Application of taraxasterol or its salt in preparing medicine for preventing or treating traumatic brain injury

Abstract

The present invention relates to the field of medicine, and specifically to the use of taraxasterol or its salts in the preparation of drugs for preventing or treating traumatic brain injury. Animal experiments have confirmed that the taraxasterol of the present invention can improve the motor ability of the balance beam experiment in the neurological function score of mice with brain trauma, improve the neurological function scores including motor tests and sensory tests; reduce the permeability of the blood-brain barrier, reduce brain edema, improve the short-term lesion volume after traumatic brain injury, and inhibit microglial activation and immune cell infiltration. Cytological experiments have confirmed that taraxasterol can reduce microglia-mediated neuroinflammation, reduce the expression of inflammatory factors such as TNF-α, IL-6, and IL-1β, and reduce neuronal damage.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to application of taraxasterol or its salt in preparing medicine for preventing or treating traumatic brain injury. Background Art

[0002] Traumatic brain injury (TBI) refers to damage to the brain caused by a sudden, violent external force. It manifests primarily as a series of neurological impairments, including cognitive impairment and impaired consciousness. It is a devastating neurological disorder with a complex pathophysiology, and the neurological impairments experienced by patients tend to progress over time. The mechanisms of neurological damage caused by TBI include neuroinflammation and cell death, which play a significant role in both acute and chronic neurological impairments that occur after TBI.

[0003] Chemokine ligand 2 (CC motif chemokine ligand 2, CCL2), also known as monocyte chemotactic protein 1 (MCP-1), consists of 76 amino acids, has a molecular weight of 13 kDa, and has two adjacent amino-terminal cysteine ​​residues. CCL2 is primarily secreted by immune cells, and its expression can be either constitutive or inducible. CCL2 expression can be induced by various mediators, such as IL-1, IL-4, IL-6, and TNF-α. CCL2 regulates the migration and infiltration of various immune cells, including monocytes, macrophages, memory T lymphocytes, and natural killer cells. Chemokine receptor 2 (CCR2) is considered the primary receptor for CCL2. Binding to CCL2 activates CCR2, subsequently initiating the transcription and expression of IL-1, IL-6, and TNF genes. After CCL2 triggers CCR2, it initiates multiple signaling pathways, including NLRP3, JAK / STAT, PI3K / MAPKs, and NF-κB, involved in cytokine production, inflammation, cell survival, migration, and apoptosis. Following traumatic brain injury, CCL2 expression is upregulated and CCR2 activation induces polarization of microglia, promoting the production of inflammatory factors such as IL-1β, IL-6, and TNFα. Furthermore, immune cells mobilize to sites of inflammation in a CCR2-dependent manner. Under the influence of CCR2, they are recruited to the site of inflammation and participate in the inflammatory response and neuronal damage. Furthermore, the binding of CCL2 to CCR2 increases neuronal oxidative stress and mitochondrial dysfunction, ultimately leading to neuronal cell death.

[0004] Activation of these signaling pathways mobilizes multiple transcription factors and genes involved in cytokine production, cell growth and differentiation, cell survival, migration and apoptosis, angiogenesis, and inflammation. CCL2 has a potential role in regulating neurons. Increased CCL2 expression after traumatic brain injury activates multiple pathways and produces a variety of inflammatory factors, leading to neurological dysfunction in patients.

[0005] Taraxasterol (TAR), also known as (3β, 18α, 19α)-Urs-20(30)-en-3-ol, is a pentacyclic triterpenoid compound with a 1,2-cyclopentenephenanthrene structure. Its molecular formula is C 30 H 50 O, with a molecular weight and melting point of 426.72 g / mol and 221-222 degrees Celsius, respectively. In animal or cell models of various diseases such as liver damage, gastritis, colitis, arthritis, pneumonia, tumors and immune system diseases, taraxasterol has been shown to have significant preventive and therapeutic effects. Summary of the Invention

[0006] The technical problem solved by the present invention is to provide the use of taraxasterol or its salt in the preparation of a drug for preventing or treating traumatic brain injury, so as to solve the problem of unsatisfactory treatment effect and poor prognosis of traumatic brain injury in the prior art.

[0007] In order to solve the technical problems of the invention, the present invention provides the following technical solutions:

[0008] The first aspect of the technical solution of the present invention provides the use of taraxasterol or its salt in the preparation of a drug for preventing or treating traumatic brain injury.

