Application of agilawood extract in preparation of medicine for resisting cerebral arterial thrombosis

Abstract

The invention discloses an application of an agilawood extract in preparation of a medicine for resisting cerebral arterial thrombosis, and belongs to the technical field of traditional Chinese medicines. A cerebral arterial thrombosis rat model is constructed by utilizing a 2-VO method, the action mechanism of the agilawood extract on cerebral arterial thrombosis (IS) is researched by analyzing the physiological status, behavioral performance and brain tissue pathological change of rats, the treatment effect of agilawood on cerebral arterial thrombosis is further evaluated, and a theoretical basis is provided for the action mechanism of agilawood. Results show that the agilawood extract can improve physiological status, balance ability and neurological function damage of IS rats; hE staining results show that the agilawood extract can improve pathological states such as abnormal neuron arrangement and neuronal nucleus fixation and contraction of IS rat brain hippocampus. And a theoretical basis is laid for treating behavioral diseases of cerebral arterial thrombosis patients and improving the life quality of the cerebral arterial thrombosis patients.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine, and in particular to application of an agarwood extract in preparing a drug for resisting ischemic stroke. Background Art

[0002] Ischemic stroke (IS) is a serious cerebrovascular disease. Its pathological characteristics are that local brain tissue ischemia and hypoxia are caused by interruption of cerebral blood flow, which in turn leads to neuronal cell death and brain dysfunction. IS is characterized by high morbidity, high disability rate and high mortality rate, and is one of the main causes of death and disability worldwide. According to statistics, about 15 million people suffer from stroke every year in the world, of which about 85% are ischemic stroke. Although some progress has been made in acute treatment (such as intravenous thrombolysis and intravascular thrombectomy) in recent years, many patients still face serious neurological deficits and sequelae due to limitations such as narrow treatment time window and reperfusion injury. Therefore, it has become an urgent need for current medical research to develop safer and more effective treatment options, reduce disease mortality, improve patient prognosis, and reduce socioeconomic burden.

[0003] Aquilaria sinensis (CX) is a traditional 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 multiple pharmacological effects such as anti-inflammatory, antioxidant, anti-tumor and neuroprotective. However, there is still a lack of research on the application of aquilaria 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 research on the intervention effect of aquilaria on ischemic stroke and its mechanism of action will 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 Chinese medicine anti-ischemic stroke drugs. It will provide new drug targets and treatment strategies for the treatment of ischemic stroke, promote the modernization and internationalization of Chinese medicine, and ultimately contribute to improving the quality of life of patients and reducing the social medical burden. Summary of the invention

[0004] The purpose of the present invention is to provide an application of agarwood extract in the preparation of an anti-ischemic stroke drug to solve the problems existing in the above-mentioned prior art. The present invention evaluates the therapeutic effect of agarwood extract on ischemic stroke from indicators such as 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 object, the present invention provides the following solutions:

[0006] Technical solution 1: Application of agarwood extract in the preparation of anti-ischemic stroke drugs. 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.

[0007] Furthermore, the volume ratio of the agarwood medicinal material to the ethanol solution is 1:10.

[0008] Furthermore, the temperature of the reflux extraction is 90°C.

[0009] Furthermore, the reflux extraction is performed twice, each time for 2 hours, and the extracts are combined.

[0010] Furthermore, the medicine also includes pharmaceutically acceptable excipients or auxiliary ingredients.

[0011] Furthermore, the in vitro activity is determined by CCK-8 method to determine the effect on the viability of PC12 cells injured by hypoxia / glucose deprivation and reperfusion.

[0012] Furthermore, the anti-ischemic stroke is achieved by improving the physiological state, balance ability and neurological function damage, as well as improving the abnormal arrangement of neurons in hippocampal tissue and neuronal nuclear condensation.

