenol and the like. The components with more targets are probably the core components of the volatile oil of the fructus gleditsiae for preventing and treating diseases.
3.2.3 construction of protein-protein interaction network and screening of core targets
Introducing 95 common targets into a STRING database, setting the interaction score of the targets to be 0.7, hiding 15 free targets, displaying the interaction of 80 proteins in the database, and introducing TSV text data into cytoscape3.7.2 software to draw a protein interaction network diagram, which is shown in figure 4. The 296 protein interaction connecting lines are generated by 78 target points in the network, the color of the target points is gradually darkened from blue to red, and the larger the target points are, the higher the specification value is. The closeness of the relation between the connecting line thickness and the target point is in positive correlation. Selecting 17 targets with the value of more than 11 (1.5 times of the average value) as core targets, wherein the number of the targets comprises VEGFA, SRC, MAPK8, PIK3CA, TNF, HSP90AA1, PTGS2, FYN, ESR1, RELA, JAK2, DRD2, MAPK14, CCND1, CASP3, BDKRB2 and KDR.
3.2.4 Gene function and pathway enrichment analysis
In this study, a total of 56 GO annotations and 71 KEGG pathways (FDR <0.05) were obtained for 17 core targets. Of the 56 GO annotations, 38 involved Biological Processes (BPs), such as cellular response to lipopolysaccharide, vascular endothelial growth factor receptor signaling, response to drugs, positive regulation of the nitric oxide biosynthetic process, modulation of inflammatory responses, angiogenesis, positive regulation of the ERK1 and ERK2 cascades, and the like. There are 12 involved Molecular Functions (MF), such as enzyme binding, protein tyrosine kinase activity, histone deacetylase binding, ATP binding, non-transmembrane protein tyrosine kinase activity, growth factor receptor binding, etc. There are 6 involved Cellular Components (CC), such as cytosol, nucleus, plasma membrane, nucleoplasm, external components on the cytoplasmic side of the plasma membrane, etc. Among 71 KEGG pathways, there are prolactin signaling pathway, proteoglycan in cancer, TNF signaling pathway, sphingolipid signaling pathway, hepatitis b, and cancer pathway according to FDR rank 6 from small to large. Fig. 5 lists the BP, the first 10 MF and 6 entries for CC functions according to FDR <0.05, and fig. 6 lists the first 20 KEGG pathways.
3.2.5 proof analysis of molecular docking
To illustrate the binding activity between target and component, the core target with top 5 of the median ranking in protein interaction network and its corresponding chemical component were selected for analytical docking and the results are shown in tables 2-2. The binding energy is less than 0, which indicates that the ligand and the receptor can be spontaneously combined, the lower the energy, the stronger the binding capacity, the binding energy in the table is less than-5 KJ/mol, and hydrogen bonds are formed, thus indicating that the protein target and the corresponding chemical component have better binding activity. All docking results were visually analyzed using pymol software, with yellow dashed lines indicating hydrogen bonds, pink indicating chemical structure, blue indicating target protein, and red indicating binding sites, as shown in fig. 7.
Tables 2 to 2: molecular docking results
Table2-2 Molecular docking results
4 summary of the invention
The research analyzes 64 components of the gleditsia sinensis volatile oil by using a GC-MS technology, and 24 active components are screened by a Swiss ADME platform, wherein the content of paeonol is the highest. According to the network diagram of volatile oil-component-target point, the first 5 components with the most matched target points are paeonol, linalool, methyl eugenol, anethole and eugenol. The research shows that the paeonol given in advance can obviously relieve inflammatory reaction and cerebral ischemia inflammation and improve the nerve defect symptoms of rats, thereby playing the role of resisting cerebral ischemia injury. Linalool has anti-inflammatory and antioxidant properties, has protective effects in vitro models of glutamate-induced oxidative stress and in vitro models of excitotoxicity, and can be used as a potential therapeutic agent against neurodegenerative brain diseases. Methyl eugenol can enter brain tissue through blood brain barrier to exert the function of treating brain diseases. The anethole can reduce the nerve function defect of mice, and reduce the cerebral infarction volume and cerebral edema, thereby having the protective effect on ischemic stroke. Eugenol can increase brain BDNF content through inhalation passage, and regulate brain function, thereby improving injury after ischemia. Based on the research reports and network pharmacological analysis, the gleditsia sinensis volatile oil is presumed to exert the treatment effect on cerebral ischemia-reperfusion through the 5 core components.
