Uses of Arisaema alkaloids in the preparation of drugs for the prevention or treatment of ischemic brain injury

Abstract

This invention belongs to the field of biomedical technology, specifically relating to the use of arisaema alkaloids in the preparation of drugs for the prevention or treatment of ischemic brain injury. The arisaema alkaloids are palmatine A or palmatine B. The ischemic brain injury is ischemic stroke. This invention constructed a rat MCAO (MCuloencephalopathy of Oddi) model of cerebral ischemia. Results showed that all treatment groups downregulated mNSS and angle-turning test scores, improved the rats' spontaneous activity, reduced the volume of cerebral infarction, alleviated pathological damage such as neuronal shrinkage and edema in the hippocampus, improved mitochondrial and blood-brain barrier ultrastructural abnormalities, and downregulated the high expression of microglial markers Iba-1 and pyroptosis-related protein NLRP3. The overall efficacy ranking was butylphthalide > palmatine A > palmatine B. Therefore, the arisaema alkaloids described in this invention can be used to prepare drugs for the prevention or treatment of ischemic brain injury.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the use of arisaema alkaloids in the preparation of drugs for the prevention or treatment of ischemic brain injury. Background Technology

[0002] Ischemic stroke (IS) is the second leading cause of death worldwide after ischemic heart disease, and also the leading cause of acquired disability in adults. It is caused by sudden occlusion of local blood vessels or long-term arterial stenosis, leading to persistent ischemia and hypoxia in brain tissue. This triggers a multi-pathological cascade of damage, including inflammatory responses, excitotoxicity, oxidative stress, and mitochondrial dysfunction. Recent studies have confirmed that microglia-mediated neuroinflammation is a core pathogenic mechanism throughout the progression of IS. As the earliest and most intrinsically linked immune cells in the central nervous system to respond to brain injury, microglia are precisely recruited to the periphery of the lesion within minutes to hours after a stroke by "help-me" signals released by damaged neurons. Overactivation leads to the release of pro-inflammatory factors, inducing peripheral immune cell infiltration into the central nervous system, further exacerbating blood-brain barrier disruption and delayed neuronal damage.

[0003] Arisaema tubers have been used medicinally in China for over two thousand years. The Chinese Pharmacopoeia (2020 edition) lists Arisaema tubers as belonging to the genus Arisaema in the family Araceae. Arisaema Mart. Plant Arisaema A. erubescens (Wall.) Schott, Arisaema heterophyllum A. heterophyllum Blume or Northeast Arisaema A. amurenseThe dried tubers of Maxim. According to the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica), it is bitter and pungent in taste, warm in nature, and enters the lung, liver, and spleen meridians. It has the functions of detoxifying and reducing swelling, dispelling wind and calming the nerves, resolving phlegm and dissipating nodules. It is mainly used to treat stubborn cough with phlegm, stroke, chest tightness, dizziness, facial paralysis, epilepsy, and tetanus. In Qingzhou Baiwanzi (a traditional Chinese medicine formula), it is combined with Pinellia ternata, Aconitum carmichaelii, and Aconitum carmichaelii to treat wind-phlegm invading the collaterals, numbness of the hands and feet, hemiplegia, facial paralysis, phlegm accumulation, as well as infantile convulsions and adult headaches. In Baijiangcan San (another traditional Chinese medicine formula), it is combined with Bombyx mori, Gastrodia elata, Angelica pubescens, Ephedra sinica, rhinoceros horn shavings, Aconitum carmichaelii, Pinellia ternata, Pogostemon cablin, and Borneol to treat stroke during pregnancy with lockjaw, phlegm accumulation in the chest and diaphragm, speech impairment, and limb rigidity. In Sansheng Yin (another traditional Chinese medicine formula), it is combined with Aucklandia lappa, Aconitum carmichaelii, and Aconitum carmichaelii to treat stroke with loss of consciousness. Tiannanxing Gao (another traditional Chinese medicine formula) treats sudden stroke with facial paralysis. In Duoming San (another traditional Chinese medicine formula), it is combined with Saposhnikovia divaricata to treat tetanus. Modern clinical and experimental studies have confirmed that Tiannanxing has a significant therapeutic effect on IS (Infantile Disorder). Tiannanxing contains structurally diverse alkaloid components, and experimental studies have confirmed that these alkaloid components have significant therapeutic effects on various central nervous system diseases, and can achieve a broad protective effect on the nervous system through multiple mechanisms of action. Alkaloid components significantly reduced the infarct area in rats with middle cerebral artery occlusion (MCAO), downregulated TRPC6 protein expression, protected neurons from hypoxia- and hypoglycemia-induced damage, and alleviated cerebral edema. However, there are currently no reports on the therapeutic effects of alkaloid monomers from Arisaema heterophyllum on ischemic brain injury. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the existing technology and realize the development and utilization of natural products using modern drug research methods. Combined with pharmacodynamic experiments, it provides a new pharmaceutical use for Arisaema alkaloids, namely, the use of Arisaema alkaloids in the prevention or treatment of ischemic brain injury.

