Use of pig's tooth saponin in preparation of drugs for treating cerebral ischemia-reperfusion injury

Abstract

The application provides application of pig's tooth saponin volatile oil in preparation of a medicine for treating cerebral ischemia-reperfusion injury, and a preparation method of the pig's tooth saponin volatile oil is as follows: pig's tooth saponin decoction pieces are taken, water and n-hexane are added, and volatile oil is obtained through steam distillation; the volatile oil is collected, anhydrous sulfuric acid is added, and water is removed; and the mixture is left overnight to obtain the pig's tooth saponin volatile oil. The prepared pig's tooth saponin volatile oil acts on key target points of VEGFA, SRC, MAPK8, PIK3CA and TNF through active ingredients of paeonol, linalool, methyl eugenol, anethole and eugenol, regulates multiple signal pathways of sphingolipids, TNF and VEGF, inhibits cell oxidative stress injury, inhibits release of inflammatory factors and regulates nerve function, so as to play a role in treating cerebral ischemia-reperfusion injury, and lay a foundation for further in-depth study on a treatment mechanism of pig's tooth saponin in treating 'opening the orifices and connecting the heart and the lung'.

Description

Technical Field

[0001] This invention relates to a novel use of volatile oil from *Polygonum cuspidatum*, specifically its application in the preparation of drugs for treating cerebral ischemia-reperfusion injury. Background Technology

[0002] The brain is the most oxygen-sensitive organ in the human body. Cerebral ischemia leads to damage to local brain tissue and its function. The degree of damage is related to the duration of ischemia and the amount of residual blood flow. Short-term incomplete ischemia only causes reversible damage, while prolonged complete or severe ischemia can cause infarction. Histological changes: The most obvious histological changes in the brain are cerebral edema and brain cell necrosis. Cerebral edema results from membrane lipid peroxidation, which damages the membrane structure and impairs sodium pump function. Pathophysiological mechanisms include: 1. Free radicals and cerebral ischemia-reperfusion injury; 2. Calcium overload and cerebral ischemia-reperfusion injury; 3. Excitatory amino acids and cerebral ischemia-reperfusion injury; 4. NO and cerebral ischemia-reperfusion injury; 5. Inflammatory response and cerebral ischemia-reperfusion injury; 6. Apoptosis. Cerebral ischemia-reperfusion injury is also called ischemic stroke in Traditional Chinese Medicine, and it is often treated with herbal remedies that open the orifices and awaken the mind.

[0003] Pig Tooth Soap is the dried, sterile fruit of the legume *Gleditsia sinensis* Lam. In late autumn, the mature fruits are harvested, impurities are removed, and they are sun-dried. It is cylindrical, slightly flattened and curved, 5–11 cm long and 0.7–1.5 cm wide. The surface is purplish-brown or purplish-brown, covered with a grayish-white waxy powder, which leaves a glossy sheen after rubbing. It has fine wart-like protrusions and linear or reticulate cracks. The apex has a beak-like style remnant, and the base has a fruit stalk scar. It is hard and brittle, easily broken, with a brownish-yellow cross-section, loose in the middle, containing pale green or pale brownish-yellow filaments, and occasionally incompletely developed seeds. It has a slight, pungent odor, and a taste that is initially sweet and then spicy. It is pungent and salty in taste, warm in nature, and slightly toxic. It enters the lung and large intestine meridians. Its functions include expectoration, opening the orifices, dispersing nodules, and reducing swelling. It is used for stroke with locked jaw, coma, epilepsy with excessive phlegm, obstruction of the orifices, sore throat with phlegm obstruction, stubborn cough with phlegm, difficulty in expectorating phlegm, and constipation; it is also used externally to treat carbuncles and boils. It is used to treat acute schistosomiasis, acute intestinal obstruction, and other diseases. Although the chemical composition and pharmacological activity of *Gleditsia sinensis* have been studied, no relevant data have been reported on its application in cerebral ischemia-reperfusion. This study used GC-MS technology to analyze 64 components in the volatile oil of *Gleditsia sinensis*, and screened 24 active ingredients using the Swiss ADME platform, among which paeonol had the highest content. According to the volatile oil-component-target network diagram, the top 5 components with the most matching targets were paeonol, linalool, methyl eugenol, anethole, and eugenol. Studies have shown that pre-administration of paeonol can significantly reduce inflammatory responses, cerebral ischemia-inducing inflammation, and improve neurological deficit symptoms in rats, thereby exerting an anti-cerebral ischemia-inducing effect. Linalool possesses anti-inflammatory and antioxidant properties, exhibiting protective effects in in vitro models of glutamate-induced oxidative stress and in vitro models of excitotoxicity, suggesting its potential therapeutic potential for neurodegenerative brain diseases. Methyleugenol must cross the blood-brain barrier to reach brain tissue and exert its therapeutic effects on brain diseases. Anethole can reduce neurological deficits in mice, decrease infarct volume and cerebral edema, thus providing protection against ischemic stroke. Eugenol can increase BDNF levels in the brain through the olfactory pathway, regulating brain region function and thereby improving post-ischemic reperfusion injury. Based on the above research reports and network pharmacology analysis, it is speculated that the volatile oil of *Gnaphalium affine* may exert its therapeutic effect on cerebral ischemia-reperfusion through these five core components. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides the preparation of *Porcine tooth soap* and its application in the treatment of cerebral ischemia-reperfusion injury, solving the problem of how to extract the volatile oil of *Porcine tooth soap* for the treatment of cerebral ischemia-reperfusion injury. To achieve the above objective, this invention is implemented through the following technical solution:

[0005] Application of volatile oil from pig tooth soap in the preparation of drugs for treating cerebral ischemia-reperfusion injury.

[0006] A drug for treating cerebral ischemia-reperfusion injury, mainly made from the volatile oil of hog tooth soap.

[0007] A drug for treating cerebral ischemia-reperfusion injury, comprising, by weight percentage, the following active ingredients: 33.2%–41.2% paeonol, 3.8%–11.8% eugenol, 0.7%–8.7% methyl eugenol, 19.0%–27.0% linalool, and 23.3%–31.3% anethole.

