Application of taraxasterol or taraxasterol salt in preparation of medicine for preventing or treating septic brain injury

Abstract

The invention relates to the field of medicines, in particular to taraxasterol and application of taraxasterol salt in preparation of drugs for preventing and / or treating septic encephalopathy. According to the taraxasterol disclosed by the invention, the movement ability of a balance beam experiment in neurological function scoring of a brain trauma mouse can be improved, and the neurological function scoring including a movement experiment and a sensory experiment can be reduced; the traditional Chinese medicine composition can reduce the permeability of Evans blue in brain tissues, improve the short-term lesion volume after sepsis brain injury, relieve encephaledema and inhibit microglial cell activation and immune cell infiltration. Cell experiments prove that the taraxasterol can reduce neuroinflammation mediated by microglial cells, reduce expression of inflammatory factors such as TNF-alpha (tumor necrosis factor-alpha), IL-6 (interleukin-6) and IL-1beta (interleukin-1beta), and reduce neuron damage.

Description

Technical Field

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

[0002] Sepsis-associated encephalopathy (SAE) is a serious central nervous system complication in patients with sepsis, which refers to diffuse or multifocal brain dysfunction caused by systemic infection without clinical or laboratory evidence of direct infection. SAE ranges from delirium to deep coma, characterized by changes in behavior, cognition, arousal, and consciousness, and a considerable proportion of patients have long-term cognitive dysfunction.

[0003] 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 mainly secreted by immune cells, and its expression can be either persistent or inducible. A variety of mediators can induce the expression of CCL2, such as IL-1, IL-4, IL-6, and TNF-a. CCL2 can regulate the migration and infiltration of a variety of immune cells, including monocytes, macrophages, memory T lymphocytes, and natural killer cells. CC motif receptor 2 (CCR2) is considered to be the main receptor of CCL2. By binding to CCL2, CCR2 is activated, which subsequently initiates the transcription and expression of IL-1, IL-6, and TNF genes. After CCL2 triggers CCR2, it will start a variety of signaling pathways, such as NLRP3, JAK / STAT, PI3K / MAPKs, NF-κB, etc., which are involved in cytokine production, inflammation, cell survival, migration and apoptosis. After septic encephalopathy occurs, CCL2 expression is upregulated and CCR2 is activated to induce microglial M1 polarization and promote the production of inflammatory factors such as IL-1β, IL-6, and TNFα. In addition, immune cells are mobilized to the site of inflammation in a CCR2-dependent manner, and under the action of CCR2, they are recruited to the site of inflammation to participate in inflammatory response and neuronal damage. In addition, after CCL2 binds to CCR2, it increases neuronal oxidative stress and mitochondrial dysfunction, ultimately leading to neuronal cell death.

[0004] The activation of these signaling pathways will mobilize a variety of transcription factors and genes, and participate in the production of cytokines, cell growth and differentiation, cell survival, migration and apoptosis, angiogenesis and inflammation. CCL2 has a potential role in regulating neurons. After septic encephalopathy, CCL2 expression increases, multiple pathways are activated, and multiple inflammatory factors are produced, causing patients to have cytokine neurological dysfunction.

[0005] Taraxasterol (TAX), 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 C30H50O, and its molecular weight and melting point are 426.72 g / mol and 221-222 degrees Celsius, respectively. Taraxasterol has been shown to have significant preventive and therapeutic effects in animal or cell models of various diseases such as liver injury, gastritis, colitis, arthritis, pneumonia, tumors, and immune system diseases. Taraxasterol can protect C6 glial cells from lipopolysaccharide-induced neuroinflammation and reduce the levels of proinflammatory cytokines. Taraxasterol can inhibit the production of ROS in hippocampal neurons induced by oxygen-glucose deprivation / reperfusion and alleviate oxygen-glucose deprivation / reperfusion-induced oxidative stress. The present invention found that taraxasterol can improve the motor ability of the balance beam experiment in the neurological function score of mice with septic encephalopathy, improve the neurological function scores including motor experiments and sensory experiments, improve the permeability of the blood-brain barrier, reduce the degree of brain edema after septic encephalopathy, and inhibit microglia activation and immune cell infiltration; in cell experiments, taraxasterol reduces microglia-mediated neuroinflammation, reduces the expression of inflammatory factors such as TNF-α, IL-6, IL-10, and IL-1β, and reduces neuronal damage. At the same time, taraxasterol can also reduce the level of neuronal oxidative stress and mitochondrial damage, and improve mitochondrial dysfunction. Taraxasterol has good application prospects in treating neuroinflammation, neuronal damage and restoring patients' neurological function after septic brain injury. 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 septic brain injury, so as to solve the problem that the treatment effect of septic brain injury in the prior art is unsatisfactory and the prognosis effect is poor.

