Application of agilawood extract in preparation of medicine for resisting cerebral arterial thrombosis
The preparation of anti-ischemic stroke drugs through agarwood extract solves the problem of the application gap of agarwood in the treatment of ischemic stroke. In vitro experiments and rat model studies have shown that it has significant therapeutic effects on ischemic stroke, improving physiological state and neurological function, and providing a basis for treatment.
Patent Information
- Application Number
- CN202511141835.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Priority Date
- 2025-03-31
- Filing Date
- 2025-08-15
- Publication Date
- 2025-10-17
AI Technical Summary
In the existing technology, there is still a gap in the research on the application of agarwood in the treatment of ischemic stroke. Its intervention effect and potential mechanism have not been systematically elucidated, and there is a lack of effective treatment plans, which leads to high disability and mortality rates.
Agarwood extract was used to prepare an anti-ischemic stroke drug, and its therapeutic effect on ischemic stroke was studied through in vitro activity experiments and rat models. The preparation method included reflux extraction of agarwood medicinal materials with 80% ethanol solution, drying to obtain the agarwood extract, and using the CCK-8 method to determine cell viability. A 2-VO method rat model was constructed for drug intervention to observe physiological status and pathological changes.
Agarwood extract has been shown to have a protective effect on cells injured by hypoxia/glucose deprivation and reperfusion in vitro, improve the physiological state and neurological function damage of rats, reduce pathological abnormalities of hippocampal neurons, improve the quality of life, and provide a theoretical basis for the treatment of ischemic stroke.
Smart Images
Figure CN120789157A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of traditional Chinese medicine, in particular to the application of aquilaria sinensis extract in the preparation of a drug for resisting ischemic stroke. BACKGROUND
[0002] Ischemic stroke (IS) is a serious cerebrovascular disease, and its pathological feature is that local brain tissue is ischemic and hypoxic due to interruption of cerebral blood flow, thereby causing nerve cell death and brain dysfunction. IS has the characteristics of high morbidity, high disability rate and high mortality, and is one of the main causes of death and disability worldwide. According to statistics, about 150 million people worldwide suffer from stroke every year, of which about 85% are ischemic stroke. Although some progress has been made in acute treatment (such as intravenous thrombolysis and endovascular thrombectomy) in recent years, due to the narrow treatment time window, reperfusion injury and other limitations, many patients still face serious neurological deficits and sequelae. Therefore, developing a safer and more effective treatment plan to reduce disease mortality, improve patient prognosis, and reduce social and economic burden has become an urgent need for current medical research.
[0003] Aquilaria sinensis (CX) is a traditional and precious Chinese medicinal material with a long history of medicinal use. Its chemical composition is complex, mainly including active ingredients such as sesquiterpenes, aromatic compounds and flavonoids, and has various pharmacological effects such as anti-inflammatory, antioxidant, anti-tumor and neuroprotective effects. However, there is currently no research on the application of aquilaria sinensis in the treatment of ischemic stroke at home and abroad, and its specific intervention effect and potential mechanism have not been systematically elucidated. Therefore, in-depth study of the intervention effect of aquilaria sinensis on ischemic stroke and its mechanism can not only help to reveal the pathophysiological process of IS, but also provide important theoretical basis and experimental basis for the research and development of new drugs for treating ischemic stroke. It provides a new drug target and treatment strategy for the treatment of ischemic stroke, promotes the modernization and internationalization process of traditional Chinese medicine, and ultimately contributes to improving the quality of life of patients and reducing the social and medical burden. SUMMARY
[0004] The purpose of the present application is to provide the application of aquilaria sinensis extract in the preparation of a drug for resisting ischemic stroke, in order to solve the problems existing in the prior art. The present application evaluates the therapeutic effect of aquilaria sinensis extract on ischemic stroke from the aspects of in vitro activity, behavioral changes, typical pathological manifestations and drug-containing serum activity, and provides a theoretical basis for the mechanism of action.
[0005] To achieve the above-mentioned purpose, the present application provides the following scheme:
[0006] Technical solution one: the application of the extract of linaloe in the preparation of the medicine for resisting ischemic stroke, the preparation method of the extract of linaloe, comprising the following steps: mixing linaloe medicinal materials with 80% ethanol solution in volume fraction, carrying out reflux extraction, and obtaining the extract of linaloe after drying.