[0009] The second aspect of the technical solution of the present invention is to provide the use of taraxasterol in the preparation of drugs for preventing and / or treating traumatic brain injury.

[0010] The traumatic brain injury described in the present invention is selected from any one of concussion, closed head injury, or open head injury. The traumatic brain injury includes neuroinflammation, neuronal damage, and neurological impairment caused by traumatic brain injury, as well as central nervous system diseases related to neuroinflammation, neuronal damage, and neurological impairment.

[0011] The traumatic brain injury described in the present invention includes neuroinflammation, neuronal damage, neurological function damage, blood-brain barrier disruption, cerebral edema caused by traumatic brain injury, as well as central nervous system diseases related to neuroinflammation, neuronal damage, neurological function damage, blood-brain barrier disruption, and cerebral edema.

[0012] Furthermore, the present invention also provides a pharmaceutical preparation comprising taraxasterol or a salt thereof and a pharmaceutically acceptable carrier; more preferably, the pharmaceutical preparation comprises taraxasterol and a pharmaceutically acceptable carrier. The dosage form of the preparation is selected from any one of tablets, granules, capsules, suspensions, oral solutions, injections, transdermal patches, inhalants, powder injections, aqueous solutions, or liposomes.

[0013] The pharmaceutically acceptable carrier includes, but is not limited to, a filler, a disintegrant, a accelerator, a lubricant, a binder, and a conditioning agent. The binder is selected from a 3% hydroxypropyl methylcellulose solution in 50% ethanol, a 10% aqueous solution of povidone K30, a 30% starch slurry, and a 10% aqueous solution of carboxymethyl cellulose, preferably a 3% hydroxypropyl methylcellulose solution in 50% ethanol. The filler is one or a combination of two or more of lactose, microcrystalline cellulose, starch, pregelatinized starch, dextrin, mannitol, glucose, sorbitol, sucrose, calcium carbonate, calcium sulfate, calcium hydrogen phosphate, calcium phosphate, and hydroxypropyl methylcellulose. The lubricant is selected from stearic acid, glycerin, palmitic acid stearate, magnesium stearate, calcium stearate, talc, micronized silica gel, hydrogenated vegetable oil, and sodium lauryl sulfate; the disintegrant is selected from hydroxypropyl cellulose, cross-linked polyvinyl pyrrolidone, cross-linked sodium carboxymethyl cellulose, cellulose acetate phthalate, and sodium carboxymethyl starch; the enhancer contains fatty acids, including but not limited to butyric acid, caproic acid, caprylic acid, nonanoic acid, caprylic acid, lauric acid, nutmeg, palmitic acid, stearic acid, peanut, oleic acid, linoleic acid, linolenic acid, their salts, derivatives, and combinations thereof. The glyceride can be a monoglyceride, a diglyceride, or a triglyceride. Cholic acid, deoxycholic acid, taurocholic acid, glycocholic acid, taurodeoxycholate, ursodeoxycholate, tauroursodeoxycholate, chenodeoxycholate, derivatives and complexes thereof, ethylenediaminetetraacetic acid, ethylene glycol ditetraacetic acid, surfactants such as sodium lauryl sulfate, polyethylene ethers or esters, polyethylene glycol-12 alkyl ether, salicylates, polysorbate 80, nonylphenoxypolyoxyethylene, sodium octyl sulfosuccinate, saponins, palmitoylcarnitine, lauroyl acetylcarnitine, lauroyl maltoside, acylcarnitine, alkanoylcholine. Other penetration enhancers include 3'nitrobenzoate, fatty acid esters of lactate, glycyrrhizate, hydroxyhydroxycyclodextrin, N-acetylated amino acids such as sodium N-[8-(2-hydroxybenzoyl)amino]octanoate, chitosan, salts, and derivatives of these compounds.

[0014] The pharmaceutical compositions and formulations of the present invention can be used by any route capable of delivering to the brain. Examples of administration routes include, but are not limited to, intravenous, intranasal, oral, topical, scalp, or by inhalation.