[0008] The aforementioned drug for treating cerebral ischemia-reperfusion injury comprises, by weight percentage, the following active ingredients: 35.2%–39.2% paeonol, 5.8%–9.8% eugenol, 2.7%–6.7% methyl eugenol, 21.0%–25.0% linalool, and 25.3%–29.3% anethole.

[0009] Specifically, the aforementioned drug for treating cerebral ischemia-reperfusion injury is composed of the following active ingredients by weight percentage: 37.2% paeonol, 7.8% eugenol, 4.7% methyl eugenol, 23.0% linalool, and 27.3% anethole.

[0010] The aforementioned method for preparing volatile oil from *Gnaphalium affine* is as follows: Take an appropriate amount of *Gnaphalium affine* slices, add water and n-hexane, and steam distill to obtain volatile oil. Collect the volatile oil, add an appropriate amount of anhydrous sodium sulfate to remove water, and let it stand overnight to obtain volatile oil from *Gnaphalium affine*.

[0011] Specifically, the aforementioned method for preparing the volatile oil of *Gnaphalium affine* is as follows: Take an appropriate amount of *Gnaphalium affine* slices, cut them into small pieces, add 8-12 times the amount of water, and take 5-15% of n-hexane from the *Gnaphalium affine* slices by g / ml. Steam distill for 6-14 hours to obtain the volatile oil. Collect the volatile oil, add an appropriate amount of anhydrous sodium sulfate to remove water, and let it stand overnight to obtain the volatile oil of *Gnaphalium affine*.

[0012] More specifically, the aforementioned method for preparing the volatile oil of *Gnaphalium affine* is as follows: take an appropriate amount of *Gnaphalium affine* slices, cut them into small pieces, add 10 times the amount of water, and take 10% of the *Gnaphalium affine* slices in n-hexane according to g / ml. Steam distill for 10 hours to obtain the volatile oil. Collect the volatile oil, add an appropriate amount of anhydrous sodium sulfate to remove water, and let it stand overnight to obtain the volatile oil of *Gnaphalium affine*.

[0013] Application of pig tooth soap in the preparation of drugs for treating cerebral ischemia-reperfusion injury.

[0014] A drug for treating cerebral ischemia-reperfusion injury, the drug comprising hog tooth soap.

[0015] The beneficial effects of this invention are:

[0016] This method optimizes the preparation method of volatile oil from pig tooth soap based on traditional methods. The obtained volatile oil from pig tooth soap, through its active ingredients paeonol, linalool, methyl eugenol, anethole, and eugenol, acts on key targets such as VEGFA, SRC, MAPK8, PIK3CA, and TNF, regulating multiple signaling pathways such as sphingolipids, TNF, and VEGF, inhibiting cellular oxidative stress damage, inhibiting the release of inflammatory factors, and regulating nerve function, thereby playing a role in treating cerebral ischemia-reperfusion injury. This lays the foundation for further in-depth research on the "opening the orifices" therapeutic mechanism of pig tooth soap. Attached Figure Description

[0017] Figure 1 EOGSL total ion chromatogram;

[0018] Figure 2 Venn diagram of the intersection genes of EOGSL and CIRI;

[0019] Figure 3 EOGSL - Active Ingredient-Target Network;

[0020] Figure 4 EOGSL protein interaction network;

[0021] Figure 5 GO enrichment analysis of core targets;

[0022] Figure 6 KEGG enrichment analysis of core target;

[0023] Figure 7 Molecular docking simulation diagram;

[0024] Figure 8 Postoperative TTC staining results of rats in each group (Note: A. Sham-operated group; B. Model group; C. Nimodipine group; D. Volatile oil group; E, F, H. Low, medium, and high dose groups of active ingredient formulations);

[0025] Figure 9 Morphological diagrams of neurons in the cortical region of the sham surgery group, model group, and nimodipine group;

[0026] Figure 10 Morphological diagrams of neurons in the cortical region of the volatile oil group, the low-dose group of the effective formula, and the medium-dose group of the effective formula;

[0027] Figure 11 Morphological diagram of neurons in the cortical region of the high-dose group of the effective formulation.

[0028] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below with reference to specific embodiments, but the embodiments are not intended to limit the present invention. Detailed Implementation

[0029] Example 1:

[0030] Process:

[0031] Take an appropriate amount of *Gnaphalium affine* slices, cut them into small pieces, and accurately weigh 100g. Add 10 times the amount of water and 10ml of n-hexane, and steam distill for 10 hours to obtain volatile oil. Collect the volatile oil, add an appropriate amount of anhydrous sodium sulfate to remove water, and let it stand overnight to obtain *Gnaphalium affine* volatile oil.

[0032] Take 25mg of volatile oil from *Polygonum cuspidatum*, add 3% Tween-80, and prepare 10ml of physiological saline to obtain *Polygonum cuspidatum* volatile oil nasal drops.

[0033] Efficacy: It can clear phlegm, open the orifices, disperse nodules and reduce swelling, and treat cerebral ischemia-reperfusion injury (ischemic stroke).

[0034] How to use: Spray into the nasal cavity.

[0035] Dosage and administration: 0.8ml / day (calculated based on the adult daily dosage of 1.5g of *Polygonum cuspidatum* and the volatile oil extraction rate of 0.132% in the 2020 edition of the Chinese Pharmacopoeia). Spray into the nasal cavity three times a day.

[0036] Example 2:

[0037] Process:

[0038] Take an appropriate amount of *Gnaphalium affine* slices, cut them into small pieces, and accurately weigh 100g. Add 8 times the amount of water and 5ml of n-hexane, and steam distill for 6 hours to obtain volatile oil. Collect the volatile oil, add an appropriate amount of anhydrous sodium sulfate to remove water, and let it stand overnight to obtain *Gnaphalium affine* volatile oil.

[0039] Take 25mg of volatile oil from *Polygonum cuspidatum*, add 3% Tween-80, and prepare 10ml of physiological saline to obtain *Polygonum cuspidatum* volatile oil nasal drops.

[0040] Efficacy: It can clear phlegm, open the orifices, disperse nodules and reduce swelling, and treat cerebral ischemia-reperfusion injury (ischemic stroke).