[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 septic brain injury.

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

[0010] The septic encephalopathy described in the present invention is caused by any one of bacterial infection, traumatic brain injury or other infections. The septic encephalopathy includes brain, epilepsy and central nervous system diseases caused by septic encephalopathy.

[0011] Furthermore, the present invention also provides a pharmaceutical preparation, which comprises taraxasterol or its salt, 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 liquids, injections, transdermal patches, inhalants, powder injections, water or liposomes.

[0012] The pharmaceutically acceptable carrier includes but is not limited to fillers, disintegrants, promoters, lubricants, binders, and regulators. Among them, the binder is selected from 50% ethanol solution of 3% hydroxypropyl methylcellulose, 10% aqueous solution of povidone K30, 30% starch slurry and 10% aqueous solution of carboxymethyl cellulose, preferably 50% ethanol solution of 3% hydroxypropyl methylcellulose. 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 stearic acid, glycerol, palmitic acid stearate, magnesium stearate, calcium stearate, talc, micro-powdered silica gel, hydrogenated vegetable oil, sodium lauryl sulfate; the disintegrant is selected from hydroxypropyl cellulose, cross-linked polyvinyl pyrrolidone, cross-linked sodium carboxymethyl cellulose, cellulose acetate phthalate, sodium carboxymethyl starch; the promoter contains fatty acids, including but not limited to butyric acid, caproic acid, caprylic acid, nonanoic acid, caprylic acid, lauric acid, nutmeg, palm, stearic acid, peanut, oleic acid, linoleic acid, linolenic acid, salts thereof and derivatives thereof. In some specific examples, the absorption promoter contains glycerides in fatty acids, including but not limited to butyric acid, caproic acid, caprylic acid, caprylic acid, lauric acid, nutmeg, palm, stearic acid, peanut, oleic acid, linoleic acid, linolenic acid, salts thereof, derivatives thereof and combinations thereof. The glyceride can be monoglyceride, diglyceride or 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 dodecyl sulfate, polyethylene ethers or esters, polyethylene glycol-12 alkyl ether, salicylate, polysorbate 80, nonylphenoxy polyoxyethylene, sodium octyl sulfosuccinate, saponin, palmitoyl carnitine, lauroyl acetyl carnitine, lauroyl maltoside, acyl carnitine, alkanoyl choline. 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.

[0013] The pharmaceutical compositions and formulations of the present invention can be used by any route capable of delivering to the brain. Examples of routes of administration include, but are not limited to, intravenous, intranasal, oral, topical, scalp, or by inhalation.

[0014] Further, according to the pharmaceutical composition of the present invention, for the pharmaceutical composition for oral administration, it is preferred that the therapeutically effective amount of one or more compounds of the present invention is mixed with a pharmaceutically acceptable carrier according to conventional quantitative drug 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 a pharmaceutical composition of an oral dosage form, any conventional 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, flavoring agents, preservatives, coloring agents 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, adhesives, 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 compounds in patients.

[0015] For parenteral preparations, the carrier generally includes sterile water or sodium chloride aqueous 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 maintained, the composition and carrier must also be sterilized. It may also be prepared as an injection suspension, in which case suitable liquid carriers, suspending agents, etc. may also be used.

[0016] Furthermore, the taraxasterol described in the present invention can be a promoter for use in the induction of M2 polarization of microglia in septic brain injury. M2 polarization is an immunoregulatory state that helps to reduce inflammatory responses and promote tissue repair. The inventors of the present application have found that taraxasterol has a significant effect on promoting the polarization of microglia M2, which can effectively promote the repair of various nerve injuries.