[0007] Further, the volume ratio of the linaloe medicinal materials to the ethanol solution is 1:10.
[0008] Further, the temperature of the reflux extraction is 90 DEG C.
[0009] Further, the reflux extraction is extracted for 2 times, 2h each time, and the extraction liquid is combined.
[0010] Further, the medicine further comprises pharmaceutically acceptable adjuvant or auxiliary ingredient.
[0011] Further, the in-vitro activity is determined by the influence on the activity of PC12 cells with hypoxia / hypoglycemia reperfusion injury through CCK-8 method.
[0012] Further, the anti-ischemic stroke is realized by improving physiological state, balance ability and nerve function injury, and improving abnormal arrangement of neurons and neuron karyopyknosis of hippocampal tissue.
[0013] The present application discloses the following technical effects:
[0014] The present application determines the influence of CX on the activity of PC12 cells with ischemia / hypoxia through CCK-8 method, and the results show that the extract of CX has certain activity in vitro; the IS rat model is constructed by 2-VO method, the physiological state of the rats, the behavior performance and the pathological changes of the brain tissue are analyzed to study the mechanism of the extract of linaloe on ischemic stroke, so as to provide theoretical basis for further evaluating the therapeutic effect of linaloe on ischemic stroke and the mechanism thereof; the influence of the medicine on the balance and the nerve function of IS is evaluated by balance beam experiment, muscle strength evaluation and nerve injury index score; the influence of the medicine on the pathological state of the brain tissue of IS rats is evaluated by brain tissue HE staining. The results show that the extract of linaloe can improve the physiological state, balance ability and nerve function injury of IS rats; the HE staining results show that the extract of linaloe can improve the pathological state such as abnormal arrangement of neurons and neuron karyopyknosis of hippocampal tissue of IS rats; the activity of the drug-containing serum is determined by CCK-8, and the results show that the extract of CX can protect the activity of cells, which further shows that the extract of CX can play a therapeutic effect after entering the blood of rats. The behavior symptoms of the patients with ischemic stroke are treated, and the theoretical basis for improving the life quality of the patients with ischemic stroke is laid. BRIEF DESCRIPTION OF DRAWINGS
[0015] In order to make the technical solutions in the embodiments of the present application or the prior art clearer, the accompanying drawings needed in the embodiments will be briefly introduced below. Obviously, the accompanying drawings in the following description only need to be some embodiments of the present application, and other drawings can be obtained by those skilled in the art without any creative work on the basis of these drawings.
[0016] Figure 1 Flow chart for construction of IS rat model;
[0017] Figure 2 Results of cell survival rate for determination of CX toxicity, Con is the control group, and the other is the CX administration concentration group;
[0018] Figure 3 Results of cell survival rate for determination of CX activity, Con is the control group, M is the model group, Y is the positive control group, and the other is the administration concentration group;
[0019] Figure 4 Body weight changes of IS rats in each group, wherein Con is the sham operation group, M is the model group, H is the high-dose group, L is the low-dose group, and Y is the positive control group;
[0020] Figure 5 Detection results of balance beam experiment of IS rats in each group, wherein Con is the sham operation group, M is the model group, H is the high-dose group, L is the low-dose group, and Y is the positive control group;
[0021] Figure 6 Detection results of muscle strength score experiment of IS rats in each group, wherein Con is the sham operation group, M is the model group, H is the high-dose group, L is the low-dose group, and Y is the positive control group;
[0022] Figure 7 Detection results of nerve function injury experiment of IS rats in each group, wherein Con is the sham operation group, M is the model group, H is the high-dose group, L is the low-dose group, and Y is the positive control group;
[0023] Figure 8 Neuronal pathological features of hippocampal tissue of IS rats in each group;
[0024] Figure 9 Analysis of related part scores of samples in each group;
[0025] Figure 10 Pathological features of hippocampal cortex of cerebral ischemic rats in each group;
[0026] Figure 11 Hippocampus and cortex injury of cerebral ischemic rats in each group;
[0027] Figure 12To determine the cell survival rate results of the activity of drug-containing serum, wherein Con is a control group; M is a model group; Y is a positive control group; and the others are drug-containing serum concentration groups. DETAILED DESCRIPTION
[0028] Various exemplary embodiments of the present application will now be described in detail, which should be considered to be illustrative of certain aspects, features and embodiments of the present application and are not intended to limit the present application, but to enable a further understanding thereof.
