Application of α-asarone in preparing medicine for preventing or treating hemorrhagic stroke

Abstract

The present invention provides the use of α-asarone in the preparation of a drug for preventing or treating hemorrhagic stroke. The structure of α-asarone is shown in Formula I. It can significantly improve short-term neurological deficits and long-term learning and memory function in model rats, reduce cerebral edema, improve blood-brain barrier permeability, and prevent or alleviate brain tissue atrophy during the recovery period. It has a definite therapeutic effect on animal models of hemorrhagic stroke without significant toxic side effects, and is expected to become a promising drug for the prevention and treatment of hemorrhagic stroke. #imgabs0#

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to the application of α-asarone in preparing a medicine for treating or preventing hemorrhagic stroke. Background Art

[0002] Stroke is the second leading cause of death worldwide. In China alone, nearly 4 million new cases occur annually, making it the world's leading cause of death. Over 2 million people die annually from stroke, and approximately two-thirds of surviving stroke patients suffer permanent disability. Stroke can be clinically categorized as either ischemic or hemorrhagic. Hemorrhagic stroke occurs when a ruptured intracranial blood vessel causes blood to leak into the brain, leading to a range of clinical manifestations including neurological dysfunction. Although hemorrhagic stroke has a relatively low incidence, it carries a high mortality and disability rate. Hemorrhagic stroke is primarily categorized into two types, intracerebral hemorrhage (ICH) and subarachnoid hemorrhage (SAH), depending on the location of the bleeding within the brain. ICH occurs within the brain, while SAH occurs between the pia mater and the arachnoid membrane. Hypertensive intracerebral hemorrhage is the most common cause of non-traumatic ICH, while SAH is often caused by intracranial aneurysms.

[0003] Brain damage caused by hemorrhagic stroke is a challenge in clinical treatment and a major cause of disability. Brain damage can be divided into primary and secondary types. Primary brain damage refers to the direct mechanical compression and ischemic changes caused by the initial hemorrhage, resulting from the hematoma and its enlargement to surrounding brain tissue. These changes include excessive glutamate release, calcium overload, and mitochondrial dysfunction. Secondary brain damage, on the other hand, is more complex, with pathological pathways including blood-brain barrier disruption and cerebral edema formation, oxidative stress and inflammation, autophagy and apoptosis, microglial activation, alterations in brain energy metabolism and proteomics, and iron deposition, ultimately leading to neurological deficits. The pathological mechanisms of hemorrhagic stroke-induced brain damage involve multiple factors and multiple links, which interact and are interrelated. Among them, neuronal excitotoxicity, caused by an imbalance in the regulation of excitatory amino acids (such as glutamate) and inhibitory amino acids (such as γ-aminobutyric acid, GABA), is a key factor in neuronal injury and death in the acute phase of hemorrhagic stroke.

[0004] At present, the clinical treatment for hemorrhagic stroke patients mainly adopts drug therapy and surgical treatment. Drug treatment is mainly symptomatic treatment of internal medicine, including reducing intracranial pressure, adjusting blood pressure, hemostasis treatment, mild hypothermia treatment, brain metabolism activators, calcium ion antagonists, etc., but the efficacy is poor. Surgical treatment does play a positive role in saving patients' lives, but the effect on patients' neurological dysfunction is not ideal, and there are relatively strict application requirements. To date, no drug treatment has been approved for the treatment of neurological damage caused by hemorrhagic stroke, thereby increasing patient survival rate or improving patient prognosis. Therefore, the development of drugs that can effectively treat hemorrhagic stroke is of extremely important clinical significance.

[0005] α-asarone is the main active ingredient of the Chinese medicine Acorus tatarinowii, which has sedative, antispasmodic and anticonvulsant effects. Studies have shown that α-asarone can block Na + channels, activates GABA A receptors and exert antiepileptic effects (see Wang ZJ, Levinson SR, Sun L, et al. Identification of both GABA A receptors and voltage-activated Na + channels as molecular targets of anticonvulsant alpha-asarone[J].Front Pharmacol,2014,5(40):5-11 and Huang C,Li WG,Zhang XB,etal.alpha-asarone from Acorus gramineus alleviates epilepsy by modulating A-type GABA receptors[J].Neuropharmacology,2013,65(2):1-11). In addition, it can also promote the proliferation of neural progenitor cells, resist oxidative stress, reduce microglial activation, alleviate neuroinflammation, improve neuronal apoptosis, etc. (see Chellian R,Pandy V,Mohamed Z.Pharmacology and toxicology ofα-andβ-Asarone:A review of preclinical evidence[J].Phytomedicine,2017:41-58). Although the above studies suggest that α-asarone has multiple neuropharmacological activities, to date, the therapeutic effect of α-asarone on hemorrhagic stroke has not been reported.

[0006] On the other hand, in clinical treatment of secondary epilepsy caused by hemorrhagic stroke, the preventive administration of antiepileptic drugs is generally not recommended (Chinese Society of Neurology, Chinese Society of Neurology Cerebrovascular Disease Group, Chinese Guidelines for the Diagnosis and Treatment of Cerebral Hemorrhage 2019 [J]. Chinese Journal of Neurology, 2019, 52(12): 994-1005.). The reason is that antiepileptic drugs have significant side effects, and the preventive administration of antiepileptic drugs may damage the neurological function of patients with hemorrhagic stroke. Summary of the Invention

[0007] In order to overcome the problem of lack of drugs for preventing or treating hemorrhagic stroke in the prior art, the present invention provides a new use of α-asarone.

[0008] To this end, the present invention provides the following technical solutions:

[0009] The present invention provides the use of a compound represented by formula I (trans-2,4,5-trimethoxy-1-propenylbenzene, also known as α-asarone or α-asarone ether) in the preparation of a drug for preventing or treating hemorrhagic stroke;

[0010]

[0011] The present invention unexpectedly discovered that the compound represented by Formula I can significantly improve short-term neurological deficits and long-term learning and memory functions in model rats, reduce brain edema, improve blood-brain barrier permeability, and prevent or alleviate brain tissue atrophy in the recovery period. It has a definite therapeutic effect on animal models of hemorrhagic stroke without significant toxic side effects. The present invention used α-asarone, the positive drugs vinpocetine injection, and nimodipine injection to treat rat models of subarachnoid hemorrhage established by intravascular puncture and rat models of intraparenchymal hemorrhage established by collagenase injection. It was found that α-asarone can significantly reduce edema in the affected side of the brain tissue of the model rats, improve their blood-brain barrier permeability, and prevent or alleviate brain tissue atrophy in the recovery period model rats, thereby significantly improving their short-term neurological function scores and long-term learning and memory functions. Furthermore, α-asarone can significantly reduce the incidence and mortality of secondary epilepsy caused by acute hemorrhagic stroke in model rats, and prolong their survival.

[0012] In some embodiments, the drug is also used to prevent or treat epilepsy secondary to hemorrhagic stroke. Preferably, the drug is used to treat hemorrhagic stroke and prevent epilepsy secondary to hemorrhagic stroke.

[0013] In some embodiments, the hemorrhagic stroke is a stroke caused by at least one of intracerebral hemorrhage (ICH) and subarachnoid hemorrhage (SAH).

[0014] In the present invention, the compound represented by formula I can have the following pharmacological effects: (1) antagonizing neurotoxicity caused by excessive glutamate; (2) reducing abnormally elevated glutamate and GABA levels; (3) inhibiting calcium influx into nerve cells and reducing intracellular calcium overload; (4) stabilizing the mitochondrial membrane potential of nerve cells and reducing neuronal apoptosis; and (5) alleviating the oxidative stress response of damaged nerve cells.

[0015] In the present invention, the drug can have the following pharmacological effects: (1) antagonizing neurotoxicity caused by excessive glutamate; (2) reducing abnormally elevated glutamate and GABA levels; (3) inhibiting calcium influx into nerve cells and reducing intracellular calcium overload; (4) stabilizing the mitochondrial membrane potential of nerve cells and reducing neuronal apoptosis; and (5) alleviating the oxidative stress response of damaged nerve cells.