[0015] Further, according to the pharmaceutical composition of the present invention, for pharmaceutical compositions for oral administration, it is preferred that a therapeutically effective amount of one or more compounds of the present invention be mixed with a pharmaceutically acceptable carrier according to conventional quantitative pharmaceutical compounding techniques. The carrier can take a variety of forms, depending on the dosage form required for administration, for example, oral or parenteral administration. When preparing pharmaceutical compositions in oral dosage forms, any commonly used pharmaceutical medium can be used. Therefore, for liquid oral preparations such as suspensions, elixirs and solutions, suitable carriers and additives include water, glycols, oils, alcohols, flavorings, preservatives, colorants and the like can be used. For solid oral preparations such as powders, tablets, capsules, and solid dosage forms such as suppositories, suitable carriers and additives include starch, sugar carriers such as glucose, mannitol, lactose and related carriers, diluents, granulating agents, lubricants, binders, disintegrants and the like can be used. If necessary, tablets or capsules can be enteric-coated for sustained release by standard techniques. The use of these dosage forms can significantly affect the bioavailability of the compound in patients.

[0016] In a preferred embodiment of the present invention, the preparation is selected from an injection, which is composed of the following ingredients: 8 parts of taraxasterol, 0.1-1 part of histidine, 10-30 parts of propylene glycol, 0.02-1 part of disodium edetate, 0.1-1 part of tartaric acid, and water for injection is added to 1000 ml; more preferably, 8 parts of taraxasterol, 0.5 part of histidine, 20 parts of propylene glycol, 0.0 part of disodium edetate, 0.5 part of tartaric acid, and water for injection is added to 1000 ml.

[0017] For parenteral formulations, the carrier typically includes sterile water or sodium chloride solution, and other ingredients such as dispersing aids may also be included. When sterile water is to be used or the sterility of sterile water is to be maintained, the composition and carrier must also be sterilized. It can also be prepared as an injectable suspension, in which case appropriate liquid carriers, suspending agents, etc. may also be used.

[0018] Furthermore, the taraxasterol described in the present invention can be used as a promoter for inducing M2 polarization of microglia in traumatic brain injury. M2 polarization is an immunoregulatory state that helps alleviate inflammatory responses and promote tissue repair. The inventors of this application have discovered that taraxasterol significantly promotes M2 polarization of microglia, effectively promoting the repair of various neural injuries.

[0019] The fifth aspect of the technical solution of the present invention is the use of taraxasterol in the preparation of drugs for preventing or treating traumatic brain injury. Its action site is CCR2 (chemokine receptor family). Microglia are a type of immune cell in the central nervous system that is mainly involved in regulating neuroinflammation and immune response. Microglia can be divided into two types: M1 type (proinflammatory) and M2 type (anti-inflammatory) according to their different activation states. M2-polarized microglia play an important role in regulating neuroinflammation, repairing tissue damage, etc. CCL2 is a chemokine that acts on the chemokine receptor CCR2, which also plays an important role in the M1 / M2 polarization process of microglia. Studies have shown that the CCL2-CCR2 signaling pathway can regulate the polarization state of microglia and promote its transformation to the M1 type. Specifically, antagonizing the CCL2-CCR2 signal affects the metabolic state of microglia and the expression of related genes by regulating the intracellular signal transduction pathway, thereby promoting the polarization of microglia to the M2 type.

[0020] The taraxasterol and its salts described in the present invention have been subjected to pharmacological experiments to explore the dosage for mice, and the specific dosage is 1-15 mg / kg; more preferably, the dosage of the taraxasterol is 1-15 mg / kg; and even more preferably, the dosage of the taraxasterol is 8 mg / kg.

[0021] Furthermore, the dosage can be administered once or twice daily. When administered once daily, the dosage is any one of 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, and 15 mg / kg. When administered twice daily, the dosage is 0.5 mg / kg, 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 2.5 mg / kg, 3 mg / kg, 3.5 mg / kg, 4 mg / kg, 4.5 mg / kg, 5 mg / kg, 5.5 mg / kg, 6 mg / kg, 6.5 mg / kg, 7 mg / kg, and 7.5 mg / kg.

[0022] More preferably, the dosage can be administered once or twice a day. When administered once a day, the dosage is 8 mg / kg; when administered twice a day, the dosage is 4 mg / kg.

[0023] Compared with the existing technology, the beneficial technical effects are:

[0024] Animal experiments have confirmed that the taraxasterol described in this invention can improve motor ability in the balance beam test, a neurological function score in mice with brain trauma, and improve neurological function scores including motor and sensory tests. It also reduces blood-brain barrier permeability, alleviates cerebral edema, improves short-term lesion volume after traumatic brain injury, and inhibits microglial activation and immune cell infiltration. Cell-based experiments have also confirmed that the taraxasterol described in this invention can reduce microglial-mediated neuroinflammation, lower the expression of inflammatory factors such as TNF-α, IL-6, and IL-10, and reduce neuronal damage.