[0029] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. In addition, for numerical ranges that include an element or a combination of elements, it is intended that every numerical value within that range is also specifically recited. For example, a range of "1 to 10" is specifically intended to include the values "1-3, 4-7, 8-10" as well as individual values like 3, 5, or 9, within the range of 1 to 10. In addition, it is intended that every intermediate value of "less than" and "greater than" for each of the elements that make up the bases of each of the ranges is also disclosed. For example, the range of "1 to 10" is specifically intended to include the intermediate values of "less than 3, less than 7, less than 9" and the intermediate values of "greater than 3, greater than 7, greater than 9" as well as the individual values like 3, 5, or 9, within the range of 1 to 10. In addition, it is intended that every intermediate range of "less than" and "greater than" for each of the elements that make up the bases of each of the ranges is also disclosed. For example, the range of "1 to 10" is specifically intended to include the intermediate value of "less than 3, less than 7, less than 9" and the intermediate values of "greater than 3, greater than 7, greater than 9" as well as the individual values like 3, 5, or 9, within the range of 1 to 10.
[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, preferred methods and materials are described. All publications mentioned in this specification are herein incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any reference in this specification is not intended to constitute an admission that the reference is prior art or that this specification is not entitled to any
[0031] Many modifications and variations of this application can be made without departing from its spirit or scope, which will be apparent to those skilled in the art. The disclosure of the present application is by way of example and not intended to be limiting of the present application. Other embodiments of the present application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given are exemplary and are not intended to be limiting of the present application.
[0032] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having" and the like are open-ended terms that are intended to permit but not to exclude additional elements, features, compositions, steps, or the like.
[0033] The present application uses 2-VO method to construct ischemic stroke (IS) rat model, and studies the mechanism of action of eaglewood extract on ischemic stroke by gavage administration of eaglewood extract.
[0034] Example 1
[0035] 1. Preparation of eaglewood extract
[0036] Eaglewood (CX) was tested according to the Chinese Pharmacopoeia 2020 edition, and the contents of chalcidiole (C 17 H 18O6) content is 0.17%, which complies with the pharmacopoeia regulations and can be used to prepare agarwood extract; CX crude drug is reflux extracted twice with 10 times the volume of 80% ethanol solution at 90°C, the two extracts are combined and concentrated, and vacuum dried to obtain the agarwood extract.
[0037] 2. Experimental Methods
[0038] 2.1 CCK-8 assay for in vitro activity of CX extracts
[0039] 2.1.1 Cell culture
[0040] After thawing, culture PC12 cells in complete medium (high glucose 1640) supplemented with 10% FBS in a cell culture incubator at 37°C, 95% humidity, and 5% CO2. Observe the cells daily and replace the culture medium. Subculture when the cells reach approximately 80% of their total cell count in the culture flask.
[0041] 2.1.2 CX toxicity test
[0042] Select cells in the logarithmic growth phase with good growth status to prepare cell suspension and count them. 3 The cell suspension (100 μL / well) was inoculated at a concentration of cells / mL, and a blank group (Con) and CX extract 1.56, 3.13, 6.25, 12.5, 25, 50, and 100 μg / mL concentration groups were set up. The culture plate was placed in an incubator for pre-culture for 12-24 hours to allow the cells to reach the exponential phase. Then, the culture medium of each well was aspirated, and different concentrations of agarwood extract were added to the culture plate for intervention. Normal culture medium was added to the NC group. The culture plate was cultured in an incubator (37°C, 5% CO2) for 24 hours. Then, 10 μL of CCK-8 reagent was added to each well under light-proof conditions and cultured for another 1-4 hours. Finally, the absorbance (OD value) at a wavelength of 450 nm was detected using a microplate reader, and the cytotoxicity was judged by the OD value.