[0016] In the present invention, the drug can (1) reduce the glutamate content in the brain of the model rat, thereby antagonizing the glutamate excitotoxicity caused by cerebral hemorrhage; (2) restore the GABA level and promote the recovery of the motor function of the model rat; (3) reduce Ca 2+ Influx, reducing Ca 2+ (4) Stabilize mitochondrial membrane potential and reduce neuronal apoptosis; (5) Reduce neuronal oxidative stress and damage, thereby alleviating cerebral edema, improving blood-brain barrier permeability, preventing or alleviating brain tissue atrophy during the recovery period, improving short-term neurological deficits and long-term learning and memory dysfunction in model rats, and playing an anti-hemorrhagic stroke role.

[0017] In some embodiments, the drug is used for at least one of the following: improving neurological or motor function damage (such as neurological or motor function damage caused by ICH or SAH), improving secondary early brain damage (such as acute brain tissue edema or blood-brain barrier dysfunction, such as acute brain tissue edema or blood-brain barrier dysfunction caused by ICH or SAH), reducing acute mortality caused by hemorrhagic stroke, prolonging survival, improving long-term learning and memory dysfunction caused by hemorrhagic stroke, and preventing or alleviating brain tissue atrophy during the recovery period of hemorrhagic stroke.

[0018] In some embodiments, the compound represented by Formula I is the only active ingredient in the drug.

[0019] In some embodiments, the drug may contain pharmaceutical excipients. Preferably, the total weight ratio of the compound of Formula I to the pharmaceutical excipient is 1:20 to 1000, for example, 1:20 to 200. More preferably, the compound of Formula I is the sole active ingredient in the drug, and the total weight ratio of the compound of Formula I to the pharmaceutical excipient is 1:20 to 1000, for example, 1:20 to 200.

[0020] In some embodiments, the subject of the drug can be a human or an animal. When the drug is used to treat a hemorrhagic stroke model rat, the daily effective dose of the compound shown in Formula I in the drug can be 5 mg to 40 mg / kg body weight. When the drug is used to treat a person suffering from hemorrhagic stroke, the daily dosage range of the compound shown in Formula I in the drug can be 0.15 mg to 5.0 mg / kg body weight, preferably 0.3 mg to 3.0 mg / kg body weight, for example, 2 to 3 times a day, and the dosage range for each administration is 0.15 mg to 1.5 mg / kg body weight, preferably 0.3 mg to 1.5 mg / kg body weight. The above dosage can be obtained based on the dosage conversion relationship between different species of animals.

[0021] In some embodiments, the administration route of the drug is injection, oral administration, subcutaneous implantation, inhalation, transdermal administration, mucosal administration, etc. Preferably, the administration route of the drug is injection (preferably intravenous administration) or oral administration.

[0022] In the present invention, the drug can be prepared into a dosage form suitable for human and / or animal use, for example, any dosage form compatible with different routes of administration, as long as the dosage form allows the compound of Formula I to enter the brain and achieve an effective therapeutic concentration. In some embodiments, the drug is an emulsion (e.g., an emulsion injection, an oral emulsion). Compared to currently available commercial injections (solution-type injections), emulsions have better safety and higher bioavailability than tablets.

[0023] In some embodiments, the emulsion may contain the compound represented by Formula I, a pharmaceutically acceptable oil, a pharmaceutically acceptable emulsifier, and water.

[0024] Wherein, the pharmaceutically acceptable oil may be composed of at least one of soybean oil, medium-chain oil, olive oil and fish oil.

[0025] Wherein, the pharmaceutically acceptable emulsifier may be composed of at least one of egg yolk lecithin, soybean lecithin, Pluronic F-68 and polyethylene glycol stearate-15 (Solutol HS15).

[0026] Wherein, the water can be water for injection or purified water.

[0027] The emulsion may further contain at least one of oleic acid and sodium oleate, depending on the emulsification performance requirements. During preparation, oleic acid dissolves in the oil phase, sodium oleate dissolves in the water phase, and a mixture of the two dissolves in both the oil and water phases.

[0028] Wherein, the emulsion may further contain glycerol.

[0029] The emulsion may further contain an antioxidant, which may be sodium bisulfite, vitamin E, pyrogallate, or the like.

[0030] When administered orally, the emulsion may further contain other suitable additives such as at least one of a preservative and a flavoring agent. The preservative may be a conventional preservative in the art, such as benzoic acid, sodium benzoate, sorbic acid, potassium sorbate, ethylparaben, propylparaben, and butylparaben. The flavoring agent may be a conventional flavoring agent in the art, such as a sweetener, a fragrance, a mucilage, or an effervescent agent. The sweetener may be monosaccharide syrup, stevioside, aspartame, or the like; the fragrance may be a fruit flavor, such as apple flavor or strawberry flavor; the mucilage may be gelatin or methylcellulose mucilage; and the effervescent agent may be a mixture of citric acid, tartaric acid, and sodium bicarbonate.

[0031] In some embodiments, the emulsion may contain, by weight percentage, 0.5% to 5% of the compound of Formula I, 5% to 30% of a pharmaceutically acceptable oil, 0.6% to 1.8% of an emulsifier, 0% to 2.5% of glycerol, and the balance water (e.g., purified water or water for injection). The concentration of the compound of Formula I in the emulsion may vary within a certain range, depending on the dosage, the dosage volume, and the solubility of the compound of Formula I in the oil phase.

[0032] In some embodiments, the emulsion is an emulsion injection. Preferably, in the emulsion injection, the total weight ratio of the compound of Formula I to pharmaceutical excipients (including water for injection) is 1:20-1000, for example 1:20-200.

[0033] The method for preparing the emulsion may include the following steps: mixing the compound represented by Formula I, a pharmaceutically acceptable oil, a pharmaceutically acceptable emulsifier and water by high-speed shearing to obtain colostrum; and homogenizing the colostrum by high pressure to obtain the emulsion.

[0034] In some embodiments, the method for preparing the emulsion may include the following steps:

[0035] Step 1: Under nitrogen or inert gas protection, dissolving the compound represented by Formula I in a pharmaceutically acceptable oil at 60-80°C to obtain an oil phase, and then dissolving or dispersing an emulsifier and glycerol in water at 60-80°C to obtain an aqueous phase; or, under nitrogen or inert gas protection, dissolving or dispersing the compound represented by Formula I and an emulsifier in a pharmaceutically acceptable oil at 60-80°C to obtain an oil phase, and then dissolving glycerol in water at 60-80°C to obtain an aqueous phase;

[0036] Step 2: The oil phase and the water phase are mixed by high-speed shearing to disperse the oil phase in the water phase to obtain colostrum;

[0037] Step 3: The colostrum is subjected to high-pressure homogenization (for example, the homogenization frequency can be 1 to 3 times) to reduce the average droplet size to no more than 0.5 μm, filtered, and filled into pharmaceutical containers such as glass ampoules, infusion bottles, vials, and soft bags under nitrogen or inert gas protection conditions; depending on the required route of administration, rotary hot press sterilization or non-sterilization with the addition of a preservative is used to obtain an emulsion.

[0038] The shear rate of the high-speed shear can be a conventional shear rate used in the field of small-scale trial production or large-scale production of emulsions, for example, a small-scale trial production in the laboratory can be 10,000 to 20,000 r·min -1 For example, the speed of large-scale production is 2000-4000 r·min -1 The actual shear rate depends on the shear radius, and the two determine the magnitude of the shear force.

[0039] The shearing time of the high-speed shearing can be the conventional shearing time used in the art for preparing emulsions, for example, 3 to 10 minutes, or 5 to 8 minutes.

[0040] The homogenization pressure of the high-pressure homogenization may be a conventional homogenization pressure used in the art for preparing emulsions, for example, 500 to 1500 bar, or 500 to 1000 bar.

[0041] The number of high-pressure homogenization cycles may be the conventional number of cycles used in the art for preparing emulsions, for example, 1 to 3 times.

[0042] The present invention also provides a pharmaceutical composition for preventing or treating hemorrhagic stroke, wherein the pharmaceutical composition contains the compound represented by formula I and pharmaceutical excipients.

[0043] In some embodiments, the pharmaceutical composition is also used to prevent or treat secondary epilepsy caused by hemorrhagic stroke.