In summary, the network pharmacology is that the gleditsia sinensis volatile oil acts on key targets such as VEGFA, SRC, MAPK8, PIK3CA and TNF through active ingredients such as paeonol, linalool, methyl eugenol, anethole and eugenol, so that multiple signal paths such as sphingolipid, TNF and VEGF are regulated, oxidative stress injury of cells is inhibited, and release of inflammatory factors and nerve function regulation are inhibited, thereby playing a role in treating CIRI and laying a foundation for further deeply researching the 'opening and closing' treatment action mechanism of the gleditsia sinensis.
Secondly, the protection effect of the active ingredient formula of the gleditsia sinensis volatile oil on cerebral ischemia reperfusion
1. Material
1.1 Experimental drugs
The extraction rate of the volatile oil of the fructus gleditsiae (extracted in the experiment is 0.132%), the daily dosage of the adult fructus gleditsiae and the daily dosage of the rat are converted by referring to the daily dosage of the fructus gleditsiae and a body surface area method in the section of Chinese pharmacopoeia of 2015 edition, and the dosage of the volatile oil of the fructus gleditsiae rat is 0.231mg/kg through conversion.
The following reference substances are extracted from decoction pieces of Chinese medicinal materials, paeonol (GZDD-0046), eugenol (GZDD-0401), methyl eugenol (C102127), linalool (GZDD-0244) and anethole (C100243) which are all purchased from Guizhou Dingzhi scientific and technical Limited liability company and have a purity of more than 98%. The effective components are as follows: 37.2 percent of paeonol, 7.8 percent of eugenol, 4.7 percent of methyl eugenol, 23.0 percent of linalool and 27.3 percent of anethole, and the effective formula (accounting for 11.86 percent of the total components of the volatile oil of the honeylocust fruit) is prepared according to the proportion, the low dose of the effective formula is 0.028mg/kg, the medium dose is 0.056mg/kg, and the high dose is 0.108 mg/kg.
Nimodipine tablet (Yabao pharmaceutical industry group, Ltd., batch number: 170606), the daily dosage of the drug is 50mg according to the instruction, and the daily dosage of the rat is 5.83mg/kg converted from 20mg per tablet.
1.2 animals
SD rat, clean, male, 180-: SCXK (Xiang) 2019-,
1.3 reagents and instruments
Hematoxylin eosin staining KIT, 4% and 10% formaldehyde fixing solution (Wuhan Seville Biotech Co., Ltd.), 2,3, 5-triphenyltetrazolium chloride (TTC, Sigma Co., Ltd., 1001011963), enzyme-linked immunosorbent assay (produced by Meigu molecular instruments Co., Ltd., SpectraMAX Plus 384), Rat IL-1. beta. ELISA KIT (produced by Shanghai Yanghi Biotech Co., Ltd., ZC-36391); rat IL-6ELISA KIT (healthy color production, ZC-36404), Rat TNF-alpha ELISA KIT (healthy color production, ZC-37624), total protein determination KIT (Nanjing institute of biological engineering, A045-2-1), total superoxide dismutase test KIT (SOD, institute of biological engineering, A001-1-1), malondialdehyde determination KIT (MDA, A003-1-1, institute of biological engineering), 10% chloral hydrate, physiological saline and Tween-80.