[0025] Experiments conducted in this study have demonstrated that taraxasterol can protect C6 glial cells from lipopolysaccharide-induced neuroinflammation and reduce proinflammatory cytokine levels. It can inhibit the production of ROS in hippocampal neurons induced by oxygen-glucose deprivation / reperfusion, alleviating oxidative stress induced by oxygen-glucose deprivation / reperfusion. The present invention has found that taraxasterol can improve motor ability in the balance beam test, a neurological function score in mice with brain trauma, improve neurological function scores including motor and sensory tests, improve blood-brain barrier permeability, reduce short-term lesion volume after traumatic brain injury, and inhibit microglial activation and immune cell infiltration. In cell-based experiments, taraxasterol competitively binds to CCR2 and inhibits downstream NLRP3 and NF-κB signaling pathways, promoting microglia / macrophage M2 polarization, reducing microglia-mediated neuroinflammation, and decreasing the expression of inflammatory factors such as TNF-α, IL-6, IL-10, and IL-1β, thereby reducing neuronal damage. At the same time, taraxasterol can also reduce neuronal oxidative stress and mitochondrial DNA damage, improve mitochondrial dysfunction, upregulate tight junction complex expression, reduce luciferase leakage, and reverse the decrease in intercellular electrical resistance. This suggests that taraxasterol has promising application prospects in treating neuroinflammation and neuronal damage after traumatic brain injury, as well as restoring neurological function in patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 : Flow chart of drug administration for animal experimental modeling.

[0027] Figure 2 : Effects of different administration times of taraxasterol on balance beam scores in mice with traumatic brain injury.

[0028] Figure 3 : Effects of different administration times of taraxasterol on mNSS neurological function scores in mice with traumatic brain injury.

[0029] Figure 4 : Effects of taraxasterol administration on neuronal damage in mice with traumatic brain injury.

[0030] Figure 5 : Effect of taraxasterol incubation on the release of inflammatory factors (IL-1β).

[0031] Figure 6 : Effect of taraxasterol incubation on the release of inflammatory factors (IL-6).

[0032] Figure 7 : Effect of taraxasterol incubation on the release of inflammatory factors (TNF-α).

[0033] Figure 8 : Effects of 10μg / ml, 25μg / ml and 50μg / ml of taraxasterol on cell expression activity.

[0034] Figure 9 : Effects of taraxasterol 25μg / ml and 50μg / ml on CCR2 protein expression.

[0035] Figure 10 : The content (%) of taraxasterol injection at 0, 1, 2, 3 and 6 months. DETAILED DESCRIPTION

[0036] The following non-limiting examples are provided to further illustrate the present invention.

[0037] 1. Brain protection of taraxasterol in mice with traumatic brain injury

[0038] 1. Experimental Animals

[0039] Male C57BL / 6 mice weighing 25–35 g were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. (SCXK (Lu) 20220006).

[0040] 2. Traumatic Brain Injury Model Preparation

[0041] Mice were anesthetized with isoflurane, and the scalp was incised 0.8 mm posterior to the right coronal suture and 1.3 mm lateral to the midline. A 2 mm diameter bone hole was drilled. Using a modified Feeney free-fall injury apparatus, a 20 g hammer was dropped from a height of 20 cm to impact a striker rod, achieving a 2 mm impact depth. The scalp was then sutured. Mice in the sham-operated group underwent only scalp incision, drill hole, and scalp suture without hammering.

[0042] 3. Animal Grouping and Dosing

[0043] 3.1 Grouping

[0044] The mice were divided into sham operation group, model group (TBI group) and drug administration group (TBI+TAX group); specifically, sham operation 1d group, model 1d group, drug administration 1d group, sham operation 3d group, model 3d group, drug administration 3d group, sham operation 7d group, model 7d group, drug administration 7d group, sham operation 14d group, model 14d group, drug administration 14d group, sham operation 28d group, model 28d group, and drug administration 28d group, with 10 mice in each group.

[0045] 3.2 Administration

[0046] Two hours after surgery, taraxasterol was injected intraperitoneally at a dose of 8 mg / (kg·d) and administered on days 1, 3, 7, 14, and 28.

