CN116019802A - Application of crocetin in preparation of medicine for treating and preventing cerebral ischemic diseases - Google Patents
Application of crocetin in preparation of medicine for treating and preventing cerebral ischemic diseases Download PDFInfo
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- CN116019802A CN116019802A CN202310208125.2A CN202310208125A CN116019802A CN 116019802 A CN116019802 A CN 116019802A CN 202310208125 A CN202310208125 A CN 202310208125A CN 116019802 A CN116019802 A CN 116019802A
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- crocetin
- medicament
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- ethyl acetate
- cerebral ischemic
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- PANKHBYNKQNAHN-MQQNZMFNSA-N crocetin Chemical compound OC(=O)C(/C)=C/C=C/C(/C)=C/C=C/C=C(\C)/C=C/C=C(\C)C(O)=O PANKHBYNKQNAHN-MQQNZMFNSA-N 0.000 title claims abstract description 42
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
Use of crocetin in the manufacture of a medicament for the treatment and prevention of cerebral ischemic diseases, comprising: taking stigma croci, sun-drying, pulverizing, and ultrasonically dispersing in methanol solution; reflux-extracting the methanol solution of the dispersed stigma croci Sativi under heating, filtering and mixing the filtrates; extracting the obtained solution with equal volume of ethyl acetate for multiple times to obtain ethyl acetate extract, mixing ethyl acetate layers in the ethyl acetate extract, concentrating under reduced pressure, and drying to obtain crocetin; fully dissolving polylactic acid-glycolic acid copolymer in a mixed solvent of tetrahydrofuran and acetone, adding crocetin, and adding distilled water under rapid stirring; continuously stirring the obtained mixture, volatilizing an organic solvent, centrifugally washing with deionized water, centrifuging, removing supernatant, collecting precipitate, and drying to obtain micro-nano particles of polylactic acid-glycolic acid copolymer supported crocetin; the micro-nano particles adopt an injection or aerosol type administration mode. The invention can be used for treating cerebral ischemia.
Description
Technical Field
The invention relates to the field of medicines for activating endogenous neural stem cells, in particular to application of crocetin in preparing medicines for treating and preventing cerebral ischemic diseases.
Background
Ischemic stroke, also known as cerebral infarction, is a disease of brain tissue injury caused by insufficient blood supply to the brain due to vessel occlusion, and serious arterial pressure and Ca2+ overload can be caused after cerebral hypoxia, resulting in massive death of cells in the brain. The global approach of 600 tens of thousands of people dying from cerebral ischemia is investigated, the number of people dying from cerebral ischemia per year in China is up to 150 tens of thousands, and because of high morbidity, high mortality, high disability rate and high recurrence rate, huge harm and heavy economic burden are brought to families and society of patients, and the diseases tend to be younger and younger, so that the life of people is seriously threatened, when cerebral ischemia occurs, the brain is lack of oxygen due to blood flow blockage, a plurality of pathological changes are caused, and finally, a large number of cells die, and cell communication is aggravated and damaged. At present, cerebral ischemia treatment modes comprise vascular recanalization and neuroprotective drugs, and the vascular recanalization drugs have the defects of short empty window period and undefined toxicity to nerve cells in cerebral ischemia treatment, so that development is hindered, curative effects are hardly observed by the neuroprotective drugs, and few reports on nerve cell regeneration are provided. Therefore, how to achieve the treatment of cerebral ischemia by activating stem cells is a new direction worthy of research, and needs to be further studied.
Disclosure of Invention
Based on the above, the invention provides the application of crocetin in preparing the medicine for treating and preventing cerebral ischemia diseases, so as to solve the technical problems that the cerebral ischemia treatment mode in the prior art adopts medicines comprising vascular recanalization and neuroprotection, the treatment effect is still lacking and needs to be further researched.