[0044] In some embodiments, the pharmaceutical composition is used to treat hemorrhagic stroke and prevent secondary epilepsy caused by hemorrhagic stroke.

[0045] In some embodiments, the compound represented by Formula I is the only active ingredient in the pharmaceutical composition.

[0046] In some embodiments, the pharmaceutical composition is an emulsion.

[0047] The present invention also provides a method for treating or preventing hemorrhagic stroke in a subject, comprising: administering a therapeutically or preventively effective amount of a compound as shown in Formula I to the subject.

[0048] Preferably, the method is used to treat or prevent hemorrhagic stroke in a subject and to treat or prevent secondary epilepsy caused by hemorrhagic stroke.

[0049] More preferably, the method is used to treat hemorrhagic stroke in a subject and prevent secondary epilepsy caused by hemorrhagic stroke.

[0050] Definition and Explanation

[0051] Unless otherwise indicated, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be construed as indefinite or unclear unless specifically defined, but rather should be understood in accordance with its ordinary meaning. When a trade name appears in this document, it is intended to refer to the corresponding commercial product or its active ingredient.

[0052] Unless otherwise indicated, the term "pharmaceutically acceptable" as used herein refers to compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0053] Unless otherwise indicated, the term "pharmaceutically acceptable amount" as used herein refers to an amount of a compound, material, composition and / or dosage form that is within the scope of sound medical judgment without excessive toxicity, irritation, allergic response or other problems or complications, and is commensurate with a reasonable benefit / risk ratio.

[0054] Unless otherwise specified, the term "pharmaceutical excipients" refers to excipients and additives used in the production of pharmaceuticals and the preparation of prescriptions. These excipients are all substances, other than the active ingredient, contained in pharmaceutical preparations. For more information, see Part IV of the Pharmacopoeia of the People's Republic of China (2020 Edition) or the Handbook of Pharmaceutical Excipients (Raymond C Rowe, 2009, Sixth Edition).

[0055] Unless otherwise indicated, the term "treat" refers to therapeutic treatment. With respect to a specific condition, treatment means: (1) ameliorating the disease or one or more biological manifestations of the condition, (2) interfering with (a) one or more points in the biological cascade that leads to or causes the condition or (b) one or more biological manifestations of the condition, (3) ameliorating one or more symptoms, effects, or side effects associated with the condition or one or more symptoms, effects, or side effects associated with the condition or its treatment, or (4) slowing the progression of the condition or one or more biological manifestations of the condition.

[0056] Unless otherwise indicated, the terms "prevent" and "prevent" refer to the reduction of the risk of acquiring or developing a disease, disorder or condition.

[0057] Unless otherwise indicated, the term "therapeutically effective amount" refers to an amount of a compound that, when administered to a subject, is sufficient to effectively treat a disease or condition described herein. The "therapeutically effective amount" will vary depending on the compound, the condition and its severity, and the age of the patient to be treated, but can be adjusted as needed by those skilled in the art. The effective amount will also vary depending on the subject being administered (e.g., a human or an animal).

[0058] Unless otherwise indicated, the term "prophylactically effective amount" refers to an amount sufficient to prevent a disease, disorder or condition, or to prevent one or more symptoms associated with a disease, disorder or condition, or to prevent recurrence of the disease, disorder or condition.

[0059] Unless otherwise indicated, the term "subject" refers to any animal, preferably a mammal, and most preferably a human, that is about to be or has been administered a compound according to the embodiments of the present invention. The term "mammal" includes any mammal. Examples of mammals include, but are not limited to, cattle, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, and humans, with humans being the most preferred.

[0060] If the reaction temperature is not specified in the present invention, the reaction temperature is room temperature, which is generally 20-35°C.

[0061] Unless otherwise specified, "secondary epilepsy caused by hemorrhagic stroke" in the present invention refers to secondary epileptic seizures caused by hemorrhagic stroke in patients with no history of epilepsy (excluding lesions unrelated to hemorrhagic stroke).

[0062] Without violating the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain preferred embodiments of the present invention.

[0063] The reagents and raw materials used in the present invention are commercially available.

[0064] The positive progress effect of the present invention is:

[0065] The present invention discloses for the first time that α-asarone has the effect of treating / preventing hemorrhagic stroke. The results of the pharmacodynamic mechanism study show that α-asarone can (1) reduce the glutamate content in the brain of model rats, thereby antagonizing the glutamate excitotoxicity caused by cerebral hemorrhage; (2) restore GABA levels and promote the recovery of motor function in model rats; (3) reduce Ca 2+ Influx, reducing Ca 2+ (4) Stabilize mitochondrial membrane potential and reduce neuronal apoptosis; (5) Reduce neuronal oxidative stress and damage, thereby reducing cerebral edema, reducing brain damage, improving blood-brain barrier permeability, preventing or alleviating brain tissue atrophy during the recovery period, and thus improving short-term neurological deficits and long-term learning and memory dysfunction in model rats, significantly reducing the incidence and mortality of acute epilepsy in model rats, prolonging their survival, increasing their survival rate and improving their prognosis, and playing an anti-hemorrhagic stroke role.

[0066] The present invention unexpectedly discovered that α-asarone is significantly more effective than vinpocetine injection in improving neurological deficits in rats in the acute phase of SAH; significantly more effective than nimodipine injection in improving learning and memory functions and preventing or alleviating brain atrophy in rats in the recovery phase of SAH; and significantly more effective than nimodipine injection and vinpocetine injection in improving neurological deficits in rats with ICH. Therefore, α-asarone is expected to become a promising preventive / therapeutic drug for hemorrhagic stroke.

[0067] α-Asarone is safe and effective, and no obvious toxic or side effects of α-asarone were observed during the entire experimental process of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 : Effects of α-asarone on learning and memory function and brain tissue atrophy during the recovery period of SAH rats. A: The latency of each group of rats to find the platform during acquisition training; B: The residence time and swimming speed of each group of rats in the target quadrant during spatial exploration; C: Activity heat map of each group of rats during spatial exploration, the circle indicates the position of the platform, and the quadrant where it is located is the target quadrant; D: After the end of the water maze experiment, the brain was obtained by cardiac perfusion, and the brain tissue atrophy of each group; P, S, M, N represent sham operation group, SAH model group, α-asarone medium dose group, and nimodipine injection group, respectively; Compared with the sham operation group, ### P<0.001, ## P<0.01, # P<0.05; compared with the model group, ** P<0.01, * P<0.05; compared with the α-asarone medium dose group, &&& P<0.001, &&P<0.01.

[0069] Figure 2 : Effects of α-asarone on brain edema and blood-brain barrier permeability in model rats. A: Water content in different parts of brain tissue of SAH rats in each group; B: Evans blue exudation in brain tissue of SAH rats in each group; C: Water content in different parts of brain tissue of ICH rats in each group; D: Evans blue exudation in brain tissue of ICH rats in each group; P, S, I, and M represent sham operation group, SAH model group, ICH model group, and α-asarone medium dose group, respectively; Compared with the sham operation group, ### P<0.001, ## P<0.01, # P<0.05; compared with the model group, * P<0.05.

[0070] Figure 3 : Effects of α-asarone on glutamate and GABA content in brain tissue of model rats. A: Glutamate content in brain tissue of SAH rats in each group; B: GABA content in brain tissue of SAH rats in each group; C: Glutamate content in brain tissue around hematoma of ICH rats in each group; D: GABA content in brain tissue around hematoma of ICH rats in each group; Compared with the sham operation group, ## P<0.01, # P<0.05; compared with the model group, * P<0.05.

[0071] Figure 4 :The effect of α-asarone on brain tissue calcium ion and mitochondrial membrane potential in model rats. A: The results of the determination of brain tissue calcium ion content in SAH rats in each group; B: The results of the determination of brain tissue mitochondrial membrane potential in SAH rats in each group; C: The statistical graph of the fluorescence intensity of brain tissue calcium ion and mitochondrial membrane potential in SAH rats in each group; D: The results of the determination of brain tissue calcium ion content in ICH rats in each group; E: The results of the determination of brain tissue mitochondrial membrane potential in ICH rats in each group; F: The statistical graph of the fluorescence intensity of brain tissue calcium ion and mitochondrial membrane potential in ICH rats in each group; Compared with the sham operation group, ### P<0.001, ## P<0.01, # P<0.05; compared with the model group, *** P<0.001, * P<0.05.