Electronic balance (SHIMADZU; AUW20D), digital slide scanner (Pannoramic 250, 3DHISTECH (Hungary)), microscope imaging system (BA200Digita), thread bolt, suture thread, ophthalmic scissors, hemostatic clip, forceps, weighing bottle.
2 method
2.1 grouping, administration, Molding
SD male rats are randomly divided into a pseudo-operation group, a model control group, a nimodipine group (5.83mg/kg), volatile oil group (0.231mg/kg), an effective group prescription low dose group (0.028mg/kg), an effective group prescription medium dose group (0.054mg/kg) and an effective group prescription high dose group (0.108mg/kg), 11 rats are adaptively fed for 7 days before administration, each group is subjected to daily dose conversion corresponding to the dose of pig soap taken by adults according to pharmacopoeia, the nimodipine group is subjected to stomach filling and administration of 2.5ml/250g, the rest of administration groups are subjected to nasal administration of 20 mu l/250g, the model group and the pseudo-operation group are subjected to nasal administration of physiological saline containing 3% of Tween-80, when the nasal administration is performed, the head is fixed, a micro-syringe connected with a PE plastic hose is inserted into the nasal cavity of the mice for about 0.2cm, and the drug liquid (dissolved in the physiological saline containing 3% of Tween-80) is administered into the mice, and observing whether the liquid medicine is lost from the nasal cavity of the rat. The preparation is administered 1 time per day for 7 days, and the final time is 1h after the preparation.
The method comprises the steps of fasting the rat for 12 hours before model building, establishing a rat middle artery blocking model by adopting a wire tying method, carrying out intraperitoneal injection by using 4% chloral hydrate (0.75mL/100g), fixing the rat on the back on an operating table after the rat is anesthetized, shearing rat hair in front of the neck, disinfecting skin by iodophors, carrying out incision from the center of the neck, separating the right Common Carotid Artery (CCA), the External Carotid Artery (ECA) and the Internal Carotid Artery (ICA), ligating the proximal common carotid artery and the external carotid artery by using a No. 1 operation wire, and shearing a small opening by using ophthalmology at a position 4mm from the inner part of the neck and the external part of the neck. Inserting a plug into the right common carotid artery from the ostium to occlude blood from the middle cerebral artery; ligating and fixing a fish wire at the internal carotid artery by using an operation wire; suturing the wound layer by layer, exposing the thread plug to the outside of the body by more than about 0.5cm, and disinfecting the wound by using iodophor; placing in a cage, after 2h of ischemia, slightly pulling out the fish wire to the neck internal bifurcation, and then perfusing for 24 h.
The sham group only isolated the vessels, without fish line insertion and ligation, and the rest of the procedure was the same as the other groups.
2.2 neurobehavioral Scoring
After 24h of ischemia, rats were scored according to the Berderson scoring method using the scoring criteria: score 0 indicates no symptoms; score 1 indicates that the contralateral forepaw can be fully extended; 2 points are expressed as turns towards the opposite side; 3 points are shown as tipping to the contralateral side; a score of 4 indicates a failure to walk and loss of consciousness. If the score is 1-3, the molding success is shown.
2.3 percent by volume of cerebral infarction
After rat reperfusion for 24h, randomly selecting 3 decapitations from each group, rapidly taking ischemic side half brain, freezing in a refrigerator at-20 ℃ for 20min, taking out, performing continuous 6 coronal slices with the thickness of 2.0mm on brain tissue from front to back on an operation table, placing the brain slices in 2% TTC phosphate buffer solution, drying in an oven at 37 ℃ in the dark for 20min, and then fixing with 4% paraformaldehyde for 24 h. After dyeing, the peduncle area is white, and the non-peduncle area is red. Taking a picture, measuring the cerebral infarction area by adopting ImageJ software, and calculating the volume percentage of cerebral infarction according to the following formula: the volume percentage of cerebral infarction (volume of normal brain tissue on the opposite side of infarction-volume of normal brain tissue on the side of infarction)/volume of normal brain tissue on the opposite side of infarction x 100%.