[0013] The present invention discloses the following technical effects:

[0014] The present invention determines the effect of CX on the activity of ischemic / hypoxic PC12 cells by CCK-8 method, and the results show that CX extract has certain activity in vitro; the 2-VO method is used to construct an ischemic stroke (IS) rat model, and the action mechanism of agarwood extract on ischemic stroke is studied by analyzing the physiological state, behavioral performance and brain tissue pathological changes of rats, so as to provide a theoretical basis for further evaluating the therapeutic effect of agarwood on ischemic stroke and its action mechanism. It includes evaluating the effect of drugs on IS balance and neurological function through balance beam experiment, muscle strength assessment and nerve injury index score; evaluating the effect of drugs on the pathological state of brain tissue of IS rats by HE staining of brain tissue. The results show that agarwood extract can improve the physiological state, balance ability and neurological function damage of IS rats; HE staining results show that agarwood extract can improve the pathological states such as abnormal arrangement of neurons and nuclear condensation of neurons in the hippocampus tissue of IS rats; the activity of drug-containing serum is measured by CCK-8, and the results show that CX extract can protect cell activity, further indicating that CX can exert a therapeutic effect after entering the blood in rats. It lays a theoretical foundation for treating behavioral symptoms of ischemic stroke patients and improving their quality of life. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0016] Figure 1 Flow chart for the construction of the IS rat model;

[0017] Figure 2 To determine the cell survival rate due to CX toxicity, Con was the control group; the others were CX administration concentration groups.

[0018] Figure 3 The results of cell survival rate obtained by measuring CX activity, Con is the control group, M is the model group; Y is the positive control group; and the others are the drug administration concentration groups.

[0019] Figure 4 The weight changes of IS rats in each group, Con is the sham operation group; M is the model group; H is the high-dose group; L is the low-dose group; Y is the positive control group;

[0020] Figure 5 The results of the balance beam test of IS rats in each group, among which Con is the sham operation group; M is the model group; H is the high-dose group; L is the low-dose group; Y is the positive control group;

[0021] Figure 6 The results of the muscle strength scoring experiment of IS rats in each group, among which Con is the sham operation group; M is the model group; H is the high-dose group; L is the low-dose group; Y is the positive control group;

[0022] Figure 7 The results of the neurological injury experiment of IS rats in each group, Con is the sham operation group; M is the model group; H is the high-dose group; L is the low-dose group; Y is the positive control group;

[0023] Figure 8 The neuronal pathological characteristics of the hippocampus tissue in the brain of IS rats in each group;

[0024] Fig. 9 Score and analyze the relevant parts of each group of samples;

[0025] Fig.10 Pathological characteristics of hippocampal cortex in rats with cerebral ischemia in each group;

[0026] Fig.11 The damage of hippocampus and cortex of rats with cerebral ischemia in each group;

[0027] Fig.12Immunohistochemical analysis of CA3 area of ​​the brain of rats in each group;

[0028] Fig.13 The expression of GAP43 and Caspase3 in CA3 area of ​​the brain of rats in each group.

[0029] Fig.14 To determine the cell survival rate results of the drug-containing serum activity, Con is the control group; M is the model group; Y is the positive control group; and the others are drug-containing serum concentration groups. DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0033] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0034] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0035] The present invention utilizes the 2-VO method to construct an ischemic stroke (IS) rat model, and administers agarwood extract by intragastric administration to study the mechanism of action of agarwood extract on ischemic stroke.

[0036] Example 1

[0037] 1. Preparation of Agarwood Extract

[0038] Agarwood (CX) is tested according to the 2020 edition of the Chinese Pharmacopoeia, among which agarwood tetraol (C 17 H 18 O6) content is 0.17%, which meets the requirements of the pharmacopoeia and can be used to prepare agarwood extract; CX crude drug is extracted twice with 10 times the volume of 80% ethanol solution at 90°C, the two extracts are combined and concentrated, and the agarwood extract is obtained by vacuum drying.