[0072] Figure 5 :The effects of different doses of α-asarone on PC12 cells damaged by 6μM oxyhemoglobin. Compared with the control group, ### P<0.001, ## P<0.01; compared with the model group, *** P<0.001,** P<0.01. DETAILED DESCRIPTION

[0073] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0074] Preparation Example 1 Preparation of α-asarone injection emulsion

[0075] Weigh 0.50-50.0g of α-asarone and 50.0-300.0g of soybean oil for injection into a suitable container. Heat to 60-80°C under nitrogen and stir to dissolve. Add 6.0-18.0g of egg yolk lecithin and stir to dissolve (if necessary, add 0.10-0.50g of oleic acid, sodium oleate, or a mixture of the two) to prepare the oil phase, which is set aside. Separately, weigh 0-3.0g of Pluronic (F68) and 0-25.0g of glycerol, and measure approximately 800mL of water. Heat to 60-80°C under nitrogen and stir to dissolve to prepare the aqueous phase. Add the oil phase to the aqueous phase and shear at high speed for 5-15 minutes. Add more water to 1000mL to prepare colostrum. The colostrum is then homogenized 1 to 3 times using a high-pressure homogenizer until the average particle size of the homogenized emulsion droplets is no more than 0.5 μm. The colostrum is then filtered through a membrane, and the filtrate is filled into a 5 mL to 20 mL glass ampoule under nitrogen protection. The ampoule is then sterilized by rotary autoclaving at 121°C for 8 to 12 minutes to obtain an α-asarone injectable emulsion containing 0.5 to 50 mg / mL of α-asarone.

[0076] Preparation Example 2 Preparation of α-asarone injection emulsion

[0077] Weigh 10.0g of α-asarone, 50.0g of soybean oil for injection, and 50.0g of medium-chain triglycerides (MCT) for injection into a suitable container. Heat to 60-80°C under nitrogen and stir to dissolve. Add 12.0g of egg yolk lecithin and 0.3g of sodium oleate to the mixture and stir to dissolve to prepare the oil phase, which is set aside. Separately, weigh 22.0g of glycerin and approximately 800mL of water. Heat to 60-80°C under nitrogen and stir to dissolve to prepare the aqueous phase. Add the oil phase to the aqueous phase and shear at high speed for 5-15 minutes. Add more water to 1000mL to prepare colostrum. The colostrum is then homogenized 1 to 3 times using a high-pressure homogenizer until the average particle size of the homogenized emulsion droplets is no greater than 0.5 μm. The colostrum is then filtered through a membrane, and the filtrate is filled into a 5 mL or 10 mL glass ampoule under nitrogen protection. The ampoule is then sterilized by rotary autoclaving at 121°C for 8 min to obtain an α-asarone injectable emulsion containing 10 mg / mL of α-asarone.

[0078] Preparation Example 3 Preparation of α-asarone injection emulsion

[0079] Weigh 20.0g of α-asarone, 100.0g of soybean oil for injection, and 100.0g of medium-chain triglycerides (MCT) for injection into a suitable container. Heat to 60-80°C under nitrogen and stir to dissolve. Add 12.0g of egg yolk lecithin and 0.3g of oleic acid and stir to dissolve to prepare the oil phase, which is set aside. Separately, weigh 22.0g of glycerol and approximately 800mL of water. Heat to 60-80°C under nitrogen and stir to dissolve to prepare the aqueous phase. Add the oil phase to the aqueous phase and shear at high speed for 5-15 minutes. Add more water to 1000mL to prepare colostrum. The colostrum is then homogenized 1 to 3 times using a high-pressure homogenizer until the average particle size of the homogenized emulsion droplets is no greater than 0.5 μm. The colostrum is then filtered through a membrane, and the filtrate is filled into a 5 mL or 10 mL glass ampoule under nitrogen protection. The ampoule is then sterilized by rotary autoclaving at 121°C for 8 min to obtain an α-asarone injectable emulsion containing 20 mg / mL of α-asarone.

[0080] Preparation Example 4 Preparation of α-asarone injection emulsion

[0081] Weigh 1.0g of α-asarone and 100.0g of soybean oil for injection into a suitable container. Heat to 60-80°C under nitrogen and stir to dissolve. Add 12.0g of egg yolk lecithin and 0.3g of oleic acid and stir to dissolve to prepare the oil phase, which is set aside. Separately, weigh 22.0g of glycerol and approximately 800mL of water. Heat to 60-80°C under nitrogen and stir to dissolve to prepare the aqueous phase. Add the oil phase to the aqueous phase and high-speed shear for 5-15 minutes. Add more water to 1000mL to prepare colostrum. The colostrum was homogenized 1 to 3 times using a high-pressure homogenizer until the average particle size of the homogenized emulsion droplets was no more than 0.5 μm. The colostrum was filtered through a filter membrane, and the filtrate was filled into a 50 mL infusion bottle under nitrogen protection. The filtrate was sterilized by rotary autoclaving at 121°C for 12 min to obtain an α-asarone injectable emulsion containing 1 mg / mL of α-asarone.

[0082] Preparation Example 5 Preparation of α-asarone oral emulsion

[0083] The preparation method is the same as that of Example 1, except that a pharmaceutically acceptable amount of antioxidants such as vitamin E and pyrogallate may be added to the oil phase, and a pharmaceutically acceptable amount of preservatives such as ethylparaben may also be added to the oil phase; a pharmaceutically acceptable amount of flavoring agent such as aromatic fruit juice syrup may be added to the aqueous phase; and a pharmaceutically acceptable amount of preservatives such as benzoic acid and sodium benzoate may also be added to the aqueous phase. Colostrum is prepared using the same method, and the colostrum is homogenized 1 to 3 times using a high-pressure homogenizer until the average particle size of the homogenized emulsion droplets is no greater than 10 μm. The colostrum is then filtered through a membrane, and the filtrate is packaged in suitable pharmaceutical packaging under nitrogen protection. The filtrate is then sterilized by circulating steam at 100°C for 30 minutes, or 121°C for 8 minutes, to obtain an α-asarone oral emulsion.

[0084] Preparation Example 6 Preparation of α-asarone injection emulsion

[0085] Weigh 1.0-20.0g of α-asarone and 50.0-200.0g of injectable soybean oil into a suitable container. Heat to 60-80°C under nitrogen, stirring to dissolve. Add 12.0g of egg yolk lecithin and 0-0.3g of oleic acid to the mixture, stirring to dissolve, to prepare the oil phase, which is set aside. Separately, weigh 22.0g of glycerol and approximately 800mL of water, heat to 60-80°C under nitrogen, stirring to dissolve, to prepare the aqueous phase. Add the oil phase to the aqueous phase, shear at high speed for 5-15 minutes, and then add water to 1000mL to prepare colostrum. The colostrum is homogenized 1 to 3 times using a high-pressure homogenizer until the average particle size of the homogenized emulsion droplets is no more than 0.5 μm. The colostrum is filtered through a filter membrane, and the filtrate is filled into 2 mL, 5 mL, and 10 mL glass ampoules under nitrogen protection. The filtrate is sterilized by rotary autoclaving at 121°C for 8 to 12 min to obtain an α-asarone emulsion injection, in which the α-asarone content is 1 mg / mL to 20 mg / mL.