In order to achieve the above object, the present invention provides an application of crocetin in preparing a medicament for treating and preventing cerebral ischemic diseases, comprising the following steps:
taking stigma croci, sun-drying, crushing, and then dispersing in a methanol solution by ultrasonic, wherein the dosage of the methanol solution is 10 times of the mass of crocus sativus, and the volume concentration of methanol in the methanol solution is 50% -70%;
extracting the methanol solution of the dispersed saffron by heating and refluxing at 70-80 ℃, filtering and merging filtrate, extracting the obtained solution with equal volume of ethyl acetate for a plurality of times to obtain ethyl acetate extract, merging ethyl acetate layers in the ethyl acetate extract, concentrating under reduced pressure, and drying to obtain crocetin;
fully dissolving polylactic acid-glycolic acid copolymer in a mixed solvent of tetrahydrofuran and acetone, adding crocetin, and adding distilled water under rapid stirring; continuously stirring the obtained mixture, volatilizing an organic solvent, centrifugally washing with deionized water, centrifuging, removing supernatant, collecting precipitate, and drying to obtain micro-nano particles of polylactic acid-glycolic acid copolymer supported crocetin;
the micro-nano particles adopt an administration mode that the dosage form is injection or aerosol.
As a further preferable embodiment of the present invention, the micro-nano particles are formulated into an injection or aerosol type drug by being mixed with physiological saline.
As a further preferable technical scheme of the invention, the concentration of the crocetin in the injection or aerosol type medicine is 5-30 mg/ml.
As a further preferable technical scheme of the invention, the extraction times of the heating reflux are at least 3 times, and each heating reflux time is 1h.
As a further preferable technical scheme of the invention, BDNF brain neurotrophic factor is also added when the polylactic acid-glycolic acid copolymer loads crocetin.
As a further preferable technical scheme of the invention, the concentration of BDNF brain neurotrophic factor in the medicine prepared into injection or aerosol is 5-20 mg/ml.
As a further preferable technical scheme of the invention, the polylactic acid-glycolic acid copolymer is fully dissolved in a mixed solvent of tetrahydrofuran and acetone, and after the crocetin is added, the polylactic acid-glycolic acid copolymer and the crocetin are combined under the assistance of ultrasound, and the ultrasonic frequency is 35-45 KHz.
When the crocetin is used for treating and preventing cerebral ischemic diseases, the crocetin can be prepared into injection or aerosol, and can be absorbed into blood through intramuscular injection or respiratory tract mucous membrane, and the crocetin takes polylactic acid-glycolic acid copolymer as a carrier to form nano particles, so that the crocetin is convenient to absorb and reach an action part.
The crocetin disclosed by the invention can be used for activating endogenous neural stem cells when being used for preparing a medicament for treating and preventing cerebral ischemia diseases, so that the problem of massive apoptosis of the neural stem cells after cerebral ischemia is solved, and the aim of treating cerebral ischemia is fulfilled. It has excellent nerve cell damage repairing function, and can promote the neuron differentiation of nerve stem cell by activating the subventricular neurogenesis of damaged cortex and striatum part, repair ischemic apoplexy and protect nerve by relieving histopathological form, nerve function defect and neuron damage.
Drawings
The invention will be described in further detail with reference to the drawings and the detailed description.
FIG. 1 is a flow of a model construction and in vivo dosing regimen;
FIG. 2 is a representation of MCAO model building identification, a) brain laser speckle imaging detection; b) Brain TTC staining;
FIG. 3 shows a Nestin/BrdU immunohistochemical staining, a) Nestin/BrdU double staining; b) Quantitative analysis of Nestin/BrdU double positive cells; p >0.05,ns,no significant difference; * P <0.001;
FIG. 4 shows Tuj1/GFAP immunohistochemical staining, a) Tuj1/GFAP double staining; b) Quantitative analysis of Tuj1 positive cells; c) Quantitative analysis of GFAP positive cells; p >0.05,ns,no significant difference; * P <0.01; * P <0.001; * P <0.0001;
FIG. 5 is MAP2 immunohistochemical staining, a) MAP2 staining; b) Quantitative analysis of MAP2 positive cells; p >0.05,ns,no significant difference; * P <0.0001;
FIG. 6 is Nissl staining, a) treatment of 7d Nissl staining; b) Treatment 7d Nissl staining quantification; c) Treatment 7d Nissl staining; d) Treatment 7d Nissl staining quantification; p >0.05,ns,no significant difference; * P <0.001; * P <0.0001;
FIG. 7 is TTC staining, a) treatment of 7d TTC staining; b) Treatment 7d TTC staining quantification; c) Treatment of 7d TTC staining; d) Treatment 7d TTC staining quantification; p >0.05,ns,no significant difference; * P <0.0001;
FIG. 8 laser speckle imaging detection rCBF, a) laser speckle imaging blood flow detection; b) rCBF relative quantitative analysis; * P < 0.0001.