[0039] 2. Experimental Methods

[0040] 2.1 CCK-8 assay for in vitro activity of CX extracts

[0041] 2.1.1 Cell culture

[0042] After thawing the cells, culture the PC12 cells in high-glucose 1640 complete medium containing 10% FBS in a cell culture incubator at 37°C, 95% humidity, and 5% CO2. Observe the cell status and replace the culture medium every day. Subculture the cells when the number of cells in the culture flask reaches about 80%.

[0043] 2.1.2 CX toxicity test

[0044] 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 1.56, 3.13, 6.25, 12.5, 25, 50, and 100 μg / mL of CX extract, and the blank group (Con) and the CX extract concentration groups of 1.56, 3.13, 6.25, 12.5, 25, 50, and 100 μg / mL 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. Next, the culture medium of each well was aspirated, and different concentrations of agarwood extract were added to the culture plate for intervention, and 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 the culture was continued for 1-4 hours. Finally, the absorbance (OD value) at a wavelength of 450 nm was detected using an enzyme reader, and the cytotoxicity was judged by the OD value.

[0045] 2.1.3 Protective effect of CX on PC12 cells induced by oxygen-glucose deprivation / reperfusion injury

[0046] PC12 cells were seeded into 96-well plates at a density of 6×103 cells / mL, with 100 μL in each well. The experimental settings 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 hours of culture, 1.56, 3.13, 6.25, 12.5, 25, 50, and 100 μg / mL of CX extract were added to the drug group, 5 μmol / L of nimodipine was added to the Y group, and the Con group and the M group were replaced with new normal culture medium. After all groups were cultured for 24 hours, the culture medium was removed, and 5 mmol / L of Na2S2O4 sugar-free balanced salt solution (EBSS) was added to the M, Y groups, and different concentrations of drug groups for 80 minutes to simulate hypoxia and glucose deprivation injury. After treatment, normal culture medium was added to each group again for 24 hours. Finally, the CCK-8 kit was used to determine the survival rate of cells in the 96-well plate.

[0047] 2.2 Construction of ischemic stroke (IS) rat model by bilateral common carotid artery ligation

[0048] 2.2.1 Establishment of IS rat model using 2-VO method

[0049] The construction process of the IS rat model is as follows Figure 1 As shown in the figure, specifically, the construction process is as follows: the SD rats are anesthetized and fixed, the bilateral common carotid arteries (CCA) are exposed through a midline incision in the neck, the bilateral CCAs are separated and permanently ligated to block cerebral blood flow, and the rats are placed in a warm environment to recover after the incisions are sutured. The model is successfully established when the rats show symptoms such as poor appetite, weight loss, unilateral eyelid ptosis, eye movement disorders, congestion symptoms, and sensory and motor loss in the contralateral limbs.

[0050] 2.2.2 Grouping

[0051] SPF male SD rats were randomly divided into 5 groups (8 rats in each 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 given oral administration for 28 consecutive days. The H and L groups were given CX (dissolved in saline containing 0.5% sodium carboxymethylcellulose) at 600 mg / kg and 150 mg / kg, respectively, the Y group was given donepezil at 0.45 mg / kg, and the Con and M groups were given the same volume of saline containing 0.5% sodium carboxymethylcellulose.

[0052] 2.3 Balance beam test, muscle strength test and nerve injury assessment were performed on each group of IS rats

[0053] The IS rats in each group were subjected to balance beam test, muscle strength test and nerve injury assessment on the 3rd, 7th, 14th, 21st and 28th days of administration.

[0054] 2.4 Pathological index detection of IS rats

[0055] After 28 days of administration, the rat plasma, brain tissue and feces were collected and stored in a -80°C refrigerator; the whole brain of the rat was obtained by cardiac perfusion with 0.01 mol / L PBS and 4% paraformaldehyde for fixation, frozen sectioning and subsequent pathological index detection.