[0005] Specifically, the present invention is achieved through the following technical solutions: This invention provides the use of arisaema alkaloids in the preparation of medicaments for the prevention or treatment of ischemic brain injury.

[0006] Alternatively, in the above-mentioned uses, the arisaema alkaloids are palmate pinellidin A or palmate pinellidin B.

[0007] Alternatively, in the above-described uses, the ischemic brain injury is ischemic stroke.

[0008] Alternatively, in the above-described uses, the ischemic stroke is selected from one or both of focal ischemic stroke and diffuse ischemic stroke.

[0009] Alternatively, in the above-described uses, the drug comprises arisaema alkaloids and a pharmaceutically acceptable carrier.

[0010] Alternatively, in the above-described uses, the arisaema alkaloids are the sole active ingredient in the drug.

[0011] Alternatively, in the above-described uses, the arisaema alkaloids are used in combination with other active ingredients, wherein the other active ingredients are selected from butylphthalide, edaravone, or nimodipine.

[0012] In the aforementioned drug, the combination of arisaema alkaloids with other active ingredients (especially butylphthalide, edaravone, or nimodipine) has a significant synergistic effect in the prevention or treatment of ischemic brain injury (especially ischemic stroke).

[0013] The synergistic effect of the combination of the above-mentioned arisaema alkaloids with other active ingredients can be verified by the experimental methods used in the specific embodiments section of this application, which is easy for those skilled in the art.

[0014] Alternatively, in the above-described uses, the dosage form of the drug is an oral dosage form.

[0015] Alternatively, in the above-described uses, the oral dosage form is selected from capsules, tablets, granules, or oral liquids.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The inventors have discovered for the first time that arisaema alkaloids (especially palmitoline A or palmitoline B) can be used to prevent or treat ischemic brain injury. This invention constructed a rat MCAO (mechanoangiogenic apoplexy) model of cerebral ischemia. Results showed that all treatment groups downregulated mNSS and angle-turning test scores, improved the rats' spontaneous activity, reduced the volume of cerebral infarction, alleviated pathological damage such as neuronal shrinkage and edema in the hippocampus, and improved mitochondrial and blood-brain barrier ultrastructural abnormalities. Simultaneously, they downregulated the high expression of microglial markers Iba-1 and pyroptosis-related protein NLRP3. The overall efficacy ranking was butylphthalide > palmitoline A > palmitoline B. Therefore, the arisaema alkaloids described in this invention can be used to prepare drugs for the prevention or treatment of ischemic brain injury. Attached Figure Description

[0017] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings: Figure 1 mNSS method score (A); movement trajectory of rats in each group during the open field experiment (B). Among them, # p<0.05 Compared to the SHAM group; ** p<0.01Compared with the MCAO group, n≥3.

[0018] Figure 2 TTC staining of rat brain tissue in each group. # p<0.05 Compared to the SHAM group; ** p<0.01 Compared with the MCAO group, n≥3.

[0019] Figure 3 HE staining (A) and Nissl staining (B) of rat brain tissue in each group (200x).

[0020] Figure 4 Transmission electron microscopy was used to detect ultrastructural changes in brain tissue (30000×).

[0021] Figure 5 Immunofluorescence staining results of Iba-1 in rat brain tissue of each group (200x).