The achievement of the objects, functional features and advantages of the present invention will be further described with reference to the accompanying drawings, in conjunction with the embodiments.
Detailed Description
The following describes specific embodiments of the present invention in detail with reference to the drawings. It should be understood that the detailed description and specific examples, while indicating and illustrating the invention, are not intended to limit the invention.
Unless defined otherwise, technical terms used in the following examples have the same meaning as commonly understood by one of ordinary skill in the art to which the inventive concepts pertain. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods are conventional methods unless otherwise specified.
The invention provides an application of crocetin in preparing a medicament for treating and preventing cerebral ischemic diseases, which comprises the following steps:
taking stigma croci, sun-drying, crushing, and then dispersing in a methanol solution by ultrasonic, wherein the dosage of the methanol solution is 10 times of the mass of crocus sativus, and the volume concentration of methanol in the methanol solution is 50% -70%;
heating and reflux-extracting the methanol solution dispersed with the crocus sativus at 70-80 ℃, filtering and combining the filtrates, wherein the extraction times of the heating and reflux are at least 3 times, and the time of each heating and reflux is 1h; extracting the obtained solution with equal volume of ethyl acetate for multiple times to obtain ethyl acetate extract, mixing ethyl acetate layers in the ethyl acetate extract, concentrating under reduced pressure, and drying to obtain crocetin;
fully dissolving polylactic acid-glycolic acid copolymer in a mixed solvent of tetrahydrofuran and acetone, adding crocetin, and adding distilled water under rapid stirring; continuously stirring the obtained mixture, volatilizing an organic solvent, centrifugally washing with deionized water, centrifuging, removing supernatant, collecting precipitate, and drying to obtain micro-nano particles of polylactic acid-glycolic acid copolymer supported crocetin;
the micro-nano particles adopt an administration mode that the dosage form is injection or aerosol. The micro-nano particles are prepared into injection or aerosol type medicines by being mixed with normal saline, namely medicines for treating and preventing cerebral ischemic diseases, and the concentration of the crocetin is 5-30 mg/ml.
The preparation forms of the medicine for treating and preventing cerebral ischemic diseases are injection and aerosol, and can be absorbed into blood through injection or respiratory tract mucous membrane.
Preferably, BDNF brain neurotrophic factors are also added into the medicine for treating and preventing cerebral ischemic diseases, and the concentration of the BDNF brain neurotrophic factors in the medicine for treating and preventing cerebral ischemic diseases is 5-20 mg/ml, so that the treatment effect can be improved by combined administration.
Further preferably, when the polylactic acid-glycolic acid copolymer and the crocetin are combined in a loaded manner, ultrasonic stirring is assisted, and the ultrasonic frequency is 35-45 KHz.
The nerve stem cells die in a large quantity after cerebral ischemia, the sensory, reflex and motor abilities of patients are seriously affected, and the medicine can activate endogenous nerve stem cells so as to treat cerebral ischemia. The influence of the drug on proliferation and differentiation fate of the neural stem cells is explored through in vitro experiments, and whether the drug can activate nerve regeneration in a subventricular zone or not is verified in vivo so as to repair cerebral ischemia injury, and an adult rat is taken as an experimental object, wherein the specific experimental means are as follows:
for convenience of description, in the following experimental tests, the drugs for treating and preventing cerebral ischemic diseases of the present invention are referred to as CRO.
1. Cerebral ischemia model construction:
rats were randomly divided into sham-operated and MCAO model groups, MCAO-modeled using the modified Zea Longa wire-plug method, as shown in figures 1 and 2, and after weighing the rats, were anesthetized with 2% sodium pentobarbital and mounted on animal operating plates in supine position, first a shallow incision was made in the middle of the neck, and tissues were blunt-isolated to expose the common carotid artery (common carotid artery, CCA) and further to isolate the external carotid artery (internal carotid arteries, ECA) and internal carotid artery (internal carotid arteries, ICA). Secondly, selecting a fine line from the bifurcation of the ECA and the ICA to form a slipknot, cutting a small opening at the proper position of the ECA, inserting a marked nylon single line from the ECA, slowly putting the ICA into the bifurcation of the CCA, fastening the slipknot, penetrating forward until the black point mark on the nylon single line is covered, dripping a proper amount of gentamicin into the neck, aiming at preventing infection, finally, respectively sewing a muscle layer and a epidermis layer by using surgical suture lines, and slightly pulling out the nylon single line of 2cm by forceps after blocking for 2 hours to recover blood flow supply. The Sham group was treated with the same surgical procedure but without occlusion of the blood supply.