[0047] 4. Behavioral research

[0048] 4.1 Tail-Lift Test: The mouse is suspended by its tail at one meter in the air, and the head and forelimbs are observed for deviation and flexion. A normal mouse is scored as 0 if the head and body vertical axis are at zero angle or ≤10° for a short period of time, and if all four limbs are extended toward the ground. Abnormalities such as forelimb flexion, hindlimb flexion, or head deviation from the vertical axis greater than 10° within 30 seconds are scored as 1.

[0049] 4.2 Walking test: The mice were placed on a large soft cushion and their free walking behavior was observed. Four behaviors were scored as 0-3 points: normal walking, inability to walk in a straight line, circling toward the paretic side, and falling toward the paretic side.

[0050] 4.3 Sensory test Animal behavior scoring was performed according to Kawamata et al., including:

[0051] ① Visual test: The experimenter holds the animal in his hand with its front paws suspended in the air. The experimenter slowly tilts the table 45° from 10 cm above the mouse to the table (the table is in front of the mouse or on the affected side). The normal reaction of the mouse is to immediately grasp the table with its forelimbs (0 points). The injured mouse shows a delayed limb reaction (1 point). If the limb reaction is delayed in any of the three tests (front, left, or right), it is considered a visual test disorder (1 point).

[0052] ② Tactile test: The mouse's head is tilted upwards 45 degrees, with its front paws suspended in the air. At this point, the mouse should be unable to see or touch the table with its whiskers. The mouse should lightly touch the table with the dorsal side of its front paws, with the stimulation depth only reaching the skin and hair. The animal's reaction and scoring are the same as those in the visual test. Tactile stimulation is also divided into frontal and lateral stimulation.

[0053] ③ Proprioception experiment: Place the mouse on the table with its head facing the edge of the table. Gently push the mouse toward the edge of the table from behind. Under normal circumstances, the mouse will grab the edge of the table and the affected limb will fall off. Alternatively, place the mouse on the table with the affected limb close to the edge of the table. When pushing the mouse toward the edge of the table, observe the placement of the forelimb and hindlimb on that side. Normal mice can grab the edge of the table, but the forelimb and hindlimb of the affected mouse cannot.

[0054] 4.4 Balance beam test: The balance beam is 170 cm long and 2 cm wide. It is placed flat on the ground 70 cm above the ground. The mice are observed to see how they balance on the balance beam and scored according to the following scoring criteria:

[0055] 0 points for stable balance posture

[0056] 1 point for holding onto the edge of the balance beam

[0057] Hold the balance beam tightly and let one limb hang down from the balance beam 2 points

[0058] Hold onto the balance beam and drop two limbs from it or rotate on it (>60 seconds) 3 minutes

[0059] 4 points for falling while attempting to balance on a beam (>40 seconds)

[0060] 5 points for falling while attempting to balance on the beam (>20 seconds)

[0061] Fall; No attempt to balance on the beam (<20 seconds) 6 points

[0062] 4.5 Loss of reflexes and abnormal movements

[0063] ① Pinna reflex: Place the mouse on a table and touch the external auditory canal with your hand. Normal mice will shake their heads (0 points), while no shaking of the head is abnormal (1 point).

[0064] ① Corneal reflex: blinking when the cornea is lightly touched with cotton (0 points), and no blinking response is abnormal (1 point);

[0065] ① Startle reflex: Place the mouse on a table and quickly flick a piece of cardboard near its ear that makes a noise. Normal mice will show an escape movement response (0 points), while no escape movement is considered abnormal (1 point).

[0066] ① The presence of any one of the three symptoms of epilepsy, myoclonus, and dystonia is considered abnormal (1 point), and the absence of any of the three symptoms is considered normal (0 point).

[0067] The lowest score is 0 and the highest score is 18, with increasing degrees of neurological dysfunction.

[0068] 5. Nissl staining of brain tissue

[0069] The brains of mice in different groups were removed and dehydrated, and then frozen sections were made. The sections were rinsed with distilled water, placed in a staining agent, and stained in a constant temperature box at 40-60℃ for 25-50 minutes. They were differentiated twice with 95% ethanol and sealed with gum.

[0070] 6. Data statistical analysis

[0071] All statistical analyses were performed using Graphpad Prism 5.0 software. Data are expressed as mean ± SD. All data were analyzed using one-way analysis of variance for multiple comparisons or Student's t-test and nonparametric tests. A p value < 0.05 was considered statistically significant.