[0041] How to use: Spray into the nasal cavity.

[0042] Dosage and administration: 0.8ml / day (calculated based on the adult daily dosage of 1.5g of *Polygonum cuspidatum* and the volatile oil extraction rate of 0.132% in the 2020 edition of the Chinese Pharmacopoeia). Spray into the nasal cavity three times a day.

[0043] Example 3:

[0044] Process:

[0045] Take an appropriate amount of *Gnaphalium affine* slices, cut them into small pieces, and accurately weigh 100g. Add 12 times the amount of water and 15ml of n-hexane, and steam distill for 14 hours to obtain volatile oil. Collect the volatile oil, add an appropriate amount of anhydrous sodium sulfate to remove water, and let it stand overnight to obtain *Gnaphalium affine* volatile oil.

[0046] Take 25mg of volatile oil from *Polygonum cuspidatum*, add 3% Tween-80, and prepare 10ml of physiological saline to obtain *Polygonum cuspidatum* volatile oil nasal drops.

[0047] Efficacy: It can clear phlegm, open the orifices, disperse nodules and reduce swelling, and treat cerebral ischemia-reperfusion injury (ischemic stroke).

[0048] How to use: Spray into the nasal cavity.

[0049] Dosage and administration: 0.8ml / day (calculated based on the adult daily dosage of 1.5g of *Polygonum cuspidatum* and the volatile oil extraction rate of 0.132% in the 2020 edition of the Chinese Pharmacopoeia). Spray into the nasal cavity three times a day.

[0050] Example 4:

[0051] Process:

[0052] Take 3 mg of active ingredients from the volatile oil of *Gnaphalium affine* (calculated based on the fact that the active ingredients from the volatile oil of *Gnaphalium affine* account for 11.86% of the total volatile oil components of *Gnaphalium affine*), including 37.2% paeonol, 7.8% eugenol, 4.7% methyl eugenol, 23.0% linalool, and 27.3% anethole. Add 3% Tween-80 to prepare 10 ml of physiological saline solution to obtain *Gnaphalium affine* volatile oil nasal drops.

[0053] Efficacy: It can clear phlegm, open the orifices, disperse nodules and reduce swelling, and treat cerebral ischemia-reperfusion injury (ischemic stroke).

[0054] How to use: Spray into the nasal cavity.

[0055] Dosage and administration: 0.8 ml / day, spray into the nasal cavity three times a day.

[0056] Example 5:

[0057] Process:

[0058] Take 6 mg of the active ingredient of volatile oil from *Gnaphalium affine*, which contains 37.2% paeonol, 7.8% eugenol, 4.7% methyl eugenol, 23.0% linalool, and 27.3% anethole. Add 3% Tween-80 to prepare 10 ml of physiological saline solution to obtain *Gnaphalium affine* volatile oil nasal drops.

[0059] Efficacy: It can clear phlegm, open the orifices, disperse nodules and reduce swelling, and treat cerebral ischemia-reperfusion injury (ischemic stroke).

[0060] Instructions for use: Spray the spray into the nasal cavity.

[0061] Dosage and administration: 0.8 ml / day, spray into the nasal cavity three times a day.

[0062] Example 6:

[0063] Process:

[0064] Take 12 mg of the active ingredient of volatile oil from *Gnaphalium affine*, which contains 37.2% paeonol, 7.8% eugenol, 4.7% methyl eugenol, 23.0% linalool, and 27.3% anethole. Add 3% Tween-80 to prepare 10 ml of physiological saline solution to obtain *Gnaphalium affine* volatile oil nasal drops.

[0065] How to use: Spray into the nasal cavity.

[0066] Dosage and administration: 0.8 ml / day, spray into the nasal cavity three times a day.

[0067] The inventors conducted numerous experiments, and the following is a study of the extraction method described in this invention:

[0068] I. Extraction of volatile oil from pig tooth soap

[0069] 1. Medicinal materials and reagents

[0070] The pig tooth soap was purchased from Beijing Tongrentang: batch number 20180408; n-hexane (Lialong Bohua Pharmaceutical Chemical Co., Ltd., batch number: 20180110); electronic temperature-controlled heating mantle (Tianjin Tester Instrument Co., Ltd., model: 98-1-B); electronic balance (SHIMADZU; AUW20D).

[0071] 2 methods

[0072] Single-factor experiment

[0073] Since there were no interactions among the factors involved in the extraction of volatile oil from *Gnaphalium affine* using steam distillation, a single-factor analysis was conducted to investigate the extraction process. The three factors were water addition, extraction time, and hexane addition. The extraction rate of volatile oil was used as the evaluation index to determine the optimal extraction process. Extraction rate = (Volatile oil extracted / Raw material amount) * 100%.

[0074] 2.1 Time consideration

[0075] Weigh 100g of *Polygonum cuspidatum* into three portions, add 10 times the amount of water and 10mL of n-hexane, and extract using steam distillation for 6, 8, 10, and 12 hours respectively; collect the n-hexane portion, dry it, weigh it, and calculate the extraction rate.

[0076] 2.2 Investigation into the amount of n-hexane added

[0077] Weigh 100g of *Polygonum cuspidatum* (a type of soap) into three portions, add 10 times the amount of water, and extract with 5, 10, and 15 mL of n-hexane respectively. Extract by steam distillation for 10 hours. Collect the n-hexane fraction, dry it, weigh it, and calculate the extraction rate.

[0078] 2.3 Investigation of water addition amount

[0079] Weigh 100g of *Gnaphalium affine* (a type of soapberry) into three portions, add 10mL of n-hexane, and add water in amounts of 8, 10, and 12 times the weight of the medicinal material, respectively. Extract by steam distillation for 10 hours. Collect the n-hexane fraction, dry it, weigh it, and calculate the extraction rate.

[0080] 3 Results

[0081] 3.1 Time consideration

[0082] As can be seen from Table 1-1, the extraction rate is the best when the extraction time is 10 hours.