2.4 determination of Biochemical indicators in brain tissue
After rat reperfusion for 24h, randomly selecting 5 decapitations in each group, rapidly taking ischemic side half brain, homogenizing 10% brain tissue in ice bath, centrifuging at 3000r/min for 10min, and determining IL-1 beta, IL-6, TNF-alpha, MDA content and SOD activity in serum and ischemic side brain tissue according to the instruction of the kit.
2.5 brain histopathological Observation (HE staining)
After rats are ischemic for 24 hours, 3 decapitations are randomly selected from each group, and the ischemic side half brain is quickly taken and fixed by 4% paraformaldehyde solution for more than 24 hours. Trimming tissue mass, marking. After dehydration, trimming, embedding, slicing, staining and mounting, image acquisition is finally carried out on the slices by adopting a digital slice scanner, all tissues are observed in each slice at the rate of 40 times, general lesions are observed, 100-time and 400-time pictures are acquired in the area to be observed, and specific lesions are observed.
3 results
3.1 comparison of neurobehavioral scores in groups of rats
Table 3-1 shows that the behavioural score of the sham group was 0, the model group was 3.18, and the difference was very significant (P <0.01),
according to the evaluation method, the rat can not walk after the model building, hemiplegia appears, the walking is in a counterclockwise tailing state, listlessness and other symptoms show that the model building is successful; compared with the model group, the scores of all the administration groups are reduced, and the differences of the other groups are very obvious (P is less than 0.01) except the low-dose group with the effective component formula, which shows that the other administration groups have the effect of improving the neurological behavior of the rat.
Table 3-1 post-operative groups of rats neurobehavioral scores (X ± S, n ═ 11)
| Group of | Dosage (mg/Kg) | Neurological behavior scoring |
| Artificial operation group | - | 0.00±00 |
| Model set | - | 3.18±0.41** |
| Nimodipine group | 5.83 | 1.09±0.54## |
| Volatile oil group | 0.231 | 1.73±0.47## |
| Effective formula low dose group | 0.028 | 2.64±0.51 |
| In the effective formulaDose groups | 0.054 | 1.73±0.47## |
| High dose group of effective formulations | 0.108 | 1.18±0.41## |
Note: p <0.05, P <0.01, compared to sham group; compared with the model group, # P <0.05, # P <0.01
3.2 comparison of cerebral infarct volume of rats in each group (see FIG. 8 and tables 3-2)
Table 3-2 percent cerebral infarction volume of each group of rats after surgery (X ± S, n ═ 3)
| Group of | Dosage (mg/Kg) | Volume percent of cerebral infarction (%) |
| Artificial operation group | - | 0.00±00 |
| Model set | - | 36.23±0.26** |
| Nimodipine group | 5.83 | 23.01±0.55## |
| Volatile oil group | 0.231 | 24.32±4.06## |
| Effective formula low dose group | 0.028 | 29.41±1.72 |
| Effective formula medium dosage group | 0.054 | 21.01±2.78## |
| High dose group of effective formulations | 0.108 | 19.58±0.76## |
Note: p <0.05, P <0.01, compared to sham group; compared with the model group, # P <0.05, # P <0.01
3.2 comparison of Biochemical indicators in brain tissues of various groups
Tables 3-3 show that the concentrations of IL-1 beta, IL-6 and TNF-alpha in the brain tissue of the rat in the model group are remarkably increased (P is less than 0.01) compared with the brain tissue of the sham operation group, which indicates that the modeling is successful; compared with the model group, the concentrations of IL-1 beta and IL-6 in the volatile oil group, the effective formula and the high-dose group of brain tissues are remarkably reduced (P is less than 0.01), and the other groups have no remarkable difference; compared with the model group, the concentration of TNF-alpha in the brain tissue of the volatile oil group and the effective formula high-dose group is obviously reduced (P is less than 0.05), and the other groups have no obvious difference.