[0017] The fifth aspect of the technical solution of the present invention is the application of taraxasterol in the preparation of drugs for preventing or treating septic brain injury. Its action site is CCR2 (chemokine receptor family). Microglia are a type of immune cells in the central nervous system, 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. CCR2 is a chemokine receptor, which also plays an important role in the polarization process of microglia M1 / M2. Studies have shown that the CCL2-CCR2 signaling pathway can regulate the polarization state of microglia and affect its transformation to M2 type. Specifically, the CCL2-CCR2 signal affects the metabolic state of microglia and the expression of M1 / M2 related genes by regulating the intracellular signal transduction pathway, thereby promoting the polarization of microglia to different inflammatory phenotypes.

[0018] The taraxasterol and its salts described in the present invention have been subjected to relevant 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.

[0019] Furthermore, the dosage can be administered once or twice a day. When administered once a day, 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 a day, 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.

[0020] 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.

[0021] Compared with the existing technology, the beneficial technical effects are:

[0022] 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 septic encephalopathy, reduce the neurological function scores including motor experiments and sensory experiments; reduce the permeability of Evans blue in brain tissue, improve the short-term lesion volume after septic brain injury, reduce brain edema, and inhibit microglia activation and immune cell infiltration. Cell experiments have confirmed that the taraxasterol of the present invention can reduce microglia-mediated neuroinflammation, reduce the expression of inflammatory factors such as TNF-α, IL-6, and IL-1β, and reduce neuronal damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Effects of different administration times of taraxasterol on the balance beam scores of mice with septic encephalopathy. As can be seen from the figure, there is a significant difference between the comprehensive doses of the model control group and the drug-treated group (P<0.05), indicating that taraxasterol has an improving effect on the motor ability of mice with septic encephalopathy.

[0024] Figure 2 The effect of different administration times of taraxasterol on the mNSS neurological function score of mice with septic encephalopathy. As can be seen from the figure, there is a significant difference between the comprehensive doses of the model control group and the drug-treated group (P<0.05), indicating that taraxasterol has an improving effect on the nervous system of mice with septic encephalopathy.

[0025] Figure 3 Effect of taraxasterol incubation on the release of inflammatory factors, where (a) represents the comparison of IL-1β expression levels among the sham operation group, control group and drug treatment group; (b) represents the comparison of TNF-α expression levels among the sham operation group, control group and drug treatment group; (c) represents the comparison of IL-6 expression levels among the sham operation group, control group and drug treatment group; It can be seen that there is a significant difference between the control group and the drug treatment group (P<0.05).

[0026] Figure 4 Effects of taraxasterol administration on neuronal damage in mice with septic encephalopathy (HE staining and Nissl staining). (a) Red is HE staining: the structure of neurons in the hippocampus of the sepsis group was disordered, with swollen vacuoles, which decreased after TAX administration; (b) Purple is Nissl staining: the number of Nissl bodies in the hippocampus of the model group decreased, indicating neuronal damage. TAX administration increased the number of Nissl bodies, suggesting that it played a neuroprotective role. A large number of neurons in the mice in the septic encephalopathy model group died.

[0027] Figure 5 Effects of taraxasterol administration on brain water content in mice with septic encephalopathy.

[0028] Figure 6 Effect of taraxasterol administration on 28-day survival rate of mice with septic encephalopathy.

[0029] Figure 7 Effects of taraxasterol administration on blood-brain barrier permeability in mice with septic encephalopathy.

[0030] Figure 8 Effects of taraxasterol administration on mitochondrial membrane potential in mice with septic encephalopathy.

[0031] Fig. 9 Effects of taraxasterol administration on ATP in mice with septic encephalopathy.

[0032] Fig.10 Effects of taraxasterol administration on mitochondrial oxidative stress in mice with septic encephalopathy.

[0033] Fig.11 Effects of taraxasterol administration on neuronal viability in mice with septic encephalopathy.

[0034] Fig.12 Effects of taraxasterol administration on CCR2 expression in mice with septic encephalopathy.