2 immunohistochemistry:
immunohistochemistry (IHC) technology is an experimental technology for determining antigens (such as polypeptides and proteins) in tissue cells by developing color developing agents (fluorescein, enzyme, metal ions and isotopes) of labeled antibodies through chemical reaction by utilizing the principle of antigen-antibody specific reaction in immunology, researching antigen localization, qualitative and quantitative, and has the characteristics of high specificity, high sensitivity, accurate localization, multi-layer multi-angle detection and observation of tissue cell structures and the like, and is widely applied to the fields of basic science research and clinical disease diagnosis at present, and the number of new-born neural stem cells and neuron precursor cells and neurons and astrocyte cells is mainly detected through immunofluorescence double-staining and single-staining in the experiment.
3. Cerebral infarction volume detection:
cerebral infarct volume was detected using 2,3, 5-triphenyltetrazolium chloride (TTC) staining. TTC is a fat-soluble light-sensitive complex, which can be used as a redox indicator, and dehydrogenase in living cells (especially succinic dehydrogenase in mitochondria) can reduce TTC, and a sub-receptor of pyridine-nucleotide structural enzyme system in respiratory chain can react with dehydrogenase in normal tissues to form red, while in ischemic tissues, due to reduced dehydrogenase activity, no reaction can form pale color, so that the infarct volume can be measured by calculating the ratio of the white area to the red area.
4. Neural ischemia score:
researchers blinded to the experiment were selected to score against the neurological score (table 1) and mNSS composite score (table 2) and statistically analyzed. The neurological score reflects the neurological deficit of the rat, with higher scores being more severe. The mNSS comprehensive scores include motor (muscle state, abnormal motor), sensory (visual, tactile and proprioceptive) and reflex tests and the like, and can reflect the sensory, motor, balance and reflex abilities of rats, and the total score is 18, and the higher score represents more serious nerve function damage.
Nissl staining:
various nerve cells contain Nib's body, which is an important site for protein synthesis in neurons, but often differ in shape, number, and location of distribution. When neurons are stimulated, there is a significant decrease in the Nib's body. Typically, nile can be stained violet by basic dyes such as thionine, methylene blue, toluidine blue, and tar violet. The Nissl staining method can dye out Nishi bodies, is used for observing the cell structure in neurons, and can also know the damage condition of the neurons through observing the Nishi bodies after the Nishi staining, and the Nissl staining solution is blue-purple, and the Nissl bodies are large and large in number, so that the function of synthesizing proteins by nerve cells is higher; in contrast, the number of Nissl bodies decreases or even disappears when the nerve cells are damaged.
6.RT-qPCR：
And (3) taking NSCs transferred to the generation P2-P5, digesting, re-suspending, blowing and beating by using a culture medium, uniformly mixing, inoculating into a 6-hole plate, culturing for 24 hours at a density of 1 multiplied by 104 per cm < 2 >, preparing CRO with a concentration of 0,5, 10 and 20 mu M by using a proliferation culture medium, and respectively collecting all cells of each group after inducing for 72 hours, so as to carry out RT-qPCR experiments.
7.Western blot：
And (3) taking NSCs transferred to the generation P2-P5, digesting, carrying out heavy suspension blowing and uniform mixing by using a culture medium, inoculating the NSCs into a 6-hole plate, culturing for 24 hours at a density of 1 multiplied by 104 per cm < 2 >, preparing CRO with a concentration of 0,5, 10 and 20 mu M by using a proliferation culture medium, and collecting all cells after 72 hours of induction by using the CRO with the concentration of 0,5 and 10 mu M, and carrying out Western Blot experiments.