Tables 3-4 show that compared with the sham operation group, the SOD activity in the brain tissue of the rat in the model group is extremely obviously reduced (P is less than 0.01), and the MAD content is extremely obviously increased (P is less than 0.01), which indicates that the model building is successful; compared with a model group, the SOD activity in the brain tissue of the volatile oil group is remarkably increased (P is less than 0.01), the SOD activity in the effective formula and the low-dose group is remarkably increased (P is less than 0.05), and the other groups have no remarkable difference; compared with the model group, the brain tissue of rats in each group has no obvious difference in MDA content.
Table 3-3 effects of inflammatory factors in brain tissue of rats in each group after surgery (X ± S, n ═ 5)
Note: p <0.05, P <0.01, compared to sham group; compared with the model group, # P <0.05, # P <0.01
Table 3-4 effects of SOD and MDA in brain tissue of rats in each group after surgery (X ± S, n ═ 5)
| Group of | Dosage (mg/Kg) | SOD(U/mgprot) | MDA(nmol/mgprot) |
| Artificial operation group | - | 1698.461±283.372 | 1.879±0.486 |
| Model set | - | 1039.620±195.065** | 3.023±0.494** |
| Nimodipine group | 5.83 | 1192.902±173.024 | 2.755±0.776 |
| Volatile oil group | 0.231 | 1433.241±252.353## | 2.346±0.627 |
| Effective formula low dose group | 0.028 | 1095.304±151.523 | 2.779±0.408 |
| Effective formula medium dosage group | 0.054 | 1463.937±168.026# | 2.512±1.522 |
| High dose composition of effective formulation | 0.108 | 1357.053±121.469# | 2.352±0.679 |
Note: p <0.05, P <0.01, compared to sham group; compared with the model group, # P <0.05, # P <0.01
3.4 comparison of histopathological manifestations in the brains of rats in each group (see FIGS. 9-11 and tables 3-5)
The sham operation group: the neuron cells in the cortical area have normal shapes, and no obvious cell degeneration and necrosis are seen; the layered structure of the hippocampus is clear, the cells are closely arranged in a band shape, the cell morphology is clear, the nucleus is large and round, and obvious degeneration necrosis, gliosis or inflammatory cell infiltration is not seen.
Model group: the neurons and nerve fibers in the half cortical area are necrosed in a sheet shape, tissues in the necrotic area are edematous, the staining is light, the nerve fibers are loose, and the local area is vacuolated or even dissolved and disappeared; the necrotic edge area has fuzzy shape and structure of great amount of nerve cells, fixed and deep nucleus, obviously reduced volume, dissolved nerve fiber and light color.
Nimodipine group: the hemicortical neuron and the nerve fiber are necrosed and edematous in a sheet shape, and the staining is light; the neuron morphological structure of the marginal zone part is relatively normal, a small amount of neuron nuclei are fixedly contracted and are irregular, and peripheral nerve fibers are relatively compact; no other obvious pathological changes are seen.
Volatile oil group: hemicortical neuron and nerve fiber sheet necrosis and edema, marginal neuron necrosis, nucleus shrinkage and lysis, and partial neuron degeneration are obvious.
The active ingredients are formulated into a low-dose group: the hemicortical neuron and nerve fiber are necrosed and edematous in a sheet shape, the staining is light, and the severe lesion area is affected by the hippocampus; the neuron in the marginal zone is necrotic, the neuron is partially neurodegenerative, the cell nucleus of the degenerative neuron is relatively normal in shape, and cavities are visible around the nucleus.
The effective components comprise the following components in the formula: the hemicortical neuron and the nerve fiber are necrosed and edematous in a sheet shape, and the staining is light; marginal neuron degeneration is obvious
The active ingredients are formulated into a high-dose group: the hemicortical region neurons and nerve fibers are necrosed and edematous in a lamellar manner, and the staining is light; the morpho-structure of marginal zone neurons is normal, and a small amount of neurons are necrotic; no other obvious pathological changes are seen.