[0083] Table 1-1 Effect of time on extraction rate

[0084]

[0085] 3.2 Investigation into the amount of n-hexane added

[0086] As can be seen from Table 1-2, the extraction rate is best when the amount of n-hexane is 10 mL.

[0087] Table 1-2 Effect of n-hexane on extraction rate

[0088]

[0089] 3.3 Investigation of water addition amount

[0090] As can be seen from Table 1-3, the extraction rate is best when the amount of water added is 10 times the amount of medicinal materials.

[0091] Table 1-3 Effect of water addition on extraction rate

[0092]

[0093] 4. Summary

[0094] Experimental results show that the optimal extraction process for extracting volatile oil from *Gnaphalium affine* by steam distillation is to add 100g of medicinal material to 10 times the amount of water and 10mL of n-hexane, and steam distill for 10 hours.

[0095] II. Network pharmacology predicts potential effective components in the volatile oil of *Gnaphalium affine* for improving cerebral ischemia-reperfusion injury.

[0096] 1. Database and software

[0097] ① Databases: Online Human Mendelian Genetics 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 Genome Annotation Database (GeneCards, https: / / www.genecards.org / ), DAVID Bioinformatics Resource Website (https: / / david.ncifcrf.gov / ), Venny 2.1.0 (http: / / bioinfogp.cnb.csic.es / tools / venny / index.html), STRING Platform (https: / / string-db.org / ), Online Plotting Website MicroBioInformatics (www.bioinformatics.com.cn / ), Protein Structure Database (PDB, http: / / www.rcsb.org / ).

[0098] ② Software: Cytoscpae 3.7.2 (network topology data analysis software), Open Babe 2.4.1 (file format conversion software), Auto Dock 4.2.6 (protein molecular docking software), and PyMOL 2.5 (molecular simulation and mapping software).

[0099] 2 methods

[0100] 2.1 Determination of active components in volatile oils

[0101] Take an appropriate amount of *Gnaphalium affine* slices, cut them into small pieces, and accurately weigh 100g. Add 10 times the amount of water and 10ml of n-hexane, and steam distill for 10 hours to obtain volatile oil. Collect the volatile oil, add an appropriate amount of anhydrous sodium sulfate to remove water, and let it stand overnight. Weigh an appropriate amount of sample into a headspace vial, seal it, and place it in an 80℃ water bath for 30 minutes to equilibrate. Maintain the temperature and extract with a solid-phase microextraction needle for 30 minutes. After extraction, aspirate the extraction needle at the injection port for 5 minutes. Analyze the mass spectrum obtained by GC-MS, search the NIST spectral library, identify the sample components, and calculate the relative content of each component.

[0102] Gas chromatography conditions: Agilent HP-5MS column (0.25mm × 30m × 0.25μm); split ratio: splitless; injection port temperature: 270℃; temperature program: initial temperature 70℃, hold for 2 min, then increase to 280℃ at a rate of 5℃ / min, hold for 10 min. Carrier gas: high-purity helium, flow rate 1.0 mL / min. Mass spectrometry conditions: ion source temperature 230℃; scan mode: Full Scan; quadrupole temperature 150℃.

[0103] 2.2 Network Pharmacological Analysis

[0104] 2.2.1 Screening of active ingredients in the volatile oil of pig tooth soap

[0105] Preliminary screening was conducted based on a matching rate greater than 90% in the NIST17 database. The chemical structures of the screened chemical components were then confirmed using the PubChem database. The active ingredients of the porcine tooth soap were then screened using the Swiss ADME online platform with the criteria of "high" for gastrointestinal absorption and two "yes" results for drug similarity.

[0106] 2.2.2 Target Query and Determination

[0107] The 2D structural formulas of the active ingredients in the volatile oil of *Gnaphalium affine* obtained through screening were submitted to the Swiss Target Prediction platform to predict potential targets of the active ingredients, with a probability set > 0.1. Using "Cerebral ischemia-reperfusion injury" as the keyword, searches were performed in the Gene Cards and OMIM databases. Genes with a relevance score greater than 10 were merged, and duplicate values ​​were removed to obtain disease gene targets. Venn diagrams were then constructed using the Venny platform to plot the obtained active ingredient targets and cerebral ischemia-reperfusion targets from the volatile oil.

[0108] 2.2.3 Construction of volatile oil, active ingredients, and targets in pig tooth soap

[0109] The active pharmaceutical ingredients and drug-disease intersection targets are imported into Cytoscape software for network visualization and data analysis.

[0110] 2.2.4 Construction of protein-protein interaction networks

[0111] To assess and integrate protein-protein interactions, a protein-protein interaction network (PPI network) was constructed. Common targets of *Homo sapiens* and cerebral ischemia-reperfusion syndrome were imported into the String database. The analyzed organism was selected as humans (Homo Sapiens), with a high confidence level > 0.7, and 15 free proteins were hidden. PPI data for the intersecting proteins were obtained and saved as a ".tsv" file. This file was then imported into Cytoscape 3.7.2 software to construct the protein-protein interaction network. The Network Analyzer function was used to analyze the target degree values, and targets with degree values ​​≥ 1.5 times the average degree value were selected as core targets.

[0112] 2.2.5 Enrichment Analysis of GO and KEGG

[0113] Core targets were entered into the DAVID database, and the organism selected for analysis was humans (Homo Sapiens). GO functional enrichment and KEGG pathway enrichment analyses were performed. A false negative rate (FDR) < 0.05 was considered significant enrichment, with lower FDR values ​​indicating higher significance. The FDR values ​​were sorted from smallest to largest, and the top 10 were used to create GO functional enrichment bar charts (i.e., secondary classification bar charts, including cellular components, molecular functions, and biological processes) and KEGG pathway enrichment bubble charts using online bioinformatics tools.

[0114] 2.2.6 Molecular docking

[0115] The 3D structures of target proteins were downloaded from the PDB database and saved in PDB format. Water molecules and ligands were then removed using PyMOL software. The 3D structures of the components were downloaded from the PubChem database and saved in SDF format. Open Babel software was used to convert the SDF format to Mol2 or PDB format. The processed target protein and compound molecular structures were imported into Autodock for routine molecular structure preprocessing. Molecular docking was then performed using the Docking module to analyze the binding activity. Finally, the docking results were visualized using PyMOL.