[0035] Fig.13 Changes in the taraxasterol content in Examples 3-11 at 0th month, 1st month, 2nd month, and 6th month. DETAILED DESCRIPTION

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

[0037] Example 1: Brain protective effect of taraxasterol on mice with septic brain injury

[0038] 1. Experimental Animals

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

[0040] 2. Preparation of Septic Encephalopathy Model

[0041] After the mouse was anesthetized with isoflurane, it was fixed in a supine position on the operating table, and the abdominal surgical area was prepared and disinfected. A vertical incision of about 1 cm was made along the linea alba at 1-1.5 cm below the xiphoid process, and the mesentery and cecum were freed under sterile conditions. The intestinal tract was ligated about 0.8 cm from the end of the cecum, and a needle was used to puncture twice to squeeze out a small amount of feces, and the cecum was wiped dry and returned, and the muscles and skin were sutured.

[0042] 3. Animal Grouping and Dosing

[0043] The mice were divided into: sham operation group, model control group (TBI group) and drug administration group (TBI+TAX group). The control group was the model control group; specifically, sham operation 1d group, TBI1d group, drug administration 1d group, sham operation 3d group, TBI3d group, drug administration 3d group, sham operation 7d group, TBI7d group, drug administration 7d group, 10 mice in each group. Two hours after surgery, taraxasterol was injected intraperitoneally at a dosage concentration of 10μg / mL, 25μg / mL, and 50μg / mL. The drugs were divided into groups according to time and administered for 1, 3, and 7 days, respectively. Among them, TBI was septic encephalopathy, the control group was septic encephalopathy group, and the drug administration group was administered on the basis of septic encephalopathy.

[0044] 4. Behavioral research

[0045] 4.1. Tail-lifting test: The mouse is suspended by its tail for one meter in the air, and the deviation and flexion of the head and forelimbs and hindlimbs are observed. Normally, if the head of a mouse has no angle with the vertical axis of the body for a short period of time or the angle is ≤10°, and the limbs are stretched toward the ground, the score is 0. If the forelimbs are flexed, the hind limbs are flexed, and the head deviates from the vertical axis by more than 10° within 30 seconds, it is abnormal and is scored 1 point respectively;

[0046] 4.2 Walking test: The mice were placed on a large soft cushion and their free walking behavior was observed. Four performances, namely, normal walking, inability to walk in a straight line, turning in circles toward the paretic side, and falling toward the paretic side, were scored 0-3 points respectively.

[0047] 4.3. Sensory test Animal behavior scoring was performed according to Kawamata et al., including:

[0048] ① Visual experiment: The experimenter holds the animal in his hand with its front paws suspended in the air, and slowly tilts the mouse 45 degrees from 10 cm above the table to the table (the table is in front of the mouse or on the affected side). The normal reaction of the mouse is that the forelimbs immediately grab the table (0 points), while the injured mouse shows delayed limb reaction (1 point). If the limb reaction is delayed in any of the three tests in front, left or right, it is regarded as visual experiment disorder (recorded 1 point);

[0049] ②Tactile experiment: lift the mouse head up 45 degrees, with the front paws hanging in the air. At this time, the mouse should be unable to see and cannot touch the table with its whiskers. Use the back of its front paws to touch the table. The stimulation depth only reaches the skin and hair. The animal reaction and scoring are the same as the visual experiment. Tactile stimulation is also divided into front and side stimulation.

[0050] 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 front and rear limbs on that side. Normal mice can grab the edge of the table, while the front and rear limbs of mice on the affected side cannot.

[0051] 4.4 Balance beam test: The balance beam is 170 cm long and 2 cm wide. It is placed flat at 70 cm from the ground. The mice are observed to balance on the balance beam and scored according to the scoring criteria:

[0052]

[0053]

[0054] 4.5. Loss of reflexes and abnormal movements

[0055] ① Auricular reflex: Place the mouse on the 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);

[0056] ① Corneal reflex: blinking when the cornea is lightly touched with cotton thread (0 points), and no blinking response is abnormal (1 point);

[0057] 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 reaction (0 points), while no escape movement is abnormal (1 point);

[0058] ① The presence of any one of the three abnormal symptoms of epilepsy, myoclonus, and dystonia is considered abnormal (1 point), and the absence of any of the three is considered normal (0 point).