Based on the experimental operation, the following research is carried out on the beneficial technical effects of the crocetin in the application of the medicine for treating and preventing cerebral ischemic diseases:
firstly, incubating CRO with different concentrations (5-30 mg/ml) and neural stem cells, and detecting the proliferation and differentiation conditions in vitro; second, ischemic stroke in adult rats was induced by occlusion of middle cerebral artery and treated with CRO in comparison to nimodipine, a pharmaceutical product. The behavioral function, infarct volume and apoptotic Nissl bodies of rats were significantly improved after CRO treatment, and in addition, the cerebral blood flow in the areas monitored by laser speckle contrast imaging was also increased. Neurogenesis and neuronal precursor differentiation in vivo was assessed by immunohistochemistry of 5-bromo-2' deoxyuridine labeling and neural cell type specific labeling, and examined brain tissue after ischemic stroke showed a significant increase in neuronal regeneration in focal ischemic injury areas. Brain tissue examined after ischemic stroke showed a significant increase in neuronal regeneration in the focal ischemic injury zone, while the length of neurons was prolonged, suggesting that CRO might promote neuronal extension to enhance intercellular communication. These findings indicate that CRO promotes neuronal differentiation of neural stem cells by activating subventricular neurogenesis in damaged cortex and striatum areas, thereby repairing ischemic stroke.
FIG. 3 shows BrdU/Nestin double positive labelling detection after 7d continuous CRO treatment, and FIG. 3 a shows that the number of BrdU/Nestin double positive cells in the subventricular zone of the control group is 61.00+ -7.34, the number of CRO double positive cells is 189.33 + -14.61, 3 times (P < 0.001) of the control group, and the number is slightly better than that of Sham double positive cells (184.00 + -15.76). Indicating that CRO can promote proliferation of the nerve stem cells in the subventricular zone and is close to the proliferation speed of the nerve stem cells of normal rats.
After 14d continuous CRO treatment, tuj1 and GFAP were double stained (fig. 4 a) in order to assess neuronal and astrocyte regeneration. Tuj1 and GFAP positive cells (b, c in FIG. 4) were present in greater numbers in the CRO group than in the MCAO treated group (P)<0.01 Indicating that CRO promotes differentiation of neural stem cells and migration thereof to the lesion area, replaces damaged neurons and astrocytes, and neurons/mm after CRO treatment 2 Increasing from 66.00 + -16.62 to 222.00 + -19.41, approaching the level of the Sham group. These results indicate that CRO promotes neuronal and astrocyte production, thereby exerting neural cell damage repair function.
The neurite may function in cellular communication, and the above experiments have found that CRO can promote the production of neurons, and after 14d continuous treatment of CRO, we further examined the extension of neurites. As shown in fig. 5a, immunohistochemical staining detection by MAP2 showed that the length of the CRO group neurons protruding significantly exceeded that of MCAO group, quantification of the neuron length showed that CRO increased the elongation of neurons by nearly 2.6-fold (b, ×p <0.001 in fig. 5). MAP2 is also a marker of mature neurons, further demonstrating that CRO promotes neuronal differentiation, maturation, and elongation of its processes.
Cerebral ischemia damage causes pathological microenvironment changes in the brain, which causes death or apoptosis of neurons, and Nib staining reflects the number of neurons according to the number of Nib bodies at the focal site. As shown in FIG. 6, the Sham group had a large number of Nissl bodies, while the morphology was normal and the color was bright, and the MCAO group had a large number of denatured Nissl bodies compared to the Sham group. Based on detection and analysis of Nissl staining on day 7 (a, b in fig. 6) and day 14 (c, d in fig. 6), CRO repaired damaged nissen (< 0.001). The above results indicate that CRO exerts neuroprotective effects by alleviating histopathological morphology, neurological deficit and neuronal damage.
TTC staining was used to observe infarct volume after treatment 7d, 14d, and MCAO rats had significantly decreased infarct volume from 39.93±0.02% to 26.20 ±0.01% (+p < 0.0001) after 7 days CRO treatment according to TTC staining analysis (a, b in fig. 7). After 14 days of treatment (c in fig. 7), the infarct volume was reduced to 12.79±0.01%, and according to quantitative analysis (d in fig. 7), the reduction of focal zone demonstrated that this is equivalent to the positive drug NMDP (9.86±0.02%) in treatment effect, and the above experimental data indicate that CRO can reduce ischemic infarct area and can achieve the effect of clinical drug NMDP.
The combinations of neurological ischemia scores are shown in tables 1 and 2.