[0072] 7. Results

[0073] Figure 2 Effects of taraxasterol administration on balance beam scores in mice with traumatic brain injury within 28 days

[0074] Figure 3 Effects of taraxasterol administration on mNSS neurological function scores in mice with traumatic brain injury within 28 days

[0075] Figure 4 Effects of taraxasterol administration on neuronal damage in mice with traumatic brain injury

[0076] Through behavioral experiments ( Figure 2 、 Figure 3 To assess the motor function and neurological impairment of mice, the TBI model group showed significantly reduced motor function, with neurological function scores ranging from 9 to 11. Continuous administration of taraxasterol for 3-7 days significantly improved the mice's motor function and alleviated neurological impairment.

[0077] The Evans Blue (Evans Blue) assay was used to assess blood-brain barrier permeability in mice. Evans Blue levels were significantly increased in the TBI model group. Continuous administration for 3-7 days demonstrated a time-dependent reduction in Evans Blue levels in the brain tissues of these mice. These results suggest that Evans Blue (Evans Blue) significantly improves blood-brain barrier impairment in TBI-induced mice.

[0078] TBI lesion volume measurements were performed to examine the brain lesion volume in each group of mice. Lesions in the TBI model group were larger, but as the duration of taraxasterol administration increased (3-7 days), the lesion volume in these mice decreased significantly. These results demonstrate that taraxasterol can significantly reduce lesion volume in mice with TBI.

[0079] Immunohistochemistry and flow cytometry were used to detect microglial activation and immune cell infiltration. In the traumatic brain injury model group, microglial activation and a large number of immune cells infiltrated the mice.

[0080] The neuronal morphology was observed by Nissl staining. Figure 4 Mice in the traumatic brain injury model group experienced significant neuronal cell death. Administration of taraxasterol for 3-7 days at a dose of 8 mg / kg per day significantly reduced neuronal death. The present invention also conducted a gradient study, demonstrating that, under safe experimental conditions, daily injection doses of 1-15 mg / kg significantly reduced neuronal death.

[0081] 2. Taraxasterol reduces microglia-mediated neuroinflammation and neuronal damage after traumatic brain injury through CCR2.

[0082] 1. Cell model construction

[0083] BV2 microglial cells were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C and 5% CO 2 . LPS was used to induce an inflammatory phenotype in microglial cells.

[0084] 2. Cell experiment grouping

[0085] BV2 microglia were inoculated into 6-well plates and divided into control group (CON), model group (LPS) and drug-treated group (TAX). The drug-treated group was divided into three doses, namely 10 μg / ml, 25 μg / ml and 50 μg / ml. BV2 cells in the control group were cultured normally without treatment. The model group was treated with LPS for 24 hours, and the drug-treated group was treated with taraxasterol (10-50 μg / ml) for 24 hours.

[0086] 3. Inflammatory factors and CCR2 detection

[0087] After the cells were collected by centrifugation, protein was extracted using a kit and the protein concentration was determined.

[0088] The expression of inflammatory factors and CCR2 was measured, the bands were visualized using an imaging system, and quantitative analysis was performed using ImageJ software.

[0089] 4. Data statistical analysis

[0090] All statistical analyses were performed using Graphpad Prism 5.0 software. Data are expressed as mean ± SD. All data were analyzed using one-way analysis of variance for multiple comparisons or Student's t-test and nonparametric tests. A p value < 0.05 was considered statistically significant.

[0091] 5. Results

[0092] Figure 5-7 Effects of taraxasterol administration on the expression of inflammatory factors in BV2 microglia

[0093] Figure 8 and 9 Effects of taraxasterol 25μg / ml and 50μg / ml on CCR2 protein expression

[0094] The inventors constructed an inflammatory phenotype of BV2 microglia and incubated them with taraxasterol to detect the effect of taraxasterol on reducing the expression level of inflammatory factors. After incubation for 24 hours, taraxasterol at various doses (10-50 μg / ml) effectively reduced the expression level of inflammatory factors ( Figure 5The effect of taraxasterol on neuronal damage was tested by treating HT22 cells with BV2 culture supernatant. Taraxasterol was effectively reduced in HT22 cells after 24 hours of incubation at all doses (10-50 μg / ml). Figure 8 The effect of 10 μg / ml of taraxasterol on CCR2 protein expression was minor and is not shown.