[0059] The lowest score is 0 and the highest score is 18, with increasing degrees of neurological dysfunction.

[0060] 5. Blood-brain barrier permeability assay

[0061] The mice were injected with 3 ml / kg of 4% Evans blue solution through the tail vein. After 2 hours of anesthesia, the heart was perfused with normal saline, the brain was removed by decapitation, and the cerebral hemispheres were quickly separated on ice, weighed, and placed in 1 ml of formamide solution. The solution was incubated in a 45°C constant temperature incubator for 48 hours, and the absorbance was measured with an enzyme marker (wavelength 632 nm). The concentration of Evans blue in the solution was calculated according to the linear regression equation.

[0062] 6. Mitochondrial Function Assay

[0063] Fluorescent dye probe method combined with flow cytometry was used to detect mitochondrial membrane potential. MitoSOX fluorescent probe method was used to detect ROS. High performance liquid chromatography was used to determine mitochondrial ATP. Electrochemical analysis was used to detect mitochondrial calcium ions.

[0064] 7. Nissl staining of brain tissue

[0065] The brains of mice in different groups were taken out and dehydrated, and then the processed mouse brain tissues were frozen and sliced. The slices were washed with distilled water, placed in a staining agent, and stained in a constant temperature box at 40-60℃ for 25-50 minutes, differentiated twice with 95% ethanol, and sealed with gum.

[0066] 8. Statistical analysis

[0067] All data were analyzed blindly using Prism software. The data of each group are expressed as mean±SD in vivo and mean±SEM in vitro. All data were analyzed by one-way ANOVA for multiple comparisons or Student's t test and nonparametric test. Differences were considered statistically significant at the p<0.05 level.

[0068] 9. Results

[0069] The inventors conducted behavioral experiments ( Figure 1 , Figure 2 ) to detect the motor ability and neurological function damage of mice. The motor ability of mice in the septic encephalopathy model group (model control group) was significantly weakened, and the neurological function score was in the range of 9-11 points. After continuous administration for 3-7 days, the comprehensive score analysis of the taraxasterol (administration group) group showed that the administration group could significantly improve the motor ability of mice, reduce neurological function damage, and reduce the degree of brain edema every day ( Figure 5 ), improve the survival rate of mice ( Figure 6 ).

[0070] The inventors tested the permeability of the blood-brain barrier of mice by the Evans blue experiment. The Evans blue content of mice in the septic encephalopathy model group increased significantly. After continuous administration for 3-7 days, taraxasterol can time-dependently reduce the Evans blue content in the brain tissue of mice in the septic encephalopathy model group. The above results show that taraxasterol can significantly improve the damage of the blood-brain barrier in mice with septic encephalopathy. ( Figure 7 )

[0071] The inventors used a fluorescent dye probe method combined with flow cytometry to detect mitochondrial membrane potential. MitoSOX fluorescent probe method was used to detect ROS. High performance liquid chromatography was used to determine mitochondrial ATP. Electrochemical analysis was used to detect mitochondrial calcium ions. The mitochondrial dysfunction in mice with septic encephalopathy was more obvious as the administration time of taraxasterol increased (3-7 days). The above results show that taraxasterol can significantly improve the mitochondrial dysfunction in mice with septic encephalopathy ( Figure 8-10 ).

[0072] The inventors detected the level of inflammatory factors by ELISA. Microglia were activated in mice in the septic encephalopathy model group, and a large number of inflammatory factors were released. The administration of taraxasterol for 3-7 days and each dose group could significantly reduce the activation of microglia and the level of inflammatory factors ( Figure 3 ).