TABLE 1 neurological score (Bederson's score)
TABLE 2mNSS comprehensive scoring criteria
After continuous administration of 7, 14d to the abdominal cavity, the neurological deficit recovery condition is predicted according to the rat behaviours, and is mainly judged by the Bederson score and the mNSS score, the positive drug group rats basically have no paralysis symptoms at 7d, the movement, balance and reaction capacity are greatly improved compared with the MCAO group rats, the movement, reaction and balance capacity of the rats treated by 14d in the CRO group are also basically recovered, and the MCAO group rats still have the phenomena of bradykinesia, reaction decline and imbalance, which indicates that the CRO has the same cerebral ischemia treatment effect as the positive drug NMDP.
For acute ischemic stroke, improvement of blood flow by thrombolysis is critical, which is associated with hemorrhagic transformation, angioedema, excessive production of oxygen and nitrogen radicals leading to cell death. The cerebral infarction area blood flow is detected by using a laser speckle imaging system, as shown in fig. 8, from the gray level diagram of the false operation, the blood vessel of the rat is normal without other damage, after modeling, the blood flow of the right area is obviously increased (a in fig. 8) as shown by continuous treatment of CRO 14d, and the quantitative analysis according to b in fig. 8 shows that the blood flow is 2.7 times (P is less than 0.0001) of the blood flow of MCAO, which indicates that CRO influences angiogenesis and promotes the recanalization of microcirculation blood flow.
While particular embodiments of the present invention have been described above, it will be appreciated by those skilled in the art that these are merely illustrative, and that many variations or modifications may be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined only by the appended claims.
Claims (7)
1. Use of crocetin in the manufacture of a medicament for the treatment and prevention of cerebral ischemic disease, comprising the steps of:
taking stigma croci, sun-drying, crushing, and then dispersing in a methanol solution by ultrasonic, wherein the dosage of the methanol solution is 10 times of the mass of crocus sativus, and the volume concentration of methanol in the methanol solution is 50% -70%;
extracting the methanol solution in which the saffron is dispersed by heating and refluxing, wherein the heating and refluxing temperature is 70-80 ℃, filtering and merging filtrate, extracting the obtained solution with equal volume of ethyl acetate for a plurality of times to obtain ethyl acetate extract, merging ethyl acetate layers in the ethyl acetate extract, concentrating under reduced pressure, and drying to obtain crocetin;
fully dissolving polylactic acid-glycolic acid copolymer in a mixed solvent of tetrahydrofuran and acetone, adding crocetin, and adding distilled water under rapid stirring; continuously stirring the obtained mixture, volatilizing an organic solvent, centrifugally washing with deionized water, centrifuging, removing supernatant, collecting precipitate, and drying to obtain micro-nano particles of polylactic acid-glycolic acid copolymer supported crocetin;
the micro-nano particles adopt an administration mode that the dosage form is injection or aerosol.
2. The use of crocetin according to claim 1 for the preparation of a medicament for the treatment and prevention of cerebral ischemic diseases, wherein the micro-nano particles are formulated into an injection or aerosol type medicament by being formulated with physiological saline.
3. The use of crocetin according to claim 1 for the preparation of a medicament for the treatment and prevention of cerebral ischemic diseases, characterized in that the concentration of crocetin in the medicament in an injection form or an aerosol form is 5-30 mg/ml.
4. Use of crocetin according to claim 1 for the preparation of a medicament for the treatment and prevention of cerebral ischemic diseases, characterized in that the number of extraction by heat reflux is at least 3, each heat reflux time being 1h.
5. The use of crocetin according to claim 1 for the preparation of a medicament for the treatment and prevention of cerebral ischemic diseases, characterized in that BDNF brain neurotrophic factor is also added when crocetin is loaded with the polylactic acid-glycolic acid copolymer.
6. The use of crocetin according to claim 5 for the preparation of a medicament for the treatment and prevention of cerebral ischemic diseases, characterized in that the concentration of BDNF brain neurotrophic factor in the medicament formulated as an injection or aerosol is 5-20 mg/ml.
7. The use of crocetin according to any one of claims 1-6 for the preparation of a medicament for the treatment and prevention of cerebral ischemic diseases, characterized in that the polylactic acid-glycolic acid copolymer is fully dissolved in a mixed solvent of tetrahydrofuran and acetone, and after crocetin is added, the polylactic acid-glycolic acid copolymer and crocetin are combined under the assistance of ultrasound with an ultrasonic frequency of 35-45 KHz.
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