[0095] 3. Preparation of preparations containing taraxasterol

[0096] Example 1: Capsules containing taraxasterol

[0097] 30g taraxasterol, 190g calcium carbonate, 21g starch

[0098] Calcium carbonate and starch are mixed evenly, sieved, mixed evenly with an extract diluted with an appropriate amount of ethanol, passed through a No. 7 sieve, dried at 60-70°C, and filled into capsules to produce 1000 capsules, each capsule containing 30 mg of taraxasterol.

[0099] Example 2: Tablets containing taraxasterol

[0100] Taraxasterol 30g Microcrystalline cellulose 150g Low-substituted hydroxypropyl cellulose 20g Magnesium stearate 5g

[0101] The above materials were passed through a No. 7 sieve, granulated by a wet method, and then compressed into 1000 tablets, each tablet containing 30 mg of taraxasterol.

[0102] Example 3: Injection containing taraxasterol

[0103] Taraxasterol 8g, histidine 0.5g, propylene glycol 20ml, edetate disodium 0.05g, tartaric acid 0.5g, add water for injection to 1000ml.

[0104] Preparation method: In a preparation container, add histidine and propylene glycol to 80% of the prepared injection volume of water, slowly add tartaric acid in batches, stir until completely dissolved, add the pre-prepared disodium edetate solution, stir evenly, adjust the pH value of the solution to 6.0-6.5, add sufficient water for injection, filter with a fused glass funnel and a membrane filter, fill the solution under a nitrogen flow, seal, and finally sterilize with circulating steam at 100°C for 15 minutes.

[0105] Example 4: Injection containing taraxasterol

[0106] Taraxasterol 8g, histidine 0.1g, propylene glycol 10ml, edetate disodium 0.02g, tartaric acid 0.1g, add water for injection to 1000ml.

[0107] Preparation method: In a preparation container, add histidine and propylene glycol to 80% of the prepared injection volume of water, slowly add tartaric acid in batches, stir until completely dissolved, add the pre-prepared disodium edetate solution, stir evenly, adjust the pH value of the solution to 6.0-6.5, add sufficient water for injection, filter with a fused glass funnel and a membrane filter, fill the solution under a nitrogen flow, seal, and finally sterilize with circulating steam at 100°C for 15 minutes.

[0108] Example 5: Injection containing taraxasterol

[0109] Taraxasterol 8g, histidine 1g, propylene glycol 30ml, edetate disodium 0.1g, tartaric acid 1.0g, add water for injection to 1000ml.

[0110] Preparation method: In a preparation container, add histidine and propylene glycol to 80% of the prepared injection volume of water, slowly add tartaric acid in batches, stir until completely dissolved, add the pre-prepared disodium edetate solution, stir evenly, adjust the pH value of the solution to 6.0-6.5, add sufficient water for injection, filter with a fused glass funnel and a membrane filter, fill the solution under a nitrogen flow, seal, and finally sterilize with circulating steam at 100°C for 15 minutes.

[0111] Example 6: Injection containing taraxasterol

[0112] Taraxasterol 8g, propylene glycol 20ml, disodium edetate 0.05g, tartaric acid 0.5g, add water for injection to 1000ml.

[0113] Preparation method: In a preparation container, add propylene glycol to 80% of the prepared injection water, slowly add tartaric acid in batches, stir until completely dissolved, add the pre-prepared disodium edetate solution, stir evenly, adjust the pH value of the solution to 6.0-6.5, add sufficient injection water, filter with a fused glass funnel and a membrane filter, fill the solution under a nitrogen flow, seal, and finally sterilize with circulating steam at 100°C for 15 minutes.

[0114] Example 7: Injection containing taraxasterol

[0115] Taraxasterol 8g, histidine 0.5g, propylene glycol 20ml, disodium edetate 0.05g, hydrochloric acid 50ml, add water for injection to 1000ml.

[0116] Preparation method: In a preparation container, add propylene glycol to 80% of the prepared injection water, slowly add hydrochloric acid in portions, stir to completely dissolve, add the pre-prepared edetate disodium solution, stir evenly, adjust the pH value of the solution to 6.0-6.5, add sufficient injection water, filter with a fused glass funnel and a membrane filter, fill the solution under a nitrogen flow, seal, and finally sterilize with circulating steam at 100°C for 15 minutes.