[0073] The inventors observed the neuronal morphology by Nissl staining ( Figure 4 ). Red is HE staining: the structure of neurons in the hippocampus of the sepsis group is disordered, and swollen vacuoles appear, which are reduced after TAX administration; purple is Nissl staining: the Nissl bodies of neurons in the hippocampus of the model group are reduced, indicating neuronal damage. TAX administration increases the number of Nissl bodies, indicating that a large number of neurons in the septic encephalopathy model group of mice die due to neuroprotection. The administration of taraxasterol for 3-7 days at a dose concentration of 25μg / mL per day can significantly reduce neuronal death. At the same time, in order to verify the dose range, the test dose concentrations of 10μg / mL and 50μg / mL per day can achieve the same or similar technical effects.

[0074] Example 2. Taraxasterol reduces microglia-mediated neuroinflammation and neuronal damage after septic encephalopathy via CCR2.

[0075] 1. Cell model construction

[0076] BV2 microglia 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 the inflammatory phenotype of microglia.

[0077] 2. Cell Experiment Grouping

[0078] BV2 microglia were inoculated into 6-well plates and divided into a control group, a model group (septic encephalopathy group) and a drug-treated group (septic encephalopathy + TAX). TAX refers to the group given taraxasterol. The BV2 cells in the control group were cultured normally without treatment. The model group was treated with LPS. The drug-treated group was treated with 10 μg / mL, 25 μg / mL, and 50 μg / mL of taraxasterol for 24 hours.

[0079] 3. Neuronal Damage Detection

[0080] Normally cultured HT22 hippocampal neurons were inoculated into 6-well plates and treated with BV-2 cells, culture supernatants of the model group, and culture supernatants of the drug-treated group for 72 hours. CCK8 was used to detect cell viability.

[0081] 4. CCR2 detection

[0082] After BV-2 cells were collected by centrifugation, proteins were extracted using a kit and the protein concentration was determined. Western blot was used to detect CCR2 expression, and the bands were visualized using an imaging system and quantitatively analyzed using ImageJ software.

[0083] 5. Statistical analysis

[0084] All data were analyzed blindly using Prism software. The data of each group are expressed as mean±SD in vivo and mean±SEM in vitro. All data were analyzed by one-way ANOVA for multiple comparisons or Student's t test and nonparametric test. Differences were considered statistically significant at the p<0.05 level.

[0085] 6. Results

[0086] Fig.11 Effects of taraxasterol administration on the survival of co-cultured neurons

[0087] The inventors constructed the inflammatory phenotype of BV2 microglia and incubated HT22 hippocampal neuron cells with the supernatant to detect the effect of taraxasterol in reducing inflammatory damage. After incubation for 24 hours, taraxasterol at each dose (10 μg / mL, 25 μg / mL, 50 μg / mL) effectively increased neuronal survival ( Fig.11 ).

[0088] Fig.12 Effects of taraxasterol administration on the expression level of CCR2 in microglia

[0089] The inventors constructed the inflammatory phenotype of BV2 microglia and incubated them with taraxasterol to detect the effect of taraxasterol on the expression level of CCR2 in microglia. After incubation with taraxasterol for 24 hours, all doses (10 μg / mL, 25 μg / mL, 50 μg / mL) of taraxasterol effectively reduced the expression level of CCR2.

[0090] 3. Preparation of preparations containing taraxasterol

[0091] Example 1: Capsules containing taraxasterol

[0092] 30g taraxasterol, 190g calcium carbonate, 21g dry starch

[0093] Calcium carbonate and dry starch are mixed evenly, sieved, mixed with an extract diluted with an appropriate amount of ethanol, sieved through a No. 7 sieve, dried at 60-70°C, and capsuled to produce 1000 capsules, each containing 30 mg of taraxasterol.

[0094] Example 2: Tablet containing taraxasterol

[0095] Taraxasterol 30g Microcrystalline cellulose 150g Low-substituted hydroxypropyl cellulose 20g Magnesium stearate 5g

[0096] The above materials were passed through a No. 7 sieve, granulated by a wet method, and then compressed into 1000 tablets, each containing 30 mg of taraxasterol.