[0022] Figure 6 NLRP3 immunofluorescence staining results of rat brain tissue in each group (200x). Detailed Implementation

[0023] This invention utilizes modern pharmaceutical research methods to develop and utilize natural products. Combined with pharmacodynamic experiments and extensive screening, it was the first time that arisaema alkaloids (e.g., palmatine A or palmatine B) have been found to have effects in preventing or treating ischemic brain injury (especially ischemic stroke). Based on this, this invention was completed.

[0024] As used herein, the dosage form of the pharmaceutical preparations of the present invention is tablets, capsules, granules, oral liquids, or injections. Preferably, the dosage form of the present invention is tablets or capsules.

[0025] As used herein, the term "pharmaceutically acceptable carrier" in this invention refers to a conventional pharmaceutical carrier in the field of pharmaceutical formulations, selected from one or more of fillers, binders, disintegrants, lubricants, suspending agents, wetting agents, pigments, flavoring agents, solvents, and surfactants.

[0026] The fillers described in this invention include, but are not limited to, starch, microcrystalline cellulose, sucrose, dextrin, lactose, powdered sugar, glucose, etc.; the lubricants include, but are not limited to, magnesium stearate, stearic acid, sodium chloride, sodium oleate, sodium lauryl sulfate, poloxamer, etc.; the binders include, but are not limited to, water, ethanol, starch paste, syrup, hydroxypropyl methylcellulose, sodium carboxymethyl cellulose, sodium alginate, polyvinylpyrrolidone, etc.; the disintegrants include, but are not limited to, starch effervescent mixtures, i.e., sodium bicarbonate and citric acid, tartaric acid, low-substituted hydroxypropyl cellulose, etc.; the suspending agents include, but are not limited to, polysaccharides such as farnesian gum, agar, alginic acid, cellulose ether, and carboxymethyl chitosan, etc.; and the solvents include, but are not limited to, water, balanced salt solutions, etc.

[0027] Preferably, the drug of the present invention can be formulated into various solid oral dosage forms, liquid oral dosage forms, etc. Pharmaceutically acceptable oral solid dosage forms include: ordinary tablets, dispersible tablets, enteric-coated tablets, granules, capsules, pellets, powders, etc., and oral liquid dosage forms include oral liquids, emulsions, etc.

[0028] The above dosage forms can be prepared using conventional processes in the pharmaceutical formulation field.

[0029] As used herein, the "palm-leaf pinellia alkaloid A", "palm-leaf pinellia alkaloid B" and other natural product extracts or natural product extract monomers used in this invention can be extracted and separated from plants containing the active ingredient by biological purification methods, or can be purchased directly from commercially available products.

[0030] In the pharmaceutical applications described above, the timing, frequency, and duration of administration of "palmophylline A," "palmophylline B," and other active ingredients need to be determined based on the specific diagnostic results of the patient's condition, which is within the technical scope of those skilled in the art. For example, when applying treatment regimens for mice or rats to humans, the effective dose of all drugs for humans can be converted from the effective dose of the drug for mice or rats, which is easily achievable by those skilled in the art.

[0031] The present invention will be further described below with reference to specific embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the invention.

[0032] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0033] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0034] Unless otherwise stated, all percentages and parts in this invention refer to weight percentages and weight parts.

[0035] Example: 1. Establishment of the MCAO rat model SPF-grade male SD rats (180-220 g) were selected and, after acclimatization, anesthetized them in an induction box using 3% isoflurane under 95% O2 / 5% CO2 guidance. In the sham-operated group, only the left common carotid artery, external carotid artery, and internal carotid artery were exposed. In the other groups, a rat MCAO model was established using a modified suture ligation method. A nylon suture (0.26 mm in diameter) with one rounded end was used to ligate the external carotid artery. The suture was then inserted into the internal carotid artery until resistance was felt, at which point the procedure was stopped. A slipknot was then applied to the left common carotid artery, fixing the nylon suture within the vessel. Excess suture ends and the nylon suture itself were trimmed, and the wound was cleaned of bleeding with sterile cotton balls. The rat's neck skin was sutured, disinfected with povidone-iodine, and the rat was placed on a warming blanket until consciousness was regained. After the rats naturally recovered from surgery, their neurological function was assessed: 0 points, no neurological damage symptoms; 1 point, unable to fully extend the contralateral forelimb; 2 points, turning the body to the opposite side while walking; 3 points, leaning the body to the opposite side while walking; 4 points, unable to walk spontaneously, loss of consciousness.