[0116] 3 Results

[0117] 3.1 Screening of active ingredients in pig tooth soap

[0118] The GC-MS analysis of the total ion chromatogram of the volatile oil of *Polygonum cuspidatum* is shown below. Figure 1 After searching, matching, and manual analysis of the NIST17 database, a total of 64 components were identified, as shown in Table 2-1. The obtained components were screened according to item 2.2.1, and a total of 24 components were obtained, mainly including aromatic aldehydes, unsaturated alcohols, esters, and terpenoids.

[0119] Table 2-1 Chemical components in EOGSL

[0120]

[0121]

[0122]

[0123] 3.2 Results of network pharmacology 3.2.1 Target screening

[0124] Twenty-four active ingredients from the volatile oil of *Polygonum cuspidatum* were used for target prediction in the Swiss Target Prediction database. After removing duplicates, a total of 311 potential targets were predicted. Using the Gene Cards and OMIM disease databases, 62 and 817 CIRI target genes were matched, respectively, resulting in 822 duplicates. The intersection targets of the active ingredients and diseases were uploaded to Venny software to construct a Venn diagram. Figure 2 The study identified 95 targets for the drugs to work together.

[0125] 3.2.2 Network construction and core component screening of volatile oil, active ingredients and targets in pig tooth soap

[0126] Network visualization of volatile oil, active ingredients, and targets in pig tooth soap (see...) Figure 3 The graph contains 122 nodes and 181 edges. Using Network Analyzer software to calculate the degree, the more edges a node has connected to, the higher its degree value. The volatile oil of *Polygonum cuspidatum* is represented by yellow hexagons, components by blue rhombuses, and related target points by red circles; the higher the degree value, the larger the shape and the darker the color. Paeonol has 33 interacting target points. Other components with high degree values ​​in the network graph include linalool, methyleugenol, and eugenol. These components with a high number of target points are likely the core components of *Polygonum cuspidatum* volatile oil for disease prevention and treatment.

[0127] 3.2.3 Construction of protein-protein interaction networks and screening of core targets

[0128] Ninety-five common target sites were imported into the STRING database. The target interaction score was set to 0.7, and 15 free target sites were hidden. The database displayed interactions between 80 proteins. The TSV text data was then imported into Cytoscape 3.7.2 software to draw a protein-protein interaction network diagram. (See...) Figure 4The network generated 296 protein-protein interaction connections across 78 target sites. The target sites' colors gradually deepened from blue to red, with larger sites indicating higher intensity values. The thickness of the connections was positively correlated with the strength of the relationship between target sites. Target sites with intensity values ​​>11 (1.5 times the average) were selected as core targets, totaling 17, including VEGFA, SRC, MAPK8, PIK3CA, TNF, HSP90AA1, PTGS2, FYN, ESR1, RELA, JAK2, DRD2, MAPK14, CCND1, CASP3, BDKRB2, and KDR.

[0129] 3.2.4 Gene function and pathway enrichment analysis

[0130] In this study, 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 (BP), such as cellular responses to lipopolysaccharide, the vascular endothelial growth factor receptor signaling pathway, drug responses, positive regulation of nitric oxide biosynthesis, regulation of inflammatory responses, angiogenesis, and positive regulation of the ERK1 and ERK2 cascades. Twelve involved molecular functions (MF), such as enzyme binding, protein tyrosine kinase activity, histone deacetylase binding, ATP binding, non-transmembrane protein tyrosine kinase activity, and growth factor receptor binding. Six involved cellular components (CC), such as cytosol, nucleus, plasma membrane, nucleoplasm, and cytoplasmic components of the plasma membrane. Of the 71 KEGG pathways, the top 6 according to FDR (Functional Directedness) from smallest to largest are the prolactin signaling pathway, proteoglycan in cancer, TNF signaling pathway, sphingolipid signaling pathway, hepatitis B, and cancer pathway. Figure 5 List the top 10 entries for BP, MF, and 6 entries for CC functions based on FDR < 0.05. Figure 6 The top 20 KEGG pathways are listed.

[0131] 3.2.5 Verification Analysis of Molecular Docking

[0132] To illustrate the binding activity between targets and components, this study selected the top 5 core targets with the highest binding intensity in the protein-protein interaction network and their corresponding chemical components for docking analysis. The results are shown in Table 2-2. A binding energy less than 0 indicates that the ligand and receptor can bind spontaneously; the lower the energy, the stronger the binding ability. All binding energies in the table are less than -5 KJ / mol, and hydrogen bonds are formed, indicating good binding activity between the protein targets and their corresponding chemical components. All docking results were visualized using PyMOL software. Yellow dashed lines represent hydrogen bonds, pink represents chemical component structures, blue represents target proteins, and red represents binding sites. Figure 7 As shown.

[0133] Table 2-2: Molecular docking results

[0134]

[0135] 4. Summary

[0136] This study used GC-MS technology to analyze 64 components in the volatile oil of *Gnaphalium affine*, and screened 24 active ingredients using the Swiss ADME platform, with paeonol having the highest content. According to the volatile oil-component-target network diagram, the top 5 components with the most target matches were paeonol, linalool, methyl eugenol, anethole, and eugenol. Studies have shown that pre-administration of paeonol can significantly reduce inflammatory responses, cerebral ischemia-inducing inflammation, and improve neurological deficit symptoms in rats, thus exerting an anti-cerebral ischemia-inducing effect. Linalool has anti-inflammatory and antioxidant properties, and exhibits protective effects in in vitro models of glutamate-induced oxidative stress and in in vitro models of excitotoxicity, suggesting it as a potential therapeutic agent for neurodegenerative brain diseases. Methyl eugenol needs to cross the blood-brain barrier to enter brain tissue to exert its therapeutic effect on brain diseases. Anethole can reduce neurological deficits in mice, decrease infarct volume and cerebral edema, thus having a protective effect against ischemic stroke. Eugenol can increase BDNF levels in the brain and regulate brain region function through the olfactory pathway, thereby improving post-ischemic reperfusion injury. Based on the above research reports and network pharmacology analysis, it is speculated that the volatile oil of *Gnaphalium affine* may exert its therapeutic effect on cerebral ischemia-reperfusion injury through these five core components.