In conclusion, in the experiment, except for the sham operation group, large-area necrotic foci can be seen in the cortical areas of other groups, marginal neurons are degenerated or necrotized in different degrees, the marginal neurons are obviously necrotized in a model group numbered, the volatile oil group and the effective component formula are inferior to the low-dose and medium-dose groups, and the effective component formula is slightly light in the high-dose group and the nimodipine group.
TABLE 3-5 brain histopathological manifestations in groups of rats after surgery
If there was no lesion, it was recorded as (-) and 4-grade methods were used, respectively, mild (+), mild (+ +), moderate (++++), and severe (++++).
Claims (10)
1. Application of fructus Gleditsiae Abnormalis volatile oil in preparing medicine for treating cerebral ischemia reperfusion injury is provided.
2. A medicine for treating cerebral ischemia reperfusion injury is mainly prepared from fructus Gleditsiae Abnormalis volatile oil.
3. A medicine for treating cerebral ischemia-reperfusion injury is characterized in that: the composition comprises the following active ingredients in percentage by weight: 33.2 to 41.2 percent of paeonol, 3.8 to 11.8 percent of eugenol, 0.7 to 8.7 percent of methyl eugenol, 19.0 to 27.0 percent of linalool and 23.3 to 31.3 percent of anethole.
4. The medicament for treating cerebral ischemia-reperfusion injury according to claim 3, wherein: the composition comprises the following active ingredients in percentage by weight: 35.2 to 39.2 percent of paeonol, 5.8 to 9.8 percent of eugenol, 2.7 to 6.7 percent of methyl eugenol, 21.0 to 25.0 percent of linalool and 25.3 to 29.3 percent of anethole.
5. The medicament for treating cerebral ischemia-reperfusion injury according to claim 3 or 4, wherein: the composition comprises the following active ingredients in percentage by weight: 37.2 percent of paeonol, 7.8 percent of eugenol, 4.7 percent of methyl eugenol, 23.0 percent of linalool and 27.3 percent of anethole.
6. The method of preparing the toilet soap of claim 1, wherein: the preparation method comprises the following steps: taking a proper amount of fructus gleditsiae decoction pieces, adding water and n-hexane, performing steam distillation to obtain volatile oil, collecting the volatile oil, adding a proper amount of anhydrous sodium sulfate to remove water, and standing overnight to obtain the fructus gleditsiae volatile oil.
7. The method of preparing the toilet soap of claim 6, wherein: the preparation method comprises the following steps: cutting appropriate amount of fructus Gleditsiae Abnormalis decoction pieces, adding 8-12 times of water, collecting 5-15% n-hexane of fructus Gleditsiae Abnormalis decoction pieces according to g/ml, steam distilling for 6-14 hr to obtain volatile oil, collecting volatile oil, adding appropriate amount of anhydrous sodium sulfate to remove water, standing overnight to obtain fructus Gleditsiae Abnormalis volatile oil.
8. The method for preparing the volatile oil of fructus gleditsiae as claimed in claim 6 or 7, characterized in that: the preparation method comprises the following steps: cutting appropriate amount of fructus Gleditsiae Abnormalis decoction pieces, adding 10 times of water, collecting 10% n-hexane of fructus Gleditsiae Abnormalis decoction pieces, steam distilling for 10 hr to obtain volatile oil, collecting volatile oil, adding appropriate amount of anhydrous sodium sulfate to remove water, and standing overnight to obtain fructus Gleditsiae Abnormalis volatile oil.
9. Application of fructus Gleditsiae Abnormalis in preparing medicine for treating cerebral ischemia reperfusion injury is provided.
10. A medicine for treating cerebral ischemia-reperfusion injury is characterized in that: the medicine comprises fructus Gleditsiae Abnormalis.
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| 刘阳欣等: "丹皮酚药理学研究进展", 《陕西中医》 * |
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