[0036] 2. Grouping and Dosing Regimen Rats with MCAO model were randomly divided into four groups: the model group (MCAO), the sham-operated group (SHAM), the positive control group (butylphthalide, NBP, 20 mg / kg), the palmatine A group (ZYBXJJ, 20 mg / kg), and the palmatine B group (ZYBXJY, 20 mg / kg), with 12 rats in each group. All groups were administered the corresponding drugs by gavage for 7 consecutive days. The sham-operated group and the model group were administered physiological saline by gavage at the corresponding doses.

[0037] 3. Behavioral evaluation According to the mNSS scoring method, the evaluation criteria are shown in Table 1. A higher score indicates a more severe degree of damage. Furthermore, the experimental animals in each group were placed in an open area for behavioral evaluation and animal sampling. The experimental results showed that compared with the Model group, each drug-treated group exhibited a certain degree of score reduction. The experimental results are as follows: Figure 1As shown in the figure. Compared with the SHAM group, the MCAO group showed a significant increase in the turning angle test score. After administration, compared with the MCAO group, the turning angle scores of each administration group were significantly reduced, indicating that the alkaloid components of Arisaema heterophyllum can improve the nerve function of rats to a certain extent and promote the recovery of nerve function. The effect of each administration group from strongest to weakest was: butylphthalide group > palmatine A group > palmatine B group. Compared with the SHAM group, the total distance traveled by rats in the MCAO group was significantly reduced, and the movement speed was slow. Compared with the MCAO group, the total distance traveled by rats in each administration group was significantly increased, and the movement speed was faster. The effect of each administration group from strongest to weakest was: palmatine A group > butylphthalide group > palmatine B group.

[0038] Table 1. mNSS scoring method 4. TTC staining Three rats were randomly selected from each group, euthanized by cervical dislocation, and the intact brain was removed. The brain was frozen at -80℃ for 2 min and then coronally sectioned (2 mm thick). The sections were immersed in 1% TTC solution and incubated in a constant temperature incubator at 37℃ in the dark for 30 min. The infarct area of ​​the rats was photographed and analyzed.

[0039] Experimental results are as follows Figure 2 As shown, no infarct foci were observed in the SHAM group, while obvious infarct foci appeared in the MCAO group, with obvious ischemic core area and penumbra. Compared with the MCAO group, all treatment groups significantly reduced the cerebral infarction volume in rats, indicating that the alkaloid components of Arisaema heterophyllum can effectively reduce the cerebral infarction volume in IS rats and reduce brain damage caused by cerebral ischemia. The effects of each treatment group from strongest to weakest were: butylphthalide group > palmarinae pinellia alkaloid A group > palmarinae pinellia alkaloid B group.

[0040] 5. Observation of pathological sections HE staining method: Wash the extracted hippocampal tissue with phosphate-buffered saline (PBS) (pH 7.4). After fixing the hippocampal tissue with 4% paraformaldehyde to an appropriate degree, immediately place it in 4% paraformaldehyde solution for 6-8 h (4 ℃), then transfer it to 30% sucrose solution (4 ℃) overnight, and then fix it with paraffin. Sequentially immerse the sections in xylene I for 20 min, xylene II for 20 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, and 75% ethanol for 5 min, then wash with tap water. Stain the sections with hematoxylin for 3-5 min, wash with tap water, differentiate with differentiation solution, wash with tap water, apply blue solution for blueing, and rinse with running water. Dehydrate the sections sequentially with 85% and 95% graded ethanol solutions for 5 min each, then stain with eosin solution for 5 min. Sections were sequentially immersed in anhydrous ethanol I for 5 min, then anhydrous ethanol II for 5 min, then anhydrous ethanol III for 5 min, then xylene I for 5 min, and finally xylene II for 5 min. After clearing, the sections were mounted with neutral resin. The sections were observed under a microscope, and images were acquired for analysis.

[0041] Nissl staining method: Rat brain sections were fixed in 4% paraformaldehyde for 30 minutes, washed two or three times with distilled water to remove paraformaldehyde, and then immersed in a dish containing Nissl dye at 40°C for 10 minutes. After staining, the sections were washed three times with distilled water, dehydrated three times with 95% ethanol, and cleared three times with xylene, and then mounted. The sections were observed and photographed under a microscope.