[0137] In summary, according to the online pharmacology, the volatile oil of *Zhuya soap*, through its active ingredients paeonol, linalool, methyl eugenol, anethole, and eugenol, acts on key targets such as VEGFA, SRC, MAPK8, PIK3CA, and TNF, regulating multiple signaling pathways including sphingolipids, TNF, and VEGF, inhibiting cellular oxidative stress damage, suppressing the release of inflammatory factors, and regulating nerve function, thereby playing a role in the treatment of CIRI. This lays the foundation for further in-depth research on the "opening the orifices" therapeutic mechanism of *Zhuya soap*.

[0138] II. Protective Effect of the Effective Component Formula of the Volatile Oil of Gleditsiae Fructus Abnormalis on Cerebral Ischemia-Reperfusion

[0139] 1. Materials

[0140] 1.1 Experimental Drugs

[0141] The volatile oil of Gleditsiae Fructus Abnormalis (extracted in this experiment, extraction rate: 0.132%). The daily dosage of Gleditsiae Fructus Abnormalis for adults in the first part of the Chinese Pharmacopoeia (2015 edition) was referred to, and the daily dosage for rats was calculated by the body surface area method. The administration dosage of the volatile oil of Gleditsiae Fructus Abnormalis for rats was 0.231 mg / kg.

[0142] The following reference substances were all extracted from traditional Chinese medicine pieces. Paeonol (GZDD-0046), Eugenol (GZDD-0401), Methyl Eugenol (C102127), Linalool (GZDD-0244), Anethole (C100243), all purchased from Guizhou Dida Technology Co., Ltd., purity > 98%. The proportion of effective components was: Paeonol 37.2%, Eugenol 7.8%, Methyl Eugenol 4.7%, Linalool 23.0%, Anethole 27.3%. According to this proportion, an effective formula was prepared (accounting for 11.86% of the total components of the volatile oil of Gleditsiae Fructus Abnormalis). The low dosage of the effective formula was 0.028 mg / kg, the medium dosage was 0.056 mg / kg, and the high dosage was 0.108 mg / kg.

[0143] Nimodipine Tablets (Yabao Pharmaceutical Group Co., Ltd., batch number: 170606). The administration dosage was calculated according to the daily dosage of 50 mg for adults in the instruction manual. Each tablet was 20 mg, and the daily administration dosage for rats was 5.83 mg / kg.

[0144] 1.2 Animals

[0145] SD rats, clean type, male, 180 - 200 g, purchased from Changsha Tianqin Biotechnology Co., Ltd., certificate of conformity: SCXK(Xiang)2019 - 0014

[0146] 1.3 Reagents and Instruments

[0147] Hematoxylin and eosin staining kit, 4% and 10% formaldehyde fixative (Wuhan Sewell Biotechnology Co., Ltd.), 2,3,5-triphenyltetrazolium chloride (TTC, Sigma, 1001011963), microplate reader (SpectraMAX Plus384, manufactured by Meigu Molecular Instruments Co., Ltd.), Rat IL-1β ELISA kit (Shanghai Zhuocai Biotechnology Co., Ltd., ZC-36391); Rat IL-6 ELISA kit (Zhuocai, ZC-36404), Rat TNF-α ELISA kit (Zhuocai, ZC-37624), total protein assay kit (Nanjing Jiancheng Bioengineering Institute, A045-2-1), total superoxide dismutase assay kit (SOD, Jiancheng Institute, A001-1-1), malondialdehyde assay kit (MDA, A003-1-1, Jiancheng Institute), 10% chloral hydrate, physiological saline, Tween-80.

[0148] Electronic balance (SHIMADZU; AUW20D), digital slide scanner (Pannoramic 250, 3DHISTECH (Hungary)), microscopic imaging system (BA200Digita), suture plugs, sutures, ophthalmic scissors, hemostatic clips, forceps, weighing bottles.

[0149] 2 methods

[0150] 2.1 Grouping, drug administration, and modeling

[0151] Male SD rats were randomly divided into four groups: sham-operated group, model control group, nimodipine group (5.83 mg / kg), volatile oil group (0.231 mg / kg), low-dose effective formulation group (0.028 mg / kg), medium-dose effective formulation group (0.054 mg / kg), and high-dose effective formulation group (0.108 mg / kg), with 11 rats in each group. Rats were acclimatized for 7 days before administration. The dosage of each group was converted to the adult daily dose of *Gnaphalium affine* as recorded in the pharmacopoeia. Dosage: Nimodipine was administered via gavage at a dose of 2.5 ml / 250 g, while all other treatment groups received 20 μl / 250 g via nasal administration. The model group and sham-operated group received nasal administration of physiological saline containing 3% Tween-80. During nasal administration, the mouse's head was fixed, and a microsyringe connected to a PE plastic tubing was inserted approximately 0.2 cm into the mouse's nasal cavity. The drug solution (dissolved in physiological saline containing 3% Tween-80) was administered into the nasal cavity, and any drug leakage from the rat's nasal cavity was observed. Administration was once daily for 7 consecutive days, with model initiation 1 hour after the last administration.

[0152] Rats were fasted for 12 hours prior to modeling but allowed free access to water. A rat model of middle cerebral artery occlusion was established using the suture method. The rats were anesthetized with an intraperitoneal injection of 4% chloral hydrate (0.75 mL / 100 g). After anesthesia, the rats were fixed supine on an operating table. The fur on the front of the neck was trimmed, and the skin was disinfected with iodine. An incision was made in the midline of the neck, and the right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were separated. The proximal end of the CCA and the external carotid artery were ligated with No. 1 surgical suture. A small incision was made with ophthalmic scissors 4 mm from the bifurcation of the CCA. The suture plug was inserted into the right common carotid artery through the incision to block blood flow from the middle cerebral artery. The suture was then ligated and fixed at the internal carotid artery. The wound was sutured layer by layer, with the suture plug exposed at least 0.5 cm outside the body. The wound was disinfected with iodine. After 2 hours of ischemia in the rats, the suture was gently pulled out to the internal carotid bifurcation, followed by 24 hours of perfusion.