[0043] 2.1.3 Protective effect of CX on PC12 cells induced by oxygen-glucose deprivation / reperfusion injury
[0044] PC12 cells were seeded into 96-well plates at a density of 6 x 103 / mL, 100 μL per well. The experimental setup included a blank control group (Con), a model group (M), a positive drug nimodipine group (Y), and drug groups treated with different concentrations of CX extract. After 24 h of culture, the drug groups were added with 1.56, 3.13, 6.25, 12.5, 25, 50, 100 μg / mL of CX extract, the Y group was added with 5 μmol / L of nimodipine, and the Con group and the M group were replaced with new normal culture medium. After 24 h of continuous culture, the culture medium was removed, and the M, Y, and different concentration drug groups were treated with 5 mmol / L of Na2S2O4 sugar-free balanced salt solution (EBSS) for 80 min to simulate hypoxic and anoxic injury. After treatment, each group was again added with normal culture medium for 24 h of culture. Finally, the survival rate of cells in the 96-well plate was determined using a CCK-8 kit.
[0045] 2.2 Construction of bilateral common carotid artery ligation ischemic stroke (IS) rat model
[0046] 2.2.1 Construction of IS rat model by 2-VO method
[0047] The construction process of the IS rat model is shown in Figure 1 , and specifically, the construction process is as follows: after the SD rats were anesthetized and fixed, a median incision was made on the neck to expose the bilateral common carotid arteries (CCA), the bilateral CCA were separated and permanently ligated to block the cerebral blood flow, and after the incision was sutured, the rats were placed in a warm environment for recovery. It was observed that the rats had symptoms such as poor appetite, weight loss, unilateral eyelid ptosis, eye movement disorder, hyperemia, and sensory and motor loss in the contralateral limbs, which indicated that the modeling was successful.
[0048] 2.2.2 Grouping
[0049] SPF level male SD rats were selected and randomly divided into 5 groups (8 rats per group): sham operation group (Con), model group (M), high-dose group (H), low-dose group (L), and positive control group (Y). After modeling, the rats were continuously administered by gavage for 28 days. The H and L groups were administered with CX at 600 mg / kg and 150 mg / kg, respectively (dissolved in 0.5% carboxymethylcellulose sodium physiological saline), the Y group was administered with donepezil at 0.45 mg / kg, and the Con and M groups were administered with the same volume of 0.5% carboxymethylcellulose sodium physiological saline.
[0050] 2.3 Balance beam experiment, muscle strength determination, and nerve injury assessment were performed on IS rats in each group
[0051] Balance beam experiment, muscle strength determination, and nerve injury assessment were performed on IS rats in each group on the 3rd, 7th, 14th, 21st, and 28th days of administration.
[0052] 2.4 Pathological index detection on IS rats
[0053] After 28 days of drug administration, the rat plasma, brain tissue and feces were taken and stored in a -80°C refrigerator. The whole brain of the rat was obtained by 0.01 mol / L PBS and 4% paraformaldehyde cardiac perfusion for fixation and subsequent pathological index detection.
[0054] 2.5 In vitro activity of CX-containing serum
[0055] The drug-containing serum obtained from the rats was prepared into 10%, 15%, and 20% serum concentrations, and the cells were cultured according to 2.1.1. The in vitro activity of the drug-containing serum was determined according to the method of 2.1.3, and the control group (Com), model group (M), 0, 10%, 15%, and 20% drug-containing serum groups, and positive control group (Y) were set.
[0056] 3. Experimental results
[0057] 3.1 CX toxicity experiment results
[0058] Through CCK-8 cell viability determination, the results are as shown in Figure 2 At a concentration gradient of 1.56-100 μg / mL, after the action of the CX extract, the cell survival rate of the administration group did not decrease significantly compared with the Con group, and was basically similar to the Con group.
[0059] 3.2 Effect of CX on PC12 cell hypoxia-glucose deprivation / reperfusion injury
[0060] The CCK-8 experiment results show that Figure 3 The cell survival rate of the M group was significantly lower than that of the Con group, and the cell survival rate of the Y group was significantly higher than that of the M group. In the different concentration CX extract treatment groups, the cell survival rate gradually increased with the increase of the CX extract concentration. Among them, the cell survival rate of the 12.5 and 25 μg / mL CX extract treatment groups was close to that of the Y group. The cell survival rate of the 50 and 100 μg / mL concentration treatment groups increased compared with the M group.