[0056] 2.5 In vitro activity of CX-containing serum

[0057] The drug-containing serum obtained from the rat experiment was mixed with cell culture medium to prepare serum concentrations of 10%, 15%, and 20%. Cells were cultured according to 2.1.1. The in vitro activity of the drug-containing serum was determined according to 2.1.3. A control group (Com), a model group (M), 0%, 10%, 15%, and 20% drug-containing serum groups, and a positive control group (Y) were set up.

[0058] 3. Experimental results

[0059] 3.1 CX toxicity test results

[0060] The cell viability was determined by CCK-8. Figure 2 It was shown that under the concentration gradient of 1.56-100 μg / mL, after the action of CX extract, the cell survival rate of the drug-treated group did not decrease significantly compared with the Con group, and was basically similar to that of the Con group.

[0061] Effects of CX on Oxygen-Glucose Deprivation / Reperfusion Injury in PC12 Cells

[0062] The results of CCK-8 assay showed that ( Figure 3 ), the cell survival rate of group M was significantly lower than that of group Con, while that of group Y was significantly higher than that of group M. In the groups treated with different concentrations of CX extract, the cell survival rate gradually increased with the increase of 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 group Y. The cell survival rate of the 50 and 100 μg / mL concentration treatment groups was higher than that of group M.

[0063] Effects of 3CX extract on neurological function damage in rats with ischemic stroke (IS) model

[0064] The effects of agarwood extract on the physiological changes of IS rats in each group were evaluated by recording the changes in rat body weight. Figure 4 ), balance beam test, muscle strength test, and neurological function injury score were used to evaluate the effect of agarwood extract on neurological function injury in IS rats ( Figure 5-Figure 7 ). The results showed that compared with the model group, the high-dose group and the low-dose group could improve the physiological state, balance ability and neurological function damage of IS rats.

[0065] Effects of 3.4CX extract on histopathological characteristics of rats in ischemic stroke (IS) rat model

[0066] After HE staining, the rat brain sections were observed and analyzed under a microscope. The pyramidal neurons in the CA1 region of the hippocampus of IS rats were disordered, the neurons were swollen or shrunken, the number of pyramidal cells decreased, the nuclei were condensed, and the chromatin was aggregated. The results of HE staining of rat brain tissue showed ( Figure 8 ), compared with the Con group, the overall structure of the hippocampal tissue in the M group was abnormal, the neurons were disordered, and some neurons had karyopyknosis (as shown by the black arrow in the figure). After CX treatment, the abnormality of the hippocampal tissue structure was alleviated, the neurons were neatly arranged and tightly packed, the karyopyknosis of neurons (as shown by the black arrow in the figure) was reduced, and the infiltration of inflammatory cells in the tissue (as shown by the red arrow in the figure) was reduced, indicating that CX can improve the pathological characteristics of neurons in the hippocampal tissue 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 Fig. 9 shown).

[0067] 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 ( Fig.10 and Fig.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.

[0068] Effects of 3.5CX on neuronal damage in mice with cerebral ischemia

[0069] To explore whether CX plays a protective role on neurons in rats with cerebral ischemia, immunohistochemical staining was used to detect the expression of GAP43 and Caspase3 in the CA3 region. The results showed that compared with the Con group, the number of GAP43 and Caspase3-positive neurons in the CA3 region of the M group rats was significantly reduced, indicating that the neurons of the M group rats were severely damaged. However, after co-treatment with CX, the loss of GAP43 and Caspase3-positive cells was significantly alleviated, which was statistically significant compared with the M group ( Fig.12 and Fig.13 ). CX appears to play a neuroprotective role in the IS model.

[0070] 3.6 Effects of CX-containing serum on oxygen-glucose deprivation / reperfusion injury in PC12 cells

[0071] To investigate whether rat serum containing CX can activate PC12 cells injured by oxygen-glucose deprivation / reperfusion, CCK-8 method was used for determination. Fig.14 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 cell hypoxia-glucose deprivation / reperfusion injury.

[0072] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined 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 the physiological state, balance ability and nerve function damage, as well as improving the abnormal arrangement of neurons in hippocampal tissue and neuronal nuclear condensation.

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