[0153] The sham surgery group only separated blood vessels, without inserting or ligating fishing line; the rest of the procedures were the same as the other groups.

[0154] 2.2 Neurobehavioral scoring

[0155] After 24 hours of ischemia in rats, the Berderson scoring system was used. The scoring criteria were as follows: 0 points indicated no symptoms; 1 point indicated the ability to fully extend the contralateral forepaw; 2 points indicated circling to the contralateral side; 3 points indicated falling to the contralateral side; and 4 points indicated inability to walk and loss of consciousness. A score of 1 to 3 indicated successful model establishment.

[0156] 2.3 Percentage of cerebral infarction volume

[0157] Twenty-four hours after reperfusion in rats, three rats from each group were randomly decapitated, and the ischemic hemisphere was rapidly harvested. The hemispheres were frozen at -20°C for 20 minutes, then removed. Six consecutive 2.0 mm thick coronal sections of brain tissue were prepared from anterior to posterior on a surgical table. The sections were then placed in 2% TTC phosphate buffer and dried in a 37°C oven in the dark for 20 minutes, followed by fixation with 4% paraformaldehyde for 24 hours. After staining, infarcted areas appeared white, and non-infarcted areas appeared red. Images were taken, and the infarct area was measured using ImageJ software. The percentage of infarct volume was calculated using the following formula: Percentage of infarct volume = (Volume of normal brain tissue contralateral to the infarct - Volume of normal brain tissue on the infarcted side) / Volume of normal brain tissue contralateral to the infarct × 100%.

[0158] 2.4 Determination of biochemical indicators in brain tissue

[0159] Twenty-four hours after reperfusion, five rats were randomly selected from each group, and the ischemic hemisphere was quickly removed. The brain tissue was homogenized into a 10% homogenate under ice bath conditions and centrifuged at 3000 r / min for 10 min. The levels of IL-1β, IL-6, TNF-α, MDA, and SOD activity in serum and ischemic brain tissue were measured according to the kit instructions.

[0160] 2.5 Pathological observation of brain tissue (HE staining)

[0161] After 24 hours of ischemia in rats, three rats were randomly selected from each group, and the ischemic hemisphere was quickly removed and fixed in 4% paraformaldehyde solution for at least 24 hours. The tissue blocks were trimmed and labeled. After dehydration, trimming, embedding, sectioning, staining, and mounting, images of the sections were finally acquired using a digital slide scanner. Each section was first examined at 40x magnification to observe the entire tissue and gross lesions. Then, images at 100x and 400x magnification were acquired for the areas to be observed to examine specific lesions.

[0162] 3 Results

[0163] 3.1 Comparison of neurobehavioral scores among different groups of rats

[0164] Table 3-1 shows that the behavioral score of the sham surgery group was 0, while that of the model group was 3.18, and the difference was extremely significant (P < 0.01).

[0165] According to the scoring method, the rats were unable to walk after the model was established, exhibited hemiplegia, walked in a counterclockwise tail-chasing manner, and showed signs of lethargy, indicating that the model was successfully established. Compared with the model group, the scores of all treatment groups were reduced. Except for the low-dose group of the effective ingredient formula, which showed no significant difference, the differences in the other groups were extremely significant (P<0.01), indicating that the other treatment groups had an improving effect on the neurological behavior of rats.

[0166] Table 3-1 Postoperative neurological behavioral scores of rats in each group (X±S, n=11)

[0167] Group Dosage (mg / Kg) Neurological behavioral score Sham surgery group - 0.00±00 Model group - <![CDATA[3.18±0.41 ** ]]> Nimodipine group 5.83 <![CDATA[1.09±0.54 ## ]]> Volatile oil group 0.231 <![CDATA[1.73±0.47 ## ]]> Effective formulation low-dose group 0.028 2.64±0.51 Medium dose group of effective formulation 0.054 <![CDATA[1.73±0.47 ## ]]> High-dose group of effective formulation 0.108 <![CDATA[1.18±0.41 ## ]]>

[0168] Note: Compared with the sham surgery group, *P<0.05, **P<0.01; compared with the model group, #P<0.05, ##P<0.01

[0169] 3.2 Comparison of cerebral infarction volume among different groups of rats (see...) Figure 8 (and Table 3-2)

[0170] Table 3-2 Percentage of cerebral infarction volume in rats after surgery (X±S, n=3)

[0171] Group Dosage (mg / Kg) Percentage of cerebral infarction volume (%) Sham surgery group - 0.00±00 Model group - <![CDATA[36.23±0.26 ** ]]> Nimodipine group 5.83 <![CDATA[23.01±0.55 ## ]]> Volatile oil group 0.231 <![CDATA[24.32±4.06 ## ]]> Effective formulation low-dose group 0.028 29.41±1.72 Medium dose group of effective formulation 0.054 <![CDATA[21.01±2.78 ## ]]> High-dose group of effective formulation 0.108 <![CDATA[19.58±0.76 ## ]]>

[0172] Note: Compared with the sham surgery group, *P<0.05, **P<0.01; compared with the model group, #P<0.05, ##P<0.01

[0173] 3.2 Comparison of biochemical indicators in brain tissue of different groups

[0174] Table 3-3 shows that, compared with the sham-operated group, the concentrations of IL-1β, IL-6, and TNF-α in the brain tissue of rats in the model group were significantly increased (P < 0.01), indicating successful modeling. Compared with the model group, the concentrations of IL-1β and IL-6 in the brain tissue of the volatile oil group, the medium- and high-dose groups of the effective formula were significantly decreased (P < 0.01), while there were no significant differences in the other groups. Compared with the model group, the concentration of TNF-α in the brain tissue of the volatile oil group and the high-dose group of the effective formula was significantly decreased (P < 0.05), while there were no significant differences in the other groups.