[0086] Preparation Example 7 Preparation of α-asarone injection emulsion (also known as emulsion injection)

[0087] Experimental Materials:

[0088] α-Asarone (2883-98-9, Wuhan Lanabai Pharmaceutical Chemical Co., Ltd.);

[0089] Soybean oil for injection (DD20200603, Shandong Ruisheng Pharmaceutical Excipients Co., Ltd.);

[0090] Egg yolk lecithin (202008013, Shanghai Taiwei Pharmaceutical Co., Ltd.);

[0091] Oleic acid (160907, Xi'an Libang Pharmaceutical Co., Ltd.);

[0092] Glycerol (20191213, Zhejiang Suichang Huikang Pharmaceutical Co., Ltd.);

[0093] Experimental Procedure: Weigh 10.0g of α-asarone and 100.0g of soybean oil for injection into a suitable container, heat to 80°C under nitrogen, and stir to dissolve. Next, weigh 12.0g of egg yolk lecithin and 0.3g of oleic acid, add them, and stir to dissolve to prepare the oil phase, which is set aside. Separately, weigh 22.0g of glycerol and approximately 800mL of water, heat to 80°C under nitrogen, and stir to dissolve to prepare the aqueous phase. Add the oil phase to the aqueous phase, and high-speed shear at 19,000 rpm for 10 minutes to disperse the oil phase in the aqueous phase. Add more water to 1000mL to prepare colostrum. The colostrum was homogenized three times using a high-pressure homogenizer at a pressure of 1000 bar until the average particle size of the homogenized emulsion droplets was no more than 0.5 μm. The colostrum was filtered through a filter membrane, and the filtrate was filled into 2 mL, 5 mL, and 10 mL glass ampoules under nitrogen protection. The filtrate was rotary autoclaved at 121°C for 8 min to obtain α-asarone emulsion injection, in which the α-asarone content was 10 mg / mL, batch number 20201228.

[0094] Preparation Example 8 Preparation of α-asarone oral emulsion

[0095] Weigh 10.0g of α-asarone and 100.0g of medicinal soybean oil into a suitable container, heat to 80°C under nitrogen, and stir to dissolve. Next, weigh 12.0g of egg yolk lecithin, 0.3g of oleic acid, 10.0g of the antioxidant vitamin E, and 2.0g of ethylparaben, add these and stir to dissolve to produce an oil phase, which is set aside. Separately, weigh 22.0g of glycerin and approximately 800mL of water, heat to 80°C under nitrogen, and stir to dissolve to produce an aqueous phase. Add the oil phase to the aqueous phase and high-speed shear at 19,000 rpm for 10 minutes to disperse the oil phase in the aqueous phase. Add more water to 1000mL to prepare colostrum. The colostrum was homogenized three times using a high-pressure homogenizer at a pressure of 1000 bar until the average particle size of the homogenized emulsion droplets was no more than 0.5 μm. The colostrum was filtered through a filter membrane, and the filtrate was filled into a 10 mL oral vial under nitrogen protection. The vial was sterilized by circulating steam at 100°C for 30 min, or by rotary autoclaving at 121°C for 8 min, to obtain an α-asarone oral emulsion with a content of 10 mg / mL of α-asarone. The batch number is 20210105.

[0096] Effect Example 1: Short-term therapeutic effect of α-asarone on SAH and ICH rats

[0097] Experimental materials: SPF-grade SD rats, half male and half female, weighing 200-240 g, were purchased from Chengdu Dashuo Experimental Animal Co., Ltd., Sichuan Province, with certificate number: SCXK(Chuan)2020-030.

[0098] Collagenase VII was purchased from Sigma-Aldrich, USA (specification: 1.5 KU; batch number: 0000111586).

[0099] α-Asarone raw material was purchased from Wuhan Lanabai Pharmaceutical Chemical Co., Ltd. (specification: 2 kg; batch number: 2883-98-9), and its emulsion injection was homemade, batch numbers 20201228 and 20210105.

[0100] Vinpocetine injection was purchased from Henan Runhong Pharmaceutical Co., Ltd. (specification: 10 mg:2 mL; batch number: 1811283).

[0101] Nimodipine injection was purchased from Bayer HealthCare (specification: 10 mg:50 mL; batch number: BXJC71).

[0102] Experimental groups: Zea Longa score was used to judge whether the model was successful 2 hours after intravascular puncture or collagenase VII injection. Rats with successful modeling were randomly divided into groups and given medication.

[0103] Rats were randomly divided into a sham operation group (P group, administered with a volume of normal saline equal to that of the high-dose emulsion injection group), a model group (S or I group, administered with a volume of blank emulsion equal to that of the high-dose emulsion injection group), a low-dose α-asarone emulsion injection group (prepared in Preparation Example 7, 7.5 mg / kg, L group), a medium-dose α-asarone emulsion injection group (prepared in Preparation Example 7, 15 mg / kg, M group), a high-dose α-asarone emulsion injection group (prepared in Preparation Example 7, 30 mg / kg, H group), α-asarone emulsion oral administration group (prepared by Preparation Example 8, 40 mg / kg, O group), β-asarone emulsion injection group (the same operation as Preparation Example 7, prepared into an emulsion injection, the concentration was 10 mg / mL, the dosage was 20 mg / kg, B group), vinpocetine injection group (commercially available, 2 mg / kg, V group), nimodipine injection group (commercially available, 1 mg / kg, N group), 12 rats in each group, except Group N which was administered by intraperitoneal injection, the other groups were administered via tail vein.

[0104] 1.1 Establishment of SAH by intravascular puncture

[0105] Rats were fasted for 12 hours before surgery. Anesthesia was induced with 4% isoflurane and maintained with 2% isoflurane. The animals were fixed in the supine position and their body temperature was maintained at approximately 37°C. The skin of the neck was prepared, and a midline incision was made. The muscles and fascia were separated along the inner edge of the sternocleidomastoid muscle. The right side was exposed and the common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were bluntly dissected. Threads were placed at the proximal end of the CCA, ICA, and ECA for later use. The proximal end of the CCA and ECA were ligated, and the ICA was temporarily clamped with an artery clamp. Then, a small hole was punctured with a needle approximately 4 mm from the bifurcation of the CCA. The puncture thread was inserted through the CCA into the ICA. The artery clamp on the ICA was released, and the puncture thread was inserted into the skull. When the puncture line tip is approximately 18–19 mm from the common carotid artery bifurcation and resistance is felt, it indicates that the tip has reached the bifurcation of the anterior and middle cerebral arteries. Applying slight force, the line is advanced approximately 2 mm further, indicating that the bifurcation has been punctured. The puncture line is completely removed, the ICA is ligated, and the wound is cleaned with saline and sutured. In the sham-operated group, the line is withdrawn when resistance is felt, leaving the bifurcation of the anterior and middle cerebral arteries unpunctured. All other procedures are the same as in the experimental group. After awakening from anesthesia, the animals are maintained as normal.

[0106] SAH severity was scored after euthanasia following the completion of short-term neurological assessment. The severity of SAH was assessed based on the presence of subarachnoid hemorrhage (SAH) within the basal cistern and on the brain surface. The basal cistern was divided into six regions by the Circle of Willis, which is comprised of the basilar artery, anterior cerebral artery, internal carotid artery, posterior cerebral artery, and posterior communicating artery. Each region was scored from 0 to 3 based on the amount of SAH clot present: 0: no SAH; 1: minimal SAH; 2: moderate SAH with discernible skull base arteries; and 3: clot covering all arteries within the region. The sum of all six scores yielded a total of 18 points. Based on the final score, SAH severity was categorized as 0-7 (mild SAH), 8-12 (moderate SAH), and 13-18 (severe SAH). Moderate to severe SAH models (score ≥ 8) were selected for inclusion in the analysis.

[0107] 1.2 Construction of ICH model by collagenase injection

[0108] Rats were fasted for 12 hours before surgery. Anesthesia was induced with 4% isoflurane and maintained with 2% isoflurane. The animals were immobilized in the prone position, and their body temperature was maintained at approximately 37°C. The head was prepared, and a midline incision was made. According to the stereotaxic atlas of the rat brain (translated by Zhuge Qichuan, George Paxinos, Charles Watson, Paxinos, Watson, & Zhuge Qichuan. Stereotaxic atlas of the rat brain [M]. People's Medical Publishing House, 2005), the right caudate nucleus of the rat was located using a stereotaxic apparatus (from bregma as the origin, 3 mm to the right, 5.5 mm deep). After marking, a skull drill was used to insert a microsyringe needle into the caudate nucleus of the brain tissue. 1 μL of 0.5 U type VII collagenase was injected over 5 minutes. The needle was retained for 8 minutes after the injection. The needle was then slowly withdrawn, the skull burr hole sealed with bone wax, the skin sutured, and the rat was returned to its cage for maintenance. Rats in the sham operation group were only injected with sterile saline without injection of drugs, and other operations remained unchanged.