[0042] In the brain tissue of rats in the MCAO group, numerous neuronal structural losses, cytoplasmic condensation, nuclear pyknosis, dissolution or fragmentation, and significant interstitial edema were observed. These severe pathological changes manifested behaviorally as a marked decline in neurological function. Compared to the MCAO group, the number of normal cells in each treatment group was significantly increased, and interstitial edema was alleviated. The experimental results are as follows: Figure 3 As shown, the effects of each treatment group, from strongest to weakest, are: butylphthalide group > palmatine A group > palmatine B group.

[0043] 6. Transmission electron microscopy Brain tissue was pre-fixed with 3% glutaraldehyde and then re-fixed with 1% osmium tetroxide. Acetone was used for stepwise dehydration, with a concentration gradient of 30%→50%→70%→80%→90%→95%→100% (the 100% concentration was changed 3 times). The dehydrating agent and Epon-812 embedding medium were applied sequentially in ratios of 3:1, 1:1, and 1:3. Embedding was performed using pure Epon-812 embedding medium. Semi-thin sections and ultrathin sections were prepared by observing the semi-thin sections under a light microscope. Areas rich in the blood-brain barrier of rat brain tissue were selected and ultrathin sections (60-90 nm) were prepared using an ultramicrotome and transferred to a copper grid. Staining was performed by staining with uranium acetate for 10-15 min, followed by lead citrate staining for 1-2 min at room temperature.

[0044] Compared with the SHAM group, the MCAO group showed abnormal morphology and structure of vascular endothelial cells in brain tissue, with irregularly shaped nuclei, slight loss of chromatin, slight decrease in electron density, and intact nuclear membranes. A few mitochondria showed mild swelling, cristae breakage and dissolution, matrix loss, and decreased electron density; most mitochondria showed swelling, extensive cristae breakage, significant matrix loss, vacuolization, and intact outer membranes. The MCAO group showed significant recovery after drug administration. (See experimental results below.) Figure 4 The effects of each treatment group, from strongest to weakest, were: butylphthalide group > palmatine A group > palmatine B group.

[0045] 7. Immunofluorescence After fixation, embedding, preparation, permeabilization, and blocking, brain sections were immersed in primary antibodies Iba-1 and NLRP3 and incubated overnight at 4 °C. After washing with PBS for 10 min, repeated three times, the samples were immersed in secondary antibodies and incubated at 37 °C for 1 h. The samples were then washed three times with PBS for 10 min each time, and the cell nuclei were stained with DAPI at 37 °C for 2 h. Fluorescence images were obtained using a fluorescence microscope.

[0046] Compared with the SHAM group, the MCAO group showed significantly increased expression of Iba-1 and NLRP3 proteins, and the Arisaema alkaloid component was able to significantly restore the expression of Iba-1 and NLRP3. Experimental results are shown below. Figure 5 and Figure 6 The effects of each treatment group, from strongest to weakest, were: butylphthalide group > palmatine A group > palmatine B group.

[0047] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. The use of Arisaema alkaloids in the preparation of drugs for the prevention or treatment of ischemic brain injury.

2. The use according to claim 1, characterized in that: The alkaloids mentioned are palmate pinellidin A or palmate pinellidin B.

3. The use according to claim 1, characterized in that: The ischemic brain injury mentioned is ischemic stroke.

4. The use according to claim 3, characterized in that: The ischemic stroke is selected from one or both of focal ischemic stroke and diffuse ischemic stroke.

5. The use according to any one of claims 1 to 4, characterized in that: The drug contains arisaema alkaloids and a pharmaceutically acceptable carrier.

6. The use according to claim 5, characterized in that: In the drug, the arisaema alkaloid is the sole active ingredient.

7. The use according to claim 5, characterized in that: In the drug, the arisaema alkaloid is used in combination with other active ingredients, wherein the other active ingredients are selected from butylphthalide, edaravone, or nimodipine.

8. The use according to claim 5, characterized in that: The drug is in oral dosage form.

9. The use according to claim 8, characterized in that: The oral dosage form is selected from capsules, tablets, granules or oral liquids.

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