[0175] Table 3-4 shows that, compared with the sham-operated group, the SOD activity in the brain tissue of the model group rats was significantly decreased (P<0.01) and the MAD content was significantly increased (P<0.01), indicating that the model was successfully established. Compared with the model group, the SOD activity in the brain tissue of the volatile oil group was significantly increased (P<0.01), and the SOD activity in the medium and low dose groups of the effective formula was significantly increased (P<0.05), while there were no significant differences in the other groups. Compared with the model group, there were no significant differences in the MDA content in the brain tissue of rats in each group.

[0176] Table 3-3 Effects of inflammatory factors in the brain tissue of rats in each group after surgery (X±S, n=5)

[0177]

[0178] Note: Compared with the sham surgery group, *P<0.05, **P<0.01; compared with the model group, #P<0.05, ##P<0.01

[0179] Table 3-4 Effects of SOD and MDA on brain tissue of rats in different groups after surgery (X±S, n=5)

[0180] Group Dosage (mg / Kg) SOD(U / mgprot) MDA (nmol / mgprot) Sham surgery group - 1698.461±283.372 1.879±0.486 Model group - <![CDATA[1039.620±195.065 ** ]]> <![CDATA[3.023±0.494 ** ]]> Nimodipine group 5.83 1192.902±173.024 2.755±0.776 Volatile oil group 0.231 <![CDATA[1433.241±252.353 ## ]]> 2.346±0.627 Effective formulation low-dose group 0.028 1095.304±151.523 2.779±0.408 Medium dose group of effective formulation 0.054 <![CDATA[1463.937±168.026 # ]]> 2.512±1.522 High-dose group of effective formulation 0.108 <![CDATA[1357.053±121.469 # ]]> 2.352±0.679

[0181] Note: Compared with the sham surgery group, *P<0.05, **P<0.01; compared with the model group, #P<0.05, ##P<0.01

[0182] 3.4 Comparison of pathological manifestations of rat brain tissue in different groups (see...) Figure 9-11 (and Table 3-5)

[0183] Sham surgery group: The morphology of neurons in the cortical area was normal, and no obvious cell degeneration or necrosis was observed; the hippocampus had a clear stratified structure, with cells arranged in tight bands, clear cell morphology, large and round nuclei, and no obvious degeneration, necrosis, glial cell proliferation or inflammatory cell infiltration was observed.

[0184] Model group: Hemicortical area showed patchy necrosis of neurons and nerve fibers, with edema and lighter staining in the necrotic area, and relatively loose nerve fibers, with some areas showing vacuolation or even dissolution and disappearance; in the necrotic edge area, a large number of neurons had blurred morphology and structure, shrunken and deeply stained nuclei, significantly reduced volume, and dissolved nerve fibers with lighter staining.

[0185] Nimodipine group: patchy necrosis and edema of neurons and nerve fibers in the hemicortical area, with lighter staining; some neurons in the marginal area have relatively normal morphology and structure, a small number of neurons have shrunken and irregular nuclei, and the surrounding nerve fibers are relatively dense; no other obvious pathological changes were observed.

[0186] Volatile oil group: patchy necrosis and edema of neurons and nerve fibers in the hemicortical area, partial necrosis of neurons in the marginal area, nuclear condensation and lysis, and more obvious degeneration of some neurons.

[0187] Low-dose group of active ingredient formulation: patchy necrosis and edema of neurons and nerve fibers in the hemicortical area, with lighter staining; the severely affected area involved the hippocampus; partial necrosis of neurons in the marginal zone, partial neuronal degeneration, the nuclei of degenerated neurons were relatively normal in morphology, and cavities were visible around them.

[0188] In the medium-dose group of the active ingredient formulation: patchy necrosis and edema of neurons and nerve fibers in the hemicortical area, with lighter staining; neuronal degeneration in the limbic area was more obvious.

[0189] High-dose group of active ingredient formulation: patchy necrosis and edema of neurons and nerve fibers in the hemicortical area, with lighter staining; neurons in the marginal area have relatively normal morphology and structure, with a small number of necrotic neurons; no other obvious pathological changes were observed.

[0190] In summary, except for the sham surgery group, large areas of necrotic foci were observed in the cortical areas of all other groups in this experiment. Neurons in the marginal zone showed varying degrees of degeneration or necrosis. The necrosis of neurons in the marginal zone was more obvious in the model group, followed by the volatile oil group, the low-dose group and the medium-dose group of the active ingredient group, and slightly less in the high-dose group of the active ingredient group and the nimodipine group.

[0191] Table 3-5 Postoperative pathological findings of rat brain tissue in each group

[0192]

[0193]

[0194] If there is no lesion, it is recorded as (—), using a 4-level system, namely mild (+), mild (++), moderate (+++), and severe (++++).

Claims

1. Use of volatile oil of Gynocardia odorata in preparation of a medicament for treating cerebral ischemia-reperfusion injury. The preparation method of the volatile oil of Gynocardia odorata is as follows: a proper amount of Gynocardia odorata decoction pieces are cut into pieces, 8-12 times of water is added, 5-15% of the Gynocardia odorata decoction pieces in terms of g / ml are taken, and the volatile oil is obtained by water vapor distillation for 6-14 hours, the volatile oil is collected, an appropriate amount of anhydrous sodium sulfate is added to remove water, and the volatile oil of Gynocardia odorata is obtained after overnight standing. The active ingredients of the volatile oil of Gynocardia odorata include aromatic aldehydes, unsaturated alcohols, esters and terpenes.

2. Use of the volatile oil of P. granulata according to claim 1 for the preparation of a medicament for the treatment of cerebral ischemia-reperfusion injury, characterized in that: The preparation method of the volatile oil of Gynocardia odorata is as follows: a proper amount of Gynocardia odorata decoction pieces are cut into pieces, 10 times of water is added, 10% of the Gynocardia odorata decoction pieces in terms of g / ml are taken, and the volatile oil is obtained by water vapor distillation for 10 hours, the volatile oil is collected, an appropriate amount of anhydrous sodium sulfate is added to remove water, and the volatile oil of Gynocardia odorata is obtained after overnight standing.

3. A medicament for treating cerebral ischemia-reperfusion injury, characterized by: The medicament is mainly prepared from the volatile oil of Gynocardia odorata in claim 1 or 2.

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