[0109] 1.3 Inclusion criteria for intracerebral hemorrhage models

[0110] The Zea Longa neurological function score was used and the rats were scored 2 hours after awakening from anesthesia. Rats with a score of 1 to 3 were included in the group.

[0111] 0 points: no neurological deficit symptoms and normal activities;

[0112] 1 point: unable to fully extend the contralateral forepaw;

[0113] 2 points: The animal appears to be moving in circles while crawling;

[0114] 3 points: the body falls to the hemiplegic side;

[0115] 4 points: Unable to walk spontaneously and has lost consciousness.

[0116] 1.4 Short-term neurological impairment score

[0117] Twenty-four hours after modeling, the rats' neurological function was comprehensively assessed using the Garcia score and balance beam test. The Garcia score criteria are shown in Table 1. Scores range from 3 to 18, with lower scores indicating more severe neurological impairment. The balance beam test scoring criteria are shown in Table 2. Proprioception and coordination are assessed, with scores ranging from 0 to 6, with higher scores indicating more severe neurological impairment. Scoring was performed independently by a blinded individual who was not involved in modeling or drug administration.

[0118] Table 1 Garcia neurological function score

[0119]

[0120]

[0121] Table 2 Balance beam scoring criteria

[0122] Evaluation Criteria Score Stable balance posture 0 points Hold on to the edge of the balance beam 1 point Holding onto the balance beam, one limb hangs down from it 2 points Holding onto the balance beam, two limbs hang from the balance beam or rotate on the balance beam (>60 seconds) 3 points Falling while attempting to balance on a beam (>40 seconds) 4 points Falling while attempting to balance on a beam (>20 seconds) 5 points Falling; not attempting to balance on the beam (<20 seconds) 6 points

[0123] As shown in Table 3, compared with the sham operation group (P group), the Garcia score of the model group (S or I group) was significantly reduced (P<0.001), and the balance beam score was significantly increased (P<0.001) 24 hours after surgery. It can be seen that the rats in the model group had obvious neurological deficits 24 hours after SAH or ICH; intravenous administration of different doses of α-asarone (L, M, H groups) and oral administration of α-asarone (O group) can improve the Garcia score and reduce the balance beam score to varying degrees, and improve the neurological deficits caused by SAH or ICH, among which the improvement effect of group M was the most significant (P<0.01). In the SAH model, the efficacy of the drug in group M was superior to that of nimodipine (group N), a drug clinically used to improve vasospasm after subarachnoid hemorrhage, and vinpocetine (group V), a drug used to treat the sequelae of cerebral hemorrhage. In the ICH model, the efficacy of the drug in groups L and M was superior to that of group V, and both groups were significantly superior to group N. In contrast, the β-asarone-administered group (B) had no significant effect on neurological function in either SAH or ICH model rats. Furthermore, after brains were harvested via cardiac perfusion in the SAH rats, no significant differences in bleeding scores were found between the model and drug-administered groups, eliminating the possibility that behavioral differences could be due to varying model severity.

[0124] Table 3 Short-term neurological deficit scores in rats

[0125]

[0126]

[0127] Note: Compared with the sham operation group (P group), ### P<0.001, ## P<0.01, # P<0.05; compared with the model group (S or I group), ** P<0.01, * P<0.05; compared with the nimodipine group (group N), & P < 0.05. Comparative data are expressed as x ± SD, and multiple group comparisons were analyzed using ANOVA and Tukey-post-hoc.

[0128] Effect Example 2: α-asarone reduces the incidence of secondary epilepsy in SAH rats

[0129] The experimental materials, grouping, modeling method, and administration regimen were the same as those in Example 1. The seizures of rats in each group were observed within 24 hours after SAH in rats. The results are shown in Table 4. Compared with the P group, the Racine score of the S group was significantly increased (P < 0.001). The Racine score of the L, M, H, and O groups was reduced to varying degrees, with the M group having the most significant effect (P < 0.05). Based on this, α-asarone can significantly reduce the incidence of secondary epilepsy caused by SAH in rats.

[0130] Table 4 Effects of α-asarone on secondary epileptic seizures induced by SAH in rats

[0131] Group Racine score Clonic seizures (%) Tonic seizures (%) P 0±0 0 0 S <![CDATA[1.72±1.87 ### ]]> 38.9％ 11.1％ L 0.53±1.46 6.7％ 6.7％ M <![CDATA[0.44±1.34 * ]]> 5.6％ 5.6％ H <![CDATA[0.73±1.44 # ]]> 8.0％ 6.7％ O <![CDATA[0.77±1.59 # ]]> 15.4％ 7.7％ N <![CDATA[0.81±1.56 # ]]> 18.8％ 6.2％

[0132] Note: Compared with group P (administered with normal saline), ### P<0.001, # P<0.05; compared with group S (administered blank emulsion), * P < 0.05. Seizure severity was determined according to the Racine classification, which categorizes seizure behavior into six levels based on the severity of the seizure: Level 0 (no response or cessation of convulsions); Level I (rhythmic mouth or facial twitching); Level II (head nodding or tail flicking); Level III (monilial twitching); Level IV (multi-limb twitching or rigidity); and Level V (generalized tonic-clonic seizure). Levels I, II, and III are clonic seizures, while levels IV and V are tonic seizures.

[0133] Effect Example 3: Long-term protective effect of α-asarone on SAH rats

[0134] 3.1 Long-term survival rate of rats

[0135] The experimental materials, grouping, and modeling methods were the same as those in Example 1. Two hours after SAH modeling, the drugs were administered according to the grouping dosing schedule and continued to be administered once daily for 14 days. The survival of the rats over 14 days was observed and recorded. The results are shown in Table 5. The 24-hour mortality rate in the S group was as high as 53.8%. The drug administration in the M, H, O, and N groups significantly reduced the 24-hour mortality rate and prolonged the survival of the SAH rats. Specifically, α-asarone significantly reduced the 24-hour mortality rate and prolonged the 14-day survival of SAH rats.

[0136] Table 5 Long-term survival rate of SAH rats

[0137]

[0138] 3.2 Evaluation of long-term learning and memory function

[0139] The Morris water maze was used to assess the long-term spatial perception and memory abilities of each group of rats. This was performed on days 15-19 after the end of the survival observation period. The water maze consisted of a circular pool with a diameter of 150 cm and a depth of 60 cm. Prior to the experiment, the pool was filled with warm water at (24 ± 2)°C to a depth of 30 cm and then dyed black. The pool was divided into four equal quadrants, each marked with a distinctive marking. A colorless, transparent platform with a diameter of 10 cm and a height of 28 cm was placed in the center of one quadrant, submerged 2 cm below the surface. Following the start of the experiment, rats were released from the designated quadrant according to the experimental instructions. On days 1-4, rats were released from each of the four quadrants for one trial each, with a 10-minute interval between trials. If a rat found the platform within 60 seconds, it was allowed to stand on it for 10 seconds. If it failed to find the platform, it was guided to the platform with a rod and allowed to stand for 10 seconds. On the fifth day, the platform was removed and the rats were allowed to swim freely for 60 seconds. The escape latency, swimming speed, and exploration time of the target quadrant were recorded using a computer tracking system (Noldus Ethovision, Tacoma, WA, USA).

[0140] The experimental results are as follows Figure 1 As shown in A, during the acquisition training period, the latency of the S and N groups to find the platform was significantly longer than that of the P group (P<0.001); while the escape latency of the M group was significantly shorter than that of the S and N groups, and even on the fourth day, there was no statistical difference between the M group and the P group. Figure 1 As can be seen from Figures B and 1C, during the spatial exploration period, compared with the P group, the S and N groups spent less time in the target quadrant, while the M group spent more time in the target quadrant, which was comparable to the P group. In addition, the swimming speed of this group was significantly faster than that of the S group (P<0.05). Figure 1 As shown in Figure D, after long-term administration, rats in groups S and N showed whitening and atrophy of the affected side of the brain tissue, while groups P and M did not. In summary, long-term administration of α-asarone not only significantly improved learning and memory function and promoted motor recovery in SAH rats during the recovery period, but also alleviated brain tissue atrophy in the recovery period.