[0117] Example 8: Injection containing taraxasterol

[0118] Taraxasterol 8g, histidine 0.5g, benzyl alcohol 20ml, edetate disodium 0.05g, tartaric acid 0.5g, add water for injection to 1000ml.

[0119] Preparation method: In a preparation container, add histidine and benzyl alcohol to 80% of the prepared injection volume of water, slowly add tartaric acid in portions, stir until completely dissolved, add the pre-prepared disodium edetate solution, stir evenly, adjust the pH value of the solution to 6.0-6.5, add sufficient water for injection, filter with a fused glass funnel and a membrane filter, fill the solution under a nitrogen flow, seal, and finally sterilize with circulating steam at 100°C for 15 minutes.

[0120] Example 9: Injection containing taraxasterol

[0121] Taraxasterol 8g, histidine 0.05g, propylene glycol 40ml, edetate disodium 1.02g, tartaric acid 0.05g, add water for injection to 1000ml.

[0122] Preparation method: In a preparation container, add histidine and propylene glycol to 80% of the prepared injection volume of water, slowly add tartaric acid in batches, stir until completely dissolved, add the pre-prepared disodium edetate solution, stir evenly, adjust the pH value of the solution to 6.0-6.5, add sufficient water for injection, filter with a fused glass funnel and a membrane filter, fill the solution under a nitrogen flow, seal, and finally sterilize with circulating steam at 100°C for 15 minutes.

[0123] Example 10: Injection containing taraxasterol

[0124] Taraxasterol 8g, histidine 2g, propylene glycol 30ml, edetate disodium 0.5g, tartaric acid 2.0g, add water for injection to 1000ml.

[0125] Preparation method: In a preparation container, add histidine and propylene glycol to 80% of the prepared injection volume of water, slowly add tartaric acid in batches, stir until completely dissolved, add the pre-prepared disodium edetate solution, stir evenly, adjust the pH value of the solution to 6.0-6.5, add sufficient water for injection, filter with a fused glass funnel and a membrane filter, fill the solution under a nitrogen flow, seal, and finally sterilize with circulating steam at 100°C for 15 minutes.

[0126] Physical and chemical properties detection: The content of taraxasterol was determined according to the high performance liquid chromatography method (General Rule 0512).

[0127] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as the filler; methanol-water-glacial acetic acid (98:2:0.15) was used as the mobile phase; the detection wavelength was 287 nm. The theoretical plate number calculated based on the taraxasterol peak should be no less than 5000.

[0128] Preparation of reference solution: Take an appropriate amount of taraxasterol reference substance, accurately weigh it, and add methanol to make a solution containing 40 μg per 1 ml as the reference solution.

[0129] Preparation of the test solution: Take 1 ml of the liquid of Example 3-10 of the present invention, dilute it to 10 ml with methanol, mix well, and then draw 1 ml. Place it in a stoppered conical flask, accurately add methanol to 25 ml, take it out, cool it, weigh it again, add methanol to make up the lost weight, filter it, and take the filtrate to obtain the product.

[0130] Determination method: Accurately aspirate 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0131] Accelerated test conditions: temperature (40 ± 2) ° C, relative humidity (75 ± 5)%, duration of 6 months. During the test, samples were taken at the end of the 0th, 1st, 2nd, 3rd, and 6th month to examine taraxasterol, dissolution time, and appearance. The results are shown in Table 1 and Figure 8 .

[0132] Table 1 Test results of Examples 3-10 at the end of 0, 1, 2, 3, and 6 months

[0133]

[0134] For other preparations containing taraxasterol, there was not much change in the active substance content before and after accelerated testing.

[0135] The above embodiments are intended only to illustrate the technical features of the present invention and to enable those skilled in the art to understand the present invention and implement it accordingly. They are not intended to limit the scope of protection of the present invention. Any equivalent changes or modifications made based on the spirit of the present invention are intended to be included within the scope of protection of the present invention.

Claims

1. Use of taraxasterol in the preparation of drugs for preventing and / or treating traumatic brain injury.

2. The use according to claim 1, characterized in that The traumatic brain injury is selected from any one of concussion, closed head injury or open head injury.

3. The use according to claim 2, characterized in that The traumatic brain injury includes neuroinflammation, neuronal damage and neurological function damage caused by traumatic brain injury.

4. The use according to claim 2, characterized in that: The dosage of taraxasterol is 1-15 mg / kg.

5. The use according to claim 2, characterized in that: The dosage of taraxasterol is 8 mg / kg.

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