[0097] Example 3: Lyophilized powder containing taraxasterol

[0098]

[0099] The preparation process is:

[0100] (1) Weigh mannitol, add 1 / 2 amount of water for injection, stir to dissolve, add tert-butyl alcohol, stir to dissolve, then add taraxasterol and stir to completely dissolve the main drug;

[0101] (2) Add an appropriate amount of activated carbon and stir for 15 to 30 minutes to remove carbon;

[0102] (3) Add water for injection to the full amount, adjust the pH value to 6.0-6.5 with citric acid, and perform nitrogen protection during the preparation process;

[0103] (4) Filling: Fill the drug solution into vials and freeze-dry;

[0104] (5) Freeze-drying curve: Freeze-drying curve: After the filled product is put into the box, it is cooled to -35°C for pre-freezing and kept for 2.5 hours; the temperature is raised to -5°C and kept for 1 hour, and then the temperature is lowered to -20°C and kept for 2.5 hours; the cold trap is opened and the vacuum is opened; the temperature of the partition is raised to -11°C and kept for 12 hours; the temperature of the partition is raised to 2°C and kept for 5 hours; the temperature of the partition is raised to 38°C and kept for 1 hour; after the freeze-drying is completed, nitrogen is filled under vacuum, the stopper is pressed, and the product is taken out of the pot;

[0105] (6) Seal: Seal the freeze-dried ampoule. The ambient humidity during the sealing process is 28% relative humidity.

[0106] Example 4: Lyophilized powder containing taraxasterol

[0107]

[0108] The preparation process is the same as that of Example 3.

[0109] Example 5: Lyophilized powder containing taraxasterol

[0110]

[0111] The preparation process is the same as that of Example 3.

[0112] Example 6: Lyophilized powder containing taraxasterol

[0113]

[0114] The preparation process is the same as that of Example 3.

[0115] Example 7: Lyophilized powder containing taraxasterol

[0116]

[0117] The preparation process is the same as that of Example 3.

[0118] Example 8: Lyophilized powder containing taraxasterol

[0119]

[0120]

[0121] The preparation process is the same as that of Example 3.

[0122] Example 9: Lyophilized powder containing taraxasterol

[0123]

[0124] The preparation process is the same as that of Example 3.

[0125] Example 10: Lyophilized powder containing taraxasterol

[0126]

[0127] The preparation process is the same as that of Example 3.

[0128] Example 11: Lyophilized powder containing taraxasterol

[0129]

[0130] The preparation process is the same as that of Example 3.

[0131] Physical and chemical property detection: The taraxasterol content was determined according to the high performance liquid chromatography method (General Rule 0512).

[0132] Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel is used as filler; methanol-water-glacial acetic acid (98:2:0.15) is used as mobile phase; detection wavelength is 287nm. The theoretical plate number calculated based on the taraxasterol peak should be no less than 5000.

[0133] Preparation of reference solution: Take an appropriate amount of taraxasterol reference substance, weigh it accurately, and add methanol to make a solution containing 40 μg per 1 ml as the reference solution.

[0134] Preparation of the test solution: Take the lyophilized powder of 3-11 of the present invention, dilute it to 10 ml with methanol, pipette 1 ml evenly, put 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 solution.

[0135] Determination method: Accurately pipette 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0136] 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 months to examine taraxasterol, dissolution time, and appearance. The results are shown in Table 1 and Figure 8 .

[0137] Table 1 Test results of Examples 3-10 at the end of 0, 1, 2, 3, and 6 months

[0138]

[0139]

[0140] For other preparations containing taraxasterol, there was not much change in the content of active substances before and after accelerated testing.

[0141] The above embodiments are only for illustrating the technical features of the present invention, and their purpose is to enable people familiar with this field to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A pharmaceutical preparation containing taraxasterol and its salt, characterized in that: The pharmaceutical preparation is used in the preparation of a drug for preventing and / or treating septic encephalopathy, and the pharmaceutical preparation may further contain a pharmaceutically acceptable carrier.

2. The pharmaceutical preparation according to claim 1, characterized in that The pharmaceutically acceptable carrier includes, but is not limited to, a filler, a disintegrant, a promoter, a lubricant, a binder, and a regulator.

3. The pharmaceutical preparation according to claim 1, characterized in that The dosage form of the preparation is selected from freeze-dried powder, which is made of the following components by weight:

4. The pharmaceutical preparation according to claim 3, characterized in that: The drug preparation has an action site of CCR2.

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