[0141] Effect Example 4: Study on the mechanism of action of α-asarone against hemorrhagic stroke

[0142] Experimental Materials:

[0143] Evans Blue (C11891158, Shanghai MacLean Biochemical Technology);

[0144] Formamide (20190716, Tianjin Bodi Chemical);

[0145] Glutamate detection kit (20210525, Beijing Solebow Technology);

[0146] GABA-ELISA kit (202101, Shanghai Jianglai Biotechnology);

[0147] DNA enzyme I (226F031, Beijing Solebow Technology);

[0148] Papain (111S022, Beijing Solebow Technology);

[0149] Calcium ion fluorescent probe (20210313, Jiangsu KeyGen Biotechnology);

[0150] Rhodamine 123 stain (119I033, Beijing Solebow Technology);

[0151] Ice-cold centrifugation buffer (20210525, Beijing Solebow Technology).

[0152] Experimental steps and results:

[0153] 4.1 Determination of brain water content and blood-brain barrier permeability

[0154] After completing the 24-hour short-term neurological function assessment, rats were injected with 4% Evans blue solution (2.5 mL / kg) into the right tail vein. One hour later, the rats were deeply anesthetized and perfused transcardially with 100 mL of normal saline. The brains were rapidly decapitated and immediately separated into the left and right hemispheres, cerebellum, and brainstem. The left and right hemispheres were divided coronally into two sections, and the brain tissue (wet weight) was weighed using a 0.1 mg scale. The samples were then dried in an oven at 105°C for 24 hours and weighed again (dry weight). Brain water content was calculated as follows: brain water content = [(wet weight - dry weight) / wet weight] × 100%. The other part of the brain tissue was weighed to obtain its wet weight, then immersed in 10 times the volume of pure formamide, incubated at 60°C for 48 h, and centrifuged at 25°C and 10,000 rpm / min for 30 min. The supernatant was aspirated and the Evans blue dye was detected by ultraviolet spectrophotometry at 622 nm. A standard curve was drawn for quantification, and the final result was displayed as the Evans blue content per gram of brain tissue (μg / g).

[0155] The results are as follows Figure 2 As shown in the results, compared with the sham operation group (P group), the brain water content of the hemorrhage side cerebral hemisphere in the model group (S or I group) was significantly increased 24 hours after surgery, the Evans blue exudation was significantly increased, and the blood-brain barrier permeability was increased. The intravenous administration of medium-dose α-asarone to the brain (M group) can significantly reduce the water content of the hemorrhage side brain tissue of rats, alleviate brain edema, reduce Evans blue exudation, and improve the permeability of the blood-brain barrier.

[0156] 4.2 Determination of glutamate and GABA content

[0157] 12 to 24 hours after modeling, the rats were deeply anesthetized, decapitated and the brains removed. Approximately 60 to 120 mg of the hemispheric cortex of the blood-side cerebral hemisphere was removed and added with 10 times the volume of ice-cold centrifugation buffer for biochemical testing. The mixture was homogenized on ice for 10 minutes and centrifuged at 4°C and 14,000 rpm / min for 30 minutes. The supernatant was collected and the glutamate and GABA contents were measured according to the instructions of the glutamate content detection kit and the rat GABA Elisa kit, respectively.

[0158] The results are as follows Figure 3 As shown in the results, compared with the P group, the glutamate and GABA contents in the hemisphere of the hemorrhage side of the rats in the S or I group were significantly increased 12 to 24 hours after surgery, while the administration of the M group could significantly reduce the glutamate and GABA contents in the brain tissue of rats with cerebral hemorrhage, which is beneficial to counteract the excitotoxicity of glutamate, restore the balance of excitatory amino acids / inhibitory amino acids (EAA / IAA) in the brain, and improve the motor function of rats.

[0159] 4.3Ca 2+ mitochondrial membrane potential assay

[0160] 12 to 24 hours after modeling, the brain was removed by decapitation, and the cerebral hemisphere cortex around the hematoma was taken. Single-cell suspension was immediately prepared by enzyme digestion (papain: 2 mg / mL; DNase I: 0.05 mg / mL) and the cell concentration was adjusted to 5×10 6 Cells were collected at 400 μL / mL and 100 μL of cell suspension was added with Fluo-3 / AM dye at a final concentration of 5 μM or Rhodamine 123 dye at a final concentration of 10 μM. The cells were incubated at 37°C for 45 min, washed twice with PBS (phosphate buffer, pH = 7.2-7.4), and resuspended in 0.5 mL of PBS. 10,000 cells were examined by flow cytometry at an excitation wavelength of 506 nm and an emission wavelength of 526 nm. The mean fluorescence intensities of Fluo-3 and Rh123 were analyzed using FlowJo software.

[0161] The results are as follows Figure 4 As shown in the data, compared with the P group, the calcium ion content in the S or I group increased significantly 12 to 24 hours after surgery, and the mitochondrial membrane potential increased, indicating that mitochondria were damaged and cell apoptosis increased; while the administration of the M group could significantly reduce the calcium ion content and stabilize the mitochondrial membrane potential, thereby reducing neuronal apoptosis and necrosis.

[0162] Effect Example 5: Protective Effect of α-asarone on Neurons Injured by Oxyhemoglobin

[0163] Experimental Materials:

[0164] PC12 cell line was purchased from Wuhan Punosai Life Science Technology Co., Ltd.;

[0165] Oxyhemoglobin (20210201, Beijing Solebow Technology Co., Ltd.);

[0166] MTT (C12029690, Sigma-Aldrich, USA);

[0167] DMEM high-glucose medium (AG29301810, Hyclone, USA);

[0168] Fetal bovine serum (20010401, Gibco, USA)

[0169] Penicillin-streptomycin solution (double antibody) (20201220, Hyclone, USA)

[0170] PBS powder (WK173618-1, Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.);

[0171] DMSO (20201220, Beijing Solebow Technology Co., Ltd.).

[0172] Experimental steps:

[0173] Complete culture medium: Mix DMEM high glucose medium, fetal bovine serum and penicillin-streptomycin solution (double antibody) in a volume ratio of 90:9:1 and store in a refrigerator at 4°C.

[0174] Serum-free culture medium: Mix DMEM high-glucose culture medium and penicillin-streptomycin solution (double antibody) in a volume ratio of 99:1 and store in a refrigerator at 4°C.

[0175] PC12 cells in the logarithmic growth phase cultured in complete medium were cultured at a concentration of 1×10 4Cells were inoculated into 96-well plates at 100 μL / well, and the edge wells were filled with sterile PBS. The plates were cultured at 37°C and 5% CO2 for 24 hours until the cells were completely attached. The supernatant was discarded, and oxyhemoglobin was added at final concentrations of 0 μM, 4 μM, 6 μM, 8 μM, and 10 μM, respectively, and the plates were cultured at 37°C and 5% CO2 for 24 hours. Subsequently, 10 μL of 5 mg / mL MTT was added to each well, and the plates were cultured at 37°C and 5% CO2 for 4 hours. The supernatant was discarded, and DMSO was added. 100 μL / well was added and shaken at 37°C, 500 rpm for 15 minutes to fully dissolve the formazan. The OD value at 570 nm was measured using a microplate reader. PC12 cell viability was calculated using the formula "abnormal cell proliferation multiple = [(average absorbance value of the experimental group - average absorbance value of the zero-adjusted well) / (average absorbance value of the control group - average absorbance value of the zero-adjusted well]". Under these experimental conditions, the maximum abnormal cell proliferation multiple was 1.5 times, corresponding to an oxyhemoglobin concentration of 6 μM. Based on this, this concentration was used as the optimal concentration for the oxyhemoglobin-induced oxidative stress injury model in the preliminary test and was used in the following cell pharmacodynamic experiments.