[0061] 3.3 Effect of CX extract on nerve function injury of rats in ischemic stroke (IS) rat model
[0062] The effect of the eaglewood extract on the physiological changes of the IS rats in each group was evaluated by recording the body weight changes of the rats Figure 4 The effect of the eaglewood extract on the nerve function injury of the IS rats was evaluated by the balance beam experiment, muscle strength determination, and nerve function injury score Figures 5-7 The results show that compared with the model group, the high-dose group and the low-dose group can improve the physiological state, balance ability, and nerve function injury of the IS rats.
[0063] Effects of 3.4CX extract on histopathological characteristics of rats in an ischemic stroke (IS) rat model
[0064] After HE staining, the rat brain sections were observed and analyzed under a microscope. IS rats had disordered pyramidal neurons in the hippocampus CA1 region, swollen or shrunken neurons, decreased pyramidal cell numbers, condensed nuclei, and chromatin aggregation. Figure 8 ), compared with the Con group, the overall structure of the hippocampus in the M group was abnormal, the neurons were disordered, and some neurons had karyopyknosis (as shown by the black arrows in the figure). After CX treatment, the abnormality of the hippocampal tissue structure was alleviated, the neurons were neatly arranged and tightly packed, the neuronal karyopyknosis (as shown by the black arrows in the figure) was reduced, and the infiltration of inflammatory cells in the tissue (as shown by the red arrows in the figure) was reduced, indicating that CX can improve the pathological characteristics of neurons in the hippocampus of IS rats. In addition, we scored and analyzed the relevant parts of the samples, and the scoring was based on the morphological structure of brain cells and the surrounding environment of the cells. 0 points, normal; 1 point, a small number of cells were damaged, and there were no vacuoles around the cells; 2 points, obvious cell damage, and a small number of vacuoles around the cells; 3 points, obvious cell damage, and obvious vacuoles around the cells; 4 points, a large number of cells were damaged (the results are shown in the table). Figure 9 shown).
[0065] Nissl staining results showed that the number of Nissl bodies in the cortex and hippocampus of the M group was significantly reduced compared with the blank group; while the number of Nissl bodies in the above areas of the CX group was significantly more than that of the model group, and the effect of the CX-H group was more significant, which was equivalent to that of the positive drug group ( Figure 10 and Figure 11 ). This indicates that CX has a significant protective effect on the brain tissue of IS rats and significantly reduces the damage of IS lesions to brain neurons.
[0066] 3.5 Effects of CX-containing serum on PC12 cells induced by oxygen-glucose deprivation / reperfusion injury
[0067] To investigate whether rat serum containing CX can activate PC12 cells injured by oxygen-glucose deprivation / reperfusion, CCK-8 method was used to determine the activity. Figure 12 The results showed that the cell survival rate increased after treatment with drug-containing serum at three concentrations. In comparison, the cell survival rate did not increase significantly in the absence of drug-containing serum, indicating that CX-containing serum has a certain protective effect on PC12 cells from hypoxia-glucose deprivation / reperfusion injury.
[0068] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.
Claims
1. The use of agarwood extract in the preparation of a drug for anti-ischemic stroke, characterized in that: The preparation method of the agarwood extract comprises the following steps: mixing agarwood medicinal materials with an ethanol solution with a volume fraction of 80%, performing reflux extraction, and obtaining the agarwood extract after drying.
2. The use according to claim 1, characterized in that The volume ratio of the agarwood medicinal material to the ethanol solution is 1:
10.
3. The use according to claim 1, characterized in that The temperature of the reflux extraction is 90°C.
4. The use according to claim 1, characterized in that The reflux extraction was performed twice, each time for 2 hours, and the extracts were combined.
5. The use according to claim 1, characterized in that The medicine also includes pharmaceutically acceptable excipients or auxiliary ingredients.
6. The use according to claim 1, characterized in that The anti-ischemic stroke is achieved by improving physiological state, balance ability and nerve function damage, as well as improving abnormal arrangement of neurons in hippocampal tissue and neuronal nuclear pyknosis.