[0176] PC12 cells in the logarithmic growth phase cultured in complete medium were taken and cultured at 1×10 4 Cells were inoculated into 96-well plates at 100 μL / well. The edge wells were filled with sterile PBS and incubated at 37°C, 5% CO2 for 24 hours until the cells were completely adhered. The supernatant was discarded, and 100 μL of different concentrations of α-asarone emulsion diluted in serum-free medium (prepared in Preparation Example 7, with final α-asarone concentrations of 1 μM, 5 μM, 10 μM, 25 μM, and 50 μM, respectively) was added to each of the treatment groups except the control and model groups. After 2 hours of incubation, 20 μL of oxyhemoglobin solution diluted in serum-free medium was added to each of the treatment groups to a final concentration of 6 μM, and incubation was continued at 37°C, 5% CO2 for up to 24 hours. The control group was treated with the same volume of serum-free medium alone; the model group was treated with 100 μL of a blank emulsion diluted in serum-free medium (the formula was the same as in Example 7 except that α-asarone was not present) and 20 μL of a serum-free medium-diluted oxyhemoglobin solution with a final concentration of 6 μM. The remaining procedures for the control and model groups were the same as those for the drug-treated group. Subsequently, 10 μL of 5 mg / mL MTT was added to each well and incubated at 37°C, 5% CO2 for 4 hours. The supernatant was discarded, and 100 μL / well of DMSO was added. The cells were shaken at 37°C, 500 rpm, for 15 minutes to fully dissolve the formazan. The OD value at 570 nm was measured using a microplate reader. The results are as follows: Figure 5 shown.

[0177] Experimental results: Figure 5As shown in the results, compared with the control group, the addition of oxyhemoglobin caused abnormal proliferation of PC12 cells in the model group, and the absorbance increased significantly, indicating that cell viability was significantly enhanced and obvious oxidative stress occurred; on the other hand, different concentrations of α-asarone significantly reduced the abnormal increase in cell viability caused by oxyhemoglobin, indicating that it can significantly reduce the oxidative stress caused by oxyhemoglobin ( Figure 5 ).

[0178] Effect Example 6: Preliminary Safety Evaluation of α-Asarone Emulsion Injection—Mouse Bone Marrow Micronucleus Experiment

[0179] Experimental Materials: Fifty SPF male Kunming mice, weighing 18–22 g, were purchased from Chengdu Dashuo Laboratory Animal Co., Ltd., license number SCXK(Chuan)2020-030. Cyclophosphamide for injection was purchased from Jiangsu Shengdi Pharmaceutical Co., Ltd.; 1,4-piperazinediethanesulfonic acid (PIPES, 715H021, Beijing Solarbio Co., Ltd.), Triton X-100 (829I0210, Beijing Solarbio Co., Ltd.), and propidium iodide (PI, 1024S043, Beijing Solarbio Co., Ltd.) were also purchased.

[0180] Experimental grouping and dosing: The animals were randomly divided into five groups, with 10 animals in each group, a blank control group (Blank group, administered with a blank emulsion of the same volume as that of the α-asarone high-dose group), a cyclophosphamide group (CTX group, 40 mg / kg), a low-dose α-asarone emulsion injection group (prepared according to Preparation Example 7, 100 mg / kg / day, ASA-L group), a medium-dose α-asarone emulsion injection group (prepared according to Preparation Example 7, 150 mg / kg / day, ASA-M group), and a high-dose α-asarone emulsion injection group (prepared according to Preparation Example 7, 200 mg / kg / day, ASA-H group).

[0181] All drugs were injected through the tail vein. Except for the positive control drug cyclophosphamide (CTX), which was injected once only 24 hours before sampling, the drugs were administered through the tail vein for 4 consecutive days in each group. 24 hours after the last administration, the mice were killed by cervical dislocation. The femurs on both sides were separated, and the femoral bone marrow cells were flushed out with PBS. The cells were passed through a 300-mesh nylon mesh to prepare a single-cell suspension, centrifuged at 1650 rpm for 5 minutes, and resuspended in PBS to adjust the cell concentration to 5×10 6 / mL, 100 μL of cell suspension was carefully added to 400 μL of PIPES-PI solution (10 mL of PIPES solution (concentration of 3.5 mg / mL) + 0.5 mg of PI + 0.01 mL of Triton X-100 (concentration of 0.1%)) for each sample, and the mixture was gently pipetted and mixed. After staining at 4°C in the dark for 30 minutes, flow cytometry was used for detection. The results are shown in Table 6. As shown in Table 6, PCE is polychromatic erythrocytes, MNPCE is polychromatic erythrocytes with micronuclei, and fMNPCE is the ratio of polychromatic erythrocytes with micronuclei to polychromatic erythrocytes, which reflects the micronucleus rate of mouse bone marrow cells. The higher the value, the stronger the genotoxicity.

[0182] The results are shown in Table 6. The micronucleus rate of the positive control drug cyclophosphamide group (CTX) was significantly higher than that of the blank emulsion group (P<0.01). There was no significant difference in the micronucleus rate between the various dose groups of α-asarone emulsion injection and the blank emulsion group. Compared with the CTX group, the micronucleus rate was significantly lower, and the differences were statistically significant (ASA-L: P<0.01; ASA-M: P<0.05; ASA-H: P<0.05).

[0183] The results of the aforementioned toxicology study on chromosomal damage in mouse hematopoietic cells showed that intravenous administration of α-asarone emulsion injection at doses up to 200 mg / kg did not significantly alter the micronucleus rate in mouse bone marrow cells. Considering the aforementioned effective dose for the treatment of hemorrhagic stroke, the drug is expected to have a good safety profile.

[0184] Table 6 Results of fMNPCE (‰) in mouse bone marrow detected by flow cytometry

[0185]

[0186] Note: Compared with the blank group (Blank), ## P<0.01; compared with the CTX group, ** P<0.01, * P<0.05.

[0187] In summary, the results of in vitro and in vivo pharmacodynamic studies showed that α-asarone could significantly improve the short-term neurobehavioral function and long-term learning and memory function of rats with hemorrhagic stroke, reduce the incidence and mortality of secondary epilepsy in SAH rats, reduce brain edema, improve blood-brain barrier permeability, prevent or alleviate brain tissue atrophy during the recovery period, antagonize glutamate excitotoxicity, restore GABA levels, and restore the balance of excitatory amino acids / inhibitory amino acids (EAA / IAA) in the brain; reduce Ca 2+ It can stabilize mitochondrial membrane potential, reduce neuronal apoptosis, and alleviate oxidative stress, thus exerting a neuroprotective effect. Therefore, α-asarone is expected to become a promising drug for the treatment of hemorrhagic stroke.

[0188] Although the above describes specific embodiments of the present invention, it should be understood by those skilled in the art that these are merely illustrative and that various changes or modifications may be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. Use of the compound represented by Formula I in the preparation of a medicament for preventing or treating hemorrhagic stroke; in, The compound represented by formula I is the only active ingredient in the medicine.

2. The use according to claim 1, characterized in that The medicine is used for treating hemorrhagic stroke and preventing secondary epilepsy caused by hemorrhagic stroke.

3. The use according to claim 1 or 2, characterized in that The hemorrhagic stroke is a stroke caused by at least one of intracerebral hemorrhage and subarachnoid hemorrhage.

4. The use according to claim 1 or 2, characterized in that The drug is used for at least one of the following: improving neurological or motor function damage caused by ICH or SAH, improving acute brain tissue edema or blood-brain barrier dysfunction caused by ICH or SAH, reducing acute mortality caused by hemorrhagic stroke, prolonging survival, improving long-term learning and memory dysfunction caused by hemorrhagic stroke, and preventing or alleviating brain tissue atrophy during the recovery period of hemorrhagic stroke.

5. The use according to claim 1 or 2, characterized in that The medicine contains pharmaceutical excipients.

6. The use according to claim 5, characterized in that The total weight ratio of the compound represented by formula I to the pharmaceutical excipients is 1:20-1000.

7. The use according to claim 1 or 2, characterized in that When the drug is used to treat people suffering from hemorrhagic stroke, the daily dosage of the compound represented by formula I in the drug is in the range of 0.15 mg to 5.0 mg / kg body weight.

8. The use according to claim 1 or 2, characterized in that When the drug is used to treat people suffering from hemorrhagic stroke, the daily dosage of the compound represented by formula I in the drug is in the range of 0.3 mg to 3.0 mg / kg body weight.

9. The use according to claim 1 or 2, characterized in that The administration route of the drug is injection or oral administration.

10. The use according to claim 1 or 2, characterized in that The medicine is an emulsion.

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